Magnetic microcarriers for image-differentiated 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 of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 193,338, filed on 26 May 2021, the entire contents of which are incorporated herein by reference.

[0002] This specification provides coded microcarriers having a substantially transparent magnetic polymer layer, useful for, for example, analyte detection in multiplex assays, as well as methods for preparing and using the same, and related kits. In some embodiments, the microcarriers include a capture agent for capturing the analyte and are coded with an analog code prepared from the two-dimensional shape of the opaque layer. [Background technology]

[0003] Immunological and molecular diagnostic assays play a crucial role in both research and clinical settings. Often, assays need to be performed against a panel of multiple targets to obtain meaningful results or an overview of results, facilitating research or clinical decision-making. This is particularly true in the age of genomics and proteomics, where a wealth of genetic and / or biomarkers are thought to influence or predict specific disease conditions. Theoretically, multi-target assays can be achieved by testing each target separately or sequentially in parallel within different reactors (i.e., multiple singleplexes). However, assays employing a singleplex strategy are often not only cumbersome but also typically require large sample volumes, especially when there are numerous targets to analyze.

[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 samples can be analyzed at once. The ability to assay many analytes simultaneously and many samples in parallel is a prominent feature of multiplex assays, and it is why such assays have become a powerful tool in fields ranging from drug discovery to functional genomics for clinical diagnosis. In contrast to singleplex assays, by combining all targets within the same reactor, only one reactor is used per sample, thus greatly reducing the complexity of the assay and making it much easier to perform. Therefore, the volume of sample required for testing can be dramatically reduced, which is particularly important when it is difficult and / or invasive to collect large quantities 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 for complex macromolecular samples consist of two steps. In the first step, a drug capable of specifically capturing the target macromolecule is attached to the surface of a solid phase. These immobilized molecules can then be used to capture the target macromolecule from the complex sample by various means, such as hybridization (e.g., in DNA and RNA-based assays) or antigen-antibody interaction (in immunoassays). In the second step, a detection molecule is incubated with the capture molecule and target complex to bind to this complex, and the detection molecule emits a signal, such as fluorescence or other electromagnetic signals. The amount of the target is then quantified by the intensity of these signals.

[0006] Multiplex assays can be performed by utilizing multiple capture agents, each specific to a different target macromolecule. In a tip-based array multiplex assay, each type of capture agent is attached to a predefined location on a tip. The amount of multiplex target 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 the 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 particles with the same ID, and the signal emitted from the detection molecule on the surface of particles with a specific ID reflects the amount of the corresponding target.

[0007] Existing systems for suspension array multiplex assays have limited resolution. Some multiplex systems use digital barcodes printed on flat microbeads using standard semiconductor manufacturing techniques. However, the number of identifiers that can be generated with a given number of digits is limited. Increasing the number of unique identifiers requires increasing the number of digits in the barcode, which in turn requires more space to print on 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 in such fluorescent systems is limited by overlaps in excitation / emission spectra, and identification errors can occur, for example, due to variations between batches of fluorescent dyes.

[0008] Therefore, there is a need for analog-encoded multiplex assay systems that are not constrained by limitations such as digital barcode size or fluorophore resolution. Such systems would enable virtually unlimited unique identifiers and minimize recognition errors due to the use of analog codes (e.g., spectral overlap or fluorophore variability between batches). Furthermore, there is a need for microcarriers for such systems that are easy to handle and / or easily separable from, for example, biological samples or other liquids.

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

[0010] To meet this need, this specification provides microcarriers encoded with analog codes, in particular, containing a capture agent for capturing the analyte. The analog codes are fabricated from a two-dimensional shape of substantially opaque layers, and the microcarriers also include a substantially transparent magnetic polymer layer. These features allow for rapid and efficient separation of the microcarriers from a solution (e.g., a biological sample or assay solution) by magnetic attraction and enable fabrication using convenient microfabrication techniques. Furthermore, the use of superparamagnetic materials, such as certain iron oxides, eliminates residual magnetism that could complicate assay procedures. These microcarriers can be used, for example, in multiplex assays, where each microcarrier contains a capture agent for capturing a specific analyte and an analog code for identification. Methods for fabricating and using such microcarriers, as well as related kits, are further provided.

[0011] Accordingly, in one embodiment, the coded microcarrier provided herein includes: (a) a substantially transparent magnetic polymer layer having a first surface and a second surface, the first and second surfaces being parallel to each other, wherein the substantially transparent magnetic polymer layer comprises 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 constituting a two-dimensional shape representing an analog code; and (c) a capture agent for capturing an analyte, the capture agent being attached to at least one of the first and second surfaces of the substantially transparent magnetic polymer layer.

[0012] In some embodiments, the magnetic nanoparticles are superparamagnetic. In some embodiments, the magnetic nanoparticles have a diameter of less than about 30 nm and a diameter of about 3 nm or more. In some embodiments, the plurality of magnetic nanoparticles comprises a mixture of the plurality of magnetic nanoparticles with a substantially transparent polymer in an amount of less than about 10% (by weight) and more than about 0.1% (by weight). In some embodiments, the substantially transparent magnetic polymer layer has a thickness between about 0.1 μm and about 50 μm. In some embodiments, the substantially transparent polymer is an epoxy polymer. In some embodiments, the epoxy 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 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 residual magnetism. In some embodiments, the substantially opaque layer comprises titanium or chromium. In some embodiments, the substantially opaque layer has a thickness between about 0.05 μm and about 2 μm. In some embodiments, the analog code includes one or more overlapping or partially overlapping arc elements that form continuous or discontinuous rings. In some embodiments, the microcarrier further includes a direction indicator for oriented the analog code of the substantially opaque layer. In some embodiments, the direction indicator includes asymmetry of the substantially opaque layer. In some embodiments, the microcarrier is a substantially circular disk. In some embodiments, the microcarrier has a diameter between about 5 μm and about 200 μm. In some embodiments, the microcarrier has a diameter of about 40 μm. In some embodiments, the microcarrier has a thickness of less than about 50 μm. In some embodiments, the microcarrier has a thickness between about 2 μm and about 10 μm. In some embodiments, the microcarrier has a thickness of about 5 μm.

[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 embodiment, the present invention provides a method for fabricating coded microcarriers, 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, wherein the substantially transparent magnetic polymer layer comprises 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 a 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 includes: (a) depositing a second polymer layer on a sacrificial layer; (b) applying a mask to the deposited second polymer layer to create a masked portion and an unmasked portion of the deposited second polymer layer; (a) irradiating the deposited and masked second polymer layer with light, the light having a wavelength sufficient to remove the unmasked portion of the deposited second polymer layer; wherein (a) includes depositing a substantially opaque layer on the sacrificial layer and the masked portion of the deposited second polymer layer; and (b) patterning the deposited substantially opaque layer by removing the deposited second polymer layer to remove 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, this method further includes depositing a sacrificial layer on a substrate carrier before (a)-(d). In some embodiments, this method further includes etching the sacrificial layer after (a)-(d). In some embodiments, this method further includes 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.

[0015] In some embodiments, before mixing with the monomer of the substantially transparent polymer, a plurality of magnetic nanoparticles, a solvent, and a dispersant are mixed to form a mixture of a plurality of magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the dispersant comprises 10% of the mixture of a plurality of magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the plurality of magnetic nanoparticles constitutes 2.5% of the mixture of a monomer of a substantially transparent polymer, a plurality of magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the solvent includes cyclopentanone. In some embodiments, the dispersant includes a phosphoric acid polymer. In some embodiments, the phosphoric acid polymer includes carboxyl-PEG-phosphoric acid. 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 includes linking a capture agent for capturing an analyte to at least one of a first and a second surface of a substantially transparent magnetic polymer layer. In some embodiments, linking the capture agent includes: reacting a substantially transparent polymer of the substantially transparent magnetic polymer layer with a photoacid generator and light to produce a crosslinked polymer, wherein the light is of a wavelength that activates the photoacid generator; reacting an epoxide of the crosslinked polymer with a compound comprising an amine and a carboxyl, wherein the amine of the compound reacts with the epoxide to form a crosslinked polymer in which the compound is linked; and reacting the carboxyl of the 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 have a diameter of less than about 30 nm and a diameter of about 3 nm or more. In some embodiments, the plurality of magnetic nanoparticles comprises a mixture of the plurality of magnetic nanoparticles with a substantially transparent polymer in an amount of less than about 10% (by weight) and more than about 0.1% (by weight). In some embodiments, the substantially transparent magnetic polymer layer has a thickness between about 0.1 μm and about 50 μm. In some embodiments, the substantially transparent polymer in the substantially transparent magnetic polymer layer is an epoxy polymer. In some embodiments, the epoxy 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 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 residual magnetism. In some embodiments, the substantially opaque layer comprises titanium or chromium. In some embodiments, the substantially opaque layer has a thickness between about 0.05 μm and about 2 μm. In some embodiments, the analog code includes one or more overlapping or partially overlapping arc elements that form continuous or discontinuous rings. 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 have a diameter between about 5 μm and about 200 μm. In some embodiments, the microcarriers have a diameter of about 40 μm. In some embodiments, the microcarriers have a thickness of less than about 50 μm. In some embodiments, the microcarriers have a thickness between about 2 μm and about 10 μm.

[0018] 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.

[0019] 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.

[0020] In one aspect, there is disclosed herein a coded microcarrier produced by any of the methods provided herein.

[0021] In another embodiment, a method for detecting multiple analytes in a solution is disclosed, comprising contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers include at least: (i) a first microcarrier according to any embodiment described herein for specifically capturing the first analyte, encoded with a first analog code; and (ii) a second microcarrier according to any embodiment described herein for specifically capturing the second analyte, encoded with a second analog code, the second analog code being different from the first analog code; (b) using analog shape recognition to decode the first analog code and the second analog code to identify the first microcarrier and the second microcarrier; and (c) detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier. 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 a first analog code and a second analog code includes (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 a surrounding solution, the light being unable to pass through a substantially opaque layer of the first and second microcarriers, and generating a first analog-encoded light pattern corresponding to the first microcarrier and a second analog-encoded light pattern corresponding to the second microcarrier; (ii) imaging the first analog-encoded light pattern to generate a first analog-encoded image and imaging the second analog-encoded light pattern to generate a second analog-encoded image; and (iii) using analog shape recognition to match the first analog-encoded image with the first analog code and the second analog-encoded image with the second analog code.

[0022] In some embodiments, detecting the amount of a first analyte bound to a first microcarrier and the amount of a second analyte bound to a second microcarrier includes (i) incubating the first and second microcarriers with a detection agent after (a), wherein the detection agent incubates the first and second microcarriers to bind with the first analyte captured by the first microcarrier and the second analyte captured by the second microcarrier; and (ii) measuring the amount of the detection agent bound to the first and second microcarriers. In some embodiments, 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. In some embodiments, 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. In some embodiments, the solution contains a biological sample. In some embodiments, the biological sample is selected from the group consisting of blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluid from skin or adipose tissue, saliva, tears, bronchoalveolar lavage, oropharyngeal secretions, intestinal fluid, transvaginal or uterine secretions, and semen.

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

[0024] In some embodiments, the kit further includes a detection agent for detecting the amount of a first analyte bound to a first microcarrier and the amount of a second analyte bound to a second microcarrier. In some embodiments, the kit further includes instructions for using the kit to detect the first and second analytes.

[0025] It should be understood that other embodiments of the invention can be formed by combining one, some, or all of the characteristics of the various embodiments described herein. These and other aspects of the invention will be apparent to those skilled in the art. [Brief explanation of the drawing]

[0026] [Figure 1A-B] Two diagrams illustrating exemplary microcarriers are shown. [Figure 1C-D] An exemplary assay for detecting the analyte using exemplary microcarriers is shown. [Figure 2A] Here are three examples of microcarriers, each with its own unique analog code. [Figure 2B] Examples of microcarriers having unique analog codes are shown in several embodiments. [Figure 2C] Examples of microcarriers having unique analog codes are shown in several embodiments. [Figure 3] This document describes a method for manufacturing exemplary microcarriers. [Figure 4] A method for manufacturing exemplary microcarriers is shown. A top view (left) and a cross-sectional view (right) are shown. [Modes for carrying out the invention]

[0027] In one embodiment, coded microcarriers for analyte detection in multiplex assays are provided herein. In some embodiments, the microcarriers include: (a) a substantially transparent magnetic polymer layer having a first surface and a second surface, the first and second surfaces being parallel to each other, wherein the substantially transparent magnetic polymer comprises 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 bonded to the first surface of the substantially transparent magnetic polymer layer, the contour of the substantially opaque layer constituting a two-dimensional shape representing an analog code; and (c) a capture agent for capturing the analyte, the capture agent being bonded to at least one of the first and second surfaces of the substantially transparent magnetic polymer layer.

[0028] In another embodiment, the present invention provides a method for fabricating coded microcarriers, 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, wherein the substantially transparent magnetic polymer layer comprises 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 a microcarrier shape.

[0029] In some embodiments, coded microcarriers manufactured by the methods disclosed herein are further provided herein.

[0030] In another embodiment, a method is provided 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 include at least: (i) a first microcarrier of the Disclosure that specifically captures the first analyte and is coded with a first analog code; and (ii) a second microcarrier of the Disclosure that specifically captures the second analyte and is coded with a second analog code, the second analog code being different from the first analog code; (b) using analog shape recognition to decode the first analog code and the second analog code to identify the first microcarrier and the second microcarrier; and (c) detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0031] In yet another embodiment, the Specified Inventions Provided herein are kits or products for performing a multiplex assay comprising a plurality of microcarriers, the plurality of microcarriers comprising at least (a) a first microcarrier of the Disclosure that specifically captures a first analyte and is encoded with a first analog code; and (b) a second microcarrier of the Disclosure that specifically captures a second analyte and is encoded with a second analog code, the second analog code being different from the first analog code.

[0032] I. General techniques The practices of the techniques described herein utilize, unless otherwise specified, polymer techniques, microfabrication, microelectromechanical system (MEMS) fabrication, photolithography, microfluidics, organic chemistry, biochemistry, oligonucleotide synthesis and modification, bioconjugate chemistry, nucleic acid hybridization, molecular biology, microbiology, genetics, recombinant DNA, and prior art in related fields within these techniques. These techniques are described in and are adequately explained in the references cited herein.

[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.See (1999), *Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology*, Wiley, and its annual updates.

[0034] For information on DNA synthesis techniques and nucleic acid chemistry, please refer to, for example, Gait, MJ (1990), Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991), Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992), The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994), Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996), Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996), Bioconjugate Techniques, Academic Press).

[0035] For information on microfabrication, please refer to, 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 should be understood that the present invention is not limited to any particular composition or biological system and is naturally subject to change. It should also be understood that the terms used herein are for the purpose of describing a particular embodiment only and are not intended to limit it.

[0037] As used herein, the term “microcarrier” may refer to a physical substrate on which a scavenger can be bound. The microcarriers of this disclosure can take any preferred geometric form or shape. In some embodiments, the microcarriers may be disk-shaped. Typically, the form or shape of the microcarriers is 10 -4 ~10 -7 It includes at least one dimension of the order of m (hence the prefix "micro").

[0038] As used herein, the term “polymer” can refer to any macromolecular structure containing repeating monomers. Polymers may be natural (e.g., found in nature) or synthetic (e.g., artificial polymers, such as polymers composed of non-natural monomers and / or polymers polymerized in configurations or combinations not found in nature).

[0039] As used herein, the terms “substantially transparent” and “substantially opaque” may refer to the ability of a substrate, such as a polymer layer, to allow light (e.g., of a specific wavelength, such as infrared, visible light, or UV) to pass through. A substantially transparent polymer may refer to a polymer that is transparent, translucent, and / or transmits light, while a substantially opaque polymer may refer to a polymer that reflects and / or absorbs light. It should be understood that whether a material is substantially transparent or substantially opaque may depend on the wavelength and / or intensity of the light illuminating 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 of 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, substantially transparent materials allow a perceptible amount of transmitted light to pass through the material 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] As used herein, the term “analog code” can refer to any code in which encoded information is represented in a non-quantized and / or non-discrete manner, as opposed to, for example, a digital code. For example, while a digital code is sampled at discrete locations with respect to a limited set of values ​​(e.g., values ​​of type 0 / 1), an analog code can be sampled at a wider range of locations (or as a continuous whole) and / or may contain a wider set of values ​​(e.g., shapes). In some embodiments, an analog code can be read or decoded using one or more analog shape recognition techniques.

[0041] As used herein, the term “scavenger” is a broad term and is used in the usual sense to refer to any compound or substance that can specifically recognize the analyte of interest. In some embodiments, specific recognition may refer to specific binding. Non-limiting examples of scavengers 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] As used herein, “analyte” is a broad term and is used in the ordinary sense to refer to any substance or chemical component in a sample, such as a biological sample or a cell or cell population, whose presence, absence, or quantity is to be determined, but which can be analyzed. An analyte may be a substance in which naturally occurring binding members exist, or a substance in which binding members 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, etc.

[0043] The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full-length antibodies with 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, “sample” refers to a composition containing material such as molecules 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, fungi, protists, viruses)). A biological sample may be in any form including solid materials (e.g., tissues, cell pellets, and biopsies) as well as biological fluids (e.g., urine, blood, saliva, lymph, tears, sweat, prostatic fluid, semen, bile, mucus, amniotic fluid, and oral lavage (containing buccal cells)). Solid materials are typically mixed with fluids. The sample may also refer to an environmental sample such as water, air, soil, or any other environmental source.

[0045] As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple subjects unless otherwise explicitly indicated in the content. Thus, for example, a reference to "molecule" optionally includes a combination of two or more such molecules.

[0046] As used herein, the term “about” refers to the normal range of error for each value, which is readily known to those skilled in the art. References to “about” a value or parameter herein include (and are described) embodiments relating to that value or parameter itself.

[0047] The aspects and embodiments of the present invention described herein are understood to include aspects and embodiments, "comprising," "consisting," and "consisting essentially of."

[0048] III. Coded Microcarriers This specification provides coded microcarriers suitable for analyte detection, such as multiplex analyte detection. Several configurations of coded microcarriers are contemplated, described, and illustrated herein.

[0049] In some embodiments, this specification provides coded microcarriers 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 contour constitutes a two-dimensional shape representing an analog code. In some embodiments, the microcarrier further comprises a capture agent for capturing an analyte bonded to the substantially transparent magnetic polymer layer (e.g., on one surface or both surfaces). In some embodiments, the magnetic nanoparticles comprise iron(II,III) oxide or iron(III) oxide. The configurations, parameters, and optional features of the coded microcarriers of this disclosure are provided in the following non-limiting description.

[0050] In some embodiments, the substantially transparent magnetic polymer layer of this 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 polymer. For example, magnetic nanoparticles can be prepared in a stable dispersion (using a solvent, e.g., containing but not limited to cyclopentanone or γ-butyrolactone, and / or a dispersant, containing but not limited to a phosphate polymer) and mixed with a soluble polymer or polymer in monomer form. Incorporating magnetic properties into a substantially transparent polymer layer is advantageous over using a dedicated magnetic layer (e.g., a magnetic ring) by allowing a reduction in the overall size (e.g., area) of microcarriers, thereby increasing the number of microcarriers that can be manufactured from a single wafer and reducing the cost per unit. In addition, the substantially transparent magnetic polymer layer does not require a layer sandwiched between polymer layers to insulate from the environment, as other dedicated magnetic layers (e.g., a nickel layer) may require. This simplified two-layer model reduces manufacturing costs and improves manufacturing consistency without sacrificing functionality. Exemplary descriptions and techniques relating to magnetic nanoparticle suspensions and their mixing with epoxy 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: pp. 433-4343.

[0051] In some embodiments, the magnetic nanoparticles are superparamagnetic nanoparticles. While we do not wish to be constrained by theory, such materials are considered advantageous for the fabrication of microcarriers because the resulting microcarriers exhibit no residual magnetism and / or low magnetic attraction to each other, thus preventing them from interacting with each other in a detrimental manner in solution (as opposed to magnetic materials such as nickel, which exhibit some residual magnetism after the removal of the magnetic field). 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 also considered particularly 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 considered easier to fabricate. In certain embodiments, the magnetic nanoparticles comprise iron(III) oxide (Fe2O3; also known as ferric oxide or maghemite) and / or iron(II,III) oxide (Fe3O4; also known as magnetite). In certain embodiments, the epoxy polymer is SU-8.

[0052] In some embodiments, the magnetic nanoparticles of the Disclosure have a diameter of less than about 30 nm. In some embodiments, the magnetic nanoparticles of the Disclosure have a diameter of about 3 nm or more. In some embodiments, the magnetic nanoparticles of the Disclosure may be any size (e.g., diameter) less than any of the following sizes (in nm): 30, 25, 20, 15, 10, or 5. In some embodiments, the magnetic nanoparticles of the Disclosure may 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 may be any size (e.g., diameter) having 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, where the lower limit is less than the upper limit.

[0053] In some embodiments, a substantially transparent magnetic polymer layer comprises a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, wherein the plurality of magnetic nanoparticles constitute 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 constitute about 2.5% (by weight) of the mixture. In some embodiments, a substantially transparent magnetic polymer layer of the present disclosure comprises a mixture containing the plurality of magnetic nanoparticles at a concentration less than 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, a substantially transparent magnetic polymer layer of the present disclosure comprises a mixture containing the plurality of magnetic nanoparticles at a concentration greater than 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. In other words, the substantially transparent magnetic polymer layer of the present disclosure may contain a mixture of multiple magnetic nanoparticles at concentrations 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 polymers of this disclosure include epoxy polymers. Suitable epoxy polymers for the preparation of the compositions described herein include, but are not limited to, the EPON® family of epoxy resins provided by Hexion Specialty Chemicals, Inc. (Columbus, OH) and any number of epoxy resins provided by The Dow Chemical Company (Midland, MI). Many examples of suitable polymers, which are generally known in the art, include, but are not limited to, SU-8, EPON1002F, EPON165 / 154, and poly(methyl methacrylate) / poly(acrylic acid) block copolymer (PMMA-co-PAA). For additional polymers, see, for example, Warad, IC Packaging: Package Construction Analysis in Ultra Small IC Packaging, LAP LAMBERT Academic Publishing (2010); The Electronic Packaging Handbook, CRC Press (edited by Blackwell), (2000); and Pecht et al., Electronic Packaging Materials and Their Properties, CCR Press, 1st edition, (1998). These types of materials have the advantage of not swelling in aqueous environments and ensure that uniform microcarrier size and shape are maintained within the microcarrier population. In some embodiments, the substantially transparent polymer is a photoresist polymer. In some embodiments, the epoxy polymer is an epoxy-based negative near-UV photoresist. In some embodiments, the epoxy polymer is SU-8.

[0055] In some embodiments, the microcarriers of the Disclosure include a substantially transparent magnetic polymer layer having a thickness of about 0.1 μm or more. In some embodiments, the microcarriers of the Disclosure include a substantially transparent magnetic polymer layer having a thickness of about 50 μm or less. In some embodiments, the microcarriers of the Disclosure include a substantially transparent magnetic polymer layer having a thickness between about 0.1 μm and about 50 μm. In some embodiments, the microcarriers of the Disclosure include a substantially transparent magnetic polymer layer having a thickness of approximately 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 or less. In some embodiments, the microcarriers of the Disclosure include a substantially transparent magnetic polymer layer having a thickness of approximately 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 greater. In other words, the microcarriers of this disclosure may include a substantially transparent magnetic polymer layer having a thickness 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 (upper limit) and 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 (independently selected lower limit), where the lower limit is less than the upper limit.

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

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

[0058] In some embodiments, the substantially opaque layer comprises a substantially opaque polymer exhibiting an absorbance greater than approximately 1.8 (OD) at wavelengths between approximately 230 nm and approximately 660 nm. For example, a polymer can exhibit an absorbance greater than approximately 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 includes a metal. In some embodiments, the metal lacks residual magnetism. In some embodiments, the substantially opaque layer includes nickel, titanium, copper, and / or chromium.

[0060] In some embodiments, the microcarriers of the Disclosure include a substantially opaque layer with a thickness of about 0.05 μm or more. In some embodiments, the microcarriers of the Disclosure include a substantially opaque layer with a thickness of about 2 μm or less. In some embodiments, the microcarriers of the Disclosure include a substantially opaque layer with a thickness between about 0.05 μm and about 2 μm. In some embodiments, the microcarriers of the Disclosure include a substantially opaque layer having a thickness of approximately 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 or less. In some embodiments, the microcarriers of the Disclosure include a substantially opaque layer having a thickness of approximately 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 or greater. In other words, the microcarriers of this disclosure 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 0.05, 0.075, 0 It may include substantially opaque layers with thicknesses having independently selected lower limits 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 this disclosure can be coded with a substantially opaque layer constituting a two-dimensional shape. For example, as described above, the two-dimensional shape may constitute the shape of a substantially opaque layer contrasting with a substantially transparent layer of the microcarrier, or it may constitute the shape of the microcarrier itself (e.g., the periphery). That is, the code is the shape of the substantially opaque layer itself (rather than a code generated by, for example, a fluorescent portion or other visible portion on the surface of the microcarrier's layer). Any two-dimensional shape can be used that can encompass multiple decomposable characteristic types. In some embodiments, the two-dimensional shape includes one or more linear, circular, elliptical, rectangular, quadrilateral, or higher-order polygonal aspects, elements, and / or shapes.

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

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

[0064] In some embodiments, the two-dimensional shape of a substantially opaque polymer layer includes a gear shape (see, for example, Figure 2A). As used herein, a gear shape can refer to a plurality of shapes (e.g., gear teeth) arranged on the periphery of a substantially spherical, elliptical, or circular body, where at least two of the plurality of shapes are spatially separated. In some embodiments, the gear shape includes a plurality of gear teeth. In some embodiments, the analog code is represented by one or more embodiments selected from the height of one or more gear teeth of the plurality of gear teeth, the width of one or more gear teeth of the plurality of gear teeth, the number of gear teeth of the plurality of gear teeth, and the arrangement of the gear teeth of one or more gear teeth of the plurality of gear teeth. Advantageously, the gear shape encompasses a plurality of embodiments including the height of the gear teeth, the width of the gear teeth, the number of gear teeth, and the arrangement of the gear teeth, which may vary to generate a wide variety of possible unique two-dimensional shapes. However, it should be recognized that the gear shapes of the disclosed gears are used for coding purposes and do not need to physically mesh with other gears (e.g., mechanical gears that transmit torque), and therefore the teeth of the disclosed gears are not constrained by the need for identical shapes or meshing shapes, either within a single gear shape or between multiple gear shapes. Consequently, the diversity of shapes that can be considered for the teeth of the disclosed gears is far greater than that for mechanical gears.

[0065] In some embodiments, the teeth of the multiple gears include one or more gear teeth with widths between approximately 1 μm and approximately 10 μm. In some embodiments, the teeth of the multiple gears include one or more gear teeth with widths of approximately 1 μm, approximately 1.5 μm, approximately 2 μm, approximately 2.5 μm, approximately 3 μm, approximately 3.5 μm, approximately 4 μm, approximately 4.5 μm, approximately 5 μm, approximately 5.5 μm, approximately 6 μm, approximately 6.5 μm, approximately 7 μm, approximately 7.5 μm, approximately 8 μm, approximately 8.5 μm, approximately 9 μm, approximately 9.5 μm, or approximately 10 μm. In some embodiments, the teeth of a plurality of gears include one or more gear teeth with approximately the following widths (in μm): less than any 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. In some embodiments, the teeth of a plurality of gears include one or more gear teeth with approximately the following widths (in μm): greater than any 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. That is, the teeth of multiple gears may include teeth of one or more gears, which may be in any width range having an upper limit of 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, or 1.5 and an independently selected lower limit of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, or 9.5, where the lower limit is less than the upper limit.

[0066] In some embodiments, the gear teeth include one or more gear teeth with heights between approximately 1 μm and approximately 10 μm. In some embodiments, the gear teeth include one or more gear teeth with heights of approximately 1 μm, approximately 1.5 μm, approximately 2 μm, approximately 2.5 μm, approximately 3 μm, approximately 3.5 μm, approximately 4 μm, approximately 4.5 μm, approximately 5 μm, approximately 5.5 μm, approximately 6 μm, approximately 6.5 μm, approximately 7 μm, approximately 7.5 μm, approximately 8 μm, approximately 8.5 μm, approximately 9 μm, approximately 9.5 μm, or approximately 10 μm. In some embodiments, the teeth of a plurality of gears include one or more gear teeth with approximately the following heights (in μm): less than any 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. In some embodiments, the teeth of a plurality of gears include one or more gear teeth with approximately the following heights (in μm): greater than any 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. That is, the teeth of multiple gears may include one or more gear teeth that are in any of the height ranges 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 the adjacent outer segments on 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 teeth of a plurality of gears include one or more gear teeth spaced apart at intervals between approximately 1 μm and approximately 10 μm. In some embodiments, the teeth of a plurality of gears include one or more gear teeth spaced apart at intervals of approximately 1 μm, approximately 1.5 μm, approximately 2 μm, approximately 2.5 μm, approximately 3 μm, approximately 3.5 μm, approximately 4 μm, approximately 4.5 μm, approximately 5 μm, approximately 5.5 μm, approximately 6 μm, approximately 6.5 μm, approximately 7 μm, approximately 7.5 μm, approximately 8 μm, approximately 8.5 μm, approximately 9 μm, approximately 9.5 μm, or approximately 10 μm. In some embodiments, the teeth of a plurality of gears include one or more gear teeth that are spaced less than 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 teeth of a plurality of gears include one or more gear teeth that are spaced more than 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 teeth of multiple gears may be spaced apart within one or more ranges of width intervals having upper limits 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 independently selected lower limits 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 includes a directional indicator for orienting the analog code of a substantially opaque layer. Any feature of the microcarrier visible and / or detectable by imaging (e.g., microscopy or other forms of imaging as described herein) and / or image recognition software can function as a directional indicator. The directional indicator can serve, for example, as a reference point for an image recognition algorithm to orient an image of the analog code in a uniform direction (i.e., the shape of the substantially opaque layer). This simplifies image recognition because the algorithm only needs to compare an image of a particular analog code with a library of analog codes in the same direction, and does not need to compare it with a library containing all analog codes in all possible directions. In some embodiments, the directional indicator includes asymmetry of the substantially opaque layer, such as discontinuities in its contour or shape. For example, the directional indicator may include a visible feature such as 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 this 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 periphery of the shape and the geometric center of the shape is substantially the same. In some embodiments, a shape is considered substantially circular if any variation in any of the radii that can connect the geometric center and a given point on the periphery 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 may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of its diameter. In certain embodiments, the thickness of a substantially circular disk may be about 20% of its diameter. It should be understood that a microcarrier of this disclosure whose profile is gear-shaped can also be considered substantially circular disks; for example, the shape of a microcarrier excluding the teeth of one or more gears can constitute a substantially circular disk.

[0070] In some embodiments, the microcarriers have a diameter of less than approximately 200 μm. For example, in some embodiments, the diameter of the microcarriers is less than approximately 200 μm, less than approximately 180 μm, less than approximately 160 μm, less than approximately 140 μm, less than approximately 120 μm, less than approximately 100 μm, less than approximately 80 μm, less than approximately 60 μm, less than approximately 40 μm, or less than approximately 20 μm. In some embodiments, the diameter of the microcarriers is greater than approximately 5 μm, greater than approximately 10 μm, greater than approximately 20 μm, greater than approximately 30 μm, greater than approximately 40 μm, greater than approximately 50 μm, greater than approximately 60 μm, greater than approximately 70 μm, greater than approximately 80 μm, greater than approximately 90 μm, greater than approximately 100 μm, greater than approximately 120 μm, greater than approximately 140 μm, or greater than approximately 150 μm. In some embodiments, the microcarriers of the Disclosure may be any size (e.g., diameter) less than any of the following approximately sizes (in μm): 200, 180, 160, 140, 120, 100, 80, 60, 40, or 20. In some embodiments, the magnetic nanoparticles of the Disclosure may be any size (e.g., diameter) greater than or equal to any of the following approximately sizes (in μm): 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, or 150. In other words, 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, where the lower limit is less than the upper limit.

[0071] In some embodiments, the diameter of the microcarriers is approximately 180 μm, 160 μm, 140 μm, 120 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In a particular embodiment, the microcarrier has a diameter of approximately 40 μm.

[0072] In some embodiments, the microcarrier has a thickness of less than approximately 50 μm. For example, in some embodiments, the thickness of the microcarrier is less than approximately 70 μm, less than approximately 60 μm, less than approximately 50 μm, less than approximately 40 μm, less than approximately 30 μm, less than approximately 25 μm, less than approximately 20 μm, less than approximately 15 μm, less than approximately 10 μm, or less than approximately 5 μm. In some embodiments, the thickness of the microcarrier is 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, or 2. In some embodiments, the thickness of the microcarrier is greater than 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. In other words, the thickness of the microcarrier can be in any of the following ranges (in μm): 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 approximately 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 19 μm, 18 μm, 17 μm, 16 μm, 15 μm, 14 μm, 13 μm, 12 μm, 11 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm. In some embodiments, the thickness of the microcarrier is between approximately 50 μm and 2 μm, between approximately 20 μm and 2 μm, or between approximately 10 μm and 2 μm. In a particular embodiment, the microcarrier has a thickness of approximately 5 μm.

[0074] In some embodiments, the microcarriers of this disclosure may include a scavenger. In some embodiments, the scavenger for a particular microcarrier species may be a “specific scavenger,” for example, by associating the scavenger with a particular microcarrier species having a specific identifier (e.g., an analog code). The scavenger may be any biomolecule or chemical compound that can bind one or more analytes (such as biomolecules or chemical compounds) present in solution. Examples of biomolecule scavengers 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 scavengers 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 scavenger is bonded to the surface of the microcarrier (in some embodiments, at least the central portion of the microcarrier surface). In some embodiments, the scavenger can be chemically attached to the microcarrier. In other embodiments, the scavenger can be physically absorbed onto the surface of the microcarrier. In some embodiments, the bond between the scavenger and the microcarrier surface may be a covalent bond. In other embodiments, the bond between the scavenger and the microcarrier surface may be a non-covalent bond, including but not limited to salt bridges or other ionic bonds, one or more hydrogen bonds, hydrophobic interactions, van der Waals forces, London dispersion forces, mechanical bonds, one or more halogen bonds, gold affinity, intercalation, or stacking.

[0076] In some embodiments, 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 the plurality of microcarriers in the composition, there can be two or more subpopulations of microcarriers with capture agents that bind 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 10 -6 M or less, by way of example 10 -7 M or 10 -8 M. In other embodiments, the dissociation constant of the analyte for any of the capture agents is from about 10 -10 M to about 10 -6 M, by way of example from about 10 -10 M to about 10 -7 M, about 10 -10 M to about 10 -8 M, about 10 -10 M to about 10 -9 M, about 10 -9 M to about 10 -6 M, about 10 -9 M to about 10 -7 M, about 10 -9 M to about 10 -8 M, about 10 -8 M to about 10 -6 M, or from about 10 -8 M to about 10 -7 M. In some embodiments, the dissociation constant of the analyte for any two capture agents is only about 3 log 10 as much, by way of example, about 2.5 log 10 、2 log 10 、1.5 log 10 、or 1 log 10 as much different. <[

[0077] In some embodiments, the analytes of this disclosure are 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 analytes may include, but are not limited to, DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and antibody fragments. In other embodiments, the analytes are compounds (such as small molecule compounds) that can be bound to a capture agent, such as individual components of a chemical library, small molecules, or environmental toxins (e.g., pesticides or heavy metals).

[0078] In some embodiments, the analyte in a sample (such as a biological sample) can be labeled with a signal-emitting entity that emits a detectable signal when bound to a capture agent. In some embodiments, the signal-emitting entity can be colorimetric-based. In other embodiments, the signal-emitting entity may be fluorescence-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 may be 32 P, 33 P, 22 Na, 36 Cl, 2 H, 3 H, 35 S, and 123The signal-emitting entities may be radioisotope-based, including but not limited to molecules labeled with I. In other embodiments, the signal-emitting entities are light-based, including but not limited to luciferases (e.g., chemiluminescent-based), horseradish peroxidases, alkaline phosphatases, and their derivatives. In some embodiments, biomolecules or compounds present in the sample can be labeled with the signal-emitting entity before contact with the microcarrier. In other embodiments, biomolecules or compounds present in the sample can be labeled with the signal-emitting entity after contact with the microcarrier.

[0079] IV. Method for creating coded microcarriers Certain aspects of this disclosure relate to coded microcarriers, for example, methods for producing the microcarriers described herein. Methods for producing coded microcarriers may include one or more features or aspects of the microcarriers described herein, for example, in Section III and / or the following examples.

[0080] In some embodiments, the method includes depositing a substantially transparent magnetic polymer layer (e.g., a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, such as magnetic nanoparticles containing iron(II,III) oxide or iron(III) oxide), patterning the deposited substantially transparent magnetic polymer layer into the microcarrier shape of the Disclosure, depositing a substantially opaque layer (e.g., on 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 the analog code of the Disclosure. For example, the substantially transparent magnetic polymer layer can be deposited on a substrate of the 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 and second surfaces being parallel to each other, wherein the substantially transparent magnetic polymer layer comprises 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 an 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: depositing a second polymer layer on a first surface of a deposited substantially transparent magnetic polymer layer before (c) and after (a); applying a mask to the deposited second polymer layer before (c) and after (a) to create a masked portion of the deposited second polymer layer and an unmasked portion of the deposited second polymer layer; shining light on the deposited and masked second polymer layer before (c) and after (a), wherein the light has a wavelength sufficient to remove the unmasked portion of the deposited second polymer layer; and (c) includes depositing a substantially opaque layer on the first surface of the deposited substantially transparent magnetic polymer layer and on the masked portion of the deposited second polymer layer; and (d) includes 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.

[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 Disclosure, depositing a substantially transparent magnetic polymer layer (e.g., a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles, such as magnetic nanoparticles containing iron(II,III) oxide or iron(III) oxide) (e.g., on at least a portion of the deposited substantially opaque layer), and patterning the deposited substantially transparent magnetic polymer layer into a microcarrier shape of the Disclosure. For example, the substantially opaque layer can be deposited on a substrate of the Disclosure.

[0083] In some embodiments, a substantially transparent or substantially opaque magnetic polymer layer of the present disclosure can be deposited on a substrate. Suitable substrates may include those used in standard semiconductor and / or microelectromechanical system (MEMS) manufacturing techniques. In some embodiments, the substrate may 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, for example, the substrate or substrate carrier described herein. In some embodiments, the sacrificial layer can be made from a polymer containing, 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 on a sacrificial layer. Using a substantially transparent magnetic polymer layer or a planar substantially opaque layer that constitutes a two-dimensional shape representing an analog code, the corresponding layer can be deposited on a planar sacrificial layer to generate a planar microcarrier surface.

[0086] In some embodiments using an optional sacrificial layer and / or substrate / substrate carrier of this disclosure, a solvent may 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 stripping solvent 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 a substantially transparent magnetic polymer layer if deposited, or on a sacrificial layer otherwise); applying a mask to the deposited second polymer layer; shining light on the deposited and masked second polymer layer; depositing the substantially opaque layer on the masked portion of the deposited second polymer layer (on a substantially transparent magnetic polymer layer if deposited, or on a sacrificial layer otherwise); and removing the deposited second polymer layer. In some embodiments, the light has 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 herein for producing coded microcarriers further include generating a mixture of a substantially transparent polymer monomer and a plurality of magnetic nanoparticles (e.g., magnetic nanoparticles containing iron(II,III) oxide or iron(III) oxide) for a 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, the method herein for producing encoded microcarriers before depositing a substantially transparent magnetic polymer layer further comprises mixing a substantially transparent polymer monomer, a plurality of magnetic nanoparticles, a solvent, and a dispersant to form a mixture of the substantially transparent polymer monomer, a plurality of magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the plurality of magnetic nanoparticles constitute 2.5% of the mixture of the substantially transparent polymer monomer, a plurality of magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the plurality of magnetic nanoparticles include concentrations (by weight %) of approximately the following: 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 less than 0.3. In some embodiments, the multiple magnetic nanoparticles include concentrations (by weight %) of approximately 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 plurality of magnetic nanoparticles of the present disclosure can have concentrations 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 a weight % relative to a mixture of a substantially transparent polymer monomer, the plurality of magnetic nanoparticles, a solvent, and a dispersant.

[0090] In some modified forms, multiple magnetic nanoparticles, a solvent, and a dispersant are mixed before being mixed with a substantially transparent polymer monomer to form a mixture of multiple magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the dispersant contains 10% of the mixture of multiple magnetic nanoparticles, a solvent, and a dispersant. In some embodiments, the dispersant contains approximately the following concentrations (by weight %) relative to the mixture of multiple magnetic nanoparticles, a solvent, and a dispersant: less than 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 multiple magnetic nanoparticles include concentrations greater than any of the following (by weight %) relative to the mixture of the multiple 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 plurality of magnetic nanoparticles of the present disclosure may have concentrations 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 a weight % relative to a mixture of the plurality of magnetic nanoparticles, solvent, and dispersant.

[0091] In some embodiments, the solvents described herein include cyclopentanone, γ-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 dispersant includes a polyethylene glycol (PEG) polymer. In some embodiments, the dispersant includes a polyester polymer. In some embodiments, the dispersant includes a phosphate polymer. In some embodiments, the dispersant includes a polymer having one or more carboxyl groups. In some embodiments, the dispersant includes a polymer having one or more phosphate groups. In some embodiments, the dispersant includes a polymer having a PEG-phosphate moiety. In some embodiments, the dispersant includes carboxyl-PEG-phosphate, for example, as described in J. Mater. Chem., 2021, 22, 19806. In some embodiments, the dispersant includes a phosphate polyester. 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 dispersant makes it possible to stably maintain the magnetic nanoparticles and the monomers of a substantially transparent polymer in the mixture, thereby reducing lot-to-lot variability of the microcarriers produced. In some embodiments, by mixing the magnetic nanoparticles with a solvent containing a phosphate polyester polymer as a dispersant, the mixture containing the magnetic nanoparticles and the monomers of a substantially transparent polymer can be stably maintained for approximately the following number of days: greater than 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

[0093] In some embodiments, the substantially transparent magnetic polymer layer of the Disclosure is deposited by spin coating. In some embodiments, the substantially transparent magnetic polymer layer of the 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 layer of the Disclosure is deposited by lithography. In some embodiments, the substantially opaque layer of the Disclosure is deposited by lift-off.

[0095] In some embodiments, the capture agent can be coupled to the microcarriers of the Disclosure, for example, the microcarriers described herein and / or microcarriers produced by any of the methods described herein. Any of the capture agents described herein, or any capture agent known in the Art that is suitable for capturing the analytes described herein, can find applications in the methods and / or microcarriers of the Disclosure.

[0096] In some embodiments, the scavenger can be bonded to a polymer layer of the Disclosure, for example, a substantially transparent magnetic polymer layer as described herein. In some embodiments, the scavenger can be bonded to one or both of the first or second surfaces of the polymer layer. In some embodiments, the scavenger can be bonded to at least the central portion of the polymer layer (for example, the central portion as described herein). In some embodiments, the polymer comprises an epoxy polymer or otherwise comprises epoxide groups.

[0097] In some embodiments, the linking of the scavenger involves 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, but is not limited to: triphenyl or triarylsulfonium hexafluoroantimonate; triarylsulfonium hexafluorophosphate; triphenylsulfonium perfluoro-1-butanesulfonate; triphenylsulfonium triflate; tris(4-tert-butylphenyl)sulfonium perfluoro-1-butanesulfonate or triflate; bis(4-tert-butylphenyl)iodonium-containing photoacid generators, for example, bis(4-tert-butylphenyl)iodonium-containing photoacid generators. 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 It includes (nyl)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 listed 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 the epoxide of the crosslinked polymer with functional groups such as amines, carboxyls, or thiols. Alternatively, the epoxy groups on the surface can be oxidized to hydroxyl groups, which are then used as starting points for graft polymerization of water-soluble polymers 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 the 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. While not wishing to be bound by theory, it is conceivable that the scavenger could be linked to the polymer before the polymer is crosslinked; however, this may reduce the uniformity of the resulting surface. Any compound having 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 is reacted with the amine of the scavenger (e.g., a primary amine) to link the scavenger to a substantially transparent polymer.

[0100] A description of various scavengers and analytes suitable for the above-described method can be found throughout this disclosure, for example, in Section III above.

[0101] V. Multiplex Assay Certain aspects of this disclosure relate to a method for detecting an analyte in solution by using coded microcarriers, for example, the microcarriers described herein. A method for detecting an analyte using coded microcarriers, comprising one or more of the features or embodiments of the microcarriers described herein, for example, in Sections III and IV above, and / or in the following examples. Advantageously, these coded microcarriers enable a reduction in 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 methods used herein can be performed in any suitable assay vessel known in the art, for example, a microplate, a Petri dish, or any number of other well-known assay vessels.

[0102] In some embodiments, a method for detecting an analyte in a solution includes contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers include at least a first microcarrier of the Disclosure that specifically captures the first analyte and is coded with a first analog code, and a second microcarrier of the Disclosure that specifically captures the second analyte and is coded with a second analog code; using analog shape recognition to decode the first analog code and the second analog code to identify the first microcarrier and the second microcarrier; and detecting the amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier.

[0103] In some embodiments, the method involves 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 Disclosure, coded with a first analog code, which specifically captures the first analyte (e.g., using a capture agent specific to the first analyte coupled to the microcarrier); and a second microcarrier of the Disclosure, coded with a second analog code different from the first analog code, which specifically captures the second analyte (e.g., using a capture agent specific to the second analyte coupled to the microcarrier). 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 recognize the same analyte redundantly (which may be useful, for example, for quality control purposes), or the first and second microcarriers may recognize different regions of the same analyte (for example, antibodies that recognize different epitopes of the same antigen).

[0104] The methods of this disclosure can be used to detect an analyte in any suitable solution. In some embodiments, the solution includes a biological sample. Examples of biological samples include, but are not limited to, blood, urine, sputum, bile, cerebrospinal fluid, interstitial fluid from skin or adipose tissue, saliva, tears, bronchoalveolar lavage, oropharyngeal secretions, intestinal fluid, transvaginal or uterine secretions, and semen. In some embodiments, the biological sample may be of human origin. In other embodiments, the solution includes non-biological samples, such as environmental samples, laboratory-prepared samples (e.g., samples containing one or more analytes that have been prepared, isolated, purified, and / or synthesized), and fixed samples (e.g., formalin-fixed samples, paraffin-embedded samples, or FFPE samples).

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

[0106] In some embodiments, the method includes using analog shape recognition to decode a first analog code and a second analog code to identify the first and second microcarriers. Conceptually, this decoding may involve imaging the analog code of each microcarrier (e.g., in a solution or sample), comparing each image with a library of analog codes, and matching each image with an image from the library, thus ensuring the identification of the codes. Optionally, if microcarriers including a directional indicator (e.g., asymmetry) are used as described herein, the decoding may further include the step of rotating each image to align it in a specific direction (partially, for example, based 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 in 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 plugins. In some embodiments, analog shape recognition may include, but are not limited to, image processing steps such as foreground extraction, shape detection, and binarization (e.g., automatic or manual image binarization).

[0108] Those skilled in the art will understand that the methods and microcarriers described herein can be adapted to a variety of imaging devices, including, but not limited to, microscopes and plate readers. In some embodiments, decoding the analog codes may include illuminating the first and second microcarriers by passing light through substantially transparent portions of the first and second microcarriers (e.g., substantially transparent polymer layers) and / or the surrounding solution. The light can then pass through substantially opaque portions of the first and second microcarriers (e.g., substantially opaque polymer layers) either with reduced intensity or with other significant differences, thereby generating a first analog-encoded light pattern corresponding to the first microcarrier and a second analog-encoded light pattern corresponding to the second microcarrier.

[0109] As described above, any type of optical microscopy can be used in the method of this disclosure, and this optical microscopy includes, but is 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, an 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-encoded light pattern to generate a first analog-encoded image, and imaging a second analog-encoded light pattern to generate a second analog-encoded image. That is, the imaged light patterns correspond to patterns of substantially transparent / substantially opaque regions of the microcarriers, and thus an image of the analog code can be generated. This imaging may include, but is not limited to, capturing an image, binarizing an image, and any other image processing steps desired to achieve a more accurate, precise, or robust image of the analog code.

[0111] In some embodiments, decoding the analog code may further include matching a first analog-coded image with a first analog code and a second analog-coded image with a second analog code using analog shape recognition. In some embodiments, the image can be matched with an 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 that allows for a predetermined amount of deviation or mismatch between the image and a typical analog-coded image. Such a threshold may be determined empirically, or, of course, based on the degree of variability between a particular type of two-dimensional shape used for the analog code and a set of possible two-dimensional shapes.

[0112] In some embodiments, the method includes detecting the amount of a first analyte bound to a first microcarrier and the amount of a second analyte bound to a second microcarrier. Any suitable analyte detection technique known in the art can be used. For example, in some embodiments, the first and second microcarriers can be incubated with one or more detection agents. In some embodiments, 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 a solution (such as a biological sample) can be labeled with a detection agent (e.g., a signal-emitting entity) that, when bound to a capture agent, emits a detectable signal. In some embodiments, the detection agent can be colorimetric-based. In other embodiments, the detection agent may be fluorescence-based, including but not limited to phycoerythrin, blue fluorescent protein, green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, and their derivatives. In other embodiments, the detection agent may 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 may be light-based, including but not limited to luciferase (e.g., chemiluminescent-based), horseradish peroxidase, alkaline phosphatase, and their derivatives. In some embodiments, biomolecules or compounds present in a solution can be labeled with the detection agent before contact with the microcarrier composition. In other embodiments, biomolecules or compounds present in a solution can be labeled with the detection agent after contact with the microcarrier composition. In yet another embodiment, the detection agent can be linked to a molecule or macromolecular structure that specifically binds to the analyte of interest, such as DNA molecules, DNA analog molecules, RNA molecules, RNA analog molecules, polynucleotides, proteins, enzymes, lipids, phospholipids, carbohydrate moieties, polysaccharides, antigens, viruses, cells, antibodies, small molecules, bacterial cells, organelles, and / or antibody fragments.

[0114] In some embodiments, 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 (e.g., a fluorescence microscope or plate reader). In other embodiments, 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 emission microscopy (e.g., an emission microscope or plate reader).

[0115] In some embodiments, each analyte / capture agent can be used with a specific detection agent. In non-limiting examples, the detection agent may be an antibody that specifically binds to the analyte; or, if the analyte is a homologous 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 a ligand-receptor pair. This technique is conceptually analogous to a sandwich ELISA or protein microarray containing a capture antibody and a detection antibody (though it should be noted that the agents in this example are not strictly limited to antibodies). In another non-limiting example, the detection agent may 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 a detection agent to one or more proteins in a solution of interest (e.g., a sample), which are then captured by a capture agent (conceptually analogous to an antigen-capturing type protein microarray).

[0116] In other embodiments, multiple specific analytes / capturers can be used with a general-purpose detection agent. In non-limiting examples, the detection agent may be a drug that binds to the Fc region of an antibody if the analyte is an antibody; or, if the analyte is a polynucleotide such as DNA or RNA, it may 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 cleaning steps, such as to remove any substances nonspecifically bound to the scavenging agent and / or microcarrier surface, for example, to reduce impurities. In some embodiments, a magnetic separation step can be used to clean the microcarriers containing the magnetic layer or material of the Disclosure. In other embodiments, other separation steps known in the Art may be used.

[0118] In some embodiments, the decoding step may be performed after the detection step. In other embodiments, the decoding step may be performed before the detection step. In yet another embodiment, the decoding step may be performed simultaneously with the detection step.

[0119] VI. Kits or manufactured products Kits or products containing multiple microcarriers of the present disclosure are further provided herein. These kits or products may find applications in particular when performing multiplex assays, such as the exemplary multiplex assays described herein (see, for example, Section V above).

[0120] In some embodiments, the kit or product may include: a first microcarrier of the Disclosure, encoded with a first analog code, which specifically captures a first analyte (e.g., using a capture agent specific to the first analyte, linked to the microcarrier); and a second microcarrier of the Disclosure, encoded with a second analog code different from the first analog code, which specifically captures a second analyte (e.g., using a capture agent specific to the second analyte, linked to the microcarrier). 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 recognize the same analyte redundantly (which may be useful, for example, 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 manufactured product may include any of the microcarriers described herein (see, for example, Section III above) or manufactured using the methods described herein (see, for example, Section IV above).

[0121] In some embodiments, the kit or product may further include one or more detection agents of the Disclosure for detecting the amount of a first analyte bound to a first microcarrier and the amount of a second analyte bound to a 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 product may further include instructions for using the kit or product to detect one or more analytes, for example, a first and a second analyte. These instructions may be for using the kit or product in any of the methods described herein.

[0123] In some embodiments, the kit or product 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 agents to one or more analytes, or to one or more macromolecules that recognize the analytes. The kit or product may further include any additional components for using microcarriers in an assay (e.g., a multiplex assay), but are not limited to, plates (e.g., 96-well or other similar microplates), dishes, microscope slides, or other suitable assay containers; non-temporary computer-readable storage media (e.g., containing software and / or other instructions for analog shape or code recognition); washing agents; buffers; plate sealers; mixing containers; diluents or storage solutions, etc. [Examples]

[0124] The present invention will be better understood by referring to the following examples. However, they should not be construed as limiting the scope of the invention. The examples and embodiments described herein are for illustrative purposes only, and it should be understood that various modifications or changes from therein are proposed to those skilled in the art and should be included within the spirit and scope of this application and the appended claims.

[0125] Here, we turn our attention to microcarriers for multiplex assays (e.g., analyte detection) and methods for producing them. The following examples illustrate exemplary embodiments of analog-coded microcarriers for analyte detection, which may find applications in the methods, assays, and kits or products 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: Microcarriers coded in two-dimensional analog code As described above, analog-encoded microcarriers are highly advantageous for multiplexed assays due to the vast number of possible unique identifiers and the reduction of recognition errors. This embodiment describes various types of microcarriers encoded in two dimensions that can be used as analog codes for identification. It should be understood that the encoded microcarriers of this disclosure may include some or all of the optional features described below in any combination.

[0127] Figures 1A and 1B show two diagrams of an exemplary microcarrier 100. The microcarrier 100 is a circular disk with a diameter of approximately 40 μm and a thickness of 2 to 10 μm. Figure 1A provides a diagram of the microcarrier 100 as seen from the circular surface of the disk, and Figure 1B shows a side view of the microcarrier 100 perpendicular to the surface shown in Figure 1A. Two components of the microcarrier 100 are shown. Firstly, a substantially transparent polymer layer 102 provides the body of the microcarrier. Layer 102 can be produced using a polymer, for example, SU-8, 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 attached to the surface of layer 102. The cross-sectional view of the microcarrier 100 shown in Figure 1B shows a discontinuous shape of layer 104, while the view shown in Figure 1A shows that layer 104 has a shape like a circular gear with multiple teeth. This is merely an illustrative shape; other shapes have been described above and are shown, for example, in Figures 2A-2C. Advantageously, the shape of layer 104 fits within the outer circumference of layer 102. This allows each of the various analog codes to represent a unique identifier for one type of microcarrier, while maintaining a uniform overall shape across multiple types of microcarriers. In other words, each microcarrier species within a population of multiple species may have a different two-dimensional gear shape (i.e., analog code), but each microcarrier has the same outer circumference, thereby improving uniformity of physical properties (e.g., size, shape, behavior in solution, etc.). Layer 104 may be produced, for example, using a polymer, such as SU-8 mixed with a dye, as described above, using a black matrix resist, or using a metal that lacks residual magnetism.

[0129] Layer 104 surrounds the central portion 106 of layer 102. A capture agent for capturing the analyte is attached to at least the central portion 106 on one or both surfaces (i.e., the top / bottom) of layer 102. Advantageously, this makes it possible to image the central portion 106 without any potential interference caused by layer 104.

[0130] Figures 1C and 1D show an exemplary assay using a microcarrier 100 for analyte detection. Figure 1C shows that the microcarrier 100 may include a capture agent 108 attached to one or more surfaces, at least in its central portion 106. The microcarrier 100 is brought into contact with a solution containing the analyte 110 captured by the capture agent 108. As described 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. Figure 1C shows a single microcarrier species (i.e., microcarrier 100) capturing the analyte 110, but multiple microcarrier species are used in multiplex assays, each having a specific capture agent that recognizes a particular analyte.

[0131] Figure 1D shows an exemplary process for “reading” the microcarrier 100. This process involves two steps that can be achieved simultaneously or separately. First, the capture of the analyte 110 by the capture agent 108 is detected. In the example shown in Figure 1D, the detection agent 114 binds to the analyte 110. Any analyte not captured by the capture agent bound to the microcarrier 100 may be washed off before detection, so that only the analyte bound to the microcarrier 100 is detected. The detection agent 114 also includes a detection reagent. For example, the detection agent 114 may include a fluorophore that emits light 118 (e.g., photons) when excited by light 116 of a wavelength in the excitation spectrum of the fluorophore. The light 118 may be detected by any suitable detection method, e.g., a fluorescence microscope, a plate reader, etc.

[0132] In addition, the microcarriers 100 are read for their unique identifiers. In the embodiment shown in Figure 1D, light 112 is used to illuminate the region containing the microcarriers 100 (in some embodiments, light 112 may have a different wavelength from light 116 and light 118). When light 112 illuminates the region 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 Figure 1D. This generates 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. The analog code represented by the image of the microcarriers 100 can be decoded using standard image recognition techniques.

[0133] The analyte detection and identifier imaging steps can be performed in any order or simultaneously. Advantageously, both detection steps, as shown in Figure 1D, can be achieved on a single imaging device. As an example, a microscope capable of both fluorescence microscopy 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 the detection agent 114) and to image the analog codes created by layers 102 and 104. This reduces the number of instruments required and enables a more efficient assay process.

[0134] Figure 2A shows three embodiments of the coding scheme for exemplary gear shapes, 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 the number of shapes (e.g., two different shapes for code ZN_3 compared to seven different shapes for code ZN_10) and / or the size of the shapes (e.g., large, small, and medium-sized shapes for code ZN_2).

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

[0136] Example 2 Method for manufacturing microcarriers using two-dimensional analog codes While an exemplary embodiment of the microcarrier has been described in Example 1, we now focus on the method for manufacturing the microcarrier. As described above, the microcarrier of this disclosure can be fabricated from one, two, or more constituent layers, depending on the desired configuration and / or optional features.

[0137] Figure 3 shows an exemplary process 300 for manufacturing the microcarriers of this disclosure having a substantially transparent magnetic polymer layer and a substantially opaque layer.

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

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

[0140] In 322, substantially opaque layers were deposited and patterned on each microcarrier (e.g., on substantially transparent magnetic polymer layers 308a, 308b, and 308c that were deposited and patterned). 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 due to the cross-section, but it could be 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 to analog codes, for example, analog codes of the exemplary type shown in Figures 2A and 2C. In this example, layers 310a, 310b, and 310c are deposited and patterned to multiple analog code shapes, each analog code shape belonging to a microcarrier. The analog shapes may be the same or different from each other (for example, sacrificial layer 306 may contain all microcarriers of one shape fabricated simultaneously for convenience, or microcarriers representing two or more different analog shapes). 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 can be fabricated from any opaque material, for example substantially opaque polymers (including, e.g., black matrix resists or polymers exhibiting absorbance greater than about 1.8 (OD) at wavelengths between about 230 nm and about 660 nm) or metals (e.g., non-residual magnetism, such as titanium or chromium).

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

[0142] Layer 402 shows the victim layer 404.

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

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

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

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

[0147] For the sake of clarity, the invention has been described in some detail by examples and embodiments, but this description and embodiment should not be construed as limiting the scope of the invention. All patent and scientific literature disclosures cited herein are expressly incorporated in their entirety by reference.

Claims

1. A coded microcarrier, (a) A substantially transparent magnetic polymer layer having a first surface and a second surface, wherein the first surface and the second surface are 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, wherein the substantially opaque layer is attached to a first surface of a substantially transparent magnetic polymer layer, and the contour of the substantially opaque layer constitutes a two-dimensional shape representing an analog code; and (c) A trapping agent for capturing an analyte, wherein the trapping agent is connected to at least one of a first surface and a second surface of a substantially transparent magnetic polymer layer. Encoded microcarriers, including those mentioned 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 approximately 30 nm and more than approximately 3 nm.

4. The microcarrier according to claim 1 or 2, comprising a mixture of a substantially transparent polymer and a plurality of magnetic nanoparticles in an amount of less than about 10% (by weight) and more than about 0.1% (by weight).

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

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

7. The microcarrier according to claim 6, wherein the epoxy 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 includes a black matrix resist.

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

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

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

13. The microcarrier according to claim 1 or 2, wherein the substantially opaque layer has a thickness between approximately 0.05 μm and approximately 2 μm.

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

15. The microcarrier according to claim 1 or 2, further comprising a direction indicator for oriented an analog code in a substantially opaque layer.

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

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

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

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

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

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

22. The microcarrier according to claim 21, wherein the microcarrier has a thickness of approximately 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 scavenger 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 for creating coded microcarriers, (a) Depositing a substantially opaque layer having a first surface and a second surface, wherein the first surface and the second surface are parallel to each other; (b) Patterning the deposited, substantially opaque layers into a two-dimensional shape representing an analog code; (c) Depositing a substantially transparent magnetic polymer layer on a first surface of a deposited substantially opaque layer, wherein the substantially transparent magnetic polymer layer comprises 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) A method comprising patterning a deposited substantially transparent magnetic polymer layer into a microcarrier shape.

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

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

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

29. (a) Before that, deposit a second polymer layer on the sacrificial layer; (a) Before this, apply a mask to the deposited second polymer layer to create a masked portion of the deposited second polymer layer and an unmasked portion of the deposited second polymer layer; (a) Before this, the deposited and masked second polymer layer is illuminated with light, the light having a wavelength sufficient to remove any unmasked portions of the deposited second polymer layer; It further includes, (a) comprises depositing a substantially opaque layer on the sacrificial layer and the masked portion of the deposited second polymer layer; The method according to claim 28, wherein (b) the deposited substantially opaque layer is patterned by removing the deposited second polymer layer, thereby removing the deposited substantially opaque layer on the masked portion of the deposited second polymer layer.

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 substantially transparent polymer monomer, a plurality of magnetic nanoparticles, a solvent, and a dispersant prior to (c) to form a mixture of a substantially transparent polymer monomer, 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 being mixed with a substantially transparent polymer monomer 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 a mixture of 10% of a plurality of magnetic nanoparticles, a solvent, and the dispersant.

36. The method according to claim 33, wherein multiple magnetic nanoparticles constitute 2.5% of a mixture of a substantially transparent polymer monomer, multiple 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 phosphate polymer.

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

40. The method according to any one of claims 25 to 30, wherein a 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 a 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 a substantially transparent magnetic polymer layer.

43. Connecting the trapping agent, The process involves reacting a substantially transparent polymer in a substantially transparent magnetic polymer layer with a photoacid generator and light to produce a crosslinked polymer, wherein the light is of a wavelength that activates the photoacid generator; Reacting a crosslinked polymer epoxide with a compound containing an amine and a carboxyl, wherein the amine of the compound reacts with the epoxide to form a crosslinked polymer in which the compound is linked; and The method according to claim 42, comprising reacting the carboxyl of a crosslinked polymer in which compounds are linked with a scavenger to link the scavenger to a substantially transparent magnetic polymer layer.

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 approximately 30 nm and more than approximately 3 nm.

46. The method according to any one of claims 25 to 30, wherein the plurality of magnetic nanoparticles comprises a mixture of substantially transparent polymer and the plurality of magnetic nanoparticles in an amount 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 in the substantially transparent magnetic polymer layer is an epoxy polymer.

49. The method according to claim 48, wherein the epoxy 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 includes a black matrix resist.

52. The method according to claim 50, wherein a substantially opaque polymer exhibits an absorbance greater than approximately 1.8 (OD) at wavelengths between approximately 230 nm and approximately 660 nm.

53. The method according to any one of claims 25 to 30, wherein the substantially opaque layer comprises a metal that lacks 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 of about 0.05 μm to about 2 μm.

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

57. The method according to any one of claims 25 to 30, for patterning a deposited substantially opaque layer to provide asymmetry.

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

59. The method according to claim 58, wherein the microcarriers have a diameter between approximately 5 μm and approximately 200 μm.

60. The method according to claim 59, wherein the microcarriers have a diameter of approximately 40 μm.

61. The method according to any one of claims 25 to 30, wherein the microcarriers have a thickness of less than approximately 50 μm.

62. The method according to claim 61, wherein the microcarriers have a thickness between approximately 2 μm and approximately 10 μm.

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 scavenger 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. A coded microcarrier manufactured by the method described in any one of claims 25 to 30.

66. A method for detecting multiple analytes in a solution, (a) Contacting a solution containing a first analyte and a second analyte with a plurality of microcarriers, wherein the plurality of microcarriers are at least: (i) a first microcarrier according to claim 1 or 2, which specifically captures a first analyte, and is encoded with a first analog code; and (ii) A second microcarrier according to claim 1 or 2, which specifically captures a second analyte, wherein the second microcarrier is coded 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 with multiple microcarriers; (b) Using analog shape recognition to decode the first analog code and the second analog code to identify the first microcarrier and the second microcarrier; and (c) A method comprising detecting the amount of a first analyte bound to a first microcarrier and the amount of a second analyte bound to a second microcarrier.

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 is (i) Irradiating the first and second microcarriers by passing light through the substantially transparent magnetic polymer layers of the first and second microcarriers and / or the surrounding solution, wherein the light cannot pass through the substantially opaque layers of the first and second microcarriers and generates a first analog-encoded light pattern corresponding to the first microcarrier and a second analog-encoded light pattern corresponding to the second microcarrier; (ii) Imaging a first analog-encoded light pattern to generate a first analog-encoded image, and imaging a second analog-encoded light pattern to generate a second analog-encoded image; and (iii) The method according to claim 66, comprising using analog shape recognition to compare a first analog encoded image with a first analog code and a second analog encoded image with a second analog code.

71. The amount of the first analyte bound to the first microcarrier and the amount of the second analyte bound to the second microcarrier are detected. (i) After (a), incubating the first and second microcarriers with a detection agent, wherein the detection agent incubates the first and second microcarriers, which bind to the first analyte captured by the first microcarrier and the second analyte captured by the second microcarrier; and (ii) The method according to claim 66, comprising measuring the amount of a 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 emission microscopy.

74. The method according to claim 66, wherein the solution comprises 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 from skin or adipose tissue, saliva, tears, bronchoalveolar lavage, oropharyngeal secretions, intestinal fluid, transvaginal or uterine secretions, and semen.

76. A kit for performing a multiplex assay involving multiple microcarriers, wherein the multiple microcarriers are at least: (a) a first microcarrier according to claim 1 or 2, which specifically captures a first analyte, and is encoded with a first analog code; and (b) A kit comprising a second microcarrier according to claim 1 or 2, which specifically captures a second analyte, wherein the second microcarrier is coded 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 a first analyte bound to a first microcarrier and the amount of a second analyte bound to a second microcarrier.

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

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