Magnetic microcarriers for image differentiation multiplex assays

Abstract

Provided herein are coded microcarriers for analyte detection in multiplex assays. The microcarriers are coded with an analog code for identification and include a capture agent for analyte detection and a substantially transparent magnetic polymer. The analog code is generated by a two-dimensional shape of a substantially opaque layer. Also provided are methods of making the coded microcarriers disclosed herein. Further provided are methods and kits for performing multiplex assays using the microcarriers described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 193,338, filed May 26, 2021, the entire contents of which are incorporated herein in their entirety.

[0002] Provided herein are encoded microcarriers having a substantially transparent magnetic polymer layer, useful for analyte detection, for example in multiplex assays, as well as methods of making and using the same, and related kits. In some embodiments, the microcarriers include a capture agent for capturing the analyte, and are encoded with an analog code generated from the two-dimensional geometry of the opaque layer. [Background technology]

[0003] Immunologic and molecular diagnostic assays play an important role in both research and clinical fields. Often, assays need to be performed on a panel of multiple targets to obtain meaningful results or a bird's-eye view of results to facilitate research or clinical decision making. This is especially true in the era of genomics and proteomics, where abundant genetic markers and / or biomarkers are believed to influence or predict specific disease states. In theory, assays for multiple targets can be achieved by testing each target separately in parallel or sequentially in different reactors (i.e., multiple singleplex). However, assays employing singleplex strategies are not only often cumbersome, but also typically require large sample volumes, especially when there are a large number of targets to be analyzed.

[0004] In a multiplex assay, multiple analytes (two or more) are assayed simultaneously in a single assay. Multiplex assays are commonly used in high-throughput screening environments where many specimens can be analyzed at once. The ability to assay many analytes simultaneously and many specimens in parallel is the hallmark of a multiplex assay and is why such assays have become powerful tools in fields ranging from drug discovery to functional genomics to clinical diagnostics. In contrast to singleplex, by combining all targets in the same reactor, the assay becomes much less cumbersome and much easier to perform, since only one reactor is handled per sample. Thus, the volume of test sample required can be dramatically reduced, which is particularly important when it is difficult and / or invasive to collect large amounts of sample (e.g., tumor tissue, cerebrospinal fluid, or bone marrow). Equally important is the fact that reagent costs can be reduced and assay throughput can be dramatically increased.

[0005] Many assays of complex macromolecular samples consist of two steps. In the first step, agents capable of specifically capturing target macromolecules are attached to a solid phase surface. These immobilized molecules can be used to capture target macromolecules from complex samples by various means, such as hybridization (e.g., in DNA, RNA-based assays) or antigen-antibody interactions (in immunoassays). In the second step, detection molecules are incubated with the complex of capture molecules and target to bind to the complex, and the detection molecules emit signals, such as fluorescence or other electromagnetic signals. The amount of target is then quantified by the intensity of these signals.

[0006] A multiplex assay can be performed by utilizing multiple capture agents, each specific for a different target macromolecule. In a chip-based array multiplex assay, each type of capture agent is attached to a predefined location on a chip. The amount of multiplex targets in a complex sample is determined by measuring the signal of the detection molecule at each location corresponding to the type of capture agent. In a suspension array multiplex assay, 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 microparticles / microcarriers. Each type of capture agent is immobilized on a particle with the same ID, and the signal emitted from the detection molecule on the surface of the particle with a particular ID reflects the amount of the corresponding target.

[0007] Existing systems for suspension array multiplex assays are limited in resolution. Some multiplex systems use digital barcodes printed on flat microbeads using standard semiconductor fabrication techniques. However, the number of identifiers that can be generated at a given digit is limited. Increasing the number of unique identifiers requires increasing the number of digits in the barcode, which requires more space to print on the already small microbeads. Another type of multiplex system uses color coding, such as fluorescent beads coded with unique fluorescent dyes. However, the number of unique identifiers available for such fluorescent systems is limited by overlapping excitation / emission spectra, which can lead to identification errors due to, for example, batch-to-batch variations in fluorescent dyes.

[0008] Thus, there is a need for an analog-coded multiplex assay system that is not constrained by limitations such as, for example, digital barcode size or fluorophore resolution. Such a system allows for nearly unlimited unique identifiers and minimizes recognition errors (e.g., spectral overlap or batch-to-batch fluorophore variation) due to the use of analog codes. Furthermore, there is a need for microcarriers for such systems that can be easily manipulated and / or easily separated from, for example, biological samples or other liquids.

[0009] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Summary of the Invention

[0010] To meet this need, the present invention provides microcarriers encoded with analog codes, which include, inter alia, a capture agent for capturing analytes. The analog code is made from a two-dimensional shape of a substantially opaque layer, and the microcarrier also includes a substantially transparent magnetic polymer layer. These features allow the microcarriers to be rapidly and efficiently separated from a solution (e.g., a biological sample or an assay solution) by magnetic attraction, and to be fabricated using convenient microfabrication techniques. Furthermore, the use of superparamagnetic materials, such as certain iron oxides, eliminates residual magnetism that can complicate assay procedures. These microcarriers can be used, for example, in multiplex assays, where each microcarrier includes a capture agent for capturing a specific analyte and an analog code for identification. Methods of making and using such microcarriers, as well as kits related thereto, are also provided.

[0011] Thus, in one aspect, provided herein is an encoded microcarrier comprising: (a) a substantially transparent magnetic polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to one another, the substantially transparent magnetic polymer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide; (b) a substantially opaque layer, the substantially opaque layer being affixed to the first surface of the substantially transparent magnetic polymer layer, the contour of the substantially opaque layer forming a two-dimensional shape representing an analog code; and (c) a capture agent for capturing an analyte, the capture agent being linked to at least one of the first surface and the second surface of the substantially transparent magnetic polymer layer.

[0012] In some embodiments, the magnetic nanoparticles are superparamagnetic. In some embodiments, the magnetic nanoparticles are less than about 30 nm in diameter and greater than or equal to about 3 nm in diameter. In some embodiments, the plurality of magnetic nanoparticles comprises less than about 10% (by weight) and greater than about 0.1% (by weight) of a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles. In some embodiments, the substantially transparent magnetic polymer layer is between about 0.1 μm and about 50 μm in thickness. In some embodiments, the substantially transparent polymer is an epoxy-based polymer. In some embodiments, the epoxy-based polymer is SU-8. In some embodiments, the substantially opaque layer comprises a substantially opaque polymer. In some embodiments, the substantially opaque layer comprises a black matrix resist. In some embodiments, the substantially opaque polymer exhibits an absorbance of greater than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm. In some embodiments, the substantially opaque layer comprises a metal lacking remanence. In some embodiments, the substantially opaque layer comprises titanium or chromium. In some embodiments, the substantially opaque layer is between about 0.05 μm and about 2 μm in thickness. In some embodiments, the analog code comprises one or more overlapping or partially overlapping arc elements forming a continuous or discontinuous ring. In some embodiments, the microcarrier further comprises a directional indicator for orienting the analog code of the substantially opaque layer. In some embodiments, the directional indicator comprises an asymmetry of the substantially opaque layer. In some embodiments, the microcarrier is a substantially circular disk. In some embodiments, the microcarrier is between about 5 μm and about 200 μm in diameter. In some embodiments, the microcarrier is about 40 μm in diameter. In some embodiments, the microcarrier is less than about 50 μm in thickness. In some embodiments, the microcarrier is between about 2 μm and about 10 μm in thickness. In some embodiments, the microcarrier is about 5 μm in thickness.

[0013] 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.

[0014] In another aspect, provided herein is a method of making an encoded microcarrier, comprising: (a) depositing a substantially opaque layer having a first surface and a second surface, the first surface and the second surface being parallel to one another; (b) patterning the deposited substantially opaque layer into a two-dimensional shape representing an analog code; (c) depositing a substantially transparent magnetic polymer layer on the first surface of the deposited substantially opaque layer, the substantially transparent magnetic polymer layer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide; and (d) patterning the deposited substantially transparent magnetic polymer layer into the microcarrier shape. In some embodiments, (a) comprises depositing the substantially opaque layer on a sacrificial layer. In some embodiments, the substantially opaque layer is deposited and patterned by lithography. In some embodiments, the deposited substantially opaque layer is deposited and patterned by a lift-off process. In some embodiments, the method further comprises: before (a), depositing a second polymer layer on the sacrificial layer; before (a), applying a mask to the deposited second polymer layer, thereby generating a masked portion of the deposited second polymer layer and an unmasked portion of the deposited second polymer layer; before (a), applying light to the deposited masked second polymer layer, the light being of a wavelength sufficient to remove the unmasked portion of the deposited second polymer layer; (a) comprises depositing a substantially opaque layer on the sacrificial layer and the masked portion of the deposited second polymer layer; (b) comprises removing the deposited second polymer layer, thereby patterning the deposited substantially opaque layer by removing the deposited substantially opaque layer on the masked portion of the deposited second polymer layer. In some embodiments, the second polymer layer is deposited by spin coating.In some embodiments, the method further comprises, prior to (a)-(d), depositing a sacrificial layer on the substrate carrier. In some embodiments, the method further comprises, after (a)-(d), etching the sacrificial layer. In some embodiments, the method further comprises, prior to (c), mixing a monomer of the substantially transparent polymer, the plurality of magnetic nanoparticles, a solvent, and a dispersant to form a mixture of monomer of the substantially transparent polymer, the plurality of magnetic nanoparticles, a solvent, and a dispersant.

[0015] In some embodiments, the magnetic nanoparticles, the solvent, and the dispersant are mixed to form a mixture of the magnetic nanoparticles, the solvent, and the dispersant prior to mixing with the monomer of the substantially transparent polymer. In some embodiments, the dispersant comprises 10% of the mixture of the magnetic nanoparticles, the solvent, and the dispersant. In some embodiments, the magnetic nanoparticles constitute 2.5% of the mixture of the monomer of the substantially transparent polymer, the magnetic nanoparticles, the solvent, and the dispersant. In some embodiments, the solvent comprises cyclopentanone. In some embodiments, the dispersant comprises a phosphate polymer. In some embodiments, the phosphate polymer comprises carboxyl-PEG-phosphate. In some embodiments, the substantially transparent magnetic polymer layer is deposited by spin coating or spray coating. In some embodiments, the deposited substantially transparent magnetic polymer layer is patterned by photolithography, lift-off, or sputtering.

[0016] In some embodiments, the method further comprises linking a capture agent to at least one of the first and second surfaces of the substantially transparent magnetic polymer layer for capturing the analyte. In some embodiments, linking the capture agent comprises reacting a substantially transparent polymer of the substantially transparent magnetic polymer layer with a photoacid generator and light to produce a crosslinked polymer, where the light is of a wavelength that activates the photoacid generator; reacting an epoxide of the crosslinked polymer with a compound that includes an amine and a carboxyl, where the amine of the compound reacts with the epoxide to form a compound-linked crosslinked polymer; and reacting the carboxyl of the compound-linked crosslinked polymer with the capture agent to link the capture agent to the substantially transparent magnetic polymer layer.

[0017] In some embodiments, the magnetic nanoparticles are superparamagnetic. In some embodiments, the magnetic nanoparticles are less than about 30 nm in diameter and greater than or equal to about 3 nm in diameter. In some embodiments, the plurality of magnetic nanoparticles comprises less than about 10% (by weight) and greater than about 0.1% (by weight) of a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles. In some embodiments, the substantially transparent magnetic polymer layer is between about 0.1 μm and about 50 μm in thickness. In some embodiments, the substantially transparent polymer of the substantially transparent magnetic polymer layer is an epoxy-based polymer. In some embodiments, the epoxy-based polymer is SU-8. In some embodiments, the substantially opaque layer comprises a substantially opaque polymer. In some embodiments, the substantially opaque layer comprises a black matrix resist. In some embodiments, the substantially opaque polymer exhibits an absorbance of greater than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm. In some embodiments, the substantially opaque layer comprises a metal lacking remanence. In some embodiments, the substantially opaque layer comprises titanium or chromium. In some embodiments, the substantially opaque layer is between about 0.05 μm and about 2 μm in thickness. In some embodiments, the analog code includes one or more overlapping or partially overlapping arc elements forming a continuous or discontinuous ring. In some embodiments, the deposited substantially opaque layer is patterned to provide asymmetry. In some embodiments, the microcarriers are patterned into substantially circular disks in (b). In some embodiments, the microcarriers are between about 5 μm and about 200 μm in diameter. In some embodiments, the microcarriers are about 40 μm in diameter. In some embodiments, the microcarriers are less than about 50 μm in thickness. In some embodiments, the microcarriers are between about 2 μm and about 10 μm in thickness.

[0018] In some embodiments, the analyte is selected from the group consisting of a DNA molecule, a DNA analog molecule, an RNA molecule, an RNA analog molecule, a polynucleotide, a protein, an enzyme, a lipid, a phospholipid, a carbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, an antibody, a small molecule, a bacterial cell, an organelle, and an antibody fragment.

[0019] In some embodiments, the capture agent for capturing the analyte is selected from the group consisting of a DNA molecule, a DNA analog molecule, an RNA molecule, an RNA analog molecule, a polynucleotide, a protein, an enzyme, a lipid, a phospholipid, a carbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, an antibody, a small molecule, a bacterial cell, an organelle, and an antibody fragment.

[0020] In one aspect, disclosed herein are encoded microcarriers produced by any of the methods provided herein.

[0021] In another aspect, a method for detecting multiple analytes in a solution is disclosed, comprising contacting the solution comprising a first analyte and a second analyte with a plurality of microcarriers, the plurality of microcarriers including at least: (i) a first microcarrier according to any of the embodiments described herein that specifically captures the first analyte, the first microcarrier being encoded with a first analog code; and (ii) a second microcarrier according to any of the embodiments described herein that specifically captures the second analyte, the second microcarrier being encoded with a second analog code, the second analog code being different from the first analog code; (b) decoding the first analog code and the second analog code using analog shape recognition to distinguish between the first microcarrier and the second microcarrier; and (c) detecting an amount of the first analyte bound to the first microcarrier and an amount of the second analyte bound to the second microcarrier. In some embodiments, (b) is performed before (c). In some embodiments, (c) is performed before (b). In some embodiments, (b) and (c) are performed simultaneously.In some embodiments, decoding the first analog code and the second analog code comprises: (i) illuminating the first and second microcarriers by passing light through a substantially transparent magnetic polymer layer of the first and second microcarriers and / or a surrounding solution, where the light cannot pass through a substantially opaque layer of the first and second microcarriers, generating a first analog coded light pattern corresponding to the first microcarrier and a second analog coded light pattern corresponding to the second microcarrier; (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 (iii) matching the first analog coded image to the first analog code and matching the second analog coded image to the second analog code using analog shape recognition.

[0022] In some embodiments, 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 (i) incubating the first and second microcarriers with a detection agent after (a), where the detection agent binds to 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 the skin or adipose tissue, saliva, tears, bronchoalveolar lavage, oropharyngeal secretions, intestinal fluids, vaginal or uterine secretions, and semen.

[0023] In one aspect, described herein is a kit for performing a multiplex assay comprising a plurality of microcarriers, the plurality of microcarriers comprising at least: (a) a first microcarrier according to any of the embodiments described herein that specifically captures a first analyte, the first microcarrier being encoded with a first analog code; and (b) a second microcarrier according to any of the embodiments described herein that specifically captures a second analyte, the second microcarrier being encoded with a second analog code, the second analog code being different from the first analog code.

[0024] In some embodiments, the kit further comprises 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, hi some embodiments, the kit further comprises instructions for using the kit to detect the first analyte and the second analyte.

[0025] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. [Brief description of the drawings]

[0026] [Figure 1A-B] Two views of an exemplary microcarrier are shown. [Figure 1C-D] 1 shows an exemplary assay for analyte detection using exemplary microcarriers. [Figure 2A] Three examples of microcarriers are shown, each with a unique analog code. [Figure 2B] 1 shows examples of microcarriers with unique analog codes, according to some embodiments. [Figure 2C] 1 shows examples of microcarriers with unique analog codes, according to some embodiments. [Diagram 3] 1 illustrates a method for producing an exemplary microcarrier. [Figure 4] 1 illustrates a method for manufacturing an exemplary microcarrier, with top view (left) and cross-sectional view (right) shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] In one aspect, the present disclosure provides coded microcarriers for analyte detection in multiplex assays. In some embodiments, the microcarriers include: (a) a substantially transparent magnetic polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, the substantially transparent magnetic polymer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide; (b) a substantially opaque layer, the substantially opaque layer being attached to the first surface of the substantially transparent magnetic polymer layer, the contour of the substantially opaque layer forming a two-dimensional shape representing an analog code; and (c) a capture agent for capturing an analyte, the capture agent being linked to at least one of the first surface and the second surface of the substantially transparent magnetic polymer layer.

[0028] In another aspect, provided herein is a method of making an encoded microcarrier comprising: (a) depositing a substantially opaque layer having a first surface and a second surface, the first surface and the second surface being parallel to one another; (b) patterning the deposited substantially opaque layer into a two-dimensional shape representing an analog code; (c) depositing a substantially transparent magnetic polymer layer on the first surface of the deposited substantially opaque layer, the substantially transparent magnetic polymer layer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide; and (d) patterning the deposited substantially transparent magnetic polymer layer into the microcarrier shape.

[0029] Further provided herein, in some embodiments, is an encoded microcarrier produced by the methods disclosed herein.

[0030] In another aspect, a method for detecting multiple analytes in a solution is provided comprising: (a) contacting the solution comprising a first analyte and a second analyte with a plurality of microcarriers, the plurality of microcarriers comprising at least: (i) a first microcarrier of the present disclosure that specifically captures the first analyte, the first microcarrier being encoded with a first analog code; and (ii) a second microcarrier of the present disclosure that specifically captures the second analyte, the second microcarrier being encoded with a second analog code, the second analog code being different from the first analog code; (b) decoding the first analog code and the second analog code using analog shape recognition to distinguish between the first microcarrier and the second microcarrier; and (c) detecting an amount of the first analyte bound to the first microcarrier and an amount of the second analyte bound to the second microcarrier.

[0031] In yet another aspect, provided herein is a kit or article of manufacture for performing a multiplex assay comprising a plurality of microcarriers, the plurality of microcarriers comprising at least: (a) a first microcarrier of the present disclosure that specifically captures a first analyte, the first microcarrier being encoded with a first analog code; and (b) a second microcarrier of the present disclosure that specifically captures a second analyte, the second microcarrier being encoded with a second analog code, the second analog code being different from the first analog code.

[0032] I. General techniques The practice of the techniques described herein employs, unless otherwise indicated, conventional techniques in 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 within the art, which techniques are described in the references cited herein and are fully explained in the literature.

[0033] Regarding molecular biology and recombinant DNA technology, 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 its annual updates.

[0034] For DNA synthesis techniques and nucleic acid chemistry, see, e.g., 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).

[0035] 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, CRC Press; Rai-Choudhury, P. (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography).

[0036] II. Definition Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0037] The term "microcarrier" as used herein can refer to a physical substrate to which a capture agent can be attached. Microcarriers of the present disclosure can take any suitable geometric form or shape. In some embodiments, the microcarrier can be disc-shaped. Typically, the form or shape of the microcarrier is less than 10 mm. -4 ~10 -7 It contains at least one dimension on the order of m (hence the prefix "micro").

[0038] 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., man-made polymers, including polymers composed of non-natural monomers and / or polymerized in configurations or combinations not found in nature).

[0039] The terms "substantially transparent" and "substantially opaque" as used herein can refer to the ability of light (e.g., of a particular 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 polymer that is transparent, translucent, and / or transmits light, 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 that illuminates the material, as well as the means of detecting the light (or its reduction or absence) traveling through the material. In some embodiments, a substantially opaque material causes a perceptible reduction in transmitted light when compared to the surrounding material or image area, for example, when imaged by optical microscopy (e.g., bright field, dark field, phase contrast, differential interference contrast (DIC), Nomarski interference contrast (NIC), Nomarski, Hoffman modulation contrast (HMC), or fluorescence microscopy). In some embodiments, a substantially transparent material allows a perceptible amount of transmitted light to pass through the material, for example, when imaged by optical microscopy (e.g., bright field, dark field, phase contrast, differential interference contrast (DIC), Nomarski interference contrast (NIC), Nomarski, Hoffman modulation contrast (HMC), or fluorescence microscopy).

[0040] The term "analog code" as used herein may refer to any code in which the coded information is represented in a non-quantized and / or non-discrete manner, as opposed to, for example, digital codes. For example, digital codes are sampled at discrete locations for a limited set of values ​​(e.g., 0 / 1 type values), while analog codes may be sampled at a larger range of locations (or as a continuous whole) and / or may contain a wider set of values ​​(e.g., shapes). In some embodiments, analog codes may be read or decoded using one or more analog shape recognition techniques.

[0041] The term "capture agent" as used herein is a broad term and is used in the usual sense to refer to any compound or substance that can specifically recognize an analyte of interest. In some embodiments, specific recognition can 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.

[0042] "Analyte" as used herein is a broad term and is used in its ordinary sense as a substance whose presence, absence, or amount is to be determined, and refers to a substance or chemical component in a sample, such as, but not limited to, a biological sample or a cell or cell population, that can be analyzed. An analyte can be a substance for which a naturally occurring binding member exists, or a substance for which a binding member can be prepared. Non-limiting examples of analytes include, for example, antibodies, antibody fragments, antigens, polynucleotides (such as DNA molecules, DNA analog molecules, RNA molecules, or RNA analog 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, and the like.

[0043] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv).

[0044] As used herein, a "sample" refers to a composition containing a material, such as a molecule, to be detected. In one embodiment, the sample is a "biological sample" (i.e., any material obtained from a biological source (e.g., human, animal, plant, bacteria, fungus, protist, virus)). Biological samples can be in any form, including solid materials (e.g., tissues, cell pellets, and biopsies) and biological fluids (e.g., urine, blood, saliva, lymph, tears, sweat, prostatic fluid, seminal fluid, semen, bile, mucus, amniotic fluid, and oral washings (containing buccal cells)). Solid materials are typically mixed with a fluid. A sample can also refer to an environmental sample, such as water, air, soil, or any other environmental source.

[0045] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and so forth.

[0046] The term "about" as used herein refers to the normal error range for the respective value, which is readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to the value or parameter itself.

[0047] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments, "comprising," "consisting of," and "consisting essentially of."

[0048] III. Encoded Microcarriers Provided herein are encoded microcarriers suitable for analyte detection, such as multiplexed analyte detection. Multiple configurations of encoded microcarriers are contemplated, described and exemplified herein.

[0049] In some aspects, provided herein is an encoded microcarrier comprising a substantially transparent magnetic polymer layer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, and a substantially opaque layer whose outline constitutes a two-dimensional shape that represents an analog code. In some embodiments, the microcarrier further comprises a capture agent for capturing an analyte linked to the substantially transparent magnetic polymer layer (e.g., on one or both surfaces). In some embodiments, the magnetic nanoparticles comprise iron (II, III) oxide or iron (III) oxide. The configuration, parameters, and optional features of the encoded microcarriers of the present disclosure are provided in the following non-limiting description.

[0050] In some embodiments, the substantially transparent magnetic polymer layer of the present disclosure comprises a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles. In some embodiments, the substantially transparent magnetic polymer layer comprises a suspension of magnetic nanoparticles in a substantially transparent polymer, such as an epoxy-based polymer. For example, the magnetic nanoparticles can be made into a stable dispersion (e.g., using a solvent, including but not limited to, cyclopentanone or g-butyrolactone, and / or a dispersant, including but not limited to, a phosphate polymer) and mixed with the dissolved or monomeric form of the polymer. Incorporating magnetic properties into the substantially transparent polymer layer is advantageous over using a dedicated magnetic layer (e.g., magnetic rings, etc.) by allowing a reduction in the overall size (e.g., area) of the microcarriers, thereby increasing the number of microcarriers that can be produced from a single wafer and reducing the cost per unit. In addition, the substantially transparent magnetic polymer layer does not need to be sandwiched between polymer layers to insulate it from the environment, as other dedicated magnetic layers (e.g., nickel layers) may require. This simplified two-layer model reduces manufacturing costs and improves manufacturing consistency without sacrificing functionality. Exemplary descriptions and techniques for magnetic nanoparticle suspensions and mixing them with epoxy-based polymers can be found, for example, in Suter, Marcel. Photopatternable superparamagnetic nanocomposite for the fabrication of microstructures. Diss. ETH Zurich, 2011; and Suter, M. (2011) Sensors and Actuators B: Chemical 156:433-43.

[0051] In some embodiments, the magnetic nanoparticles are superparamagnetic nanoparticles. Without wishing to be bound by theory, such materials are believed to be advantageous for the fabrication of microcarriers because the resulting microcarriers exhibit no residual magnetism and / or low magnetic attraction to each other (as opposed to magnetic materials such as nickel, which have some residual magnetism after removal of the magnetic field), thus preventing them from detrimentally interacting with each other in solution. It is also believed that such materials (e.g., SU-8 / iron (II, III) oxide or iron (III) oxide nanoparticle mixtures or suspensions, which can replace the use of nickel or rare earth metals) are inherently suitable for analog coding (e.g., as described herein) because they allow for very thin magnetic layers with improved image recognition (e.g., when imaged as described herein). These microcarriers are also believed to be easier to fabricate. In certain embodiments, the magnetic nanoparticles include iron (III) oxide (Fe2O3; also known as ferric oxide or maghemite) and / or iron (II, III) oxide (Fe3O4; also known as magnetite). In one particular embodiment, the epoxy-based polymer is SU-8.

[0052] In some embodiments, the magnetic nanoparticles of the present disclosure are less than about 30 nm in diameter. In some embodiments, the magnetic nanoparticles of the present disclosure are greater than or equal to about 3 nm in diameter. In some embodiments, the magnetic nanoparticles of the present disclosure can be any size (e.g., diameter) less than about any of the following sizes (in nm): 30, 25, 20, 15, 10, or 5. In some embodiments, the magnetic nanoparticles of the present disclosure can be any size (e.g., diameter) greater than or equal to any of the following sizes (in nm): 3, 5, 10, 15, 20, or 25. That is, the magnetic nanoparticles can be any size (e.g., diameter) with an upper limit of 30, 25, 20, 15, 10, or 5 nm and an independently selected lower limit of 3, 5, 10, 15, 20, or 25 nm, which is less than the upper limit.

[0053] In some embodiments, the substantially transparent magnetic polymer layer comprises a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the plurality of magnetic nanoparticles comprising less than about 10% (by weight) and / or more than about 0.1% (by weight) of the mixture. In some embodiments, the plurality of magnetic nanoparticles comprises about 2.5% (by weight) of the mixture. In some embodiments, the substantially transparent magnetic polymer layer of the present disclosure comprises a mixture comprising a plurality of magnetic nanoparticles at a concentration of less than about any of the following concentrations (by weight): 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3. In some embodiments, the substantially transparent magnetic polymer layer of the present disclosure comprises a mixture comprising a plurality of magnetic nanoparticles at a concentration of more than about any of the following concentrations (by weight): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, or 8. That is, a substantially transparent magnetic polymer layer of the present disclosure can include a mixture including a plurality of magnetic nanoparticles in a concentration having an upper limit of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3% and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, or 8%, where the lower limit is less than the upper limit.

[0054] In some embodiments, the substantially transparent polymer of the present disclosure comprises an epoxy-based polymer. Epoxy-based polymers suitable for fabricating the compositions described herein include, but are not limited to, the EPON™ family of epoxy resins offered by Hexion Specialty Chemicals, Inc. (Columbus, OH) and any number of epoxy resins offered by The Dow Chemical Company (Midland, MI). Many examples of suitable polymers are commonly known in the art and include, but are not limited to, SU-8, EPON1002F, EPON165 / 154, and poly(methyl methacrylate) / poly(acrylic acid) block copolymers (PMMA-co-PAA). For additional polymers, see, e.g., Warad, IC Packaging: Package Construction Analysis in Ultra Small IC Packaging, LAP LAMBERT Academic Publishing (2010); The Electronic Packaging Handbook, CRC Press (Blackwell, ed.), (2000); and Pecht et al., Electronic Packaging Materials and Their Properties, CCR Press, 1st ed., (1998). These types of materials have the advantage of not swelling in aqueous environments, ensuring uniform microcarrier size and shape within a population of microcarriers. In some embodiments, the substantially transparent polymer is a photoresist polymer. In some embodiments, the epoxy-based polymer is an epoxy-based negative near-UV photoresist. In some embodiments, the epoxy-based polymer is SU-8.

[0055] In some embodiments, microcarriers of the present disclosure comprise a substantially transparent magnetic polymer layer having a thickness of about 0.1 μm or more. In some embodiments, microcarriers of the present disclosure comprise a substantially transparent magnetic polymer layer having a thickness of about 50 μm or less. In some embodiments, microcarriers of the present disclosure comprise a substantially transparent magnetic polymer layer having a thickness between about 0.1 μm and about 50 μm. In some embodiments, microcarriers of the present disclosure comprise a substantially transparent magnetic polymer layer having a thickness of about or less than any of the following thicknesses (in μm): 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3. In some embodiments, microcarriers of the present disclosure comprise a substantially transparent magnetic polymer layer having a thickness of approximately any of the following (in μm): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 or more. That is, microcarriers of the present disclosure can comprise a substantially transparent magnetic polymer layer of a thickness having an upper limit of 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 μm and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 μm, where the lower limit is less than the upper limit.

[0056] In some embodiments, the microcarriers of the present disclosure include a substantially opaque layer. For example, the substantially opaque layer can be made from a substantially opaque polymer or metal.

[0057] In some embodiments, the substantially opaque layer comprises a polymer (e.g., SU-8) described herein mixed with one or more opaque or colored pigments. In other embodiments, the substantially opaque layer comprises a black matrix resist. Any black matrix resist known in the art can be used; see, for example, U.S. Pat. No. 8,610,848 for exemplary black matrix resists and related methods. In some embodiments, the black matrix resist may be a photoresist colored with a black pigment, for example, patterned on the color filter of an LCD as part of the black matrix. Black matrix resists can include, but are not limited to, those sold by Toppan Printing Co. (Tokyo), Tokyo OHKA Kogyo (Kawasaki), and Daxin Materials Corp. (Taichung City, Taiwan).

[0058] In some embodiments, the substantially opaque layer comprises a substantially opaque polymer that exhibits an absorbance of greater than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm. For example, the polymer may exhibit an absorbance of greater than about 1.8 (OD) at one or more wavelengths selected from the group consisting of 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, and 660 nm.

[0059] In some embodiments, the substantially opaque layer comprises a metal. In some embodiments, the metal lacks remanence. In some embodiments, the substantially opaque layer comprises nickel, titanium, copper, and / or chromium.

[0060] In some embodiments, microcarriers of the present disclosure comprise a substantially opaque layer having a thickness of about 0.05 μm or more. In some embodiments, microcarriers of the present disclosure comprise a substantially opaque layer having a thickness of about 2 μm or less. In some embodiments, microcarriers of the present disclosure comprise a substantially opaque layer having a thickness between about 0.05 μm and about 2 μm. In some embodiments, microcarriers of the present disclosure comprise a substantially opaque layer having a thickness of about no more than any of the following thicknesses (in μm): 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.125, 0.1, or 0.075. In some embodiments, microcarriers of the present disclosure comprise a substantially opaque layer having a thickness of at least any of the following approximately (in μm): 0.05, 0.075, 0.1, 0.125, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8. That is, the microcarriers of the present disclosure may have upper limits of 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.125, 0.1, or 0.075 μm, and upper limits of 0.05, 0.075, 0 and an independently selected lower limit of 0.1, 0.125, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 μm, where the lower limit is less than the upper limit.

[0061] In some embodiments, the microcarriers of the present disclosure can be coded with a substantially opaque layer that constitutes a two-dimensional shape. For example, as described above, the two-dimensional shape may constitute the shape of the substantially opaque layer that contrasts with the substantially transparent layer of the microcarrier, or may constitute the shape (e.g., the perimeter) of the microcarrier itself. That is, the code is the shape of the substantially opaque layer itself (e.g., rather than a code generated by a fluorescent or other visible moiety on the surface of the layer of the microcarrier). Any two-dimensional shape that can encompass multiple resolvable characteristic types can be used. In some embodiments, the two-dimensional shape includes one or more of linear, circular, elliptical, rectangular, quadrilateral, or higher order polygonal aspects, elements, and / or shapes.

[0062] In some embodiments, the two-dimensional shape of the substantially opaque polymer layer includes one or more rings surrounding a central portion of the substantially transparent polymer layer. In some embodiments, at least one of the one or more rings includes a discontinuity. Exemplary, non-limiting two-dimensional shapes formed using one or more rings (e.g., two rings) with various numbers and configurations of discontinuities are shown in FIG. 2B.

[0063] In some embodiments, the analog code comprises one or more overlapping or partially overlapping arc elements that form a continuous or discontinuous loop (e.g., surrounding a central portion of the microcarrier). The two-dimensional shape is decoded by imaging the microcarrier (e.g., by optical microscopy) such that an image of the code is formed by the pattern produced by light passing through the substantially transparent magnetic polymer layer and light blocked from passing through the substantially opaque layer. A non-limiting example of a two-dimensional shape composed of overlapping arc elements that form discontinuous loops is shown in Figure 2C.

[0064] In some embodiments, the two-dimensional shape of the substantially opaque polymer layer comprises a gear shape (see, e.g., FIG. 2A). A gear shape, as used herein, can refer to a plurality of shapes (e.g., gear teeth) arranged on the periphery of a substantially spherical, elliptical, or circular body, at least two of the plurality of shapes being spatially separated. In some embodiments, the gear shape comprises a plurality of gear teeth. In some embodiments, the analog code is represented by one or more aspects selected from a gear tooth height of one or more of the plurality of gear teeth, a gear tooth width of one or more of the plurality of gear teeth, a number of gear teeth of the plurality of gear teeth, and a gear tooth arrangement of one or more of the plurality of gear teeth. Advantageously, the gear shape encompasses a plurality of aspects including a gear tooth height, a gear tooth width, a number of gear teeth, and a gear tooth arrangement, which may be varied to generate a large variety of possible unique two-dimensional shapes. However, it should be appreciated that because the gear shapes of the present disclosure are used for encoding purposes and do not need to physically mesh with other gears (e.g., with mechanical gears that transmit torque), the teeth of the gears of the present disclosure are not constrained by the need for identical or intermeshing shapes, either within a gear shape or between multiple gear shapes. Thus, the variety of shapes that can be considered as gear teeth of the present disclosure is much greater than that of mechanical gears.

[0065] In some embodiments, the plurality of gear teeth comprises one or more gear teeth between about 1 μm and about 10 μm wide, in some embodiments, the plurality of gear teeth comprises one or more gear teeth about 1 μm wide, about 1.5 μm wide, about 2 μm wide, about 2.5 μm wide, about 3 μm wide, about 3.5 μm wide, about 4 μm wide, about 4.5 μm wide, about 5 μm wide, about 5.5 μm wide, about 6 μm wide, about 6.5 μm wide, about 7 μm wide, about 7.5 μm wide, about 8 μm wide, about 8.5 μm wide, about 9 μm wide, about 9.5 μm wide, or about 10 μm wide. In some embodiments, the plurality of gear teeth includes one or more gear teeth that are less than about any of the following widths (in 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 that are greater than about any of the following widths (in 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 can include one or more gear teeth that can be any of a range of widths 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, where the lower limit is less than the upper limit.

[0066] In some embodiments, the plurality of gear teeth comprises one or more gear teeth between about 1 μm and about 10 μm in height, in some embodiments, the plurality of gear teeth comprises one or more gear teeth about 1 μm in height, about 1.5 μm in height, about 2 μm in height, about 2.5 μm in height, about 3 μm in height, about 3.5 μm in height, about 4 μm in height, about 4.5 μm in height, about 5 μm in height, about 5.5 μm in height, about 6 μm in height, about 6.5 μm in height, about 7 μm in height, about 7.5 μm in height, about 8 μm in height, about 8.5 μm in height, about 9 μm in height, about 9.5 μm in height, or about 10 μm in height. In some embodiments, the plurality of gear teeth includes one or more gear teeth that are less than about any of the following heights (in 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 that are greater than about any of the following heights (in 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 can include one or more gear teeth that can be any of a range of heights 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, where the lower limit is less than the upper limit. It should be understood that if adjacent circumferential segments through which the gear teeth extend are non-uniform, the gear teeth may have different measurable heights depending on the reference point.

[0067] In some embodiments, the plurality of gear teeth comprises one or more gear teeth spaced apart at intervals between about 1 μm and about 10 μm, hi some embodiments, the plurality of gear teeth comprises one or more gear teeth spaced apart at intervals 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 that are less than about any of the following width intervals (in 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 that are more than about any of the following width intervals (in 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 can include one or more gear teeth that may be spaced apart in any of a range of width spacings 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, where the lower limit is less than the upper limit.

[0068] In some embodiments, the microcarrier further comprises an orientation indicator for orienting the analog code of the substantially opaque layer. Any feature of the microcarrier that is visible and / or detectable by imaging (e.g., microscopy or other forms of imaging as described herein) and / or image recognition software may serve as an orientation indicator. The orientation indicator may, for example, serve as a reference point for an image recognition algorithm to orient the image of the analog code in a uniform orientation (i.e., the shape of the substantially opaque layer). Advantageously, this simplifies image recognition, since the algorithm only needs to compare the image of a particular analog code to a library of analog codes in the same orientation, and not to a library containing all analog codes in all possible orientations. In some embodiments, the orientation indicator comprises an asymmetry of the substantially opaque layer, such as a discontinuity in its contour or shape. For example, the orientation indicator may comprise a visible feature, such as an asymmetry in the two-dimensional shape representing the analog code of the microcarrier (e.g., as shown in Figures 2A-2C).

[0069] In some embodiments, the microcarriers of the present disclosure are substantially circular disks. As used herein, a substantially circular shape can refer to any shape in which the distance between all points on the circumference of the shape and the geometric center of the shape is approximately the same. In some embodiments, a shape is considered to be substantially circular if any variation in the radius that may connect the geometric center to a given point on the circumference exhibits a variation of 10% or less of the length. As used herein, a substantially circular disk can refer to any substantially circular shape in which the thickness of the shape is significantly less than its diameter. For example, in some embodiments, the thickness of a substantially circular disk can 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 certain embodiments, the thickness of a substantially circular disk can be about 20% of its diameter. It should be understood that microcarriers of the present disclosure that are gear-shaped in profile can also be considered to be substantially circular disks; for example, the shape of a microcarrier excluding one or more gear teeth can constitute a substantially circular disk.

[0070] In some embodiments, the microcarriers are less than about 200 μm in diameter. For example, in some embodiments, the microcarriers are 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 in diameter. In some embodiments, the microcarriers are more than about 5 μm, more than about 10 μm, more than about 20 μm, more than about 30 μm, more than about 40 μm, more than about 50 μm, more than about 60 μm, more than about 70 μm, more than about 80 μm, more than about 90 μm, more than about 100 μm, more than about 120 μm, more than about 140 μm, or more than about 150 μm in diameter. In some embodiments, microcarriers of the present disclosure can be any size (e.g., diameter) less than approximately any of the following sizes (in μm): 200, 180, 160, 140, 120, 100, 80, 60, 40, or 20. In some embodiments, magnetic nanoparticles of the present disclosure can be any size (e.g., diameter) greater than or equal to approximately any of the following sizes (in μm): 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, or 150. That is, the magnetic nanoparticles can be of any size (e.g., diameter) having an upper limit of 200, 180, 160, 140, 120, 100, 80, 60, 40, or 20 μm and an independently selected lower limit of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, or 150 μm, which is less than the upper limit.

[0071] In some embodiments, the microcarriers have a diameter of 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 certain embodiments, the microcarriers are about 40 μm in diameter.

[0072] In some embodiments, the microcarrier is less than about 50 μm thick. For example, in some embodiments, the microcarrier is less than about 70 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than 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 thick. In some embodiments, the microcarrier is less than about any of the following thicknesses (in μm): 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2. In some embodiments, the microcarrier is more than about any of the following thicknesses (in μm): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45. That is, the thickness of the microcarriers can be any of a range of thicknesses (in μm) having an upper limit of 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, or 45, where the lower limit is less than the upper limit.

[0073] 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 between about 50 μm and about 2 μm, between about 20 μm and about 2 μm, or between about 10 μm and about 2 μm. In certain embodiments, the microcarrier is about 5 μm in thickness.

[0074] In some aspects, the microcarriers of the present disclosure may include a capture agent. In some embodiments, a capture agent for a particular microcarrier species may be a "unique capture agent", e.g., the capture agent is associated with a particular microcarrier species having a specific identifier (e.g., an analog code). A capture agent may be any biomolecule or chemical compound capable of binding one or more analytes (such as biomolecules or chemical compounds) present in a solution. Examples of biomolecular capture 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 compound capture agents include, but are not limited to, individual components of a chemical library, small molecules, or environmental toxins (e.g., pesticides or heavy metals).

[0075] In some embodiments, the capture agent is linked to the surface of the microcarrier (in some embodiments, at least a central portion of the microcarrier surface). In some embodiments, the capture agent may be chemically attached to the microcarrier. In other embodiments, the capture agent may be physically absorbed to the surface of the microcarrier. In some embodiments, the attachment bond between the capture agent and the microcarrier surface may be a covalent bond. In other embodiments, the attachment bond between the capture agent and the microcarrier surface may be a non-covalent bond, including but not limited to a salt bridge or other ionic bond, one or more hydrogen bonds, hydrophobic interactions, van der Waals forces, London dispersion forces, mechanical bonds, one or more halogen bonds, gold affinity, intercalation, or stacking.

[0076] In some aspects, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) capture agents for the same analyte can each be associated with a microcarrier as 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 a composition, there may be two or more subpopulations of microcarriers with capture agents that bind to the same analyte, but the capture agents associated with each subpopulation bind to the analyte with different affinities. In some embodiments, the dissociation constant of the analyte for any of the capture agents is greater than 10 -6 M or less, for example 10 -7 M or 10 -8 In other embodiments, the dissociation constant of the analyte for any of the capture agents is about 10 -10 M ~ about 10 -6 M, for example, about 10 -10 M ~ about 10 -7 M, about 10 -10 M ~ about 10 -8 M, about 10 -10 M ~ about 10 -9 M, about 10 -9 M ~ about 10 -6 M, about 10 -9 M ~ about 10 -7 M, about 10 -9 M ~ about 10 -8 M, about 10 -8 M ~ about 10 -6 M, or about 10 -8 M ~ about 10 -7 M. In some embodiments, the dissociation constant of the analyte for any two capture agents is about 3 log 10 For example, about 2.5 log 10 , 2log 10 , 1.5log 10 , or 1log 10 differs by the same amount.

[0077] In some embodiments, the analyte of the present disclosure is linked to a microcarrier to capture 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 analyte may include, but is not limited to, a DNA molecule, a DNA analog molecule, an RNA molecule, an RNA analog molecule, a polynucleotide, a protein, an enzyme, a lipid, a phospholipid, a carbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, an antibody, a small molecule, a bacterial cell, an organelle, and an antibody fragment. In other embodiments, the analyte is a compound (such as a small molecule compound) that can bind to a capture agent, such as an individual component of a chemical library, a small molecule, or an environmental toxin (e.g., a pesticide or heavy metal).

[0078] In some aspects, the analyte in the sample (such as a biological sample) can be labeled with a signal emitting entity that can emit a detectable signal upon binding to the capture agent. In some embodiments, the signal emitting entity can be colorimetric based. In other embodiments, the signal emitting entity can be fluorescent based, including but not limited to phycoerythrin, blue fluorescent protein, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and derivatives thereof. In other embodiments, the signal emitting entity can be 32 P, 33 P, 22 Na, 36 Cl, 2 H, 3 H, 35 S, and 123The signal emitting entity may be radioisotope-based, including but not limited to, I-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 compounds present in the sample may be labeled with a signal emitting entity prior to contacting with the microcarriers. In other embodiments, biomolecules or compounds present in the sample may be labeled with a signal emitting entity after contacting with the microcarriers.

[0079] IV. Methods of Making Encoded Microcarriers Certain aspects of the present disclosure relate to methods for making encoded microcarriers, such as the microcarriers described herein. The methods for making encoded microcarriers may include one or more of the features or aspects of the microcarriers described herein, such as in Section III above and / or in the Examples below.

[0080] In some embodiments, the method includes depositing a substantially transparent magnetic polymer layer (e.g., comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, such as magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide), patterning the deposited substantially transparent magnetic polymer layer into a microcarrier shape of the present disclosure, depositing a substantially opaque layer (e.g., over at least a portion of the deposited substantially transparent magnetic polymer layer), and patterning the deposited substantially opaque layer into a two-dimensional shape representing an analog code of the present disclosure. For example, a substantially transparent magnetic polymer layer can be deposited on a substrate of the present disclosure.

[0081] In some embodiments, the method includes: (a) depositing a substantially transparent magnetic polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, the substantially transparent magnetic polymer layer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide; (b) patterning the deposited substantially transparent magnetic polymer layer into a microcarrier shape; (c) depositing a substantially opaque layer on the first surface of the deposited substantially transparent magnetic polymer layer; and (d) patterning the deposited substantially opaque layer into a two-dimensional shape representing the analog code. In some variations, step (a) includes depositing the substantially transparent magnetic polymer layer on a sacrificial layer. In some variations, the deposited substantially opaque layer is patterned by a lift-off process. In some variations, the method includes, before (c) and after (a), depositing a second polymer layer on a first surface of the deposited substantially transparent magnetic polymer layer; before (c) and after (a), applying a mask to the deposited second polymer layer, thereby producing a masked portion of the deposited second polymer layer and an unmasked portion of the deposited second polymer layer; before (c) and after (a), applying light to the deposited masked second polymer layer, where the light is of a wavelength sufficient to remove the unmasked portion of the deposited second polymer layer; (c) includes depositing a substantially opaque layer on the first surface of the deposited substantially transparent magnetic polymer layer and the masked portion of the deposited second polymer layer; and (d) includes removing the deposited second polymer layer, thereby patterning the deposited substantially opaque layer by removing the deposited substantially opaque layer on the masked portion of the deposited second polymer layer.

[0082] In some embodiments, the method includes depositing a substantially opaque layer, patterning the deposited substantially opaque layer into a two-dimensional shape representing an analog code of the present disclosure, depositing (e.g., over at least a portion of the deposited substantially opaque layer) a substantially transparent magnetic polymer layer (e.g., comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, such as magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide), and patterning the deposited substantially transparent magnetic polymer layer into a microcarrier shape of the present disclosure. For example, the substantially opaque layer can be deposited on a substrate of the present disclosure.

[0083] In some embodiments, the substantially transparent magnetic polymer layer or the substantially opaque layer of the present disclosure can be deposited on a substrate. Suitable substrates can include substrates used in standard semiconductor and / or microelectromechanical systems (MEMS) manufacturing techniques. In some embodiments, the substrate can include glass, silicon, quartz, plastic, polyethylene terephthalate (PET), indium tin oxide (ITO) coatings, and the like.

[0084] In some embodiments, the sacrificial layer can be deposited on a substrate, such as a substrate or substrate carrier described herein. In some embodiments, the sacrificial layer can be made from a polymer, including, but not limited to, polyvinyl alcohol (PVA) or OmniCoat™ (MicroChem; Newton, MA). The sacrificial layer can be applied, used, dissolved, or peeled off, for example, according to the manufacturer's instructions.

[0085] In some embodiments, a substantially transparent magnetic polymer layer or a substantially opaque layer of the present disclosure is deposited onto a sacrificial layer. A corresponding layer can be deposited onto a planar sacrificial layer using a substantially transparent magnetic polymer layer or a planar substantially opaque layer that comprises a two-dimensional shape representing an analog code to generate a planar microcarrier surface.

[0086] In some embodiments using the optional sacrificial layer and / or substrate / substrate carrier of the present disclosure, a solvent can be used to dissolve, etch, or strip the sacrificial layer and / or remove the substrate / substrate carrier. A variety of solvents useful for fabrication (e.g., standard semiconductor or MEMS fabrication processes such as photoresist removal) are known in the art. In some embodiments, the solvent is a photoresist stripper, such as a DMSO-based or 1-methyl-2-pyrrolidone (NMP)-based solvent. In some embodiments, the solvent is an AZ® photoresist stripper, such as AZ® 300T (AZ Electronic Materials; Somerville, NJ).

[0087] In some embodiments, the deposited substantially opaque layer is patterned by a lift-off process. In some embodiments, the substantially opaque layer is patterned by depositing a second polymer layer (e.g., on the substantially transparent magnetic polymer layer if deposited, or on the sacrificial layer if not); applying a mask to the deposited second polymer layer; exposing the deposited masked second polymer layer to light; depositing a substantially opaque layer on the masked portion of the deposited second polymer layer (on the substantially transparent magnetic polymer layer if deposited, or on the sacrificial layer if not); and removing the deposited second polymer layer. In some embodiments, the light is of a wavelength sufficient to remove the unmasked portion of the deposited second polymer layer. Thus, the deposited substantially opaque layer is patterned by removing the deposited substantially opaque layer on the masked portion of the deposited second polymer layer. In some embodiments, the second polymer layer is deposited by spin coating.

[0088] In some embodiments, the methods described herein for making encoded microcarriers further include generating a mixture of a substantially transparent polymer monomer and a plurality of magnetic nanoparticles (e.g., magnetic nanoparticles comprising iron (II, III) oxide or iron (III) oxide) for the substantially transparent magnetic polymer layer of the present disclosure. For example, the substantially transparent polymer monomer and the plurality of magnetic nanoparticles can be mixed by vortexing or sonication.

[0089] In some embodiments, prior to depositing the substantially transparent magnetic polymer layer, the methods described herein for making encoded microcarriers further include mixing the substantially transparent polymer monomer, the plurality of magnetic nanoparticles, the solvent, and the dispersant to form a mixture of the substantially transparent polymer monomer, the plurality of magnetic nanoparticles, the solvent, and the dispersant. In some embodiments, the plurality of magnetic nanoparticles constitutes 2.5% of the mixture of the substantially transparent polymer monomer, the plurality of magnetic nanoparticles, the solvent, and the dispersant. In some embodiments, the plurality of magnetic nanoparticles comprises less than about any of the following concentrations (wt % of the mixture of the substantially transparent polymer monomer, the plurality of magnetic nanoparticles, the solvent, and the dispersant): 10, 9, 8, 7, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3. In some embodiments, the plurality of magnetic nanoparticles comprises greater than about any of the following concentrations (weight % relative to the mixture of substantially transparent polymer monomer, plurality of magnetic nanoparticles, solvent, and dispersant): 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, or 8. That is, the magnetic nanoparticles of the present disclosure can have a concentration having an upper limit of 10, 9, 8, 7, 6, 5, 4, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3% and an independently selected lower limit of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, or 8%, where the lower limit is less than the upper limit, and the concentration is in weight percent relative to the mixture of substantially transparent polymer monomer, magnetic nanoparticles, solvent, and dispersant.

[0090] In some variations, the magnetic nanoparticles, solvent, and dispersant are mixed to form a mixture of magnetic nanoparticles, solvent, and dispersant prior to mixing with the substantially transparent polymer monomer. In some embodiments, the dispersant comprises 10% of the mixture of magnetic nanoparticles, solvent, and dispersant. In some embodiments, the dispersant comprises less than about any of the following concentrations (wt % of the mixture of magnetic nanoparticles, solvent, and dispersant): 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1. In some embodiments, the magnetic nanoparticles comprise greater than any of the following concentrations (weight % of the mixture of magnetic nanoparticles, solvent, and dispersant): 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, or 90. That is, the magnetic nanoparticles of the present disclosure can have a concentration having an upper limit of 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1% and an independently selected lower limit of 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, or 90%, where the lower limit is less than the upper limit and the concentration is in weight percent of the mixture of the magnetic nanoparticles, solvent, and dispersant.

[0091] In some embodiments, the solvent described herein comprises cyclopentanone or g-butyrolactone, or a combination thereof.

[0092] In some embodiments, the dispersants described herein include a phosphate polymer or contain phosphate groups. In some embodiments, the dispersants include a polyethylene glycol (PEG) polymer. In some embodiments, the dispersants include a polyester polymer. In some embodiments, the dispersants include a phosphate polymer. In some embodiments, the dispersants include a polymer with one or more carboxyl groups. In some embodiments, the dispersants include a polymer with one or more phosphate groups. In some embodiments, the dispersants include a polymer with PEG-phosphate moieties. In some embodiments, the dispersants include carboxyl-PEG-phosphate as described, for example, in J. Mater. Chem., 2021, 22, 19806. In some embodiments, the dispersants include phosphate polyesters. In some embodiments, 100% of the dispersant is carboxyl-PEG-phosphate. In some embodiments, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, or 0.1% of the dispersant is carboxyl-PEG-phosphate. In some embodiments, the dispersing agent allows the magnetic nanoparticles and monomers of the substantially transparent polymer to remain stable in the mixture, thereby reducing lot-to-lot variability of the microcarriers produced. In some embodiments, mixing the magnetic nanoparticles with a solvent comprising a phosphate polyester polymer as a dispersing agent allows the mixture comprising the magnetic nanoparticles and monomers of the substantially transparent polymer to remain stable for more than approximately any of the following days: 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days.

[0093] In some embodiments, the substantially transparent magnetic polymer layer of the present disclosure is deposited by spin coating. In some embodiments, the substantially transparent magnetic polymer layer of the present disclosure is deposited by spray coating. In some embodiments, the substantially transparent magnetic polymer layer is deposited and / or patterned by photolithography. In some embodiments, the substantially transparent magnetic polymer layer is deposited and / or patterned by lift-off. In some embodiments, the substantially transparent magnetic polymer layer is deposited and / or patterned by sputtering.

[0094] In some embodiments, the substantially opaque layers of the present disclosure are deposited by lithography, hi some embodiments, the substantially opaque layers of the present disclosure are deposited by lift-off.

[0095] In some embodiments, a capture agent can be linked to a microcarrier of the present disclosure, e.g., a microcarrier described herein, and / or a microcarrier produced by any of the methods described herein. Any of the capture agents described herein, or any capture agent known in the art suitable for capturing an analyte described herein, can find use in the methods and / or microcarriers of the present disclosure.

[0096] In some embodiments, the capture agent can be linked to a polymer layer of the present disclosure, such as a substantially transparent magnetic polymer layer as described herein. In some embodiments, the capture agent can be linked to one or both of the first or second surfaces of the polymer layer. In some embodiments, the capture agent can be linked 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 comprises an epoxide group.

[0097] In some embodiments, linking the scavenger comprises reacting the polymer with a photoacid generator and light to produce a crosslinked polymer. In some embodiments, the light is of a wavelength that activates the photoacid generator, such as UV light or near UV light. Photoacid generators are commercially available from Sigma-Aldrich (St. Louis) and BASF (Ludwigshafen).Any suitable photoacid generator known in the art may be used, including but not limited to triphenyl or triaryl sulfonium hexafluoroantimonate; triaryl sulfonium hexafluorophosphate; triphenyl sulfonium perfluoro-1-butanesulfonate; triphenyl sulfonium triflate; tris(4-tert-butylphenyl)sulfonium perfluoro-1-butanesulfonate or triflate; bis(4-tert-butylphenyl)iodonium-containing photoacid generators, such as bis(4-tert-butylphenyl)iodonium. (rt-butylphenyl)iodonium perfluoro-1-butanesulfonate, p-toluenesulfonate, and triflate;Boc-methoxyphenyldiphenylsulfonium triflate;(tert-butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate;(4-tert-butylphenyl)diphenylsulfonium triflate;Diphenyliodonium hexafluorophosphate, nitrate, perfluoro-1-butanesulfonate, triflate, or p-toluenesulfonate;(4-fluorophenyl)iodonium hexafluorophosphate, nitrate, perfluoro-1-butanesulfonate, triflate, or p-toluenesulfonate; (4-methylphenyl)diphenylsulfonium triflate; N-hydroxynaphthalimide triflate; N-hydroxy-5-norbornene-2,3-dicarboximide perfluoro-1-butanesulfonate; (4-iodophenyl)diphenylsulfonium triflate; (4-methoxyphenyl)diphenylsulfonium triflate; 2-(4-methoxystyryl)-4,6-bis(trichloromethyl)-1,3,5-triazine; (4-methylphenyl)diphenylsulfonium triflate; (4-methylthiophenyl)methylphenylsulfonium triflate; (4-phenoxyphenyl)diphenylsulfonium triflate; (4-phenylthiophenyl)diphenylsulfonium triflate; or any of the photoacid generators described in product-finder.basf.com / group / corporate / product-finder / de / literature-document: / Brand+Irgacure-Brochure--Photoacid+Generator+Selection+Guide-English.pdf.In some embodiments, the photoacid generator is a sulfonium-containing photoacid generator.

[0098] In some embodiments, linking the scavenger involves reacting an epoxide on the crosslinked polymer with a functional group such as an amine, carboxyl, or thiol. Alternatively, the surface epoxy groups can be oxidized to hydroxyl groups, which are then used as initiation sites for the graft polymerization of a water-soluble polymer such as poly(acrylic acid). The carboxyl groups of poly(acrylic acid) are then used to form covalent bonds with the amino or hydroxyl groups of the scavenger.

[0099] In some embodiments, linking the scavenger involves reacting an epoxide of the crosslinked polymer with a compound containing an amine and a carboxyl. In some embodiments, the amine of the compound reacts with the epoxide to form a crosslinked polymer with the compound linked. Without wishing to be bound by theory, it is believed that the scavenger can be linked to the polymer before the polymer is crosslinked; however, this may result in a less uniform surface. Any compound with a primary amine and a carboxyl group can be used. Compounds may include, but are not limited to, glycine, aminoundecanoic acid, aminocaproic acid, acrylic acid, 2-carboxyethylacrylic acid, 4-vinylbenzoic acid, 3-acrylamido-3-methyl-1-butanoic acid, glycidyl methacrylate, and the like. In some embodiments, the carboxyl of the crosslinked polymer with the compound linked reacts with the amine (e.g., primary amine) of the scavenger to link the scavenger to the substantially transparent polymer.

[0100] Descriptions of various capture agents and analytes suitable for the above-described methods can be found throughout this disclosure, for example, in Section III above.

[0101] V. Multiplex Assays Certain aspects of the present disclosure relate to methods for detecting analytes in a solution by using coded microcarriers, such as the microcarriers described herein. The method for analyte detection using coded microcarriers includes one or more of the features or aspects of the microcarriers described herein, such as in Sections III and IV above, and / or in the Examples below. Advantageously, these coded microcarriers allow for reduced analyte detection and recognition errors in improved multiplex assays with a large number of possible unique microcarriers compared to conventional multiplex assays. The analyte detection method used herein can be carried out in any suitable assay vessel known in the art, such as a microplate, a Petri dish, or any number of other known assay vessels.

[0102] In some embodiments, a method of detecting an analyte in a solution includes contacting the solution containing a first analyte and a second analyte with a plurality of microcarriers, the plurality of microcarriers including at least a first microcarrier of the present disclosure that specifically captures the first analyte and is encoded with a first analog code, and a second microcarrier of the present disclosure that specifically captures the second analyte and is encoded with a second analog code; decoding the first analog code and the second analog code using analog shape recognition to distinguish between the first microcarrier and the second microcarrier; and detecting an amount of the first analyte bound to the first microcarrier and an amount of the second analyte bound to the second microcarrier.

[0103] 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 the present disclosure that specifically captures the first analyte (e.g., using a capture agent specific for the first analyte linked to the microcarrier), the first microcarrier being encoded with a first analog code; and a second microcarrier of the present disclosure that specifically captures the second analyte (e.g., using a capture agent specific for the second analyte linked to the microcarrier), the second microcarrier being encoded with a second analog code different from the first analog code. In some embodiments, the first and second analytes may be different. In other embodiments, the first and second analytes may be the same, for example the first and second microcarriers may overlap and recognise the same analyte (which may be useful, for example, for quality control purposes), or the first and second microcarriers may recognise different regions of the same analyte (e.g. antibodies recognising different epitopes of the same antigen).

[0104] The disclosed method can be used to detect an analyte 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, oropharyngeal secretions, intestinal fluid, vaginal or uterine secretions, and semen. In some embodiments, the biological sample can be from a human. In other embodiments, the solution comprises a non-biological sample, such as an environmental sample, a laboratory-prepared sample (e.g., a sample containing one or more analytes that have been prepared, isolated, purified, and / or synthesized), a fixed sample (e.g., a formalin-fixed sample, a paraffin-embedded sample, or a FFPE sample), etc.

[0105] In some embodiments, the analysis is multiplexed, i.e., each solution (e.g., sample) is analyzed such that signals from the signal emitting entities are detected by the reaction detection system for at least two analytes of interest, at least three analytes of interest, at least four analytes of interest, at least five analytes of interest, at least ten analytes of interest, at least fifteen analytes of interest, at least twenty analytes of interest, at least twenty-five analytes of interest, at least thirty analytes of interest, at least thirty-five analytes of interest, at least forty analytes of interest, at least forty-five analytes of interest, or at least fifty analytes of interest, or more.

[0106] In some embodiments, the method includes using analog shape recognition to decode the first and second analog codes to identify the first and second microcarriers. Conceptually, this decoding can 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 with an image from the library, thus positively identifying the code. Optionally, when using microcarriers that include a directional indicator (e.g., asymmetry), as described herein, the decoding can further include rotating each image to align it in a particular direction (based in part, e.g., on the directional indicator). For example, if the directional indicator includes a gap, the image can be rotated until the gap reaches a predetermined position or direction (e.g., the 0° position of the image).

[0107] Various shape recognition software, tools and methods are known in the art. Examples of such APIs and tools include, but are not limited to, Microsoft® Research FaceSDK, OpenBR, Face and Scene Recognition from ReKognition, Betaface API, and various ImageJ plug-ins. In some embodiments, analog shape recognition may include, but is not limited to, image processing steps such as foreground extraction, shape detection, binarization (e.g., automatic or manual image binarization), etc.

[0108] Those skilled in the art will appreciate that the methods and microcarriers described herein may be compatible with a variety of imaging devices, including, but not limited to, microscopes, plate readers, and the like. In some embodiments, decoding the analog code may include illuminating the first and second microcarriers by passing light through a substantially transparent portion of the first and second microcarriers (e.g., a substantially transparent polymer layer) and / or a surrounding solution. The light may then be unable to pass through, or pass with a lower intensity or other significant difference through, a substantially opaque portion of the first and second microcarriers (e.g., a substantially opaque polymer layer) to generate a first analog-coded light pattern corresponding to the first microcarrier and a second analog-coded light pattern corresponding to the second microcarrier.

[0109] As explained above, any type of optical microscopy can be used in the methods of the present disclosure, including, but not limited to, one or more of the following: bright field, dark field, phase contrast, differential interference contrast (DIC), Nomarski interference contrast (NIC), Nomarski, Hoffman modulation contrast (HMC), or fluorescence microscopy. In certain embodiments, the analog code can be decoded using bright field microscopy and the analyte can be detected using fluorescence microscopy.

[0110] In some embodiments, decoding the analog code may further include imaging a first analog coded light pattern to generate a first analog coded image and imaging a second analog coded light pattern to generate a second analog coded image. That is, the imaged light pattern corresponds to a pattern of substantially transparent / substantially opaque regions of the microcarrier, thus generating an image of the analog code. This imaging may include steps including, but not limited to, capturing an image, binarizing the image, and any other image processing steps desired to achieve more accurate, precise, or robust imaging of the analog code.

[0111] In some embodiments, decoding the analog code may further include matching the first analog coded image to the first analog code and the second analog coded image to the second analog code using analog shape recognition. In some embodiments, the image may be matched to the analog code (e.g., an image file from a library of image files, each image file corresponding to a unique two-dimensional shape / analog code) within a predetermined threshold, for example, that allows a predetermined amount of deviation or mismatch between the image and a typical analog code image. Such a threshold may be determined empirically and, of course, based on the particular type of two-dimensional shape used for the analog code and the degree of variation among the set of possible two-dimensional shapes.

[0112] In some embodiments, the method includes detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier. Any suitable analyte detection technique known in the art can be used. For example, in some embodiments, the first and second microcarriers can be incubated with one or more detection agents. In some embodiments, the one or more detection agents bind to 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.

[0113] In some embodiments, the analyte in the solution (such as a biological sample) can be labeled with a detection agent (e.g., a signal-emitting entity) that can emit a detectable signal upon binding to the capture agent. In some embodiments, the detection agent can be colorimetric-based. In other embodiments, the detection agent can be fluorescent-based, including but not limited to phycoerythrin, blue fluorescent protein, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and derivatives thereof. In other embodiments, the detection agent can be radioisotope-based, including but not limited to molecules labeled with 32P, 33P, 22Na, 36Cl, 2H, 3H, 35S, and 123I. 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 biomolecule or compound present in the solution can be labeled with a detection agent prior to contact with the microcarrier composition. In other embodiments, the biomolecule or compound present in the solution can be labeled with a detection agent after contact with the microcarrier composition. In yet other embodiments, the detection agent can be linked to a molecule or macromolecular structure that specifically binds to an analyte of interest, e.g., a DNA molecule, a DNA analog molecule, an RNA molecule, an RNA analog molecule, a polynucleotide, a protein, an enzyme, a lipid, a phospholipid, a carbohydrate moiety, a polysaccharide, an antigen, a virus, a cell, an antibody, a small molecule, a bacterial cell, an organelle, and / or an antibody fragment.

[0114] 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 (e.g., a fluorescence microscope or a plate reader). In other 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 (e.g., a luminescence microscope or a plate reader).

[0115] In some embodiments, each analyte / capture agent can be used with a specific detection agent. As a non-limiting example, the detection agent can be an antibody that specifically binds to the analyte; or, if the analyte is the cognate ligand / receptor of a ligand-receptor pair, a detection agent (e.g., fluorescent, luminescent, enzymatic, or other detection agent) linked to the ligand or receptor of the ligand-receptor pair. This technology is conceptually similar to a sandwich ELISA or protein microarray that includes a capture antibody and a detection antibody (although it should be noted that in this case the agents in this example are not strictly limited to antibodies). As another non-limiting example, the detection agent can be a fluorescent or other detectable probe linked to a protein of interest, such as a labeled analyte of interest. For example, a reaction can be used to link the detection agent to one or more proteins in a solution of interest (such as a sample), which are then captured by a capture agent (conceptually similar to an antigen capture type of protein microarray).

[0116] In other embodiments, multiple specific analytes / capture agents can be used with a universal detection agent. As a non-limiting example, the detection agent can be an agent that binds to the Fc region of an antibody if the analyte is an antibody; if the analyte is a polynucleotide such as DNA or RNA, it can be a fluorescent or other detectable probe linked to an oligonucleotide (e.g., a single-stranded oligonucleotide that hybridizes with the analyte). The latter scenario is conceptually similar to microarray technology.

[0117] In some embodiments, the detection step may include one or more washing steps, such as to remove any material non-specifically bound to the capture agent and / or the microcarrier surface, for example to reduce contaminants. In some embodiments, a magnetic separation step can be used to wash the microcarriers containing the magnetic layer or material of the present disclosure. In other embodiments, other separation steps known in the art can be used.

[0118] In some embodiments, the decoding step may occur after the detecting step, in other embodiments, the decoding step may occur before the detecting step, and in yet other embodiments, the decoding step may occur simultaneously with the detecting step.

[0119] VI. Kits or Articles of Manufacture Further provided herein are kits or articles of manufacture that contain a plurality of the microcarriers of the present disclosure.These kits or articles of manufacture may find use, in particular, in carrying out multiplex assays, such as the exemplary multiplex assays described herein (see, e.g., Section V above).

[0120] 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 for the first analyte linked to the microcarrier), the first microcarrier being 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 for the second analyte linked to the microcarrier), the second microcarrier being encoded with a second analog code that is different from the first analog code. In some embodiments, the first and second analytes may be different. In other embodiments, the first and second analytes may be the same, e.g., the first and second microcarriers may overlap and recognize the same analyte (which may be useful, e.g., for quality control purposes), or the first and second microcarriers 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 of the microcarriers described herein (see, e.g., Section III above) or produced using the methods described herein (see, e.g., Section IV above).

[0121] In some embodiments, the kit or article of manufacture may further comprise one or more detection agents of the present disclosure 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 detection agent for the first analyte may be the same as the detection agent for the second analyte. In other embodiments, the detection agent for the first analyte may be different from the detection agent for the second analyte.

[0122] In some embodiments, the kit or article of manufacture may further include instructions for using the kit or article of manufacture to detect one or more analytes, e.g., a first and a second analyte. These instructions may be for using the kit or article of manufacture, e.g., in any of the methods described herein.

[0123] In some embodiments, the kit or article of manufacture may further include one or more detection agents (e.g., as described above), along with any instructions or any reagents suitable for linking the detection agent to one or more analytes or to one or more macromolecules that recognize the analytes. The kit or article of manufacture may further include any additional components for using the microcarriers in an assay (e.g., a multiplex assay), including, but not limited to, a plate (e.g., a 96-well or other similar microplate), dish, microscope slide, or other suitable assay container; a non-transitory computer-readable storage medium (e.g., containing software and / or other instructions for analog shape or code recognition); a washing agent; a buffer; a plate sealer; a mixing vessel; a diluent or storage solution, and the like. EXAMPLES

[0124] The present invention will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes from the standpoint of these will be suggested to those skilled in the art and should be included within the spirit and scope of this application and the scope of the appended claims.

[0125] Attention is now directed to microcarriers for multiplex assays (e.g., analyte detection) and their methods of manufacture. The following examples provide exemplary embodiments of analog-encoded microcarriers for analyte detection that may find use in particular in the methods, assays, and kits or articles of manufacture described herein. It should be noted that these exemplary embodiments are not intended to be limiting in any way, but are provided to illustrate some of the aspects and features described herein.

[0126] Example 1: Two-dimensional analogue coded microcarriers As mentioned above, analog-coded microcarriers are highly advantageous for multiplexed assays due to the large number of possible unique identifiers and reduced recognition errors. In this example, various types of two-dimensional shape-coded microcarriers that can be used as analog codes for identification are described. It should be understood that the coded microcarriers of the present disclosure may include some or all of the optional features described below in any combination.

[0127] FIG. 1A and FIG. 1B show two views of an exemplary microcarrier 100. The microcarrier 100 is a circular disk with a diameter of about 40 μm and a thickness of 2-10 μm. FIG. 1A provides a view of the microcarrier 100 from the circular face of the disk, and FIG. 1B shows a side view of the microcarrier 100 perpendicular to the face shown in FIG. 1A. 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 using a polymer, e.g., SU-8, for example, as described above. In some embodiments, layer 102 is a substantially transparent magnetic polymer, for example, as described above.

[0128] A substantially opaque layer 104 is applied to the surface of layer 102. The cross-sectional view of microcarrier 100 shown in FIG. 1B shows a discontinuous view of layer 104, while the view shown in FIG. 1A shows layer 104 to be shaped like a circular gear with multiple teeth. This is merely an exemplary shape; other shapes are described above and shown, for example, in FIGS. 2A-2C. Advantageously, the shape of layer 104 fits within the perimeter of layer 102. This allows each of the different analog codes to represent a unique identifier of one microcarrier species while maintaining a uniform overall shape across the multiple species of microcarriers. Stated differently, each microcarrier species within a multiple species population may have a different two-dimensional gear shape (i.e., analog code), but each microcarrier has the same perimeter, which increases uniformity in physical properties (e.g., size, shape, behavior in solution, etc.). Layer 104 may be produced, for example, as described above, using a polymer, such as SU-8 mixed with a dye, using a black matrix resist, or using a metal lacking remanence.

[0129] Layer 104 surrounds a central portion 106 of layer 102. A capture agent for capturing an analyte is coupled to at least central portion 106 on one or both surfaces (i.e., top / bottom) of layer 102. Advantageously, this allows imaging of central portion 106 without any potential interference from layer 104.

[0130] 1C and 1D show an exemplary assay using a microcarrier 100 for analyte detection. FIG. 1C shows that the microcarrier 100 may include a capture agent 108 linked to one or more surfaces at least at a central portion 106. The microcarrier 100 is contacted with a solution containing an analyte 110 that is captured by the capture agent 108. As mentioned above, various capture agents may be used to capture different types of analytes ranging from small molecules, nucleic acids and proteins (e.g., antibodies) to organelles, viruses and cells. Although FIG. 1C shows a single microcarrier species (i.e., microcarrier 100) capturing the analyte 110, multiple microcarrier species are used in a multiplex assay, with each species having a specific capture agent that recognizes a particular analyte.

[0131] FIG. 1D shows an exemplary process for "reading" the microcarriers 100. This process includes two steps that can be accomplished simultaneously or separately. First, the capture of the analyte 110 by the capture agent 108 is detected. In the example shown in FIG. 1D, the detection agent 114 binds to the analyte 110. Analytes that are not captured by the capture agent linked to the microcarriers 100 may be washed away before detection, so that only analytes bound to the microcarriers 100 are detected. The detection agent 114 also includes a reagent for detection. As an example, the detection agent 114 may include a fluorophore that emits light 118 (e.g., photons) when excited by light 116 of a wavelength within the excitation spectrum of the fluorophore. The light 118 may be detected by any suitable detection method, such as a fluorescent microscope, a plate reader, etc.

[0132] In addition, the microcarriers 100 are read for their unique identifier. In the example shown in FIG. 1D, light 112 is used to illuminate the area containing the microcarriers 100 (in some embodiments, light 112 can have a different wavelength than light 116 and light 118). When light 112 illuminates the area containing the microcarriers 100, the light passes through the substantially transparent polymer layer 102 but is blocked by the substantially opaque polymer layer 104, as shown in FIG. 1D. This produces a light pattern that can be imaged, for example, by optical microscopy (e.g., using differential interference contrast or DIC microscopy). This light pattern is based on the two-dimensional shape (i.e., analog code) of the microcarriers 100. Standard image recognition techniques can be used to decode the analog code represented by the image of the microcarriers 100.

[0133] The analyte detection and identifier imaging steps can be performed in any order or simultaneously. Advantageously, both detection steps shown in FIG. 1D can be accomplished on one imaging device. As an example, a microscope capable of both fluorescence and optical (e.g., bright field) microscopy can be used to quantify the amount of analyte 110 bound to the microcarrier 100 (e.g., as detected by detection agent 114) and image the analog code created by layers 102 and 104. This allows for less equipment to be required and a more efficient assay process.

[0134] Figure 2A shows three embodiments of an exemplary gear shape coding scheme, namely microcarriers 200, 202, and 204. Unique codes for microcarriers 200, 202, and 204 are generated using a simple "filled or unfilled" scheme. Figure 2B shows ten exemplary embodiments of codes using number of shapes (e.g., two different shapes for code ZN_3 compared to seven different shapes for code ZN_10) and / or size of shapes (e.g., large, small, and medium sized shapes for code ZN_2).

[0135] 2C illustrates another code format that uses continuous or discontinuous ring shapes, where the code can be represented by the overall difference in number, pattern, two-dimensional width, and / or thickness throughout the shape (e.g., composed of one or more arc elements). The discontinuity of the ring shapes provides an asymmetry that can be used, for example, to orient the image of the code as part of decoding. Importantly, as discussed above, the use of analog image recognition allows for more complex coding schemes, greatly increasing the number of possible unique codes.

[0136] Example 2 Method for manufacturing microcarriers using two-dimensional analogue codes Having described exemplary embodiments of microcarriers in Example 1, attention is now directed to methods of making the microcarriers. As noted above, microcarriers of the present disclosure can be made from one, two, or more constituent layers depending on the desired configuration and / or optional features.

[0137] FIG. 3 illustrates an exemplary process 300 for manufacturing a microcarrier of the present disclosure having a substantially transparent magnetic polymer layer and a substantially opaque layer.

[0138] At 302, a substantially transparent magnetic polymer layer 304 is deposited on a sacrificial layer 306. In some embodiments, the layer 304 is deposited by spin coating. The layer 304 can be made of any substantially transparent polymer having superparamagnetic elements, such as magnetic nanoparticles.

[0139] At 312, the deposited layer 304 was patterned into a microcarrier shape to produce microcarrier shape 308b (partial views of microcarrier shapes 308a and 308c are also shown). That is, the deposited layer 304 was patterned to produce a plurality of microcarrier shapes, each shape including a substantially transparent magnetic polymer layer patterned into a microcarrier shape. In some embodiments, the layer 304 is patterned using photolithography. In some embodiments, the layer 304 is patterned using a lift-off process.

[0140] At 322, a substantially opaque layer was deposited and patterned on each microcarrier (e.g., on the deposited and patterned substantially transparent magnetic polymer layers 308a, 308b, and 308c). In this figure, substantially opaque layer 310a was deposited and patterned on substantially transparent magnetic polymer layer 308a, substantially opaque layer 310b was deposited and patterned on substantially transparent magnetic polymer layer 308b (in this figure, layer 310b appears discontinuous because of the cross section, but can be either continuous or discontinuous), and substantially opaque layer 310c was deposited and patterned on substantially transparent magnetic polymer layer 308c. Layers 310a, 310b, and 310c were patterned into an analog code, for example an analog code of the exemplary type shown in Figures 2A-2C. In this example, layers 310a, 310b, and 310c were deposited and patterned into multiple analog code shapes, each of which belonged to a microcarrier. The analog shapes may be the same as or different from each other (e.g., sacrificial layer 306 may contain all of the microcarriers of one shape, or microcarriers representing two or more different analog shapes, fabricated at the same time for convenience). In some embodiments, layers 310a, 310b, and 310c are deposited by lithography. In some embodiments, layers 310a, 310b, and 310c are patterned by a lift-off process. Layers 310a, 310b, and 310c may be made of any opaque material, such as a substantially opaque polymer (e.g., including a black matrix resist or a polymer that exhibits an absorbance greater than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm) or a metal (e.g., lacking remanence, such as titanium or chromium).

[0141] Alternatively, the order of deposition and patterning of the substantially transparent magnetic polymer layer and the substantially opaque layer can be reversed, as shown in Figure 4. Figure 4 shows a process 400 for manufacturing a microcarrier of the present disclosure having a substantially transparent magnetic polymer layer and a substantially opaque layer.

[0142] At 402, a sacrificial layer 404 is shown.

[0143] At 412, a substantially opaque layer 406 was deposited and patterned on the sacrificial layer 404. The layer 406 was patterned into an analog code, for example the exemplary type of analog code shown in Figures 2A-2C. In this example, the layer 406 was deposited and patterned into a number of analog code shapes, each analog code shape belonging to a microcarrier. The analog shapes may be the same as each other or different (e.g., the sacrificial layer 404 can contain all microcarriers of one shape fabricated at the same time for convenience, or microcarriers representing two or more different analog shapes). In some embodiments, the layer 406 is deposited by lithography. In some embodiments, the layer 406 is patterned by a lift-off process. Layer 406 can be made from any opaque material, such as a substantially opaque polymer (including, for example, a black matrix resist or a polymer that exhibits an absorbance greater than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm) or a metal (e.g., lacking remanent magnetism, such as titanium or chromium).

[0144] At 422, a substantially transparent magnetic polymer layer 408 is deposited on layer 406 and sacrificial layer 404. In some embodiments, layer 408 is deposited by spin coating. Layer 408 can be made of any substantially transparent polymer having superparamagnetic elements, such as magnetic nanoparticles.

[0145] At 432, layer 408 is patterned into a microcarrier shape 410 to produce microcarrier shapes 410a, 410b, and 410c. That is, deposited layer 408 is patterned to produce a plurality of microcarrier shapes, each shape including a substantially transparent magnetic polymer layer patterned into a microcarrier shape. In some embodiments, layer 408 is patterned using photolithography, for example using mask 414. In some embodiments, layer 304 is patterned using a lift-off process.

[0146] Optionally, after 322 and / or 432, the sacrificial layers 306 and / or 404 may be removed, for example by etching.

[0147] For clarity of understanding, the foregoing invention has been described in some detail by way of illustration and example, but the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. A coded microcarrier comprising: (a) a substantially transparent magnetic polymer layer having a first surface and a second surface, the first surface and the second surface being parallel to each other, the substantially transparent magnetic polymer comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles comprising iron (II,III) oxide or iron (III) oxide; (b) a substantially opaque layer affixed to the first surface of the substantially transparent magnetic polymer layer, the outline of the substantially opaque layer constituting a two-dimensional shape representing an analog code; and (c) a capture agent for capturing an analyte, the capture agent being linked to at least one of the first and second surfaces of the substantially transparent magnetic polymer layer. The coded microcarrier as described above.

2. The microcarrier according to Claim 1, wherein the magnetic nanoparticles are superparamagnetic.

3. The microcarrier according to Claim 1 or 2, wherein the magnetic nanoparticles have a diameter of less than about 30 nm and greater than or equal to about 3 nm.

4. The microcarrier according to Claim 1 or 2, wherein the plurality of magnetic nanoparticles comprise a mixture of a substantially transparent polymer and the plurality of magnetic nanoparticles, with less than about 10% (by weight) and more than about 0.1% (by weight) of the mixture being magnetic nanoparticles.

5. The microcarrier according to Claim 1 or 2, wherein the substantially transparent magnetic polymer layer has a thickness between about 0.1 μm and about 50 μm.

6. The microcarrier according to Claim 1 or 2, wherein the substantially transparent polymer is an epoxy-based polymer.

7. The microcarrier according to Claim 6, wherein the epoxy-based polymer is SU-8.

8. The microcarrier according to Claim 1 or 2, wherein the substantially opaque layer comprises a substantially opaque polymer.

9. The microcarrier according to Claim 8, wherein the substantially opaque layer comprises a black matrix resist.

10. The microcarrier according to Claim 8, wherein the substantially opaque polymer exhibits an absorbance of more than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm.

11. The microcarrier according to Claim 1 or 2, wherein the substantially opaque layer comprises a metal lacking remanent magnetism.

12. The microcarrier according to claim 11, wherein the substantially opaque layer contains titanium or chromium.

13. The microcarrier according to claim 1 or 2, wherein the substantially opaque layer is between about 0.05 μm and about 2 μm in thickness.

14. The microcarrier according to claim 1 or 2, wherein the analog code comprises one or more overlapping or partially overlapping arc elements forming a continuous or discontinuous ring.

15. The microcarrier according to claim 1 or 2, further comprising a direction indicator for orienting the analog code of the substantially opaque layer.

16. The microcarrier according to claim 15, wherein the direction indicator comprises an asymmetry of the substantially opaque layer.

17. The microcarrier according to claim 1 or 2, wherein the microcarrier is a substantially circular disk.

18. The microcarrier according to claim 1 or 2, wherein the microcarrier has a diameter between about 5 μm and about 200 μm.

19. The microcarrier according to claim 18, wherein the microcarrier has a diameter of about 40 μm.

20. The microcarrier according to claim 1 or 2, wherein the microcarrier has a thickness of less than about 50 μm.

21. The microcarrier according to claim 20, wherein the microcarrier has a thickness between about 2 μm and about 10 μm.

22. The microcarrier according to claim 21, wherein the microcarrier has a thickness of about 5 μm.

23. The microcarrier according to claim 1 or 2, 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.

24. The microcarrier according to claim 1 or 2, wherein 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.

25. A method of producing a coded microcarrier, comprising (a) depositing a substantially opaque layer having a first surface and a second surface, the first and second surfaces being parallel to each other; (b) patterning the deposited substantially opaque layer into a two-dimensional shape representing an analog code; (c) depositing a substantially transparent magnetic polymer layer on the first surface of the deposited substantially opaque layer, the substantially transparent magnetic polymer layer including a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, the magnetic nanoparticles including iron (II, III) oxide or iron (III) oxide, depositing the substantially transparent magnetic polymer layer; and (d) patterning the deposited substantially transparent magnetic polymer layer into a microcarrier shape.

26. The method according to claim 25, wherein (a) includes depositing the substantially opaque layer on a sacrificial layer.

27. The method according to claim 26, wherein the substantially opaque layer is deposited and patterned by lithography.

28. The method according to claim 26, wherein the deposited substantially opaque layer is deposited and patterned by a lift-off process.

29. depositing a second polymer layer on the sacrificial layer before (a); applying a mask to the deposited second polymer layer before (a), thereby generating a masked portion and an unmasked portion of the deposited second polymer layer; irradiating the deposited and masked second polymer layer with light before (a), the light having a wavelength sufficient to remove the unmasked portion of the deposited second polymer layer; further comprising (a) including depositing the substantially opaque layer on the sacrificial layer and on the masked portion of the deposited second polymer layer; (b) including patterning the deposited substantially opaque layer by removing the deposited second polymer layer, thereby removing the deposited substantially opaque layer on the masked portion of the deposited second polymer layer, the method according to claim 28.

30. The method according to claim 29, wherein the second polymer layer is deposited by spin coating.

31. The method according to any one of claims 26 to 30, further comprising depositing a sacrificial layer on a substrate carrier before (a) to (d).

32. The method according to any one of claims 26 to 30, further comprising etching the sacrificial layer after (a) to (d).

33. The method according to any one of claims 25 to 30, further comprising mixing a monomer of a substantially transparent polymer, a plurality of magnetic nanoparticles, a solvent, and a dispersant before (c) to form a mixture of a monomer of a substantially transparent polymer, a plurality of magnetic nanoparticles, a solvent, and a dispersant.

34. The method according to claim 33, wherein a plurality of magnetic nanoparticles, a solvent, and a dispersant are mixed before mixing with the monomer of the substantially transparent polymer to form a mixture of a plurality of magnetic nanoparticles, a solvent, and a dispersant.

35. The method according to claim 34, wherein the dispersant comprises 10% of the mixture of a plurality of magnetic nanoparticles, a solvent, and a dispersant.

36. The method according to claim 33, wherein the plurality of magnetic nanoparticles constitute 2.5% of the mixture of a monomer of a substantially transparent polymer, a plurality of magnetic nanoparticles, a solvent, and a dispersant.

37. The method according to claim 33, wherein the solvent comprises cyclopentanone.

38. The method according to claim 33, wherein the dispersant comprises a phosphoric acid polymer.

39. The method according to claim 38, wherein the phosphoric acid polymer comprises carboxyl-PEG-phosphoric acid.

40. The method according to any one of claims 25 to 30, wherein the substantially transparent magnetic polymer layer is deposited by spin coating or spray coating.

41. The method according to any one of claims 25 to 30, wherein the deposited substantially transparent magnetic polymer layer is patterned by photolithography, lift-off, or sputtering.

42. The method according to any one of claims 25 to 30, further comprising linking a capture agent for capturing an analyte to at least one of a first surface and a second surface of the substantially transparent magnetic polymer layer.

43. Linking the capture agent is reacting the substantially transparent polymer of the substantially transparent magnetic polymer layer with a photoacid generator and light to generate a crosslinked polymer, the light being of a wavelength that activates the photoacid generator, to generate a crosslinked polymer; Reacting the epoxide of the crosslinked polymer with a compound containing an amine and a carboxyl group, reacting the amine of the compound with the epoxide to form a crosslinked polymer to which the compound is linked; and Reacting the carboxyl group of the crosslinked polymer to which the compound is linked with a scavenger to link the scavenger to a substantially transparent magnetic polymer layer, the method according to claim 42.

44. The method according to any one of claims 25 to 30, wherein the magnetic nanoparticles are superparamagnetic.

45. The method according to any one of claims 25 to 30, wherein the magnetic nanoparticles have a diameter of less than about 30 nm and greater than or equal to about 3 nm.

46. The method according to any one of claims 25 to 30, wherein a plurality of magnetic nanoparticles comprise a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles of less than about 10% (by weight) and about 0.1% (by weight).

47. The method according to any one of claims 25 to 30, wherein the substantially transparent magnetic polymer layer has a thickness between about 0.1 μm and about 50 μm.

48. The method according to any one of claims 25 to 30, wherein the substantially transparent polymer of the substantially transparent magnetic polymer layer is an epoxy-based polymer.

49. The method according to claim 48, wherein the epoxy-based polymer is SU-8.

50. The method according to any one of claims 25 to 30, wherein the substantially opaque layer comprises a substantially opaque polymer.

51. The method according to claim 50, wherein the substantially opaque layer comprises a black matrix resist.

52. The method according to claim 50, wherein the substantially opaque polymer exhibits an absorbance of more than about 1.8 (OD) at a wavelength between about 230 nm and about 660 nm.

53. The method according to any one of claims 25 to 30, wherein the substantially opaque layer comprises a metal lacking residual magnetism.

54. The method according to claim 53, wherein the substantially opaque layer comprises titanium or chromium.

55. The method according to any one of claims 25 to 30, wherein the substantially opaque layer has a thickness between about 0.05 μm and about 2 μm.

56. The method according to any one of claims 25 to 30, wherein the analog code comprises one or more overlapping or partially overlapping arc elements forming a continuous or discontinuous ring.

57. The method according to any one of claims 25 to 30, wherein the deposited substantially opaque layer is patterned to provide asymmetry.

58. The method according to any one of claims 25 to 30, wherein the microcarriers are patterned into substantially circular disks in (b).

59. The method according to claim 58, wherein the microcarriers are between about 5 μm and about 200 μm in diameter.

60. The method according to claim 59, wherein the microcarriers are about 40 μm in diameter.

61. The method according to any one of claims 25 to 30, wherein the microcarriers are less than about 50 μm thick.

62. The method according to claim 61, wherein the microcarriers are between about 2 μm and about 10 μm thick.

63. The method according to claim 42, 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.

64. The method according to claim 42, wherein 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.

65. An encoded microcarrier produced by the method according to any one of claims 25 to 30.

66. A method for detecting a plurality of analytes in a solution, 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 claim 1 or 2 that specifically captures the first analyte and is encoded with a first analog code; and (ii) a second microcarrier according to claim 1 or 2 that specifically captures the second analyte and is encoded with a second analog code, the second analog code being different from the first analog code. Contacting a solution containing a first analyte and a second analyte, including (b) using analog shape recognition to decode a first analog code and a second analog code to identify a first microcarrier and a 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, a method comprising

67. The method according to claim 66, wherein (b) is performed before (c).

68. The method according to claim 66, wherein (c) is performed before (b).

69. The method according to claim 66, wherein (b) and (c) are performed simultaneously.

70. Decoding the first analog code and the second analog code (i) irradiating the first and second microcarriers by passing light through a substantially transparent magnetic polymer layer of the first and second microcarriers and / or the surrounding solution, the light being unable to pass through a substantially opaque layer of the first and second microcarriers, generating a first analog coded light pattern corresponding to the first microcarrier and a second analog coded light pattern corresponding to the second microcarrier; (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 (iii) using analog shape recognition to match the first analog coded image with the first analog code and the second analog coded image with the second analog code, the method according to claim 66

71. Detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier (i) after (a), incubating the first and second microcarriers with a detection agent, the detection agent binding to the first analyte captured by the first microcarrier and the second analyte captured by the second microcarrier, incubating the first and second microcarriers; and The method according to claim 66, comprising: (ii) measuring the amount of the detection agent bound to the first and second microcarriers.

72. The method according to claim 71, wherein the detection agent is a fluorescent detection agent, and the amount of the detection agent bound to the first and second microcarriers is measured by fluorescence microscopy.

73. The method according to claim 71, wherein the detection agent is a luminescent detection agent, and the amount of the detection agent bound to the first and second microcarriers is measured by luminescence microscopy.

74. The method according to claim 66, wherein the solution contains a biological sample.

75. The method according to claim 74, 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, oropharyngeal secretions, intestinal fluid, vaginal or uterine secretions, and semen.

76. A kit for performing a multiplex assay comprising a plurality of microcarriers, the plurality of microcarriers comprising at least: (a) a first microcarrier according to claim 1 or 2 that specifically captures a first analyte, the first microcarrier being encoded with a first analog code; and (b) a second microcarrier according to claim 1 or 2 that specifically captures a second analyte, the second microcarrier being encoded with a second analog code, the second analog code being different from the first analog code.

77. The kit according to claim 76, further comprising 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.

78. The kit according to claim 76, further comprising instructions for using the kit to detect the first analyte and the second analyte.

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