CN110891684A

CN110891684A - QMAX card-based measurement device and method

Info

Publication number: CN110891684A
Application number: CN201880023331.3A
Authority: CN
Inventors: 斯蒂芬·Y·周; 丁惟; 戚骥; 张玙璠
Original assignee: Essenlix Corp
Current assignee: Shanghai Yisheng Biotechnology Co ltd; Yewei Co ltd
Priority date: 2017-02-16
Filing date: 2018-02-13
Publication date: 2020-03-17
Anticipated expiration: 2038-02-13
Also published as: CN110891684B; CN115634721A

Abstract

More particularly, the present invention relates to devices and methods for performing biological and chemical assays, devices and methods for performing biological and chemical extractions from liquids and performing assays such as, but not limited to, immunoassays and nucleic acid assays.

Description

QMAX card-based measurement device and method

Cross-referencing

The benefit of U.S. provisional patent application No. 62/456,488 filed on 8.2.2017, U.S. provisional patent application No. 62/456,612 filed on 8.2.2017, U.S. provisional patent application No. 62/456,504 filed on 8.2.2017, U.S. provisional patent application No. 62/456,988 filed on 9.2.2017, U.S. provisional patent application No. 62/457,133 filed on 9.2.2017, U.S. provisional patent application No. 62/457,103 filed on 9.2.2017, and U.S. provisional application No. 62/460,062 filed on 16.2.16.2017 are claimed and are incorporated herein by reference in their entirety for all purposes.

Technical Field

Background

In many biological/chemical test processes (e.g., immunoassays, nucleotide determinations, blood cell counts, etc.), chemical reactions, and other processes, there is a need for methods, kits, and systems that can accelerate the process (e.g., binding, mixing reagents, etc.), quantify parameters (e.g., analyte concentration, sample volume, etc.), and do so in the case of small sample volumes.

On the other hand, it is desirable to separate components from a complex liquid sample, such as plasma separation. In general, centrifugation is the most common technique for separating components from a composite liquid sample based on the difference in centrifugal force. This method is laborious, requires complex equipment and specialized handling. It is particularly unsuitable for small volume samples, but becomes increasingly desirable in point-of-care settings and personal health management in the context of rapidly developing and commercializing miniaturized testing equipment. Other prior art in this field involves the use of microfluidic channels, eliminating the need for large volumes of sample. However, the fabrication of microfluidic channels is technically challenging and cost-effective little. Some other techniques utilize various filter media, which are composed primarily of porous materials (e.g., filter paper) or fiberglass, in conjunction with a housing and a support device. This method is generally cost effective and easy to handle, but generally requires the discharge or transfer of the filtered product for further analysis or processing.

Disclosure of Invention

The following summary is not intended to include all features and aspects of the present invention.

The present invention relates to methods, devices and systems that enable biological/chemical sensing (including but not limited to immunoassays, nucleic acid assays, electrolyte analysis, etc.) to be much faster, more sensitive, with much fewer steps and ease of performance, with much less sample size required, with much more ease of use, with much less or no professional assistance, and/or at much lower cost than the many current sensing currently in use.

In particular, the invention relates to a device and a method for the determination of QMAX ("QMAX" (q.: quantification; m. amplification, a. addition of reagents, X: acceleration), also known as "CROF" (compression regulated open flow) -based) cards. More particularly, the present invention relates to a compressed open flow assay method, device, kit and system for performing squeeze washing, dilution calibration, component separation and multi-plate sample analysis.

Improved measurement-accurate measurement of sample volume

One aspect of the present invention is a method and apparatus for converting at least a portion of a droplet of a liquid sample deposited on a plate into a thin film having a precisely controlled, predetermined and uniform thickness over a large area. The uniform thickness may be less than 1 μm. Furthermore, the present invention allows the same uniform thickness to be maintained for a long period of time without evaporation in the open surface.

Another aspect of the present invention is a method and apparatus for determining the precise volume of a portion or all of a sample using the uniformly thin sample thickness formed by the present invention without the use of any pipette or the like.

Efficient method for improved assay-reduction of non-specific binding

Another aspect of the invention is a method and apparatus for squeeze/sponge washing with a QMAX apparatus.

Easy calibration of improved assay-dilution factor

Another aspect of the invention is a method for conveniently calibrating the dilution factor of any sample (e.g., blood or plasma) using QMAX cards.

Component separation using QMAX device

Yet another aspect of the present invention is a method and apparatus for separating certain components from a composite liquid sample using QMAX cards and obtaining a liquid sample without the components therein and/or extracting the components from the sample.

Drawings

Those skilled in the art will appreciate that the drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present invention in any way. The figures are not drawn to scale. In the figures giving experimental data points, the lines connecting the data points are used only to guide the observed data, and have no other purpose.

Fig. 1 is a schematic diagram of an example of an assay method according to the present disclosure.

Fig. 2 is a schematic view of an assay plate according to the present disclosure.

Fig. 3 is a schematic view of a second plate according to the present invention.

Fig. 4 is a schematic view of a wash pad according to the present disclosure.

FIG. 5 is a schematic view of a sample and assay plate.

Fig. 6 is a schematic (exploded) view of the assay assembly.

FIG. 7 is a schematic view of a squeezed assay assembly.

FIG. 8 is a schematic view of a wash pad for use with an assay plate.

Fig. 9 is a graph comparing the results of measurements performed by various techniques. "No wash" is a determination without a wash step. "sponge wash" is the same assay performed with the squeeze wash according to the present disclosure. "Normal wash" is the same assay performed with conventional washing steps. The measurements and washing parameters are given in table 1.

Fig. 10 is a schematic of a kit and kit components according to the present disclosure.

FIG. 11 is a schematic side view of a wash pad.

FIG. 12 is a flow chart of an exemplary embodiment of a method of determining a sample dilution factor provided by the present invention.

FIG. 13 is a flow chart of another exemplary embodiment of a method of determining a dilution factor of a sample provided by the present invention.

Fig. 14 shows an embodiment of a QMAX apparatus.

Fig. 15 is a flow chart of an exemplary embodiment of a method of determining a dilution factor of a blood sample according to the present invention.

Fig. 16 shows representative images of undiluted (a) and 10X diluted (b) samples acquired in bright field mode.

Fig. 17 schematically illustrates an exemplary embodiment of an apparatus and method for separating components from a composite liquid sample provided by the present invention.

FIG. 18 is a flow chart of an exemplary embodiment of a method disclosed in the present invention.

Figure 19 shows representative images of the filtration products produced by different experimental configurations of the device when used for plasma separation.

Fig. 20 shows the results of Triglyceride (TG) determination using the filtered product from the experimental filtration apparatus as the measurement sample and using the QMAX apparatus as the measurement apparatus.

Figure 21 shows a QMAX (Q: dosing; M: magnification, a. adding reagent, X: acceleration; also known as a compression-regulated open flow (CROF) device comprising a first plate, a second plate and a third plate figure (a) shows a perspective view of the plates in an open configuration when the plates are separated and figure (B) shows a cross-sectional view of the plates in an open configuration.

Fig. 22 shows an exemplary embodiment of a QMAX device and a flow to be used for filtering and analyzing a liquid sample with the QMAX device. Figure (a) shows the QMAX device in an open configuration, with a sample deposited on a filter placed on top of the first plate, figure (B) shows a cross-sectional view of the QMAX device when the third plate is pressed on top of the filter, pushing a portion of the sample through the filter, figure (C) shows a cross-sectional view of the QMAX device when the third plate 30 is open after filtration and before the second plate is pivoted towards the first plate, and figure (D) shows a cross-sectional view of the QMAX device in a closed configuration when the portion of the sample flowing through the filter is pressed into a layer of uniform thickness.

Fig. 23 illustrates an exemplary embodiment of a QMAX device. Figure (a) shows a top view of a QMAX device containing a notch in a closed configuration; figure (B) shows a top view of QMAX device containing the notch in a closed configuration when the filter is placed on top of the first plate.

Detailed description of example embodiments

The following detailed description illustrates some embodiments of the invention by way of example and not by way of limitation. The section headings and any sub-headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. The content under the chapter titles and/or sub-titles is not limited to the chapter titles and/or sub-titles but applies to the entire specification of the present invention.

The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Definition of

The following definitions are set forth to illustrate and describe the meaning and scope of (a) certain examples of the invention and (b) certain terms used in the detailed description section.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, some exemplary methods and materials are now described.

If any patent, patent application, or other reference is incorporated by reference herein and (1) the manner in which a term is defined is inconsistent with an unincorporated portion of this disclosure or other incorporated reference and/or (2) is otherwise inconsistent with an unincorporated portion of this disclosure or other incorporated reference, the unincorporated portion of this disclosure shall control and the term or disclosure incorporated therein shall only control the reference in which the first definition of the term and/or the incorporated disclosure first appears.

The terms used to describe the devices, systems and methods disclosed herein are defined in the present application or in PCT applications (assigned US) No. PCT/US2016/045437 and No. PCT/US0216/051775, filed on day 7 on month 2 of 2017, and No. 62/456065, filed on day 8 on month 2 of 2017, and No. 62/456287, filed on day 8 on month 2 of 2017, filed on day 10 on month 8 of 2016 and 14 on month 9 of 2016, respectively, all of which are incorporated herein in their entirety for all purposes.

"QMAX" (Q.: quantitation; M: amplification, A. addition of reagents, X: acceleration; also known as self-calibrating compressed open flow (SCOF) apparatus, assay, method, kit and system are described in US provisional patent application No. 62/202,989 filed 8/10/2015, US provisional patent application No. 62/218,455 filed 14/2015, US provisional patent application No. 62/293,188 filed 2016, 2/2016, 9/2016, US provisional patent application No. 62/305,123 filed 3/8/2016, US provisional patent application No. 62/369,181 filed 2016, US provisional patent application No. 62/394,753 filed 15/2016, PCT application No. PCT/US/045437 filed 2016 (assigned US) filed 8/10/2016, PCT application No. PCT/US/045437 filed 2016, 14/2016 (assigned US) filed 2016, PCT application Ser. No. PCT/US2016/051794, filed on 9/15/2016, and PCT application Ser. No. PCT/US2016/054025, filed on 27/9/2016, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

The terms "CROF card (or card)", "COF card", "QMAX card", "Q card", "CROF device", "COF device", "QMAX device", "CROF board", "COF board", and "QMAX board" are interchangeable, except that in some embodiments the COF card does not contain a spacer; and the term refers to a device comprising first and second plates that are movable relative to each other into different configurations (including open and closed configurations) and comprising spacers (except for some embodiments of COFs) that adjust the spacing between the plates. The term "X-board" refers to one of the two boards in a CROF card, with the spacer fixed to the method board. Further descriptions of COF cards, CROF cards and X boards are in provisional application serial No. 62/456065 filed on 7.2.2017, provisional application serial No. 62/456065 filed on 7.2.2017 and us provisional application No. 62/456287 filed on 8.2.2017, which are incorporated herein in their entirety for all purposes.

1 extrusion/sponge wash assay methods, kits and systems

FIGS. 1-11 illustrate a squeeze-wash self-calibrating compressed open flow assay method, kit and system. Generally, in the drawings, optional or alternative elements are shown in phantom. However, the elements shown in solid lines are not essential to all embodiments of the disclosure, and elements shown in solid lines are omitted from particular embodiments without departing from the scope of the disclosure. Elements that serve a similar or at least substantially similar purpose are identified with a common numeral in the figures. The same numbers are used in each of the figures and not all corresponding elements will be discussed in detail herein with reference to each figure. Similarly, not all elements are labeled or shown in each figure, but rather the reference numbers associated therewith are used for consistency. Elements, components, and/or features discussed with reference to one or more of the figures are included in and/or used with any of the figures without departing from the scope of the present disclosure.

In general, reference is made to the figure elements according to the following table.

The squeeze or sponge wash technique (also known as the S technique) can be used for QMAX (Q: quantitation; M: amplification, A. addition of reagents, X: acceleration; also known as SCOF: self-calibrating compressed open flow) assays. In particular, the squeeze wash technique is used to reduce non-specific binding and improve the specificity of the assay. It should also be noted that the squeeze-wash technique can be used for other assays besides the QMAX assay. In the QMAX assay, a sample containing an analyte is squeezed between two plates. At least one of the plates or the sample has a spacer configured to adjust a thickness of the sample when compressed between the plates. The compression causes the sample to spread between the plates and limits diffusion to less than unconstrained three-dimensional diffusion (three-dimensional brownian motion). In some embodiments, the extruded thickness is sufficiently small that the diffusion is substantially two-dimensional. The limited thickness improves (accelerates) the incubation time of the reagent through the thickness (the reagent mixes relatively quickly through the thickness). Constrained lateral diffusion isolates assay sites along the plate surface (reagent lateral mixing (transverse to thickness) is relatively slow).

Many assays are suitable for self-calibrating compressed open flow technology. Some assays benefit from or require a washing step. The assay washing step is generally designed to remove unbound assay components and reduce non-target binding. Conventional cleaning techniques include rinsing (allowing excess solution to drain), soaking, and cycles of suction and dispensing. In self-calibrating compressed open flow technology, some of the benefits of increasing assay speed and efficiency may be lost by conventional washing.

In some embodiments of the sponge washing technique, any of the following is implemented or described:

(1) a sponge sheet (or any porous and absorbent material) is used with a wash solution (e.g., water) to ash the assay surface.

(2) The sponge is a flexible porous material; the pore size can be reduced under compressive pressure and returned to the original size when the pressure is removed.

(3) When the sponge sheet covers the assay surface, the pressing of the sponge causes the washing solution in the sponge to contact and wash the assay surface. The pressure was then released to allow the spent wash solution to reabsorb back into the sponge, leaving the assay surface washed and almost free of spent wash solution.

(4) The assay washed in this way is ready for the next step, e.g. reading or subsequent reagent interaction.

(5) The S-technique wash can be reused if desired.

(6) FIG. 1(A) provides an example of a sponge having dimensions of 1cm by 1 cm. + -. 0.5 cm.

(7) The sponge may have a plastic bottom plate that is easy to handle and easy to wash.

As shown in fig. 1(B), in the squeeze-and-purge self-calibrating compressed open flow technique, the plates are separated (e.g., opened) after the self-calibrating compressed open flow squeeze step. The initial pressing step mixes and/or reacts the assay components and bonds at least some of the assay components to at least one of the plate surfaces. Washing is performed by separating the plate and by contacting the assay sites (sites with bound analyte) with a wash pad loaded with a wash solution. In some embodiments, the wash pad is pre-loaded with wash solution; the wash pad is loaded (filled) with wash solution just prior to contacting the assay site, and/or loaded after contacting the assay site. Washing was continued by pressing the wash pad on the assay site. Squeezing the loaded wash pad causes the wash solution to drain from the wash pad and contact/rinse the assay site. In some embodiments, the wash program includes releasing the force pressing the wash pad, in which case the wash pad expands to its original shape and draws in adjacent fluid (e.g., wash solution mixed with unbound assay components). In certain embodiments, the used wash pad is removed from the assay site to prepare the plate for subsequent measurement or assay steps (e.g., further assay additives and/or washing with different reagents). Additionally or alternatively, the wash pad is reused in place (e.g., by reloading with wash solution and squeezing again). The dimensions of the wash pad shown in fig. 1(a) (e.g., 1cm × 1cm × 0.5cm) are merely illustrative and do not represent limitations or restrictions on dimensions.

In some embodiments, the wash pad is pressed by one plate 20. In some embodiments, the wash pad is pressed with an object that is not part of the assay part. In some embodiments, the wash pad is squeezed by a human hand.

Fig. 2 shows an assay plate 22 (also referred to as a first plate). Assay plate 22 includes an assay surface 28 and assay sites 30 on the assay surface. The assay site 30 has a capture agent 54 bound thereto. The capture agent 54 is schematically illustrated as an antibody, although the capture agent need not be an antibody. In some embodiments, the assay site 30 includes a blocking agent 56 to reduce non-specific and non-target binding at the assay site. In some embodiments, the capture agent 54 is bound to the assay site 30 via a linker peptide 58 (e.g., protein a, avidin, etc.). Additionally or alternatively, the capture agent 54 is covalently bound (directly or via a linker peptide 56) to the assay surface 28 at the assay site 30. The capture agent 54 is bound to the assay site 30 in a dry and/or environmentally stable form. In some embodiments, the capture agent 54 and/or blocking agent 56 is dried and/or coated on the assay sites 30 of the first plate 22.

In some embodiments, assay plate 22 includes a plurality of assay sites 30. Each assay site 30 includes the same or different type of capture agent 54. For example, each assay site 30 has a different type of capture agent 54 for analysis of a different type of analyte, or each assay site 30 has the same type of capture agent 54 but at a different concentration. As another example, the assay plate 22 includes one or more duplicate assay sites (e.g., replicates), each assay site 30 of which has the same type of capture agent 54 to perform the same assay.

Fig. 3 shows the second plate 24. In the example of fig. 3, the second plate 22 comprises a reagent 60 on the assay surface 28. The reagent 60 in this embodiment is a detection agent 62. In some embodiments, the detection agent 62 includes a label 64 and is referred to as a labeled detection agent. The detection agent 62 is schematically illustrated as an antibody, although the detection agent need not be an antibody. The detection agent 62 binds, adheres, and/or binds to the assay surface 28. Typically, the detection agent 62 is disposed on the assay surface 28 in a form that allows the detection agent to dissociate from the assay surface 10 and diffuse to the assay sites 30 of the detection agent 22. In some embodiments, the detection agent 62 is dried onto the assay surface 28 and is in a dry and/or environmentally stable form.

Referring to fig. 2-3, assay plate 22 and second plate 24 are components of plate combination 20. In some embodiments, assay plate 22, second plate 24, or both plates comprise spacers affixed to respective surfaces of the plates. When the plates are pressed together, the measuring surfaces face each other, and the spacer controls the spacing between the plates 20. In addition, if the plate 20 is pressed after depositing the sample, the spacer controls the thickness of the sample, resulting in a thin and uniform thickness.

Fig. 4 shows a wash pad 40. The wash pad 40 includes a porous media 42 and, at least when ready for use, a wash solution 44. The wash pad 40 is configured, selected and/or adapted to hold (retain) the wash solution 44 in an uncompressed state and drain at least some of the wash solution when compressed. As also shown in fig. 8, 10 and 11, in some embodiments, the wash pad includes a backing 140 and/or tabs (not shown). Wash pad 40 has a wash surface 144 configured to contact assay surface 28 and/or assay sites 30 of assay plate 22.

The porous media 42 of the wash pad 40 is absorbent and includes and/or is a foam (reticulated and/or open-celled), fibrous material, gel, sponge, or the like. Examples of materials include cellulose, polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, and combinations thereof. Generally, porous medium 42 is selected and/or configured to avoid specific binding of analyte molecules 52, sample 50, and/or assay reagents 60. However, in some embodiments, porous medium 42 is selected and/or configured to preferentially and/or specifically bind certain assay components (e.g., components of sample 50).

In some embodiments, the wash pad 40 includes a backing 140 to facilitate handling and/or to facilitate pressing. In certain embodiments, the backing 140 comprises and/or is a non-absorbent layer and/or an impermeable layer. In certain embodiments, the backing 140 is rigid and/or resilient. Typically, the porous medium 42 is bonded or otherwise attached to the backing 140 with the washing surface 144 of the porous medium facing away from the backing (i.e., one side of the washing pad is the backing and the other side includes the washing surface). In certain embodiments, backing 140 (and/or generally wash pad 40) includes tabs (not shown) to aid in handling wash pad 40 and/or to aid in separating the wash pad from assay plate 22.

The porous media 42 and the pores in the porous media are configured to hold a wash solution 44. Typically, porous medium 42 has a substantial open volume, e.g., greater than 50%, greater than 80%, or greater than 90% open volume, that holds wash solution 44. Typically, the average effective pore size is from about 0.1 μm to about 1,000 μm, such that capillary forces retain the wash solution 44 within the pores. Porous medium 42 is configured to reduce open volume when subjected to compression (compressive force). The reduced volume due to the squeezing causes at least some of the wash solution 44 to drain from the wash pad 40 when the wash solution 44 was previously loaded. In addition, when the previously compressed (squeezed) wash pad 44 is released from compression, the wash pad relaxes, returning substantially to its original shape, causing the pores to expand and the open volume to increase. When the compressive force is released, this action draws fluid into the wash pad 44.

When used as described herein, the compression of the wash pad 40 causes wash solution 44 to wash the assay surface 28 and/or assay sites 30 of the assay plate 22. When used as described herein, the release of the squeezing of the wash pad 40 causes the wash solution (wash solution 44 and unbound sample 50) to be substantially drawn into the wash pad 40.

Fig. 5 shows a sample 50 associated with assay plate 22. The sample 50 typically includes one or more analytes, each of which is found as an analyte molecule 52. Because the assay is configured to detect the presence, quantity, and/or activity of an analyte species, certain samples 50 have little or no analyte molecules 52 (or no analyte molecules of a particular analyte species). In some embodiments, the sample 50 is placed in contact with the assay site 30. In some embodiments, the sample 50 is placed on or near the assay site 30 on the assay plate 22. Additionally or alternatively, when the plates 20 are placed together, the sample 50 is placed on the second plate 24 at a location that will be above or near the assay site 30. In some embodiments, the sample 50 is drawn to a location at or near the assay site 30 by capillary action of the sample between the plates 20. For example, the plates 20 are spaced apart at a spacing sufficient to allow capillary action of the sample 50, and the sample 50 is introduced into the plates 20 at the open edges of the spaced apart plates 20. As described above, in some embodiments, the capture agent (e.g., antibody) and/or blocking agent are dried and coated onto the assay sites of the plate 20.

The plates 20 are movable relative to each other into different configurations; one of the configurations is an open configuration in which the two plates 20 are partially or fully separated, the spacing between the plates not being adjusted by spacers, allowing the liquid sample to be deposited on one or both of the plates; and the other of the configurations is a closed configuration, which is configured after deposition of the sample in the open configuration, and in which at least part of the spacing between the two plates 20 is adjusted by the plates and spacers, and at least part of the sample is compressed into a layer of uniform thickness, which layer is in contact with the capture agent.

As shown in fig. 6, the measuring surface 28 of each plate 20 is the operating surface of the plate. The sample 50 contacts the assay surface 28. Typically, when assembled in the assay assembly 10, the sample 50 is sandwiched between the plates 20, with the assay surfaces 28 of each plate 20 facing each other. In some embodiments, detector 22 and second plate 24 are connected by a rotational structure, such as one or more hinges, that allow plates 20 to pivot relative to each other. The boards 20 connected by a structure such as a hinge are called QMAX cards.

When the plate 20 is assembled in the assay assembly 10, precise alignment is generally not required. The sample 50 between the plates 20 is squeezed by the pressing of the plates so that the sample expands laterally across the assay surface 28. The extension of the sample 50 when squeezed allows the sample to be placed on the assay surface 28 of the receiving plate 26 with a rough accuracy and allows the plates 20 to be brought together with a rough accuracy. The extension of the sample 50 will substantially fill the assay sites 30 on the assay plate 22 even if the sample is not initially aligned with the assay sites 30. In some embodiments, receiving plate 26 includes sample alignment marks 110 to guide placement of sample 50. In some embodiments, one or more plates 20 include plate alignment fiducials 112 (e.g., markings or physical structures as shown in fig. 10) to guide the plate placement together. For example, the plate 20 has one or more edges that align when the plates are sufficiently aligned.

As shown in fig. 6, one or more of the measurement surface 28 of the measurement plate 22, the measurement surface 28 of the second plate 24, and the sample 50 typically include spacers 70 (not shown). The spacer is configured, sized, selected, and/or adapted to define a minimum distance (also referred to as an adjustment distance and/or a threshold thickness) between the assay plate 22 and the second plate 24. In some embodiments, the minimum distance is a non-zero distance and is the same as the height of the spacer. In certain embodiments, the minimum distance between the plates 20 is also the same as the thickness of the sample 50 when the plates are pressed together, making the sample 50 a thin layer. This distance is the minimum distance between plates 20 in a local neighborhood. In some embodiments, a separate spacer 70 contacts both plates 20 (e.g., the spacer is integral with one plate and contacts the other plate when the plates are pressed together). Generally, the height of the spacer 70 (the length of the dimension between the plates) determines the minimum distance. In some embodiments, the minimum distance is the height of the spacer 70 plus the residual height of the sample between the spacer and the plate. The minimum distance, spacer height, and/or sample 50 thickness is typically 3nm or less, 10nm or less, 50nm or less, 100nm or less, 200nm or less, 500nm or less, 800nm or less, 1000nm 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, 1mm or less, 2mm or less, 4mm or less, or within a range between any two of these values.

Fig. 7 shows the assay assembly 10 when extruded in a closed configuration. The assay plate 22 and the second plate 24 are pressed together with the sample 50 between the plates. The sample 50 contacts the assay site 30 (rehydrating the assay site and/or the capture agent 54, if desired). The sample 50 also contacts the assay surface 28 of the second plate 24, allowing reagents 60 (e.g., detection agents 62 as shown) to mix in the sample and migrate to the assay site 30. Contact of the sample 50 with the reagent 60 on the assay surface 28 of the second plate 24 releases the reagent from the assay surface and rehydrates and/or dissolves the reagent.

In the closed configuration (squeezed state), as shown in fig. 7, the assay assembly 10 is incubated to allow the capture agents 54, sample 50, analyte molecules 52, detection agents 62, and/or other reagents 60 to mix and/or react. Due to the reduced thickness of the sample 50 between the plates (distance adjusted by the spacer 70), the time for the molecules or other assay components to diffuse through the thickness is greatly reduced compared to the original sample thickness. Sample thicknesses less than about 200 μm strongly influence molecular diffusion. A sample thickness of less than about 20 μm limits diffusion to substantially two dimensions (motion in the thickness direction is more ballistic than diffusion). The incubation time can be significantly reduced from that required when performing similar assays in bulk (e.g., in multi-well plates). Useful incubation times in the squeeze-wash QMAX assay format are less than 500 seconds, less than 100 seconds, less than 50 seconds, less than 20 seconds, less than 5 seconds, or less than 2 seconds, or within a range between any two values. Useful incubation times are substantially instantaneous with respect to the manual handling time of plate 20. The assay assembly 10 is held in the squeezed state for a period of time longer than the time required for the assay components to mix and react.

FIG. 8 shows a wash pad 40 for washing assay plate 22. The wash pad 40 is placed in contact with the assay surface 28 and/or the assay site 30. The wash pad 40 is pre-loaded with wash solution 44 and/or wash solution 44 is added to the wash pad 40. The wash pad 40 and assay plate 22 are pressed together to drain the wash solution 44 from the wash pad onto the assay plate 22. In some embodiments, the pressing of the wash pad 40 is facilitated by the optional backing 140 and/or the second sheet 24. In some embodiments, the second plate 24 is used to press the wash pad 40. In some embodiments, the assay assembly 10 comprises one or more hinges connecting the detection agent 22 and the second plate 24, with the plates 20 pivoting relative to each other, switching between open and closed configurations. In certain embodiments, after incubation in the closed configuration, second plate 24 is opened and wash pad 40 is placed against the assay surface on the assay plate, then second plate 24 is pressed against wash pad 40, and wash solution 44 is deposited on assay plate 22 to wash the assay site while second plate 24 is released. The scrubbing solution 44 is reabsorbed into the scrubbing pad 40.

After incubation and switching to the open configuration, the wash pad is placed on assay plate 22 such that wash pad 40 contacts assay plate 22 (plate 20 with capture agent 54 and assay sites 30), typically without any precise alignment. The size of the wash pad 40 is typically larger than the area covered by all relevant assay sites 30. For example, in some embodiments, the lateral dimension of wash pad 40 is substantially the same as the dimension of assay surface 28 of assay plate 22. In addition, the wash pad 40 is sized to hold enough wash solution 44 to wash the associated assay site 30. Thus, when the wash pad 40 is squeezed, excess wash solution 44 flows out of the periphery of the wash pad.

In some preferred embodiments, there is a spacer (also referred to as a "wash spacer") between the wash surface 144 of the wash pad 40 and the detector 22, the spacer configured to maintain a non-zero spacing between the wash surface 144 and the assay site 30. To prevent direct contact therebetween during compression and thereby prevent possible physical removal by direct contact of the reagents 60 (e.g., capture agents 54, detection agents 62) and/or analytes 52 bound thereto in the associated assay site 30. In some embodiments, the wash spacer is part of the spacer 70 of the assay plate 22 and is within and/or adjacent to the assay site 30. Additionally or alternatively, the spacer is part of the spacer 70 of the sample 50 and is located within the assay site 30 and/or adjacent to the assay site 30 after separation of the assay plate 22 and the second plate 24 after the assay. Additionally or alternatively, the wash spacer is a portion of the wash surface 144 of the wash pad 40 (referred to as a "wash pad spacer") and is within and/or adjacent to the assay site 30 after contact between the wash pad 40 and the assay plate 22.

In these preferred embodiments, the wash surface 144 is configured (e.g., sufficiently rigid) to engage with the wash spacer, preventing direct contact with the assay site 30 during pressing, while the wash pad 40 as a whole is configured, selected, and/or adapted to hold (retain) the wash solution 44 in an uncompressed state and expel at least some of the wash solution when compressed, as described above.

1.3 experiment

Fig. 9 and table 1 summarize the experimental implementation of an exemplary embodiment of the present disclosure and indicate the relative performance of the squeeze wash QMAX assay (samples 3 and 4 in table 1) versus the QMAX assay without wash (

samples

1 and 2 in table 1) and the QMAX assay with conventional wash (samples 5 and 6 in table 1) according to this embodiment.

Table 1:

in the experiment, to prepare the samples, one plate was coated with: (1) protein a for 2 hours, (2) CAb for 2 hours, (3) blocker and stabilizer for 2 hours, another plate was coated with dAb-L and stabilizer for 2 hours; the sample, which included 1. mu.g/ml of human IgG antigen, was incubated in the closed configuration for 5 minutes, and the assay plate was then washed. As shown in fig. 9, the sponge wash gave the same signal as the conventional wash. It should be noted, however, that sponge washing is performed faster and easier/simpler than conventional washing. In the experiment shown in fig. 9, the sponge wash took less than 30 seconds, and the conventional wash took about 10 minutes. Unwashed samples showed high signal but varied widely (too high background signal) making the results unreliable.

FIG. 10 shows a squeeze wash SCOF assay kit 12. The kit 12 includes an assay plate 22, one or more second plates 24, and a wash pad 40. Assay plate 22, second plate 24, and wash pad 40 are sealed and/or environmentally stable (e.g., reagents are dried on the respective plates and/or contained in an environmentally stable layer). As shown in FIG. 11, the wash pad 40 is sealed with a wash pad seal 146. The wash pad seal 146 is configured (in combination with the optional backing 140) to retain the wash solution 44 within the wash pad 40, which is useful, for example, when dispensing wash pads in the kit 12.

In some embodiments, an apparatus for cleaning a surface of a plate, comprising: a first plate and a second plate, wherein:

i. the first plate is the plate with the sample surface to be washed,

the second plate is a plate made of a porous material having at least a part of pores which are deformable and capable of absorbing a solution by capillary force,

the plates may be moved relative to each other into different configurations;

one or both plates are flexible;

v. one or both of the plates comprises spacers fixed with the respective plate, wherein the spacers have a predetermined substantially uniform height and a predetermined constant spacer pitch of up to 250 μm;

wherein one of the configurations is an open configuration, wherein: the two plates are separated, the spacing between the plates is not adjusted by spacers, and a sample is deposited on one or both of the plates, an

Wherein the other of the configurations is a closed configuration configured after deposition of the sample in the open configuration; and in the closed configuration: at least a portion of the spacing between the two plates is adjusted by the plates and spacers.

During operation, a wash solution is first filled into the pores of the porous material, and then the two plates are brought into a closed configuration and the porous material is deformed to release the solution. The solution will be in the space between the plates and will be absorbed back by the porous material when the pressure is released and the pores will turn to their original shape (same or similar shape before pressing).

This space can reduce contact between the two surfaces of the plate in the closed configuration, thereby reducing damage to the sample surface to be washed.

In some embodiments, the spacer pitch is in a range of 1 μm to 400 μm (e.g., 1 μm to 10 μm, 10 μm to 50 μm, 50 μm to 100 μm, 100 μm to 200 μm, 200 μm to 300 μm, or 300 μm to 400 μm).

In some embodiments, the spacers have a height in a range of 1 μm to 250 μm (e.g., 1 μm to 10 μm, 10 μm to 50 μm, 50 μm to 100 μm, 100 μm to 200 μm, or 200 μm to 250 μm); and a lateral dimension from 1 μm to 300 μm (e.g., 1 μm to 10 μm, 10 μm to 50 μm, 50 μm to 100 μm, 100 μm to 200 μm, or 200 μm to 300 μm), wherein the spacers will each select one of these values.

In some embodiments, the spacers are fixed on the plate by directly stamping the plate or injection molding the plate.

In some embodiments, the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

In some embodiments, the spacer has at least 100/mm²At least 1000/mm²Or at least 10000/mm²The density of (c).

In some embodiments, wherein the mold used to fabricate the spacer is fabricated from a mold containing features fabricated by (a) direct reactive ion etching or ion beam etching or (b) repeating the reactive ion etching or ion beam etching features one or more times.

Example overview of 2 squeezing/sponge Wash assay methods, kits and systems

The present invention includes various embodiments that can be combined in various ways as long as the various components are not contradictory to each other. The embodiments should be considered as a single invention file: each application has other applications as references and is also incorporated by reference in its entirety for all purposes rather than as a discrete, independent document. These embodiments include not only the disclosure in the present document, but also documents that are referenced, incorporated or claim priority herein.

Embodiments of the inventive subject matter according to the present disclosure are described in the paragraphs listed below.

2.1 squeeze/sponge Wash assay

Example 1: an assay method comprising, in order:

(a) placing a biological sample between an assay surface of an assay plate and an assay surface of a second plate, wherein the biological sample comprises analyte molecules, wherein the assay surface of the assay plate comprises assay sites comprising a capture agent that binds to the assay sites, wherein the capture agent is configured to specifically bind to the analyte molecules, and wherein at least one of the assay surface of the assay plate, the assay surface of the second plate, and the biological sample comprises a spacer sized to separate the assay plate and the second plate by a threshold thickness,

(b) the measuring plate and the second plate are pressed together to a pressing thickness adjusted by the spacer,

(c) the assay plate and the second plate are separated,

(d) contacting the assay plate with a wash pad, the wash pad having a wash surface, loaded with a wash solution, wherein the wash surface is the surface of the wash pad that is in contact with the assay plate, and

(e) the wash pad and assay plate are pressed together to drain the wash solution from the wash pad onto the assay sites of the assay plate.

In the method of example 1, (a) placing comprises placing the biological sample on at least one of the assay surface of the assay plate and the assay surface of the second plate.

In the method of example 1, (a) placing comprises placing the biological sample on at least one of the assay surface of the assay plate and the assay surface of the second plate and closing the biological sample between the assay plate and the second plate.

In the method of any preceding embodiment, the method further comprises, after (a) placing and before (c) separating, incubating the biological sample contacted with the capture agent for a period of time correlated to a saturation binding time of the analyte molecule to the capture agent.

In the method of any preceding embodiment, the period of time is less than 500 seconds, less than 100 seconds, less than 50 seconds, less than 20 seconds, less than 5 seconds, or less than 2 seconds.

In a method as in any preceding embodiment, the assay plate comprises a plurality of assay sites spaced apart by a minimum site spacing, and the time period is less than the average lateral diffusion time for an analyte molecule to traverse the minimum site spacing.

In a method as in any preceding embodiment, wherein (b) pressing comprises pressing the assay plate and the second plate together to accelerate a diffusion-limited reaction time of the analyte molecules to the capture agent relative to the non-pressed sample.

In a method as in any preceding embodiment, the assay surface of the second plate comprises a detection agent adhered to the assay surface, and the detection agent is configured to specifically associate with at least one of analyte molecules and analyte molecules bound to the capture agent.

In a method as in any preceding embodiment, wherein (b) pressing comprises pressing the assay plate and the second plate together to accelerate a diffusion-limited reaction time of the detector for the analyte molecules relative to the non-pressed sample.

In a method as in any preceding embodiment, wherein (b) pressing comprises pressing the assay plate and the second plate together to accelerate a diffusion-limited reaction time of the detection agent for analyte molecules bound to the capture agent relative to the non-pressed sample.

In a method as in any preceding embodiment, the (d) contacting comprises contacting at least a portion of the spacer with a wash pad loaded with a wash solution, wherein the portion of the spacer and the wash surface are configured to prevent direct contact between the wash surface and the assay site.

In the method of any preceding embodiment, the portion of the spacer is within and/or adjacent to the assay site prior to (d) contacting.

In the method of any preceding embodiment, the washing surface is rigid.

In a method as in any preceding embodiment, the wash pad comprises a wash pad spacer on the wash surface, the wash surface and the wash pad spacer configured to prevent direct contact between the wash surface and the assay site.

In the method of any preceding embodiment, after the contacting of (d), the wash pad spacer is within and/or adjacent to the assay site.

In the method of any preceding embodiment, the washing surface is rigid.

In a method as in any preceding embodiment, wherein (d) contacting comprises placing a wash pad between the assay surface of the assay plate and the assay surface of the second plate.

In a method as in any preceding embodiment, wherein (e) pressing comprises pressing a wash pad between the second plate and the assay plate.

In a method as in any preceding embodiment, the method further comprises removing the wash pad from the assay plate after (e) pressing.

In the method of any preceding embodiment, the method further comprises covering the assay surface of the assay plate after removing the wash pad, optionally by covering the assay plate with at least one of a second plate and a cover plate.

In the method of any preceding embodiment, the method further comprises, after (e) pressing, detecting the analyte molecule bound to the capture agent.

In the method of any preceding embodiment, detecting comprises measuring at least one of fluorescence, luminescence, scattering, reflection, absorption, and surface plasmon resonance associated with the analyte molecule bound to the capture agent.

In a method as in any preceding embodiment, the assay surface of the assay plate at the assay site comprises a signal amplification surface, such as a metal and/or dielectric microstructure (e.g., a disk coupled spot post antenna array).

In a method as in any preceding embodiment, the (d) contacting comprises contacting the assay site with the wash pad without the wash solution, and adding the wash solution to the wash pad while in contact with the assay site to load the wash pad with the wash solution.

In the method of any preceding embodiment, the method further comprises, prior to the contacting of (d), adding a wash solution to the wash pad to load the wash pad with the wash solution.

In the method of any preceding embodiment, the wash pad comprises a porous medium configured to hold a wash solution.

In the method of any preceding embodiment, the porous medium is configured to hold the wash solution in an open volume of the porous medium.

In the method of any preceding embodiment, the open volume of the porous medium decreases when the porous medium is compressed.

In a method as in any preceding embodiment, the porous medium is resiliently compressible, configured to return to an uncompressed shape and an uncompressed open volume after application and subsequent release of compression.

In the method of any preceding embodiment, the (e) squeezing comprises diluting the sample and unbound analyte molecules with the drained wash solution.

In a method as in any preceding embodiment, (e) pressing comprises draining drained wash solution from the wash pad and assay plate.

In a method as in any preceding embodiment, the method further comprises stopping (e) the pressing to allow the scrubbing pad to absorb excess fluid into the porous medium of the scrubbing pad.

In the method of any preceding embodiment, the threshold thickness is at least 0.1 μ ι η, at least 0.5 μ ι η, or at least 1 μ ι η.

In the method of any preceding embodiment, the extruded thickness is at most 1mm or at most 200 μm.

In the method of any preceding embodiment, the extruded thickness is at most 20 μm, at most 10 μm, or at most 2 μm.

In the method of any preceding embodiment, the assay plate comprises a spacer.

In the method of any preceding embodiment, the second plate comprises spacers.

In the method of any preceding embodiment, the biological sample does not comprise a spacer.

A multi-step assay comprising:

performing the method of any preceding embodiment, the second plate is a first reagent plate, the first reagent plate comprises a first reagent on the assay surface, and the wash pad is a first wash pad;

removing the first wash pad from the assay plate, and

performing the method of any preceding embodiment, the second plate is a second reagent plate comprising a second reagent on the analysis surface, and the wash pad is a second wash pad.

2.2 extrusion/sponge Wash assay kit

Example 2: 2. a kit for assaying a sample comprising:

first board, second board, and sponge between, wherein:

i. the plates may be moved relative to each other into different configurations,

the first plate comprising on its inner surface a sample contacting area for contacting a sample comprising the analyte,

the sponge is made of a flexible porous material having flexible pores, the shape of the flexible pores being changeable under force and the flexible porous material being capable of absorbing or releasing liquid into or from the sponge when the shape of the pores is changed;

wherein one of the configurations is an open configuration, wherein: the two plates are partially or completely separated, allowing the sample to be deposited on one or both of the plates,

wherein the other of the configurations is a closed configuration, the closed configuration being configured after deposition of the sample in the open configuration; and in the closed configuration: at least a portion of the sample is compressed into a layer by the two plates and is substantially stagnant with respect to the plates, wherein the layer is bounded by the inner surfaces of the two plates, and

wherein the sponge is configured to deposit the sponge-filled wash solution on the sample contact area when the sponge is pressed and to reabsorb the wash solution when the pressing force is released.

Example 3: a kit for assaying a sample comprising:

a first plate, a second plate, a spacer, and a sponge, wherein:

spacers are fixed on the respective surfaces of one or both of the plates,

wherein the spacers have a predetermined substantially uniform height and a predetermined fixed spacer spacing, and

wherein one of the configurations is an open configuration in which the two plates are partially or fully separated, the spacing between the plates not being adjusted by spacers, allowing a sample to be deposited on one or both of the plates;

wherein the other of the configurations is a closed configuration configured after deposition of the sample in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant with respect to the plates, wherein the uniform thickness of the layer is bounded by the inner surfaces of the two plates and is regulated by the plates and the spacer; and is

In the kit of example 2 or 3, the kit further comprises a sponge container configured to hold a sponge.

In a kit as in any preceding embodiment, the sponge comprises a closure wall having a sealed bottom that retains the solution inside the sponge container.

In a kit as in any preceding embodiment, the bottom of the plate and sponge container is used as a press.

In a kit as claimed in any preceding embodiment, the kit comprises a plurality of sponges.

In a kit as claimed in any preceding embodiment, the kit comprises a plurality of containers.

In a kit as claimed in any preceding embodiment, the kit comprises a plurality of sponges configured to be received by one container.

In a kit as claimed in any preceding embodiment, the kit comprises a separate dry sponge to absorb only liquid.

In a kit as claimed in any preceding embodiment, the kit comprises a separate sponge for releasing liquid only.

In a kit as claimed in any preceding embodiment, the sponge container further comprises a lid.

2.3 methods for squeeze/sponge Wash assays

Example 4: a method of sample analysis comprising:

(a) obtaining a QMAX device comprising a first plate and a second plate, the first plate and the second plate being movable into different configurations, including an open configuration and a closed configuration,

(b) depositing a liquid sample on the sample contacting area of the first plate in an open configuration, wherein the two plates are partially or completely separated, (c) pressing the plates into a closed configuration, wherein at least a portion of the sample is compressed into a layer of uniform thickness and incubating the sample for a predetermined period of time,

(d) the second plate is removed and the first plate is removed,

(e) a sponge containing a wash solution is placed on the sample contacting area of the first plate,

(f) the sponge is pressed to deposit the wash solution onto the sample contact area, held in the pressed position for a period of time, and released to reabsorb the wash solution.

In the method of embodiment 4, the first plate or the second plate includes a spacer fixed on the corresponding surface.

In the method of embodiment 4, the first plate or the second plate comprises a plurality of spacers fixed on corresponding surfaces and configured to adjust the thickness of the sample between the first plate and the second plate when the sample is compressed.

In the method of any preceding embodiment, the incubation time is less than 500 seconds, less than 100 seconds, less than 50 seconds, less than 20 seconds, less than 5 seconds, or less than 2 seconds, or within a range between any two values.

In a method as in any preceding embodiment, the inner surface of the second plate comprises a detection agent adhered to the assay surface, and the detection agent is configured to specifically associate at least one of analyte molecules and analyte molecules bound to the capture agent.

In a method as in any preceding embodiment, the compressing in step (f) comprises compressing a sponge between the second plate and the first plate.

In the method of any preceding embodiment, the method further comprises removing the sponge from the first plate after step (f).

A method as in any preceding embodiment, further comprising repeating step (f) one or more times.

In the method of any preceding embodiment, the method further comprises reloading the sponge with fresh wash solution and repeating steps (e) and (f) one or more times.

In a method as in any preceding embodiment, the sponge is made of a flexible porous material having flexible pores, the shape of the flexible pores being changeable under force, and the porous material being capable of absorbing or releasing liquid into or from the sponge when the shape of the pores changes.

2.4 device for squeeze/sponge Wash assays

Example 5: an apparatus for sample analysis, comprising:

a first plate, a second plate, a third plate, and a spacer, wherein:

i. the second plate and the third plate are respectively connected with the first plate, the second plate and the third plate are configured to respectively pivot against the first plate without interfering with each other,

the second or third plate is movable relative to the first plate into a different configuration by pivoting against the first plate,

the first plate comprises an inner surface having a sample contacting area for contacting a component-containing liquid sample, and

spacers are affixed to one or more plates or mixed in a sample, and one of the configurations is an open configuration, wherein: all three plates are partially or completely separated and the spacing between the plates is not adjusted by spacers, and the sample is deposited on the first plate, the second plate, or both; and

wherein another of the configurations is a closed configuration configured after deposition of the sample in the open configuration, and in the closed configuration: at least a portion of the deposited sample is compressed by the first and second plates into a layer of very uniform thickness that is confined by the inner surfaces of the first and second plates and conditioned by the plates and spacers.

In the device of example 5, the device further comprises a sponge made of a flexible porous material.

In a device as in any preceding embodiment, the flexible porous material has pores, the shape of the pores being changeable under force, and the pores being capable of absorbing or releasing liquid into or from the sponge when the shape of the pores is changed.

In a device as in any preceding embodiment, the third plate is configured to press against the sponge when the third plate is pivoted towards the first plate.

In an apparatus as claimed in any preceding embodiment, one edge of the second panel is connected to the inner surface of the first panel by a first hinge.

In a device as claimed in any preceding embodiment, one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In a device as claimed in any preceding embodiment, one edge of the second panel is connected to the inner surface of the first panel by a first hinge and one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In a device as claimed in any preceding embodiment, between the first and second plates, in the closed configuration, the third plate is adjustable to pivot against the first and second plates.

In a device as claimed in any preceding embodiment, the first plate comprises one or more notches on one or more edges thereof, the notches being positioned such that the second plate and/or the third plate are juxtaposed over the notches to facilitate manipulation of pivoting of the second plate and the third plate.

In an apparatus as in any preceding embodiment, the second plate includes a plate tab configured to facilitate switching the plate between different configurations.

In a device as in any preceding embodiment, the sponge includes a sponge tab, and the sponge tab is configured to facilitate removal of the sponge from the plate.

2.5 kits for sample washing and analysis

Example 6: a kit for sample washing and analysis comprising: the apparatus as described in example 5, and

a sponge made of a flexible porous material having flexible pores, the shape of the flexible pores being changeable by a force, and the flexible porous material being capable of absorbing or releasing a liquid into or from the sponge when the shape of the pores is changed.

In the kit as described in example 6, the sponge is configured to be pressed by the third plate when the sponge is positioned on the first plate.

In a kit as described in example 6 or any derived example, wherein:

i. the sample contains the analyte(s) and,

coating a capture agent on the sample contact area in the first plate, and

the capture agent is configured to specifically bind to the analyte.

In a kit as in example 6 or any derivative thereof, one edge of the second panel is connected to the inner surface of the first panel by a first hinge and one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In a kit as in example 6 or any derivative thereof, the third plate can be adjusted to pivot against the first and second plates in a closed configuration between the first and second plates.

In the kit of example 6 or any derivative thereof, the kit further comprises a container configured to hold a sponge.

In a kit as described in example 6 or any derived example, the container contains a wash medium.

In a kit as described in example 6 or any derived example, the sponge comprises an enclosing wall with a sealed bottom, which keeps the solution inside the sponge container.

2.6 sample analysis method

Example 7: a method of sample analysis comprising:

(a) obtaining the device of example 5;

(b) depositing a liquid sample on the inner surface of the first plate in an open configuration,

(c) the second panel is pressed into a closed configuration,

(d) the second plate is opened and the second plate is opened,

(e) a sponge containing a washing solution is placed on the inner surface of the first plate,

(f) pressing the sponge with the third plate to deposit the wash solution on the inner surface of the first plate, holding the sponge in the pressed position for a period of time, releasing the sponge to reabsorb the wash solution.

In the method of embodiment 7, one edge of the second panel is connected to the inner surface of the first panel by a first hinge, and one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In the method of example 7 or any derivative thereof, the first plate comprises at least one assay site, and the sample and the spacer deposited on the assay site are immobilized to the assay site.

In a method as described in example 7 or any derivative example, the first plate comprises a capture reagent coated on an inner surface of the first plate, the capture reagent configured to specifically bind to an analyte in the sample.

In a method as described in example 7 or any derivative example, the first plate comprises a plurality of assay sites separated by a minimum site spacing.

In the method of example 7 or any derivative thereof, further comprising: after step (f), detecting the analyte bound to the capture agent.

In the method of example 7 or any derivative example, the detecting comprises measuring at least one of fluorescence, luminescence, scattering, reflection, absorption, and surface plasmon resonance associated with the analyte bound to the capture agent.

In a method as described in example 7 or any derivative thereof, the inner surface of the first plate at the assay site comprises a signal amplification surface, such as a metal and/or dielectric microstructure (e.g., a disk coupled point post antenna array).

2.7 methods for performing assays

Example 8: a method for performing an assay, comprising:

(a) obtaining a first plate comprising on its inner surface a sample contacting area having a first reagent site, wherein the first reagent site comprises a first reagent that bio/chemically interacts with a target analyte in a sample,

(b) obtaining a second plate comprising on its inner surface a sample contacting area having a second reagent site, wherein the second reagent site comprises a second reagent capable of diffusing in the sample when contacting the sample,

(c) obtaining a third plate comprising on its inner surface a sample contacting area having third reagent sites comprising a third reagent capable of diffusing in the transfer liquid upon contacting the transfer liquid,

(d) in the open configuration, a sample is deposited on one or both of the sample contacting regions of the first and second plates,

(e) after (d), placing the first and second panels in a closed configuration;

(f) after (e) separating the first and second sheets,

(g) after (f) depositing the transfer liquid in an open configuration on one or both of the sample contacting regions of the second and third plates,

(h) after (g), placing the second and third panels in a closed configuration; and is

(i) Detecting a signal associated with the target analyte,

wherein the first plate, the second plate, and the third plate are movable relative to one another into different configurations, including an open configuration and a closed configuration;

wherein, in the open configuration, the sample contact area separation of the two plates is greater than 200 μm; and

wherein, in the closed configuration, at least a portion of the sample deposited in (d) or the transfer liquid deposited in (g) is confined between the sample contacting areas of the two plates and has an average thickness in the range of 0.01 μm to 200 μm.

2.8 kits, devices and methods for sample analysis

Example 9: the kit, device and method of any preceding embodiment wherein the sponge comprises a porous substrate and the porous substrate contains pores having diameters in the range of 10nm to 100nm, 100nm to 500nm, 500nm to 1 μ ι η,1 μ ι η to 10 μ ι η,10 μ ι η to 50 μ ι η,50 μ ι η to 100 μ ι η, 100 μ ι η to 500 μ ι η, 500 μ ι η to 1 mm.

In the kit, device and method as described in example 9, the sponge comprises a porous substrate and the porous substrate contains pores having a diameter in the range of 500nm to 1 μm, 1 μm to 10 μm, 10 μm to 50 μm, 50 μm to 100 μm, 100 μm to 500 μm.

In the kits, devices, and methods of any preceding embodiment, the sponge comprises a porous substrate, and the porous substrate has a porosity in the range of 10 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 to 90%, 90 to 99%.

In the kits, devices, and methods of any preceding embodiment, the sponge comprises a porous substrate, and the porous substrate has a porosity in the range of 70 to 80%, 80 to 90%, 90 to 99%.

In the kit, device and method of any preceding embodiment, the sponge comprises a porous substrate, and the material of the porous substrate comprises rubber, cellulose wood fibers, foamed plastic polymers, low density polyethers, polyvinyl alcohol (PVA), polyester, poly (methyl methacrylate) (PMMA), polystyrene, and the like.

In the kits, devices, and methods of any preceding embodiment, the sponge comprises a porous substrate, and the porous substrate is hydrophilic, meaning that the contact angle of a sample droplet (e.g., water) on the substrate is 0-15 degrees, 15 to 30 degrees, 30 to 45 degrees, 45 to 60 degrees, 60 to 90 degrees, preferably the contact angle is 15 to 30 degrees, 30 to 45 degrees, 45 to 60 degrees.

In the kits, devices, and methods of any preceding embodiment, the porous substrate is hydrophobic, and the contact angle of the sample drop (e.g., water) on the substrate is 90 to 105 degrees, 105 to 120 degrees, 120 to 135 degrees, 135 to 150 degrees, 150 to 180 degrees, preferably the contact angle is 105 to 120 degrees, 120 to 135 degrees, 135 to 150 degrees.

In the kits, devices, and methods of any preceding embodiment, the porous substrate is hydrophilic, meaning that the contact angle of a sample droplet (e.g., water) on the substrate is 0 to 15 degrees, 15 to 30 degrees, 30 to 45 degrees, 45 to 60 degrees, 60 to 90 degrees.

In the kits, devices, and methods of any preceding embodiment, wherein:

applying a capture agent to the sample contact area, and

v. the capture agent is configured to specifically bind to the analyte.

In the kit, device and method of any preceding embodiment, the wash solution is deposited on the sample contact area after the binding of the analyte and the capture agent reaches equilibrium.

In the kits, devices, and methods of any preceding embodiment, the capture agent is an antibody, a DNA molecule, or an RNA molecule.

In the kit, device and method of any preceding embodiment, any one of the plates comprises at least one assay site on the respective sample contacting area, the sample and the spacer deposited on the assay site being fixed to the assay site.

In the kits, devices, and methods of any preceding embodiment, the second panel comprises a panel tab configured to facilitate switching the panel between different configurations.

In the kits, devices, and methods of any preceding embodiment, the sponge comprises a sponge tab configured to facilitate removal of the sponge from the plate.

In the kit, device and method of any preceding embodiment, the sponge is configured to:

(i) before being pressed, the sponge contains a washing solution inside,

(ii) when pressed, at least a portion of the wash solution is released, and

(iii) when the pressing is complete, at least a portion of the released liquid is absorbed.

In the kit, device and method of any preceding embodiment, the spacer is fixed to the first plate.

In the kit, device and method of any preceding embodiment, the spacer is affixed to the first and second panels.

In the kit, device and method of any preceding embodiment, the sample is whole blood and the components are blood cells.

In the kits, devices, and methods of any preceding embodiment, the first plate comprises reagent sites on its sample contacting region.

In the kits, devices, and methods of any preceding embodiment, the second plate comprises reagent sites on its sample contacting area.

In the kits, devices, and methods of any preceding embodiment, the sponge contains a wash solution.

In the kits, devices, and methods of any preceding embodiment, the sponge comprises a solution.

In the kits, devices, and methods of any preceding embodiment, the sponge contains a liquid reagent.

3 measurement of dilution calibration

FIG. 12 is a flow chart of an exemplary embodiment of a method of determining a sample dilution factor provided by the present invention. The method comprises the following steps:

(i) providing a sample containing a calibration marker having a concentration known as the preset value Cp,

(ii) providing a diluent having an unknown volume of the diluent,

(iii) diluting the sample with a diluent to form a diluted sample;

(iv) (iv) after (iii), obtaining a second value C using a concentration measurement tool₂The second value is the concentration of the calibration marker in the diluted sample; and

(v) by comparing a predetermined value Cp with a second value C₂Determining dilution factor of diluted sampleAnd (4) adding the active ingredients.

In some embodiments, the preset value Cp may be a predetermined value for the actual concentration of the calibration marker in the sample. In other embodiments, the preset value Cp may be a normal value assumed based on past experience, standards in the art, or other reasons, and such normal value does not differ too much from the actual concentration of the calibration marker in the sample. In some embodiments, the difference between the preset value and the actual concentration is 20% or less, 15% or less, 10% or less, 5% or less, 2.5% or less.

FIG. 13 is a flow chart of another exemplary embodiment of a method of determining a dilution factor of a sample provided by the present invention. The method comprises the following steps:

(i) providing a sample comprising a calibration marker, the calibration marker having an unknown concentration;

(ii) providing a diluent having an unknown volume of the diluent,

(iii) obtaining a first value of area C using a concentration measurement tool₁The first value is the concentration of the calibration marker in the sample,

(iv) diluting the sample with a diluent to form a diluted sample;

(v) (iv) after (iv), obtaining a second value C using a concentration measurement tool₂The second value is the concentration of the calibration marker in the diluted sample; and

(vi) by comparing the first value C₁And a second value C₂To determine the dilution factor.

As shown in the sequence of fig. 13, in some embodiments, the method may comprise: first, a first value C1 is obtained, and then the sample is diluted with a diluent to form a diluted sample.

However, it should be noted that in other embodiments, the method may be included in obtaining the first value C₁And a step preceding the step of diluting the sample: the sample is divided into at least two parts:

a first part for obtaining a first value C and a second part₁And (c) diluting the second portion with a diluent to form a diluted sample.

Although the present invention may be particularly useful when the volume of diluent is not known to the user of the method, in some embodiments it is also applicable where the volume of diluent is known to the user of the method.

In some embodiments, the step of diluting the sample may be a single step of mixing the sample with a diluent, which may be a single impurity or a mixture of multiple impurities. In other embodiments, the dilution step may be a series of dilution steps in which the sample is mixed with a plurality of impurities in sequence.

3.1 definition of

Sample (I)

The term "sample" as used herein generally refers to a material or mixture of materials that contains one or more analytes of interest. In some embodiments of the invention, the sample may be one of a biological sample, an environmental sample, and a food sample, or any combination thereof.

In particular embodiments, the sample may be obtained from a biological sample, such as cells, tissues, bodily fluids, and stool. Typically, a sample in a non-liquid form is converted to a liquid form prior to analysis of the sample by the method of the invention. Bodily 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), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled condensate. In particular embodiments, the sample can be obtained from a subject (e.g., a human), and it can be processed prior to use in a subject assay. For example, prior to analysis, proteins/nucleic acids may be extracted from tissue samples prior to use, methods of which are known. In particular embodiments, the sample may be a clinical sample, e.g., a sample collected from a patient.

In other embodiments, the sample may be obtained from an environmental sample, including but not limited to: liquid samples from rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, and the like; from soil, compost,

Solid samples such as sand, rock, concrete, wood, brick, dirt, and the like; and gas samples from air, underwater heat sinks, industrial exhaust, vehicle exhaust, and the like. Typically, a sample that is not in liquid form is converted to liquid form prior to analysis of the sample by the method of the invention.

In other embodiments, the sample can be obtained from a food sample suitable for consumption by an animal (e.g., huinaconsumputo). Food samples may include, but are not limited to, raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and the like. Typically, a sample in a non-liquid form is converted to a liquid prior to analysis of the sample by the method of the invention.

Calibration marker

The term "calibration marker" as used herein refers to any analyte contained in a sample whose detectable amount is not affected by the addition of a diluent. Here, the term "detectable amount" refers to the amount of analyte detected by the calibration measurement tool provided in the present method. Thus, in some embodiments, in certain instances when the diluent is neutral to the sample (i.e., distinct from the sample and its components and has no physical, chemical, or biological effect on the sample), the calibration marker can be any analyte contained in the sample, such as, but not limited to, proteins, peptides, DNA, RNA, nucleic acids, inorganic molecules and ions, small organic molecules, cells, tissues, viruses, nanoparticles of different shapes, and any combination thereof.

In other embodiments, if the diluent is not neutral to the sample, the calibration marker may be selected from the analytes contained in the sample based on the physical, chemical, and/or properties of both the analyte and the diluent.

More details of analytes that can be used as calibration markers have been filed in U.S. provisional application serial No. 62/202,989, 2015, 8/10, 62/218,455, 2015, 9/14, 62/293,188, 2016, 2/9, and 62/305,123, 2016, 3/8, the entire disclosures of which are incorporated herein by reference for all purposes.

Use of 3.2QMAX device

The concentration measuring means in the method of the invention may be any type of device or apparatus which determines the concentration of the calibration marker in the sample or diluted sample, respectively. In some embodiments, it may comprise determining a first portion of a volume (V) of a portion or all of a sample to be analyzed, determining a second portion of the sample in which the portion or all contains an amount of a Calibration Marker (CM), and configured to calculate a concentration of the calibration marker (ICM) based on the determined values of V and CM, CM) ═ CM/V.

In some embodiments of the invention, the concentration measurement tool may be a CROF (compressed regulated open flow) device, or otherwise designated QMAX (Q: quantitative, M. multiplexed, A. reagent addition, and X: accelerated) device, such as, but not limited to, U.S. provisional patent application Ser. No. 62/202,989 filed on 8/10/2015, U.S. provisional patent application Ser. No. 62/218,455 filed on 14/2015 9/2015, U.S. provisional patent application Ser. No. 62/293,188 filed on 9/2016, U.S. provisional patent application Ser. No. 62/305,123 filed on 8/2016, U.S. provisional patent application Ser. No. 62/369,181 filed on 31/2016, U.S. provisional patent application Ser. No. 62/394,753 filed on 15/2016, PCT application (assigned USA) filed on 10/2016 (U.S.) 2016, CROF devices and QMAX devices are disclosed in PCT application No. PCT/US2016/051775 filed on 14/9/2016 (assigned U.S.), PCT application No. PCT/US2016/051794 filed on 15/9/2016 (assigned U.S.), and PCT application No. PCT/US2016/051794 filed on 27/9/2016 (assigned U.S.), PCT/US2016/054025, all of which are incorporated herein by reference in their entirety for all purposes.

In some embodiments, the QMAX device comprises:

a first plate and a second plate, wherein:

i. the plates are movable relative to each other into different configurations;

one or both plates are flexible;

each plate having on its respective surface a sample contacting area for contacting a sample with an analyte;

one or both of the plates includes a spacer secured to the respective plate,

wherein the spacers have a predetermined substantially uniform height and a predetermined constant spacer pitch, and wherein at least one of the spacers is within the sample contact area; and

a detector for detecting an analyte;

wherein one of the configurations is an open configuration, wherein: the two plates are separated, the spacing between the plates is not adjusted by spacers, and the sample is deposited on one or both plates; and

wherein the other of the configurations is a closed configuration configured after deposition of the sample in the open configuration; and in the closed configuration: at least one part of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant with respect to the plates, wherein the uniform thickness of the layer is limited by the inner surfaces of the two plates and is regulated by the plates and the spacers and has a small variation of average thickness equal to or less than 5 μm; and wherein in the closed configuration, the detector detects an analyte in at least a portion of the sample.

Fig. 14 shows an embodiment of a QMAX apparatus comprising a first board 10 and a second board 20. In particular, fig. (a) shows a perspective view of a first plate 10 and a second plate 20, wherein the first plate has spacers. It should be noted, however, that the spacers may also be fixed on the second plate 20 (not shown) or on both the first plate 10 and the second plate 20 (not shown). Figure (B) shows a perspective view and a cross-sectional view of a sample deposited on the first plate in an open configuration; it should be noted, however, that the sample 90 may also be deposited on the second plate 20 (not shown), or on both the first plate 10 and the second plate 20 (not shown). Graph (C) shows (i) spreading of sample 90 (sample flowing between the inner surfaces of the plates) and reduction of sample thickness using first plate 10 and second plate 20, and (ii) adjustment of sample thickness using spacers and plates in the closed configuration of the QMAX device. The inner surface of each panel may have one or more binding sites and/or storage sites (not shown).

In some embodiments, the spacers 40 have a predetermined uniform height and a predetermined uniform spacer spacing. In the closed configuration, as shown in fig. 14(C), the spacing between the plates, and thus the thickness of the sample 910, is adjusted by the spacer 40. In some embodiments, the uniform thickness of the sample 910 is substantially similar to the uniform height of the spacer 40.

In some embodiments of the present invention, when the QMAX means is adapted to obtain the first value, the obtaining step may comprise:

(a) acquiring a concentration measurement tool, namely a QMAX device;

(b) depositing a sample on the sample contact area of one or both plates in an open configuration;

(c) compressing the relevant volume of deposited sample into a layer of uniform thickness by bringing the two plates into a closed configuration;

(d) determining an amount of the calibration marker in a portion or all of the thickness layer by detecting the calibration marker using a detector;

(e) estimating a volume of part or all of the thickness layer by timing a predetermined uniform height of the spacer and a lateral area of part or all of the thickness uniform layer;

(f) obtaining a first value by dividing the amount of the calibration marker determined in step (d) by the volume estimated in step (e).

In some embodiments, when a QMAX apparatus is used to obtain the second value, the obtaining step may comprise similar steps as described above, except that the diluted sample is the material to be deposited, compressed, and analyzed rather than the sample.

3.3 determination of dilution factor of blood samples

Fig. 15 is a flow chart of an exemplary embodiment of a method of determining a dilution factor of a blood sample according to the present invention. The method comprises the following steps:

(i) providing a blood sample containing a calibration marker, the calibration marker having an unknown concentration;

(ii) obtaining a first concentration using a concentration measurement toolValue C₁The first value is the concentration of the calibration marker in the blood sample;

(iii) providing a diluent having an unknown volume of the diluent,

(iv) diluting the sample with a diluent to form a diluted blood sample;

(v) (iv) after (iv), obtaining a second value C using a concentration measurement tool₂The second value is the concentration of the calibration marker in the diluted blood sample; and

As described above, when determining the dilution factor of a blood sample, the calibration marker may be selected from any analyte contained in the blood sample, as long as the addition of the diluent has no physical, chemical or biological effect on the detectable amount of the calibration marker. One or any combination of the groups comprises Red Blood Cells (RBCs), White Blood Cells (WBCs), and Platelets (PLTs).

According to some embodiments of the invention, a QMAX device may be used to measure the concentration of RBCs, WBCs, and/or PLTs before and after diluting a blood sample. Methods for determining RBC, WBC and/or PLT concentrations using QMAX devices include, but are not limited to, US provisional patent application No. 62/202,989 filed on day 8/10 2015, US provisional patent application No. 62/218,455 filed on day 9/2015, US provisional patent application No. 62/293,188 filed on day 9/2016, US provisional patent application No. 62/305,123 filed on day 8/2016, US provisional patent application No. 62/369,181 filed on day 31/7/2016, US provisional patent application No. 62/394,753 filed on day 15/9/2016, PCT application No. PCT/US2016/045437 filed on day 10/2016 (assigned US), PCT application No. PCT/US2016/051775 filed on day 14/2016, PCT application No. PCT/US2016 (assigned US) filed on day 15/9/2016, and those disclosed in PCT/US2016/054.025 filed on 9, 27, 2016, the entire disclosures of which are incorporated herein by reference for all purposes.

3.4 example: determination of dilution factor of human blood samples using RBC and WBC

As disclosed in the experiments below, exemplary devices and methods for determining the dilution factor of a human blood sample have been implemented. In these experiments, fresh human blood samples were taken and diluted in saline solution by different predetermined dilution factors. RBCs and WBCs were used as calibration markers, respectively, to determine the dilution factor in each diluted blood sample. Briefly, their concentrations were measured in all samples (including undiluted and diluted blood samples) using a QMAX instrument.

Thus, the measured RBC and WBC concentrations were used to determine the dilution factor for each diluted sample separately. Finally, to check the quality of the calculated dilution factors, they are compared with the predetermined dilution factor for each diluted sample. The effectiveness of the method and apparatus provided by the present invention is clearly demonstrated by the fact that the calculated dilution factors all show a close resemblance to the predetermined dilution factor for each diluted sample.

E-1. materials and methods

QMAX device: the QMAX apparatus used in this experiment contained: 1) a planar glass substrate (25.4mm by 25.4mm surface, 1mm thick), and 2) an X plate, which is a planar PMMA plate (25.4mm by 25.4mm surface, 175 μm thick), having on one of its surfaces a regular array of spaced pillars spaced apart by a distance of 80 μm. Each spacer pillar was rectangular in shape, having an almost uniform cross section and rounded corners (side surface: 30 μm. times.40 μm, height: 2 μm).

Acridine orange dye: acridine Orange (AO) is a stable dye with natural affinity for nucleic acids. When bound to DNA, AO intercalates into DNA as a monomer and produces intense green fluorescence under blue excitation. (for White Blood Cells (WBC), 470nm excitation, 525nm green emission). When bound to RNA and proteins, they form electrostatic complexes in the form of polymers, which produce red fluorescence under blue excitation. (WBC and Platelets (PLT), 470nm excitation, 685nm red emission). As a result, Red Blood Cells (RBCs) do not stain because they have no nuclei and therefore few nucleic acids; WBCs stain strongly because they have significant amounts of nucleic acid; PLTs weakly stained the small amount of RNA they had.

Sample processing, dilution and imaging: fresh human blood samples were obtained by piercing the fingers of human subjects and then staining with AO dye. Briefly, it was mixed with AO (100. mu.g/mL in PBS) at a ratio of 1:1 for 1 minute.

After staining, the sample was divided into 5 portions, one of which was labeled "undiluted sample", and the remaining portions were each diluted with 0.9% sodium chloride solution in one of the following ratios: 1:2 ("2X diluted sample"), 1:5 ("5X diluted sample"), 1:10 ("10X diluted sample"), 1:20 ("20X diluted sample").

Using an Eppendorf pipette, 1 μ Ι _ of each blood sample was transferred to the center of the substrate, and then an X-plate was placed on top of the substrate with the blood drop, with the spacer pillars facing the blood drop on the substrate, covering most of the area of the substrate. The two plates are then pressed evenly against each other with a human hand for 10 seconds and then released, after which the two plates self-hold in the same configuration, possibly due to forces between the two plates, such as capillary forces. An imaging system consisting of a commercial DSLR camera (Nikon), two filters, a light source and a magnifying/focusing lens set was used to take pictures of the blood sample deposited between the two plates in bright field mode and fluorescence mode, counting RBCs and WBCs, respectively. In bright field mode, a broadband white light xenon lamp light source without any filters is used. In the fluorescent mode, the excitation source is a xenon lamp with an excitation filter of 470 + -20 nm

And the emission filter is a 500nm long pass filter

E-2. results and discussion

Here, the dilution factor of each diluted human blood sample was determined using the method and QMAX apparatus provided by some embodiments of the present invention.

1. The concentrations of RBC and WBC in each sample (including undiluted and serially diluted samples) were measured using a QMAX instrument.

Number of: RBCs deposited in a QMAX device correlate in bright field modeThe product was counted, while WBCs were counted in fluorescence mode. Fig. 16 shows representative images of undiluted (a) and 10X diluted (b) samples acquired in bright field mode. From the image, RBCs are readily identifiable as defined by their darker circular borders and relatively lighter centers of contrast, while the fixed-interval aligned circular rectangles are the spacer pillars on the X-plate. It should be noted that the number of RBCs in fig. 16(a) is significantly less than fig. 16(b), indicating that the 10X diluted sample is indeed more diluted and the RBC concentration is lower than the undiluted sample.

Volume of: given that the distance between the two plates is the height of the posts when the two plates are manually pressed to enter the closed configuration of the device, the relevant volume of deposited sample is easily calculated based on the predetermined size, height and pattern of the spaced post array.

Concentration of: the concentration of RBCs in each sample was then quantified as the quotient of the measured number of RBCs and the associated volume, as summarized in table a1, and the concentration of WBCs in each sample was similarly quantified using the count of WBCs in the associated volume (table a 2).

2. The concentration of RBC and WBC was used to determine the dilution factor for each diluted sample separately (tables a1 and a 2). Specifically, to calculate the dilution factor based on RBC, the measured concentration of RBC in each diluted sample was compared to its concentration in the undiluted sample (table a1, N/a ═ not applicable). For the dilution factors from WBCs, the measured concentration of WBCs in each diluted sample was compared to its concentration in the undiluted sample (table a2, N/a not applicable).

3. The dilution factors calculated from RBC and WBC are then compared to predetermined dilution factors in each sample, respectively. For each diluted sample, the percentage difference of the method using RBCs (dilution factor calculated from RBCs-predetermined dilution factor/predetermined dilution factor x 100%) was calculated (table a 2). The percentage difference of the method using WBCs (dilution factor calculated from WBCs-predetermined dilution factor/predetermined dilution factor x 100%) was also calculated (table a 2). As shown in tables a1 and a2, no percent difference exceeded 5%, demonstrating the effectiveness of the method and apparatus for determining dilution factors provided by the present invention.

Table a1. concentration of rbc and calculated dilution factor

TABLE A2. concentration of WBC and calculated dilution factor

In summary, the methods and devices for determining dilution factor in human blood samples were examined in the above exemplary experiments involving the use of RBC and WBC as calibration markers, respectively, and a QMAX device. The resulting dilution factors showed significant similarity to the predetermined dilution factor for each diluted sample, demonstrating the effectiveness of the methods and devices provided by the present invention.

Example overview for assay dilution calibration

4.1 method for determining dilution factor of diluted sample

Example 10: a method for determining a dilution factor of a diluted sample, comprising the steps of:

(i) providing an initial sample containing a calibration marker, the calibration marker having a first concentration of a known preset value;

(ii) diluting the initial sample with an unknown volume of diluent to form a diluted sample;

(iii) (iii) after (ii), obtaining a second concentration of the calibration marker in the diluted sample using a concentration measurement device; and

(iv) determining a dilution factor for the diluted sample by comparing the first concentration and the second concentration.

In the method as described in example 10, the preset value is an estimated normal value that differs by less than 5% from the true value of the first concentration.

Example 11: a method for determining a dilution factor of a diluted sample, comprising the steps of:

(i) providing an initial sample containing a calibration marker, the calibration marker having an unknown concentration;

(ii) a first concentration of a calibration marker in an initial sample is obtained using a concentration measurement device,

(iii) diluting the initial sample with an unknown volume of diluent to form a diluted sample;

(iv) (iv) after (iii), obtaining a second concentration of the calibration marker using the concentration measurement device; and

(v) the dilution factor is determined by comparing the first concentration and the second concentration.

The method as in example 10 or example 11, the initial sample being made of a material selected from the group consisting of: cells, tissues, stool, amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, inflammatory secretions, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled condensate.

In the method of any preceding embodiment, the sample is an environmental liquid sample from a source selected from the group consisting of: a river, lake, pond, sea, glacier, iceberg, rain, snow, sewage, reservoir, tap or potable water, a solid sample from soil, compost, sand, rock, concrete, wood, brick, sewage, and any combination thereof.

In the method of any preceding embodiment, the sample is an ambient gas sample from a source selected from the group consisting of: air, underwater heat rejection, industrial waste gas, vehicle waste gas, and any combination thereof.

In the method of any preceding embodiment, the sample is a food selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, and partially or fully processed foods, and any combination thereof.

In the method of any preceding embodiment, the calibration marker is selected from the group consisting of: proteins, peptides, DNA, RNA, nucleic acids, inorganic molecules and ions, small organic molecules, cells, tissues, viruses, nanoparticles having different shapes, and any combination thereof.

In the method of any preceding embodiment, the concentration measurement device comprises: a first plate and a second plate, wherein:

v. the plates may be moved relative to each other into different configurations;

one or both plates are flexible;

each plate having on its respective surface a sample contacting area for contacting a sample containing an analyte;

one or both of the plates includes a spacer secured to the respective plate,

a detector for detecting an analyte;

wherein one of the configurations is an open configuration in which the two plates are partially or fully separated, the spacing between the plates is not adjusted by spacers, and the sample is deposited on one or both of the plates; and is

Wherein the other of the configurations is a closed configuration configured after deposition of the sample in the open configuration; and in the closed configuration: at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant with respect to the plates, wherein the layer of uniform thickness is bounded by the inner surfaces of the two plates and is conditioned by the plates and spacers and has a small variation of average thickness equal to or less than 5 μm;

wherein in the closed configuration, the detector detects an analyte in at least a portion of the sample and calculates a concentration of the analyte in the sample.

In the method of any preceding embodiment, the step of obtaining the first concentration comprises:

(a) acquiring a concentration measuring device;

(b) depositing an initial sample on the sample contact area of one or both plates in an open configuration;

(c) compressing the relevant volume of the deposited initial sample into a layer of uniform thickness by bringing the two plates into a closed configuration;

(e) estimating a volume of part or all of the thickness layer by timing a lateral region of the spacer of a predetermined uniform height and part or all of the thickness layer;

(f) obtaining a first concentration by dividing the amount of the calibration marker determined in step (d) by the volume estimated in step (e).

In the method of any preceding embodiment, the step of obtaining the second concentration comprises:

(a) acquiring a concentration measuring device;

(b) depositing the diluted sample on the sample contact area of one or both plates in an open configuration;

(c) compressing the relevant volume of the deposited diluted sample into a layer of uniform thickness by bringing the two plates into a closed configuration;

(f) obtaining a second concentration by dividing the amount of the calibration marker determined in step (d) by the volume estimated in step (e).

4.2 method for determining dilution factor of blood sample

Example 12: a method for determining a dilution factor of a blood sample, comprising:

(i) providing an initial blood sample containing a calibration marker, the calibration marker having an unknown concentration;

(ii) a first concentration of a calibration marker in an initial blood sample is obtained using a concentration measurement device,

(iii) diluting an initial blood sample with an unknown volume of diluent to form a diluted blood sample;

(iv) (iii) after (iv), obtaining a second concentration of the calibration marker in the diluted blood sample using a concentration measurement device; and

In the method of example 12, the calibration marker is selected from the group consisting of: red blood cells, white blood cells, platelets, and any combination thereof.

In the method of embodiment 12 or any derivative thereof, the concentration measurement device comprises:

a first plate and a second plate, wherein:

i. the plates are movable relative to each other into different configurations;

one or both plates are flexible;

one or both of the plates includes a spacer secured to the respective plate,

wherein the spacers have a predetermined substantially uniform height and a predetermined constant spacer pitch in the range of 7 μm to 200 μm, and wherein at least one of the spacers is within the sample contact area and has a small variation of an average thickness equal to or less than 5 μm; and

a detector for detecting an analyte;

wherein the other of the configurations is a closed configuration configured after deposition of the sample in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant with respect to the plates, wherein the uniform thickness of the layer is bounded by the inner surfaces of the two plates and is regulated by the plates and the spacer; and

wherein in the closed configuration, the detector detects an analyte in at least a portion of the sample.

In the method of example 12 or any derivative thereof, the step of obtaining a first concentration comprises:

(a) acquiring a concentration measuring device;

(b) depositing an initial blood sample on the sample contacting area of one or both plates in an open configuration;

(c) compressing the relevant volume of deposited initial blood sample into a layer of uniform thickness by bringing the two plates into a closed configuration;

In the method of example 12 or any derivative thereof, the step of obtaining a second concentration comprises:

(a) acquiring a concentration measuring device;

(b) depositing a diluted blood sample on the sample contacting area of one or both plates in an open configuration;

(c) compressing the relevant volume of deposited diluted blood sample into a layer of uniform thickness by bringing the two plates into a closed configuration;

In the method of embodiment 12 or any derivative thereof, wherein the spacer that adjusts the uniform thickness layer has a fill factor of at least 1%, the fill factor is the ratio of the area of the spacer in contact with the uniform thickness layer to the total plate area in contact with the uniform thickness layer.

In the method as in embodiment 12 or any derivative thereof, wherein for tuning the spacer of the uniform thickness layer, the young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the area of the spacer in contact with the uniform thickness layer to the total plate area in contact with the uniform thickness layer.

In the method as in embodiment 12 or any derivative thereof, wherein for the flexible sheet, the thickness of the flexible sheet times the young's modulus of the flexible sheet is in a range from 60 to 750GPa- μm.

In the method of embodiment 12 or any derivative thereof, wherein for the flexible sheet, the fourth power of the spacer spacing (ISD) divided by the thickness (h) of the flexible sheet and the Young's modulus (E) of the flexible sheet, ISD4/(Δ E), is equal to or less than 106 μm3/GPa,

in the method of example 12 or any derivative thereof, one or both plates comprise position markers located on or within the surface of the plate, the position markers providing information on the position of the plate.

In a method as described in example 12 or any derivative thereof, one or both plates comprise graduation marks on or within the surface of the plate, the graduation marks providing information on the lateral dimensions of the sample and/or the structure of the plate.

In the method of example 12 or any derivative thereof, one or both plates comprise imaging indicia on or within the surface of the plate, the imaging indicia facilitating imaging of the sample.

In the method of embodiment 12 or any derivative thereof, the spacer is used as a position mark, a scale mark, an imaging mark, or any combination thereof.

In the method of example 12 or any derivative thereof, wherein the average thickness of the thickness uniformity layer is in the range of 2 μ ι η to 2.2 μ ι η and the sample is blood.

In the method of example 12 or any derivative thereof, wherein the average thickness of the thickness uniformity layer is in the range of 2.2 μm to 2.6 μm and the sample is blood.

In the method of example 12 or any derivative thereof, wherein the average thickness of the thickness uniformity layer is in the range of 1.8 μm to 2 μm and the sample is blood.

In the method of example 12 or any derivative thereof, wherein the average thickness of the thickness uniformity layer is in the range of 2.6 μm to 3.8 μm and the sample is blood.

In the method as described in example 12 or any derivative thereof, the uniform thickness layer has an average thickness in the range of 1.8 μm to 3.8 μm, and the sample is whole blood without dilution with another liquid.

In the method of example 12 or any derivative thereof, the average thickness of the thickness uniformity layer is about equal to the smallest dimension of the analyte in the sample.

In the method of embodiment 12 or any derivative thereof, the spacer pitch is in a range from 7 μm to 50 μm.

In the method of embodiment 12 or any derivative thereof, the spacer pitch is in a range from 50 μm to 120 μm.

In the method of embodiment 12 or any derivative thereof, the spacer pitch is in a range from 120 μm to 200 μm.

In the method of embodiment 12 or any derivative thereof, the spacer pitch is substantially fixed.

In the method of embodiment 12 or any derivative thereof, the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

In the method of embodiment 12 or any derivative thereof, wherein the spacers have a columnar shape and have a substantially flat top surface, wherein for each spacer, a ratio of a lateral dimension of the spacer to a height thereof is at least 1.

In the method of embodiment 12 or any derivative thereof, each spacer has a ratio of a lateral dimension of the spacer to a height thereof of at least 1.

In the method of example 12 or any derivative thereof, the smallest lateral dimension of the spacer is less than or substantially equal to the smallest dimension of the analyte in the sample.

In the method of embodiment 12 or any derivative thereof, the minimum lateral dimension of the spacer is in a range from 0.5 μm to 100 μm.

In the method of embodiment 12 or any derivative thereof, the minimum lateral dimension of the spacer is in a range from 0.5 μm to 10 μm.

In the method of example 12 or any derivative thereof, wherein the sample layer having a uniform thickness is at least 1mm²Is uniform in lateral area.

5 composite liquid sample separation device and method

5.1 composite liquid sample separation device

In one aspect, the present invention also provides a device for separating components from a composite liquid sample, comprising: a collecting plate having a plurality of columnar spacers on one surface thereof; and a filter having a sample receiving surface and a sample ejection surface, wherein at least a portion of the cylindrical spacers of the collection plate are in contact with and directed toward the sample ejection surface, forming a microcavity bounded by the sample ejection surface and the portion of the cylindrical spacers, wherein the microcavity provides a capillary force that is at least a first portion of a driving force for flowing at least a portion of the sample deposited on the sample receiving surface through the filter toward the collection plate, and wherein the filter is configured to separate the component from the portion of the sample.

Fig. 17(a) shows an exemplary embodiment of an apparatus, wherein the apparatus comprises a collection plate 10 and a filter 70. As shown in fig. (a), in some embodiments, the collection sheet 10 has an inner surface 11, an outer surface 12, and a plurality of columnar spacers 41 on its inner surface 11. The filter 70 has a sample receiving surface 71 and a sample discharge surface 72. In some embodiments, the cylindrical spacers 41 are fixed to the inner surface 11. At least a portion of the cylindrical spacer 41 is directed toward and in contact with the sample discharge surface 72 of the filter 70, forming a microcavity 107 defined by the sample discharge surface 72 and the portion of the cylindrical spacer 41.

Fig. 17(B) further shows an exemplary embodiment of a device in which a composite liquid sample 90 containing components to be removed 901 is deposited on the sample receiving surface 71 of the filter 70. According to the present invention, filter 70 is configured to separate components 901 from portions of sample 90 as sample 90 flows from sample receiving surface 7110 through filter 70 to the collection plate. As shown in figure (B), in some embodiments, at least a portion of the sample 90 is driven by a driving force to flow through the filter 70 in a direction from the sample receiving surface 71 toward the sample discharge surface 72 and the collection plate 10. As a portion of sample 90 flows through filter 70, component 901 is retained by filter 70 and/or removed from filtered product 900, which filtered product 900 is a portion of the sample that exits filter 70. In some embodiments, the microcavities 107 and/or filters 70 provide a capillary force that is at least a portion of the driving force. In some embodiments, the capillary force provided by microcavities 107 and/or filters 70 is the only and all part of the driving force. However, in other embodiments, the capillary force from the microcavities 107 and/or filters 70 is only a fraction of the driving force, sometimes even a negligible fraction of the driving force.

As shown in fig. 17(a) and (B) and described herein, the features described for the common device also apply to the embodiments shown and described in all of the other figures 17, 18 to 20. Furthermore, it should be noted that this device serves as an example of the features shown and described in all the figures.

Fig. 17(C1) through (C4) schematically illustrate various embodiments of the device disclosed herein, wherein the device further comprises a source that provides at least a portion of the driving force for causing at least a portion of the sample 90 to flow through the filter 70 toward the collection plate 10. Different exemplary embodiments of such sources are shown from fig. (C1) to fig. (C4), respectively. The exemplary sources disclosed herein are in no way meant to be exclusive of other possible embodiments and combinations of any of these sources with other embodiments. The sources disclosed herein are deployed separately, alternatively, sequentially or in combination, or in any other manner so long as they serve their primary function, i.e., providing at least a portion of the driving force for causing sample flow for component separation by filter 70.

As shown in fig. 17(C1), in some embodiments, the device further comprises a source (not shown) providing a first liquid 81, the first liquid 81 having low, if not zero, miscibility with the sample 90 and being configured to provide at least a portion of the driving force. For example, where sample 90 is a water-based solution, first liquid 81 may be selected from various types of hydrocarbon oils, including but not limited to mineral oil, gasoline and related products, vegetable oils, and any mixtures thereof. In some embodiments, the first liquid 81 has a higher density than the sample 90, and it drives the sample flow due to its own weight. In some embodiments, the first liquid 81 is subjected to the greater capillary forces provided by the microcavity 107 and/or the filter 70, and is thus capable of driving the sample 90 flow. In other embodiments, the first liquid 81 is pressurized and pressure is applied to the filter 70 and the collection plate 10, thus forcing the sample 90 to flow toward the collection plate.

In other embodiments, first liquid 81 has high miscibility with sample 90, as long as it is configured to drive a portion of sample 90 through filter 70, e.g., it can be highly pressurized. It should be noted, however, that this type of configuration may compromise the quality of the filtered product 900, e.g., the filtered product 900 may be contaminated by the first liquid 81, and thus the analyte in the filtered product 900 may be contaminated by the first liquid 81, which may be undesirable in most applications, physically or chemically diluted and/or altered.

As shown in fig. 17(C2), in some embodiments, the device further comprises a source (not shown) providing a pressurized gas 82, the pressurized gas 82 configured to provide at least a portion of the driving force. As shown, in some embodiments, the pressurized gas 82 is applied over at least a portion of the sample 90 in a direction from the sample receiving surface 71 toward the sample discharge surface 72.

In some embodiments, the device further comprises a sponge for providing at least a portion of the driving force. As used herein, the term "sponge" refers to a flexible porous material that has pores that change their shape under force, and when the shape of the pores changes, the flexible porous material can absorb or release liquid into or from the material. Sponges typically have an uncompressed state and a compressed state. In the uncompressed state, the porous structure of the sponge reaches its maximum internal dimension, i.e. in the absence of major external influences, the internal pores are in their maximum shape with their maximum possible volume therein, while in the compressed state, in some embodiments, the sponge is subjected to an external compression force and, as a result, the internal pores of the sponge are compressed and deformed into a shape with a dimension smaller than the maximum internal dimension. The main external influence is any external influence that deforms the inner pores of the sponge. When the sponge is deformed in a direction from its compressed to uncompressed state, the sponge can absorb any liquid in fluid communication therewith; when the sponge is deformed in the opposite direction from its uncompressed to compressed state, the sponge releases the liquid it contains.

For example, fig. 17(C3) shows some embodiments of the device, wherein the device further comprises a sponge 50. As described above, the sponge 50 has an uncompressed state and a compressed state. In some embodiments, the sponge 50 is movable into different configurations relative to the collection plate and filter:

(i) one of the configurations is a deposition configuration (not shown), in which: the sponge 50 is in an uncompressed state and is partially or completely separated from the collection plate 10 and the filter 70, the distance between the collection plate 10 and the sponge 50 is not adjusted by the spacer 41, the filter 70 or the deposited sample 90,

(ii) another of the configurations is a filtration configuration, wherein: as shown in (C3), the filter 70 is located between the sponge 50 and the collection sheet 10, the distance between the collection sheet 10 and the sponge 50 is adjusted by the spacer 41, the filter 70, and the deposited sample 90, and the sponge 50 is in a compressed state, which is configured to provide at least a portion of the driving force.

According to these embodiments, in the deposition configuration, when placed in contact with the sample 90, the sponge 50 absorbs the liquid sample such that a portion or all of the sample 90 enters the sponge 50, as shown. When the sponge 50, collection sheet 10 and filter 70 enter their filtering configuration (i.e., the sponge 50 is compressed to its compressed state by the compressive force, and the distance between the collection sheet 10 and the sponge is adjusted 50 by the spacer 41, the filter 70 and the deposited sample 90), a portion of the sample 90 absorbed in the sponge 50 is forced to exit the sponge 50 and flow through the filter 70 toward the collection sheet 10. Thus, assembly 901 is retained and/or removed from filtered product 900. In some embodiments, a compressive force is exerted on the sponge 50 in a direction against the filter 70. In other embodiments, the compressive force is applied to the sponge 50 in any other direction so long as the sample 90 is forced through the filter 70 toward the collection plate 10.

Fig. 17(C4) shows an additional embodiment of the apparatus, wherein the apparatus further comprises a platen 20, the platen 20 having a plurality of spacers 42 on one surface thereof. In some embodiments, the pressure plate 20 is relatively movable with respect to the collection plate 10 and the filter 70 into different configurations:

(i) one of the configurations is a deposition configuration in which the platen 20 is partially or completely separated from the collection plate 10 and filter 70, and the distance between the collection plate 10 and platen 7 is not adjusted by their

spacers

41 and 42, filter 70, or deposition sample 90.

(ii) Another of the configurations is a filtration configuration, wherein: as shown in fig. 1(C4), the filter 70 is located between the pressing plate 20 and the collection plate 10, the distance between the collection plate 10 and the pressing plate 20 is adjusted by their

spacers

41 and 42, the filter 70, and the deposited sample 90, and at least a portion of the columnar spacer 42 and the inner surface 21 of the pressing plate press at least a portion of the deposited sample 90 against the filter 70, providing at least a portion of the driving force.

Fig. 17(C4) shows that, in some embodiments, collection sheet 10, filter 70, and pressure plate 20 are brought into a filtering configuration by a compressive force exerted on pressure plate outer surface 22 and collection sheet outer surface 12. In the filter configuration, platen cylindrical spacers 42 are directed toward and in contact with filter 70 and at least a portion of deposited sample 90. The distance between platen inner surface 11 and sample receiving surface 71 is reduced to about the height of cylindrical spacer 42. In some embodiments, in the filtering configuration of the device, at least a portion of the deposited sample 90 is forced to flow through the filter 70 toward the collection plate 10 for one of the following reasons, any combination thereof, or any other possibility: (a) the height of the columnar spacer 42 is configured to be smaller than the unlimited height of the deposition sample 90; (b) the filter 70 is configured to have a relatively low obstruction such that the deposited sample 90 flows through it in a direction from the sample receiving surface 71 towards the sample discharge surface 72; (c) microcavity 107 is configured to provide a relatively high capillary force to draw the sample flow toward collection plate 10, and (d) column spacer 42 is configured to provide a relatively high resistance to lateral flow of deposited sample 90.

X plate

In some embodiments of the invention, the collection plate is also referred to as an "X-plate". It is a plate comprising on its surface (i) spacers having a predetermined spacer pitch and a predetermined height and being fixed on the surface, and (ii) a sample contact area for contacting a sample to be deposited, wherein at least one spacer is located within the sample contact area.

In some embodiments, the second plate is an X plate. Thus, in these embodiments, in the filtration configuration of the device, the pressure plate, filter and collection plate become a sandwich-like structure, with the filter being compressed in the center by two X-plates.

The details of the X plate, including but not limited to the thickness, shape and area of the plate, flexibility, surface flatness and wettability, height, lateral dimensions, voids of the columnar spacers, materials and mechanical strength of the plate and columnar spacers, are predetermined to provide the appropriate partial driving force for the flow of the deposited sample from the platen side through the filter to the collector side.

In some embodiments, the X-plates include, but are not limited to, the embodiments described in U.S. provisional patent application No. 62/202,989 filed on 8/10/2015, U.S. provisional patent application No. 62/218,455 filed on 9/2015 9/14/2015, U.S. provisional patent application No. 62/293,188 filed on 2/9/2016, U.S. provisional patent application No. 62/305,123 filed on 8/2016 3/2016, U.S. provisional patent application No. 62/369,181 filed on 31/2016, U.S. provisional patent application No. 62/394,753 filed on 15/2016, PCT application No. 5639/2016 filed on 10/2016, PCT application No. PCT/US2016/051775 filed on 14/2016 (assigned US) filed on 9/15/2016, and PCT/US 2016/2016, the entire disclosures of these documents are incorporated herein by reference in their entirety for all purposes.

Filter

As used herein, the term "filter" refers to a device having at least a sample receiving surface and a sample discharge surface, and which removes certain components from a composite liquid sample as the liquid sample flows through the filter in a direction transverse to both the first and sample discharge surfaces. According to the invention, the filter may be a mechanical, chemical or biological filter, or any combination thereof.

In some embodiments of the invention, the filter may be a mechanical filter. Mechanical filters mechanically remove, trap or block certain solid components from the composite liquid sample as the sample flows through the filter in a certain direction. Typically made of a porous material, and the pore size determines the size of the solid particles that can flow through the filter and the size of the solid particles that are removed from the sample flowing through the filter. The components of the mechanical device are inert so that they do not affect or interfere with the sample. Examples of mechanical filters include, but are not limited to, foam (reticulated and/or open-celled), fibrous material (e.g., filter paper), gel, sponge. Examples of materials include cellulose acetate, cellulose esters, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, any other material that can form a porous structure, and any combination thereof.

In some embodiments of the invention, the pore size of the mechanical filter is uniform or varies within a range having a predetermined distribution. In some embodiments, the average pore size of the mechanical filter is 10nm, 20nm, 40nm, 80nm, 100nm, 200nm, 400nm, 800nm, 1 μm, 2 μm, 4 μm, 8 μm, 10 μm, 20 μm, 40 μm, 80 μm, 100 μm, 500 μm, 1mm to 1cm, 5mm, or a range between any two values.

In some embodiments of the invention, the filter is a chemical filter that removes a particular component from the composite liquid sample when the sample flows through the composite liquid sample in a direction. In some embodiments, it comprises a chemical reactant and a housing for the chemical reactant. The chemical reactant reacts specifically with certain components to be removed from the sample. It is capable of binding and immobilizing the components, or transforming the components into other materials that are retained in the housing or released to the exterior of the housing and the filtration product. In some embodiments, the chemical reactant is an inorganic chemical, an organic chemical, or any combination thereof. In some embodiments, the chemical reactant is a biological material, including but not limited to antibodies, oligonucleotides, other biological macromolecules having affinity for components to be removed from the sample.

In some embodiments of the invention, the filter may be a mechanical filter. The biofilter includes a biologically active substance and a housing for the active substance. In some embodiments, the active agent specifically ingests, engulfs, or binds and immobilizes certain components in the sample. Exemplary active substances that can be used in the biofilter include, but are not limited to, bacteria, fungi, viruses, mammalian cells with phagocytic function or affinity binding properties, such as macrophages, T cells, and B cells.

5.2 composite liquid sample separation method

In another aspect, the present invention provides a method for complex liquid sample separation, comprising the steps of:

(1) providing a collection plate having a plurality of cylindrical spacers on one surface thereof, and a filter having a sample receiving surface and an opposing sample exit surface, wherein at least a portion of the cylindrical spacers of the collection plate are in contact with the sample exit surface and are directed toward the sample exit surface, thereby forming a microcavity bounded by the sample exit surface and the portion of the cylindrical spacers of the collection plate,

(2) depositing a sample on the sample receiving surface of the filter, an

(3) Driving at least a portion of the deposited sample through the filter toward the collection plate with a driving force, wherein the filter is configured to separate the component from the portion of the deposited sample, and wherein at least a first portion of the driving force is a capillary force provided by the microcavity.

FIG. 18 is a flow chart of an exemplary embodiment of a method disclosed in the present invention. In this embodiment, an exemplary apparatus as shown in fig. 17(a) is used.

First, the user of the device obtains a collection plate 10 having a plurality of cylindrical spacers 41 on one surface thereof, and a filter 70 having a sample receiving surface 71 and a sample discharge surface 72, wherein at least a portion of the cylindrical spacers 41 contact and are directed toward the sample discharge surface 72, forming a microcavity 107 defined by the sample discharge surface 72 and the collection plate 10. Next, a composite liquid sample 90 having components 901 to be separated from the sample is deposited on the sample receiving surface 71 of the filter 70. After the depositing step, at least a portion of sample 90 is driven by a driving force to flow through filter 70 to collection plate 10, wherein filter 70 is configured to separate component 901 from portion 90, producing filtered product 900, and wherein microcavity 107 is configured to provide a portion of the driving force.

In some embodiments, the partial driving force provided by microcavity 107 is the full driving force. In these embodiments, the driving step is actually letting the microcavity pull a portion of the sample 90 toward the collection plate 10 by capillary force, without any external influence.

In other embodiments, the partial driving force provided by microcavity 107 is only a fraction thereof, and thus another source is required to provide another partial driving force. For example, in some embodiments, gravity participates in driving the sample 90 through the filter 70 when the sample receiving surface 71 is farther from the ground than the sample discharge surface 72 and the collection plate 10. Or in other cases, another source is part of the apparatus described above, including but not limited to a source that provides the first liquid 81, a source that provides the pressurized gas 82, the sponge 50, and the platen 20. The driving force provided by these sources, as well as gravity, may be utilized individually, alternatively, sequentially, or in combination, or in any other manner so long as they serve their primary function, i.e., providing at least a portion of the driving force for flowing the sample for component separation through the filter 70. According to these embodiments, the driving step of the method further comprises providing and operating a source for providing at least a portion of the driving force.

In some embodiments, the driving step of the method comprises depositing a first liquid to contact the deposited sample, the first liquid having low miscibility with the sample and being configured to provide at least a part of the driving force.

In other embodiments, the driving step of the method comprises applying a pressurized gas to the deposited sample, the pressurized gas being configured to provide at least a portion of the driving force.

In other embodiments, the driving step of the method comprises: (a) contacting the sponge with the deposited sample; (b) the sponge is pressed against the filter to provide at least a portion of the driving force.

In other embodiments, the driving step of the method comprises: (a) placing a platen having a plurality of columnar spacers on one surface thereof in contact with the deposited sample, wherein at least a portion of the columnar spacers of the platen are directed toward the sample receiving surface of the filter and are in contact with the deposited sample; (b) after the placing step (a), pressing the pressure plate against the filter to reduce the distance between the pressure plate and the filter and to provide at least a portion of the driving force.

5.3 samples

The composite liquid sample according to the present invention comprises one or more components separated from the sample by the devices and methods provided herein.

The devices and methods disclosed herein are used with samples such as, but not limited to, diagnostic samples, clinical samples, environmental samples, and food samples. Types of samples include, but are not limited to, those described and outlined in PCT application No. PCT/US2016/045437 (assigned U.S.) filed on 10/8/2016, and incorporated herein by reference in its entirety.

In particular embodiments, the sample is obtained from a biological sample such as cells, tissue, body fluids, and stool. Typically, a sample in a non-liquid form is converted to a liquid form prior to analysis of the sample by the method of the invention. Bodily 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), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, sebum (skin oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine, and exhaled condensate. In particular embodiments, the sample is obtained from a subject, e.g., a human. In some embodiments, the subject is treated prior to use in an assay. For example, prior to analysis, proteins/nucleic acids are extracted from tissue samples prior to use, methods of which are known. In particular embodiments, the sample is a clinical sample, e.g., a sample collected from a patient.

In particular embodiments, the sample is obtained from an environmental sample, including, but not limited to, a liquid sample from a river, lake, pond, ocean, glacier, iceberg, rain, snow, sewage, reservoir, tap water, drinking water, etc., a solid sample from soil, compost, sand, rock, concrete, wood, brick, dirt, etc., and a gas sample from air, underwater heat sinking, industrial waste, vehicle waste, etc. Typically, a sample, if not in liquid form, is converted to liquid form prior to analysis of the sample by the method of the invention. In particular embodiments, the sample is obtained from a food sample suitable for consumption by an animal (e.g., hunnar corpumpitor). Food samples include, but are not limited to, raw materials, cooked foods, foods of plant and animal origin, pre-processed foods, partially or fully processed foods, and the like. Typically, a sample in a non-liquid form is converted to a liquid form prior to analysis of the sample by the method of the invention.

According to the present invention, the component to be separated from the sample may be solid, liquid, gaseous or any combination thereof. Components isolated from the sample include, but are not limited to ces, tissue, virus, bacteria, protein, DNA, RNA, gas bubbles, lipids.

In a preferred embodiment of the invention, the sample is a whole blood sample and the component separated from the whole blood sample is blood cells (red blood cells, white blood cells, platelets, etc.). Thus, if a preferred embodiment, the device and method are particularly configured for plasma separation.

According to the invention, the sample volume is 1 μ L or less, 2 μ L or less, 5 μ L or less, 10 μ L or less, 20 μ L or less, 50 μ L or less, 100 μ L or less, 200 μ L or less, 1mL or less, 2mL or less, 5mL or less, 10mL or less, 20mL or less, 50mL or less, 100mL or less, 200mL or less, 500mL or less, 1L or less, or a range between any two values.

5.4 filtration of the product

In some embodiments of the invention, the collection plate is an X-plate that is used for QMAX processing in addition to complex sample separation, further sensing the assay processing of the filtered product.

In QMAX (Q: quantitative; M: magnification; a: addition of reagents; X: acceleration; also known as compression-regulated open flow (CROF) processing or assay platform, the QMAX apparatus uses two plates to manipulate the shape of the sample into a thin layer (e.g., by compression).

In QMAX assays, one of the plate configurations is an open configuration, where the two plates are fully or partially separated (the spacing between the plates is not controlled by spacers) and a sample can be deposited. Another configuration is a closed configuration, in which at least a portion of the sample deposited in the open configuration is compressed by the two plates into a layer of very uniform thickness, the uniform thickness of the layer being defined by the inner surfaces of the plates and adjusted by the plates and spacers.

In some embodiments of the invention, after filtering the sample, the filter and the source providing the second portion of the driving force are separated from the collection plate. The filtration product is retained on the collection plate at least in part due to capillary forces and surface tension.

In some embodiments, the collection plate carrying the filtered product is combined with a capture plate to form a QMAX device: the collection and capture plates are movable relative to each other into different configurations, wherein one of the configurations is an open configuration, wherein the collection and capture plates are separated, the spacing between the plates is not adjusted by the spacers, wherein the other of the configurations is a closed configuration, wherein the plates face each other, the spacers and the filtration product are located between the plates, the thickness of the filtration product is adjusted by the plates and spacers and is thinner than when the plates are in the open configuration, and at least one of the spacers is located inside the sample.

In some embodiments of the invention, the capture plate is a flat glass plate and/or comprises binding sites or storage sites for binding agents, respectively, for detection agents for detection of the filtered product, and in some embodiments, the collection plate further comprises binding sites or storage sites for detection of the filtered product.

In some embodiments, QMAX devices formed of collection and capture plates after a filtration process include, but are not limited to, US provisional patent application No. 62/202,989 filed on day 8/10 2015, US provisional patent application No. 62/218,455 filed on day 9/2015 14, US provisional patent application No. 62/293,188 filed on day 9/2016, US provisional patent application No. 62/305,123 filed on day 8/2016, US provisional patent application No. 62/369,181 filed on day 31/7/2016, US provisional patent application No. 62/394,753 filed on day 15/9/2016, PCT application No. PCT/US2016/045437 filed on day 10/2016 (assigned US), PCT application No. PCT/US2016/051775 filed on day 14/2016, PCT application No. PCT/US2016 (assigned US) 2016, and PCT/US2016/054025 filed on 9, 27, 2016, the entire disclosures of which are hereby incorporated by reference in their entirety for all purposes.

5.5 advantageous effects of application

The apparatus and methods provided by the present invention may be used in a variety of different applications in a variety of fields where undesired components separated from a given complex liquid sample and/or desired components extracted from a given sample are supplied. For example, the devices and methods of the present invention may be used in assays involving plasma where separation of blood cells is desired, in applications where pure water without contaminating particles is desired, in applications involving the study of contaminating bacteria in drinking water and the like. Various fields include, but are not limited to, human, veterinary, agricultural, food, environmental, pharmaceutical testing, and the like.

The devices and methods provided by the present invention have many advantages over the prior art for complex liquid sample separation for a variety of reasons, including but not limited to: the apparatus and methods provided in some preferred embodiments may be relatively much simpler and easier to operate, do not require trained professionals, require much less time and lower cost, and in some particular embodiments. This is particularly good when dealing with small liquid samples.

In addition, the devices provided in some preferred embodiments of the present invention may be used to form QMAX devices, which may be used in a wide range of applications.

These applications include, but are not limited to, biochemical assays, quantitative sampling of liquid samples, biochemical processing, and biomarker detection.

The devices and methods disclosed herein have various types of biological/chemical sampling, sensing, assays, and applications, including but not limited to PCT application No. (assigned US) No. PCT/US2016/045437, filed 2016, 8, 10, and PCT/US16/51794, filed 2016, 9, 14, which are incorporated herein by reference in their entirety.

The devices and methods disclosed herein are used to detect, purify, and/or quantify analytes, such as, but not limited to, biomarkers. Examples of biomarkers include, but are not limited to, PCT application No. PCT/US2016/045437 (assigned U.S.) filed on 10/8/2016, incorporated herein by reference in its entirety.

The apparatus and methods disclosed herein are used with the facilitation and enhancement of mobile communication devices and systems, including those listed, described and outlined in PCT application No. (assigned US) PCT/US2016/045437, filed on 10/8/2016, and which is hereby incorporated by reference in its entirety.

5.6 example 1

Exemplary devices and methods for separating plasma from a whole blood sample according to the present invention have been implemented experimentally herein. In order to test and compare different experimental conditions for plasma separation, experiments were performed.

For this experiment, two different types of X-plates were used as collection plates according to the invention. Both made of PMMA, 175 μm thick and 1 inch by 1 inch wide. Type 1X plates have cubic column spacers on their surfaces that are 30X 40 μm wide and 30 μm high and spaced apart by 80 μm pitch (ISD). The type 2X plate has cubic columnar spacers on its surface, which have all the same parameters as type 1 except that the height is 2 μm.

Under some experimental conditions, a different X-plate selected from one of the two types was also used as the platen. Four types of filter membranes (all purchased from sterlett corp., Kent, WA and made of polycarbonate) with different pore sizes (0.4 μm, 1 μm, 2 μm and 3 μm) were used as filters for separating blood cells from plasma in a blood sample.

Whole blood samples were purchased commercially or freshly obtained by puncturing the fingers of human subjects. For all experimental conditions, during plasma separation, the filter membrane was placed on top of the collection plate, the collection plate was placed on a table with its column spacers pointing upwards, and then a drop of whole blood sample (1 μ L, using a platen with 2 μm high spacers and 3 μ L when using flat glass plates, sponges or platens with 30 μm high spacers) was deposited on top of the filter membrane for plasma separation. A flat glass plate, sponge or pressure plate is used as a pressure medium for providing a driving force to flow the blood sample through the filter membrane to the collection plate. The pressing medium is placed on top of the deposited blood sample and then manually pressed against the collection plate for a certain amount of time (30 or 180 seconds), thereby forcing the blood sample to flow through the filter membrane for plasma separation.

After manual pressurization for plasma separation, the top pressurizing medium and the filtration membrane were peeled off while the filtered product was left on the collection plate. Different flat glass plates ("capture plates", 1mm thick and 1 inch by 1 inch wide) were then placed in contact with the collection plate. Here, sample observation and quantification were then performed using QMAX treatment. The collection plate and capture plate were hand pressed against each other for 30 seconds and then "self-held" to form a QMAX device. The resulting QMAX apparatus carrying the filtered product was then imaged under an optical microscope and the volume of the filtered product was estimated accordingly.

11 different experimental conditions were tested in this experiment and the details of each condition are summarized in Table 2.

Figure 19 shows representative images of the filtration products produced by different experimental configurations of the device when used for plasma separation. The numbers in the upper left corner of each image represent the experimental set of numbers for it as listed in table 2, and the regularly arranged circular rectangles shown in each image are the columnar spacers of the collector plates. As the images show, the glass plate (group 1) apparently lysed the red blood cells in the sample, leaving a visible red filter product, group 11 showed blood cells in the filter product, indicating that the pore size (5 μm) was insufficient to filter out the blood cells, group 7 showed a small amount of plasma or blood, probably due to the over-sized sponge, which absorbed and retained most, if not all, of the blood sample. Plasma was obtained in all other groups: as can be seen from the images, panels 5 and 6 give the best results because the filtered product (plasma) forms a continuous membrane in the QMAX apparatus, and

panels

2, 3, 4, 8, 9 and 10 show mainly plasma droplets and sometimes a small amount of flaky plasma membrane, probably due to the 30 μm column height of the collection plate, compared to the 2 μm column height in panels 5 and 6.

TABLE 2 Experimental conditions

The estimation of the volume of the filtration product was performed by timing the height of the columnar spacer from the total area of the plasma calculated from the image, and the filtration efficiency was calculated by dividing the volume of the filtration product by the volume of the whole blood sample. The total data are summarized in table 3.

TABLE 3 filtration product quantification

This example illustrates the effectiveness of the apparatus and method provided by the present invention. The advantages of plasma separation achieved using the present invention are also demonstrated: the exemplary device has a relatively much simpler structure and is easier to handle than many other prior art in the field; this method takes shorter time, possibly within 1 minute from the acquisition device and sample to the completion of plasma separation; the method can treat very small amounts of blood samples, and reduces the burden on the subject, particularly the patient, by avoiding the invasive extraction of large amounts of blood.

5.7 example 2

Here, plasma isolated by the exemplary apparatus and method shown in example 1 has been demonstrated for Triglyceride (TG) determination as part of routine laboratory testing. TG is a fat found in blood, and high levels of TG may increase the risk of coronary artery disease. Therefore, the TG test is part of a blood lipid panel used to assess the risk of an individual developing heart disease. Typically, TG assays are colorimetric and are performed with plasma instead of a whole blood sample to avoid color interference from hemoglobin in red blood cells. The exemplary apparatus and methods are used herein to separate plasma from a whole blood sample and the resulting plasma is used as a substrate for TG determination.

In this experiment, for plasma separation, an X plate (PMMA, 175 μm thick and 1 inch by 1 inch wide, cubic columnar spacers: 3030X 40 μm wide, 30 μm high and 80 μm ISD) was used as the collection plate. A filter membrane (sterlett corp., Kent, WA) with 0.4 μm pores WAs used as filter. Different X plates (PMMA, cubic cylindrical spacers 175 μm thick and 1 inch by 1 inch wide: 30X 40 μm wide, 30 μm high and 80 μm ISD) were used as platens. Approximately 2 μ l of whole blood sample was freshly obtained by pricking the patient's finger and deposited on the filter membrane placed on top of the cylindrical spacer of the collection plate, then the pressure plate was placed on top of the deposited sample and the collection plate was pressed by hand for 30 s. Thereby forcing a portion of the whole blood sample through the filter membrane to the collection plate to effect plasma separation.

For TG assay, after plasma separation, the filter and press plates were then peeled off from the collection plate, leaving the plasma (filtered product) on the collection plate. Next, 0.5 μ L of TG assay reagent (Express Biotech International inc., Frederick, MD) was deposited on a capture plate (a planar plastic plate, made of PMMA, 1mm thick and 3 inches by 1 inch wide) and then transferred to plasma on a collection plate. Capture plates were pressed by hand onto collection plates to form QMAX devices, and TG was incubated for 1 minute assay. The assay images were then read by an iPhone, which was pre-configured to capture and analyze images from a QMAX device.

Fig. 20 shows the results of Triglyceride (TG) determination using the filtered product from the experimental filtration apparatus as the measurement sample and using the QMAX apparatus as the measurement apparatus. The lower panel shows pictures of a QMAX apparatus for TG determination and imaging. As shown, a long flat glass plate was used to contact and press against all three collection plates tested, forming three separate QMAX devices. The TG assay here is a colorimetric assay because the assay solution changes color (to pink) when TG is detected, and a higher color intensity indicates a higher level of TG in the assay sample. The upper panel shows a graph of the color intensity results under three different experimental conditions. The color intensity is close to zero when only plasma (filtration product) is present, and at a very low level when only reagent is present. However, when both plasma and reagent were present, the color intensity reached the highest level, indicating the presence of TG in the plasma.

This example again illustrates the effectiveness of the apparatus and method provided by the present invention. It also clearly demonstrates the ease of combining the invention with QMAX processing, which will significantly speed up the sampling/sensing/analysis/processing of samples and extend the applicability of QMAX devices.

6 overview of examples for separating complex liquid samples

6.1 device for separating Components from Complex liquid samples

Example 13: an apparatus for separating components from a composite liquid sample, comprising:

a collection plate having a plurality of spacers affixed to one surface thereof, and a filter having a sample receiving surface and a sample exit surface,

wherein at least a portion of the spacer is directed toward and in contact with the sample discharge surface of the filter to form a microcavity defined by the sample discharge surface and the portion of the spacer, and

wherein the filter is configured to separate the component from the portion of the sample flowing from the sample receiving surface through the filter to the collection plate.

In a device as described in example 13, the microcavity provides a capillary force that constitutes at least a portion of the driving force for causing at least a portion of the sample deposited on the sample receiving surface to flow through the filter toward the collection plate.

In the device of embodiment 13 or any derivative thereof, the device further comprising a force source providing a first liquid configured to provide at least a portion of the driving force, the first liquid having low miscibility with the sample.

The apparatus of embodiment 13 or any derivative thereof, further comprising a source of force providing pressurized gas configured to provide at least a portion of the driving force.

In the device of example 13 or any derivative thereof, the device further comprises a sponge,

wherein the sponge has a compressed state and an uncompressed state,

wherein the sponge is movable to different configurations relative to the collection plate and the filter,

wherein one of the configurations is a deposition configuration, wherein: the sponge is in an uncompressed state and is partially or completely separated from the collection plate and the filter, the distance between the collection plate and the sponge is not adjusted by spacers, filters or deposited samples, and

wherein another of the configurations is a filtration configuration, wherein: the filter is positioned between the sponge and the collection plate, the distance between the collection plate and the sponge is regulated by the spacer, the filter, and the deposited sample, and the sponge is converted from an uncompressed state to a compressed state during which the sponge is configured to provide at least a portion of the driving force.

In the apparatus of embodiment 13 or any derivative thereof, the apparatus further comprising a platen having a plurality of spacers on one surface thereof,

wherein the pressure plate is movable to different configurations relative to the collection plate and the filter,

wherein one of the configurations is a deposition configuration in which the platen is partially or completely separated from the collection plate and filter, the distance between the collection plate and platen is not adjusted by their spacers, filters, or deposited samples, and

wherein another of the configurations is a filtration configuration, wherein: the filter is positioned between the pressure plate and the collection plate, the distance between the collection plate and the pressure plate is regulated by their spacers, the filter, and the deposited sample, and at least a portion of the spacers and the inner surface of the pressure plate press at least a portion of the deposited sample against the filter, thereby providing at least a portion of the driving force.

In the apparatus of example 13 or any derivative thereof, the platen spacers have a uniform height in the range of 0.5 μm to 100 μm and the constant spacer pitch is in the range of 5 μm to 200 μm.

In the apparatus of example 13 or any derivative thereof, the platen spacers have a uniform height in the range of 1 μm to 50 μm and the constant spacer pitch is in the range of 7 μm to 50 μm.

6.2 method of separating Components from Complex liquid sample

Example 14: a method of separating a component from a composite liquid sample, comprising the steps of:

(1) providing a collection plate having a plurality of spacers on one surface thereof, and a filter having a sample receiving surface and a sample exit surface, wherein at least a portion of the spacers are directed toward and in contact with the sample exit surface of the filter, thereby forming a microcavity bounded by the sample exit surface and said portion of the spacers,

(2) depositing a sample on the sample receiving surface of the filter, an

(3) Driving at least a portion of the deposited sample with a driving force to flow through the filter toward the collection plate, wherein the filter is configured to separate a component from a portion of the deposited sample flowing through the filter from the sample receiving surface toward the collection plate.

In the method as described in example 14, the microcavity provides a capillary force, which constitutes at least a part of the driving force in step (3).

In the method of example 14 or any derivative thereof, step (3) comprises depositing a first liquid to contact the deposited sample, the first liquid having low miscibility with the sample and being configured to provide at least a portion of the driving force.

In the method of embodiment 14 or any derivative thereof, step (3) comprises applying a pressurized gas against the deposited sample, the pressurized gas configured to provide at least a portion of the driving force.

In the method of example 14 or any derivative thereof, step (3) comprises:

(a) contacting the sponge with the deposited sample, and

(b) the sponge is pressed against the filter to provide at least a portion of the driving force.

In the method of example 14 or any derivative thereof, step (3) comprises:

(a) placing a platen having a plurality of spacers on one surface thereof in contact with the deposited sample, at least a portion of the spacers of the platen being directed toward the sample receiving surface of the filter and in contact with the deposited sample;

(b) after the placing step (a), pressing the pressure plate against the filter to reduce the distance between the pressure plate and the filter and to provide at least a portion of the driving force.

In the method of example 14 or any derivative thereof, the platen spacers have a uniform height in the range of 0.5 μm to 100 μm and the constant spacer pitch is in the range of 5 μm to 200 μm.

In the method of example 14 or any derivative thereof, the platen spacers have a uniform height in the range of 1 μm to 50 μm and the constant spacer pitch is in the range of 7 μm to 50 μm.

In the method of example 14 or any derivative thereof, the compressing step is performed by a human hand.

6.3 devices or methods for separating components from Complex liquid samples

Example 15: the apparatus or method of any of the preceding embodiments, wherein the collection plate spacers have a predetermined substantially uniform height and a predetermined substantially constant spacer spacing.

In the apparatus or method as described in example 15, the uniform height is in the range of 0.5 to 100 μm and the constant spacer pitch is in the range of 5 to 200 μm.

In the apparatus or method as described in example 15, the uniform height is in the range of 0.5 to 20 μm and the constant spacer pitch is in the range of 7 to 50 μm.

In the device or method of example 15 or any derivative thereof, the filter is a mechanical filter, a chemical filter, a biological filter, or any combination thereof.

In an apparatus or method as described in example 15 or any derivative thereof, the filter is made from a material selected from the group consisting of: silver, fiberglass, ceramic, cellulose acetate, cellulose ester, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, many other materials that can form a porous structure, and any combination thereof.

In the device or method of example 15 or any derivative thereof, the filter has an average pore size in the range of 10nm to 500 μm.

In the apparatus or method of example 15 or any derivative thereof, the filter has an average pore size in the range of 0.1 μm to 5 μm.

6.4 device for extracting plasma from blood samples

Example 16: a device for extracting plasma from a blood sample, comprising: a collecting plate having a plurality of spacers fixed on one surface thereof; and a filter having a sample receiving surface and a sample discharge surface,

wherein at least a portion of the spacer is directed toward and in contact with the sample ejection surface of the filter, forming a microcavity defined by the sample ejection surface and the portion of the spacer;

wherein the spacers have a uniform height in a range of 1 μm to 50 μm and a constant spacer pitch in a range of 7 μm to 50 μm; and

wherein the filter is configured to separate blood cells from a portion of the blood sample flowing from the sample receiving surface through the filter to the collection plate, and the filter is made of a material selected from the group consisting of: silver, glass fiber, ceramic, cellulose acetate, cellulose ester, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, any other material that can form a porous structure, and any combination thereof, and has an average pore diameter in the range of 0.1 to 5 μm.

In the device of example 16, the microcavity provides a capillary force that includes at least a portion of the driving force for causing at least a portion of the sample deposited on the sample receiving surface to flow through the filter to the collection plate.

6.5 method for extracting plasma from blood samples

Example 17: a method of extracting plasma from a blood sample comprising the steps of:

(1) providing a collection plate having a plurality of spacers on one surface thereof, and a filter having a sample receiving surface and a sample exit surface,

wherein the spacers have a uniform height in a range of 1 μm to 50 μm and a constant spacer pitch in a range of 7 μm to 50 μm;

(2) depositing a blood sample on a sample receiving surface of the filter, and

(3) driving at least a portion of the sedimented blood sample through the filter toward the collection plate with a driving force,

wherein the filter is configured to separate blood cells from a portion of the deposited blood sample flowing from the sample receiving surface through the filter to the collection plate, and the filter is made of a material selected from the group consisting of: silver, glass fiber, ceramic, cellulose acetate, cellulose ester, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, any other material that can form a porous structure, and any combination thereof, and has an average pore diameter in the range of 0.1 to 5 μm.

In the method as described in example 17, the microcavity provides a capillary force that consists of at least a portion of the driving force in step (3).

In the method of example 17 or any derivative thereof, the depositing step comprises: (a) piercing the skin of a person to release a drop of blood onto the skin; and (b) contacting the drop of blood with the filter without the use of a blood transfer means.

6.6 device for separating plasma from blood samples

Example 18: a device for separating plasma from a blood sample, comprising: a collecting plate and a pressing plate both having a plurality of spacers fixed on one surface thereof; and a filter having a sample receiving surface and a sample discharge surface, wherein at least a portion of the collection plate spacer is directed toward and in contact with the sample discharge surface of the filter forming a microcavity defined by the sample discharge surface and the portion of the spacer,

wherein the spacers of the collection plate and the pressure plate have a uniform height in a range of 1 μm to 50 μm and a constant spacer pitch in a range of 7 μm to 50 μm, respectively;

wherein the filter is configured to separate blood cells from a portion of the blood sample flowing from the sample receiving surface through the filter to the collection plate, and the filter is made of a material selected from the group consisting of: silver, glass fiber, ceramic, cellulose acetate, cellulose ester, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, any other material that can form a porous structure, and any combination thereof, and has an average pore diameter in the range of 0.1 to 5 μm;

wherein another of the configurations is a filtration configuration, wherein: the filter is positioned between the platen and the collection plate, the distance between the collection plate and the platen is regulated by their spacers, the filter, and the deposited sample, at least a portion of the spacers and the inner surface of the platen press at least a portion of the deposited sample against the filter, providing at least a portion of the driving force.

6.7 method of extracting plasma from blood samples

Example 19: a method of extracting plasma from a blood sample comprising the steps of:

(1) providing a collection plate and a pressure plate both having a plurality of spacers on one surface thereof, and a filter having a sample receiving surface and a sample exit surface, wherein at least a portion of the spacers of the collection plate are directed toward and in contact with the sample exit surface of the filter, forming a microcavity bounded by the sample exit surface and the portion of the spacers, and

(2) depositing a sample on the sample receiving surface of the filter, an

(3) Placing a platen in contact with the deposited blood sample, the platen having a plurality of spacers on one surface thereof, wherein at least a portion of the spacers of the platen are directed toward the sample receiving surface of the filter and are in contact with the deposited sample, and

(4) after the placing step, pressing the platen against the filter to reduce a distance between the platen and the filter and force at least a portion of the deposited blood sample to flow through the filter to the collection plate, wherein the filter is configured to separate blood cells from the portion of the deposited blood sample that flows from the receiving surface through the filter to the collection plate and is made of a material selected from the group consisting of: silver, glass fiber, ceramic, cellulose acetate, cellulose ester, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, any other material that can form a porous structure, and any combination thereof, and has an average pore diameter in the range of 0.1 to 5 μm.

In the method of example 19, the compressing step is performed by a human hand.

In the method of embodiment 19 or any derivative thereof, the depositing step comprises: (a) piercing the skin of a person to release a drop of blood onto the skin; and (b) contacting the drop of blood with the filter without the use of a blood transfer means.

6.8 devices or methods for extracting plasma from blood samples

Example 20: the apparatus or method of any of the preceding embodiments, wherein each plate has a thickness of less than 200 μ ι η.

In the method as described in example 20, each plate had a thickness of less than 100 μm.

In the method of example 20 or any derivative thereof, each plate has an area of less than 5cm²。

In the method of example 20 or any derivative thereof, each plate has an area of less than 2cm²。

In the method of embodiment 20 or any derivative thereof, at least one plate is made of a flexible polymer.

In the method of embodiment 20 or any derivative thereof, at least one of the plates is a flexible plate, and the thickness of the flexible plate times the young's modulus of the flexible plate is in the range of 60 to 75GPa- μm.

In the method of embodiment 20 or any derivative thereof, the spacer is secured to the inner surface of the second panel.

In the method of embodiment 20 or any derivative thereof, the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

In the method of embodiment 20 or any derivative thereof, wherein the spacers have a columnar shape and a substantially flat top surface, wherein for each spacer, a ratio of a lateral dimension of the spacer to a height thereof is at least 1.

In the method of embodiment 20 or any derivative thereof, each spacer has a ratio of a lateral dimension of the spacer to a height thereof of at least 1.

In the method of embodiment 20 or any derivative thereof, the smallest lateral dimension of the spacer is less than or substantially equal to the smallest dimension of the analyte in the sample.

In the method of embodiment 20 or any derivative thereof, the spacers have a cylindrical shape and the sidewall angles of the spacers have a rounded shape with a radius of curvature of at least 1 μm.

In the method of embodiment 20 or any derivative thereof, the spacer has at least 100/mm²The density of (c).

In the method of embodiment 20 or any derivative thereof, the spacer has a thickness of at least 1000/mm²The density of (c).

In the method of embodiment 20 or any derivative thereof, the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in contact with the uniform thickness layer to the total plate area in contact with the uniform thickness layer.

In the method of embodiment 20 or any derivative thereof, the young's modulus of the spacer multiplied by a fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the area of the spacer in contact with the uniform-thickness layer to the total plate area in contact with the uniform-thickness layer.

In the method of embodiment 20 or any derivative thereof, at least one of the plates is flexible, and for the flexible plate, the fourth power of the spacer spacing (ISD) divided by the thickness (h) of the flexible plate and the young's modulus (E) of the flexible plate, ISD/(hE), is equal to or less than 106 μm³/GPa。

In the method of embodiment 20 or any derivative thereof, the spacer is secured to the plate by directly stamping the plate or injection molding the plate.

In the method of example 20 or any derivative thereof, the material of the plates and spacers is independently selected from polystyrene, PMMG, PC, COC, COP, or other plastic.

7 multi-plate QMAX device with hinge and filter

Fig. 21 shows an embodiment of a QMAX apparatus. QMAX (Q: quantitative; M: amplification, a. addition of a reagent, X: acceleration; also called a compression-regulated open flow (CROF) device, which comprises a first plate 10, a second plate 20, a third plate 30 and a spacer 40.

Figure (a) shows a perspective view of the panel in an open configuration, wherein: the plates are partially or completely separated, the spacing between the plates not being adjusted by spacers 40, thereby allowing the sample to be deposited on one or more of the plates or on a structure (e.g., a filter) placed on top of one of the plates, fig. (B) showing a cross-sectional view of the plates in an open configuration.

As shown in fig. 21(a) and (B), in some embodiments, both the second plate 20 and the third plate 30 are connected to the first plate 10. In certain embodiments, the second panel 20 is connected to the first panel 10 by a hinge 103 and the third panel 30 is connected to the first panel 10 by another hinge 103. The second plate 20 and the third plate 30 are configured such that each can pivot toward and away from the first plate 10 without interfering with each other. In some embodiments, the surfaces of the first plate 10 facing the second plate 20 and the third plate 30 are defined as inner surfaces, and the surfaces of the second plate 20 and the third plate 30 facing the first plate 10 are also defined as inner surfaces of the respective plates.

In some embodiments, the hinge 103 is placed partially on top of the inner surface of the first panel 10 and connects the second panel 20 and the third panel 30 to the first panel 10. In certain embodiments, the edge of the second plate 20 and/or the edge of the third plate 30 are not closely aligned with the edge of the first plate 10. In some embodiments, the hinge 103 does not wrap around any edge of the first panel 10. However, it should also be noted that the second plate 20 and the third plate 30 need not be connected to the first plate 10. In certain embodiments, the second plate 20 and/or the third plate 30 are completely separate from the first plate 10.

In some embodiments, the hinges are configured such that one or more hinges can be torn to cause the panels to become disconnected. In some embodiments, one panel is torn before the other two panels are closed. In some embodiments, the panels are not connected by hinges.

Fig. 21(a) and (B) also show the spacer 40 fixed on the first plate 10. However, it should also be noted that spacer 40 may be secured to third plate 30, second plate 20, or any selection and combination of the three plates. In certain embodiments, spacers 40 are secured to the inner surfaces of first plate 10 and third plate 30. In certain embodiments, the spacer 40 is fixed to the inner surfaces of the first and

second panels

10 and 20. In certain embodiments, spacers 40 are secured to the inner surfaces of second plate 20 and third plate 30. In certain embodiments, the spacer 40 is fixed only to the first plate 10. In certain embodiments, the spacer 40 is secured only to the second plate 20. In certain embodiments, the spacer 40 is fixed to the third plate 30 only. In certain embodiments, the spacer 40 is fixed to all three plates. When the spacer 40 is secured to more than one plate, the spacer height may be the same or different on different plates. In some embodiments, the spacer 40 is not fixed to any plate, but rather is mixed in the sample.

It should be noted that in some embodiments, the spacer 40 is not a required structure. In certain embodiments, none of the plates contain spacers that are fixed on the plate or added to the sample.

Fig. 22 shows an exemplary embodiment of a QMAX device and a flow to be used for filtering and analyzing a liquid sample with the QMAX device. In certain embodiments, the elements shown in fig. 22 are organized into kits. For example, in certain embodiments, a cartridge comprises a QMAX device comprising a first plate 10, a second plate 20, a third plate 30, and a spacer 40, wherein the second plate 20 and the third plate 30 are connected to the first plate 10, e.g., by a hinge 103, and a filter.

Fig. 22(a) shows a cross-sectional view of a QMAX apparatus in an open configuration, where sample 90 is deposited on filter 70 placed on top of first plate 10. As shown in figure (a), in some embodiments, the filter 70 is actually placed on top of the spacer 40, leaving a cavity between the filter 70 and the inner surface of the first plate 10. In some embodiments, a sample 70 is placed on top of the filter, wherein the sample comprises a plurality of components. In certain embodiments, the sample comprises at least one component that is separable by the filter from the remainder of the sample, and in certain embodiments, the component of the sample is blocked or absorbed by the filter 70 and separated from the portion of the sample 90 that flows through the filter 70 and into the cavity. In some embodiments, the sample 90 is whole blood. In certain embodiments, the components of sample 90 enclosed or absorbed by filter 70 comprise blood cells; the portion of sample 90 that flows through filter 70 contains plasma.

The component shown in fig. 22(a) may be an element of a cartridge containing the first plate 10, the second plate 20, the third plate 30, the spacer 40, and the filter 70, wherein the second plate 20 and the third plate 30 are connected to the first plate 10 so that the second plate 20 and the third plate 30 can pivot toward and away from the first plate 10. As shown in fig. (a), in some embodiments, second panel 20 and third panel 30 are connected to first panel 10 by hinges 103. In some embodiments, the kits of the present invention further comprise a wash pad and a wash solution, wherein the wash pad and wash solution can be used to wash the inner surface of the first plate 10 after depositing the sample 90 on the first plate 10. In certain embodiments, certain components in sample 90 may be incubated after second plate 20 has been pressed against first plate 10 for a period of time, followed by washing.

Fig. 22(B) shows a cross-sectional view of the QMAX device when the third plate is pressed against the top of the filter, pushing a portion of the sample through the filter. In some embodiments, the filter covers all of the spacers 40. In some embodiments, the filter covers only a portion of the spacer 40. As shown in fig. (a) and (B), after sample 90 is deposited on top of filter 70, third plate 30 may be pressed toward the filter such that third plate 30 is substantially parallel to first plate 10, such that a portion of sample 90 flows through filter 70 when one or more components of sample 90 are captured or absorbed in filter 70. As shown in fig (B), the portion of the sample 90 that flows through the filter 70 may be referred to as a filtered sample 900. In certain embodiments, a portion of sample 90 flows through filter 70 due to capillary forces in filter 70 and in the cavity formed between filter 70 and first plate 10.

In some embodiments, the spacer 40 is fixed only to the first plate 10, not to the third plate 30. In some embodiments, the spacer 40 is fixed only to the third plate 30, not to the first plate 10. In some embodiments, the spacer 40 is fixed to both the first plate 10 and the third plate 30. In certain embodiments, using the third plate 30 to press against the filter 70 may prevent damage to certain components of the sample 90 when the spacer 40 is secured to the third plate 30. For example, in certain embodiments, when the sample 90 is whole blood, pressing the sample 90 with the third plate 30 having the spacer 40 may prevent lysing of some cells (e.g., red blood cells) in the blood. In some embodiments, lysis of the cells is undesirable, at least in part, because elements in the cells may be released into the plasma and flow through filter 70, leading to confusion in the results of the analysis. It should also be noted that in certain embodiments, any components of the sample 90 may not be lysed or damaged when the properties of the spacer 40 are appropriately selected.

In some embodiments of the invention, the filter may be a mechanical filter. Mechanical filters mechanically remove, absorb, trap, or block certain components from the composite liquid sample as the sample flows through the filter in a certain direction. Typically made of a porous material, and the pore size determines the size of the solid particles that can flow through the filter and the size of the solid particles that are removed from the sample flowing through the filter. The components of the mechanical device are inert so that they do not affect or interfere with the sample. Examples of mechanical filters include, but are not limited to, foam (reticulated and/or open-celled), fibrous material (e.g., filter paper), gel, sponge. Examples of materials include cellulose acetate, cellulose esters, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, any other material that can form a porous structure, and any combination thereof.

Fig. 22(C) shows a cross-sectional view of the QMAX device when the third plate 30 is opened after filtration and before the second plate 20 is pivoted towards the first plate 10. As shown in fig. B and (C), after the sample 90 is pressed with the third plate 30, a part of the sample 90 (filtered sample 900) flows through the filter 70 and enters the cavity between the filter 70 and the first plate 10. In some embodiments, after filtration is complete or after a predetermined period of time, the third plate 30 and filter 70 are opened so that the second plate 20 can be used. In some embodiments, the combined filter 70 and third plate 30 may be removed from the first plate 10 by one manipulation motion, although the filter 70 is adhered to the third plate 30 by capillary effect or other mechanism. In some embodiments, filter 70 is not attached to third panel 30; in certain embodiments, a user may first open the third panel 30 and then remove the filter 70 from the first panel 10.

After opening the third plate 30 and the filter 70, the filtered sample 900 remains on the first plate 10. In some embodiments, when the spacers 40 are secured on the first plate 10, the filtered sample 900 is positioned above the spacers 40 and/or between the spacers 40. In some embodiments, the second plate 20 may be pressed against the second plate 20. In some embodiments, there are no spacers 40 on the second plate 20; in certain embodiments, spacers 40 are present on the second plate 20.

Fig. 22(D) shows a cross-sectional view of the QMAX device in a closed configuration when the portion of the sample flowing through filter 70 (filtered sample 900) is pressed into a layer of uniform thickness by second plate 20. As shown, the plates may be moved relative to each other into different configurations. One of the configurations between the second panel 20 and the first panel 10 is a closed configuration, in which: the first plate 10 and the second plate 20 are pressed together, and the interval between the second plate 20 and the first plate 10 is adjusted by the height of the spacer 40; at least a portion of the filtered sample 900 is pressed into a layer of uniform thickness. In certain embodiments, an external force F is used to press the first plate 10 and the second plate 20 together. In certain embodiments, after the force is removed, the

panels

10 and 20 may remain in the closed configuration and the spacing between the panels is well maintained. In some embodiments, the spacing between the plates, the thickness of the filtered sample layer, and the height of the spacer 40 are the same.

After the first and

second panels

10, 20 are converted to the closed configuration, the filtered sample 900 in the uniform thickness layer can be analyzed and measured. In some embodiments, the thickness is less than 0.2 μm, 0.5 μm, 1 μm, 1.5 μm, 2 μm, 3 μm, 5 μm, 10 μm, 20 μm, 30 μm, 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 375 μm, or 500 μm, or within a range between any two values. In some embodiments, the measurement and analysis can be performed accurately and quickly due to the uniformity and limited thickness of the filtered sample.

In some embodiments, the sample is a food product. After filtration with filter 70, blood cells, such as red blood cells and white blood cells, are captured, absorbed, or blocked by filter 70. The filtered sample 900 contains plasma. In some embodiments, the plasma may be analyzed using various types of biological and/or chemical assays. For example, a colorimetric assay may be used to analyze the glucose level in plasma.

Fig. 23 illustrates an exemplary embodiment of a QMAX device. Fig. (a) shows a top view of a QMAX device containing a notch. Fig (B) shows a top view of a QMAX device containing a notch when filter 70 is placed on top of first plate 10, and for clarity, second plate 20 is not shown in fig (B). In some embodiments, it is convenient and/or necessary to include structure to facilitate pivoting of the second and

third plates

20, 30. In other words, in some embodiments, it may be convenient and/or necessary to include structure that allows a user to adjust the angle between the first and

second panels

10, 20, the angle between the first and

third panels

10, 30, and the positioning of the filter 70 relative to the first and

third panels

10, 30. An example of such a structure is provided in fig. 23.

As shown in fig. 23(a) and (B), the first plate 10 includes a first notch 1051, a second notch 1052, and a third notch 1053. It should be noted that in some embodiments, the first plate 10 may contain only one of the three recesses, and in some embodiments, the first plate 10 may contain only two (any two) of the three recesses.

In some embodiments, the notches are the same size. In some embodiments, the dimensions of the notches are different. The size of the recess is adjusted according to the size of the plate and the specific needs of the user. For example, in some embodiments, the length of the notch (which is defined as the length of the widest opening on the edge of the notch) is less than 1mm, 2.5mm, 5mm, 10mm, 15mm, 20mm, 25mm, 30mm, 40mm, 50mm, or ranges between any two values. In some embodiments, the length of the notch is less than 1/10, 1/9, 1/7, 1/6, 1/5, 1/4, 1/3, 2/5, 1/2, 3/5, 2/3, 3/4, 4/5, 5/6, or 9/10 of the length of the edge of the notch, or within a range between any two values. In some embodiments, when the notch is in the shape of a portion of a circle, such circle has a radius of less than 1mm, 2.5mm, 5mm, 10mm, 15mm, 20mm, 25mm, 30mm, 40mm, 50mm, or a radius within a range between any two values.

Fig. 23 shows a semicircular recess. It should be noted, however, that the recess may be of any shape as long as an opening is provided in the first plate 10 below the second plate 2 to facilitate opening of the first plate 1 and the second plate 2. For example, in some embodiments, the notch has the shape of any portion of a circle. In some embodiments, the recess has a shape of a portion or all of a square, a rectangle, a triangle, a hexagon, a polygon, a trapezoid, a sector, or any combination thereof. The recesses on the same plate may have the same or different shapes.

As shown in fig. 23(a), the first plate 10 includes a first notch 1051, and the first notch 1051 is positioned and sized such that when one edge of the third plate 30 is partially juxtaposed over the first notch 1051, no edge of the second plate 20 is juxtaposed over the first notch 1051. In certain embodiments, the first notch 1051 is located on the first panel 10 distal to the hinge 103 relative to the third panel 30. Conversely, the first notch 1051 may be positioned on the third plate 30 rather than the first plate 10 such that one edge of the first plate 10 is juxtaposed over the first notch 1051 and manipulation of the relative positioning between the first plate 10 and the third plate 30 is facilitated.

As shown in fig. 23(a) and (B), in some embodiments, the first plate 10 includes a first notch 1051 and a third notch 1053. In certain embodiments, when the filter 70 is positioned on top of the first plate 10, one edge of the filter 70 is juxtaposed over the first notch 1051, but not over the second notch 1053. In certain embodiments, the third plate 30 is juxtaposed over both the first and

second recesses

1051, 1053. With this design, when a user wishes to manipulate the position of the third panel 30 and the filter 70 together (e.g., to change from a closed position to an open position), the user can push the third panel 30 and the filter 70 over the first recess 1051, and when the user wishes to manipulate only the position of the third panel 30, the user can push the third panel 30 over the third recess 1053. It should also be noted that in some embodiments, the first plate 10 contains only the first notch 1051, and not the third notch 1053; the edges of the third panel 30 and the filter 70 on the first recess 1051 do not completely overlap and the user may choose to manipulate the third panel 30 alone or to manipulate the third panel 30 and the filter 70 together by changing the position of the force prior to manipulation. In certain embodiments, the third notch 1053 is located on the first panel 10 distal to the hinge 103 relative to the third panel 30. Furthermore, due to the positioning of the first notch 1051, the third notch 1053 may be positioned on the third plate 30, rather than the first plate 10.

As shown in fig. 23(a), in some embodiments, the first plate 10 includes a second notch 1052. In some embodiments, the second notch 1052 is located on the first plate 10 distal to the hinge 103 relative to the second plate 20. In some embodiments, juxtaposing one edge of the second plate 20, but not the edge of the first plate 10, over the second notch 1052 facilitates changing the relative positioning of the second plate 20 and the first plate 10. Conversely, in some embodiments, the second notch 1052 is placed on the second plate 20 rather than the first plate 10.

Other structures besides recesses may be used to facilitate handling of the first, second, third and

filter plates

10, 20, 30 and 70. For example, in some embodiments, any one, or two, or all three of the panels include a tab attached to the body of the panel. The user can manipulate the positioning of the plate by pulling on the tab.

For example, in some embodiments, the second plate 20 includes plate tabs configured to facilitate switching the plate between different configurations between the second plate 20 and the second plate 20. In certain embodiments, the third panel 30 contains press tabs configured to facilitate switching the panel between different configurations between the third panel 30 and the first panel 10. Further, in some embodiments, filter 70 also includes a tab. For example, in certain embodiments, the filter 70 includes filter tabs configured to facilitate removal of the filter from the plate.

Overview of an embodiment of a multi-plate QMAX device with hinge and filter

Other embodiments of the inventive subject matter in accordance with the present disclosure are described in the paragraphs listed below.

8.1 measurement method Using Multi-plate QMAX device

Transfer of reagents with two plates

Example 21: a method for performing an assay comprising

(a) Obtaining a first plate comprising on its inner surface a sample contacting area having a first reagent site, wherein the first reagent site comprises an immobilized first reagent,

(b) obtaining a second plate comprising on its inner surface a sample contacting area with a storage site, wherein the storage site comprises a reagent capable of diffusing in the transfer liquid upon contacting the transfer liquid, wherein the second reagent binds or reacts with the first reagent,

wherein the first and second panels are movable relative to each other into different configurations, including an open configuration and a closed configuration;

(c) depositing a transfer liquid onto one or both of the sample contacting regions of the plate in an open configuration,

(d) after (c), bringing the two panels into a closed configuration; wherein in the open configuration, the sample contact areas of the two plates are spaced more than 200 μm apart; wherein, in the closed configuration, at least a portion of the transfer liquid deposited in (c) is confined between the sample contacting areas of the two plates and has an average thickness in the range of 0.01 μm to 200 μm.

Using three plates

Example 22: a method for performing an assay, comprising:

(d) depositing a sample on one or both of the sample contacting regions of the first plate and the second plate in an open configuration,

(e) after (d), placing the first and second panels in a closed configuration;

(f) after (e) separating the first and second sheets,

(i) Detecting a signal associated with the target analyte,

wherein in the open configuration, the sample contact areas of the two plates are spaced more than 200 μm apart;

8.2 Multi-Board QMAX device

Example 23: an apparatus for performing an assay, comprising:

a first plate comprising on its inner surface a sample contacting area having first reagent sites comprising a first reagent that bio/chemically interacts with a target analyte in a sample,

a second plate comprising on its inner surface a sample contacting area having a second reagent site, wherein the second reagent site comprises a second reagent capable of diffusing in the sample when contacting the sample,

a third plate comprising on its inner surface a sample contacting area having third reagent sites, wherein the third reagent sites comprise a third reagent that is capable of diffusing in the transfer liquid upon contact with the transfer liquid, wherein the first, second and third plates are movable relative to each other into different configurations comprising an open configuration and a closed configuration, wherein in the open configuration the sample contacting areas of the two plates are spaced more than 200 μm apart;

wherein in the closed configuration at least a portion of the sample or of the transfer liquid is confined between the sample contacting areas of the two plates and has an average thickness in the range of 0.01 μm to 200 μm.

Wherein in the open configuration the sample is deposited on one or both of the sample contacting regions of the first and second plates; and

wherein in the open configuration transfer liquid is deposited on one or both of the sample contacting regions of the second and third plates.

8.3 multi-board QMAX device and measuring method thereof

Example 24: the method or device of any preceding embodiment, wherein one or both of the sample contact areas comprise a spacer, wherein the spacer modulates a spacing between the sample contact areas of the plates when the plates are in the closed configuration.

In a method or apparatus as described in example 24, the spacing between the sample contacting regions is adjusted by a spacer when the plates are in the closed configuration.

In the method or device of embodiment 24 or any derivative thereof, the device further comprises a spacer that adjusts the spacing between the sample contacting regions when the plates are in the closed configuration.

In a method or device as described in example 24 or any derivative thereof, the storage site comprises another reagent in addition to the competitor.

In a method or device as described in example 24 or any derivative thereof, the binding site comprises, in addition to the immobilised capture reagent, another reagent which is capable of diffusing through the sample when contacted therewith.

In a method or device as described in example 24 or any derivative thereof, the binding site faces the storage site when the panel is in the closed configuration.

In a method or device as described in example 24 or any derivative thereof, the first panel comprises a plurality of binding sites and the second panel comprises a plurality of corresponding storage sites, wherein each binding site faces a corresponding storage site when the panels are in the closed configuration.

In a method or device as described in example 24 or any derivative thereof, the detection agent is dried on the storage site.

In a method or device as described in example 24 or any derivative thereof, the capture agent at the binding site is on an amplification surface that amplifies the optical signal of the analyte or captured competitor in examples 1, 2 and 3.

In a method or device as described in example 24 or any derivative thereof, the capture agent at the binding site is on an amplification surface that amplifies the optical signal of the analyte or captured competitor in examples 1, 2 and 3, wherein the amplification is proximity dependent in that the amplification decreases significantly as the distance between the capture agent and the analyte or competitor increases.

In a method or apparatus as described in example 24 or any derivative thereof, the detection of the signal is electrical, optical, fluorescent, SPR, or the like.

In a method or device as described in example 24 or any derivative thereof, the sample is a blood sample (whole blood, plasma or serum).

In the method or apparatus of example 24 or any derivative thereof, the material of the fluorescent microspheres is dielectric (e.g., SiO2, polystyrene) or a combination of dielectric materials thereof.

In the method or device of example 24 or any derivative thereof, the method further comprises the step of adding a fluorescently labeled detection agent to the first plate to bind to the competitor.

In the method or device of embodiment 24 or any derivative thereof, the method further comprises the step of washing after the detection agent is added to the first plate.

8.4 device for sample analysis

Example 25: an apparatus for sample analysis, comprising:

a first plate, a second plate, a third plate, and a spacer, wherein:

i. the second plate and the third plate are each connected to the first plate, wherein the second plate and the third plate are configured to each pivot against the first plate without interfering with each other,

spacers are fixed on one or more of the plates or mixed in the sample, and

In the device of example 25, the device further comprises a filter made of a porous material.

In the apparatus of embodiment 25 or any derivative thereof, the filter is configured to separate a component from a portion of the sample flowing through the filter.

In the apparatus of embodiment 25 or any derivative thereof, the third plate is configured to press the sample against the filter when the third plate is pivoted toward the first plate.

In the apparatus of embodiment 25 or any derivative thereof, an edge of the second panel is connected to the inner surface of the first panel by a first hinge.

In the apparatus of embodiment 25 or any derivative thereof, an edge of the third plate is connected to the inner surface of the first plate by a second hinge.

In the apparatus of embodiment 25 or any derivative thereof, an edge of the second panel is connected to the inner surface of the first panel by a first hinge and an edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In the device of embodiment 25 or any derivative thereof, the third plate can be adjusted to pivot against the first and second plates in a closed configuration between the first and second plates.

In the apparatus of embodiment 25 or any derivative thereof, the first plate comprises one or more notches on one or more edges thereof, wherein the notches are positioned such that the second plate and/or the third plate are juxtaposed over the notches to facilitate manipulation of pivoting of the second plate and the third plate.

In the apparatus of embodiment 25 or any derivative thereof, the second plate includes a plate tab configured to facilitate switching the plate between different configurations.

In the apparatus of embodiment 25 or any derivative thereof, the filter includes a filter tab configured to facilitate removal of the filter from the plate.

The apparatus of embodiment 25 or any derivative thereof, wherein the spacer is fixed to the first plate.

The apparatus of embodiment 25 or any derivative thereof, wherein the spacer is affixed to the first and second plates.

In the device of embodiment 25 or any derivative thereof, the sample is whole blood and the component is blood cells.

8.5 kits for sample washing and analysis

Example 26: a kit for sample washing and analysis comprising:

a first plate, a second plate, a third plate, a spacer, and a filter, wherein:

the first plate comprises an inner surface having a sample contacting area for contacting a liquid sample comprising a component, an

Spacers are fixed on one or more of the plates or mixed in the sample,

wherein one of the configurations is an open configuration, wherein: the three plates are partially or completely separated, the spacing between the plates not being adjusted by spacers, thereby allowing the liquid sample to be deposited on the first plate, the second plate, or both; wherein another of the configurations is a closed configuration, the closed configuration being configured after deposition of the sample in the open configuration, and in the closed configuration: at least a portion of the deposited sample is compressed by the first and second plates into a layer of very uniform thickness, the layer being bounded by the inner surfaces of the first and second plates and conditioned by the plates and spacers, and

wherein the filter is made of a porous material and is configured to separate a component from a portion of the sample flowing through the filter.

In the kit of example 26, the filter is configured to be pressed by the third panel when the filter is positioned on the first panel.

In a kit as described in example 26:

i. the sample contains the analyte(s) and,

coating a capture agent on the sample contact area in the first plate, and

the capture agent is configured to specifically bind to the analyte.

In the kit of example 26 or any derivative thereof, the filter is made of a material selected from the group consisting of: silver, fiberglass, ceramic, cellulose acetate, cellulose ester, nylon, polytetrafluoroethylene polyester, polyurethane, gelatin, agarose, polyvinyl alcohol, polysulfone, polyester sulfone, polyacrylonitrile, polyvinylidene fluoride, polypropylene, polyethylene, polyvinyl chloride, polycarbonate, many other materials that can form a porous structure, and any combination thereof.

In a kit as in example 26 or any derivative thereof, one edge of the second panel is attached to the inner surface of the first panel by a first hinge.

In a kit as in example 26 or any derivative thereof, one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In a kit as in example 26 or any derivative thereof, one edge of the second panel is connected to the interior surface of the first panel by a first hinge and one edge of the third panel is connected to the interior surface of the first panel by a second hinge.

In a kit as in example 26 or any derivative thereof, the third plate can be adjusted to pivot against the first and second plates in a closed configuration between the first and second plates.

In a kit as described in example 26 or any derivative thereof, the first panel comprises one or more notches on one or more edges thereof, wherein the notches are positioned such that the second panel and/or the third panel are juxtaposed over the notches to facilitate manipulation of pivoting of the second panel and the third panel.

In a kit as in example 26 or any derivative thereof, the second panel comprises a panel tab configured to facilitate switching the panel between different configurations.

In a kit as in embodiment 26 or any derivative thereof, the filter includes a filter tab configured to facilitate removal of the filter from the plate.

In a kit as described in example 26 or any derivative thereof, the spacer is affixed to the first plate.

In a kit as described in example 26 or any derivative thereof, the spacer is affixed to both the first and second plates.

In the kit of example 26 or any derivative thereof, the sample is whole blood and the components are blood cells.

8.6 methods of analyzing Components in samples

Example 27: a method of analyzing a component in a sample, comprising:

(a) a sample is taken that contains the components,

(b) an acquisition device comprising a first plate, a second plate, a third plate, a filter, and

a spacer, wherein:

i. the second plate and the third plate are connected to the first plate respectively,

wherein the second plate and the third plate are configured to each pivot against the first plate without interfering with each other,

the second or third plate is movable relative to the first plate into a different configuration by pivoting relative to the first plate,

the first plate comprises an inner surface having a surface for contacting a liquid sample containing a component,

spacers are fixed on one or more of the plates or mixed in the sample, and

v. a filter is placed on top of the first plate,

(c) the sample is deposited on top of the filter,

(d) pressing the third plate against the sample and forcing a portion of the sample to flow through the filter onto the first plate, wherein the filter is configured to separate the component from the portion of the sample flowing through the filter,

(e) removing the third plate and the filter from the first plate, and

(f) by pressing the first and second plates together, the portion of the sample flowing onto the first plate is compressed into a layer of uniform thickness.

In the method of embodiment 27, the second plate and the third plate are each connected to the first plate, wherein the second plate and the third plate are configured to each pivot against the first plate without interfering with each other,

in the method of embodiment 27 or any derivative thereof, one edge of the second panel is connected to the inner surface of the first panel by a first hinge.

In the method of embodiment 27 or any derivative thereof, an edge of the third panel is connected to the inner surface of the first panel by a second hinge.

In the method of embodiment 27 or any derivative thereof, one edge of the second panel is connected to the interior surface of the first panel by a first hinge and one edge of the third panel is connected to the interior surface of the first panel by a second hinge.

In the method of embodiment 27 or any derivative thereof, the first plate includes one or more notches on one or more edges thereof, wherein the notches are positioned such that the second plate and/or the third plate are juxtaposed over the notches to facilitate manipulation of pivoting of the second plate and the third plate.

In the method of embodiment 27 or any derivative thereof, the second panel comprises a panel tab configured to facilitate switching the panel between different configurations.

In the method of embodiment 27 or any derivative thereof, the filter includes a filter tab configured to facilitate removal of the filter from the plate.

In the method of example 27 or any derivative thereof, the first plate comprises at least one assay site, wherein the sample and the spacer deposited on the assay site are immobilized to the assay site.

In the method of embodiment 27 or any derivative thereof, the first plate comprises a capture reagent coated on an inner surface of the first plate, wherein the capture reagent is configured to specifically bind to an analyte in the sample.

In the method of example 27 or any derivative thereof, the first plate comprises a plurality of assay sites separated by a minimum site spacing.

In the method of embodiment 27 or any derivative thereof, the second plate contacts the sample with an interior surface of the second plate, and the interior surface of the second plate comprises an adhered detection agent, wherein the detection agent is configured to specifically associate with at least one of the analyte and the analyte bound to the capture agent.

In the method of embodiment 27 or any derivative thereof, the spacer is secured to the first plate.

In the method of embodiment 27 or any derivative thereof, the spacer is secured to both the first and second plates.

In the method of example 27 or any derivative thereof, the sample is whole blood and the component is blood cells.

In the method of embodiment 27 or any derivative thereof, the filter comprises a filter spacer on the washing surface, wherein the washing surface and the filter spacer are configured to prevent direct contact between the washing surface and the assay site.

In the method of embodiment 27 or any derivative thereof, the method further comprising: after step (f), detecting the analyte bound to the capture agent.

In the method of example 27 or any derivative thereof, the detecting comprises measuring at least one of fluorescence, luminescence, scattering, reflection, absorption, and surface plasmon resonance associated with the analyte bound to the capture agent.

In the method of embodiment 27 or any derivative thereof, the inner surface of the first plate at the assay site comprises a signal amplification surface, such as a metal and/or dielectric microstructure (e.g., a disk coupled spot column antenna array).

In the method of embodiment 27 or any derivative thereof, the uniform thickness is at most 1mm, at most 800 μ ι η, at most 600 μ ι η, at most 500 μ ι η, at most 400 μ ι η, at most 200 μ ι η, at most 150 μ ι η, at most 100 μ ι η, at most 75 μ ι η, at most 50 μ ι η, at most 20 μ ι η, at most 10 μ ι η, or at most 2 μ ι η, or within a range between any two values.

In the method of example 27 or any derivative thereof, the biological sample does not include a spacer.

9 additional features

9.1Q cards, spacers and uniform sample thickness

The devices, systems, and methods disclosed herein may include or use Q-card, spacer, and uniform sample thickness embodiments for sample detection, analysis, and quantification. In some embodiments, the Q-card includes a spacer that helps make at least a portion of the sample a very uniform layer. The structure, materials, function, variation and dimensions of the spacer and uniformity of the spacer and sample layers are listed, described and summarized herein or in PCT applications (assigned US) No. PCT/US2016/045437 and No. PCT/US0216/051775, filed on PCT 8/2016 and 9/14/2016, respectively, No. 62/456065, filed on US provisional application No. 2/7/2017, filed on US provisional application No. 62/456287, filed on US 2/8/2017, all of which are incorporated herein in their entirety for all purposes.

9.2 other examples

(1) Size of

The devices, apparatus, systems, and methods disclosed herein may include or use a QMAX device, which may include a plate and a spacer. In some embodiments, the dimensions of the various components of a QMAX device and its adapter are listed, described and/or outlined in PCT application No. (assigned US) PCT/US2016/045437 filed on 8/10/2016 and in US provisional application No. 62,431,639 filed on 12/9/2016 and in US provisional application No. 62/456,287 filed on 2/8/2017, which are all incorporated herein by reference.

In some embodiments, the dimensions are listed in the following table:

plate:

hinge:

notch:

groove:

socket slot

(2)Applications of

The devices/apparatus, systems, and methods disclosed herein may be used in a variety of applications (fields and samples). Applications are disclosed herein or listed, described and summarized in PCT applications (assigned US) No. PCT/US2016/045437 and No. PCT/US0216/051775, filed 2016, 8, 9, 14, 2017, US provisional application No. 62/456065, filed 2017, 2, 8, 2017, US provisional application No. 62/456287, filed 2017, 2, 8, and US provisional application No. 62/456504, filed 2017, 2, 8, all of which are incorporated herein in their entireties for all purposes.

In some embodiments, the devices, apparatuses, systems, and methods disclosed herein are used in a variety of different applications in a variety of fields where it is desirable to determine the presence or absence, quantification, and/or amplification of one or more analytes in a sample. For example, in certain embodiments, the subject devices, systems, and methods are used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds, organic compounds, bacteria, viruses, cells, tissues, nanoparticles, and other molecules, compounds, mixtures, and substances. Various areas in which the subject devices, apparatus, systems and methods may be used include, but are not limited to: diagnosis, management and/or prevention of human diseases and conditions, diagnosis, management and/or prevention of animal diseases and conditions, diagnosis, management and/or prevention of plant diseases and conditions, agricultural uses, veterinary uses, food testing, environmental testing and decontamination, pharmaceutical testing and prevention, and the like.

Applications of the present invention include, but are not limited to: (a) detection, purification, quantification and/or amplification of compounds or biomolecules associated with certain diseases or certain stages of diseases, such as infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric disorders, neuropsychiatric disorders and organic diseases, such as lung diseases, kidney diseases, (b) detection, purification, quantification and/or amplification of cells and/or microorganisms (e.g., viruses, fungi and bacteria) from the environment (e.g., water, soil) or biological samples (e.g., tissues, body fluids), (c) detection, quantification of the hazards posed by compounds or biological samples (e.g., toxic waste, anthrax) to food safety, human health or national safety, (d) detection and quantification of vital parameters in medical or physiological monitors, such as glucose, blood oxygen levels, total blood cell counts, (e) detection and quantification of specific DNA or RNA from biological samples (e.g., cells, viruses, body fluids), (f) sequencing and comparison of genetic sequences of DNA in chromosomes and mitochondria for genomic analysis, or (g) detection and quantification of reaction products, e.g., during synthesis or purification of drugs.

In some embodiments, subject devices, apparatuses, systems, and methods are used to detect nucleic acids, proteins, or other molecules or compounds in a sample. In certain embodiments, the devices, apparatuses, systems and methods are used for rapid clinical detection and/or quantification of one or more, two or more, or three or more disease biomarkers in a biological sample, e.g., for diagnosis, prevention and/or control of a disease condition in a subject. In certain embodiments, devices, apparatuses, systems, and methods are used to detect and/or quantify one or more, two or more, or three or more environmental markers in environmental samples, such as samples obtained from rivers, oceans, lakes, rain, snow, sewage treatment runoff, agricultural runoff, industrial runoff, tap water, or potable water. In certain embodiments, devices, apparatuses, systems, and methods are used to detect and/or quantify one or more, two or more, or three or more food markers from a food sample obtained from tap water, drinking water, prepared food, processed food, or raw food.

In some embodiments, the subject device is part of a microfluidic device. In some embodiments, subject devices, apparatus, systems, and methods are used to detect fluorescent or luminescent signals. In some embodiments, subject devices, apparatus, systems, and methods include or are used with communication devices such as, but not limited to: mobile phones, tablet computers and portable computers. In some embodiments, subject devices, apparatuses, systems, and methods include or are used with an identifier, such as, but not limited to, an optical barcode, a radio frequency ID tag, or a combination thereof.

In some embodiments, the sample is a diagnostic sample obtained from a subject, the analyte is a biomarker, and the measured amount of the analyte in the sample is diagnostic of the disease or disorder. In some embodiments, the subject devices, systems, and methods further comprise receiving or providing a report to the subject indicating the measured amount of the biomarker and the measured value range for the biomarker in an individual who does not have or is at low risk of having a disease or disorder, wherein the measured amount of the biomarker relative to the measured value range is used to diagnose the disease or disorder.

In some embodiments, the sample is an environmental sample, and wherein the analyte is an environmental marker. In some embodiments, subject devices, systems, and methods include receiving or providing a report indicating the safety or hazardousness of the subject's exposure to the environment from which the sample was taken. In some embodiments, subject devices, systems, and methods include sending data containing measured quantities of environmental indicia to a remote location and receiving a report indicating the safety or hazard of the subject's exposure to the environment from which the sample was taken.

In some embodiments, the sample is a food sample, wherein the analyte is a food marker, and wherein the amount of the food marker in the sample correlates with safety of food consumption. In some embodiments, subject devices, systems, and methods include receiving or providing a report indicating the safety or hazardousness of a subject to consume food from which a sample is obtained. In some embodiments, subject devices, systems, and methods include sending data containing measured amounts of food markers to a remote location and receiving a report indicating the safety or hazardousness of the subject to consume the food from which the sample was obtained.

9.2 hinge, open notch, groove edge and slider

The devices, systems, and methods disclosed herein may include or use a Q-card for sample detection, analysis, and quantification. In some embodiments, the Q-card includes hinges, notches, recesses, and sliders that help facilitate the handling of the Q-card and the measurement of the sample. The structure, materials, functions, variations and dimensions of the hinges, notches, grooves and sliders are disclosed herein or listed, described and summarized in PCT applications (assigned US) nos. PCT/US2016/045437 and PCT/US0216/051775 filed 2016, 10/8/9/14/2016, 2017, 62/456065, 2017, 2/7, and 62/456287, 2017, 2/8, all of which are incorporated herein in their entirety for all purposes.

9.3Q card, slider and cell-phone detecting system

The devices, systems, and methods disclosed herein may include or use a Q-card for sample detection, analysis, and quantification. In some embodiments, a Q-card is used with a slider of the card that allows the smartphone detection system to read. The structure, materials, functions, variations, dimensions and connections of the Q-card, slider and cell phone detection system are disclosed herein or listed, described and summarized in PCT application (assigned US) numbers PCT/US2016/045437 and PCT/US0216/051775, US provisional application number 62/456065 filed on 2/7/2017, US provisional application number 62/456287 filed on 2/8/2017, and US provisional application number 62/456504 filed on 2/8/2017, respectively, all of which are incorporated herein by reference in their entirety for all purposes.

9.4 detection method

The devices, systems, and methods disclosed herein may include or be used with various types of detection methods. The detection methods are disclosed herein or listed, described and summarized in PCT applications (assigned US) No. PCT/US2016/045437 and No. PCT/US0216/051775, U.S. provisional application No. 62/456065 filed on 7/2/2017, U.S. provisional application No. 62/456287 filed on 8/2/2017, all of which are incorporated herein in their entirety for all purposes.

9.5 Label

The devices, systems, and methods disclosed herein may employ various types of labels for analyte detection. The labels are disclosed herein or listed, described and summarized in PCT applications (assigned US) nos. PCT/US2016/045437 and PCT/US0216/051775, filed on PCT application days 8/10/2016 and 9/14/2016, respectively, US provisional application nos. 62/456065 filed on US 2/7/2017, and US provisional application No. 62/456287 filed on US 2/8/2017, all of which are incorporated herein in their entireties for all purposes.

9.6 analytes

The devices, systems, and methods disclosed herein can be used to manipulate and detect various types of analytes, including biomarkers. Analytes are disclosed herein or listed, described and summarized in PCT applications (assigned US) nos. PCT/US2016/045437 and PCT/US0216/051775, filed on

PCT application days

8, 10, 2016, 9, 14, respectively, 2017, US provisional application No. 62/456065, filed on US2, 7, 2017, US provisional application No. 62/456287, filed on US2, 8, 2017, all of which are incorporated herein in their entireties for all purposes.

Applications (fields and samples) 9.7

The devices, systems, and methods disclosed herein may be used in a variety of applications (fields and samples). Applications are disclosed herein or listed, described and summarized in PCT applications (assigned US) nos. PCT/US2016/045437 and PCT/US0216/051775, filed on PCT applications 8/10/2016 and 9/14/2016, respectively, US provisional application nos. 62/456065 filed on US 2/7/2017, and US provisional application No. 62/456287 filed on US 2/8/2017, all of which are incorporated herein in their entireties for all purposes.

9.8 cloud

The devices, systems, and methods disclosed herein may employ cloud technology for data transmission, storage, and/or analysis. Related cloud technologies are disclosed herein or listed, described and summarized in PCT applications (assigned US) nos. PCT/US2016/045437 and PCT/US0216/051775, filed on days 8/10 and 2016 9/14, respectively, US provisional application nos. 62/456065, filed on days 2/7, 2017, and US provisional application No. 62/456287, filed on days 2/8, 2017, all of which are incorporated herein in their entireties for all purposes.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise, e.g., when the word "single" is used. For example, reference to "an analyte" includes a single analyte and a plurality of analytes, reference to "a capture agent" includes a single capture agent and a plurality of capture agents, reference to "a detection agent" includes a single detection agent and a plurality of detection agents, and reference to "a reagent" includes a single reagent and a plurality of reagents.

As used herein, the terms "adapted" and "constructed" mean that an element, component, or other subject matter is designed and/or intended to perform a given function. Thus, the use of the terms "adapted" and "configured" should not be construed to mean that a given element, component, or other subject matter is simply "capable" of performing a given function. Similarly, subject matter recited as configured to perform a particular function may additionally or alternatively be described as operable to perform that function.

As used herein, the phrase "for example," when used in reference to one or more components, features, details, structures, embodiments, and/or methods in accordance with the present disclosure, is intended to convey that the described components, features, details, structures, embodiments, and/or methods are illustrative, non-exclusive examples of components, features, details, structures, embodiments, and/or methods in accordance with the present disclosure. Accordingly, the described components, features, details, structures, embodiments, and/or methods are not intended to be limiting, required, or exclusive/exhaustive; as well as other components, features, details, structures, embodiments, and/or methods, including structurally and/or functionally similar and/or equivalent components, features, details, structures, embodiments, and/or methods, are also within the scope of the present disclosure.

As used herein, the phrases "at least one" and "one or more" in reference to a list of more than one entity refer to any one or more of the entities in the list of entities and are not limited to at least one of each (each) and each (every) entity specifically listed in the list of entities. 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") can refer to a alone, B alone, or a combination of a and B.

As used herein, the term "and/or" as placed between a first entity and a second entity refers to one of (1) the first entity, (2) the second entity, and (3) the first entity and the second entity.

The use of "and/or" listed plural entities should be construed in the same way, i.e., "one or more" of the entities so combined. In addition to the entities specifically identified by the "and/or" clause, other entities, whether related or unrelated to those specifically identified, may optionally be present.

When numerical ranges are mentioned herein, the invention includes embodiments in which endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other endpoint is excluded. Both endpoints should be assumed to be included unless otherwise stated. Moreover, unless otherwise indicated or apparent from the context and understanding to one of ordinary skill in the art.

It is believed that the following claims particularly point out certain combinations and subcombinations directed to one of the disclosed inventions and are novel and non-obvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed through amendment of the present claims or presentation of new claims in this or a related application. Such amended or new claims, whether they are directed to a different invention or directed to the same invention, whether different, broader, narrower or equal in scope to the original claims, are also regarded as included within the subject matter of the inventions of the present disclosure.

Claims

1. A device for assaying a sample, comprising:

a first plate, a second plate, a spacer, and a sponge, wherein:

the first plate comprising on its inner surface means for contacting a sample containing or suspected of containing an analyte,

the spacers are fixed on respective surfaces of one or both of the plates, the spacers having a predetermined substantially uniform height and a predetermined fixed spacer spacing, and

the sponge is made of a flexible porous material that can absorb or release liquid;

wherein one of the configurations is an open configuration, wherein:

the two plates are partially or completely separated,

the spacing between the plates is not adjusted by the spacers, allowing the sample to be deposited on one or both of the plates,

wherein another of the configurations is a closed configuration that is configured after deposition of the sample in the open configuration; and in the closed configuration:

at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness, and the uniform thickness of the layer is limited by the inner surfaces of the two plates and is adjusted by the plates and spacers, and

wherein a washing configuration is configured when the second plate is separated from the first plate after the closed configuration; and in the washing configuration:

the sponge containing a wash solution is placed on the sample contact area of the first plate and when pressed, the sponge fills the sample contact area with the wash solution and when the press is released, the wash solution is resorbed.

2. A method of assaying a sample comprising

(a) Obtaining a first plate, a second plate, and a spacer, wherein:

i. the plates are movable relative to each other into different configurations;

the first plate comprising on its inner surface a sample contacting area for contacting a sample comprising an analyte,

one or both of the plates comprises a spacer secured to an inner surface of the respective plate;

the spacers have a predetermined substantially uniform height and a predetermined spacer spacing;

(b) depositing a liquid sample on the sample contacting area of the first plate in an open configuration in which the two plates are partially or fully separated;

(c) pressing the plates into a closed configuration in which at least a portion of the sample is compressed into a layer of uniform thickness by the first and second plates and incubating the sample for a predetermined period of time,

(d) the second plate is removed from the first plate,

(e) placing a sponge containing a wash solution on the sample contacting area of the first plate,

(f) pressing the sponge to deposit the wash solution onto the sample contact area, holding the sponge in the pressed position for a period of time, and releasing the sponge to reabsorb the wash solution.

3. The device of any preceding device claim, wherein the liquid sample comprises a bodily fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

4. The device of any preceding device claim, wherein the sample is blood.

5. The device of any preceding device claim, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

6. The device of any preceding device claim, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and any combination thereof.

7. The device of any preceding device claim, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

8. The device of any preceding device claim, wherein the young's modulus of the spacer multiplied by the fill factor of the spacer, which is the ratio of the spacer area in the sample contact surface to the total area of the sample contact surface, is equal to or greater than 10 MPa.

9. The device of any preceding device claim, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

10. The device of any preceding device claim, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

11. The device of any preceding device claim, wherein the spacer has at least 100/mm²The density of (c).

12. The device of any preceding device claim, wherein the spacer has at least 1000/mm²The density of (c).

13. The device of any preceding device claim, wherein the spacer is a post having a cross-sectional shape selected from a circle, polygon, perfect circle, square, rectangle, oval, ellipse, or any combination thereof.

14. The device of any preceding device claim, wherein the average thickness of the layer of uniform thickness has a value equal to or less than 1 μ ι η.

15. The device of any preceding device claim, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η.

16. The device of any preceding device claim, wherein the average thickness of the uniform thickness layer has a value in the range of 10 μ ι η to 30 μ ι η.

17. The device of any preceding device claim, wherein the average thickness of the uniform thickness layer is a value in the range of 2 μ ι η to 3.8 μ ι η, and the sample is blood.

18. The device of any preceding device claim, wherein the average thickness of the uniform thickness layer has a value in the range 1 μm to 10 μm, and the sample is exhaled breath condensate.

19. The device of any preceding device claim, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

20. The device of any preceding device claim, wherein the first and second panels are connected and configured to be changed from the open configuration to the closed configuration by folding the panels.

21. The device of any preceding device claim, wherein the first and second panels are connected by a hinge and are configured to change from the open configuration to the closed configuration by folding the panels along the hinge.

22. The device of any preceding device claim, wherein the first and second panels are connected by a hinge and are configured to change from the open configuration to the closed configuration by folding the panels along the hinge, the hinge being a separate material from the panels.

23. The device of any preceding device claim, wherein the sponge comprises a porous substrate and the porous substrate comprises pores having diameters in the range of 10nm to 100nm, 100nm to 500nm, 500nm to 1 μ ι η, 1 μ ι η to 10 μ ι η, 10 μ ι η to 50 μ ι η, 50 μ ι η to 100 μ ι η, 100 μ ι η to 500 μ ι η, 500 μ ι η to 1 mm.

24. The device of any preceding device claim, wherein the sponge comprises a porous substrate and the porous substrate contains pores having diameters in the range of 500nm to 1 μ ι η, 1 μ ι η to 10 μ ι η, 10 μ ι η to 50 μ ι η, 50 μ ι η to 100 μ ι η, 100 μ ι η to 500 μ ι η.

25. The device of any preceding device claim, wherein the sponge comprises a porous substrate, and the porous substrate has a porosity in the range of 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 99%.

26. The device of any preceding device claim, wherein the sponge comprises a porous substrate, and the porous substrate has a porosity in the range of 70% to 80%, 80% to 90%, 90% to 99%.

27. The device of any preceding device claim, wherein the sponge comprises a porous substrate and the material of the porous substrate comprises rubber, cellulose wood fibers, foamed plastic polymers, low density polyethers, polyvinyl alcohol (PVA), polyester, poly (methyl methacrylate) (PMMA), polystyrene, and the like.

28. The method of any preceding method claim, further comprising: after said step (f), detecting said analyte bound to said capture agent.

29. The method of any preceding method claim, wherein the detecting comprises measuring at least one of fluorescence, luminescence, scattering, reflection, absorption, and surface plasmon resonance associated with an analyte bound to the capture agent.

30. The method of any preceding method claim, wherein the inner surface of the first plate at the assay site comprises a signal amplification surface, such as a metal and/or dielectric microstructure.

31. The method of any preceding method claim, wherein the sample comprises a bodily fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

32. The method of any preceding method claim, wherein the sample is blood.

33. The method of any preceding method claim, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

34. The method of any preceding method claim, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and any combination thereof.

35. The method of any preceding method claim, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

36. The method of any preceding method claim, wherein the young's modulus of the spacer multiplied by the fill factor of the spacer, which is the ratio of the spacer area in the sample contact surface to the total area of the sample contact surface, is equal to or greater than 10 MPa.

37. The method of any preceding method claim, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

38. The method of any preceding method claim, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

39. The method as claimed in any of the preceding method claimsThe method of (1), wherein the spacer has at least 100/mm²The density of (c).

40. The method of any preceding method claim, wherein the spacer has at least 1000/mm²The density of (c).

41. The method of any preceding method claim, wherein the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

42. The method of any preceding method claim, wherein the average thickness of the uniform thickness layer has a value equal to or less than 1 μ ι η.

43. The method of any preceding method claim, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η.

44. The method of any preceding method claim, wherein the average thickness of the uniform thickness layer has a value in the range of 10 μ ι η to 30 μ ι η.

45. The method of any preceding method claim, wherein the average thickness of the uniform thickness layer has a value in the range of 2 μ ι η to 3.8 μ ι η, and the sample is blood.

46. The method of any preceding method claim, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η, and the sample is exhaled breath condensate.

47. The method of any preceding method claim, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

48. The method of any preceding method claim, wherein the first and second panels are connected and configured to be changed from the open configuration to the closed configuration by folding the panels.

49. The method of any preceding method claim, wherein the first and second panels are connected by a hinge and are configured to change from the open configuration to the closed configuration by folding the panels along the hinge.

50. The method of any preceding method claim, wherein the first and second panels are connected by a hinge and are configured to change from the open configuration to the closed configuration by folding the panels along the hinge, the hinge being a separate material from the panels.

51. The method of any preceding method claim, wherein the sponge comprises a porous substrate and the porous substrate contains pores having diameters in the range of 10nm to 100nm, 100nm to 500nm, 500nm to 1 μ ι η, 1 μ ι η to 10 μ ι η, 10 μ ι η to 50 μ ι η, 50 μ ι η to 100 μ ι η, 100 μ ι η to 500 μ ι η, 500 μ ι η to 1 mm.

52. The method of any preceding method claim, wherein the sponge comprises a porous substrate and the porous substrate contains pores having diameters in the range of 500nm to 1 μ ι η, 1 μ ι η to 10 μ ι η, 10 μ ι η to 50 μ ι η, 50 μ ι η to 100 μ ι η, 100 μ ι η to 500 μ ι η.

53. The method of any preceding method claim, wherein the sponge comprises a porous substrate, and the porous substrate has a porosity in the range of 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 99%.

54. A method according to any preceding method claim, wherein the sponge comprises a porous substrate having a porosity in the range 70% to 80%, 80% to 90%, 90% to 99%.

55. The method of any preceding method claim, wherein the sponge comprises a porous substrate and the material of the porous substrate comprises rubber, cellulose wood fibers, foamed plastic polymers, low density polyethers, polyvinyl alcohol (PVA), polyester, poly (methyl methacrylate) (PMMA), polystyrene, and the like.

56. A method for determining a dilution factor of a diluted sample, comprising the steps of:

(a) providing an initial sample containing a calibration marker;

(b) obtaining a first concentration of a calibration marker in the initial sample;

(c) diluting the initial sample with an unknown volume of diluent to form a diluted sample;

(d) after (c), obtaining a second concentration of the calibration marker using a concentration measurement device; and

(e) determining the dilution factor by comparing the first concentration and the second concentration,

wherein the concentration measuring device comprises:

a first plate, a second plate, a spacer, and a detector, wherein:

i. the plates are movable relative to each other into different configurations;

one or both plates are flexible;

each of said plates having on its respective surface a sample contacting region for contacting a sample containing an analyte;

v. the spacers have a predetermined substantially uniform height and a predetermined constant inter-spacer distance, at least one of the spacers being within the sample contact area, an

A detector for detecting an analyte;

wherein one of the configurations is an open configuration, wherein:

the two plates are partially or completely separated,

the spacing between the plates is not adjusted by the spacers, depositing the sample on one or both of the plates; and

at least a portion of the sample is compressed by the two plates into a layer of uniform thickness,

a layer of uniform thickness defined by the inner surfaces of the two plates is regulated by the plates and spacers and has a small variation of average thickness equal to or less than 5 μm, and

the detector detects the analyte and calculates the concentration of the analyte in the sample.

57. The method of claim 56, wherein in step (b) a first concentration of the calibration marker, if unknown, is obtained using the concentration measurement device.

58. The method of claim 56 or any claim dependent therefrom, wherein the sample comprises a body fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

59. The method of claim 56 or any claim dependent thereon, wherein the sample is blood.

60. The method of claim 56 or any claim dependent thereon, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

61. The method of claim 56 or any claim dependent thereon, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and combinations thereof.

62. The method of claim 56 or any claim dependent thereon, wherein one or both plates comprise position markings on or within the surface of the plate, the position markings providing information of the position of the plate.

63. The method of claim 56 or any claim dependent thereon, wherein one or both plates comprise graduation markings on or within the surface of the plate, which provide information on the lateral dimensions of the sample and/or plate structure.

64. The method of claim 56 or any claim dependent thereon, wherein one or both plates comprise imaging indicia on or within a surface of the plate, the imaging indicia facilitating imaging of the sample.

65. The method of claim 56 or any claim dependent thereon, wherein the spacer is used as a position mark, a scale mark, an imaging mark, or any combination thereof.

66. The method of claim 56 or any claim dependent thereon, wherein the uniform thickness layer has an average thickness in the range of 2 μ ι η to 2.2 μ ι η and the sample is blood.

67. The method of claim 56 or any claim dependent thereon, wherein the uniform thickness layer has an average thickness in the range of 2.2 μ ι η to 2.6 μ ι η and the sample is blood.

68. The method of claim 56 or any claim dependent thereon, wherein the uniform thickness layer has an average thickness in the range of 1.8 μ ι η to 2 μ ι η and the sample is blood.

69. The method of claim 56 or any claim dependent thereon, wherein the uniform thickness layer has an average thickness in the range of 2.6 μ ι η to 3.8 μ ι η and the sample is blood.

70. The method of claim 56 or any claim dependent thereon, wherein the uniform thickness layer has an average thickness in the range of 1.8 μ ι η to 3.8 μ ι η and the sample is whole blood that is not diluted with another liquid.

71. The method of claim 56 or any claim dependent thereon, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

72. The method of claim 56 or any claim dependent thereon, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

73. The method of claim 56 or any claim dependent thereon, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

74. The method of claim 56 or any claim dependent thereon, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

75. The method of claim 56 or any claim dependent thereon, wherein the spacer has at least 100/mm²The density of (c).

76. The method of claim 56 or any claim dependent thereon, wherein the spacer has at least 1000/mm²The density of (c).

77. The method of claim 56 or any claim dependent thereon, wherein the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

78. The device of claim 56 or any claim dependent thereon, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

79. An apparatus for sample analysis, comprising:

a first plate, a second plate, a spacer, and a filter, wherein:

i. the plates are movable relative to each other into different configurations;

the spacers are fixed on the inner surface of one or more of the plates, the spacers having a predetermined substantially uniform height and a predetermined spacer spacing;

placing the filter having a sample receiving surface and a sample discharge surface on top of the first plate, wherein the sample discharge surface faces the inner surface of the first plate; and

a sample receiving surface of the filter for depositing a liquid sample comprising one or more components;

wherein one of the configurations is a deposition configuration, wherein:

the second plate is partially or completely separate from the first plate and filter;

the sample is deposited on a sample receiving surface of the filter; and

the distance between the first and second plates is not adjusted by their spacers, the filter or the deposited sample; and

wherein another of the configurations is a filtration configuration, wherein:

the filter is positioned between the first plate and the second plate,

the distance between the first plate and the second plate is adjusted by their spacers, the filter, and the deposited sample, and

the inner surface of the second plate presses the deposited sample against the filter, forcing at least one component of the sample through the filter towards the first plate, thereby separating the at least one component from the sample.

80. A method for sample analysis, comprising the steps of:

(a) obtaining a liquid sample;

(b) a first plate, a second plate, a spacer, and a filter, wherein:

i. the plates are movable relative to each other into different configurations;

placing the filter having a sample receiving surface and a sample discharge surface on top of the first plate, wherein the sample discharge surface faces the inner surface of the first plate;

(c) depositing the sample on a sample receiving surface of the filter when the plate is in a deposition configuration, wherein:

the two plates are partially or completely separate, and

the spacing between the plates is not adjusted by spacers, the filter or the deposited sample; and (d) after (c), bringing the two plates together; and

conforming, in parallel or in sequence, regions of at least one of the plates to press the plates into a filtering configuration, wherein:

The conforming press produces a substantially uniform pressure on the panel,

the conformal compression is such that the pressure exerted on an area is substantially constant regardless of changes in the shape of the outer surface of the plate.

The parallel conformal compression simultaneously applies pressure on the desired area,

the conformal compression sequentially applies pressure on a portion of the intended region and gradually moves to other regions, and

in the filtration configuration, the spacing between plates in a layer of uniform thickness regions is adjusted by the spacer, the filter, and the deposited sample.

81. The device of claim 79, wherein the liquid sample comprises a bodily fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

82. The device of claim 79 or any claim dependent thereon, wherein the sample is blood.

83. The device of claim 79 or any claim dependent thereon, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

84. The device of claim 79 or any claim dependent thereon, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and combinations thereof.

85. The device of claim 79 or any claim dependent therefrom, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

86. The device of claim 79 or any claim dependent thereon, wherein the young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

87. The device of claim 79 or any claim dependent thereon, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

88. The device of claim 79 or any claim dependent thereon, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

89. The device of claim 79 or any claim dependent thereon, wherein the spacer has at least 100/mm²The density of (c).

90. The device of claim 79 or any claim dependent thereon, wherein the spacer has at least 1000/mm²The density of (c).

91. The device of claim 79 or any claim dependent thereon, wherein the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

92. The device of claim 79 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value equal to or less than 1 μ ι η.

93. The device of claim 79 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η.

94. The device of claim 79 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 10 μ ι η to 30 μ ι η.

95. The device of claim 79 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 2 μ ι η to 3.8 μ ι η and the sample is blood.

96. The device of claim 79 or any claim dependent thereon, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

97. The method of claim 80 or any claim dependent thereon, wherein the sample comprises a body fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

98. The method of claim 80 or any claim dependent thereon, wherein the sample is blood.

99. The method of claim 80 or any claim dependent thereon, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

100. The method of claim 80 or any claim dependent thereon, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and combinations thereof.

101. The method of claim 80 or any claim dependent thereon, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

102. The method of claim 80 or any claim dependent thereon, wherein the young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

103. The method of claim 80 or any claim dependent thereon, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

104. The method of claim 80 or any claim dependent thereon, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

105. The method of claim 80 or any claim dependent thereon, wherein the spacer has at least 100/mm²The density of (c).

106. The method of claim 80 or any claim dependent thereon, wherein the spacer has at least 1000/mm²The density of (c).

107. The method of claim 80 or any claim dependent thereon, wherein the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

108. The method of claim 80 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value equal to or less than 1 μ ι η.

109. The method of claim 80 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η.

110. The method of claim 80 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 10 μ ι η to 30 μ ι η.

111. The method of claim 80 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 2 μ ι η to 3.8 μ ι η and the sample is blood.

112. The method of claim 80 or any claim dependent thereon, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

113. An apparatus for sample analysis, comprising:

a first plate, a second plate, a third plate, and a spacer, wherein:

i. the second plate and the third plate are respectively connected to the first plate, wherein the second plate and the third plate are configured to each pivot against the first plate without interfering with each other,

the second or third plate is movable into a different configuration relative to the first plate by pivoting against the first plate,

the first plate comprises an inner surface having a sample contacting area for contacting a liquid sample, and

the spacers are fixed on the inner surface of one or more of the plates or mixed in the sample, the spacers having a predetermined substantially uniform height and a predetermined spacer pitch; and

wherein one of the configurations is an open configuration, wherein:

all three plates are partially or completely separated,

the spacing between the plates is not adjusted by the spacers, and

the sample is deposited on an inner surface of the first plate, the second plate, or both; and

wherein another of the configurations is a closed configuration configured after deposition of the sample in the open configuration, and in the closed configuration:

at least a portion of the deposited sample is compressed into a layer of uniform thickness by the first and second plates, and

the uniform thickness of the layer is defined by the inner surfaces of the first and second plates and is adjusted by the plates and spacers.

114. The apparatus of claim B1, further comprising a filter, wherein:

the filter, having a sample receiving surface and a sample discharge surface, is placed on top of the first plate, wherein the sample discharge surface is directed towards the inner surface of the first plate;

in the open configuration:

all three plates are partially or completely separated,

the spacing between the plates is not adjusted by the spacers, and

depositing a sample comprising one or more components on a sample receiving surface of the filter;

a filtration configuration is configured after the sample is deposited in the open configuration, in which:

the filter is positioned between the first plate and the third plate,

the distance between the first plate and the third plate is adjusted by their spacers, the filter, and the deposition sample, and

the inner surface of the third plate pressing the deposited sample against the filter forcing at least one component of the sample through the filter towards the first plate, thereby separating the at least one component from the sample; and

the closed configuration is configured after removing the third plate, the press sample, and the filter from the first plate, and in the closed configuration:

the filtered at least one component remaining on the first sheet is compressed into a layer of uniform thickness by the first sheet and the second sheet, and

the uniform thickness of the layer is defined by the inner surfaces of the first and second plates and is regulated by the plates and spacers.

115. A method for sample analysis, comprising:

(a) obtaining a liquid sample comprising one or more components,

(b) obtaining a device comprising a first plate, a second plate, a third plate, a filter, and a spacer, wherein:

i. the second and third plates are respectively connected to the first plate, wherein the second and third plates are configured to each pivot against the first plate without interfering with each other,

the first plate comprises an inner surface having a sample contacting area for contacting a liquid sample;

the spacer is fixed on one or more of the plates or mixed in the sample, and

v. placing the filter having a sample receiving surface and a sample discharge surface on top of the first plate, wherein the sample discharge surface faces the inner surface of the first plate,

(c) depositing a sample comprising one or more components on a sample receiving surface of the filter,

(d) pressing the third plate against the deposited sample against the filter, forcing at least one component of the sample through the filter towards the first plate, thereby separating the at least one component from the sample,

(e) removing the third plate, the pressing sample and the filter from the first plate, and

(f) compressing the filtered at least one component remaining on the first plate into a layer of uniform thickness by pressing the first plate and the second plate together.

116. The device of claim 113 or any claim dependent thereon, wherein the sample comprises a bodily fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

117. The device of claim 113 or any claim dependent thereon, wherein the sample is blood.

118. The device of claim 113 or any claim dependent thereon, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

119. The device of claim 113 or any claim dependent thereon, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and combinations thereof.

120. The device of claim 113 or any claim dependent thereon, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

121. The apparatus of claim 113 or any claim dependent thereon, wherein the young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

122. The device of claim 113 or any claim dependent thereon, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

123. The device of claim 113 or any claim dependent thereon, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

124. The device of claim 113 or any claim dependent thereon, wherein the spacer has at least 100/mm²The density of (c).

125. The device of claim 113 or any claim dependent thereon, wherein the spacer has at least 1000/mm²The density of (c).

126. The device of claim 113 or any claim dependent thereon, wherein the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

127. The device of claim 113 or any claim dependent thereon, wherein the average thickness of the uniform-thickness layer has a value equal to or less than 1 μ ι η.

128. The device of claim 113 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η.

129. The device of claim 113 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 10 μ ι η to 30 μ ι η.

130. The device of claim 113 or any claim dependent thereon, wherein the average thickness of the uniform-thickness layer has a value in the range of 2 μ ι η to 3.8 μ ι η and the sample is blood.

131. The device of claim 113 or any claim dependent thereon, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

132. The device of claim 113 or any claim dependent thereon, wherein the third plate is configured to press the sample against the filter when the third plate is pivoted towards the first plate.

133. The device of claim 113 or any claim dependent thereon, wherein one edge of the second panel is connected to the inner surface of the first panel by a first hinge.

134. The device of claim 113 or any claim dependent thereon, wherein an edge of the third plate is connected to the inner surface of the first plate by a second hinge.

135. The device of claim 113 or any claim dependent thereon, wherein one edge of the second plate is connected to the inner surface of the first plate by a first hinge and one edge of the third plate is connected to the inner surface of the first plate by a second hinge.

136. The device of claim 113 or any claim dependent thereon, wherein in the closed configuration between the first plate and the second plate, the third plate is adjustable to pivot against the first plate and the second plate.

137. The device of claim 113 or any claim dependent thereon, wherein the first plate comprises one or more notches on one or more edges thereof, wherein the notches are positioned such that the second plate and/or the third plate are juxtaposed over the notches to facilitate manipulation of pivoting of the second plate and the third plate.

138. The method of claim 115 or any claim dependent thereon, wherein the sample comprises a bodily fluid selected from the group consisting of: amniotic fluid, aqueous humor, vitreous humor, blood (e.g., whole blood, fractionated blood, plasma, or serum), breast milk, cerebrospinal fluid (CSF), cerumen (cerumen), chyle, chyme, endolymph, perilymph, stool, breath, gastric acid, gastric juice, lymph, mucus (including nasal drainage and sputum), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatic fluid, saliva, exhaled breath condensate, sebum, semen, sputum, sweat, synovial fluid, tears, vomit, urine, and combinations thereof.

139. The method of claim 115 or any claim dependent thereon, wherein the sample is blood.

140. The method of claim 115 or any claim dependent thereon, wherein the sample is an environmental sample from an environmental source selected from the group consisting of: rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, sewage, air, underwater heat dissipation, industrial waste gas, vehicle waste gas, and combinations thereof.

141. The method of claim 115 or any claim dependent thereon, wherein the sample is a food sample selected from the group consisting of: raw materials, cooked foods, plant and animal food sources, pre-processed foods, partially or fully processed foods, and combinations thereof.

142. The method of claim 115 or any claim dependent thereon, wherein the spacer has a fill factor of at least 1%, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

143. The method of claim 115 or any claim dependent thereon, wherein the young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 10MPa, the fill factor being the ratio of the spacer area in the sample contacting surface to the total area of the sample contacting surface.

144. The method of claim 115 or any claim dependent thereon, wherein the spacer pitch is in the range of 1 μ ι η to 200 μ ι η, and the spacer pitch is substantially fixed.

145. The method of claim 115 or any claim dependent thereon, wherein the spacer pitch is in the range of 7 μ ι η to 200 μ ι η and the sample is blood.

146. The process of claim 115 or any one thereofThe method of the dependent claim, wherein the spacer has at least 100/mm²The density of (c).

147. The method of claim 115 or any claim dependent thereon, wherein the spacer has at least 1000/mm²The density of (c).

148. The method of claim 115 or any claim dependent thereon, wherein the spacer is a post having a cross-sectional shape selected from a circle, a polygon, a perfect circle, a square, a rectangle, an oval, an ellipse, or any combination thereof.

149. The method of claim 115 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value equal to or less than 1 μ ι η.

150. The method of claim 115 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 1 μ ι η to 10 μ ι η.

151. The method of claim 115 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 10 μ ι η to 30 μ ι η.

152. The method of claim 115 or any claim dependent thereon, wherein the average thickness of the uniform thickness layer has a value in the range of 2 μ ι η to 3.8 μ ι η and the sample is blood.

153. The method of claim 115 or any claim dependent thereon, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP or other plastics.

154. The method of claim 115 or any claim dependent thereon, wherein the third plate is configured to press the sample against the filter when the third plate is pivoted towards the first plate.

155. The method of claim 115 or any claim dependent thereon, wherein one edge of the second panel is connected to the inner surface of the first panel by a first hinge.

156. The method of claim 115 or any claim dependent thereon, wherein one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

157. The method of claim 115 or any claim dependent thereon, wherein one edge of the second panel is connected to the inner surface of the first panel by a first hinge and one edge of the third panel is connected to the inner surface of the first panel by a second hinge.

158. The method of claim 115 or any claim dependent thereon, wherein the third plate is adjustable to pivot against the first and second plates in a closed configuration between the first and second plates.

159. The method of claim 115 or any claim dependent thereon, wherein the first plate comprises one or more notches on one or more edges thereof, wherein the notches are positioned such that the second plate and/or the third plate are juxtaposed over the notches to facilitate manipulation of pivoting of the second plate and the third plate.