JP2020098202A - Assay system and cartridge device - Google Patents

Abstract

To provide a method, a system, and a device for detecting and measuring one or plural kinds of analytes.SOLUTION: In one embodiment, the present invention provides an assay system comprising a cartridge device and a measuring device. The cartridge device includes reservoir portions and assay portions, that consist of one or plural reservoir portions for retaining one or plural kinds of liquids, and at least one assay portion for receiving the one or plural kinds of liquids from the at least one reservoir portion. Further, the assay portion has plural binding sites over which the one or plural kinds of liquids from the one or plural reservoir portions can flow repeatedly (twice or more). The measuring device is intended to measure binding of one or plural kinds of analytes in the one or plural kinds of liquids to the plural binding sites.SELECTED DRAWING: Figure 1

Description

Cross-reference to related application

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

The life sciences industry relies on the accurate and timely development and processing of tests known as bioassays to discover new drugs, identify new biomarkers for diagnosing and monitoring disease, or measure biomarker levels. There is.

However, known assay methods suffer from some limitations. One such limitation is that the specific binding of the target analyte to the capture reagent in the assay cannot be distinguished from the non-specific binding of the non-target substance, molecule, or analyte to the capture reagent in the assay. .. Any non-specific binding can result in unnaturally high measurements of analyte concentration and inaccurate quantitation. In fact, in some cases, the signal generated by the non-specific interaction gives a false positive test result even though the analyte is not present in the test sample.

A further significant limitation of the known assays is their inability to accurately detect and measure different analytes, which may be present in the at least one sample being analyzed at concentrations that differ by orders of magnitude. .. The limitations on quantifying analyte concentrations in a single test over such a wide dynamic range have limited the biological relevance of many currently available multiplex assays, and in addition to the large number of sequential sequencing of samples. Dilution is also required, which requires additional time, expense and use of valuable sample.

Moreover, the use of many assays is limited to well-equipped laboratories with trained and trained staff, as most known assays require relatively complex, user-intensive protocols. It

International Patent Application Publication No. WO 2012/071044 (the “'044 application”), 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 mentioned 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 incorporating and constructing from long-term assay screens.

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

In one embodiment, the present invention is an assay system comprising a cartridge device and a measurement device, said cartridge device comprising one or more reservoir portions for holding one or more liquids, and at least At least one assay portion for receiving one or more liquids from one reservoir portion, onto which one or more liquids from one or more reservoirs are repeatedly (two or more times) flowed An assay moiety having a plurality of binding sites to obtain, the measuring device for measuring the binding of one or more analytes in one or more liquids to a plurality of binding sites. , Provides an assay system.

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

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 analyzed and a target in the sample analyzed. Obtaining a plurality of signal intensity measurements of the sample to be analyzed, during repeated interactions of the analyte with the capture agent; a plurality of signal intensity measurements of the sample to be analyzed and the capture agent Plotting as a function of cumulative duration of 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 components of the sample exhibit binding to a capture agent capable of binding to the analyte. A plurality of signal intensity measurements are performed at known time intervals for a known duration of interaction between the sample and the capture agent; Plotting values as a function of cumulative duration of sample-capture agent interaction; calculating one or more first derivative or tangent (initial velocity) to the plotted signal intensity measurements. One or more first derivatives or tangents (initial velocities) each are calculated using signal intensity measurements plotted in close proximity; for one or more standard analyte concentrations The first derivative or tangent (initial velocity) of is then used to provide a method that can later be used to determine the fit of the back curve. If the enzyme-substrate complex is formed prior to the reaction, the back-calculation fit is based on the enzyme-catalytic model. The first derivative or tangent (initial velocity) of the unknown sample is then used to back calculate the analyte concentration from the analytical concentration of one or more standards.

In yet another embodiment, the invention provides at least one reservoir portion for holding 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 on which fluid is flowed and 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 conjunction with the accompanying drawings, which illustrate various embodiments of the invention. Ah

FIG. 3 is an exploded perspective view of an assay cartridge according to an embodiment of the present invention. FIG. 6 is a simplified schematic diagram of four assay portions of an assay cartridge according to an embodiment of the invention. FIG. 6 shows a simplified schematic diagram of one assay portion of an assay cartridge with a simplified schematic diagram of the reservoir and valve elements of the system and assay cartridge according to embodiments of the present invention. FIG. 3 is a simplified diagram of assay steps according to embodiments of the present invention. FIG. 6 is a simplified diagram of another step of the assay method according to an embodiment of the present invention. FIG. 6 is a simplified diagram of yet another step of an assay method according to an embodiment of the present invention. FIG. 6 is a simplified diagram of another step of an assay method according to an embodiment of the present invention. FIG. 6 is a simplified diagram of analyte-capture agent and enhancer interaction according to another embodiment of the invention. FIG. 6 is a simplified diagram of analyte-capture agent and enhancer interaction according to another embodiment of the invention. FIG. 6 is a simplified diagram of analyte-capture agent and enhancer interaction according to another embodiment of the invention. FIG. 6 is a graph of signal intensity of binding of target and non-target analytes. FIG. 6 shows binding curves for triplicate assays targeting IL-6 according to embodiments of the present invention. FIG. 6 shows binding curves for triplicate assays targeting CRP, according to embodiments of the invention. FIG. 6 shows binding curves for 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 meanings in the context of the claimed invention, as described herein. The term "fluid" is intended to broadly encompass the condition of a body that is or is capable of undergoing fluid movement, including liquids, gases, and finely divided particulate solids, and combinations and colloidal suspensions thereof. Includes suspensions such as liquids. In the context of the present invention, "liquid" means animal (including but not limited to) blood, serum, saliva, urine, plasma, tracheal alveolar lavage fluid, tracheal lavage fluid, tissue, tumor, and tissue/tumor. Homogenates, as well as plant extracts, liquefied foods, ascites, organic fluids, inorganic fluids, buffers, labeled buffers, wash fluids, and the like, but are not limited thereto.

The term "analyte" is intended to mean anything that is or can be detected or measured. An analyte is a biological entity such as a protein, hormone, antibody, antigen, virus, antibody complex, peptide, cell, cell fragment, aptamer, cell lysate, DNA, RNA, mRNA, gene, gene expression product, And chemical entities such as, but not limited to, chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, pollutants and the like.

The term "detector reagent" (or detection reagent) is intended to mean anything used as a mechanism that allows visualization (detection) and/or measurement of an analyte. To be done. It is a biological product of proteins, hormones, antibodies, antigens, viruses, antibody complexes, antibody fragments, peptides, cells, cell fragments, aptamers, cell lysates, DNA, RNA, mRNA, genes, gene expression products, etc. Entities, as well as chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, pollutants, etc.

Detection reagents can also be labeled with fluorescent, luminescent or colorimetric labels to facilitate visualization (detection) and/or measurement, which are further complexed with affinity tag labels such as biotin. You can also This allows the use of fluorescent, luminescent or colorimetrically labeled streptavidin for the visualization (detection) and measurement of the detection antibody.

The terms “detecting” and “measuring” and variants thereof are intended to refer to the identification of the presence of an article and the evaluation of a changeable characteristic of the article such as concentration, strength, strength, etc. The term "analyze" and variants thereof refers more broadly to such detection and measurement, as well as other or different assessments of events.

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

The phrase "signal strength" is intended to refer to the measurement of interaction at a binding site, such as by interaction of a capture agent with 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, while the binding interaction between the capture agent and the analyte at an unknown concentration. Can be compared. The terms "display of a kinetic binding curve" and "display of a kinetic binding curve" are intended to refer to a display of the kinetics or kinetics of a binding interaction between a capture agent and an analyte. These differ from the typical and more traditional endpoint standard curves, which are an indication of the signal intensity from the assay at a particular time point in the dilution of a given analyte (ie, at the completion of the assay). These can include data obtained from all or part of the steps of the binding assay, and whether the detected or measured interaction and/or the corresponding signal intensity can be attributed to the specific binding interaction. It can be used to determine if. "Binding curve" or "kinetic curve" refers to a signal that becomes strong from low to high due to the exposure (association) of the binding site to the sample and the detection reagent and in the absence of the sample and detection reagent (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 invention are described in the '044 application. These include, for example, devices and devices for controlling the movement of liquids 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 liquid flow rate, liquid flow duration, or the number of times a certain amount of liquid is flowed over the binding site. Enable control of.

As mentioned above and as described in more detail below, in some embodiments of the assay system according to the invention, both the inclusion of the sample and the supply of other assay reagents and the area in which the assay itself is performed. The cartridge is used for. Thus, 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 sample and reagents can be flowed. including. As described with respect to FIG. 3, the assay portion includes a plurality of binding sites (containing the capture agent) upon which liquid is flowed, 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 and capture agent can be detected and/or measured, and more typically, a plurality of capture agent printed spots (or dots). 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 the detection/measurement of binding events can take place. If the assay system uses a fluorescence detection device, the excitation beam can pass through the transparent surface to the binding site and excite the fluorescently labeled analyte or analyte-capture agent complex. The luminescence from the fluorescently labeled analyte or complex of analyte and capture agent can then be detected/measured through the same or different transparent surfaces.

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

Imaging is performed from just below the assay solution through the bottom of the cartridge. The assay surface is a printed capture agent (typically a protein or antibody, but in some embodiments hormones, viruses, antibody complexes, antibody fragments, peptides, cells, cell fragments, aptamers, cell lysates. Other biological entities such as substances, DNA, RNA, mRNA, genes, gene expression products, and chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, pollutants, etc. , Which are used for quantitatively measuring analyte concentrations in a fluorescently labeled sandwich-type binding assay.

Fluorescence emitted from the assay at the cartridge surface is collected retrograde to the excitation light path and directed to a photomultiplier tube (PMT) through a series of spatial filters and mirrors. The "scan" in the system is accomplished by a biaxial galvanometer driven mirror assembly and a telecentric lens system that is capable of producing a high resolution image that is time aligned pixels, but other mirrors. And lens systems can also be used. In some embodiments, 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, however, these In other and other embodiments, the assay portion of the cartridge can be smaller than the allowed scan area and can range from 1 mm x 1 mm to 100 mm x 100 mm or more. In some embodiments, there are four assay parts available per cartridge. See, for example, FIGS. However, in other embodiments, the number of assay moieties may range from 1-100.

Fluorescent lasers scan the assay through the underside of the cartridge as the sample is repeatedly flowed during the assay; binding data can be collected over time, a sensitive kinetic binding curve. To be edited. The binding curve for each assay on the cartridge is processed by the analytical software to deliver data on the presence and concentration of the analyte to the user.

System components 1. Detector In some embodiments, a detector can include an excitation light path and an emission light path that are combined to include a detector. The optical path of the excitation light may include a light source, which in some embodiments may be a 532 nm laser tuned by a beam expander and an aperture assembly that sizes the laser light source to collimate the light. Good.

In one embodiment, the light source can 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 fixed mounting of the target sample, thus eliminating the need for post-processing multiple image alignment. The fixed mounting of the sample also allows the coupling of the flow control module to the sample cartridge, which typically eliminates noise in the fluid control system caused by vibrations resulting from the motion of the precision flow system.

In certain embodiments, the detection light path includes a detector (eg, photomultiplier tube (PMT)), bandpass filter, pinhole aperture, focusing lens, dichroic beam splitter and telecentric lens. In such an embodiment, when the sample is excited by a light source, the sample emits photons that are collected by a telecentric lens and retrograde in the excitation path to a dichroic beam splitter. The beam splitter allows the emission wavelengths to pass through the focusing lens, focus at one point, and pass through the pinhole aperture. The pinhole aperture excludes 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 wavelengths of interest to interact with the PMT. The PMT intensity value can then be recorded as a single pixel and can be spatially assigned based on the galvanometer controlled mirror xy position. In this way, an image including pixel location and intensity can be created.

2. Flow Control Module The 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 Figure 2), the microfluidic controller is a computer controlled pressure controller, a three-way valve, pressurized. It can include a sample container, eight inlet valves, four outlet valves, and in some embodiments, a micromanifold that can direct flow from the four outlet valves to a precision flow sensor. The controller can then allow fine timing of flow events up to milliseconds, as well as slower 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 to provide accurate flow and flow rates independent of flow path characteristics. A closed loop flow control system can be created that guarantees 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 valves and four outlet valves, the number of valves being proportional to the number of assay portions contained in the cartridge, eg In certain embodiments, each assay portion requires two inlet valves and one outlet valve (see Figure 3).

3. Assay Cartridge In some embodiments, such as shown in Figure 1, the assay cartridge 100 is a capture agent (typically a protein or antibody, although some embodiments, as described in further detail below). , Hormones, viruses, antibody complexes, antibody fragments, peptides, cells, cell fragments, aptamers, cell lysates, other biological entities such as DNA, RNA, mRNA, genes, gene expression products, and chemicals. Printed with elements, compounds, pharmaceutically active compounds or their chemical entities such as metabolites, minerals, pollutants, etc.) and molded with flow channels and assay moieties (assay cells) And a treated glass surface 20 bonded to a polydimethylsiloxane (PDMS) block 12. PDMS is just one material that can be used and, needless to say, should not be considered as limiting the scope of the invention. Other suitable materials include, for example, chemically treated glass, nanoparticle-coated glass, metal-coated glass, glass treated with other forms of material, carbon (all forms), silicon. , Rubber, plastics, metals, crystals, polymers, semiconductors, organic materials and inorganic materials.

In some embodiments, as shown in FIG. 1, the assay cartridge 100 includes a bottom plate 10 having a transparent portion 14 through which analyte-capture agent interactions can be detected/measured, an assay reservoir coupled to a PDMS block 12. Further included is a mating body 30 and treated glass surface 20, and optionally a top plate 60 on the body 30. 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 invention. Other suitable materials include, for example, PDMS, nanoparticle coated glass, metal coated glass, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastic. , Metals, crystals, polymers, semiconductors, organic materials and inorganic materials.

The 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 portion 30 can further include a transport region 50 having a plurality of channels 52 through which a certain amount of test sample and assay reagents can flow to and from the PDMS block 12 and the treated glass surface 20. obtain.

In some embodiments, the body 30 and PDMS block 12 are coated with molded or otherwise PDMS or other suitable material, such as chemically treated glass, nanoparticle coated glass, metal. Glass, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastics, metals, crystals, polymers, semiconductors, organic and inorganic materials, etc. It can be replaced by the main body/block. In such as well as 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, coated with nanoparticles. Glass, metal-coated glass, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastics, metals, crystals, polymers, semiconductors, organic materials and inorganic materials You can replace them all with one good surface.

In yet a further embodiment, the reservoir may be separate from the assay cartridge and connected to the PDMS block (or other material as described above) through 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 required to form the assay cartridge. ..

FIG. 2 shows a detailed view of PDMS block 12 and treated glass surface 20 of FIG. 1 and other embodiments described above. In this embodiment, there are four assay parts included in the assay cartridge. In this view, the 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 one outlet 28A, 28B, 28C, 28D, and allows for rapid exchange of multiple assay reagents as described further below.

As noted previously, 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 invention. Other suitable materials include, for example, PDMS, nanoparticle coated glass, metal coated glass, glass treated with other forms of material, carbon (all forms), silicon, rubber, plastic. , Metals, crystals, polymers, semiconductors, organic materials and inorganic materials.

As noted previously, in certain embodiments, such as those shown in Figures 1 and 2, the cartridge may comprise four such assay portions (cells), each with a very high number of bindings per cell. Sites (capture spots) are possible (the number of spots per cell can range from 2 to over 20,000). The binding site or capture spot is used herein to describe the area on the surface that contains the 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 method apparent to those skilled in the art. In some embodiments, the assay cartridge may be connected to the flow control system through the multi-pin connection manifold using peak tubing (connecting tubing) to and 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 the fluid control system 200. Only the PDMS block 12 of the cartridge and the treated glass surface 20 are shown for the sake of simplicity.

In this figure, test sample 212 and assay reagent 214 are contained within a pressurized reservoir (introduction well) 210. Opening the first inlet valve 220 allows the test sample 212 to flow to the assay portion of the cartridge (including the PDMS block 12 and treated glass surface 20) and onto the binding site (capture spot) 22. be introduced. The drain valve 230 is then opened to allow the test sample 212 to advance to the waste chamber 240.

The assay reagent 214 can then be introduced into the assay portion by opening the inlet valve 222, allowing the assay reagent 214 to flow over the binding site (capture spot) 22. The assay reagent 214 can then be discharged to the waste chamber 240 as described above. This iterative flushing process can then be repeated multiple times.

In some embodiments after exiting the assay portion, both sample 212 and assay reagent 214 are first flushed past the precision flow sensor and then discharged into waste chamber 240. The controller allows precise timing of flow events up to milliseconds, as well as slower 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 to provide accurate flow and flow rates independent of flow path characteristics. To create a closed loop flow control system that guarantees

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

In other embodiments, the cartridge may incorporate a passive valve and a liquid (sample, labeled detection antibody, buffer solution, etc.) is placed in a reservoir on the cartridge itself to assay the cartridge from the reservoir. The flow of liquid 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 1. Assay Process In one embodiment, the total processing time for each cartridge and associated assay can be 2-200 minutes depending on the analyte concentration detected and measured. Once the sample is injected, the processing of at least one sample and the collection/processing of relevant data over at least one assay can be fully automated. The user only has to select the assay protocol to use (due to the system's software interface) and the system will automatically perform the assay and collect and process all relevant data. ..

An example process is detailed in FIGS. FIG. 4 shows a schematic diagram of the assay portion of an assay cartridge containing multiple binding sites (capture spots) 22, each containing multiple capture agents 23. Some embodiments of the present invention may include multiple capture spots containing different capture agents. In other embodiments, each capture spot 22 may contain the same (and only type of) capture agent, and different capture spots may contain different or the same capture agent, so that the assay portion of the cartridge is 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 blocked with capture agent 23, the sample is flushed over the assay portion of the cartridge A, as shown in FIG. When the sample contains the target analyte (analyte of interest) 70, the analyte 70 binds to the immobilized capture agent 23, but the other analytes 72, 74 in the sample do not bind and the cartridge Over the assay portion of and will flow through it.

As shown in FIG. 6, after a predetermined volume of sample has been flown for a predetermined time (optionally 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 flown for a predetermined time, the sample may be flowed through the assay portion (chamber) again, as shown in FIG. 5, these steps being a circuit that can be repeated any number of times. .. The binding site (capture spot) is imaged (visualized) by the measuring device during these repeating circuits and a representation of the kinetic or dynamic binding curve for the capture agent of the target analyte is drawn.

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

8-10 show the steps of another process according to the present invention capable of amplifying a signal. In FIG. 8, the fluorescently labeled streptavidin 76 binds to the biotinylated capture agent 71. Streptavidin is tetravalent and can bind to 4 molecules of biotin. Also present in the detection reagent cocktail is biotinylated dendrimer 78, which contains 1-8 molecules of biotin/dendrimer and is capable of binding to fluorescently labeled streptavidin 76.

9 and 10 show that the binding of additional fluorescently labeled streptavidin to the biotinylated dendrimer shows a large increase in signal and greatly improved sensitivity and the ability to detect very low levels of at least one target analyte 70. Indicates that it is caused. As the reaction proceeds, multiple layers of fluorescently labeled streptavidin and biotinylated dendrimer are captured in a analyte-dependent manner (ie, directly correlated to the concentration of target analyte 70 in the sample). Can accumulate on top.

Although this description and the related figures briefly describe the whole process as if in separate steps, in reality there are only two steps in the 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, biotinylated detection reagent and biotinylated dendrimer are flushed across the assay surface. If there are immobilized analytes, biotinylated detection reagents will bind to these analytes, and if there is immobilized streptavidin, biotinylated dendrimers will bind to the immobilized streptavidin. These two steps are repeated for a predetermined number of cycles, and the assay surface is visualized at the completion of each step in every cycle. The result of this assay framework is a highly specific and very sensitive sandwich immunoassay capable of specifically detecting 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 scavenger 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 run in a separate experiment or sample. An example experimental design is shown below, however, this is for example purposes only and other experiments that vary the time, sequence and/or presence of some or more of the parameters included below. It should be noted that plans can also be used and are incorporated herein by reference.

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

Device connection and blockage: The test device is removed and a user-specific assay cartridge is placed on the pedestal of the system disclosed herein and the connection manifold is the inlet/outlet of the cartridge (cartridge with four assay portions). Flow inlets and outlets, for example eight inlets and four outlets, which may be more or less). The assay portion is filled with blocking solution and air is expelled in a short (2-15 minutes) filling process. In some embodiments, liquids (samples, reagents, detection antibodies, labels, buffers, etc.) can also be placed directly into the reservoir contained within the disposable assay cartridge, thus eliminating flow tubing.

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

Assay Incubation and Data Collection: The process of flowing sample and/or detection reagents over the assay portion for the desired incubation time. After each incubation time, an imaging event is performed to capture the signal intensity due to binding of the analyte to the capture agent. Many such imaging events occur automatically throughout the assay flow process, drawing a binding curve from multiple measurements of fluorescence over time.

System flush: Performed using a test device and flushing buffer. Prepare the system for the next assay run by removing assay reagents from the flow controller by flushing the system.

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 instantaneously from the PMT at desired time intervals.

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

Analyte Concentration Calculations : Standards of known concentration are used to generate a time course curve of standards specific to the assay. Then, in addition to these known standards, the rate equations and accompanying proprietary algorithms disclosed herein were used to analyze the kinetic curves of the assay processed by each user, thereby Precise measurement of analyte concentration is achieved.

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

4. Detailed Analytical Procedures Analysis of data generated from the systems and methods disclosed herein may involve capturing reagents, which in some embodiments are printed or otherwise arranged in microarray form (which in some embodiments are It is based on measuring the initial binding rate between the target monoclonal antibody) and the analyte of interest or the target analyte (which in some embodiments is the target protein). The quantitative analysis method is based on the measured initial binding rates and back calculation from known standards. Although the initial binding rate is a delta measurement between two time points and is therefore not susceptible to absolute signal intensity results associated with variable background, detection limit determination is a trend of variable background. Affected by. As such, the data can be corrected by simply subtracting the signal intensity from the low signal background region at each image and 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-lapse images are collected at fixed time intervals (which can be anywhere from a few seconds to tens of minutes). Prior to each image, the volume of sample (flowing solution 1), followed by the volume of detection reagent (flowing solution 2) is sequentially 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 certain embodiments, these are microarray spots) are extracted from the image and expressed as a number as the average of several replicates. Over time as the sample concentration remains fixed (by flushing virgin or fresh solution throughout the assay process) and because the surface concentration of capture reagent is relatively high (compared to the analyte concentration in the sample) A linear analysis of the data gives the slope (used directly as the initial velocity in units of signal/time). The 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 imager saturation (outside the PMT) or surface saturation (loss of linearity), linear fitting is only done at earlier time points. At moderate and low concentrations, additional or all time points can be used for linear fitting.

In some embodiments, "non-linear data" is generated by the systems and methods disclosed herein and analyzed as follows. An alternative to the linear data described above 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 the binding of the analyte as in the case of linear data, but then the catalyst, for generating additional signals from the "set" of facilitating reagents. (Described in more detail below). Typically, the set of facilitating reagents comprises two reagents, one contained in reservoir 1 (tube 1) and the other in reservoir 2 (tube 2), where the facilitated signal is in the flow cycle. It is possible to be a measure of the number of iterations and the amplification properties of the facilitating reagent. If the signal for the generated time data is linear it fits as above, if it is non-linear it is a fit to a polynomial equation and the first derivative extracts the slope at a particular analysis time. Used for. Regardless of how long the time course of the binding experiment preceded, any earlier analysis time can be used for the back calculation. Early analysis times typically result in less sensitive results and, as such, are useful for measuring high-concentration analytes simultaneously with low-concentration analytes using two different analysis times. is there.

Standardization and back calculation. As mentioned above, initial velocities are measured for both standards and unknowns. In general, the unknown sample concentration is back calculated from a standard of known concentration. During the development of the systems and methods disclosed herein, analysis of initial rate experiments with varying analyte concentrations showed that the initial rate followed an equilibrium or saturation model where the rate of binding reaction approaches maximum at increased concentrations. It was decided. Although several assay parameters were found to significantly affect maximal rate, maximal rate effectively determines the highest quantifiable dose for any given method (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 equation below known as the Michaelis-Menten model to explain their kinetic properties (V _max is the Represents the maximum velocity, S represents the substrate concentration, and K _m is known as the Michaelis constant).
Speed=(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 is based on the formation of enzyme-substrate complexes that reach equilibrium or saturation. The model also describes the 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 is effective at equilibrium or saturation at higher concentrations. Several other models for reactions that reach saturation or equilibrium have been found to be equally useful in processing 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 and 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)))
Equilibrium model of hyperbola: Fit = (C+(BLmax ^* (l-(x/(Kd+x)))))
Eadie-Hofstee model: Fit = (A+((B ^* x)/((C ^* (D+l))+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 enzyme-type reactions for many years. The most important novelties and breakthroughs described herein are 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) are used to describe.

Such an approach to calculate target analyte concentration from a complex (or single) sample facilitates time-based analysis, which is important for the systems and methods disclosed herein, which comprises: It produces the unique ability of the system to accurately quantify the concentration of different target analytes present in a sample at very different concentrations in a progressive manner. The reason for this is that these target analytes, which are present in very high concentrations, are detected and measured early in the assay process (calculated concentrations) whereas those targets which are present in very low concentrations are present. This is because the analyte is detected and measured (calculated concentration) later in the same assay process (when the system detects binding curves generated from these relatively low concentrations of 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) within the assay described in more detail below.

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

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

The slope, and therefore the analyte concentration, can be determined after the two signal intensities have been measured, but such a determination can be made after the third or subsequent measurement of the signal, in particular the third, It is even more accurate if the fourth, fifth or subsequent signals are at least twice 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) versus the signal of the IL-1b antibody (capture agent) binding to streptavidin. Shown alongside intensity plots (representing simulated non-target binding signals). In this example, in addition to the non-linearity from the third measurement, the first measured rate is significantly outside the calculated V _max for IL-1b. Each of these observations provides a basis for classifying the IL-1b antibody and the associated plot for the streptavidin signal as originating from a non-specific (non-target) binding event to the binding site (and the capture agent there). give.

Alternatively, if the signal is the result of a relatively high concentration of non-specific interactions, it may be observed that the initial velocity is very high at early time points and approaches zero at late time points. Generally, however, the distinction between true and non-true signals is due to the binding curve from a given experiment for the presence and concentration of an unknown analyte and the standard analyte with a known presence and concentration. It is found by comparative analysis with binding curves.

An additional distinction of specific binding events from non-specific binding events (true or false signals) can be made as follows (for the purposes of sandwich-type assays, specific signals are , The target analyte is a signal bridging 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 Cross-linking between the capture agent and the detection reagent or directly adhering to the surface of the assay portion of the cartridge, followed by attachment of the detection reagent and/or facilitating reagents such as biotinylated dendrimers, or streptavidin. Note that the signal is either attached directly to the surface of the cartridge).

Option 1: At the end of the assay run, the sample and detection reagents are exchanged and replaced with assay buffer and assay buffer containing a concentration of capture agent.

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

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

In each of the options detailed above, several cycles of repeated 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 diminished, so in this case it can be assumed that the signal and the binding curve involved are specific signals from the target analyte. However, if the signal is virtually non-specific then it should remain almost stable as it does not depend on the presence of the analyte of interest (target analyte). In such a situation, the signal and associated binding curve can be presumed to be the non-specific signal from the non-target substance in the sample.

Proposed another way, the system disclosed herein detects the signal in real-time, so that at the end of the assay run, a moderate to high concentration of something specific (chosen by above options). Described) can be inserted into the reservoir of the cartridge and system to flow over the assay portion of the cartridge. If a decrease in signal is observed, this is a further indication that the signal is effectively specific (true and analyte dependent), whereas the signal is roughly constant in its intensity. If observed to remain, this indicates that the observed signal is non-specific (pseudo).

Additional differentiation of specific binding events from non-specific binding events (true or false signals) can be done as follows. The analyte or sample and/or detection reagent is simply removed to measure dissociation, rather than adding additional reagents as described above. The subsequently measured kinetic binding signature provides further insight as to whether the signal was generated from a specific binding event or a non-specific binding event. Fast and low signal intensity changes indicate non-specific binding events, while slow and high signal intensity changes indicate specific binding events. This difference in "dissociation rate" between non-specific and specific binding is due to the difference in "association rate" previously described (from higher to lower rather than from lower to higher signal intensities). Reflect to). 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 distinguished from specific (target analyte) binding interactions to further enhance the signal formed by mixing both specific and non-specific components. Additional techniques and methods for adjusting and correcting relate to 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 show rheumatoid arthritis and hypersensitivity. Derived from autoimmune patients such as GI bowel syndrome and similar conditions. These heterophilic antibodies are capable of cross-linking between the capture and detection antibodies, yielding a signal that appears to be analyte-dependent (ie, based on target analyte concentration), but is not.

An antibody (goat, rabbit, mouse, etc.) that is the same species as the capture agent (capture antibody in this example) used in at least one assay but is not directed to any of the target analytes measured in the assay. By including a "negative control spot" containing a mixture of, the system disclosed herein contains a heterophilic antibody as this "negative control spot" produces a signal in a sample-dependent manner. The sample to be detected can be detected. Thus, the signal from this "negative control spot" adapts to the "concentration" of heterophilic antibodies present to adjust the signal in the spot specific for the analyte, and thus of the target analyte concentration. It can be used to provide a more accurate measurement. Moreover, samples containing heterophilic antibodies can be easily identified using this novel method, so that such samples "flag" when they have a higher level of uncertainty in the quantification process. be able to.

6. Promoter Method In some embodiments of the invention, the intentional use of a facilitating reagent to increase assay sensitivity and kinetic range allows non-linear kinetic data to be obtained for a given assay protocol. It can be characteristic. For example, a facilitating reagent that promotes or amplifies the signal intensity resulting from the binding of antigen and capture agent can be performed.

The facilitator typically comprises two reagents and is repeatedly passed over the bound analyte. The first reagent can interact with the analyte and/or the detection reagent and/or other facilitating reagent. The second facilitating reagent interacts with the first facilitating reagent to form a secondary network of facilitator-only reagents, the concentration of which directly reflects the analyte concentration. Examples of these types of reagents are: 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 macromolecule capable of forming an avidin network. More broadly, promoters include: biotinylated proteins, biotinylated antibodies, biotinylated peptides, biotinylated strands of DNA, biotinylated dendrimers, anti-species antibodies, and antibodies and detection species that are captured in an analyte-independent manner. Can include, but is not limited to, any active agent capable of being crosslinked.

This enhancement or amplification of signal intensity is therefore a function of the number of repeating streams of the first and second liquids (sample and reagents). The resulting data, if nonlinear, can be fitted to a polynomial equation, the first derivative of which 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 sample initial velocity or slope during standardization and analysis of unknown samples. In addition, the linear portion of the binding curve can be utilized to calculate the binding rate.

7. Exemplary Assay Parameters The systems and methods disclosed herein allow processing of assays and generation of relevant assay data. Typical assay parameters used to exemplify 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 a confidence of 60-90%. The LDD can be calculated as the initial velocity and subsequently the back-calculated concentration in relation to either the signal above the average background signal by one standard deviation or just above the measured zero dose signal. .. The background signal typically shows a signal obtained from the binding site before the analyte sample and/or the detection reagent flows or even a very low signal intensity even after the analyte sample and/or 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 in some cases 3 times) the observed signal intensity at the lowest non-zero analyte concentration. To be done. The LDD can be verified by running a 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 a confidence of 90-95%. LQD is associated with a signal that is either an initial velocity followed by either two standard deviations above background signal or twice the measured zero dose signal, both of which give the least perceived LQD. It can be calculated as the concentration to be calculated backward. Again, the background signal may be based on the signal obtained from the binding site prior to running the analyte sample and/or the detection reagent or from a region unrelated to the binding site. The standard deviation of the background signal can be calculated from the 5 signal intensities taken at the end of the assay run. The standard deviation is divided by the total incubation time to determine the initial rate and the associated LQD in concentration units. If the zero dose method is used, a value equal to twice the rate observed with the zero dose can be used. Regardless of the method chosen, the LQD is set not to exceed a value that is three times the lowest non-zero standard concentration measured. The LQD can be verified by running a concentration of a known standard on the 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 concentration of standard. Not defined. The HQD can be verified by running a concentration of a known standard on the HQD and calculating the percent recovery.

Dynamic Range : High to low concentration range that can be quantified during the assay. It is defined by HQD as the high end and LQD as the low end.

Data variability/accuracy : Intrinsic variability in signal intensity measured for experiments performed under essentially the same experimental conditions but over different time frames. Intra- and inter-experimental variability (CV) of analyte quantitation is obtained passively by multiple experiments showing LDD, LQD, and HQD. Reagents were kept constant, causing spot-to-spot, experiment-to-experiment (intraday), assay cell-to-cartridge, and day-to-day variations.

Example Experimental Design, Runs and Data This section summarizes one possible approach to perform an assay based on the systems disclosed herein and also incorporates some example data.

Overview : The experimental procedure in this example is referred to herein as "real-time ELISA" or "real-time protein quantification." Real-time ELISA is performed using standard commercial ELISA kits and reagents reconstituted into the systems and methods disclosed herein for comparison reference data, as well as the systems and methods disclosed herein. It is a great explanation because it provides complementary data only available by.

Real-time ELISA method : A typical ELISA kit is complete 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, the ELISA assay involves an additional step in which biotin is further reacted with another enzyme (SA-HRP), and the signal is developed by the catalytic action of HRP on the dye substrate (“enzyme ligation”).

In a real-time ELISA, capture antibody (Reagent A) is printed (arrayed) or otherwise placed on the flow cartridge disclosed herein. Like the ELISA assay, real-time methods use the protein/antigen target (reagent B) as a standard of 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 develop a signal from the captured/measured antigen target. Let In the two flow method, which involves repetitive flow of reagent 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. It is placed in channel 1 (reservoir 1). A repeating flow of channel 1 and channel 2 on the printed capture antibody produces 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, a triplicate assay is used to analyze three human analytes: C-reactive protein (CRP), interleukin-1β (IL-1b), and interferin. Used to test for Leukin-6 (IL-6). Although any other analyte and any number thereof may be used, the three species used herein are included only as examples to exemplify assay processes, systems, methods and devices. A study designed to test the potency and accuracy of the triplicate assay was conducted with the following study design and associated procedure 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 used.

These binding sites include capture agents that are printed or otherwise applied to the assay moieties 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 typical for many, but not all, embodiments, the cartridge is intended to exemplify predictable results when each sample is tested, with positive controls and negative controls. A control was also included. In addition, the control features incorporated into the system ensured that the appropriate reagents were added at near-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 diluent was added to the sample reservoir of the cartridge along with other required buffers, reagents, etc. contained in another reservoir. In some cases, the cartridge can include multiple sample reservoirs, and different dilutions of the sample 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 8 (7 sample dilutions and 1 zero dose) concentrations. In this study, the volume of sample containing unlabeled analyte was flowed first from the sample reservoir portion through the assay portion of the cartridge, followed by the volume of detection reagent containing the detection label. Fluorescent detection labels were used in this study, but as noted previously, 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 flowing the detection reagent, a detection device, here a fluorescence detector, detects the presence of the detection label in the assay portion and the signal intensity is recorded by imaging. As described above and in more detail in the '044 application, as will be apparent to those skilled in the art, fluorescence detectors include 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 standard binding curves for IL-6, CRP, and IL-1b, respectively. Binding curves are fitted to individual signal intensities as indicated. The initial velocity of each binding curve was calculated from the slope of the linear part of the resulting binding curve. If the characteristic binding curve is non-linear, higher order rate equations or polynomial derivatives at a given time can be used to determine the overall rate of reaction. A specific gradient (or initial velocity) was determined for each standard concentration. The initial rates for the subsequent concentration data fitted the equation describing enzyme catalysis, the mode of which was previously disclosed. Following normalization, the unknown slope (or initial velocity) can be back-calculated to a concentration based rate equation and 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。 It is preferred to generate a matching standard curve using at least 3 runs of each concentration standard. The accuracy of the method can be run iteratively and evaluated from either initial velocity, back calculated concentration, or percent recovery. The accuracy of the method can be assessed over the concentration range by looking at the back calculated concentration or percent recovery. Table 1 is a summary of the data from the example experiments.

[Invention Item]
[Item 1]
An assay system comprising a cartridge device and a measuring device, comprising:
The cartridge device is
One or more reservoir portions for holding one or more liquids;
One or more assay parts for receiving the one or more liquids from the one or more reservoir parts, wherein the one or more liquids from the one or more reservoir parts are on top of the assay parts. One or more assay moieties having multiple binding sites that can be repeatedly (two or more times) flowed
Including,
An assay system, wherein the measuring device is for measuring the binding of one or more analytes in the one or more liquids to a plurality of binding sites.
[Item 2]
An interface through which the cartridge device is received and withdrawn to control the flow or movement of the one or more liquids from the one or more reservoir sections through the one or more assay sections. The assay system of item 1, further comprising an interface comprising.
[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 flow of said one or more liquids over said one or more assay parts,
The number of times an amount of the one or more liquids is flowed over the one or more assay portions
The assay system according to item 2, wherein one or more of the above can be independently controlled.
[Item 4]
4. The assay system of item 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 fluorescent labels, luminescent labels, and colorimetric labels,
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 colorimetric measurement device.
[Item 6]
A system in which the measuring device is capable of directing a laser or other form of labeled stimulus, under computer control, 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, which is configured in a system capable of performing detection and/or measurement of.
[Item 7]
Item 1. 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 signals from binding sites in the assay portion of the cartridge device are performed under computer control and the resulting signal is one of the plurality of binding sites in the one or more assay portions. The assay system of item 1, further comprising being used to generate a representation of a dynamic or kinetic binding curve for those analytes that bind to multiple.
[Item 9]
3. The assay system of item 2, wherein the interface provides a variable pressure (positive pressure and/or negative pressure) that controls the flow rate and duration of the one or more liquids over the one or more assay portions.
[Item 10]
An analysis of one or more binding curve representations of one or more analytes that bind to one or more of the plurality of binding sites in the one or more assay moieties is analyzed for the one or more analytes. Item 8 further comprising computer software for comparing the assay 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. The assay system according to.
[Item 11]
The assay system of item 1, wherein the one or more reservoir portions are covered by a thin membrane that encloses the liquid in the reservoir.
[Item 12]
One or more facilitator molecules or entities in the liquid that provide additional binding sites for detection reagents to facilitate and/or amplify the increase in intensity of the fluorescent, luminescent or colorimetric signal The assay system of item 1, wherein the species contains.
[Item 13]
Streptavidin, avidin, dye-labeled streptavidin, avidin; dimerized biotin molecule, PAMAM dendrimer partially or completely labeled with biotin, or biotin-avidin network. In a manner independent of any biotin-containing molecule or macromolecule capable of forming a biotinylated protein, biotinylated antibody, biotinylated peptide, biotinylated strand of DNA, biotinylated dendrimer, anti-species antibody, and analyte. 13. The assay system according to item 12, which is selected from the group consisting of any active substance capable of cross-linking 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 detector reagent, some enhancer bound to some detector reagents. 13. The assay system according to item 12, wherein the enhanced signal effect is provided by providing multiple additional sites to which further detection reagents bind.
[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, gene expression products,
With chemical entities such as chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals or pollutants
The assay system of item 1, which comprises one or more of:
[Item 16]
11. The assay system of paragraph 10, wherein the display of kinetic or kinetic binding curves is used to calculate the concentration of one or more target analytes.
[Item 17]
Display of a kinetic or kinetic binding curve is used to distinguish the signal resulting from the specific interaction between the target analyte and the capture agent within the binding site from the signal resulting from the non-specific interaction, Item 11. The assay system according to Item 10.
[Item 18]
The use of the assay system according to item 15, comprising:
Determination of analyte concentration in a fluid by analysis of time-dependent binding curves and associated velocities was performed using the same test and the same cartridge as the very high concentration of analyte (eg, above 500,000 pg/mL). Ultra low concentrations (eg less than 10 pg/mL) can 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 ultra high concentration analyte binding curve. ), while ultra low concentration analytes are detected/measured later in the assay process (eg, after 30 minutes) when these analyte binding curves are visible in the system.
[Item 19]
A method of calculating an analyte concentration using the assay system of item 15, comprising:
The binding velocity data (initial velocity) is obtained by calculating the slope of the binding curve of each binding site or, in the case of non-linear data, the slope (first derivative or tangent) at a particular analysis time, and then the A method wherein the binding rate data is applied to an enzyme catalyzed reaction equation and compared to rate-based binding curves for known standards developed in advance or simultaneously for these analytes.
[Item 20]
A method of distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the assay system of item 15.
The shape of the binding curve is either the result observed or the signal is the result of a binding event of the specific capture agent to the target analyte or the result of non-specific interaction of the capture agent with one or more non-target analytes. A method used to know what is.
[Item 21]
The distinction between specific and non-specific binding is based on the initial binding rate of the selected binding site (calculated from the first two or three detection/measurement events) from the subsequent detection or measurement events (3 Or after 4 detections or measurements, and/or by comparison with the calculated binding rate (from detection or measurement events after 10-15 minutes), after 3 or 4 events, and/or 10 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 represent non-specific binding.
[Item 22]
A method of distinguishing specific binding from non-specific binding to a capture agent of a target analyte in a binding site using the assay system of item 15.
The method is observed in which the initial velocity is very high at early time points and approaches zero at late time points when the signal is the result of a relatively high concentration of non-specific interactions.
[Item 23]
A method of distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the assay system of item 15.
The distinction between the specific and non-specific binding signals is due to the binding curve from a given experiment with unknown analyte presence and concentration and the standard analyte with known presence and concentration. A method achieved by comparative analysis with a binding curve.
[Item 24]
A method of distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the assay system of item 15.
Replacing the test sample and detection reagent with a first assay buffer and a second assay buffer containing a concentration of capture agent at the end of the assay run, or
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 at the end of the assay run, 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 diminished, in which case the signal and associated binding curve can be presumed to be the specific signal from the target analyte,
If the signal is virtually non-specific then it remains substantially stable as the signal does not depend on the presence of the analyte of interest (target analyte) and the signal and associated binding curve is A method that can be presumed to be a non-specific signal from the non-target substance of.
[Item 25]
A method of distinguishing non-specific binding from specific binding of a target analyte to a capture agent in a binding site using the assay system of item 15.
In the case where the non-specific signal results from the cross-linking of the heterophilic antibody between the capture agent and the detection antibody, it is of the same species as the capture agent (in this example the capture antibody) used in at least one assay but at least 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, And
It is inferred from the observation whether or not the "negative control spot" gives a signal in a sample-dependent manner, which sample contains the heterophilic antibody, and if so, the signal is Considered to contain specific components, or
The signal from the “negative control spot” is used to adjust the signal at the analyte-specific spot (binding site) to take into account the “concentration” of the heterophilic antibody present and thus the target analysis. A method that provides a more accurate measurement of substance concentration.
[Item 26]
A method of discovering new candidate biomarkers for drug development, drug discovery, diagnostics or other uses using the assay system of item 15.
Each binding site is analyzed for specific vs. non-specific binding signals and only those binding sites that give a specific signal are present in additional fractions and/or mass spectrometry or which analytes are present at these binding sites (by The method is further analyzed by another technique that can be used to confirm that it has been captured).
[Item 27]
One of the fluids in which the binding sites contain fractionated cell lysates, tissues or other biological samples and are taken from one or more subjects with a particular disease or condition and processed during the assay. Is from either a healthy control or a subject having the particular disease or condition,
Data from a fluid run assay from a healthy control is compared with data from a fluid run assay from a subject having the particular disease or condition to obtain fluid from the subject having the particular disease or condition. The above binding sites, which show a specific binding signal at, but not a fluid specific binding signal from a healthy control, give rise to subsequent fractionation and/or mass spectrometry or said specific binding signal. 27. The method of item 26, which is further analyzed by any of the other analytical procedures that isolate and identify candidate biomarkers.
[Item 28]
16. The assay system of item 15, wherein the flow rate can be controlled by dynamically adjusting 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]
The laser or other labeling stimulus/interaction process is not working and the user sees the fluid movement to see that the cartridge is properly flowing and free of bubbles or other visible contaminants. 16. The assay system of item 15, wherein the cartridge can be illuminated from the back if it is transparent enough to allow visual confirmation.
[Item 30]
A removable waste container may be incorporated into the system that collects all the fluid processed by the cartridge, with a sensor to inform the user when the waste container should be emptied near full. The assay system according to item 15, which may also be:
[Item 31]
A user with a removable tablet computer that provides instructions for setting up and performing the assay and providing analysis and result 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 both in the user interface and the system itself to guide the user through the steps involved in setting up and performing the assay, eg, the interface presenting a visual representation of the cartridge to the user. Item 16. The assay system according to Item 15, which requests the fluid 1 to be inserted into the reservoir 1 when is illuminated by green (or other color) light or the like.
[Item 33]
Items 15, 28-32, wherein the assay system according to any one of items 15, 28-32 is used for detection and measurement of diagnostic biomarkers in a laboratory, near care or in a care setting. Use of the assay system according to any one of 32.
[Item 34]
A receptacle 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 cartridge for a fluid 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 fluorescent, luminescent or colorimetric labels and the cartridge is further capable of transferring one or more fluids from one compartment of the cartridge to another compartment or compartments under computer control. A cartridge comprising fluid channels, the assay portion of the cartridge comprising an array of two or more binding sites each containing one or more specific capture agents;
A device for measuring the intensity of a fluorescent or luminescent or colorimetric signal in time series at each such site, interacting with and/or stimulating a fluorescent or luminescent or colorimetric label,
Device for incorporating such a time series of measurements to create a display of dynamic or kinetic binding curves between non-target substances/molecules/analytes and capture agents or other substances in the binding site When
A system equipped with.
[Item 35]
The reservoir is covered by a membrane that seals the fluid in the reservoir, and the membrane can be pierced by a hypodermic needle or similar device to add or withdraw fluid from the reservoir, Item 34. The system according to Item 34.
[Item 36]
One of the fluids contains one or more facilitator molecules or entities to provide additional binding sites for the detection reagent to facilitate increased intensity of fluorescence, luminescence or colorimetric signals, and/or A system according to item 34, which facilitates amplification.
[Item 37]
The one or more promoter molecules or entities are streptavidin, avidin, a dye standard type 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 macromolecule capable of forming a biotinylated protein, biotinylated antibody, biotinylated peptide, biotinylated strand of DNA, biotinylated dendrimer, anti-species antibody, and analyte. 37. The system according to item 36, which is selected from the group comprising any active substance capable of cross-linking the captured active substance and 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 detector reagent, some enhancer bound to some detector reagents. 38. The system of paragraphs 36 and 37, thus providing a multitude of additional sites to which further detection reagents bind to provide an enhanced signal effect.
[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, 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 34-38, containing one or more of:
[Item 40]
40. The system of item 39, wherein the display of kinetic or kinetic binding curves is used to calculate the concentration of one or more target analytes.
[Item 41]
Display of a kinetic or kinetic binding curve is used to distinguish the signal resulting from the specific interaction between the target analyte and the capture agent in the binding site from the signal resulting from the non-specific interaction, Item 39. The system according to Item 39.
[Item 42]
Using the system according to item 39,
Determination of analyte concentration in a fluid by analysis of time-dependent binding curves and associated velocities was performed using the same test and the same cartridge as the very high concentration of analyte (eg, above 500,000 pg/mL). Ultra low concentrations (eg less than 10 pg/mL) can 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 ultra high concentration analyte binding curve. ), while ultra low concentration analytes are detected/measured later in the assay process (eg, after 30 minutes) when these analyte binding curves are visible in the system.
[Item 43]
A method of calculating an analyte concentration using the system of item 39, comprising:
The binding velocity data (initial velocity) is obtained by calculating the slope of the binding curve of each binding site or, in the case of non-linear data, the slope (first derivative or tangent) at a particular analysis time, and then the A method wherein data is applied to an enzyme catalyzed reaction equation and compared to rate-based binding curves for known standards developed in advance or simultaneously for these analytes.
[Item 44]
A method of distinguishing non-specific binding from specific binding of a target analyte to a capture agent in the 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 a specific capture agent is the result of a binding event for 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 that the initial binding rate of the selected binding site (calculated from the first two or three detection or measurement events) is calculated from the subsequent detection or measurement events (3 Or after 4 and/or 10-15 minutes after detection/measurement events) by comparing with the calculated binding rate, after 3 or 4 events, and/or after 10-15 minutes 45. The method of paragraph 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 to a capture agent of a target analyte in a binding site using the system according to item 39,
The method is observed in which the initial velocity is very high at early time points and approaches zero at late time points when the signal is the result of a relatively high concentration of non-specific interactions.
[Item 47]
A method of 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 true and non-true signals is unknown. The method is accomplished by comparative analysis of the binding curve from a given experiment with the presence and concentration of the analyte in question and the binding curve of a standard analyte with the known presence and concentration.
[Item 48]
A method for distinguishing non-specific binding from specific binding to a capture agent of a target analyte in a binding site using the system according to item 39,
Replacing the test sample and detection reagent with a first assay buffer and a second assay buffer containing a concentration of capture agent at the end of the assay run, or
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 at the end of the assay run, 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 diminished, in which case the signal and associated binding curve can be presumed to be the specific signal from the target analyte,
If the signal is virtually non-specific then it remains substantially stable as the signal does not depend on the presence of the analyte of interest (target analyte) and the signal and associated binding curve is A method that can be presumed to be a non-specific signal from the non-target substance of.
[Item 49]
A method for distinguishing non-specific binding from specific binding to a capture agent of a target analyte in a binding site using the system according to item 39,
The same species as the capture agent (in this example the capture antibody) used in at least one assay, but at least one if the non-specific signal results from the heterophilic antibody cross-linking of the capture and detection antibodies. 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. ,
It is inferred from the observation whether or not the "negative control spot" gives a signal in a sample-dependent manner, which sample contains the heterophilic antibody, and if so, the signal is Considered to contain specific components, or
The signal from the “negative control spot” is used to adjust the signal at the analyte-specific spot (binding site) to take into account the “concentration” of the heterophilic antibody present and thus the target analysis. A method that provides a more accurate measurement of substance concentration.
[Item 50]
A method for discovering new candidate biomarkers for drug development, drug discovery, diagnostics or other uses using the system according to item 39,
Each binding site is analyzed for specific vs. non-specific binding signals and only those binding sites that give a specific signal are present in additional fractions and/or mass spectrometry or which analytes are present at these binding sites (by The method is further analyzed by another technique that can be used to confirm that it has been captured).
[Item 51]
One of the fluids in which the binding sites contain fractionated cell lysates, tissues or other biological samples and are taken from one or more subjects with a particular disease or condition and processed during the assay. Is from either a healthy control or a subject having the particular disease or condition,
Data from a fluid run assay from a healthy control is compared with data from a fluid run assay from a subject having the particular disease or condition to obtain fluid from the subject having the particular disease or condition. The above binding sites, which show a specific binding signal at, but not a fluid specific binding signal from a healthy control, give rise to subsequent fractionation and/or mass spectrometry or said specific binding signal. The method of item 50, which is further analyzed by any of the other analytical procedures for isolating and identifying candidate biomarkers.
[Item 52]
40. The system of item 39, wherein the pressure can be controlled by monitoring the flow rate of fluid exiting the cartridge and by feedback establishing the pressure required to set the desired flow rate.
[Item 53]
The laser or other labeling stimulus/interaction process is not working and the user sees the fluid movement to see that the cartridge is properly flowing and free of bubbles or other visible contaminants. 40. The system of item 39, wherein the cartridge can be illuminated from the back if it is sufficiently transparent to allow visual confirmation.
[Item 54]
40. The system of item 39, wherein a removable waste container may be incorporated into the system to collect all the fluid processed by the cartridge.
[Item 55]
A user with a removable tablet computer that provides instructions for setting up and performing the assay and providing analysis and result details 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 both in the user interface and the system itself to guide the user through the steps involved in setting up and performing the assay, eg, the interface presenting a visual representation of the cartridge to the user. 40. A system according to item 39, wherein the system requests the fluid 1 to be inserted into the reservoir 1 when is illuminated by green (or other color) light or the like.
[Item 57]
Items 39 and 52-56, wherein the system according to any one of items 39 and 52-56 is used for 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.
[Item 58]
A method of distinguishing between specific and non-specific binding in an assay, comprising:
Each of the plurality of signal intensity measurements is made during repeated interactions of the sample to be analyzed with a capture agent of the target analyte in the sample to be analyzed, and the plurality of signal intensity measurements of the sample to be analyzed To get
Plotting multiple signal intensity measurements as a function of cumulative duration of sample-capture agent interaction analyzed.
Determining that the signal intensity measurements indicate specific binding between the target analyte and the capture agent if the signal intensity measurements plotted are characteristic of a known standard.
Including the method.
[Item 59]
A method of calculating an analyte concentration in a sample containing an analyte, comprising:
A step of obtaining a plurality of signal intensity measurements representative of binding of a component of a sample to a capture agent capable of binding to an analyte, the plurality of signal intensity measurements being at known time intervals between the sample and the capture agent. The steps performed during a known duration of interaction,
Plotting multiple signal intensity measurements as a function of cumulative duration of sample-capture agent interaction;
Calculating one or more first derivatives or tangents (initial velocities) for the plotted signal strength measurements, each of the one or more first derivatives or tangents (initial velocities) being plotted in close proximity. And the steps calculated using the measured signal strength
Including,
The first derivative or tangent (initial velocity) for one or more standard analyte concentrations can later be used to determine the fit of the back curve and the enzyme substrate complex is formed prior to the reaction. In some cases, the back-calculation fit is based on an enzyme-catalyst model and the first derivative or tangent (initial velocity) of an unknown sample is used to back-calculate the concentration 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 moieties for receiving one or more fluids from the one or more reservoir moieties, having a plurality of binding sites over which fluids are flowed, the fluid channels or tubing. One or more assay parts connected to said one or more reservoir parts through
A cartridge device comprising.
[Item 61]
61. The cartridge device of item 60, further comprising a channel that provides an outlet for fluid to enter the assay portion without leaking fluid.
[Item 62]
Peripheries 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 that forms a hermetic seal around the portion.
[Item 63]
One or more attachment points for a mechanism that can vary the pressure exerted on each of the reservoir portions to allow for controlled and repetitive flow of fluid from the reservoir portions through the assay portion of the cartridge device. 63. The cartridge device of item 62, further comprising:
[Item 64]
61. The cartridge of item 60, further comprising in the assay portion of the cartridge device a plurality of binding sites containing a selected capture agent specific for selecting a target analyte from a fluid to be analyzed. device.
[Item 65]
A controlled, repetitive flow of fluid across the binding site can be used to form an indication of the kinetic binding curve of these analytes in the fluid that binds these capture agents in the binding site. 60. The cartridge device according to item 60.
[Item 66]
The cartridge device is
A base portion on which a binding site containing a capture agent can be located;
An upper or body portion of the cartridge device comprising the reservoir portion, the fluid channel and the assay portion;
Item 61. The cartridge device of Item 60, comprising:
[Item 67]
The base is made of the following materials:
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, plastics, metals , Crystals, polymers, semiconductors, organic materials or inorganic materials
67. The cartridge device of item 66, which comprises one or more of:
[Item 68]
The upper part or the main body part of the cartridge device is made of the following materials:
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, plastics, metals Item 67. The cartridge device of Item 66, which comprises one or more of: crystals, polymers, semiconductors, organic materials or inorganic materials.
[Item 69]
The base is hydrophobic,
The base comprises an array of binding sites containing the desired capture agent or active agent,
The base portion comprising the array of binding sites is plasma etched,
The body is composed of a molded material or a material constructed to include the fluid channel, the assay portion and the reservoir portion,
The body portion is joined to the base portion by adjusting its position,
67. The cartridge device of claim 66.
[Item 70]
The hermetic seal around the upper perimeter of each of the reservoir portions is achieved by a raised ring placed over the perimeter of the perimeter, which seal is of any gas or other form of pressure. 63. The cartridge device of item 62, which mates with an interface device that provides pressure to the top of the reservoir portion having a seal that prevents fluid leakage.
[Item 71]
Further comprising a membrane overlying and sealing the top of each of said reservoir portions,
The membrane can be pre-filled with fluid without leaking into the cartridge device, and the fluid can be easily inserted or removed during any storage by a hypodermic needled or similar device. Seal around the perimeter of the reservoir to allow fluid flow, and to allow the fluid to flow under the control of applied pressure when the cartridge device is inserted into the system. 60. The cartridge device according to item 60.
[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 that does not interfere with the detection and measurement of binding events during the assay, Allows the user to see the movement of fluid through the cartridge device, visually determine that there is momentum in the flow, and observe any potential irregularities such as bubbles or other contaminants Item 60. The cartridge device according to Item 60.
[Item 73]
The flow rate of the liquid through the cartridge device is either behind the assay portion of the cartridge device or behind the outlet of the cartridge device, placed in the system in which the cartridge device is inserted or otherwise connected. 61. A 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 have a height of 150 microns or less, and preferably 50 microns or less.
[Item 75]
61. The cartridge device of item 60, wherein the assay portion produces a turbulent flow of fluid.

Claims (13)

An assay system comprising a cartridge device, comprising:
The cartridge device is
A pressurized sample reservoir,
A sample reservoir,
A membrane covering the sample reservoir,
A ring surrounding the periphery of the sample reservoir,
An interface device that engages with the ring,
A reservoir of pressurized sample having
A pressurized reagent reservoir,
A reagent reservoir,
A membrane covering the reagent reservoir,
A ring surrounding the periphery of the reagent reservoir,
An interface device that engages with the ring,
A reservoir of pressurized reagent having a
An assay portion for receiving liquid from the pressurized sample reservoir,
An inlet connected to the pressurized sample reservoir for introducing the liquid into the assay portion;
A drain for draining the liquid from the assay portion,
An assay part having a plurality of binding sites provided between the introducing part and the discharging part, wherein the liquid can be repeatedly flown from the introducing part to the discharging part via the binding sites. When,
A measuring device for measuring the binding of the one or more analytes in the liquid to the binding sites, after each of the flows of the liquid from the inlet to the outlet. Including an assay system. An interface through which the cartridge device is received and removed, further comprising an interface for controlling the flow or movement of the liquid from the pressurized sample reservoir through the assay portion;
The device for controlling the flow of the liquid comprises at least one of the following: a flow rate of the liquid across the assay portion,
The duration of the flow of the liquid across the assay portion, and
The assay system of claim 1, wherein at least one of a number of times a quantity of the liquid is flowed through the assay portion can be independently controlled. Further equipped with a flow velocity sensor,
The interface provides a variable positive pressure and/or a negative pressure to control the flow rate and/or duration of the liquid across the assay portion based on the output from the flow rate sensor. The assay system according to claim 2. The liquid contains one or more labels selected from the group consisting of fluorescent labels, luminescent labels, and colorimetric labels,
The assay system according to claim 1, wherein the measuring device is selected from the group consisting of a fluorescence measuring device, a luminescence measuring device, and a colorimetric measuring device. Such that the measuring device, under computer control, can direct a laser or other form of labeled stimulus to the assay portion of the cartridge device, and subsequently detect fluorescence, luminescence or colorimetric signals and/or Alternatively, the assay system according to claim 4, which is configured in the assay system so that the measurement can be performed. With additional computer software,
When the computer software is executed,
Analyzing the display of one or more binding curves of one or more analytes that bind 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,
2. The assay system of claim 1, operative to determine the presence and/or concentration of the one or more analytes in the liquid. 2. The assay system of claim 1, wherein the pressurized sample reservoir is covered by a thin membrane enclosing the liquid in the pressurized sample reservoir. If the liquid is
Streptavidin, streptavidin species containing avidin and dye-labeled streptavidin,
Dimerized biotin molecule, PAMAM (polyamidoamine) dendrimer partially or completely labeled with biotin, or any biotin-containing molecule or macromolecule capable of forming a biotin-avidin network, biotinylated protein, biotinylated antibody, biotin Biotinylated species, including biotinylated peptides, biotinylated strands of DNA, and biotinylated dendrimers, or
The assay system of claim 1, comprising at least one of any active substance and anti-species antibody capable of cross-linking the captured active substance and the detection reagent in an analyte independent manner. 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,
10. The assay system of claim 9, wherein the chemical entity is selected from the group consisting of chemical elements, compounds, pharmaceutically active compounds or their metabolites, minerals, and contaminants. The assay system of claim 1, wherein the cartridge device further comprises a waste chamber connected to the outlet. A pressurized sample reservoir,
A sample reservoir,
A membrane covering the sample reservoir,
A ring surrounding the periphery of the sample reservoir,
An interface device that engages with the ring,
A pressurized sample reservoir,
A reservoir of pressurized reagent,
A reagent reservoir,
A membrane covering the reagent reservoir,
A ring surrounding the periphery of the reagent reservoir,
An interface device that engages with the ring,
A reservoir of pressurized reagent having a
An assay portion for receiving fluid from the pressurized sample reservoir, comprising:
An inlet connected to the pressurized sample reservoir for introducing the fluid into the assay portion;
A drainage for draining the fluid from the assay portion;
An assay part having a plurality of binding sites provided between the introduction part and the discharge part, the binding site being capable of repeatedly flowing the fluid from the introduction part to the discharge part via the binding sites. When,
A cartridge device comprising. 13. The cartridge device of claim 12, further comprising a waste chamber connected to the outlet.

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