RU2810819C2 - Device and system for analyzing sample, in particular blood, as well as methods of their use - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: group of inventions relates to the field of sampling, detection, analysis in biochemical research and options of their use. The following is presented: a device for analyzing an analyte in a sample comprising a first plate and a second plate moved relative to each other in different configurations. One or both plates are flexible, on the corresponding surface of each plate there is a contact area with the sample containing the analyte. The plates contain spacers fixed to a corresponding plate, wherein the spacers have a column shape, a flat top surface, a predetermined uniform height and a predetermined constant distance between the spacers. At least one of the separators is located inside the area of contact with the sample. The fourth power of the interspacer distance (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate, ISD⁴/(hE), is equal to or less than 5x10⁶ mcm³/GPa; and the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is between 60 and 750 GPa-mcm. One configuration is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is applied either to one or both plates. Another configuration is a closed configuration, which is configured after application of the sample in an open configuration. At least part of the sample is compressed by two plates into a layer of uniform thickness and is motionless with respect to the plates. The uniform layer thickness is limited to the inner surfaces of the two plates and is controlled by the plates and spacers and has an average thickness equal to or less than 200 mcm. The following is also presented: a system for accelerated analysis of a sample using a mobile communication device, a method of accelerated analysis of an analyte in a sample, a method of accelerated analysis of an analyte in a sample using a mobile communication device, and a method of analyzing a sample.

EFFECT: simplification, acceleration and increased reliability of analysis with high sensitivity are achieved.

154 cl, 2 tbl, 34 dwg

Description

This application claims priority to provisional applications 62/218455 filed September 14, 2015, 62/293188 filed February 9, 2016, 62/305123, filed March 8, 2016, 62/369181, filed July 31, 2016, PCT application serial number PCT/US16/46437 filed August 10, 2016, which PCT application claims priority to provisional applications serial number 62/202989 filed August 10, 2015 ., 62/218455, filed September 14, 2015, 62/293188, filed February 9, 2016, 62/305123, filed March 8, 2016, and 62/369181, filed July 31, 2016, all applications are incorporated herein in their entirety for all purposes.

Field of invention

The present invention relates to the field of sampling, detection, analysis in biochemical research and options for their application.

Prior Art

When analyzing samples in biology/chemistry, in particular blood, there is a need for methods and devices that can speed up the process (for example, binding, mixing reagents, etc.) and quantitatively measure parameters (for example, the concentration of analytes, sample volume and etc.), which can simplify sampling and measurement processes, which can use small sample volumes, which can complete the entire analysis in less than a minute, which can be used to perform the analysis using a smartphone (e.g. mobile phone) that allow a non-professional to perform the analysis themselves and that allow test results to be transmitted locally, remotely or wirelessly to various interested parties. The present invention relates to methods, devices and systems that can meet these needs.

Brief description of the invention

The following brief description is not intended to include all features and aspects of the present invention. The present invention relates to methods, devices and systems that make detection in biology/chemistry (including, but not limited to, immunoassays, nucleic acid assays, electrolyte assays, etc.) faster, more sensitive, provide fewer steps, more ease of implementation, fewer samples required, contribute to less or reduced need (or lack thereof) for professional assistance and/or lower cost compared to many current detection methods and devices.

The goal of many modern laboratory tests is to accurately determine the absolute concentration of an analyte in a sample. For example, a red blood cell (RBC) test involves counting the number of red blood cells in a specified amount of whole blood and then calculating the number of red blood cells per microliter of whole blood. However, such measurements may be difficult to perform without the use of a specialized diagnostic facility (e.g., in a “home,” “pharmacy,” or “point-of-care” setting) because such tests often require a specialized instrument and/or a precision measuring device, which is capable of accurately measuring a relatively small volume (such as a precision pipette or the like) of biological fluid.

Measuring the right volume

Many assays determine the absolute concentration of an analyte in a sample. However, the results of such analyzes become quite inaccurate if only a small volume is analyzed (particularly, for example, from 100 nl to 10 μl). This is because small volumes are difficult to dose and/or measure accurately.

In some assays, a liquid sample can be placed between two plates that are separated by spacers and analyzed. Theoretically, the volume of the analyte can be calculated by multiplying the area of the sample being analyzed by the thickness of the sample being analyzed. However, in practice such estimates are not easy to perform and are quite inaccurate for a number of reasons. As an example, some devices use granules to separate the plates, and either the granules or one of the plates is deformable. Such devices may be prone to inaccuracy for the following reasons.

- Spherical spacers have a much smaller contact area (almost a point) with the plates. In such devices, due to the much smaller contact area for each unit to which a compressive force is applied, much greater pressure is applied to the contact area from both the plate and sphere sides. This greater pressure causes the spheres and/or plates (if they are flexible) to deform, distorting any measurements.

- Spherical spacers usually end up being evenly distributed between two plates. Because the spherical spacers are uniformly distributed, the distances between the spacers can vary significantly and some of the distances are quite large. This causes the spacers and/or plates (if flexible) to deform to a significantly greater extent in some areas than others, which also skews the results.

- Randomly placed separators that are close to each other can become obstructions that block the movement of analytes (eg, cells), thereby potentially leading to the formation of analyte or cell “clumps” that can cause even more complications.

- Significant deformation of one of the plates can lead to cell lysis, which can cause errors in cell counting.

- Volume calculations are imprecise because the number of spherical spacers in the analyzed area, as well as the degree to which the spacers and/or one of the plates is deformed, vary from sample to sample.

- The deformation causes a difference in the time required for the molecules to diffuse to the surface of one of the plates.

In devices that use spherical separators, the volume of the portion of the sample that was analyzed can potentially be estimated by a) counting the spheres in the volume of the sample being analyzed and b) experimentally estimating the layer thickness of the sample (e.g., adding an internal standard such as an immiscible liquid that contains a known concentration of the calibration agent, which can be used to calculate the distance between the plates). However, the additional steps are inconvenient, and since the top plate and/or spacers deform significantly during use, the readings obtained from such devices are still not very accurate.

In contrast, embodiments of the method and apparatus of the present invention are based on dividers that have substantially uniform heights, nearly uniform lateral (eg, straight sidewall column), and perfectly flat (eg, "flat") surfaces that secured to one or more plates in a regular manner in which the spacers are separated from each other by a continuous defined distance (eg, not in random positions as governed by Poisson statistics). During use of some of the embodiments of the method and apparatus of the present invention, the separators and plates are not compressed or deformed to a significant extent in any dimension, at least while the plates are in the closed position and pushed together by capillary force. The device of the present invention may have many advantages in that when using the device of the present invention, the volume of the portion of the sample from which data is obtained (for example, the "suitable volume" or the volume of the portion of the sample in the analyzed area) can be easily calculated very accurately and in some cases it can even be calculated before starting the analysis, even if an unknown amount of sample is placed in the device. Since the plates are largely flat in the closed position (meaning the thickness of the sample is uniform) and the number and dimensions of spacers in the area being analyzed are known, the volume of the sample in the area can be easily calculated with high accuracy. The appropriate sample volume can be determined without counting spacers in the area or estimating sample layer thickness after the analysis is performed. There is also a need to place a certain amount of sample into the device. In addition, at the beginning of incubation, it is necessary that the analyte molecules are evenly distributed in a suitable volume (to the extent possible according to Poisson statistics) to prevent greater concentration in one area compared to others.

Reduced reaction time

It is known that the diffusion constant of many analytes in aqueous media is very low, and therefore many assays require long incubation times (often hours and in some cases days), mixing, and the use of mixing agents or forces. Such assays are designed to allow lateral diffusion of the analyte from an initial location to a distant location on one of the plates (see, for example, Wei et al, Nucl. Acids Res. 33: e78, and Toegl et al, J Biomol Tech 2003 14: 197-204). Such systems are limited because they can take several hours to produce results. In addition, when obtaining a result, it is often difficult to state with any certainty that the reaction reached equilibrium at the time the reaction was completed. This uncertainty, along with other factors, makes it impossible to estimate the absolute concentration of the analyte in a sample.

As will be explained in more detail below, in some embodiments of the method and apparatus of the present invention, the height of the separator and the end point of the analysis can be selected to limit the amount of horizontal diffusion of analytes during analysis. In these cases, such analysis (usually binding analysis) can be performed within a very short period of time. In addition, the concentration of an analyte in a sample can be estimated very accurately, even though the entire sample has not been analyzed or may be of unknown volume.

In these embodiments, the analysis can be stopped and/or the results of the analyzes can be read during the time that i. equal to or greater than the time it would take for the target to diffuse through a uniform thickness layer in a closed configuration (i.e., less than the time it would take for the analyte to diffuse vertically from one plate to another ); and ii. less than the time it would take for the target to diffuse horizontally in a linear dimension of the predetermined area of the binding site (e.g., less than the time it would take for the analyte to diffuse horizontally from one side of the binding site to the other). In such "local coupling" configurations, the volume of the portion of the sample from which data is obtained (the "suitable volume") can be estimated quite accurately because it represents the volume of the sample that lies directly above the region being analyzed. Indeed, the volume of the portion of the sample from which data is obtained may be known before analysis begins. Such "local binding" embodiments have the additional advantage that the sample and optionally any detection reagents are pressed into a thin layer along the binding site and thus binding between any analytes and/or detection reagents should reach equilibrium more quickly, than in embodiments in which the sample is not pressed into the thin layer, for example, if a drop of sample is simply placed on the surface of the binding site plate. Thus, in many cases, equilibrium binding can be achieved in seconds rather than minutes, and thus many assays, particularly binding assays, can be performed very quickly, for example in less than a minute.

Multiplexing

In addition, the "local coupling" configuration allows multiplexed assays to be performed without liquid separation of the various reactions from each other. In other words, multiple assays can be performed in an open environment without the need to separate assays from each other (i.e., without liquid isolation). For example, in local coupling embodiments, two different analytes in the same sample can be analyzed together and, because the assay must be stopped and/or the assay results read before one analyte diffuses from one assay to another, the absolute concentrations of these analytes in the sample can be determined separately from each other, even if they are not liquid separated from each other.

The ability to perform multiple analyzes on a single sample without liquid isolation, simply by sandwiching the sample between two plates and performing the analysis while limiting diffusion, has several advantages. For example, assays can be performed by simply dropping a drop of an unknown volume of sample (eg, blood), spreading the sample onto plates by pressing the plates together, incubating the sample for a period of time, and taking a reading from multiple areas of the device. In the practical implementation of this method, there is no need to transfer certain quantities of the sample into several chambers, which is difficult to do without precise transfer of liquid and/or a measuring device. Moreover, the analysis is extremely fast for the reasons outlined above. In addition, since there is no need to produce plates with "walls", the manufacture of the device is simple. Ultimately, there is no requirement for ports in any of the plates, i.e. ports that could potentially be used to add or remove sample or reagent while the device is in the closed position.

Surface reinforcement

Additionally, in some embodiments of the device and method of the present invention, the device may include a “gain surface,” see, for example, a surface that enhances a signal, such as fluorescence or luminescence, that is produced by the detection means. In some cases, the signal can be enhanced as a result of a nanoplasmonic effect (for example, surface enhanced Raman scattering). Examples of signal amplification using a gain surface are described, for example, in Li et al., Optics Express 201 1 19: 3925-3936 and WO 2012/024006, incorporated herein by reference. In some cases, the gain surface may be a disk dot coupled antenna (D2PA), which was described in US Pat. No. 9,013,690. When used, a device containing an amplification surface can amplify the signal by a factor of 103 or more compared to a detector that is not configured to amplify the signal, thereby allowing analytes to be detected with extremely high sensitivity. In some embodiments, the amount of analyte in a suitable volume of sample, in particular non-cellular analytes that are detected using a sandwich assay, can be counted digitally, for example, using the methods disclosed in WO 2014144133. Use of surface enhancement in some cases allows the analysis to be read using a smartphone or the like. Other characteristics

In embodiments of the device of the present invention, the spacers are attached to one or more plates and are not capable of changing position or being displaced if the plate is immersed in an aqueous environment. The spacers are not spherical and are not attached to the surface of the plate by a small force such as electrostatic force, gravity, or the like. In some embodiments, the plate having the spacers may be a monolith. In many embodiments, the spacers are not prefabricated and then secured to the plate (eg, glued to it or the like). In contrast, spacers can be grown and/or etched using an embossing and/or micromachining process (eg, photolithography).

The parameters of the spacers (e.g. their cross-section, space between them and density, etc.) can be optimized so that, in the closed position, the top plate (which may be flexible) does not deform significantly throughout the portion of the sample to be analyzed (“ suitable sample volume). In some cases, the parameters of the spacers can be adjusted depending on the flexibility of the top plate. For example, if the top plate is more flexible, the spacers may be positioned closer together. Likewise, if the top plate is less flexible, the spacers may be spaced further apart.

Moreover, with many embodiments of the device of the present invention, analytes do not migrate directly into the device after the device is closed. As such, a closed configuration may lack both sorting or fractionation of analytes and directional forced flow of analytes into the device (eg, by gravity or electrophoresis), as described by Austin (US 6,632,652). In many cases, there is no need for the device to be coupled to an energy source to generate the electromotive force. In many embodiments, there are no "obstacles" that impede the passage of the analyte (cell) during sample distribution, resulting in the analytes being evenly distributed in a suitable volume (to the extent possible according to Poisson statistics), to prevent greater concentration in one area compared to another. Moreover, in other devices, the function of the cover plate is to seal the device to prevent leakage of liquid, and thus the cover plate is placed on top of the substrate plate at a time when the sample is not on any of the plates. Such devices do not force liquid onto the surface of an open plate to form a thin layer of sample that can be analyzed. Additionally, in other devices, the main function of the pillars is to “filter” or sort nanoparticles (eg, cells or the like). Therefore, the distance between the pillars is determined using the nanoparticles to be sorted, without the goal of obtaining a uniform space between the cover plate and the substrate plate. Ultimately, in devices such as the Austin device, sorting accuracy is primarily controlled by the space between the posts, and control of the space between the posts is not considered significant. Thus, such openings would not result in modification of the uniformity of the space between the plates as a result of the size, shape of the posts, spacing of the posts, etc.

Based on the foregoing, it is believed that the device and method of the present invention provides an easy to use, inexpensive, easy to manufacture, and extremely rapid method for determining the absolute concentration of an analyte (or analytes, if the device and method is used in multiplex form) in a liquid sample.

One aspect of the present invention provides means that employ a pair of plates that are movable relative to each other for the purpose of converting a small volume sample or one or more reagents or both for easier, faster and/or better analysis. Conversion includes, but is not limited to, changing the shape of the sample, pushing out the sample flow, creating contact between the sample and the reagent, measuring the volume of the samples, decreasing the diffusion distance, increasing the collision frequency, etc., all of which have advantageous effects for certain assays. In the present invention, special characteristics and properties of the plates, special methods of handling the plates, and special methods of handling reagents and samples provide advantages in the analysis.

One aspect of the present invention provides means that cause at least a portion of a small drop of a liquid sample placed on a plate to become a thin layer of thickness that is controlled, predetermined, and uniform over a large area. The uniform thickness can be less than 1 µm. In addition, the present invention ensures that the same uniform thickness is maintained over a long period of time without evaporation into the medium.

Another aspect of the present invention provides means that utilize a predetermined uniform thin sample thickness obtained by the present invention to determine the volume of part or all of the sample without the use of a pipette or the like.

Another aspect of the present invention provides an embodiment of spacers (to control the space between two plates) that provide a column shape with a flat top surface and a nearly uniform cross-section in horizontal projection. Such separators provide many advantages in controlling sample thickness in spherical separators (pellets).

Another aspect of the present invention provides embodiments of spacers (to control the space between two plates) that provide a column shape with a flat top surface and a nearly uniform cross-section in horizontal projection. Such separators provide many advantages in controlling sample thickness in spherical separators (pellets).

Another aspect of the present invention provides means that cause certain chemical reactions (or mixing) to occur predominantly in only a small portion of the sample rather than in another portion of the sample, without liquid isolation of the two portions of the sample.

Another aspect of the present invention provides means that ensure that multiple chemical reactions (or mixing) occur preferentially only in each prospective small portion of the sample and not in another portion of the sample, without liquid isolation between the different portions of the sample. Thus, the present invention provides multiplexed parallel analysis using a single small drop of sample, without liquid isolation of different reaction sites.

Another aspect of the present invention provides means that make analysis (eg, immunoassay, nucleic acid analysis, etc.) faster. For example, incubation time to saturation (the time it takes for molecules to bind to reach equilibrium) is reduced from several hours to less than 60 seconds.

Another aspect of the present invention provides means that significantly increase detection sensitivity using one or a combination of several methods that include surface enhancement, large or bright marks, etc.

Another aspect of the present invention provides means that provide analysis using a very small amount of sample, for example, in the range of 0.5 μl (microliter) or less.

Another aspect of the present invention provides means that simplify the analysis by allowing a small amount of biological fluid to be placed directly from the subject to the test area or sample area.

Another aspect of the present invention provides means that simplify and speed up the analysis by precoating the reagents on the plates. For example, the capture agent and the detection agent are pre-applied and dried on the plates. Another example is where all necessary detection reagents are pre-coated onto the plates and detection is performed by placing the sample on the pre-coated plates without the need to place other reagents.

Another aspect of the present invention provides means that allow the results of an analysis to be read using a mobile phone.

Another aspect of the present invention provides means that allow an individual to examine his/her own biomarkers on their own within 60 seconds by directly placing a drop of their own body fluid (eg, saliva) between a pair of plastic articles and capturing the image using a mobile phone.

A device for analyzing an analyte in a sample may have on one or both plates a dry binding site having a predetermined area, wherein the dry binding site binds and immobilizes the analyte in the sample.

The device may have one or more dry binding sites and/or one or more reagent sites on one or both plates.

The average thickness of the uniform layer can range from 1.8 to 2.6 µm and the sample is whole blood without dilution with other fluid.

The device may contain a dry reagent applied to one or both plates and the reagent may contain an anticoagulant.

The dividers have a columnar shape and a substantially flat top surface, wherein for each divider the ratio of the horizontal dimension of the divider to its height is at least 1.

For each separator, the ratio of the horizontal size of the separator to its height is at least 1.

The separators have a columnar shape with a substantially flat top surface, a specified substantially uniform height, and a specified constant distance between the separators that is at least 2 times the size of the analyte, wherein the product of the Young's modulus of the separators and the fill factor of the separators is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the contact area of the spacers to the total area of the plate, and for each spacer the ratio of the horizontal dimension of the spacer to its height is at least 1, the fourth power of the distance between the spacers (ISD) divided by the thickness (h) and modulus Young's (E) of the flexible plate (ISD ⁴ /(hE)) is 5×10 ⁵ µm ³ /GPa or less.

The spacers have a density of at least 100/mm ² .

The substantially uniform sample layer thickness has less than 30% variation.

The sample layer of uniform thickness is uniform over a horizontal area that is at least 1 mm ² .

In a closed configuration, the finite sample thickness device is capable of performing sample analysis in 10 seconds or less.

The detector may be an optical detector that detects an optical signal.

The dividers are shaped like columns, and the ratio of the width to the height of the columns is greater than or equal to one.

The detector may be an electrical detector that detects an electrical signal.

The spacers can be fixed to the plate by direct plate embossing or plate injection molding.

At least one of the sample detection and/or imaging cameras may sense a signal from a compressed controlled open flow (CROF) device.

The analysis includes a differential analysis of leukocytes.

The mobile communication device may receive a prescription, diagnosis, or recommendation from a medical professional located in a remote location.

The device may contain at least a pair of scale marks spaced at a known distance in a plane parallel to the horizontal area.

The distance between the separators can be approximately equal to the average thickness of the red blood cells (RBC).

The sample may be a blood sample and the analysis step involves visualizing the cells in the blood.

The sample may be a blood sample, and analysis of the blood sample includes measurement of hemocytes, which includes determining the thickness of the sample using a separator, determining horizontal area by imaging, and calculating the area of red blood cells using two-dimensional imaging.

The sample may be blood, the analytes may be blood cells, and the uniform layer thickness may be between 1.9 and 2.2 µm.

The dividers are shaped like columns and have essentially the same cross-section.

The separators have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant spacing between the separators that is at least twice the size of the analyte, wherein the product of the separators' Young's modulus and the separator filling factor is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the contact area of the spacers to the total area of the plate, and for each spacer the ratio of the horizontal size of the spacer to its height is at least 1.

The separators have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant spacing between the separators that is at least about 2 times the size of the analyte, wherein the product of the separators' Young's modulus and the separator filling factor is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the contact area of the separators to the total area of the plate, wherein for each separator the ratio of the horizontal size of the separator to its height is at least 1, and the sample is blood; the analyte is an erythrocyte, leukocyte or platelet; and the uniform layer thickness is in the range of 1.8 to 2.6 μm.

The analysis step can be the identification of proteins, peptides, nucleic acids, artificial compounds and inorganic compounds.

The samples may be samples that are used to analyze blood sugar, oxygen levels, general blood composition (red blood cells, white blood cells, platelets).

Brief description of graphic materials

One skilled in the art will appreciate that the graphics described below are presented for illustrative purposes only. These drawings are in no way intended to limit the scope of the teachings of the present invention. Graphic materials may not be provided to scale. In the figures that represent experimental points, the lines that connect the experimental points are provided only for the purpose of guiding the display of the data and have no other meaning.

In fig. 1 is an illustration of an embodiment of CROF (Compressed Regulated Open Flow). Panel (a) shows a first plate and a second plate, the first plate having spacers. Panel (b) shows the placement of the sample on the first plate (shown) or the second plate (not shown) or both (not shown) in an open configuration. Panel (c) shows (i) the use of two plates to distribute the sample (sample flow between the plates) and reduce sample thickness and (ii) the use of spacers and a plate to control sample thickness in a closed configuration. The inner surface of each plate may have one or multiple bonding and storage areas (not shown).

In fig. 2 shows plates with a binding section or a storage section. Panel (a) shows a plate having a binding site. Panel (b) shows a plate having a reagent storage area. Panel (c) shows a first plate having a binding portion and a second plate having a reagent storage portion. Panel (d) shows a plate having several sections (binding sections and/or storage section).

In fig. Figure 3 shows a technological map and a diagram of a method for reducing incubation time in an analysis by reducing the thickness of the sample. Panel (a) shows the first plate, which has at least one binding site on the surface of the substrate. Panel (b) shows a second plate (which may have a different size than the first plate). Panel (c) shows the placement of the sample (containing the binding target) on the surface of the substrate (shown) or the cover plate (not shown) or both (not shown). Panel (d) shows the movement of the first and second plates so that they face each other, and the reduction in the thickness of the sample as a result of the reduction in the internal space between the plates. The reduced thickness sample is incubated. Reduced sample thickness speeds up incubation time. Some embodiments of the method provide space control spacers, which are not shown in the illustration.

In fig. Figure 4 shows the reduction in binding or mixing time by reducing sample thickness using two plates, spacers and compression (shown in cross section). Panel (a) shows the reduction in the binding time of objects in the sample to the binding site on a solid surface (X-(volume to surface)). Panel (b) illustrates the reduction in time when binding objects (eg, reagent) stored on the surface of a plate to a binding site on the surface of another plate (X-(surface to surface)). Panel (c) illustrates the reduction in time when adding surface-stored reagents to a sample that is placed midway between a plate and another plate (X-(surface to bulk)).

In fig. Figure 5 shows how to avoid or reduce local bending of a flexible plate. Panel (a) shows that if the distance between the spacers is too large for a flexible plate (a second plate, such as a plastic film) under a certain sample set and compression conditions, then the plate has, in a closed configuration, a local deflection (for example, bending inwards) between two adjacent spacers, provided that the first plate is rigid. The sample between the plates is not shown. Panel (b) shows that local bending in the flexible plate in panel (a) is reduced or virtually eliminated by appropriate spacer spacing and appropriate compression force. The sample between the plates is not shown.

In fig. Figure 6 shows the weakening effect of large dust particles on the regulation of the space between the plates (sample thickness). Panel (a) shows that when two rigid plates are used, a dust particle with a thickness greater than the height of the spacer can disrupt the control of the intended space between the plates by the spacers (i.e., disrupt the control of the intended thickness of the sample). The sample between the plates is not shown. Panel (b) shows the use of an appropriate flexible plate and appropriate spacing between the plates, where the influence of dust is limited to a small area surrounding the dust particle, while in other areas the space between the plates (i.e. the sample thickness) is controlled by the spacers rather than the dust . This illustration shows that the first plate is rigid, the second plate is flexible, and the spacers are initially secured to the first plate. Panel (c) is an illustration showing the use of a suitable flexible plate and an appropriate distance between the plates, with the effect of dust being limited to a small area surrounding the dust particle, while in other areas the space between the plates (i.e. thickness of the sample) is controlled by spacers , not dust. This illustration shows that the first plate is rigid, the second plate is flexible, and the spacers are initially attached to the second plate.

In fig. 7 shows the dampening effects of variations in plate surface flatness as a result of appropriately adjusting the distance between the spacers and the flexible plate(s). Panel (a) shows that the variation in surface flatness can be significantly large compared to the desired sample thickness, leading to errors in determining the sample thickness. In this illustration, only one plate has a large variation in flatness (virtually both plates can have a large variation in flatness). The sample between the plates is not shown. Panel (b) represents that the variation distance of plate surface flatness, λ, is the distance from a local maximum to an adjacent local minimum in surface height. Panel (c) shows how a small variation in surface flatness can be achieved by making one or both plates flexible and by applying appropriate spacer spacing and appropriate compression force to correct, in a closed configuration, the original variation in surface flatness of a plate in an open configuration. . The sample between the plates is not shown. Panel (d) illustrates obtaining a sample thickness variation less than the original plate surface flatness variation using a second flexible plate and appropriate spacer spacing. The flexible plate follows the contour of the rigid plate. The sample between the plates is not shown.

In fig. Figure 8 shows plates and separators of the enclosing type (holes) for regulating the thickness of the sample. Panel (a) shows a first plate and a second plate, the first plate having a barrier-type separator (well). Panel (b) shows the placement of the sample on the first plate (shown) or the second plate (not shown) or both (not shown) in an open configuration. Panel (c) shows (i) the use of two plates to distribute the sample (sample flow between the plates) and reduce sample thickness and (ii) the use of spacers and a plate to control sample thickness in a closed configuration.

In fig. 9 shows another embodiment that uses guard-type spacers (wells) to control the thickness of the sample. Panel (a) shows a first plate and a second plate, the first plate having a barrier type separator (well) and at least one spacer within the well. Panel (b) shows the placement of the sample on the first plate (shown) or the second plate (not shown) or both (not shown) in an open configuration. Panel (c) shows (i) the use of two plates to distribute the sample (sample flow between the plates) and reduce sample thickness and (ii) the use of spacers and a plate to control sample thickness in a closed configuration. Panel (d) shows another embodiment of the first and second plates, wherein the first plate does not have a spacer within the well.

In fig. 10 schematically illustrates an exemplary embodiment of the present invention that is multiplex detection in a single device for CROF using one binding site on one wafer and multiple storage sites on another wafer. Panels (a) and (b) are perspective views and cross-sections of the illustrative device, respectively.

In fig. 11 is a schematic diagram of a further exemplary embodiment of the present invention that provides multiplex detection in a single device for CROF using one storage site on one wafer and multiple binding sites on another wafer. Panels (a) and (b) are perspective views and cross-sections of the illustrative device, respectively.

In fig. 12 is a schematic representation of a further exemplary embodiment of the present invention that is multiplex detection in a single device for CROF with multiple binding sites on one wafer and multiple corresponding storage sites on another wafer. Panels (a) and (b) are perspective views and cross-sections of the illustrative device, respectively.

In fig. 13A is a schematic representation of a QMAX assay that uses a CROF with a 30 µm spacer array to perform an analysis with saturation incubation times of less than 30 seconds.

In fig. 13B shows a measurement of the captured label signal versus incubation time, showing that the incubation time to saturation is less than 30 seconds for the QMAX assay described in FIG. 13a.

In fig. 14 shows the experimentally measured LoD (limit of detection) value for the QAX & QMAX assay at 30 µm spacing (for CROF device) with wash (heterogeneous analysis) and without wash (homogeneous analysis).

In fig. 15 is an overhead and cross-sectional view of (i) a small volume sample being excavated onto a glass substrate, (ii) an expanded area of the sample in a closed CROF configuration.

In fig. Figure 16 provides a definition of some of the terms used in this document.

Fig. 17. Separators on the plate. Top view of the photograph: (a) the size of the column spacers is 46 μm × 46 μm and the distance between the columns is 54 μm and (b) the size of the column spacers is 10 μm × 70 μm and the distance between the columns is 10 μm; and SEM perspective view: (c) pillar spacer size 30 μm × 40 μm and spacer height 2 μm and (d) spacer size 30 μm × 40 μm and spacer height 30 μm.

In fig. Figure 18 shows the effect of IDS and plate thickness and materials on sample thickness. Measured sample thickness variation and uniformity as a function of interspacer distance (IDS) for different plates and materials, different plate thicknesses and different samples. CROF device substrates are untreated, perfectly flat 250 μm thick PMMA (dimensions 25.4 mm × 25.4 mm). The X-plate contains an array of periodic column spacers with a column height of 5 μm of a rectangular shape (horizontal column size 10x10 μm, almost uniform cross section and rounded corners), and distance between spacers of 20 μm, 50 μm, 100 μm, 200 μm, 500 µm, made of PMMA or PS with dimensions of 25.4 mm × 25.4 mm. The sample was 2 μl of blood (dropped by direct finger contact), saliva, or PBS (dropped by pipette), and the CROF devices were manually compressed by applying pressure with the hand and stopper over a 1-inch by 1-inch area, and they self-retained once pressed. There is a mark on the figure - designed for 175 µm thick PMMA when using a blood sample, tag designed for 175 µm thick PMMA when using a saliva sample, mark designed for 125 µm PS when using PBS sample, mark designed for 50 µm thick PMMA when using a blood sample, tag Designed for 25 µm thick PS when using a blood sample.

Fig. 19. Deviation of the measured sample thickness and homogeneity depending on the value of ISD ⁴ /(h ^x E) (x=1 on the graph) for X-plates. ISD is the space between spacers, h is the height (thickness) of the material, E is the Young's modulus of the material, x is an adjustable parameter with a typical range of 1 to 3. In the study, the CROF device substrates are 250 µm thick untreated PMMA (size 25.4 mm × 25.4 mm), X-plates are 175 µm thick untreated PMMA, 125 µm thick untreated PS, 50 µm thick untreated PMMA and 25 µm thick untreated PS (size 25.4 mm × 25.4 mm ), containing an array of periodic column separators with a column height of 5 µm of a rectangular shape (horizontal column size 10×10 µm, almost uniform cross-section and rounded corners) and distance between the separators 20 µm, 50 µm, 100 µm, 200 µm, 500 µm , the sample was 2 µl of blood (dropped by direct finger contact), saliva or PBS (dropped by pipette), and the CROF devices were compressed by hand by squeezing with the hand and stopper over a 1 inch by 1 inch area, and they self-retained after squeezing . When calculating ISD ⁴ /h ^x=1 /E, Young's modulus is 2.5 GPa in the case of PMMA and 3.3 GPa in the case of PS. If the value of ISD ⁴ /(hE) is greater than 10 ⁶ μm ³ /GPa, then the performance of the device for CROF deteriorates. There is a mark on the figure designed for 175 µm thick PMMA when using a blood sample, tag designed for 175 µm thick PMMA when using a saliva sample, mark designed for 125 µm thick PS when using a PBS sample, the tag is designed for 50 µm thick PMMA when using a blood sample, tag Designed for 25 µm thick PS when using a blood sample.

Fig. 20. Deviation of measured sample thickness and uniformity depending on the distance between spacers for different sizes of spacer columns and height of X-plates. The substrate wafers of the CROF devices are 1 mm thick raw glass (size 25.4 mm × 25.4 mm), X-wafers are 125 μm thick untreated PS (size 25.4 mm × 25.4 mm) containing an array periodic separators-columns with a separator height of 5 microns of a rectangular shape with a horizontal column size of 10×10 microns (almost uniform cross-section and rounded corners) with a distance between separators of 20 microns, 50 microns, 100 microns, 200 microns, 500 microns (mark ); horizontal column size 40×40 µm with distance between separators 60 µm, 150 µm and 200 µm (label ); an array of periodic column separators with a separator height of 12 microns of a rectangular shape with a horizontal column size of 40×40 microns with a distance between separators of 150 microns and 200 microns (label ); an array of periodic column separators with a separator height of 22 microns of a rectangular shape with a horizontal column size of 40×40 microns with a distance between separators of 150 microns and 200 microns (label ); the sample was 2 μl of blood in the case of 5 μm thick CROF, 5 μl in the case of 12 μm thick CROF, and 9 μl of PBS in the case of 22 μm thick CROF (pipetted), and the CROF devices were compressed by hand by squeezing with the hand and a stopper over an area of 1 inch by 1 inch and they self-restrained after pressing. (The lines in the figures are for visual guidance.)

Fig. 21. Deviation of measured sample thickness and uniformity as a function of different column width to column height ratios, with ISD remaining less than 150 µm for all samples. The substrates of CROF devices are 1 mm thick raw glass (dimensions 25.4 mm × 25.4 mm). CROF devices were manually compressed by applying pressure between the hand and the plug over a 1-inch by 1-inch area and self-retained once pressed. An example of the above figures with a label is given below.

A. X-plate made of 125 µm thick PS (with mark ), from left to right: 1: X-plate pillar size 10×10 µm, height 22 µm, ISD 100 µm, 9 µl PBS buffer, ratio (width/height) = 0.45; 2: X-plate pillar size 10×10 µm, height 12 µm, ISD 100 µm, 5 µl PBS buffer, ratio (width/height) = 0.83; 3: X-plate pillar size 40×40 µm, height 22 µm, ISD 150 µm, 9 µl PBS buffer, ratio (width/height) = 1.81; 4: X-plate pillar size 40×40 µm, height 5 µm, ISD 100 µm, 2 µl PBS buffer, ratio (width/height) = 2; 5: X-plate pillar size 40×40 µm, height 12 µm, ISD 150 µm, 5 µl PBS buffer, ratio (width/height) = 3.33; 6: X-plate pillar size 40×40 µm, height 5 µm, ISD 150 µm, 2 µl PBS buffer, ratio (width/height) = 8; 7: X-plate pillar size 70×70 µm, height 5 µm, ISD 150 µm, 2 µl PBS buffer, ratio (width/height) = 14.

B. X-plate made of PMMA, 175 µm thick (with mark ), from left to right: 1: X-plate column size 10×10 µm, height 22 µm, ISD 100 µm, 5 µl blood, ratio (width/height) = 0.45; 2: X-plate column size 10×10 µm, height 5 µm, ISD 50 µm, 2 µl blood, ratio (width/height) = 2; 3: X-plate column size 30×30 µm, height 30 µm, ISD 80 µm, 12 µl blood, ratio (width/height) = 1; 4: X-plate column size 30×30 µm, height 10 µm, ISD 80 µm, 1 µl blood, ratio (width/height) = 3; 5: X-plate pillar size 30 × 30 µm, height 2 µm, ISD 80 µm, 1 µl blood, ratio (width/height) = 15.

C. X-plate made of PMMA 50 µm thick (with mark ), from left to right: 1: X-plate column size 10 × 10 µm, height 5 µm, ISD 50 µm, 2 µl blood, ratio (width/height) = 2.

X-plate made of 25 µm thick PS (with mark ), from left to right: 1: X-plate column size 10 × 10 µm, height 5 µm, ISD 50 µm, 2 µl blood, ratio (width/height) = 2.

Fig. 22. Deviation of measured sample thickness and uniformity as a function of spacer spacing and pillar size/height of X-plates, with CROF device substrates being 1 mm thick untreated glass (dimensions 25.4 mm × 25.4 mm), X -plates are 125 µm thick untreated PS (25.4 mm × 25.4 mm in size) containing an array of periodic column spacers with 5 µm spacer height rectangular in shape with a horizontal column size of 10 × 10 µm (almost uniform cross-section and rounded corners) with distance between separators 20 µm, 50 µm, 100 µm, 200 µm, 500 µm (mark ), the lateral size of the columns is 40 × 40 µm with the distance between the separators 60 µm, 150 µm and 200 µm (label ); an array of periodic column separators with a separator height of 12 µm of a rectangular shape with a horizontal column size of 40 × 40 µm with a distance between separators of 60 µm, 150 µm and 200 µm (label ); an array of periodic column separators with a separator height of 22 µm of a rectangular shape with a horizontal column size of 40 × 40 µm with a distance between the separators of 150 µm and 200 µm (label ); the sample was 2 μl of blood for 5 μm thick CROF, 5 μl for 12 μm thick CROF, and 9 μl of PBS for 22 μm thick CROF (pipetted), and the CROF devices were compressed by hand by pressing with the hand and a stopper over an area of 1 inch by 1 inch and they self-retained after being squeezed. (The lines in the figures are for visual guidance.)

Fig. 23. Deviation of measured sample thickness and uniformity as a function of varying X-plate thickness (from 25 µm to 525 µm), but with a fixed column size (30 × 38 µm), column height (2 µm) and spaces between columns (80 × 82 µm) made from untreated PMMA, where the substrate is 1 mm thick untreated glass (size 25.4 mm × 25.4 mm), the sample is 1 µl of blood dripped by direct contact with the finger, and the CROF device were squeezed by hand by squeezing the hand and the plug in an area measuring 1 inch by 1 inch, and they self-retained after being squeezed.

In fig. 24 shows the deviation of the measured space size/uniformity of the device for CROF (various combined hydrophilic-hydrophilic pairs with a label hydrophobic-hydrophobic with label ) with a blood volume of 0.1 µl to 0.5 µl, but the same X-plate column size (30 × 38 µm), column height (2 µm) and spacer distances (80 × 82 µm), where the substrate is 1 mm thick glass (dimensions 25.4 mm × 25.4 mm) and an X-plate made of 175 μm thick PMMA (dimensions 25.4 mm × 25.4 mm). Blood was instilled by direct finger contact and CROF devices were compressed by hand using hand and plug pressure in a 1 inch by 1 inch area.

Fig. 25. Deviation of measured sample thickness and uniformity depending on substrates of untreated glass 1 mm thick with a mark or untreated PMMA 250 microns thick with a mark (size 25.4 mm × 25.4 mm), where the X-plate is a raw PMMA 175 μm thick (size 25.4 mm × 25.4 mm), containing an array of periodic column spacers with a column height of 5 μm in a rectangular shape (horizontal bar size of 10 × 10 µm, almost uniform cross-section and rounded corners), and spacers of 50 µm, 100 µm, 200 µm and 500 µm, the sample was 2 µl of blood, dripped by direct finger contact, and CROF devices were hand compressed by applying pressure between the hand and the plug over a 1 inch by 1 inch area and self-retained after compression.

Fig. 26. Deviation of measured sample thickness and uniformity as a function of studies at different hand pressure times from 0 seconds to 60 seconds, where the substrate of CROF devices is untreated 250 μm thick PMMA (size 25.4 mm × 25.4 mm), X -the plate is a 175 µm thick untreated PMMA (25.4 mm × 25.4 mm in size) containing an array of periodic column separators with a 2 µm rectangular separator height (horizontal column size 30 × 38 µm, almost uniform cross-section and rounded corners) and 80 μm spacer spacing, the sample was 1 μL of blood placed by direct contact, and the CROF devices were compressed by hand by squeezing the hand and plug over a 1-inch by 1-inch area and self-retained after compression.

Fig. 27. Deviation of measured sample thickness and uniformity as a function of average IDS in case of using a random spherical spacer or a regular column spacer (X-plate), where the substrate of the CROF devices is 1 mm thick untreated glass (size 25.4 mm × 25 .4 mm), the X-plate is a 175 µm thick untreated PMMA (25.4 mm × 25.4 mm in size) containing an array of periodic column spacers with a 5 µm spacer height of a rectangular shape (horizontal column size 10 × 10 µm, nearly uniform cross-section and rounded corners), and spacers of 20 μm, 50 μm, and 100 μm, the sample was 2 μL of PBS, and the CROF devices were compressed by hand by applying pressure with the hand and stopper over a 1-inch by 1-inch area, and they self-restrained after compression. The sphere is a soda lime microsphere with an average diameter of 4 µm (5% size variation) in PBS. The microspheres were distributed in PBS at concentrations of 4 × 105/μL, 0.9 × 105/μL, and 0.2 × 105/μL, corresponding to the average separation distance after squeezing corresponding to 20 μm, 50 μm, and 100 μm. Two types of cover plates are used, 220 μm thick untreated glass (25.4 mm × 25.4 mm in size) and 175 μm untreated PMMA (25.4 mm × 25.4 mm in size). All CROF devices were pressed using hand and plug pressure over a 1 inch by 1 inch area and were self-retaining once pressed. Label provided when using an X-plate, mark provided in the case of using granules as a separator, and a glass coating with a thickness of 220 microns as a cover plate, mark is provided in the case of using granules as a separator, and a 175 micron thick PMMA film as a cover plate.

Fig. 28. Deviation of measured sample thickness and uniformity depending on the thickness of the X-plate (from 25 µm to 350 µm) and the thickness of the substrate (from 25 µm to 750 µm). X-plates have a fixed pillar size (30 × 38 µm), pillar height (10 µm) and spaces between spacers (80 × 82 µm) made of untreated PMMA in thicknesses of 25 µm, 175 µm and 350 µm, where the substrate is made of untreated PMMA (size 25.4 mm × 25.4 mm) with thicknesses of 25 µm, 50 µm, 175 µm, 250 µm and 750 µm. The sample was 4 μl of blood drawn by direct finger contact, and the CROF devices were compressed by hand by applying pressure with the hand and plug over a 1-inch by 1-inch area, and they self-retained once pressed. There is a mark on the figure provided in the case of using an X-plate with a thickness of 25 microns, mark provided in the case of using an X-plate with a thickness of 175 microns, mark provided when using an X-plate with a thickness of 350 microns.

In fig. 29 shows (a) a micrograph (40x) of blood cells in X-devices with interplate space (ie sample thickness) of 1 µm, 2 µm, 3 µm and 5 µm. The 1 μm X-device lyses the majority (99%) of RBCs, leaving platelets unlysed. The 2 μm X-device separates each RBC well and makes a single layer of RBC. Some stacked RBCs are observed in the 3 μm X-device, and many more stacked RBCs are observed in the 5 μm X-device. For counting, a single cell layer (X-device with 2 µm space) is preferred. And (b) the ratio of the RBC area (measured from the 2D top view image) and the total area in the horizontal projection of the plate for CROF. The maximum value is at 2 µm interplate space (i.e. sample thickness) because below 2 µm some RBCs are lysed and above 2 µm RBCs overlap and rotate, all of which produce less RBC area in the 2D image.

Fig. 30. Schematic of BCI (blood cell counting using CROF and imaging) using a smartphone (a) and photos of the device (b). In a smartphone-BCI blood test, the individual first receives a card (1) and pricks their finger (2), then places a small amount of blood directly from the finger onto the CROF card by touching the card (2), closes the card (3), squeezes finger (4) and releases it (5), inserts the card into the optical adapter (5), ultimately takes a photo of the card using a smartphone (6), and based on the photos taken, the computer program measures blood volume, counts blood cells and other parameters (6). (b) Photo of the actual smartphone and r-BCI adapter.

Fig. 31. Bright-field optical microscope images of (a) fresh and (b) stored undiluted whole blood in a CROF card at various endpoints, and illustration of RBC behavior for different endpoints. Fresh blood contains an anticoagulant and was collected from a pricked finger, while stored blood contains an anticoagulant and was obtained from a commercial supplier, (a-1 to a-6) and (b-1 to b-6): for g = 2 . (2) RBCs overlap each other in CROF with a gap greater than 2 μm.

Fig. 32. Brightfield (1) and fluorescence (2) images of the same sample (fresh blood in a harvested CROF card) using a smartphone with an optical adapter (a) and using a high-resolution microscope with a DSLR camera (b). The images show that a smartphone with an optical adapter has the same photo quality of blood cells as a high-resolution microscope with a camera.

In fig. Figure 33 shows the measured optical intensity of one typical WBC and PLT as a function of the locations of these separated cells. WBC has a diameter (FWHM) of approximately 12 µm, while PLT has a diameter (FWHM) of approximately 2 µm. The maximum intensity of WBC is approximately 3 times that of PLT. Both intensity and area produce a total WBC intensity approximately 108 times greater than PLT. Thus, when lower magnifications are applied (eg 4x), the WBC area becomes smaller and their total intensity becomes lower. In such a case, the PLT signal may be negligible.

In fig. 34 shows (a) a scatter plot of green light channel intensities versus red light channel intensities and (b) a histogram of the red/green light channel intensity ratio for 594 WBCs in the images. Based on this image, it can be clearly seen that the cells are grouped into three distinct areas (shaded areas represented as eye landmarks), corresponding to the three major subpopulations of leukocytes.

Detailed Description of Illustrative Embodiments

The following detailed description shows some embodiments of the present invention by way of example and not by way of limitation. Section titles and any subheadings used herein are for organizational purposes only and are not intended to limit the subject matter of the invention in any way. The information contained under a section title and/or subtitle is not limited to the section title and/or subtitle, but applies to the entire disclosure of the present invention.

Citation of any publication constitutes disclosure prior to the filing date and should not be construed as an admission that the claims of the present invention are not entitled to backdate such publication by virtue of the fact that it constitutes a prior invention. In addition, published publication dates may differ from actual publication dates, which may require independent confirmation.

The present invention relates, among other things, to methods, devices and systems that can improve and/or accelerate the quantitation, binding and/or detection of an analyte and/or object in a sample.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein may also be used in practicing or testing the ideas of the present invention, some illustrative methods and materials are described below.

The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" are used interchangeably and may also include the plural of each, respectively, depending on the context, in which terms are used.

The term "capture agent" as used herein refers to a binding element, e.g., a nucleic acid molecule, a polypeptide molecule, or any other molecule or compound that can specifically bind to its binding partner, e.g., a second nucleic acid molecule containing sequences complementary to a first nucleic acid molecule, an antibody that specifically recognizes an antigen, an antigen specifically recognized by an antibody, a nucleic acid aptamer that specifically binds to a target molecule, etc.

The term “secondary capture agent,” which may also be referred to as a “detection agent,” refers to a group of biomolecules or chemical compounds that have a highly specific affinity for an antigen. The secondary capture agent may be tightly coupled to an optical detection label, such as an enzyme, a fluorescent label, or may itself be detected by another detection agent that binds to the optical detection label during bioconjugation (Hermanson, "Bioconjugate Techniques" Academic Press, 2nd Ed., 2008).

The term "capture agent-reactive group" refers to a moiety of a chemical functional element in a molecule that is reactive to the capture agents, i.e. can react with a moiety (eg hydroxyl, sulfhydryl, carboxyl or amino group) in the capture agent to form a stable, strong, eg covalent bond.

The terms “specific binding” and “selective binding” refer to the ability of a capture agent to preferentially bind to a particular target analyte that is present in a heterogeneous mixture of different target analytes. Specific or selective binding interactions will discriminate between desired (eg, active) and undesired (eg, inactive) target analytes in a sample, typically by a factor of more than about 10-100 times or more (eg, by a factor of more than about 1000 or 10,000 times).

The term "sample" as used herein refers to a material or mixture of materials containing one or more analytes or object of interest. In certain embodiments, the sample may be obtained from a biological sample, such as cells, tissues, body fluids, and feces. Biological fluids of interest include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF) , earwax (cerumen plug), chyle, chyme, endolymph, perilymph, feces, gastric acids, gastric juice, lymph, mucus (including nasal discharge and mucous sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus , watery discharge, saliva, secretion of the sebaceous glands (sebum), sperm, sputum, sweat, synovial fluid, tears, vomit, urine and exhaled condensate, cerebrospinal fluid, vaginal discharge, interstitial fluid obtained from tumor tissues, intraocular fluid , dermis. In certain embodiments, the sample may be obtained from a subject, such as a human, and may be processed prior to use in the subject's assay. For example, prior to analysis, the protein/nucleic acid can be extracted from a tissue sample prior to use using methods that are known in the art. In certain embodiments, the sample may be a clinical sample, such as a patient sample, a biological sample, an environmental sample, a chemical sample, or a clinical specimen.

The term "analyte" refers to a molecule (eg, protein, peptides, DNA, RNA, nucleic acid or other molecules), cells, tissues, viruses and nanoparticles of various forms.

The term "analysis" refers to testing a sample to determine the presence and/or content of an analyte.

As used herein, the terms “definition,” “measurement,” and “assessment” and “analysis” are used interchangeably and include both quantitative and qualitative definitions.

As used herein, the term “light-emitting tag” refers to a tag that can emit light when externally stimulated. This may represent luminescence. Fluorescent labels (which include dye molecules or quantum dots) and luminescent labels (eg, electro- or chemiluminescent labels) are types of light-emitting label. The external excitation is light (photons) in the case of fluorescence, electric current in the case of electroluminescence, and a chemical reaction in the case of chemiluminescence. External stimulation may be a combination of the above.

The phrase “labeled analyte” refers to an analyte that is labeled in a detectable manner with a light-emitting label such that the analyte can be detected by assessing the presence of the label. The labeled analyte may be directly labeled (i.e., the analyte itself may be directly conjugated to a label, e.g., via a strong bond, such as a covalent or non-covalent bond), or the labeled analyte may be indirectly labeled (i.e., the assay bound by a secondary capture agent that is directly labeled).

The terms "hybridization" and "linking" in relation to nucleic acids are used interchangeably.

The term "capture agent/analyte complex" is a complex that is formed by the specific binding of a capture agent to an analyte. The capture agent and the capture agent analyte will generally bind specifically to each other under "specific binding conditions" or "specific binding conditions", such conditions being the conditions (in terms of salt concentration, pH, detergent content , protein concentration, temperature, etc.) that allow binding to occur between the capture agents and the analytes to be bound in solution. Such conditions, particularly with respect to antibodies and their antigens and nucleic acid hybridization, are well known in the art (see, for example, Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) ), and Ausubel, et al., Short Protocols in Molecular Biology, 5th ed., Wiley & Sons, 2002).

The subject may be either a human or a non-human animal. The subject may be an individual performing the method of the present invention, a patient, a client at a testing center, etc.

An "analyte" as used herein can be any substance that is suitable for testing in the present method.

As used herein, the term “diagnostic sample” refers to any biological sample that is a by-product of an organism, such as body fluids, that has been obtained from a subject. The diagnostic sample may be obtained directly from the subject in the form of a liquid or may be obtained from the subject by initially disposing of the body byproduct into a solution such as a buffer. Exemplary diagnostic samples include, but are not limited to, saliva, serum, blood, sputum, urine, sweat, tears, seminal fluid, feces, breath air, biopsies, mucus, etc.

As used herein, the term “environmental sample” refers to any sample that is obtained from the environment. Environmental sample includes fluid samples from river, lake, pond, ocean, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, etc.; solid samples from soil, compost, sand, stones, concrete, wood, brick, waste, etc. and gaseous samples from air, underground hydrothermal vents, industrial emissions, traffic emissions, etc. Typically, samples that are not in liquid form are converted to liquid form before the sample is analyzed using the method of the present invention.

As used herein, the term “food sample” refers to any sample that is suitable for animal consumption, such as human consumption. The food sample may include raw ingredients, cooked food, plant and animal food sources, pre-processed food, and partially or fully processed food, etc. Typically, samples that are not in liquid form are converted to liquid form before the sample is analyzed using the method of the present invention.

The term “diagnostic” as used herein refers to the use of a method or analyte to identify, predict outcome, and/or predict response to treatment of a disease or condition of interest. The diagnosis may include predicting the likelihood or susceptibility of having a disease or condition, assessing the severity of the disease or condition, determining the risk of progression of the disease or condition, assessing the clinical response to treatment, and/or predicting the response to treatment.

The term "biomarker" as used herein is any molecule or compound that occurs in a sample of interest and that is known to have diagnostic value or is associated with the presence of or susceptibility to a disease or condition of interest in a subject from from which the sample was received. Biomarkers include, but are not limited to, polypeptides or complex thereof (e.g., antigen, antibody), nucleic acids (e.g., DNA, miRNA, mRNA), drug metabolites, lipids, carbohydrates, hormones, vitamins, etc., which are known to be associated with a disease or condition of interest.

The term "condition" as used herein in relation to the diagnosis of a health condition refers to a physiological state of the mind or body that is distinct from other physiological conditions. In some cases, a health condition may not be diagnosed as a disease. Exemplary health conditions of interest include, but are not limited to, nutritional health; aging; exposure to environmental toxins, pesticides, herbicides, synthetic hormone analogues; pregnancy; menopause; andropause; dream; stress; prediabetes; physical exercise; fatigue; acid-base balance, etc. The term "biotin moiety" refers to an affinity agent that includes biotin or a biotin analog such as desthiobion, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, etc. Biotin moieties bind to streptavidin with an affinity of at least 10-8 M. The biotin affinity agent may also include a linker, for example, -LC-biotin, -LC-LC-biotin, -SLC-biotin or -PEGn-biotin, where n equals 3-12.

The term "amplify" refers to increasing the magnitude of the signal, for example, at least 10 times the increase, at least 100 times the increase, at least 1000 times the increase, at least 10,000 times the increase, or at least 100,000 times signal increase.

The term "object" refers, without limitation, to proteins, peptides, DNA, RNA, nucleic acid, molecules (small or large), cells, tissues, viruses, nanoparticles of various shapes that will bind to the "binding site". The object includes gripping means, detecting means and blocking means. The term "object" includes "analyte", and the two terms are used interchangeably.

The term "binding site" refers to a location on a solid surface that can immobilize an "object" in a sample.

The term "object partners" refers, without limitation, to proteins, peptides, DNA, RNA, nucleic acid, molecules (small or large), cells, tissues, viruses, nanoparticles of various forms that are at the "binding site" and will bind to the object. Subject matter includes, but is not limited to, binding agents, detection agents, secondary detection agents, or a “capture agent/analyte complex.”

The term "smartphone" or "mobile phone", which are used interchangeably, refers to a type of telephone that has a camera, data communications hardware and software that can take an image using the camera, convert an image captured using the camera, and transfer data to a remote location. In some embodiments, the smartphone has a photoflash.

The term "average linear size" of an area is defined as the length that is equal to the area multiplied by 4, then divided by the perimeter of the area. For example, if an area is a rectangle that has width w and length L, then the average of the linear size of the rectangle is 4*W*L/(2*(L+W)) (where "*" means multiply and "/" means to divide). According to this definition, the average linear dimension is respectively W for a square of width W and d for a circle with diameter d. The region includes, without limitation, the region of the binding portion or the storage portion.

The term "period" of a periodic structure array refers to the distance from the center of the structure to the center of the nearest adjacent identical structure.

The term “storage portion” refers to a portion of an area on the wafer, wherein the portion contains reagents to be added to the sample, and wherein the reagents are capable of dissolving in the sample that is in contact with the reagents and diffusing into the sample.

The term "suitable" means that it is suitable for detecting analytes, quantifying and/or monitoring an analyte or object in a sample or on a plate, or quantifying or monitoring a reagent to be added to a sample or on a plate.

The term "hydrophilic", "wettable" or "wet" with respect to a surface means that the contact angle of the sample on the surface is less than 90 degrees.

The term "hydrophobic", "non-wettable" or "non-wet" with respect to a surface means that the contact angle of the sample on the surface is equal to or greater than 90 degrees.

The term "variation" in relation to quantity refers to the difference between the actual value and the required value or the average value of the quantity. The term "relative variation" in relation to quantity refers to the ratio of the variation to the required value or average value of the quantity. For example, if the required quantity value is Q and the actual quantity is (Q+Δ), then Δ represents the variation, and Δ/(Q+Δ) represents the relative variation. The term "relative sample thickness variation" refers to the ratio of the sample thickness variation to the average sample thickness.

The term "optically transparent" refers to a material that facilitates the transmission of an optical signal, where the term "optical signal" refers, unless otherwise specified, to the optical signal that is used to examine the properties of the sample, plate, spacers, scale divisions, any structures used or any combinations thereof.

The term “non-sample volume” refers, in a closed CROF process configuration, to the volume between the plates that is occupied not by the sample but by other objects that are not the sample. Objects include, but are not limited to, separators, air bubbles, dust particles, or any combination thereof. Often a non-sample volume(s) is mixed within the sample.

The term “incubation time to saturation” refers to the time required for two types of molecules (e.g., capture agents and analytes) to bind to reach equilibrium. In surface immobilization assays, "incubation time to saturation" refers to the time required for the binding between the target analyte (object) in the sample and the binding site on the surface of the plate to reach equilibrium, namely, the time after which the average number of target molecules (objects) ) captured and immobilized by the binding site is statistically almost constant.

In some cases, "analyte", "linking entity" and "object" are used interchangeably.

The terms "processor", "communication device", "mobile device" refer to computer systems that contain basic electronic elements (including one or more of memory, input/output interface, central processing unit, instructions, network interface, power supply and etc.) to perform computational tasks. A computer system may be a general purpose computer that contains instructions to perform a specific task, or may be a specialized computer.

"Site" or "location" as used herein when describing signal or data transmission refers to the local area in which the device or entity is located. An area may be a room within a building, such as a hospital, or a smaller geographically defined area within a larger geographically defined area. A remote site or remote location with respect to a first site that is remote from a second site is a first site that is physically separated from the second site by distance and/or a physical obstruction. The remote site may be a first site that is in a separate room in a building relative to the second site, a first site that is in a different building from the second site, a first site that is in a different city from the second site, etc.

As used herein, the term “sample collection site” refers to the location at which a sample can be obtained from a subject. The sample collection site may be, for example, a retail location (eg, a retail store, pharmacy, supermarket, or department store), a supplier's office, a doctor's office, a hospital, a subject's home, a military installation, an employer's site, or another site or combination of sites. As used herein, the term “sampling site” may also refer to the owner or representative of a business, service, or institution located at or having a branch within the site.

As used herein, the term “raw data” includes signals and direct readings from sensors, cameras, and other components and instruments that detect or measure properties or characteristics of a sample.

"Process management" as used herein refers to any number of methods and systems for planning and/or monitoring the performance of a process, such as a sample analysis process.

One skilled in the art will appreciate that the present invention is not limited in its application to the design details, component ordering, category selection, measurements, predefined signal boundaries, or steps set forth in the specification or drawings herein. The present invention may be implemented in accordance with other embodiments and may be practiced or carried out in many different ways.

It should be noted that singular noun forms as used herein and in the accompanying claims include plural forms unless the context clearly indicates otherwise, such as when the word “one” is used. For example, a reference to "analyte" includes one analyte and multiple analytes, a reference to "capture means" includes one capture means and multiple capture means, a reference to "detection means" includes one detection means and multiple detection means, and a reference to “means” includes one means and multiple means.

Apparatus and system for analyzing a sample, in particular blood, and methods of using them

Provided herein is a device for analyzing an analyte in a sample, particularly blood. In some embodiments, the device includes a first plate and a second plate, where:

the plates are movable relative to each other in different configurations (for example, by means of a hinge);

one or both plates are flexible;

each of the plates has on its respective surface a sample contact region for effecting contact with the sample;

one or both plates include spacers that are secured to a corresponding plate, where the spacers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the spacers, which ranges from 7 μm to 200 μm (for example, from 7 μm to 50 μm (micron ), from 50 µm to 120 µm or from 120 µm to 200 µm), and where at least one of the separators is located inside the sample contact area;

a detector that detects the analyte in the sample;

where one of the configurations is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates; And

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer with a high degree of uniformity in thickness, and it is substantially stationary (i.e., characterized by virtually no movement or directional flow) with respect to the plates, where the uniform thickness of the layer (which may have a horizontal area of at least 0.1 mm ² , at least 0.5 mm ² or at least 1 mm ² ) limited by the inner surfaces of the two plates, and adjusted by plates and spacers, and characterized by an average thickness equal to 5 µm or less (for example, 1.8 µm to 2 µm, 2 µm to 2.2 µm, 2.2 µm to 2.6 µm, or 2.6 µm to 3.8 µm) with slight variation (eg, a variation of less than 10%, less than 5% or less than 1%); and wherein, in a closed configuration, the detector detects the analyte in at least a portion of the sample.

As described below, the device can be used to analyze an analyte that contains a molecule (eg, protein, peptides, DNA, RNA, nucleic acid or other molecule), cells, tissues, viruses and nanoparticles of various shapes; and, for example, to count cells (eg, red blood cells and white blood cells) in a blood sample that is placed in the device.

In some embodiments, the device may include a dry reagent applied to one or both plates. In some embodiments, the dry reagent may bind to an analyte in the sample and immobilize the analyte on the surface of one or both plates. In these embodiments, the reagent, for example, may be an antibody or other specific binding agent. This dry reagent may have a predetermined area. In other embodiments, the device may comprise a releasable dry reagent on one or more plates, for example, a labeled reagent such as a cell dye or a labeled detection agent such as an antibody or the like. In some cases, the plate that contains the releasable dry reagent may have a release time control material, where the release time control material delays the time at which the releasable dry reagent is released into the sample. In some cases, the release time control material delays the time at which the dry reagent begins to be released into the sample by at least 3 seconds, such as at least 5 seconds or at least 10 seconds. In some embodiments, the assembly may contain multiple dry binding sites and/or multiple reagent sites, thereby enabling multiplex assays. In some cases, areas occupied by dry binding sites may be located opposite areas occupied by reagent sites when the plates are in the closed position.

In some embodiments, the reagent comprises an anticoagulant and/or staining reagent(s).

In some embodiments, the analyte may be a molecule (eg, protein, peptides, DNA, RNA, nucleic acid, or other molecule), cell, tissue, virus, or nanoparticles of various forms. In some embodiments, the analytes may be white blood cells, red blood cells, and platelets. In some embodiments, the analyte is colored.

In some embodiments, the spacers that control a layer of uniform thickness (i.e., the spacers that separate the plates from each other in a layer) have a "fill factor" of at least 1%, such as at least 2%, or at least 5%, where the fill factor is the ratio of the area of the spacer in contact with a layer of uniform thickness and the total area of the plate in contact with a layer of uniform thickness. In some embodiments, for spacers that control a layer of uniform thickness, the product of the Young's modulus of the spacers and the spacer fill factor is 10 MPa or more, such as at least 15 MPa or at least 20 MPa, where the fill factor is the ratio of the area of the spacer located in contact with a layer of uniform thickness, and the total area of the plate in contact with a layer of uniform thickness. In some embodiments, the product of the flex plate thickness and the flex plate's Young's modulus is in the range of 60 to 750 GPa-μm, such as 100 to 300 GPa-μm, 300 to 550 GPa-μm, or 550 to 750 GPa-μm. In some embodiments, for a flex plate, the fourth power of the interspacer distance (ISD) divided by the thickness of the flex plate (h) and the Young's modulus (E) of the flex plate ISD ⁴ /(hE) is equal to 10 ⁶ µm ³ /GPa or less, for example , less than 10 ⁵ µm ³ /GPa, less than 10 ⁴ µm ³ /GPa or less than 10 ³ µm ³ /GPa.

In some embodiments, one or both wafers include a location mark either on the surface or within the wafer that provides information about a location on the wafer, such as the location to be analyzed or the location at which the sample is to be placed. In some cases, one or both plates may contain a scale mark, either on the surface or within the plate, which provides information about the size of the sample structure and/or plate in horizontal projection. In some embodiments, one or both wafers include an imaging mark either on the surface or within the wafer to facilitate visualization of the sample. For example, the imaging cue may assist in focusing the imaging device or directing the imaging device to the location of the device. In some embodiments, the delimiters may function as a location mark, a scale mark, a rendering mark, or any combination thereof.

In some embodiments, the average thickness of the uniform thickness layer is in the range of 2 μm to 2.2 μm, and the sample is blood. In some embodiments, the average thickness of the uniform thickness layer is in the range of 2.2 μm to 2.6 μm, and the sample is blood. In some embodiments, the average thickness of the uniform thickness layer is in the range of 1.8 μm to 2 μm, and the sample is blood. In some embodiments, the average thickness of the uniform thickness layer is in the range of 2.6 μm to 3.8 μm, and the sample is blood. In some embodiments, the average thickness of the uniform thickness layer is in the range of 1.8 μm to 3.8 μm, and the sample is whole blood without dilution with another fluid.

In some cases, the average thickness of a layer of uniform thickness is approximately equal to the minimum size of the analyte in the sample, such as a red blood cell or other cell.

In some embodiments, the spacing between the separators may be substantially regular. In some cases, the delimiters may be in a regular pattern, and the space between adjacent delimiters may be approximately equal. The dividers may be posts with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination thereof, and in some embodiments, the dividers may have a substantially flat top surface, where for each divider a ratio of the size of the divider in horizontal projection and its height is at least 1. In some cases, the minimum size of the separator in the horizontal projection is less than or almost equal to the minimum size of the analyte in the sample. The minimum size of the spacer in horizontal projection is in the range from 0.5 μm to 100 μm, for example in the range from 2 μm to 50 μm or from 0.5 μm to 10 μm.

In some embodiments, the sample may be whole blood, such as blood from a clinical sample. In some cases, blood may be obtained by drawing blood from an individual, for example by piercing the individual's skin and touching the drawn blood (without the aid of a blood supply device) to one of the plates. In some embodiments, the sample is undiluted whole blood.

In some embodiments, the spacers are column-shaped and the corners of the side walls of the spacers are rounded with a radius of curvature of at least 1 μm, such as at least 1.2 μm, at least 1.5 μm, or at least 2 .0 µm. The spacers may have any suitable density, for example, a density of at least 1000/mm ² , for example, a density of at least 1000/mm ² , a density of at least 2000/mm ² , a density of at least 5000/ ^mm2 , or a density of at least 10000/ ^mm2 .

In this device, at least one of the plates may be transparent, thereby providing optical reading of the analysis. Similarly, in this device, at least one of the plates may be made of a flexible polymer, thereby allowing efficient distribution of the sample by pressing the plates together. In some embodiments, with respect to the pressure that compresses the plates, the spacers are incompressible and/or independently only one of the plates is flexible. The flexible plate may have a thickness in the range of 10 μm to 200 μm, for example 10 μm to 50 μm, 50 μm to 150 μm, or 150 μm to 200 μm. As noted above, in the closed position the thickness of the layer of uniform thickness may vary slightly. In some embodiments, the variation may be less than 30%, less than 20%, less than 10%, less than 5%, or less than 2%, meaning that the thickness of the region does not exceed +/- 30%, +/- 20%, +/- 10 %, +/- 5% or +/- 2% average thickness.

In some embodiments, the first and second plates are connected and configured to transition from an open configuration to a closed configuration by folding the plates. In some embodiments, the first and second plates are connected by a hinge and are configured to transition from an open configuration to a closed configuration by folding the plates such that the device flexes along the hinge. The hinge may be of a different material, that is, attached to the plates, or in some cases the plates may be integral with the plates. In some cases, the first and second plates are made from a single piece of material and are configured to transition from an open configuration to a closed configuration by folding the plates, such as along a hinge.

In some embodiments, the device is configured to perform sample analysis very quickly. In some cases, the analysis can be completed in 60 seconds or less, in 30 seconds, in 20 seconds or less, or in 10 seconds or less.

In certain embodiments, the dry binding site may comprise a capture agent, such as an antibody or nucleic acid. In some embodiments, the released dry reagent may be a labeled reagent, such as a fluorescently labeled reagent, such as a fluorescently labeled antibody, or a cell stain, such as Romanowsky stain, Leishman stain, May-Gruenwald stain, Giemsa stain, Jenner stain, Wright stain, or any combination of them (for example, Wright-Giemsa stain). Such a dye may contain eosin Y or eosin B with methylene blue. In certain embodiments, the dye may be an alkaline dye such as hematoxylin.

In some embodiments, the detector may be an optical detector that detects an optical signal. In some embodiments, the detector may be an electrical detector that detects an electrical signal

In some embodiments, the space is secured to the plate by directly embossing the plate or injection molding the plate.

In some embodiments, the plate and spacers are made of polystyrene, PMMA, PC, COC, COP, or other plastic.

A system is also provided for accelerated sample analysis using a mobile phone. In certain embodiments, the system may comprise (a) the apparatus described above; (b) a mobile communications device (for example, a mobile phone such as an iPhone or the like) containing: i. one or more cameras for detecting and/or visualizing a sample; ii. integrated circuits, signal processors, hardware and software for receiving and/or processing the detected signal and/or image of the sample and for remote data transmission and (c) a light source, either in a mobile communication device, or an external source. In some cases, where the detector in the device may be provided by a mobile communication device and detects the analyte in the sample in a closed configuration.

In this system, one of the plates may have a binding site that binds the analyte, wherein at least a portion of a layer of uniform thickness of the sample is located on the binding site and is substantially smaller than the average linear dimension in the horizontal projection of the binding site.

In some embodiments, the system may further comprise a housing (d) configured to hold the sample and be secured to the mobile communication device. The housing may include optical means to facilitate visualization and/or processing of a signal from the sample by a mobile communication device, and a mounting system configured to support the optical means on the mobile communication device. In some cases, a device optical element (eg, a lens, filter, mirror, prism, or beam splitter) in the housing may be movable relative to the housing such that the sample can be imaged in at least two channels.

In some embodiments, the mobile communication device may be configured to transmit test results to a medical professional (eg, an MD), a healthcare facility (eg, a hospital or clinical laboratory), or an insurance company. In addition, the mobile communication device may be configured to transmit information regarding the subject (for example, the subject's age, gender, weight, address, first and last name, previous test results, medical history, etc.) to a medical professional, medical facility, or insurance company. company. In certain embodiments, the mobile communication device may be configured to receive a prescription, diagnosis, or recommendation from a medical professional. For example, in some embodiments, the mobile communication device may send test results to a remote location where a medical professional makes a diagnosis. The diagnosis can be transmitted to the subject via a mobile communication device. In some embodiments, the mobile communication device may be configured to transmit information regarding the test and the subject to a cloud network, and the information is processed in the cloud network to refine the test results. In some embodiments, the mobile communication device may be configured to transmit information regarding the test and the subject to a cloud network, and the information is processed in the cloud network to refine the test results, and the refined test results will be sent back to the subject.

In some embodiments, the mobile communication device may include hardware and software that allows (a) to capture an image of a sample; (b) analyze the test location and the reference location in the image; and (c) compare the value obtained from the analysis of the test location with a threshold value that characterizes the accelerated diagnostic test. In some cases, the mobile communication device communicates with a remote location through technology wireless access or mobile cellular network. In any embodiment, the mobile communication device may be a mobile phone.

This system can be used in a method that involves (a) placing a sample on a device of the system; (b) performing an analysis of the analyte in the sample placed on the device and obtaining a result; and (c) transmitting the result from the mobile communication device to a location remote from the mobile communication device. The method may include analyzing the results at a remote location to obtain an analyzed result and transmitting the analyzed result from the remote location to a mobile communication device. As noted above, the test may be performed by a medical professional at a remote location. And in some embodiments, the mobile communication device may receive a prescription, diagnosis, or recommendation from a medical professional located in a remote location.

In this method, the analyte may, for example, be a molecule (eg, protein, peptides, DNA, RNA, nucleic acid or other molecule), cell, tissue, virus or nanoparticles of various shapes. In some embodiments, the analytes may be white blood cells, red blood cells, and/or platelets.

In this method, the sample may be undiluted whole blood, which can be transferred directly to the device from the blood collection site. In some embodiments, the blood sample is a clinical sample.

In some embodiments, the analysis step may include detecting an analyte in the sample, for example, a biomarker such as a protein, nucleic acid, cell or metabolite, that is, found in the blood. The assay may, for example, be a binding assay or a biochemical assay.

In some embodiments, the method includes counting the number of red blood cells and/or counting the number of white blood cells. In some cases, the method may include staining cells in the sample and counting the number of one or more of neutrophils, lymphocytes, monocytes, eosinophils and basophils in the sample.

In some embodiments, the method can be used to differentiate leukocytes (at least neutrophils, eosinophils and lymphocytes) to obtain a probable diagnosis of infection, inflammation, allergies, asthma, immunological disorders (for example, autoimmune disorders or immunodeficiencies), leukemia (for example, chronic myeloid leukemia, chronic lymphocytic leukemia), myelodysplastic syndrome or myeloproliferative neoplasms (eg myelofibrosis).

A method for analyzing the sample is also provided. In some embodiments, the method may include preparing the device described above, placing a sample on one or both plates of the device; placing the plates in a closed configuration, and applying an external force over at least a portion of the plates; and analyzing the analyte in the sample in a layer of uniform thickness while the plates are in a closed configuration.

In some embodiments, the method may include:

(a) obtaining a sample;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein each plate has a sample contact surface that is substantially flat, one or both plates are flexible, and one or both plates includes spacers that fixed to a suitable surface for contact with the sample, and where the separators are characterized by:

i. preset almost uniform height,

ii. column shape with an almost uniform cross-section and a flat top surface;

W. ratio of width to height equal to one or more;

iv. a predetermined constant distance between the separators, which ranges from 10 µm to 200 µm;

v. a fill factor of 1% or more; And

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) using two plates to compress at least a portion of the sample into a layer of substantially uniform thickness, which is limited by the surfaces of the plates for contact with the sample, where the uniform layer thickness is controlled by spacers and plates and has an average value in the range of 1.8 µm to 3 µm with a variation of less than 10%, where compression provides:

bringing the two plates together and

form-matching compression, either simultaneously or sequentially, in the region of at least one of the plates, squeezing the plates together into a closed configuration, wherein the shape-matching compression creates substantially uniform pressure on the plates over at least a portion of the sample and the compression extends over less at least in part of the sample horizontally between the surfaces of the plates for contact with the sample, and where the closed configuration is a configuration in which the space between the plates in a layer of a region of uniform thickness is controlled by spacers; And

(f) blood testing in a layer of uniform thickness while the plates are in a closed configuration;

where the fill factor is the ratio of the contact area with the separator and the total area of the plate, where shape-matching compression is a method that makes the pressure applied throughout the area practically constant regardless of variation in the shape of the outer surfaces of the plates; And

where in simultaneous squeezing, pressure is simultaneously applied to a given area, and in sequential squeezing, pressure is applied to part of a given area and gradually moves to another area.

In some embodiments, this method may involve removing the external force after the plates have been brought into a closed configuration; imaging blood cells in a layer of uniform thickness while the plates are in a closed configuration; and counting the number of analytes, such as cells, in an area in the image. As noted above, in these embodiments, the distance between the spacers may vary in the range from 20 μm to 200 μm or from 5 μm to 20 μm. In these embodiments, the result of the product of the fill factor and the Young's modulus of the separator is 2 MPa or more. In some embodiments, the surface variation is less than 30 nm.

In some embodiments, the sample may be undiluted whole blood to which no anticoagulant has been added. In these embodiments, the placement step (b) can be performed by i. piercing human skin to release a drop of blood onto the skin and ii. bringing a drop of blood into contact with one or both plates without the use of a blood transfer instrument.

The analysis step can be carried out, for example, by counting the number of red blood cells and/or counting the number of white blood cells. In some embodiments, the method may include staining cells in the sample and counting the number of neutrophils, lymphocytes, monocytes, eosinophils and basophils, or any combination thereof.

In any of these embodiments, visualization and counting may be accomplished by: i. illumination of cells in a layer of uniform thickness; ii. obtaining one or more images of blood cells using a CCD or CMOS sensor; iii. identification of cells in the image using a computer and iv. counting the number of cells in an area in the image.

In some embodiments, the external force may be provided by a person's hand, such as by applying pressure with a finger, such as a thumb, or pinching between a thumb and another finger, such as an index finger, of the same hand.

In some embodiments, the method may include measuring sodium, potassium, chloride, bicarbonate, blood urea, nitrogen, magnesium, creatinine, glucose, calcium, HDL-cholesterol and LDL-cholesterol levels and/or triglyceride levels in a layer of uniform thickness. Details regarding how to perform such assays may be adapted from known methods.

In some embodiments, one or more of the plates may contain a dry reagent applied to one or both of the plates (eg, a binding agent, a coloring agent, a detection agent, or an assay reagent).

In some embodiments, the sample layer of uniform thickness may have a thickness uniformity of no more than +/-5%, such as no more than +1-2% or no more than +/-1%.

In some embodiments, the dividers are columns with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination thereof. In some embodiments, the space between the spacers represents approximately the average thickness of the red blood cells.

A sample can be analyzed using a number of different methods. For example, in some embodiments, the analysis step involves visualizing cells, such as red blood cells, white blood cells, or platelets, in the blood. This test may include visualization of cancer cells, viruses or bacteria in the blood. In some embodiments, the method may include a blood test that detects proteins or nucleic acids.

In some embodiments, the blood test may involve measuring blood cells, which may involve determining the thickness of the sample using a separator, determining horizontal area using imaging, and performing a calculation of red blood cell area using 2D imaging. The method may include measuring the concentration of red blood cells, the concentration of white blood cells and/or the concentration of platelets in the blood.

In any of the embodiments described above, the sample may be whole blood.

Immunohistochemistry

In immunohistochemical staining (IHC) methods, the tissue sample is fixed (eg, in paraformaldehyde), optionally embedded in wax, cut into thin sections that are less than 100 μm thick (eg, 2 μm to 6 μm thick), and then prepared for microscopy preparation on a support such as a glass slide. Once a microscopic slide has been prepared, tissue sections can be dehydrated using increasing concentrations of alcohol washes and cleared using a detergent such as xylene.

Most IHC methods can use primary and secondary antibodies. In such methods, the primary antibody binds to an antigen (eg, biomarker) of interest, and it is unlabeled. The secondary antibody binds to the primary antibody and is directly conjugated to either a reporter molecule or a linker molecule (eg, biotin), which may involve a reporter molecule that is in solution. Alternatively, the primary antibody itself may be directly conjugated to either a reporter molecule or a linker molecule (eg, biotin), which may involve a reporter molecule that is in solution. Reporter molecules include fluorophores (e.g. FITC, TRITC, AMCA, fluorescein and rhodamine) and enzymes such as alkaline phosphatase (AP) and horseradish peroxidase (HRP), for which a number of fluorogenic, chromogenic and chemiluminescent substrates such as DAB or BCIP are available /NBT.

In direct methods, the tissue section is incubated with a labeled primary antibody (eg, FITC-conjugated antibody) in binding buffer. The primary antibody binds directly to the antigen in the tissue section and, after the tissue section has been washed to remove any unbound primary antibody, the section is analyzed using microscopy.

In indirect methods, a tissue section is incubated with an unlabeled primary antibody that binds to the target antigen in the tissue. After washing the tissue section to remove unbound primary antibody, the tissue section is incubated with a labeled secondary antibody that binds to the primary antibody.

After immunohistochemical antigen staining, the tissue sample can be stained with another stain, such as hematoxylin, Hoechst stain and DAPI, which provide contrast and/or identification of other features.

The device of the present invention can be used for immunohistochemical (IHC) staining of a tissue sample. In these embodiments, the device may comprise

the first plate and the second plate, where:

the plates are movable relative to each other in different configurations; one or both plates are flexible;

each of the plates has on its respective surface a sample contact region for contacting the tissue sample or IHC staining liquid;

the sample contact area in the first plate is smooth and flat;

the sample contact area in the second plate contains separators that are fixed to the surface and characterized by a predetermined practically uniform height, and a predetermined constant distance between the separators is in the range from 7 μm to 200 μm;

where one of the configurations is an open configuration in which the two plates are either completely or partially separated from each other, the space between the plates is not controlled by spacers, and

where the other of the configurations is a closed configuration, which is brought about after placing the sample and IHC staining fluid in an open configuration; and in a closed configuration, at least a portion of the sample is disposed between two plates, and a layer of at least a portion of the coloring fluid is disposed between at least a portion of the sample and the second plate, wherein the thickness of at least a portion of the layer of coloring fluid is controlled by the plates, the sample, and the spacers , and is characterized by an average distance between the surface of the sample and the surface of the second sample, which is 250 μm or less with a slight variation.

In some embodiments, the device may comprise a dry IHC staining agent applied to the sample contact area of one or both plates.

In some embodiments, the device may comprise a dry IHC stain agent applied to the sample contact area of the second plate, and the IHC stain fluid provides a liquid that dissolves the dry IHC stain agent. The device according to claim 1, where the thickness of the sample is from 2 μm to 6 μm.

There is also a system for accelerated staining and analysis of a tissue sample using a mobile phone, which contains:

(a) a sample, a dyeing liquid and the device described above, (b) a mobile communication device containing:

i. one or more cameras for detecting and/or visualizing a sample;

ii. integrated circuits, signal processors, hardware and software for receiving and/or processing the detected signal and/or image of the sample and for remote interaction; And

(c) a light source either in a mobile communication device or an external source. There is also a method for accelerated staining and analysis of a tissue sample using a mobile phone, which includes:

(a) placing the tissue sample and dyeing fluid on the apparatus of the system described above and placing the two plates in a closed configuration;

(b) obtaining a mobile phone that has hardware and software for visualization, data processing and data transmission;

(c) analyzing a tissue sample placed on the CROF device using a mobile phone and obtaining the result; and

(c) transmitting the result from the mobile communication device to a location remote from the mobile phone.

There is also a method for dyeing a fabric sample, comprising:

(a) obtaining a tissue sample;

(b) obtaining a liquid for dyeing;

(b) obtaining a first plate and a second plate, where:

the plates are movable relative to each other in different configurations;

one or both plates are flexible;

each of the plates has on its respective surface a sample contact region for contacting the tissue sample or IHC staining liquid;

the sample contact area in the first plate is smooth and flat; the sample contact area in the second plate contains separators that are fixed to the surface and characterized by a predetermined practically uniform height, and a predetermined constant distance between the separators is in the range from 7 μm to 200 μm;

(c) placing the fabric sample and dyeing fluid on the plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) using two plates to compress at least a portion of the fabric sample and at least a portion of the dyeing fluid into a closed configuration;

wherein in a closed configuration, at least a portion of the sample is located between two plates, and a layer of at least a portion of the coloring fluid is located between at least a portion of the sample and the second plate, wherein the thickness of at least a portion of the layer of coloring fluid is controlled by the plates, the sample, and the spacers and is characterized by an average distance between the surface of the sample and the surface of the second sample, which is equal to or less than 250 μm with a slight variation.

All benefits and advantages (eg, faster response, faster results, etc.) of other embodiments may be applicable to the present device, system and method.

In addition, all of the parameters described above in the context of other embodiments (e.g., size, space and shape of spacers, flexibility of spacers and plates, and device and system use cases, etc.) can be implemented in the AL embodiments described in this section.

For example, in some embodiments, the spacers that control a layer of uniform thickness (i.e., the spacers that separate the plates from one another in a layer) have a "fill factor" of at least 1%, such as at least 2%, or at least 5%, where the fill factor is the ratio of the area of the spacer in contact with a layer of uniform thickness and the total area of the plate in contact with a layer of uniform thickness. In some embodiments, for spacers that control a layer of uniform thickness, the product of the Young's modulus of the spacers and the spacer fill factor is 10 MPa or more, such as at least 15 MPa or at least 20 MPa, where the fill factor is the ratio of the area of the spacer located in contact with a layer of uniform thickness, and the total area of the plate in contact with a layer of uniform thickness. In some embodiments, the product of the flex plate thickness and the flex plate's Young's modulus is in the range of 60 to 550 GPa-μm, such as 100 to 300 GPa-μm. In some embodiments, for a flex plate, the fourth power of the interspacer distance (ISD) divided by the thickness of the flex plate (h) and the Young's modulus (E) of the flex plate ISD ⁴ /(hE) is equal to 10 ⁶ µm ³ /GPa or less, for example , less than 10 ⁵ µm ³ /GPa, less than 10 ⁴ µm ³ /GPa or less than 10 ³ µm ³ /GPa.

In some embodiments, one or both wafers include a location mark either on the surface or within the wafer that provides information about a location on the wafer, such as the location to be analyzed or the location at which the sample is to be placed. In some cases, one or both wafers may contain a scale mark, either on the surface or within the wafer, that provides horizontal dimension information about the slice structure and/or wafer. In some embodiments, one or both wafers include an imaging mark, either on the surface or within the wafer, to facilitate visualization of the sample. For example, the imaging cue may assist in focusing the imaging device or directing the imaging device to the location of the device. In some embodiments, the delimiters may function as a location mark, a scale mark, a rendering mark, or any combination thereof.

In some embodiments, the spacing between the spacers may be substantially regular. In some cases, the delimiters may be in a regular pattern, and the space between adjacent delimiters may be approximately the same. The dividers may be posts with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination thereof, and in some embodiments, the dividers may have a substantially flat top surface, where for each divider a ratio of the size of the divider in horizontal projection and its height is at least 1. In some cases, the minimum size of the separator in the horizontal projection is less than, or almost equal to, the minimum size of the analyte in the sample. The minimum dimension of the spacer in horizontal projection is in the range from 0.5 µm to 100 µm, for example in the range from 2 µm to 50 µm or from 0.5 µm to 10 µm.

In some embodiments, the spacers are column-shaped and the corners of the side walls of the spacers are rounded with a radius of curvature of at least 1 μm, such as at least 1.2 μm, at least 1.5 μm, or at least 2 .0 µm. The spacers may have any suitable density, for example, a density of at least 1000/mm ² , for example, a density of at least 1000/mm ² , a density of at least 2000/mm ² , a density of at least 5000/ ^mm2 , or a density of at least 10000/ ^mm2 .

In this device, at least one of the plates may be transparent, thereby providing optical reading of the analysis. Similarly, in this device, at least one of the plates may be made of a flexible polymer, thereby allowing efficient distribution of the sample by pressing the plates together. In some embodiments, with respect to the pressure that compresses the plates, the spacers are incompressible and/or independently only one of the plates is flexible. The flexible plate may have a thickness in the range from 20 µm to 200 µm, for example from 50 µm to 150 µm. As noted above, in the closed position the thickness of the layer of uniform thickness may vary slightly. In some embodiments, the variation may be less than 10%, less than 5%, or less than 2%, meaning that the thickness of the region does not exceed +/-10%, +/- 5%, or +/- 2% of the average thickness.

In some embodiments, the first and second plates are connected and the device can be converted from an open configuration to a closed configuration by folding the plates. In some embodiments, the first and second plates may be connected by a hinge, and the device may be configured to transition from an open configuration to a closed configuration by folding the plates such that the device flexes along the hinge. The hinge may be of a different material, that is, attached to the plates, or in some cases the plates may be integral with the plates.

In some embodiments, the device may be capable of very fast slice analysis. In some cases, the analysis can be completed in 60 seconds or less, in 30 seconds, in 20 seconds or less, or in 10 seconds or less.

In certain embodiments, the dry binding site may comprise a capture agent, such as an antibody or nucleic acid. In some embodiments, the released dry reagent may be a labeled reagent, such as a fluorescently labeled reagent, such as a fluorescently labeled antibody, or a cell stain, such as Romanowsky stain, Leishman stain, May-Gruenwald stain, Giemsa stain, Jenner stain, Wright stain, or any combination of them (for example, Wright-Giemsa stain). Such a dye may contain eosin Y or eosin B with methylene blue. In certain embodiments, the dye may be an alkaline dye such as hematoxylin.

In some embodiments, the system may further comprise a housing (d) configured to hold the sample and be secured to the mobile communication device. The housing may include optical means to facilitate visualization and/or processing of a signal from the sample by a mobile communication device, and a mounting system configured to support the optical means on the mobile communication device. In some cases, an optical element of the device (eg, a lens, filter, mirror, prism, or beam splitter) may be movable such that the sample can be imaged in at least two channels.

In some embodiments, the mobile communication device may be configured to transmit test results to a medical professional (eg, an MD), a healthcare facility (eg, a hospital or clinical laboratory), or an insurance company. In addition, the mobile communication device may be configured to transmit information regarding the subject (for example, the subject's age, gender, weight, address, first and last name, previous test results, medical history, etc.) to a medical professional, medical facility, or insurance company. company. In certain embodiments, the mobile communication device may be configured to receive a prescription, diagnosis, or recommendation from a medical professional. For example, in some embodiments, the mobile communication device may send test results to a remote location where a medical professional makes a diagnosis. The diagnosis can be transmitted to the subject via a mobile communication device.

In some embodiments, the mobile communication device may include hardware and software that allows (a) to capture an image of a sample; (b) analyze the test location and the reference location in the image; and (c) compare the value obtained from the analysis of the test location with a threshold value that characterizes the accelerated diagnostic test. In some cases, a mobile communication device communicates with a remote location using wireless technology or a mobile cellular network. In any embodiment, the mobile communication device may be a mobile phone.

The system may be used in a method that includes (a) a sample on a device of the system; (b) performing an analysis of the sample placed on the device and obtaining a result; and (c) transmitting the result from the mobile communication device to a location remote from the mobile communication device. The method may include analyzing the results at a remote location to obtain an analyzed result and transmitting the analyzed result from the remote location to a mobile communication device. As noted above, the test may be performed by a medical professional at a remote location. And in some embodiments, the mobile communication device may receive a prescription, diagnosis, or recommendation from a medical professional located in a remote location.

A method for analyzing a tissue section is also provided. In some embodiments, the method may include obtaining the device described above, placing a slice on one or both plates of the device; placing the plates in a closed configuration and applying an external force over at least a portion of the plates; and analyzing the sample in a layer of uniform thickness while the plates are in a closed configuration.

In some embodiments, the method may include:

(a) obtaining a tissue sample;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein each plate has a sample contact surface that is substantially flat, one or both plates are flexible, and one or both plates includes spacers that fixed to a suitable surface for contact with the sample, and where the separators are characterized by:

i. preset almost uniform height,

ii. column shape with an almost uniform cross-section and a flat top surface;

iii. a width to height ratio of one or more;

iv. a predetermined constant distance between the separators, which ranges from 10 µm to 200 µm;

v. a fill factor of 1% or more; And

(c) placing a cut on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) the use of two plates to compress at least part of the section into a layer of substantially uniform thickness, which is limited by the surfaces of the plates for contact with the sample, where the uniform layer thickness is controlled by spacers and plates and has an average value in the range of 1.8 µm to 3 µm with a variation of less than 10%, where compression involves

bringing the two plates together and

form-matching compression, either simultaneously or sequentially, in the region of at least one of the plates, squeezing the plates together into a closed configuration, wherein the shape-matching compression creates substantially uniform pressure on the plates over at least a portion of the sample and the compression extends over less at least in part of the sample horizontally between the surfaces of the plates for contact with the sample, and where the closed configuration is a configuration in which the space between the plates in a layer of a region of uniform thickness is controlled by spacers; And

(e) slice analysis in a layer of uniform thickness while the plates are in a closed configuration;

where the fill factor is the ratio of the contact area with the spacer and the total area of the plate;

wherein shape-matching compression is a technique that makes the pressure applied over an area substantially constant regardless of variation in the shape of the outer surfaces of the plates; And

where in simultaneous squeezing, pressure is simultaneously applied to a given area, and in sequential squeezing, pressure is applied to part of a given area and gradually moves to another area.

In some embodiments, the method may involve removing the external force after the plates have been brought into a closed configuration; imaging blood cells in a layer of uniform thickness while the plates are in a closed configuration. As noted above, in these embodiments, the distance between the spacers may vary in the range from 20 μm to 200 μm or from 5 μm to 20 μm. In these embodiments, the result of the product of the fill factor and the Young's modulus of the separator is 2 MPa or more. In some embodiments, the surface variation is less than 30 nm.

In any of these embodiments, visualization and counting may be accomplished by: i. illumination of cells in a layer of uniform thickness; ii. obtaining one or more images of blood cells using a CCD or CMOS sensor.

In some embodiments, the external force may be provided by a person's hand, such as by applying pressure with a finger, such as a thumb, or pinching between a thumb and another finger, such as an index finger, of the same hand.

In some embodiments, one or more of the plates may contain a dry reagent applied to one or both of the plates (eg, a binding agent, a coloring agent, a detection agent, or an assay reagent).

In some embodiments, the sample layer of uniform thickness may have a thickness uniformity of no more than +/-5%, such as no more than +1-2% or no more than +/-1%.

In some embodiments, the dividers are columns with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination thereof.

Compressed Regulated Open Flow (CROF)

In many embodiments of the present invention, geometric size, location, contact areas, and sample and/or reagent mixing are performed using a method referred to as "compressed controlled open flow (CROF)" and a device that performs CROF.

The term "compressed open flow (COF)" refers to a method in which the shape of a fluid sample placed on a plate is changed by (i) placing another plate on top of at least a portion of the sample and (ii) then compressing the sample between the two plates as a result pushing two plates towards each other; wherein compression reduces the thickness of at least a portion of the sample and allows the sample to move into the open space between the plates.

The term "compressed controlled open flow" or "CROF" (or "self-calibrating compressed open flow" or "SCOF" or "SCCOF") refers to a specific type of COF where the final thickness of part or all of the sample after compression is "adjusted" by spacers, in this case, the separators are placed between two plates.

The term "the final thickness of part or all of the sample is controlled by spacers" in CROF means that during CROF, once a certain thickness of the sample is reached, the relative movement of the two plates and thus the change in thickness of the sample is stopped, with the specific thickness being determined by the spacer.

One embodiment of the CROF method, as shown in FIG. 1 provides:

(a) obtaining a sample that is fluid;

(b) providing a first plate and a second plate that are movable relative to each other in various configurations, each plate having a sample contact surface that is substantially flat, wherein one or both plates include spacers, the spacers being pre-characterized a given height, and the separators are located on the corresponding surface of contact with the sample;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers; And

(d) after (c) distributing the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, with spacers and the corresponding volume of the sample being between the plates, wherein the thickness of the suitable sample volume is controlled by the plates and spacers, where the corresponding the volume constitutes at least part or all of the volume of the sample, and where during distribution of the sample the sample flows in a horizontal projection between two plates.

The term "plate" refers, unless specifically stated otherwise, to the plate used in the CROF process, which is a solid substance that has a surface that can be used in conjunction with another plate to compress a sample placed between the two plates to reduce the thickness of the sample .

The terms "wafer" or "wafer pair" refer to two plates in a process

CROF.

The terms "first wafer" or "second wafer" refer to the wafer used in the CROF process.

The term "plates facing each other" refers to cases where a pair of plates are located at least partially relative to each other.

The terms "spacers" or "stoppers" refer, unless otherwise specified, to mechanical objects that, when placed between two plates, limit the minimum space between the two plates that can be achieved when the plates are pressed together. Namely, when compressed, the spacers will stop the relative movement of the two plates to prevent the space between the plates from becoming less than a preset (ie, predetermined) value. There are two types of dividers: “open type dividers” and “enclosed type dividers”.

The term "open type separator" means that the separator is shaped to allow liquid to flow around the entire perimeter of the separator and to flow beyond the separator. For example, a column represents an open type separator.

The term "containment type separator" means a separator having a shape such that liquid cannot flow around the entire perimeter of the separator and cannot flow beyond the separator. For example, a ring-shaped separator is a containment-type separator for liquid inside the ring, wherein the liquid inside the ring separator remains inside the ring and cannot escape outside (outside the perimeter).

The terms "separator has a predetermined height" and "dividers have a predetermined distance between the dividers" mean, respectively, that the value of the height of the divider and the distance between the dividers are known before the CROF process. They are not predefined if the height of the separator and the distance between the separators are not known before the CROF process. For example, in the case where the granules are sprayed onto a plate in the form of spacers, where the granules are placed at random locations on the plate, the distance between the spacers is not predetermined. Another example of non-preset space between spacers is when the spacers move during CROF processes.

The term "spacer is attached to its corresponding wafer" during the CROF process means that the spacer is attached to a wafer location, and the attachment to that location is maintained during CROF (ie, the location of the spacer on the corresponding wafer does not change). An example of the "spacer being secured to its respective plate" is where the spacer is formed monolithically as one piece of material with the plate, and the location of the spacer relative to the surface of the plate does not change during CROF. An example of "spacer not secured to its respective wafer" is where the spacer is bonded to the wafer with an adhesive, but as the wafer is applied during CROF, the adhesive is unable to hold the spacer in its original location on the wafer surface and the spacer becomes dislodged from its original location on the surface of the plate.

The term "separator is monolithically attached to the wafer" means that the separator and the wafer behave as a single part of the object without the separator moving or separating from its original location on the wafer during use.

The term "open configuration" of two plates in the CROF process means a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers.

The term "closed configuration" of two plates in the CROF process means a configuration in which the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the sample sample volume is controlled by the plates and spacers, and the suitable volume constitutes at least a portion of the total volume sample.

The term "sample thickness controlled by plate and spacers" in the CROF process means that for a particular condition of the plates, sample, spacers, and method of plate compression, the thickness of at least a portion of the sample in a closed plate configuration can be preset based on the properties of the spacers and plate.

The term "inner surface" or "sample surface" of a wafer in a CROF device refers to the surface of the wafer that is in contact with the sample, while the other surface (which is not in contact with the sample) of the wafer is called the "outer surface".

The term "X-plate" of a CROF device refers to a plate that contains spacers that are located on the sample surface of the plate, where the spacers are characterized by a predetermined distance between the plates and the height of the spacers, and where at least one of the spacers is located inside the contact area with a sample.

The term "CROF device" refers to the device that performs the CROF process. The term “subjected to the CROF process” means that the CROF process is used. For example, the term “sample has been subjected to the CROF process” means that the sample was placed inside the CROF device, the CROF process was performed, and the sample was held, unless otherwise noted, in the final CROF configuration.

The term “CROF plates” refers to the two plates used during the CROF process.

The term "surface smoothness" or "surface smoothness variation" of a flat surface refers to the average deviation of a flat surface from an ideal flat surface over a short distance, which is approximately a few micrometers or less. Surface smoothness is different from variation in surface evenness. A flat surface may have good surface flatness but poor surface smoothness.

The term “surface flatness” or “surface flatness variation” of a flat surface refers to the average deviation of a flat surface from an ideal flat surface over a long distance, which is approximately 10 µm or more. Variation in surface flatness is different from surface smoothness. A flat surface may have good surface smoothness but poor surface flatness (i.e., high variation in surface flatness).

The term "relative surface flatness" of a plate or specimen is the ratio of the variation in the surface flatness of the plate to the final thickness of the specimen.

The term "final sample thickness" in the CROF process refers, unless otherwise specified, to the thickness of the sample in a closed plate configuration in the CORF process.

The term "compression method" in CROF refers to a method that forces two plates from an open configuration to a closed configuration.

The term "region of interest" or "region of interest" of a plate refers to the area of the plate that corresponds to the function performed by the plates.

The term “not more than” means “equal or less.” For example, if the height of the spacer is 1 µm or less, this means that the height of the spacer is 1 µm or less.

The term "sample area" means the area of the sample in a direction approximately parallel to the space between the plates and perpendicular to the thickness of the sample.

The term "sample thickness" refers to the size of the sample in the direction perpendicular to the surface of the plates that face each other (for example, the direction of the space between the plates).

The term "plate space" refers to the distance between the inner surfaces of two plates.

The term "sample final thickness deviation" in CROF means the difference between the predetermined height of the spacer (specified when the spacer was manufactured) and the average of the final sample thickness, where the average final sample thickness is averaged within a certain area (for example, the average of 25 different points (on distance 4 mm) within an area of 1.6 cm by 1.6 cm).

The term "measured final sample thickness uniformity" in the CROF process refers to the standard deviation of the measured final sample thickness within a defined area of the sample (eg, standard deviation relative to the mean).

The terms "suitable sample volume" and "suitable sample area" in the CROF process refer, respectively, to the volume and area of the portion or all of the sample volume placed on the plates during the CROF process that corresponds to the function performed by the suitable method or apparatus, where function includes, but is not limited to, reduction of analyte or target binding time, analyte detection, analyte quantification, volume quantification, concentration quantification, reagent mixing, or concentration control (of analytes, target, or reagents).

The term “certain embodiments”, “in some embodiments”, “in some embodiments of the present invention”, “embodiment”, “one embodiment”, “another embodiment”, “certain embodiments”, “many embodiments” " and so on. refers, unless specifically stated otherwise, to embodiment(s) that are applicable to the entire disclosure (i.e., the entire present invention).

The terms "height" or "thickness" of an object in the CROF process refer, unless specifically stated otherwise, to the size of the object that is in the direction perpendicular to the surface of the wafer. For example, spacer height is the size of the spacer in the direction perpendicular to the surface of the plate, and spacer height and spacer thickness mean the same thing.

The term "area" of an object in the CROF process refers, unless specifically stated otherwise, to the area of the object that is parallel to the surface of the plate. For example, the spacer area is the area of the spacer that is parallel to the surface of the plate.

The term "horizontal" or "horizontal" in the CROF process refers, unless specifically stated otherwise, to the direction that is parallel to the surface of the plate.

The term "width" of the divider in the CROF process refers, unless specifically stated otherwise, to the size of the divider in horizontal projection.

The term "spacer inside the sample" means that the spacer is surrounded by the sample (for example, the column spacer is inside the sample).

The term "critical bending span" of a plate in a CROF process refers to the span (i.e., distance) of a plate between two supports at which the bending of the plate, for a given flexible plate, specimen, and compression force, is equal to the allowable bending. For example, if the allowable bending is 50 nm and the critical bending range is 40 µm for a particular flexible plate, sample and compression force, then the bending of the plate between two adjacent spacers spaced 40 µm apart will be 50 nm and the bending will be less than 50 nm if two adjacent separators are located less than 40 µm apart.

The term “fluid” in the case of a sample means that if the thickness of the sample decreases, the size in horizontal projection increases. For example, a stool sample is considered fluid.

In some embodiments of the present invention, the sample under CROF process conditions does not need to be fluid in order to benefit from the process, as long as the thickness of the sample can be reduced under CROF process conditions. For example, to dye fabric, by placing dye on the surface of a CROF plate, the CROF process can reduce the thickness of the fabric and therefore speed up the incubation time to saturation in the case of dye dyeing.

1. Reduced (shortened) binding or mixing time (X) When performing assays or other chemical reactions, it is desirable to reduce the incubation/reaction time. For example, in surface immobilization assays where the target analyte in a sample is detected by capture by capture agents immobilized on the surface of the plate (i.e., solid phase), it is often desirable to have a short incubation time to saturation to capture the target analytes in the sample or immobilize the agents for capture in solution on the surface of the plate, or both. Another example is the need to shorten the application time of the gripping agent to the surface of the plate. Another example is the need to shorten the mixing time of a reagent in a sample.

The present invention provides methods and apparatus that reduce (ie, shorten) the saturation incubation time required to bind an object in a sample to a binding site on a solid surface (ie, the time to bind a bulk object to a surface). Another aspect of the present invention is to reduce the time required to bind an object stored on the surface of a wafer to a binding site on the surface of another wafer (ie, the time to bind an object from one surface to another surface). Another aspect of the present invention is to reduce the time required to add/mix a surface reagent to/with a sample volume (ie, time to add/mix a surface reagent to a sample volume).

The present invention provides for reducing the saturation incubation time for binding and/or mixing in an assay using devices and methods that distribute the sample (or liquid) to a thinner thickness, thereby reducing the time it takes for an object to diffuse through the thickness of the sample. The diffusion time of an object in a material (such as liquid, solid, or semi-solid) is proportional to the square of the diffusion distance, so reducing sample thickness can reduce the diffusion distance, resulting in a significant reduction in diffusion time and incubation time to saturation. Thinner thickness (such as a dense enclosed space) also increases the frequency of an object's collisions with other objects in the material, further enhancing binding and mixing. The means of the present invention also make sample thickness reduction precise, uniform, fast, simple (fewer processing steps), and useful for reducing sample thickness to micrometers or nanometers. The present invention has significant utility in rapid, low-cost PoC diagnostics and chemical/biological analysis. Several embodiments of the present invention are shown in FIGS. 1-4.

1.1. Reducing the incubation time to saturation when binding an object in a sample to a binding site on a solid surface by reducing the thickness of the sample

X1. A method for reducing the incubation time to saturation when binding a target object in a sample to the binding site of the plate surface, as shown in Figs. 1-2, 3a and 4a, which includes:

(a) obtaining a sample that is fluid and contains a target object that is capable of diffusion in the sample;

(b) providing a first plate and a second plate that are movable relative to each other into different configurations, wherein the first plate has on its surface a coupling portion that is capable of being coupled to a target object, wherein one or both plates comprise spacers, and each of the dividers are fixed to their respective plate and have a predetermined height;

(c) placing the sample on one or both plates when the plates are in an open configuration; where an open configuration is a configuration in which two

the plates are either completely or partially separated from each other, and the space between the plates is not regulated by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, with the separators and a suitable volume of the sample being between the plates, the binding site being in contact with the suitable volume, and the thickness the appropriate sample volume is adjusted using plates and spacers, and is thinner than the maximum sample thickness when the plates are in an open configuration;

where the suitable volume is part or all of the sample volume; And

where the reduced sample thickness reduces the incubation time to saturation when binding the target object in a suitable volume to the binding site.

For a given sample volume, CROF reduces the thickness of the sample but increases the horizontal dimension of the sample. The present invention utilizes this fact to effect (a) local binding or mixing in a portion of the sample and (b) multiplexing multiple binding or mixing regions without a liquid barrier to fluidly separate the sample into different pockets to separate liquids.

X2. A device for reducing the incubation time to saturation when binding the target object in a suitable sample volume to the surface, as shown in Figs. 1-2, 3a and 4a, which contains:

a first plate and a second plate that (a) are movable relative to each other in various configurations, (b) each plate has a sample contact area for contacting a sample that has a target object within a suitable sample volume, (c) one of of the plates has a bonding area that binds the target object, and (d) at least one of the plates includes spacers that have a predetermined distance between the spacers and a height and are secured to their respective surface, wherein at least one of the spacers is within the area contact with the sample;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers,

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the binding site is in contact with the suitable volume, and the thickness of the suitable sample volume is adjusted by the plates and spacers, and is thinner than the maximum thickness of the sample, when the plates are in an open configuration; where the suitable volume is part or all of the sample volume; and wherein the reduced sample thickness reduces the incubation time to saturation when the target is bound in a suitable volume to the binding site.

1.2. Reduced incubation time to saturation when binding an object stored on the surface of one plate to a binding site on the surface of another plate

X3. A method for reducing the incubation time to saturation when binding an object stored in a storage area of one plate to a corresponding binding area in another plate, as shown in FIGS. 1, 3c and 4b, which comprises:

(a) providing a first plate and a second plate that are movable relative to each other in different configurations, where the surface of the first plate has a bonding area; and the surface of the second plate has a storage portion that contains an object to be bonded to the bonding portion; where the area of the binding area and the area of the storage area are less than the area of the corresponding plates; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(b) obtaining a transmission medium, where the object in the storage area is capable of dissolving in the transmission medium and diffusing in the transmission medium;

(c) placing, when the plates are in an open configuration, the transfer medium on one or both plates; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the transmission medium by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, spacers, a bonding area, a storage area and at least a portion of the transmission medium are between the plates, the binding area and the area storages are at least partially on the surface of each other, the transfer medium is in contact with at least a part of the binding portion and the storage portion, the thickness of the transmission medium is controlled by the plates and spacers, and is thinner than the maximum thickness of the transmission medium in the case where the plates are in an open configuration;

wherein the reduced thickness of the transmission medium reduces the binding time of an object stored on the second wafer to the binding site on the first wafer.

X4. A device for reducing the incubation time to saturation when binding an object stored on a storage area of one plate to a binding area on another plate, as shown in FIGS. 1, 3c and 4b, comprising:

a first plate and a second plate that are movable relative to each other in different configurations, where the surface of the first plate has a bonding portion; and the surface of the second plate has a storage portion that contains an object to be bonded to the bonding portion; wherein the area of the binding portion and the area of the storage portion are smaller than the corresponding plates; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the transmission medium can be placed on one or both plates, where the object in the storage area is capable of dissolving into the transmission medium environment and diffuse in the transmitting medium,

where the other of the configurations is a closed configuration, which is brought about after placing the transmission medium in an open configuration; and in a closed configuration, the plates face each other, the spacers, the bonding portion, the storage portion, and at least a portion of the transmission medium are between the plates, the bonding portion and the storage portion are at least partially on top of each other, the transmission medium is in contact with at least part of the binding portion and the storage portion, the thickness of the transmission medium is controlled by the plates and spacers, and is thinner than the maximum thickness of the transmission medium in the case where the plates are in an open configuration;

wherein the reduced thickness of the transfer medium reduces the incubation time to saturation when binding an object in the storage area of the second plate to the binding area of the first plate.

In the method of item X3 and the device of item X4, in some embodiments, the transfer medium contains a liquid that allows for the diffusion of the object or the reagent or both.

In the method of item X3 and the device of item X4, in some embodiments, the transfer medium is a sample, where the sample contains an analyte (also called a target analyte) that binds to a binding site.

In the method of item X3 and the apparatus of item X4, in some embodiments, the transfer medium is a sample, where the sample contains an analyte (also called a target analyte) that binds to a binding site, and the reagent is a detection agent that binds to the analyte.

1.3. Reducing the time it takes to add (mix) a reagent stored on the surface to a liquid sample

Many tests require the addition of reagents (including liquid) to the sample. It is often necessary to control the concentration of reagents added to a sample or liquid. There is a need for new methods for adding such reagents and controlling their concentrations that are simple and/or low cost. Two examples where the addition of reagents are required are (a) red blood cell counts, where anticoagulant and/or staining reagent(s) can be added to the blood sample, and (b) immunoassays, where detection agents are added to bind the target analyte in solution.

One aspect of the present invention provides methods, devices and systems that make adding a reagent and controlling the concentration of a reagent simple and/or low cost. In one embodiment of the present invention, a layer of reagent (eg, a layer of dried reagent) is first applied to the surface of the wafer in a CROF device, and then the sample is placed in the CROF device, and during the CROF process, the sample is brought into contact with the reagent, and the thickness sample is thinner than the thickness when the sample is in an open plate configuration for CROF. By reducing the thickness of the sample, the diffusion time during which the reagent diffuses from the surface into the entire sample will be reduced, and, consequently, the time for mixing the reagent with the sample will be reduced.

X5. A method for reducing the mixing time of a reagent stored on the surface of a plate in a sample, as shown in Figs. 1, 3b and 4c, comprising:

(a) providing a first plate and a second plate that are movable relative to each other into different configurations, wherein the first plate has on its surface a storage portion that contains reagents to be added to the sample, and wherein the reagents are capable of dissolving in the sample and diffusing into sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(b) obtaining a sample;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the sample by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, the separators, the storage area and at least a portion of the sample are between the plates, the sample is in contact with at least a portion of the storage area , the thickness of the sample in the storage area is controlled by plates and spacers, and is thinner than the maximum thickness of the sample when the plates are in an open configuration;

where the reduced thickness of the sample reduces the time for mixing reagents in the storage area with the sample.

In the method of item X5, while the plates are in a closed configuration, an additional incubation step is provided, where the incubation time is chosen such that it results in a significant amount of reagents soluble in the sample being contained in a suitable volume of the sample, where the suitable volume is the volume of the sample that is located at the binding site, and incubation is the process that allows the reagent to dissolve and diffuse into the sample.

In the method according to point X5, after (d) and while the plates are in a closed configuration, there is additionally a stage in which incubation is carried out for a time equal to or less than the time that is a multiple of the time of diffusion of the reagent in the sample through the thickness of the sample, controlled by the plates in a closed configuration, and then the incubation stops; in this case, incubation provides the possibility of diffusion of the reagent in the sample; and the coefficient is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000 , 10000, or between any of these values. For example, if the coefficient is 1.1 and the diffusion time is 20 seconds, then the incubation time is 22 seconds or less. In one preferred embodiment, the coefficient is 0.1, 1, 1.5, or a range between any of these values.

X6. A device for reducing the time of adding a reagent stored on the surface of a plate to a sample, as shown in Figures 1, 3b and 4c, which contains:

a first plate and a second plate that are movable relative to each other in different configurations, wherein the first plate has on its surface a storage portion that contains reagents to be added to the sample, the reagents being capable of dissolving in the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is brought about after placing the transmission medium in an open configuration; and in a closed configuration, the plates face each other, the spacers, the storage area and at least a portion of the sample are between the plates, the sample is in contact with at least a portion of the storage area, the thickness of the sample in the storage area is controlled by the plates and spacers and is thinner , than the maximum sample thickness when the plates are in an open configuration;

where the reduced thickness of the sample reduces the time for mixing reagents in the storage area with the sample.

In the method or devices of any one of claims X1-6, in some embodiments, a suitable sample volume is a sample volume that is placed on (ie, on the surface of) the binding site or storage site.

In the method or devices of any one of claims X1-6, in some embodiments, a suitable sample volume is a sample volume that is placed on (ie, on the surface of) all or part of the area of the binding area or storage area.

In the method or devices of any one of items X1-6, in some embodiments, the ratio of the horizontal dimension of the binding area or storage area and the thickness of the sample in a closed configuration is 1.5 3 or more, 3 or more, 5 or more, 10 or more , 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 1000 or more, 10000 or more, or is between any two of these values.

In the method or devices of any one of items X1 to 6, the ratio of the horizontal dimension of the binding section or storage section and the thickness of the sample in a closed configuration is from 3 to 20 in a preferred embodiment, from 20 to 100 in another preferred embodiment, and from 100 up to 1000 in another preferred embodiment and from 1000 to 10000 in another preferred embodiment.

In the method of any one of X1 and X3, in some embodiments, the final reduced thickness of the sample is significantly less than the area of the binding site, such that an object in the sample area that is outside the binding site will take longer to contact the binding site. With proper incubation timing, the object that binds to the binding sites will preferentially be an object in the sample volume that is located at the binding site (ie, in the sample volume that is directly above the binding region). The calculation of the concentration of the object in the sample will then be based on the thickness of the sample and the area of the binding site.

In the method of item X5, in some embodiments, the final reduced thickness of the sample is significantly less than the area of the storage area, so that the object

in a sample region that is outside the binding site, it will take longer to contact the binding site. With proper incubation timing, the object that binds to the binding sites will preferentially be an object in the sample volume that is located at the binding site (ie, in the sample volume that is directly above the binding region). The calculation of the concentration of the object in the sample will then be based on the thickness of the sample and the area of the binding site.

The method according to any one of items X2, X4, X6 additionally provides a compression device that brings the plates from an open configuration to a closed configuration. In some embodiments, the compression device is one embodiment or any combination thereof described in the present invention.

The method according to any one of items X2, X4, X6 is further provided with a compression device that drives the plates from an open configuration to a closed configuration, and a holding device that is configured to hold the plates in a closed configuration. In some embodiments, the retention device is one or any combination thereof described in the present invention.

The method of any one of X2, X4, X6 further includes a compression device that drives the plates from an open configuration to a closed configuration, and a holding device that is designed to hold the plates in a closed configuration for a time of 0.001 s or less , 0.01 s or less, 0.1 s or less, 1 s or less, 5 s or less, 10 s or less, 20 s or less, 30 s or less, 40 s or less, 1 min. or less, 2 min. or less, 3 min. or less, 5 min. or less, 10 min. or less, 20 min. or less, 30 min. or less, 60 min. or less, 90 min. or less, 120 min. or less, 180 min. or less, 250 min. or less than or in the range between any two of these values.

The method according to any one of items X2, X4, X6 is further provided with a compression device that drives the plates from an open configuration to a closed configuration, and a holding device that is configured to hold the plates in a closed configuration for a time of, in the preferred embodiment, 0.001 s or less, 0.01 s or less, 0.1 s or less, 1 s or less, 5 s or less, 10 s or less, 20 s or less, 30 s or less, 40 s or less, 1 min. or less, 2 min. or less, 3 min. or less than or in the range between any two of these values.

Final sample thickness. The final sample thickness in a closed plate configuration can be a significant factor in reducing incubation time to saturation. The final thickness after thickness reduction/strain, depending on object and specimen properties and applications, is discussed in relation to the adjustable plate spacing.

In some embodiments, the final sample thickness is less than about 0.5 μm (micrometer), less than about 1 μm, less than about 1.5 μm, less than about 2 μm, less than about 4 μm, less than about 6 μm, less than about 8 μm, less than about 10 microns, less than about 12 microns, less than about 14 microns, less than about 16 microns, less than about 18 microns, less than about 20 microns, less than about 25 microns, less than about 30 microns, less than about 35 microns, less than about 40 microns, less than about 45 microns, less than about 50 microns, less than about 55 microns, less than about 60 microns, less than about 70 microns, less than about 80 microns, less than about 90 microns, less than about 100 microns, less than about 110 microns, less than about 120 microns, less than about 140 microns, less than about 160 microns, less than about 180 microns, less than about 200 microns, less than about 250 microns, less than about 300 microns, less than about 350 microns, less than about 400 microns, less than about 450 microns, less than about 500 microns, less than about 550 µm, less than about 600 µm, less than about 650 µm, less than about 700 µm, less than about 800 µm, less than about 900 µm, less than about 1000 µm (1 mm), less than about 1.5 mm, less than about 2 mm , less than about 2.5 mm, less than about 3 mm, less than about 3.5 mm, less than about 4 mm, less than about 5 mm, less than about 6 mm, less than about 7 mm, less than about 8 mm, less than about 9 mm, less than approximately 10 mm or between any two of these values.

In certain embodiments, the final sample thickness in a closed configuration is less than 0.5 μm (micron), less than 1 μm, less than 5 μm, less than 10 μm, less than 20 μm, less than 30 μm, less than 50 μm, less than 100 μm, less than 200 µm, less than 300 µm, less than 500 µm, less than 800 µm, less than 200 µm, less than 1 mm (millimeters), less than 2 mm (millimeters), less than 4 mm (millimeters), less than 8 mm (millimeters) or between any two of these values.

In certain embodiments, Q-methods make the final sample thickness uniform and employ flat surfaces of the first plate and the second plate.

In the present invention, sample incubation is performed at different temperatures, humidity, gas environment and different durations of time, with or without shaking.

Incubation time. In the method according to any of points XI and X3, after (d) and while the plates are in a closed configuration, there is additionally a stage in which incubation is carried out for a time equal to or less than the time that is a multiple of the time of diffusion of the object in the sample, diffusing through the thickness of the sample, adjustable plates in a closed configuration, and then incubation stops; wherein incubation allows the object to bind to the binding site; and the coefficient is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000 , 10000, or between any of these values. For example, if the coefficient is 1.1 and the diffusion time is 20 seconds, then the incubation time is 22 seconds or less. In one preferred embodiment, the coefficient is 0.1, 1, 1.5, or a range between any of these values.

In the method according to point X5, after (d) and while the plates are in a closed configuration, there is additionally a stage in which the incubation is carried out for a time equal to or less than the time of diffusion of the reagents diffusing through the thickness of the sample, controlled by the plates in a closed configuration, and then the incubation stops; wherein incubation allows the object to bind to the binding site; and the coefficient is 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000 , 10000, or between any of these values. For example, if the coefficient is 1.1 and the diffusion time is 20 seconds, then the incubation time is 22 seconds or less. In one preferred embodiment, the coefficient is 0.1, 1, 1.5, or a range between any of these values.

The method according to any of paragraphs X1, X3 and X5 or the device according to any of paragraphs X2, X4 and X6, where at least one of the separators is located inside the contact area with the sample.

The method of any one of X1, X3 and X5 or the device of any of X2, X4 and X6, wherein the spacers have a predetermined distance between the spacers.

In the method of any one of X1, X3, X5, while the plates are in a closed configuration, an incubation step is additionally provided, the incubation time to saturation at reduced sample thickness is 0.001 s or less, 0.01 s or less, 0.1 s or less, 1 s or less, 5 s or less, 10 s or less, 20 s or less, 30 s or less, 40 s or less, 1 min. or less, 2 min. or less, 3 min. or less, 5 min. or less, 10 min. or less, 20 min. or less, 30 min. or less, 60 min. or less, 90 min. or less, 120 min. or less, 180 min. or less, 250 min. or less than or in the range between any two of these values.

In the method of any one of X1, X3, X5, the incubation time to saturation at reduced sample thickness in a closed configuration is 0.001 s or less, 0.01 s or less, 0.1 s or less, 1 s or less, 5 s or less, 10 s or less, 20 s or less, 30 s or less, 40 s or less, 1 min. or less, 2 min. or less, 3 min. or less, 5 min. or less, 10 min. or less, 20 min. or less, 30 min. or less, 60 min. or less, 90 min. or less, 120 min. or less, 180 min. or less, 250 min. or less than or in the range between any two of these values.

In some embodiments, the capture means is first immobilized at the binding site, then the sample is brought into contact with the binding site and the object in the sample is captured by the capture means, and finally the detection means to be associated with the captured object and the signal from the detection means are added. will be read (for example, using optical methods, electrical methods, or a combination thereof). In some embodiments, other reagents (eg, a blocking agent) are added in addition to capture agents and detection agents.

In many applications, such as PoC, it is desirable to have simple and/or inexpensive devices and methods for adding additional reagents to the sample. One aspect of the present invention relates to simple and/or inexpensive devices and methods for adding additional reagents to a sample. Additional reagents added include detection agents, blocking agents, light signal enhancers, light signal suppressors, or others. In some embodiments of the present invention, assay processes are controlled by using different release times for reagents stored at the same location. Different release times can be achieved by adding other materials that have different dissolution rates.

In certain embodiments, the concentration of the reagent mixed with the sample can be controlled by controlling the thickness of the sample (eg, controlling the ratio of sample thickness to storage area and/or mixing time).

2. Plates, separators, scale divisions, regulation of sample thickness

2.1. Plate configurations and sample thickness adjustment

Open configuration. In some embodiments, in an open configuration, the two plates (ie, the first plate and the second plate) are separated from each other. In certain embodiments, the two plates have one side locked together during all plate operations (including open and closed configurations), with the two plates opening and closing like a book. In some embodiments, the two wafers are rectangular (or square) in shape, and the two sides of the rectangle are joined together during all wafer operations.

In some embodiments, an open configuration is one in which the plates are spaced far apart such that the sample is placed on one plate of a pair without being obstructed by the other plate of the pair.

In some embodiments, an open configuration provides a configuration in which the plates are placed far apart from each other, whereby the sample is placed directly on one plate as if the other plate did not exist.

In some embodiments, the open configuration includes a configuration in which a pair of plates are separated from each other by a distance of at least 10 nm, at least 100 nm, at least 1000 nm, at least 0.01 cm, at least 0 .1 cm, at least 0.5 cm, at least 1 cm, at least 2 cm or at least 5 cm or between any two of these values.

In some embodiments, the open configuration provides a configuration in which the pair of plates are oriented in different directions. In some embodiments, the open configuration provides a configuration that defines an access gap between a pair of wafers into which they are driven to effect sample addition.

In some embodiments, the open configuration provides a configuration where each wafer has a sample contact surface, and where at least one of the wafers' contact surfaces is uncovered when the wafers are in the open configuration.

Closed configuration and sample thickness adjustment. In the present invention, the closed two-plate configuration is a configuration in which the space (ie, distance) between the inner surfaces of the two plates is controlled by spacers between the two plates. Since the inner surfaces (also called "sample surface") of the plates are in contact with the sample during the compression stage of the CROF process, in a closed configuration the thickness of the sample is controlled using spacers.

During the process of transferring the plates from an open configuration to a closed configuration, the plates are facing each other (at least a portion of the plates are facing each other), and a force is applied to transfer the two plates together. If two plates are brought from an open configuration to a closed configuration, then the inner surfaces of the two plates compress the sample placed on the plate(s) to reduce the thickness of the sample (with the sample having open flow in the lateral direction between the plates), and the thickness of the suitable volume of the sample is determined using spacers, plates and the method to be used, as well as the mechanical/fluidic properties of the sample. The closed configuration thickness can be preset for a specific sample and specific spacers, plates, and plate compression method.

The terms “control of the space between the inner surfaces of the plates using spacers”, or “control of the thickness of the sample using plates and a spacer”, or “the thickness of the sample is controlled using spacers and plates” mean that the thickness of the sample in the CROF process is set using specific plates, separators, sample and compression method.

In some embodiments, the adjustable sample thickness in a closed configuration is the same as the height of the spacer; in this case, in a closed configuration, the spacers are in direct contact with both plates (with one plate being the plate on which the spacer is secured and the other plate being the plate that is brought into contact with the spacer).

In certain embodiments, the adjustable sample thickness in a closed configuration is greater than the height of the spacer; in this case, in a closed configuration, the spacers directly contact only the plate on which the spacers are attached or attached to its surface, and indirectly contact the other plate (ie, indirect contact). The term "indirect contact" with the plate means that the spacer and the plate are separated by a thin layer of the sample, which is called the "residual sample layer", and its thickness is called the "residual thickness". In the case of certain spacers and plates, a certain method of pressing the plates and a certain sample, the residual thickness may be preset (preset means before reaching the closed configuration), which results in a preset thickness of the sample at the closed configuration. This is due to the fact that the residual layer is the same under certain conditions (sample, separators, plates and compression force) and can be pre-calibrated and/or calculated. The adjustable thickness of the sample is approximately equal to the height of the separator together with the remaining thickness of the sample.

In many embodiments, the size and shape of the posts are pre-characterized (ie, predetermined) before they are used. Information about the preset parameters is used for further analysis, such as determining the sample volume (or suitable volume), etc.

In some embodiments, adjusting the thickness of the sample includes applying a closing force (compressive force) to the plates to maintain space between the plates.

In some embodiments, adjusting the thickness of the sample includes establishing a space between the plates using spacers, a closing force is applied to the plates and the physical properties of the sample, and optionally the physical properties of the sample include at least one of viscosity and compressibility.

2.2. Plates

In the present invention, in general, CROF plates are made from any material that (i) is capable of being used, in conjunction with spacers, to regulate the thickness of part or all of the sample volume and (ii) does not have significant adverse effects on the sample, assay, or purpose. , which is supposed to be carried out using plates. However, in some embodiments, specific materials (due to their properties) are used to achieve certain purposes for the plate.

In some embodiments, the two wafers have the same or different properties for each of the following: wafer material, wafer thickness, wafer shape, wafer area, wafer flexibility, wafer surface properties, and wafer optical transparency.

Plate materials. The plates are made from a single material, composite materials, multiple materials, multi-layer materials, alloys, or a combination of these. Each of the plate materials is an inorganic material, an organic material, or a mixture, where examples of materials are provided in Mat-1 and Mat-2.

Mat-1. Inorganic wafer materials include, but are not limited to, glass, quartz, oxides, silicon dioxide, silicon nitride, hafnium oxide (HfO), aluminum oxide (AIO), semiconductors (silicon, GaAs, GaN, etc.), metals (e.g. gold, silver, copper, aluminum, Ti, Ni, etc.), ceramic materials or any compositions thereof.

Mat-2. Organic separator materials include, but are not limited to, polymers (eg, plastics) or amorphous organic materials. Polymeric release materials include, but are not limited to, acrylate polymers, vinyl polymers, olefin polymers, cellulose polymers, non-cellulosic polymers, polyester polymers, nylon, cyclic olefin copolymer (COC), poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefin polymer (COP), liquid crystal polymer (LCP), polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP), poly(phenyl ether) (PPE), polystyrene (PS), polyoxymethylene (POM), simple polyether oxyketone (PEEK), polyethersulfone (PES), poly(ethylene phthalate) (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polydimethylsiloxane (PDMS), rubber or any combination thereof.

In some embodiments, each of the plates is independently made from at least one of glass, plastic, ceramic material, and metal. In some embodiments, each plate independently includes at least one of glass, plastic, ceramic material, and metal.

In some embodiments, one wafer differs from another wafer in plan area, thickness, shape, materials, or surface finish. In some embodiments, one wafer is the same as the other wafer in plan area, thickness, shape, materials, or surface finish.

Plate materials are rigid, flexible, or have any flexibility in between. Stiffness (i.e., inelasticity) or flexibility is determined relative to certain compressive forces applied when moving the plates into a closed configuration.

In some embodiments, the choice of rigid or flexible plate is determined based on the requirements of controlling sample thickness uniformity in a closed configuration.

In some embodiments, at least one of the two plates is transparent (to light). In some embodiments, at least a portion or more portions of one plate or both plates are transparent. In some embodiments, the plates are opaque.

Plate thickness. In some embodiments, the average thickness for at least one of the wafers is 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less, 1000 nm or less, 2 μm (microns) or less, 5 μm or less, 10 µm or less, 20 µm or less, 50 µm or less, 100 µm or less, 150 µm or less, 200 µm or less, 300 µm or less, 500 µm or less, 800 µm or less, 1 mm ( millimeter) or less, 2 mm or less, 3 mm or less, or between any two of these values.

In some embodiments, the average thickness for at least one of the plates is no more than 3 mm (millimeters), no more than 5 mm, no more than 10 mm, no more than 20 mm, no more than 50 mm, no more than 100 mm, no more than 500 mm or between any two of these values.

In some embodiments, the thickness of the plate is not uniform throughout the entire length of the plate. The use of different plate thicknesses in different locations can be used to control plate bending, folding, adjusting sample thickness, etc.

Shape and area of plates. In general, the plates can be of any shape as long as the shape provides compression of the open flow of the sample and control of the thickness of the sample. However, in certain embodiments, a particular form may be preferred. The shape of the plate can be circular, elliptical, rectangular, triangular, polygonal, annular, or any overlap of these shapes.

In some embodiments, the two plates may be the same size or shape, or they may be different. The area of the plates depends on the application. The plate area is no more than 1 mm2 (square millimeter), no more than 10 mm2, no more than 100 mm2, no more than 1 cm2 (square centimeter), no more than 5 cm2, no more than 10 cm2, no more than 100 cm2, no more than 500 cm2 , not more than 1000 cm2, not more than 5000 cm2, not more than 10000 cm2 or more than 10000 cm2 or is in any range between any two of these values. The shape of the plate can be rectangular, square, round or other.

In certain embodiments, at least one of the plates has a strip (or tape) shape that is characterized by width, thickness, and length. The width is no more than 0.1 cm (centimeter), no more than 0.5 cm, no more than 1 cm, no more than 5 cm, no more than 10 cm, no more than 50 cm, no more than 100 cm, no more than 500 cm, no more than 1000 cm or between any two of these values. The length can be as required. The strip can be rolled up.

Flatness of the plate surface. In many embodiments, the interior surface of the plates is flat or substantially flat. In certain embodiments, the two inner surfaces are arranged in a closed configuration parallel to each other. Smooth interior surfaces make it easy to quantify and/or control sample thickness simply by applying preset heights of spacers in a closed configuration. For wafer interior surfaces that are not smooth, quantifying and/or monitoring sample thickness in a closed configuration requires knowledge of not only the height of the spacer, but also the exact topology of the interior surface. Determining surface topology requires additional measurements and/or corrections, which can be complex, time-consuming and expensive.

The surface flatness of a wafer is relative to the final thickness of the specimen (the final thickness of the specimen is the thickness in a closed configuration) and is often characterized by the term "relative surface flatness", which is the ratio of the variation in flatness of the wafer surface to the final thickness of the specimen.

In some embodiments, the relative surface area is less than 0.01%, 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 30%, less than 50 %, less than 70%, less than 80%, less than 100%, or between any two of these values.

Parallelism of the surface of the plates. In some embodiments, the two surfaces of the plate are substantially parallel to each other. In certain embodiments, the two surfaces of the plate are not parallel to each other.

Plate flexibility. In some embodiments, the plate is flexible under the compression conditions of the CROF process. In some embodiments, both plates are flexible under the compression conditions of the CROF process. In some embodiments, one plate is rigid and the other plate is flexible under the compression conditions of the CROF process. In some embodiments, both plates are rigid. In some embodiments, both plates are flexible, but have different flexibility.

Optical transparency of the plates. In some embodiments, the plate is optically transparent. In some embodiments, both plates are optically transparent. In some embodiments, one plate is optically transparent and the other plate is opaque. In some embodiments, both plates are opaque. In some embodiments, both plates are transparent, but have different optical clarity. The optical transparency of a wafer refers to part or all of the area of the wafer.

Surface wetting properties. In some embodiments, the plate has an interior surface that wets (ie, the contact angle is less than 90 degrees) the sample, the transfer fluid, or both. In some embodiments, both plates have an inner surface that is wetted by the sample, the transfer fluid, or both; in this case, the wettability is either the same or different. In some embodiments, the plate has an interior surface that wets the sample, the transfer fluid, or both; and the other plate has an inner surface that does not wet (ie, the contact angle is equal to or greater than 90 degrees). Wetting of the inside of the plate refers to part or all of the plate area.

In some embodiments, the inner surface of the wafer has other nano- or microstructures to control the horizontal flow of the sample during CROF. Nano- or microstructures include, but are not limited to, channels, pumps, etc. Nano- or microstructures are also used to control the wetting properties of the inner surface.

2.3. Separators

Separator function. In the present invention, the separators are configured to provide one or any combination of the following functions and properties: the separators are configured to (1) control, in conjunction with the plates, the thickness of the sample or a suitable volume of the sample (preferably, the thickness control is accurate or uniform, or both, in a suitable area); (2) providing compressed controlled open flow (CROF) of the sample on the surface of the plate; (3) not occupying a significant surface area (volume) in a certain area (volume) of the sample; (4) reducing or increasing the effect of precipitation of particles or analytes in the sample; (5) changing and/or controlling the wetting properties of the inner surface of the plates; (6) identifying a location on the wafer, a size scale, and/or information associated with the wafer, or (7) doing any combination of the foregoing.

Design and shape of dividers. To achieve the desired reduction and control of sample thickness, in certain embodiments, spacers are attached to a suitable plate. Generally, the spacer can be of any shape as long as the spacers are capable of adjusting sample thickness during the CROF process, however, shapes are preferred to achieve certain features such as increased uniformity, less overpressure, etc.

The delimiter(s) represent(s) a single delimiter or a plurality of delimiters (eg, an array). In some embodiments, the plurality of spacers is an array of spacers (eg, pillars), wherein the spacing between the spacers is periodic or non-periodic, or is periodic or non-periodic in certain regions of the plates, or has different spacing in different regions of the plates.

There are two types of dividers: open type dividers and enclosing type dividers. An open-type separator is a separator that allows the sample to flow through the separator (i.e., the sample flows around and past the separator. For example, the separator is a stand), and a barrier-type separator is a separator that stops the flow of the sample (i.e. the sample cannot flow outside the separator (for example, the separator is ring-shaped and the sample is inside the ring). Both types of spacers use their height to control the final thickness of the sample in a closed configuration.

In some embodiments, the separators are only open type separators. In some embodiments, the separators are only fence-type separators. In some embodiments, the separators are a combination of open-type separators and enclosing-type separators.

The term "boom separator" means that the separator is shaped like a column, and a bollard separator refers to an object that has a height and shape in horizontal projection that allows a sample to flow around it during compressed open flow.

In some embodiments, the plan view of the column dividers is a shape selected from the group consisting of (i) circular, elliptical, rectangular, triangular, polygonal, ring, star, letter shape (e.g., L-shaped , C-shaped, in the form of letters from A to Z), number-shaped (for example, shapes like 0, 1, 2, 3, 4, ... to 9); (ii) shapes from group (i) with at least one rounded corner; (iii) shapes from group (i) with zigzag or rough edges and (iv) any overlap of (i), (ii) and (iii). In the case of multiple separators, different separators may have different shapes in horizontal projection, as well as different sizes and different distances from adjacent separators.

In some embodiments, the spacers may be and/or may include posts, columns, granules, spheres, and/or other suitable geometric shapes. The shape and horizontal size (i.e., intersection with the corresponding surface of the plate) of the spacers can be any, with the following limitations in certain embodiments: (i) the geometric characteristics of the spacer will not cause significant error in the measurement of the thickness and volume of the sample; or (ii) the geometric characteristics will not prevent the sample from flowing between the plates (i.e. they do not have a confining shape). However, some embodiments require that some separators be closed separators to restrict sample flow.

In some embodiments, the separator shapes have rounded corners. For example, a rectangle-shaped separator has one, some, or all rounded corners (like a circle, instead of a 90-degree angle). A round corner often makes the separator easier to make, and in some cases it is less damaging to biological material.

The sidewall of the posts can be straight, curved, inclined, or have different shapes in different areas of the sidewall. In some embodiments, the dividers are columns having different planar shapes, sidewalls, and column height to column area ratios in horizontal projection.

In a preferred embodiment, the separators are shaped like columns, which allows for open flow.

Separator materials. In the present invention, the spacers are generally made of any material that can be used in conjunction with two plates to regulate the thickness of a suitable sample volume. In some embodiments, the materials for the spacers are different from the materials for the plates. In some embodiments, the materials for the spacers are at least the same as a portion of the materials of the at least one plate.

Separators are made from a single material, composite materials, multiple materials, multi-layer materials, alloys, or a combination of these. Each of the spacer materials is an inorganic material, an organic material, or a mixture, with examples of materials provided in Mat-1 and Mat-2. In a preferred embodiment, the spacers are made of the same material as the plate used in the CROF.

Mechanical strength and flexibility of separators. In some embodiments, the mechanical strength of the spacers is strong enough such that, during compression and in a closed plate configuration, the height of the spacers is the same or substantially the same as when the plates are in an open configuration. In some embodiments, the delimiter differences between an open configuration and a closed configuration may be characterized and predefined.

The spacer material is rigid, flexible, or has any flexibility in between. Stiffness is relative to produce the compressive forces applied to force the plates into a closed configuration: if the spacer does not deform more than 1% of its height under conditions of compression force, then the spacer material is considered rigid, otherwise flexible. If the spacer is made of a flexible material, the final thickness of the sample in a closed configuration can still be predetermined based on the compressive force and mechanical properties of the spacer.

Separator inside the sample. To achieve the desired control and reduction in sample thickness, in particular to achieve high uniformity of sample thickness, in certain embodiments, separators are placed within the sample or a suitable volume of the sample. In some embodiments, there are one or more separators within the sample or a suitable sample volume with appropriate spacing between the separators. In certain embodiments, at least one of the spacers is within the sample, at least two of the spacers are within the sample or a suitable sample volume, or at least "n" spacers are within the sample or a suitable sample volume, where "n" can be defined using the uniformity of sample thickness or the desired flow property of the sample during CROF.

Height of dividers. In some embodiments, all dividers have the same predetermined height. In some embodiments, the dividers have different preset heights. In some embodiments, the dividers may be divided into groups or areas, where each group or area has its own divider height. In certain embodiments, the predetermined height of the dividers is the average height of the dividers. In some embodiments, the dividers are approximately the same height. In some embodiments, the percentage of the number of separators is the same height.

The height of the spacers is selected using the desired adjustable final thickness of the sample and the remaining thickness of the sample. The spacer height (predetermined spacer height) and/or sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 µm or less, 2 µm or less, 3 µm or less, 5 µm or less, 10 µm or less, 20 µm or less, 30 µm or less, 50 µm or less, 100 µm or less, 150 µm or less, 200 µm or less, 300 µm or less, 500 µm or less, 800 µm or less, 1 mm or less, 2 mm or less, 4 mm or less, or between any two of these values.

The spacer height and/or sample thickness is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a particular preferred embodiment, from 1 μm (i.e. e. 1000 nm) to 2 µm in another preferred embodiment, from 2 µm to 3 µm in a particular preferred embodiment, from 3 µm to 5 µm in another preferred embodiment, from 5 µm to 10 µm in a particular preferred embodiment, from 10 µm to 50 µm in another preferred embodiment and from 50 µm to 100 µm in a particular preferred embodiment.

In some embodiments, the spacer height and/or sample thickness is (i) equal to or slightly greater than the minimum analyte size, or (ii) equal to or slightly greater than the maximum analyte size. “A little more” means from about 1% to 5% or more and is any number between these two values.

In some embodiments, the height of the separator and/or the thickness of the sample is greater than the minimum analyte size (e.g., the analyte has an anisotropic shape) but is less than the maximum analyte size.

For example, a red blood cell is disc-shaped with a minimum size of 2 µm (disc thickness) and a maximum size of 11 µm (disc diameter). In one embodiment of the present invention, the spacers are selected to provide a spacing between the inner surfaces of the plates in the respective area of 2 µm (equal to the minimum size) in one embodiment, 2.2 µm in another embodiment, or 3 µm (50% greater than than the minimum size) in another embodiment, however, less than the maximum size of the red blood cell. This embodiment has certain advantages when counting blood cells. In one embodiment, when counting red blood cells, by obtaining a gap between the inner surfaces of 2 or 3 μm and any number between these two values, the undiluted whole blood sample is contained in a gap such that on average each red blood cell (RBC) does not overlap with others, facilitating accurate visual red blood cell counting . (Too much overlap between RBCs can cause serious counting errors.)

In some embodiments of the present invention, the plates and spacers are used to control not only the thickness of the sample, but also the orientation and/or surface density of the analytes/objects in the sample when the plates are in a closed configuration. If the plates are in a closed configuration, then the thinner sample thickness results in fewer analytes/objects per surface area (i.e., lower surface concentration).

The size in the horizontal projection of the separator. In the case of an open type divider, the horizontal dimensions can be characterized by their horizontal dimension (sometimes called width) along the x and y axis - two orthogonal directions. The size of the separator in horizontal projection in each direction is the same or different. In some embodiments, the horizontal dimension for each direction (x or y) is ....

In some embodiments, the ratio of horizontal dimension x to dimension y is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10000, or between any two of these values. In some embodiments, a different ratio is used to control the direction of movement of the sample; the larger the ratio, the more flow is directed along one direction (the larger direction).

In some embodiments, spacers are used with different horizontal dimensions in the x- and y-directions to (a) use the spacers as scale marks to indicate plate orientation, (b) use the spacers to create more sample flow in a preferred direction, or for both.

In a preferred embodiment, the period, width and height.

In some embodiments, all of the separators are of the same shape and size. In some embodiments, each divider has different dimensions in horizontal projection.

In the case of boundary-type separators, in some embodiments, the internal shape and horizontal dimension are selected based on the total volume of the sample to be confined by the containment-type separator(s), where the volume size has been described in the present invention; and in certain embodiments, the external shape and horizontal dimension are selected based on the force required to restrain fluid pressure in the direction of the separator and the compression pressure that compresses the plates.

Aspect ratio of height and average size in the horizontal projection of the separator-column.

In certain embodiments, the aspect ratio of the height and average size in the horizontal projection of the column separator is 100000, 10000, 1000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or in the range between any two of these values.

Divider height precision. The height of the dividers can be controlled precisely. The relative precision of the spacer (i.e., the ratio of the deviation to the desired height of the spacer) is 0.001% or less, 0.01% or less, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or between any of these values.

Distance between separators. The spacers may be a single spacer or multiple spacers on a wafer or in a suitable area of the sample. In some embodiments, the spacers on the wafers are configured and/or arranged in the form of an array, and the array is periodic, non-periodic, or periodic at some locations of the wafer while being non-periodic at other locations.

In some embodiments, the periodic array of spacers has a lattice pattern of square, rectangle, triangle, hexagon, polygon, or any combination thereof, where the combination means that different wafer locations have different spacer arrays.

In some embodiments, the distance between spacers in an array of spacers is periodic (ie, a uniform distance between spacers) in at least one direction of the array. In some embodiments, the spacing between the spacers is configured to improve plate spacing uniformity in a closed configuration.

The distance between adjacent spacers (i.e., distance between spacers) is 1 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 40 μm or less, 50 μm or less, 60 µm or less, 70 µm or less, 80 µm or less, 90 µm or less, 100 µm or less, 200 µm or less, 300 µm or less, 400 µm or less, or between any two of these values.

In certain embodiments, the distance between the spacers is 400 μm or less, 500 μm or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm or less, 10 mm or less, or is in any range between these values. In certain embodiments, the distance between the spacers is 10 mm or less, 20 mm or less, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, or any range between these values.

The distance between adjacent spacers (i.e., spacing between spacers) is selected such that, for certain properties of the wafers and sample, in a closed wafer configuration, the variation in sample thickness between two adjacent spacers is, in some embodiments, no more than 0.5%, 1 %, 5%, 10%, 20%, 30%, 50%, 80%, or any other range in between; or in certain embodiments, no more than 80%, 100%, 200%, 400%, or a range between any two of these values.

Obviously, to maintain a certain variation in sample thickness between two adjacent spacers, if a more flexible plate is used, a smaller distance between the spacers is required.

- It is necessary to determine the accuracy of the distance between the dividers.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 2 to 4 μm, an average horizontal dimension of 5 to 20 μm, and a space between the spacers of 1 μm to 100 μm.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 2 to 4 μm, an average horizontal dimension of 5 to 20 μm, and a space between the spacers of 100 μm to 250 μm.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 4 to 50 μm, an average horizontal dimension of 5 to 20 μm, and a space between spacers of 1 μm to 100 μm.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 4 to 50 μm, an average horizontal dimension of 5 to 20 μm, and a space between the spacers of 100 μm to 250 μm.

The spacing in the spacer array is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a particular preferred embodiment, from 1 μm (i.e. 1000 nm) to 2 µm in another preferred embodiment, from 2 µm to 3 µm in a particular preferred embodiment, from 3 µm to 5 µm in another preferred embodiment, from 5 µm to 10 µm in a particular preferred embodiment, from 10 µm to 50 µm in another preferred embodiment, from 50 µm to 100 µm in a particular preferred embodiment, from 100 µm to 175 µm in a particular preferred embodiment, and from 175 to 300 µm in a particular preferred embodiment.

Separator density. The separators are arranged on the corresponding plates with a surface distribution density of more than one per µm ² , more than one per 10 µm ² , more than one per 100 µm ² , more than one per 500 µm ² , more than one per 1000 µm ² , more than one per 5000 µm ² , more than one per 0.01 mm ² , more than one per 0.1 mm ² , more than one per 1 mm ² , more than one per 5 mm ² , more than one per 10 mm ² , more than one per 100 mm ² , more one per 1000 mm ² , more than one per 10,000 mm ² or a range between any two of these values.

(3) the separators are designed to not occupy a significant surface area (volume) in a certain area (volume) of the sample;

The ratio of separator volume to sample volume. In many embodiments, the ratio of separator volume (i.e., separator volume) to sample volume (i.e., sample volume) and/or the ratio of the volume of separators that are contained within a suitable sample volume to a suitable sample volume is controlled to achieve certain benefits. Benefits include, but are not limited to, control of sample thickness uniformity, analyte uniformity, sample flow properties (eg, flow rate, flow direction, etc.).

In certain embodiments, the ratio of the volume of separator d) to the sample volume and/or the ratio of the volume of separators that are contained within a suitable sample volume to a suitable sample volume is less than 100%, not more than 99%, not more than 70%, not more than 50% , no more than 30%, no more than 10%, no more than 5%, no more than 3%, no more than 1%, no more than 0.1%, no more than 0.01%, no more than 0.001% or is in the range between any from these values.

Separators mounted on plates. The distance between the spacers and the orientation of the spacers, which play a key role in the present invention, are preferably maintained during the process of converting the plates from the open configuration to the closed configuration, and/or preferably they are predetermined before the conversion process from the open configuration to the closed configuration.

Some embodiments of the present invention provide that the spacers are secured to one of the plates before converting the plates into a closed configuration. The term "spacer is secured to its respective plate" means that the spacer is attached to the plate and the attachment is maintained during use of the plate. An example of a "spacer being secured to its respective plate" is where the spacer is formed monolithically as one piece of plate material and the position of the spacer relative to the surface of the plate does not change. An example of "the spacer not being secured to its respective plate" is where the spacer is adhered to the plate by an adhesive, but during use of the plate the adhesive fails to hold the spacer in its original location on the surface of the plate (i.e. the separator moves from its original location on the surface of the plate).

In some embodiments, at least one of the spacers is secured to its respective plate. In certain embodiments, two spacers are secured to their respective plates. In certain embodiments, most of the spacers are secured to their respective plates. In certain embodiments, all of the spacers are secured to their respective plates.

In some embodiments, the separator is mounted monolithically to the plate.

In some embodiments, the spacers are secured to their respective plate using one or any combination of the following methods and/or configurations: attached, bonded, fused, imprinted, and etched.

The term "imprinted" means that the spacer and plate are secured monolithically by imprinting (eg, embossing) an area of material to form the spacer on the surface of the plate. The material may be a single layer of material or multiple layers of material.

The term "etched" means that the spacer and wafer are attached monolithically by etching a section of material to form the spacer on the surface of the wafer. The material may be a single layer of material or multiple layers of material.

The term "fused" means that the spacer and plate are secured monolithically by attaching the spacer and plate together, wherein the source materials for the spacer and plate are fused to each other, and there is a clear material boundary between the two materials after fusion.

The term "bonded" means that the spacer and plate are secured monolithically by bonding the spacer and plate through adhesion.

The term "attached" means that the spacer and plate are attached together.

In some embodiments, the spacers and plate are made from the same materials. In another embodiment, the spacers and plate are made of different materials. In another embodiment, the spacer and plate are formed as one piece. In another embodiment, one end of the spacer is fixed to its corresponding plate, with the end open to accommodate different configurations of the two plates.

In another embodiment, each of the spacers is independently at least attached, bonded, fused, imprinted or etched into a respective plate. The term "independently" means that one spacer is secured to its respective plate using the same or a different method, which is selected from the methods associated with attachment, bonding, merging, imprinting and etching in the corresponding plate.

In some embodiments, at least the distance between two spacers is predetermined (“preset distance between spacers” means that the distance is known when the user applies the plates.).

In some embodiments of all of the methods and devices described herein, there are additional spacers in addition to the fixed spacers.

Specimen specific thickness. In the present invention, it has been observed that greater plate holding force (i.e., the force that holds two plates together) can be achieved by using a smaller space between the plates (for a certain sample area) or a larger sample area (for a certain space between plates), or both.

In some embodiments, at least one of the plates is transparent in a portion covering a corresponding area, each plate having an internal surface configured to contact the sample in a closed configuration; the inner surfaces of the plates are substantially parallel to each other in a closed configuration; the internal surfaces of the plates are substantially flat, apart from the locations where the spacers are located; or any combination thereof.

2.4. Final sample thickness and uniformity

In some embodiments, substantially flat is defined relative to the final sample thickness and is characterized, depending on embodiments and applications, by having a ratio to sample thickness of less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5 % or less than 10% or between any two of these values.

In some embodiments, the flatness relative to sample thickness may be less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 50%, or less than 100 % or is in the range between any two of these values.

In some embodiments, substantially flat may mean that the surface flatness variation itself (measured based on average thickness) is less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, or less than 10 % or is in the range between any two of these values. Generally, the flatness with respect to plate thickness can be less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%, less than 50% or less than 100%. or is in the range between any two of these values.

2.5. Methods for making separators

Spacers can be fabricated on the wafer in a variety of ways, using lithography, etching, embossing (nanoimprinting), deposition, explosion lithography, fusion, or a combination thereof. In some embodiments, the separators are directly imprinted or imprinted on the plates. In some embodiments, the separators are imprinted into a material (eg, plastics) that is placed on the plates. In certain embodiments, the spacers are manufactured directly by embossing onto the surface of the CROF plate. Nanoimprinting can be done using roll-by-roll technology using a roller imprinter or applied through planar nanoimprinting. This process has a great economic advantage and therefore reduces the cost.

In some embodiments, the separators are placed on plates. The placement may be evaporation, pasting or blast lithography. When pasting, the separator is first made on a carrier, then the separator is transferred from the carrier to the plate. In explosive lithography, the material to be removed is first placed on a plate and holes are created in the material; the bottom of the hole is oriented towards the surface of the plate and then the spacer material is placed in the hole and then the material to be removed is removed, leaving only the spacers on the surface of the plate. In some embodiments, the separators placed on the wafer are fused to the wafer. In some embodiments, the separator and plates are manufactured in the same process. One process involves imprinting (eg, embossing, molding) or synthesis.

In some embodiments, the at least two spacers are secured to a respective plate through different manufacturing methods, and optionally the different manufacturing methods include at least one of placement, bonding, merging, imprinting, and etching.

In some embodiments, one or more spacers are secured to the respective plate(s) by a manufacturing process involving bonding, fusing, imprinting or etching, or any combination thereof.

In some embodiments, manufacturing methods for forming such monolithic spacers on a wafer include a method involving bonding, merging, imprinting or etching, or any combination thereof.

2.6. Scale marks

The term “scale mark(s)” refers to scale mark(s) that are capable of facilitating quantification (eg, dimensional measurement) or control of the relevant area and/or relative volume of a sample. In some embodiments, the scale marks are on the first plate or the second plate, on both plates, on one surface of the plate, on both surfaces of the plate, between the plates, near the plates, or any combination thereof. In some embodiments, the scale marks are attached to the first plate or the second plate, to both plates, to one surface of the plate, to both surfaces of the plate, between the plates, near the plates, or any combination thereof. In some embodiments, the scale marks are located on the first plate or the second plate, on both plates, on one surface of the plate, on both surfaces of the plate, between the plates, near the plates, or any combination thereof. In some embodiments, some dividers are secured and some dividers are positioned.

In some embodiments, the scale marks are etched scale marks, applied materials, or imprinted materials. In certain embodiments, the materials absorb light, reflect light, emit light, or any combination thereof.

In some embodiments, the scale marks are a single object or multiple objects with known dimensions and/or known spacing. Examples of objects include, but are not limited to, rectangles, cylinders, or circles.

In some embodiments, the scale marks are in the range of nanometers (nm), microns (μm), or millimeters (mm), or other sizes.

In some embodiments, the scale marks are a ruler that has a scale of scale marks that are configured to measure the size of an object. In some embodiments, the scale marks are on the scale of nanometers (nm), microns (μm), or millimeters (mm), or other sizes. In some embodiments, the scale marks are etched scale marks, applied materials, or imprinted materials. In some embodiments, the scale mark materials are materials that absorb light, reflect light, scatter light, interfere with light, diffuse light, emit light, or any combination thereof.

In some embodiments, the marks are spacers that perform the dual functions of “adjusting sample thickness” and “providing scale marks and/or dimensional scale measurement.” For example, a rectangular spacer with a known size or two spacers with a known separation distance can be used to measure the size associated with the sample around the spacer(s). Based on the measured sample size, a suitable sample volume can be calculated.

In some embodiments, the scale marks are configured to at least partially define the boundary of a suitable sample volume.

In some embodiments, the at least one scale mark is configured to produce a known dimension that is parallel to the area plane of a suitable sample volume in horizontal projection.

In some embodiments, at least a pair of scale marks are separated by a known distance that is parallel to the area plane in the horizontal projection.

In some embodiments, the scale marks are optically detectable.

In some embodiments, each scale mark independently provides at least one of light absorption, light reflection, light scattering, light diffraction, and light emission.

In some embodiments, the scale marks are arranged in a regular array with a known horizontal projection space.

In some embodiments, each scale mark independently has a horizontal profile that is at least one of square, rectangular, polygonal, and circular.

In some embodiments, at least one of the scale marks is attached, connected, fused, imprinted and etched onto one of the plates.

In some embodiments, at least one of the scale marks is one of the delimiters.

In some embodiments, some separators also act as a scale mark to quantify the appropriate sample volume.

In certain embodiments, the binding site(s) (which immobilizes the analytes), storage sites, or the like serve as the scale mark(s). In one embodiment, an area of known lateral size interacts with light to generate a detectable signal that gives the known size of the area in a horizontal projection, thereby serving as the scale mark(s).

In another embodiment, the size of the patches is predetermined before the CROF process, and the thickness of the portion of the sample placed on the patch when the plates are in a closed configuration is substantially less than the average horizontal dimension of the patch, thereby controlling the incubation time such that after incubation, (1) most of the analytes/object that binds to the binding site exits the sample volume, which is located on the surface of the binding site, or (2) most of the reagent that mixes (diffuses) in the sample volume, which is located on the surface of the binding site, exits the binding site. In these cases, a suitable volume of sample for binding or mixing the reagent is a volume that is approximately equal to the predetermined area of the site multiplied by the thickness of the sample at the site. The main reason for this to be possible is that, given a certain incubation time, the analytes/object in the sample volume outside the appropriate volume do not have enough time to diffuse into the binding site, or the reagents in the storage site do not have enough time to diffuse into the sample volume beyond suitable volume.

An example to illustrate a method for measuring and/or monitoring the appropriate area and volume using a region of known size and with an incubation time limit is that the assay has a binding site (e.g., a region with capture means) measuring 1000 μm by 1000 μm on the first plate in the CROF process (which has a surface area larger than the bonding area); In a closed plate configuration, the sample with analytes is located above the binding site, has a thickness of approximately 20 μm (in the region of the binding site) and an area that is larger than the binding site, and is incubated for a time equal to the time of diffusion of the target analyte/object through the thickness of the sample. In this case, most of the analytes/object that binds to the binding site comes out of the volume of the sample that is located on the surface of the binding site, which is 1000 µm by 1000 µm by 20 µm = 0.02 p, since the analytes are in the part of the sample that located at a distance of 20 µm from the binding site, do not have time to diffuse to the binding site (statistically). In this case, if the signal resulting from the capture of analytes/object by the binding site is measured after incubation, the concentration of the analyte/object in the corresponding area and suitable volume of the sample can be determined based on the information (obtained from the binding site) about the corresponding area and suitable volume . Analyte concentration is quantified using the amount of analytes captured by the binding site divided by an appropriate volume.

In some embodiments, the suitable volume is approximately equal to the area of the binding site multiplied by the thickness of the sample, and the concentration of the target analyte in the sample is approximately equal to the amount of analyte captured by the binding site divided by the suitable sample volume. This accuracy of the method for quantifying the volume of the target analyte increases if the ratio of the size of the binding site to the thickness of the sample becomes large (assuming that the incubation time is approximately equal to the time of diffusion of the target analyte in the sample over a distance equal to the thickness of the sample).

Distribution time in CROF. In the present invention, in the methods and devices of all points in which the sample is distributed by two plates, the time of distribution of the sample to the final thickness in the closed configuration is 0.001 s or less, 0.01 s, 0.1 s, 1 s, 5 s, 10s, 20s, 30s, 60s, 90s, 100s, 150s, 200s, 300s, 500s, 1000s or between any two of these values.

In the methods and devices of all items that distribute the sample using two plates, in a preferred embodiment, the time of distribution of the sample to the final thickness in a closed configuration is 0.001 s or less, 0.01 s, 0.1 s, 1 s, 3 s , 5 s, 10 s, 20 s, 30 s, 60 s, 90 s, 100 s, 150 s, or between any two of these values.

In the methods and devices of all items that distribute the sample using two plates, in a preferred embodiment, the time of distribution of the sample to the final thickness in a closed configuration is 0.001 s or less, 0.01 s, 0.1 s, 1 s, 3 s , 5 s, 10 s, 20 s, 30 s, 60 s, 90 s, or between any two of these values.

In the methods and devices of all items that distribute the sample using two plates, in a preferred embodiment, the time of distribution of the sample to the final thickness in a closed configuration is 0.001 s or less, 0.01 s, 0.1 s, 1 s, 3 s , 5 s, 10 s, 20 s, 30 s, or between any two of these values.

In the methods and devices of all items that distribute the sample using two plates, in a preferred embodiment, the time of distribution of the sample to the final thickness in a closed configuration is 0.001 s or less, 0.01 s, 0.1 s, 1 s, 3 s , 5 s, 10 s, or between any two of these values.

In the methods and devices of all items that distribute the sample using two plates, in a preferred embodiment, the time of distribution of the sample to the final thickness in a closed configuration is 0.001 s or less, 0.01 s, 0.1 s, 1 s, 3 s or is in the range between any two of these values.

The embodiments and any combinations thereof described in section 3 are applicable (ie, combined) to other embodiments throughout the specification of the present invention.

In one preferred embodiment, the spacers are manufactured monolithically on the X-plate by embossing (eg, nanoimprinting) a thin plastic film using a mold and are made from the same materials.

In one preferred embodiment, the spacers are monolithically manufactured on the X-plate by embossing (eg, nanoimprinting) a thin plastic film using a mold and are made from the same materials, and the thickness of the X-plate is from 50 μm to 500 μm.

In one preferred embodiment, the spacers are monolithically manufactured on the X-plate by embossing (eg, nanoimprinting) a thin plastic film using a mold, and are made from the same materials, and the thickness of the X-plate is from 50 μm to 250 μm.

In one preferred embodiment, the spacers are monolithically manufactured on the X-plate and are made from the same materials, and the thickness of the X-plate is from 50 μm to 500 μm.

In one preferred embodiment, the spacers are monolithically manufactured on the X-plate using a thin plastic film using a mold, and are made from the same materials, and the thickness of the X-plate is from 50 μm to 250 μm.

In one preferred embodiment, the spacers are monolithically manufactured on an X-plate by embossing (eg, nanoimprinting) a thin plastic film using a mold and made from the same materials, where the plastic film is either PMMA (polymethyl methacrylate) or PS (polystyrene).

In one preferred embodiment, the spacers are monolithically manufactured on an X-plate by embossing (e.g., nanoimprinting) a thin plastic film using a mold and made from the same materials, where the plastic film is either PMMA (polymethyl methacrylate) or PS (polystyrene), and the thickness X-plates range from 50 µm to 500 µm.

In one preferred embodiment, the spacers are monolithically manufactured on an X-plate by embossing (e.g., nanoimprinting) a thin plastic film using a mold and made from the same materials, where the plastic film is either PMMA (polymethyl methacrylate) or PS (polystyrene), and the thickness X-plates range from 50 µm to 250 µm.

In one preferred embodiment, the spacers are monolithically manufactured on an X-plate by embossing (e.g., nanoimprinting) a thin plastic film using a mold and made from the same materials, where the plastic film is either PMMA (polymethyl methacrylate) or PS (polystyrene), and the spacers are either square or rectangular in shape and have equal height dividers.

In one preferred embodiment, the dividers are square or rectangular in shape (with or without round corners).

In one preferred embodiment, the spacers are in the form of square or rectangular columns, wherein the column width (the width of the spacer in each horizontal direction) is from 1 μm to 200 μm; the bar spacing (ie, space between spacers) is from 2 μm to 2000 μm, and the bar height (ie, spacer height) is from 1 μm to 100 μm.

In one preferred embodiment, the spacers made of PMMA or PS are in the form of square or rectangular pillars, wherein the column width (the width of the spacer in each horizontal direction) is from 1 μm to 200 μm; the bar spacing (ie, space between spacers) is from 2 μm to 2000 μm, and the bar height (ie, spacer height) is from 1 μm to 100 μm.

In one preferred embodiment, the spacers are monolithically manufactured on an X-plate and made of plastic materials, and the spacers are in the form of square or rectangular posts, the width of the post (the width of the spacer in each horizontal direction) being from 1 μm to 200 μm; the bar spacing (ie, space between spacers) is from 2 μm to 2000 μm, and the bar height (ie, spacer height) is from 1 μm to 100 μm.

In one preferred embodiment, the spacers are monolithically manufactured on the X-plate and made from the same materials, and the spacers are in the form of square or rectangular pillars, wherein the column width (the width of the spacer in each horizontal direction) is from 1 μm to 200 μm; the bar spacing (ie, space between spacers) is from 2 μm to 2000 μm, and the bar height (ie, spacer height) is from 1 μm to 10 μm.

In one preferred embodiment, the spacers are monolithically manufactured on an X-plate and made of uniform materials selected from PS or PMMA or other plastic materials, and the spacers are in the form of square or rectangular posts, wherein the post width (the width of the spacer in each horizontal direction) is from 1 µm to 200 µm; the bar spacing (ie, space between spacers) is from 2 μm to 2000 μm, and the bar height (ie, spacer height) is from 10 μm to 50 μm.

In one preferred embodiment of the CROF device, one plate is an X plate and the other plate is a flat thin film, wherein the thickness of at least one of the plates is in the range of 10 μm to 250 μm; wherein the spacers are secured to an X-plate, and where the plates and spacers may be of the same or different materials and are made of PMMA (polymethyl methacrylate), PS (polystyrene) or a material with similar mechanical properties to those of PMMA or PS.

In one preferred embodiment of the CROF device, one plate is an X plate and the other plate is a flat thin film, wherein the thickness of at least one of the plates is in the range of 250 μm to 500 μm; wherein the spacers are secured to an X-plate, and where the plates and spacers may be of the same or different materials and are made of PMMA (polymethyl methacrylate), PS (polystyrene) or a material with similar mechanical properties to those of PMMA or PS.

In one preferred embodiment of the CROF device, one plate is an X plate and the other plate is a perfectly flat thin film, wherein the thickness of at least one of the plates is in the range of 10 μm to 250 μm; where the spacers are fixed to the X-plate, and they are an array of square or rectangular pillars with a column width (the width of the spacer in each horizontal direction) from 1 μm to 200 μm; a post spacing (i.e. space between spacers) from 2 µm to 2000 µm and a post height (i.e. spacer height) from 1 µm to 100 µm, and where the plates and spacers may be of the same or different materials, and they are made of PMMA (polymethyl methacrylate), PS (polystyrene) or a material with similar mechanical properties to PMMA or PS.

“Similar” in the above points means that the difference in mechanical properties is within 60%.

Ferrule. In some embodiments, a safety ring is provided to prevent the sample from leaking from the surface of the plate. In some embodiments, the safety ring is a boundary wall around the sample area. The height of the wall is equal to the height of the separator or different from the height of the separator. The wall may be located at a considerable distance from the sample measurement area.

The movable plates in the CROF process may include a hinge, post, or some other orientation system that is configured to allow the plates to transition between and/or be associated with an open configuration and a closed configuration. The movable plates may be connected together by one or more connections such that an opening remains for access to the space between the plates (for example, for insertion and/or removal of a sample), provided that at least one of the connections and/or at least At least one of the plates is flexible enough to achieve the open and closed configurations described. The diaphragm pump is not considered to be moving plate(s).

3. Uniform space between plates and sample thickness (U)

In many applications of the CROF process, it is desirable to increase the uniformity of the space between the plates and thereby the thickness of the sample in a closed configuration, in particular when the space is in the micron and/or nanoscale. High homogeneity can improve the homogeneity of the analysis. The present invention provides means for increasing uniformity.

Factors that can disrupt the uniformity of the space between plates in CROF include (a) local plate bending, (b) roughness of the internal surface of the plate, and (c) dust particles. The smaller the final space between the plates, the worse the effects of these factors become.

To increase the uniformity of the spaces (thus the thickness of the sample), the present invention applies a certain pattern in the plates (mechanical force, thickness, etc.), size of the spaces, number of spacers, location of spacers, spacing between spacers, precision of spacer height, among others, to overcome these factors that cause heterogeneity.

Smoothness of internal surfaces

3.1 Apply spacer spacing to achieve uniform sample thickness for flexible plate

Some applications require that one or both CROF plates be flexible. However, as shown in FIG. 5a, in the case of a flexible plate (e.g., a plastic thin plate), if the distance between the spacers is too large, then during the CROF process the flexibility of the plate(s) may cause local bending (e.g., bowing, namely inward bending) of the plate at locations , which are located between two adjacent spacers, resulting in low uniformity of sample thickness. Low uniformity of sample thickness has many disadvantages, such as significant errors in determining sample volume and/or analyte concentrations, variations in incubation time, etc.

One embodiment of the present invention provides a solution that reduces local bending, and therefore the variation in the final thickness of the sample, by using proper spacing between spacers. As shown in FIG. 5, the CROF device has one rigid plate with a flat sample surface and one flexible plate that has a local bend between two adjacent spacers if the distance between the spacers is too large (Fig. 5a). To reduce local bending, the distance between the spacers is set so that it is equal to or less than the critical bending span of the flexible plate (Fig. 5b). If both plates are flexible, the distance between the spacers must be less than the lesser of the critical bending span of the two plates.

U1. A method for regulating the thickness uniformity of a suitable sample volume using two plates, comprising:

(a) obtaining a sample, where the thickness of a suitable volume of the sample is subject to regulation;

(b) providing two plates that are movable relative to each other in different configurations; where one or both plates are flexible; and where one or both of the plates comprise spacers, the spacers are characterized by a predetermined distance between the plates and a height, and each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted using the plates and spacers;

wherein, in the case of certain plates, the spacers are configured to obtain a thickness of a suitable sample volume having a variation within a certain area that is less than a predetermined value; and wherein the suitable volume is part or all of the sample volume.

In the method of item U1, the configuration of the separators involves selecting the appropriate distance between the separators. In some embodiments, the distance between the spacers is selected such that, given the allowable variation in sample thickness, the specified two plates, and the compression method, the deflection of the two plates under the conditions of the compression method is equal to or less than the allowable variation in sample thickness. The adjustable specimen thickness in a closed configuration may be thinner than the maximum specimen thickness when the plates are in an open configuration.

U2. A device for adjusting the thickness of a suitable sample volume, containing

a first plate and a second plate that are movable relative to each other in different configurations;

where one or both plates are flexible, and where one or both plates contain spacers, the spacers are characterized by a predetermined distance between the plates and height, and each of the spacers is fixed to its respective plate;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted using the plates and spacers;

wherein, in the case of certain plates, the spacers are configured to obtain a thickness of a suitable volume of the sample having a thickness variation within a certain area less than a predetermined value; and wherein the suitable volume is part or all of the sample volume.

In the device according to item U2, the configuration of the spacers and plates allows for the selection of the appropriate distance between the spacers. In some embodiments, the distance between the spacers is selected such that, given the allowable variation in sample thickness, the specified two plates, and the compression method, the deflection of the two plates under the conditions of the compression method is equal to or less than the allowable variation in sample thickness. The adjustable specimen thickness in a closed configuration may be thinner than the maximum specimen thickness when the plates are in an open configuration.

In some embodiments, the small space between the spacers also allows the use of flexible thin films (eg, 100 μm thick plastic film) by providing a space between the spacers that is less than the bend of the plate between the two spacers.

In some embodiments, to obtain uniform sample thickness over a large area in a closed configuration, in the case of a certain allowable maximum bending of the flexible plate, the ratio of the distance between the spacers and the span of the critical bending of the plate is no more than 0.001%, no more than 0.001%, no more than 0.001%, no more than 0.01%, no more than 0.1%, no more than 1%, no more than 10%, no more than 20%, no more than 50%, no more than 70%, no more than 100% or is in the range between any two of these values.

3.2. Application of flexible plate(s) and separators to overcome the influence of dust in CROF

One problem that must be overcome in the CROF process is that dust thicker than the spacer height can disrupt the regulation of the spacers to achieve the intended final space between the plates (thus the final sample thickness) (shown in Fig. 6a). When using two rigid plates, one such dust particle can disrupt the regulation of the separators within the entire plate area.

Certain embodiments of the present invention solve the problem of using proper flex plate(s) and spacer spacing to limit dust exposure to a small area around the dust, while ensuring that the area outside the small area has a finite space between plates and sample thickness, set (adjustable) using spacers).

For example, in FIG. 6b shows that to overcome the effects of dust, one flexible plate with proper flexibility is used to limit the dust area and is used in conjunction with a rigid plate that has fixed separators. In fig. 6c shows another embodiment of dust mitigation where the separators are secured to a flexible plate. Obviously there is another solution to make both plates flexible.

The appropriate wafer flexibility to minimize dust effects during the CROF process can be selected based on the thickness and mechanical properties of the wafer. Based on the study shown in the example, the preferred embodiments are as follows.

U3. A method of minimizing the influence of dust on the thickness control of a suitable sample volume, comprising:

(a) obtaining a sample, where the thickness of a suitable volume of the sample is subject to regulation;

(b) providing two plates that are movable relative to each other in different configurations; where one or both plates are flexible; and where one or both of the plates comprise spacers, the spacers are characterized by a predetermined distance between the plates and a height, and each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, separators, a suitable volume of the sample and one or more of the dust particles thicker than the height are located between the plates, with In this case, the thickness of the appropriate sample volume is adjusted using plates and dividers;

wherein the separators and plates are configured to minimize the area between the two plates that is susceptible to dust; wherein the dust affected area is the area where dust prevents the separators from adjusting the final space between the plates in the area in a closed plate configuration in the same manner as if there was no dust present; and wherein the suitable volume is part or all of the sample volume.

In the method of claim U3, configuring the separators and plates to minimize the dust-influenced area comprises selecting an appropriate thickness and mechanical property of the flexible plate.

In some embodiments, the distance between the spacers is selected such that, given the allowable variation in sample thickness, the specified two plates, and the compression method, the deflection of the two plates under the conditions of the compression method is equal to or less than the allowable variation in sample thickness. The adjustable specimen thickness in a closed configuration may be thinner than the maximum specimen thickness when the plates are in an open configuration.

It is necessary to determine the flexibility of the plate.

U4. A device for minimizing the effects of dust on thickness control of a suitable sample volume, comprising:

a first plate and a second plate that are movable relative to each other in different configurations, and each plate has a sample contact surface that contacts the sample, wherein one or both plates are flexible;

spacers on the sample contact surface of one or both of the plates, where the spacers are characterized by a predetermined distance between the plates and height, and each of the spacers is fixed to its respective plate;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, the plates face each other, separators, a suitable sample volume and one or a plurality of dust particles with a thickness greater than the height of the separator are located between the plates, the thickness of the suitable sample volume is controlled by the plates and separators; And

wherein the separators and plates are configured to minimize the area between the two plates that is susceptible to dust; where the area affected by dust is the area of the inner surface of the plates where the plates and spacers are no longer able to regulate the thickness of the sample because the area no longer contains dust; and wherein the suitable volume is part or all of the sample volume.

3.3. Using spacers to reduce the effects of variations in surface flatness

In fact, no surface of the plate is completely flat. As shown in FIG. 7a, in CROF the variation in surface evenness can be significantly greater compared to the desired sample thickness, which can lead to large errors in determining the sample thickness. As the final sample thickness in CROF becomes very thin (e.g. in the micro- or nanometer range), variations in surface flatness can increasingly lead to significant errors.

The surface flatness variation can be characterized by the plate surface flatness variation distance, X, which is the distance from the local maximum of the surface height to the adjacent local minimum (shown in Fig. 7b).

The present invention provides means that make the variation in the final sample thickness in the closed configuration during the CROF process less than the variation in surface flatness on the surface of the wafers sample that existed when the wafers were in the open configuration. The main approach in the present invention to achieve uniform thickness of the final sample is the use of a flexible plate, proper distance between spacers and proper compression force (shown in Fig. 7c and d).

When considering the case where one rigid plate and a flexible plate are used in the CROF process, in an open plate configuration, the surface of the rigid plate sample has high flatness, but the surface of the flexible plate sample has a large variation in flatness (i.e., significant compared to the expected final thickness of the sample). , as shown in Fig. 7a and b. The present invention corrects the initial surface flatness variation of a sample in an open configuration (eg, obtaining a smaller flatness variation) by (i) a spacing distance that is smaller than the initial surface flatness variation distance; (ii) proper compressive force and/or proper capillary force between the sample and the plates in a closed configuration with deformation of the flexible plate; and (iii) proper flexibility of the flexible plate such that in the final configuration of the plates the surface of the flexible plate sample is deformed and follows the contour of the flat surface of the rigid one. plates (Fig. 7c). In addition, in order to reduce the variation in the final thickness of the sample, the distance between the spacers should also be less than the critical bending range of the flexible plate.

The above method for correcting surface flatness variation also works in cases where (a) the rigid plate has an initial large variation in the flatness of the specimen surface while the flexible plate has a flat specimen surface, (b) both the flexible plate and the rigid plate have significant variation flatness on their respective specimen surface, and (c) both plates are flexible and the specimen surface(s) of one or both plates have significant variation in surface flatness (FIG. 7d).

U5. A method for reducing the effect of variation in plate surface flatness on the final thickness uniformity of a suitable sample volume in the CROF process, comprising:

(a) obtaining a sample, where the thickness of a suitable volume of the sample is subject to regulation;

(b) providing two plates that are movable relative to each other in different configurations; where one or both plates are flexible; where one or both plates have a variation in surface flatness, and where one or both plates include spacers, the spacers are characterized by a predetermined height, and each of the spacers is fixed to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted using the plates and spacers;

wherein the spacers and plates are configured to produce a thickness variation of a suitable sample volume in a closed configuration that is less than a variation in the surface flatness of the plate(s) in an open configuration, and where the suitable volume is part or all of the sample volume.

U6. A device for reducing the effect of variation in the flatness of a plate surface on the uniformity of thickness control of a suitable sample volume, comprising:

a first plate and a second plate that are movable relative to each other into different configurations, where one or both plates are flexible and one or both plates have surface flatness variations;

dividers that are fixed to one or both plates and that have a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted using the plates and spacers;

wherein the spacers and plates are configured to produce a variation in the thickness of a suitable sample volume in a closed configuration that is less than the variation in the surface flatness of the plate(s) in an open configuration, and where the suitable volume is part or all of the volume of the sample, and where the average size of the suitable volume is greater than the variation in plate surface flatness in an open configuration.

In the method of item U5 and the device of item U6, the configuration of the spacers and plates to reduce the effect of variation in the flatness of the plate surface on the uniformity of the final thickness of the suitable sample volume provides for an appropriate distance between the spacers (IDS). One preferred embodiment is that the IDS is equal to or less than the original plate surface flatness distance in an open configuration.

In the method and apparatus of claims U5 and U6, in some embodiments, (1) the spacers are located within the sample in a closed configuration, (2) the spacers are secured to a suitable plate, (3) there is a short distance between the spacers, or (4) any combination thereof.

The methods and devices in U1 to U8 for configuring spacers and plates that make the thickness of a suitable sample volume uniform are provided in the embodiment described above. In some embodiments, a predetermined distance between the spacers is configured to limit local bending of the plates between two spacers, and where the suitable volume is part or all of the volume of the sample.

This includes cases where one or both plates are flexible and have different flexibility (eg 100 µm thick PMMA or PS).

In one preferred embodiment, one plate is PMMA. In one preferred embodiment, one plate (first plate) is 0.5 to 1.5 mm thick glass and does not have any separator, and the other (second) plate is 175 µm thick PMMA film and has an array of separators, where the separator is a rectangular shaped column (40 µm in size in the x-direction and 30 µm in the y-direction) with round corners and a period of 120 µm in the x-direction and 110 µm in the y-direction (leading to the formation of space between spacers, being 80 µm in both x- and y-directions).

In the methods and devices of section 3, some embodiments in the case of spacers within the sample in a closed configuration, the materials of the spacers and plates are provided in the embodiments of section 2.

In the methods or devices of claims U1-6, in some embodiments, the ratio of column width (or average horizontal dimension) to column height is 0.01 or greater, 0.1 or greater, 1 or greater, 1.5 or greater, 2 or more, 3 or more, 5 or more, 10 or more, 50 or more, 100 or more, or between any two of these values.

In the methods and devices of paragraphs U1-U6, in a preferred embodiment, the ratio of the column width (or average size in horizontal projection) and the column height is 1, 1.5, 2, 10, 20, 30, 50, 100 or is in the range between any two of these values.

In the methods and devices of paragraphs U1-6, in some embodiments, the ratio of column spacing to column width (or average horizontal dimension) is 1, 0.1, 1.1, 1.2, 1.5, 1.7 , 2, 3, 5, 7, 10, 20, 50, 100, 500, 1000, 10000 or is in the range between any two of these values.

The ratio of the distance between the separators (columns) to the average width of the separators is equal to or more than 2.

In the methods and devices of paragraphs U1-U6, in a preferred embodiment, the ratio of column spacing to column width (or average horizontal dimension) is 1.2, 1.5, 1.7, 2, 3, 5, 7, 10, 20, 30, or between any two of these values.

In the methods and devices of paragraphs U1 to U6, in a preferred embodiment, the ratio of column spacing to column width (or average horizontal dimension) is 1, 2.1, 5, 1.7, 2, 3, 5, 7, 10, or is in the range between any two of these values.

c) For example, when used in blood cell counting, the preferred X-plate column height is from 1 µm to 5 µm, the column width is from 2 µm to 30 µm, the column spacing is from 4 µm to 300 µm.

d) For example, when used in an immunoassay, the preferred height of the X-plate pillars is from 5 µm to 50 µm, the width of the pillars is from 10 µm to 250 µm, the spacing between pillars is from 20 µm to 2500 µm.

The embodiments and any combinations thereof described in section 3 are applicable (ie, combined) to other embodiments throughout the specification of the present invention.

In some embodiments, other factors are also used to control sample thickness uniformity, these factors include, but are not limited to, sample area, mechanical properties of the wafer, final thickness of the sample in a closed configuration, and wetting properties of the wafer surface.

Below are some preferred embodiments for the methods and devices in section 1 and the remainder of the disclosures.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 2 to 4 μm, an average horizontal dimension of 5 to 20 μm, and a space between the spacers of 1 μm to 100 μm.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 2 to 4 μm, an average horizontal dimension of 5 to 20 μm, and a space between the spacers of 100 μm to 250 μm.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 4 to 50 μm, an average horizontal dimension of 5 to 20 μm, and a space between spacers of 1 μm to 100 μm.

In a preferred embodiment, the spacer is a periodic array of squares, where the spacer is a column that has a height of 4 to 50 μm, an average horizontal dimension of 5 to 20 μm, and a space between the spacers of 100 μm to 250 μm.

The spacing in the spacer array is from 1 nm to 100 nm in one preferred embodiment, from 100 nm to 500 nm in another preferred embodiment, from 500 nm to 1000 nm in a particular preferred embodiment, from 1 μm (i.e. 1000 nm) to 2 µm in another preferred embodiment, from 2 µm to 3 µm in a particular preferred embodiment, from 3 µm to 5 µm in another preferred embodiment, from 5 µm to 10 µm in a particular preferred embodiment, from 10 µm to 50 µm in another preferred embodiment, from 50 µm to 100 µm in a particular preferred embodiment, from 100 µm to 175 µm in a particular preferred embodiment, and from 175 to 300 µm in a particular preferred embodiment.

4. Sample and placement

In the methods and devices of the present invention, which use the CROF process, the sample is placed using several methods or a combination of methods. In one placement embodiment, the sample is placed on only one plate. In certain embodiments, the sample is placed on both plates (ie, the first plate and the second plate).

The sample is placed with the plates in an open configuration. In some embodiments, the first plate and the second plate are carefully separated from each other during sample placement so that the sample can be easily placed on one plate without obstructing the other plate. For example, the first plate and the second plate may be spaced far apart, such that the sample is directly dropped onto the first plate or the second plate as if the second plate did not exist. In certain embodiments of sample placement, the first plate and the second plate are spaced apart in an open plate configuration, then the sample is placed onto the plates (eg, using horizontal flow or other excavation techniques). In certain embodiments, two wafers have one side (eg, an edge) bonded together during all wafer operations (FIG. 30); and the opening and closing of the two plates is like the opening and closing of a book.

The sample placement may be a single droplet or multiple droplets. Multiple droplets may be at one location or multiple locations on either one or both plates. The drops can be carefully separated from each other, connected, or a combination of these.

In some embodiments, the sample contains more than one material, and the materials are placed together or separately. Materials are placed separately, either in parallel or in series.

Placing the sample on the plates (ie, the first plate and the second plate) can be done using the device or directly from the test subject onto the plates. In some embodiments, the sample is positioned using the device. The device includes, but is not limited to, pipettes, needles, sticks, swabs, tubes, nozzles, liquid dispensers, tips, rods, spray nozzles, printers, spray devices, etc. In certain embodiments, the sample is placed by direct contact between the sample at the sample source and the CROF plate without the use of any device (ie, bringing the sample and the plate into contact to obtain contact between both). This is called "direct sample placement."

Examples of direct placement of the sample onto the plate(s) are (a) direct contact between the pricked finger (or other body parts) and the plate, (b) spitting saliva onto the plate(s), (c) taking tears from a person's eye as a result of direct contact between the tear and the plate(s), (d) direct contact between the sweat and the plate(s), and (e) direct exhalation onto the plate(s) to accommodate condensation, etc. This method of direct placement can be used for both humans and animals.

In some embodiments, both direct and indirect (using a device) sample placement are used.

In the present invention, the volume of the sample that is placed on the plate or plates (“sample volume”) is not more than 0.001 pl (picoliter), not more than 0.01 pl, not more than 0.1 pl, not more than 1 pl, not more than 10 pl , no more than 100 pl, no more than 1 nl (nanoliter), no more than 10 nl, no more than 100 nl, no more than 1 µl (microliter), no more than 10 µl, no more than 100 µl, no more than 1 ml (milliliter), not more than 10 ml or between any two of these values.

In some embodiments, sample placement involves the steps of (a) placing the sample on one or both plates and (b) distributing the sample by means other than compressing the second plate during the CROF process. Means of sample distribution include the use of another device (e.g., stick, blade), air flow, or other.

Sample deformation

During the CROF process, in some embodiments, the samples behave like an incompressible fluid (which is called a fluid that maintains a constant volume under conditions of shape deformation), such that changing the thickness of the sample would result in a change in the area of the sample. In some embodiments, the samples behave like an incompressible fluid, with their horizontal area still increasing while their thickness is decreasing during the CROF process. In certain embodiments, the sample is a liquid, gel, and semi-solid, provided that during the CROF process their lateral area increases while their thickness decreases.

In the present invention, the disclosed term "turning the first plate and the second plate toward each other" is a process that affects the position and orientation of the first plate or the second plate or both such that the sample is located between the internal surfaces of the first plate and the second plate. In some embodiments, the action of "turning the first plate and the second plate toward each other" is performed by human hands, by human hands using certain devices, or by automatic devices without involving human hands.

In some embodiments, the thickness is no more than 1 mm, no more than 100 microns, no more than 20 microns, no more than 10 microns, or no more than 2 microns. In some embodiments, the thickness is at least 0.1 microns. In some embodiments, a thickness measurement is additionally provided.

In some embodiments, the variation in thickness of a suitable sample volume is no more than 300%, no more than 100%, no more than 30%, no more than 10%, no more than 3%, no more than 1%, no more than 0.3%, or no more than 0 .1% of the effective diameter of the corresponding area.

In some embodiments, the thickness is at least partially determined by a predetermined height.

5. Analytes, facility, binding site, storage site and transfer media

In the present invention, the subject includes, without limitation, one of a protein, amino acid, nucleic acid, lipid, carbohydrate, metabolite, cell or nanoparticle.

In some embodiments, the binding site includes a binding partner configured to bind a corresponding entity.

In some embodiments, the binding site includes an object associated with the binding site.

In some embodiments, placing the sample includes placing the sample within the binding site.

In some embodiments, the reagent includes at least one of a protein, an amino acid, a nucleic acid, a lipid, a carbohydrate, and a metabolite.

In certain embodiments, the storage area includes dried reagent.

In some embodiments, the storage area includes a reagent configured to be released from the storage area upon contact with the sample.

In some embodiments, the first storage area and the second storage area are located in a common storage area.

In some embodiments, the transmission medium is a sample. In some embodiments, the transmission medium is a liquid, and the reagent or object can dissolve and diffuse in the liquid.

In some embodiments, the wafer contains multiple storage areas. In another embodiment, one storage area contains multiple reagents.

Various release times. In some embodiments, the wafer has multiple storage areas at different locations on the wafer, or one storage area contains multiple reagents and upon contact with a sample of the storage areas, the reagents are released, but are released over different times for different reagents of the same storage area or reagents. various storage areas.

In some embodiments, the first reagent is configured to be released from the first storage area after contact with the sample at a first average release time, and the second reagent is configured to be released from the second storage area after contact with the sample at a second average release time, and where the first average time release is less than the second average release time.

In some embodiments, the first reagent is configured to be released from the first storage area upon contact with the sample, and wherein the second reagent is a bound reagent.

In some embodiments, placement includes associating at least one of the reagents with a corresponding plate.

In some embodiments, contacting includes releasing at least one of the reactants from the corresponding plate.

In some embodiments, placement includes placing a first reactant and a second reactant, and wherein bringing into contact includes releasing the first reactant before the second reactant.

In some embodiments, at least one of the plates contains a storage area that includes a reagent to be added to a suitable sample volume.

In some embodiments, the reagent includes at least one of a protein, an amino acid, a nucleic acid, a lipid, a carbohydrate, and a metabolite.

In some embodiments, the storage area includes a dried reagent.

In some embodiments, the storage area includes a reagent configured to be released from the storage area upon contact with the sample.

In some embodiments, the storage area is a first storage area and the reagent is a first reagent, where the device includes a second storage area including a second reagent to be added to a suitable sample volume, where the second storage area is located on one of the plates.

In some embodiments, the first storage area and the second storage area are located in a common storage area.

In some embodiments, the first reagent is configured to be released from the first storage area after contact with the sample at a first average release time, and the second reagent is configured to be released from the second storage area after contact with the sample at a second average release time, and where the first average time release is less than the second average release time.

In some embodiments, at least one of the reagents is dried on a suitable plate.

In some embodiments of the kit, at least one of the reagents is associated with a corresponding plate.

In some embodiments of the kit, at least one of the reagents is configured to be released from the corresponding plate upon contact with the sample.

In some embodiments of the kit, a first reagent is on one or both of the plates and a second reagent is on one or both of the plates, wherein the first reagent is configured to be released from the respective plate upon contact with the sample at a first average release time and the second reagent is configured to being capable of being released from the respective plate upon contact with the sample in a second average release time, and wherein the first average release time is less than the second average release time.

In some embodiments of the devices, the storage area is a first storage area and the reagent is a first reagent, where the device includes a second storage area including a second reagent to be added to a suitable sample volume, where the second storage area is located on one of the plates.

6. Local binding or mixing in part of the sample (P)

In some applications, it is desirable to have a binding site to capture (ie, bind) analytes in only a portion of the sample rather than in the entire sample. Also in some cases it is desirable that the reagent be added (i.e., mixed) to a portion of the sample rather than to the entire sample. It is often desirable that there is no liquid separation between part of the sample and the rest of the sample. Such requirements are preferred or necessary for certain multiplex detections.

The present invention provides a solution to the above requirements through the use of a CROF method and a CROF apparatus by converting the sample into an ultra-thin film, the thickness of which is less than the horizontal dimension of a portion of the sample, where only the analyte within that portion of the sample will be captured or only a portion of the sample will be mixed with the reagent. The operating principle for this approach is that if the sample thickness is less than the horizontal dimension of the sample, then the capture of the analyte by the surface or the mixing of the reagent placed on the surface may be primarily limited by the diffusion of the analytes and reagent in the thickness direction, where diffusion in the form diffusion in horizontal projection is relatively insignificant. For example, if the sample is reduced to a thin film 5 µm thick, if the part of the sample in which the analyte is to be entrapped or which is to be mixed with the reagent has a horizontal dimension of 5 mm by 5 mm, and if the diffusion time of the analyte or reagent through the 5 µm thickness is 10 s, then the diffusion in a horizontal projection of an analyte or reagent over a distance of 5 mm is 1,000,000 s (since the diffusion time is proportional to the square of the distance over which diffusion occurs). This means that by selecting the correct ratio of the horizontal size of the sample portion of interest to the sample thickness at a given time interval, the captured analytes primarily originate from the sample portion of interest, or the reagent is mixed primarily with the sample portion of interest.

6.1. Local binding of an object in part of the sample to the surface (P: volume to surface)

P1. A method for local binding of target objects in a suitable sample volume with a binding site on the surface, comprising:

(i) carrying out steps (a) to (d) in the method of item X1, where the thickness of the sample in a closed configuration is significantly less than the average linear dimension of the binding site; and wherein the suitable volume is the volume of the sample that is located at the binding site when the plates are brought into a closed configuration;

(ii) after (i) and while the plates are in a closed configuration, one of:

(1) incubating the sample for a suitable period of time and then stopping the incubation; or

(2) incubating the sample for a time that is equal to or greater than the minimum suitable time interval, and then assessing for a period of time that is equal to or less than the maximum suitable time interval, while binding the target object at the binding site;

where is the appropriate time interval:

i. equal to or greater than the time required for the target object to diffuse through a layer of uniform thickness in a closed configuration; And

ii. significantly shorter than the time required for horizontal diffusion of the target object through the binding site with a minimum size in the horizontal projection;

where, at the end of incubation (1) or during evaluation (2), the majority of targets associated with the binding site originate from a sample of suitable volume;

wherein the incubation allows the target to bind to the binding site, and where the suitable volume is the portion of the sample that is located above the binding site in a closed configuration.

The method of claim P2, wherein the term "the thickness of the suitable volume of the sample is significantly less than the minimum average size of the binding site" means that the ratio of the minimum average size of the binding site and the thickness of the sample (referred to as the "length-to-thickness ratio") is at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 10000, at least 100000 or any range between these values. In preferred embodiments, the length to thickness ratio is at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or any range between these values.

The method of claim P2, where the term "substantially shorter than the time required for the target object to diffuse horizontally through the minimum horizontal dimension of the binding site" means that the ratio of the time for diffusion through the minimum horizontal dimension of the binding site and the time diffusion through the thickness of the sample (referred to as the ratio of length and time of diffusion through thickness) is at least 3, at least 10, at least 50, at least 10, at least 100, at least 1000, at least at least 10,000, at least 100,000, at least 100,000, or anywhere in between. In preferred embodiments, the ratio of length to time of diffusion through thickness is at least 3, at least 10, at least 50, at least 10, at least 100, at least 1000, at least 10000, or any range between these values.

P2. A device for locally binding an object in a suitable sample volume with a binding site on the surface, containing

a first plate and a second plate that are movable relative to each other in different configurations,

wherein the first plate has on its surface a binding site that has an area less than that area of the plate and is configured to bind a target object in the sample, wherein the target object is capable of diffusing in the sample, and where one or both of the plates includes spacers and each of the spacers fixed to its corresponding plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates,

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, the plates face each other, the spacers, the binding site and at least a portion of the sample are between the plates, the sample is in contact with at least a portion of the bond site, the thickness of the suitable volume of the sample is controlled by the plates and spacers and is thinner, than the maximum sample thickness when the plates are in an open configuration; where the suitable sample volume is the volume of the sample that is placed at the binding site;

wherein the height of the spacer is selected to regulate the thickness of a suitable volume in a closed configuration that is at least 3 times smaller than the average linear dimension of the binding site.

Adjusting the thickness of the suitable volume so that it is 3 times smaller than the average linear size of the binding site results in the time of diffusion of the object through the thickness of the sample being 9 times less than the time of diffusion over a distance equal to the average linear size of the binding site. This thickness control makes it possible to select the incubation time such that the incubation results in (i) a significant amount of the target object in a suitable volume being associated with the binding site and (ii) a significant amount of the target object associated with the binding site originating from the suitable volume sample, and incubation is a process that promotes the binding of the target object to the binding site.

For example, if the incubation time is set so that the time is equal to the time of diffusion of the object through the thickness of the suitable volume of the sample, then after incubation most of the object within the suitable volume has already reached the binding site and is bound in accordance with the reaction rate equation, with the object that initially (i.e. before incubation) is outside the suitable volume, can diffuse only into the peripheral part of the suitable volume (relatively small volume), and such volume becomes less significant as the ratio of the average linear size of the binding site to the thickness of the suitable volume becomes large.

6.2. Local binding of an object stored on a wafer surface to a binding site on the surface of another wafer (surface to surface)

P3. A method for locally linking an object stored in a storage area of one wafer with a linkage area on another wafer, comprising:

(a) providing a first plate and a second plate that are movable relative to each other in different configurations, where the surface of the first plate has a bonding area; and the surface of the second plate has a storage portion that contains an object to be bonded to the bonding portion; where the area of the binding site and the area of the reagent site are less than the area of the corresponding plates; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(b) obtaining a transmission medium, where the object is able to dissolve in the transmission medium and diffuse in the transmission medium;

(c) placing, when the plates are in an open configuration, the transfer medium on one or both plates; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the transmission medium by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, separators, a bonding area, a storage area and at least a portion of the transmission medium are between the plates; at least a portion of the storage portion directly faces the coupling portion with a portion of the transfer medium therebetween, and the thickness of a suitable volume of the transfer medium is adjusted by means of plates and spacers and is thinner than the maximum thickness of the sample when the plates are in an open configuration and significantly less than the average linear dimension of the suitable volume in the direction of the plate surface; And

(e) after (d) and while the plates are in a closed configuration, incubating for some time and stopping the incubation, where the incubation time is chosen such that it causes a significant amount of the object associated with the binding site to originate from the site storage, where the suitable volume is the volume of the transmission medium that is located at the binding site, and incubation is the process that allows the object to bind to the binding site.

The term “at least a portion of the storage portion directly faces the binding portion” means that the shortest distance from a point in the portion to the binding portion coincides with the thickness of the suitable volume in a closed plate configuration.

P4. A device for locally linking an object stored on a storage area of one wafer with a corresponding linkage area on another wafer, comprising:

a first plate and a second plate that are movable relative to each other in different configurations, where the surface of the first plate has a bonding portion; and the surface of the second plate has a storage portion that contains an object to be bonded to the bonding portion; wherein the area of the binding portion and the area of the storage portion are smaller than the corresponding plates; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the transfer medium is placed on one or both plates, where the object in the storage area is capable of dissolving into the transfer medium and diffuse in the transmitting medium,

where the other of the configurations is a closed configuration, which is brought about after placing the transmission medium in an open configuration; and in a closed configuration, the plates face each other, the separators, the coupling portion, the storage portion, and at least a portion of the transmission medium are located between the plates; at least a portion of the storage portion directly faces the coupling portion with a portion of the transfer medium therebetween, and the thickness of a suitable volume of the transfer medium is adjusted by the plates and spacers and is thinner than the maximum thickness of the sample when the plates are in an open configuration;

where the suitable volume is the volume of the transmission medium that is located in the storage area when the plates are in a closed configuration; And

wherein the height of the spacer is selected to regulate the thickness of a suitable volume in a closed configuration that is at least 3 times smaller than the average linear dimension of the binding site,

where at least one of the separators is located inside the contact area with the sample;

and the separators have a preset separator spacing and height.

6.3. A method for locally binding an object on several storage areas of one wafer corresponding to binding areas on another wafer

P5. A method for locally binding an object stored on several storage areas of one wafer corresponding to binding areas on another wafer, comprising:

(a) providing a first plate and a second plate that are movable relative to each other in different configurations, where the surface of the first plate has multiple binding sites; and the surface of the second plate has multiple corresponding storage areas; wherein each respective storage portion is located at a location on the second plate that corresponds to the location of the binding portion such that when two plates are positioned opposite each other, each binding portion overlaps only one storage portion; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(b) obtaining a transmission medium, where the object in the storage areas is capable of dissolving in the transmission medium and diffusing in the transmission medium;

(c) placing, when the plates are in an open configuration, the transfer medium on one or both plates; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distributing the transmission medium by bringing the plates into contact in a closed configuration, wherein in the closed configuration the two plates face each other, separators, coupling areas, storage areas and at least a portion of the transmission medium are between the plates, each the bonding portion directly faces only one corresponding storage portion, the transfer medium is in contact with at least a portion of each of the bond portions and a portion of each of the storage portions, the thickness of the suitable volume of the transfer medium is adjusted by means of plates and spacers, and is thinner than the maximum thickness of the transmission medium in the case when the plates are in an open configuration, and is significantly less than the average linear size of the bonding areas; And

(e) after (d) and while the plates are in a closed configuration, incubate for a specified time and stop the incubation, where the incubation time is chosen such that it causes a significant amount of the object bound to each binding site to originate from corresponding storage area, where the suitable volume is the volume of the transfer medium that is located at the binding sites, and incubation is a process that promotes the binding of the object to the binding site.

In some embodiments, the gap is limited by the area of the binding sample.

In some embodiments of method P5, the transfer medium is a sample with a target analyte, the binding site comprises a capture agent, and the entity in the binding site is a detection agent, where the target analyte binds to the capture agent and the detection agent to form a sandwich. capture agent - analyte - detection agent. The P5 method simplifies the analysis steps and can reduce analysis time by using lower separator heights to obtain thinner sample thicknesses and shorter vertical diffusion times of both analytes and detection agents for shorter analysis times to saturation.

P6. A device for locally linking an object stored on several storage areas of one wafer with several corresponding linking areas on another wafer, comprising:

a first plate and a second plate that are movable relative to each other in different configurations;

where the surface of the first plate has several binding sites; and the surface of the second plate has several corresponding storage areas; wherein each respective storage portion is located at a location on the second plate that corresponds to the location of the binding portion such that when two plates are positioned opposite each other, each binding portion overlaps with only one storage portion; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the spacing between the plates is not controlled by spacers, and the transfer medium is placed on one or both plates, where the object in the storage area is capable of dissolving into the transfer medium and diffuse in the transmitting medium,

where the other of the configurations is a closed configuration, which is brought about after placing the transmission medium in an open configuration; and in a closed configuration, the two plates face each other, the spacers, bonding areas, storage areas and at least a portion of the transfer medium are between the plates, each binding area directly faces only one corresponding storage area, the transfer medium is in contact with at least part of each of the bonding portions and part of each of the storage portions, the thickness of the suitable volume of the transmission medium is adjusted by the plates and spacers, and is thinner than the maximum thickness of the transmission medium when the plates are in an open configuration;

where the suitable volume is the volume of the transmission medium that is located in the storage area when the plates are in a closed configuration; And

wherein the predetermined height of the spacer is selected to regulate the thickness of the suitable volume in the closed configuration to be significantly less than the average linear dimension of the binding sites.

6.4. Local addition of a surface-stored reagent to a portion of the sample (surface to bulk)

P7. A method of locally adding a reagent to a suitable volume of sample, comprising:

(a) providing a first plate and a second plate that are movable relative to each other in different configurations, wherein the first plate has on its surface a storage portion that contains reagents to be added to a suitable volume of the sample, the reagents being capable of dissolving in the sample and diffusing in the sample, and the area of the storage area is smaller than the area of the plate; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(b) obtaining a sample;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration; where in a closed configuration the plates face each other; separators, a storage area and at least part of the sample are located between the plates; the sample is in contact with at least a portion of the storage area and is in contact with the plates within an area that is larger than that of the storage area; the thickness of the suitable sample volume is controlled by the plates and spacers, and is thinner than the maximum thickness of the sample when the plates are in an open configuration, and significantly less than the average linear dimension of the suitable volume in the direction of the surface of the plate; And

(e) after (d) and while the plates are in a closed configuration, incubation for some time and termination of incubation, where the incubation time is chosen such that it causes (i) a significant number of reagents dissolved in the sample to be contained in a suitable sample volume, and (ii) the reagents are contained in a significant portion of the suitable volume, and where the suitable volume is the volume of the sample that is located in the storage area when the plates are in a closed configuration, and incubation is a process that promotes dissolution and diffusion reagent in the sample.

P8. A device for locally adding a reagent stored on the surface of a plate to a suitable sample volume containing

a first plate and a second plate that are movable relative to each other in different configurations,

wherein the first plate has on its surface a storage portion which contains reagents to be added to a suitable volume of the sample, the reagents being capable of dissolving in the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, the plates face each other, the spacers, the storage area, and at least a portion of the sample are between the plates, the sample is in contact with at least a portion of the storage area and at least a portion of the binding surface outside the storage area, the thickness of a suitable volume of the sample is adjusted by the plates and spacers and is thinner than the maximum thickness of the sample when the plates are in an open configuration, and where the suitable sample volume is the volume of the sample that is placed in the storage area when the plates are in a closed configuration;

where the height of the separator is chosen to regulate the thickness of the suitable volume in a closed plate configuration, which is at least 3 times smaller than the average linear dimension of the suitable volume in the direction of the surface of the plate.

7. Formation of a “sandwich” of capture agent-analyte-detection agent at the binding site (W)

One aspect of the present invention provides for the formation of a capture agent-analyte-detection agent sandwich at a binding site on a solid surface in one step using the CROF process and by placing the binding site on a single plate and a storage region that contains the detection agent. , at the corresponding location of the other plate.

7.1. Formation of a capture agent-analyte-detection agent sandwich at the binding site in one incubation step (total) (W)

W1. A method for forming a “sandwich” of capture means-analyte-detection means at the plate binding site, comprising:

(a) obtaining a sample that contains a target analyte, where the target analyte is capable of diffusion in the sample;

(b) obtaining the capture means and obtaining the detection means, where the capture means and the detection means are capable of binding to the target analyte to form a capture means-target analyte-detection means sandwich;

(c) providing a first plate and a second plate that are movable relative to each other in different configurations; wherein the first plate has a binding portion that contains binding means to be immobilized onto the portion, and the second plate has a storage portion that contains detection means; wherein, in the case where the storage area is in contact with the sample, the detection agents are capable of dissolving in the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(d) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(e) after (d) distribution of the sample by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is controlled by the plates and spacers and is thinner than the thickness of the sample in the case when the plates are in an open configuration; and the sample is in contact with the binding portion and the storage portion;

(f) after (e), while the plates are in a closed configuration, incubating for a period of time that allows the formation of a capture agent-target analyte-detection agent sandwich;

where the suitable volume is at least part or all of the volume of the sample.

W2. Device for forming a “sandwich” capture means-analyte-detection means at the plate binding site, containing

a first plate and a second plate that are movable relative to each other in different configurations;

wherein the first plate has a binding portion that contains capture means immobilized thereon, and the second plate has a storage portion that contains detection means; wherein the capture agents and detection agents (capable of binding) bind to a target analyte in the sample to form a capture agent-target analyte-detection agent sandwich; wherein, when the storage area is in contact with the sample, the detection agents are able to dissolve in the sample and diffuse in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other configuration is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is controlled by the plates and spacers, and is thinner than the thickness of the sample in the case where the plates are brought into the open configuration; and the sample is in contact with the binding portion and the storage portion;

and where the suitable volume is at least part or all of the volume of the sample.

7.2. Formation of a capture agent-analyte-detection agent sandwich at the binding site in a single incubation step using an analyte that originates from a portion of the sample (i.e., locally).

W3. A method for forming a “sandwich” of capture means-analyte-detection means at the binding site of a plate using analytes that originate from a portion of the sample, comprising:

(a) obtaining a sample that contains a target analyte, where the target analyte is capable of diffusion in the sample;

(b) obtaining the capture means and obtaining the detection means, where the capture means and the detection means are capable of binding to the target analyte to form a capture means-target analyte-detection means sandwich;

(c) providing a first plate and a second plate that are movable relative to each other in different configurations; wherein the first wafer has a binding portion which contains binding means to be immobilized to the area, and the second wafer has a storage portion which contains detection means which, when the storage portion is in contact with a sample, are capable of dissolving into the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(d) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(e) after (d) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, the spacers, the binding area and the storage area are between the plates, the binding area and the storage area are in contact with a suitable sample volume , and the thickness of the suitable sample volume is controlled by the plates and spacers and is thinner than the thickness of the sample when the plates are in an open configuration and significantly less than the average linear dimension of the binding site; And

(f) after (e) and while the plates are in a closed configuration, incubate for a specified time and stop the incubation, where the incubation time is chosen such that it results in a significant amount of capture agent-analyte-agent sandwich for detection formed at the binding site contains analytes that originate from a suitable sample volume, where the suitable volume is the volume of the sample that is located at the binding site, and incubation is a process that promotes the formation of a capture agent-analyte sandwich. detection tool.

In some embodiments, the space to plot size ratio may be no more than 1/5.

W4. A device for forming a "sandwich" capture means-analyte-means for detection at the binding site of the plate using analytes that originate from a portion of the sample, containing:

a first plate and a second plate that are movable relative to each other in different configurations;

wherein the first plate has a binding portion that contains capture means immobilized thereon, and the second plate has a storage portion that contains detection means; wherein the capture agents and detection agents (capable of binding) bind to a target analyte in the sample to form a capture agent-target analyte-detection agent sandwich; wherein, when the storage area is in contact with the sample, the detection agents are able to dissolve in the sample and diffuse in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers, the binding portion and the storage portion are between the plates, the binding portion and the storage portion are in contact with the suitable sample volume, and the thickness of the suitable sample volume is adjusted by the plates and spacers and is thinner than the thickness of the sample in the case where the plates are in an open configuration; and where the suitable volume is the volume of the sample that is placed at the binding site; And

wherein the height of the spacer is selected to regulate the thickness of a suitable volume in a closed configuration that is significantly smaller than the average linear dimension of the binding site.

7.3. A method for reducing the formation time of a “sandwich” of capture agent-analyte-detection agent at the binding site as a result of reducing the diffusion distance (W, X)

W5. A method for reducing the formation time of a “sandwich” of capture means-analyte-detection means at the plate binding site, comprising:

(a) obtaining a sample that contains a target analyte, where the target analyte is capable of diffusion in the sample;

(b) obtaining the capture means and obtaining the detection means, where the capture means and the detection means are capable of binding to the target analyte to form a capture means-target analyte-detection means sandwich;

(c) providing a first plate and a second plate that are movable relative to each other in different configurations; wherein the first wafer has a binding portion which contains binding means to be immobilized to the area, and the second wafer has a storage portion which contains detection means which, when the storage portion is in contact with a sample, are capable of dissolving into the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(d) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(e) after (d) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, the separators, the binding area and the storage area are between the plates, the binding area overlaps the storage area, the binding area and the storage area are in contact with a suitable sample volume, and the thickness of the suitable sample volume is controlled by the plates and spacers and is thinner than the thickness of the sample when the plates are in an open configuration, and thereby the reduced sample thickness reduces the time for vertical diffusion of the analytes and means for detection within the sample, where the suitable volume is at least a portion of the total volume of the sample.

where the period of time allowing the target entity to adequately communicate with the binding site is shorter than that in the absence of a closed configuration.

- The method may further include a washing step to remove the sample between the plates, and the washing step is performed while the plates are in either a closed configuration or an open configuration.

- The methods further include a readout step during which the signal from the capture agent-analyte-detection agent sandwich immobilized at the binding site is read. The reading is performed either after washing or without any washing.

The method may further be multiplexed, as described above or below.

W6. Device for reducing the formation time of a “sandwich” capture means-analyte-detection means at the plate binding site, containing

a first plate and a second plate that are movable relative to each other in different configurations;

wherein the first plate has a binding portion that contains capture means immobilized thereon, and the second plate has a storage portion that contains detection means; wherein the capture agents and detection agents (capable of binding) bind to a target analyte in the sample to form a capture agent-target analyte-detection agent sandwich; wherein, when the storage area is in contact with the sample, the detection agents are able to dissolve in the sample and diffuse in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers, the binding portion and the storage portion are between the plates, the binding portion overlaps the storage portion, the binding portion and the storage portion are in contact with a suitable sample volume, and the thickness of the suitable sample volume is adjusted by the plates and separators and is thinner than the thickness of the sample when the plates are in an open configuration, and thereby the reduced sample thickness reduces the time for vertical diffusion of analytes and detection agents within the sample where the suitable volume is at least a portion of the total volume sample.

In these embodiments, the method may include attaching the gripper to a wafer, where the attachment is accomplished by chemically reacting the gripper with a reactive group on the wafer. The other plate may contain a spot of dried detection agent in position such that upon closing of the plates, the attached capture means and the spot of detection agent face each other. Additionally, the method may include contacting a sample containing the target analyte with the device and closing the plates as described above. The detection agent dissolves and diffuses into the sample. Since the target analyte is in solution, the target analyte will be bound by the capture agent and immobilized on the surface of one of the plates. The detection agent may bind to the target analyte before or after it binds to the capture agent. In some cases, the method may involve removing any target analytes that are not bound to the capture agent or any unbound detection reagent (eg, by washing the plate surface in binding buffer). The detection agent can be conjugated to an optical detectable label, thereby providing a method for detecting the target analyte. After optionally removing the detection agent that is not bound to the target analyte, the system can be subjected to reading, for example, using a reading system to read the light signal (for example, light at a wavelength that is in the range from 300 nm to 1200 nm ) from the detection agent that contacted the plate. In addition, as mentioned above, the detection agent may be directly labeled (in which case the detection agent can be firmly bound to the light-emitting tag prior to placement on one of the plates), or indirectly labeled (i.e., by binding the detection agent to a second capture agent, for example, a secondary antibody that is labeled, or a labeled nucleic acid that specifically binds to the detection agent, and which is associated with a light-emitting label). In some embodiments, the method may include a blocking agent, thereby preventing nonspecific binding of capture agents to non-target analytes. Suitable conditions for specific binding of target analytes to other agents, including appropriate temperature, time, pH of the solution, level of natural light, humidity, concentration of chemical reagents, antigen-antibody ratio, etc., are well known or can be easily achieved. obtain based on the present invention. General methods for molecular interaction methods between capture agents and their binding partners (including analytes) are well known in the art (see, for example, Harlow et al. Antibodies: A Laboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.; Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995). The methods described above and below are illustrative; The methods in this document do not represent the only ways to perform analysis.

In certain embodiments, a nucleic acid-based capture agent can be used to capture a protein analyte (eg, a DNA or RNA binding protein). In alternative embodiments, a protein capture agent (eg, a DNA or RNA binding protein) can be used to capture a nucleic acid analyte.

The sample may be a clinical sample obtained from cells, tissues or body fluids. Biological fluids of interest include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF) , earwax (cerumen plug), chyle, chyme, endolymph, perilymph, feces, gastric acids, gastric juice, lymph, mucus (including nasal discharge and mucous sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus , watery discharge, saliva, secretion of the sebaceous glands (sebum), semen, sputum, sweat, synovial fluid, tears, vomit, urine and condensation of exhaled air.

In one embodiment of this assay, the plate is brought into contact with a sample containing the target analyte (eg, the target protein) and the plates are closed. The sample contains all necessary reagents or is modified to contain them (eg, salts, etc.) under conditions suitable for specific binding. The capture agents (eg, antibodies) and the detection agent specifically bind to the target analyte in the sample, thereby causing a spot of labeled analyte to be detected.

As in any embodiment, the amount of the target analyte in the sample can be measured to provide a qualitative or quantitative measure of the amount of the target analyte in the sample. In some embodiments, the magnitude of the signal provides a quantitative determination of the magnitude of the target analyte in the sample. In some cases, the estimate can be compared to a standard curve, for example, obtained for a second analyte or a precisely measured analyte, which in certain cases may be at a known concentration. This comparison can be facilitated by placing the capture means at different densities (eg, different concentrations) and reading the signal from each capture means insert.

8. Binding and Addition Using Low Volume Samples and Reagents (V)

In many applications, it is highly desirable to use as little sample or reagent volume as possible. However, in microfluidic channel devices (the most popular approach currently when using small samples), a significant volume of the sample is consumed when moving from the inlet to the testing (detection) site of the device, which as a result entails the need for a sample volume larger than the volume in the test location. One aspect of the present invention is to significantly reduce the volume of sample or reagent used in testing by placing a small volume of sample or reagent on a plate and then converting the volume into a thin film with less thickness but a larger surface area than previously. This transformation also promotes a faster reaction.

8-1. Binding of a target object in a small volume sample at the surface binding site as a result of sample distribution

V1. A method of binding a target object in a sample to a binding site, comprising:

(a) providing a first plate and a second plate that are movable relative to each other into different configurations, where the first plate has a bonding portion on its surface, and where one or both plates include spacers, and each of the spacers is secured to its respective plate and has preset height;

(b) obtaining a sample that contains a target object to be bound to the binding site;

(c) placing the sample on one or both plates when the plates are in an open configuration; wherein in an open configuration, the two plates are partially or completely separated from each other, the gap between the plates is not adjustable using spacers, and once placed, the sample either does not cover the area or partially covers the area of the binding site;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, separators and a suitable sample volume are between the plates, the sample is in contact with a larger area of the storage area than when the plates are in the open configuration, and the thickness of the suitable sample volume is adjusted using a divider, where the suitable volume is a portion of the sample in the storage area.

V2. A device for binding a target object in a sample to the surface of a binding site, comprising a first plate and a second plate that are movable relative to each other in different configurations;

where the first plate has on its surface a binding site that binds the target object in the sample, and where the binding site has an area larger than the contact area with the sample when the sample is placed on only one of the plates and is not in contact with the other plate;

wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates, and once placed, either does not cover the area or partially covers the area of the binding site;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the sample are between the plates, the sample contacts a larger area of the binding site than when the plates are in the open configuration, and the thickness of a suitable volume of the sample at the binding site is controlled by the plates and spacers.

8-2. Adding reagents to a small sample volume by spreading the sample

V3. A method of binding a target object in a sample to a binding site, comprising:

(a) providing a first plate and a second plate that are movable relative to each other into different configurations, where the first plate has on its surface a storage area that contains reagents to be added to the sample, and where one or both plates contain spacers, and each of the dividers is fixed to its respective plate and has a predetermined height;

(b) placing the sample on one or both plates when the plates are in an open configuration; where in an open configuration the two plates are partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample, once placed, either does not contact the area or partially covers the area of the storage area;

(c) after (b) sample distribution by placing the plates in a closed configuration, where in the closed configuration the plates are facing each other, separators and a suitable sample volume are between the plates, the sample is in contact with a larger area of the storage area than when the plates are in the open configuration, and the thickness of the suitable sample volume is adjusted using a divider, where the suitable volume is a portion of the sample in the storage area.

V4. A device for binding a target object in a sample to a binding site, comprising

a first plate and a second plate that are movable relative to each other in different configurations,

where the first plate has on its surface a storage area that contains reagents, and the reagents are to be added to the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates,

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration the plates face each other, the separators and the suitable sample volume are between the plates, the sample is in contact with a larger area of the storage area than when the plates are in the open configuration, and the thickness of the suitable sample volume is adjusted by the separator, where the suitable volume is part of the sample in the storage area.

In the methods of points VI and V2 and the devices of points V3 and V4, in some cases, even if the sample is placed in the region of the binding area or storage area, due to the small volume of the sample and the wettability of the surface, the contact area with the sample so placed with the plate will be less than the area of the binding area or storage area. Thus, distribution, especially accurate distribution, is required.

Sample droplets can be multiple droplets, and in a closed configuration the droplets coalesce into a film thickness less than the maximum thickness.

In the present invention, in the method according to points V1-V7 and devices according to points V2-V8, the volume of the sample that is placed on the plate or plates (“sample volume”) is not more than 0.001 pl (picoliter), not more than 0.01 pl, not more than 0.1 pl, no more than 1 pl, no more than 10 pl, no more than 100 pl, no more than 1 nl (nanoliter), no more than 10 nl, no more than 100 nl, no more than 1 μl (microliter), no more than 10 µl, not more than 100 µl, not more than 1 ml (milliliter), not more than 10 ml, or between any two of these values.

9. Uniformity of binding or addition of reagents using uniform sample thickness (UAB)

In the case of analyzes and chemical reactions, it is preferable to obtain a thin film of the sample that is uniform over a large area. Examples include binding an object in a sample to a surface binding site, adding reagents to a sample, quantifying an appropriate sample volume, quantifying analytes, etc. In the case of methods in which two plates are used to reduce and control the thickness of a suitable volume (part or all volume) of the sample, it is necessary that they be accurate, uniform and easy to use.

One aspect of the present invention is to improve the accuracy, consistency, or ease of use of methods and/or devices that control the thickness of a suitable sample volume by compressing the sample using two plates.

9.1. Method for uniformly binding an object in a sample in the binding area of a plate

UAB1. A method for uniformly binding an object in a sample in the binding area of a plate, comprising:

(a) obtaining a sample that contains a target object that is capable of diffusion in the sample;

(b) providing a first plate and a second plate that are movable relative to each other into different configurations, wherein the first plate has on its surface a coupling portion that is capable of being coupled to a target object, wherein one or both plates comprise spacers, and each of the dividers are fixed to their respective plate and have a predetermined height;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, the separators and a suitable sample volume are between the plates, the binding site is in contact with the suitable volume, the thickness of the suitable sample volume adjustable using plates and spacers, and compared to plates that are in an open configuration, it is thinner than the maximum thickness of the sample and more uniform within the binding site;

wherein the spacers and plate are configured to produce a suitable volume adjustable thickness in a closed plate configuration that is more uniform than that thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

- Additionally, there is a storage area on the plate opposite the binding area to form a homogeneous “sandwich”.

UAB2. A device for uniformly binding an object in a sample in a plate binding area, comprising

a first plate and a second plate that are movable relative to each other in different configurations;

wherein the first plate has on its surface a coupling portion which is configured to bind a target object, wherein one or both plates comprise separators and each of the separators is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the binding site is in contact with the suitable volume, the thickness of the suitable sample volume is adjusted using the plates and spacers, and compared to the plates which are in the open configuration , it is thinner than the maximum sample thickness and more uniform within the binding site;

wherein the spacers and plates are configured to produce a more uniform adjustable thickness of suitable volume in a closed plate configuration than that in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

9.2. Method for uniform addition of reagent on a plate to a sample

UAB3. A method for uniformly adding a reagent to a suitable volume of sample, comprising:

(a) providing a first plate and a second plate that are movable relative to each other in different configurations, wherein the first plate has on its surface a storage portion that contains reagents to be added to a suitable volume of the sample, the reagents being capable of dissolving in the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(b) obtaining a sample;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, the separators and a suitable sample volume are between the plates, the storage area is in contact with the suitable volume, and the thickness of the suitable volume The sample is adjusted using plates and spacers and is thinner than the maximum thickness of the sample when the plates are in an open configuration;

wherein the spacers and plates are configured to produce a more uniform thickness of a suitable sample volume over a region of suitable volume in a closed plate configuration than a thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

UAB4. A device for uniformly adding a reagent to a suitable volume of sample, containing

a first plate and a second plate that are movable relative to each other in different configurations;

wherein the first plate has on its surface a storage portion which contains reagents to be added to a suitable volume of the sample, the reagents being capable of dissolving in the sample and diffusing in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the separators and the suitable sample volume are between the plates, the storage area is in contact with the suitable volume, and the thickness of the suitable sample volume is adjusted by the plates and separators, and it is thinner than the maximum thickness of the sample, when the plates are in an open configuration;

wherein the spacers and plates are configured to produce a more uniform thickness of a suitable sample volume over a region of suitable volume in a closed plate configuration than a thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

9.3. A method for uniformly forming a “sandwich” of capture agent-analyte-detection agent at the binding site

UAB5. A method for uniformly forming a “sandwich” of capture means-analyte-detection means at the plate binding site, comprising:

(a) obtaining a sample that contains the target analyte;

(b) obtaining the capture means and obtaining the detection means, where the capture means and the detection means are capable of binding to the target analyte to form a capture means-target analyte-detection means sandwich;

(c) providing a first plate and a second plate that are movable relative to each other in different configurations; wherein the first wafer has a binding portion that contains binding means to be immobilized onto the portion, and the second wafer has a storage portion that contains detection means that, when the storage portion is in contact with the sample, are capable of dissolving into the sample and diffuse in the sample; and wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

(d) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(e) after (d) distributing the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is controlled by the plates and spacers and is thinner, than the thickness of the sample in the case when the plates are in an open configuration; and the sample is in contact with the binding portion and the storage portion;

wherein the spacers and plates are configured to produce a more uniform thickness of a suitable sample volume over a region of suitable volume in a closed plate configuration than a thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

UAB6. A device for homogeneous formation of a “sandwich” - a capture means - an analyte - a detection means at the plate binding site, containing:

a first plate and a second plate that are movable relative to each other in different configurations;

where the first plate has a binding site that contains capture means to be immobilized on the site, and the capture means are capable of binding to a target analyte in the sample;

wherein the second plate has a binding site that contains detection agents that are capable of (a) upon bringing the storage site into contact with a sample, dissolving in the sample and diffusing into the sample and (b) binding to the target analyte and forming a capture agent-target multilayer structure detection analyte;

wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other configuration is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is controlled by the plates and spacers, and is thinner than the thickness of the sample in the case where the plates are in the open configuration; and the sample is in contact with the binding portion and the storage portion;

wherein the spacers and plates are configured to produce a more uniform thickness of a suitable sample volume over a region of suitable volume in a closed plate configuration than a thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

9.4. Uniform thickness regulation of a suitable sample volume between two plates

UAB7. A method for regulating the thickness of a suitable sample volume, comprising:

(a) obtaining a sample, where the thickness of a suitable volume of the sample is subject to regulation;

(b) providing two plates that are movable relative to each other in different configurations, wherein one or both plates contain spacers, and wherein the spacers have a predetermined distance between the spacers and height, and wherein each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted using the plates and spacers, and it is thinner than the maximum sample thickness when the plates are in an open configuration;

wherein the spacers and plates are configured to produce a more uniform thickness of a suitable sample volume over a region of suitable volume in a closed plate configuration than a thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

UAB8. A device for adjusting the thickness of a suitable sample volume, comprising a first plate and a second plate that are movable relative to each other in different configurations;

wherein one or both of the plates comprise spacers, the spacers having a predetermined distance between the spacers and height, and each of the spacers is secured to its respective plate;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates;

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted by the plates and spacers, and is thinner than the maximum sample thickness when the plates are in the open configuration;

wherein the spacers and plates are configured to produce a more uniform thickness of a suitable sample volume over a region of suitable volume in a closed plate configuration than a thickness in an open plate configuration; and wherein the suitable volume is part or all of the sample volume.

In the methods and apparatus of claims U1 to U8, the configuration of spacers and plates that make the thickness of a suitable sample volume uniform is provided by the embodiment described in the present disclosure.

Uniformity of sample thickness. In the methods and devices of paragraphs U1-U8, the uniformity of the thickness of a suitable volume of the sample is such that the thickness of the sample in a closed configuration has a relative variation of no more than 0.001%, no more than 0.01%, no more than 0.05%, no more than 0.1 %, no more than 0.5%, no more than 1%, no more than 2%, no more than 5%, no more than 10%, no more than 20%, no more than 30%, no more than 50%, no more than 75%, not more than 90%, no more than 100%, or between any two of these values.

In a preferred embodiment of the methods and devices in items U1 to U8, the thickness uniformity of a suitable sample volume is such that the thickness of the sample in a closed configuration exhibits a relative variation of no more than 0.1%, no more than 0.5%, no more than 1%, no more than 2%, no more than 5%, no more than 10%, no more than 20%, no more than 30%, no more than 50%, or between any two of these values.

Another parameter that may be important in reducing incubation time to saturation is the uniformity of sample thickness. If the thickness has a large variation within the binding site, then the incubation time to saturation can vary from location to location within the binding site, precipitating longer incubation times to saturation to ensure that all locations within the binding site reach saturation.

10. Surface reinforcement

One of the current major obstacles for PoC diagnostics, and for any assay that uses a small sample volume, is low sensitivity. It is advisable to amplify the analysis signal. One aspect of the present invention relates to devices and methods that place a binding site on a signal amplification surface (SAS) to amplify a signal to achieve higher sensitivity. Signal enhancement surfaces may also be called signal enhancement layers (SALs).

Common SAL structures involve nanoscale metal-insulator/semiconductor-metal structures that enhance the electric field and local surface gradient as well as light signals. The enhancement is strong at a location where there are sharp (eg, highly curved) edges of metal structures and small gaps between two metal structures. The areas with the greatest enhancement are those that have both sharp edges and small gaps. In addition, the sizes of all metallic and nonmetallic micro/nanostructures are typically smaller than the wavelength of light that SAL amplifies (i.e., subwavelength).

In some embodiments, the SAL layer has as many metal sharp edges and small gaps as possible. This requires a dense group of metal nanostructures with small spaces between the nanostructures. SAL structures can include several different layers. In addition, the SAL layer itself can be further improved by a method in which parts of metallic materials that do not have sharp edges and small gaps can be further coated, as described in US Provisional Application Serial No. 61/801,424, filed March 15, 2013 and PCT Application WO2014197096 filed March 15, 2014, which are incorporated by reference for all purposes, and PCT/US2014/028417 (Chou et al, "Analyte Detection Enhancement By Targeted Immobilization, Surface Amplification, And Pixelated Reading And Analysis"), which is incorporated herein by reference for all purposes.

One particular embodiment of a signal amplification surface is a D2PA array (Disk Post Dot Antenna Arrays), which may also comprise a molecular adhesive layer that covers a portion of said metal dot structure, said metal disk and/or said metal backplate, and optionally an entrapment agent that specifically binds to the analyte, wherein said entrapment agent is bound to a molecular adhesive layer of the D2PA array. The nanosensor can amplify the light signal from an analyte if said analyte is coupled to a capture agent. In some embodiments, the size of one, more, or all of the critical metal and dielectric components of the SAL is smaller than the detection wavelength of the light. Details of the physical structure of disk antenna device arrays of pillar point disk antennas, methods of their manufacture, methods of associating capture means with disk antenna arrays paired with column points, and methods of using pillar disk antenna arrays for detection of analytes are described in a number of publications, including WO2012024006, WO2013154770, Li et al (Optics Express 201 1 19, 3925-3936), Zhang et al (Nanotechnology 2012 23: 225-301); and Zhou et al (Anal. Chem. 2012 84: 4489), which are incorporated by reference for all purposes.

10.1. Signal amplification when analyzing a target object in a suitable sample volume

M1. A method of signal amplification when analyzing a target object in a suitable sample volume, providing:

(a) obtaining a sample that contains the target object;

(b) providing two plates that are movable relative to each other into different configurations, where one plate contains on its surface one coupling site that contains a signal amplification surface that is brought into configuration for the purpose of coupling the target object and amplifying the optical signal that is located on or near the signal amplification surface; and wherein one or both of the plates comprise spacers, and each of the spacers is located on its respective plate and has a predetermined height;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, wherein in the closed configuration the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is controlled by the plates and spacers and is thinner than this thickness when the plates are in an open configuration; and a suitable volume of sample is in contact with the binding site; And

(f) after (f) incubation, with the plates in a closed configuration, for a period of time that ensures binding of the target object in a suitable sample volume to the binding site; where the suitable volume is the portion of the sample that is in contact with the binding site while the plates are in a closed configuration.

M2. A device for amplifying the signal when determining a target object in a suitable sample volume, containing

a first plate and a second plate that are movable relative to each other in different configurations,

wherein the first plate contains on its surface one binding site, and the binding site contains a signal amplification surface that is configured to (i) bind a target object in the sample and (ii) amplify an optical signal that is located on or near the signal amplification surface ;

where one or both plates contain spacers, and each of the spacers is on its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates,

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in the closed configuration, the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted by the plates and spacers, and is thinner than that thickness when the plates are in the open configuration;

where the suitable volume is the portion of the sample that contacts the binding site when the plates are in a closed configuration.

In some embodiments, the signal amplification surface includes at least one of a metal-dielectric nanostructure, a metal-semiconductor nanostructure, and a disk antenna array coupled to points on the posts.

In some embodiments, the signal amplification surface includes a metal layer.

11. Preservation of reagent volume during analysis with fast binding (S)

In the situation of binding an object in a reagent to a binding site on a surface (eg, coating a plate with a capture agent or staining the surface with a biological sample), it is desirable to have a short incubation time. One approach for short incubation times is to significantly increase the concentration of the target in the reagent. However, this approach is wasteful of the target and therefore expensive because with short incubation times, only a small portion of the target in the reagent that is near the binding site can reach the binding site to bind, and the remaining portion is too far away to diffuse to the binding site for the purpose of binding and is useless and wasted. For typical diffusion constants of common reagents in common solutions, typical diffusion lengths are approximately 10 µm, 33 µm and 100 µm, respectively, for incubation times of 1 s (second), 10 s and 100 s. The typical thickness of a liquid droplet on a typical surface is 2.5 mm, which is at least 25 times thicker than the above diffusion length values, resulting in significant waste generation (cost) if incubation times are 100 s or less. One aspect of the present invention provides for distributing the reagent drop(s) over a larger area, but at a very thin thickness (thinner than a natural pinprick) to conserve reagents and thereby reduce cost.

11-1. A method of storing a reagent that contains a target entity in reagents that binds to a surface binding site as a result of the distribution of the reagent. (The volume has a natural contact area that is smaller than the binding site)

S1. A method for storing a reagent that contains a target object that binds to a surface binding site, comprising:

(a) providing a first plate and a second plate that are movable relative to each other into different configurations, where the first plate has a bonding portion on its surface, and where one or both plates include spacers, and each of the spacers is secured to its respective plate and has preset height;

(b) providing a reagent that (i) contains a target entity capable of binding the binding site, and (ii) has a volume and wetting property such that the area of contact with the reagent placed at the binding site without contact with another plate is smaller, than the area of the binding site;

(c) placing the sample on one or both plates when the plates are in an open configuration; where in an open configuration the two plates are partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates are facing each other, the spacers and the sample are between the plates, the sample is in contact with a larger area of the binding site than when the plates are in the open configuration, and the thickness of the sample at the bonding site is controlled by the plates and spacers and is thinner than that when the plates are in an open configuration.

The method of item S1 further provides a step after (d), and when the plates are in a closed configuration, incubation for a certain time and stopping the incubation, where the incubation time is approximately equal to the time during which the target object diffuses within the maximum thickness of the sample, until the plates are in a closed configuration and where incubation is a process that promotes the binding of an object to the binding site.

S2. A device for storing a reagent that contains a target object that binds to a surface binding site, comprising:

a first plate and a second plate that are movable relative to each other in different configurations,

where the first plate has on its surface a binding site that binds the target object in the reagent, and where the binding site has an area larger than the area of contact with the reagent if the reagent is placed only on one of the plates, without contact with the other plate;

wherein one or both of the plates comprise spacers and each of the spacers is secured to its respective plate and has a predetermined height;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the reagent is placed on one or both plates,

where the other of the configurations is a closed configuration, which is achieved after placing the reagent in the open configuration; and in the closed configuration, the plates face each other, the spacers and reagent are between the plates, the reagent contacts a larger area of the binding site than when the plates are in the open configuration, and the thickness of the suitable volume of reagent at the binding site is adjusted by the plates and spacers, and it thinner than this thickness when the plates are in an open configuration.

12. Detection and/or quantification of volume and/or concentration (Q)

Quantification and/or control of a suitable sample volume is useful for quantifying and/or controlling the concentration of chemical compounds (including analytes, object, reagents, etc.) in a sample.

Common methods for quantifying sample volume include the use of a dropper pipette (eg, Research plus Eppendorf variable volume pipette, 0.5-10 µL, SKU #3120000020) or the use of geometric analysis. For PoC (point of care) or home use, such dosing devices are inconvenient and/or expensive to use. There is a need for simpler and cheaper methods and devices to quantify and/or control sample volume and/or concentration.

One aspect of the present invention relates to methods, devices and systems that quantify and/or control a suitable volume of sample that is placed on a plate without the use of a pipette and/or a fixed microfluidic channel. A suitable volume, which may represent part or all of the sample volume, relates to the quantification and/or control of the concentration of the target analyte and/or object in the sample. The methods, devices and systems of the present invention are easy to use and inexpensive.

12.1. Method for quantifying a suitable sample volume

Q1. A method for quantifying a suitable sample volume, comprising:

(a) obtaining a sample in which a suitable volume of the sample is to be quantified;

(b) providing two plates that are movable relative to each other in different configurations, wherein one or both plates contain spacers, and wherein the spacers have a predetermined distance between the spacers and height, and wherein each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample by placing the plates in a closed configuration, where in the closed configuration the plates face each other, the spacers and the suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted using the plates and spacers, and it is thinner than the maximum thickness of the sample when the plates are in an open configuration and at least one of the spacers is inside the sample;

(f) quantifying the appropriate sample volume while the plates are in a closed configuration;

where the suitable volume is at least a portion of the total volume of the sample.

Q2. In some embodiments, a method for quantifying a suitable volume in a sample involves:

(a) obtaining a first plate and a second plate;

(b) obtaining a sample to be quantified between two plates;

(c) deformation of the sample shape as a result of compression of the two plates, which reduces the thickness of the sample, and the distribution of the sample between the plates in the lateral direction; And

(d) quantifying the appropriate sample volume while the plates are in a closed configuration;

where the suitable volume is at least a portion of the total volume of the sample.

12.2. Plate for use in quantifying the appropriate volume in a sample

Q3. Plate for use in quantifying a suitable volume in a sample, providing

a plate that contains on its surface (i) spacers that have a predetermined distance between the spacers and a height and are fixed to the surface, and (ii) a contact surface with a sample of the corresponding volume to be quantified, where at least one of the spacers is located inside contact area with the sample.

12.3. Device for use in quantifying a suitable volume in a sample

Q4. A device for quantifying a suitable volume in a sample, comprising

a first plate and a second plate that (a) are movable relative to each other into different configurations, and (b) each has a sample contact area for the purpose of contacting the sample with a suitable volume to be quantified,

wherein one or both plates comprise spacers on their surface(s), the spacers have a predetermined spacer spacing and height, and the spacers are secured to their respective plates;

where one of the configurations is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates,

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted by the plates and spacers, and is thinner than that when the plates are in an open configuration, and at least one of the separators is located inside the sample; And

wherein the suitable volume of the sample is quantified in a closed configuration, and the suitable volume is at least a portion of the total volume of the sample.

12-5. Measuring the appropriate sample volume

MS1. In the present invention, quantifying the appropriate sample volume when the plates are in a closed configuration includes, without limitation, each of the following five embodiments:

(a) measuring a suitable volume of the sample by a method that is mechanical, optical, electrical, or any combination thereof;

(b) measuring one or more parameters associated with a suitable sample volume independently using a method selected from a method that is mechanical, optical, electrical, or any combination thereof;

(c) applying a predetermined parameter(s) associated with a suitable sample volume (ie, sample parameter(s) specified before the plates are in a closed configuration);

(d) determining a suitable sample volume by (i) measuring one or more parameters associated with the suitable volume while the plates are in a closed configuration, and (ii) pre-determining other parameters associated with the suitable volume before the plates are in closed configuration;

(e) determination of non-sample volume;

(f) any combination of the above (i.e. a, b and c).

In the method of MS1, mechanical methods include, without limitation, the use of spacers (i.e., a mechanical device that adjusts the space between the internal surfaces of the substrate and the cover plate to a predetermined value), a mechanical probe or scale rulers, sound waves (e.g., reflections and/or or ultrasonic wave interference to measure space) or any combination thereof.

In the method of MS1, optical methods include, without limitation, the use of light interference or optical imaging (e.g., obtaining a 2D/30D image of a sample, multiple optical imaging (at different viewing angles, different wavelengths, different phase and/or different polarization), image processing or any combination thereof.

Electrical methods include, but are not limited to, determination of capacitance, resistance or impedance or any combination thereof.

In some embodiments of the method of MS1, the sample thickness measurement is a measurement of the space between the inner surfaces of two plates.

In the method of MS1, in some embodiments, the use of a predetermined one or more parameters associated with a suitable sample volume, where the predetermined parameter is a predetermined sample thickness that is adjusted by the spacers when the plates are in a closed configuration.

In some embodiments of the method of MS1, the use of a predetermined one or more parameters associated with a suitable sample volume, where the predetermined parameter is a predetermined height of the divider.

In some embodiments of the method of claim MS1, the parameters associated with a suitable sample volume are parameters in a closed configuration, which include, but are not limited to (i) the spacing between the internal surfaces of the first plate and the second plate (in CROF), (ii) the thickness of the sample, (iii) all or a suitable portion of the sample area, (iv) all or a suitable portion of the volume, or (v) any combination thereof.

In some embodiments of the method of MS1, quantifying the sample volume or suitable sample volume involves the steps of (i) multiplying the thickness of the sample by the area of the entire sample to obtain the volume of the entire sample, (ii) multiplying the thickness of the sample by the corresponding area of the sample to obtain the appropriate sample volume, or (iii) multiplying the appropriate sample thickness by the entire or suitable area of the sample to obtain the appropriate sample volume.

In some embodiments of the method of MS1, the measurement is a 3D (three-dimensional) image of a suitable volume.

In some embodiments of the method of MS1, the quantitative determination of a suitable sample volume is accomplished by measuring the area of the suitable sample volume in a horizontal projection, and then using it together with the thickness of the suitable volume to determine the volume of the suitable sample volume, where the thickness of the suitable volume is determined based on information about separator, and the separator information includes the height of the separator.

In some embodiments, the method of MS1 quantifies a suitable sample volume by measuring the horizontal area of the suitable sample volume and separator together, then applying it to the thickness of the suitable volume and the volume of the separators to determine the volume of the suitable sample volume, wherein the thickness of the suitable volume is determined based on from separator information.

In some embodiments, the method of MS1 quantifies a suitable sample volume by measuring the horizontal area and thickness of the suitable sample volume.

In some embodiments of the method of MS1, the quantification of the appropriate sample volume is accomplished by measuring the volume of the appropriate sample volume optically.

In some embodiments of the method of MS1, scale marks are used to facilitate quantification of a suitable sample volume while the plates are in a closed configuration, where some embodiments of scale marks, their use, as well as measurements, etc. are described in section 2.

In some embodiments of the method of MS1, quantifying a suitable sample volume involves the step of subtracting a non-sample volume, where in some embodiments, the non-sample volume is determined using the embodiments described in the present invention.

12-4. Method for quantifying the concentration of analytes in a suitable sample volume

Q5. A method for the quantitative determination of analytes in a suitable volume of sample, comprising:

(a) performing the steps in the method of paragraph Q1 and

(b) after step (a) measuring the signal associated with the analytes from a suitable volume, where the suitable volume represents at least a portion of the total volume of the sample.

Q6. A method for the quantitative determination of analytes in a suitable volume of sample, comprising:

(a) performing the steps in the method of paragraph Q2 and

(b) after step (a) measuring the signal associated with the analytes from a suitable volume, where the suitable volume represents at least a portion of the total volume of the sample.

In some embodiments, the method of any one of Q5-6 further includes the step of calculating the concentration of the analytes by dividing the signal value associated with the analytes from a suitable sample volume by the volume of a suitable volume.

In the method of any one of items Q5-6, one or both plates further comprise a binding portion, a storage portion, or both.

In some embodiments of the method of any one of Q5-6, the signal associated with the analyte is a direct signal of the analytes or a label attached to the analyte.

Q7. A method for the quantitative determination of analytes in a suitable volume of sample, comprising:

(a) performing the steps in the method of item Q1, wherein one or both plates further comprise a binding site; And

(b) measuring, after step (a), the signal associated with the analytes from a suitable volume,

where the suitable volume is at least a portion of the total volume of the sample.

Q8. A method for the quantitative determination of analytes in a suitable volume of sample, comprising:

(a) performing the steps in the method of item Q2, wherein one or both plates further comprise a binding site; And

(b) measuring, after step (a), the signal associated with the analytes from a suitable volume,

where the suitable volume is at least a portion of the total volume of the sample.

In some embodiments of the method of any one of Q7-8, the signal associated with the analytes is a direct signal from the analytes that bind to the binding site, or a label attached to the analyte that binds to the binding site.

12.5. Plate for use in quantifying the concentration of analytes at a suitable volume in a sample

Q9. A plate for use in quantifying the concentration of analytes in a suitable volume in a sample, providing

a plate that contains on its surface (i) separators that have a predetermined distance between the separators and height, and (ii) a sample contact area for the purpose of bringing the sample into contact with a concentration of analytes in a suitable volume to be quantified, where at least At least one of the separators is located inside the contact area with the sample.

12.6. Plate for use in quantifying the concentration of analytes at a suitable volume in a sample

The concentration of target analytes and/or entities in a sample can be quantified or controlled if the amount of target analytes and/or entities in the sample is quantified and an appropriate sample volume is quantified.

Q10. A device for quantitatively determining the concentration of analytes in a suitable volume in a sample, comprising:

a first plate and a second plate that (a) are movable relative to each other in various configurations, and (b) each has a sample contact area for the purpose of bringing the sample into contact with a concentration of analytes in a suitable volume to be quantified, wherein one or both the plates contain on their surface(s) separators that have a predetermined distance between the separators and a height, and each of the separators is attached to the corresponding plates;

where one of the configurations is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates,

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted by the plates and spacers, and is thinner than that when the plates are in an open configuration, and at least one of the separators is located inside the sample; And

wherein the concentration of the analyte in a suitable sample volume is quantified in a closed configuration, and the suitable volume represents at least a portion of the total volume of the sample.

In the device of any one of Q9 and Q10, the plate further comprises a binding portion or a storage portion, or both. One embodiment of the binding site provides a binding site that binds analytes in the sample.

In the device of any one of claims Q9 and Q10, the plate further includes one or a plurality of scale marks, where some embodiments of the scale marks are described in section 2.

In the method or apparatus of any one of claims Q1-10, in some embodiments, the measurement device includes at least one of an imaging device and a camera.

In the method or apparatus of any one of paragraphs Q1-10, in some embodiments, the measurement device is configured to visualize the horizontal projection area of a suitable volume of the sample.

In the method or apparatus of any one of paragraphs Q1-10, in some embodiments, the measurement device includes a light source for illuminating a horizontal projection area of a suitable volume of the sample.

In the method or apparatus of any one of paragraphs Q1-10, in some embodiments, the step of calculating the concentration involves dividing all target analytes or object by an appropriate sample volume.

In the method or apparatus of any one of Q1-10, in some embodiments, signal measurement involves using an optical imaging device to count the amount of target analytes or object. For example, the measurement may involve using an optical microscope to measure blood cells (red blood cells, white blood cells, platelets) in a blood sample.

In the method or apparatus of any one of claims Q1-10, in some embodiments, measuring the amount of target analytes or object in a sample may involve an embodiment of a surface immobilization assay in which the target analytes or object are captured on a surface.

In some embodiments, the apparatus for quantifying sample volume or detecting/quantitating an analyte in a sample includes any of the devices in items Q1-10 in conjunction with (1) optical imaging devices and/or (2) a light source and optical imaging devices, etc. d. An optical imaging device includes a photosensor, optical lenses, filters, polarizers, wave plates, beam splitters, mechanical stands, or any combination thereof.

In some embodiments, measuring a suitable area or volume of a sample involves (i) having a mark on the first plate, the cover plate, between them, or any combination thereof, (ii) obtaining an optical image (e.g., obtaining a 2D/3D image sample, and image acquisition can be performed several times at different viewing angles, different wavelengths, different phases and/or different polarizations) and (iii) image processing based on the mark and sample images. "Appropriate" means related to the determination of the concentration of the target analyte.

Scanning. In some embodiments, when reading a signal from a sample, a scanning technique is used in which the reader (for example, photodetectors or a camera) reads part of the sample (or wafer) and then moves to another part of the sample (or wafer), and this process continues until until a certain pre-specified portion of the sample (or plate) is read. The scanning readout of a sample covers all parts of the sample (or wafer) or a small fraction of the sample (or wafer). In some embodiments, scanning readout is facilitated by location marks that indicate the location of the sample (or wafer). One example of location marks are periodic spacers, which have a fixed spacing and location, or corresponding area marks, which also have a predefined location and size to indicate the location of the sample or wafer.

13. Identification and quantification of analytes, etc. (D)

In certain embodiments, the analyte is detected and/or quantified (ie, analyzed) by measuring a signal associated with the analyte, where the signal is an optical signal, an electrical signal, a mechanical signal, a chemical-physical signal, or any combination thereof. In some embodiments, analyte analysis is performed while the two plates in the CROF device are closed relative to each other. In some embodiments, the analyte analysis is performed while the two plates in the CROF device are separated from each other.

An optical signal includes, but is not limited to, reflection, scattering, transmission, light absorption, spectrum, light color, emission, light intensity, light wavelength, light location, polarization, luminescence, fluorescence, electroluminescence, chemiluminescence, electrochemoluminescence, or any combination thereof. The optical signal is in the form of an optical image (i.e., a light signal depending on the location of the sample or device) or the total sum of all photons arriving from a specified area or volume. Preferred wavelengths of light are in the range of 400 nm to 1100 nm, in the range of 50 nm to 400 nm, in the range of 1 nm to 50 nm, or in the range of 1100 nm to 30,000 nm. Another preferred wavelength is in the terahertz range.

An electrical signal includes, but is not limited to, charge, current, impedance, capacitance, resistance, or any combination thereof. A mechanical signal includes, but is not limited to, a mechanical wave, a sound wave, a shock wave, or vibration. The chemical-physical signal includes, but is not limited to, the pH value, ions, heat, gas bubbles, and color changes that are produced during the reaction.

For example, the label is a bead, and the label is attached to the label during a process of specifically binding the analyte (e.g., using a detection agent to bind the bead to the analyte, using a capture agent to capture the analyte on the bead, using the capture agent to bind the analyte, and then using detection means for bead attachment or other approaches It should be noted that capture and detection means bind the analyte in a specific manner), measurement is then used to identify each of the beads that have attached to the analytes and they are counted.

In some embodiments, each of the analyte or beads is detected and counted using optical means (such as (i) optical labeling and label reading, (ii) surface plasmon resonance, (iii) optical interference, (iv) electrical methods (e.g., capacitance , resistance, impedance, etc.) etc. The sensors may be located on the surface of the first plate and/or the second plate.

Certain embodiments may include determining the concentration of an analyte in (a) a surface immobilization assay, (b) a volumetric assay (eg, blood cell counting), and (c) others. In some embodiments, methods for determining sample volume, appropriate sample volume, or concentration utilize a smartphone.

In some embodiments of the method or apparatus of any one of Q1-10, the signal measurement involves measuring a series of analytes in a sample, or measuring a series of tracers attached to analytes in the sample. In another embodiment of Q5, "signal measurement" is (a) identifying each of the analytes or a label attached to each analyte, and (b) counting their quantity.

In some embodiments, the detection of analytes is an electrical method in which electrodes are placed on one or both of the first and second plates (this is applicable to any of the methods and devices that employ CROF). Electrodes measure the charge, current, capacitance, impedance or resistance of a sample, or any combination thereof. Electrodes are used to measure the electrolyte content of a sample. The electrodes have a thickness equal to or less than the thickness of the separator. In some embodiments, the electrode acts as part of the separators. Electrodes are made from various conductive materials. The preferred electrode material is gold, silver, aluminum, copper, platinum, carbon nanotubes, or any combination thereof.

In the method or apparatus of any one of Q1-10, in some embodiments, devices are used during the measurement, i.e. a camera or photodetector together with an optical processor configured to carry out measurements.

In some embodiments of the method or apparatus of any one of Q1-10, the concentration determining devices comprise a processor configured to determine concentration based on measurements (volume, area, thickness, number of analytes, intensity).

The method or apparatus of any one of Q1 to 10, in some embodiments, further provides a concentration-determining device that is configured to determine the concentration of target analytes in a suitable volume based on measured horizontal area, thickness, and measured number of target molecules.

On the issue of signal detection using pixel reading and analysis

In some embodiments of the present invention, signals obtained from the sample, analytes, as well as the object, binding sites, reagents, CROF plates, or any combination thereof are detected and analyzed. Some embodiments of signal detection using pixel reading and analysis are described in the present disclosure, while other embodiments are described in publication number WO 2014144133 A and application number PCT/US2014/028417 (Chou et al, "Analyte Detection Enhancement By Targeted Immobilization, Surface Amplification, And Pixelated Reading And Analysis"), which are incorporated herein by reference for all purposes.

In some embodiments, the signal is an electromagnetic signal, including electrical and optical signals with various frequencies, light intensities, fluorescence, chromaticity, luminescence (electro- and chemiluminescence), Raman scattering, time-resolved signal (including flicker). The signals can often be forces due to local electrical, local mechanical, local biological, or local optical interactions between the wafer and the reader. The signal also includes the spatial (eg, positional), temporal, and spectral distribution of the signal. The detected signal may also be an absorption.

The analyte includes proteins, peptides, DNA, RNA, nucleic acid, small molecules, cells, nanoparticles of various shapes. The target analyte can be either in solution, in air, or in the gaseous phase. Determination involves identifying the existence, quantifying the concentration, and determining the states of the target analyte.

In some embodiments, an electric field is used to assist molecular selectivity or binding as well as detection.

Detection/reading methods

In some embodiments of optical detection (i.e., detection using electromagnetic radiation), the methods include, but are not limited to, far-field optical techniques, near-field optical techniques, epifluorescence spectroscopy, confocal microscopy, confocal microscopy, two-photon microscopy, and total internal reflection microscopy. where target analytes are labeled using an electromagnetic radiation emitter, and the signal for these types of microscopy can be amplified using the enhancement surface of the CROF plate.

In some embodiments, the signal contains information about the position, local intensity, local spectrum, local polarization, local phase, local Raman signature of these signals, or any combination thereof.

In some embodiments, signal detection involves measuring the overall signal from a site (eg, the signal from a site, regardless of location within the site).

In certain embodiments, signal detection is a measurement by imaging a signal over an area (ie, a signal versus location); namely, an area is divided into pixels and the signal from each pixel in the area is measured separately, also called “PIX” or “Pixel Image Identification”. Obtaining a separate measurement of each pixel can be simultaneous, or sequential, or mixed.

In some embodiments, the readout employs appropriate detection systems so that the signal is detected sequentially or simultaneously, or a combination thereof. In sequential detection, one or more pixels are detected at a time, and the scanner will be used to continue detection in other areas of the SAL. In simultaneous detection, a multi-pixel detector array such as an imaging camera (eg CCD) will be used to detect signals from different pixels at the same time. The scanner can be single-line or multi-line with a different pixel size for each line. In fig. 2C PCT/US2014/028417 schematically shows pixel readout on a three-axis table.

The readout/detection pixel size will be adjusted to balance optical resolution and overall readout time. A smaller pixel size will take longer to read/scan all or part of the SAL. Typical pixel sizes range from 1 µm to 10 µm. Pixel has different shapes: round, square and rectangular. The lower limit of pixel size is determined by the optical resolution of the microscopic system, and the upper limit of pixel size is determined to avoid readout error from incorrect optical signal of the imaging device (optical aberration, illumination uniformity, etc.).

Reading system

As indicated in the figures in PCT/US2014/028417, an embodiment of the reading system provides (a) a plate or plates used for CROF; (b) a reader 205 for the purpose of obtaining an image of signals emanating from the surface of the specified plate, where the signals represent individual binding events of target analytes; (c) an assembly device 300 that holds the plate and the imaging device; (d) electronic devices and data storage 301 for the purpose of storing said images; and (f) a computer executing a program to identify and count individual binding events in a region in the image.

The assembly 300 controls or changes the relative position between the wafer and the reader in at least one of three (x, y, z) orthogonal directions in the event of a signal being read. An embodiment of the assembly includes a scanner 301. In some embodiments, the scanner 301 scans in at least one of three (x, y, z) orthogonal directions.

In some embodiments, the reader 302 is a CCD camera. In some embodiments, the reader 302 is a photodetector comprising one or more optical devices selected from optical filters 303, a spectrometer, lenses 304, apertures, a beam splitter 305, mirrors 306, polarizers 307, waveplates, and shutters. In some embodiments, the reader 302 is a smartphone or mobile phone that has local or remote communication capabilities. The reader collects information about the position, local intensity, local spectrum, local Raman signature of said signals, or any combination thereof.

In some embodiments, optical filters 303, light beam splitters 305, optical fibers, a photodetector (e.g., a junction, diode, PMT (photomultimer tube) or APD (avalanche photodiode), an imaging camera (e.g., a CCD or cell phone camera), and a spectrometer together with a scanner equipped with an assembly device 301, are connected to a microscope system that uses a far-field confocal setup or a wide-field landscape setup.

In some embodiments, in a confocal setup, the readout is accomplished by capturing the brightness, temporal variation, and spectral variation of one or more pixels at a time and raster scanning the entire SAL region of interest. In some embodiments, in a wide-area landscape installation, the camera is used to record the brightness and temporal change of all or part of the SAL at a point in time. In some embodiments, appropriate optical filters and light beam handlers (polarizer, beam splitters, optical fibers, etc.) are required to ensure that only the desired signal is collected and detected. In fig. 9 PCT/US2014/028417 schematically shows one component diagram for such a system. In some embodiments, the analysis involves image processing methods, including but not limited to methods in Open-CV or Image-J.

Pixel analysis (PIX). In some embodiments, PIX signals detected on a pixel basis are analyzed to determine the number and/or types of specific molecules in a particular pixel or multiple pixels, which in turn is used to quantify the type and/or concentration of target analytes. The term "pixel detected signal" refers to a method in which an area that has signal(s) is divided into pixels and the signal from each pixel of the area is measured separately, also called "PIX" or "pixel-in-image detection". Obtaining a separate measurement of each pixel can be simultaneous, or sequential, or mixed.

In some embodiments, the analysis involves analyzing spatial, temporal, spectral information about the signal. In some embodiments, the analysis includes, but is not limited to, statistical analysis, comparison, integration, etc. In fig. 5 in PCT/US2014/028417 presents a flow chart for one such embodiment of this method.

14. Tags

One or any combinations of optical tag embodiments described throughout the present invention are applicable to all methods and devices described throughout the present invention.

In some embodiments, the label(s) are attached to the detection agent(s), analyte(s), or object(s). In certain embodiments, the label is an optical label, an electrical label, enzymes that can be used to generate an optical or electrical signal, or any combination thereof. In certain embodiments, the detection agent(s), analyte(s), or entity(ies) are attached to a connecting molecule (eg, a protein, nucleic acid, or other compounds), which is subsequently attached to a label. In certain embodiments, cells (eg, blood cells, bacteria, etc.) or nanoparticles are stained with labels. In some embodiments, an optical tag is an object that can generate an optical signal, where the generation of an optical signal includes, but is not limited to, reflection, scattering, transmission, absorption, spectrum, color, emission, intensity, wavelength, location, polarization of light (e.g., photons ), luminescence, fluorescence, electroluminescence, photoluminescence (fluorescence), chemiluminescence, electrochemiluminescence, or any combination thereof. In some embodiments, the optical signal is in the form of an optical image (ie, a light signal depending on the location of the sample or device) or the total sum of all photons arriving from a specified area or volume. Preferred wavelengths of light are in the range of 400 nm to 1100 nm, in the range of 50 nm to 400 nm, in the range of 1 nm to 50 nm, or in the range of 1100 nm to 30,000 nm. Another preferred wavelength is in the terahertz range.

Granules, nanoparticles and quantum dots. In some embodiments, the optical tag is beads, nanoparticles, quantum dots, or any combination thereof.

In some embodiments, the diameter of the beads, nanoparticles, or quantum dots is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 20 nm or less, 30 nm or less, 40 nm or less, 50 nm, or less, 60 nm or less, 70 nm or less, 80 nm or less, 100 nm or less, 120 nm or less, 200 nm or less, 300 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less than, 1500 nm or less, 2000 nm or less, 3000 nm or less, 5000 nm or less, or a range between any two of these values.

In some embodiments, beads or quantum dots are used as tags and are precoated on CROF wafers, and the internal space between two wafers is 1 μm or less, 10 μm or less, 50 μm or less, or a range between any two of these values.

In some embodiments, separation between beads in solution

- Diffusion time. (The thickness of a suitable volume of transmission medium results in a diffusion time of the optical mark through the thickness of less than 1 ms.

- You can control the dissolution time. Photon, heat or other types of excitation and their combinations can be used to carry out control. Dissolution will not begin until excitation energy is applied.

In some embodiments, the tags are nanoparticles having a diameter of 10 nm or greater. Nanoparticles with such a large diameter have a lower diffusion constant than small molecules (mass <1000 Da) and large molecules (mass = 1000-1000000 Dalton (Da), resulting in longer diffusion times for a given solution and distance. To reduce diffusion times, it is necessary to reduce the diffusion distance.

They offer particular advantages over the prior art if the optical tags are beads or other nanoparticles with a diameter of more than a few nanometers. This is because the diffusion constant of an object in a liquid for the first order approximation is inversely proportional to the diameter of the object (according to the Einstein-Stokes equation).

For example, an optical tag that is a bead with a diameter of 20 nm, 200 and 2000 nm, respectively, has a diffusion constant and therefore a diffusion time that is 10, 100 and 1000 times larger and longer than in the case of a bead with a size of 2 nm. As for the typical diffusion distance used in these assays, this will result in long incubation times to saturation, which is impractical for Point of Care (Point of Care) applications.

However, the present invention solves the problem of long incubation times for optical tags with a diameter greater than a few nanometers. In the present invention, the optical mark is stored on the surface of the wafer, and then the storage surface is placed adjacent to the bonding site with a separation distance (between them) of the order of sub-millimeters, micrometers or even nanometers, and the separation gap is filled with a transmission medium (wherein the stored optical mark is dissolved in the transmission medium and diffuses to the binding site). With the present invention, it is also possible to control the uniformity of such a small distance over a large area of the binding site and do this easily using spacer technologies.

The introduction of a label into the analyte may include the use, for example, of a labeling agent, such as an analyte-specific binding element that contains the detectable label. Detection tags include, but are not limited to, fluorescent tags, colorimetric tags, chemiluminescent tags, enzyme-linked reagents, multicolor reagents, avidin-streptavidin-associated detection reagents, and the like. In certain embodiments, the detectable label is a fluorescent label. Fluorescent tags are labeling fragments that are detected using a fluorescence detector. For example, binding a fluorescent label to an analyte of interest may enable detection of the analyte of interest using a fluorescence detector. Examples of fluorescent labels include, but are not limited to, fluorescent molecules that fluoresce upon contact with a reagent, fluorescent molecules that fluoresce when irradiated with electromagnetic radiation (e.g., UV, visible light, X-ray, etc.), and the like.

In certain embodiments, suitable fluorescent molecules (fluorophores) for labeling include, but are not limited to, IRDye800CW, Alexa 790, Dylight 800, fluorescein, fluorescein isothiocyanate, carboxyfluorescein succinimidyl esters, fluorescein succinimidyl esters, fluorescein dichlorothiazine 5-isomer, scaffold car boxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green 514; Lucifer yellow, acridine orange, rhodamine, tetramethylrhodamine, Texas red, propidium iodide, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazoylcarbocyanine iodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (Tetramethylrhodamine methyl ester), TMRE (Tetramethylrhodamine ethyl ester), Tetramethylrosamine, Rhodamine B and 4-dimethylaminotetramethylrosamine, Green Fluorescent Protein, Blue Shift Green Fluorescent Protein, Green Shift Green Fluorescent Protein, Red Shift Green Fluorescent Protein, yellow-shifted green fluorescent protein, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and derivatives such as acridine, acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonylphenyl]naphthalimide-3,5-disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; 4,4-difluoro-5-(2-thienyl)-4-boron-3a,4a diaza-5-indacene-3-propionic acid BODIPY; cascading blue; brilliant yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5''-dibromopyrogallol-sulfonaphthalein (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-(dimethylamino)naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin and its derivatives: eosin, eosin isothiocyanate, erythrosine and derivatives: erythrosine B, erythrosine, isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE) , fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; malachite green isothiocyanate; 4-methylumbelli-feroneorthocresolphthalein; nitrotyrosine; pararosaniline; phenyl red; B-phycoerythrin; o-phthaldialdehyde; pyrene and its derivatives: pyrene, pyrene butyrate, succinimidyl-1-pyrene; butyrate quantum dots; reactive red 4 (Cibacron™ Brilliant Red 3 BA), rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxy-rhodamine (R6G), lissamine rhodamine B sulfonyl chloride (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CAL Fluor Orange 560; terbium chelate derivatives; Su 3; Su 5; Su 5.5; Su 7; IRD 700; IRD 800; La Jolla Blue; phthalocyanine and naphthalocyanine, coumarins and related dyes, xanthene dyes such as rhodoles, resorufins, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquino us, fluorescent complexes europium and terbium; their combinations, etc. Suitable fluorescent proteins and chromogenic proteins include, but are not limited to, green fluorescent protein (GFP), including without limitation GFP derived from Aequoria victoria, or a derivative thereof, for example, a “humanized” derivative such as enhanced GFP; GFP from another species such as Renilla reniformis, Renilla mulleri or Ptilosarcus guernyi; "humanized" recombinant GFP (hrGFP); any of a variety of fluorescent and colored proteins from Anthozoa species; their combinations, etc.

In certain embodiments, dyes that can be used to stain blood cells include Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue and Azure B), May-Gruenwald stain, Leishman's stain ("polychrome" methylene blue). i.e., demethylated into various azures) and eosin), erythrosin B dye (erythrosine B) and other fluorescent dye, including but not limited to acridine orange dye, 3,3-dihexyloxycarbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC ) and basic orange dye 21 (B021), ethidium bromide, brilliant sulfaflavin and stilbene disulfonic acid derivative, erythrosine B or trypan blue, Hoechst 33342, trihydrochloride, trihydrate and DAPI (4',6-diamidino-2-phenylindole, dihadrochloride) .

In certain embodiments, the labeling agent is designed to specifically bind to an analyte of interest. In certain embodiments, the labeling agent may be present in the CROF device before the sample is applied to the CROF device. In other embodiments, the labeling agent may be applied to the CROF device after the sample has been applied to the CROF device. In certain embodiments, after a sample is applied to the CROF device, the CROF device can be washed to remove any unbound components, such as unbound analyte and other non-analyte components in the sample, and the labeling agent can be applied to device for CROF after washing to label the bound analyte. In some embodiments, the CROF device may be washed after the labeling agent has bound to the analyte-capture complex to remove any excess labeling agent from the CROF device that is not bound to the analyte-capture complex.

In certain embodiments, the analyte is labeled after the analyte has bound to the CROF device, for example, by using a labeled coupling agent that can bind to the analyte at the same time as the capture agent to which the analyte is bound in the CROF device, i.e., the assay "sandwich" type. In some embodiments, the nucleic acid analyte can be captured on the CROF device, and the labeled nucleic acid can hybridize to the analyte at the same time as the capture agent to which the nucleic acid analyte binds in the CROF device.

In certain aspects, the CROF device amplifies a light signal, such as fluorescence or luminescence, that is generated by a detectable label directly or indirectly associated with an analyte, which in turn is associated with the CROF device. In certain embodiments, the signal is amplified using a physical signal amplification process. In some embodiments, the light signal is amplified by a nanoplasmonic effect (eg, surface-enhanced Raman scattering). Examples of signal amplification using nanoplasmonic effects are described, for example, in Li et al, Optics Express 201 1 19: 3925-3936 and WO 2012/024006, incorporated herein by reference. In certain embodiments, signal enhancement is achieved without the use of biological/chemical signal enhancement. Biological/chemical signal amplification may include enzymatic signal amplification (eg, used in enzyme-linked immunosorbent assays (ELISA)) and polymerase chain reaction (PCR) signal amplification. In other embodiments, signal amplification can be achieved through a physical process and biological/chemical amplification.

Sensitivity. In certain embodiments, the CROF device is configured to provide a detection sensitivity of 0.1 nM or less, such as 10 pM or less, or 1 pM or less, or 100 fM or less, such as 10 fM or less, including 1 fM or less, or 0.5 fM or less, or 100 aM or less, or 50 aM or less, or 20 aM or less. In certain embodiments, the CROF device is configured to provide detection sensitivity in the range of 10 aM to 0.1 nM, such as 20 aM to 10 pM, 50 aM to 1 pM, including 100 aM to 100 fM. In some cases, the CROF device is configured to detect analytes at a concentration of 1 ng/mL or less, such as 100 pg/mL or less, including 10 pg/mL or less, 1 pg/mL or less, 100 fg/mL or less, 10 fg/mL or less, or 5 fg/mL or less. In some cases, the CROF device is configured to detect analytes at concentrations ranging from 1 fg/mL to 1 ng/mL, such as 5 fg/mL to 100 pg/mL, including 10 fg/mL to 10 pg/mL ml. In certain embodiments, the CROF apparatus is configured to provide a dynamic range of 5 orders of magnitude or greater, such as 6 orders of magnitude or greater, including 7 orders of magnitude or greater.

Reading. In certain cases, the time period from application of the sample to the CROF device to the reading of the CROF device may range from 1 second to 30 minutes, such as from 10 seconds to 20 minutes, from 30 seconds to 10 minutes, including from 1 minute to 5 minutes. In some cases, the period of time from application of the sample to the signal amplification detector to generation of an output signal that can be received by the device may be 1 hour or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less, 1 minute or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, 1 second or less, or even less. In some cases, the period of time from application of the sample to the signal amplification detector to generation of an output signal that can be received by the device may be 100 milliseconds or more, including 200 milliseconds or more, such as 500 milliseconds or more, 1 second or more, 10 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, or longer.

Any suitable method can be used to read the CROF device to obtain a measurement of the amount of analyte in the sample. In some embodiments, sensing a CROF device includes receiving an electromagnetic signal from a detectable label associated with an analyte in the CROF device. In certain embodiments, the electromagnetic signal is a light signal. The received light signal may include light intensity, light wavelength, light source location, and the like. In specific embodiments, the light signal generated by the tag has a wavelength that is in the range of 300 nm to 900 nm. In certain embodiments, the light signal is read as a visual image of the device for CROF.

In certain embodiments, sensing a CROF device includes providing an electromagnetic radiation source, such as a light source, as an excitation source for a detectable label associated with a biomarker in the CROF device. The light source may be any suitable light source for exciting the detectable mark. Examples of light sources include, but are not limited to, sunlight, natural light, UV lamps, fluorescent lamps, light-emitting diodes (LEDs), photodiodes, incandescent lamps, halogen lamps, and the like.

Reading of the CROF device can be accomplished using any suitable method for measuring the amount of analyte present in the sample and associated with the CROF device. In certain embodiments, the CROF device is read by a device configured to capture a light signal from a detectable label associated with an analyte in the CROF device. In some cases, the device is a handheld device such as a cell phone or smartphone. Any suitable handheld device capable of reading a device for CROF can be used in the devices, systems and methods of the present invention. Devices configured to read a device for CROF are described, for example, in US provisional patent application Serial No. 62/066777, filed October 21, 2014, which is incorporated herein by reference.

In some embodiments, the device includes an optical recording apparatus that is configured to capture a light signal from the CROF device, such as capturing an image of the CROF device. In certain cases, the optical recording apparatus is a camera, such as a digital camera. The term "digital camera" means any camera that contains as a main component an image capturing apparatus equipped with an optical imaging system for forming an optical image, an image sensor for converting the optical image into an electrical signal and other components, examples of such cameras include digital cameras, digital cinema cameras and webcams (i.e. cameras that are connected, either publicly or privately, to a device connected to a computer network for the exchange of images, including those connected directly to a computer network and those connected to a computer network network via a device, such as a personal computer, capable of processing information). In one example, device sensing for CROF may involve obtaining a video image that can record changes over time. For example, video may be acquired to evaluate dynamic changes in a sample applied to a CROF device.

In certain embodiments, the optical recording apparatus has a sensitivity that is weaker than the sensitivity of a high-sensitivity optical recording apparatus used in a research/clinical laboratory setting. In certain cases, the optical recording apparatus used in the inventive method is characterized by a sensitivity that is 10 times or more, for example 100 times or more, including 200 times or more, 500 times or more, or 1000 times or more, weaker than the sensitivity of high-sensitivity optical recording equipment used in research/clinical laboratory settings.

In certain embodiments, the device may have a video display. Video displays may include components on which a display page can be displayed in a manner perceivable by a user, such as a computer monitor, cathode ray tube, liquid crystal display, LED display, touch panel or touch display, and/or other means known in the art for emitting visually perceived output signal. In some embodiments, the device is equipped with a touch screen for displaying information, such as an image obtained from the detector and/or a report generated from the processed data, and allowing the subject to enter the information.

15. Multiplexing

In any embodiment described herein, the system may be designed to perform a multiplex assay and thus may comprise multiple storage sites, multiple binding sites, or multiple storage sites and multiple binding sites, whereby different assays can be performed in different areas on the surface one of the plates. For example, in one embodiment, one of the plates may contain multiple binding sites, each containing a different capture means, thereby allowing multiple analytes in a sample to be detected in the same assay. The plots can be spatially separated, although located next to each other.

In fig. 10 schematically illustrates an exemplary embodiment of the present invention that is multiplex detection in a single device for CROF using one binding site on one wafer and multiple storage sites on another wafer. Panels (a) and (b) are perspective views and cross-sections of the illustrative device, respectively. In this illustrative case, the multiplex device for CROF includes a first plate and a second plate, where one surface of the first plate has one binding site; wherein one surface of the second plate has a plurality of storage areas; and where different storage areas may have the same detection agent but at different concentrations, or may have different detection agents at the same or different concentrations. In some embodiments, the area of the binding area is larger than the area of each storage area. In some embodiments, the area of the binding area is greater than the total area of all storage areas, and/or the area of the binding area is aligned across the storage areas (that is, they are located on top of each other, namely the shortest distance between the binding area and a point on the storage units is the same or substantially the same).

In fig. 11 is a schematic diagram of a further exemplary embodiment of the present invention that provides multiplex detection in a single device for CROF using one storage site on one wafer and multiple binding sites on another wafer. Panels (a) and (b) are perspective views and cross-sections of the illustrative device, respectively. In an illustrative case, a multiplex device for CROF includes a first wafer and a second wafer, where one surface of the first wafer has multiple binding sites; wherein one surface of the second plate has one storage area; and where different binding sites may have the same entrapment agent but at different concentrations, or they may have different entrapment agents at the same or different concentrations. In some embodiments, the area of the storage area is greater than the area of each binding area. In some embodiments, the area of the storage area is greater than the total area of all binding sites, and/or is aligned across the binding sites (ie, they are located on top of each other).

In fig. 12 is a schematic representation of a further exemplary embodiment of the present invention, which is multiplex detection in a single device for CROF with multiple binding sites on one wafer and multiple corresponding storage sites on another wafer. Panels (a) and (b) are perspective views and cross-sections of the illustrative device, respectively. In an illustrative case, a multiplex device for CROF includes a first wafer and a second wafer, where one surface of the first wafer has a plurality of bonding sites; wherein one surface of the second plate has a plurality of corresponding storage areas; wherein each respective storage portion is located at a location on the second wafer that corresponds to the location of the binding portion on the first wafer such that, when the plates are placed opposite each other, each binding portion overlaps with only one storage portion, and each storage portion overlaps with only one portion binding; where different storage areas may have the same detection agent but at different concentrations, or may have different detection agents at the same or different concentrations; and where different storage areas may have the same capture agent but at different concentrations, or may have different capture agents at the same or different concentrations.

In certain embodiments, a device according to any of FIGS. 10, 11 and 12, wherein the first plate further comprises on its surface a first predefined analysis area and a second predefined analysis area, wherein the distance between the edges of several adjacent analysis areas is significantly greater than the thickness of the layer of uniform thickness when the plates are brought into a closed position where at least a portion of a layer of uniform thickness of the sample is located over predetermined areas for analysis, and where the sample contains one or more analytes that are capable of diffusion in the sample. By providing a distance between the edges of adjacent multiple assay sites that is greater than the thickness of the sample, it is possible to obtain multiple binding sites without liquid isolation of a separate portion of the sample, since incubation to assay saturation can occur with significant interdiffusion between two adjacent sites. By appropriately selecting the ratio of adjacent distance to sample thickness and appropriately selecting the measurement time between a time greater than the saturation incubation time for the assay but less than the time for significant interdiffusion between two adjacent sites, it is possible to perform multiplexing with CROF without isolating a single parts of the sample. In some embodiments, the ratio of adjacent distance to sample thickness in a closed configuration is 1.5 or greater, 3 or greater, 5 or greater, 10 or greater, 20 or greater, 30 or greater, 50 or greater, 100 or greater, 200 or more, 1000 or more, 10,000 or more, or a range between any two of these values. The ratio is 3 or more for a preferred embodiment, 5 or more for another preferred embodiment, 10 or more for a certain preferred embodiment, 30 or more for another preferred embodiment, and 100 or more for another preferred embodiment.

In certain embodiments, a device according to any of FIGS. 10, 11 and 12, where the first plate has on its surface at least three areas for analysis of the analyte, and the distance between the edges of any two adjacent areas for analysis is significantly greater than the thickness of the layer of uniform thickness when the plates are brought to the closed position, wherein at least a portion of a layer of uniform thickness is located above the areas to be analyzed, and where the sample contains one or more analytes that are capable of diffusion in the sample.

In certain embodiments, a device according to any of FIGS. 10, 11 and 12, where the first plate has on its surface at least two adjacent areas for analysis of the analyte, which are not separated by a distance that is significantly greater than the thickness of the layer of uniform thickness when the plates are brought to the closed position, where at least part a layer of uniform thickness located above the areas to be analyzed, and where the sample contains one or more analytes that are capable of diffusion in the sample.

The method or devices according to any of paragraphs U1-6, X-6, P1-8, W1-6, V1-4, UAB1-8, M1-2, S1-2, Q1-10 and H1, as well as any combination thereof wherein the first and second plates further comprise a binding portion(s) and a storage portion, as described in FIG. 10, fig. 11 or fig. 12 for multiplex detection.

In these embodiments, the device can perform simultaneous multiplex analysis of a liquid sample without liquid isolation (ie, without a physical barrier between analysis sites). This device may include a first plate and a second plate, where i. the plates are movable relative to each other in different configurations; wherein one or both plates are flexible; ii. one or both plates contain spacers that are fixed to the corresponding plate; and the dividers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the dividers; iii. each of the plates on its respective surface has a sample contact region for contacting the sample, which contains a sample that contains one or more target analytes that are capable of diffusion in the sample, iii. the first plate has on its surface one or a plurality of binding sites, each of which is characterized by a predetermined area and contains a capture means that binds and immobilizes the corresponding target analyte in the sample; and iv. the second plate has on its surface one or a plurality of corresponding storage areas, each of which is characterized by a predetermined area and contains a detection agent in a certain concentration, which, after contact with the sample, dissolves in the sample and diffuses into the sample, where each capture agent, the target analyte and the corresponding detection agent are capable of forming a capture agent-target analyte-detection agent sandwich at a binding site on the first plate; where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates, and where the other configuration is a closed configuration in which lead after placing the sample in an open configuration; and in a closed configuration: i. at least a portion of the sample is compressed into a layer of uniform thickness that is in contact with and bounded by the inner surfaces of the two plates, and that covers one or more bonding areas and one or more storage areas, ii one or more corresponding storage areas are located above one or more multiple binding sites, and iii. a layer of uniform thickness, which is regulated by separators and plates, is less than 250 microns, and it is significantly smaller than the linear dimension of the predetermined area of each storage area; and iv. there is no liquid isolation between the binding site and/or storage areas where the separation between the edges of adjacent storage sites and the separation between the edges of adjacent binding sites is greater than the distance over which the target analyte or detection agent can diffuse over a suitable period of time, and where there is no liquid isolation isolation between binding areas and/or storage areas.

In some embodiments, the first plate has multiple (at least 2, at least 4, or at least 16 or more) binding sites on its surface.

In some embodiments, each of the plurality of binding sites binds to a separate target analyte.

In some embodiments, the second plate has a plurality of (at least 2, at least 4, or at least 16 or more) corresponding storage areas on its surface.

In some embodiments, each of the plurality of respective storage sites is associated with a separate target analyte.

In some embodiments, the first wafer has on its surface a plurality of said bonding sites, and the second wafer has on its surface a plurality of said corresponding storage regions, with each bonding site facing a corresponding storage region when the wafers are in a closed configuration.

In some embodiments, the first plate has on its surface a plurality of said bonding sites, and the second plate has on its surface a storage region where at least some of the bonding sites face a region in the storage region when the plates are brought into a closed configuration.

In some embodiments, the first wafer has a bonding portion on its surface and the second wafer has a plurality of storage portions on its surface, where at least some of the storage portions face a region within the bonding portion when the plates are in a closed configuration.

In some embodiments, the first wafer has a plurality of binding sites on its surface, where the binding sites comprise different entrapment agents that bind and immobilize the same target analyte.

In some embodiments, the first plate has a plurality of bonding sites on its surface, where the bonding sites comprise the same gripping means.

In some embodiments, the capture agent is present at different densities at different binding sites. These embodiments can be used to provide a method for quantifying the amount of an analyte in a sample.

In some embodiments, there is separation between two adjacent binding sites or two adjacent storage sites, and wherein the ratio of separation to specimen thickness in a closed configuration is at least 3, such as at least 5, at least 10, at least 20, or at least 50.

In some embodiments, the distance between the spacers is in the range of 1 μm to 120 μm.

In some embodiments, the flexible plates have a thickness in the range of 20 µm to 250 µm (for example, in the range of 50 µm to 150 µm) and a Young's modulus in the range of 0.1 to 5 GPa (for example, in the range of 0.5-2 GPa).

In some embodiments, the product of the flex plate thickness and the flex plate's Young's modulus is in the range of 60 to 750 GPa-μm.

In some embodiments, the method may comprise (a) obtaining a sample that contains one or more target analytes that are capable of diffusing in the sample; (b) providing first and second plates that are movable relative to each other into different configurations, where i. one or both plates include spacers that are fixed to the corresponding plate, and one or both plates are flexible, ii. The dividers are characterized by a predetermined practically uniform height and a predetermined constant distance between the dividers, iii. the first plate has on its surface one or a plurality of binding sites, each of which has a predetermined area containing a capture means that binds and immobilizes the corresponding target analyte of (a); and iv. the second plate has on its surface one or a plurality of corresponding storage areas, each of which has a predetermined area and contains a detection agent in a concentration that, upon contact with the sample, dissolves in the sample and diffuses into the sample, where each capture agent, target analyte and the corresponding detection agent is capable of forming a capture agent-target analyte-detection agent sandwich at the binding site of the first plate; (c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers; (d) after (c) compression of the sample by bringing the two plates into a closed configuration, where the closed configuration is the configuration in which i. at least a portion of the sample is compressed into a layer of uniform thickness that is in contact with and limited by the internal surfaces of both plates, and which is in contact with one or more bonding areas and one or more storage areas, wherein the one or more corresponding storage areas are located above one or more binding sites, and iii. uniform layer thickness, controlled by separators and plates, is less than 250 microns and is significantly less than the linear dimension of the predetermined area of each storage area; (e) after (d) and while the plates are in a closed configuration, or (1) incubating the sample for a suitable period of time and then stopping the incubation; or (2) incubating the sample for a time that is equal to or greater than the minimum suitable time interval, and then assessing for a period of time that is equal to or less than the maximum suitable time interval, binding each target analyte to the binding site; where the appropriate time interval is: i. equal to or greater than the time it takes for the target analyte (a) to diffuse through a layer of uniform thickness in a closed configuration; and ii. significantly less time taken for lateral diffusion of the target analyte (a) through the smallest linear dimension of a predetermined area of the storage site or binding site; whereby, at the end of the incubation (1) or during the evaluation (2), the majority of the capture agent-target analyte-detector sandwich associated with each binding site is derived from a corresponding suitable volume of sample ; wherein the incubation allows each target analyte to bind to a binding site and detection means, wherein the corresponding suitable volume is the portion of the sample that is located above the corresponding storage site in a closed configuration, where the separation between the edges of adjacent storage sites and the separation between the edges of adjacent binding sites is greater , than the distance over which the target analyte or detection agent can diffuse over a suitable period of time, and where there is no liquid isolation between the binding sites and/or storage sites.

Any embodiment of the multiplex assay device described above can be used in this method.

16. Small volume samples or wide well reagent (E)

In some applications, a well on a plate will be used to test a sample with a sample volume that is small relative to the area of the well that the sample must cover. One aspect of the present invention is methods and devices that enable analysis and other chemical reactions with small volume samples or reagent in a wide well. The term "well" refers to a concave cell, recessed area or depression on the surface that prevents liquid contained within the well from flowing out of the well due to the solid bottom of the well and the enclosing side wall (Fig. 8). Hole area means the area enclosed by the side wall. The terms “small sample volume” and “wide well” mean that if the sample is dropped onto the bottom of the hole and without any device for distributing the sample, then the volume of the sample at the bottom of the hole is characterized by an area of contact with the bottom of the hole that is less than the area of the hole (i.e. small and wide is a comparison of the natural contact area of the sample and the area of the bottom of the hole). The hole plays the role of an enclosing separator (E).

In fig. 8 and 9 illustrate certain embodiments of guard-type plates and spacers (wells) for controlling sample thickness. Two exemplary embodiments are shown: (a) the first plate has a barrier-type separator (well) and at least one spacer within the well (Fig. 9), and (b) the first plate does not have a spacer within the well (Fig. 8). Other embodiments are that before the first and second plates are brought into a closed configuration, a barrier-type separator is located on one of the plates and an insulated separator(s) are located on the other plate; and in a closed plate configuration, the insulated spacer(s) are within the well.

In one embodiment, the volume of the sample placed in the well of the plate may be characterized by a predetermined volume (i.e., the volume is dispensed to a certain volume) that is approximately equal to the internal volume of the well (i.e., the product of the internal surface area of the well and the height of the well), so that when the plates are brought into a closed configuration, the sample substantially completely fills the well with little or no sample flowing out of the well.

In another embodiment, the volume of sample placed in the plate well is not dosed, and in a closed plate configuration, part of the sample fills the well almost completely, while another portion of the sample flows out of the well.

In other embodiments, a plurality of wells constitute one plate. In some embodiments, there are grooves (overflow points) between the wells for samples to overflow from the wells. Overflow points prevent sample from flowing from one well to another well(s).

E1. As shown in FIG. 8, a method for analyzing and/or carrying out chemical reactions in a small volume sample in a wide well involves:

(a) providing a first plate and a second plate that are movable relative to each other in different configurations, where the first plate has on its surface a dimple that is characterized by a predetermined size (including the bottom of the dimple, the depth and the rim), and a binding site at the bottom holes;

(b) obtaining a sample that (i) contains a target object capable of binding to a binding site and diffusion in the sample, and (ii) has a volume and wetting property such that the contact area of the sample placed only at the bottom of the well is without contact with a second plate, less than the area of the hole bottom;

(c) placing the sample within the well or on an appropriate area on a second plate, or both when the plates are in an open configuration;

where in an open configuration the two plates are either partially or completely separated from each other, and the space between the second plate and the bottom of the well is not controlled by the rim of the well (i.e., the depth of the well);

(d) after (c) distribution of the sample by placing the plates in a closed configuration; wherein in a closed configuration the second plate covers the well, the thickness of the sample at the binding site is controlled by the well and the second plate, and the sample has a larger contact area with the bottom of the well than when the plates are in an open configuration;

where the corresponding area of the second plate is the area along the top edge of the socket and inside the rim of the socket in a closed configuration.

In the method of item E1, the plate further comprises at least one insulated spacer within the well (ie, a well spacer).

In the method of item E1, in some embodiments, the volume of the sample is measured (eg, to a specific volume). The measured volume is approximately less than, greater than or equal to the volume of the well.

In some embodiments of the method of E1, the compression force on the outside of the plates is adjusted to hold the plates in a closed configuration.

In some embodiments of the method of E1, the capillary force is adjusted to hold the plates in a closed configuration.

As shown in FIG. 8d, in some embodiments of the method of paragraph E1, the bottom of the well, the corresponding area of the latter, or both the first and second are attached to spacers of a predetermined height, where in a closed configuration the thickness of the sample is controlled by the spacers, the rim, or both the first and second.

In some embodiments, the height of the spacer is equal to, less than, or greater than the depth of the well. The bottom of the hole is flat (i.e. level) or curved. In some embodiments, the spacers are (1) characterized by a predetermined space between the spacers, (2) an internal space of the sample, (3) secured to respective plates, or any combination thereof.

In some embodiments, the volume of the sample is approximately equal to the volume of the well minus the volume of the separators. In some embodiments, the second plate is configured to seal the well.

In some embodiments, the ratio of socket area to square of socket depth is 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 1000 or more, 10,000 or greater than, or a range between any two of these values.

The ratio of the socket area to the square of the socket depth is from 3 to 20 in a preferred embodiment, from 20 to 100 in another preferred embodiment, and from 100 to 1000 in another preferred embodiment, and from 1000 to 10000 in another preferred embodiment. 17. Quantification by correcting for effects produced by non-sample volume (C)

In the CROF process, the sample is often mixed with non-sample volume(s) that are caused by non-sample objects, which include, but are not limited to, separators, air bubbles, dust, or any combination thereof. Air bubbles or dust may be introduced during sample application or other processes in the CROF process. These non-sample objects occupy the volume and interior space of the sample, which must be adjusted for when determining the appropriate volume (volume of interest) of the sample. One aspect of the present invention is to correct the effects created by a non-sample volume in the interior space of a suitable sample volume between two plates, where the thickness of the suitable volume is controlled by spacers.

C1. A method of adjusting for effects caused by non-specimen material when determining an appropriate sample volume between two plates, comprising:

(a) obtaining a sample in which a suitable volume of the sample is to be quantified;

(b) providing two plates that are movable relative to each other in different configurations, wherein one or both plates contain spacers, and wherein the spacers have a predetermined distance between the spacers and height, and wherein each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) bringing the plates into a closed configuration, where in the closed configuration the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the sample of the suitable volume is controlled by the plates and spacers and is thinner than the maximum thickness of the sample when the plates are in open configuration, and a suitable volume may contain a volume of non-sample material;

(e) measuring, while the plates are in a closed configuration, (i) the horizontal surface area of a suitable sample volume and (ii) the volume of non-specimen material; And

(f) calculating a suitable sample volume using the thickness of the suitable volume adjusted by the spacers and correcting for effects due to non-sample material;

where the relevant volume is at least a portion of the total volume of the sample, and non-sample materials are materials that do not originate from the sample.

- Non-sample volume measurement is carried out by visualizing the sample between two plates.

18. Accurate quantification by double checking space

Given a given set of CROF conditions, even spacers and plates may provide a preset specimen thickness in a closed configuration, the actual set of conditions during a particular CROF may differ from those expected, resulting in errors in the preset final specimen thickness. To reduce such errors, one aspect of the present invention is to double check the final sample thickness in a closed configuration.

C2. A method for determining and checking the thickness of a suitable sample volume between two plates involves:

(a) obtaining a sample in which a suitable volume of the sample is to be quantified;

(b) providing two plates that are movable relative to each other in different configurations, wherein one or both plates contain spacers, and wherein the spacers have a predetermined distance between the spacers and height, and wherein each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) bringing the plates into a closed configuration, where in the closed configuration the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the sample of the suitable volume is controlled by the plates and spacers and is thinner than the maximum thickness of the sample when the plates are in open configuration, and a suitable volume may contain a volume of non-sample material;

(e) measuring, while the plates are in a closed configuration, (i) the horizontal surface area of a suitable sample volume and (ii) the volume of non-specimen material; And

(f) calculating an appropriate sample volume while correcting for effects due to non-sample material;

where the relevant volume is at least a portion of the total volume of the sample, and non-sample materials are materials that do not originate from the sample.

19. Flushing (WS)

In the present invention, one or any combinations of plate compression and retention embodiments described herein are used in all methods and devices described throughout the specification of the present invention.

A method for the washing step during analysis, comprising:

(a) carrying out the steps in one or any combination of the methods described above, and

(b) washing the sample or transfer medium between the plates.

In the CROF method, washing is accomplished by holding the plate in a closed configuration.

In the CROF method, washing is carried out by separating the plates from a closed configuration.

20. Multistep Assays (MA)

In the present invention, the embodiments described in the disclosure (i.e., all sections) can be used in combination (a) by combining one embodiment with other embodiment(s), (b) by using the same embodiment(s) more than once, and (c) any combination of (a) and (b).

MA1. A method for analyzing an analyte in a sample, comprising:

(a) obtaining a sample containing the analyte;

(b) carrying out a process using CROF; And

(c) separating the plates and carrying out the process using CROF. The method according to paragraph MA1 in some embodiments additionally

provides after stage (c) MA1 a stage of repeating the same stages from all stages

method according to point MA1 at least once.

MA2. A method for analyzing an analyte in a sample, comprising:

(a) obtaining a sample containing the analyte;

(b) carrying out a process using CROF;

(c) separating the plates and carrying out the process (washing) in which CROF is used; And

(d) carrying out a process using CROF.

The method of MA2 in some embodiments further comprises, after step (c) in MA2, the step of repeating the same steps from all steps of the method of MA2 at least once.

The method of item MA2 in some embodiments further includes, after step (c) in MA2, the step of repeating the same steps from all stages of the method of item MA1 at least once.

MA3. A kit for analyzing an analyte in a sample that contains;

the first CROF device to use CROF; And

a third plate that is combined with one of the plates of the first CROF device when the plates of the first CROF device are separated to form a second CROF device.

MA4. A kit for analyzing an analyte in a sample that contains:

the first CROF device to use CROF;

at least one binding site or storage site that is located on the sample contact area on the plate of the CROF device; And

a third plate which, when the plates of the first CROF device are separated, is combined with one of the plates of the first CROF device to form a second CROF device; wherein the binding site binds the target analyte to the surface of the plate, and wherein the storage region contains a reagent that, upon contact with the sample, can dissolve in the sample and diffuse into the sample.

Acquiring the image may involve the use of a smartphone. The methods of this section may further include the step of illuminating using a light source. The light source can be a laser, LED, lamp or camera flash.

Kit (MQXA) for analysis to identify the target object in the sample

The sample target assay kit may contain:

A. a first plate, where one surface of the first plate has one or more binding sites that can immobilize a target object, and the binding site contains a binding partner that binds the target object;

b. cover plate;

c. sample in the internal space between the cover plate and the first plate, where the sample contains the specified target object, which is movable in the sample, the shape of the sample is deformable, the first plate and the second plate are movable relative to each other, the shape of the sample practically corresponds to the internal surfaces, according to at least a portion of the sample is in contact with the binding site, and the internal space during incubation is less than a certain distance, while the sample is in contact with the specified binding sites;

d. an imaging device that can acquire an image of the surface of the first plate and/or the surface of the cover plate; And

e. a measuring device that can measure the space of the internal gap. Methods in this section may involve using a smartphone. The methods in this section may include the use of a lighting device. The lighting device may include a laser, LED, lamp, or camera flash.

21. Squeezing and holding plates (H)

Compressive forces. The CROF method uses forces to compress two plates, bringing the plates from an open configuration to a closed configuration. Compressive forces reduce the distance between the inner surfaces of the plates and thus the thickness of the sample that is located between the plates. In the present invention, compression forces include, but are not limited to, mechanical force, capillary forces (due to surface tension), electrostatic force, electromagnetic force (including light), and any combination thereof.

In some embodiments, when moving the plates from an open configuration to a closed configuration, an external force is applied to force the first plate and the second plate toward each other.

In some embodiments, when moving the plates from an open configuration to a closed configuration, external pressure is applied from the outside of the first plate and the second plate to force the plates toward each other, and the pressure is greater than the pressure between the plates. A device is used to create higher pressure on the outside of the plate than on the inside of the plate. The device includes in particular a sealing device.

In some embodiments, the compressive force is at least partially provided by capillary force, which is caused by the fluid between the first plate and the second plate and the corresponding surface tensions and interactions with the plates. In some embodiments, the liquid is the sample itself or a sample mixed with the liquid. In certain embodiments, capillary force is used in conjunction with other forces. In many cases, the sample is often in a liquid state and the surface tensions are suitable for introducing capillary force. In some embodiments, deformation of the sample by the plates may automatically stop when capillary force equals the force required to deform the sample.

In certain embodiments, the compressive force (hence the deformation of the sample) is created by isolating the pressure between the first plate and the second plate (internal pressure) from the pressure outside the plates (external pressure), and then reducing the internal pressure below the external pressure. Isolation can be accomplished using a vacuum seal or other devices.

In some embodiments, a combination of the methods described above is provided.

Gradual compression. In certain embodiments, the compressive force to bring the plates into a closed configuration is applied in a process called "gradual compression", which involves the following: compression (i.e., application of a compressive force) is first applied at one location of the plate(s), then gradually applied to other sample locations. In some embodiments of progressive squeezing, the compressive force (excluding capillary forces of the sample itself) at one location, after deforming the sample to the desired thickness at that location (i) is maintained throughout the process of squeezing and deforming the sample, (ii) is released when squeezing other locations, or (iii) apply (i) to a certain part of the plates and apply (ii) to another part of the sample.

In one embodiment of progressive compression, a roller is used to press a first plate and a second plate (the sample is located between the plates and the plates are slightly flexible) against another roller or a flat surface.

In another embodiment, the human fingers are a tool for compressing the plates (thus the sample). The compression is performed by one part of a person's hand relative to another part of the person's body (including another part of the person's hand) or by a person's hand relative to an object (for example, a table surface). In one embodiment, compression begins at one location on the sample and is gradually moved to other locations on the sample.

In one embodiment of progressive compression, a jet of compressed air is first directed to a location (eg, the center) of a pair of plates (which is between the first plate and the second plate, with one of the plates being slightly flexible) and the pressure is gradually applied to the other part of the pair of plates.

In another embodiment, one or both of the first plate and the second plate are flexible and in contact with one location of the sample, and then capillary force at that given location moves the pair of plates (towards each other) to deform the sample.

The advantages of gradual squeezing include the following: the ability to use less force to deform the sample (because for the same force, the smaller the squeezing area, the greater the pressure); assisting movement (deformation) of the sample and/or reducing the content of air bubbles in the sample. The greater the pressure, the greater the deformation of the sample. Gradual compression can improve the thickness uniformity of the deformed specimen.

Compression devices. Devices for controlling the compressive force(s) for sample deformation in CROF have several practical implementations. In some embodiments, a human hand is used to perform the compression, for example, the compression is performed by a person's fingers. In certain embodiments, a squeezing device is used, where the squeezing device includes, but is not limited to, a human hand(s), a mechanical clip, a mechanical press, a mechanical clamp, a mechanical slider, a mechanical device, an electromagnetic device, a roller that is rolled over a surface, two rollers, each other. opposite each other, a jet press, a hydraulic device, or any combination thereof. In certain embodiments, a pressurized fluid (including compressed air) is used to directly or indirectly compress the first plate and/or the second plate. "Directly" means that the pressurized liquid is applied directly to the first plate and/or the second plate; and “indirectly” means that it is applied through a third object. In certain embodiments, squeezing uses a combination of the above embodiments of squeezing devices and methods.

Additionally, in some embodiments, sample deformation is monitored for compression and deformation of the sample. Monitoring can be used to control compression and deformation of the sample. Strain monitoring includes, but is not limited to, mechanical, electrical, optical, chemical, magnetic, and any combination thereof. Mechanical methods include, but are not limited to, mechanical sensors, spacers (mechanical stops, discussed in more detail below), and sound waves.

In a CROF, the space control device includes a mechanical press, mechanical stages for translational motion, human fingers, a fluid that provides capillary forces that move the plates relative to each other, a fluid (including air) that applies pressure to the plates, or a combination thereof. .

In certain embodiments, mechanical stages (translational and/or rotational) are used to monitor sample deformation and sample thickness, and they operate in conjunction with monitoring systems.

In some embodiments, the compressive force is at least partially provided by a press (which is a device that forces the plates into a closed configuration) configured to press the plates together into a closed configuration.

In some embodiments, compression of the plates is accomplished by a human hand. The person may be the subject being tested, the subject performing the test, or the subject collecting the sample.

In some embodiments, plate compression involves holding two plates together to apply capillary force. Capillary force is generated by imparting hydrophilicity to at least a portion of the inner surface of one or both plates. With the proper capillary force, the two plates are able to maintain the same space between the plates and the same thickness of the suitable sample volume, the same as when the plates were initially converted to a closed configuration, even if some or all of the forces (except capillary force) that were used to compress are removed plates into a closed configuration.

In some embodiments, a device that applies a compressive force to the outer surface of the plates to reduce the distance between the inner surfaces of the plates includes a contact surface corresponding to the outer surfaces of the plate, where the device contact surface is a surface of the device that contacts the outer surface of the plates, and "matching the outer surface of the plate" means that the surface of the device can be deformed during compression to conform to the shape of the outer surface of the plate. In one illustrative embodiment, the compression device is a person's fingers. In another illustrative embodiment, the compression device has a contact surface made of soft plastic or rubber types.

Self-retention (maintaining the final thickness of the sample after the compression forces are removed). In some CROF compression embodiments, after the sample is deformed in a closed configuration, some of the compression forces are eliminated and the sample maintains the same final sample thickness as if the compression forces were still present. This situation is called “self-restraint.” One reason for self-retaining is that after the compressive forces that were applied on the outside of the plate pair are removed, other forces still exist between the inner surfaces of the plates, such as capillary force, which holds the plate pair together. Capillary force is due to the wetting properties of the sample on the plates.

For self-retention to occur, it is necessary to control the wetting properties of the surface of the plates, the total contact area of the sample with the plates, the final thickness of the sample in a closed configuration, or a combination of these.

In some embodiments, to achieve self-retention, one or both of the inner surfaces of the plates are hydrophilic. Namely, either one of the plates has an inner surface that is hydrophilic, or both plates have an inner surface that is hydrophilic.

Capillary force depends on the radius of curvature of the liquid surface, and the smaller the curvature, the higher the capillary force. Less curvature can be achieved by using less space between two plates (i.e. a pair of plates) and thus less sample thickness. In some embodiments, the final sample thickness to achieve self-retention is 10 nm or less, 100 nm or less, 100 nm or less, 500 nm or less, 1 μm (micrometer) or less, 2 μm or less, 3 μm or less, 5 µm or less, 10 µm or less, 20 µm or less, 50 µm or less, 70 µm or less, 100 µm or less, 150 µm or less, 300 µm or less, 500 µm or less, 700 µm or less, 1000 µm or less, 1200 µm or less, or a range between any two of these values.

In some embodiments, the area of the sample in contact with the self-retaining plates is no more than 10 µm ² , no more than 100 µm ² , no more than 200 µm ² , no more than 500 µm ² , no more than 1000 µm ² , no more than 2000 µm ² , no more than 5000 µm ² , no more than 8000 µm ² , no more than 0.01 mm ² , no more than 0.05 mm ² , no more than 0.1 mm 2, no more than 0.5 ^{mm 2 ,} no more than 1 mm ² , not more than 5 mm ² , no more than 10 mm ² , no more than 50 mm ² , no more than 100 mm ² , no more than 500 mm ² , no more than 1000 mm ² , no more than 2000 mm ² , no more than 5000 mm ² , no more than 10000 mm ² , not more than 100,000 mm ² or a range between any two of these values.

In some embodiments, the wetting properties of the inner surface of one or both plates are modified to improve self-retention.

HS.1 In some embodiments, the CROF method uses a device to apply a compressive force to force the wafers into a closed configuration, and once the closed configuration is achieved, the compressive force of the device is released and the sample thickness and the internal space between the surfaces of the wafers remains approximately the same as before removing the compression force of the device. In some embodiments, the methods of the preceding claim further include the step of reading a signal from or between plates, wherein the signal includes, but is not limited to, a signal associated with analytes, subject, labels, sample volume, concentration of material (i.e., chemicals), or any their combination.

In the method of point SH.1, the device is a human hand(s), a mechanical clip, a mechanical press, a mechanical clamp, a mechanical slider, a mechanical device, an electromagnetic device, a roller that is rolled over the surface, two rollers opposite each other, a jet press, a hydraulic device or any combination thereof.

In the method of paragraph SH.1, in some embodiments, the expression "the thickness of the sample and the space between the internal surfaces of the plates remains approximately the same as before the compression force of the device was removed" means that the relative difference in the thickness of the sample and the space between the internal surfaces of the plate before and after the compression force release is 0.001% or less, 0.01% or less, 0.1% or less; 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or a range between any of these values.

In some embodiments of the method of SH.1, the thickness of the sample and the space between the inner surfaces of the plates after the compression force is removed by the device are predetermined, where the expression "preset" means that the thickness and the space after the compression force is removed are known before the compression force is applied. for given compression conditions.

H1. A method of reducing the thickness of a suitable sample volume and maintaining the reduced thickness, comprising:

(a) obtaining a sample in which it is necessary to reduce the thickness of a suitable volume of the sample;

(b) providing two plates that are movable relative to each other in different configurations, wherein one or both plates contain spacers, and wherein the spacers have a predetermined distance between the spacers and height, and wherein each of the spacers is secured to its respective plate;

(c) placing the sample on one or both plates when the plates are in an open configuration; where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) distribution of the sample using a compression device that forces the plates into a closed configuration, where in the closed configuration the plates face each other, spacers and a suitable sample volume are between the plates, the thickness of the suitable sample volume is adjusted by the plates and spacers and is thinner the maximum thickness of the sample when the plates are in an open configuration, and at least one of the spacers is inside the sample; And

(e) after (d) release of the device, where, after releasing the compression device, the space between the plates remains the same or approximately the same as during use of the device,

where the suitable volume is at least a portion of the total volume of the sample.

In the method according to point HI, it is approximately the same as the space between the plates is no more than 1%, no more than 2%, no more than 5%, no more than 10%, no more than 20%, no more than 50%, no more than 60%, no more than 70%, no more than 80%, no more than 90%, or a range between any two of these values.

For example, in CROF, a person's hand or hands are used to compress two plates into a closed position, then the hand(s) and thus the compression force from the hand(s) is removed, but the final thickness of the sample is still the same as when the existence of compression force due to the hands.

22. Other combinations

In the present invention, each of the embodiments in the disclosures (i.e., all sections) may be used (a) alone, (b) in combination with other embodiment(s), (c) multiple times, and (d) in any combinations (a) - (c).

The methods and devices disclosed in the present invention can be used individually or in any combination thereof. The term "QMAX" method or device refers to embodiments of the method or device described herein.

In some embodiments, the methods and devices disclosed in the present invention may be used in the form of Q, X, A, M, QX, QA, QM, XA, XM, AM, QXA, QAM, XAM, and QXAM.

Some embodiments of the use of Q, X, A and M for surface immobilization analysis include:

a. providing a first plate, wherein the surface of the first plate has at least one well of known depth and volume, and the bottom surface of the well has one or more binding sites that can immobilize the target object in the sample;

b. placement in a well of a sample with a volume approximately the same as the volume of the well, where the sample contains the target object, the target object is movable in the sample, the shape of the sample is deformable, and the sample covers only part of the well (hence, characterized by a conventional thickness that is greater than than the depth of the hole);

c. presence of a cover plate;

d. causing the first plate and the cover plate to face each other, where the sample is located between the inner surfaces of the first plate and the second plate;

e. reducing the thickness of the sample by reducing the space between the internal surfaces of the first plate and the second plate and

f. incubating the sample at reduced sample thickness for a period of time.

One variation of these methods is to apply one or more of the above steps to 96-well plates or other well plates.

The methods and devices of the present invention disclosed in sections 1, 2, 3 and 5 can be used individually or in any combination thereof. In particular, Q is used for the inventions disclosed in sections 1 and 2, A for the inventions disclosed in sections 3 and 5, X for the inventions disclosed in sections 4 and 5, and M for the inventions disclosed in section 6. Therefore, the methods and the devices of the present invention disclosed in sections 1, 2, 3 and 5 can be used in the form of Q, X, A, M, QX, QA, QM, XA, XM, AM, QXA, QAM, XAM and QXAM.

Some embodiments of the use of Q, X, A and M for surface immobilization analysis include:

a. providing a first plate, wherein the surface of the first plate has at least one well of known depth and volume, and the bottom surface of the well has one or more binding sites that can immobilize the target object in the sample;

b. placement in a well of a sample with a volume approximately the same as the volume of the well, where the sample contains the target object, the target object is movable in the sample, the shape of the sample is deformable, and the sample covers only part of the well (hence, characterized by a conventional thickness that is greater than than the depth of the hole);

c. presence of a cover plate;

d. causing the first plate and the cover plate to face each other, where the sample is located between the inner surfaces of the first plate and the second plate;

e. reducing the thickness of the sample by reducing the space between the internal surfaces of the first plate and the second plate and

f. incubating the sample at reduced sample thickness for a period of time.

One variation of these methods is to apply one or more of the above steps to 96-well plates or other well plates.

Several embodiments of methods, devices, and systems combine one or more features of sample volume quantification (Q), reagent addition (A), and/or analysis acceleration (X) (and may be referred to by the corresponding acronyms QA, QX, AX, and QAX). Below are some experimental demonstrations of Q, A, X, QA, QX, AX and QAX methods and devices.

23. Reagents

The term "reagents" refers, unless otherwise specified, to one or more biological agents, biochemical agents and/or chemical agents. For example, reagents may include capture agents, detection agents, chemical compounds, optical tags, radioactive tags, enzymes, antibodies, proteins, nucleic acids, DNA, RNA, lipids, carbohydrates, salts, metals, surfactants, solvents or any combination of them.

In some embodiments, the reactants on the plate are present in the form of a liquid, a solid, a molecular vapor, or a combination thereof. Reagent placement includes, but is not limited to, placement, placement, printing, stamping, liquid dispensing, evaporation (thermal evaporation, vapor evaporation, human respiration), chemical vapor deposition and/or sputtering. Different reagents may be in different locations. Reagents may be printed and/or applied as small dots of reagents.

In some embodiments, the reactants are first applied to the wafer in liquid or vapor form and then dried to form dry reactants on the wafer prior to the CROF process.

Regulating the release time of reagents. Methods A may further include the step of adjusting the release time of the reagent (ie, a time that determines how quickly the reagent can dissolve in the sample). Some embodiments of controlling the release time of a reagent include the step of mixing or applying over the reagent one or more “release control materials” that affect the release (into the sample) of the reagent. In some embodiments, the release control material may be another reagent. For example, if there are two reagents A and B, and reagent A is applied on top of reagent B, then under certain conditions reagent A will dissolve in the sample before reagent B.

In addition, the surface properties of the first plate and the second plate can be used to control the release of the reagent. One example is the regulation of surface wetting properties. For many reagents, a hydrophobic surface binds the reagent well, which consequently results in slow or no release of the reagent into the sample (depending on the thickness of the reagent layer), while a hydrophilic surface binds the reagent less well, which consequently results in a rapid release into the sample. sample.

Drying reagents. In some embodiments, after step (c) of applying the reagent, but before step (d) of applying the sample, Methods A further includes the step of drying some or all of the reagents applied in step (c).

Location of reagents. Reagents can be applied and/or arranged on one or both plates. The reagents may be located in storage areas on the plate(s), where each storage area contains one or more reagents. Different storage areas may include different reagents, the same reagents, or one or more conventional reagents.

Monitoring the concentration of added reagents. In some embodiments, the methods may further include the step of controlling the concentration of added reagents by monitoring the thickness of the samples over the storage areas (ie, the reagent surfaces).

The reagent used in the present invention may be any suitable reagent required for the assay, for example, a labeled or unlabeled antibody, a labeled or unlabeled nucleic acid, an enzyme that may or may not contain an affinity moiety, etc. In some embodiments, and as discussed above, the stored reagent may be a component of an assay designed to test blood or other fluid sample for the presence of an analyte. For example, chloride ions can be measured according to any of the following protocols, and components of these assays may be present in the storage area: Colorimetric methods: Chloride ions displace thiocyanate from mercuric thiocyanate. Free thiocyanate reacts with ferric ions to form a colored ferric thiocyanate complex, which is measured photometrically. Coulometric methods: When a direct current is passed between silver electrodes, silver ions are formed, which react with chloride to form silver chloride. Once all the chloride has combined with the silver ions, free silver ions accumulate, causing an increase in electrical current at the electrodes, indicating the end point of the reaction. Mercurimetric methods: The chloride is titrated with a standard solution of mercuric ions and they form a soluble HgCl2 complex. The end point of this reaction is detected colorimetrically when an excess of mercury ions reacts with the indicator dye diphenylcarbazone to form a blue color. Similarly, magnesium can be measured colorimetrically using calmagite, which when reacting with magnesium gives a red-violet color; in the test with formazan dye, emission occurs at 600 nm in reaction with magnesium; or using methylthymol blue, which binds to magnesium to form a blue-colored complex. Similarly, calcium can be detected using a colorimetric technique using O-cresolphthalein, which produces a purple color when O-cresolphthalein complexone reacts with calcium. Similarly, bicarbonate can be tested bichromatically because bicarbonate (HCO3 ^- ) and phosphoenolpyruvate (PEP) are converted to oxaloacetate and phosphate in a reaction catalyzed by phosphoenolpyruvate carboxylase (PEPC). Malate dehydrogenase (MD) catalyzes the reduction of oxalacetate to malate while simultaneously oxidizing reduced nicotinamide adenine dinucleotide (NADH). This oxidation of NADH results in a decrease in the absorbance of the reaction mixture, measured bichromatically at 380/410 nm, in proportion to the bicarbonate content of the sample. Blood urea nitrogen can be detected in a colorimetric test in which diacetyl or pearone forms a yellow chromogen with urea and can be quantified using photometry or repeated use of the enzyme urease, which converts urea into ammonia and carbon dioxide, which can be analyzed by e.g. ) decreasing the absorbance at 340 nm when ammonia reacts with alpha-ketoglutaric acid, ii) measuring the rate of increase in conductivity of the solution in which urea is hydrolyzed. Similarly, creatinine can be measured colorimetrically by treating the sample with an alkaline picrate solution to obtain a red complex. In addition, creatinine can be measured using a reaction other than the Jaffe reaction, which measures the ammonia produced by the hydrolysis of creatinine by creatinine iminohydrolase. Glucose can be measured in an assay in which blood is exposed to a specific amount of glucose oxidase for a limited period of time to estimate the concentration. After this time, excess blood is removed and color is allowed to develop, which is used to assess glucose concentration. For example, the reaction of glucose oxidase with glucose produces oxygen, which converts potassium iodide (on filter paper) to iodine, producing a brown color. Glycated hemoglobin concentration as an indirect estimate of blood glucose levels. When erythrocyte hemolysates are chromatographed, three or more minor peaks corresponding to hemoglobin A1, A1 and A1c elute before the main peak of hemoglobin A. These “fast” hemoglobins are formed by the irreversible addition of glucose to hemoglobin in a two-step reaction. Hexokinase can be measured in an assay in which glucose is phosphorylated by hexokinase (HK) in the presence of adenosine triphosphate (ATP) and magnesium ions to produce glucose-6-phosphate and adenosine diphosphate (ADP). Glucose-6-phosphate dehydrogenase (G6P-DH) specifically oxidizes glucose-6-phosphate to gluconate-6-phosphate while reducing NAD+ to NADH. The increase in absorbance at 340 nm is proportional to the glucose concentration in the sample. HDL, LDL, triglycerides can be measured using the Abel-Kendall protocol, which involves the development of color by the Lieberman-Burkhardt reagent (a mixed reagent of acetic anhydride, glacial acetic acid and concentrated sulfuric acid) at 620 nm after hydrolysis and extraction of cholesterol . Fluorometic analysis can be used to determine triglyceride reference values. Determination of high-density lipoprotein cholesterol (HDL-C) in plasma is carried out using the same procedures used to measure total plasma cholesterol after precipitation of apoprotein B-containing lipoproteins from whole blood plasma (LDL and VLDL) using heparin. manganese chloride. These compounds can also be detected colorimetrically in an assay based on an enzymatic reaction that quantifies both cholesterol esters and free cholesterol. Cholesterol esters are hydrolyzed by cholesterol esterase to cholesterol, which is then oxidized by cholesterol oxidase to the ketone cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide is then detected using a highly specific colorimetric probe. Horseradish peroxidase catalyzes the reaction of the probe and hydrogen peroxide, which bind in a 1:1 ratio. The samples can be compared to a known concentration of a cholesterol standard.

24. Applications, samples and range of bio/chemical biomarkers

Applications of the present invention include, without limitation, (a) the identification, purification and quantitation of chemical compounds or biomolecules that correlate with the stage of certain diseases, for example, infectious and parasitic diseases, trauma, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders and organic diseases, e.g. lung diseases, kidney diseases, (b) detection, purification and quantification of microorganism, e.g. virus, fungus and bacteria, from the environment, e.g. water, soil or biological samples, e.g. tissues, body fluids, (c) identification, quantification of chemical compounds or biological samples that pose a threat to food safety or national security, e.g. toxic waste, anthrax, (d) quantification of vital parameters during medical or physiological monitoring, e.g. glucose, blood oxygen level, complete blood count, (f) identification and quantification of specific DNA or RNA from biological samples, e.g. cells, viruses, body fluids, (f) sequencing and comparison of genetic sequences in DNA in chromosomes and mitochondria for genome analysis, or (g) to identify reaction products, for example during the synthesis or purification of pharmaceuticals. The present invention may also be used in a variety of fields including, but not limited to, medicine, veterinary medicine, agriculture, food, environment, drug testing, and others.

Detection can be performed in a variety of sample matrices such as cells, tissues, body fluids, and feces. Biological fluids of interest include, but are not limited to, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF) , earwax (cerumen plug), chyle, chyme, endolymph, perilymph, feces, gastric acids, gastric juice, lymph, mucus (including nasal discharge and mucous sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus , watery discharge, saliva, secretion of the sebaceous glands (sebum), semen, sputum, sweat, synovial fluid, tears, vomit, urine and condensation of exhaled air. In some embodiments, the sample comprises a biological fluid from a human. In some embodiments, the sample comprises at least one of cells, tissue, body fluids, stool, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid , earwax, chyle, chyme, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal discharge, mucous sputum, pericardial fluid, peritoneal fluid, pleural fluid, pus, watery discharge, saliva, sebum , semen, sputum, sweat, synovial fluid, tears, vomit, urine and exhaled air.

In embodiments, the sample is at least one of a biological sample, an environmental sample, and a biochemical sample.

In some embodiments, the sample is at least one of a biological sample, an environmental sample, and a biochemical sample.

In any embodiment, the CROF device may be placed in the microfluidic device and application step b) may include applying the sample to the microfluidic device containing the CROF device.

In any embodiment, readout step d) may include detecting a fluorescent or luminescent signal from the CROF device.

In any embodiment, reading step d) may include reading the CROF device using a portable device configured to read the CROF device. The portable device may be a mobile phone, such as a smartphone.

In any embodiment, the CROF device may include a labeling agent that can bind to an analyte-capture complex on the CROF device.

In any embodiment, the devices, systems and methods of the present invention may further include, between steps c) and d), the steps of applying to the CROF device a labeling agent that binds to the analyte-capture complex on the CROF device, and rinsing the CROF device. CROF.

In any embodiment, read step d) may include reading the device identifier for the CROF. The identifier may be an optical bar code, an RFID tag, or combinations thereof.

In any embodiment, the devices, systems and methods of the present invention may further include applying a control sample to a CROF control device containing a capture agent that binds to an analyte, where the control sample contains a known detectable amount of the analyte, and reading the CROF control device from thereby obtaining a reference value for a known detectable amount of analyte in the sample.

In any embodiment, the sample may be a diagnostic sample obtained from the subject, the analyte may be a biomarker, and the measured amount of analyte in the sample may be diagnostic of a disease or condition.

The amount of sample may be approximately a drop of sample. The amount of sample may be the amount obtained from a pricked finger or from a single finger prick. The amount of sample can be the amount obtained from a microneedle or from a single venous blood draw.

The sample may be used without further processing once obtained from the source or may be processed, for example, to enrich the analyte of interest, remove large solids, dissolve or resuspend the solid sample, etc.

Any suitable method for applying the sample to the CROF device may be used. Suitable methods may include the use of a pipette, dropper, syringe, etc. In certain embodiments, when the CROF device is positioned on a substrate in a test strip format, as described below, the sample can be applied to the CROF device by immersing the sample receiving area of the test strip in the sample.

The sample can be collected in one time or over several times. Samples collected over time can be pooled and/or processed (by applying to a CROF device and obtaining a measure of the amount of analyte in the sample as described herein) individually. In some cases, indicators obtained over time can be combined and can be used for extended analysis over time to facilitate screening, diagnosis, treatment and/or prevention of diseases.

Washing the CROF device to remove unbound sample components can be done in any convenient manner as described above. In certain embodiments, the surface of the CROF device is washed using a binding buffer to remove unbound sample components.

Labeling the analyte with a detectable label can be done in any convenient way. The analyte can be labeled directly or indirectly. In direct labeling, the analyte in the sample is labeled before the sample is applied to the CROF device. In indirect labeling, the unlabeled analyte in the sample is labeled after the sample is applied to the CROF device to capture the unlabeled analyte, as described below.

Data processing

In certain embodiments, the inventive device is configured to process data obtained from reading the device for CROF. The device may be configured to process data in any suitable manner for use in the claimed methods. In certain embodiments, the device has a memory cell for storing data, and/or storing instructions for processing data, and/or storing a database. The data may be stored in memory in any suitable format.

In certain embodiments, the device has a processor for processing data. In certain embodiments, instructions for processing data may be stored in the processor or may be stored in a separate memory location. In some embodiments, the device may include software to implement the processing.

In certain embodiments, a device configured to process data received from a device for CROF includes software-implemented methods for performing the processing. The software-implemented methods may include one or more of image acquisition algorithms; image processing algorithms; methods for implementing a user interface that facilitate interaction between the user and a computing device and serve as tools for collecting, transmitting and analyzing data, communication protocols; and data processing algorithms. In certain embodiments, the image processing algorithms include one or more of a particle counter, LUT (lookup table) filter, particle filter, pattern recognition, morphological detection, histogram, line profile, topographic representation, binary transform, or color matching profile.

In certain embodiments, the device is configured to display information on a video display or touch display, wherein the display page is interpreted by software residing in the memory of the device. The display pages described herein may be created using any suitable programming language, such as, for example, HyperText Markup Language (“HTML”), Dynamic HyperText Markup Language (“DHTML”), Extensible HyperText Markup Language (“XHTML”) ), eXtensible Markup Language (“XML”), or other programming language that can be used to create a computer file displayed on a video or other display in a user-perceivable manner. Any machine-readable medium containing logic, code, data, instructions can be used to implement any software or steps or methodology. If the network includes the Internet, then the display page may include a suitable type of web page.

The display page of the present invention may include embedded functions providing software products stored on a storage device, such as, for example, VBScript routines, JScript routines, JavaScript routines, Java utilities, ActiveX components, ASP.NET, AJAX, Flash utilities, Silverlight utilities or AIR.

The display page may include well-known graphical user interface technology features such as, for example, frames, windows, scroll bars, buttons, icons, and hyperlinks, as well as well-known features such as a pointer interface or a touch screen interface. Pointing and clicking on a GUI button, icon, menu choice, or hyperlink is also referred to as “selecting” the button, choice, or hyperlink. The display page in accordance with the present invention may also include multimedia functions, multi-touch, pixel-sensitive, IR LED-based surfaces, video interactions with or without cameras.

The user interface may be displayed on a video display and/or display page. The user interface may display a report generated from the analyzed data related to the sample, as further described below.

The processor may be configured to process data in any suitable manner for use in the claimed methods. The data is processed, for example, into grouped data, transformed data (eg, spatio-temporal data is transformed by Fourier transform into spatial-frequency data), or it may be combined with other data. Processing can put the data into the required form, and this may involve changing the format of the data. Processing may include detecting a signal from the sample, adjusting the raw data based on mathematical manipulations or corrections and/or calibrations specific to the device or reagents used to examine the sample; calculating a value, such as a concentration value, comparing (for example, to a baseline, threshold, standard curve, historical data, or data from other sensors), determining whether a test is accurate, highlighting values or results that are outliers or may be cause for concern (eg, above or below the normal or acceptable range or indicative of an abnormal condition), or combinations of results that together may indicate the presence of an abnormal condition, fitting a curve, using data as a basis for mathematical or other analytical reasoning (including deductive, inductive, Bayesian or other reasoning) and other appropriate forms of processing. In certain embodiments, the processing may include comparing the data being processed to a database stored on the device to extract instructions regarding a course of action to be performed by the subject.

In certain embodiments, the device may be configured to process input data by comparing the input data to a database stored in memory to obtain instructions regarding the order of actions to be performed by the subject. In some embodiments, the database may contain stored information that contains a threshold value for an analyte of interest. The threshold value may be useful to determine the presence or concentration of one or more analytes. A threshold can be useful to identify situations where a warning might be useful. The data storage unit may include records or other information that may be useful for generating a report related to the sample.

In certain embodiments, the device may be configured to receive data that is received from the device for CROF. Thus, in certain cases, the device may be configured to receive data that is not related to the sample provided by the subject, but may still be relevant to the diagnosis. Such data includes, without limitation, age, gender, height, weight, personal and/or family history, etc. In certain embodiments, the device is configured to process data obtained from a sample, or independent of a sample applied to the CROF device.

Net. In certain embodiments, the device may be configured to communicate over a network, such as a local area network (LAN), a wide area network (WAN) such as the Internet, a personal area network, a telecommunications network such as a telephone network, a mobile phone network, a mobile network, wireless network, data network or any other type of network. In certain embodiments, the device may be configured to use wireless technology, such as Bluetooth or RTM technology. In some embodiments, the device may be configured to use a variety of communication methods, such as a dial-up wire connection to a modem, a point-to-point connection such as TI, an integrated services digital network (ISDN), or a cable line. In some embodiments, the wireless connection may utilize exemplary wireless networks such as cellular, satellite, or paging networks, packet radio service (GPRS), or a local area transfer system such as Ethernet or Token Ring over a LAN. In some embodiments, the device may communicate wirelessly using infrared optical communications components.

In certain embodiments, the device is configured to receive a computer file, which may be stored in memory, transferred from a server over a network. A device may receive tangible computer-readable media that may contain instructions, logic, data, or code that may be stored in permanent or temporary memory of the device or may influence or initiate the operation of the device. One or more devices may transmit computer files or links that may provide access to other computer files.

In some embodiments, the device is a personal computer, server, laptop, mobile device, tablet, mobile phone, cellular phone, satellite phone, smartphone (e.g., iPhone, Android, Blackberry, Palm, Symbian, Windows), personal digital assistant, Bluetooth device, pager, landline phone, or other network device. Such devices may be communication-enabled devices. As used herein, the term "mobile telephone" refers to a handset that may operate on a mobile cellular network, a Voice over IP (VoIP) network such as Session Initiation Protocol (SIP), or a wireless local area network (WLAN) with using the 802.11x protocol, or any combination thereof. In certain embodiments, the device may be hand-held and compact so that it can fit into a consumer's wallet and/or pocket (eg, pocket size).

Environmental testing. As summarized above, the devices, systems and methods of the present invention may find use in analyzing an environmental sample, such as a sample from water, soil, industrial waste, etc., for the presence of environmental markers. The environmental marker may be any suitable marker that can be captured by a capture means that specifically binds the environmental marker in a CROF device configured to include the capture means. The environmental sample can be obtained from any suitable source such as river, ocean, lake, rain, snow, sewage, wastewater treatment effluent, agricultural effluent, industrial effluent, tap water or drinking water, etc. In some embodiments, the devices and systems of the present invention detect the concentration of lead or toxins in water. In some embodiments, the presence or absence or quantitative level of an environmental marker in a sample may indicate the condition of the environment from which the sample was obtained. In some cases, the environmental marker may be a substance that is toxic or harmful to an organism, such as a human, pet, plant, etc., that is exposed to that environment. In some cases, the environmental marker may be an allergen, which may cause allergic reactions in some individuals who are exposed to that environment. In some cases, the presence or absence or quantitative level of an environmental marker in a sample may correlate with the overall health of the environment. In such cases, the overall health of the environment can be measured over a period of time, such as a week, months, years or decades.

In some embodiments, the devices, systems and methods of the present invention further provide for obtaining or providing a report that indicates safety or harm to a subject exposed to the environment from which the sample is obtained based on information including a measured amount of an environmental marker. Information used to assess a safety hazard or environmental condition may include data other than the type and measured amount of the environmental marker. This additional data may include location, altitude, temperature, time of day/month/year, pressure, humidity, wind direction and speed, weather, etc. The data may represent an average or trend over a period (minutes, hours, days, weeks, months, years, etc.) or the current value over a shorter period (milliseconds, seconds, minutes, etc.).

The report may be generated by a device configured to read the device for the CROF, or may be generated at a remote location after data has been sent, including the measured amount of the environmental marker. In some cases, the expert may be located in a remote location or have access to data sent to a remote location and can analyze or review the data to produce a report. The expert may be a researcher or administrator at a government agency such as the US Centers for Disease Control and Prevention (CDC) or the US Environmental Protection Agency (EPA), a research institution such as a university, or a private company. In certain embodiments, the expert may send instructions or recommendations to the user based on data transmitted by the device and/or analyzed at a remote location.

Food testing. As summarized above, the devices, systems and methods of the present invention may find use in analyzing a sample of food, such as a sample of unprocessed food, processed food, cooked food, drinking water, etc., for the presence of food markers. The food marker may be any suitable marker, such as shown in Table B9 below, that can be captured by the capture means, specifically binding the food marker in the CROF device configured with the capture means. The environmental sample can be obtained from any suitable source such as tap water, drinking water, cooked food, processed food or unprocessed food, etc. In some embodiments, the presence or absence or quantitative level of a food marker in a sample may indicate whether the food is safe or unsafe for a subject to consume. In some embodiments, a food marker is a substance derived from a pathogenic organism or microorganism that indicates the presence of the organism in the food product from which the sample is obtained. In some embodiments, the food marker is a substance that is toxic or harmful when consumed by a subject. In some embodiments, the food marker is a biologically active compound that may inadvertently or unexpectedly alter physiology when consumed by a subject. In some embodiments, the food marker is an indicator of how the food product was produced (grown, purchased, caught, harvested, processed, prepared, etc.). In some embodiments, the nutrition marker indicates the nutritional composition of the food. In some embodiments, the food marker is an allergen that may cause an allergic reaction if the food from which the sample is obtained is consumed by the subject.

In some embodiments, the devices, systems and methods of the present invention further provide for obtaining or providing a report that indicates the safety or harmfulness for a subject to consume the food from which the sample is obtained based on information including a measured level of a food marker. Information used to evaluate the safety of foods for consumption may include data other than the type and measured amount of the food marker. This additional data may include any health condition associated with the consumer (allergies, pregnancy, chronic or acute illnesses, prescription medications, etc.).

The report may be generated by a device configured to read the device for the CROF, or may be generated at a remote location by sending data including the measured amount of a food marker. In some cases, a food safety expert may be located in a remote location or have access to data sent to a remote location and may analyze or review the data to produce a report. A food safety expert may be a researcher or administrator at a government agency such as the US Food and Drug Administration (FDA) or CDC, a research institution such as a university, or a private company. In certain embodiments, the food safety expert may send instructions or recommendations to the user based on data transmitted by the device and/or analyzed at a remote location.

25. Blood test

Some exemplary embodiments of the present invention are simple, rapid blood cell counts using a smartphone.

In some embodiments, the first plate and the second plate are selected from a thin glass slide (eg, 0.2 mm thick) or thin plastic film (eg, 15 mm thick) with a relatively flat surface, and each has an area with a length and width from approximately 0.5 cm to 10 cm. Dividers are made of glass, plastic or other materials that will not deform significantly when squeezed. Before applying the sample, the separator is placed on the first plate, the second plate, or both the first and second; or both are optionally coated with a reagent that facilitates blood counting (coloring dyes and/or anticoagulant). The first plate and the second plate may optionally be sealed in a bag for ease of transportation and longer shelf life.

Blood cell count testing requires only approximately 1 μL (microliter) (or approximately 0.1 μL to 3 μL) of blood, which can be collected from a finger or other location on the person's body. The blood sample can be applied directly from the human body (such as a finger) onto the first plate and the second plate without any dilution. The first plate and the second plate are then turned toward each other such that the blood sample is located between the inner surfaces of the first plate and the second plate. If optional reagents (staining dyes or anticoagulants) are pre-applied, they are applied to the inner surface to mix with the sample. The first plate and the second plate are then compressed by fingers or a simple mechanical device (eg a clip that is compressed by a spring). When compressed, the internal space is reduced, and the reduction will eventually stop at a value given by the height of the spacers, and a final thickness of the sample will be reached, which is generally equal to the final internal space. Since the final internal space is known, the final thickness of the sample becomes known, namely quantified (measured) using this method.

If the blood sample is not diluted, then after compression (deformation of the sample) the separators and therefore the final thickness of the sample may be thin, for example less than 1 µm, less than 2 µm, less than 3 µm, less than 4 µm, less than 5 µm, less than 7 µm , less than 10 µm, less than 15 µm, less than 20 µm, less than 30 µm, less than 40 µm, less than 50 µm, less than 60 µm, less than 80 µm, less than 100 µm, less than 150 µm or any range between any of the two numbers. A thin final sample may be useful because if the final sample is thick, many red blood cells may overlap during imaging, which may result in incorrect cell counts. For example, an undiluted whole blood sample approximately 4 µm thick will yield approximately one layer of red blood cells.

After compression, the sample can be visualized using a smartphone either directly or through additional optical elements (e.g. lenses, filters or light sources if necessary). The image of the sample will be processed to identify the cell types as well as the number of cells. Image processing can be done locally on the same smartphone that receives the image, or remotely, but the final result is transferred back to the smartphone (where the image is transferred to a remote location and processed there). The smartphone will display the cell count for a specific cell type. In some cases, specific recommendations will be shown. Recommendations can be stored on the smartphone before testing or come from remote computers or specialists.

In certain embodiments, the reagents will be placed on the internal surfaces of the first plate and/or the second plate using the methods and devices described in section 5 (mixing reagents).

An apparatus or method for blood testing comprises (a) the apparatus or method as described herein, and (b) an interplate space (that is, the space between the inner surfaces of two plates) in a closed configuration, or the use of such a space in which undiluted whole the blood in the space between the plates has an average intercellular distance for red blood cells (RBCs) in the horizontal direction greater than the average diameter of the RBC disk.

An apparatus or method for arranging the orientation of a non-spherical cage, which includes (a) an apparatus or method as described herein, and (b) an interplate space (that is, a space between the interior surfaces of two plates) in a closed configuration, or the use of such a space where the space less than the average size of the cell in its longitudinal direction (the longitudinal direction represents the maximum side size of the cell). This arrangement can improve sample volume measurements (eg, red blood cell volume).

In the present invention, the analytes in blood tests include protein markers, a list of which can be found on the website of the American Association of Clinical Chemistry).

26. Types of packaging

Another aspect of the present invention relates to packaging that will extend the life of the reagent used and facilitate ease of use.

In some embodiments, wafers in CROF, with or without reagents, are placed inside a package, either one wafer per pack, or multiple wafers per pack. In one embodiment, the first wafer and the second wafer are packaged in a different package prior to use. In some embodiments, different assays share a common first wafer or a common second wafer.

In some embodiments, each of the packages is sealed. In some embodiments, sealing is necessary to prevent air, chemicals, moisture, contaminants, or any combination thereof from entering the package from outside the package. In some embodiments, the packaging is vacuum sealed or filled with nitrogen gas or inert gases. In some embodiments, material that can extend the shelf life of the plate and/or reagents (including capture agents, detection agents, etc.) is packaged within the package along with the plate.

In some embodiments, the packaging materials are in the form of a thin layer such that the packaging can be easily torn by a person by hand.

27. RoS, smartphone and network

One aspect of the present invention is directed to a method for monitoring the health of a subject, the method comprising: applying a sample provided by the subject to a CROF-based detector configured to indicate an output signal that is characteristic of the sample; processing the output signal of the detector with a device configured to receive the output signal of the detector as input data and analyze the input data to generate a report; and receiving a report. The Signal Enhancement Detector offers the benefits of fast detection, simplified sensing (e.g., replacing a large conventional sensing device with a smartphone), and reduced cost.

Biological fluid. In certain embodiments, the sample may include various liquid or solid samples. In some cases, the sample may be a biological fluid sample from the subject. In some cases, solid or semi-solid samples may be obtained. The sample may include tissues and/or cells collected from the subject. The sample may be a biological sample. Examples of biological samples may include, but are not limited to, blood, serum, blood plasma, nasal swab, nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, ear plug, fat, glandular secretions, cerebrospinal fluid, tissue, semen, vaginal fluid, interstitial fluids derived from tumor tissue, ocular fluids, cerebrospinal fluid, throat swab, breath air, hair, fingernails, skin, biopsy, placental fluid, amniotic fluid, umbilical cord blood, lymphatic fluids, fluids from cavities, sputum, pus, microbiota, meconium, breast milk and/or other secretions. Samples may include nasopharyngeal swabs. Nasal swabs, throat swabs, stool samples, hair, fingernail, ear plug, exhaled breath and other solid, semi-solid or gaseous samples can be processed in the extraction buffer, for example, for a fixed or variable amount of time before being analyzed. The extraction buffer or an aliquot of it can then be processed similarly to other fluid samples if necessary. Examples of tissue samples from a subject may include, but are not limited to, connective tissue, muscle tissue, neural tissue, epithelial tissue, cartilage, a cancer tumor sample, or bone.

In certain embodiments, the subject may be a human or non-human animal. The subject may be a mammal, a vertebrate such as mice, monkeys, humans, farm animals, competitive animals, or pets. In some embodiments, the subject may be a patient. In other embodiments, the subject may be diagnosed with a disease, or the subject may not be diagnosed with a disease. In some embodiments, the subject may be a healthy subject.

Read device

As summarized above, aspects of the method include processing the output of a signal amplification detector with an apparatus configured to receive the output of the detector as input and process the input to produce a report. Any device suitable for receiving the detector output as input and processing the input to produce a report can be used. In some embodiments, the device includes an optical recorder that is configured to receive the output of the optical detector as input. In certain cases, the optical recording apparatus is a camera, such as a digital camera. The term "digital camera" means any camera that contains as a main component an image capturing apparatus equipped with an optical imaging system for forming an optical image, an image sensor for converting the optical image into an electrical signal and other components, examples of such cameras include digital cameras, digital cinema cameras and webcams (i.e. cameras that are connected, either publicly or privately, to a device connected to a computer network for the exchange of images, including those connected directly to a computer network and those connected to a computer network network through a device, such as a personal computer, with the ability to process information). In one example, the input data may contain a video image that can record changes over time. For example, video may be acquired to evaluate dynamic changes in a sample.

In certain embodiments, the device receives the output of the detector through an adapter that forms an interface between the device and the detector. In certain embodiments, the interface is generic for compatibility with any device suitable for performing the claimed method. Interfaces of interest include, but are not limited to, USB (Universal Serial Bus), FireWire (Firewire), Ethernet, etc. In certain embodiments, the device receives the detector output via wireless communications, including cellular, Bluetooth, Wi-Fi, etc.

In certain embodiments, the device may have a video display. Video displays may include components on which a display page can be displayed in a manner perceivable by a user, such as a computer monitor, cathode ray tube, liquid crystal display, LED display, touch panel or touch display, and/or other means known in the art for emitting visually perceived output signal. In some embodiments, the device is equipped with a touch screen for displaying information, such as input data received from the detector and/or a report generated from the processed data, and allowing the subject to enter the information.

In certain embodiments, the device is equipped with vibration capabilities as a method of alerting a subject, for example, to a report generated while processing the detector output or in preparation for receiving output from the detector.

In certain embodiments, the device is configured to display information on a video display or touch display, wherein the display page is interpreted by software residing in the memory of the device. The display pages described herein may be created using any suitable programming language, such as, for example, HyperText Markup Language (“HTML”), Dynamic HyperText Markup Language (“DHTML”), Extensible HyperText Markup Language (“XHTML”) ), eXtensible Markup Language (“XML”), or other programming language that can be used to create a computer file for display on a video or other display in a user-perceivable manner. Any machine-readable medium containing logic, code, data, instructions can be used to implement any software or steps or methodology. If the network includes the Internet, then the display page may include a suitable type of web page.

The display page of the present invention may include embedded functions providing software products stored on a storage device, such as, for example, VBScript routines, JScript routines, JavaScript routines, Java utilities, ActiveX components, ASP.NET, AJAX, Flash utilities, Silverlight utilities or AIR.

The display page may include well-known graphical user interface technology features such as, for example, frames, windows, scroll bars, buttons, icons, and hyperlinks, as well as well-known features such as a pointer interface or a touch screen interface. Pointing and clicking on a GUI button, icon, menu choice, or hyperlink is also referred to as “selecting” the button, choice, or hyperlink. The display page in accordance with the present invention may also include multimedia functions, multi-touch, pixel-sensitive IR LED-based surfaces, video interaction with or without cameras.

In certain embodiments, the device may be configured to receive data that is not output from the signal amplification detector. Thus, in certain cases, the device may be configured to receive data that is not representative of the sample provided by the subject, but may still be representative of the subject. Such data includes, but is not limited to, age, gender, height, weight, personal and family history, etc. In certain embodiments, the device is configured to process input data received from the detector output in combination with data that is received independently of the detector output.

In certain embodiments, the device may be configured to communicate over a network, such as a local area network (LAN), a wide area network (WAN) such as the Internet, a personal area network, a telecommunications network such as a telephone network, a mobile phone network, a mobile network, wireless network, data network or any other type of network. In certain embodiments, the device may be configured to use wireless technology, such as Bluetooth or RTM technology. In some embodiments, the device may be configured to use a variety of communication methods, such as a dial-up wired connection to a modem, a point-to-point connection such as TI, ISDN, or a cable line. In some embodiments, the wireless connection may utilize exemplary wireless networks such as cellular, satellite, or paging networks, GPRS, or a local area transfer system such as Ethernet or Token Ring over a LAN. In some embodiments, the device may communicate wirelessly using infrared optical communications components.

In certain embodiments, the device is configured to receive a computer file, which may be stored in memory, transferred from a server over a network. A device may receive tangible computer-readable media that may contain instructions, logic, data, or code that may be stored in permanent or temporary memory of the device or may otherwise influence or initiate the operation of the device. One or more devices may transmit computer files or links that may provide access to other computer files.

In some embodiments, the device is a personal computer, server, laptop, mobile device, tablet, mobile phone, cellular phone, satellite phone, smartphone (e.g., iPhone, Android, Blackberry, Palm, Symbian, Windows), personal digital assistant, Bluetooth device, pager, landline phone, or other network device. Such devices may be communication-enabled devices. As used herein, the term "mobile telephone" refers to a handset that may operate on a mobile cellular network, a Voice over IP (VoIP) network such as Session Initiation Protocol (SIP), or a wireless local area network (WLAN) with using the 802.11x protocol, or any combination thereof. In certain embodiments, the device may be hand-held and compact so that it can fit into a consumer's wallet and/or pocket (eg, pocket size).

In certain embodiments, the method involves transmitting data obtained from a sample to a remote location where the transmitted data is analyzed. The remote location may be a location that is different from the location where the device is located. The remote location may include, but is not limited to, a hospital, doctor's office or other medical facility or research laboratory. In some cases, there may be a computer at a remote location, such as a server, that is configured to communicate (eg, receive information and transmit information) with a device over a network. In some embodiments, the device may transmit data to a cloud computing infrastructure. The device may have access to cloud computing infrastructure. In some embodiments, on-demand provisioning of computing resources (data, software) may be accomplished over a computer network rather than from a local computer. The device may contain very little software or data (perhaps only a minimal operating system and a web browser), acting as a basic display terminal connected to the Internet. Because the cloud can be the primary delivery mechanism, cloud applications and services can support any type of software application or service. Information provided by a device and/or accessible to devices may be distributed across various computing resources. Alternatively, the information may be stored in one or more fixed storage units or a database.

In certain embodiments, the remote location contains a central database stored in a data storage unit that receives and analyzes data transmitted from the device. The data storage units may be capable of storing computer-readable media that may contain code, logic, or instructions to a processor to perform one or more steps. In some embodiments, the resulting data is analyzed against data contained in a central database and the result is sent back to the subject. The analysis may include identifying the signal from the sample, adjusting the raw data based on mathematical manipulations or corrections and/or calibrations specific to the device or reagents used to test the sample; calculating a value, such as a concentration value, comparing (for example, to a baseline, threshold, standard curve, historical data, or data from other sensors), determining whether a test is accurate, highlighting values or results that are outliers or may be cause for concern (eg, above or below the normal or acceptable range or indicative of an abnormal condition), or combinations of results that together may indicate the presence of an abnormal condition, fitting a curve, using data as a basis for mathematical or other analytical reasoning (including deductive, inductive, Bayesian or other reasoning) and other appropriate forms of processing.

In certain embodiments, the analysis may include comparing the analyzed data to a database stored in a data storage unit at a remote location to extract instructions regarding a course of action to be performed by the subject. In some embodiments, the database may contain stored information that contains a threshold value for an analyte of interest. The threshold value may be useful to determine the presence or concentration of one or more analytes. A threshold can be useful to identify situations where a warning might be useful. The data storage unit may contain other information related to sample preparation or clinical tests that may be performed on the sample. The data storage unit may contain records or other information that may be useful in generating a report related to the data being analyzed.

In certain embodiments, the healthcare professional is located at a remote location. In other embodiments, the healthcare professional has access to data transmitted by the device at a third location that is different from the remote location or location of the device. A health care professional may be a person or entity who is associated with the health care system. The health care professional may be a health care provider in the health care industry. The health care professional may be a physician. A health care professional may be an individual or institution that systematically provides preventive, curative, promotional, or rehabilitative health services to individuals, families, and/or communities. Examples of health care professionals may include doctors (including general practitioners and specialists), dentists, physician assistants, nurses, midwives, pharmacists/pharmacists, nutritionists, therapists, psychologists, chiropractors, clinical staff, physical therapists, exfusionists, occupational therapists diseases, optometrists, paramedics, emergency responders, laboratory technicians, radiologists, social workers and a wide range of other personnel trained to provide specific types of health care services. A healthcare professional may or may not be certified to prescribe. A health care professional may work in or be associated with hospitals, medical centers, and other service delivery locations, as well as academic training, research, and management. Some health care professionals may provide care and treatment to patients in private homes. Community health workers may work outside of formal health care settings. Health service managers, medical records and health information technicians, and other support workers may also be health care professionals or associated with a health care provider.

In some embodiments, the health care professional may already be familiar with or associated with the subject. The subject may be a patient of a health care professional. In some cases, a health care professional may order the subject to undergo a clinical test. In one example, the health care professional may be the primary care physician for the subject. A health care professional can be any type of physician for a given entity (including general practitioners and specialists).

Thus, a healthcare professional can analyze or view data transmitted from the device and/or the results of analyzes performed at a remote location. In certain embodiments, the healthcare professional may send instructions or recommendations to the subject based on data transmitted by the device and/or analyzed at a remote location.

28. Control and measurement of sample thickness without using separators

In some embodiments of the present invention, the spacers that are used to adjust the sample or appropriate sample volume are replaced with: (a) position sensors that can measure the internal space of the plates, and (b) devices that can monitor the positions of the plates and move the plates to the desired the internal space of the plates based on information provided by the sensors. In some embodiments, all dividers are replaced with mechanical translational stages, monitor sensors, and a feedback system.

Measuring the space and/or thickness of a sample using an optical method. In some embodiments, the measurement (f) of the space between the internal surfaces involves the use of optical interference. Optical interference can use multiple wavelengths. For example, a light signal, as a result of the interference of light reflected from the inner surface of the first plate and the second plate, oscillates at a certain wavelength of light. By vibration, you can determine the space between the internal surfaces. To enhance the interference signal, one or both of the internal surfaces can be coated with reflective material.

In some embodiments, the measurement (f) of the space between the internal surfaces involves obtaining optical imaging (for example, obtaining a 2D (two-dimensional)/3D (three-dimensional) image of the sample, where the image acquisition may occur multiple times with different viewing angles, different wavelengths, different phase and/or different polarization) and image processing.

Measuring the area or volume of an entire sample using optical methods. In some embodiments, measurement (f) of the area or volume of an entire sample involves optical imaging (e.g., obtaining a 2D/3D image of the sample, which may be imaged multiple times at different viewing angles, different wavelengths, different phase and/or different polarization) and image processing. The sample area means the area in a direction approximately parallel to the first plate and the second plate. For 3D imaging, you can use projection fringe profilometry (FPP), which is one of the most common methods for obtaining three-dimensional (3D) images of objects.

In some embodiments, measuring the area or volume of a sample by imaging involves (a) calibrating the image scale by using a sample of known area or volume (e.g., the imaging device is a smartphone, and the dimensions of the image acquired using the phone can be calibrated by comparison with an image of a sample with a known size obtained by the same phone); (b) comparing the image with scale marks (rulers) placed on or adjacent to the first plate and the second plate (discussed further herein), and (c) a combination thereof.

Light used herein may include visible light, ultraviolet light, infrared light, and/or near-infrared light. Light can include wavelengths ranging from 20 nm to 20,000 nm.

29. Other Descriptions of Embodiments

The following methods, devices and systems are provided. These embodiments may be implemented using any of the components, materials, parameters, or steps described above or below. In the following embodiments, a CROF plate is used.

Embodiment 1. A method for analyzing a liquid sample, comprising:

(a) obtaining a sample that contains the analyte;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein each plate has a sample contact surface that is substantially flat, one or both plates are flexible, and one or both plates includes spacers that mounted on a suitable surface for contact with the sample, and wherein the separators are characterized by a predetermined substantially uniform height and a predetermined constant distance between the separators, which is at least approximately 2 times the size of the analyte, but not more than 200 μm (micrometers);

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) using two plates to compress at least a portion of the sample into a layer of substantially uniform thickness, which is limited by the surfaces of the plates for contact with the sample, where the uniformity of the layer thickness is controlled by spacers and plates, where compression is provided

bringing the two plates together and

form-matching compression, either simultaneously or sequentially, in the region of at least one of the plates, squeezing the plates together into a closed configuration, wherein the shape-matching compression creates substantially uniform pressure on the plates over at least a portion of the sample and the compression extends over less at least in part of the sample horizontally between the surfaces of the plates for contact with the sample, and where the closed configuration is a configuration in which the space between the plates in a layer of a region of uniform thickness is controlled by spacers; And

(e) performing the analysis of the analyte in a layer of uniform thickness while the plates are in a closed configuration;

wherein shape-matching compression is a technique that makes the pressure applied over a region substantially constant regardless of variation in the shape of the outer surfaces of the plates; And

where in simultaneous squeezing, pressure is simultaneously applied to a given area, and in sequential squeezing, pressure is applied to part of a given area and gradually moves to another area.

Embodiment 2: A device for analyzing a liquid sample, comprising

the first plate and the second plate, where:

i. the plates are movable relative to each other in different configurations;

ii. one or both plates are flexible;

iii. each of the plates has on its respective surface a sample contact area for contacting the sample which contains the analyte,

iv. one or both plates contain spacers that are fixed to a suitable area for contact with the sample, where the spacers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the spacers, which is at least approximately 2 times the size of the analyte, but not more than 200 μm , and where at least one of the separators is located inside the contact area with the sample;

where one of the configurations is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates; And

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of highly uniform thickness, where the uniform thickness of the layer is limited by the sample contact surfaces of the plates and controlled by the plates and spacers.

Embodiment 3: A method for analyzing a blood sample, comprising:

(a) obtaining a blood sample;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein each plate has a sample contact surface that is substantially flat, one or both plates are flexible, and one or both plates includes spacers that fixed to a suitable surface for contact with the sample, and where the separators are characterized by:

i. a predetermined practically uniform height, a straight column shape with a practically uniform cross-section and a flat top surface;

iii. a width to height ratio equal to one or greater;

iv. a predetermined constant distance between the separators, which ranges from 10 μm to 200 μm;

v. a fill factor of 1% or more; And

vi. the result of the product of the fill factor and the Young's modulus of the separator being 2 MPa or more; And

(c) placing a blood sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) using two plates to compress at least a portion of the blood sample into a layer of substantially uniform thickness, which is limited by the surfaces of the plates for contact with the sample, where the uniformity of the layer thickness is controlled by the separators and plates and is characterized by an average value in the range of 1, 8 µm to 3 µm with less than 10% variation where compression involves

bringing the two plates together and

form-matching compression, either simultaneously or sequentially, in the region of at least one of the plates, squeezing the plates together into a closed configuration, wherein the shape-matching compression creates substantially uniform pressure on the plates over at least a portion of the sample and the compression extends over less at least in part of the sample horizontally between the surfaces of the plates for contact with the sample, and where the closed configuration is a configuration in which the space between the plates in a layer of a region of uniform thickness is controlled by spacers; And

(f) blood testing in a layer of uniform thickness while the plates are in a closed configuration;

where the fill factor is the ratio of the contact area with the spacer and the total area of the plate;

wherein shape-matching compression is a technique that makes the pressure applied over an area substantially constant regardless of variation in the shape of the outer surfaces of the plates; And

where in simultaneous squeezing, pressure is simultaneously applied to a given area, and in sequential squeezing, pressure is applied to part of a given area and gradually moves to another area.

Embodiment 4: A device for analyzing a liquid sample, comprising

the first plate and the second plate, where:

v. the plates are movable relative to each other in different configurations;

vi. one or both plates are flexible;

vii. each of the plates has on its respective surface a sample contact area for contacting the blood sample;

viii. one or both plates contain spacers that are fixed to a corresponding plate, where the spacers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the spacers, which is in the range from 7 μm to 200 μm, and where at least one of the spacers is located inside area of contact with the sample;

where one of the configurations is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates; And

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of highly uniform thickness, wherein the uniform thickness of the layer is limited by the inner surfaces of the two plates, and is controlled by the plates and spacers, and has an average value in the range of 1.8 μm to 3 μm with slight variation.

Embodiment 5. A method for locally binding a target object to a portion of a liquid sample, comprising:

(a) obtaining a sample that contains a target object capable of diffusion in the sample;

(b) providing first and second plates that are movable relative to each other in different configurations, where one or both plates contain spacers that are fixed to the corresponding plate, where the spacers are characterized by a predetermined substantially uniform height, and where the first plate contains on its surface a binding site, which is characterized by a predetermined area and binds to the target object and immobilizes it;

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) compressing the sample by bringing the two plates into a closed configuration, where the closed configuration is a configuration in which at least a portion of the sample is compressed into a layer of uniform thickness that is in contact with and limited by the internal surfaces of the two plates, and which contacts the bonding site where the uniform layer thickness, controlled by spacers and plates, is less than 250 μm and is substantially less than the linear dimension for a predetermined area of the bonding site;

(e) after (d) and while the plates are in a closed configuration, or:

(1) incubating the sample for a suitable period of time and then stopping the incubation, or

(2) incubating the sample for a time that is equal to or greater than the minimum suitable time interval, and then assessing, for a period of time that is equal to or less than the maximum suitable time interval, binding of the target object at the binding site;

where is the appropriate time interval:

i. equal to or greater than the time required for the target object to diffuse through a layer of uniform thickness in a closed configuration; And

ii. significantly shorter than the time required for horizontal diffusion of the target object through the binding site with a minimum size in the horizontal projection;

where, at the end of incubation (1) or during evaluation (2), the majority of targets associated with the binding site originate from a sample of suitable volume;

wherein the incubation allows the target to bind to the binding site, and where the suitable volume is the portion of the sample that is located above the binding site in a closed configuration.

Embodiment 6: A device for locally binding a target object to a portion of a liquid sample, comprising

the first plate and the second plate, where:

i. the plates are movable relative to each other in different configurations; one or both plates are flexible;

iii. each of the plates has on its respective surface a sample contact region for contacting the sample, which contains an object capable of diffusion in the sample,

iv. one of the plates in its area of contact with the sample has a binding site, which is characterized by a predetermined area, and binds and immobilizes the target object;

v. one or both plates contain spacers that are fixed to a corresponding plate, where the spacers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the spacers, and where at least one of the spacers is located inside the contact area with the sample;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates, and

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of uniform thickness, wherein at least a portion of the layer of uniform thickness is located above the bonding area, and where the uniform thickness of the layer, limited by the internal surfaces of the two plates, which is controlled by the plates and spacers, is less than 250 μm and is substantially smaller than the average linear dimension for a predetermined area of the binding site.

Embodiment 7. A method for locally releasing a reagent into a portion of a liquid sample, comprising:

(a) obtaining a sample;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein:

(i) one or both plates contain spacers which are fixed to the corresponding plate,

(ii) the dividers are characterized by a predetermined uniform height, and

(iii) the first plate contains on its surface a storage area, which is characterized by a predetermined area and which contains a reagent that, upon contact with the sample, dissolves in the sample and diffuses in the sample;

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) compressing the sample by bringing the two plates into a closed configuration, where the closed configuration is a configuration in which at least a portion of the sample is compressed into a layer of uniform thickness that is limited by the internal surfaces of the two plates and that covers the storage area, where the uniform layer thickness, controlled by spacers and plates, is less than 250 μm and is practically less than the linear dimension for a predetermined area of the binding site;

(e) after (d) and while the plates are in a closed configuration, incubating the sample for a suitable period of time and then stopping the incubation,

where is the appropriate time interval:

i. approximately equal to or longer than the time required for the target object to diffuse through a layer of uniform thickness in a closed configuration; And

ii. shorter than the time required for horizontal diffusion of the target object through a predetermined area of the binding site with a minimum linear dimension;

as a result, after incubation, most of the reagent, which was initially located in the storage area, is located in a suitable sample volume,

wherein incubation is a process that allows the reagent to bind or mix with the sample, and where the appropriate volume is the portion of the sample located above the binding site in a closed configuration.

Embodiment 8: Device for locally releasing a reagent into a portion of a liquid sample containing

the first plate and the second plate, where:

i. the plates are movable relative to each other in different configurations;

ii. one or both plates are flexible;

vi. each of the plates has on its respective surface a sample contact region for effecting contact with the sample;

vii. one of the plates contains, in its area of contact with the sample, a storage area, which is characterized by a predetermined area and which contains a reagent that, upon contact with the sample, dissolves in the sample, diffuses in the sample and binds to the target object;

viii. one or both plates comprise spacers that are secured to a corresponding plate, wherein the spacers are characterized by (a) a predetermined substantially uniform height of 250 μm or less and which is substantially less than the average linear dimension for the predetermined area of the reagent site, and ( b) a predetermined constant distance between the spacers, which is 200 μm or less, and where at least one of the spacers is located within the contact area with the sample;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates, and

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of uniform thickness, wherein at least a portion of the layer of uniform thickness is located above the bonding area, and where the uniform thickness of the layer, defined by the inner surfaces of the two plates, is controlled by the plates and spacers.

Embodiment 9. A method for reducing the time for binding a target object in a suitable sample volume to a binding site on the surface of a plate, comprising:

(a) obtaining a sample that contains a target object capable of diffusion in the sample;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein one or both plates include spacers that are fixed to the corresponding plate, and one or both plates are flexible, where the spacers have a predetermined substantially uniform height and a predetermined constant distance between the spacers, and where the first plate contains on its surface a binding site, which is characterized by a predetermined area and binds to the target object and immobilizes it;

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) compressing the sample by bringing the two plates into a closed configuration, where the closed configuration is a configuration in which the thickness of the sample of suitable volume is reduced compared to that of the open plate configuration, providing a layer of substantially uniform thickness, characterized by an area of a horizontal projection of at least 1 mm2, which is limited by the inner surfaces of the two plates and which covers the bonding site, where the uniform layer thickness, controlled by the spacers and plates, is less than 250 μm and is substantially less than the linear dimension for the predetermined area of the bonding site; where the suitable volume is the entire volume of the sample or a portion thereof;

wherein reducing the thickness of the suitable volume of the sample reduces the binding time between the binding site and the target object in the suitable volume to achieve equilibrium.

Embodiment 10. A device for locally binding a target object to a portion of a liquid sample, comprising

the first plate and the second plate, where:

i. the plates are movable relative to each other in different configurations; one or both plates are flexible;

iii. each of the plates has on its respective surface a sample contact region for contacting the sample, which contains an object capable of diffusion in the sample,

iv. one of the plates has, in its area of contact with the sample, a binding site, which is characterized by a predetermined area and binds and immobilizes the target object;

v. one or both plates contain spacers that are fixed to a corresponding plate, where the spacers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the spacers, and where at least one of the spacers is located inside the contact area with the sample;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates, and

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of uniform thickness, wherein at least a portion of the layer of uniform thickness is located above the bonding area, and where the uniform thickness of the layer, limited by the internal surfaces of the two plates, is controlled by the plates and spacers, is less than 250 µm and is practically smaller than the average linear size for a predetermined area of the binding site.

wherein reducing the thickness of the suitable volume of the sample reduces the binding time between the binding site and the target object in the suitable volume to achieve equilibrium.

Embodiment 11. A method for simultaneous multiplex analysis of a liquid sample without liquid isolation, comprising:

(a) obtaining a sample that contains one or more target analytes that are capable of diffusion in the sample;

(b) providing first and second plates that are movable relative to each other into different configurations, wherein:

i. one or both plates contain spacers that are fixed to the corresponding plate, and one or both plates are flexible,

ii. the dividers are characterized by a preset, almost uniform height and a preset constant distance between the dividers,

iii. the first plate has on its surface one or a plurality of binding sites, each of which is characterized by a predefined region containing a capture means that binds and immobilizes the corresponding target analyte(s); And

iv. the second plate has on its surface one or a plurality of corresponding storage areas, each of which is characterized by a predetermined area and contains a detection agent in a concentration that, upon contact with the sample, dissolves in the sample and diffuses in the sample,

wherein each capture agent, target analyte, and corresponding detection agent are capable of forming a capture agent-target analyte-detection agent sandwich at the binding site of the first wafer;

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) compression of the sample by bringing the two plates into a closed configuration, where the closed configuration is the configuration in which:

i. at least a portion of the sample is compressed into a layer of uniform thickness that is in contact with and limited by the internal surfaces of the two plates, and which is in contact with one or more binding areas and one or more storage areas,

ii. one or a plurality of respective storage sites are located throughout the one or a plurality of binding sites, and

iii. the uniform layer thickness, controlled by spacers and plates, is less than 250 microns and is practically less than the linear dimension for a predetermined area of each storage area;

(e) after (d) and while the plates are in a closed configuration, or:

(1) incubating the sample for a suitable period of time and then stopping the incubation, or

(2) incubating the sample for a time that is equal to or greater than the minimum suitable time interval, and then assessing, for a period of time that is equal to or less than the maximum suitable time interval, binding of the target object at the binding site;

where is the appropriate time interval

i. equal to or greater than the time required for the target analyte (a) to diffuse through a layer of uniform thickness in a closed configuration; And

ii. substantially less time required for horizontal diffusion of the target analyte (a) through a predetermined area of the storage site or binding site with the smallest linear dimension;

whereby, upon completion of incubation in (1) or evaluation in (2), the majority of the capture agent-target analyte-detection agent sandwich associated with each binding site is derived from the corresponding suitable sample volume;

wherein the incubation allows each target analyte to bind to a binding site and detection means, wherein the corresponding suitable volume is a portion of the sample located above the corresponding storage site in a closed configuration, where the separation distance between the edges of adjacent storage sites and the separation between the edges of adjacent binding sites is greater , than the distance that the target analyte or detection agent can travel by diffusion in a suitable period of time, and where there is no liquid isolation between binding sites and/or storage sites.

Embodiment 12. A device for simultaneous multiplex analysis of a liquid sample without liquid insulation, comprising a first plate and a second plate, where:

i. the plates are movable relative to each other in different configurations; one or both plates are flexible;

ii. one or both plates contain spacers that are fixed to the corresponding plate; and wherein the dividers are characterized by a predetermined substantially uniform height and a predetermined constant distance between the dividers;

iii. each of the plates has on its respective surface a sample contact region for contacting the sample, which contains one or more target analytes capable of diffusing in the sample,

iv. the first plate on its surface has one or a plurality of binding sites, each of which is characterized by a predefined region containing a capture agent that binds and immobilizes the corresponding target analyte in the sample; And

v. the second plate on its surface has one or a plurality of corresponding storage areas, each of which is characterized by a predetermined area and contains a detection agent in a certain concentration, which, upon contact with the sample, dissolves in the sample and diffuses in the sample,

wherein each capture agent, target analyte, and corresponding detection agent are capable of forming a capture agent-target analyte-detection agent sandwich at the binding site of the first wafer;

where one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates, and

where the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration:

i. at least a portion of the sample is compressed into a layer of uniform thickness that is in contact with and limited to the inner surfaces of the two plates, and that covers one or more binding areas and one or more storage areas,

ii. one or a plurality of respective storage sites are located throughout the one or a plurality of binding sites, and

iii. the uniform layer thickness, controlled by spacers and plates, is less than 250 μm and is practically less than the linear dimension for a predetermined area of each storage area; And

iv. there is no liquid insulation between the binding areas and/or storage areas,

wherein the separation distance between the edges of adjacent storage sites and the separation distance between the edges of adjacent binding sites is greater than the distance that the target analyte or detection agent can traverse by diffusion over a suitable period of time, and where there is no liquid isolation between the binding sites and/or storage sites.

Embodiment 13A. System for accelerated sample analysis using a mobile phone, containing:

(a) a CROF device, wherein one or both plates of the CROF device are movable relative to each other in different configurations; Where:

i. one of the configurations is an open configuration in which the two plates are either partially or completely separated from each other, the space between the plates is not controlled by spacers, and the sample is placed on one or both plates; And

ii. the other of the configurations is a closed configuration, which is achieved after placing the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of uniform thickness, and wherein the layer of uniform thickness that is in contact with and limited by the internal surfaces of the two plates is controlled by the plates and spacers;

(b) a mobile communication device containing:

i. one or more cameras for detecting and/or visualizing a sample;

ii. integrated circuits, signal processors, hardware and software for receiving and/or processing the detected signal and/or image of the sample and for remote interaction; And

(c) a light source either in a mobile communication device or an external source.

Embodiment 13 B. Method for accelerated sample analysis using a mobile phone, comprising:

(a) placing the sample on the device for the CROF system according to embodiment 13A;

(b) carrying out the analysis of the sample placed on the CROF device, obtaining a result and

(c) transmitting the result from the mobile communication device to a location remote from the mobile communication device.

Embodiment 14. A method for analyzing a liquid sample, comprising:

(a) obtaining a sample that contains an analyte capable of diffusion in the sample;

(b) providing first and second plates that are movable relative to each other in different configurations, where one or both plates contain spacers that are fixed to a corresponding plate, where the spacers are characterized by a predetermined uniform height, and where the first plate contains on its surface an area analysis of the analyte, which is characterized by a predetermined area;

(c) placing the sample on one or both plates when the plates are in an open configuration, where the open configuration is a configuration in which the two plates are either partially or completely separated from each other and the space between the plates is not controlled by spacers;

(d) after (c) using two plates to compress at least a portion of the sample into a layer of uniform thickness that is limited by the inner surfaces of the two plates, where at least a portion of the layer is located throughout the analyte analysis region, where the uniform layer thickness is controlled by spacers and plates and is practically smaller than the linear size of a predetermined area in the horizontal projection of the area of analysis of the analyte, where compression involves

bringing the two plates together and

applying an external force to the outer surfaces of the plates to compress the plates together into a closed configuration, wherein the force creates a pressure on the plates over at least a portion of the sample, and the pressure extends over at least a portion of the sample horizontally between the inner surfaces of the plates, and where the closed configuration is a configuration in which the space between the plates in a layer of a region of uniform thickness is controlled by spacers;

(e) incubating the sample while the plates are in a closed configuration for a time that is (i) approximately equal to or greater than the time required for the analyte to diffuse through a layer of uniform thickness, and (i) substantially less than the time required for the analyte to diffuse through a certain area in the area of analysis of the analyte; And

(f) immediately after (e) either stopping the incubation and measuring the analyte in the assay area, or continuing the incubation while the plates are in a closed configuration and measuring the analyte in the assay area in a time that is substantially less than the time required for the analyte to diffuse through some region in the region of analysis of the analyte.

The following descriptions may be applied to embodiments 1-14 as set forth above.

In any embodiment in which CROF is used, spacers may be located within the sample region and within a suitable sample region to ensure proper uniformity of sample thickness control.

In any embodiment in which CROF is used, at least one of the two plates may be a plastic film with a thickness of from 1 μm to 50 μm.

In any embodiment in which CROF is used, at least one of the two plates may be a plastic film with a thickness of 50 μm to 100 μm.

In any embodiment in which CROF is used, at least one of the two plates may be a plastic film with a thickness of 100 μm to 150 μm.

In any embodiment in which CROF is used, at least one of the two plates may be a plastic film with a thickness of 150 μm to 250 μm.

In any embodiment in which CROF is used, both of these plates may be a plastic film thickness, each independently selected from 10-300 microns.

In any embodiment in which CROF is used, both of these plates may be a plastic film thickness, each independently selected from 100-200 microns.

In any embodiment in which CROF is used, both of these plates may be a plastic film thickness, each independently selected from 10-100 microns.

In any embodiment in which CROF is used, the height of the spacer on the wafer can be in the range of 5 nm to 100 nm.

In any embodiment in which CROF is used, the height of the spacer on the wafer can be in the range of 100 nm to 500 nm.

In any embodiment in which CROF is used, the height of the spacer on the wafer can be in the range of 500 nm to 1 μm.

In any embodiment in which CROF is used, the height of the spacer on the plate may be in the range of 1 to 2 μm.

In any embodiment in which CROF is used, the height of the spacer on the plate can be in the range of 2 to 5 μm.

In any embodiment in which CROF is used, the height of the spacer on the plate can be in the range of 5 to 10 μm.

In any embodiment in which CROF is used, the height of the spacer on the plate can be in the range of 10 to 30 μm.

In any embodiment in which CROF is used, the height of the spacer on the plate can be in the range of 30 to 50 μm.

In any embodiment in which CROF is used, the height of the spacer on the plate can be in the range of 50 to 100 μm.

In any embodiment in which CROF is used, the distance between spacers (IDS) is no more than 200 μm.

In any embodiment in which CROF is used, the distance between spacers (IDS) is no more than 150 μm.

In any embodiment in which CROF is used, the distance between spacers (IDS) is no more than 100 μm.

In any embodiment in which CROF is used, the distance between spacers (IDS) is no more than 80 μm, such as no more than 60 μm, no more than 40 μm, or no more than 20 μm.

In any embodiment in which CROF is used, the ratio of the width to height of the dividers is at least 1.5 (eg, at least 2, at least 3, at least 4, or at least 5).

In any embodiment in which CROF is used, the column width to column height ratio may be at least 1, at least 2, at least 5, or at least 10.

In any embodiment in which CROF is used, the plate spacing may be in the range of 2-50 μm, and any assay may have a saturation time of less than 1 minute.

In any embodiment in which CROF is used, the method involves washing.

In any embodiment in which CROF is used, the method does not involve washing.

In any embodiment in which CROF is used, the method has a sensitivity of less than 1 nM, for example, 0.1 nmol, 10 pmol, 1 pmol, 0.1 pmol, 10 fmol, 1 fmol, or 0.1 fmol after incubation for less than 1 minute .

In any embodiment in which CROF is used, the ratio of spacer spacing to spacer width may be less than about 7.0 (eg, about 7.0-1.0), particularly when the column height is less than about 100 μm.

In any embodiment in which CROF is used, the plate may have a thickness of 20-200 microns, for example 10-50 or 50-200 microns.

In any embodiment in which CROF is used, the sample volume may be less than 0.5 μm, such as less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 µm.

In any embodiment in which CROF is used, the distance between the spacers may be less than 200 μm, such as 20-200 μm, 20-50 μm, or 50-200 μm.

Other embodiments. In a preferred embodiment, to reduce incubation time to saturation for a binding process, reagent mixing process, combination of the two, or other process, the final sample thickness in a closed configuration is less than 0.5 μm (micron). In another preferred embodiment, the final sample thickness is in the range of 0.5 μm to 1 μm. In another preferred embodiment, the final sample thickness is in the range of 1 µm to 4 µm. In another preferred embodiment, the final sample thickness is in the range of 4 µm to 10 µm. In another preferred embodiment, the final thickness of the sample is in the range from 10 μm to 30 μm. In another preferred embodiment, the final thickness of the sample is in the range from 30 μm to 100 μm.

In a preferred embodiment, to reduce the saturation incubation time for a binding process, a reagent mixing process, a combination of the two, or another process, the final sample thickness is selected to obtain a saturation incubation time of less than 2 seconds. In other preferred embodiments, the final sample thickness is selected to provide an incubation time to saturation of less than 4 seconds, less than 8 seconds, less than 12 seconds, less than 20 seconds, less than 30 seconds, less than 40 seconds, less than 60 seconds, less than 120 seconds , less than 300 s, less than 420 s, or a range between any two of these values.

In any embodiment in which CROF is used, the device can be compressed manually for a period of less than 1 minute, for example less than 10 seconds.

In certain embodiments, the CROF device is integrated with a microfluidic platform or device. The microfluidic device may be configured to have different areas to receive a sample, detect analytes in the sample using a CROF device, collect waste in a reservoir, etc. Thus, in certain embodiments, the microfluidic channel platform may include fluid manipulation components to direct a sample applied to the sample receiving area of the microfluidic device into a CROF device configured to detect an analyte as described above. The fluid handling components may be configured to direct one or more fluids through the microfluidic device. In some cases, the fluid handling components are configured to route fluids, such as, but not limited to, sample solution, buffers, and the like. Fluid handling components may include, but are not limited to, passive pumps and microfluidic channels. In some cases, passive pumps are capable of microfluidic manipulation using capillary action and directing fluids through a microfluidic device disclosed herein. In certain cases, microfluidic liquid manipulation components are configured to deliver small volumes of liquid, such as 1 mL or less, such as 500 μL or less, including 100 μL or less, such as 50 μL or less, or 25 μL or less , or 10 µl or less, or 5 µl or less, or 1 µl or less. Thus, in certain embodiments, no external power source is required to operate the microfluidic device and operate the devices, systems, and methods of the present invention.

In certain embodiments, the microfluidic device has dimensions ranging from 5 mm x 5 mm to 100 mm x 100 mm, including dimensions of 50 mm x 50 mm or smaller, such as 25 mm x 25 mm or smaller, or 10 mm x 10 mm or less. In certain embodiments, the microfluidic device has a thickness in the range of 5 mm to 0.1 mm, such as 3 mm to 0.2 mm, including 2 mm to 0.3 mm or 1 mm to 0.4 mm.

In certain embodiments, the CROF device is located within a container, such as a well of a multiwell plate. The CROF device can also be built into the bottom or wall of a multiwell plate well.

In some embodiments, the media containing the CROF device, such as a microfluidic device or multiwell plate, may have a CROF device identifier contained in the media. The identifier may be a physical object formed on a carrier such as a microfluidic device. For example, the identifier can be read using a handheld device such as a mobile phone or smartphone, as described above.

In some embodiments, the camera may capture an image of the identifier, and this image may be analyzed to identify the device for CROF contained in the microfluidic device. In one example, the identifier may be a barcode. The barcode can be a 1D or 2D barcode. In some embodiments, the identifier may emit one or more signals that the signal amplification detector can identify. For example, the identifier may provide an infrared, ultrasonic, optical, audio, electrical, or other signal that may indicate the identity of the device to the CROF. The identifier may use a radio frequency identification (RFID) tag.

The identifier may contain information that identifies the specific device type for CROF present in the microfluidic device or multiwell plate. In certain embodiments, the identifier provides a key to a database that associates each identifier key with information specific to the device type for CROF present in the microfluidic device or multiwell plate. Information specific to the type of CROF device may include, but is not limited to, the identifier of the analytes that the CROF device is configured to detect, the coordinates of the position at which a particular analyte can communicate with the CROF device, the detection sensitivity for each analyte, etc. . The database may contain other information related to a specific CROF device, including expiration date, lot number, etc. The database may be present on a handheld device provided on a machine-readable medium, or may reside on a remote server accessible by the handheld device.

In certain embodiments, when the CROF card (e.g., a CROF wafer) is in a closed configuration, the overall thickness of the CROF card is in the range of 10 μm to 3 mm (e.g., in the range of 10 μm to 100 μm, 100 to 500 µm, from 500 µm to 1 mm, from 1 mm to 2 mm or from 2 mm to 3 mm); and the plate size for CROF in horizontal projection is from 2 mm to 50 mm (for example, from 2 mm to 5 mm, from 5 mm to 10 mm, from 10 mm to 20 mm, from 20 mm to 30 mm, from 30 mm to 40 mm or from 40 mm to 50 mm), where the x and y directions respectively take the value in this range.

In certain embodiments, the CROF wafer is inserted into and removed from the optical adapter for testing.

In a certain embodiment, the optical adapter has a thickness in the range of 2 mm to 40 mm (e.g., 2 to 5 mm, 5 to 10 mm, 10 to 20 mm, 20 to 30 mm, or 30 to 40 mm), and the horizontal dimension from 10 mm to 100 mm (for example, from 10 to 20 mm, from 20 to 30 mm, from 30 to 40 mm, from 40 to 50 mm, from 50 to 60 mm, from 50 to 60 mm, 60 to 70 mm, 70 to 80 mm or 80 to 100 mm), where the determined thickness, the horizontal dimension in the x direction and the horizontal dimension in the y direction take one of the values in a given range, respectively.

In certain embodiments, leukocyte testing separators are in the range of 2 μm to 40 μm (e.g., 2 to 10 μm, 10 to 20 μm, 20 to 30 μm, or 30 μm to 40 μm).

30. Homogeneous Analysis Using a Signal Enhancement Surface In many assay applications, particularly in PoC or other rapid assays, it is desirable to avoid washing steps. One aspect of the present invention relates to devices, systems and methods in which washing can be avoided during analysis.

By incorporating and/or applying a signal amplification surface, the disclosed devices, systems and methods can facilitate wash-free assays. The signal amplification surface can only amplify light emitted at a small distance from the surface (for example, 20 nm, or 50 nm, or 100 nm). One example of a signal amplification layer is D2PA.

31. An example of speeding up analysis using CROF with a ring (closed type) separator

An analysis acceleration experiment was conducted in which a polystyrene thin film was used as one of the CROF plates, a glass thin film was used as the other plate, and a wax ring was used as a separator and was fixed on the polystyrene plate. In the CROF method, 2 μL (microliters) of sample was dropped into a ring spacer (and into the center to form a small drop) and compressed into a thinner film by two plates, with the distance between the plates controlled by the ring spacer (i.e., a closed configuration of two plates for CROF). The plates were compressed manually. The sample thickness was uniform in the closed plate configuration. One of the main reasons for uniformity is that the volume of the sample corresponds to the volume between the ring spacers and the two plates. Both immunoassay and DNA hybridization assay were tested.

When testing the immunoassay (wax ring spacer ∼40 μm high and 0.8 cm in diameter), Protein A was used as the capture agent and applied to a polystyrene surface, and labeled IgG was used as the analyte. After incubation for binding between protein A and labeled IgG, unbound IgG was washed and the label of captured IgG was measured. Various incubation times were tested. In our experiment, the binding reaction was shown to saturate in less than 1 minute of incubation (ie, after 1 minute or less, the captured IgG signal would not change with incubation time). This short incubation time to saturation is expected for a space of 40 µm (hence the thickness of the sample), since the diffusion time for IgG in solution over a distance of 40 µm is approximately a few seconds.

We also tested the incubation of a similar direct assay on a conventional 96-well plate with a sample thickness of 3 mm and found that the typical incubation time to saturation was approximately 120 min. If the incubation process is limited by the diffusion of labeled IgG, then when the sample thickness is reduced from 3 mm to 40 μm, the incubation time is reduced from -120 min. to 1.28 s, which is consistent with our observation of an incubation time to saturation of less than 1 minute.

In DNA hybridization testing (wax ring spacer ∼52 μm in height and 0.7 cm in diameter), streptavidin-BSA presented a molecular linking layer on a polystyrene support and was coupled to a biotinylated capture strand, with the capture strand capturing the labeled target strand via hybridization. After incubation, the unhybridized target strand was washed and the label signal was tested. Various incubation times were tested. In this experiment, it was shown that the binding reaction saturates within 30 s of incubation time (ie, after 1 min or less, the captured IgG signal will not change with incubation time). This short incubation time to saturation is expected for a space of 52 µm (hence the sample thickness), since the diffusion time for a target probe in solution over a distance of 52 µm is approximately a few seconds. (The experiments are disclosed in more detail, for example, in the preliminary application with serial number 62/202989

For comparison, we tested the same assays at a larger sample thickness and found that a 1 mm thick sample would require approximately 20 minutes of incubation to reach saturation.

(The experiments are disclosed in more detail, for example, in the preliminary application with serial number 62/202989

32. An example of speeding up analysis (QAX and QMAX) using CROF with column delimiters

E-1.1 QAX assay with a CROFF device with an array of 30 µm column spacers to achieve saturation incubation times of less than 30 s

We tested QAX using CROF and reached a saturation time of ~30 s. The experiment is illustrated in Fig. 13a and b. In this experiment, a capture agent and a labeled detection agent were pre-applied to one of a pair of CROF plates prior to the CROF method and dried, then the sample was dropped onto the plate and covered with the other plate using the CROF method. Excavation of the sample took a few seconds, the CROF method took less than 10 seconds. This experiment showed that for a separator height of 30 µm, the incubation time until saturation is no more than 30 seconds.

Plates, samples, reagents. (1) CROF uses a self-retaining CROF device that contains (i) a 2.5 cm by 2.5 cm X-plate made of 175 µm thick PMMA film with an array of spacers in the sample contact area where the array of the separators has a lattice with a structure in the form of a rectangle with a constant interval of 120 μm/110 μm (in the horizontal directions x and y, respectively), while all separators are columns and have the same rectangular shape with the same separator height of 30 μm and width of 40 μm in x and 30 µm in y, and the spacers are made of the same material (PMMA) as the plate and are made by nanoimprinting a PMMA film using a mold (hence, the spacers are fixed to the plate with a predetermined height of the spacer and the space between separators 80 microns); and/or a glass plate with a flat surface (1 mm thick, 3 cm by 5 cm). The surfaces of the X-plate and glass plate are untreated and hydrophilic to the sample. (2) Dry capture agent (cAb), which is an anti-IgG antibody, was pre-coated onto the glass plate before sample dissection and CROF procedure; (3) a dry detection agent (dAb), which is an anti-IgG antibody, was pre-applied to the X-plate before the sample was sampled and the CROF method was performed; and (4) the sample is human IgG in BSA buffer at different nominal concentrations.

Stages of the experiment and results. A small volume of the analyte sample (human IgG) was dropped onto the surface of one of the plates of the CROF devices described in E2-1. Initially, the sample on the plate forms a puddle, but by placing another plate of the CROF device on the pool and pressing the two plates together, the original pool of blood is distributed into a sample film of a large area, but ultra-thin (~30 µm), controlled by an array of separators that are within a distributed sample. The person then used their hands to press the X-plate evenly against the drop (center to center) on the glass plate for 5-10 seconds, released their hands, waited 30 seconds, and the plates remained in the closed configuration.

Different samples (with different CROF devices) were then incubated for different times, washed and measured (optical signal). The results are shown in Fig. 13.b, which shows incubation times to saturation of less than 30 seconds for the QAX assay described in FIG. 13.a.

E.1.2. QMAX analysis and homogeneous analysis

QMAX was experimentally tested using an M-plate (i.e. D2PA) to amplify the signal. In addition, the QMAX assay was compared with the QAX assay, where the M-plate for signal amplification was omitted. They tested both heterogeneously (with washing) and homogeneously (without washing). The assay tested is a human IgG fluorescence immunoassay using QAX & QMAX.

Materials and methods: X-plate (pillar height 30 µm, column size 30 µm × 40 µm, ISD 80 µm), 25 mm × 25 mm; M-plate, size 25 mm × 25 mm; and assay reagents (in order of application) (a) DSU, Protein A, Anti-Human IgG (applied and dried on a backing plate), (b) Human IgG (analyte), and (c) Anti-Human IgG-IR800 Reagents ( applied and dried on X-plate storage areas).

Results (also shown in Fig. 14). These experiments show that for a CROF device with a 30 µm space in a closed configuration, incubation to saturation is within 1 min, and the sensitivity for a single reading is LoD=2 pM for QMAX with wash, LoD=10 pM for QMAX without wash (homogeneous); LoD=200 pM for QAX with wash and for QAX without wash (homogeneous) LoD= (cannot be read, no difference for different analyte concentration).

33. Additional Illustrative Test Experiments and Preferred Embodiments

This section provides additional illustrative experimental testing and observations and additional preferred embodiments of the present invention, which were carried out using the following conditions and carrying out the following general work together.

Volume of applied sample. Unless otherwise stated, all samples applied to the CROF plate are of unknown volume, meaning the exact volume is not known at the time of application.

Plates. In the CROF devices used in this section, unless otherwise noted, one of the two plates, referred to as the “X-plate,” is the only plate that has spacers. The other wafer, called the "substrate" wafer, has a flat surface and no spacers. Various materials (including glass, PMMA (polymethacrylate) and PS (polystyrene)) for plates and spacers, different plate thicknesses and spacer geometric characteristics (shapes and sizes) were tested. The sample contact surface of each wafer is a flat surface (except for the raised spacers) with surface smoothness variation typically less than 30 nm, but many of the flat surfaces had variation in surface flatness that was caused by the flexibility of the wafer, a flatness characteristic of the surfaces (not related to plate flexibility), or both. Some of the wafers have a variation in inner surface smoothness of more than 30 nm. Typical dimensions of the plates used in these examples are, unless otherwise noted, at least 25 mm wide and at least 25 mm long.

Separators. Unless otherwise noted, all spacers in this section are: (i) secured to the sample surface of the X-plate and manufactured by surface embossing (hence the material of the spacers is the same as in the X-plate); (ii) consist of an array of posts that have a substantially uniform cross-section of a rectangular or square shape with rounded corners, a substantially straight side wall with an angle of inclination from the normal of less than 5 degrees, a flat top surface, and a uniform height of the divider; and (iii) had a fixed internal spacer distance (ISD) in each X and Y direction (note that the X space may be different from the Y space) (see Fig. 17.b). In addition, the shape in the horizontal projection of the column dividers is either square or rectangular with rounded corners; tested dividers with different heights, sizes, distances between dividers, shapes and materials.

Manufacturing of separators. The separators embossed on the surface of the X-plate were manufactured using the nanoimprint technique, where the mold was pressed directly into the plate and embossing was carried out on an initially completely flat surface to obtain a flat surface, but with the presence of separators-columns protruding from the surface. The embossing process used a temperature above the glass transition temperature of the plastic, where the plastic may flow during the embossing process. The mold was made by lithographing and etching, and in some cases by electroplating the original mold. The mold was made of Si, silicon dioxide or nickel.

In fig. 17 shows examples of spacers manufactured on a plate. The separators were made by directly imprinting on the surface of a plastic plate using a mold. In fig. 17(a) and (b) show a top view of an optical micrograph of a square spacer array. Top view of the photograph: (a) the size of the column spacers is 46 μm × 46 μm and the distance between the columns is 54 μm and (b) the size of the column spacers is 10 μm × 70 μm and the distance between the columns is 10 μm; and SEM perspective view: (c) pillar spacer size 30 μm × 40 μm and spacer height 2 μm and (d) spacer size 30 μm × 40 μm and spacer height 30 μm. The micrographs show that (1) the top of the post spacer is very smooth, (2) the spacer has a nearly uniform cross-section, and (3) the corners of the post spacer are rounded with a radius of curvature of approximately 1 μm. A large radius curvature (eg, less sharp edge) is preferred because a sharp edge may cause cell lysis or affect fluid flow more than a rounded edge.

Using a surface profilometer, the height of the pillars was measured over a 2 cm by 2 cm area of the X-plate. The typical height uniformity of the pillar spacers of an X-plate manufactured using the methods described above was found to have an average variation of 4 nm, 10 nm, and 100 nm and relative average variation of 0.2%, 0.2% and 0.33%, respectively, for spacer heights of 2 µm, 5 µm and 30 µm.

Typical experimental procedure. As illustrated in FIG. 15, first, a small volume (a few microliters or less) of the sample was applied to either the substrate or the X-plate to form a small puddle(s). Second, the plates were brought together with an overlapping surface for the sample plate. Third, hands were used to compress the plates into a closed configuration, where the sample became a thin film with a much larger area than the original puddle. Fourthly, the hand(s) were released. And fifth, various measurements were carried out in a closed configuration. Below are specific details of the stages.

Methods for applying samples. Two methods of applying samples were used. In one method, the sample was applied to the plates using a pipette. In another method, blood samples were directly applied from the subject's finger (punctured with an instrument), bringing blood on the subject and the plate into contact. Blood was directly applied from a finger onto the plate without dilution. These experiments found that the final experimental results, unless otherwise stated, were independent of the sample application methods.

Sample application was carried out indoors and under standard room conditions without any special temperature control or the use of dust filters. It was found that under such conditions, dust falling on the samples did not affect the final measurement results, since (1) the flexible plate was used to take into account the falling dust, allowing the thickness of the sample in other areas to still be adjusted by the spacers and not affect the self-retention of the sample thickness , and (2) the area covered by dust was only a very small part of the total accessible area of the sample, with measurements taken in areas that were not exposed to dust. Selection of the dust-free area was carried out using optical imaging.

In some situations, the two plates have protective coatings to reduce the amount of dust that gets onto the plates. In some situations, two plates are placed along with the sample surfaces in the interior to prevent the entry of dust and other contaminants.

Wetting properties of the plate surface. The wetting properties of the surfaces of various plates used in these illustrative experiments were measured. The table below shows the contact angle measurements of a 5 µL sample on untreated or treated surfaces of various plate materials (glass, PMMA and PS) and with various surface geometries (flat surface and X-plate sample surface) for different sample types ( water, PBS (phosphate buffered saline) buffer and blood), where the X-plate is 175 µm thick PMMA and its sample surface has an array of pillars (i.e. spacers) 2 µm high with a horizontal dimension of 30 µm × 40 µm and spacing 110 µm/120 µm (i.e. the distance between the separators is 80).

Experiments have shown that (1) all untreated glass, PMMA, and PS surfaces have a hydrophilic surface (i.e., contact angle less than 90 degrees); (2) the untreated glass surface has a lower contact angle (more hydrophilic) than untreated PMMA and PS; (3) the contact angles for water, PBS, and blood are similar and blood is slightly better at wetting than water and PBS; (4) the untreated PMMA X-plate has almost the same contact angle as the untreated PMMA plate; and (5) surface wetting properties can, as expected, change significantly when the surface is treated to be more hydrophilic or more hydrophobic.

The hydrophobicity of the wafer surface can be modified by surface treatment. For example, the PMMA X-plate was made more hydrophilic by exposing the surface to oxygen plasma, and more hydrophobic by treating the surface with tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane. The contact angles were 25, 27, 28 degrees for the hydrophobically treated X-plate and 105, 104 and 103 degrees for the hydrophilically treated X-plate, respectively, for water, PBS buffer and blood samples.

In the discussion below, unless otherwise noted, the entire sample surface (i.e., the inner surface that contacts the sample) of the plates is untreated.

Area and height of the applied sample. The area and height of the sample on the plate were measured when the water sample was pipetted onto the plates, which were in an open configuration.

Experiments have shown that a typical sample deposited on a wafer in an open configuration is much thicker than in a closed configuration.

It was noted that with a closed plate configuration (1) the total area of the sample expands from a few millimeters in diameter to several centimeters (depending on the height of the spacer), and (2) if the array of spacers has a grid with a square structure, then the area of the sample in a closed plate configuration, it is also essentially square with the square edge of the specimen aligned in the grid direction with a spacer-square structure. Therefore, this demonstrates that the final sample area in a closed configuration can be controlled by using spacers in different spatial arrangements. If the separator has a grid with a rectangular structure, then the resulting sample area in a closed configuration should be rectangular. If the separator has an annular circular pattern, then the resulting sample area in a closed configuration may be circular.

Manual compression. In all experiments in Section 30, the plates in the CROF method were brought together and compressed into a closed plate configuration using a human hand(s). In final compression to ensure uniform sample thickness over a large area of the CROF plates, the thumb is often compressed in one area and then rubbed into different areas of the CROF plates. The process of using hand(s) to compress the CROF device(s) into a closed configuration is called "hand pressing".

Self-restraint. It was noted, unless otherwise noted, that after compressing the CROF plates into the final configuration and removing the compressive force (e.g., hand pressure), the thickness of the sample between the two plates was still controlled by the height of the spacers and held at a constant thickness for an extended period of time (until the sample will not dry out as a result). This result is called “self-restraint.” Self-retention is the capillary forces between the plates, the fluid sample and the surrounding medium (e.g. air) caused by surface tension. It was noted that manual compression and self-retaining of the CROF device produced excellent sample thickness as shown in E-1.

Measurements of the space between the plates and the thickness of the sample. In all experiments below, the space between the inner surfaces (i.e., sample surfaces) of two plates in a closed configuration was measured using the Fabry-Perot cavity resonance (FP resonance) due to the inner surfaces of the plates. Due to differences in optical index between the sample (and air) and the inner surface, each of the inner surfaces of the plates acts as an optical reflector, and the two inner surfaces form an optical cavity. The FP resonance spectra are periodic, and the space between the internal surfaces, h, (hence the sample thickness) at the optical measurement point can be calculated using the formula:

where c represents the speed of light, Av represents the period in the frequency domain, and n represents the refractive index of the sample between the plates.

In this FP resonance test, the light source had an area of approximately 2 µm by 2 µm. Typically, the space between the inner surfaces of the plates was measured at 25 different points over a 1.6 cm by 1.6 cm area around the center of the CROF device, where the 25 points constituted a 5 × 5 square array with spacing (i.e. e. the distance between two adjacent points) is 4 mm. The measurements did not affect the areas that were occupied by separators (i.e., bars).

Since the internal surfaces and the sample are in contact in a closed plate configuration, the measured space between the internal surfaces coincides with the thickness of the sample at the measurement point.

Average sample thickness, N. The average sample thickness, N, was calculated using the space between the plates, measured at 25 points, and the formula:

The sample thickness deviation refers to the deviation of the average sample thickness, H, over a given area, based on the predetermined height of the divider, H ₀ : (H - H ₀ ). In this case, the relative deviation of the sample thickness is the deviation divided by the predetermined height of the separator: [(H - H ₀ )/H ₀ ]. A positive thickness deviation means that the sample is on average thicker than the height of the spacer, and a negative thickness deviation means that the sample is on average thinner than the height of the spacer.

Uniformity of sample thickness. The sample thickness uniformity, Δ, over a given area is defined as the standard deviation of the sample thickness over a given area.

32.1 Specimen thickness variation and uniformity in CROF with manual compression and self-retaining

Parameters in the CROF devices and method that may influence sample thickness variation and uniformity in the closed plate configuration after hand release were studied experimentally. Parameters have been found to include, but are not limited to, inter-divider spacing (IDS), separator shape and dimensions (e.g., separator horizontal dimension, separator height, separator width to height ratio, space fill factors for separator (divider area to total area ratio or spacing ratio spacers and width), the mechanical strength of the material (Young's modulus) of the spacers and the plate, the thickness of the plate and the surface flatness of each plate. Below are some results and preferred embodiments obtained from the experiments. The determination of the spacer height, IDS, spacing and horizontal dimension of the spacers is given in Fig. 16.

E-1.1 Effect of IDS (Interspace Distance) and Thickness and Plate Materials on Specimen Thickness. The experiment noted that the interspacer distance (ISD) of a periodic spacer array can significantly influence the sample thickness deviation (based on spacer height) and its uniformity in a closed process configuration for CROF.

In fig. Figure 18 shows the effect of IDS, thickness, and plate materials on the sample thickness. Measured sample thickness variation and uniformity as a function of interspacer distance (IDS) for different plates and materials, different plate thicknesses and different samples. The separators are presented in the form of a periodic array and have a separator height of 5 µm, a flat top surface and a square shape (horizontal column size 10 × 10 µm, almost uniform cross-section and rounded corners). The IDS was 20 μm, 50 μm, 100 μm, 200 μm, and 500 μm, respectively. The substrate was a 250 µm thick untreated PMMA wafer with a flat surface (1 inch × 1 inch area). The X-plates on which the spacers were directly manufactured were, respectively, an untreated PMMA plate of 175 μm and 50 μm thickness and an untreated PS plate of 125 μm and 25 μm thickness, respectively. The sample was, respectively, 2 μl of blood (dropped by direct contact with a finger), saliva or PBS (dropped with a pipette). CROF devices were compressed using hand compression and rubbing over an area of 1 inch by 1 inch and were self-retaining after compression. The sample thickness was measured with the CROF devices in a closed configuration.

In fig. Figure 18 shows that for the given experimental conditions and for a square-shaped separator (horizontal column size 10×10 µm, almost uniform cross-section and rounded corners):

(1) If the ISD is 20 µm, 50 µm, 100 µm, then the average final thickness of the sample is 5.1 µm to 5.2 µm, which is very close to the preset spacer height of 5 µm, and is characterized by thickness deviation and uniformity less than 4% (namely, if the ISD is equal to or less than about 120 μm, then the deviation and uniformity may be less than 4%).

(2) But if the ISD is 200 µm and 500 µm, then the average final sample thickness is 4.3 µm and 3.5 µm, respectively, which is significantly less than the preset height of the spacers (5 µm) and has a thickness deviation of -13.9% and -30.9% and homogeneity 10.9% and 27.7%, respectively. This means that if the ISD is greater than approximately 200 µm, not only does the average thickness decrease significantly, but also the uniformity becomes unsatisfactory.

If for an array of spacers with a horizontal column size of 40 µm by 40 µm (Fig. 18) the ISD is 60 µm, 150 µm, 100 µm, then the average final thickness of the sample is from 5.1 µm to 5.2 µm, which is very close to the predetermined spacer height of 5 µm and has a thickness deviation and uniformity of less than 4% (namely, if the ISD is equal to or less than approximately 100 µm, then the deviation and uniformity may be less than 4%).

E-1.2 Influence of IDS/(Eb ^∧ 3) on sample thickness

These experiments show (for example, Fig. 19) that in order to achieve a small deviation in the thickness of the sample and proper homogeneity, the value of SD ⁴ /(h ^x E) (x=1 on the graph) of the X-plates should be less than 10 ^∧ 6 μm Λ3 /GPa, where ISD is the distance between the spacers, h is the height (thickness) of the material, and E is the Young's modulus of the material.

In all methods and devices that use CROF, in certain embodiments, the value of SD ⁴ /(h ^x E) (x=1 on the graph) is less than 10 ^∧ 6 μm Λ3/GPa, less than 5×10 ^∧ 5, less than 1 ×10 ^∧ 6, less than 5×10 ^∧ 6, etc.

In any embodiment, the flexible plates may have a thickness in the range of 20 μm to 250 μm (for example, in the range of 50 μm to 150 μm) and a Young's modulus in the range of 0.1 to 5 GPa (for example, in the range of 0.5 - 2 GPa).

In any embodiment, the product of the flex plate thickness and the flex plate's Young's modulus is in the range of 60 to 750 GPa-μm.

E-1.3 Effect of Spacer Size and Height on Specimen Thickness These experiments show (e.g., FIG. 20) that to achieve small specimen thickness variation and for a given plate, specimen, and pressure thickness, the IDS must be approximately 150 µm or less.

E-1.4 Effect of Spacer Width to Height Ratio on Specimen Thickness These experiments show (e.g., Fig. 21) that to achieve a small deviation in specimen thickness for a given plate thickness, specimen, and pressure, and for ISD from 20 µm to 150 µm, the ratio column width and thickness (WRH) must be greater than 1, and in certain embodiments preferably greater than 2.

This indicates that at WHR ~1 or higher, the separators are strong enough to withstand the pressure and friction of hand squeezing, otherwise the overall deviation will be large and the uniformity weak for all ISDs.

E-1.5 Influence of filling factors for the separator on the thickness of the sample

These experiments show (eg, FIG. 22) that to achieve small sample thickness variation and proper thickness uniformity for a given sample plate thickness, the fill factor for the spacer must be approximately 2.3 or greater.

For example, the achieved variance and uniformity of less than 4% shown in FIG. 22 suggest that for a given post area and IDS, and for a given spacer fill factor (i.e., the ratio of horizontal post area to total area), PS posts are strong enough to withstand crushing and rubbing when squeezed by hand. The deformation of a PS post can be estimated as follows: the thumb pressure is approximately 1-10 kg/cm2 (10 ^∧ 5 Pa), the Young's modulus for PS is ~3 GPa, and the fill factor for a post spacer of 20 µm width and ISD is 100 µm is ~4%, resulting in a relative deformation (stretch) of the column under thumb pressure of 1% to 0.1%, which is consistent with our experimental observation.

E-1.6 Effect of plate thickness on sample thickness

These experiments show (e.g., FIG. 23) that (i) to achieve small sample thickness variation (equal to or less than 5%) and proper thickness uniformity for a given plate thickness, sample, at least one of the plates must have a plate thickness less than 200 microns; and (ii) if the X-plate and substrate are thicker than 200 µm, they are too rigid to overcome dust contamination, resulting in poor spacer uniformity/deviation.

E-1.7. Influence of the substrate plate on the thickness of the sample

These experiments found (e.g., FIG. 25) that if a thicker (1 mm) glass substrate was used, the maximum IDS for less sample thickness variation and proper sample thickness uniformity could range from 150 µm to 200 µm for a glass substrate. PMMA.

E-1.8 Modification of plate surface wetting properties and influence on self-retention

These experiments found (eg, FIG. 24) that: (1) Proper self-retention in a CROF device requires that at least one of the two internal surfaces of the CROF device be hydrophilic. (2) If both internal surfaces of the CROF device are hydrophilic, this provides the best self-retention and control and uniformity of sample thickness. (3) If one internal surface of the CROF device is hydrophilic and the other internal surface is hydrophobic, then the sample area must be greater than 0.5 cm2 to obtain proper self-confinement. (4) If both inner surfaces are hydrophobic, then self-retention is either weak or insufficient (unstable). (The lines in the figures are for visual guidance.)

E-1.9 Effect of manual compression time on sample thickness

In these experiments, it was found that the CROF device could self-retain at compression times ranging from 1 s to 60 s and had similar good performance. The CROF device has poor performance and cannot self-retain unless compressed (0 s compression).

E-1.10. Comparison of the influence of periodic spacer posts and random spherical spacers on sample thickness

Measurements in Fig. 27 show that, for given experimental conditions, a CROF device with periodic spacer posts has much less sample thickness variation and better uniformity (both less than 5%) than with random spherical spacers (i.e., beads). Specifically, for 20 μm, 50 μm, and 100 μm ISDs, the average thickness deviation and uniformity using periodic spacer posts with a uniform cross-section were 2.3% and ~3.4%. However, when using random spherical spacers with an average ISD of 20 µm, 50 µm and 100 µm, the average thickness deviation and uniformity were 11.2% and 12.5% when using a 220 µm thick glass cover plate and 10.8% and 20% when using PMMA cover plate with a thickness of 175 μm, while these results are approximately 5 times larger in the deviation of the sample thickness and express weaker homogeneity.

E1.12. Other results

In fig. Figure 28 shows the effect of different X-Plate thickness and substrate thickness on sample thickness.

In these experiments, it was found that the liquid pipetted immediately upon sampling from a finger has similar characteristics of the final sample thickness and homogeneity.

These experiments also show that the liquid dropped onto the substrate or onto the X-plate has similar measured sample thickness and uniformity characteristics.

32.2 CBC in undiluted whole blood using CROP with self-retention

E2.1 Devices used for CROF

The CROF devices used in all tests in Example 32.2 contained an X-plate and a glass plate with a flat surface. The X-plate was a PMMA film 175 μm thick, with an area of 2.5 cm by 2.5 cm, with an array of periodic spacers in the area of contact with the sample. The glass plate had a thickness of 1 mm and was characterized by a flat surface and an area of 3 cm by 5 cm. The spacers on the X plate were directly embossed on PMMA film with an initially flat surface, therefore they were made of PMMA (the same material as for X -plates) and were attached to the X-plate.

Each spacer is a column that has a nearly uniform horizontal cross-section, a smooth top surface, and a rectangular shape with a width of 40 μm and 30 μm in the horizontal x and y directions, respectively. All spacers on a given X-plate have the same spacer height. The periodic spacer array was characterized by a grid with a rectangular structure with a constant spacing of 120 μm and 1 10 μm (in x and y, respectively), resulting in a constant interspacer space (IDS) of 80 μm.

The surfaces of the X-plate and glass plate are untreated and are hydrophilic to human blood drawn onto the surface with a contact angle of approximately 40-50 degrees. Both plates are transparent to visible light.

E2.2 Sample, preparation, application, CROF method, self-retention

Unless otherwise stated, all blood samples were obtained from healthy subjects, which were fresh and directly applied to the CROF plate without dilution and without the addition of anticoagulants.

In all experiments reported in Example 3, unless specifically stated otherwise, blood was obtained from a human finger prick and the blood was applied to a CROF plate by directly contacting the blood and the plate. Typically, a volume of blood of approximately 0.1-1 µl is applied in direct contact with the surface of the plate. Within approximately 60 seconds of applying the blood, the CROF method was used to compress the blood sample into a thin film and then take measurements. Unless specifically stated, no antiagglutination agents or liquid diluents were added to the blood sample.

In certain experiments where it was necessary to stain the WBC before blood analysis, the reagent, a layer of dry acridine orange dye, was first applied to the inner surface (sample contact surface) of one of the plates of the CROF device. Coating of the dry dye layer involved the following steps: (a) dropping 30 μl of acridine orange dye in water at a concentration of 20 μg/ml onto a glass plate, (b) spreading it over an area of ~ 1 ^cm2 , and (c) drying for approximately 1 hour.

Regardless of the methods used to apply approximately 1 μl or less of blood to one of the CROF plates, the blood applied to the plate will form a pool of a few millimeters or less in diameter on the plate. The two plates of the CROF device were then manually brought into a closed configuration and manually compressed for several seconds, where the original pool of blood was compressed by the two plates into a large area thin film of blood (with a horizontal dimension of approximately 1-3 cm). It was found that throughout Example 3, unless otherwise noted, the manually compressed CROF devices had uniform sample thickness controlled by spacers and were able to independently maintain uniform specimen thickness once manual compression was removed. The samples were applied under normal room conditions. It was found that dust had no effect on achieving the predetermined final sample thickness over a large sample area. We also found that the final blood sample was distributed in a rectangular shape with rounded corners, which we believe is due to a lattice with a rectangular structure of periodic separators. This stage is illustrated in FIG. 15.

For the sample application using dry dye on a plate, this sample was held for 30 seconds before any measurements were taken.

The blood sample directly applied to the plate was not diluted with liquid at all (i.e., no dilution with liquid), but was mixed with the dry reagent coating the plate.

All spacers had the same rectangular shape with the same spacer height of 2 μm.

The 2 µm separator height was chosen so that the resulting blood sample size in the closed configuration of the CROF device was approximately 2 µm, which is approximately equal to the thickness of the red blood cells (RBC) (2-2.5 µm), but much smaller than the diameter of the RBC (6.2 -8.2 µm). This resulting sample thickness resulted in a closed CROF configuration where each RBC was well separated from the others and there was no overlap or columnar formation between different RBCs, allowing RBCs to be accurately counted when imaging the sample area.

E2.3 Visualization of blood cells

Unless otherwise noted, imaging of blood samples in Example 3 was performed with samples between two CROP plates that were in a closed configuration and using a commercial DSLR camera (Nikon) and iPhone, respectively. The results from each type of camera are comparable. Unless otherwise noted, the images are top views of the samples through one of the plates, which is transparent (ie, two-dimensional images of the sample in a plane parallel to the surface of the plates).

Nikon camera. Samples are observed using a conventional commercial DSLR camera (Nikon) with two filters (470 ± 20 nm bandpass filter as excitation filter and 500 nm high-pass filter as emission filter), one light source (xenon lamp) and a set of magnification lenses. focus. Brightfield mode produces a broadband white light source without the use of any filters. In fluorescence mode, a 470 ± 20 nm filter was placed in front of the xenon lamp to create a narrow-band excitation source with a wavelength of approximately 470 nm, and a high-pass 500 nm filter was placed in front of the chamber to block light entering the chamber with a wavelength of less than 500 nm.

Mobile phone. For our experiments we used an iPhone-6.

E2.4 Height of the separator (thickness of the sample). Effect on blood cells and RBC count

In these experiments, the thickness of the samples was controlled so that it was the same as the height of the spacer. The effect of the separator height (and, consequently, sample thickness) in the CROF method on blood cells, as well as on their visualization and counting, was experimentally studied. The devices for CROF and the method used and application of blood were as described at the beginning of this section in Example 2. The blood samples were from the same healthy subject. In one of our experiments, four different spacer heights were tested (1 μm, 2 μm, 3 μm, and 5 μm).

In fig. 29 shows optical micrographs of a top view (bright field optical microscope) of a blood sample that has been subjected to the CROF process within four different CROF devices, where each CROF device has a grid with a rectangular structure of periodic spacers and a different constant spacer height: 1 µm (a), 2 µm (b), 3 µm (c) and 5 µm (d). The blood sample was directly applied from the subject's finger to the plate of the CROF device and no antiagglutination agent or liquid diluent was added to the blood.

With bright-field optical microscopy, RBC cells are much easier to see than WBC cells. Red blood cells (RBCs), also called red blood cells, have a disc diameter of approximately 6.2-8.2 µm, and a thickness at its thickest point of 2-2.5 µm (near the edge of the disc), and a minimum thickness at the center of 0. 8-1 microns.

Optical microscope observation showed that at a spacer height of 1 μm, ~99% of RBCs were lysed. For example, in FIG. 29(a) shows that only RBCs remained in the observation field. The 1 µm spacer height is significantly less than the average RBC thickness. This experiment demonstrated that the CROF devices and method can be used to lyse a cell by making the final plate separator (by controlling the height of the separator) smaller than the minimum cell size.

Observation in an optical microscope (for example, in Fig. 29) showed that with a separator height of 2 μm (sample thickness), the RBCs are separated from each other, practically do not overlap each other, and have an almost round and symmetrical shape. The separation between each RBC is clearly visible by the completely circular dark border line of each RBC (i.e. the border line completely surrounds each (and only one) cell) in the 2D microscope image

In addition, microscopic observation also showed that the RBC is darker at the center of the cell than at the edge, indicating that at a spacer height of 2 μm (sample thickness), the RBC at the center is still thinner than at the edge.

Optical microscope observation showed (e.g., FIG. 29) that if the spacer height (hence the sample thickness) was 3 μm, the image of the blood sample was dramatically different from the image with a spacer height of 2 μm for several reasons, including but not limited to: (1 ) The RBCs were largely overlapping and most RBCs lacked the completely circular dark boundary line that separates each cell that exists at the 2 µm divider height, but a few RBCs together shared one dark boundary line that was no longer circular; and (2) some of the RBCs did not have the nearly rounded shape of the 2 μm spacer height, but rather were ellipsoidal in shape, and the central dark disk of each RBC, which was transparent at the 2 μm spacer height, was difficult to visualize. When the spacer height was 5 μm, the RBCs had more overlap and more RBCs had a non-circular shape and a virtually invisible dark center (eg, FIG. 29).

It is well known that in the blood, without spatial limitation, RBCs can overlap each other (forming, for example, columns). When confining a blood sample between two flat plates with a 2 µm space using the height of the spacers and plates, the thickness of the blood is approximately equal to the thickness at the point of greatest thickness of the RBC (which is 2-2.5 µm), so at this location on the surface of the plate the restriction forces only one RBC to pass between the two plates and forces the RBC to orient its disk parallel to the surface of the plate, resulting in proper separation of the RBC, formation of a complete round dark boundary line and a nearly circular shape when viewed from above using optical microscopy.

As the spacer height and therefore the sample thickness becomes larger, such as at 3 µm and 5 µm spacer heights, the sample thickness allows more than one RBC to be between the two plates at the plate location, causing the RBCs to overlap and the disappearance of distinct boundaries each RBC; and allows the RBC to rotate between the two plates and rotate parallel to the location of the plate surface, resulting in a non-circular shape in the top view image of the RBC.

To count the number of RBCs (for example, to measure the RBC concentration), our experiment clearly shows that by making the sample thickness up to 2 μm (for example, using a 2 μm spacer height), it can be counted more easily and more accurately than with a sample thickness of 3 μm and 5 µm.

With the height of the separator (hence the space between the two plates and the thickness of the blood sample) being approximately 2 µm, which is approximately equal to the thickness of the red blood cells (RBC) (2-2.5 µm), but much smaller than the diameter of the RBC (6.2-8.2 µm), counting blood cells is much simpler and more accurate than with a larger sample thickness.

At the final sample thickness (2-2.5 µm) or preferably 1.9 to 2.2 µm, which occurred with the closed CROF configuration, each RBC was well separated from the others and there was no overlap or columnar formation between different RBCs, which allowed RBC to be accurately calculated while imaging the sample area.

On the other hand, it was observed that in the CROF device with a spacer height of 1 μm, most of the RBCs were lysed, but not the WBCs or platelets. In these experiments, using optical imaging from above the CROF (i.e., the CROF plates are essentially parallel to the imaging plane of the microscope or camera imaging system), (a) the number of cells per area and (b) the exact horizontal size of the area were determined. The horizontal dimension of the CROF device can be determined by pre-calibration. Or, the horizontal size of the device area for CROF can be determined during rendering by using the horizontal size of the separator as a marker. Both options were used in these experiments.

In the experiment of Example 2, for these CROF plates, the space between the two CROF plates (hence the thickness of the blood sample) was the same as the height of the spacer within 5% or better. Using this sample thickness information along with the horizontal dimensions of a given area determined by optical imaging, the volume of the sample associated with a given area was determined, which is equal to the cross-sectional area of the sample multiplied by the thickness of the sample. Knowing the volume of the sample and the number of cells in the volume (determined by imaging), the concentration of cells in this volume of the sample could be determined.

In fig. 29b shows (b) the ratio of red blood cell area (measured from the 2D top view image) and total planar area of the plate for CROF. The maximum value is at 2 μm interplate space (i.e., sample thickness) because below 2 μm some RBCs are lysed and above 2 μm RBCs overlap and rotate, all of which produce less RBC area in the 2D image.

One of the results from the following experiments is that the optimized space size in the CROF device for counting blood cells (RBC and WBC) is 1.9 µm - 2.2 µm, or 2 µm to 2.2 µm, or 2 µm to 2.1 µm.

Another experimental finding is that in a CROF device with 1 µm space between the two plates, most RBCs are lysed but WBCs are not lysed: CROF device

It has been found that if the size of the space in the CROF device is much smaller than the thickness of the RBCs (eg, 1 μm plate spacing), the RBCs are lysed. WBC is more elastic, most of them can still be observed and may not be lysed.

E3.5 RBC (Red Blood Cell) counting

In one embodiment, RBCs were counted in bright field mode without any filter. Magnification factors of 4x, 10x, 20x or 40x were used to obtain images. Since both the size of the X-plate space (t) and the field of view (A) are known for each magnification (separators and their periods were used as scale marks (i.e. marks), the concentration of RBC in the blood sample was calculated. For example , to calculate N RBC in one field of view, the concentration of RBC (C) in the blood was C=N/t/A. This calculation method was the same for measuring the concentration of WBC, PLT.

E2.6. WBC and platelet count

Each white blood cell (WBC), also called a white blood cell, has a typical disc diameter of approximately 10-15 µm. A typical platelet (PLT) has a typical size of 1-2 µm. Because WBC and PLT have no visible pigment themselves, they are more difficult to detect with conventional microscopy than RBC. To make the WBC and PTL more visible when counted, in one embodiment they are stained with acridine orange (AO) dye.

Acridine orange is a stable dye that has a natural affinity for nucleic acids. When bound to DNA, AO intercalates with DNA as a monomer and produces intense green fluorescence when excited with blue light (excitation 470 nm, green emission 525 nm for WBC). When bound to RNA and proteins, it forms an electrostatic complex in polymeric form, which produces red fluorescence when excited by blue light (excitation 470 nm, red emission 685 nm for WBC, PLT). RBCs lack nucleic acids and therefore cannot be stained. WBCs have both DNA and RNA cores, so they stain intensely. PLTs have a small amount of RNA, so they stain weakly. See fig. 33.

WBCs were counted in fluorescence mode using an excitation filter of 470 ± 20 nm and an emission filter of 500 nm high-pass filter, and magnification factors of 4x, 10x, 20x, or 40x were selected for image acquisition. By using these embodiments, WBC and PLT were properly calculated.

E2.7 Measurements of different WBCs

WBC can be divided into five main subclasses: neutrophils, eosinophils, basophils, lymphocytes, and monocytes; or sometimes three main classes: granulocytes, lymphocytes and monocytes. The amount of each class in a subject's blood may have clinical significance, since various infections with viruses, bacteria, or fungi, or allergies, may alter the amount of a particular WBC subclass.

WBCs contain a nucleus, which distinguishes them from anucleate red blood cells and platelets. In addition, different subclasses of WBC are characterized by different DNA/RNA and protein ratios, and they can be differentiated according to this by using a suitable dye to stain DNA and RNA separately.

For example, the AO dye intercalates with DNA as a monomer and produces intense green fluorescence when excited with blue light (excitation 470 nm, green emission 525 nm for WBC). When bound to RNA and proteins, it forms an electrostatic complex in polymeric form, which produces red fluorescence when excited by blue light (excitation 470 nm, red emission 685 nm for WBC, PLT). Thus, different WBCs will have different R/G color ratios (red emission versus green emission) after staining with AO dye.

Using the AO dye, 3 types of WBC can potentially be differentiated:

granulocytes (neutrophils, eosinophils, basophils), lymphocytes, monocytes. Additionally, you can directly use the camera's (or iPhone's) built-in RGB filter to distinguish between green and red emission from the G channel and R channel of a single captured photo. So there is no need to use 2 separate filter sets (like 525nm and 685nm bandpass filter).

As shown in FIG. 34, a total of 594 WBCs counted and displayed. It can be clearly seen that the cells are grouped into three distinct areas (shaded areas represented as landmarks for the eye), corresponding to the three major subpopulations of leukocytes. The percentage of each subpopulation is given in the table and corresponds well to normal values in human blood.

E2.8. Hematocrit measurements

Hematocrit (Ht or HCT), also known as pelleted cell volume (PCV) or red blood cell volume fraction (EVF), is the percentage volumetric content (%) of red blood cells in the blood. When setting up the X-SBC, a space size of 2 μm was used, in which each RBC would be tightly packed using the substrate and X-plate. Thus, NBT in this case is equal to the volume of RBC in the total blood volume.

E2.10. Dried AO dye quickly stains WBC

WBC was stained with dry AO dye after 30 s, 10 min, 30 min, 90 min. In the CROF method with dried AO dye on the surface of the plate, the AO dye can completely stain WBC in less than 1 min. and will not affect others or over-stain another area after a long time. In addition, since bound AO fluoresces more intensely than unbound dye, a washing step is not required.

E2.11. Other non-fluorescent dyes for WBC staining

A non-fluorescent dye for WBC staining can simplify the WBC counting procedure. Crystal violet or gentian violet (also known as methyl violet 10B or hexamethyl pararosaniline chloride) is a triarylmethane dye that can be used to stain WBC core. Similar to the AO dye, 30 µl at 1 mg/ml acridine orange dye in water was dried on a 1 cm ² glass slide for 1 hour. The process of the X-CBC experiment was then repeated. The WBCs would turn purple. One of the disadvantages of this method is the difficulty in differentiating WBC subpopulations.

E2.12. No anticoagulants were required in the CROP blood test

One of the advantages of the present invention is that there is no need to use anticoagulant reagents for counting, as was observed during the experiment. In these experiments, X-plates with spacings of 2 μm, 3 μm, and 10 μm and a blood area of 1 cm x 1 cm were tested in a CROF blood sample device. For durations ranging from 0 min to 80 min, photography was taken every 10 min at 5 typical points from the center to the edge of the sample. All tested samples did not contain anticoagulant reagents. It was noted that under these experimental conditions, there was no agglutination of the blood sample in the closed configuration during the observation period. This is because (1) during CROF with a space of ~2 μm (the thickness of the sample in a closed configuration), the blood cells are separated from each other and (2) the CROF plates protect most of the blood cells from oxygen.

E2.13. Additional blood cell counting experiments using CROF and iPhone.

Other experiments tested and validated the technology and a compact, easy-to-use device that allows a person to independently perform a complete blood cell count in less than 20 seconds using a smartphone with a sample volume smaller than a drop of blood (<1 μL). All the person has to do is allow a tiny amount (random unknown volume) of blood from a pricked finger to touch the card, close the card, and take a photo with the smartphone.

One aspect of the present invention is the observation that by precisely rearranging a drop of blood into a uniform layer of blood that is one red blood cell (~2 µm) thick and sandwiched between two plates, it offers unprecedented advantages in blood cell counting. The advantages include the following: (i) for fresh undiluted whole blood without the addition of any anticoagulant, the blood cells will be well separated from each other, not coagulated, and thus easily identified by imaging; and (ii) the sample exhibits virtually zero evaporation (in the test area) with a constant blood cell concentration over an extended period of time. The second key technology that has been developed is called Compressed Regulated Open Flow (CROF), which uses a CROF card (foldable, disposable, postage stamp-sized (1-inch wide, paper-thin) hand-actuated plastic film) to perform blood transformation, measure the thickness (hence volume) of the transformed blood sample and mix (if necessary) pre-applied dry reagents in the blood (and perform all functions in one motion and in less than 5 seconds). The last two techniques described in this paper involve a small, matchbox-sized optical adapter for smartphone imaging, as well as software for smartphone control and image analysis. The method (“blood cell counting using CROF and imaging” or BCI) using a smartphone was validated against an industry standard, a commercial handheld hemocytometer, and imaging using a microscope (instead of a smartphone). For each method, over 42 tests were performed using two types of blood (stored and fresh from the subject) and the number of red blood cells (RBC), white blood cells (WBC), platelets, three different types of WBC, hematocrit (HCT) and mean cell volume (MCV) were measured. . Validation shows that smartphone-based BCI has the same accuracy as or better than the accuracy of a commercially available hand-held hemocytometer (can be further improved) and the same day-to-day stability as commercially available measurement instruments. It is clear that BCI technology has broad and significant applications in cell imaging, immunoassays, nucleic acid assays, personal health monitoring and other biochemical diagnostics.

The BCI device contains three hardware components: a disposable, postage stamp-sized plastic CROF card (1" x 1" square, paper-thin), a smartphone, and a matchbox-sized optical adapter (1.5" x 1.5" x 0. 7 inches (LxWxH)); and software that controls the smartphone, creates the user interface and analyzes blood cells. All of them (except for the smartphone) were designed and developed by the authors of this application. An optical adapter ("adapter"), which contains lenses, mirrors and filters; and mounted on a smartphone, makes the flash and smartphone camera the light source and imaging system for testing, respectively. The optical adapter also has a slot for moving the CROF-Card into the correct position in front of the camera (Fig. 30). In our current tests we used an iPhone-6.

In a blood test using a BCI (FIG. 30), the individual first pricks their finger, then applies a small amount (random unknown volume) of blood (e.g., less than one drop (<1 μL)) directly from the finger to the CROF card, touching the card, closing card, inserts the card into the optical adapter and finally takes a photo of the card using a smartphone. From the received pictures, the software performs analysis and gives the result of the number of blood cells and other parameters. Total time from applying blood to the CROF card to displaying the results of the blood test on the smartphone ranges from ~12 to 19 seconds, where 1-2 s - to apply blood to the CROF-Card, 3-5 s - to close the card, ~2-4 s - to insert the card into the adapter, ~3- 5 s for shooting and 3 s for completing the analysis to show the results of the blood cell count.

One of the key innovations of BCI is the CROF-Card technology developed by the authors of the present invention [link]. The CROF card consists of two pieces of thin plastic, each approximately 1" x 1" square, paper-thin, hinged to another piece at one end (Fig. 30) (note that the hinge is not necessary but is a convenience) . The CROF card offers the following key features when processing a blood sample: (i) rapid distribution (e.g. 1 sec) of the blood sample from an applied form (e.g. puddles of 2 mm diameter and 0.4 mm height) onto a uniform film of 2 µm thickness (~1 /200 of the original thickness) over a large area (~ -500 mm2) and limited to two plates for CROF; (ii) stopping further reduction in sample thickness after reaching a thickness of 2 µm; (iii) maintaining a uniform thickness of 2 µm even if the hands are released from compression (ie self-retention, which is caused by capillary forces between the blood and the plates); and (iv) preventing evaporation of the sample at such a thin thickness (i.e., when held by two plates, evaporation occurs only from the edge of the blood film, and the sample testing area exhibits zero evaporation over a very long period of time). Experiments using optical interference (i.e. the Fabry-Perot resonator effect of the two internal surfaces of the CROF card) found that the Essenlix CROF card can maintain a uniform thickness of 2 µm with a uniformity of 5% (i.e. 100 nm) at least over an area of 20 mm by 20 mm.

The CROF card offers several key and unprecedented advantages for blood cell counting over existing methods. The most significant observation of the present inventors is that when a drop of blood is transformed into a uniform layer of blood that is only one red blood cell (~2 µm) thick and is confined between two plates, (i) then the blood cells in fresh undiluted whole blood without any anticoagulant are well separated from each other, have zero coagulation, are characterized by much lower blood cell mobility and are easily identified by visualization; and (ii) the blood sample has almost zero evaporation in the testing area, hence keeping the blood cell concentration in that area constant over a long period of time.

The second key advantage of the CROF card is the "automatic" measurement of the volume of the blood sample (as the thickness of the sample is determined). The third advantage is that the smallest amount of blood sample is used (since there is no fluid inlet or outlet, or any channels and/or devices to transfer the sample). Other advantages are that: (i) the dry reagent on the surface of the CROF card can be mixed with the sample within seconds; (ii) the card is simple and fast and manually operated, and (iii) it is convenient and inexpensive.

Although the method of analyzing blood by visualizing a blood sample enclosed between two plates has a history of more than 150 years and is the basis of the commercially available hand-held hemocytometer, no one has been reported to count blood cells using a plate-confined layer of blood of uniform thickness, which is equal to the thickness of just one red blood cell, and no one has examined the behavior of blood cells in a homogeneous, confined sample of blood that has a thickness exactly or approximately equal to one red blood cell. In previous imaging-based approaches, because the confinement space of the blood sample is greater than the thickness of the red blood cells, the blood sample must be diluted (often an anticoagulant is used) to avoid overlapping (hence erroneous counting) of the red blood cells. Our study observed an unusual pattern of blood cell behavior in a whole blood sample that was sandwiched between two plates and characterized by a uniform sample thickness of only one red blood cell or slightly more or less than that thickness. The behavior of blood cells differs dramatically depending on the CROF-Card's boundary span (i.e., sample thickness).

Now consider whole blood, which is undiluted, freshly drawn from a finger prick onto a CROF card without the addition of any anticoagulant (Fig. 31.a). For a confinement gap within 2 µm, the optical microscopy image shows that all blood cells (RBC, WBC, PLT) are separated from each other in the sample plane (i.e. do not overlap) and that each RBC has a clearly defined a defined border surrounding each cell with a shaded center, and each border does not intersect the border of another RBC. In addition, virtually no cell movement was observed during imaging. One explanation for this behavior is that since the 2 μm confining space is marginally smaller than the average thickness of a red blood cell, each RBC is slightly pinched by the confining plates, leaving no room for other cells to overlap and preventing movement.

Clearly, the cell behavior at 2 μm spacing provides the optimal condition for cell counting by imaging.

However, at 2.2 μm spacing, some RBCs begin to overlap with other RBCs, but there is no observable platelet overlap. A possible reason is that there is insufficient space for platelets to overlap with the PLT. For gaps of 2.6 μm and 3 μm, more RBC overlaps were observed, with triple RBC overlaps becoming visually distinguishable and platelets overlapping with RBCs. These overlaps increase with increasing spacing. Blood cell counting by imaging is possible at 2.2, 2.6, and 3 µm spacing, but accuracy deteriorates as the spacing increases. At 5 μm and 10 μm spacing, a significant number of cells overlap (eg, coagulate), pillars of RBCs are visible, and many RBCs have a narrow elliptical shape, which is caused by rotation of the RBC relative to the imaging plane (the larger spacing allows rotation to occur). It is clear that it is extremely difficult, if not impossible, to accurately count the blood cells in these gaps.

Now consider stored undiluted anticoagulant whole blood (collected from subjects by a commercial service (Bioreclamation Inc.)). The study of the present inventors showed that (Fig. 31.b) it has a different response to the limiting gap of the CROF card compared to fresh undiluted blood without anticoagulant. For a gap of 2 µm, the behavior of blood cells in stored blood is the same as for fresh blood without an anticoagulant. But for larger gaps, stored blood with anticoagulant shows a different response in 2D imaging compared to fresh blood without anticoagulant. When using an anticoagulant and with a limiting gap greater than 2 µm, although the RBCs do not coagulate together, they may (a) overlap each other and (b) rotate into a narrow ellipsoidal shape in 2D top view imaging, all of which significantly degrade counting accuracy cells.

In the BCI smartphone blood cell counting presented in this paper, the cutoff gap for the CROF card (hence the sample thickness) was set to 2 μm with an accuracy of better than 5%. The sample volume was determined from the sample thickness specified by the CROF-Card and images of the relevant area obtained using a smartphone. Blood cell concentrations (RBC, WBC, PLT) were determined by counting cells in a suitable area from a smartphone image and then dividing by the appropriate volume. The mean cellular volume (MCV) of RBCs was determined by measuring the area of each RBC in the 2D top view image and the mean total volume associated with each RBC using a preset sample thickness of 2 μm. Hematocrit was determined by the product of MCV and RBC concentration.

To count three different types of WBC (granudocytes, lymphocytes, monocytes), a blood sample was stained by placing a dried layer of acridine orange (AO) dye on one of the surfaces of the CROF card. Because AO stains nucleic acids and stains DNA and RNA differently, only WBCs and PLTs are stained, and they stain differently depending on the amount and ratio of DNA to RNA in each cell, whereas RBCs are not stained. The difference in staining produces different fluorescence wavelengths (eg, green emission at 525 nm for stained DNA and red emission at 685 nm for stained RNA) and intensities, allowing each of the three different WBCs and PLTs to be identified. It was found that when using the CROF-Card, WBCs were colored with a pre-applied layer of AO dye in less than 5 seconds due to the small thickness of the sample and thus the short dye diffusion time. WBC dye staining and fluorescence represents, in addition to bright-field microscopy, another way to measure WBC and was used in the validation below.

The optical adapter provides an effective field of view of 0.84 mm x 0.63 mm for RBC, 2.8 mm x 2.1 mm for WBC and a radius of 0.2 mm for PLT. Currently, the optical adapter must move in the slider to receive RBC and WBC separately, adding ~5 sec of additional run time. The next generation will develop a combined optical adapter without using a slider. All of the software for image analysis, user interface, and control of the iPhone was built by writing custom codes and using some open source code. All blood cell analysis presented herein was performed using proprietary software in less than 2 seconds from image analysis to blood cell counting, with the exception of PLT analysis, which would be less than 5 seconds in the next generation adapter.

To validate the BCI with a smartphone, it was compared with the following four different reference methods (RM). The RM-1 used a high-resolution microscope (Nikon Diaphot Inverted Microscope) and a DSLR camera (Nikon D5100) instead of an iPhone and an optical adapter to read a CROF card for the same reading area as the current BCI iPhone. The RM-2 is the same as the RM-1, except that the read area on the CROF card is expanded to a 3 x 3 array with 8mm spacing (9 read areas in total), evenly distributed over the area of the CROF-Card 16 mm by 16 mm. RM-3 used a commercially available hand-held hemocytometer (purchased from Sigma-Aldrich, Z3 59629) and imaged with the same microscope and camera as RM 1 and 2, but with an imaging area of 3 mm by 3 mm. The handheld hemocytometer has two chambers, each 3 mm by 3 mm, in the measurement area and a gap of 100 µm). This requires diluting the blood 100-fold and lysing the RBCs to measure PLT. The RM-4 used a commercially available PoC blood cell counting instrument (manufactured by one of the largest blood testing instrument companies); it involves a flow cytometer measuring ~1 cubic foot, weighing ~20 pounds, and costing ~$20,000. The PoC device requires at least 10 mL of blood (more than 10 drops), blood dilution, three liquid reagents (for lysis, dilution, and purification), a run time of 5 minutes, and 30 minutes daily for calibration. The comparison allows us to evaluate each individual function as well as the combined effects of the CROF-Card, optical adapter and smartphone imaging, and microscope imaging regarding their effectiveness in blood cell counting.

Two types of blood were used in the validation: (i) stored blood purchased from a commercial supplier (Boreclamation.inc) that was mixed with an anticoagulant (EDTA); and (ii) fresh blood, which was blood collected from the finger prick of two volunteers (during each test, fresh finger prick blood was immediately and directly applied from the finger onto (a) the CROF test card and into (b) the tube, coated with EDTA for analysis using a commercial PoC instrument and a hand-held hemocytometer. A total of 42 samples were tested for each method over several days.

A total of 24 samples were tested on 4 different days (3, 3, 3 and 15 samples), and the blood samples were from the same test batch on the first three days, but from a different batch on the last day. In fresh blood samples, a total of 18 samples were tested on 3 different days (6, 6 and 6 samples).

The test results revealed a number of important facts. (1) For a given blood sample, the average daily blood cell count for the smartphone BCI (r-BCI) and all four reference methods are consistent with each other within their prospective daily CV (coefficient of variation, standard deviation to mean ratio).

(2) Comparison of p-BCI with RM-1 showed that for a given CROF sample, blood cell counting using an iPhone and the optical adapter that was developed had the same accuracy (CV) as using a high-resolution microscope and a DSLR camera (e.g. both have CV ~12% for RBC) (Fig. 32).

(3) A comparison of RM-1 and RM-2 showed that visualization of multiple map fields for CROF provided better accuracy than the current visualization of a single field. CV for RBC improved from ~12% to ~6%. The ability to view multiple fields will be implemented in our new generation BCI with smartphone for higher accuracy.

(4) A comparison of RM-2 and RM-3 showed that (i) the CROF card is not only much easier to use, but also provides cell counting accuracy that is equal to or better than a commercially available handheld hemocytometer for cell counting blood, and, (ii) given the fact in (i) and the comparison of RM1 and RM2, this leads to the conclusion that the smartphone multi-pole BCI should have the same or even better accuracy than a commercially available hand-held hemocytometer in blood cell counting. We would also like to point out that the source of variability, although the same for the real CROF-Card and the handheld hemocytometer, comes from different sources. For the hemocytometer, variability is provided by dilution, lysis, and manual counting. But for the CROF map, the existing variability (~7% for RBC) is mainly due to the sample thickness variability (~5%), which can be further improved.

(5) In smartphone BCI, each of the three different types of WBC can be identified by staining and measuring the intensity value for each WBC cell based on the ratio of green to red fluorescence intensity. The standard deviation is similar to the standard deviation for other blood cell measurements. This is because each subtype of leukocyte has a specific ratio of fluorescence colors (depending on the relative amount and ratio of RNA (red fluorescence) and DNA (green fluorescence); the granulocyte is characterized by a large amount of RNA and granules (thus high red fluorescence and weak green fluorescence), a lymphocyte is characterized by a low amount of RNA and a high amount of DNA (thus weak red but strong green fluorescence), and a monocyte is characterized by a red to green ratio between granulocyte and lymphocyte.

(6) Within the limits of statistical significance, the interday (i.e., daily) variability for all five methods tested is essentially the same, indicating that smart-BCI is very stable over the multi-day period over which the tests were run.

Finally, (7) using our existing optical imaging equipment and software, blood cell counts by imaging are not yet as accurate as a commercially available PoC sample flow cytometer instrument (e.g., ~7% vs. 1% for RBC).

However, two important facts must be recognized: (i) with the current accuracy, the r-BCI already demonstrated herein has significant implications for health monitoring and clinical value in a remote area or in developing countries, and (ii) the accuracy of the r-BCI can be further improved with obtaining better accuracy. Undoubtedly, BCI technology has broad and significant applications in cell imaging, immunoassays, nucleic acid assays, personal health monitoring and other biochemical diagnostics.

Certain aspects of the present invention have been described in the following documents, all of which are incorporated by reference for all purposes:

US Patent Application Serial No. 13/838600, filed March 15, 2013 (NSNR-003), which claims priority to U.S. Provisional Patent Application Serial No. 61/622226, filed April 10, 2012, and is a continuation portion of the patent application US Serial No. 13/699270, filed June 13, 2013, which is § 371, filed US 2011/037455, filed May 20, 2011, and claims priority to the US Provisional Patent Application Serial No. 61/347178, filed May 21 2010;

US Patent Application Serial No. 13/699270, filed June 13, 2013 (NSNR-001), which is § 371, filed in International Application Serial No. US 2011/037455, filed May 20, 2011, which claims provisional application priority US Patent Serial No. 61/347178, filed May 21, 2010; And

US Provisional Application Serial No. 61/801424, filed 15/3/2013 (NSNR-004PRV), Provisional Application Serial No. 61/801096, filed 3/15/2013 (NSNR-005PRV), Provisional Application Serial No. 61/800915, filed 3/15/2013 (NSNR-006PRV), provisional application serial No. 61/793092, filed 3/15/2013 (NSNR-008PRV), provisional application serial No. 61/801933, filed 3/15 /2013 (NSNR-009PRV), provisional application with serial No. 61/794317, filed on 3/15/2013 (NSNR-010PRV), provisional application with serial No. 61/802020, filed on 3/15/2013 (NSNR-011PRV) and provisional application with serial number 61/802223, filed on 3/15/2013 (NSNR-012PRV).

Other examples of subject matter according to the present invention are described in the following paragraphs.

As used herein, the expressions “adapted” and “capable” mean that an item, component, or other object is designed and/or intended to perform a specific function. Thus, the use of the expressions "adapted" and "capable" should not be construed to mean that a particular element, component or other object is merely "capable" of performing a certain function, but also that the element, component and/or other object specifically selected, created, implemented, used, programmed and/or intended to perform a given function. Moreover, within the scope of the present disclosure, elements, components, and/or other enumerated items that are listed as being adapted to perform a particular function may additionally or alternatively be described as being configured to perform that function, and vice versa. Likewise, an object that is listed as being capable of performing a particular function may be additionally or alternatively described as performing that function.

As used herein, the phrase “for example,” the phrase “as an example,” and/or simply the terms “example” and “illustrative” when used with reference to one or more components, features, details, structures, embodiments, and/or methods in in accordance with the present invention are intended to convey that the described component, feature, part, structure, embodiment and/or method is an illustrative, non-exclusive example of components, features, parts, structures, embodiments and/or methods in accordance with the present invention. Thus, the described component, feature, part, structure, embodiment and/or method is not intended to be limiting, necessary or exclusive/exhaustive; and wherein other components, features, parts, structures, embodiments and/or methods, including structurally and/or functionally similar and/or equivalent components, features, parts, structures, embodiments and/or methods, are also within the scope of this inventions.

As used herein, the phrases "at least one of" and "one or more of" with respect to a list of more than one object mean any one or more objects in the list of objects and are not limited to at least one of each object and each object specifically indicated in the list of objects. For example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and/or B") may refer to only to A, only to B, or to a combination of A and B.

As used herein, the combination of “and/or” placed between a first entity and a second entity means one of (1) the first entity, (2) the second entity, and (3) the first entity and the second entity. Several items listed using "and/or" are to be interpreted in the same way, i.e. "one or more" objects combined in this way. Another object may not necessarily be present other than the object specifically indicated in the clause using "and/or", whether or not they are related to the specifically specified object. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with a non-limiting expression such as “comprising”, may refer in some embodiments only to A (not necessarily including an entity other than B); in certain embodiments, only to B (optionally including an entity other than A); in other certain embodiments, for both A and B (optionally including the other entity). These objects can refer to elements, actions, structures, stages, workflows, values and the like

To the extent that any patents, patent applications, or other references are incorporated herein by reference and (1) define a term in a manner that is inconsistent with and/or (2) otherwise conflict with or omit a portion of this disclosure or any other included references, then the non-included portion of the present invention should be controlled, and the term or included disclosure therein should be governed only with respect to the reference in which the term is defined and/or the included disclosure was originally present.

It is believed that the following claims specifically identify certain combinations and subcombinations that are directed to one of the disclosed inventions and are novel and not obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements and/or properties may be claimed by amending the claims of the present invention or by introducing new claims in this or a related application. Such amended or new claims, whether directed to a different invention or directed to the same invention, whether different, broader, narrower or equal in scope to the original claims, also are considered to be included within the scope of the disclosure of the present inventions.

Claims (212)

1. A device for analyzing an analyte in a sample, containing: the first plate and the second plate, and: i. the plates are movable relative to each other in different configurations; ii. one or both plates are flexible; iii. each of the plates has on its respective surface a sample contact region for contacting the sample containing the analyte; iv. one or both plates include spacers that are secured to a corresponding plate, wherein the spacers have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant distance between the spacers, wherein at least one of the spacers is located within the contact area with sample; v. the fourth power of the interspacer distance (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate, ISD ⁴ /(hE), equal to or less than 5x10 ⁶ µm ³ /GPa; And vi. the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is from 60 to 750 GPa-μm, wherein one of the configurations is an open configuration in which the two plates are separated from each other, the space between the plates is not controlled by spacers, and the sample is applied to one or both plates; the other of the configurations is a closed configuration, which is configured after applying the sample in an open configuration; and in a closed configuration, at least a portion of the sample is compressed by two plates into a layer of substantially uniform thickness and is substantially stationary with respect to the plates, wherein said substantially uniform layer thickness is limited by the inner surfaces of the two plates and is controlled by the plates and spacers, and has an average thickness equal to or less than 200 microns. 2. The device according to claim 1, which further comprises a detector that detects the analyte. 3. The device of claim 1, further comprising on one or both plates a dry binding site having a predetermined area, wherein the dry binding site binds and immobilizes the analyte in the sample. 4. The apparatus of claim 1, further comprising on one or both plates a release dry reagent and a release time control material that delays the time at which the release dry reagent is released into the sample. 5. The apparatus of claim 4, wherein the release time control material delays the time at which the dry reagent begins to be released into the sample by at least 3 seconds. 6. The device of claim 4, which further comprises a dry reagent applied to one or both plates and wherein the reagent comprises a coloring reagent(s). 7. The device according to claim 4, which further has on one or both plates one or more dry binding sites and/or one or more reagent sites. 8. The device according to claim 1, which additionally contains a dry reagent applied to one or both plates. 9. The device according to claim 1, in which the ratio of the distance between the spacers to the average width of the spacers is equal to or more than 2, and the filling factor of the spacers, multiplied by the Young's modulus of the spacers, is equal to or more than 2 MPa. 10. The device according to claim 1, in which the analyte is colored. 11. The device according to claim 1, in which for the separators that regulate a layer of uniform thickness, the product of the Young's modulus of the separators and the filling factor of the separators is equal to or greater than 10 MPa, and the filling factor is the ratio of the contact area of the separators to the total area of the plate. 12. The device according to claim 1, wherein the average thickness of said layer of uniform thickness is from 1.8 to 2.6 microns and the sample is whole blood without dilution with another liquid. 13. The device according to claim 1, which further contains a dry reagent applied to one or both plates and in which the reagent contains an anticoagulant. 14. The apparatus of claim 1, wherein for the flexible plate, the fourth power of the interspacer distance (ISD) divided by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate, ISD ⁴ /(hE), is equal to or less than 10 ⁵ µm ³ /GPa. 15. The apparatus of claim 1, wherein one or both of the wafers includes a location mark either on the surface or within the wafer providing location information on the wafer. 16. The device of claim 1, wherein one or both of the plates contains a scale mark either on the surface or inside the plate providing information about the size of the sample structure and/or plate in horizontal projection. 17. The device of claim 1, wherein one or both of the wafers includes an imaging mark either on the surface or within the wafer to facilitate visualization of the sample. 18. The apparatus of claim 1, wherein the separators function as a location mark, a scale mark, a rendering mark, or any combination thereof. 19. The device according to claim 1, in which the average thickness of the layer of uniform thickness is in the range from 2 to 5 μm. 20. The device according to claim 1, in which the average thickness of the layer of uniform thickness is in the range from 5 to 10 μm. 21. The device according to claim 1, in which the average thickness of the layer of uniform thickness is in the range from 10 to 40 μm. 22. The device according to claim 1, in which the average thickness of the layer of uniform thickness is 30 μm. 23. The device of claim 1, wherein the average thickness of the uniform thickness layer is 100 µm or less. 24. The device according to claim 1, in which the average thickness of the layer of uniform thickness is equal to the minimum size of the analyte in the sample. 25. The device according to claim 1, in which the distance between the separators is in the range from 7 to 50 μm. 26. The device according to claim 1, in which the distance between the separators is in the range from 50 to 120 μm. 27. The device according to claim 1, in which the distance between the separators is in the range from 120 to 200 μm. 28. The device of claim 1, wherein the distance between adjacent spacers is substantially the same. 29. The device of claim 1, wherein the separators are columns with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination thereof. 30. The device of claim 1, wherein the separators are column-shaped and have a substantially flat top surface, wherein for each separator the ratio of the horizontal dimension of the separator to its height is at least 1. 31. The device according to claim 1, in which for each separator the ratio of the horizontal size of the separator to its height is at least 1. 32. The device of claim 1, wherein the minimum horizontal size of the separator is less than or substantially equal to the minimum size of the analyte in the sample. 33. The device according to claim 1, in which the minimum horizontal size of the separator is in the range from 0.5 to 100 microns. 34. The device according to claim 1, in which the minimum horizontal size of the separator is in the range from 0.5 to 10 microns. 35. The device of claim 1, wherein the separators are shaped like a column with a substantially flat top surface, a specified substantially uniform height, and a predetermined constant distance between the separators that is at least 2 times the size of the analyte, wherein the product of the Young's modulus of the separators and the filling factor of the spacers is equal to or greater than 2 MPa, wherein the filling factor is the ratio of the contact area of the spacers to the total area of the plate, and for each spacer the ratio of the horizontal dimension of the spacer to its height is at least 1, and the fourth power of the distance between the spacers (ISD ), divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD ⁴ /(hE)) is 5x10 ⁵ µm ³ /GPa or less. 36. The device according to claim 1, in which the sample is stained, and Romanovsky stain, Leishman stain, May-Grunwald stain, Giemsa stain, Jenner stain, Wright stain, or any combination thereof are used for staining. 37. The device of claim 1, wherein the sample is stained, the stain being an immunohistochemical stain. 38. The device of claim 1, wherein the sample is a biological sample, an environmental sample, a chemical sample, or a clinical sample. 39. The device according to claim 1, in which the separators have the shape of columns, and the corners of the side walls of the separators have a rounded shape with a radius of curvature of at least 1 μm. 40. The device according to claim 1, in which the separators have a density of at least 100/mm ² . 41. The device according to claim 1, in which the separators have a density of at least 1000/mm ² . 42. The device according to claim 1, in which at least one of the plates is transparent. 43. The device according to claim 1, in which at least one of the plates is made of a flexible polymer. 44. The device according to claim 1, wherein with respect to the pressure that compresses the plates, the separators are incompressible and/or, independently, only one of the plates is flexible. 45. The device according to claim 1, in which the flexible plate has a thickness in the range from 10 to 200 microns. 46. The device of claim 1, wherein said substantially uniform layer thickness has a variation of less than 30%. 47. The device of claim 1, wherein said substantially uniform layer thickness has a variation of less than 10%. 48. The device of claim 1, wherein said substantially uniform layer thickness has a variation of less than 5%. 49. The device of claim 1, wherein the first and second plates are connected and are configured to change from an open configuration to a closed configuration by folding the plates. 50. The device of claim 1, wherein the first and second plates are connected by a cylindrical hinge and are configured to transition from an open configuration to a closed configuration by folding the plates as they rotate around the axis of the cylindrical hinge. 51. The device of claim 1, wherein the first and second plates are connected by a cylindrical hinge, which is made of a material different from the plates, and are configured to transition from an open configuration to a closed configuration by folding the plates as they rotate around the axis of the cylindrical hinge. 52. The apparatus of claim 1, wherein the first and second plates are made from a single piece of material and are configured to transition from an open configuration to a closed configuration by folding the plates. 53. The device of claim 1, wherein said sample layer of uniform thickness is uniform over a horizontal area that is at least 1 mm ² . 54. The device of claim 1, which is configured to analyze a sample in 60 seconds or less. 55. The device of claim 1, wherein in a closed configuration, the finite sample thickness device is configured to perform analysis of the sample in 60 seconds or less. 56. The device of claim 1, wherein, in a closed configuration, the device with a finite sample thickness is configured to perform analysis of the sample in 10 seconds or less. 57. The device of claim 3, wherein the dry binding site comprises a gripping means. 58. The device of claim 3, wherein the dry binding site comprises an antibody or nucleic acid. 59. The device of claim 4, wherein the released dry reagent is a labeled reagent. 60. The device of claim 4, wherein the released dry reagent is a fluorescently labeled reagent. 61. The device of claim 4, wherein the released dry reagent is a fluorescently labeled antibody. 62. The device of claim 4, wherein the released dry reagent is a cell dye. 63. The device of claim 2, wherein the detector is an optical detector that detects an optical signal. 64. The device according to claim 1, in which the separators have the shape of columns, and the ratio of the width to the height of the columns is greater than or equal to one. 65. The apparatus of claim 2, wherein the detector is an electrical detector that detects an electrical signal. 66. The device of claim 1, wherein the spacers are fixed to the plate by directly embossing the plate or injection molding the plate. 67. The device according to claim 1, in which the materials of the plate and spacers are selected from polystyrene, PMMA, PC, COC, COP. 68. A system for accelerated sample analysis using a mobile communication device, containing: (a) a device according to any of the preceding paragraphs; (b) a mobile communication device containing: i. one or more cameras for detecting and/or imaging a sample; ii. electronics, signal processors, hardware and software for receiving and/or processing the detected signal and/or image of the sample and for remote communication; And (c) a light source in a mobile communication device or an external light source; wherein the detector in the device according to any one of the preceding claims is provided with a mobile communication device and detects the analyte in the sample in a closed configuration. 69. The system of claim 68, wherein one of the plates has a binding site for binding the analyte, at least a portion of the sample layer of uniform thickness is located on the binding site and is substantially less than the average linear horizontal dimension of the binding site. 70. The system according to claim 68, additionally containing: a housing configured to hold a sample and for mounting on a mobile communication device. 71. The system of claim 70, wherein the housing contains optical means that facilitate visualization and/or processing of the signal from the sample by the mobile communication device, and a mount configured to hold the optical means on the mobile communication device. 72. The system according to claim 71, in which the optical element in the housing is movable relative to the housing. 73. The system of claim 68, wherein the mobile communication device is configured to transmit test results to a medical specialist, medical facility, or insurance company. 74. The system of claim 68, wherein the mobile communication device is further configured to transmit test and subject information to a medical professional, medical facility, or insurance company. 75. The system according to claim 68, in which the mobile communication device is additionally configured to transmit information about the test to the cloud network, and the cloud network processes the information to clarify the test results. 76. The system according to claim 68, in which the mobile communication device is additionally configured to transmit information about the test and the subject to the cloud network, wherein the cloud network processes information to clarify the test results, and the updated test results are sent back to the subject. 77. The system of claim 68, wherein the mobile communication device is configured to receive an appointment, diagnosis, or recommendation from a medical professional. 78. The system according to claim 68, in which the mobile communication device is configured to use hardware and software for: (a) taking an image of the sample; (b) analyzing the test location and the reference location in the image; And (c) comparing the value obtained from the analysis of the test location with a threshold value that characterizes the accelerated diagnostic test. 79. The system of claim 68, wherein at least one of the plates has a storage area in which assay reagents are stored. 80. The system of claim 68, wherein at least one of the cameras reads a signal from a compressed controlled open flow (CROF) device. 81. The system of claim 68, wherein the mobile communication device communicates with a remote location via Wi-Fi or a mobile cellular network. 82. A method for accelerated analysis of an analyte in a sample, including: (a) obtaining a sample; (b) providing first and second plates movable relative to each other into various configurations, each plate having a substantially flat contact surface with the sample, one or both plates being flexible, and one or both plates including spacers firmly coupled with the corresponding surface of contact with the sample, while the separators have: i. a predetermined substantially uniform height; ii. a column shape with a substantially flat top surface; iii. a specified constant interval between separators, which is at least 2 times the size of the analyte and is up to 200 μm; iv. the Young's modulus of the separators multiplied by the filling factor of the separators is equal to or greater than 2 MPa; And (c) applying the sample to one or both plates when the plates are in an open configuration, wherein the open configuration is a configuration in which both plates are partially or completely separated from each other and the distance between the plates is not controlled by spacers; (d) following step (c), bringing the two plates together into a closed configuration in which at least a portion of the sample is converted into a layer of substantially uniform thickness defined by the contact surfaces of the plates with the sample, said uniform layer thickness being controlled by spacers and plates and having average value in the range from 1.8 to 3 µm; And (e) analyzing the analyte in said layer of uniform thickness while the plates are in a closed configuration; wherein the fill factor is the ratio of the contact area of the spacers to the total area of the plate, wherein the fourth power of the interspacer distance (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate, ISD ⁴ /(hE), is equal to or less than 5x10 ⁵ μm ³ /GPa; and the thickness of the flexible plate multiplied by the Young's modulus of the flexible plate is from 60 to 750 GPa-μm. 83. A method for accelerated analysis of an analyte in a sample using a mobile communication device, including: (a) applying a sample to a device in the system according to any of the preceding paragraphs relating to the system; (b) analyzing the analyte in the sample applied to the device to obtain a result; And (c) transmitting the result from the mobile communication device to a location remote from the mobile communication device. 84. The method according to claim 82 or 83, in which the analyte contains a molecule, cells, tissues, viruses and nanoparticles of various shapes. 85. The method according to claim 82 or 83, in which the analyte contains a leukocyte, an erythrocyte and platelets. 86. The method of claim 83, wherein performing the analysis includes performing a differential analysis of leukocytes. 87. The method according to claim 82 or 83, including: analysis of results at a remote location to obtain an analyzed result; And transferring the analyzed result from a remote location to a mobile communication device. 88. The method of claim 82 or 83, wherein the medical professional performs the analysis at a remote location. 89. The method of claim 83, wherein the mobile communication device receives a prescription, diagnosis, or recommendation from a medical professional located at a remote location. 90. The method according to claim 82 or 83, in which the sample is a physiological fluid or exhaled air. 91. The method of claim 90, wherein the physiological fluid is blood, saliva or urine. 92. The method of claim 82 or 83, wherein the sample is whole blood without dilution with another liquid. 93. The method of claim 83, wherein the step of performing the analysis includes identifying an analyte in the sample. 94. The method of claim 93, wherein the analyte is a biomarker. 95. The method of claim 93, wherein the analyte is a protein, nucleic acid, cell, or metabolite. 96. The method according to claim 92, including counting the number of red blood cells. 97. The method according to claim 92, including counting the number of leukocytes. 98. The method according to claim 92, including staining the cells in the sample and counting the number of neutrophils, lymphocytes, monocytes, eosinophils and basophils. 99. The method of claim 83, wherein the assay performed in step (b) is a binding assay or a biochemical assay. 100. A method of analyzing a sample, including: obtaining a device under any of the preceding paragraphs relating to a device or system; applying the sample to one or both plates of the device; converting the plates into a closed configuration and applying an external force to at least a portion of the plates; And Carrying out analysis in a layer of uniform thickness when the plates are in a closed configuration. 101. The method according to claim 100, including: (a) obtaining a sample; (b) providing first and second plates movable relative to each other in various configurations, each plate having a substantially flat surface contacting the sample, one or both plates being flexible, wherein one or both plates include spacers that are firmly connected to the corresponding surface of contact with the sample, while the separators have: i. given essentially the same height, ii. the shape of columns with a flat top surface; iii. ratio of width to height equal to or greater than one; iv. a given constant distance between the separators, which ranges from 10 to 200 μm; v. the product of Young's modulus and the fill factor of the spacers, equal to or greater than 2 MPa, with the fill factor being the ratio of the contact area of the spacers to the total area of the plate; And (c) applying the sample to one or both plates when the plates are in an open configuration, wherein the open configuration is a configuration in which both plates are partially or completely separated from each other and the space between the plates is not controlled by spacers; (d) after step (c) compressing the sample by bringing the two plates together into a closed configuration in which at least a portion of the sample forms a layer of substantially uniform thickness defined by the contact surfaces of the plates with the sample, said uniform layer thickness being controlled by spacers and plates and has an average value in the range from 1.8 to 3 µm; And (e) analyzing the analyte in a layer of uniform thickness when the plates are in a closed configuration; in this case, the filling factor is the ratio of the contact area of the spacers to the total area of the plate. 102. The method of claim 100, wherein the sample is a blood sample and which includes removing the external force after placing the plates in a closed configuration; And visualization of blood cells in a layer of uniform thickness when the plates are in a closed configuration; And counting the number of blood cells in the image area. 103. The method according to claim 101, in which the distance between the separators is in the range from 20 to 200 μm. 104. The method according to claim 101, in which the distance between the separators is in the range from 5 to 20 μm. 105. The method of claim 101, wherein the product of the filling factor and the Young's modulus of the separators is 2 MPa or greater. 106. The method of claim 100, wherein said layer of substantially uniform thickness has a surface variation of less than 30 nm. 107. The method of claim 100, wherein the sample is undiluted whole blood to which no anticoagulant has been added. 108. The method according to claim 100, in which the application step is carried out by: i. piercing human skin to release a drop of blood onto the skin, and ii. bringing a drop of blood into contact with one or both plates without the use of a blood transfer instrument. 109. The method of claim 100, wherein the analysis step includes counting the number of red blood cells. 110. The method of claim 100, wherein the device further comprises at least a pair of scale marks spaced at a known distance in a plane parallel to the horizontal area. 111. The method of claim 100, wherein the analysis step includes staining the cells in the sample and counting the number of neutrophils, lymphocytes, monocytes, eosinophils and basophils. 112. The method according to claim 102, in which visualization and counting of quantities is carried out by: i. illumination of cells in a layer of uniform thickness; ii. obtaining one or more images of cells using a CCD or CMOS sensor; iii. identifying cells in an image using a computer; And iv. counting the number of cells in the image area. 113. The method according to claim 100, in which the external force is created by a human hand. 114. The method of claim 100, further comprising measuring sodium, potassium, chloride, bicarbonate, blood urea, nitrogen, magnesium, creatinine, sugar, calcium, HDL and LDL cholesterol levels and/or triglyceride levels in a layer of uniform thickness. 115. The method according to claim 100, in which a dry reagent is additionally applied to one or both plates. 116. The method according to claim 100, in which the specified layer of the sample of uniform thickness is characterized by a thickness uniformity of up to +/-5%. 117. The method of claim 101, wherein the dividers are columns with a cross-sectional shape selected from round, polygonal, circular, square, rectangular, oval, elliptical, or any combination thereof. 118. The method of claim 101, wherein the distance between the separators is equal to the average thickness of red blood cells (RBC). 119. The method of claim 100, wherein the sample is a blood sample and the analysis step includes imaging cells in the blood. 120. The method of claim 119, wherein the cells are red blood cells, white blood cells or platelets. 121. The method of claim 100, wherein the sample is a blood sample and the blood test includes visualizing cancer cells, viruses or bacteria in the blood. 122. The method of claim 100, wherein the analysis of the blood sample includes detecting proteins or nucleic acids. 123. The method of claim 100, wherein the sample is a blood sample and the analysis of the blood sample includes measuring hemocytes, including determining the thickness of the sample using a separator, determining a horizontal area by imaging, and calculating the area of red blood cells using a two-dimensional image. 124. The method of claim 100, wherein the sample is a blood sample and the blood test includes measuring the concentration of red blood cells in the blood. 125. The method of claim 100, wherein the sample is a blood sample and the analysis of the blood sample includes measuring the concentration of white blood cells in the blood. 126. The method of claim 100, wherein the sample is a blood sample and the blood test includes measuring the concentration of platelets in the blood. 127. The system of claim 68, wherein the mobile communication device is a mobile telephone. 128. The method of claim 82, wherein the sample is blood, the analytes are blood cells, and the uniform layer thickness is from 1.9 to 2.2 microns. 129. The method according to claim 101, in which the compression is performed by a human hand. 130. The method of claim 101, wherein the compression is performed by a human hand and the sample thickness has a variation of 50% or less. 131. The method of claim 100, wherein at least a portion of the inner surface of one or both plates is wettable. 132. The method of claim 100, wherein the sample is applied to the plate directly from the subject without the use of a transfer device. 133. The method of claim 101, wherein after deforming the sample in a closed configuration, the sample retains the same final thickness when some or all of the compressive force is removed. 134. The method of claim 101, wherein the separators are column-shaped and have a substantially uniform cross-section. 135. The method of claim 101, wherein the separators have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant distance between the separators that is at least twice the size of the analyte, wherein the product of the Young's modulus of the separators and the fill factor of the spacers is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the contact area of the spacers to the total area of the plate, and for each spacer the ratio of the horizontal size of the spacer to its height is at least 1. 136. The method of claim 101, wherein the separators have a columnar shape, a substantially flat top surface, a predetermined substantially uniform height, and a predetermined constant distance between the separators that is at least 2 times the size of the analyte, wherein the product of the Young's modulus of the separators and the filling factor of the separators is equal to or greater than 2 MPa, wherein the filling factor is the ratio of the contact area of the separators to the total area of the plate, and for each separator the ratio of the horizontal size of the separator to its height is at least 1, and the sample is blood; the analyte is an erythrocyte, leukocyte or platelet; and the uniform layer thickness is in the range of 1.8 to 2.6 μm. 137. The method of claim 101, wherein the analysis step is the detection of proteins, peptides, nucleic acids, artificial compounds and inorganic compounds. 138. The method according to any one of paragraphs. 82, 83 and 100, in which the sample applied to one or both plates has an unknown volume. 139. The method according to any one of paragraphs. 82, 83, 100, in which the sample is intended to detect, purify and quantify a chemical compound or biomolecule that is associated with a disease. 140. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used for infectious and parasitic diseases, trauma, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, lung diseases or kidney diseases. 141. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used for detection, purification and quantification of the microorganism. 142. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that contain virus, fungi and bacteria from the environment. 143. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used for the detection and quantification of chemical compounds or biological samples that pose a risk to food or national security. 144. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used to quantify vital parameters in medical or physiological monitoring. 145. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used to analyze blood sugar, oxygen levels, total red blood cells, white blood cells, platelets. 146. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used to detect and quantify specific DNA or RNA from biological objects. 147. The method according to any one of paragraphs. 82, 83, 100, wherein said sample refers to samples that are used to sequence and compare genetic sequences in DNA in chromosomes and mitochondria for genome analysis. 148. The method according to any one of paragraphs. 82, 83, 100, in which the specified sample refers to samples that are used to detect reaction products during the synthesis or purification of pharmaceuticals. 149. The method according to any one of paragraphs. 82, 83, 100, in which the sample is cells, tissues, body fluids and feces. 150. The method according to any one of paragraphs. 82, 83, 100, in which the sample is a sample in the determination of protein, peptides, nucleic acids, artificial compounds and inorganic compounds. 151. The method according to any one of paragraphs. 82, 83, 100, wherein the sample is a sample relevant to human, veterinary, agricultural, food, environmental and drug testing. 152. The method according to any one of paragraphs. 82, 83, 100, in which the sample is a biological sample selected from blood, serum, plasma, nasal swab, nasopharyngeal wash, saliva, urine, gastric juice, cerebrospinal fluid, tears, feces, hair, mucus, sweat, earwax, fat, gland secretions, cerebrospinal fluid, tissue, sperm, vaginal discharge, interstitial fluid obtained from tumor tissues, intraocular fluid, cerebrospinal fluid, throat swab, exhaled air, dermis, biopsy, hair, fingernails, placental fluid, amniotic fluid, umbilical cord blood, lymphatic fluids, intracavitary fluids, sputum, pus, microbiota, meconite, human milk, cerebrospinal fluid, exhaled condensate, nasopharyngeal wash, nasal swab, throat swab, stool samples, hair, fingernail, earwax , exhaled air, connective tissue, muscle tissue, nerve tissue, epithelial tissue, cartilage, cancer cell sample, or bone. 153. The method according to any one of paragraphs. 82, 83, 100, in which the sample is blood. 154. The method according to any one of paragraphs. 82, 83, 100, wherein the sample is a biological sample, an environmental sample, a chemical sample, or a clinical specimen.

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