JP6625519B2 - Assay systems and cartridge devices - Google Patents

Description

Cross-reference to related application

  This application claims the benefit of co-pending US Provisional Patent Application No. 61 / 800,101, filed March 15, 2013, which is incorporated herein.

  The life sciences industry relies on the accurate and timely development and processing of tests known as bioassays for the discovery of new drugs, the identification of new biomarkers or the measurement of biomarker levels for diagnosing and monitoring disease. I have.

  However, known assays suffer from several limitations. One such limitation is that specific binding of the target analyte to the capture reagent during the assay cannot be distinguished from non-specific binding of the non-target substance, molecule, or analyte to the capture reagent during the assay. . Any non-specific binding can result in unnaturally high measurements of analyte concentrations and inaccurate quantification. Indeed, in some cases, even if the analyte is not present in the test sample, the signal generated by the non-specific interaction results in a false positive test result.

  A further significant limitation of the known assays is that they cannot accurately detect and measure different analytes that can be present in the at least one sample to be analyzed at orders of magnitude different concentrations. . The limitations on quantifying analyte concentrations in a single test over such a wide dynamic range limit the biological validity of many currently available multiplex assays, and furthermore, the multiple serial number of samples. Dilutions also need to be performed, which requires additional time, expense and use of valuable samples.

  In addition, most known assays require relatively complex user-intensive protocols, thus limiting the use of many assays to well-equipped laboratories with trained and skilled staff. You.

  International Patent Application Publication No. WO 2012/071044 (“'044 Application”), which is incorporated herein by reference in its entirety, describes a long-term assay method that overcomes many of the limitations of known assays. However, the systems and methods disclosed herein further address the limitations set forth above and provide a unique and novel approach to accurate detection and measurement of multiple analytes. .

  The invention disclosed herein relates to systems and related methods for integrating and constructing from long-term assay screening.

  Important to the disclosed systems and methods is the ability to collect image data during the bioassay incubation process. This allows for the collection of data over time or long term used to generate real-time kinetic binding curves.

  In one embodiment, the invention is an assay system comprising a cartridge device and a measurement device, wherein the cartridge device comprises one or more reservoir portions for holding one or more liquids, at least At least one assay portion for receiving one or more liquids from one reservoir portion, wherein one or more liquids from one or more reservoirs are flowed over (twice or more) repeatedly; An assay moiety having a plurality of binding sites to be obtained, wherein the measurement device is for measuring binding of one or more analytes in one or more liquids to the plurality of binding sites. Provide an assay system.

  In another embodiment, the present invention is a receiver for receiving a cartridge for a fluid, the cartridge controlling a flow rate of one or more fluids from one or more reservoirs in the cartridge. A cartridge having one or more reservoirs containing a fluid, wherein the cartridge has one or more reservoirs containing a fluid, wherein at least one of the fluids contains one or more target analytes. The fluid to be analyzed, wherein at least one fluid contains a fluorescent, luminescent or colorimetric label, and the cartridge further comprises one or more fluids that can be transferred from one compartment of the cartridge to another under computer control. The assay portion of the cartridge comprises one or more fluid channels that can be transferred to at least one such compartment. A cartridge comprising an array of two or more binding sites each containing a specific capture agent, each site containing one or more specific capture agents; and interacting with a fluorescent or luminescent or colorimetric label; A device for stimulating and / or stimulating the intensity of a fluorescent or luminescent or colorimetric signal at each such site in a time series; incorporating such a measurement of the time series to a non-target substance within the binding site Apparatus for generating a representation of a dynamic or kinetic binding curve between a / molecule / analyte and a capture agent or other substance.

  In another embodiment, the invention is a method of discriminating between specific and non-specific binding in an assay, wherein each of a plurality of signal intensity measurements comprises a sample to be analyzed and a target in the sample to be analyzed. Obtaining during the iterative interaction of the analyte with the capture agent to obtain a plurality of signal intensity measurements of the sample to be analyzed; and analyzing the plurality of signal intensity measurements with the sample to be analyzed and the capture agent. Plotting as a function of the cumulative duration of the interaction.

  In yet another embodiment, the invention is a method of calculating an analyte concentration in a sample containing an analyte, wherein the plurality of analytes represent binding of a component of the sample to a capture agent capable of binding the analyte. Obtaining a signal intensity measurement of the plurality of signal intensity measurements at a known time interval for a known duration of interaction between the sample and the capture agent; and Plotting the values as a function of the cumulative duration of the interaction of the sample with the capture agent; calculating one or more first derivatives or tangents (initial velocities) to the plotted signal intensity measurements. Calculating each of the one or more first derivatives or tangents (initial velocities) using closely plotted signal intensity measurements; one or more standards Analyte concentration first derivative or tangent for (initial velocity) can be used to determine the suitability of the back-calculated curves later, it provides methods. If the enzyme-substrate complex is formed prior to the reaction, the back-calculated fit is a fit based on an enzyme-catalyzed model. The first derivative or tangent (initial velocity) of the unknown sample is then used to calculate the analyte concentration back from the analytical concentration of one or more standards.

  In yet another embodiment, the invention includes at least one reservoir portion for retaining one or more fluids; and at least one assay for receiving one or more fluids from at least one reservoir portion. A cartridge device having a plurality of binding sites through which fluid is flowed, the at least one assay portion being connected to at least one reservoir portion through a fluid channel or tubing.

  These and other features of the present invention will be more readily understood from the following detailed description of various aspects and embodiments of the invention in connection with the accompanying drawings, which illustrate various embodiments of the present invention. There will be.

1 is an exploded perspective view of an assay cartridge according to an embodiment of the present invention. FIG. 3 is a simplified schematic diagram of four assay portions of an assay cartridge according to an embodiment of the present invention. FIG. 3 shows a simplified schematic diagram of one assay portion of an assay cartridge, plus a simplified schematic diagram of the reservoir and valve components of the system and assay cartridge according to embodiments of the present invention. FIG. 4 is a simplified diagram of the steps of an assay according to an embodiment of the present invention. FIG. 4 is a simplified diagram of another step of an assay according to an embodiment of the present invention. FIG. 4 is a simplified diagram of yet another step in an assay according to an embodiment of the present invention. FIG. 4 is a simplified view of another step of an assay according to an embodiment of the present invention. FIG. 5 is a simplified diagram of an analyte-capture agent and enhancer interaction according to another embodiment of the present invention. FIG. 5 is a simplified diagram of an analyte-capture agent and enhancer interaction according to another embodiment of the present invention. FIG. 5 is a simplified diagram of an analyte-capture agent and enhancer interaction according to another embodiment of the present invention. Figure 4 is a graph of the signal intensity of binding of a target analyte and a non-target analyte. FIG. 4 shows binding curves for triplicate assays targeting IL-6, according to embodiments of the present invention. FIG. 4 shows binding curves for triplicate assays targeting CRP, according to embodiments of the present invention. FIG. 4 shows binding curves of triplicate assays targeting IL-1b, according to embodiments of the present invention.

  It is noted that the drawings are not drawn to scale and are intended to depict only typical aspects of the invention. Therefore, the drawings should not be considered as limiting the scope of the invention. In the drawings, like numbering represents like elements in the drawings.

DEFINITIONS Various terms and phrases used herein are intended to have meaning in the context of the claimed invention, as set forth herein. The term "fluid" is intended to broadly encompass the state of matter that is or is capable of flowing fluid, and includes liquids, gases, and fine particulate solids, and combinations and colloidal suspensions thereof. Including suspensions such as liquids. In the context of the present invention, "fluid" refers to animal (including but not limited to, human) blood, serum, saliva, urine, plasma, tracheal alveolar lavage, tracheal lavage, tissue, tumor, and tissue / tumor. As well as plant extracts, liquefied foods, ascites, organic fluids, inorganic fluids, buffers, labeled buffers, washings, and the like, but is not limited thereto.

  The term "analyte" is intended to mean anything to be detected or measured or capable of doing so. Analytes include proteins, hormones, antibodies, antigens, viruses, antibody complexes, peptides, cells, cell fragments, aptamers, cell lysates, DNA, RNA, mRNA, genes, biological entities such as gene expression products, And chemical entities such as, but not limited to, chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, contaminants, and the like.

  The term "detector reagent" (or detection reagent) is intended to mean anything used as a mechanism to enable visualization (detection) and / or measurement of an analyte. Is done. This includes proteins, hormones, antibodies, antigens, viruses, antibody complexes, antibody fragments, peptides, cells, cell fragments, aptamers, cell lysates, DNA, RNA, mRNA, genes, gene expression products, etc. Includes, but is not limited to, chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, contaminants, and the like.

  The detection reagents can also be labeled with a fluorescent, luminescent or colorimetric label to facilitate visualization (detection) and / or measurement, which are further complexed with an affinity tag label such as biotin. You can also. This makes it possible to use fluorescent, luminescent or colorimetrically labeled streptavidin for the visualization (detection) and measurement of the detection antibody.

  The terms "detect" and "measuring" and variants thereof are intended to refer to the identification of the presence of an object and the assessment of the variable characteristics of an object, such as concentration, strength, intensity, respectively. The term "analyze" and its variants refer more broadly to such detection and measurement, and other or different evaluations of the event.

  The phrase "binding site" is intended to refer to where interaction with an analyte is possible or intended and detection or measurement can be made. Typically, the binding site comprises a capture agent. The phrase “capture agent” (or capture reagent) in sequential order is intended to refer to any structure, compound, system, or device with which the analyte can interact. Often, such interactions include structural or chemical interactions, but this is not required. Capture agents include proteins, hormones, antibodies, antigens, viruses, antibody complexes, antibody fragments, peptides, cells, cell fragments, aptamers, cell lysates, DNA, RNA, mRNA, genes, gene expression products, etc. Including, but not limited to, chemical entities, chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, contaminants, and the like.

  The phrase "signal intensity" is intended to refer to the measurement of an interaction at a binding site, such as by an interaction between a capture agent and an analyte. Such measurements can be made directly or indirectly by any number or combination of techniques such as fluorescence, luminescence, or colorimetric labeling.

  The phrase "standard binding curve" is intended to refer to the binding curve of the binding interaction between the capture agent and the analyte at a known concentration, as opposed to the binding interaction between the capture agent and the analyte at an unknown concentration. Can be compared. The phrases "displaying a dynamic binding curve" and "displaying a kinetic binding curve" are intended to refer to displaying the kinetics or kinetics of the binding interaction between a capture agent and an analyte. These differ from typical and more conventional endpoint standard curves, which are an indication of the intensity of the signal from the assay at a particular time point (ie, at the completion of the assay) at a given analyte dilution. These can include data obtained from all or some of the steps of the binding assay, and whether the detected or measured interaction and / or the corresponding signal intensity can be attributed to a specific binding interaction. Can be used to determine if. A "binding curve" or "kinetic curve" is a signal that increases from low to high due to exposure (association) of the binding site to the sample and the detection reagent and when no sample and detection reagent are present (dissociation). Signals that weaken from high to low can be described.

Assay System or Platform The components of the assay system according to some embodiments of the present invention are described in the '044 application. These include, for example, devices and devices for controlling the movement of liquid across binding sites. These may include, for example, pumps or vacuum devices that can apply positive or negative pressure to the liquid, respectively. In other cases, the device or apparatus may include a capillary or similar structure through which liquid can move by capillary action. In any case, such devices and apparatus may include one or more aspects of liquid flow, such as the flow rate of the liquid, the duration of the liquid flow, or the number of times a quantity of liquid is flowed over the binding site, etc. Control.

  As mentioned above and as described in more detail below, in some embodiments of the assay system according to the present invention, both the inclusion of a sample and the supply of other assay reagents and the area in which the assay itself is performed The cartridge is used for Accordingly, some embodiments of such cartridges include one or more reservoir portions for holding sample and / or assay reagents and another assay portion into which and through which samples and reagents may flow. including. As described with respect to FIG. 3, the assay portion includes a plurality of binding sites (containing a capture agent) over which liquid is flown, where the target analyte and the capture agent interact.

  The assay portion of the cartridge includes at least one location where binding of the target analyte to the capture agent can be detected and / or measured, and more typically a plurality of spots (or dots), such as printed spots (or dots) of the capture agent. There is a place. Each printed spot can contain one or more types of capture agents, and each printed spot in the assay portion contains the same or a different type of capture agent. In some embodiments of the invention, the assay portion of the cartridge comprises a transparent surface, often glass, through which binding events can be detected / measured. If the assay system uses a fluorescence detection device, the excitation beam can pass through the transparent surface to the binding site to excite the fluorescently labeled analyte or the analyte-capture agent complex. The luminescence by the fluorescently labeled analyte or the complex of analyte and capture agent can then be detected / measured through the same or a different transparent surface.

  In certain embodiments, the system incorporates a scanner that uses a confocal approach where the 532 nm laser beam is focused and incident on the surface of the assay portion of the cartridge.

  Imaging is performed just below the assay solution and through the bottom of the cartridge. The assay surface may be a printed capture agent (typically a protein or antibody, but in some embodiments, a hormone, virus, antibody conjugate, antibody fragment, peptide, cell, cell fragment, aptamer, cell lysis And other biological entities such as DNA, RNA, mRNA, genes, gene expression products, and chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, contaminants, etc. Which are used to quantitatively measure analyte concentration in a fluorescently labeled sandwich-type binding assay.

  Fluorescence emitted from the assay at the cartridge surface is collected back in the excitation light path and directed through a series of spatial filters and mirrors to a photomultiplier tube (PMT). The "scan" in the system is performed by a two-axis galvanometer driven mirror assembly and a telecentric lens system that can produce a high resolution image, which is a pixel aligned in time, while the other mirrors And lens systems can also be used. In one embodiment, the scan area allowed for the system is 25 × 25 mm; in other embodiments, this can be reduced or increased to 100 mm × 100 mm or more; In and in other embodiments, the assay portion of the cartridge may be smaller than an acceptable scan area and may range from 1 mm x 1 mm to 100 mm x 100 mm or more. In some embodiments, there are four assay portions available per cartridge. See, for example, FIGS. However, in other embodiments, the number of assay portions may range from 1 to 100.

  The fluorescent laser scans the assay through the underside of the cartridge as the sample is repeatedly flowed through the assay; binding data can be collected over time, providing a sensitive kinetic binding curve Edited to The binding curve for each assay on the cartridge is processed by the analysis software to deliver data on analyte presence and concentration to the user.

Components of the system Detector In some embodiments, the detector can include an excitation light path and an emission light path that are combined to include the detector. The path of the excitation light may include a light source, which in one embodiment is a 532 nm laser tuned by a beam expander and an aperture assembly to size the laser light source and collimate the light. Good.

  In some embodiments, the light source may then be directed to the sample by a dichroic beam splitter, a focusing lens assembly, an XY axis galvanometer driven mirror assembly, and a telecentric lens. The galvanometer driven mirror and telecentric lens assembly allows for a fixed installation of the target sample, thus eliminating the need to align multiple images in post-processing. Fixed mounting of the sample also allows coupling of the flow control module to the sample cartridge, eliminating noise in the fluid control system typically caused by vibrations coming from the movement of the precision flow system.

  In some embodiments, the detection beam path includes a detector (eg, a photomultiplier tube (PMT)), a bandpass filter, a pinhole aperture, a focusing lens, a dichroic beam splitter, and a telecentric lens. In such an embodiment, when the sample is excited by the light source, the sample emits photons, which are collected by a telecentric lens and directed back to the excitation light path to a dichroic beam splitter. The beam splitter allows the emission wavelength to pass through the focusing lens, focus at one point, and pass through the pinhole aperture. The pinhole aperture filters out any light that is out of focus, thus making the detector virtually confocal. The focused light can then pass through a bandpass filter, allowing only the wavelength of interest to interact with the PMT. The value of the PMT intensity can then be recorded as a single pixel and assigned spatially based on the galvanometer controlled xy position of the mirror. In this way, an image containing pixel locations and intensities can be created.

2. The flow control module system can incorporate a microfluidic controller to control the flow of liquid through multiple assay portions (assay cells) of the cartridge. In embodiments where the assay cartridge comprises four such assay portions (or assay cells-see FIG. 2), the microfluidic controller is a computer controlled pressure controller, three-way valve, pressurized. The sample container, eight inlet valves, four outlet valves, and in some embodiments, can include a micromanifold that can direct flow from the four outlet valves to a precision flow sensor. The controller can then allow precise timing of flow events to milliseconds, as well as later before / after assay operation. A dynamically controlled controller (at the beginning of the flow path) paired with a precision flow sensor (at the end of the flow path) works in tandem and provides accurate flow rates and flow rates independent of flow path characteristics A closed loop flow control system that ensures The controller also provides the user with real-time feedback on the status of all valves. The flow control module is optionally incorporated into the body of the system and can be designed to allow the user to efficiently insert and replace samples and reagents. In certain embodiments, the flow control module may have more or less than eight inlet and four outlet valves, the number of valves being proportional to the number of assay portions included in the cartridge, e.g., In certain embodiments, each assay portion requires two inlet valves and one outlet valve (see FIG. 3).

3. Assay Cartridges In some embodiments, such as shown in FIG. 1, the assay cartridge 100 is a capture agent (typically a protein or antibody, but in some embodiments, as described in more detail below). Include other biological entities such as hormones, viruses, antibody complexes, antibody fragments, peptides, cells, cell fragments, aptamers, cell lysates, DNA, RNA, mRNA, genes, gene expression products, and chemicals. Element, compound, pharmaceutically active compound or a chemical entity thereof such as metabolites, minerals, contaminants, etc.) and molded including flow channels and assay portions (assay cells). A treated glass surface 20 bonded to a modified polydimethylsiloxane (PDMS) block 12. PDMS is only one material that can be used and, of course, should not be considered as limiting the scope of the present invention. Other suitable materials include, for example, chemically treated glass, glass covered with nanoparticles, glass coated with metal, glass treated with other forms of material, carbon (all forms), silicon , Rubber, plastics, metals, crystals, polymers, semiconductors, organic and inorganic materials.

  In some embodiments, such as shown in FIG. 1, the assay cartridge 100 includes a bottom plate 10 having a transparent portion 14 through which the interaction of an analyte with a capture agent can be detected / measured, and an assay reservoir connected to a PDMS block 12. It further includes a body 30 to be fitted and the treated glass surface 20 and, optionally, an upper plate 60 on the body 30. Treated glass is only one material that can be used for surface 20 and should not be considered as limiting the scope of the present invention. Other suitable materials include, for example, PDMS, glass covered with nanoparticles, glass coated with metal, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastic , Metals, crystals, polymers, semiconductors, organic and inorganic materials.

  Body portion 30 can include a reservoir portion 40 having a plurality of reservoirs 42 for holding test samples and / or assay reagents (including detection reagents and optionally enhancers). The body 30 may further include a transport region 50 having a plurality of channels 52 through which certain amounts of test samples and assay reagents flow to and from the PDMS block 12 and the treated glass surface 20. obtain.

  In certain embodiments, the body 30 and the PDMS block 12 are coated with molded or otherwise PDMS or other suitable material, such as chemically treated glass, glass covered with nanoparticles, metal. One piece constructed from broken glass, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastics, metals, crystals, polymers, semiconductors, organic and inorganic materials, etc. Can be replaced with the main body / block. In such embodiments, as well as in other embodiments, the bottom plate 10, the transparent portion 14, and the treated glass surface portion 20 may be treated glass, or in some embodiments, covered with nanoparticles. Glass, glass coated with metal, glass treated with other forms of materials, carbon (all forms), silicon, rubber, plastics, metals, crystals, polymers, semiconductors, organic and inorganic materials One good surface can replace everything.

  In still further embodiments, the reservoir may be separate from the assay cartridge and connected to the PDMS block (or other material as described above) through the connecting tubing. In such embodiments, only the PDMS block reservoir (or other material as described above) and the treated glass surface 20 (or other material as described above) are needed to form an assay cartridge. .

  FIG. 2 shows a detailed view of the PDMS block 12 and the treated glass surface 20 of FIG. 1 and the other embodiments described above. In this embodiment, there are four assay portions provided on the assay cartridge. In this view, multiple binding sites 22 (or capture spots or dots) on the treated glass surface 20 can be more easily seen. In some embodiments as shown in FIGS. 1 and 2, each assay portion 24A, 24B, 24C, 24D has two inlets 26A1-2, 26B1-2, 26C1-2, 26D1-2, and 1 It has two outlets 28A, 28B, 28C, 28D and allows for rapid exchange of multiple assay reagents as described further below.

  As noted earlier, the treated glass is only one material that can be used for the surface 20 and should not be considered as limiting the scope of the present invention. Other suitable materials include, for example, PDMS, glass covered with nanoparticles, glass coated with metal, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastic , Metals, crystals, polymers, semiconductors, organic and inorganic materials.

  As noted above, in certain embodiments, such as those shown in FIGS. 1 and 2, the cartridge may include four such assay portions (cells), each with a very large number of bindings per cell. Sites (capture spots) are possible (the number of spots per cell can range from 2 to over 20,000). A binding site or capture spot is used herein to describe an area on a surface that contains a group of capture agents. Such capture spots can be placed on the treated glass surface 20 using a microarray printer device or by any alternative apparent to one skilled in the art. In some embodiments, the assay cartridge may be connected to the reservoir and to the flow control system through a multi-pin connection manifold using peak tubing (connecting tubing) through the reservoir.

  FIG. 3 shows a simplified view of a cartridge according to an embodiment of the invention, for example, with various assay reagents as part of a fluid control system 200. For simplicity, only the PDMS block 12 of the cartridge and the treated glass surface 20 are shown.

  In this figure, a test sample 212 and an assay reagent 214 are contained in a pressurized reservoir (introduction well) 210. Opening the first inlet valve 220 causes the test sample 212 to flow into the assay portion of the cartridge (including the PDMS block 12 and the treated glass surface 20) such that the test sample flows over the binding site (capture spot) 22. be introduced. Next, opening the discharge valve 230 allows the test sample 212 to proceed to the waste chamber 240.

  Next, by opening the introduction valve 222, the assay reagent 214 can be introduced into the assay portion, and the assay reagent 214 can flow over the binding site (capture spot) 22. The assay reagent 214 can then be drained into the waste chamber 240 as described above. This repeating flow process can then be repeated multiple times.

  In some embodiments after exiting the assay portion, both the sample 212 and the assay reagent 214 are first flowed over the precision flow sensor and then discharged into the waste chamber 240. The controller allows precise timing of flow events up to milliseconds, as well as later / before / after assay operation. A dynamically controlled controller (at the beginning of the flow path) paired with a precision flow sensor (at the end of the flow path) works in tandem and provides accurate flow rates and flow rates independent of flow path characteristics Create a closed loop flow control system that ensures

  One skilled in the art will, of course, recognize that embodiments of the present invention can include multiple test samples and assay reagents. The embodiment shown in FIG. 3 is for illustrative purposes only.

  In other embodiments, the cartridge can incorporate a passive valve, and liquid (sample, labeled detection antibody, buffer solution, etc.) is placed in a reservoir on the cartridge itself, and the cartridge is assayed from the reservoir. Liquid flow through the portion can be controlled by adjusting the pressure through an interface built into the system attached to the disposable cartridge.

System method and procedure Assay Process In one embodiment, the overall processing time for each cartridge and associated assay can be 2-200 minutes, depending on the analyte concentration to be detected and measured. Once the sample has been injected, the processing of at least one sample beyond the at least one assay and the collection / processing of relevant data can be fully automated. The user only has to select the assay protocol to be used (via the software interface of the system), and the system can automatically run the assay and collect and process all relevant data .

  An example process is shown in detail in FIGS. FIG. 4 shows a schematic diagram of an assay portion of an assay cartridge that includes a plurality of binding sites (capture spots) 22 each including a plurality of capture agents 23. Some embodiments of the present invention can include multiple capture spots containing different capture agents. In other embodiments, each capture spot 22 may contain the same (only one type) capture agent, and different capture spots may contain different or the same capture agent, such that the assay portion of the cartridge There are multiple capture spots, each containing one type of capture agent, but many different capture agents are represented by many different capture spots across the surface.

  Once the assay portion of the cartridge surface has been properly printed and sealed with capture agent 23, the sample is flowed over the assay portion of the cartridge, as shown in FIG. If the sample contains a target analyte (the analyte of interest) 70, that analyte 70 will bind to the immobilized capture agent 23, but the other analytes 72, 74 in the sample will not Will flow past it beyond the assay portion of the sample.

  As shown in FIG. 6, after a predetermined volume of sample has been flowed for a predetermined time (possibly controlled by computer software), the detection reagent 71 is flowed through the assay portion (assay chamber) of the cartridge B . The detection reagent 71 causes the sample to flow out of the chamber and specifically binds to any analyte 70 immobilized by the capture agent 23. After a predetermined volume of detection reagent has been flowed for a predetermined time, the sample may be flowed again through the assay portion (chamber), as shown in FIG. 5, and these steps result in a circuit that is repeated any number of times. . The binding sites (capture spots) are imaged (visualized) by the measurement device during these iterative circuits, and a representation of the kinetic or dynamic binding curve of the target analyte to the capture agent is drawn.

  In some embodiments of the invention as shown in FIG. 7, the sample can also include fluorescently labeled streptavidin 76 or a similar compound. In such embodiments, the fluorescently labeled streptavidin 76 binds to the biotinylated detection agent 23 and can be measured to quantify the level of at least one target analyte 70 present in the sample. This produces a signal that can be used for In addition, in such embodiments, more of the at least one analyte of interest (the target analyte) 70 may be bound to the immobilized capture agent 23, and the subsequent repeated flow cycles Provides an additional site for binding of the detection reagent.

  8 to 10 show the steps of another process according to the invention in which the signal can be amplified. In FIG. 8, streptavidin 76 labeled with fluorescence binds to biotinylated capture agent 71. Streptavidin is tetravalent and can bind to four molecules of biotin. Also present in the detection reagent cocktail is a biotinylated dendrimer 78, which contains 1-8 molecules of biotin / dendrimer and can bind to fluorescently labeled streptavidin 76.

  Figures 9 and 10 show that the binding of additional fluorescently labeled streptavidin to the biotinylated dendrimer demonstrates a large increase in signal and greatly enhanced sensitivity and the ability to detect very low levels of at least one target analyte 70. To cause As the reaction progresses, multiple layers of fluorescently labeled streptavidin and biotinylated dendrimer form the capture spot in an analyte-dependent manner (ie, directly correlating to the concentration of target analyte 70 in the sample). May accumulate on top.

  Although this description and the associated figures briefly describe the entire process as if in separate steps, in practice only two steps are in reaction. In the first step, a sample containing the analyte of interest and fluorescently labeled streptavidin is flowed over the surface. The analyte of interest binds to the immobilized capture reagent and the fluorescently labeled streptavidin binds to any biotinylated detection reagent or dendrimer present on the surface. In the second step of the reaction, a biotinylated detection reagent and a biotinylated dendrimer are flowed across the assay surface. If there is an immobilized analyte, the biotinylated detection reagent binds to these analytes, and if there is immobilized streptavidin, the biotinylated dendrimer binds to the immobilized streptavidin. These two steps are repeated a predetermined number of cycles, and the assay surface is visualized (imaged) at the completion of each step in every cycle. The result of this assay framework is a highly specific and very sensitive sandwich immunoassay that can specifically detect very low levels of analyte.

2. Performing Assays In certain embodiments for performing assays based on the systems disclosed herein, typical design factors include assay type, assay reagents and concentrations, number of assays per assay portion of the cartridge, and microarray design. (Pattern of the capture agent on the surface). Each assay portion in the cartridge can be prepared for the same or different assays. In addition, each assay portion (or cell) can be performed on a separate experiment or sample. An exemplary experimental design is shown below, however, for the purpose of example only, and other experiments that vary the time, sequence and / or presence of some or more parameters included below. It should be noted that the scheme can also be used and is incorporated herein by reference.

Startup test of the system : This is the beginning of the short 2-15 minute process of the day, which is the task of flushing the system with flushing buffer and verifying the performance of the system. The "test device" has been connected to the system the day before. The user collects and measures the amount of flow to verify system performance. Typically, the flow variation is around 1-2% CV on a 5% -10% pass / fail basis. However, these criteria may optionally be set as desired by the user and / or suitable for any given assay or experiment. Testing of the system flow may be performed automatically by a computer controlled flow control module.

Device connection and closure: The test device is removed and the user-specific assay cartridge is placed on the platform of the system disclosed herein, and the connection manifold is connected to the entrance of the cartridge (cartridge with four assay portions). For example, eight inlets and four outlets, but there may be more or less). Fill the assay portion with the blocking solution and expel air in a short (2-15 minutes) filling process. In some embodiments, there is no flow tubing as liquids (samples, reagents, detection antibodies, labels, buffers, etc.) can also be placed directly into a reservoir contained within a disposable assay cartridge.

Sample injection: The blocking solution can be replaced by a sample vial and a rapid sample injection through the device is performed to allow the maximum sample and reagent concentrations to flow into the inlet of the device (pipe flushed or If injected) can be ensured to be present.

Assay incubation and data collection: The process of flowing sample and / or detection reagent onto the assay portion for a desired incubation time. After each incubation time, an imaging event is performed to capture the signal intensity resulting from binding of the analyte to the capture agent. Many such imaging events occur automatically throughout the assay flow process and draw binding curves from multiple measurements of the time course of fluorescence.

Flushing the system: performed using test devices and flushing buffer. The system is flushed to remove the assay reagents from the flow controller and prepare the system for the next assay run.

3. Data collection and analysis (for certain system embodiments)
Raw data : In one embodiment, in its most raw form, data can be collected as a 16-bit grayscale tiff image. Each pixel in the image corresponds to a preselected image resolution. Data is collected instantly from the PMT at the desired time intervals.

Time lapse data : The signal intensity over a period of time is the first level of processing performed. This type of data can be derived using several methods (chosen by the user). In all cases, it is the variation in signal intensity number over time for each assay.

Calculation of analyte concentration : A known concentration of standard is used to generate a time course curve of the assay-specific standard. Each known target is then used in addition to the kinetic equations disclosed herein and the accompanying proprietary algorithm to analyze the kinetic curves of the assay processed by each user to allow each target to be analyzed. A precise measurement of the analyte concentration is achieved.

Statistical reliability : This is the confidence level of the threshold for the sample curve relative to the expected curve. Once the confidence of the unknown curve (over all of the multiple assays) is met, the assay incubation process can be stopped to optimize data collection while minimizing processing time.

4. Detailed Analysis Procedures Analysis of the data generated from the systems and methods disclosed herein requires that the capture reagent, which in some embodiments be printed or otherwise arranged in a microarray format (which in some embodiments is It is based on measuring the initial binding rate between the target monoclonal antibody) and the analyte of interest or target analyte (which in some embodiments is the target protein). Quantitative analysis methods are based on measured initial binding rates and back calculations from known standards. The initial binding rate is a delta measurement between two time points and is therefore not susceptible to the consequences of absolute signal intensity associated with a variable background, but the determination of the limit of detection is dependent on the variable background trend. Affected by As such, the data can be corrected by simply subtracting the signal intensity from each image and the low signal background area at each time point.

  In some embodiments, “linear data” is generated by the systems and methods disclosed herein and analyzed as follows. During the assay process, time course images are collected at fixed time intervals (can be anywhere from a few seconds to tens of minutes). Prior to each image, the volume of the sample (flowing solution 1), followed by the volume of the detection reagent (flowing solution 2), is in turn over the assay portion of the cartridge using the control of the system and the associated flow cartridge. Passed or shed. The signal intensities of the assay binding sites (in one embodiment, these are microarray spots) are extracted from the image and are numerically expressed as the average of several repetitions. Since the sample concentration remains fixed (by flowing fresh or fresh solution throughout the assay process) and because the surface concentration of the capture reagent is relatively high (compared to the analyte concentration in the sample), Linear analysis of the data gives the slope (used directly as initial velocity in units of signal / time). Initial velocity is directly proportional to the amount of antigen presented by the sample (more antigen equals greater initial velocity). For very high antigen concentrations that result in either saturation of the imager (outside of the PMT) or surface saturation (loss of linearity), a linear fit is performed only at an earlier point in time. For moderate and low concentrations, additional or all time points can be used for a linear fit.

  In some embodiments, "non-linear data" is generated by the systems and methods disclosed herein and analyzed as follows. An alternative to the above linear data is one that typically utilizes a form of signal enhancement or amplification that results in the generation of non-linear data. In such cases, the signal initially depends on analyte binding, as in the case of linear data, but then, like a catalyst to generate an additional signal from a "set" of facilitating reagents. (Described in more detail below). Typically, the set of enhancing reagents includes two reagents, one in reservoir 1 (tube 1) and the other in reservoir 2 (tube 2), and the enhanced signal is used for the flow cycle. It allows to be a measure of the number of iterations and the nature of the amplification of the facilitating reagent. If the signal for the generated time data fits linearly as above, if it is non-linear it is a fit to a polynomial type equation and the first derivative extracts the gradient at a particular analysis time Used for Regardless of how long the time lapse binding experiment was preceded, any earlier analysis time can be used for the back calculation. An early analysis time typically results in lower sensitivity results, and as such is useful for measuring high analytes simultaneously with low analytes using two different analysis times. is there.

  Standardization and back calculation. As described above, the initial velocity is measured for both standards and unknowns. Generally, unknown sample concentrations are back calculated from known concentrations of standards. During the development of the systems and methods disclosed herein, empirical analysis of initial rates with varying analyte concentrations followed an equilibrium or saturation model where the rates of binding reactions approached a maximum at increased concentrations. It was decided. Several assay parameters have been found that significantly affect the maximum rate, but for any given method, the maximum rate effectively determines the highest quantifiable dose (defined later in this section). .

For many enzymes, the rate of catalysis or rate (V) varies with substrate concentration in a linear fashion at lower concentrations and reaches a maximum at higher concentrations. In 1913, Leonor Michaelis and Maud Menten proposed a simple model described by the following equation, known as the Michaelis-Menten model, to explain their kinetic properties (V _max is the catalysis represents the maximum speed, S is represents the substrate concentration, and _{K m} is known as the constant of Michaelis).
Rate ^{_{= (V max * S n)}} / (S n + K m n)

The Michaelis-Menten model was developed to describe enzyme catalyzed reactions, and the reasoning behind the observation of V _max in such reactions was based on the formation of an enzyme-substrate complex that reached equilibrium or saturation. The model also accounts for data generated by the systems and methods disclosed herein. One possible explanation for the observation relates to the solid phase nature of the binding reaction, in which the solution phase analyte must first interact with the surface phase capture reagent before a reaction or binding event can occur. This first step is probably "corresponding" to the formation of the enzyme-substrate complex in the Michaelis-Menten model formula. This surface association step has effect at equilibrium or saturation at higher concentrations. Several other models of the reaction reaching saturation or equilibrium have been found to be equally useful in processing the data. In fact, the Michaelis-Menten equation above represents only one type of equation that can be used to fit the initial velocity data. Other embodiments use other similar equations. By way of example, but not limitation,
Wagner model: fit = exp ((A + (B ^* ln (x))) + (C ^* (ln (x) ² )))
One site saturation model for non-specific binding: fit = (C + ((BLmax ^* x) / (Kd + x)))
Hyperbolic equilibrium model: fit = (C + (BLmax ^* (l− (x / (Kd + x))))))
Eadie-Hofstee model: fit = (A + ((B ^* x) / ((C ^* (D + 1)) + x)))
Hyperbolic model from C: fit = (C + ((A ^* x) / (B + x)))
Michaelis - Menten model: fit ^{= ((Vmax * (x n} )) / ((x n) + (K m n)))
Integral Michaelis-Menten model: fit = ((l / Vmax) ^* ((S0-x) + (Km ^* ln (S0 / x))))
Michaelis-Menten steady state model: fit = (Vmax / (l + (Km / x)))
Michaelis-Menten equation: Fit = ((BLmax ^* x) / (Kd + x))
MMF model: fit ^{= (((A * B)} + (C * (x D))) / (B + (x D)))
As well as many other similar such models known in the art.

  It should be noted that the rate equations themselves are not new and they have been used for enzymatic reactions for many years. The most important novelties and breakthroughs described here are the assays of these equations (reaction equations based on enzyme catalysis) processed using the systems and methods disclosed herein. Typically, biological assays, and in some embodiments, protein-based biological assays).

  Such an approach for calculating the target analyte concentration from a complex (or single) sample facilitates a time-based analysis that is important to the systems and methods disclosed herein, Progression from a single sample creates the unique ability of the system to be able to accurately quantify different target analyte concentrations present in very different concentrations in that sample. The reason for this is that those target analytes that are present at very high concentrations are detected and measured early in the assay process (calculated concentrations), whereas those targets that are present at very low concentrations The analyte is detected and measured (the calculated concentration) later in the same assay process (when the system detects the binding curve generated from these relatively low concentrations of the analyte).

  This rate-based analytical approach also facilitates the unique ability of the system to distinguish specific signals from non-specific signals (target binding from non-target binding) in the assays described in more detail below.

5. Specific versus non-specific signal The specific signal (the signal from the binding of the target analyte to the capture agent) is derived from the non-specific signal (the signal generated by the binding of the non-target substance / molecule / analyte to the capture agent). Distinguishing has significant benefits in many areas of assay development, validation and processing.

  The systems and methods disclosed herein provide these benefits. In summary, in order to differentiate non-target binding from target binding or to validate a signal as a target signal, the sample being tested must follow the binding rate / curve trajectory observed during the standardization process. Must.

The slope, and thus the analyte concentration, can be determined after the two signal intensities have been measured, but such a determination is made after the measurement of the third or subsequent signal, especially the third, It is more accurate if the fourth, fifth or subsequent signal is at least twice as strong as the standard deviation of the background signal. For example, FIG. 11 shows a plot of the signal intensity of the IL-1b antibody (capture agent) binding to the target IL-1b antigen (specific analyte), and the signal of the IL-1b antibody (capture agent) binding to streptavidin. Shown side by side with the intensity plot (representing the simulated non-target binding signal). In this example, in addition to the non-linear from the third measurement, a first measured speed lies in significantly outside the V _max calculated for IL-1b. Each of these observations provides a basis for classifying IL-1b antibodies to streptavidin signal and related plots as resulting from non-specific (non-target) binding events to the binding site (and capture agent there). give.

  Alternatively, if the signal is the result of a relatively high concentration of non-specific interactions, it will be observed that the initial rate is very high at early time points and approaches zero at late time points. In general, however, the distinction between true and non-true signals is based on the binding curves from a given experiment for the presence and concentration of an unknown analyte and the standard analyte using the known presence and concentration. It becomes clear from a comparative analysis with a binding curve.

  Additional distinction of a specific binding event from a non-specific binding event (true signal or false signal) can be performed as follows (for the purpose of a sandwich-type assay, the specific signal is , The target analyte is the signal bridged between the capture agent and the detection reagent, whereas the non-specific signal is that some other substance / molecule / analyte other than the target analyte is A bridge between the capture agent and the detection reagent or adheres directly to the surface of the assay portion of the cartridge, followed by the attachment and / or enhancement of a detection reagent, such as a biotinylated dendrimer, or streptavidin. Note that the signal is either directly attached to the cartridge surface).

  Option 1: At the end of the assay run, replace the sample and detection reagent with assay buffer and assay buffer containing a certain concentration of capture agent.

  Option 2: At the end of the assay run, replace the sample and detection reagent with assay buffer and assay buffer containing a certain concentration of detection reagent.

  Option 3: At the end of the assay run, replace the sample and detection reagents with assay buffer and assay buffer containing a concentration of at least one target analyte.

  In each of the options described in detail above, several cycles of a repetitive flow of each of these reagents are performed to image the assay portion of the cartridge. In the case of specific binding, the signal is reduced, so that in this case the signal and the associated binding curve can be assumed to be a specific signal from the target analyte. However, if the signal is effectively non-specific, then the signal should remain almost stable since it does not depend on the presence of the analyte of interest (the target analyte). In such a situation, the signal and associated binding curve can be deduced to be a non-specific signal from a non-target in the sample.

  In another approach, the systems disclosed herein detect the signal in real time, so that at the end of the assay run, medium to high concentrations of a specific entity (as outlined in the options above) Described) can be inserted into the reservoir of the cartridge and system and flow over the assay portion of the cartridge. If a decrease in signal is observed, this further indicates that the signal is virtually specific (true and analyte dependent), whereas the signal is roughly constant in its intensity. If it is observed that it remains, this is an indication that the signal observed is non-specific (false).

  Additional distinction of a specific binding event from a non-specific binding event (true or false signal) can be made as follows. To measure dissociation rather than adding additional reagents as described above, the analyte or sample and / or detection reagent is simply removed. The subsequently measured kinetic binding signature provides further insight as to whether the signal was generated from a specific or non-specific binding event. A fast and low signal intensity change indicates a non-specific binding event, whereas a slow and high signal intensity change indicates a specific binding event. This difference in "off rate" between non-specific and specific binding is due to the difference in "association rate" described earlier (from higher to lower rather than lower to higher signal intensities). To reflect). Since the association and dissociation rates can be different, this approach complements the previously described methods for distinguishing specific binding events from non-specific binding events and related signals.

  In the assay, non-specific (non-target) binding is discriminated against specific (target analyte) binding interactions to further enhance the signal formed by mixing both the specific and non-specific components. A further technique and method for adjusting and correcting involves the use of control spots contained in the assay portion of the cartridge. The most common type of background signal is elicited by heterophilic antibodies, which are essentially anti-species antibodies found in human serum samples, and most of these serum samples have rheumatoid arthritis and hypersensitivity. Derived from autoimmune patients such as genital bowel syndrome and similar such conditions. These heterophilic antibodies are capable of cross-linking between the capture and detection antibodies, resulting in a signal that is likely to be analyte dependent (ie, based on the target analyte concentration) but is not.

  An antibody (goat, rabbit, mouse, etc.) of the same species as the capture agent (capture antibody in this example) used in at least one assay, but not directed to any of the target analytes measured in the assay By including a "negative control spot" containing a mixture of the following, the system disclosed herein contains heterophilic antibodies because this "negative control spot" produces a signal in a sample-dependent manner. Sample to be detected. Thus, the signal from this “negative control spot” adjusts the signal in the spot specific for the analyte to adapt to the “concentration” of the heterophilic antibody present, and thus the target analyte concentration Can be used to provide more accurate measurements. In addition, since samples containing heterophilic antibodies can be easily identified using this novel method, such samples "flaunt" if they have a higher level of uncertainty in the quantification process. be able to.

6. In some embodiments of the present invention, non-linear kinetic data can be obtained by the deliberate use of enhancing reagents to increase assay sensitivity and kinetic range, and Can be characteristic. For example, a facilitating reagent that promotes or amplifies the signal intensity resulting from the binding of the antigen and the capture agent can be implemented.

  The enhancer typically contains two reagents and is repeatedly passed over the bound analyte. The first reagent can interact with the analyte and / or detection reagent and / or other facilitating reagent. The second facilitating reagent interacts with the first facilitating reagent to create a secondary network of facilitator-only reagents, the concentration of which directly reflects the analyte concentration. Examples of these types of reagents; Reagent 1: streptavidin, avidin, dye-labeled streptavidin, avidin; Reagent 2: dimerized biotin molecule, PAMAM dendrimer partially or completely labeled with biotin, or biotin -Including, but not limited to, any biotin-containing molecule or polymer capable of forming an avidin network. More broadly, enhancers include: biotinylated proteins, biotinylated antibodies, biotinylated peptides, biotinylated strands of DNA, biotinylated dendrimers, anti-species antibodies, and antibodies captured and detected in an analyte-independent manner. May be included, but is not limited to, any active substance capable of cross-linking.

  This enhancement or amplification of the signal strength is therefore a function of the number of repetitive flows of the first and second liquids (sample and reagent). The resulting data, if non-linear, can be fitted to a polynomial equation, and its first derivative can be used to calculate the slope of the binding curve at a particular point in time. The most important feature is the consistent determination of the initial velocity or slope of the sample during normalization and analysis of the unknown sample. In addition, the linear part of the binding curve can be used to calculate the binding rate.

7. Exemplary Assay Parameters The systems and methods disclosed herein allow for processing of an assay and generation of related assay data. Typical assay parameters used to illustrate and present the data include, but are not limited to:

Minimum detectable dose (LDD) : defined as the lowest concentration of analyte that can be observed above the background signal with 60-90% confidence. LDD can be calculated as the initial rate and subsequently the back-calculated concentration associated with either the signal exceeding the mean of the background signal by one standard deviation or the signal just above the measured zero dose signal. . The background signal typically indicates a signal obtained from the binding site before the analyte sample and / or the detection reagent has flowed or a very low signal intensity even after the analyte sample and / or the detection reagent has flowed. It may be based on the signal obtained from the region between the binding sites. Regardless of the method chosen, the LDD is typically set to not exceed a value that is a given multiple (and possibly three) of the signal intensity observed at the lowest non-zero analyte concentration. Is done. LDD can be verified by running the concentration of a known standard on the LDD and calculating the percent recovery.

Minimum quantifiable dose (LQD) : defined as the lowest concentration of analyte that can be observed above the background signal with 90-95% confidence. LQD is related to the signal that is either the initial rate and subsequently twice the standard deviation over the background signal or twice the measured zero-dose signal, both of which give the least perceived LQD. Can be calculated as the back calculated concentration. Again, the background signal may be based on a signal obtained from the binding site before the analyte sample and / or the detection reagent flows or from a region not associated with the binding site. The standard deviation of the background signal can be calculated from the five signal intensities taken at the end of the assay run. The standard deviation is divided by the total incubation time to determine the initial velocity and associated LQD in concentration units. If a zero dose method is used, a value equal to twice the rate observed at zero dose can be used. Regardless of the method selected, the LQD is set to not exceed a value that is three times the lowest non-zero standard concentration measured. LQD can be verified by running the concentration of a known standard on LQD and calculating the percent recovery.

Highest Quantifiable Dose (HQD) : Calculated maximum rate or rate of catalysis, 5% below V _max (disclosed previously), but typically above the highest measured standard concentration Not defined. HQD can be verified by running the concentration of a known standard on HQD and calculating the percent recovery.

Dynamic range : the higher to lower concentration range that can be quantified during the assay. Defined by HQD as high termination and LQD as low termination.

Data variability / accuracy : Intrinsic variability of signal intensity measured for experiments performed at essentially the same experimental conditions but for different time frames. Intra- and inter-experimental variability (CV) of analyte quantification is obtained passively by multiple experiments showing LDD, LQD, and HQD. Reagents are kept constant, causing variations between spots, between experiments (intraday), between assay cells, between cartridges, and between days.

Example Experimental Designs, Runs and Data This section summarizes one possible approach for performing assays based on the systems disclosed herein, and also incorporates some example data.

Summary : The experimental procedure in this example is referred to herein as "real-time ELISA" or "real-time protein quantification." Real-time ELISAs use standard commercial ELISA kits and reagents reconstituted into the systems and methods disclosed herein, as well as reference data for comparison, as well as the systems and methods disclosed herein. Provides a supplementary data that can only be obtained by

Method of real-time ELISA : A typical ELISA kit is completed with the following reagents: A) capture antibody (target specific), B) protein / antigen target (as standard of known concentration), C) Labeled secondary antibody (specific for biotin-labeled target). Typically, an ELISA assay includes an additional step ("enzyme ligation") in which biotin is further reacted with another enzyme (SA-HRP) to generate a signal by catalysis of HRP on a dye substrate.

  In a real-time ELISA, capture antibodies (Reagent A) are printed (arrayed) or otherwise placed on a flow cartridge as disclosed herein. Real-time methods, such as ELISA assays, use a protein / antigen target (Reagent B) as a standard at a known concentration. Unlike the ELISA assay, a secondary antibody (Reagent C) is used in combination with a selected streptavidin dye reagent (SA-Cy3 or equivalent) to generate a signal from the captured / measured antigen target. Let it. In the two-flow method (including the repetitive flow of reagents and sample through the two flow channels), the antigen target and SA-dye are combined into flow channel 2 (reservoir 2) and the secondary antibody flows. Channel 1 (Reservoir 1). The repeating flow of channel 1 and channel 2 on the printed capture antibody generates a real-time signal that is used to quantify the antigen target concentration.

1. EXPERIMENTAL In this embodiment of the systems and methods disclosed herein, the triplicate assay comprises three human analytes: C-reactive protein (CRP), interleukin-1β (IL-1b), and Used to test for Leukin-6 (IL-6). Although any other analytes and any number thereof may be used, the three used herein are included only as examples illustrating assay processes, systems, methods and devices. A study designed to test the efficacy and accuracy of the triplicate assay was performed with the following study design and related procedures and system information.

  Each cartridge was equipped with an assay portion having binding sites for each of CRP, IL-1b and IL-6 and a reservoir portion for containing the test sample and various buffers and reagents to be used.

  These binding sites include capture agents printed or otherwise applied to the assay portion at known concentrations and amounts. In this case, the capture agent was a known antibody against human CRP, IL-6, and IL-1b analytes. Here, as is typical of many, but not all embodiments, the cartridge has a positive control and a negative control which are intended to illustrate the predictable results when each sample is tested. Controls were included. In addition, the characteristics of the controls incorporated into the system ensured that the appropriate reagents were added at approximately the correct concentrations.

  The cartridge was first used to prepare a standard binding curve for each analyte. In practice, binding curves for such standards are provided or can be obtained separately. For each analyte, serial dilutions were prepared from known concentrations of standards and analyzed along with a "zero dose" sample containing no analyte. The sample dilution was added to the cartridge sample reservoir along with any other necessary buffers, reagents, etc. contained in another reservoir. In some cases, the cartridge can have multiple sample reservoirs, and samples at different dilutions or different samples can be analyzed using the same cartridge.

  When generating binding curves for triplicate standards, sample detection was performed in triplicate for each of the eight (seven sample dilutions and one zero dose) concentrations. In this study, the volume of the sample containing the unlabeled analyte was first flowed from the sample reservoir portion through the assay portion of the cartridge, followed by the volume of the detection reagent containing the detection label. Fluorescent detection labels were used in this study, but as noted above, this is only an example illustrating one of the experiments using the systems and methods disclosed herein, and other detection labels and Corresponding detection devices can also be used.

  After running the detection reagent, a detection device, here a fluorescence detector, detects the presence of the detection label in the assay portion and records the signal intensity by imaging. As previously described, as well as in more detail in the '044 application, as will be apparent to those skilled in the art, a fluorescence detector includes an excitation device, usually a laser, for exciting the detection label at a particular wavelength. In response to such excitation, the detection label fluoresces at different wavelengths and its signal intensity is measured.

2. Data Figures 12-14 show plots of the binding curves of the standards for each of IL-6, CRP, and IL-lb. The binding curves are fitted to the individual signal intensities, as indicated. The initial velocity of each binding curve was calculated from the slope of the linear portion of the resulting binding curve. If the characteristic binding curve is non-linear, higher order rate equations or polynomial differentiation at a given time can be used to determine the overall rate of the reaction. A specific slope (or initial velocity) was determined for each standard concentration. The initial rate for the subsequent concentration data was fitted to an equation describing the enzyme catalysis, the format of which was previously disclosed. Following normalization, the unknown slope (or initial velocity) can be back calculated to the rate equation and concentration based on the fit.

［発明の項目］
［項目１］
カートリッジデバイスと、測定デバイスとを備えるアッセイシステムであって、
前記カートリッジデバイスが、
１種以上の液体を保持するための１つ以上のリザーバ部分と、
前記１つ以上のリザーバ部分からの前記１種以上の液体を受けるための１つ以上のアッセイ部分であり、前記１つ以上のリザーバ部分からの前記１種以上の液体が該アッセイ部分の上に繰り返し（２回以上）流され得る複数の結合部位を有する、１つ以上のアッセイ部分と
を含み、
前記測定デバイスが、前記１種以上の液体中の１種以上の分析物の複数の結合部位への結合を測定するためのものである、アッセイシステム。
［項目２］
前記カートリッジデバイスが受け入れられたり取り出されたりするインターフェースであって、前記１つ以上のリザーバ部分から前記１つ以上のアッセイ部分を通る前記１種以上の液体の流れ又は移動を制御するための装置を含む、インターフェースをさらに備える、項目１に記載のアッセイシステム。
［項目３］
前記１種以上の液体の流れを制御するための前記装置がコンピューター制御され、該装置が、以下の事項の少なくとも１つ以上、すなわち
前記１つ以上のアッセイ部分を越える前記１種以上の液体の流速、
前記１つ以上のアッセイ部分を越える前記１種以上の液体の流れの持続時間、
ある量の前記１種以上の液体が、前記１つ以上のアッセイ部分の上に流される回数
の１つ以上を独立に制御することができる、項目２に記載のアッセイシステム。
［項目４］
前記インターフェースが、前記１つ以上のアッセイ部分から前記１種以上の液体を取り出す又は流出させるための装置を備える、項目３に記載のアッセイシステム。
［項目５］
前記１種以上の液体が、蛍光標識、発光標識、及び比色標識からなる群から選択される１種以上の標識を含み、
前記測定デバイスが、蛍光測定装置、発光測定装置、及び比色測定装置からなる群から選択される、項目１に記載のアッセイシステム。
［項目６］
前記測定デバイスが、コンピューター制御下で、レーザー又は他の形態の標識刺激を、前記カートリッジデバイスの前記１つ以上のアッセイ部分に向けることができるシステムであって、続いて蛍光、発光又は比色シグナルの検出及び／又は測定を実施できるシステムに構成されている、項目５に記載のアッセイシステム。
［項目７］
前記１種以上の液体が、検出、測定及び／又は分析しようとする１種以上の分析物を含有し得る、項目１に記載のアッセイシステム。
［項目８］
前記カートリッジデバイスの前記アッセイ部分中の結合部位からのシグナルの複数の時系列測定がコンピューター制御下で実施され、生じたシグナルが、前記１つ以上のアッセイ部分中の複数の結合部位の１つ又は複数に結合するこれらの分析物の動的又は速度論的結合曲線の表示を生成するために使用されることをさらに含む、項目１に記載のアッセイシステム。
［項目９］
前記インターフェースが、前記１つ以上のアッセイ部分を越える前記１種以上の液体の流速及び持続時間を制御する可変圧力（陽圧及び／又は陰圧）を提供する、項目２に記載のアッセイシステム。
［項目１０］
前記１つ以上のアッセイ部分中における複数の結合部位の１つ又は複数に結合する１種以上の分析物の１つ以上の結合曲線の表示を分析して、前記１種以上の分析物について該分析を既知の標準物質の時間経過（速度論的）結合曲線と比較して、液体中の前記１種以上の分析物の存在及び／又は濃度を決定するためのコンピューターソフトウェアをさらに備える、項目８に記載のアッセイシステム。
［項目１１］
前記１つ以上のリザーバ部分が、該リザーバ中の液体を封じる薄い膜により覆われている、項目１に記載のアッセイシステム。
［項目１２］
検出用試薬に対する追加の結合部位を提供して、蛍光、発光又は比色シグナルの強度の増大を促進及び／又は増幅することを助長する１種以上の促進剤分子又は実体を、前記液体の１種が含有する、項目１に記載のアッセイシステム。
［項目１３］
前記１種以上の促進剤分子又は実体が、ストレプトアビジン、アビジン、色素標識型のストレプトアビジン、アビジン；二量化ビオチン分子、ビオチンで部分的に若しくは完全に標識されたＰＡＭＡＭデンドリマー、又はビオチン−アビジンネットワークを形成し得る任意のビオチンを含有する分子若しくは高分子、ビオチン化タンパク質、ビオチン化抗体、ビオチン化ペプチド、ＤＮＡのビオチン化ストランド、ビオチン化デンドリマー、抗種抗体、及び分析物とは独立した様式で捕捉された活性物質と検出試薬とを架橋させることができる任意の活性物質からなる群から選択される、項目１２に記載のアッセイシステム。
［項目１４］
増幅の効果が、少なくとも１種の促進剤、少なくとも１種の分析物及び少なくとも１種の検出用試薬の循環的処理によって得ることができ、一部の促進剤が、一部の検出試薬に結合して、さらなる検出試薬が結合する多数の追加の部位を提供することにより、促進されたシグナル効果がもたらされる、項目１２に記載のアッセイシステム。
［項目１５］
結合部位が、以下の実体の１つ又は複数、すなわち、
タンパク質、ホルモン、抗体、抗原、ウイルス、抗体複合体、抗体断片、ペプチド、細胞、細胞断片、アプタマー、細胞溶解物、分画された細胞溶解物、分画された細胞、ＤＮＡ、ＲＮＡ、ｍＲＮＡ、遺伝子、遺伝子発現生成物などの生物学的実体と、
化学元素、化合物、薬学的に活性な化合物若しくはそれらの代謝物、鉱物、又は汚染物質などの化学的実体と
の１つ又は複数を含有する、項目１に記載のアッセイシステム。
［項目１６］
動的又は速度論的結合曲線の表示が、１種以上の標的分析物濃度を計算するために使用される、項目１０に記載のアッセイシステム。
［項目１７］
動的又は速度論的結合曲線の表示が、結合部位内における標的分析物と捕捉剤との特異的相互作用から生ずるシグナルを、非特異的相互作用から生ずるシグナルから区別するために使用される、項目１０に記載のアッセイシステム。
［項目１８］
項目１５に記載のアッセイシステムの使用であって、
時間依存性の結合曲線及び関係する速度の分析による流体中の分析物濃度の決定は、分析物の超高濃度（例えば５００，０００ｐｇ／ｍＬを超える）と同じ試験及び同じカートリッジで、分析物の超低濃度（例えば１０ｐｇ／ｍＬ未満）を流体から検出及び／又は測定することを可能にし、該決定は、超高濃度分析物の結合曲線の分析によりアッセイプロセスにおける初期（例えば最初の２０分以内）に達成され、一方、これらの分析対象の結合曲線がシステムで見える場合に、超低濃度分析物は、アッセイプロセスの後期（例えば３０分後）に検出／測定される、使用。
［項目１９］
項目１５に記載のアッセイシステムを使用して分析物濃度を計算する方法であって、
各結合部位の結合曲線の勾配、又は非線形のデータの場合には特定の分析時間における勾配（１次微分又は接線）を計算することにより結合速度のデータ（初速度）を得て、次にその結合速度のデータを酵素触媒作用型の反応方程式に適用して、これらの分析物のための前もって又は同時に開発された既知の標準物質に関する速度に基づく結合曲線と比較する、方法。
［項目２０］
項目１５に記載のアッセイシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
結合曲線の形状が、観察された結果又はシグナルが特異的捕捉剤の標的分析物に対する結合事象の結果であるか又は捕捉剤の１種以上の非標的分析物に対する非特異的相互作用の結果であるかを知るために使用される、方法。
［項目２１］
特異的結合と非特異的結合との区別が、選択された結合部位の初期結合速度（最初の２又は３回の検出／測定事象から計算される）を、その後の検出又は測定事象から（３又は４回の検出若しくは測定後、及び／又は１０〜１５分後の検出又は測定事象から）計算された結合速度と比較することにより行われ、３又は４回の事象後、及び／又は１０〜１５分後の検出又は測定事象の後に変化する（非線形になる）結合速度が、非特異的結合を表している可能性が高い、項目２０に記載の方法。
［項目２２］
項目１５に記載のアッセイシステムを使用して結合部位中における標的分析物の捕捉剤に対する非特異的結合から特異的結合を区別する方法であって、
シグナルが非特異的相互作用の比較的高い濃度の結果である場合に、初速度が早期の時点で非常に高く、後期の時点でゼロに近づくことが観察される、方法。
［項目２３］
項目１５に記載のアッセイシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
特異的結合のシグナルと非特異的結合のシグナルとの区別が、未知の分析物の存在及び濃度を用いた所与の実験からの結合曲線と、既知の存在及び濃度を用いた標準分析物の結合曲線との比較分析により達成される、方法。
［項目２４］
項目１５に記載のアッセイシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
アッセイ実行の終了時に、試験試料及び検出試薬を、ある濃度の捕捉剤を含有する第１のアッセイ緩衝液及び第２のアッセイ緩衝液で置き換えるステップ、又は
アッセイ実行の終了時に、試験試料及び検出試薬を、ある濃度の検出試薬を含有する第１のアッセイ緩衝液及び第２のアッセイ緩衝液で置き換えるステップ、又は
アッセイ実行の終了時に、試験試料及び検出試薬を、ある濃度の少なくとも１つの標的分析物を含有する第１のアッセイ緩衝液及び第２のアッセイ緩衝液で置き換えるステップと、
第１のアッセイ緩衝液及び第２のアッセイ緩衝液の反復する流れを数サイクル実行するステップと、
カートリッジのアッセイ部分を撮像するステップと
を含み、
特異的結合であれば、シグナルが減少するため、その場合、シグナル及び関係する結合曲線が標的分析物からの特異的シグナルであると推定でき、
シグナルが事実上非特異的であれば、その場合、シグナルが目的の分析物（標的分析物）の存在に依存しないので実質的に安定なままであり、シグナル及び関係する結合曲線が、試料中の非標的物質からの非特異的シグナルであると推定できる、方法。
［項目２５］
項目１５に記載のアッセイシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
非特異的シグナルが捕捉物と検出抗体との異好性抗体の架橋により生ずる場合について、少なくとも１つのアッセイで使用される捕捉剤（この例では捕捉抗体）と同じ種であるが、少なくとも１つのアッセイで測定される標的分析物のいずれにも向けられていない抗体（ヤギ、ウサギ、マウス、その他）の混合物を含有する「陰性対照」スポット（結合部位）が、カートリッジのアッセイ部分に加えられ、そして
どの試料が異好性の抗体を含有するかを、「陰性対照スポット」が試料に依存する様式でシグナルを生じるか否かの観察から推定して、ここで生じるのであれば、該シグナルが非特異的成分を含むと考え、又は
「陰性対照スポット」からのシグナルを使用して分析物に特異的なスポット（結合部位）におけるシグナルを調整して、存在する異好性の抗体の「濃度」を考慮に入れ、したがって、標的分析物濃度のより正確な測定を提供する、方法。
［項目２６］
項目１５に記載のアッセイシステムを使用して、薬剤開発、薬剤発見、診断の又は他の用途のための新しい候補バイオマーカーを発見する方法であって、
各結合部位を特異的対非特異的結合シグナルについて分析し、特異的シグナルをもたらす結合部位のみが、追加の分画及び／若しくは質量分析法又はどの分析物がこれらの結合部位に存在する（により捕捉された）かを確認するために使える別の技法によりさらに分析される、方法。
［項目２７］
結合部位が、分画された細胞溶解物、組織又は他の生物学的試料を含有し、特定の疾患又は状態を有する１名又は複数の対象から採取され、アッセイ中に処理される流体の１つが、健常な対照又は前記特定の疾患又は状態を有する対象のいずれか由来であり、
健常な対照からの流体で実行したアッセイからのデータが、前記特定の疾患又は状態を有する対象からの流体で実行したアッセイからのデータと比較されて、特定の疾患又は状態を有する対象からの流体で特異的結合のシグナルを示し、健常な対照からの流体で特異的結合のシグナルを示さない上記の結合部位が、その後の分画及び／若しくは質量分析法又は前記特異的結合のシグナルを生じさせる候補バイオマーカーを単離して同定する他の分析手順のいずれかによりさらに分析される、項目２６に記載の方法。
［項目２８］
流速が、カートリッジの出口又は該出口付近にあるカートリッジの前記アッセイ部分に続く流速センサーからのフィードバックに基づいて圧力を動的に調整することにより制御され得る、項目１５に記載のアッセイシステム。
［項目２９］
レーザー又は他の標識刺激／相互作用プロセスが作用しておらず、使用者が流体の移動を見て、カートリッジが、適切に流れていること及び泡又は他の可視的汚染物質のないことを視覚的に確認することを可能にするのに十分透明である場合に、前記カートリッジが後方から照明され得る、項目１５に記載のアッセイシステム。
［項目３０］
取り外し可能な廃棄物容器が、カートリッジにより処理された全ての流体を集めるシステムに組み込まれていてもよく、前記廃棄物容器が満杯に近く空にすべきときに使用者に知らせるセンサーを備えていてもよい、項目１５に記載のアッセイシステム。
［項目３１］
取り外し可能なタブレットがアッセイを遂行してモニターするために遠隔で使用できるように、取り外し可能なタブレットコンピューターが、アッセイを設定して遂行する指示を提供して、分析及び結果の詳細を提供するユーザーインターフェースとして使用される、項目１５に記載のアッセイシステム。
［項目３２］
着色光がユーザーインターフェース及びシステム自体の両方で使用されて、アッセイを設定して遂行するために関係するステップを通して使用者を導く、例えばインターフェースがカートリッジの視覚的表示を示して、使用者に前記リザーバが緑色（又は他の色）の光等で点灯されたときに、流体１をリザーバ１に挿入するように依頼する、項目１５に記載のアッセイシステム。
［項目３３］
項目１５、２８〜３２のいずれか一項に記載のアッセイシステムが実験室、介護の近く又は介護環境の場で診断用のバイオマーカーの検出及び測定のために使用される、項目１５、２８〜３２のいずれか一項に記載のアッセイシステムの使用。
［項目３４］
流体用のカートリッジを受けるための受け部であって、前記カートリッジは、前記カートリッジ中の１つ以上のリザーバからの１種以上の流体の流速を制御するためのインターフェースを有する、受け部と、
流体を含有する１つ又は２つ以上のリザーバを有する流体用のカートリッジであって、前記流体の１つ以上は１種以上の標的分析物を含有し得る分析しようとする流体であり、１つ以上の前記流体が蛍光、発光又は比色標識を含有し、該カートリッジはさらに、１種以上の流体をコンピューター制御下で該カートリッジの１つの区画から別の１つ以上の区画へ移すことができる流体チャネルを備え、該カートリッジのアッセイ部分は、１種以上の特異的捕捉剤を各々含有する２箇所以上の結合部位のアレイを備える、カートリッジと、
蛍光又は発光又は比色標識と相互作用して及び／又は刺激して、蛍光又は発光又は比色シグナルの強度を、各々のそのような部位において時系列で測定するためのデバイスと、
そのような時系列の測定を取り入れて、結合部位中における非標的物質／分子／分析物と捕捉剤又は他の物質との間の動的又は速度論的結合曲線の表示を作製するためのデバイスと
を具備するシステム。
［項目３５］
前記リザーバが、該リザーバ中の流体を封じる薄膜により覆われて、該薄膜が皮下注射用の針又は類似のデバイスにより穴を開けられて、該リザーバからの流体を添加又は引き出しが可能である、項目３４に記載のシステム。
［項目３６］
流体の１種が、１種以上の促進剤分子又は実体を含有し、検出用試薬に追加の結合部位を提供して、蛍光、発光又は比色シグナルの強度の増大を促進し、及び／又は増幅することを助長する、項目３４に記載のシステム。
［項目３７］
前記１種以上の促進剤分子又は実体が、ストレプトアビジン、アビジン、色素標準型のストレプトアビジン、アビジン；二量化ビオチン分子、ビオチンで部分的に若しくは完全に標識されたＰＡＭＡＭデンドリマー、又はビオチン−アビジンネットワークを形成し得る任意のビオチンを含有する分子若しくは高分子、ビオチン化タンパク質、ビオチン化抗体、ビオチン化ペプチド、ＤＮＡのビオチン化ストランド、ビオチン化デンドリマー、抗種抗体、及び分析物とは独立した様式で捕捉された活性物質と検出試薬とを架橋させることができる任意の活性物質を含む群から選択される、項目３６に記載のシステム。
［項目３８］
増幅の効果が、少なくとも１種の促進剤、少なくとも１種の分析物及び少なくとも１種の検出用試薬の循環的処理によって得ることができ、一部の促進剤が、一部の検出試薬に結合して、さらなる検出試薬が結合する多数の追加の部位を提供することにより、促進されたシグナル効果がもたらされる、項目３６及び３７に記載のシステム。
［項目３９］
結合部位が、以下の実体の１つ又は複数、すなわち
タンパク質、ホルモン、抗体、抗原、ウイルス、抗体複合体、抗体断片、ペプチド、細胞、細胞断片、アプタマー、細胞溶解物、分画された細胞溶解物、分画された細胞、ＤＮＡ、ＲＮＡ、ｍＲＮＡ、遺伝子、遺伝子発現生成物などの生物学的実体と、
化学元素、化合物、薬学的に活性な化合物若しくはそれらの代謝物、鉱物、又は汚染物質などの化学的実体と
の１種又は複数を含有する、項目３４〜３８のいずれか一項に記載のシステム。
［項目４０］
動的又は速度論的結合曲線の表示が、１種以上の標的分析物濃度を計算するために使用される、項目３９に記載のシステム。
［項目４１］
動的又は速度論的結合曲線の表示が、結合部位中における標的分析物と捕捉剤との特異的相互作用から生ずるシグナルを、非特異的相互作用から生ずるシグナルから区別するために使用される、項目３９に記載のシステム。
［項目４２］
項目３９に記載のシステムの使用であって、
時間依存性の結合曲線及び関係する速度の分析による流体中の分析物濃度の決定は、分析物の超高濃度（例えば５００，０００ｐｇ／ｍＬを超える）と同じ試験及び同じカートリッジで、分析物の超低濃度（例えば１０ｐｇ／ｍＬ未満）を流体から検出及び／又は測定することを可能にし、該決定は、超高濃度分析物の結合曲線の分析によりアッセイプロセスにおける初期（例えば最初の２０分以内）に達成され、一方、これらの分析対象の結合曲線がシステムで見える場合に、超低濃度分析物は、アッセイプロセスの後期（例えば３０分後）に検出／測定される、使用。
［項目４３］
項目３９に記載のシステムを使用して分析物濃度を計算する方法であって、
各結合部位の結合曲線の勾配、又は非線形のデータの場合には特定の分析時間における勾配（１次微分又は接線）を計算することにより結合速度のデータ（初速度）を得て、次にそのデータを酵素触媒作用型の反応方程式に適用して、これらの分析物のために前もって又は同時に開発された既知の標準物質に関する速度に基づく結合曲線と比較する、方法。
［項目４４］
項目３９に記載のシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、結合曲線の形状は、観察又は測定されたシグナルが特異的捕捉剤の標的分析物に対する結合事象の結果であるか又は捕捉剤の１種以上の非標的分析物に対する非特異的相互作用の結果であるかを知るために使用される、方法。
［項目４５］
特異的結合と非特異的結合との区別が、選択された結合部位の初期結合速度（最初の２又は３回の検出又は測定事象から計算される）を、その後の検出又は測定事象から（３又は４回の後、及び／又は１０〜１５分後の検出／測定事象から）計算された結合速度と比較することにより行われ、３又は４回の事象後、及び／又は１０〜１５分後の検出事象後に変化する（非線形になる）結合速度が、非特異的結合を表している可能性が高い、項目４４に記載の方法。
［項目４６］
項目３９に記載のシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
シグナルが非特異的相互作用の比較的高い濃度の結果である場合に、初速度が早期の時点で非常に高く、後期の時点でゼロに近づくことが観察される、方法。
［項目４７］
項目３９に記載のシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、真のシグナルと真でないシグナルとの区別が、未知の分析物の存在及び濃度を用いた所与の実験からの結合曲線と、既知の存在及び濃度を用いた標準分析物の結合曲線との比較分析により達成される、方法。
［項目４８］
項目３９に記載のシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
アッセイ実行の終了時に、試験試料及び検出試薬を、ある濃度の捕捉剤を含有する第１のアッセイ緩衝液及び第２のアッセイ緩衝液で置き換えるステップ、又は
アッセイ実行の終了時に、試験試料及び検出試薬を、ある濃度の検出試薬を含有する第１のアッセイ緩衝液及び第２のアッセイ緩衝液で置き換えるステップ、又は
アッセイ実行の終了時に、試験試料及び検出試薬を、ある濃度の少なくとも１つの標的分析物を含有する第１のアッセイ緩衝液及び第２のアッセイ緩衝液で置き換えるステップと、
第１のアッセイ緩衝液及び第２のアッセイ緩衝液の反復する流れを数サイクル実行するステップと、
カートリッジのアッセイ部分を撮像するステップと
を含み、
特異的結合であれば、シグナルが減少するため、その場合、シグナル及び関係する結合曲線が標的分析物からの特異的シグナルであると推定でき、
シグナルが事実上非特異的であれば、その場合、シグナルが目的の分析物（標的分析物）の存在に依存しないので実質的に安定なままであり、シグナル及び関係する結合曲線が、試料中の非標的物質からの非特異的シグナルであると推定できる、方法。
［項目４９］
項目３９に記載のシステムを使用して結合部位中における標的分析物の捕捉剤に対する特異的結合から非特異的結合を区別する方法であって、
非特異的シグナルが捕捉物と検出抗体との異好性の抗体架橋により生ずる場合について、少なくとも１つのアッセイで使用される捕捉剤（この例では捕捉抗体）と同じ種であるが、少なくとも１つアッセイで測定される標的分析物のいずれにも向けられていない抗体（ヤギ、ウサギ、マウス、その他）の混合物を含有する「陰性対照」スポット（結合部位）が、カートリッジのアッセイ部分に加えられて、
どの試料が異好性の抗体を含有するかを、「陰性対照スポット」が試料に依存する様式でシグナルを生じるか否かの観察から推定して、ここで生じるのであれば、該シグナルが非特異的成分を含むと考え、又は
「陰性対照スポット」からのシグナルを使用して分析物に特異的なスポット（結合部位）におけるシグナルを調整して、存在する異好性の抗体の「濃度」を考慮に入れ、したがって、標的分析物濃度のより正確な測定を提供する、方法。
［項目５０］
項目３９に記載のシステムを使用して、薬剤開発、薬剤発見、診断の又は他の用途のための新しい候補バイオマーカーを発見する方法であって、
各結合部位を特異的対非特異的結合シグナルについて分析し、特異的シグナルをもたらす結合部位のみが、追加の分画及び／若しくは質量分析法又はどの分析物がこれらの結合部位に存在する（により捕捉された）かを確認するために使える別の技法によりさらに分析される、方法。
［項目５１］
結合部位が、分画された細胞溶解物、組織又は他の生物学的試料を含有し、特定の疾患又は状態を有する１名又は複数の対象から採取され、アッセイ中に処理される流体の１つが、健常な対照又は前記特定の疾患又は状態を有する対象のいずれか由来であり、
健常な対照からの流体で実行したアッセイからのデータが、前記特定の疾患又は状態を有する対象からの流体で実行したアッセイからのデータと比較されて、特定の疾患又は状態を有する対象からの流体で特異的結合のシグナルを示し、健常な対照からの流体で特異的結合のシグナルを示さない上記の結合部位が、その後の分画及び／若しくは質量分析法又は前記特異的結合のシグナルを生じさせる候補バイオマーカーを単離して同定する他の分析手順のいずれかによりさらに分析される、項目５０に記載の方法。
［項目５２］
圧力が、カートリッジを出る流体の流速のモニタリングにより、及び所望の流速を設定するのに必要な圧力を確立させるフィードバックにより制御され得る、項目３９に記載のシステム。
［項目５３］
レーザー又は他の標識刺激／相互作用プロセスが作用しておらず、使用者が流体の移動を見て、カートリッジが、適切に流れていること及び泡又は他の可視的汚染物質のないことを視覚的に確認することを可能にするのに十分透明である場合に、カートリッジを後方から照明することができる、項目３９に記載のシステム。
［項目５４］
取り外し可能な廃棄物容器がカートリッジにより処理された全ての流体を集めるためにシステムに組み込まれていてもよい、項目３９に記載のシステム。
［項目５５］
取り外し可能なタブレットがアッセイを遂行してモニターするために遠隔で使用できるように、取り外し可能なタブレットコンピューターが、アッセイを設定して遂行する指示を提供して、分析及び結果の詳細を提供するユーザーインターフェースとして使用される、項目３９に記載のシステム。
［項目５６］
着色光がユーザーインターフェース及びシステム自体の両方で使用されて、アッセイを設定して遂行するために関係するステップを通して使用者を導く、例えばインターフェースがカートリッジの視覚的表示を示して、使用者に前記リザーバが緑色（又は他の色）の光等で点灯されたときに、流体１をリザーバ１に挿入するように依頼する、項目３９に記載のシステム。
［項目５７］
項目３９及び５２〜５６のいずれか一項に記載のシステムが実験室、介護の近く又は介護環境の場で診断用のバイオマーカーの検出及び測定のために使用される、項目３９及び５２〜５６のいずれか一項に記載のシステムの使用。
［項目５８］
アッセイにおいて特異的結合と非特異的結合を識別する方法であって、
複数のシグナル強度測定の各々が、分析される試料と分析される試料中の標的分析物の捕捉剤との反復する相互作用の間に行われて、分析される試料の複数のシグナル強度測定値を得るステップと、
複数のシグナル強度測定値を分析される試料と捕捉剤の相互作用の累積持続時間の関数としてプロットするステップと、
プロットされたシグナル強度測定値が既知の標準物質の特性を示している場合に、シグナル強度測定値が標的分析物と捕捉剤の特異的結合を示していると判定するステップと
を含む、方法。
［項目５９］
分析物を含有する試料中の分析物濃度を計算する方法であって、
試料の成分の分析物に結合することができる捕捉剤への結合を表す複数のシグナル強度測定値を得るステップであって、複数のシグナル強度測定が既知の時間間隔で試料と捕捉剤の間の相互作用の既知の持続時間の間に行われるステップと、
複数のシグナル強度測定値を試料と捕捉剤の相互作用の累積持続時間の関数としてプロットするステップと、
プロットされたシグナル強度測定値に対する１つ以上の１次微分又は接線（初速度）を計算するステップであって、１つ以上の１次微分又は接線（初速度）の各々が近接してプロットされたシグナル強度測定値を使用して計算されるステップと
を含み、
１つ以上の標準分析物濃度についての１次微分又は接線（初速度）は、後で逆算曲線の適合を決定するために使用することができ、酵素基質複合体が反応に先立って形成される場合、逆算の適合は、酵素触媒モデルに基づき、未知の試料の１次微分又は接線（初速度）が、１つ以上の標準物質の分析濃度から濃度を逆算するために使用される、方法。
［項目６０］
１種以上の流体を保持するための１つ以上のリザーバ部分と、
前記１つ以上のリザーバ部分からの１種以上の流体を受けるための１つ以上のアッセイ部分であって、該アッセイ部分の上に流体が流される複数の結合部位を有し、流体チャネル又は配管を通して前記１つ以上のリザーバ部分に接続された、１つ以上のアッセイ部分と
を備えるカートリッジデバイス。
［項目６１］
流体を漏洩することなく前記アッセイ部分に入る流体のための出口を提供するチャネルをさらに備える、項目６０に記載のカートリッジデバイス。
［項目６２］
前記リザーバ部分の各々における上部の空気の圧力の制御で該リザーバ部分から出て当該カートリッジデバイスの前記１つ以上のアッセイ部分を通る流体の流れを直接制御するように、前記リザーバ部分の各々の周縁部の周りに気密シールを形成する機構をさらに備える、項目６１に記載のカートリッジデバイス。
［項目６３］
前記リザーバ部分から当該カートリッジデバイスの前記アッセイ部分を通る流体の制御された反復する流れを可能にするために、前記リザーバ部分の各々にかけられる圧力を変化させることができる機構に対する１つ以上の取り付け箇所をさらに備える、項目６２に記載のカートリッジデバイス。
［項目６４］
当該カートリッジデバイスの前記アッセイ部分中に、分析しようとする流体から標的の分析物を選択するために特異的な選択された捕捉剤を含有する複数の結合部位をさらに備える、項目６０に記載のカートリッジデバイス。
［項目６５］
結合部位を越える流体の制御された反復する流れが、結合部位中においてこれらの捕捉剤と結合する流体中のこれらの分析物の速度論的結合曲線の表示を形成するために使用され得る、項目６０に記載のカートリッジデバイス。
［項目６６］
当該カートリッジデバイスが、
捕捉剤を含有する結合部位が上に所在し得るベース部と、
前記リザーバ部分、前記流体チャネル及び前記アッセイ部分を備える当該カートリッジデバイスの上部又は本体部と
を具備する、項目６０に記載のカートリッジデバイス。
［項目６７］
前記ベース部が、以下の材料：
ガラス、ＰＤＭＳ、化学処理されたガラス、ナノ粒子で覆われたガラス、金属でコートされたガラス、他の形態の材料で処理されたガラス、炭素（全ての形態）、ケイ素、ゴム、プラスチック、金属、結晶類、ポリマー、半導体、有機材料又は無機材料
の１つ又は複数からなる、項目６６に記載のカートリッジデバイス。
［項目６８］
当該カートリッジデバイスの前記上部又は前記本体部が、以下の材料：
ガラス、ＰＤＭＳ、化学処理されたガラス、ナノ粒子で覆われたガラス、金属でコートされたガラス、他の形態の材料で処理されたガラス、炭素（全ての形態）、ケイ素、ゴム、プラスチック、金属、結晶類、ポリマー、半導体、有機材料又は無機材料の１つ又は複数からなる、項目６６に記載のカートリッジデバイス。
［項目６９］
前記ベース部が疎水性であり、
前記ベース部が、所望の捕捉剤又は活性物質を含有する結合部位のアレイを含み、
前記結合部位のアレイを備える前記ベース部が、プラズマでエッチングされており、
前記本体部が、成形された材料、又は、前記流体チャネル、前記アッセイ部分及び前記リザーバ部分を含むように構築された材料から構成され、
前記本体部が前記ベース部と位置を調整して結合されている、
請求項６６に記載のカートリッジデバイス。
［項目７０］
前記リザーバ部分の各々における上部の周縁部周りの前記気密シールが、前記周縁部の周りの上に置かれた隆起したリングにより達成され、そのシールが、圧力をかける任意の気体又は他の形態の流体の洩れを防止するシールを有する該リザーバ部分の上部に圧力を提供するインターフェースデバイスとかみ合う、項目６２に記載のカートリッジデバイス。
［項目７１］
前記リザーバ部分の各々における上部を覆って封じる薄膜をさらに備え、
該薄膜は、流体を当該カートリッジデバイスに漏洩を生ずることなく予め充填できて、皮下注射用の針の付いた又は同様な装置により、流体を任意の保存中に容易に挿入し又はそこから取り出すことができるように、かつ、当該カートリッジデバイスをシステムに挿入するとこのシールが自動的に破れて流体が適用された圧力の制御下で流れることを可能にするように、リザーバの周縁部の周りを封じている、項目６０に記載のカートリッジデバイス。
［項目７２］
当該カートリッジデバイスが透明な材料で作製されており、当該カートリッジデバイスが挿入されるシステムが、可視性のバックライトを提供して、この点灯がアッセイ中に結合事象の検出及び測定に干渉しない場合、使用者が当該カートリッジデバイスを通る流体の移動を見て、流れに勢いがあることを視覚で判定し、泡又は他の汚染物質などの任意の潜在的な不規則性を観察することを可能にする、項目６０に記載のカートリッジデバイス。
［項目７３］
当該カートリッジデバイスを通る液体の流速を、当該カートリッジデバイスにおける前記アッセイ部分の後ろ又は当該カートリッジデバイスの出口の後ろで、当該カートリッジデバイスが挿入されるシステム内に置かれた又はそれ以外で接続された流れセンサーにより測定することができる、項目６０に記載のカートリッジデバイス。
［項目７４］
前記アッセイ部分が高さ１５０ミクロン以下、及び好ましくは５０ミクロン以下となるサイズである、項目６０に記載のカートリッジデバイス。
［項目７５］
前記アッセイ部分が流体の乱流を生じさせる、項目６０に記載のカートリッジデバイス。
  Preferably, at least three runs of each concentration standard are used to generate a matching standard curve. The accuracy of the method can be run repeatedly and evaluated from either initial velocity, back calculated concentration, or recovery (percent). The accuracy of the method can be evaluated over the entire concentration range by looking at the back calculated concentration or recovery (percent). Table 1 summarizes the data from the example experiments.

[Items of Invention]
[Item 1]
An assay system including a cartridge device and a measurement device,
Wherein the cartridge device is
One or more reservoir portions for holding one or more liquids;
One or more assay portions for receiving the one or more liquids from the one or more reservoir portions, wherein the one or more liquids from the one or more reservoir portions are over the assay portions. One or more assay portions having multiple binding sites that can be flowed repeatedly (two or more times);
Including
An assay system, wherein said measurement device is for measuring binding of one or more analytes in said one or more liquids to a plurality of binding sites.
[Item 2]
An interface for receiving and withdrawing the cartridge device, wherein the apparatus controls the flow or movement of the one or more liquids from the one or more reservoir portions through the one or more assay portions. The assay system of claim 1, further comprising an interface.
[Item 3]
The device for controlling the flow of the one or more liquids is computer controlled, the device comprising at least one or more of the following:
A flow rate of the one or more liquids over the one or more assay portions;
The duration of the one or more liquid flows over the one or more assay portions;
Number of times that an amount of the one or more liquids is flowed over the one or more assay portions
3. The assay system of claim 2, wherein one or more of the following can be independently controlled.
[Item 4]
4. The assay system of claim 3, wherein the interface comprises a device for removing or draining the one or more liquids from the one or more assay portions.
[Item 5]
The one or more liquids include one or more labels selected from the group consisting of a fluorescent label, a luminescent label, and a colorimetric label,
Item 2. The assay system according to item 1, wherein the measurement device is selected from the group consisting of a fluorescence measurement device, a luminescence measurement device, and a colorimetry device.
[Item 6]
A system wherein the measurement device is capable of directing, under computer control, a laser or other form of labeled stimulus to the one or more assay portions of the cartridge device, followed by a fluorescent, luminescent or colorimetric signal. Item 6. The assay system according to Item 5, wherein the assay system is configured to be capable of performing detection and / or measurement of S.
[Item 7]
Item 2. The assay system of item 1, wherein the one or more liquids may contain one or more analytes to be detected, measured and / or analyzed.
[Item 8]
A plurality of time series measurements of a signal from a binding site in the assay portion of the cartridge device are performed under computer control, and the resulting signal indicates one or more of a plurality of binding sites in the one or more assay portions. The assay system of claim 1, further comprising: being used to generate a representation of a kinetic or kinetic binding curve of these analytes binding to a plurality.
[Item 9]
The assay system of claim 2, wherein the interface provides variable pressures (positive and / or negative) to control the flow rate and duration of the one or more liquids over the one or more assay portions.
[Item 10]
Analyzing one or more binding curve representations of one or more analytes binding to one or more of a plurality of binding sites in the one or more assay portions, and Item 8 further comprising computer software for comparing the analysis to a time course (kinetic) binding curve of a known standard to determine the presence and / or concentration of said one or more analytes in a liquid. An assay system according to claim 1.
[Item 11]
The assay system of claim 1, wherein the one or more reservoir portions are covered by a thin film that seals liquid in the reservoir.
[Item 12]
One or more enhancer molecules or entities that provide additional binding sites for the detection reagent to facilitate and / or amplify the increase in the intensity of the fluorescent, luminescent, or colorimetric signal are added to one or more of the liquids. 2. The assay system according to item 1, wherein the species is contained.
[Item 13]
The one or more promoter molecules or entities are streptavidin, avidin, dye-labeled streptavidin, avidin; a dimerized biotin molecule, a PAMAM dendrimer partially or completely labeled with biotin, or a biotin-avidin network In a manner independent of any biotin-containing molecule or polymer, biotinylated protein, biotinylated antibody, biotinylated peptide, biotinylated strand of DNA, biotinylated dendrimer, anti-species antibody, and analyte capable of forming 13. The assay system according to item 12, wherein the assay system is selected from the group consisting of any active substance capable of crosslinking the captured active substance and the detection reagent.
[Item 14]
The effect of amplification can be obtained by cyclic treatment of at least one enhancer, at least one analyte and at least one detection reagent, wherein some enhancers bind to some detection reagents. Item 13. The assay system of item 12, wherein providing an additional number of additional sites for additional detection reagents results in an enhanced signal effect.
[Item 15]
The binding site is one or more of the following entities:
Protein, hormone, antibody, antigen, virus, antibody complex, antibody fragment, peptide, cell, cell fragment, aptamer, cell lysate, fractionated cell lysate, fractionated cell, DNA, RNA, mRNA, Biological entities such as genes and gene expression products;
With chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals or pollutants
Assay system according to item 1, comprising one or more of the following.
[Item 16]
Item 11. The assay system of item 10, wherein the display of a kinetic or kinetic binding curve is used to calculate one or more target analyte concentrations.
[Item 17]
The display of a kinetic or kinetic binding curve is used to distinguish signals resulting from specific interactions between the target analyte and the capture agent within the binding site from signals resulting from non-specific interactions. Item 11. The assay system according to item 10.
[Item 18]
Use of an assay system according to item 15, comprising:
Determination of the analyte concentration in the fluid by analysis of the time-dependent binding curve and the associated velocities is based on the same test and the same cartridge as the very high concentration of the analyte (eg, above 500,000 pg / mL). Allows ultra-low concentrations (eg, less than 10 pg / mL) to be detected and / or measured from the fluid, the determination being made early in the assay process (eg, within the first 20 minutes) by analysis of the binding curve of the ultra-high analyte. ), While the ultra-low concentration analyte is detected / measured later in the assay process (eg, after 30 minutes), while the binding curves of these analytes are visible in the system.
[Item 19]
A method of calculating an analyte concentration using an assay system according to item 15, comprising:
The binding rate data (initial rate) is obtained by calculating the slope of the binding curve for each binding site, or in the case of non-linear data, the slope (first derivative or tangent) at a particular analysis time. A method wherein the binding rate data is applied to an enzyme-catalyzed reaction equation and compared to a rate-based binding curve for a known standard previously or simultaneously developed for these analytes.
[Item 20]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using an assay system according to item 15, comprising:
The shape of the binding curve indicates whether the observed result or signal is the result of a binding event of the specific capture agent to the target analyte or the result of a non-specific interaction of the capture agent with one or more non-target analytes. The method used to know if there is.
[Item 21]
The distinction between specific and non-specific binding is achieved by determining the initial binding rate of the selected binding site (calculated from the first two or three detection / measurement events) from subsequent detection or measurement events (3 Or after 3 or 4 events, and / or 10 to 15 minutes, and / or from the detection or measurement event after 10 to 15 minutes. 21. The method of item 20, wherein the binding rate that changes (becomes non-linear) after a detection or measurement event after 15 minutes is likely to indicate non-specific binding.
[Item 22]
A method for distinguishing specific binding from non-specific binding of a target analyte to a capture agent in a binding site using an assay system according to item 15, comprising:
The method wherein the initial rate is observed to be very high at an early time point and approach zero at a late time point when the signal is the result of a relatively high concentration of non-specific interactions.
[Item 23]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using an assay system according to item 15, comprising:
The distinction between the specific binding signal and the non-specific binding signal is determined by comparing the binding curve from a given experiment with the presence and concentration of the unknown analyte to the standard analyte using the known presence and concentration. A method achieved by comparative analysis with a binding curve.
[Item 24]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using an assay system according to item 15, comprising:
Replacing the test sample and the detection reagent with a first assay buffer and a second assay buffer containing a certain concentration of a capture agent at the end of the assay run; or
At the end of the assay run, replacing the test sample and the detection reagent with a first assay buffer and a second assay buffer containing a concentration of the detection reagent, or
At the end of the assay run, replacing the test sample and detection reagent with a first assay buffer and a second assay buffer containing a concentration of at least one target analyte;
Performing several cycles of a repetitive flow of a first assay buffer and a second assay buffer;
Imaging the assay portion of the cartridge;
Including
With specific binding, the signal is reduced, in which case the signal and the associated binding curve can be assumed to be a specific signal from the target analyte,
If the signal is effectively non-specific, then it remains substantially stable since the signal does not depend on the presence of the analyte of interest (the target analyte), and the signal and associated binding curves are The method can be presumed to be a non-specific signal from a non-target substance.
[Item 25]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using an assay system according to item 15, comprising:
For the case where the non-specific signal is caused by cross-linking of a heterophilic antibody between the capture and the detection antibody, it is of the same species as the capture agent (the capture antibody in this example) used in at least one assay, but at least one A “negative control” spot (binding site) containing a mixture of antibodies (goat, rabbit, mouse, etc.) that is not directed to any of the target analytes measured in the assay is added to the assay portion of the cartridge, And
Which samples contain heterophilic antibodies is inferred from observations of whether the "negative control spot" produces a signal in a sample-dependent manner, and if so, if the signal is non- Considered to contain specific components, or
The signal from the “negative control spot” was used to adjust the signal at the spot (binding site) specific for the analyte to take into account the “concentration” of the heterophilic antibody present and therefore target analysis A method that provides a more accurate measurement of a substance concentration.
[Item 26]
A method for discovering a new candidate biomarker for drug development, drug discovery, diagnostic or other use using the assay system of item 15, comprising:
Each binding site is analyzed for specific versus non-specific binding signals, and only those binding sites that yield a specific signal are subjected to additional fractionation and / or mass spectrometry or which analyte is present at these binding sites (by Method that is further analyzed by another technique that can be used to determine whether
[Item 27]
The binding site contains a fractionated cell lysate, tissue or other biological sample, is taken from one or more subjects with a particular disease or condition, and is one of the fluids to be processed during the assay. One is from either a healthy control or a subject having said particular disease or condition,
Data from an assay performed with fluid from a healthy control is compared to data from an assay performed with fluid from a subject having the particular disease or condition, and fluid from a subject having a particular disease or condition is compared. The binding site described above, which shows a signal of specific binding in the fluid from a healthy control and no signal of specific binding in a fluid from a healthy control, gives rise to subsequent fractionation and / or mass spectrometry or the signal of said specific binding. 27. The method according to item 26, wherein the method is further analyzed by any of the other analytical procedures for isolating and identifying candidate biomarkers.
[Item 28]
Item 16. The assay system of item 15, wherein the flow rate can be controlled by dynamically adjusting the pressure based on feedback from a flow rate sensor following the assay portion of the cartridge at or near the outlet of the cartridge.
[Item 29]
No laser or other beacon stimulation / interaction process is working, and the user sees the movement of the fluid and sees that the cartridge is flowing properly and free of bubbles or other visible contaminants. Item 16. The assay system according to item 15, wherein the cartridge can be illuminated from behind when it is sufficiently transparent to allow for visual confirmation.
[Item 30]
A removable waste container may be incorporated into the system that collects all fluids processed by the cartridge and includes a sensor that informs a user when the waste container is nearly full and should be emptied. 16. The assay system according to item 15, wherein
[Item 31]
A user who provides instructions for setting up and performing the assay and provides analysis and results details so that the removable tablet can be used remotely to perform and monitor the assay Item 16. The assay system according to item 15, used as an interface.
[Item 32]
Colored light is used in both the user interface and the system itself to guide the user through the steps involved in setting up and performing the assay, e.g., the interface shows a visual indication of the cartridge and provides the user with the reservoir. Item 16. An assay system according to item 15, wherein when is lit with green (or other color) light or the like, the fluid 1 is requested to be inserted into the reservoir 1.
[Item 33]
Items 15, 28-32, wherein the assay system according to any one of items 15, 28-32 is used for the detection and measurement of a diagnostic biomarker in a laboratory, near care or in a care setting. 32. Use of an assay system according to any one of the items 32.
[Item 34]
A receiver for receiving a cartridge for fluid, the cartridge having an interface for controlling a flow rate of one or more fluids from one or more reservoirs in the cartridge;
A fluid cartridge having one or more reservoirs containing a fluid, wherein one or more of the fluids is a fluid to be analyzed that may contain one or more target analytes, The above fluids contain a fluorescent, luminescent or colorimetric label, and the cartridge is further capable of transferring one or more fluids from one compartment of the cartridge to one or more compartments under computer control. A cartridge comprising a fluidic channel, wherein the assay portion of the cartridge comprises an array of two or more binding sites each containing one or more specific capture agents; and
A device for interacting with and / or stimulating a fluorescent or luminescent or colorimetric label to measure the intensity of the fluorescent or luminescent or colorimetric signal in time at each such site;
Device for incorporating such time series measurements to create a representation of a dynamic or kinetic binding curve between a non-target substance / molecule / analyte and a capture agent or other substance in the binding site When
A system comprising:
[Item 35]
The reservoir is covered by a membrane enclosing the fluid in the reservoir, the membrane being pierced by a hypodermic needle or similar device to allow for addition or withdrawal of fluid from the reservoir; Item 34. The system according to Item 34.
[Item 36]
One of the fluids contains one or more promoter molecules or entities to provide additional binding sites for the detection reagent to facilitate an increase in the intensity of the fluorescent, luminescent or colorimetric signal, and / or 35. The system according to item 34, which facilitates the amplification.
[Item 37]
The one or more promoter molecules or entities are streptavidin, avidin, dye-standard streptavidin, avidin; a dimerized biotin molecule, a PAMAM dendrimer partially or completely labeled with biotin, or a biotin-avidin network In a manner independent of any biotin-containing molecule or polymer, biotinylated protein, biotinylated antibody, biotinylated peptide, biotinylated strand of DNA, biotinylated dendrimer, anti-species antibody, and analyte capable of forming 37. The system according to item 36, wherein the system is selected from the group comprising any active substance capable of cross-linking the captured active substance with the detection reagent.
[Item 38]
The effect of amplification can be obtained by cyclic treatment of at least one enhancer, at least one analyte and at least one detection reagent, wherein some enhancers bind to some detection reagents. 38. The system of paragraphs 36 and 37, wherein the enhanced signal effect is provided by providing a number of additional sites to which additional detection reagents bind.
[Item 39]
The binding site is one or more of the following entities:
Protein, hormone, antibody, antigen, virus, antibody complex, antibody fragment, peptide, cell, cell fragment, aptamer, cell lysate, fractionated cell lysate, fractionated cell, DNA, RNA, mRNA, Biological entities such as genes and gene expression products;
With chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals or pollutants
39. A system according to any one of items 34 to 38, comprising one or more of the following.
[Item 40]
40. The system of claim 39, wherein display of a kinetic or kinetic binding curve is used to calculate one or more target analyte concentrations.
[Item 41]
The display of a kinetic or kinetic binding curve is used to distinguish signals resulting from specific interactions of the target analyte with the capture agent in the binding site from signals resulting from non-specific interactions. Item 39. The system according to Item 39.
[Item 42]
Use of the system of item 39, wherein
Determination of the analyte concentration in the fluid by analysis of the time-dependent binding curve and the associated velocities is based on the same test and the same cartridge as the very high concentration of the analyte (eg, above 500,000 pg / mL). Allows ultra-low concentrations (eg, less than 10 pg / mL) to be detected and / or measured from the fluid, the determination being made early in the assay process (eg, within the first 20 minutes) by analysis of the binding curve of the ultra-high analyte. ), While the ultra-low concentration analyte is detected / measured later in the assay process (eg, after 30 minutes), while the binding curves of these analytes are visible in the system.
[Item 43]
A method of calculating an analyte concentration using the system of item 39, comprising:
The binding rate data (initial rate) is obtained by calculating the slope of the binding curve for each binding site, or, in the case of non-linear data, the slope (first derivative or tangent) at a particular analysis time. A method wherein the data is applied to an enzyme-catalyzed reaction equation and compared to a rate-based binding curve for a known standard previously or simultaneously developed for these analytes.
[Item 44]
39. A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the system of item 39, wherein the shape of the binding curve is such that the observed or measured signal is A method used to know whether it is the result of a binding event of a specific capture agent to a target analyte or a non-specific interaction of a capture agent with one or more non-target analytes.
[Item 45]
The distinction between specific and non-specific binding is achieved by determining the initial binding rate of the selected binding site (calculated from the first two or three detection or measurement events) from subsequent detection or measurement events (3 Or after 4 and / or 10/15 minutes of detection / measurement event) by comparing with the calculated binding rate, after 3 or 4 events and / or after 10-15 minutes 45. The method of claim 44, wherein the binding rate that changes (becomes non-linear) after the detection event is likely to represent non-specific binding.
[Item 46]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the system of item 39, comprising:
The method wherein the initial rate is observed to be very high at an early time point and approach zero at a late time point when the signal is the result of a relatively high concentration of non-specific interactions.
[Item 47]
39. A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the system of item 39, wherein the distinction between a true signal and a non-true signal is unknown. A method achieved by comparative analysis of the binding curve from a given experiment using the presence and concentration of an analyte with the binding curve of a standard analyte using a known presence and concentration.
[Item 48]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the system of item 39, comprising:
Replacing the test sample and the detection reagent with a first assay buffer and a second assay buffer containing a certain concentration of a capture agent at the end of the assay run; or
At the end of the assay run, replacing the test sample and the detection reagent with a first assay buffer and a second assay buffer containing a concentration of the detection reagent, or
At the end of the assay run, replacing the test sample and detection reagent with a first assay buffer and a second assay buffer containing a concentration of at least one target analyte;
Performing several cycles of a repetitive flow of a first assay buffer and a second assay buffer;
Imaging the assay portion of the cartridge;
Including
With specific binding, the signal is reduced, in which case the signal and the associated binding curve can be assumed to be a specific signal from the target analyte,
If the signal is effectively non-specific, then it remains substantially stable since the signal does not depend on the presence of the analyte of interest (the target analyte), and the signal and associated binding curves are The method can be presumed to be a non-specific signal from a non-target substance.
[Item 49]
A method for distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the system of item 39, comprising:
In cases where the non-specific signal is caused by heterophilic antibody cross-linking of the capture with the detection antibody, it is of the same species as the capture agent (the capture antibody in this example) used in at least one assay, but at least one A "negative control" spot (binding site) containing a mixture of antibodies (goat, rabbit, mouse, etc.) not directed to any of the target analytes measured in the assay was added to the assay portion of the cartridge. ,
Which samples contain heterophilic antibodies is inferred from observations of whether the "negative control spot" produces a signal in a sample-dependent manner, and if so, if the signal is non- Considered to contain specific components, or
The signal from the “negative control spot” was used to adjust the signal at the spot (binding site) specific for the analyte to take into account the “concentration” of the heterophilic antibody present and therefore target analysis A method that provides a more accurate measurement of a substance concentration.
[Item 50]
39. A method of discovering a new candidate biomarker for drug development, drug discovery, diagnostic or other uses using the system of item 39, comprising:
Each binding site is analyzed for specific versus non-specific binding signals, and only those binding sites that yield a specific signal are subjected to additional fractionation and / or mass spectrometry or which analyte is present at these binding sites (by Method that is further analyzed by another technique that can be used to determine whether
[Item 51]
The binding site contains a fractionated cell lysate, tissue or other biological sample, is taken from one or more subjects with a particular disease or condition, and is one of the fluids to be processed during the assay. One is from either a healthy control or a subject having said particular disease or condition,
Data from an assay performed with fluid from a healthy control is compared to data from an assay performed with fluid from a subject having the particular disease or condition, and fluid from a subject having a particular disease or condition is compared. The binding site described above, which shows a signal of specific binding in the fluid from a healthy control and no signal of specific binding in a fluid from a healthy control, gives rise to subsequent fractionation and / or mass spectrometry or the signal of said specific binding. 51. The method of item 50, further analyzed by any of the other analytical procedures for isolating and identifying candidate biomarkers.
[Item 52]
40. The system according to item 39, wherein the pressure can be controlled by monitoring the flow rate of the fluid exiting the cartridge and by feedback establishing the pressure required to set the desired flow rate.
[Item 53]
No laser or other beacon stimulation / interaction process is working, and the user sees the movement of the fluid and sees that the cartridge is flowing properly and free of bubbles or other visible contaminants. Item 40. The system according to item 39, wherein the cartridge can be illuminated from behind when it is sufficiently transparent to allow for visual confirmation.
[Item 54]
40. The system of item 39, wherein a removable waste container may be incorporated into the system to collect all fluids processed by the cartridge.
[Item 55]
A user who provides instructions for setting up and performing the assay and provides details of the analysis and results so that the removable tablet can be used remotely to perform and monitor the assay 40. The system according to item 39, used as an interface.
[Item 56]
Colored light is used on both the user interface and the system itself to guide the user through the steps involved in setting up and performing the assay, e.g., the interface shows a visual indication of the cartridge and provides the user with the reservoir. 40. The system of item 39, wherein when is illuminated with green (or other color) light or the like, a request is made to insert the fluid 1 into the reservoir 1.
[Item 57]
Items 39 and 52-56, wherein the system according to any one of items 39 and 52-56 is used for the detection and measurement of diagnostic biomarkers in a laboratory, near care or in a care setting. Use of the system according to any one of the preceding claims.
[Item 58]
A method for distinguishing specific binding from non-specific binding in an assay,
Each of the plurality of signal intensity measurements is performed during a repeated interaction of the sample to be analyzed with a capture agent of a target analyte in the sample to be analyzed to provide a plurality of signal intensity measurements of the sample to be analyzed. The step of obtaining
Plotting a plurality of signal intensity measurements as a function of the cumulative duration of the interaction of the capture agent with the sample being analyzed;
Determining that the signal intensity measurement is indicative of specific binding of the target analyte to the capture agent if the plotted signal intensity measurement is characteristic of a known standard;
Including, methods.
[Item 59]
A method for calculating an analyte concentration in a sample containing an analyte, the method comprising:
Obtaining a plurality of signal intensity measurements representing binding of a component of the sample to a capture agent capable of binding to the analyte, wherein the plurality of signal intensity measurements are performed between the sample and the capture agent at known time intervals. Steps performed during a known duration of the interaction;
Plotting a plurality of signal intensity measurements as a function of the cumulative duration of the interaction of the sample with the capture agent;
Calculating one or more first derivatives or tangents (initial velocities) for the plotted signal intensity measurements, each of the one or more first derivatives or tangents (initial velocities) being plotted in close proximity Steps calculated using the measured signal strength measurements
Including
First order derivatives or tangents (initial velocities) for one or more standard analyte concentrations can be used later to determine the fit of the back calculated curve, where the enzyme-substrate complex is formed prior to the reaction. In some cases, the inverse calculation fit is based on an enzyme-catalyzed model and the first derivative or tangent (initial velocity) of the unknown sample is used to calculate the concentration back from the analytical concentration of one or more standards.
[Item 60]
One or more reservoir portions for holding one or more fluids;
One or more assay portions for receiving one or more fluids from the one or more reservoir portions, the plurality of binding sites for fluid to flow over the assay portions, and a fluid channel or tubing. One or more assay portions connected to said one or more reservoir portions through
A cartridge device comprising:
[Item 61]
61. The cartridge device of item 60, further comprising a channel that provides an outlet for fluid entering the assay portion without leaking fluid.
[Item 62]
The periphery of each of the reservoir portions such that control of the pressure of the upper air in each of the reservoir portions directly controls the flow of fluid out of the reservoir portion and through the one or more assay portions of the cartridge device. 62. The cartridge device of item 61, further comprising a mechanism for forming a hermetic seal around the portion.
[Item 63]
One or more attachment points to a mechanism capable of varying the pressure applied to each of the reservoir portions to allow for controlled and repetitive flow of fluid from the reservoir portion through the assay portion of the cartridge device 63. The cartridge device of item 62, further comprising:
[Item 64]
61. The cartridge of claim 60, further comprising a plurality of binding sites in the assay portion of the cartridge device containing a selected capture agent specific for selecting a target analyte from a fluid to be analyzed. device.
[Item 65]
The controlled repetitive flow of fluid over the binding site can be used to form a kinetic binding curve representation of these analytes in the fluid that binds these capture agents in the binding site. 60. The cartridge device according to 60.
[Item 66]
The cartridge device is
A base portion on which the binding site containing the capture agent may be located,
An upper or body portion of the cartridge device comprising the reservoir portion, the fluid channel and the assay portion;
61. The cartridge device according to item 60, comprising:
[Item 67]
The base portion is made of the following material:
Glass, PDMS, chemically treated glass, glass covered with nanoparticles, glass coated with metal, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastic, metal , Crystals, polymers, semiconductors, organic or inorganic materials
67. The cartridge device of claim 66, comprising one or more of the following.
[Item 68]
The top or body portion of the cartridge device is made of the following material:
Glass, PDMS, chemically treated glass, glass covered with nanoparticles, glass coated with metal, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastic, metal 67. The cartridge device of claim 66, comprising one or more of: a crystal, a crystal, a polymer, a semiconductor, an organic or inorganic material.
[Item 69]
The base portion is hydrophobic,
The base comprises an array of binding sites containing a desired capture agent or active substance,
The base portion comprising the array of binding sites is etched with plasma;
The body portion is comprised of a molded material or a material constructed to include the fluid channel, the assay portion, and the reservoir portion;
The main body portion is adjusted and combined with the base portion,
70. The cartridge device of claim 66.
[Item 70]
The hermetic seal around the upper perimeter in each of the reservoir portions is achieved by a raised ring placed over the perimeter, the seal being formed by any gas or other form of applying pressure. 63. The cartridge device of claim 62, wherein the cartridge device engages an interface device that provides pressure on top of the reservoir portion having a seal that prevents leakage of fluid.
[Item 71]
Further comprising a thin film covering and sealing the top of each of the reservoir portions;
The membrane can be pre-filled with fluid into the cartridge device without leaking, allowing the fluid to be easily inserted or removed during any storage by means of a needle or similar device for hypodermic injection. And seal around the perimeter of the reservoir so that when the cartridge device is inserted into the system, the seal will automatically break and allow fluid to flow under control of the applied pressure. 61. The cartridge device of item 60, wherein
[Item 72]
If the cartridge device is made of a transparent material and the system into which the cartridge device is inserted provides a visible backlight and this lighting does not interfere with the detection and measurement of binding events during the assay, Allows a user to view fluid movement through the cartridge device, visually determine that the flow is vigorous, and observe any potential irregularities, such as bubbles or other contaminants. 61. The cartridge device of item 60.
[Item 73]
The flow rate of the liquid through the cartridge device is controlled by a flow located behind or connected to the system into which the cartridge device is inserted, behind the assay portion of the cartridge device or after the outlet of the cartridge device. 61. The cartridge device according to item 60, which can be measured by a sensor.
[Item 74]
61. The cartridge device of item 60, wherein the assay portion is sized to be no more than 150 microns in height, and preferably no more than 50 microns.
[Item 75]
61. The cartridge device according to item 60, wherein the assay portion causes turbulence of a fluid.
 

Claims (13)

An assay system comprising a cartridge device, comprising:
Wherein the cartridge device is
A reservoir for a pressurized sample ,
A sample reservoir;
A membrane covering the reservoir of the sample,
A ring surrounding the periphery of the reservoir of the sample;
An interface device that engages the ring;
A pressurized sample reservoir having:
A reservoir for pressurized reagents,
A reagent reservoir;
A membrane covering the reservoir of the reagent,
A ring surrounding the periphery of the reagent reservoir;
An interface device that engages the ring;
A reservoir of a pressurized reagent having:
- wherein a assay portion for receiving the liquid material from Riza server pressurized sample,
To introduce the liquid to the assay portion, the inlet portion connected to said Riza bar pressurized sample,
An outlet for discharging the liquid from the assay portion;
An assay portion comprising: a plurality of binding sites provided between the introduction portion and the discharge portion; and a binding site through which the liquid can be repeatedly flowed from the introduction portion to the discharge portion. When,
A measuring device for measuring the binding of one or more analytes in the liquid to the plurality of binding sites after each of the plurality of flows of the liquid from the inlet to the outlet. Assay system, including. An interface to the cartridge device or retrieved or accepted, the containing device for controlling the flow or movement of the liquid through the assay portion from Riza server pressurized sample, further comprising an interface ,
The apparatus for controlling the flow of the liquid may include at least one of the following: a flow rate of the liquid across the assay portion;
The duration of the liquid flow across the assay portion; and
The assay system of claim 1, wherein at least one of the number of times a volume of the liquid is flowed through the assay portion can be controlled. Further equipped with a flow rate sensor,
The interface providing a variable positive pressure and / or a variable negative pressure for controlling at least one of a flow rate and a duration of the liquid across the assay portion based on an output from the flow rate sensor. The assay system according to claim 2. The liquid includes one or more labels selected from the group consisting of a fluorescent label, a luminescent label, and a colorimetric label,
The assay system according to claim 1, wherein the measurement device is selected from the group consisting of a fluorescence measurement device, a luminescence measurement device, and a colorimetric measurement device.   The measuring device is capable of directing a laser or other form of labeled stimulus under computer control to the assay portion of the cartridge device, and subsequently detecting and / or detecting a fluorescent, luminescent or colorimetric signal. 5. The assay system according to claim 4, wherein the assay system is configured to perform the measurement. Further equipped with computer software,
When the computer software is executed,
Analyzing a representation of one or more binding curves of one or more analytes binding to one or more of the plurality of binding sites in the assay portion;
Comparing the assay for one or more analytes to a time course binding curve of one or more known standards;
The assay system of claim 1, operable to determine at least one of the presence and concentration of the one or more analytes in the liquid. It said Riza bar pressurized sample is covered with a thin film for sealing the liquid in Riza in bus of the pressurized samples, the assay system according to claim 1. The liquid
Streptavidin species, including streptavidin, avidin and dye-labeled streptavidin,
Dimerized biotin molecule, PAMAM (polyamidoamine) dendrimer partially or completely labeled with biotin, or any biotin-containing molecule or polymer capable of forming a biotin-avidin network, biotinylated protein, biotinylated antibody, biotin Biotinylated species, including biotinylated strands of DNA, biotinylated strands of DNA, and biotinylated dendrimers, or
2. The assay system of claim 1, comprising at least one of an active agent and an anti-species antibody capable of cross-linking the captured active with the detection reagent in a manner independent of the analyte.   The assay system of claim 1, wherein the plurality of binding sites comprises one or both of a biological entity and a chemical entity. The biological entity is a protein, hormone, antibody, antigen, virus, antibody complex, functional antibody fragment, peptide, cell, cell fragment, aptamer, cell lysate, fractionated cell lysate, fractionated Selected from the group consisting of cells, DNA, RNA, mRNA, genes, and gene expression products,
The assay system according to claim 9, wherein the chemical entity is selected from the group consisting of a chemical element, a compound, a pharmaceutically active compound or a metabolite thereof, a mineral, and a contaminant.   The assay system of claim 1, wherein the cartridge device further comprises a waste chamber connected to the outlet. A reservoir for the pressurized sample ,
A sample reservoir;
A membrane covering the reservoir of the sample,
A ring surrounding the periphery of the reservoir of the sample;
An interface device that engages the ring;
A reservoir for the pressurized sample ;
A reservoir of pressurized reagent,
A reagent reservoir;
A membrane covering the reservoir of the reagent,
A ring surrounding the periphery of the reagent reservoir;
An interface device that engages the ring;
A reservoir of a pressurized reagent having:
Wherein an assay portion for receiving fluid from Riza server pressurized sample,
To introduce the flow body to the assay portion, the inlet portion connected to said Riza bar pressurized sample,
A discharge unit for discharging the flow body from the assay portion,
Wherein a plurality of binding sites disposed between the inlet portion and the discharge portion, the flow body through the binding site can be repeatedly flowed into the discharge portion from said inlet portion, and a binding site, assays Part and
A cartridge device comprising:   13. The cartridge device according to claim 12, further comprising a waste chamber connected to the outlet.

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