Decoding methods for multiplexed assays and related fluidic devices, kits and solid supports

Abstract

Decoding methods for identifying populations in assays, particularly multiplexed assays and those associated with fluidic devices, are provided. Populations of beads are encoded with different signals (e.g., dyes with different light stability). Changes, such as photobleaching, allow for differentiating different populations in kinetic assays that measure fluorescence decay.

Description

[0001] This application is a divisional application of Chinese patent application No. 202110961051.0, whose application date is October 5, 2016 and whose invention name is “Decoding method and related fluid equipment, kit and solid support for multiplex determination”.

[0002] Federal Support Statement

[0003] This invention was made with Government support under Grant No. HR0011-12-2-0001 awarded by the U.S. Department of Defense (DARPA). Field of the Invention

[0004] The present invention relates to sample analysis and may be adapted therefrom for analysis performed using a fluidic device. background

[0006] Being able to measure low-abundance analytes from biological samples is very important for several fields including clinical diagnosis. See, for example, Rissin et al., Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat. Biotechnol. 2010, 28, 595-599.; Walt, DR, Optical Methods for Single Molecule Detection and Analysis, Anal. Chem. (Washington, DC, US) 2013, 85, 1258-1263; and Witters et al., Digital microfluidics-enabled single-molecule detection by printing and sealing single magnetic beads in femtoliter droplets, Lab Chip 2013, 13, 2047-2054.

[0007] Many protein and nucleic acid diagnostic biomarkers exist at low endogenous concentrations, which requires analytical methods with very low detection limits. Increasingly, a common strategy for making such sensitive measurements is to use beads functionalized with biorecognition elements (e.g., antibodies, oligonucleotides, etc.). See, Rissin et al., Simultaneous Detection of SingleMolecules and Singulated Ensembles of Molecules Enables Immunoassays withBroad Dynamic Range, Anal. Chem. (Washington, DC, US) 2011, 83, 2279-2285; Song et al., Direct Detection of Bacterial Genomic DNA at Sub-FemtomolarConcentrations Using Single Molecule Arrays, Anal. Chem. (Washington, DC, US) 2013, 85, 1932-1939; Woolley et al., Emerging technologies for biomedical analysis, Analyst (Cambridge, UK) 2014, 139, 2277-2288; and Culbertson et al., Micro Total Analysis Systems: Fundamental Advances and Biological Applications, Anal. Chem. (Washington, DC, US) 2014, 86, 95-118.

[0008] Adding thousands to millions of microspheres per sample provides a high surface area for target capture, thereby effectively and selectively pre-concentrating target analytes from large sample volumes. See, Chang et al., Single molecule enzyme-linked immunosorbent assays: Theoretical considerations, J. Immunol. Methods 2012, 378, 102-115; and Walt, D. R, Protein measurements in microwells, Lab Chip 2014, 14, 3195-3200.

[0009] Although this method is conducive to measuring multiple individual analytes, the key advantage of this bead-based method is the ability to multiplex and quantify multiple targets from a single sample. See, Rissin et al., Multiplexed singlemolecule immunoassays, Lab Chip 2013, 13, 2902-2911; and Kingsmore, SF, Multiplexed protein measurement: technologies and applications of protein andantibody arrays, Nat. Rev. Drug Discovery 2006, 5, 310-320. This is particularly valuable in biological testing applications (e.g., clinical and point-of-care testing (POC) diagnostics), where a group of disease biomarkers can be screened in a single test. See, Nie et al., An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples, Lab Chip2014, 14, 1087-1098; Shen et al., Multiplexed Quantification of Nucleic Acids with Large Dynamic Range Using Multivolume Digital RT-PCR on a Rotational SlipChipTested with HIV and Hepatitis C Viral Load, Journal of the American ChemicalSociety 2011, 133, 17705-17712; and Chin et al. Commercialization of microfluidicpoint-of-care diagnostic devices, Lab Chip 2012, 12, 2118-2134.

[0010] Typical bead-based multiplex diagnostic methods encode multiple types of affinity beads with different combinations of dyes at different intensity levels. See, Manesse et al., Dynamic microbead arrays for biosensing applications. Lab Chip 2013, 13, 2153-2160; and Nie et al., Multiplexed Salivary Protein Profiling for Patients with Respiratory Diseases Using Fiber-Optic Bundles and Fluorescent Antibody-Based Microarrays. Anal. Chem. (Washington, DC, US) 2013, 85, 9272-9280.

[0011] The beads can be decoded by acquiring images with the excitation / emission wavelengths suitable for each coding dye used. See, Rissin et al., Multiplexed single molecule immunoassays. Lab Chip 2013, 13, 2902-2911. The beads are then assigned to a population based on a predetermined fluorescence emission intensity threshold. See, for example, Nie et al., Multiplexed Salivary Protein Profiling for Patients with Respiratory Diseases Using Fiber-Optic Bundles and Fluorescent Antibody-Based Microarrays, Anal. Chem. (Washington, DC, US) 2013, 85, 9272-9280. Although the method can produce several populations of coded beads, it may require the spectral properties of different coding dyes and each intensity level to be well resolved to ensure that each bead population can be distinguished from each other. Therefore, in the case of the method, the number of multiplex levels that can be used has a practical upper limit, even when using specialized equipment such as flow cytometers. There remains a need for alternative encoding / decoding systems that may allow for increased multiplexing and / or detection speeds.

[0012] The contents of the documents cited in the above Background section of this application are hereby incorporated by reference in their entirety.

[0013] Summary of Embodiments of the Invention

[0014] Embodiments of the present invention provide decoding measures for encoded devices that can provide additional levels of multipath that are believed to be unachievable through conventional encoding methods.

[0015] In contrast to using only a single time point encoding measurement (as conventional decoding measurements usually do), embodiments of the present application provide methods that utilize the time domain to obtain multiple decoding measurements taken at different times to distinguish and / or identify (decode) different populations. The decoding measurements can be based on encoding media (e.g., dyes), such as, for example, encoding media with similar spectral properties.

[0016] The coded media can have similar spectral properties so that they are indistinguishable in the first image (e.g., before a defined event), and can include stable coded media (e.g., producing the same fluorescence intensity at different time points before and after a defined event) and coded media that physically and / or chemically change in response to a defined event (e.g., displaying increased or decreased fluorescence intensity at different time points after a defined event compared to the fluorescence intensity before the defined event and / or being selectively activated).

[0017] Encoded media may include stable, partially stable, relatively stable, and unstable media.

[0018] Some or all of the coded media may contain similar spectral properties such that they may be indistinguishable in the first image and unstable coded media may produce decreased or increased fluorescence intensity due to physical and / or chemical changes, which may be caused by exposure to a defined event (e.g., thermal, optical, chemical, magnetic and / or electrochemical events).

[0019] Decoding measurements can be performed at different time points, including before, during and / or after a defined event that causes a physical and / or chemical change associated with the corresponding solid support (which may be all or part thereof), which may result in a change in the coded signal of the corresponding solid support (which may be all or part thereof) (e.g., the luminescence intensity generated by each solid matrix (which may be all or part thereof) having at least one coded medium may change). In some embodiments, the luminescence intensity can be measured, which may include fluorescence and / or phosphorescence intensity. Other decoding measurements of solid supports include detecting changes in magnetic behavior, pH, light scattering, physical size, shape, refractive index, solubility, light absorption and / or emission intensity or maximum wavelength shift, conductivity, dielectric constant, viscosity and radioactive decay events, and combinations thereof.

[0020] For example, in some embodiments, solid supports such as, but not limited to, beads, can be encoded with a coding medium (e.g., a dye or dyes), and in the case of using more than one coding medium, the coding medium can have similar spectral properties (e.g., similar initial spectral properties), but different stability. Although providing conventional decoding methods is indistinguishable, the method of the present invention can allow different populations of solid supports to be distinguished. In some embodiments, in order to identify at least two different populations, at least two different coding media can be used. Alternatively or in addition, in some embodiments, a coding medium (e.g., the same dye and / or active part) can be used to identify at least two different populations, but for at least one population, the coding medium, in response to a defined event, provides a coding signal different from the other so that the population can be identified, which can be, for example, that the coding medium includes an element that does not exist in the other and is selectively activated or changed in response to a defined event. For example, the coding medium for two different populations can include the same dye, but it can be connected to the solid support in different ways and a connection method can include a combination destroyed by a defined event, which changes the fluorescence intensity of the coding medium or the solid support.

[0021] The solid supports of the present invention can generate many more different encoding states than can be achieved using static single time point measurements.

[0022] The methods of the present invention can identify increased populations using additional coded signals provided by physical and / or chemical changes associated with the solid support, which changes can result in changes in the coded signal of the corresponding solid support and / or a portion thereof over time, typically based on defined events, such as, for example, defined events that result in changes in the chemical and / or physical properties of the encoding medium and / or solid matrix.

[0023] The added multiplexing capability may not require additional filter sets or other expensive optical instrumentation, which can provide a low-cost, relatively simple analysis platform.

[0024] While there are many ways to perform time-dependent measurements in and / or on a solid matrix (eg, beads), photostability and / or thermal stability may be parameters particularly suitable for development to identify multiplexed beads.

[0025] In some embodiments, the first and second coded signals of corresponding solid supports (which can be all or part thereof) can be obtained and analyzed to identify different colonies. In some embodiments, the first coded signal can be based on and / or obtained from the first image, wherein different solid supports are in different positions. The second coded signal can be based on and / or obtained from the second image, wherein different solid supports are in corresponding (same) positions. In some embodiments, the first coded signal can be based on and / or obtained from the first image, wherein the coded molecular recognition elements are in different positions on and / or in the sample on the solid support, and the second coded signal can be based on and / or obtained from the second image, wherein different coded molecular recognition elements are in corresponding (same) positions. The actual number of different coded signals in the first and second images can be the same. However, the coded signal of the corresponding solid support can vary over time (for example, migrate from one level to another level, such as high to low, or vice versa), which can increase the number of detectable coded states.

[0026] For example, in some embodiments, the analysis system can be configured to detect the same number of defined intensity levels between the first and second images, such as, for example, three (3) intensity levels (low, medium, high). However, due to changes in intensity levels between the first and second images of at least some of the populations, additional populations can be defined based on the population that remains unchanged and the population that changes (1. Initial low intensity and remains at low intensity, 2. Initial medium intensity and fades to low intensity, 3. Initial medium intensity and remains at medium intensity, 4. Initial high intensity and fades to low intensity, 5. Initial high intensity and fades to medium intensity, 6. Initial high intensity and remains at high intensity).

[0027] In addition, using the ability to become brighter when the quencher is eliminated or the sequestered dye is released, the analysis system can be configured to detect all of 1-6 above, plus 7. Initial low intensity increases to medium intensity, 8. Initial low intensity increases to high intensity, 9. Initial medium intensity increases to high intensity. That is, 9 populations are generated from only 3 intensity levels. If a second dye set (e.g., a coded medium) is used, 81 populations can be generated.

[0028] Other encoding signals may be used, including physical and / or chemical changes of the solid substrate as discussed further below.

[0029] In some embodiments, a method of decoding a solid support is provided, the method comprising: detecting a first coded signal and a second coded signal of a solid support in a plurality of solid supports (e.g., a multiplex bead set); comparing the first coded signal and the second coded signal of a corresponding solid support in the plurality of solid supports, wherein the second coded signal of the corresponding solid support is detected during or after at least one chemical and / or physical change associated with the solid support and the first and second coded signals of the corresponding solid support are different; and decoding the plurality of solid supports based at least in part on the comparison of the first and second coded signals.

[0030] In some embodiments, a method for decoding a solid support is provided, the method comprising providing a plurality of molecular recognition elements (such as, for example, antibodies, aptamers, and / or nucleic acid probes), wherein at least some of the plurality of molecular recognition elements include one or more encoding media; binding at least a portion of the plurality of molecular recognition elements to at least a portion of the sample on the solid support; detecting a first encoding signal and a second encoding signal of a molecular recognition element in the plurality of molecular recognition elements; comparing a first encoding signal and a second encoding signal of a corresponding molecular recognition element in the plurality of molecular recognition elements, wherein the second encoding signal is detected during or after at least one chemical and / or physical change associated with the molecular recognition element and the first and second encoding signals of the corresponding molecular recognition element are different; and decoding a plurality of molecular recognition elements based at least in part on the comparison of the first and second encoding signals. The solid support can be a microscope slide carrying a biological sample (such as, for example, a tissue section). In some embodiments, the solid support can be a glass, silicon or plastic slide carrying an array of proteins and / or nucleic acids involved in a direct or sandwich assay.

[0031] Some embodiments include a method for decoding a sample, the method comprising detecting a first encoding signal and a second encoding signal of an encoded element (e.g., an encoded solid support and / or an encoded molecular recognition element) among a plurality of encoded elements; comparing the first encoding signal and the second encoding signal of corresponding encoded elements among the plurality of encoded elements, wherein the second encoding signal of the corresponding encoded element is detected during or after at least one chemical and / or physical change associated with the encoded element and the first and second encoding signals of the corresponding encoded element are different; and decoding the plurality of encoded elements based at least in part on the comparison of the first and second encoding signals.

[0032] Other embodiments relate to analysis systems. A determination decoding system can be provided, comprising a circuit comprising at least one processor; a fluid analysis chip comprising a plurality of solid supports (e.g., a multiplex bead set); and a source in communication with a plurality of solid supports, wherein the circuit activates the source to cause at least one detectable physical and / or chemical change associated with at least some of the plurality of solid supports and compares the first and second coded signals of the corresponding solid supports in the plurality of solid supports to decode the solid supports of different populations in the plurality of solid supports. The source may include at least one of a heat source or a light source in communication with a plurality of solid supports, wherein the circuit activates at least one of the heat source or the light source to cause at least one detectable physical and / or chemical change associated with at least some of the plurality of solid supports.

[0033] In some embodiments, a system may include a controller, a detector connected to the controller, an image processor having a module configured to identify signals present in a signal detection section of a reaction well of a device having reaction wells at different time points for a single sample, and a dynamic decoding module.

[0034] A reaction well may have at least one bead retention section.

[0035] The module may be configured to identify a positive assay signal after the reaction and thereafter before and after at least one defined event.

[0036] The modules can be configured to perform multiplex bead decoding based on identification of positive assay signals after a reaction event and before and after a defining event.

[0037] The centerlines of the bead wells holding the individual beads can be spaced at distances ranging from 0.2 to 1000 µm apart.

[0038] Further aspects of the invention include a plurality of solid supports (e.g., multiplexed bead sets) comprising a first population of solid supports comprising a first concentration and / or ratio of at least one encoding medium and a second population of solid supports comprising a second concentration and / or ratio of the at least one encoding medium and at least one additional encoding medium, wherein the fluorescence excitation and emission wavelengths of the at least one encoding medium and the at least one additional encoding medium overlap, and wherein the first and second concentrations of the at least one encoding medium are different and the different concentrations and / or ratios of the at least one encoding medium on the respective solid supports in the first and second populations provide a plurality of encoding states.

[0039] Another aspect of the present invention includes a plurality of encoded molecular recognition elements, wherein the plurality of encoded molecular recognition elements includes a first population of molecular recognition elements comprising a first concentration and / or ratio of at least one encoding medium; and a second population of molecular recognition elements comprising a second concentration and / or ratio of at least one encoding medium and at least one additional encoding medium, wherein the fluorescence excitation and emission wavelengths of the at least one encoding medium and the at least one additional encoding medium overlap, and wherein the first and second concentrations of the at least one encoding medium are different and the different concentrations and / or ratios of the at least one encoding medium on the corresponding molecular recognition elements in the first and second populations provide multiple encoding states.

[0040] In some embodiments, the encoded molecular recognition element comprises two or more (eg, 2, 3, 4 or more) encoding media.

[0041] Another aspect of the invention relates to a kit comprising a plurality of solid supports as described herein.

[0042] It should be noted that, although not specifically described, the aspects of the present invention described in one embodiment can be combined in different embodiments. That is, all embodiments and / or the features of any embodiment can be combined in any manner and / or combination. The applicant reserves the right to change any originally submitted claims and / or submit any new claims, and therefore includes the right to modify any originally submitted claims to make them subordinate to any other claims and / or combine any features of any other claims (although not initially claimed in the manner described). These and other objects and / or aspects of the present invention are described in detail in the following specification. Those skilled in the art will understand the additional features, advantages and details of the present invention after reading the subsequent drawings and preferred embodiment details (such description is only an explanation of the present invention). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0045] Figure 1A is a schematic diagram of an analysis system according to an embodiment of the present invention.

[0046] Figure 1B is a schematic diagram of another analytical system according to an embodiment of the present invention.

[0047] Figure 1Cis a schematic diagram of a timing diagram of acquired images according to an embodiment of the present invention, showing an image I1 at a first time before a defined event ("DE") and an image I2 at a second time T2 after the defined event (DE).

[0048] Figure 1D is another schematic diagram of a timing diagram of acquired images according to an embodiment of the present invention, showing image I1 at a first time T2 before a defined event ("DE") and image I2 at a second time T2 after the defined event (DE).

[0049] Figure 2A is a graph of the temperature of a solid support over time according to an embodiment of the present invention showing the time points at which first, second and third images I1, I2 and I3 can be obtained with respect to at least one defined event (DE) (involving appropriately timed photobleaching).

[0050] Figure 2B is a graph of the temperature of a solid support over time according to an embodiment of the present invention showing the time points at which first, second and third images I1, I2 and I3 can be obtained with respect to at least one defined event (DE) (involving thermal quenching).

[0051] Figure 3A -C shows three images of different encoded bead populations (i.e., Figure 3A , 3B and 3C), the bead populations are loaded into a single fluidic device and electronically / optically distinguished in the acquired images by fluorescence changes caused by photobleaching as a defining event. Figure 3A It appears that the initial fluorescence intensity of each bead population is similar. Figure 3B It was shown that the populations were readily distinguishable after prolonged light exposure. Figure 3A and 3B Displayed at same camera brightness / contrast settings. Figure 3C Shown is a localization map depicting the position and identity of individual phycoerythrin PE (solid), quantum dots QD (small dots) and mixtures (stripes) in the array wells.

[0052] Figure 4 is a graph of the photobleaching profiles (normalized fluorescence vs. time / second) of bead populations encoded with PE, QD, or a mixture of PE and QD dyes. Five replicate microchips loaded with each bead population are depicted.

[0053] Figure 5 is a plot of the frequency (%) of each bead population sorted into the histogram after photobleaching versus endpoint-normalized fluorescence intensity. Each bead group (PE, QD, and Mix) is well distinguished from neighboring populations, which may allow for high discrimination accuracy.

[0054] Figure 6 is a plot of normalized fluorescence (%) versus temperature (Celsius) of encoded beads sealed within a microwell array of a fluidic device heated to a specified temperature for a thermally defined event. Elevated temperatures above 80°C preferentially quench fluorescence from phycoerythrin (PE).

[0055] Figure 7 is a graph showing the frequency (%) of each bead population after heating versus normalized fluorescence intensity. Thermal decoding allows different bead populations with similar initial fluorescence to be distinguished from each other.

[0056] Figure 8 is a top view of a microfluidic device with an exemplary bead well array according to an embodiment of the present invention.

[0057] Fig.9A is a top view of a microfluidic device with a conventional bead-well array.

[0058] Fig. 9B yes Fig.9A A side perspective view of a conventional bead hole is shown in FIG.

[0059] Fig. 10A is a top view of an example beadhole array according to an embodiment of the present invention.

[0060] Fig. 10B yes Fig. 10A A side perspective view of the bead hole shown in .

[0061] Fig.11A is a top view of another example beadhole array according to an embodiment of the present invention.

[0062] Fig. 11B yes Fig.11A A side perspective view of the bead hole shown in .

[0063] Fig. 12A is a top view of another beadhole array according to an embodiment of the present invention.

[0064] Fig. 12B yes Fig. 12A A side perspective view of the bead hole shown in .

[0065] Figures 13A-13C is a schematic diagram of an example analysis system according to an embodiment of the present invention.

[0066] Fig.14A is a top view of an example microfluidic chip according to an embodiment of the present invention.

[0067] Fig. 14B and 14C According to an embodiment of the present invention Fig.14AAn example of a microfluidic chip shown in Figure 1 is a top view of a connectable substrate.

[0068] Fig.14D is a cross-sectional view of a microfluidic chip showing optional spacing according to an embodiment of the present invention.

[0069] Fig.14E is a cross-sectional view of an example microfluidic chip showing the use of a sealant between adjacent reaction wells according to an embodiment of the present invention.

[0070] Fig.15 is a schematic diagram of an analysis system according to an embodiment of the present invention.

[0071] Figure 16-18 is a flow chart representing an example method of an embodiment of the present invention.

[0072] Fig.19 Shown is the packaging of a kit according to an embodiment of the present invention.

[0073] Fig. 20 is a schematic diagram of an example flow cytometry system according to an embodiment of the present invention.

[0074] Fig.21 is another schematic diagram of an example flow cytometry system according to an embodiment of the present invention.

[0075] Fig. 22 is a graph of the mean fluorescence values ​​for each population over time, with the top and bottom 5% of fluorescence values ​​plotted using dashed lines.

[0076] Fig.23 is a plot of raw fluorescence intensity data over time from six coded bead populations. The initial fluorescence value distinguishes the "high" and "low" populations from one another, while photobleaching identifies the specific coding dye used.

[0077] Detailed description of the embodiments of the invention

[0078] The present invention will now be described hereinafter with respect to the accompanying drawings and examples in which embodiments of the present invention are shown. However, the present invention can be implemented in a variety of different forms and should not be considered to be limited to the embodiments described herein. On the contrary, these embodiments are provided so that this disclosure is sufficient and complete, and will fully convey the scope of the present invention to those skilled in the art.

[0079] Like numbers refer to like elements throughout. In the accompanying drawings, the thickness of certain lines, layers, components, elements or features may be exaggerated for clarity. In the text and drawings, the abbreviations "FIG." and "Fig." for "figure" may be used interchangeably.

[0080] The term used in this article only for the purpose of describing specific embodiments is not intended to limit the present invention.As used in this article, the singular form "one", "a kind of" and "said" are also intended to include plural forms, unless the context clearly indicates otherwise.It is further understood that when used in this specification, the term "comprising" and / or "including" specifies the existence of the features, steps, operations, elements, and / or components described, but does not exclude the existence or addition of one or more other features, steps, operations, elements, components, and / or their groups.As used in this article, the term "and / or" includes any one and all combinations of one or more related enumeration items.As used in this article, phrases such as "between X to Y" and "between about X to Y" should be interpreted as including X and Y.As used in this article, phrases such as "between about X to Y" mean "between about X to about Y".As used in this article, phrases such as "from about X to Y" mean "from about X to about Y".

[0081] Unless otherwise defined, all terms (including technical and scientific terms) used in this article have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs. It is further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having meanings consistent with their meanings in the context of the specification and the relevant field, and should not be interpreted in an idealized or overly formal sense unless explicitly defined as such in this article. For the sake of brevity and / or clarity, well-known functions or structures may not be described in detail.

[0082] It should be understood that when an element is referred to as being "on," "attached to," "connected to," "coupled to," "in contact with," etc., another element, it can be directly on, attached to, connected to, coupled to, or in contact with another element, or there can be intervening elements. Conversely, when, for example, an element is referred to as being "directly on," "directly attached to," "directly connected to," "directly coupled to," or "in direct contact with" another element, there are no intervening elements. It will also be understood by those skilled in the art that reference to a structure or feature being disposed "adjacent" to another feature can have portions that overlap or are located below the adjacent feature.

[0083] For convenience of description, spatially related terms such as "below", "below", "lower", "above", "upper", etc. may be used herein to describe the relationship between an element or feature and another one or more elements or one or more features as shown in the drawings. It should be understood that, in addition to the directions depicted in the drawings, spatially related terms are intended to include different directions of the device in use or operation. For example, if the device in the drawings is inverted, the element described as being "below" or "below" other elements or features is oriented "above" another element or feature after it. Therefore, the exemplary term "below" can include both the directions of "above" and "below". The device can be oriented otherwise (rotated 90 degrees or in other orientations) and the spatially related descriptors interpreted accordingly. Similarly, the terms "upward", "downward", "vertical", "horizontal", etc. are used herein only for explanation purposes unless specifically noted otherwise.

[0084] It should be understood that although the terms "first", "second", etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another element. Therefore, the "first" element discussed below may also be referred to as the "second" element without violating the teachings of the present invention. The order of operations (or steps) is not limited to the order given in the claims or drawings unless specifically noted otherwise.

[0085] The term "microchip" and "microfluidic chip" are used interchangeably and refer to a substantially planar, thin device. A microfluidic chip can be rigid, semi-rigid or flexible. The term "thin" refers to less than 10 mm such as between 10 mm and 0.1 mm and can be a thickness dimension of about 3 mm, about 2.5 mm, about 2 mm, about 1.5 mm, about 1 mm or about 0.5 mm. A microchip typically has a width and length less than about 6 inches, more typically between about 1 inch and 6 inches. A microchip can have a width dimension less than a length dimension. In some embodiments, a microfluidic chip can have a width dimension of about 2.13 inches (54 mm) and a length dimension of about 3.4 inches (85.5 mm). A microchip can include a micron-sized and / or nano-sized fluid channel.

[0086] The term "substantial dimension" refers to the width and / or depth dimensions of a fluid channel.

[0087] With respect to fluid channels, the terms "micrometer-sized" and "microfluidics" refer to fluid flow channels having widths and / or depths of sub-millimeter or smaller dimensions (e.g., the term includes micrometer- and nanometer-sized channels) and include channels having at least one segment having a width and / or depth in the size range of hundreds of micrometers or less, typically less than 900 micrometers and greater than 1 nm. Fluid channels or portions thereof may include nanochannels having a substantial dimension of 1 nm to about 900 nm, more typically about 10 nm to 500 nm.

[0088] The channels of the bead holes of the bead hole array can have side walls and a bottom plate formed as one or more substrates, thereby having an open upper surface and a closed lower surface and a side wall extending therebetween. One or more spacers, top substrates, membranes or covers can be used. The top substrate, membrane or cover can seal, cover or close the one or more fluid channels of the reaction hole and / or the upper surface of the array.

[0089] The term "about" refers to a parameter that may vary by less than + / - 20%, such as + / - 10% or 5%.

[0090] The term "solid support" includes one or more objects such as, for example, microbeads, chips, plates, slides, needles, plate wells, beads, microspheres, nanoparticles, micropores or other members that can be used to provide probes and / or primers and / or members that can be connected to or labeled with coding media (such as, for example, fluorescent compounds, chemiluminescent compounds, radioactive elements, and / or enzymes) and / or dynamic elements. The coding medium and / or dynamic element can be directly or indirectly connected to the solid support, such as, for example, by covalent bonding, non-covalent bonding, and / or physical bonding. In some embodiments, the solid support can be an array printed on a solid surface. In some embodiments, the solid support can be a microsphere. In some embodiments, the solid support can be a solid surface, on which a sample is disposed, such as, for example, a chemical and / or biological component (e.g., a tissue sample), and a molecular recognition element comprising one or more coding media can be connected to a portion of the sample. The molecular recognition element can include or can be an antibody, an aptamer, a DNA hybridization probe, an oligonucleotide, a peptide, a protein, and / or a combination thereof. In some embodiments, solid support can include variable reagent and / or support binding, each of which can allow separation from solid support. In some embodiments, one or more encoding media can be connected to solid support (for example, beads or microspheres) to provide coded solid support (for example, coded beads or coded microspheres). In some embodiments, one or more encoding media can be connected to molecular recognition elements (for example, antibodies) to provide coded molecular recognition elements (for example, coded antibodies). In some embodiments, the coded molecular recognition elements can be connected to particles and / or particles can be connected, such as, for example, nanoparticles. In some embodiments, the coded molecular recognition elements can be connected to solid surface, and samples are provided on the solid surface, due to the binding affinity of the coded molecular recognition elements and the sample on the solid surface and / or the element sample on the sample. Therefore, in some embodiments, the encoding media can not be connected to the solid support until the determination starts or completes or unless the coded molecular recognition elements are attached to the sample on the solid support and / or the element in the sample. Therefore, the method of the present invention can include providing or combining one or more encoding media on the solid support. In some embodiments, the method of the present invention includes providing a plurality of coded molecular recognition elements and making at least part of the coded molecular recognition elements be bound to the sample on the solid support. In some embodiments, the method of the present invention can be used for fluorescence microscopy applications, including, for example, immunofluorescence localization and / or fluorescence in situ hybridization (FISH) of tissue, cell and / or receptor.

[0091] The term "bead" or "bead" refers to a solid phase element such as a particle, granule or microsphere, typically a magnetic microsphere, which can be a porous, superficially porous or non-porous material, such as, for example, a polymer, plastic, glass, silica, metal or semi-metal oxides (including but not limited to aluminum oxide, titanium oxide, zirconium oxide or other oxides), quantum dots, metal particles, etc., which can be suitable for use in a reaction well. In some embodiments, a multiplex bead set comprising a plurality of beads (e.g., microspheres) can be provided, the beads being uniquely encoded to distinguish between bead populations.

[0092] The term "loop" refers to a complete hardware implementation or an implementation that combines software and hardware.

[0093] The analyte in the sample can be any target analyte from the sample, including, for example, a variety of mixtures comprising synthetic and / or biological macromolecules, nanoparticles, small molecules, DNA, nucleic acids / polynucleic acids, peptides, proteins, etc. The analyte can be one or more analyte molecules. The sample or the analyte of the sample can include one or more polar metabolites, such as, for example, amino acids and / or charged molecules, peptides and / or proteins. The sample and / or analyte can also or alternatively include molecules extracted from biological fluids, blood, serum, urine, dried blood, cell growth medium, cracked cells, beverages and / or food. The sample can also or alternatively include environmental samples such as water, air and / or soil.

[0094] The term "oligonucleotide" refers to a nucleic acid sequence of at least about five nucleotides to about 500 nucleotides (e.g., 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 21, 22, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 nucleotides). In some embodiments, for example, an oligonucleotide can be about 15 nucleotides to about 50 nucleotides, or about 20 nucleotides to about 25 nucleotides, which can be used, for example, as a primer in a polymerase chain reaction (PCR) amplification assay and / or a probe in a hybridization assay or a microarray. The oligonucleotides of the present invention can be natural or synthetic, for example, DNA, RNA, PNA, LNA, a modified backbone, etc., or any combination thereof, as known in the art.

[0095] Probes and primers (including those used for amplification and / or detection) can comprise oligonucleotides (including naturally occurring oligonucleotides such as DNA and synthetic and / or modified oligonucleotides) of any suitable length, but typically are from 5, 6 or 8 nucleotides in length to 40, 50 or 60 nucleotides in length, or longer.

[0096] The probes and / or primers can be immobilized or attached to a solid support such as, for example, a bead, a chip, a pin or a microtiter plate well, and / or linked to or labeled with an encoding medium such as, for example, a fluorescent compound, a chemiluminescent compound, a radioactive element, and / or an enzyme.

[0097] The term "coding medium" refers to one or more media (e.g., chemicals, proteins, etc.) that are combined with (e.g., applied to, attached to, combined with, combined with, used to make or produce, etc.) a solid support and / or a material of a solid support and / or a material in contact with a solid support, and the medium provides and / or will produce a coding signal for the corresponding solid support (which may be all or part thereof). In some embodiments, one or more coding media provide and / or produce a detectable coding signal that allows a population or subpopulation of solid supports (e.g., beads) to be distinguished. For example, in some embodiments, one or more coding media can provide and / or produce a detectable coding signal for an individual solid support (e.g., beads) connected to one or more coding media and / or one or more coding media can provide and / or produce a detectable coding signal for a specific portion of a solid support connected to one or more coding media (e.g., a portion of a solid support bound to a molecular recognition element comprising one or more coding media).

[0098] The coded signal can be provided and / or generated by one or more coded media combined with a solid support and / or by the solid support itself and / or the material (e.g., compound) combined with the solid support. In some embodiments, the coded signal is a signal (e.g., light signal and / or electrical signal) generated by one or more coded media (e.g., chemicals, proteins, etc.) combined with a solid support (e.g., applied thereto, attached thereto, combined thereto, combined thereto, used to make or generate it, etc.) or produced by a solid support. Detectable coded signals can be optically and / or electrically detectable, which can be observed with the naked eye and / or read, detected and / or obtained electronically using the human eye. Detectable coded signals can include intensity, typically at or above a defined threshold, color (e.g., color hue, color intensity, and / or color value), color and intensity, and / or size, shape and / or radioactive changes. In some embodiments, detectable coded signals include luminous intensity, which can include fluorescence, phosphorescence and / or chemiluminescence intensity.

[0099] The coded signal of the corresponding solid support (which may be all or part thereof) may be detectable (e.g., light detectable, electronic detectable, electrochemical detectable, electrostatic detectable, magnetic detectable, etc.) or may not be detectable. In some embodiments, the coded signal of the corresponding solid support (which may be all or part thereof) may change. For example, the coded signal of the corresponding solid support may change from detectable to undetectable, from undetectable to detectable, from a larger value to a lower value (e.g., by a larger signal amplitude, fluorescence intensity value, diameter, etc.), and / or from a lower value to a higher value. The change of the coded signal of the corresponding solid support may occur over time and / or may occur due to at least one chemical and / or physical change associated with the solid support. In some embodiments, the coded signal of the solid matrix may change and / or may be provided and / or generated by a change in the environment in which the solid support is present (e.g., a change in the solution in which the solid support and / or the coding medium are in contact). For example, the coded signal of the solid support can vary and / or can be provided and / or generated by changes in magnetic behavior, pH, light scattering, physical size (increase or decrease), shape, refractive index, solubility, light absorption and / or emission intensity or maximum wavelength shift, conductivity, dielectric constant, viscosity, and radiation emission from isotope decay, and combinations of the above.

[0100] "Encoding state" refers to a specific combination of encoding signals (detectable or undetectable) of corresponding solid supports in a plurality of solid supports, such as, for example, two or more encoding signals of a specific solid support at two or more time points, and / or a specific combination of encoding signals (detectable or undetectable) of a specific portion of a solid support (e.g., a specific region or portion of a sample on a solid support provides a specific combination of encoding signals). For corresponding solid supports, specific encoding signals at two or more time points can allow for the identification of specific populations or subpopulations (e.g., specific solid support (e.g., bead) populations or subpopulations) respectively. In some embodiments, the specific encoding signals of corresponding solid supports at two or more time points can be the same and / or can be different. Each encoding state can be unique and / or detectable (e.g., optically and / or electrically detectable) and distinguishable from each other to provide distinguishable populations. However, the encoding signal of each population at a first time point can be the same as the encoding signal of a different population at a first time point, and the encoding signals of the two populations at a second time point can be different, so that the two populations can be distinguished and / or identified. In some embodiments, the encoded signals of the first population and the second population may start at different levels (eg, have different signal amplitudes or values) but end at the same level.

[0101] The term "decoding" refers to different populations of each sample (including, for example, a sample of a solid support and / or a sample comprising a plurality of coded molecular recognition elements) that are at least partially based on coded signals (typically electronic / program) from coded solid supports and / or coded molecular recognition elements. In some embodiments, decoding is based at least in part on two or more coded signals of a corresponding solid support (which can be all or part thereof) before, during and / or after a chemical and / or physical change related to a corresponding solid support and / or by comparing two or more coded signals at different time points. In some embodiments, decoding is based at least in part on two or more coded signals of an individual solid support before, during and / or after a chemical and / or physical change related to an individual solid support and / or by comparing two or more coded signals at different time points of an individual solid support. For example, a sample comprising a plurality of solid supports can comprise 6 different populations, and therefore comprises 6 different coded states. Detecting and / or distinguishing one or more coded signals can allow decoding samples to determine the identity of a specific solid support population. In some embodiments, decoding is based at least in part on comparing two or more coded signals of the portion of the solid support before, during and / or after at least one chemical and / or physical change associated with a specific portion of the solid support (e.g., a specific region or portion of a sample on the solid support) and / or by comparing two or more coded signals of the portion of the solid support at different time points. Different coded molecular recognition elements can provide a variety of coded states and detecting and / or identifying one or more coded signals can allow decoding of the sample to determine the identity and / or position of a specific population, such as, for example, the identity and / or position of an element of the coded molecular recognition element that binds the specific population.

[0102] The terms "dynamic decoding" or "time domain encoding" referred to interchangeably herein refer to the coded signal of a sample (including, for example, a sample of a solid support and / or a coded molecular recognition element) analyzed (typically automatically) based on multiple (e.g., more than 2) measurements (typically from an image) to obtain the coded signal of the solid support over time. Different populations in the sample (e.g., a population of a solid support or a molecular recognition element) can have each coded signal that can change over time. The change can be used to identify the population. For example, in some embodiments, time domain encoding can be combined with photobleaching kinetics to decode different populations. By combining the time domain, time domain encoding can unlock additional multiplex levels that are not available through conventional decoding methods.

[0103] Two or more measurements (e.g., 2, 3, 4, 5, 6, etc.) can be performed to obtain two or more coding signals (e.g., 2, 3, 4, 5, 6, etc.) of the corresponding solid supports and / or the solid supports of at least a portion thereof in the sample comprising a plurality of solid supports. In some embodiments, the first coding signal measurement can be performed at a first time point and the second coding signal measurement can be performed at a second time point. The first coding signal measurement can be based on the first image obtained at a first time point (e.g., before, during or after the determination, e.g., after the hybridization reaction) and / or based on the detection of the optical or electrical signal obtained at the first time point. The second coding signal measurement can be obtained after the first time point or later than the first time point. In some embodiments, the second coding signal measurement and one or more subsequent measurements can be performed at a time greater than 1 ms (e.g., 10 ms, 100 ms, or 1, 2, 3, 4, 5 seconds) after any exit state life of the compound (e.g., coding medium) bound to the solid support and / or immediately after the previous measurement. The second coded signal measurement can be based on a second image taken at a second time point and / or based on detecting an optical or electrical signal obtained at the second time point. Two or more coded signal measurements can be performed and / or obtained to detect a coded signal at a specific time point (e.g., a first and a second time point) of a specific solid support in a sample of solid supports.

[0104] In some embodiments, the coded signal measurement can be performed and / or obtained before, after and / or during the defined event. The term "defined event" means an event that can cause or result in a planned physical and / or chemical change associated with a solid support. A defined event can be passive or dynamic. In some embodiments, a defined event can cause or result in a physical and / or chemical change of a dynamic element. In some embodiments, a defined event can cause or result in a physical and / or chemical change of a coded medium (e.g., dye, albumen, etc.) and / or a solid support (e.g., microsphere).

[0105] As used herein, "dynamic element" refers to a chemical and / or biological compound that provides or displays a physical and / or chemical change in response to a defined event. The physical and / or chemical change can cause the coded signal of the corresponding solid support (which can be all or part thereof) at one time point to change compared to the coded signal at a different (e.g., earlier) time point and can be detectable (e.g., optically and / or electrically detectable). The "dynamic element" may include a coding medium (e.g., a dye, a protein, etc.) and / or a solid support (e.g., a microsphere). The physical and / or chemical change may be a physical and / or chemical change that affects a solid support (e.g., the change may be a change in the charge and / or color of the solid support and / or may be a change in the size and / or shape of the solid support). Therefore, the dynamic element may be a solid support and / or a compound connected and / or bound to a solid support. In some embodiments, the physical and / or chemical change is a physical and / or chemical change that affects the encoding medium bound to the solid support (e.g., the change may be in the stability of the linker that binds the encoding medium to the solid support, the solvent accessibility of the encoding medium, and / or the presence and / or absence of a quencher). Thus, the dynamic element may be an encoding medium and / or a compound that is connected and / or bound to an encoding medium (e.g., a quencher). The physical and / or chemical change may be reversible or irreversible. In some embodiments, the dynamic element may be an encoding medium. In some embodiments, a defined event may selectively activate or inactivate the encoding medium. Example defined events are provided in Table 1.

[0106] Table 1. Instance-qualifying events.

[0107]

[0108] One or more limiting events can be used to decode a solid support and / or multiple solid supports, and the coded signal of the corresponding solid support (which can be all or part thereof) may or may not change in response to each of the one or more limiting events. A limiting event may not be an event that typically occurs in a determination and / or analysis using a solid support. In some embodiments, a limiting event is an event (e.g., heating or light exposure) that occurs during a determination using a solid support. A limiting event can result in a chemical and / or physical change in a part of the solid support (e.g., optionally, a coding medium connected and / or combined with a solid support), which can result in a change in the coded signal of the part of the solid support associated with a specific coding state. Example static coded signals and time domain coded signals are provided in Table 2. In some embodiments, in addition to one or more time domain coded signals, a static coded signal can also be used to decode at least one population in a sample.

[0109] Table 2. Example statically coded signals and time-domain coded signals.

[0110]

[0111] Do not confuse the coding signal with the assay dye signal (if present), and the dynamic element, coding medium, and qualifying events should be configured and / or selected so that they do not interfere with the assay. In some embodiments, if a dye is used for an assay using dynamic decoding, the assay dye may be distinguishable from one or more dynamic elements and / or coding media used for dynamic decoding. For example, the assay dye signal may be spectrally displaced from a signal associated with the signal of the dynamic element and / or the signal of the coding medium and / or the assay dye may use a detectable parameter different from that of the coding medium and / or the dynamic element. Alternatively, the coding medium and / or the dynamic element may be spectrally similar to the assay dye and / or have the same amplitude and / or intensity as the assay dye; however, in this case, the qualifying event may cause a change in the coding medium signal and / or the dynamic element signal so that it is no longer spectrally similar to the assay dye and / or has the same amplitude and / or intensity as the assay dye and is therefore distinguishable from the assay dye and / or no longer interferes with the assay.

[0112] The same solid support in the same device (e.g., a fluidic device, typically the same beads, which may be in the same reaction well or virtual array) may be used for the first and second images or for detecting the first and second light and / or electrical signals. The encoding media and / or dynamic elements of the different images may have similar spectral properties.

[0113] A defined event can cause and / or result in one or more physical and / or chemical changes associated with a solid support. In some embodiments, a defined event can cause and / or result in a change in the environment of a solid support, which in turn can cause or induce a physical and / or chemical change in a dynamic element (e.g., a coded medium and / or a solid support) to provide a change in the coded signal of the solid support (e.g., a change in the coded signal of a specific solid support among a plurality of solid supports or a change in the coded signal of a specific portion of a solid support, wherein the solid support comprises a plurality of coded signals at different portions of the solid support), which change can be visually and / or electronically detectable (if thereafter, detection can be via an optical or electrical detector).

[0114] Time domain coding can include comparing the coded signal of the corresponding solid support at a specific time point (e.g., before the defined event) with the coded signal of the corresponding solid support at a different time point (e.g., after the defined event) to identify the specific population to which the coded signal and / or solid support belongs. In some embodiments, the first coded signal of the corresponding solid support is obtained in the first reading (reading) (e.g., detecting optical or electrical signals) and / or the first image before or during the defined event and at least one second reading and / or second image is obtained after the first image to obtain the second coded signal of the corresponding solid support. The first and / or second reading and / or image can include a plurality of different coded signals, which correspond to the discontinuous positions and / or entities on and / or in the sample of the discontinuous solid support or the solid support. Different coded signal measurements and / or images can be obtained from common detectors, for example, when the solid support of the fluidic device and / or the fluidic device is kept at the same temperature and the detector is operated with the same wavelength during sample analysis. Different encoded signal measurements and / or images can be obtained within about 1 microsecond to about 50 hours of each other, and in some embodiments, within about 1 ms to about 10 minutes of each other (e.g., within 1 second to 1 minute or 1 second to about 10 minutes of each other).

[0115] Two or more (e.g., 2, 3, 4, 5, 6, etc.) coded signal measurements and / or images can be obtained before, during, and / or after one or more defined events and / or one or more physical and / or chemical changes associated with a solid support. Coded signal measurements and / or images can be obtained using one or more wavelength bands (e.g., 1, 2, 3, 4, 5, etc.). In some embodiments, two or more coded signal measurements and / or images can be obtained before defining an event and / or at least one physical and / or chemical change associated with a solid support. For example, a first image can be obtained in a blue excitation wavelength band, a second image can be obtained in a green excitation wavelength band, and a third image can be obtained in a red excitation wavelength band, each of the first, second, and third images being obtained before defining an event and / or at least one physical and / or chemical change associated with a solid support. In some embodiments, two or more coded signal measurements and / or images can be obtained during and / or after defining an event and / or at least one physical and / or chemical change associated with a solid support. For example, an image can be obtained in each of the following: a blue excitation wavelength band, a green excitation wavelength band, and a red excitation wavelength band, each of the three images being obtained during and / or after a defined event and / or at least one physical and / or chemical change associated with a solid support. Therefore, embodiments of the present invention may include obtaining multiple coded signal measurements and / or images, which can be obtained in multiple wavelength bands. In some embodiments, two or more coded signal measurements and / or images can be obtained before, during and / or after two or more defined events and / or two or more physical and / or chemical changes associated with a solid support. For example, a first image can be obtained before a first defined event and / or a first physical and / or chemical change associated with a solid support, a second image can be obtained after a first defined event and / or a first physical and / or chemical change associated with a solid support, and a third image can be obtained after the second image and after a second defined event and / or a second physical and / or chemical change associated with a solid support (optionally the same or different solid supports).

[0116] Defined events can provide physical and / or chemical changes in dynamic elements, such as heat-induced changes (which can also be used for determination), pressure-induced changes, light-induced changes, natural radioisotope decay, chemically induced changes, or any combination of the above. Time domain encoding can exploit defined stable, partially stable, selectively activated, and / or unstable properties of each encoding medium (e.g., denaturation of organic protein dyes and / or changes in chemical structure due to, for example, pH, heat or light-sensitive labile binding) to cause changes in the encoding signal (such as, for example, the encoding signal of the solid support connected to each encoding medium or the encoding signal of the molecular recognition element connected to each encoding medium). For example, when the encoding signal is an intensity value, the physical and / or chemical change can cause the intensity value to increase or decrease, for example, by at least 0.01% between the first and second images and / or readings, more typically by at least 1-20% between the first and second images and / or readings. The increase or decrease may be sufficient to make the intensity level, for example, go from high to low, from low to high, from medium to low, from low to medium, from high to medium or from medium to high, from zero to low, from high or medium or high, low or medium to zero. The terms high, medium and low refer to a first, second and third level, respectively, wherein the first is greater than the second and third, the second is between the first and third, and the third is less than the first and second. In some embodiments, more than two different levels may be set between the high and low levels (i.e., there is more than one level between the high and low levels).

[0117] The coded medium may comprise an encapsulated dye that is photosensitive and selectively activated upon sufficient light exposure to increase intensity.

[0118] As used herein, the term "similar spectral properties" or "spectrally similar" refers to coded media (e.g., dyes) and / or dynamic elements that have similar excitation and / or emission wavelengths and produce detectable fluorescence intensity in response to a light source operating at a single wavelength or a single wavelength range or passband such that they are indistinguishable in imaging and / or reading.

[0119] The term "quenching" refers to the deactivation of an excited molecular entity (e.g., an encoded mediator) either intermolecularly by external environmental influences (such as a quencher) or intramolecularly by substitution by non-radioactive means. A quencher is a molecular entity that deactivates (quenches) the excited state of the encoded mediator, either by energy transfer, electron transfer, or by a chemical mechanism.

[0120] The fluorescence intensity of at least one coded mediator and / or dynamic element may decrease after a quenching event as a defined event. Alternatively, the fluorescence intensity of at least one coded mediator and / or dynamic element may increase after a defined event.

[0121] The term "bleaching" refers to a loss in absorption or emission intensity.

[0122] Polymerase chain reaction (PCR) can be performed according to known techniques. See, for example, U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. Typically, PCR includes, first, treating a nucleic acid sample (e.g., in the presence of a thermostable DNA polymerase) with an oligonucleotide primer for each strand of a specific sequence to be detected under hybridization conditions, thereby synthesizing an extension product of each primer complementary to each nucleic acid strand, and the primer is sufficiently complementary to each strand of the specific sequence to hybridize therewith, so that the extension product synthesized by each primer can serve as a template for the synthesis of an extension product of another primer when separated from its complementary sequence, and then treating the sample under these denaturing conditions to separate the primer extension product from its template in the presence of one or more sequences to be detected. These steps are repeated cyclically until the desired degree of amplification is obtained. By adding the oligonucleotide probe (for example, oligonucleotide probe of the present invention) that can hybridize with the reaction product in the reaction product, the probe carries a detectable label, and then according to known technology detection label, or by direct visualization on gel, the detection of the sequence of amplification can be carried out. It is also possible to detect the sequence of amplification by adding an intercalating dye to the reaction mixture and monitoring the fluorescence signal intensity that will be proportional to the gross mass of the double-stranded DNA. Although embodiments according to the present invention are described for PCR reaction or nucleic acid and protein ELISA, it should be understood that other nucleic acid amplification methods can be used, such as reverse transcription PCR (RT-PCR), including isothermal amplification techniques such as rolling circle amplification or loop-mediated isothermal amplification (LAMP).

[0123] DNA amplification techniques, as described above, can include the use of a probe, a pair of probes, or two pairs of probes, which specifically bind to a DNA containing a target polymorphism or mutation, but do not bind to a DNA that does not contain a target polymorphism under the same hybridization conditions, and which serve as one or more primers for the amplification of the DNA or a portion thereof in an amplification reaction. Such probes are sometimes referred to as amplification probes or primers herein.

[0124] The term "reagent" refers to any substance or compound added to a system to produce a chemical reaction or added to see if a reaction occurs, including primers, nucleic acid templates, and amplification enzymes. Amplification reagents refer to those reagents (deoxyribonucleoside triphosphates, buffers, etc.) that are commonly used for amplification except primers, nucleic acid templates, and amplification enzymes. Typically, amplification reagents are placed together with other reaction components and contained in a reaction vessel (test tube, microwell, etc.).

[0125] The term "magnetic" as used herein includes ferromagnetic, paramagnetic and superparamagnetic properties.

[0126] Usually, the oligonucleotide probe for detecting the DNA containing the target polymorphism or mutation is to be combined with the DNA encoding the mutation or polymorphism, but under the same hybridization conditions, it is not combined with the DNA containing the mutation or polymorphism. Oligonucleotide probes are suitable for detectable groups such as those detectable group marks given below. Such probes are sometimes referred to as detection probes or primers in this article.

[0127] Figure 1A is a schematic diagram of a fluid analysis system 100 having a control loop 100c including a controller 12, a detector 150 having at least one optical filter set 18, and a decoding module 40 configured to analyze an encoded signal from a solid support 10 associated with an assay using a microfluidic device 20. Figure 1B 1 is another exemplary embodiment of a fluid analysis system 100 having a control loop 100c including a controller 12, a detector 150 having at least one optical filter set 18, and a decoding module 40, wherein the detector 150 can generate a defined event (e.g., optical excitation, photobleaching, etc.). This embodiment shows that no additional hardware components are required compared to conventional assay systems relative to other embodiments. At least one optical filter set 18 can be configured to operate at a desired emission wavelength, such as, but not limited to, a single discrete wavelength and / or passband range, such as about 10 nm to 1000 nm for excitation and imaging, and in a specific embodiment about 545 / 30 nm for excitation and about 620 / 60 nm for imaging. In some embodiments, the optical filter set 18 can be configured to operate at wavelengths in the ultraviolet (UV) range (e.g., at wavelengths in the range of 400 nm to 10 nm), in the infrared (IR) range (e.g., at wavelengths in the range of 700 nm to 1 mm), and / or in the visible range (e.g., at wavelengths in the range of 390 nm to 700 nm), and / or any range and / or individual values ​​therein. In some embodiments, the optical filter set 18 may not be fluorescence based and may be optical, such as, for example, a polarizing filter to detect changes in light scattering. If the changes are size based, filters may not be needed, but rather the ability to image and measure size may be needed.

[0128] The microfluidic device 20 may have a bead well array 30 arranged with bead wells 10 that accommodate individual beads 25. The controller 12 is also in communication with at least one device 125 (e.g., a heat source and / or a light source) that is capable of generating a defined event that may alter the environment of the beads 25 in the bead wells 10 for dynamic decoding and / or may cause physical and / or chemical changes in the dynamic element.

[0129] The decoding module 40 may include or be in communication with a correlation table and / or database of different populations associated with different encoding media for a defined population of beads.

[0130] The apparatus 125 may include a light source and / or a heat source and / or a liquid source to change the well environment, the light source being configured to emit light of a defined wavelength for a defined time to selectively activate, photoquench and / or photobleach beads 25 having one or more dynamic elements (e.g., encoding media) responsive to the light source, the heat source being configured to raise the beads 25 to a critical temperature, the liquid source being in liquid communication with the beads 25 in the bead well 10, etc. The system 100 may be configured to assess the recovery of at least one detectable parameter after photobleaching. This recovery may be different for different materials / fluorophores in different environments and the differences may be exploited for decoding.

[0131] like Figure 1C As shown, the defined event (DE) may occur after the first image I1 is obtained by the detector 150 and before the second image I2 is obtained. Figure 1D As shown, the act of acquiring a first image I1 may be within and itself be a defined event that results in a measurable change for obtaining a second image I2 subsequent to the first image I1.

[0132] In some embodiments, the defined event DE can cause a defined environmental change in, within, and / or about the bead array 30, such as at least one actively induced or applied thermal change T (which can be a temperature increase or decrease, although typically an increase and, for example, develops thermal instability of the dynamic element (e.g., encoding medium) at a defined temperature), a chemical change C, (e.g., a pH change that can be introduced in a variety of ways), and / or an optical change O, such as the introduction of light, optionally at a defined wavelength and / or intensity of light, for example, for sufficient exposure to provide quenching to develop photoinstability. A second image I2 can be obtained after the defined event DE. The system 100 can compare the intensity values ​​32s from the first and second images I1, I2 to identify a solid support (e.g., bead) population / subpopulation. As shown, the second image I2 can include a stable (substantially unchanged) encoding signal 32s s ) and varying intensity values ​​32sc And this "fingerprint" or coded signal can be used to multiplex the assay. The first image I1, the second image I2 and / or the other images subsequently obtained in response to another defined event DE can be used for multiplex analysis to identify the target of each individual sample. The other defined events can be of the same type, for example, if it is temperature, a temperature higher than the temperature at the time of the first defined event can be used. The other defined events can be defined events of different types from the first or previous defined events, such as temperature changes after photobleaching. The other defined events can cause changes of the same nature as the previous defined events, for example, a temperature change that causes a decrease in the fluorescence intensity of some populations can be followed by a photobleaching event that causes a decrease in the fluorescence intensity of the same and / or different populations of solid supports. Alternatively or in addition, the other defined events can cause changes in the properties of the solid support that are different from the previous defined events, for example, a temperature change that causes a change in the size of some populations of solid supports by swelling or dissolution can be followed by a photobleaching event that causes a decrease in the fluorescence intensity of the same and / or different populations of solid supports.

[0133] In some embodiments, the difference in photostability between quantum dots (QDs) and fluorescent organic dyes and / or fluorescent proteins can be used to encode solid supports, e.g., beads, particularly if the two have similar spectral properties. For example, photobleaching kinetics can be used to decode solid supports. Organic dyes can produce high fluorescence intensity but may be prone to photobleaching after extended light exposure. However, quantum dots generally do not photobleach after additional light exposure but tend to maintain or increase fluorescence emission. See, e.g., Bouzigues et al., Biological Applications of Rare-EarthBased Nanoparticles. ACS Nano 2011, 5, 8488-8505; and Wang et al., Luminescentnanomaterials for biological labelling. Nanotechnology 2006, 17, R1-R13. The contents of these documents are incorporated herein by reference in their entirety.

[0134] As known to those skilled in the art, quantum dots are nanometer-scale particles of semiconductor materials that can be used for a variety of experimental purposes, such as labeling proteins in conventional use. In some embodiments, QDs with limited wavelengths (e.g., about 605 nm) can be used as encoding media together with organic dyes (OD) and / or fluorescent proteins (FP). Organic dyes (OD) can be small molecules. QD, organic dyes and / or fluorescent proteins can have different thermal stabilities. QD, OD and / or FP can have similar fluorescence excitation / emission wavelengths. Non-limiting examples of excitation / emission wavelengths include 545 / 30 nm and 620 / 60 nm and can be imaged using a single filter set. Embodiments of the present invention contemplate any quantum dots, fluorescent proteins and / or organic dyes that can be used for luminescence (e.g., fluorescence) or other detectable parameters for time domain encoding. Examples of quantum dots that can be used for the method of the present invention include, but are not limited to, those containing cadmium, indium, lead, sulfur, selenium, tellurium, zinc and / or phosphorus, such as, for example, CdS, CdSe, CdTe, InP, PbS, PbSe and ZnS. Examples of organic dyes that can be used in the methods of the present invention include, but are not limited to, fluorescein, Cy3 (Cy3.18), TAMRA, Texas Red (sulforhod-amine 101), Nile Red, Cy5 (Cy5.18), Atto740, Cy7, Alexa Fluor 750, IR125 (ICG). See, for example, http: / / www.fluorophores.tugraz.at / Substance Examples of fluorescent proteins (FP) include, but are not limited to, phycoerythrin, allophycocyanin, and green fluorescent protein (GFP).

[0135] The beads or other solid supports can be encoded with organic dyes, fluorescent proteins (or protein dyes), QDs, or a mixture of two or more of the same or different types to produce comparable initial fluorescent signals. The beads or other solid supports can then be used in an assay, such as, for example, the beads can be loaded into a microfluidic microwell array device, with oil seals to separate individual bead wells from their neighboring wells, and successfully imaged before, after, and / or during a defined event. See, for example, Figure 2A , the temperature of the solid support (e.g., beads) can be maintained at a defined temperature, and a first image I1 can be taken before a defined event DE1 (e.g., before 1 microsecond-50 hours photobleaching (typically about 1 second to about 3 minutes photobleaching)) and a second and third image I2 and I3 can be taken after a defined event, such as, for example, after DE1 and / or DE2. Photobleaching can be based on light energy, which can be delivered on any relevant or practical time scale, depending on the assay.

[0136] Figure 2BAnother embodiment of an exemplary thermal quenching and imaging sequence is shown, wherein the temperature of the solid support (e.g., beads) is increased at least once to a defined threshold (critical temperature) value defining an event, and a first image I1 can be taken before the defined event (e.g., the first increase in temperature) and a second image I2 can be taken after the temperature returns to the set value (thus having the same relevant operating parameters for the detector). The term "critical temperature" refers to the temperature at which a dynamic element (e.g., an encoding medium) undergoes a physical and / or chemical change. Figure 2B A second defined event of a higher threshold temperature is also shown, after which a third image I3 can be obtained. In some embodiments, the defined event can include light exposure (e.g., photobleaching) the encoded solid support for a specified time, such as, for example, more than 1 microsecond, typically about 1 second to 10 minutes, such as more than 5 seconds, such as, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 seconds or more than 1, 2, 3, 4, 5 minutes.

[0137] In some embodiments, obtaining a first image, readout, and / or detection of an optical or electrical signal of a solid support (e.g., a plurality of solid supports) that may include a first image I1 may be obtained prior to the assay and the assay itself may produce a defined event (e.g., heat and / or light exposure during the assay). In some embodiments, the first image, readout, and / or detection of an optical or electrical signal of a solid support may be obtained after the assay is complete and before the defined event, and the image, readout, and / or detection may be used to determine the initial fluorescence intensity and / or serve as a reference for data normalization. Comparing images before (including reference images) and after a defined event, readouts, and / or signals may show whether a chemical and / or physical change associated with the solid support has occurred after the defined event ( Figure 3A -C).

[0138] like Figure 3A As shown in , before light exposure, in the images, beads encoded with FP, QD, or a mixture of both produced comparable initial fluorescence signals that were indistinguishable because of their similar spectral properties. Figure 3B As shown in , upon light exposure, beads encoded with FP undergo significant bleaching over the course of 3 min, whereas beads encoded with QD are unaffected by prolonged illumination, as evidenced by their stable fluorescence over the same time period. Figure 3B It was also shown that beads labeled with a mixture of the two dyes exhibited moderate bleaching, not as wide as the range of the FP-encoded population. Thus, different mixtures of the two dyes can provide beads that exhibit different levels of bleaching, thereby providing a variety of different fluorescence-encoded states that can be used to identify each bead population ( Figure 3C ). Plotting the fluorescence decay characteristic curves showed that the bead groups were well distinguished from each other ( Figure 4The accuracy of this dynamic decoding method was also demonstrated to be high, given the similar bleaching rates observed within each bead population (n = 5 replicate microchips each, >50k beads total).

[0139] When used herein with respect to coded media, dynamic elements and / or signals (e.g., fluorescent signals), "indistinguishable" refers to signals and / or parameters (e.g., color, size, and / or intensity values ​​at a given wavelength (e.g., fluorescence intensity values)) within the standard deviation, confidence interval, and / or standard error of a population of supports for a given measurement system. Therefore, when two coded media and / or dynamic elements are indistinguishable (e.g., the fluorescent signals of two coded media and / or dynamic elements are indistinguishable), the signals or parameters of the two coded media and / or dynamic elements (e.g., the intensity values ​​of the fluorescent signals) cannot be used to identify and / or distinguish which coded media and / or dynamic elements the signal or parameter corresponds to. Therefore, it is impossible to determine which of the two coded media and / or dynamic elements the signal (e.g., fluorescent signal) is from. There may be a distribution range within which the coded signals of the coded elements (e.g., coded beads or coded molecular recognition elements) in a particular population may fall. As long as the distribution range of another coded element does not overlap or fall within the distribution range of another population, the coded states of the two populations may be determined so that they can be distinguished. In some embodiments, due to the variation in measurements of encoded elements in a single population, each encoding state may be defined by a range of values. The range may not be the standard deviation of the measurements of a single encoded element.

[0140] In some embodiments, in the sample comprising a plurality of solid supports, the solid supports of part can have indistinguishable initial intensity and the solid supports of another part can have distinguishable initial intensity.In some embodiments, even if two solid supports can have distinguishable intensity, if the intensity value falls in the solid support colony, then the solid support can encode the same solid support colony.Some embodiments include more than two different colonies, wherein subpopulations have discontinuous and distinguishable different intensity levels, but these subpopulations can contain only distinguishable other subpopulations after limiting the event.

[0141] Endpoint fluorescence decay values ​​can be measured for each individual bead population. For example, endpoint fluorescence decay values ​​are measured for beads encoded with FP, OD, QD, or a mixture of reagents, and these values ​​are then normalized to the initial bead intensity and sorted into a histogram. Figure 5The intensity distribution of the bead types is shown. The three different populations measured in this example were well distinguished from each other, yielding a >99.98% identification accuracy. This high resolution will allow beads that merge together in a single sample to be easily and unambiguously identified after being loaded into the array and dynamically decoded.

[0142] In some embodiments, the solid support and / or molecular recognition element may include one, two or more encoding media to provide two or more, typically about 2 to about 10,000 (or more) different encoding signals representing different populations (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 or more) that can be distinguished and / or identified using one or more defined events. Some embodiments include using one or more defined events with respect to each other at or capable of producing different fluorescence levels of different populations (e.g., different solid support populations and / or different molecular recognition element populations). For example, a solid support may include a first encoding medium corresponding to a first population and a second encoding medium corresponding to a second population, and the first and second encoding media may be the same or different and each may have an initial fluorescence intensity level. In some embodiments, the initial fluorescence intensity levels of the first and second coded media are the same or substantially the same. In other embodiments, the initial fluorescence intensity level of the first coded media is different from the initial fluorescence intensity level of the second coded media. It is not necessary for all solid substrates to have coded media or some populations may have a single dye. The dye may be stable and may not produce and / or show physical and / or chemical changes for some solid substrate populations.

[0143] Each of the first and second populations can include more than one subpopulation, as described above, wherein each subpopulation can have the same or substantially the same initial fluorescence intensity level. Recognizing that different systems will operate with different resolutions and capabilities, in some specific embodiments, the term "same or substantially the same" with respect to fluorescence intensity levels can refer to normalized fluorescence intensity values ​​and / or a value of ± 0.15 or less (e.g., ± 0.1 or 0.05) changes in some embodiments so that it remains within a defined range of a defined level (e.g., high, medium, low). For example (and without limitation), beads with an encoded medium can have a normalized fluorescence intensity value of 0.7 and beads with the same or substantially the same fluorescence intensity level can have a normalized fluorescence intensity value of 0.55 to 0.85.

[0144] As described above, beads encoded with FP or QD or a mixture are used to generate proteins that are substantially similar to each other (cannot be reliably or accurately distinguished) but different from Figure 3A The initial fluorescence intensity levels (brighter / lower intensity) are shown in FIG. 1 . It was shown that the beads photobleached similarly into brighter beads, resulting in three more bleach-distinguishable subpopulations or encoding states, as compared to Figure 3A This ultimately provides additional multiplexing capabilities, where a single wavelength discriminates three distinguishable intensity variations (FP, QD and mixture).

[0145] Therefore, incorporating the time domain into decoding and / or multiplexing assays (e.g., multiplexed bead-based bioassays) can greatly increase the number of different multiplexed encoding states. While only two encoding levels can be achieved using conventional methods, the dynamic decoding method produces at least two distinguishable populations or encoding states. These benefits can significantly enhance the multiplexing capabilities of bead-based POC assays while also minimizing the cost and complexity of the required analytical instrumentation.

[0146] In some embodiments, additional solid supports (e.g., beads) populations can be provided at a single initial fluorescence intensity level. In some embodiments, more bead populations can be provided at a single initial fluorescence intensity level by pre-sorting the encoded beads. In some embodiments, the method can be generalized to other wavelengths (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different wavelengths or spectral bands or ranges) without using a single, shared detection wavelength for the analysis to increase the amount of multiplexing levels that can be achieved.

[0147] Incorporating the time domain into a solid support decoding strategy can be performed using strategies that are different from the photostability differences of different coding dyes. In some embodiments, an alternative or additional dynamic encoding strategy is to utilize the thermal stability of the coding medium. For example, proteins and / or organic dyes can be sensitive to thermal denaturation at elevated temperatures. However, QDs are semiconductor materials that are stable at elevated temperatures. These properties of these dyes can be used in dynamic decoding embodiments.

[0148] The microspheres were labeled with FP or QD so that each population showed indistinguishable initial fluorescence. Each bead population was then loaded into a microchip for imaging as described above. Images were first acquired at 25 °C to determine the initial fluorescence intensity and also serve as a reference for data normalization. The chip was then heated to an elevated temperature to denature the FP, then cooled back to 25 °C and imaged again. The loss of fluorescence intensity between the images before and after heating allowed the identity of the bead population to be determined. Figure 6The decrease in normalized fluorescence signal after heating the beads is shown. Since the streptavidin-biotin binding is destroyed at temperatures ≥70 °C, a decrease in signal is observed with increasing temperature. Both FP and QD are chemically conjugated to the beads via streptavidin-biotin. However, at higher temperatures, FP begins to denature and its fluorescence decreases significantly. This differential quenching can be used to identify differently encoded beads with good resolution ( Figure 7 ).

[0149] like Figure 6 and 7 As shown, incorporating dynamic elements (i.e., fluorescent proteins (FPs) that denature when temperature changes over time) allows for previously unobtainable levels of multiplexing without complicating the imaging system. In some embodiments, incorporating temperature changes in the time domain can be used for PCR-based assays, where heating elements are already in place for thermal cycling, and therefore no additional components are required for thermal resolution multiplexing. In some embodiments, the concentration of dyes on the solid support (e.g., beads) and / or buffer compositions can be varied to enhance and / or increase the resolution between solid support populations and / or allow for the generation and identification of additional distinguishable populations.

[0150] According to some embodiments, time domain measurements can be used to decode a coded solid support (e.g., a bead set), wherein one or more compounds (e.g., a dynamic element such as a coding medium) have been added to a portion of the solid support (e.g., a subgroup of beads). The one or more compounds can be dynamic elements that provide physical and / or chemical changes in response to a defined event. In some embodiments, the physical and / or chemical changes can be observable changes. For example, in some embodiments, the one or more compounds can provide changes in fluorescence signals observed during temperature changes caused by changes in the quantum efficiency of one or more compounds (e.g., coding dyes). In some embodiments, adding a heat-labile or light-activated compound (e.g., a light-activated acid such as a triarylsulfonium / hexafluoroantimonate) to one or more portions of the solid support after activation can result in changes in the chemical properties (e.g., pH) of the solid support, which changes in the chemical properties affect changes in the fluorescence properties of the coding medium (e.g., fluorescein). Alternatively, a solid support contained in a reaction well (e.g., Ion Torrent platform, https: / / www.thermofisher.com / us / en / home / brands / ion-torrent.html) having or constituting a sensor or electrode or electrode array sensitive to changes in pH or conductivity can directly measure these changes.

[0151] In some embodiments, a portion of the plurality of solid supports can include a labile attachment chemistry for one or more dynamic elements that provides a physical and / or chemical change upon activation (e.g., temperature change, pH change, solvent addition / removal, etc.) that produces a result when one or more encoding compounds disappear from the surrounding solution (e.g., FITC-biotin, released from streptavidin-coated beads upon heating and detected in the liquid contained in the assay well). In some embodiments, a mixture of labile and non-labile (e.g., heat-labile or photodegradable streptavidin-biotin binding and stable covalent chemical binding, respectively) encoding media can be used.

[0152] Some embodiments include using a coded medium (e.g., a dye) as a dynamic element with varying degrees of sensitivity to a quencher (oxygen, dabsyl, BlackHole Quenchers, Iowa Black, etc.) such that upon addition or removal of the quencher, different populations (e.g., bead populations) can be identified. In some embodiments, the dynamic element can include a quencher and one or more compounds can be protected from the action of the quencher (e.g., by encapsulation, binding to a protein, etc.) and this can be used to enhance the differences between dyes and allow identification of different populations. In some embodiments, this can be accomplished in a flow cytometry format by adding a quencher in the sheath flow fluid upstream of the system after an initial read. An example flow cytometry system is shown in Fig. 20 , which depicts measurements before and after photobleaching. Another example flow cytometry system is shown in Fig.21 , where measurements before and after a pH shift are depicted. Alternatively, a detectable change in the encoding properties of the solid support can be achieved by adding a sheath flow to change the pH, organic solvent strength, ionic strength or other such properties.

[0153] In some embodiments, methods for dynamically decoding a solid support population can employ the time domain in chemical and / or biochemical assays. Example assays include, but are not limited to, immunoassays, nucleic acid hybridization arrays, PCR, RT-PCR, RT-qPCR, etc.

[0154] The methods of the present invention can be used with solid supports (such as, for example, beads and non-bead matrices, such as arrays printed on solid surfaces). In some embodiments, the methods of the present invention can include a photoresist or "printed" barcode reagent, wherein the strips or cross-sections of each particle can be varied and thus produce different populations. Compounds that can be used in the methods of the present invention include, but are not limited to, fluorophores, chromophores, photodegradable or photoactivated compounds, thermally activated or thermally degradable compounds or functional groups, and / or compounds that bind radioactive isotopes or magnetic or conductive elements. The solid support can also be altered to change conformation, shape, and / or other physical properties in response to a defined event, by methods such as, but not limited to, using different polymers with different physical and thermal properties or varying degrees of cross-linking between polymer chains for a particular population.

[0155] In some embodiments, the method of the present invention is used to detect more than two different nucleic acid populations after PCR. PCR has many applications, such as detecting trace nucleic acids to determine the presence of pathogenic organisms, gene expression, genotype, genetic modification or modification, and forensic science applications. PCR amplification provides excellent target identification and quantification over a large analyte concentration range. However, simultaneous and quantitative analysis of multiple analytes by PCR has proven to be challenging. Detection based on intercalating dye fluorescence can only determine total dsDNA concentration and therefore it is impossible to use this detection method to simultaneously analyze multiple templates in a single reaction vessel. Fluorescent probe technology (e.g., Taqman, molecular beacons or other chemistries) can be used for low-level multiplexing of reactions because different color fluorescent probes can be used as signal reporters to amplify each target. Probes are also sequence-specific, which reduces false positives from primer dimer formation or nonspecific amplification. A typical multiplexing method utilizing conventional microtiter plates or microfluidic real-time PCR (rt-PCR) is to use a small amount of reaction wells, each of which contains three different color probes. However, designing multiplexed primer and probe sets is generally considered challenging, requiring additional levels of careful design and optimization of primers to ensure compatibility with each other. Multiplexing by this approach is ultimately limited by instrumentation and spectral overlap between dyes, limited to four-color detection, where one color is typically reserved as an internal standard dye. However, the present invention can provide methods for increasing assay multiplexing.

[0156] Figure 8 A microfluidic device 20 having a bead well array 30 is shown according to an embodiment of the present invention. The device 20 may include a freeze-thaw valve 22 having a microfluidic channel 20ch in fluid communication with the bead well array 30.

[0157] See also Fig.9A, 9B, 10A, 10B, 11A, 11B, 12A and 12B show examples of bead wells 10 having geometric structures for improved signal detection. Fig.9A and 9B As shown in , the solid substrate 25 can be combined with a conventional bead hole 10 having a circular circumference (typically a cylindrical or conical hole) that is configured to accommodate beads. In other embodiments, such as Fig. 10A , 10B, 11A, 11B, 12A and 12B, for example, the bead hole 10 according to an embodiment of the present invention has a geometric structure in which the bead retention section 11 and the signal detection section 15 adjacent thereto are fluidically connected.

[0158] The bead wells 10 may be arranged in a relatively dense array 30 having closely spaced bead wells 10. The bead wells 10 may be arranged in rows and columns or offset from one another. The bead wells 10 may be arranged in a regularly repeating pattern, an irregularly repeating pattern, or in other patterns. The term "dense" means that the footprint of the fluid analysis device may have about 100-6000 wells 10 / mm 2 and / or 10,000 to 5,000,000 pores 10 / cm 2 , typically about 6,000 to about 2,000,000 pores 10 / cm 2 , more typically about 500,000 to about 2,000,000 pores 10 / cm 2 .

[0159] Some or all of the adjacent holes 10 may have a spacing distance of: 0.11-1,000 µm, such as about 1 µm, 2 µm, 3 µm, 4 µm, 5 µm, 6 µm, 7 µm, 8 µm, 9 µm, 10 µm, 11 µm, 12 µm, 13 µm, 14 µm, 15 µm, 16 µm, 17 µm, 18 µm, 19 µm, 20 µm, 100 µm, 200 µm, 300 µm, 400 µm, 500 µm, 600 µm, 700 µm, 800 µm, 900 µm, 1000 µm or any fraction therebetween (measured from centerline to centerline of adjacent bead retention segments 11).

[0160] Some or all of the wells 10 may be reaction wells 10 that incorporate amplification reagents. Some or all of the wells 10 may process sub-pL reaction volumes.

[0161] The signal detection segment 15 may optionally include a short and / or long fluid channel 15ch that connects the end 15e of the signal detection segment to the bead retention segment 11. The signal detection segment 15 may include an end 15e separated from the bead retention segment 11 by a distance "L", typically L is 30% to about 10,000% (.3X to about 100X) of the target bead diameter, more typically about .3X to about 10X of the target bead diameter. In some embodiments, L can be 1X to 5X of the target bead diameter. For example, for 3.2 μm diameter beads, the length L can be about 1 to about 320 μm, more typically 1 to 32 μm, and in a specific embodiment can be about 3.2 to 16 μm. For clarity, numbers with the letter "X" refer to multipliers, one times (1X), ten times (10X), etc.

[0162] In some embodiments, L can be from about 1 µm to about 100 µm, more typically from 1 to 20 µm, such as about 1 µm, about 2 µm, about 3 µm, about 4 µm, about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, about 10 µm, about 11 µm, about 12 µm, about 13 µm, about 14 µm, about 15 µm, about 16 µm, about 17 µm, about 18 µm, about 19 µm, and about 20 µm. The length L can be measured from the centerline of the bead retention segment 11 or from a line drawn on the channel 15c connecting the bead retention segment 11 to the end 15e of the signal detection segment that completes the diameter of the bead retention segment.

[0163] Fig. 10A and 10B The display signal detection section 15 may optionally be configured as a narrow channel or “slit”, typically having a width that is 25%-75% less than the width of the bead retention section 11 .

[0164] Fig.11A and 11B The display signal detection section 15 may have an arcuate end portion 15a having a radius of curvature smaller than that of the bead retaining section 11. The distance "d" from the bead retaining section midline to the arcuate end 15e may be the same as the above-mentioned length "L".

[0165] Fig. 12A and 12B The signal detection section 15 can be shown to have a distal end having an annular channel 17 surrounding an internal upwardly protruding element 19. The protruding element 19 can have a height sufficient to extend above the liquid in the annular channel 17 of the signal detection section 15. This structure can define a detectable signal including a portion having an annular shape.

[0166] It is believed that the novel geometry of bead well 10 improves detection in microbead array-based technologies. These well geometries can be configured so that one area of ​​well 11 is used for magnetic loading and bead retention, while another area of ​​well 15 can be used (primarily or only) for detection of signals. In some embodiments, after placing beads 25 (in Fig. 10A Once loaded into the bead region 11 of well 10 (see each of 11A and 12A, exemplarily shown as a solid circle in one well), small volumes of reagent fluid can be separated in well 10 using methods known to those skilled in the art such as sealing with immiscible fluids or pressing against another surface such as a gasket or other matrix.

[0167] Although some example embodiments are shown in Fig. 10A , 10B, 11A, 11B, 12A and 12B, other embodiments may include other geometries, typically configured such that the bead retention area 11 has pockets or receptacles having an opening diameter of about 101-195%, and in some embodiments about 105% to 150%, of the diameter of the bead 25. The depth of the holes in the bead retention area 11 may be about 50% to 185% of the bead diameter.

[0168] The depth of the well 10 at some or all of the signal detection zone 15 may be the same as, deeper than, or shallower than the well at the bead retention section 11. The depth may decrease as the channel 15ch extends away from the bead retention section 11. The depth may increase as the channel 15ch extends away from the bead retention section 11 and toward the end of the signal detection section 15. The wall of the well 10 may taper outwardly or inwardly in the direction of entry into the well 10.

[0169] Different wells 10 may have different volumetric capacities, geometric patterns and / or sizes.

[0170] In some embodiments, the signal detection section or region 15 may have a channel 15ch such as a pocket or slit or other geometric shape that is physically inaccessible to the target beads. The regions 11, 15 are fluidly connected so that the reagents and / or analytes released by the beads 25 can diffuse or otherwise mix into the entire common solution volume of the well 10. By spatially separating the beads 25 from the detection zone 11, the contribution of the bead fluorescence to the signal can be reduced or eliminated, improving the signal-to-noise ratio.

[0171] The geometric structure of the hole 10 can allow high single occupancy loading of the reaction hole 10 while increasing the corresponding reaction volume, thereby potentially improving the reaction efficiency.

[0172] Fig.13AAn analysis system 100 is shown having a defined event device 125, which includes at least one heater input 125h as a dynamic event applicator. The heater 125h can be a heating element, an oven or a heat gun or other suitable heater located below and / or above the array 30. The heat defined event input can be configured to increase the temperature to above ambient temperature, typically to at least 1 degree higher than 25°C, typically about 35 ºC to 200 ºC. The heater 125h can apply the critical temperature to the array 30 for 1 microsecond-50 hours, typically about 1 second to about 5 minutes in some embodiments. The solid support 10 can be returned to ambient temperature or room temperature and / or about 25 ºC or other temperature, typically to a temperature approximately the same as the temperature used for the decoded image obtained before the defined event before the detector 150 obtains the image after the defined event. After removing the heat input, forced cooling or passive cooling can be used.

[0173] Fig. 13B The analysis system 100 is shown, which has a defined event device 125 comprising at least one light source 125l as a dynamic event applicator. The light source 125l can be a single emitter emitting light at a defined wavelength or wavelength range or can include a plurality of emitters of different wavelengths, wavelength ranges or a plurality of emitters of the same wavelength or wavelength range to, for example, cover a larger area. The defined event input 125l can be configured to actively emit light to the solid support 10 for a suitable time, such as about 1 microsecond to 50 hours, typically 1 second to 5 minutes, as described above.

[0174] Fig. 13C An analytical system 100 is shown having a circuit 100c, wherein the detector 150 is a defined event device including an associated light emission source as known to those skilled in the art.

[0175] like Figures 14A-14E As shown in , the microfluidic device 20 may include a well array 30 having a plurality of wells 10. The wells 10 may hold beads 25 in a bead retention section 11. The beads 25 may optionally include primers attached thereto and may optionally be preloaded into the bead retention section 11 of the wells 10. The device 20 may include upper and lower matrices 50u, 50b ( Fig. 14B, 14C). The upper substrate 50u can be the same as or different from the lower substrate 50b. One or both of the substrates 50u can be rigid and include, for example, glass, quartz, silicon or a suitable metal. One or both of the substrates 50b can be polymeric, such as silicone or other polymeric materials (especially such as PMMA, COC, COP, PDMS, PP, PE, PTFE or Kapton (polyamide)), and it can provide an array of bead holes 10. The upper or lower substrate 50u can have at least one port 50p in fluid communication with the hole 10. During reagent filling and / or oil sealing, the reaction hole 10 can optionally be perpendicular to the flow through the array chamber having the hole array 30, for example: However, other arrangement structures can be used.

[0176] Fig.14A The microfluidic chip 20 is shown to be configured such that the bead well array 30 occupies a footprint "F" (typically 1 mm to 10 cm) with densely packed array wells. The fluidic microchip 20 may include transport channels for sample and reagent or other chemical additions that may be in fluid communication with the array 30, as known to those skilled in the art.

[0177] Fig.14D Optional spacers 50s are shown which may be located between the upper and lower substrates 50u, 50b. The spacers 50s may define some or all of the sidewalls of each hole 10. The spacers 50s may comprise a photoresist that functions as both a liner and a spacer.

[0178] Fig.14E The sealant 75 such as sealing oil is shown to separate adjacent holes 10 with beads 25, as known to those skilled in the art. A hydrophobic membrane may also or alternatively be used. The sealant 75 (e.g., oil) may extend over the top of the bottom substrate 50b. The sealant 75 may have a thickness or depth associated with the spacer / pad 50s. The sealant 75 and may cover all except the beads / reaction holes 10. The sealant 75 may contain mineral, silicone, hydrocarbon or fluorocarbon-based oils and / or waxes. The reaction solution 23, e.g., an aqueous solution, is typically not in contact with the top substrate 50u.

[0179] This technology can be particularly advantageous for bead-based assays involving amplification methods such as polymerase chain reaction (PCR) or enzyme-linked immunosorbent assay (ELISA) that use fluorescent signal readout in analog or digital detection modes. In these assays, zero, one or more analyte molecules are captured by a solid support, which may optionally include beads such as magnetic beads. The solid support may then be loaded into a microwell array 30, the geometry of which limits the loading of solid supports 25 to one or zero / hole 10. A small volume of amplification reagent may be sealed in each reaction well 10 with a solid support 25, and a chemical reaction may be performed to produce a fluorescent signal (if an analyte molecule is present). The fluorescent signal may then be measured, processed and used to determine the analyte concentration in the sample.

[0180] Single-channel reactions in dense arrays (SiRCA) are massively parallel amplification and detection methods utilizing a microbead-array format that can potentially perform hundreds, thousands, millions, or billions of separate single-channel rt-PCR reactions for multiple different target sequences simultaneously. See, e.g., U.S. Patent Application Serial No. 14 / 402,565, which describes, e.g., bead-based delivery of reagents for parallel assays on multiplexed-in-space microfluidic devices; the contents of which are incorporated herein by reference in their entirety.

[0181] The ability of single-copy digital quantification of low analyte concentration and multi-copy analog rt-PCR quantification of high analyte concentration is given in the case of aM LOD.In some embodiments, array 30 can be used together with individual groups of biotinylated primers connected to dye coding, streptavidin labeling, magnetic microspheres or beads.A bead group library containing as many as tens to hundreds of bead types can be prepared, each of which has different primer sets for different target sequences, and new bead groups can be added to bead mixtures at will.When bead mixtures are incubated with analyte DNA or RNA, the primers connected to beads serve as hybridization probes, capture and purify nucleic acid sequences specific to the beads.Sample matrix interfering substances (exogenous DNA, RNA, cell membrane components, etc.) can be removed by magnetic separation and washing beads.After clearing, add polymerase, dNTP and intercalating dye, and beads are loaded into individual holes 10 and sealed to each other using immiscible oil or hydrophobic film. Random loading of individual encoded primer beads into separate microreaction wells can be accomplished in seconds to minutes, unlike manual pipetting or reagent printing. Magnetic loading of beads into optimized geometries can be more efficient than random separation by dilution into droplets or reaction wells that do not contain areas designed for bead capture and / or separate detection zones for well 10. The streptavidin / biotin interaction is very stable at temperatures below 50 °C; however, the elevated temperatures used for denaturation during PCR can allow for subsequent release of the primers during the first rt-PCR cycle that begins amplifying the target DNA.

[0182] To evaluate the performance of SiRCA in sub-pL reactions, preliminary tests using primers and gDNA were performed in the absence of beads in a silicon fluidic chip prepared by deep reactive ion etching (DRIE) in silicon with an array of wells (3.7 µm diameter, 5 µm depth (cylindrical, volume ≈ 50 fL), with a center-to-center spacing of 10 µm). Thus, rt-PCR was performed in ≈ 50 fL cylindrical reaction wells with primers and gDNA free in solution without beads. The chip was pretreated with octyltrichlorosilane in heptane to make the surface hydrophobic. The chip was wetted with ethanol, then with water, and then blocked with loading buffer (20 mM Trizma, 50 mM KCl, 2.5 mM MgCl2, 1% BSA, and 0.1% Tween 20). A master mix (1x Platinum Quantitative PCR SuperMix-UDG with 0.0625 units / µL extra Platinum Taq DNA polymerase, 0.5% BSA, 2.7 µM primers, gDNA from S. mutans, and 3x SYBR Green) was added to the chip and Krytox GPL104 perfluorinated oil was then pulled through the chip to seal the reaction volumes to each other.

[0183] Embodiments of the present invention include reaction wells 10 having a geometry configured to contain more than one bead so that reactions between reagents released from more than one bead and reacting in solution or on the surface of more than one bead or any combination thereof can be studied in a designated detection zone as shown or alternatively on the surface of the bead. The bead well array 30 may include variations in which some wells 10 accommodate one bead 25, some wells 10 accommodate two beads 25, and some wells 10 accommodate more than two beads 25 on a single substrate or fluid device or on a separate substrate or device. When the well 10 includes more than one bead retention section 11, multiple (typically each) bead retention sections 11 can be fluidically connected to each other via a signal detection section such as a fluid channel 15ch. A kit that can be used to perform the method of the present invention can generally include one or more beads having an encoding medium and optionally reagents for performing the method as described above, such as restriction enzymes, connected thereto, optionally packaged together with instructions for use for performing the method. The kit can also include a container for accommodating the elements contained therein. Such containers include, but are not limited to, vials, microfluidic chips with bead well arrays and cartridges, including pre-loaded bead devices. Fig.19The packaging of a kit 500 having encoded beads 25 suitable for a dynamic decoding scheme is shown. The kit 500 may include one type / population of encoded beads 25 (i.e., beads that all have the same encoding signal or "fingerprint") or may include two or more different types / populations of encoded beads 25 (i.e., at least two different bead populations, one having a first encoding signal and one having a second encoding signal different from the first encoding signal). When the kit 500 includes two or more different types of encoded beads 25, the kit 500 may include the encoded beads 25 as a mixture in a single package or may package each encoded bead population separately (e.g., a bead population having a first encoding signal is packaged separately and a bead population having a second encoding signal is packaged separately).

[0184] In some embodiments, beads 25 may have primers connected thereto. Primers may be biotinylated primer pairs, and beads 25 may be streptavidin-labeled so that biotinylated primers and beads are combined. In some embodiments, beads 25 may include markers, such as optical markers, which may be used to identify primers anchored to each bead 25 during analysis. For example, beads with different encodings of the same or different sizes may be labeled for identifying different connected primer sets during analysis. Multiple precoding methods may be used to provide markers on beads 25, including predefined sizes, shapes, magnetic properties and / or fluorescent doping, which may be used alone or in combination with other coding methods. Custom sequence biotinylated primers and streptavidin-labeled paramagnetic beads may be easily purchased from commercial sellers or prepared in a laboratory with suitable equipment in a suitable amount.

[0185] As described above, although embodiments according to the present invention are also described herein with respect to PCR reactions, it should be understood that the microfluidic devices, beads, and reaction methods described herein can be used for a variety of other reactions, for example, when reagents are separated from beads and enter wells to participate in the reaction. For example, any nucleic acid transcription and / or amplification-related reaction is within the scope of the present invention, including but not limited to PCR reactions, real-time PCR (rt-PCR), digital PCR (dPCR), RNA reverse transcription into cDNA (RT), PCR of cDNA from a previous RT step (RT-PCR), RT-PCR using real-time or digital quantification, immuno-PCR (iPCR) and variants thereof, loop-mediated isothermal amplification (LAMP), rolling cycle replication, and / or non-enzymatic nucleic acid amplification methods (e.g., "DNA loops"). Other reactions included within the scope of the invention include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), single molecule arrays (SiMoA) or digital ELISAs, ELISAs in which at certain points a fluorescent substrate is bound to a support surface to be decomposed for subsequent reactions, reactions in which multiple beads are used to deliver different reagents for combinatorial chemistry, reactions in which the beads deliver catalytic reagents, and / or reactions in which "click" chemical reagents are delivered in stoichiometries determined by random bead loadings.

[0186] Fig.151 is a schematic diagram of an analysis system 100. The system 100 may include at least one controller 12 (typically including at least one processor) in communication with a signal detector 150 such as a camera or other imaging device. The signal detector 150 may be configured to detect a signal (e.g., a determination signal and / or a coding signal) of a solid matrix such as beads 25 in a bead hole array 30 of each microfluidic chip 20 having a hole array 30. The controller 12 may be in communication with an input device 125. Although shown as a separate device, the input device 125 may exist as part of a system device. For example, the input device 125 may be an optical system, an imaging device, a thermal device, etc. and / or a part thereof. The controller 12 may be in communication with an image processor 211 having a signal detection utilizing an analysis module 221 (e.g., a computer program) and a decoding module 40, the analysis module 221 being configured to identify whether a determination signal exists in a signal detection section of a reaction hole, and the decoding module 40 being configured to obtain a coding signal. The image processor module 211 may be airborne in whole or in part or may be remote from the controller and / or the signal detector 150. The image processor module 211 can be configured to merge multiple post-assay images of a well to assess whether the well has a positive readout. The image processor module 211 can remove background noise from the bead retention zone of each well. The module 211 can be configured to identify the presence of a signal from the detection zone of each well to identify whether a positive assay reaction has occurred based on the assay readout 222. The detector 150 can also detect the encoded signal using a decode readout 40R.

[0187] Fig.16 2 is a flow chart that can be used to perform an example run of an analytical method according to an embodiment of the present invention. A fluidic device having a bead hole is provided (frame 250). A coded signal is obtained from the hole while the beads are held in the bead hole, which may include obtaining a first image of the beads held in the bead hole (frame 260). Then at least some of the beads undergo a physical and / or chemical change (frame 272). A second image is then obtained (frame 274). Different bead populations are distinguished by comparing the coded signal from each bead in the first and second images (frame 276).

[0188] In these and / or other embodiments, the coded signal can be read from the section containing beads (i.e., read directly from the beads) to determine what reagents and / or analytes the beads may contain. In this way, the array can be decoded to determine the meaning of the positive or negative assay signal. The beads can be encoded by means known to those skilled in the art, including fluorescent dye staining of more than one dye with more than one intensity level, bead diameter, bead shape, or any combination of defined and / or observable or detectable properties. The beads can also be encoded by any method described herein.

[0189] The bead wells may be arranged into a dense array of bead wells defining a reaction well (block 252). The bead well may have a bead retention section that may have a geometry and a diameter equal to or greater than the bead diameter, and a signal detection section, when used, having a geometry with a width less than the bead diameter (block 254).

[0190] The signal detection segment can include an elongated channel narrower than the diameter of the bead and the bead hole geometry can be configured to have one or more of the following: (a) a slit, (b) an asymmetric circular perimeter separated by elongated channels, (c) a bull's eye form (i.e., an elongated channel merged into an annular channel), (d) multiple separated circular and / or curvilinear perimeters joined together by at least one elongated channel extending therebetween (box 256).

[0191] The bead retention zone may be sized and configured to accommodate only a single magnetic bead (block 257).

[0192] The bead wells may contain more than one amplification reagent (block 258).

[0193] The bead well can have a detection segment that can be separated from a corresponding bead retention segment by a distance L that is .3X to 100X the target bead diameter, e.g., 1 to 1000 μm (box 259).

[0194] If the analyte molecule is present, the signal may include a fluorescent signal (with a tail) (block 261).

[0195] Fig.17 is a flow chart of a method of making an example device according to an embodiment of the present invention. An array of wells is arranged (e.g., formed) in a matrix; the wells have a volume capacity of 1 aL to 100 µL, more typically 1 fL to 1 µL, and have one or more defined geometries in a defined footprint with a linear dimension of 1 mm to 10 cm, the defined geometry including a bead retention segment in fluid communication with a signal detection segment spatially separated from the bead retention segment (block 300).

[0196] An array of holes may be formed by etching in a substrate (block 302).

[0197] The array of holes can be molded into the substrate (block 304). Other fabrication methods are contemplated, for example, photolithography, FIB milling, embossing, stamping, etc.

[0198] The bead well has a bead retention segment having a diameter that can be between about 10 nm and 5,000 µm, typically about 3.1-3300 µm incorporated into the channel, a width of about 2-3 µm, and a length L that is between .3X and 100X the target bead diameter, e.g., 1-100 µm and / or 1-10 µm, in some embodiments, forming an optional signal detection segment (block 306).

[0199] The matrix is ​​sterilized and provided in packaging and / or kits for use in fluid analysis of target analytes using the magnetic beads bound to the wells (block 308).

[0200] The well may be a reaction well having more than one amplification reagent.

[0201] Hole array can be 6000 / mm 2 To about 10,000 / mm 2 , typically about 6,000 / mm 2 The density arrangement is performed (block 312).

[0202] 6,000 / cm 2 to about 5,000,000 / cm 2 , more typically 6,000 / cm 2 to about 2,000,000 / cm 2 A hole array is provided at a density of (block 314).

[0203] The bead retention segment may have a perimeter having a width that is the same as or within about 195% of the diameter of the target (e.g., magnetic) bead, e.g., 101% to 195% or 150% of the target bead diameter (box 316).

[0204] In the case of a bead volume of 20 fL, the well may have a reaction volume of 100-150 fL (block 318).

[0205] Fig.18 3 is a flow chart of a method for evaluating an assay according to an embodiment of the present invention. The assay signal is electronically detected by a plurality of reaction wells with beads (frame 350). The term "electronic" refers to all forms of machine-based detection (non-human eyes) such as cameras, e.g., CCD cameras, CMOS cameras, electrode arrays, SEMs, etc. The signal can be an increase in intensity, fluorescence, more than one defined color, defined pixel parameters, etc., including changes in light scattering properties (transparency), changes in transmission or absorption, chemiluminescence, or a combination of different parameters. Before, during, and / or after the assay is completed, multiple images are obtained, including a first image, and subsequent images are obtained after a defined event to identify a coded signal to decode the assay to identify a population of beads in the assay sample (frame 370).

[0206] The reaction wells may optionally have a signal readout segment, and after the reaction has occurred the method may electronically detect the assay signal from the array of bead wells, each having a signal readout segment spatially separated from the bead retention segment, to identify positives (block 352).

[0207] The signal readout section comprises an elongated channel having a width and / or depth that is less than the diameter of the bead retention section (block 354).

[0208] The signal readout section includes an annular fluid channel in fluid communication with the bead retention section (block 356).

[0209] The signal is determined based on a PCR or ELISA assay (block 358).

[0210] Multiple primer sets can be screened against each other or against different reference genomes using beads of the same size or beads of different sizes, with at least two beads contained by a single reaction well having two (optionally different sized) bead retention segments and at least one separate readout segment (block 360). The beads can be encoded by any of the methods listed herein or in those sources referenced herein.

[0211] The assay signal may be characterized by nucleic acid from a clinical or laboratory sample using a defined set of beads (block 362).

[0212] Pre-Example 1-10

[0213] The following pre-illustrative embodiments are described about a plurality of beads that can be decoded using the method of the present invention to identify different bead populations. However, other types of solid supports can be used. In addition, each of the following pre-illustrative embodiments can be combined with one or more embodiments utilizing the time domain of the same and / or different limiting events. For example, a plurality of beads can include more than one bead population, and the more than one bead population can be identified using a coding medium, and the coding medium is physically and / or chemically changed in response to light exposure of a specific wavelength, and a plurality of beads can include more than one bead population, and the more than one bead population can be identified using a coding medium, and the coding medium is physically and / or chemically changed in response to light exposure of the same and / or different wavelengths and / or changes in response to temperature and / or chemical environment. For example, the coded signal generated by the coding medium of each bead before the physical and / or chemical change can be different from the coded signal generated by the coding medium of each bead after the physical and / or chemical change.

[0214] In each of Examples 1-10, a plurality of coded beads may be provided, the plurality of coded beads comprising different bead populations / subgroups, the different bead populations / subgroups may be identified by comparing the fluorescence intensity of the first and / or second coded media connected to each bead at a first time point before a defined event and at another time point during and / or after the defined event. The first bead population may include a first coded media (e.g., FP, organic dye and / or quantum dot) connected thereto and the second bead population may include a second coded media (e.g., FP, organic dye and / or quantum dot) connected thereto. The other bead populations may include both the first and second coded media connected thereto at different ratios (e.g., the first coded media: the ratio of the second coded media is 25:75, 50:50, or 75:25). For example, the third population may have a ratio of 30:70, the fourth population may have a ratio of 50:50, etc. The first and second coded media may have the same and / or similar fluorescence excitation / emission properties and / or intensities such that the first, second and other bead populations are indistinguishable at the first time point before the defined event. However, at least one of the coded media may not be stable relative to the defined event. Therefore, during and / or after exposure to the defined event, the first and second coded media may display different fluorescence intensities, which allows the two coded media to be distinguished. Therefore, the first and second bead populations can be identified by comparing the fluorescence intensity before the defined event and the fluorescence intensity during and / or after the defined event. It is also possible to identify additional bead populations by the fluorescence intensity or change in fluorescence intensity after the defined event, because the ratio provides different fluorescence intensity levels or gradients between the intensities of the first and second coded media. The number of additional bead populations that can be provided can depend on the number of additional fluorescence intensities that can be identified (i.e., distinguishable) at a time point during and / or after the defined event.

[0215] In these examples, it should be noted that advantageously there are a plurality of substrate populations that can be distinguished based on the initial reading or measurement of its property, but in each initially distinguishable population, there are initially indistinguishable different subpopulations.After limiting an event (or a series of limiting events or a plurality of simultaneous limiting events), by comparing the initial signal measurement of different subpopulations and the signal measurement that occurs after and / or during the limiting event, different subpopulations will be distinguishable and identifiable. In this way, all multi-path populations are distinguished from each other by decoding. Although hereinafter the population can be described as being indistinguishable, it should be understood that it can describe the initial indistinguishable subpopulation of the larger substrate group of the distinguishable properties that can have some initial classifications. Although hereinafter the limiting event can be described as causing the physical and / or chemical changes that produce different intensity levels for a given subpopulation, it should be understood that the intensity levels of these gained are "different" compared to the intensity level measured for the specific subpopulation before limiting an event, and can correspond to and before limiting an event in some cases, after or during the initial intensity level or the similar or identical intensity level of the final intensity level of other subpopulations that are distinguished from a given subpopulation by another distinguishable property. Thus, comparing the encoded signal of a particular bead at different time points (which may include time points before, after or during a defined event) can be used to distinguish the beads from one another.

[0216] Example 1

[0217] The photostability of organic dyes in the time domain can be used to decode multiple encoded beads. The first bead population can include a first organic dye connected thereto and the second bead population can include a second organic dye connected thereto. Additional bead populations can include both the first and second organic dyes connected thereto in different ratios, as described above. The first and second organic dyes can have the same and / or similar fluorescence excitation / emission properties and / or intensities so that the first, second and additional bead populations are indistinguishable at a first time point before a defined event. After exposure to light for a defined time, typically about 30 seconds to 5 minutes, such as about 1 minute, about 2 minutes, about 3 minutes or longer, the first and second organic dyes can be susceptible to different levels of photobleaching. Therefore, after photobleaching for a defined time (e.g., at least one microsecond), different bead populations have different fluorescence intensities. For example, compared to the second organic dye (i.e., dynamic element), the first organic dye can be less susceptible to photobleaching (i.e., more stable), and the fluorescence intensity of the first organic dye can only decrease by 10% after 1 minute, while the fluorescence intensity of the second organic dye can decrease by 90% after 1 minute. Thus, the first and second bead populations can be identified by the fluorescence intensity after a defined event (i.e., photobleaching). Additional bead populations can also be identified by the fluorescence intensity after a defined event, since the ratio provides different levels or gradients of fluorescence intensity between the intensities of the first and second organic dyes.

[0218] Example 2

[0219] The photostability of organic dyes and quantum dots in the time domain can be used to decode multiple encoded beads. The first bead population can include an organic dye connected thereto and the second bead population can include quantum dots connected thereto. The other bead population can include both the first organic dye and the organic dots connected thereto in different ratios, as described above. The first organic dye and the quantum dots can have the same and / or similar fluorescence excitation / emission properties and / or intensities so that the first, second and other bead populations are indistinguishable at the first time point before the defined event. The first organic dye (i.e., dynamic element) can be susceptible to photobleaching after exposure to light for more than 1 minute, while the quantum dots are not susceptible to photobleaching. Therefore, after photobleaching for at least one minute, different bead populations have different fluorescence intensities. For example, the fluorescence intensity of beads having only the first organic dye can be significantly reduced after a defined time (e.g., 1 minute), while the fluorescence intensity of beads having only quantum dots can not be reduced after 1 minute. Therefore, the first and second bead populations can be identified by the fluorescence intensity after a defined event (i.e., photobleaching). Additional bead populations can also be identified by the fluorescence intensity after a defined event, as the ratio provides different levels or gradients of fluorescence intensity between the intensity of the first organic dye and the quantum dots.

[0220] Example 3

[0221] The thermal stability of the coding medium can be used to decode multiple coded beads by comparing the fluorescence intensity of the first and / or second coding medium connected to each bead at a first time point before the thermal defined event and another time point during and / or after the thermal defined event. The first bead population can include a first coding medium (e.g., an organic dye and / or quantum dots) connected thereto and the second bead population can include a second coding medium (e.g., an organic dye and / or quantum dots) connected thereto. The other bead population can include both the first and second coding media connected thereto at different ratios, as described above. At least one of the coding media may not be stable for an increase or decrease in temperature. For example, the coding medium (i.e., a dynamic element) may undergo a structural and / or charge change in response to a defined thermal event, which may provide a change in the fluorescence intensity of the coding medium. In some embodiments, a thermal defined event may cause changes associated with the coding medium and / or beads and / or destroy chemical bonds associated with the coding medium and / or beads, such as, for example, chemical bonds of a binding pair (e.g., streptavidin-biotin) connecting the coding medium to the beads. Thus, during and / or after exposure to a temperature change, the first and second coded media can display different fluorescence intensities, which allows for differentiation of the two coded media. Thus, the first and second bead populations can be identified by the fluorescence intensity after a defined event (i.e., an increase or decrease in temperature). Additional bead populations can also be identified by the fluorescence intensity after a defined event, as the ratio provides different levels or gradients of fluorescence intensity between the intensities of the first and second coded media.

[0222] Example 4

[0223] A thermally defined event can activate or inactivate a reagent (e.g., a quencher) that affects the fluorescence intensity of at least one coded medium (i.e., a dynamic element). For example, a thermally defined event of a temperature rise can activate a heat-labile compound. The reagent can interact with the coded medium in response to the thermally defined event, no longer interact with the coded medium, and / or can affect the chemical properties of the environment in which the coded medium is present (e.g., changing pH), which can provide a change in the fluorescence intensity of the coded medium. Therefore, during and / or after exposure to a thermally defined event, the first and second coded media can display different fluorescence intensities, which allows the two coded media to be distinguished. Therefore, the first and second bead populations can be identified by the fluorescence intensity after the defined event (i.e., an increase or decrease in temperature). Additional bead populations can also be identified by the fluorescence intensity after the defined event, because the ratio provides different levels or gradients of fluorescence intensity between the intensities of the first and second coded media.

[0224] Example 5

[0225] An optically defined event can activate or inactivate a reagent (e.g., a quencher) that affects the fluorescence intensity of at least one coded medium (i.e., a dynamic element). For example, a light-activated compound can be present with the coded beads and can be activated by an optically defined event that exposes the coded beads and reagents to light. The reagent can interact with the coded medium in response to the defined event, no longer interact with the coded medium, and / or can affect the chemical properties of the environment in which the coded medium is present (e.g., changing pH), which can provide a change in the fluorescence intensity of the coded medium. Therefore, during and / or after exposure to an optically defined event, the first and second coded media can display different fluorescence intensities, which allows the two coded media to be distinguished. Therefore, the first and second bead populations can be identified by the fluorescence intensity after a defined optical event (e.g., exposure to light of a given wavelength and / or wavelength range). Another bead population can also be identified by the fluorescence intensity after the defined event because the ratio provides different levels or gradients of fluorescence intensity between the intensities of the first and second coded media.

[0226] Example 6

[0227] Chemically defined events can be used to identify different bead populations / subgroups. Changes in the chemical environment can cause changes in the fluorescence intensity of at least one coded medium. For example, chemically defined events can cause and / or provide changes in the pH of the bead environment, change the composition (e.g., remove and / or add chemicals / ingredients and / or change solvents), activate or inactivate reagents (e.g., quenchers) that affect the fluorescence intensity of at least one coded medium, and / or change the stability of the binding complex (which can cause changes in the fluorescence intensity of the coded medium). Therefore, during and / or after the defined event, the first and second coded media can display different fluorescence intensities, which allows two types of coded media to be distinguished. Therefore, the first and second bead populations can be identified by the fluorescence intensity after the defined event. Other bead populations can also be identified by the fluorescence intensity after the defined event, because the ratio provides different levels or gradients of fluorescence intensity between the intensity of the first and second coded media.

[0228] Example 7

[0229] In some embodiments, if a defining event provides a fluorescence intensity that does not exist before the defining event and is displayed after the defining event and is different from the fluorescence intensity of other bead populations, then for those described in Examples 1-6 above, additional populations (e.g., a third population) can be identified. For example, the third bead population can include a coded medium that does not display fluorescence at a first time point before the defining event. Therefore, the first, second, and third bead populations are indistinguishable using fluorescence intensity at the first time point. However, the defining event can activate the fluorescence of the coded medium so that after the defining event, a third fluorescence intensity different from the fluorescence intensity of the first and second bead populations is passed.

[0230] Example 8

[0231] Table 3 shows a conventional fluorescence encoding scheme where three intensity levels (low, medium, high) of two different color dyes (red and blue) are used to encode nine populations of solid supports.

[0232] Table 3.

[0233]

[0234] Table 4 shows how encoding with four dyes (red stable, red unstable, blue stable, blue unstable, i.e., only two colors total) using a mixture to obtain 6 different dynamic properties for each color (low stays low, medium stays medium, medium bleaches to low, high stays high, high bleaches to medium, high bleachs to low) results in 36 different populations that can be distinguished using measurements of only two colors (red and blue) and three intensities (low, medium, high) before and after a defining event.

[0235] Table 4.

[0236]

[0237] Example 9

[0238] Table 5 shows a conventional coding scheme in which two colors (red and blue), three intensities (low, medium, high) and two sizes (small and large) are used to encode 18 substrate populations. It should be noted that the populations in Table 5 are similar to those in Table 3, but by adding a bead size dimension with two levels (small and large), the obtainable conventional coding levels are doubled from 9 to 18.

[0239] Table 5.

[0240]

[0241] Table 6 shows how the dynamic decoding method described herein can be used to further increase the number of distinguishable populations or encoding states by adding two dynamic dimensions related to bead size (swelling resulting in size increase or dissolution resulting in size decrease). This results in 54 different populations that can be distinguished based on changes observed (or not observed to occur) after a defined event. This increase in potential multiplexing can be achieved by measuring only two colors (red and blue) at three intensities (low, medium, high) and four sizes (large, larger, small, dissolved / not detected). This encoding scheme can be further expanded by combining with any other method described herein.

[0242] Table 6.

[0243]

[0244] Example 10

[0245] Fig. 20 and 21 An example of a device that is not array based but rather utilizes flow cytometry to perform bead measurements before or after a defined event is shown. Fig. 20 In the present invention, the solid support is measured before and after exposure to light to photobleach the one or more encoding mediators. Fig.21 Such an embodiment is shown in which the solid support is measured before and after mixing a sheath stream or an additional reagent stream into the bead-containing stream. The stream may contain reagents such as (but not limited to) buffers of different pH, solvents or ligands that affect at least one of the encoding medium or the solid support structure to cause a physical and / or chemical change. Comparison of the optical properties of the solid support before and after a defined event can be used to distinguish populations or subpopulations from each other.

[0246] Embodiment 11

[0247] Multiplexed bead-based bioassays typically require that each type of microsphere be uniquely encoded to distinguish the types from each other. Microspheres are typically encoded using fluorescent dyes with different spectral properties and varying concentrations. However, there are practical limits on the number of spectrally resolvable dyes or the distinguishable intensity levels of each dye. In order to expand the number of encoding levels, a method was developed that incorporates photobleaching kinetics into bead decoding, thereby unlocking additional multiplexing levels that are not available through conventional decoding methods. To demonstrate the technology, beads were encoded with two dyes with overlapping fluorescence excitation and emission wavelengths and different photostabilities. All beads initially displayed similar fluorescence intensities; however, after appropriate light exposure, the dye with lower photostability had a reduced emission intensity due to photobleaching. By comparing the original fluorescence emission intensity with the fluorescence intensity obtained after photobleaching, multiple different populations can be reliably identified. Using only a single excitation / emission band, two different initial intensity levels were optimized to produce six uniquely identifiable bead populations, while only two could be obtained using conventional decoding methods. Incorporating this encoding strategy into bead-based microwell array assays significantly increases the number of encoding levels that can be used for multiplexed assays without increasing the complexity of the imaging equipment.

[0248] The method incorporates a dynamic element into the decoding method. Instead of using a single time point to encode measurements, the method described herein exploits photobleaching kinetics to differentiate bead populations. Two dyes with overlapping spectral properties and different photostabilities are used to encode beads, which can then be differentiated based on a simple photobleaching rate measurement. The method reported here monitors photobleaching, which for these experiments occurs on a time scale of seconds, significantly increasing the multiplexing capacity of bead-based assays without increasing the complexity of the analytical platform.

[0249] Materials and methods

[0250] Bead Coding Method

[0251] Streptavidin-functionalized ProMag magnetic microspheres, typically 3 µm in diameter, were purchased from Bangs Laboratories (Fishers, IN). R-phycoerythrin (PE) biotin conjugates and 605 Qdot (QD) biotin conjugates (Life Technologies; Carlsbad, CA) were used as encoding dyes in this study. "High" level beads were labeled with 5 nM PE, 250 nM QD, or 18 nM PE / 22 nM QD mixtures. "Low" level beads were labeled with 0.6 nM PE, 4 nM QD, or 0.45 nM PE / 2 nM QD mixtures. The buffer used in this encoding method consisted of 1x Tris-buffered saline, pH 7.2 (Thermo Scientific; Waltham, MA) and 0.1% Tween 20 (Sigma Aldrich; St. Louis, MO).

[0252] A 10 µL aliquot of the bead stock solution (2.6% solids) was used for each labeling reaction. The beads were first washed 3x with a 100 µL aliquot of buffer, collected magnetically, and then resuspended in 10 µL. Encoding was performed by adding 15 µL of dye solution to the beads and incubating at ambient temperature for 1 h. The dye-labeled bead solution was protected from light and placed on a rotator (Labnet International; Edison, NJ) to ensure that the beads remained suspended during the labeling reaction. After functionalization, the beads were washed 3x and then resuspended in 20 µL of buffer and stored at 4 °C.

[0253] Microwell array preparation

[0254] Micropore array chips for these studies were prepared using silicon and glass. Silicon wafers were purchased from UniversityWafer (South Boston, MA) and coated with chromium and positive photoresist by NanoFilm (Westlake Village, CA). An array containing 3.5 μm diameter beads was photoetched on the wafer using a Heidelberg Instruments DWL FS66 laser printer (Heidelberg, Germany). After photoresist development and removal of exposed chromium, the wafer was etched to a depth of ≈5 μm in an Alcatel AMS 100 deep reactive ion etcher. 23 After etching, the remaining photoresist and chromium were removed from the wafer. Then SU-8 2050 (MicroChem; Westborough, MA) was formed on the wafer to form the external fluid boundary of the device. In order to complete the microchip, the vials containing glass matrix for fluid and the beads inlet were aligned on the silicon array and glued in place with epoxy glue (Loctite 120 HP). After bonding, the chips were treated with 1% trichloro(octyl)silane (Sigma Aldrich) in heptane (Fisher Scientific; Pittsburgh, PA) to render the surface hydrophobic.

[0255] Coding Experiments

[0256] The microchip was treated with a buffer solution containing 20 mM Tris, pH 8.0 (Life Technologies), 50 mM KCl (Life Technologies), 2.5 mM MgCl2 (Fluka; St. Louis, MO), 0.1% Tween 20, and 1% fetal bovine serum (Thermo Scientific). The beads were pipetted into a vial and placed on the array by magnetic force, wherein uniform loading was achieved across the chip. After loading, the chip was sealed with Krytox oil (DuPont; Wilmington, DE) to separate individual wells from each other. The sealed chip was kept in the dark until used for imaging.

[0257] The imaging system used in these experiments is composed of the following: Nikon AZ100 fluorescence microscope (Melville, NY), Prior Scientific Lumen 200Pro fluorescence illumination system (Rockland, MA) and Hamamatsu ORCAFlash 4.0 CMOS camera (Middlesex, NJ). The loaded chip is placed on the microscope platform and 2 s exposure is obtained every 2.5 s using Micro-Manager software. The excitation source shutter is fully opened only during exposure. The data points subsequently in this artwork show real-time x-axis time points (i.e. 2.5 s / image) rather than exposure time (i.e. 2 s / image). The image of the beads PE-encoded and QD-encoded is obtained using a single filter set (545 / 30 nm excitation, 620 / 60 nm emission).

[0258] In order to evaluate the bleaching performance, repeated chips were loaded with a single bead type and repeated imaging. This was done for all bead populations. The resulting images were processed with FIJI software. 26 After determining the position of each bead in the array well, the fluorescence signal of the entire group of images was determined. The fluorescence intensity was plotted as a function of the optical luminescence exposure time to compare the population decay characteristic curve. For histogram analysis, the normalized intensity values ​​from the last three images terminated at a given time point (e.g., when evaluating 90 s of exposure, t=80 s, 85 s, 90 s images were used) were averaged to minimize signal fluctuations and reduce noise. These endpoint decay values ​​were then sorted to determine the appropriate histogram bin range specific to each bead population.

[0259] Results and discussion

[0260] Photobleaching rate

[0261] The difference in the photostability of quantum dots and organic dyes provides an attractive opportunity for encoding microspheres when they have sufficiently overlapping excitation or emission maxima. The fluorescent protein phycoerythrin has a high fluorescence quantum yield but is susceptible to photobleaching after extended light exposure. In contrast, quantum dots are generally tolerant to photobleaching and maintain (or even increase) their observed emission after additional exposure. By encoding a small bead group with PE and another bead group with QD, it is assumed that the initial fluorescence between the two populations will appear the same, but it can be distinguished after extended exposure. Introducing photobleaching kinetics into such encoded measurements will therefore enable additional encoding levels that cannot be obtained by conventional single image decoding methods for increased multiplexing.

[0262] Sets of beads encoded with PE, QD, or a mixture of the two dyes were loaded into a microwell array chip and imaged repeatedly. Inspection of the resulting images revealed that the beads underwent different changes after prolonged imaging (see, e.g., Figure 3A , 3B and 3C). As predicted, beads encoded with PE experienced a significant decrease in fluorescence signal when the dye was bleached for 3 min, while the same time of light exposure did not substantially affect the fluorescence signal from QD-encoded beads. Beads labeled with a mixture of the two dyes showed a moderate decrease in fluorescence signal, which was not as severe as the PE-encoded population. The fluorescence decay characteristic curve plots showed good resolution between each bead group. Very similar bleaching rates were observed within each bead population, indicating that the dynamic decoding method has high accuracy.

[0263] Bleaching duration and population overlap

[0264] Although the mean fluorescence decay profiles of each bead group were quite different from each other, each population showed variation in photobleaching. Therefore, the boundaries of each population were checked to verify that they did not overlap with each other to avoid misidentification of beads. Fig. 22 The photobleaching profiles from the top and bottom 5% of beads in each population are shown. These traces show that not only can the average decays of each bead population be well distinguished from each other, but also the ranges within each group can be distinguished. Fig. 22 It also suggests that 3 min of photobleaching under these conditions is somewhat excessive, as the populations appear to be identifiable in a shorter time.

[0265] Knowing that both the throughput and performance of bioassays will benefit from a reduction in decoding time, the minimum photobleaching duration required to obtain acceptable inter-population resolution was sought within the parameters of our experimental settings. The endpoint fluorescence values ​​of individual beads after different lengths of photobleaching were placed in a histogram, where bleaching was characterized as "fast" (PE beads), "medium" (mixed beads), and "slow" (QD beads) (Table 7). Each population in the data set had >50k beads (n=5 chips) to provide a large enough sample size to accurately represent the population distribution. The decoded data showed that different bead groups were fairly well resolved within 30 s, although some overlap was still observed between mixed beads and other populations. The maximum misidentification after 30 s of bleaching was 1.7%, occurring between PE and mixed beads. Although this error is acceptable for certain applications that can tolerate a small amount of misidentification, it is too high for many digital low LOD assays. Generally, the percentage of bead misidentification should be less than the noise of the bioassay to ensure that it does not significantly affect the measurement accuracy. Furthermore, this short bleaching time necessitated the exclusion of 2.5% of the PE and mixed beads from the data set because they displayed fluorescence in the overlapping regions between adjacent bead populations. It was found that removing this relatively small percentage of beads significantly improved identification accuracy and removed ambiguity from the analysis.

[0266] Prolonging the photobleaching duration improved the resolution between bead populations (Table 7). After 1.5 min of bleaching, identification accuracy increased to >99.9% with <0.8% of beads excluded due to ambiguity. However, diminishing returns were observed when the bleaching time was further extended. Identification accuracy after 3 min of bleaching was >99.98% with an exclusion rate of <0.3%. The additional resolution, while better, is considered redundant for most bioassays where nonspecific binding causes 0.1-1% background signal. 7 Due to the combination of high encoding accuracy, low exclusion rate, and faster throughput, a 1.5 min light exposure time is considered optimal for bead decoding under these experimental parameters. Other systems with higher intensity lamps, wider bandpass filter sets, etc. can be used to further reduce this time.

[0267] Table 7: Percentage of beads sorted into each of the three photobleaching groups ("fast", "medium", "slow") based on endpoint fluorescence decay. The percentage of misidentified beads decreased as the bleaching time increased. Beads showing decay values ​​in the vague overlap region between populations were omitted from the data set ("excluded").

[0268]

[0269] Other encoding states

[0270] In order to further increase the number of encoding states using this dynamic decoding technology, new groups of beads ("low" beads) at lower initial intensity levels were developed using lower dye concentrations. The amount of each dye was optimized to obtain similar initial fluorescence values ​​for the three bleaching distinguishable bead types (PE, QD and mixtures, as described above), while also ensuring that they underwent a distinguishable degree of photobleaching attenuation. After optimizing the dye concentration, the bleaching characteristic curves of the three populations were measured. Each bead population showed a normalized photobleaching similar to the characteristic curve corresponding to the "high" intensity beads described in the previous section) with high accuracy. A histogram was constructed to measure the overlap between these "low" intensity bead populations after bleaching. The overlap between the three bead groups indicates that the "low" intensity level beads perform as well as the "high" intensity beads, and both can be easily implemented into multiplexed bead assays.

[0271] While optimizing the dye concentration, care should be taken to ensure that the initial intensity of the "low" intensity population does not overlap with the "high" encoding population. This is important because the normalized decay cannot be used to distinguish beads encoded with the same dye. Sorting the data into a histogram confirmed that the "high" and "low" intensity levels show no overlap. It is also visible that the wide range of fluorescence values ​​of the "high" population makes it difficult to achieve intermediate encoding levels (a common limitation that hinders a high degree of multiplexing in the case of conventional bead encoding methods). However, in the case of combining the time domain, multiple additional encoding levels are obtained, although the difference in initial intensity is large.

[0272] Finally, beads with two intensity levels (“high” and “low”) each with three distinguishable subpopulations (PE, QD, and mixed) were combined, enabling a total of six different encoding states to be achieved using single wavelength detection. Individual beads were first assigned to the “high” or “low” group based on t=0 fluorescence, and then imaging was repeated to identify the specific encoding dye used ( Fig.23 ). Compared to conventional encoding methods, this approach is demonstrated to significantly increase the multiplexing capacity of bead-based POC assays while also minimizing the cost and complexity of the assay optics.

[0273] Theoretical considerations

[0274] Standard encoding is typically based on a single time point observation of wavelength and intensity. The number of achievable encoding states is determined by the number of distinguishable populations for each excitation / emission band and can be expressed for three bands according to the following equation:

[0275]

[0276] Where T conventional is the total number of encoding states, n i , n j and nk are the numbers of intensity levels encoded in excitation / emission bands I, J, and K, respectively. The lowest level of fluorescence is level 0, or no encoding, where the presence of the encoding dye cannot be detected.

[0277] Mixing two or more dyes with similar spectral properties and different stabilities provides additional coding states within distinguishable fluorescence intensity levels. Figure S3 shows a diagram of a method of mixing two spectrally similar dyes together in different ratios to achieve 0-5 coding levels. The lowest level (0) uses no dye. The next level, 1, can use one equivalent of a stable or labile dye. Level 2 can use two equivalents of either dye or one equivalent of a mixture of each dye. It can be observed that for band I, for each intensity level i, there are i+1 dye combinations that react differently to light exposure. The total number of achievable coding states (T dynamic ) can be expressed as a function of the number of distinguishable fluorescence levels:

[0278]

[0279] where i, j, and k represent the intensity levels in excitation / emission bands I, J, and K. For five different encoding levels and three excitation / emission bands, a conventional single time point measurement would yield 216 unique encoding states. Using the time domain method, 21 different types of beads with unique encodings are generated at five different intensity levels and the theoretical number of unique encoding states for three excitation / emission bands is 9,261.

[0280] Pre-Example 12

[0281] The photostability of organic dyes in the time domain can be used to decode biological samples. The first antibody can include a first organic dye connected thereto and the second antibody can include a second organic dye connected thereto. The third antibody can include both the first and second organic dyes connected thereto in different ratios, as described above. The first and second organic dyes can have the same and / or similar fluorescence excitation / emission properties and / or intensity so that the first, second and other antibodies are indistinguishable at the first time point before the defined event. The first and second organic dyes can be susceptible to different levels of photobleaching after exposure to light for a defined time (typically about 30 seconds to five minutes, such as about 1 minute, about 2 minutes, about 3 minutes or more). Therefore, after photobleaching for a defined time (e.g., at least one microsecond), different antibodies have different fluorescence intensities. For example, compared with the second organic dye (i.e., dynamic element), the first organic dye can be less susceptible to photobleaching (i.e., more stable) and the fluorescence intensity of the first organic dye can only be reduced by 10% after 1 minute, while the fluorescence intensity of the second organic dye can be reduced by 90% after 1 minute. Therefore, the first and second antibody populations can be identified by the fluorescence intensity after the defined event (i.e., photobleaching). The third antibody colony can also be identified by the fluorescence intensity after the limiting event, because the ratio provides different levels or gradients of the fluorescence intensity between the intensity of the first and second organic dyes. Biological sample (for example, tissue) can be contacted with the first, second and third antibodies. Each encoded antibody can be combined with the sample of part. The fluorescence intensity at different positions can be compared on the sample at different time points to identify the position of each antibody in and / or on the sample, and can be used to identify which element is present in the sample and / or where the element is in the sample.

[0282] The foregoing is illustrative of the present invention and should not be construed as limiting it. Although some exemplary embodiments of the present invention have been described, it will be readily appreciated by those skilled in the art that many modifications are feasible in the exemplary embodiments without departing from the novel teachings and advantages of the present invention in essence. Therefore, all such modifications are intended to be included within the scope of the present invention as defined in the claims. Therefore, it should be understood that the foregoing is illustrative of the present invention and should not be construed as being limited to the specific embodiments disclosed, and modifications to the disclosed embodiments and other embodiments are intended to be included within the scope of the appended claims. The present invention is defined by the following claims and the equivalents of the claims included therein.

Claims

1. A determination decoding system, comprising: a circuit, the circuit comprising at least one processor; A fluid analysis chip comprising a plurality of solid supports (e.g., a multiplex bead set); and at least one of a heat source or a light source in communication with the plurality of solid supports, wherein the circuit activates at least one of the heat source or the light source to cause at least one detectable physical and / or chemical change associated with at least some of the plurality of solid supports and compares first and second coded signals of corresponding solid supports in the plurality of solid supports to decode different populations of solid supports in the plurality of solid supports.

2. The system of claim 1, wherein at least one of said heat source or said light source is a photobleaching light source.

3. The system of claim 1, wherein at least one of the heat source or the light source is a heat source.

4. The system of claim 1, wherein a portion of the plurality of solid supports comprises solid supports having a first dye and a second dye at different concentrations and / or ratios, optionally wherein the first and second dyes have different photostabilities and / or thermostabilities.

5. The system of claim 4, wherein different concentrations and / or ratios of said first and second dyes provide at least five different populations.

6. The system of claim 5, wherein the at least five different populations are optically and / or electrically detectable.

7. The system of claim 4, wherein the first dye is photostable and the second dye is photolabile (labile).

8. The system of claim 1, further comprising an optical detector in communication with the circuit and the fluid analysis chip, the optical detector being configured to emit light of one or more defined wavelengths.

9. The system of claim 8, wherein the circuit instructs the optical detector to obtain a first image of the plurality of solid supports and to obtain a second image of the plurality of solid supports, wherein the at least one detectable physical and / or chemical change causes at least one difference in the first image compared to the second image.

10. The system of claim 9, wherein the first image is before the at least one detectable physical and / or chemical change occurs and the second image is after or simultaneously with the at least one detectable physical or chemical change occurs.

11. The system of claim 9, wherein said second image is after said circuit activates at least one of said heat source or said light source.

12. The system of claim 9, wherein the optical detector emits light of a first wavelength onto the plurality of solid supports to obtain the first and second images, and wherein a subset of solid supports in the plurality of solid supports produces different fluorescence intensities in the first and second images to provide a decoding signal.

13. The system of claim 1, wherein for said at least one detectable physical or chemical change, at least one of said heat source or said light source causes an irreversible, detectable physical and / or chemical change in a subset of said plurality of solid supports.

14. The system of claim 9, wherein the optical detector emits light at an excitation wavelength to the plurality of solid supports to obtain the first and / or second images and transmits light of a different wavelength that causes the at least one detectable physical or chemical change.

15. The system of claim 1, wherein at least some of the plurality of solid supports comprise first and second spectrally similar encoding media (e.g., dyes) at a first point in time, wherein one of the first and second spectrally similar encoding media is stable and the other is unstable (e.g., labile), and Wherein different said solid supports have different ratios and / or concentrations of said first and second encoding media to provide a plurality of (e.g., 1-5) different distinguishable fluorescence intensity levels before and / or after said at least one detectable physical and / or chemical change to provide a plurality of encoding states.

16. The system of claim 15, wherein a photobleaching event using the light source causes the at least one detectable physical and / or chemical change.

17. The system of claim 15, wherein the plurality of encoding states ranges from about 2 to about 10,000.

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