Image differentiated multiplex assays

Abstract

The invention relates to image differentiated multiplex assays. Provided herein are encoded microcarriers for analyte detection in multiplex assays. The microcarrier is encoded using an analog code for identification and includes a capture agent for analyte detection. Also provided are methods of making the encoded microcarriers disclosed herein. Further provided are methods and kits related to multiplex assays using the microcarriers described herein.

Description

[0001] This application is a divisional application of the invention application filed on June 10, 2016, with Chinese application number 201680002147.1 and title "Image Differential Multiple Measurement".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 174,401, filed June 11, 2015, which is incorporated herein by reference in its entirety. Technical Field

[0004] This document provides coded microcarriers for analyte detection in multiplex assays, methods for preparing and using said microcarriers, and kits associated with said microcarriers. The microcarriers are coded using analog codes for identification and include a trapping agent for capturing the analyte. Background Technology

[0005] Immunological and molecular diagnostic assays play crucial roles in both research and clinical settings. Often, it is necessary to perform assays on a set of multiple targets to obtain a meaningful or overarching view of the results to aid research or clinical decision-making. This is especially true in the era of genomics and proteomics, where a multitude of genetic and / or biomarkers are believed to influence or predict specific disease states. Theoretically, assays on multiple targets can be performed by testing each target separately or sequentially in different reaction vessels (i.e., multiple singleplexing). However, assays employing singleplexing strategies are not only often cumbersome, but they also typically require large sample volumes, particularly when the number of targets to be analyzed is large.

[0006] Multiplex assays measure multiple analytes (two or more) simultaneously in a single assay. Multiplex assays are commonly used in high-throughput screening environments where many samples can be analyzed at once. The ability to simultaneously measure many analytes and to measure many samples in parallel is a hallmark of multiplex assays and the reason why such assays have become powerful tools in fields ranging from drug discovery to functional genomics to clinical diagnostics. Compared to singlex assays, these assays are less cumbersome and easier to perform by combining all targets in the same reaction vessel, as only one reaction vessel is operated per sample. Therefore, the volume of the required test sample can be significantly reduced, which is especially important when samples (e.g., tumor tissue, cerebrospinal fluid, or bone marrow) are difficult to obtain in large quantities and / or invasive for large-scale acquisition. Equally important is the fact that reagent costs are reduced and assay throughput is significantly increased.

[0007] Many assays of complex macromolecular samples consist of two steps. In the first step, a reagent capable of specifically capturing the target macromolecule is attached to a solid surface. These immobilized molecules can be used to capture the target macromolecule from the complex sample through various mechanisms such as hybridization (e.g., in DNA- or RNA-based assays) or antigen-antibody interactions (in immunoassays). In the second step, the detection molecule is incubated with the complex of the captured molecule and the target and binds to the complex, thereby emitting a signal, such as fluorescence or other electromagnetic signals. The amount of the target is then quantified by the intensity of those signals.

[0008] Multiplex assays can be performed using a variety of trapping agents, each specific to different target macromolecules. In chip-based array multiplex assays, each type of trapping agent is attached to a predetermined location on the chip. The amount of multiple targets in a complex sample is determined by measuring the signal of the detection molecule at each location corresponding to one type of trapping agent. In suspension array multiplex assays, microparticles or microcarriers are suspended in an assay solution. These microparticles or microcarriers contain identification elements that can be embedded, printed, or otherwise generated by one or more elements of the microparticle / microcarrier. Each type of trapping agent is immobilized to particles with the same ID, and the signal emitted from the detection molecule on the surface of the particle with the specific ID reflects the amount of the corresponding target.

[0009] Existing systems for multiplexing of suspended arrays are limited in resolution. Some multiplexing systems use digital barcodes printed on flat microbeads using standard semiconductor manufacturing techniques. However, the number of identifiers that can be generated from a certain number of digits is limited. Increasing the number of unique identifiers requires increasing the number of barcode digits, thus requiring more space for printing on already tiny microbeads. Another type of multiplexing system uses color encoding, such as fluorescent beads encoded with unique fluorescent dyes. However, the number of unique identifiers available for such fluorescent systems is limited by overlapping excitation / emission spectra, and identification errors can arise from, for example, batch-to-batch variations in the fluorescent dye.

[0010] Therefore, there is a need for an analog-coded multiplexing system, such as one that is not constrained by limitations such as digital barcode size or fluorophore resolution. Such a system would allow for a virtually unlimited number of unique identifiers and minimize identification errors caused by the use of analog codes (e.g., from overlapping spectra or batch-to-batch fluorophore variations).

[0011] For all purposes, all publications, patents and patent applications cited herein are hereby incorporated in their entirety by reference. Summary of the Invention

[0012] To meet this need, this document provides, in particular, microcarriers encoded with analog codes, wherein the microcarriers include a trapping agent for capturing analytes. These microcarriers can be used, for example, in multiplex assays, where each microcarrier includes a trapping agent for capturing a specific analyte and an analog code for identification. Methods for preparing and using such microcarriers, as well as kits associated with said methods, are also provided.

[0013] Therefore, in one aspect, this document provides an encoded microcarrier comprising (a) a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (b) a substantially opaque polymer layer wherein the substantially opaque polymer layer is fixed to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; and (c) a trapping agent for trapping analytes, wherein the trapping agent is coupled in at least the central portion of the substantially transparent polymer layer to at least one of the first surface and the second surface of the substantially transparent polymer layer.

[0014] In some embodiments, the microcarrier further includes (d) a substantially opaque magnetic layer surrounding the central portion of the substantially transparent polymer layer between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer, wherein the substantially opaque magnetic layer is fixed to a first or second surface of the substantially transparent polymer layer. In some embodiments, the microcarrier further includes (e) a second substantially transparent polymer layer aligned with the first substantially transparent polymer layer, the second substantially transparent polymer layer having a central portion aligned with the central portion of the first substantially transparent polymer layer, wherein the second substantially transparent polymer layer is fixed to a second surface of the first substantially transparent polymer layer and does not extend beyond the two-dimensional shape of the first substantially transparent polymer layer; and (f) a substantially opaque magnetic layer surrounding the central portion of the first substantially transparent polymer layer between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer, wherein the substantially opaque magnetic layer is fixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, the microcarrier further includes orientation indicators for orienting analog codes of the substantially opaque polymer layer. In some embodiments, the orientation index includes the asymmetry of the substantially opaque magnetic layer. In some embodiments, the substantially opaque magnetic layer comprises nickel. In some embodiments, the thickness of the substantially opaque magnetic layer is between about 50 nm and about 10 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 0.1 μm. In some embodiments, the two-dimensional shape of the substantially opaque polymer layer includes a gear shape comprising a plurality of gear teeth, and wherein the analog code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes two or more gear teeth spaced between about 1 μm and about 10 μm.In some embodiments, the microcarrier further includes (g) one or more pillars protruding from a first surface of the first substantially transparent polymer layer, wherein the one or more pillars are not within the central portion of the first substantially transparent polymer layer; and / or (h) one or more pillars protruding from a second surface of the first substantially transparent polymer layer or from a surface of the second substantially transparent polymer layer not attached to the first substantially transparent polymer layer, wherein the one or more pillars are not within the central portion of the first substantially transparent polymer layer or the second substantially transparent polymer layer. In some embodiments, the microcarrier is a substantially circular disk. In some embodiments, the central portion of the first substantially transparent polymer layer occupies between about 5% and about 90% of the surface area of ​​the first substantially transparent polymer layer. In some embodiments, the central portion of the first substantially transparent polymer layer occupies about 25% of the surface area of ​​the first substantially transparent polymer layer. In some embodiments, the diameter of the microcarrier is less than about 200 μm. In some embodiments, the diameter of the microcarrier is about 50 μm. In some embodiments, the thickness of the microcarrier is less than about 50 μm. In some embodiments, the thickness of the microcarrier is about 10 μm. In some embodiments, the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In some embodiments, the capturing agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In some embodiments, the substantially transparent polymer of the first substantially transparent polymer layer or the second substantially transparent polymer layer comprises an epoxy-based polymer. In some embodiments, the epoxy-based polymer is SU-8.

[0015] On the other hand, this document provides an encoded microcarrier comprising: (a) a substantially opaque polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; and (b) a trapping agent for trapping analytes, wherein the trapping agent is coupled in at least a central portion of the substantially opaque polymer layer to at least one of the first surface and the second surface of the substantially opaque polymer layer.

[0016] In some embodiments, the microcarrier further includes (c) one or more pillars protruding from a first and / or second surface of the substantially opaque polymer layer, wherein the one or more pillars comprise a magnetic material. In some embodiments, the height of the one or more pillars is between about 1 μm and about 10 μm. In some embodiments, the diameter of the one or more pillars is between about 1 μm and about 10 μm. In some embodiments, the microcarrier further includes (d) a magnetic layer comprising a magnetic material, the magnetic layer being fixed to a second surface of the substantially opaque polymer layer, wherein the magnetic layer does not extend beyond the central portion of the substantially opaque polymer layer, and wherein the trapping agent is coupled to at least the first surface of the substantially opaque polymer layer. In some embodiments, the microcarrier further includes (e) a second substantially opaque polymer layer aligned with the first substantially opaque polymer layer, wherein the second substantially opaque polymer layer is fixed to a second surface of the first substantially transparent polymer layer and does not extend beyond the outline of the first substantially transparent polymer layer, and wherein the magnetic layer is fixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, the magnetic material comprises nickel. In some embodiments, the microcarrier further includes orientation indicators for orienting the analog codes of the substantially opaque polymer layer. In some embodiments, the orientation indicators include the asymmetry of the contour of the substantially opaque polymer layer. In some embodiments, the contour of the substantially opaque polymer layer comprises a two-dimensional gear shape including a plurality of gear teeth, and wherein the analog codes are represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth, the width of one or more gear teeth, the number of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes two or more gear teeth spaced between about 1 μm and about 10 μm. In some embodiments, the microcarrier is a substantially circular disk. In some embodiments, the central portion of the first substantially opaque polymer layer accounts for between about 5% and about 90% of the surface area of ​​the first substantially opaque polymer layer. In some embodiments, the central portion of the first substantially opaque polymer layer accounts for about 25% of the surface area of ​​the first substantially opaque polymer layer. In some embodiments, the diameter of the microcarrier is less than about 200 μm.In some embodiments, the diameter of the microcarrier is about 60 μm. In some embodiments, the thickness of the microcarrier is less than about 50 μm. In some embodiments, the thickness of the microcarrier is about 10 μm. In some embodiments, the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In some embodiments, the capturing agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In some embodiments, the substantially opaque polymer comprises an epoxy-based polymer. In some embodiments, the substantially opaque polymer comprises a black matrix resist.

[0017] On the other hand, this document provides a method for preparing coded microcarriers, the method comprising: (a) depositing a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (b) depositing a substantially opaque magnetic layer on the first surface of the substantially transparent polymer layer; (c) etching the substantially opaque magnetic layer to remove a portion of the substantially opaque magnetic layer deposited on a central portion of the substantially transparent polymer layer; (d) depositing a second substantially transparent polymer layer on the substantially opaque magnetic layer, wherein the second substantially transparent polymer layer has a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the substantially opaque magnetic layer, and wherein the second substantially transparent polymer layer is aligned with the first substantially transparent polymer layer and has a central portion aligned with the central portion of the substantially transparent polymer layer; and (e) depositing a substantially opaque polymer layer on the first surface of the second substantially transparent polymer layer, wherein the substantially opaque polymer layer surrounds the first substantially transparent polymer layer and the central portion of the second substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code.

[0018] In some embodiments, the substantially opaque magnetic layer is etched by wet etching. In some embodiments, the substantially opaque magnetic layer comprises nickel. In some embodiments, the thickness of the substantially opaque magnetic layer is between about 50 nm and about 10 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is less than about 0.1 μm. In some embodiments, the substantially opaque magnetic layer includes asymmetry for oriented analog codes of the substantially opaque polymer layer. In some embodiments, the two-dimensional shape of the substantially opaque polymer layer is produced by lithography. In some embodiments, the two-dimensional shape of the substantially opaque polymer layer includes a gear shape comprising a plurality of gear teeth, and wherein the analog codes are represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes two or more gear teeth spaced between about 1 μm and about 10 μm. In some embodiments, the method further includes: (f) depositing a sacrificial layer on a substrate prior to step (a); (g) using photolithography to create one or more columnar holes in the sacrificial layer; (h) depositing a third substantially transparent polymer layer in the one or more columnar holes in the sacrificial layer, wherein the first substantially transparent polymer layer was deposited on top of the third substantially transparent polymer layer and the sacrificial layer in step (a); (i) after step (e), using photolithography to deposit one or more pillars comprising the substantially transparent polymer on a first surface of the second substantially transparent polymer layer at portions not covered by the substantially opaque polymer layer; (j) dissolving the sacrificial layer in a solvent; and (k) removing the substrate. In some embodiments, the method further includes: (f) depositing a sacrificial layer on a substrate prior to step (a); (g) depositing the substantially transparent polymer layer on the sacrificial layer as part of step (a); (h) dissolving the sacrificial layer in a solvent after step (e); and (i) removing the substrate. In some embodiments, the encoded microcarrier is a substantially circular disk.In some embodiments, the central portion of the first substantially transparent polymer layer occupies between about 5% and about 90% of the surface area of ​​the first substantially transparent polymer layer. In some embodiments, the central portion of the first substantially transparent polymer layer occupies about 25% of the surface area of ​​the first substantially transparent polymer layer. In some embodiments, the diameter of the encoded microcarrier is less than about 200 μm. In some embodiments, the diameter of the encoded microcarrier is about 50 μm. In some embodiments, the thickness of the encoded microcarrier is less than about 50 μm. In some embodiments, the thickness of the encoded microcarrier is about 10 μm. In some embodiments, the method further includes: (f) coupling a capture agent for capturing the analyte in at least the central portion to at least one of a first surface of the second substantially transparent polymer layer and a second surface of the first substantially transparent polymer layer. In some embodiments, the substantially transparent polymer of the first substantially transparent polymer layer or the second substantially transparent polymer layer comprises an epoxide, and coupling the trapping agent comprises: (i) reacting the substantially transparent polymer of the first substantially transparent polymer layer and / or the second substantially transparent polymer layer with a photoacid-producing agent and light to produce a crosslinked polymer, wherein the light has a wavelength that activates the photoacid-producing agent; and (ii) reacting the epoxide of the crosslinked polymer with a compound comprising an amine and a carboxyl group, wherein the amine of the compound reacts with the epoxide to form a compound-coupled crosslinked polymer; and (iii) reacting the carboxyl group of the compound-coupled crosslinked polymer with the trapping agent to couple the trapping agent at least in the central portion to at least one of a first surface of the second substantially transparent polymer layer and a second surface of the first substantially transparent polymer layer. In some embodiments, the carboxyl group of the compound-coupled crosslinked polymer reacts with a primary amine of the trapping agent. In some embodiments, the substantially transparent polymer of the first substantially transparent polymer layer and / or the second substantially transparent polymer layer comprises SU-8. In some embodiments, the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In some embodiments, the capture agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0019] On the other hand, this document provides an encoded microcarrier generated by any of the methods described in the above embodiments.

[0020] On the other hand, this article provides a method for preparing coded microcarriers, the method comprising: (a) depositing a sacrificial layer on a substrate; (b) depositing a substantially opaque polymer layer on the sacrificial layer, the substantially opaque polymer layer having a contour, a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the sacrificial layer; (c) shaping the contour of the substantially opaque polymer layer by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code; (d) dissolving the sacrificial polymer layer in a solvent; and (e) removing the substrate. On the other hand, this document provides a method for preparing coded microcarriers, the method comprising: (a) depositing a sacrificial layer on a substrate; (b) depositing a magnetic layer comprising a magnetic material on the sacrificial layer; (c) depositing a substantially opaque polymer layer on the magnetic layer, the substantially opaque polymer layer having a contour, a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the magnetic layer; (d) shaping the contour of the substantially opaque polymer layer by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code; (e) dissolving the sacrificial polymer layer in a solvent; and (f) removing the substrate.

[0021] In some embodiments, the method further includes (g) shaping the magnetic layer by photolithography after step (b) and before step (c). In some embodiments, the magnetic material comprises nickel. In some embodiments, the microcarrier includes orientation indicators for orienting analog codes of the substantially opaque polymer layer. In some embodiments, the orientation indicators include the asymmetry of the contour of the substantially opaque polymer layer. In some embodiments, the two-dimensional shape of the substantially opaque polymer layer includes a gear shape comprising a plurality of gear teeth, and wherein the analog code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes two or more gear teeth spaced between about 1 μm and about 10 μm. In some embodiments, the microcarrier is a substantially circular disk. In some embodiments, the diameter of the microcarrier is less than about 200 μm. In some embodiments, the diameter of the microcarrier is about 60 μm. In some embodiments, the thickness of the microcarrier is less than about 30 μm. In some embodiments, the thickness of the microcarrier is about 10 μm. In some embodiments, the method further includes (h) coupling a trapping agent for capturing the analyte to at least one of a first surface and a second surface of the substantially opaque polymer layer. In some embodiments, the substantially opaque polymer of the substantially opaque polymer layer comprises an epoxide, and coupling the trapping agent comprises: (i) reacting the substantially opaque polymer of the substantially opaque polymer layer with a photoacid-producing agent and light to produce a crosslinked polymer, wherein the light has a wavelength that activates the photoacid-producing agent; and (ii) reacting the epoxide of the crosslinked polymer with a compound comprising an amine and a carboxyl group, wherein the amine of the compound reacts with the epoxide to form a compound-coupled crosslinked polymer; and (iii) reacting the carboxyl group of the compound-coupled crosslinked polymer with the trapping agent to couple the trapping agent at least in the central portion to at least one of a first surface of the second substantially transparent polymer layer and a second surface of the first substantially transparent polymer layer.In some embodiments, the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In some embodiments, the capture agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0022] On the other hand, this document provides an encoded microcarrier generated by any of the methods described in the above embodiments.

[0023] On the other hand, this document provides a method for detecting two or more analytes in a solution, the method comprising: (a) contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers comprises at least: (i) a first microcarrier according to any of the above embodiments, the first microcarrier specifically capturing the first analyte, wherein the first microcarrier is encoded with a first analog code; and (ii) a second microcarrier according to any of the above embodiments, the second microcarrier specifically capturing the second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code; (b) decoding the first analog code and the second analog code using analog shape recognition to identify the first microcarrier and the second microcarrier; and (c) detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0024] In some embodiments, step (b) occurs before step (c). In some embodiments, step (c) occurs before step (b). In some embodiments, step (b) and step (c) occur simultaneously. In some embodiments, decoding the first analog code and the second analog code includes: (i) illuminating the first microcarrier and the second microcarrier by passing light through substantially transparent portions and / or surrounding solution of the first microcarrier and the second microcarrier, wherein the light fails to pass through substantially opaque portions of the first microcarrier and the second microcarrier to generate a light pattern corresponding to a first analog code of the first microcarrier and a light pattern corresponding to a second analog code of the second microcarrier; (ii) imaging the first analog code light pattern to generate a first analog code image and imaging the second analog code light pattern to generate a second analog code image; and (iii) using analog shape recognition to match the first analog code image with the first analog code and the second analog code image with the second analog code. In some embodiments, detecting the amount of a first analyte bound to the first microcarrier and the amount of a second analyte bound to the second microcarrier comprises: (i) after step (a), incubating the first and second microcarriers together with a detection agent, wherein the detection agent binds the first analyte captured by the first microcarrier and the second analyte captured by the second microcarrier; and (ii) measuring the amount of detection agent bound to the first and second microcarriers. In some embodiments, the detection agent is a fluorescent detection agent, and the amount of detection agent bound to the first and second microcarriers is measured by fluorescence microscopy. In some embodiments, the detection agent is a luminescent detection agent, and the amount of detection agent bound to the first and second microcarriers is measured by luminescence microscopy. In some embodiments, the solution comprises a biological sample. In some embodiments, the biological sample is selected from the group consisting of: blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluid of skin or adipose tissue, saliva, tears, bronchoalveolar lavage fluid, oropharyngeal secretions, intestinal fluid, cervical, vaginal, or uterine secretions, and semen.

[0025] On the other hand, this document provides a kit or article comprising a plurality of microcarriers, wherein the plurality of microcarriers comprises at least: (a) a first microcarrier according to any of the above embodiments, the first microcarrier specifically capturing a first analyte, wherein the first microcarrier is encoded with a first analog code; and (b) a second microcarrier according to any of the above embodiments, the second microcarrier specifically capturing a second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code.

[0026] In some embodiments, the kit or article of manufacture further includes a detection reagent for detecting the amount of a first analyte bound to the first microcarrier and the amount of a second analyte bound to the second microcarrier. In some embodiments, the kit or article of manufacture also includes instructions for using the kit to detect the first analyte and the second analyte.

[0027] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. Attached Figure Description

[0028] Figure 1A and 1B Two views of an exemplary microcarrier are shown.

[0029] Figure 1C and 1D An exemplary assay using an exemplary microcarrier is shown for the detection of an analyte.

[0030] Figure 2A and 2B Two views of an exemplary microcarrier are shown.

[0031] Figure 3 An exemplary analog coding scheme is shown, which includes multiple shape change points used to generate unique analog codes.

[0032] Figure 4A Three examples of microcarriers are shown, each with a unique simulation code.

[0033] Figure 4B Examples of microcarriers with unique simulation codes are shown according to some implementation schemes.

[0034] Figure 5A and 5B Two views of an exemplary microcarrier are shown.

[0035] Figure 6A and 6B Two views of an exemplary microcarrier are shown.

[0036] Figure 6C The dimensions of an example simulation code are shown. Dimensions are based on μm units.

[0037] Figure 7 An exemplary microcarrier is shown.

[0038] Figure 8A An exemplary microcarrier is shown, including an asymmetric starting position as an orientation indicator.

[0039] Figure 8B An exemplary analog coding scheme is shown, which includes multiple shape change points used to generate unique analog codes.

[0040] Figures 9A-9C Two views of an exemplary microcarrier are shown (Fig. 9A and Fig. 9B), along with a depiction of optional features (Fig. 9C).

[0041] Figure 10 A method for producing exemplary microcarriers is shown.

[0042] Figure 11A and 11B A method for producing exemplary microcarriers is shown.

[0043] Figures 12A-12E A method for producing exemplary microcarriers is shown.

[0044] Figures 13A-13C A method for producing exemplary microcarriers is shown. Detailed Implementation

[0045] On one hand, this document provides coded microcarriers for analyte detection in multiplex assays. In some embodiments, the microcarrier comprises (a) a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (b) a substantially opaque polymer layer, wherein the substantially opaque polymer layer is anchored to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing an analog code; and (c) a trapping agent for capturing the analyte, wherein the trapping agent is coupled in at least the central portion of the substantially transparent polymer layer to at least one of the first surface and the second surface of the substantially transparent polymer layer. In other embodiments, the microcarrier comprises (a) a substantially opaque polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional shape representing an analog code; and (b) a trapping agent for capturing the analyte, wherein the trapping agent is coupled in at least the central portion of the substantially opaque polymer layer to at least one of the first surface and the second surface of the substantially opaque polymer layer.

[0046] On the other hand, this document provides a method for preparing coded microcarriers. In some embodiments, the method includes: (a) depositing a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (b) depositing a substantially opaque magnetic layer on the first surface of the substantially transparent polymer layer; (c) etching the substantially opaque magnetic layer to remove a portion of the substantially opaque magnetic layer deposited on a central portion of the substantially transparent polymer layer; (d) depositing a second substantially transparent polymer layer on the substantially opaque magnetic layer, wherein the second substantially transparent polymer layer has a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the substantially opaque magnetic layer, and wherein the second substantially transparent polymer layer is aligned with the first substantially transparent polymer layer and has a central portion aligned with the central portion of the substantially transparent polymer layer; and (e) depositing a substantially opaque polymer layer on the first surface of the second substantially transparent polymer layer, wherein the substantially opaque polymer layer surrounds the first substantially transparent polymer layer and the central portion of the second substantially transparent polymer layer, and wherein the substantially opaque polymer layer includes a two-dimensional shape representing analog code. In other embodiments, the method includes: (a) depositing a sacrificial layer on a substrate; (b) depositing a substantially opaque polymer layer on the sacrificial layer, the substantially opaque polymer layer having a contour, a first surface, and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the sacrificial layer; (c) shaping the contour of the substantially opaque polymer layer by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code; (d) dissolving the sacrificial polymer layer in a solvent; and (e) removing the substrate. In other embodiments, the method includes: (a) depositing a sacrificial layer on a substrate; (b) depositing a magnetic layer comprising a magnetic material on the sacrificial layer; (c) depositing a substantially opaque polymer layer on the magnetic layer, the substantially opaque polymer layer having a contour, a first surface, and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the magnetic layer; (d) shaping the contour of the substantially opaque polymer layer by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code; (d) dissolving the sacrificial polymer layer in a solvent; and (e) removing the substrate. This paper further provides microcarriers encoded using the methods disclosed herein.

[0047] In another aspect, this document provides a method for detecting two or more analytes in a solution by means of the following steps: (a) contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers comprises at least: (i) a first microcarrier of the present disclosure, the first microcarrier specifically capturing the first analyte, wherein the first microcarrier is encoded with a first analog code; and (ii) a second microcarrier of the present disclosure, the second microcarrier specifically capturing the second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code; (b) decoding the first analog code and the second analog code using analog shape recognition to identify the first microcarrier and the second microcarrier; and (c) detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0048] On the other hand, this document provides kits or articles for performing multiple assays, the kits or articles comprising a plurality of microcarriers. The plurality of microcarriers includes at least (a) a first microcarrier of this disclosure, the first microcarrier specifically capturing a first analyte, wherein the first microcarrier is encoded with a first analog code; and (b) a second microcarrier of this disclosure, the second microcarrier specifically capturing a second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code.

[0049] I. General Technology

[0050] Unless otherwise stated, the practice of the techniques described herein will employ conventional techniques from polymer technology, microfabrication, microelectromechanical systems (MEMS) fabrication, photolithography, microfluidics, organic chemistry, biochemistry, oligonucleotide synthesis and modification, bioconjugate chemistry, nucleic acid hybridization, molecular biology, microbiology, genetics, recombinant DNA, and related fields as described in this art. The techniques are described in the references cited herein and are fully explained therein.

[0051] For molecular biology and recombinant DNA techniques, see, for example, (Maniatis, T. et al. (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Ausubel, F.M. (1987), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F.M. (1989), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Innis, M.A. (1990), PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F.M. (1995), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995), PCR Strategies, Academic Press; Ausubel, F.M. (1999), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and updated annually.

[0052] For information on DNA synthesis techniques and nucleic acid chemistry, see, for example, Gait, MJ (1990), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992), The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994), Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996), Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996), Bioconjugate Techniques, Academic Press.

[0053] Regarding microfabrication, see for example (Campbell, SA (1996), The Science and Engineering of Microelectronic Fabrication, Oxford University Press; Zaut, PV (1996), Microarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, MJ (1997), Fundamentals of Microfabrication, CRCPress; Rai-Choudhury, P. (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography).

[0054] II. Definition

[0055] Before describing the invention in detail, it should be understood that the invention is not limited to a particular composition or biological system, which can certainly be varied. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0056] As used herein, the term "microcarrier" can refer to a physical substrate to which a trapping agent can be coupled. The microcarriers of this disclosure can take any suitable geometry or shape. In some embodiments, the microcarrier may be disc-shaped. Typically, the form or shape of the microcarrier will include at least one approximately 10 -4 Up to 10 -7 The size of m (hence the prefix "micro").

[0057] As used herein, the term "polymer" can refer to any macromolecular structure that includes repeating monomers. Polymers can be natural (e.g., found in nature) or synthetic (e.g., artificial, such as polymers composed of non-natural monomers and / or polymerized in structures or combinations not found in nature).

[0058] As used herein, the terms “substantially transparent” and “substantially opaque” can refer to the ability of light (e.g., light of a specific wavelength, such as infrared, visible, UV, etc.) to pass through a substrate (such as a polymer layer). A substantially transparent polymer can refer to a transparent, translucent, and / or light-transmitting polymer, while a substantially opaque polymer can refer to a polymer that reflects and / or absorbs light. It should be understood that whether a material is substantially transparent or substantially opaque can depend on the wavelength and / or intensity of the light illuminating the material, and the means by which the light traveling through the material (or its reduction or absence) is detected. In some embodiments, such as imaging by optical microscopy (e.g., bright-field, dark-field, phase contrast, differential interference difference (DIC), Nomarski interference difference (NIC), Nomarski, Hoffman modulation phase contrast (HMC), or fluorescence microscopy), a substantially opaque material causes a perceptible reduction in transmitted light compared to the surrounding material or image field. In some implementations, such as imaging by optical microscopy (e.g., bright-field, dark-field, phase-contrast, differential interference difference (DIC), Nomarsky interference difference (NIC), Nomarsky, Hoffman modulation phase difference (HMC), or fluorescence microscopy), substantially transparent materials allow a perceptible amount of transmitted light to pass through the material.

[0059] As used herein, the term "analog code" can refer to any code that represents encoded information in a non-quantized and / or non-discrete manner (e.g., as opposed to digital code). For example, digital code is sampled at discrete locations within a finite set of values ​​(e.g., 0 / 1 type values), while analog code may be sampled at a wider range of locations (or as a continuous whole) and / or may include a wider set of values ​​(e.g., shapes). In some embodiments, one or more analog shape recognition techniques may be used to read or decode analog code.

[0060] As used herein, the term "capture agent" is a broad term and is used in its usual sense to refer to any compound or substance capable of specifically recognizing a target analyte. In some embodiments, specific recognition may refer to specific binding. Non-limiting examples of capture agents include, for example, DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0061] As used herein, "analyte" is a broad term and, in its usual sense, refers to a substance whose presence, absence, or quantity is to be determined, including but not limited to substances or chemical components in a sample such as a biological sample or cells or cell populations that can be analyzed. An analyte can be a substance with a naturally occurring binding member present thereto, or a substance with a binding member that can be prepared to bind to it. Non-limiting examples of analytes include, for example, antibodies, antibody fragments, antigens, polynucleotides (such as DNA molecules, DNA mimic molecules, RNA molecules, or RNA mimic molecules), polypeptides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, small molecules, organelles, hormones, cytokines, growth factors, steroids, vitamins, toxins, drugs, and metabolites of the above substances, as well as cells, bacteria, viruses, fungi, algae, fungal spores, etc.

[0062] The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full-length antibodies with the Fc region of an immunoglobulin), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv).

[0063] As used herein, “sample” refers to a composition containing material (such as molecules) to be tested. In one embodiment, a sample is a “biological sample” (i.e., any material obtained from a living source (e.g., human, animal, plant, bacteria, fungi, protozoa, virus). Biological samples can be in any form, including solid materials (e.g., tissue, cell pellets, and biopsies) and biological fluids (e.g., urine, blood, saliva, lymph, tears, sweat, prostatic fluid, semen, bile, mucus, amniotic fluid, and mouthwash (containing oral cells)). Solid materials are typically mixed with fluids. A sample can also refer to an environmental sample, such as water, air, soil, or any other environmental source.

[0064] As used in this specification and the appended claims, unless the content expressly indicates otherwise, the singular forms "a / an" and "the" include a plural of indicators. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, etc.

[0065] As used herein, the term "about" refers to the typical range of error for a corresponding value that is readily known to those skilled in the art. References to "about" a value or parameter herein include (and describe) embodiments for said value or parameter itself.

[0066] It should be understood that the aspects and embodiments of the invention described herein include "comprising", "consisting of", and / or "mainly composed of" aspects and embodiments.

[0067] III. Encoded Microcarriers

[0068] This article provides encoded microcarriers suitable for analyte detection, such as multiplex analyte detection. Various constructions of these encoded microcarriers are covered, described, and illustrated.

[0069] In some aspects, this document provides coded microcarriers comprising: a substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; a substantially opaque polymer layer, wherein the substantially opaque polymer layer is anchored to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; and a trapping agent for trapping analytes, wherein the trapping agent is coupled in at least the central portion of the substantially transparent polymer layer to at least one of the first surface and the second surface of the substantially transparent polymer layer. Thus, the microcarrier contains at least two layers: one of the layers is substantially transparent, and the other of the layers is a substantially opaque two-dimensional shape representing analog code. Advantageously, these microcarriers can adopt a variety of two-dimensional shapes while still maintaining a uniform overall form (e.g., the periphery of the substantially transparent polymer layer) to achieve homogeneity in aspects including, for example, overall size, physical properties, and / or behavior in solution. Examples of this type of microcarrier and its aspects are discussed in... Figure 1A-5B As shown in the image.

[0070] In some embodiments, the microcarrier further includes a substantially opaque magnetic layer fixed to the surface of a substantially transparent polymer layer, the magnetic layer surrounding a central portion of the substantially transparent polymer layer. In some embodiments, the substantially opaque magnetic layer is located between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer.

[0071] In some embodiments, the microcarrier further includes a second substantially transparent polymer layer aligned with and fixed to the first substantially transparent polymer layer. In some embodiments, the first and second substantially transparent polymer layers each have a central portion, and the central portions of both are aligned. In some embodiments, the microcarrier further includes a substantially opaque magnetic layer surrounding the central portions of both the first and second substantially transparent polymer layers. In some embodiments, the substantially opaque magnetic layer is fixed between the first and second substantially transparent polymer layers. In some embodiments, the substantially opaque magnetic layer is located between the substantially opaque polymer layer and the central portions of both the first and second substantially transparent polymer layers.

[0072] In some embodiments, the thickness of the substantially opaque magnetic layer is between about 50 nm and about 10 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is less than any of the following thicknesses (nm): 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100. In some implementations, the thickness of the substantially opaque magnetic layer is greater than any of the following thicknesses (nm): 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500. That is, the thickness of the substantially opaque magnetic layer can be selected independently, having an upper limit of 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100. The selected lower limit is any one of the following thickness (nm) ranges: 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, or 9500, wherein the lower limit is less than the upper limit.

[0073] In some implementations, the thickness of the substantially opaque magnetic layer is about 0.1 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about 800 nm, about 850 nm, about 900 nm, about 950 nm, about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the thickness of the substantially opaque magnetic layer is about 0.01 μm, about 0.02 μm, about 0.03 μm, about 0.04 μm, about 0.05 μm, about 0.06 μm, about 0.07 μm, about 0.08 μm, about 0.09 μm, about 0.1 μm, about 0.11 μm, about 0.12 μm, about 0.13 μm, about 0.14 μm, about 0.15 μm, about 0.16 μm, about 0.17 μm, about 0.18 μm, about 0.19 μm, about 0.20 μm, about 0.25 μm, about 0.30 μm, about 0.35 μm, about 0.40 μm, about 0.45 μm, or about 0.50 μm.

[0074] In some embodiments, the microcarrier further includes an orientation index for orienting the analog codes of the substantially opaque polymer layer. Any features of the microcarrier that are visible and / or detectable by imaging (e.g., a form of microscopy or other imaging described herein) and / or by image recognition software can be used as the orientation index. The orientation index can be used as a reference point, for example, in image recognition algorithms, to orient the image of the analog codes in a uniform orientation (i.e., the shape of the substantially opaque polymer layer). Advantageously, this simplifies image recognition because the algorithm will only need to compare the image of a particular analog code with a library of analog codes in the same orientation, rather than with a library that includes all analog codes in all possible orientations. In some embodiments, the orientation index may be independent of the substantially opaque polymer layer. For example, it may be formed as part of a magnetic layer and / or a substantially transparent polymer layer. In other embodiments, the orientation index may be formed as part of a substantially opaque polymer layer. In some embodiments, the orientation index includes the asymmetry of the substantially opaque magnetic layer (e.g., as shown by...). Figure 2A (as shown in gap 210).

[0075] In some embodiments, the microcarrier further includes one or more pillars projecting from the surface of the microcarrier (e.g., the top and / or bottom surfaces of the microcarrier). As used herein, "pillar" can refer to any geometry projecting from the surface of the microcarrier and does not necessarily indicate any regularity in size or any cylindrical feature. For example, the outer surface of the pillar may or may not be parallel to the surface of the microcarrier. Examples of pillar-like shapes that may project from the microcarrier include, but are not limited to, rectangular prisms, triangles, pyramids, cubes, cylinders, spheres or hemispheres, cones, etc. In some embodiments, the one or more pillars are not located within the central portion of the first and / or second substantially transparent polymer layers. In some embodiments, the one or more pillars may project from the outward-facing surface (e.g., a surface not attached to another layer) of one or more of the first substantially transparent polymer layers and the second substantially transparent polymer layers. It should be noted that any description of the microcarrier thickness herein does not include the one or more pillars in the stated dimensions. In other words, the thickness of the microcarrier as described herein is independent of any optional pillars projecting from it.

[0076] In some embodiments, the height of the one or more pillars is between about 1 μm and about 10 μm. In some embodiments, the one or more pillars are about 1 μm high, about 1.5 μm high, about 2 μm high, about 2.5 μm high, about 3 μm high, about 3.5 μm high, about 4 μm high, about 4.5 μm high, about 5 μm high, about 5.5 μm high, about 6 μm high, about 6.5 μm high, about 7 μm high, about 7.5 μm high, about 8 μm high, about 8.5 μm high, about 9 μm high, about 9.5 μm high, or about 10 μm high. In some embodiments, the one or more pillars are less than any of the following heights (μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the one or more pillars are greater than any of the following heights (μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one or more pillars can be any of a height range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit.

[0077] In some embodiments, the one or more pillars may be cylindrical. In some embodiments, the one or more pillars have a diameter between about 1 μm and about 10 μm. In some embodiments, the one or more pillars have a diameter of about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the one or more pillars have a diameter less than any of the following lengths (μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the one or more pillars have a diameter greater than any of the following lengths (μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one or more pillars may have any of the following diameter ranges with an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit. In other embodiments, the one or more pillars may have a width approximately the same as any diameter described above, or a width range approximately the same as any diameter range described above, but the one or more pillars may be elliptical pillars, parabolic pillars, hyperbolic pillars, or any other cylindrical or polyhedral shape described herein or known in the art.

[0078] In other respects, this document provides coded microcarriers comprising: a substantially opaque polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional shape representing an analog code; and a trapping agent for trapping an analyte, wherein the trapping agent is coupled in at least a central portion of the substantially opaque polymer layer to at least one of the first surface and the second surface of the substantially opaque polymer layer. Thus, the microcarrier is encoded by the shape (e.g., outline) of the microcarrier itself: a two-dimensional shape representing an analog code. Advantageously, these microcarriers can be manufactured efficiently and with high precision, thereby allowing for highly accurate decoding and cost-effective production. Examples of this type of microcarrier and its aspects are discussed in... Figure 6A As shown in -9C.

[0079] In some embodiments, the microcarrier further includes one or more pillars protruding from the surface of the substantially opaque polymer layer. As described in more detail above, "pillar" can refer to any geometry protruding from the surface of the microcarrier and does not necessarily indicate any regularity in the pillar's size. Any of the exemplary pillar shapes described above may be used.

[0080] In some embodiments, the height of the one or more pillars is between about 1 μm and about 10 μm. In some embodiments, the one or more pillars are about 1 μm high, about 1.5 μm high, about 2 μm high, about 2.5 μm high, about 3 μm high, about 3.5 μm high, about 4 μm high, about 4.5 μm high, about 5 μm high, about 5.5 μm high, about 6 μm high, about 6.5 μm high, about 7 μm high, about 7.5 μm high, about 8 μm high, about 8.5 μm high, about 9 μm high, about 9.5 μm high, or about 10 μm high. In some embodiments, the one or more pillars are less than any of the following heights (μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the one or more pillars are greater than any of the following heights (μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one or more pillars can be any of a height range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit.

[0081] In some embodiments, the one or more pillars may be cylindrical. In some embodiments, the one or more pillars have a diameter between about 1 μm and about 10 μm. In some embodiments, the one or more pillars have a diameter of about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the one or more pillars have a diameter less than any of the following lengths (μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the one or more pillars have a diameter greater than any of the following lengths (μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the one or more pillars may have any of the following diameter ranges with an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit. In other embodiments, the one or more pillars may have a width approximately the same as any diameter described above, or a width range approximately the same as any diameter range described above, but the one or more pillars may be elliptical pillars, parabolic pillars, hyperbolic pillars, or any other cylindrical or polyhedral shape described herein or known in the art.

[0082] In some embodiments, the microcarrier further includes a magnetic layer attached to the surface of a substantially opaque polymer layer, the magnetic layer comprising a magnetic material. In some embodiments, the magnetic layer does not extend beyond the two-dimensional shape of the substantially opaque polymer layer. In other words, if the outline of the substantially opaque polymer layer is imaged, the resulting image will not be altered by the presence or absence of the magnetic layer. In some embodiments, the magnetic layer may include one or more pillars as described above. That is, the one or more pillars described above may be made of the magnetic material described herein.

[0083] In some embodiments, the microcarrier further includes an orientation index for orienting the analog code of the substantially opaque polymer layer. Any feature of the microcarrier that is visible and / or detectable by imaging (e.g., a form of microscopy or other imaging described herein) and / or by image recognition software can be used as an orientation index. The orientation index can be used as a reference point, for example, in image recognition algorithms, to orient the image of the analog code in a uniform orientation (i.e., the shape of the substantially opaque polymer layer). Advantageously, this simplifies image recognition because the algorithm will only need to compare the image of a particular analog code with a library of analog codes in the same orientation, rather than with a library that includes all analog codes in all possible orientations. In some embodiments, the orientation index includes the asymmetry of the contour of the substantially opaque polymer layer. For example, the orientation index may include visible features, such as the asymmetry of the microcarrier's contour (e.g., as seen by imaging). Figure 8A (As shown in the starting positions 804 and 904 in 9A).

[0084] Any microcarrier described herein may include one or more of the features, elements, or aspects described below. Furthermore, one or more of the features, elements, or aspects described below may employ different features depending on the implementation of the microcarrier, for example, as described above.

[0085] In some embodiments, the substantially transparent polymers of this disclosure include epoxy-based polymers. Suitable epoxy-based polymers for manufacturing the compositions described herein include, but are not limited to, EPON epoxy resins supplied by Hexion Specialty Chemicals, Inc. (Columbus, OH). TMThe family of epoxy resins and any number of epoxy resins supplied by The Dow Chemical Company (Midland, MI). Many examples of suitable polymers are commonly known in the art, including but not limited to SU-8, EPON 1002F, EPON 165 / 154, and poly(methyl methacrylate) / poly(acrylic acid) block copolymers (PMMA-co-PAA). For other polymers, see, for example, Warad, IC Packaging: Package Construction Analysis in Ultra Small IC Packaging, LAP LAMBERT Academic Publishing (2010); The Electronic Packaging Handbook, CRC Press (Blackwell, editor), (2000); and Pecht et al., Electronic Packaging Materials and Their Properties, CCRPress, 1st edition, (1998). These types of materials have the advantage of not swelling in aqueous environments, which ensures uniform microcarrier size and shape within the microcarrier population. In some embodiments, the substantially transparent polymer is a photoresist polymer. In some embodiments, the epoxy polymer is a negative near-UV photoresist with epoxy groups. In some implementations, the epoxy polymer is SU-8.

[0086] In some embodiments, the substantially opaque polymer is a polymer described herein (e.g., SU-8) mixed with one or more opaque or colored dyes. In other embodiments, the substantially opaque polymer is a black matrix resist. Any black matrix resist known in the art can be used; see, for example, U.S. Patent No. 8,610,848, for examples of exemplary black matrix resists and related methods. In some embodiments, the black matrix resist can be colored with black pigment, such as a photoresist patterned on a color filter of an LCD that is part of a black matrix. Black matrix resists may include, but are not limited to, those sold by Toppan Printing Co. (Tokyo), Tokyo OHKA Kogyo (Kawasaki), and Daxin Materials Corp. (Taichung, Taiwan).

[0087] In some embodiments, reference may be made to the central portion of one or more polymer layers. The central portion of this disclosure may take any shape. In some embodiments, the shape of the central portion may reflect or correspond to the shape (e.g., profile) of the corresponding polymer layer. In other embodiments, the shape of the central portion may be independent of the shape (e.g., profile) of the corresponding polymer layer. For example, the central portion of a circular microcarrier surface may be circular in some embodiments and square in other embodiments. The central portion of a square microcarrier surface may be square in some embodiments and circular in other embodiments.

[0088] In some embodiments, the central portion of the polymer layer of this disclosure is about 5%, about 7%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% of the surface area of ​​the polymer layer. In some embodiments, the central portion of the polymer layer of this disclosure is smaller than about one of the following fractions (%) of the substantially transparent polymer layer: 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 7. In some embodiments, the central portion of the polymer layer of this disclosure is larger than approximately one of the following percentages (%) of the substantially transparent polymer layer: 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85. That is, the percentage of the polymer layer surface area included in the central portion can be any of a percentage range having an upper limit of 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 7 and an independently selected lower limit of 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85, wherein the lower limit is less than the upper limit. In some embodiments, the central portion of the polymer layer accounts for approximately 25% of the surface area of ​​the polymer layer. In some implementations, the central portion of the microcarrier surface comprises the entire surface of the microcarrier minus the contour portion.

[0089] As described above, the microcarriers of this disclosure may also include a magnetic layer, which may take the form of a variety of shapes as described herein. In some embodiments, the magnetic layer may be a substantially opaque layer. In some embodiments, the magnetic layer may include a magnetic material. The magnetic layer of this disclosure may be made of any suitable magnetic material, such as materials having paramagnetic, ferromagnetic, or ferrimagnetic properties. Examples of magnetic materials include, but are not limited to, iron, nickel, cobalt, and some rare earth metals (e.g., gadolinium, dysprosium, neodymium, etc.) and their alloys. In some embodiments, the magnetic material includes nickel, including but not limited to elemental nickel and magnetic nickel alloys such as AlNiCo and permalloy. Including a magnetic layer in the microcarriers of this disclosure may be advantageous, for example, in facilitating magnetic separation, and may be suitable for washing, collecting, and otherwise manipulating one or more microcarriers.

[0090] As described above, in some embodiments, the magnetic layer may be fixed to the surface of a substantially transparent polymer layer and surround the central portion of the substantially transparent polymer layer. In other embodiments, as described above, the magnetic layer may include one or more pillars; that is, the one or more pillars described above may be made of the magnetic material described herein.

[0091] In some embodiments, the microcarriers of this disclosure may be encoded using substantially opaque layers that constitute a two-dimensional shape. For example, as described above, the two-dimensional shape may constitute the shape of a substantially opaque layer that contrasts with the substantially transparent layer of the microcarrier, or it may constitute the shape of the microcarrier itself (e.g., its perimeter). Any two-dimensional shape that can encompass multiple distinguishable and unique variations may be used. In some embodiments, the two-dimensional shape includes one or more of linear, circular, elliptical, rectangular, quadrilateral, or higher polygonal aspects, units, and / or shapes.

[0092] In some embodiments, the two-dimensional shape of the substantially opaque polymer layer includes a gear shape. As used herein, a gear shape can refer to a plurality of shapes (e.g., gear teeth) arranged around the periphery of a substantially circular, elliptical, or toroidal body, wherein at least two of the plurality of shapes are spatially separated. In some embodiments, the gear shape includes a plurality of gear teeth. In some embodiments, the simulation code is represented by one or more aspects selected from: the height of one or more of the plurality of gear teeth, the width of one or more of the plurality of gear teeth, the number of gear teeth in the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth. Advantageously, the gear shape encompasses multiple aspects, including the height of the gear teeth, the width of the gear teeth, the number of gear teeth, and the arrangement of the gear teeth, which can be varied to produce a wide variety of potentially unique two-dimensional shapes. However, it should be understood that because the gear shapes of this disclosure are used for encoding and do not need to physically mesh with another gear (e.g., as with a mechanical gear that transmits torque), the gear teeth of this disclosure are not constrained by the need for identical or mutually meshing shapes within or between a plurality of gear shapes. Therefore, the diversity of the shape of the gear teeth in this disclosure can be considered to be significantly greater than the diversity of the shape of mechanical gears.

[0093] In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width of about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width (μm) smaller than any of the following: 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a width (μm) greater than any of the following: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may include one or more gear teeth having any one of the following width ranges: an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2 or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5, wherein the lower limit is less than the upper limit.

[0094] In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height of about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height (μm) less than any of the following: 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the plurality of gear teeth includes one or more gear teeth with a height (μm) greater than any of the following: 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may include one or more gear teeth having any one of the following height ranges: an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit. It should be understood that if the gear teeth are not uniform from their adjacent peripheral segments, the gear teeth may have different measurable heights depending on a reference point (see, for example, Figure 6C The gear tooth 602 in the middle can be 4 or 6.5 μm high depending on the reference point.

[0095] In some embodiments, the plurality of gear teeth includes one or more gear teeth spaced between about 1 μm and about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth spaced between about 1 μm, about 1.5 μm, about 2 μm, about 2.5 μm, about 3 μm, about 3.5 μm, about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, about 6 μm, about 6.5 μm, about 7 μm, about 7.5 μm, about 8 μm, about 8.5 μm, about 9 μm, about 9.5 μm, or about 10 μm. In some embodiments, the plurality of gear teeth includes one or more gear teeth spaced less than any of the following widths (μm): 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5. In some embodiments, the plurality of gear teeth includes one or more gear teeth spaced at intervals greater than any of the following widths (μm): 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5. That is, the plurality of gear teeth may include one or more gear teeth spaced at intervals having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, wherein the lower limit is less than the upper limit.

[0096] In some embodiments, the microcarriers of this disclosure are substantially circular disks. As used herein, a substantially circular shape may refer to any shape with approximately equal distances between all points on the periphery of the shape and the geometric center of the shape. In some embodiments, a shape is considered substantially circular if the variation between any potential radius connecting the geometric center and a given point on the periphery exhibits a length variation of 10% or less. As used herein, a substantially circular disk may refer to any substantially circular shape in which the thickness is significantly less than its diameter. For example, in some embodiments, the thickness of a substantially circular disk may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of its diameter. In some embodiments, the thickness of a substantially circular disk may be about 20% of its diameter. It should be understood that microcarriers with a gear-shaped outline of this disclosure may also be considered substantially circular disks; for example, the shape of the microcarrier, other than one or more gear teeth, may include a substantially circular disk.

[0097] In some embodiments, the diameter of the microcarrier is less than about 200 μm. For example, in some embodiments, the diameter of the microcarrier is less than about 200 μm, less than about 180 μm, less than about 160 μm, less than about 140 μm, less than about 120 μm, less than about 100 μm, less than about 80 μm, less than about 60 μm, less than about 40 μm, or less than about 20 μm.

[0098] In some embodiments, the diameter of the microcarrier is about 180 μm, about 160 μm, about 140 μm, about 120 μm, about 100 μm, about 90 μm, about 80 μm, about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, about 20 μm, or about 10 μm. In some embodiments, the diameter of the microcarrier is about 60 μm.

[0099] In some embodiments, the thickness of the microcarrier is less than about 50 μm. For example, in some embodiments, the thickness of the microcarrier is less than about 70 μm, about 60 μm, about 50 μm, about 40 μm, about 30 μm, less than about 25 μm, less than about 20 μm, less than about 15 μm, less than about 10 μm, or less than about 5 μm. In some embodiments, the thickness of the microcarrier is less than about any of the following thicknesses (μm): 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2. In some embodiments, the thickness of the microcarrier is greater than any of the following thicknesses (μm): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65. That is, the thickness of the microcarrier can be any one of the following ranges (μm) having an upper limit of 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 and an independently selected lower limit of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65, wherein the lower limit is less than the upper limit.

[0100] In some embodiments, the thickness of the microcarrier is about 50 μm, about 45 μm, about 40 μm, about 35 μm, about 30 μm, about 25 μm, about 20 μm, about 19 μm, about 18 μm, about 17 μm, about 16 μm, about 15 μm, about 14 μm, about 13 μm, about 12 μm, about 11 μm, about 10 μm, about 9 μm, about 8 μm, about 7 μm, about 6 μm, about 5 μm, about 4 μm, about 3 μm, about 2 μm, or about 1 μm. In some embodiments, the thickness of the microcarrier is about 10 μm.

[0101] In some aspects, the microcarriers disclosed herein may include trapping agents. In some embodiments, the trapping agent for a particular microcarrier species may be a “unique trapping agent,” for example, the trapping agent is associated with a particular microcarrier species having a specific identifier (e.g., analog code). The trapping agent may be any biomolecule or chemical compound capable of binding to one or more analytes (such as biomolecules or chemical compounds) present in solution. Examples of biomolecule trapping agents include, but are not limited to, DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. Examples of chemical compound trapping agents include, but are not limited to, individual components of a chemical library, small molecules, or environmental toxins (e.g., pesticides or heavy metals).

[0102] In some embodiments, the trapping agent is coupled to the surface of the microcarrier (in some embodiments, at least in the central portion of the microcarrier surface). In some embodiments, the trapping agent may be chemically linked to the microcarrier. In other embodiments, the trapping agent may be physically absorbed to the surface of the microcarrier. In some embodiments, the connecting bond between the trapping agent and the microcarrier surface may be a covalent bond. In other embodiments, the connecting bond between the trapping agent and the microcarrier surface may be a non-covalent bond, including but not limited to salt bridges or other ionic bonds, one or more hydrogen bonds, hydrophobic interactions, van der Waals forces, London dispersion forces, mechanical bonds, one or more halogen bonds, aurophilicity, intercalation, or stacking interactions.

[0103] In some aspects, more than one (e.g., two, three, four, five, six, seven, eight, nine, or ten) capture agents for the same analyte can each associate with the microcarriers described herein. In this embodiment, each capture agent for a particular analyte binds to the analyte with a different affinity, as measured by the dissociation constant of the analyte / capture agent binding. Thus, within a plurality of microcarriers in the composition, there may be two or more subgroups of microcarriers having capture agents bound to the same analyte, but wherein the capture agents associated with each subgroup bind to the analyte with different affinities. In some embodiments, the dissociation constant of the analyte for any of the capture agents is not greater than 10. -6 M, such as 10 - 7 M or 10 -8 M. In other embodiments, the dissociation constant of the analyte with respect to any of the trapping agents is about 10. -10 M to approximately 10 -6 M, as per approx. 10 -10 M to approximately 10-7 M, approximately 10 -10 M to approximately 10 -8 M, approximately 10 -10 M to approximately 10 -9 M, approximately 10 -9 M to approximately 10 -6 M, approximately 10 -9 M to approximately 10 -7 M, approximately 10 -9 M to approximately 10 -8 M, approximately 10 -8 M to approximately 10 -6 M or approximately 10 -8 M to approximately 10 -7 M. In some implementations, the dissociation constants of the analyte with respect to any two trapping agents differ by up to approximately 3 log. 10 If the difference is as high as approximately 2.5 log 10 2log 10 1.5log 10 or 1log 10 .

[0104] In some embodiments, the analytes of this disclosure are coupled to microcarriers for capturing one or more analytes. In some embodiments, one or more analytes can be captured from a sample, such as a biological sample as described herein. In some embodiments, the analytes may include, but are not limited to, DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In other embodiments, the analytes are chemical compounds (such as small molecule chemical compounds) capable of binding to capture agents such as individual components of a chemical library, small molecules, or environmental toxins (e.g., pesticides or heavy metals).

[0105] In some aspects, analytes in samples (such as biological samples) can be labeled with signal-emitting entities capable of emitting a detectable signal upon binding to a trapping agent. In some embodiments, the signal-emitting entity can be colorimetric. In other embodiments, the signal-emitting entity can be fluorescence-based, including but not limited to phycoerythrin, blue fluorescent protein, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and their derivatives. In other embodiments, the signal-emitting entity can be radioisotope-based, including but not limited to those using... 32 P, 33 P, 22 Na、 36 Cl、 2 H, 3 H, 35 S and 123I-labeled molecules. In other embodiments, the signal-emitting entity is light-based, including but not limited to luciferase (e.g., chemiluminescence-based), horseradish peroxidase, alkaline phosphatase, and derivatives thereof. In some embodiments, biomolecules or chemical compounds present in the sample may be labeled with the signal-emitting entity prior to contact with the microcarrier. In other embodiments, biomolecules or chemical compounds present in the sample may be labeled with the signal-emitting entity after contact with the microcarrier.

[0106] IV. Methods for preparing encoded microcarriers

[0107] Certain aspects of this disclosure relate to methods for preparing coded microcarriers (e.g., the microcarriers described herein). Methods for preparing coded microcarriers may include one or more of the microcarrier features or aspects described herein, for example, in Section III above and / or the examples below.

[0108] In some embodiments, the method includes depositing a substantially transparent polymer layer having a first surface and a second surface, the first and second surfaces being parallel to each other. In some embodiments, the parallel first and second surfaces may be a top and bottom surface of a single layer. Any suitable substantially transparent polymer known in the art or described herein may be used. In some embodiments, spin coating is used to deposit the substantially transparent polymer layer.

[0109] In some embodiments, a substantially transparent polymer layer may be deposited on a substrate. Suitable substrates may include those used in standard semiconductor and / or microelectromechanical systems (MEMS) fabrication techniques. In some embodiments, the substrate may include glass, silicon, quartz, plastic, polyethylene terephthalate (PET), indium tin oxide (ITO) coating, etc.

[0110] In some embodiments, the sacrificial layer may be deposited on a substrate (e.g., a substrate as described above). In some embodiments, the sacrificial layer may be made of a polymer, including but not limited to polyvinyl alcohol (PVA) or OmniCoat. TM (MicroChem; Newton, MA). The sacrificial layer can be applied, used, dissolved, or peeled off, for example, according to the manufacturer's instructions.

[0111] In some embodiments, a substantially transparent polymer layer of this disclosure is deposited on a sacrificial layer. To create a planar microcarrier surface using the substantially transparent polymer layer, the substantially transparent polymer layer may be deposited on the planar sacrificial layer. To create a microcarrier surface having one or more pillars protruding from it, the sacrificial layer (e.g., a sacrificial layer deposited on a substrate) may be patterned to have one or more columnar aperture or void regions, for example, using a standard photolithography process. In some embodiments, a substantially transparent polymer layer may be deposited on the sacrificial layer and optionally on a substrate, such that the layer is deposited in one or more columnar aperture or void regions. In some embodiments, another substantially transparent polymer layer may then be deposited on the sacrificial layer and on the one or more columnar aperture or void regions filled with the first substantially transparent polymer layer.

[0112] In some embodiments, the substantially opaque magnetic layer of this disclosure is deposited on a first surface of a substantially transparent polymer layer. In some embodiments, the substantially opaque magnetic layer is deposited by sputtering. The substantially opaque magnetic layer may be made of any magnetic material, such as those described herein. For example, in some embodiments, the substantially opaque magnetic layer comprises nickel (e.g., elemental nickel or alloys thereof).

[0113] In some embodiments, the substantially opaque magnetic layer may be etched to remove portions of the substantially opaque magnetic layer deposited on the central portion of the substantially transparent polymer layer. The substantially opaque magnetic layer may be etched by any method known in the art. For example, in some embodiments, the substantially opaque magnetic layer is etched by conventional wet etching. Exemplary dimensions, shapes, and optional asymmetries of the substantially opaque magnetic layer have been provided above.

[0114] In some embodiments, a second substantially transparent polymer layer of this disclosure is deposited on a substantially opaque magnetic layer. In some embodiments, the second substantially transparent polymer layer has a first surface and a second surface (e.g., a top surface and a bottom surface of a single layer) parallel to each other. In some embodiments, the second surface is anchored to the substantially opaque magnetic layer. In some embodiments, the second substantially transparent polymer layer is aligned with the first substantially transparent polymer layer and has a central portion aligned with the central portion of the substantially transparent polymer layer. Exemplary dimensions of the central portion of the substantially transparent polymer layer have been provided above.

[0115] In some embodiments, the substantially opaque polymer layer of this disclosure is deposited on the first surface of the second substantially transparent polymer layer. In some embodiments, the substantially opaque polymer layer surrounds the central portion of the first substantially transparent polymer layer and the second substantially transparent polymer layer. In some embodiments, the substantially opaque polymer layer comprises a two-dimensional shape representing analog code. Any two-dimensional shape described or illustrated herein, such as the gear shape of this disclosure, can be used. In some embodiments, the substantially opaque polymer layer is deposited on the second substantially transparent polymer layer and etched (e.g., using standard photolithography processes) into the desired two-dimensional shape.

[0116] In some embodiments, one or more pillars may be deposited on a substantially transparent polymer, for example, deposited on a first surface of a second substantially transparent polymer layer at portions not covered by a substantially opaque polymer layer. The one or more pillars may be deposited as described herein, for example using standard photolithography processes.

[0117] In some embodiments employing the optional sacrificial layer and / or substrate of this disclosure, the sacrificial layer and / or removable substrate can be dissolved or stripped by using a solvent. A variety of solvents suitable for fabrication (e.g., in standard semiconductor or MEMS fabrication processes, such as photoresist removal) are known in the art. In some embodiments, the solvent is a photoresist stripping agent solvent, such as a solvent based on DMSO or 1-methyl-2-pyrrolidone (NMP). In some embodiments, the solvent is... Photoresist stripper, such as 300T (AZ Electronic Materials; Somerville, NJ).

[0118] In some embodiments, the method includes depositing the sacrificial layer of this disclosure on a substrate of this disclosure. The sacrificial layer, the substrate, and a suitable deposition method are, for example, as described above.

[0119] In some embodiments, a substantially opaque polymer layer of this disclosure is deposited on a sacrificial layer. In some embodiments, the substantially opaque polymer layer has a first surface and a second surface parallel to each other (e.g., a top surface and a bottom surface of a single layer). In some embodiments, the second surface is anchored to the sacrificial layer.

[0120] In some embodiments, the outline of the substantially opaque polymer layer is shaped into a two-dimensional shape representing analog code, for example, as described herein. The substantially opaque polymer layer can be shaped by any method known in the art or described herein, such as using standard photolithography processes, including but not limited to spin coating, soft baking, UV exposure, etching, and hard baking.

[0121] In some implementations, the sacrificial layer and / or removable substrate can be dissolved or stripped by using a solvent (e.g., as described above).

[0122] In other embodiments, the magnetic layer comprising a magnetic material disclosed herein is deposited on a sacrificial layer. Exemplary magnetic materials, magnetic layer shapes / sizes, and associated deposition methods have been provided above. For example, in some embodiments, the magnetic layer may be shaped into one or more pillars, such as as shown by pillar 906. In other embodiments, the magnetic layer may be (e.g., embedded) between two opaque polymer layers, as shown by magnetic layer 704. The magnetic material may comprise any magnetic material described herein. For example, in some embodiments, the magnetic material comprises nickel (e.g., elemental nickel or alloys thereof).

[0123] In some embodiments, a substantially opaque polymer layer of this disclosure is deposited on a magnetic layer. In some embodiments, the substantially opaque polymer layer has a first surface and a second surface parallel to each other (e.g., a top surface and a bottom surface of a single layer). In some embodiments, the surface of the substantially opaque polymer layer (e.g., the second surface) is anchored to the magnetic layer.

[0124] In some implementations, the outline of the substantially opaque polymer layer is shaped into a two-dimensional shape representing analog code, for example, as described above.

[0125] In some implementations, the sacrificial layer and / or removable substrate can be dissolved or stripped by using a solvent (e.g., as described above).

[0126] Exemplary microcarrier shapes, sizes, and optional features suitable for the methods described above are provided throughout this disclosure.

[0127] In some embodiments, the capture agent may be coupled to the microcarriers of this disclosure, such as the microcarriers described herein and / or microcarriers produced by any of the methods described herein. Any capture agent described herein or any capture agent known in the art suitable for capturing the analytes described herein may be used in the methods and / or microcarriers of this disclosure.

[0128] In some embodiments, the trapping agent may be coupled to a polymer layer of this disclosure, such as a substantially transparent or substantially opaque polymer layer as described herein. In some embodiments, the trapping agent may be coupled to one or both of the first or second surfaces of the polymer layer. In some embodiments, the trapping agent may be coupled to at least a central portion of the polymer layer (e.g., a central portion as described herein). In some embodiments, the polymer comprises an epoxy-based polymer or otherwise contains epoxy groups.

[0129] In some embodiments, the coupling trapping agent involves reacting the polymer with a photoacidogen and light to produce a crosslinked polymer. In some embodiments, the light has a wavelength that activates the photoacidogen, such as UV or near-UV light. Photoacidogens are commercially available from Sigma-Aldrich (St. Louis) and BASF (Ludwigshafen). Any suitable photoacidogen known in the art can be used, including but not limited to triphenyl or triarylsulfonium hexafluoroantimonate; triarylsulfonium hexafluorophosphate; triphenylsulfonium perfluoro-1-butanesulfonate; triphenylsulfonium trifluoromethanesulfonate; tri(4-tert-butylphenyl)sulfonium perfluoro-1-butanesulfonate or tri(4-tert-butylphenyl)sulfonate; photoacidogens containing bis(4-tert-butylphenyl)iodonium, such as perfluoro-1-butanesulfonate, p-toluenesulfonate, and tri... bis(4-tert-butylphenyl)iodonium fluoromethanesulfonate; Boc-methoxyphenyl diphenylsulfonium trifluoromethanesulfonate; (tert-butoxycarbonylmethoxynaphthyl)-diphenylsulfonium trifluoromethanesulfonate; (4-tert-butylphenyl)diphenylsulfonium trifluoromethanesulfonate; hexafluorophosphate, nitric acid, perfluoro-1-butanesulfonic acid, trifluoromethanesulfonate or p-toluenesulfonic acid diphenyliodonium; (4-fluorophenyl)diphenylsulfonium trifluoromethanesulfonate; N-fluoromethanesulfonate -Hydroxynaphthalene dicarboximide; Perfluoro-1-butyric acid N-hydroxy-5-norbornene-2,3-dicarboximide; Trifluoromethanesulfonic acid (4-iodophenyl)diphenylsulfonium; Trifluoromethanesulfonic acid (4-methoxyphenyl)diphenylsulfonium; 2-(4-methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5-triazine; Trifluoromethanesulfonic acid (4-methylphenyl)diphenylsulfonium; Trifluoromethanesulfonic acid (4-methylthiophenyl)methylphenylsulfonium; Trifluoromethanesulfonic acid (4-phenoxyphenyl)diphenylsulfonium; Trifluoromethanesulfonic acid (4-phenylthiophenyl)diphenylsulfonium; or in product-finder.basf.com / group / corporate / product-finder / de / literature-document: / Bran Any photoacid generator described in d+Irgacure-Brochure--Photoacid+Generator+Selection+Guide-English.pdf. In some embodiments, the photoacid generator is a sulfonium-containing photoacid generator.

[0130] In some embodiments, the coupling scavenger comprises reacting the epoxide of the crosslinked polymer with functional groups such as amines, carboxyl groups, thiols, etc. Alternatively, the epoxy groups on the surface may be oxidized to hydroxyl groups, which are then used as initiation sites for the graft polymerization of water-soluble polymers such as poly(acrylic acid). The carboxyl groups in the poly(acrylic acid) are then used to form covalent bonds with the amino or hydroxyl groups in the scavenger.

[0131] In some embodiments, coupling the trapping agent involves reacting an epoxide of a crosslinked polymer with a compound containing an amine and a carboxyl group. In some embodiments, the amine of the compound reacts with the epoxide to form a compound-coupled crosslinked polymer. It is not desirable to be bound by theory, but it is believed that the trapping agent can be coupled to the polymer prior to crosslinking; however, this may reduce the uniformity of the resulting surface. Any compound having a primary amine and a carboxyl group can be used. The compound may include, but is not limited to, glycine, aminoundecanoic acid, aminohexanoic acid, acrylic acid, 2-carboxyethyl acrylic acid, 4-vinylbenzoic acid, 3-acrylamido-3-methyl-1-butyric acid, glycidyl methacrylate, etc. In some embodiments, the carboxyl group of the compound-coupled crosslinked polymer reacts with an amine (e.g., a primary amine) of the trapping agent to couple the trapping agent to a substantially transparent polymer.

[0132] Descriptions of various capture agents and analytes suitable for the methods described above can be found throughout this disclosure, for example in Part III above and / or the examples below.

[0133] V. Multiple determination

[0134] Certain aspects of this disclosure relate to methods for detecting analytes in solution using coded microcarriers (such as those described herein). Methods for analyte detection utilize coded microcarriers comprising one or more of the microcarrier features or aspects described herein, for example, in sections III and IV above and / or the examples below. Advantageously, these coded microcarriers allow for improved analyte detection and reduced identification errors in improved multiplexing using a large number of potentially unique microcarriers, compared to conventional multiplexing assays. The analyte detection methods used herein can be performed in any suitable assay vessel known in the art, such as microplates, petri dishes, or any number of other well-known assay vessels.

[0135] In some embodiments, a method for detecting an analyte in a solution includes contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers includes at least a first microcarrier of the present disclosure, the first microcarrier specifically capturing the first analyte and encoded with a first analog code, and a second microcarrier of the present disclosure, the second microcarrier specifically capturing the second analyte and encoded with a second analog code; decoding the first analog code and the second analog code using analog shape recognition to identify the first microcarrier and the second microcarrier; and detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0136] In some embodiments, the method includes contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers. In some embodiments, the plurality of microcarriers may include a first microcarrier of this disclosure that specifically captures the first analyte (e.g., using a capture agent specific to the first analyte conjugated to the microcarrier), wherein the first microcarrier is encoded with a first analog code; and a second microcarrier of this disclosure that specifically captures the second analyte (e.g., using a capture agent specific to the second analyte conjugated to the microcarrier), wherein the second microcarrier is encoded with a second analog code different from the first analog code. In some embodiments, the first analyte and the second analyte may be different. In other embodiments, the first analyte and the second analyte may be the same; for example, the first microcarrier and the second microcarrier may redundantly recognize the same analyte (which can be useful, for example, for quality control purposes), or they may recognize different regions of the same analyte (e.g., antibodies recognizing different epitopes of the same antigen).

[0137] The methods disclosed herein can be used to detect analytes in any suitable solution. In some embodiments, the solution comprises a biological sample. Examples of biological samples include, but are not limited to, blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluid of skin or adipose tissue, saliva, tears, bronchoalveolar lavage fluid, oropharyngeal secretions, intestinal fluid, cervical, vaginal, or uterine secretions, and semen. In some embodiments, the biological sample may be derived from a human. In other embodiments, the solution comprises samples that are not biological samples, such as environmental samples, samples prepared in a laboratory (e.g., samples containing one or more analytes that have been prepared, isolated, purified, and / or synthesized), fixed samples (e.g., formalin-fixed, paraffin-embedded, or FFPE samples), etc.

[0138] In some implementations, the analysis is multiplexed, i.e., each solution (e.g., sample) is analyzed such that a signal from a signal emitting entity is detected by a reaction detection system for at least two, three, four, five, ten, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, or fifty or more target analytes.

[0139] In some embodiments, the method includes using analog shape recognition to decode a first analog code and a second analog code to identify a first microcarrier and a second microcarrier. Conceptually, decoding may involve imaging the analog code of each microcarrier (e.g., in a solution or sample), comparing each image to a library of analog codes, and matching each image to images from said library to explicitly identify the code. Optionally, as described herein, when using microcarriers that include orientation indicators (e.g., asymmetry), decoding may also include the step of rotating each image to align with a specific orientation (e.g., partially based on the orientation indicator). For example, if the orientation indicator includes a gap, the image may be rotated until the gap reaches a predetermined position or orientation (e.g., the 0° position of the image).

[0140] Various shape recognition software, tools, and methods are known in the art. Examples of such APIs and tools include, but are not limited to, those from ReKognition. Research FaceSDK, OpenBR, Face and SceneRecognition, Betaface API, and various ImageJ plugins. In some implementations, simulated shape recognition may include, but is not limited to, image processing steps such as foreground extraction, shape detection, and thresholding (e.g., automatic or manual image thresholding).

[0141] Those skilled in the art will understand that the methods and microcarriers described herein are applicable to a variety of imaging devices, including but not limited to microscopes, plate readers, etc. In some embodiments, decoding the analog code may include illuminating the first and second microcarriers by passing light through substantially transparent portions (e.g., substantially transparent polymer layers) and / or the surrounding solution of the first and second microcarriers. The light may then fail to penetrate or penetrate at a lower intensity or otherwise significantly different intensity through substantially opaque portions (e.g., substantially opaque polymer layers) of the first and second microcarriers to produce a light pattern corresponding to a first analog code on the first microcarrier and a second analog code on the second microcarrier.

[0142] As described above, any type of optical microscopy can be used in the methods of this disclosure, including but not limited to one or more of the following: bright-field, dark-field, phase contrast, differential interference contrast (DIC), Nomarsky interference contrast (NIC), Nomarsky, Huffman modulation phase contrast (HMC), or fluorescence microscopy. In some embodiments, bright-field microscopy can be used to decode analog codes, and fluorescence microscopy can be used to detect analytes.

[0143] In some embodiments, decoding the analog code may further include imaging a light pattern of a first analog code to produce an image of the first analog code, and imaging a light pattern of a second analog code to produce an image of the second analog code. In other words, the pattern of the imaged light may correspond to a pattern of substantially transparent / substantially opaque regions of the microcarrier, thereby producing an image of the analog code. Such imaging may include multiple steps, including but not limited to capturing the image, thresholding the image, and any other image processing steps required to achieve more accurate, precise, or robust imaging of the analog code.

[0144] In some embodiments, decoding the analog code may further include using analog shape recognition to match a first analog-coded image with a first analog code and a second analog-coded image with a second analog code. In some embodiments, an image may be matched with an analog code within a predetermined threshold (e.g., image files from an image file library, where each image file corresponds to a unique two-dimensional shape / analog code), said predetermined threshold, for example, allowing a predetermined amount of deviation or mismatch between the image and an example analog code image. The threshold may be determined empirically and may naturally be based on the specific type of two-dimensional shape used for the analog code and the degree of variation between the potential groups of two-dimensional shapes.

[0145] In some embodiments, the method includes detecting the amount of a first analyte bound to a first microcarrier and the amount of a second analyte bound to a second microcarrier. Any suitable analyte detection technique known in the art can be used. For example, in some embodiments, the first and second microcarriers may be incubated with one or more detection agents. In some embodiments, the one or more detection agents bind the first analyte captured by the first microcarrier and the second analyte captured by the second microcarrier. In some embodiments, the method further includes measuring the amount of detection agent bound to the first and second microcarriers.

[0146] In some embodiments, the analyte in the solution (such as a biological sample) may be labeled with a detector (e.g., a signal-emitting entity) capable of emitting a detectable signal upon binding to a trapping agent. In some embodiments, the detector may be colorimetric. In other embodiments, the detector may be fluorescence-based, including but not limited to phycoerythrin, blue fluorescent protein, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and their derivatives. In other embodiments, the detector may be radioisotope-based, including but not limited to... 32 P, 33 P, 22 Na、 36 Cl、 2 H, 3 H, 35 S and 123I-labeled molecules. In other embodiments, the detection agent is light-based, including but not limited to luciferase (e.g., chemiluminescence-based), horseradish peroxidase, alkaline phosphatase, and derivatives thereof. In some embodiments, the detection agent may label biomolecules or chemical compounds present in the solution prior to contact with the microcarrier composition. In other embodiments, the detection agent may label biomolecules or chemical compounds present in the solution after contact with the microcarrier composition. In other embodiments, the detection agent may be coupled to a molecular or macromolecular structure that specifically binds to a target analyte, such as DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and / or antibody fragments.

[0147] In some embodiments, the detection agent is a fluorescent detection agent, and the amount of detection agent bound to the first microcarrier and the second microcarrier is measured by fluorescence microscopy (e.g., fluorescence microscope or ELISA reader). In other embodiments, the detection agent is a luminescent detection agent, and the amount of detection agent bound to the first microcarrier and the second microcarrier is measured by luminescence microscopy (e.g., luminescence microscope or ELISA reader).

[0148] In some embodiments, each analyte / capture agent may be used in conjunction with a specific detection agent. As a non-limiting example, the detection agent may be a detection agent conjugated to an antibody that specifically binds to the analyte (e.g., a fluorescent, luminescent, enzyme, or other detection agent; or, if the analyte is a homologous ligand / receptor of a ligand-receptor pair, the detection agent may be the ligand or receptor of said ligand-receptor pair). This technique is conceptually similar to a sandwich ELISA or protein microarray that includes the capture and detection of antibodies (however, in the context of this invention, it should be noted that the reagents in this example are not strictly limited to antibodies). As another non-limiting example, the detection agent may be a fluorescent or other detectable probe conjugated to a target protein (such as a labeled target analyte). For example, a reaction may be used to conjugate the detection agent to one or more proteins in a target solution (e.g., a sample), and then said one or more proteins are captured by a capture agent (conceptually similar to the antigen capture type of protein microarrays).

[0149] In other implementations, a variety of unique analytes / capture agents can be used with a universal detection agent. As a non-limiting example, if the analyte is an antibody, the detection agent can be a reagent that binds to the Fc region of the antibody; if the analyte is a polynucleotide such as DNA or RNA, the detection agent can be a fluorescent or other detectable probe conjugated to an oligonucleotide (e.g., a single-stranded oligonucleotide hybridized to the analyte). The latter case is conceptually similar to microarray technology.

[0150] In some embodiments, the detection step may include one or more washing steps, such as to reduce contaminants, remove any substances nonspecifically bound to the capture agent and / or the surface of the microcarrier, etc. In some embodiments, a magnetic separation step may be used to wash the microcarrier containing the magnetic layer or material of this disclosure. In other embodiments, other separation steps known in the art may be used.

[0151] In some implementations, the decoding step may occur after the detection step. In other implementations, the decoding step may occur before the detection step. In still other implementations, the decoding step may occur simultaneously with the detection step.

[0152] VI. Reagent kits or products

[0153] This document also provides kits or articles containing multiple microcarriers disclosed herein. These kits or articles are particularly suitable for performing multiplex assays, such as the exemplary multiplex assays described herein (see, for example, section V above).

[0154] In some embodiments, the kit or article of manufacture may include a first microcarrier of the present disclosure that specifically captures a first analyte (e.g., using a capture agent specific to the first analyte conjugated to the microcarrier), wherein the first microcarrier is encoded with a first analog code; and a second microcarrier of the present disclosure that specifically captures a second analyte (e.g., using a capture agent specific to the second analyte conjugated to the microcarrier), wherein the second microcarrier is encoded with a second analog code different from the first analog code. In some embodiments, the first analyte and the second analyte may be different. In other embodiments, the first analyte and the second analyte may be the same; for example, the first microcarrier and the second microcarrier may redundantly recognize the same analyte (this can be useful, e.g., for quality control purposes), or they may recognize different regions of the same analyte (e.g., antibodies that recognize different epitopes of the same antigen). The kit or article of manufacture may include any microcarrier described herein (see, for example, Section III above and the examples below) or any microcarrier produced using the methods described herein (see, for example, Section IV above and the examples below).

[0155] In some embodiments, the kit or article may further include one or more detection reagents of this disclosure for detecting amounts of a first analyte bound to a first microcarrier and amounts of a second analyte bound to a second microcarrier. In some embodiments, the detection reagent for the first analyte may be the same as the detection reagent for the second analyte. In other embodiments, the detection reagent for the first analyte may be different from the detection reagent for the second analyte.

[0156] In some embodiments, the kit or article may also include instructions for using the kit or article to detect one or more analytes (e.g., first and second analytes). These instructions may be for use with the kit or article, for example, in any of the methods described herein.

[0157] In some embodiments, the kit or article may also include one or more detection reagents (e.g., as described above), along with any instructions or reagents suitable for coupling the detection reagent to one or more analytes or for coupling the detection reagent to one or more macromolecules that identify the analyte. The kit or article may also include any additional components for using the microcarrier in an assay (e.g., multiplex assay), including but not limited to plates (e.g., 96-well or other similar microplates), petri dishes, microscope slides or other suitable assay containers; non-transitory computer-readable storage media (e.g., including software and / or other instructions for simulating shape or code recognition); detergents; buffers; plate sealants; mixing containers; diluents or storage solutions, etc.

[0158] Example

[0159] The invention will be more fully understood by referring to the following embodiments. However, these should not be construed as limiting the scope of the invention. It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or variations will be suggested to those skilled in the art based on them, and these modifications or variations are included within the spirit and scope of this application and the appended claims.

[0160] Attention now turns to microcarriers for multiplex assays (e.g., analyte detection) and methods for their production. The following examples illustrate exemplary embodiments of analog-coded microcarriers for analyte detection, which can be used, in particular, in the methods, assays, kits, or articles described herein. It should be noted that these exemplary embodiments are by no means intended to be limiting, but are provided to illustrate some aspects and features set forth herein.

[0161] Example 1: Microcarrier with two-dimensional analog code and uniform shape encoding

[0162] As described above, analog-coded microcarriers are highly advantageous for multiple determinations due to the large number of potentially unique identifiers and reduced identification errors. This embodiment describes various types of microcarriers encoded with two-dimensional shapes, which can be used as analog codes for identification. It should be understood that the encoded microcarriers of this disclosure may include some or all of the optional features set forth below in any combination.

[0163] Figure 1A and 1BTwo views of an exemplary microcarrier 100 are shown. The microcarrier 100 is a circular disk with a diameter of approximately 50 μm and a thickness of 10 μm. Figure 1A A view of the microcarrier 100 as observed from the circular surface of the disk is provided, while Figure 1B The microcarrier 100 is shown with Figure 1A The diagram shows an orthogonal side view of the surfaces. Two components of the microcarrier 100 are shown. First, a substantially transparent polymer layer 102 provides the body of the microcarrier. Layer 102 can be produced, for example, using a polymer such as SU-8, as described above.

[0164] A substantially opaque polymer layer 104 is fixed to the surface of layer 102. Although Figure 1B The cross-section of the microcarrier 100 shown illustrates a discontinuous view of layer 104, but... Figure 1A The view shown illustrates that layer 104 is shaped like a circular gear with multiple teeth. The shape, number, size, and spacing of these teeth constitute a two-dimensional shape, and one or more of these aspects of the teeth can be modified to produce multiple two-dimensional shapes for analog coding. Advantageously, the outer edges of the gear teeth of layer 104 fit within the periphery of layer 102. This allows for multiple analog codes, each representing a unique identifier for a microcarrier species, while maintaining a uniform overall shape across multiple microcarrier species. In other words, each microcarrier species within a group of multiple species can have different two-dimensional gear shapes (i.e., analog codes), but each microcarrier will have the same periphery, resulting in greater uniformity of physical properties (e.g., size, shape, behavior in solution, etc.). Layer 104 can be produced, for example, using a polymer such as SU-8 mixed with a dye or using a black matrix resist, as described above.

[0165] Layer 104 surrounds the central portion 106 of layer 102. A trapping agent for capturing the analyte is coupled to at least the central portion 106 on one or both surfaces (i.e., the upper / lower surfaces) of layer 102. Advantageously, this allows the central portion 106 to be imaged without any possibility of interference generated by layer 104.

[0166] Figure 1C and 1D An exemplary assay using microcarrier 100 for the detection of analytes is shown. Figure 1C The microcarrier 100 is shown to include a capture agent 108 coupled to one or more surfaces in at least a central portion 106. The microcarrier 100 is contacted with a solution containing an analyte 110, which is captured by the capture agent 108. As described above, various capture agents can be used to capture different types of analytes, from small molecules, nucleic acids, and proteins (e.g., antibodies) to organelles, viruses, and cells. Figure 1CA single microcarrier species (i.e., microcarrier 100) is shown for capturing analyte 110, but multiple microcarrier species are used in multiple assays, each species having a specific capture agent that identifies a particular analyte.

[0167] Figure 1D An exemplary method for “reading” microcarrier 100 is shown. The method includes two steps that can be performed simultaneously or separately. First, the capture of analyte 110 by the trapping agent 108 is detected. Figure 1D In the illustrated embodiment, the detection agent 114 is bound to the analyte 110. Analytes not captured by the trapping agent coupled to the microcarrier 100 can be washed away prior to detection, so that only the analyte bound to the microcarrier 100 is detected. The detection agent 114 also includes reagents for detection. As one embodiment, the detection agent 114 may include a fluorophore that emits light 118 (e.g., photons) when excited by light 116 at a wavelength within the excitation spectrum of the fluorophore. Light 118 can be detected by any suitable detection method, such as fluorescence microscopy, ELISA reader, etc.

[0168] In addition, the unique identifier of the microcarrier 100 is read. Figure 1D In the illustrated embodiment, light 112 is used to irradiate the field containing microcarriers 100 (in some embodiments, light 112 may have a different wavelength than light 116 and 118). When light 112 irradiates the field containing microcarriers 100, the light passes through the substantially transparent polymer layer 102 but is blocked by the substantially opaque polymer layer 104, as... Figure 1D As shown in the figure. This produces a light pattern that can be imaged, for example, by optical microscopy (e.g., using differential interference difference or DIC microscopy). The light pattern is based on the two-dimensional shape of the microcarrier 100 (i.e., analog code). Standard image recognition techniques can be used to decode the analog code represented by the image of the microcarrier 100.

[0169] The analyte detection step and the identifier imaging step can occur in any order or simultaneously. Advantageously, Figure 1D The two detection steps shown can be performed on a single imaging device. As an example, a microscope capable of fluorescence and optical (e.g., bright-field) microscopy can be used to quantify the amount of analyte 110 bound to microcarrier 100 (e.g., as detected by detector 114) and to image the analog codes generated by layers 102 and 104. This allows for a more efficient assay with fewer equipment requirements.

[0170] Now go to Figure 2A and 2BThis illustrates another exemplary microcarrier 200. Similar to microcarrier 100, microcarrier 200 includes a substantially transparent polymer layer 202 and a substantially opaque polymer layer 204. Furthermore, microcarrier 200 includes a magnetic layer 206. Figure 2A As shown, the magnetic layer 206 can be formed as a ring between the central portion 208 and the substantially opaque layer 204.

[0171] Figure 2B The magnetic layer 206 is shown to be embedded within layer 202. Layer 202 may also include more than one layer such that the magnetic layer 206 is sandwiched between two substantially transparent polymer layers (e.g., as shown in the diagram). Figure 2B Alternatively, magnetic layer 206 may be fixed to the same surface of layer 202 as layer 204, or magnetic layer 206 may be fixed to the surface of layer 202 opposite to layer 204. In some embodiments, magnetic layer 206 may include nickel.

[0172] The magnetic layer 206 imparts magnetic properties to the microcarrier 200, which can be advantageously used in a variety of applications. For example, the microcarrier 200 can be held to a surface by magnetic attraction during a washing step, thereby allowing for effective washing without loss or otherwise damage to the microcarrier.

[0173] Besides its magnetic properties, layer 206 is also essentially opaque. When... Figure 1D When imaging as shown (e.g., using light 112), layer 206 will partially or completely block the transmitted light, thereby creating a pattern for imaging. Figure 2A As shown, layer 206 is also asymmetric—in this embodiment, it includes gap 210. This asymmetry produces, for example, a... Figure 1D The orientation index shown uses light 112 for imaging. Advantageously, the orientation index can be used during image recognition to orient the two-dimensional shape generated by the imaging layer 204 in a uniform orientation, enabling easier recognition of analog codes. This allows decoding of microcarriers imaged in any orientation.

[0174] Figure 3 Showing the use Figure 1A-2B The gear shape shown may have a large number of potential simulation codes. Figure 3 An exemplary coding scheme is shown, in which multiple shape change points are marked, for example, at positions 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, and 328 on the exemplary microcarrier 300. Even using a simple "filled or unfilled" scheme, based on using 14 shape change points, up to 2 14A unique code is also possible. This approach is convenient for both manufacturing and generating two-dimensional shapes that are easily distinguishable for image recognition analysis. However, due to the use of analog encoding, more than two possibilities are used (e.g., in cases such as...). Figure 3 More complex schemes are possible (at each point of shape change marked in the diagram), thus exponentially expanding the number of unique identifiers. For example, multiple gear tooth shapes and / or multiple gear tooth sizes are possible. Figure 1A-3 The two-dimensional gear shape shown facilitates a wide range of unique analog codes while providing a large central portion (e.g., central portions 106 and 208) for analyte detection.

[0175] Figure 4A Show Figure 3 Three exemplary implementations of the encoding scheme shown are microcarriers 400, 402, and 404. The unique code for microcarriers 400, 402, and 404 is... Figure 3 It is generated by a simple "fill or not fill" scheme. Figure 4B Ten exemplary implementations of codes (cod) are shown, particularly in terms of the number of shapes (e.g., two different shapes in code ZN_3 compared to seven different shapes in code ZN_10) and / or the size of the shapes (e.g., large, small, and medium-sized shapes in code ZN_2). Importantly, as mentioned above, using analog image recognition yields more complex encoding schemes, thereby greatly expanding the number of potentially unique codes.

[0176] Now go to Figure 5A and 5B This illustrates another exemplary microcarrier 500. Similar to microcarrier 200, microcarrier 500 includes a substantially transparent polymer layer 502, a substantially opaque polymer layer 504, a magnetic layer 506, and a central portion 508. Furthermore, microcarrier 500 has four pillars including pillars 510, which may have any shape extending from the surface of layer 502. Figure 5A As shown, these pillars can be aligned with the magnetic layer 506 to prevent any possibility of interfering with the detection of analytes in the central portion 508 or the reading of the two-dimensional shape (i.e., analog code) of the layer 504. Figure 5B These pillars are shown to extend from the upper and lower surfaces of the microcarrier 500. Pillars 510 may be made, for example, using the same substantially transparent polymer as layer 502 (exemplary manufacturing methods are described below). Advantageously, one or more pillars such as pillar 510 may be used, for example, by optical contact bonding to prevent the microcarriers from adhering to each other and / or containers (e.g., the sides of pores in a porous plate).

[0177] Example 2: Microcarrier with two-dimensional analog code encoded in the shape of the microcarrier

[0178] The preceding embodiments illustrate several exemplary implementations of microcarriers in which analog codes are provided by an opaque layer attached to a transparent polymer layer. This is advantageous, for example, in allowing greater uniformity between different types of microcarriers (i.e., each having the same peripheral shape provided by the transparent polymer layer).

[0179] However, for other reasons, it may be advantageous to use the periphery of the microcarrier itself as the two-dimensional shape for analog coding. For example, if the analog code is provided by the shape of the microcarrier itself, only one layer is needed, thus simplifying the manufacturing process. Furthermore, shaping the periphery of the microcarrier can be achieved through highly precise manufacturing techniques, allowing for highly reproducible shapes for more accurate image recognition.

[0180] Figure 6A and 6B An exemplary microcarrier 600 of this type is shown. The microcarrier 600 is a gear-shaped disk with a diameter of approximately 80 μm and a height of 15 μm, including optional pillar elements (similar to pillar 510 as described above). Instead of separate transparent and opaque polymer layers, the microcarrier 600 is made of a single opaque polymer layer 602. The microcarrier 600 can be as follows: Figure 1D The imaging shown is based on the entire shape of the microcarrier (e.g., the periphery of the opaque polymer layer). One or both surfaces of the microcarrier 600 can be used to couple the trapping agent as described above, and either the central portion or the entire surface can be used.

[0181] Figure 6C The dimensions of the gear teeth 604 of the microcarrier 600 are shown. As illustrated, in this embodiment, the gear teeth 604 are 4 μm wide and spaced 4 μm apart from adjacent gear teeth 606. Because the two-dimensional shape of the microcarrier 600 is analog-coded, the perimeter between adjacent gear teeth can be variable, thus allowing for various gear tooth shapes. For example, the gear teeth 604 may extend in height by 4 or 6.5 μm relative to the adjacent peripheral segments immediately to the left or right, respectively.

[0182] Figure 7 Another embodiment of this type of microcarrier, microcarrier 700, is shown. Similar to microcarrier 600, microcarrier 700 is made of an opaque polymer layer 702. Furthermore, the microcarrier includes a magnetic layer 704. The magnetic layer 704 may be fixed to one of the surfaces of the microcarrier 700, or it may be embedded within the microcarrier 700 (e.g., between two opaque polymer layers). The magnetic layer 704 may be produced, for example, by depositing nickel. As described above, the magnetic layer allows for additional functionality, such as the option to clean the microcarrier 700 while it is magnetically attached to another surface.

[0183] Now go to Figure 8A Another exemplary microcarrier 800 is shown. Similar to microcarrier 700, microcarrier 800 includes an opaque polymer layer 802 (and optionally, a magnetic layer such as layer 704). Furthermore, microcarrier 800 includes a starting position 804 having a shape different from the rest of the periphery of microcarrier 800. The starting position 804 can be used as an orientation indicator for image recognition, as described above regarding... Figure 2A The gap 210 shown in the figure is described.

[0184] Figure 8B The available encoding schemes are shown. Figure 8B Microcarrier 810 is shown, similar to microcarrier 800, comprising an opaque polymer layer 812 and a starting position 814 (and optionally, a magnetic layer such as layer 704). In this scheme, potential shape change points around the gear are marked, for example, at positions 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, and 844. Figure 8B As shown, although only two potential shapes are available for positions 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, and 844, this implementation allows for up to 2 13 A unique code. Furthermore, as mentioned above, using analog encoding allows for encoding at any or all indicated locations around the perimeter (e.g., in situations like...). Figure 8B The number is greatly expanded by using more than two potential shapes at each shape change point marked in the middle.

[0185] Figures 9A-9C Another potential embodiment of microcarrier 900 is shown. Similar to microcarrier 800, microcarrier 900 is a gear-shaped microcarrier comprising an opaque polymer layer 902 and an initiation site 904 (and optionally, a magnetic layer such as layer 704). Furthermore, microcarrier 900 may have one or more pillars (e.g., pillar 906) fixed to one or both surfaces of microcarrier 900. As shown in the cross-section of FIG9B, pillar 906 extends from the surface of layer 902. Advantageously, pillar 906 helps to reduce the possibility of optical contact adhesion (as described above with reference to pillar 510).

[0186] Figure 9C shows the dimensions of post 906. In this embodiment, post 906 is a cylinder with a height of 3 μm and a diameter of 3 μm, but as mentioned above, such posts are by no means limited to a cylindrical shape. In some embodiments, post 906 is made of a magnetic material (such as nickel). This allows post 906 to also be used as a magnetic element for the magnetic manipulation of microcarrier 900, as described above.

[0187] Example 3: A method for generating microcarriers with two-dimensional analog codes encoded in the shape of the microcarrier.

[0188] Exemplary embodiments of various types of microcarriers have been described in the foregoing embodiments; now, attention is focused on methods for producing microcarriers. As mentioned above, depending on the desired construction and / or optional features, the microcarriers of this disclosure may be made of one, two, or more constituent layers.

[0189] Figure 10 The process 1000 shown illustrates an exemplary workflow for fabricating monolayer microcarriers as described in Example 2 above. At block 1002, a sacrificial layer 1006 is constructed on a substrate 1004. In some embodiments, the substrate 1004 may be a glass substrate. At block 1010, a layer 1012 is deposited on the sacrificial layer 1006. In some embodiments, layer 1012 is an opaque polymer layer. At block 1020, photolithography is used to shape the periphery of layer 1012 into a gear shape (as described above) to produce a gear-shaped layer 1022. At block 1030, the entire structure (i.e., layer 1022, sacrificial layer 1006, and substrate 1004) is immersed in a solvent. This solvent treatment dissolves the sacrificial layer 1006 and releases the gear-shaped layer 1022 from the substrate 1004, thereby producing a microcarrier 1032. In some embodiments, the microcarrier 1032 may be further modified, for example, by coupling a trapping agent to one or both surfaces.

[0190] As described in Embodiment 2 above, the gear-shaped microcarrier may include optional elements such as magnetic components (e.g., pillars and / or magnetic layers). Figure 11A and 11B The process 1100 shown illustrates an exemplary workflow for manufacturing a gear-shaped microcarrier having one or more magnetic components.

[0191] like Figure 11A As shown, at frame 1102, a sacrificial layer 1106 is constructed on a substrate 1104. In some embodiments, the substrate 1104 may be a glass substrate. At frame 1110, a magnetic layer 1112 is deposited on the sacrificial layer 1106. In some embodiments, the magnetic layer 1112 comprises nickel. At frame 1120, the magnetic layer 1112 is shaped into a shaped magnetic layer 1122 by photolithography. The shaped magnetic layer 1122 may take any desired shape; for example, it may be shaped into one or more pillars, as shown by pillar 906 in FIG. 9A.

[0192] like Figure 11BAs shown, at frame 1130, a substantially opaque polymer layer 1132 is deposited on the formed magnetic layer 1122 and sacrificial layer 1106. At frame 1140, the periphery of layer 1132 is formed into a substantially opaque gear-shaped layer 1142 (e.g., ...) by photolithography. Figure 6A -One of the gear shapes shown in -9A). At frame 1150, the entire structure (i.e., layer 1142, the shaped magnetic layer 1122, the sacrificial layer 1106, and the substrate 1104) is immersed in a solvent. This solvent treatment dissolves the sacrificial layer 1106 and releases the gear-shaped layer 1142 and the shaped magnetic layer 1122 from the substrate 1104, thereby producing microcarrier 1152. In some embodiments, the microcarrier 1152 may be further modified, for example, by coupling a trapping agent to one or both surfaces.

[0193] Example 4: A method for generating microcarriers with two-dimensional analog codes and uniform shapes

[0194] Now, attention is focused on methods for producing encoded microcarriers having one or more substantially transparent polymer layers and one or more substantially opaque polymer layers, such as those described in Example 1. Figures 12A-12E Process 1200 is shown as an exemplary workflow for manufacturing a microcarrier having a substantially transparent polymer layer, a substantially opaque polymer layer (whose two-dimensional shape constitutes analog code), and one or more pillars.

[0195] by Figure 12A Beginning at frame 1202, a sacrificial layer 1206 is deposited (e.g., by spin coating) onto a substrate 1204. In some embodiments, the substrate 1204 may be a glass substrate. At frame 1208, a mask 1210 is applied, and the sacrificial layer 1206 is exposed with UV light. UV light is applied through the mask 1210, thereby allowing UV light segments 1212 and 1214 to pass through and process the sacrificial layer 1206. At frame 1216, after the structure has been developed by standard photolithography, the sacrificial layer 1206 is shaped into a shaped sacrificial layer 1218 due to the masking effect of the UV treatment.

[0196] Process 1200 in box 1220 ( Figure 12B The process continues at frame 1226, where the masked holes in the shaped sacrificial layer 1218 are filled with a substantially transparent polymer to create pillars 1222 and 1224. At frame 1226, a substantially transparent polymer layer 1228 is deposited on pillars 1222 and 1224 and on the shaped sacrificial layer 1218.

[0197] Process 1200 in box 1230 ( Figure 12CThe process continues at block 1228, where a magnetic layer 1232 is deposited on layer 1228. In some embodiments, the magnetic layer 1232 comprises nickel. In some embodiments, the magnetic layer 1232 is deposited by sputtering. At block 1234, an etch barrier layer, as indicated by etch blocks 1236 and 1238, is deposited on the magnetic layer 1232. At block 1240, the unbarriered portions of the magnetic layer 1232 are etched away, thereby producing a shaped magnetic layer 1242. In some embodiments, the shaped magnetic layer 1242 may be shaped to surround layer 1228 (see, for example, ...). Figure 2A The ring shape of the central portion of layer 206 (with optional asymmetry for indicating orientation). At box 1244, the etch barrier layer (as indicated by etch blocks 1236 and 1238) is removed.

[0198] Process 1200 in box 1246 ( Figure 12D The process continues at point 1248, where a substantially transparent polymer layer 1248 is deposited on layers 1228 and 1242 (thus filling any pores in layer 1242 created by etching). At point 1250, a substantially opaque layer 1252 is deposited and shaped on top of layer 1248 by photolithography. In some embodiments, layer 1252 is shaped to have one or more gear teeth in a ring surrounding magnetic layer 1242 (see, for example, regarding...). Figure 2A Layers 202 and 206 and layer 204 of the central portion 208).

[0199] Process 1200 in box 1254 ( Figure 12E The process continues at ( ), where pillars 1256 and 1258 are formed on top of layer 1248 by photolithography. In some embodiments, pillars 1256 and 1258 are made of a substantially transparent polymer. In some embodiments, such as Figure 5A and 5B The pillars are positioned as shown in the diagram. At frame 1260, the substrate 1204 is cut into one or more microcarriers of the same shape (i.e., although for the sake of simplicity, in...). Figures 12A-12E Only one microcarrier is depicted in the diagram, but more than one microcarrier may be constructed on the substrate 1204 in process 1200. Also at block 1260, the entire structure (i.e., including 1204, 1218, 1222, 1224, 1228, 1242, 1248, 1252, 1256, and 1258) is immersed in a solvent. This solvent treatment dissolves the sacrificial layer 1218 and releases the microcarrier 1262 from the substrate 1204. In some embodiments, the microcarrier 1262 may be further modified, for example, by coupling a trapping agent to one or both surfaces.

[0200] Figures 13A-13C Process 1300 is shown, which is an exemplary workflow for producing different types of multilayer microcarriers. Figure 13A Beginning at frame 1302, a sacrificial layer 1306 is deposited on a substrate layer 1304. In some embodiments, the substrate 1304 is a glass substrate. At frame 1308, a substantially transparent layer 1310 is deposited on the sacrificial layer 1306. At frame 1312, a magnetic layer 1314 is deposited on the layer 1310. In some embodiments, the magnetic layer 1314 comprises nickel.

[0201] Process 1300 in box 1316 ( Figure 13B Continuing at ), where magnetic layer 1314 is defined as a shaped magnetic layer 1318. In some embodiments, the shaped magnetic layer 1318 is defined surrounding layer 1310 (see, for example, Figure 2A The ring shape of the central portion of layer 206 (with optional asymmetry for indicating orientation). At frame 1320, a substantially transparent layer 1322 is deposited on layers 1318 and 1310, thereby filling any pores created by the defined shaped layer 1318. At frame 1324, a substantially opaque polymer layer 1326 is deposited on layer 1322.

[0202] Process 1300 in box 1328 ( Figure 13C The process continues at [location missing], where the substantially opaque polymer layer 1326 is photolithographically formed into a gear-shaped substantially opaque polymer layer 1330. In some embodiments, layer 1330 is formed around the formed magnetic layer 1318 (see, for example, regarding [location missing]). Figure 2A The ring of layers 202 and 206, and layer 204 of the central portion 208, has one or more gear teeth. At frame 1332, the entire structure (i.e., including 1304, 1306, 1310, 1318, 1322, and 1330) is immersed in a solvent. This solvent treatment dissolves the sacrificial layer 1306 and releases the microcarrier 1334 from the substrate 1304. In some embodiments, the microcarrier 1334 may be further modified, for example, by coupling a trapping agent to one or both surfaces.

[0203] This article relates to the following implementation plan.

[0204] 1. An encoded microcarrier, comprising:

[0205] (a) A substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other;

[0206] (b) A substantially opaque polymer layer, wherein the substantially opaque polymer layer is fixed to the first surface of the substantially transparent polymer layer and surrounds a central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; and

[0207] (c) A trapping agent for capturing analytes, wherein the trapping agent is coupled in at least the central portion of the substantially transparent polymer layer to at least one of the first surface and the second surface of the substantially transparent polymer layer.

[0208] 2. The microcarrier as described in Implementation Scheme 1, further comprising:

[0209] (d) A substantially opaque magnetic layer surrounding the central portion of the substantially transparent polymer layer between the substantially opaque polymer layer and the central portion of the substantially transparent polymer layer, wherein the substantially opaque magnetic layer is fixed to the first surface or the second surface of the substantially transparent polymer layer.

[0210] 3. The microcarrier as described in Implementation Scheme 1, further comprising:

[0211] (e) a second substantially transparent polymer layer aligned with the first substantially transparent polymer layer, the second substantially transparent polymer layer having a central portion aligned with the central portion of the first substantially transparent polymer layer, wherein the second substantially transparent polymer layer is fixed to the second surface of the first substantially transparent polymer layer and does not extend beyond the two-dimensional shape of the first substantially transparent polymer layer; and

[0212] (f) A substantially opaque magnetic layer surrounding the central portion of the first substantially transparent polymer layer between the substantially opaque polymer layer and the central portion of the second substantially transparent polymer layer, wherein the substantially opaque magnetic layer is fixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer.

[0213] 4. The microcarrier as described in any one of embodiments 1-3, further comprising orientation indicators for orienting the analog code of the substantially opaque polymer layer.

[0214] 5. The microcarrier as described in embodiment 4, wherein the orientation index includes the asymmetry of the substantially opaque magnetic layer.

[0215] 6. The microcarrier as described in any one of embodiments 2-5, wherein the substantially opaque magnetic layer comprises nickel.

[0216] 7. The microcarrier as described in any one of embodiments 2-6, wherein the thickness of the substantially opaque magnetic layer is between about 50 nm and about 10 μm.

[0217] 8. The microcarrier as described in embodiment 7, wherein the thickness of the substantially opaque magnetic layer is about 0.1 μm.

[0218] 9. The microcarrier as described in any one of embodiments 1-8, wherein the two-dimensional shape of the substantially opaque polymer layer comprises a gear shape, the gear shape comprising a plurality of gear teeth, and wherein the analog code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth.

[0219] 10. The microcarrier as described in embodiment 9, wherein the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm.

[0220] 11. The microcarrier as described in embodiment 9 or embodiment 10, wherein the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm.

[0221] 12. The microcarrier as described in any one of embodiments 9-11, wherein the plurality of gear teeth comprises two or more gear teeth spaced between about 1 μm and about 10 μm.

[0222] 13. The microcarrier as described in any one of embodiments 1-12, further comprising:

[0223] (g) One or more pillars protruding from the first surface of the first substantially transparent polymer layer, wherein the one or more pillars are not located within the central portion of the first substantially transparent polymer layer; and / or

[0224] (h) One or more pillars protruding from the second surface of the first substantially transparent polymer layer or from the second substantially transparent polymer layer not fixed to the surface of the first substantially transparent polymer layer, wherein the one or more pillars are not within the central portion of the first substantially transparent polymer layer or the second substantially transparent polymer layer.

[0225] 14. The microcarrier as described in any one of embodiments 1-13, wherein the microcarrier is a substantially circular disk.

[0226] 15. The microcarrier as described in any one of embodiments 1-14, wherein the central portion of the first substantially transparent polymer layer accounts for between about 5% and about 90% of the surface area of ​​the first substantially transparent polymer layer.

[0227] 16. The microcarrier as described in embodiment 15, wherein the central portion of the first substantially transparent polymer layer accounts for about 25% of the surface area of ​​the first substantially transparent polymer layer.

[0228] 17. The microcarrier as described in any one of embodiments 1-15, wherein the diameter of the microcarrier is less than about 200 μm.

[0229] 18. The microcarrier as described in embodiment 17, wherein the diameter of the microcarrier is about 50 μm.

[0230] 19. The microcarrier as described in any one of embodiments 1-18, wherein the thickness of the microcarrier is less than about 50 μm.

[0231] 20. The microcarrier as described in embodiment 19, wherein the thickness of the microcarrier is about 10 μm.

[0232] 21. The microcarrier according to any one of embodiments 1-20, wherein the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate fractions, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0233] 22. The microcarrier as described in any one of embodiments 1-21, wherein the capturing agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0234] 23. The microcarrier as described in any one of embodiments 1-22, wherein the substantially transparent polymer of the first substantially transparent polymer layer or the second substantially transparent polymer layer comprises an epoxy-based polymer.

[0235] 24. The microcarrier as described in embodiment 23, wherein the epoxy polymer is SU-8.

[0236] 25. An encoded microcarrier, comprising:

[0237] (a) A substantially opaque polymer layer having a first surface and a second surface parallel to each other, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; and

[0238] (b) A trapping agent for capturing an analyte, wherein the trapping agent is coupled in at least a central portion of the substantially opaque polymer layer to at least one of the first surface and the second surface of the substantially opaque polymer layer.

[0239] 26. The microcarrier as described in embodiment 25, further comprising:

[0240] (c) One or more pillars protruding from the first and / or second surfaces of the substantially opaque polymer layer, wherein the one or more pillars comprise a magnetic material.

[0241] 27. The microcarrier as described in embodiment 26, wherein the height of the one or more pillars is between about 1 μm and about 10 μm.

[0242] 28. The microcarrier as described in embodiment 26 or embodiment 27, wherein the diameter of the one or more pillars is between about 1 μm and about 10 μm.

[0243] 29. The microcarrier as described in any one of embodiments 25-28, further comprising:

[0244] (d) A magnetic layer comprising a magnetic material, the magnetic layer being fixed to a second surface of the substantially opaque polymer layer, wherein the magnetic layer does not extend beyond the central portion of the substantially opaque polymer layer, and wherein the trapping agent is coupled to at least the first surface of the substantially opaque polymer layer.

[0245] 30. The microcarrier as described in embodiment 29, further comprising:

[0246] (e) A second substantially opaque polymer layer aligned with the first substantially opaque polymer layer, wherein the second substantially opaque polymer layer is fixed to the second surface of the first substantially transparent polymer layer and does not extend beyond the outline of the first substantially transparent polymer layer, and wherein the magnetic layer is fixed between the first substantially transparent polymer layer and the second substantially transparent polymer layer.

[0247] 31. The microcarrier as described in any one of embodiments 26-30, wherein the magnetic material comprises nickel.

[0248] 32. The microcarrier as described in any one of embodiments 25-30, further comprising orientation indicators for orienting the analog code of the substantially opaque polymer layer.

[0249] 33. The microcarrier as described in embodiment 32, wherein the orientation index includes the asymmetry of the contour of the substantially opaque polymer layer.

[0250] 34. The microcarrier as described in any one of embodiments 25-33, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional gear shape, the gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: the height of one or more of the plurality of gear teeth, the width of one or more of the plurality of gear teeth, the number of gear teeth in the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth.

[0251] 35. The microcarrier as described in embodiment 34, wherein the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm.

[0252] 36. The microcarrier as described in embodiment 34 or embodiment 35, wherein the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm.

[0253] 37. The microcarrier as described in any one of embodiments 34-36, wherein the plurality of gear teeth comprises two or more gear teeth spaced between about 1 μm and about 10 μm.

[0254] 38. The microcarrier as described in any one of embodiments 25-37, wherein the microcarrier is a substantially circular disk.

[0255] 39. The microcarrier as described in any one of embodiments 25-38, wherein the central portion of the first substantially opaque polymer layer accounts for between about 5% and about 90% of the surface area of ​​the first substantially opaque polymer layer.

[0256] 40. The microcarrier as described in embodiment 39, wherein the central portion of the first substantially opaque polymer layer accounts for about 25% of the surface area of ​​the first substantially opaque polymer layer.

[0257] 41. The microcarrier as described in any one of embodiments 25-40, wherein the diameter of the microcarrier is less than about 200 μm.

[0258] 42. The microcarrier as described in embodiment 41, wherein the diameter of the microcarrier is about 60 μm.

[0259] 43. The microcarrier as described in any one of embodiments 25-42, wherein the thickness of the microcarrier is less than about 50 μm.

[0260] 44. The microcarrier as described in embodiment 43, wherein the thickness of the microcarrier is about 10 μm.

[0261] 45. The microcarrier according to any one of embodiments 25-44, wherein the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0262] 46. ​​The microcarrier as described in any one of embodiments 25-45, wherein the capturing agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0263] 47. The microcarrier as described in any one of embodiments 25-46, wherein the substantially opaque polymer comprises an epoxy polymer.

[0264] 48. The microcarrier as described in any one of embodiments 25-46, wherein the substantially opaque polymer comprises a black matrix resist.

[0265] 49. A method for preparing coded microcarriers, the method comprising:

[0266] (a) Depositing a substantially transparent polymer layer, wherein the substantially transparent polymer layer has a first surface and a second surface, the first surface and the second surface being parallel to each other;

[0267] (b) Depositing a substantially opaque magnetic layer on the first surface of the substantially transparent polymer layer;

[0268] (c) Etching the substantially opaque magnetic layer to remove the portion of the substantially opaque magnetic layer deposited on the central portion of the substantially transparent polymer layer;

[0269] (d) Depositing a second substantially transparent polymer layer on the substantially opaque magnetic layer, wherein the second substantially transparent polymer layer has a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the substantially opaque magnetic layer, and wherein the second substantially transparent polymer layer is aligned with the first substantially transparent polymer layer and has a central portion aligned with the central portion of the substantially transparent polymer layer; and

[0270] (e) Deposit a substantially opaque polymer layer on the first surface of the second substantially transparent polymer layer, wherein the substantially opaque polymer layer surrounds the central portion of the first substantially transparent polymer layer and the second substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code.

[0271] 50. The method of embodiment 49, wherein the substantially opaque magnetic layer is etched by wet etching.

[0272] 51. The method of embodiment 49 or embodiment 50, wherein the substantially opaque magnetic layer comprises nickel.

[0273] 52. The method of any one of embodiments 49-51, wherein the thickness of the substantially opaque magnetic layer is between about 50 nm and about 10 μm.

[0274] 53. The method of embodiment 52, wherein the thickness of the substantially opaque magnetic layer is less than about 0.1 μm.

[0275] 54. The method of any one of embodiments 49-53, wherein the substantially opaque magnetic layer includes an asymmetry for orienting the analog code of the substantially opaque polymer layer.

[0276] 55. The method of any one of embodiments 49-54, wherein the two-dimensional shape of the substantially opaque polymer layer is produced by photolithography.

[0277] 56. The method of any one of embodiments 49-55, wherein the two-dimensional shape of the substantially opaque polymer layer comprises a gear shape, the gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth.

[0278] 57. The method of embodiment 56, wherein the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm.

[0279] 58. The method of embodiment 56 or embodiment 57, wherein the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm.

[0280] 59. The method of any one of embodiments 56-58, wherein the plurality of gear teeth comprises two or more gear teeth spaced between about 1 μm and about 10 μm.

[0281] 60. The method as described in any one of embodiments 49-59, further comprising:

[0282] (f) Prior to step (a), a sacrificial layer is deposited on the substrate;

[0283] (g) Using photolithography to create one or more columnar holes in the sacrificial layer;

[0284] (h) Deposit a third substantially transparent polymer layer in the one or more columnar pores in the sacrificial layer, wherein the first substantially transparent polymer layer is deposited on top of the third substantially transparent polymer layer and the sacrificial layer in step (a);

[0285] (i) After step (e), one or more pillars comprising the substantially transparent polymer are deposited on the first surface of the second substantially transparent polymer layer in the portion not covered by the substantially opaque polymer layer using photolithography;

[0286] (j) Dissolving the sacrificial layer in a solvent; and

[0287] (k) Remove the substrate.

[0288] 61. The method as described in any one of embodiments 49-59, further comprising:

[0289] (f) Prior to step (a), a sacrificial layer is deposited on the substrate;

[0290] (g) As part of step (a), the substantially transparent polymer layer is deposited on the sacrificial layer;

[0291] (h) After step (e), the sacrificial layer is dissolved in a solvent; and

[0292] (i) Remove the substrate.

[0293] 62. The method of any one of embodiments 49-61, wherein the encoded microcarrier is a substantially circular disk.

[0294] 63. The method of any one of embodiments 49-62, wherein the central portion of the first substantially transparent polymer layer accounts for between about 5% and about 90% of the surface area of ​​the first substantially transparent polymer layer.

[0295] 64. The method of embodiment 63, wherein the central portion of the first substantially transparent polymer layer occupies about 25% of the surface area of ​​the first substantially transparent polymer layer.

[0296] 65. The method of any one of embodiments 49-64, wherein the diameter of the encoded microcarrier is less than about 200 μm.

[0297] 66. The method of embodiment 65, wherein the diameter of the encoded microcarrier is about 50 μm.

[0298] 67. The method of any one of embodiments 49-66, wherein the thickness of the encoded microcarrier is less than about 50 μm.

[0299] 68. The method of embodiment 67, wherein the thickness of the encoded microcarrier is about 10 μm.

[0300] 69. The method as described in any one of embodiments 49-68, further comprising:

[0301] (f) A capture agent for capturing the analyte is coupled in at least the central portion to at least one of the first surface of the second substantially transparent polymer layer and the second surface of the first substantially transparent polymer layer.

[0302] 70. The method of embodiment 69, wherein the substantially transparent polymer of the first substantially transparent polymer layer or the second substantially transparent polymer layer comprises an epoxide, and wherein coupling the trapping agent comprises:

[0303] (i) reacting the substantially transparent polymer of the first substantially transparent polymer layer and / or the second substantially transparent polymer layer with a photoacid-producing agent and light to produce a crosslinked polymer, wherein the light has a wavelength that activates the photoacid-producing agent; and

[0304] (ii) reacting the epoxide of the crosslinked polymer with a compound containing an amine and a carboxyl group, wherein the amine of the compound reacts with the epoxide to form a compound-coupled crosslinked polymer; and

[0305] (iii) The carboxyl group of the crosslinked polymer to which the compound is coupled reacts with the trapping agent to couple the trapping agent in at least the central portion to at least one of the first surface of the second substantially transparent polymer layer and the second surface of the first substantially transparent polymer layer.

[0306] 71. The method of embodiment 70, wherein the carboxyl group of the crosslinked polymer coupled with the compound reacts with the primary amine of the trapping agent.

[0307] 72. The method of embodiment 70 or embodiment 71, wherein the substantially transparent polymer of the first substantially transparent polymer layer and / or the second substantially transparent polymer layer comprises SU-8.

[0308] 73. The method of any one of embodiments 49-72, wherein the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate fractions, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0309] 74. The method of any one of embodiments 49-73, wherein the capturing agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0310] 75. An encoded microcarrier produced by any one of embodiments 49-74.

[0311] 76. A method for preparing coded microcarriers, the method comprising:

[0312] (a) Depositing a sacrificial layer on the substrate;

[0313] (b) Depositing a substantially opaque polymer layer on the sacrificial layer, the substantially opaque polymer layer having a profile, a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the sacrificial layer;

[0314] (c) The contour of the substantially opaque polymer layer is shaped by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code;

[0315] (d) Dissolving the sacrificial polymer layer in a solvent; and

[0316] (e) Remove the substrate.

[0317] 77. A method for preparing coded microcarriers, the method comprising:

[0318] (a) Depositing a sacrificial layer on the substrate;

[0319] (b) Depositing a magnetic layer containing magnetic material on the sacrificial layer;

[0320] (c) Depositing a substantially opaque polymer layer on the magnetic layer, the substantially opaque polymer layer having a profile, a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the magnetic layer;

[0321] (d) The contour of the substantially opaque polymer layer is shaped by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code;

[0322] (e) Dissolving the sacrificial polymer layer in a solvent; and

[0323] (f) Remove the substrate.

[0324] 78. The method of embodiment 77, further comprising:

[0325] (g) After step (b) and before step (c), the magnetic layer is formed by photolithography.

[0326] 79. The method as described in embodiment 77 or 78, wherein the magnetic material comprises nickel.

[0327] 80. The method of any one of embodiments 76-79, wherein the microcarrier includes orientation indicators for orienting the analog code of the substantially opaque polymer layer.

[0328] 81. The method of embodiment 80, wherein the orientation index includes the asymmetry of the contour of the substantially opaque polymer layer.

[0329] 82. The method of any one of embodiments 76-81, wherein the two-dimensional shape of the substantially opaque polymer layer comprises a gear shape, the gear shape comprising a plurality of gear teeth, and wherein the simulation code is represented by one or more aspects selected from the group consisting of: the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of one or more gear teeth within the plurality of gear teeth.

[0330] 83. The method of embodiment 82, wherein the plurality of gear teeth includes one or more gear teeth with a width between about 1 μm and about 10 μm.

[0331] 84. The method of embodiment 82 or embodiment 83, wherein the plurality of gear teeth includes one or more gear teeth with a height between about 1 μm and about 10 μm.

[0332] 85. The method of any one of embodiments 82-84, wherein the plurality of gear teeth comprises two or more gear teeth spaced between about 1 μm and about 10 μm.

[0333] 86. The method of any one of embodiments 76-85, wherein the microcarrier is a substantially circular disk.

[0334] 87. The method of embodiment 86, wherein the diameter of the microcarrier is less than about 200 μm.

[0335] 88. The method of embodiment 87, wherein the diameter of the microcarrier is about 60 μm.

[0336] 89. The method of any one of embodiments 76-88, wherein the thickness of the microcarrier is less than about 30 μm.

[0337] 90. The method of embodiment 89, wherein the thickness of the microcarrier is about 10 μm.

[0338] 91. The method as described in any one of embodiments 76-90, further comprising:

[0339] (h) A capture agent for capturing analytes is coupled to at least one of the first and second surfaces of the substantially opaque polymer layer.

[0340] 92. The method of embodiment 91, wherein the substantially opaque polymer of the substantially opaque polymer layer comprises an epoxide, and wherein coupling the trapping agent comprises:

[0341] (i) reacting the substantially opaque polymer layer with a photoacid-producing agent and light to produce a crosslinked polymer, wherein the light has a wavelength that activates the photoacid-producing agent; and

[0342] (ii) reacting the epoxide of the crosslinked polymer with a compound containing an amine and a carboxyl group, wherein the amine of the compound reacts with the epoxide to form a compound-coupled crosslinked polymer; and

[0343] (iii) The carboxyl group of the crosslinked polymer to which the compound is coupled reacts with the trapping agent to couple the trapping agent in at least the central portion to at least one of the first surface of the second substantially transparent polymer layer and the second surface of the first substantially transparent polymer layer.

[0344] 93. The method of embodiment 91 or embodiment 92, wherein the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate fractions, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0345] 94. The method of any one of embodiments 91-93, wherein the capturing agent for capturing the analyte is selected from the group consisting of: DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate fractions, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments.

[0346] 95. An encoded microcarrier produced by any one of embodiments 76-94.

[0347] 96. A method for detecting two or more analytes in a solution, the method comprising:

[0348] (a) Contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers comprises at least:

[0349] (i) a first microcarrier according to any one of embodiments 1-48, 75 and 95, wherein the first microcarrier specifically captures the first analyte, wherein the first microcarrier is encoded with a first analog code; and

[0350] (ii) A second microcarrier according to any one of embodiments 1-48, 75 and 95, the second microcarrier specifically capturing the second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code;

[0351] (b) Decoding the first analog code and the second analog code using analog shape recognition to identify the first microcarrier and the second microcarrier; and

[0352] (c) Detect the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0353] 97. As described in implementation scheme 96, step (b) occurs before step (c).

[0354] 98. As described in implementation scheme 96, step (c) occurs before step (b).

[0355] 99. As described in implementation scheme 96, steps (b) and (c) occur simultaneously.

[0356] 100. The method as described in any one of embodiments 96-99, wherein decoding the first analog code and the second analog code comprises:

[0357] (i) Irradiating the first microcarrier and the second microcarrier by passing light through the substantially transparent portions of the first microcarrier and the second microcarrier and / or the surrounding solution, wherein the light fails to pass through the substantially opaque portions of the first microcarrier and the second microcarrier to produce a light pattern corresponding to a first analog code of the first microcarrier and a light pattern corresponding to a second analog code of the second microcarrier.

[0358] (ii) imaging the first analog-coded light pattern to generate a first analog-coded image and imaging the second analog-coded light pattern to generate a second analog-coded image; and

[0359] (iii) Using analog shape recognition to match the first analog-coded image with the first analog code and to match the second analog-coded image with the second analog code.

[0360] 101. The method of any one of embodiments 96-100, wherein detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier comprises:

[0361] (i) Following step (a), the first microcarrier and the second microcarrier are incubated together with a detection agent, wherein the detection agent binds the first analyte captured by the first microcarrier and the second analyte captured by the second microcarrier; and

[0362] (ii) Measure the amount of the detection agent bound to the first microcarrier and the second microcarrier.

[0363] 102. The method of embodiment 101, wherein the detection agent is a fluorescent detection agent, and wherein the amount of the detection agent bound to the first microcarrier and the second microcarrier is measured by fluorescence microscopy.

[0364] 103. The method of embodiment 101, wherein the detection agent is a luminescent detection agent, and wherein the amount of the detection agent bound to the first microcarrier and the second microcarrier is measured by luminescence microscopy.

[0365] 104. The method of any one of embodiments 96-103, wherein the solution comprises a biological sample.

[0366] 105. The method of embodiment 104, wherein the biological sample is selected from the group consisting of: blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluid of skin or adipose tissue, saliva, tears, bronchoalveolar lavage fluid, oropharyngeal secretions, intestinal fluid, cervical, vaginal or uterine secretions, and semen.

[0367] 106. A kit for performing multiplex assays, the kit comprising a plurality of microcarriers, wherein the plurality of microcarriers comprises at least:

[0368] (a) A first microcarrier according to any one of embodiments 1-48, 75 and 95, wherein the first microcarrier specifically captures a first analyte, wherein the first microcarrier is encoded with a first analog code; and

[0369] (b) A second microcarrier according to any one of embodiments 1-48, 75 and 95, the second microcarrier specifically capturing a second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code.

[0370] 107. The kit according to embodiment 106 further includes a detection agent for detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0371] 108. The kit as described in embodiment 106 or embodiment 107, further comprising instructions for using the kit to detect the first analyte and the second analyte.

[0372] Although the foregoing invention has been described in detail by way of illustration and examples for purposes of clarity, such description and examples should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. An encoded microcarrier, comprising: (a) A substantially transparent polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other; (b) A substantially opaque polymer layer, wherein the substantially opaque polymer layer is fixed to the first surface of the substantially transparent polymer layer and surrounds the central portion of the substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; as well as (c) A trapping agent for capturing analytes, wherein the trapping agent is coupled in at least the central portion of the substantially transparent polymer layer to at least one of the first surface and the second surface of the substantially transparent polymer layer.

2. An encoded microcarrier, comprising: (a) A substantially opaque polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the outline of the substantially opaque polymer layer comprises a two-dimensional shape representing analog code; as well as (b) A trapping agent for capturing an analyte, wherein the trapping agent is coupled in at least a central portion of the substantially opaque polymer layer to at least one of the first surface and the second surface of the substantially opaque polymer layer.

3. A method for preparing encoded microcarriers, the method comprising: (a) Depositing a substantially transparent polymer layer, wherein the substantially transparent polymer layer has a first surface and a second surface, the first surface and the second surface being parallel to each other; (b) Depositing a substantially opaque magnetic layer on the first surface of the substantially transparent polymer layer; (c) Etching the substantially opaque magnetic layer to remove the portion of the substantially opaque magnetic layer deposited on the central portion of the substantially transparent polymer layer; (d) Depositing a second substantially transparent polymer layer on the substantially opaque magnetic layer, wherein the second substantially transparent polymer layer has a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the substantially opaque magnetic layer, and wherein the second substantially transparent polymer layer is aligned with the first substantially transparent polymer layer and has a central portion aligned with the central portion of the substantially transparent polymer layer. as well as (e) Deposit a substantially opaque polymer layer on the first surface of the second substantially transparent polymer layer, wherein the substantially opaque polymer layer surrounds the central portion of the first substantially transparent polymer layer and the second substantially transparent polymer layer, and wherein the substantially opaque polymer layer comprises a two-dimensional shape representing analog code.

4. An encoded microcarrier, generated by the method of claim 3.

5. A method for preparing coded microcarriers, the method comprising: (a) Depositing a sacrificial layer on the substrate; (b) Depositing a substantially opaque polymer layer on the sacrificial layer, the substantially opaque polymer layer having a profile, a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the sacrificial layer; (c) The contour of the substantially opaque polymer layer is shaped by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code; (d) Dissolving the sacrificial polymer layer in a solvent; and (e) Remove the substrate.

6. A method for preparing encoded microcarriers, the method comprising: (a) Depositing a sacrificial layer on the substrate; (b) Depositing a magnetic layer containing magnetic material on the sacrificial layer; (c) Depositing a substantially opaque polymer layer on the magnetic layer, the substantially opaque polymer layer having a profile, a first surface and a second surface, the first surface and the second surface being parallel to each other, wherein the second surface is fixed to the magnetic layer; (d) The contour of the substantially opaque polymer layer is shaped by photolithography, wherein the contour is shaped into a two-dimensional shape representing analog code; (e) Dissolving the sacrificial polymer layer in a solvent; and (f) Remove the substrate.

7. An encoded microcarrier, generated by the method of claim 5 or 6.

8. A method for detecting two or more analytes in a solution, the method comprising: (a) Contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers comprises at least: (i) the first microcarrier according to any one of claims 1, 2, 4, and 7, wherein the first microcarrier specifically captures the first analyte, wherein the first microcarrier is encoded with a first analog code; and (ii) The second microcarrier according to any one of claims 1, 2, 4 and 7, wherein the second microcarrier specifically captures the second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code; (b) Decoding the first analog code and the second analog code using analog shape recognition to identify the first microcarrier and the second microcarrier; and (c) Detect the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

9. A kit for performing multiplex assays, the kit comprising a plurality of microcarriers, wherein the plurality of microcarriers comprises at least: (a) The first microcarrier according to any one of claims 1, 2, 4 and 7, wherein the first microcarrier specifically captures the first analyte, wherein the first microcarrier is encoded with a first analog code; as well as (b) The second microcarrier according to any one of claims 1, 2, 4 and 7, wherein the second microcarrier specifically captures the second analyte, wherein the second microcarrier is encoded with a second analog code, and wherein the second analog code is different from the first analog code.

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