Carboxylated solid support, methods of modifying same, liquid phase chip kits and uses thereof

Abstract

This application relates to the field of in vitro diagnostic technology, and particularly to carboxylated solid-phase supports and their modification methods, liquid-phase chip kits, and applications. The carboxylation modification method for the solid-phase support includes: reacting a solid-phase support with epoxy groups on its surface with a solution of a polycarboxylated polymer, thereby grafting the polycarboxylated polymer onto the surface of the solid-phase support through the epoxy groups; the concentration of the polycarboxylated polymer in the solution is 0.01 wt% to 10 wt%, the reaction temperature is 40℃ to 100℃, and the reaction time is 1 h to 8 h. This modification method can obtain solid-phase supports with high carboxyl grafting density, and its application in liquid-phase chips can improve the coupling efficiency of BMB-based liquid-phase chips.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic technology, and in particular to carboxylated solid-phase carriers and their modification methods, liquid-phase chip kits and their applications. Background Technology

[0002] High-throughput multi-target detection technology is a key support for precision medicine and biomolecular diagnostics. While current mainstream liquid-phase chip technologies (such as the xMAP® system based on fluorescently encoded microspheres) achieve rapid responses in a liquid environment by coupling probes to the microsphere surface, they are still limited by inherent bottlenecks in optical encoding, including: limited encoding capacity: encoding methods relying on the ratio of fluorescent dye intensities, under the technical constraint of avoiding spectral overlap, can only identify an upper limit of approximately 500 microsphere categories (such as the Luminex® system), making it difficult to support the simultaneous analysis of over a thousand targets; signal crosstalk risk: high-density multicolor fluorescent encoding is susceptible to spectral cross-interference from detection channels, especially as encoding complexity increases, leading to a sharp drop in the signal-to-noise ratio and an increased microsphere classification error rate.

[0003] Barcoded Magnetic Beads (BMB) technology offers a new path to overcome the aforementioned limitations. This technology assigns each microsphere a unique magnetic characteristic code by controlling the sequential arrangement or gradient distribution of magnetic materials within the microsphere (such as differences in magnetic core size, position, and concentration). Compared to optical coding, BMB possesses fundamental advantages.

[0004] However, existing BMB-based liquid-phase chip technology still faces core challenges, exhibiting the following drawbacks: poor surface functionalization compatibility, the magnetic core embedding process easily leading to a reduction in active sites on the microsphere surface, and probe coupling efficiency significantly lower than that of fluorescent microspheres. These shortcomings result in insufficient detection sensitivity, low signal-to-noise ratio, and a tendency to produce false positive or false negative results. Summary of the Invention

[0005] Therefore, it is necessary to provide a carboxylated solid-phase support and its modification method, a liquid-phase chip kit, and its application.

[0006] In a first aspect, a method for carboxylation modification of a solid support is provided, the method comprising: reacting a solid support with epoxy groups on its surface with a solution of a polycarboxylated polymer, thereby grafting the polycarboxylated polymer onto the surface of the solid support through epoxy groups;

[0007] The concentration of the polycarboxylated polymer in the solution is 0.01 wt% to 10 wt%, the reaction temperature is 40℃ to 100℃, and the reaction time is 1 h to 8 h.

[0008] In an optional embodiment, the epoxy groups are provided by at least one epoxy resin.

[0009] In an optional embodiment, the epoxy resin comprises a multifunctional epoxy resin containing a glycidyl ether structure.

[0010] In an optional embodiment, the epoxy resin includes at least one of phenolic epoxy resin, bisphenol type epoxy resin, and alicyclic epoxy resin.

[0011] In an optional embodiment, the epoxy resin includes one or more of SU-8 photoresist, ECN-type o-cresol epoxy resin, E-51 / E-44 bisphenol A epoxy resin, 3,4-epoxycyclohexylmethyl-3,4-epoxycyclohexylcarboxylate, and glycidyl methacrylate (GMA).

[0012] In an optional embodiment, the polycarboxylated polymer includes at least one selected from polyacrylic acid, polymethacrylic acid, polymaleic anhydride, polyaspartic acid, polyglutamic acid, carboxymethyl cellulose, and carboxymethyl dextran.

[0013] In an optional embodiment, the polycarboxylated polymer is polyacrylic acid, and the viscosity-average molecular weight of the polyacrylic acid is 1,000 to 5,000,000.

[0014] In an optional embodiment, the viscosity-average molecular weight of the polyacrylic acid is 20,000 to 1,000,000.

[0015] In a further optional embodiment, the polyacrylic acid has a viscosity-average molecular weight of 450,000.

[0016] In optional embodiments, the solid support includes microparticles, nanoparticles, membranes, sheets, microporous plates, columns, polymer chains, and microfluidic chips.

[0017] In an optional implementation, the particles include magnetic particles.

[0018] In an optional embodiment, the solid carrier includes a magnetic barcode; more preferably, it includes a nickel barcode.

[0019] In an optional embodiment, the surface of the solid support contains SU-8 photoresist.

[0020] In an optional embodiment, the reaction system further contains a surfactant.

[0021] In an optional embodiment, the surfactant includes Tween-20.

[0022] In an optional embodiment, the working concentration of Tween-20 in the reaction system is 0.002wt%~0.05wt%.

[0023] In an optional embodiment, the reaction conditions of the carboxylation modification method satisfy at least one of (a) to (d):

[0024] (a) The concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.9 wt% to 1.1 wt%;

[0025] (b) The reaction temperature is 55~65℃;

[0026] (c) The reaction time is 3 h to 4 h;

[0027] (d) The polycarboxylated polymer is polyacrylic acid, the concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.1 wt%~1 wt%, the reaction temperature is 60℃~90℃, and the reaction time is 1 h~6 h.

[0028] Secondly, a carboxylated solid support prepared by the carboxylation modification method described in the first aspect is provided.

[0029] Thirdly, a functional molecule-modified solid support is provided, which is formed by linking the carboxylated solid support described in the second aspect with the functional molecule.

[0030] In optional embodiments, the functional molecules include nucleic acids, peptides, sugars, lipids, metals, and small molecule compounds.

[0031] In optional embodiments, the functional molecules include probes, signaling molecules, drug molecules, and antibodies.

[0032] Fourthly, a liquid-phase chip kit is provided, comprising combinations of carboxylated solid-phase supports with different encodings as described in the second aspect, and capture probes for different target analytes.

[0033] In an optional embodiment, the solid support includes microparticles, nanoparticles, and polymer chains.

[0034] In an optional embodiment, the solid-phase support obtains magnetic feature encoding by controlling the magnetic material.

[0035] In an optional embodiment, a solid-phase support modified with the functional molecules described in the third aspect, with different encodings, wherein the functional molecules are the capture probes.

[0036] Fifthly, the application of the carboxylated solid-phase support described in the second aspect, the functionally modified solid-phase support described in the third aspect, or the liquid-phase chip kit described in the fourth aspect in the preparation of in vitro diagnostic products is provided.

[0037] The carboxylation modification method for solid-phase supports provided in this application modifies solid-phase supports with epoxy groups on their surface using multi-carboxyl polymers, thereby achieving a high carboxyl density surface. This method is simple in its process and reaction conditions, can be directly applied to existing epoxy-containing solid-phase supports, and uses readily available and inexpensive raw materials. Furthermore, it can stably and repeatedly achieve a high carboxyl density surface on solid-phase supports, increasing the grafting density of biomolecules. Moreover, by utilizing multi-carboxyl polymers with varying numbers or lengths of carboxyl groups, the carboxyl modification density on the solid-phase support surface can be controlled, significantly increasing the carboxyl density and enhancing the immobilization ability of functional molecules. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0039] Figure 1 Image of carboxyl-modified BMB prepared in Example 1 after modification with streptavidin-Alexa Fluor 532 Dye under natural light;

[0040] Figure 2 Image of carboxyl-modified BMB prepared in Example 1 after modification with streptavidin-Alexa Fluor 532 Dye under 530nm excitation;

[0041] Figure 3 The image shows the carboxyl-modified BMB prepared for Comparative Example 2 after being modified with streptavidin-Alexa Fluor 532 Dye and excited at 530 nm.

[0042] Figure 4 The results show the fluorescence intensity of BMB modified in Examples 1-9 and Comparative Examples 1-2. Detailed Implementation

[0043] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0045] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0046] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0047] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0048] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0049] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0050] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0051] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0052] In this application, the term "solid support" refers to a solid support used in detection technology to bind and immobilize reactants. It fixes reactants to its surface through physical adsorption or chemical coupling, and is not easily detached after repeated washing and long-term storage, without affecting the reactivity of the immobilized substances.

[0053] In this application, a group of solid-phase carriers constituting a "barcode" refers to a collection of at least two solid-phase carriers with the same or similar structures but different codes. "Different codes" refer to the fact that each or several solid-phase carriers in the combination carry a distinguishable identification feature that can be recognized by a detection system. This feature allows different solid-phase carriers to be identified individually or assigned to their corresponding groups even in a mixed state, and thus linked to a specific target analyte. A "magnetic barcode" refers to the feature that utilizes the differences in the magnetic properties of the solid-phase carriers, such as differences in the size, position, and concentration of the magnetic core, to give the solid-phase carriers different codes.

[0054] In this application, the term "multi-carboxyl polymer" refers to a polymer containing multiple carboxyl (-COOH) functional groups in a single polymer molecule. These polymers are typically produced by polymerization of carboxyl-containing monomers (such as acrylic acid, methacrylic acid, etc.), and their molecular chains have two or more carboxyl groups distributed on them.

[0055] In this application, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acids include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. Nucleic acids can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably.

[0056] In this application, peptides, polypeptides, and proteins are not strictly distinguished and can be used interchangeably in some cases. Generally, peptides refer to polyamino acids linked by peptide bonds, and are not limited to being naturally occurring or synthetic. Polypeptides may also contain non-amino acid components as modifying groups, such as carbohydrate groups, metal ions, or carboxylic acid esters. Non-amino acid components can be added by the cells expressing the polypeptide and can vary depending on the cell type. Polypeptides are defined herein based on their amino acid backbone structure or the nucleic acid encoding them.

[0057] In this application, the term "signaling substance" refers to a substance capable of providing a detectable signal that can be directly observed with the naked eye or detected by conventional instruments acceptable in the art. A signaling substance can directly provide a signal, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can indirectly provide a signal through a subsequent reaction involving the signaling substance, such as a signal generated by an electrochemiluminescence reaction or a signal generated by an enzyme-catalyzed chemiluminescence reaction. Examples of signaling substances include, but are not limited to, one or more of chemiluminescent reagents, fluorescent labels, quantum dots, radionuclides, and paramagnetic ions.

[0058] In this application, the term "small molecule compound" refers to a chemical substance with a small molecular weight and relatively simple structure, with a molecular weight of less than 900 Da.

[0059] In a first aspect, some embodiments provide a method for carboxylating a solid support, the method comprising: reacting a solid support with epoxy groups on its surface with a solution of a polycarboxylated polymer, thereby grafting the polycarboxylated polymer onto the surface of the solid support via epoxy groups.

[0060] The concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.01 wt% to 10 wt%, for example, but not limited to 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, or 10 wt%; the reaction temperature is 40℃ to 100℃, for example, but not limited to 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, 85℃, 90℃, 95℃, or 100℃; and the reaction time is 1 h to 8 h, for example, but not limited to 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, or 8 h.

[0061] The first aspect provides a carboxylation modification method for solid-phase supports. This method modifies solid-phase supports with epoxy groups on their surface by reacting the carboxyl groups on a polycarboxylated polymer with the epoxy groups on the solid-phase support surface. The polycarboxylated polymer is grafted onto the solid-phase support surface via these epoxy groups. Because the side chains of the polycarboxylated polymer have a large number of carboxylic acid groups (-COOH), each epoxy group, after grafting the polycarboxylated polymer, extends and expands to create more reaction sites on the solid-phase support surface. This allows for high-density modification of the bioactive interface using a polymer brush. For example, a solid-phase support with a surface of SU-8 photoresist modified with polyacrylic acid (PAA) is described.

[0062] SU-8 is a negative epoxy resin photoresist with the structural formula shown in formula (I). Its core component is a bisphenol A type epoxy oligomer (epoxy resin monomer / prepolymer), which typically contains two or more highly reactive epoxy groups (-CH3-O-CH2). SU-8 itself can undergo cationic photopolymerization (epoxy ring-opening polymerization) under ultraviolet light in the presence of a photoinitiator to form a crosslinked network. After curing, some unreacted epoxy groups remain inside or on the surface of the material.

[0063] Equation (Ⅰ).

[0064] Polyacrylic acid (PAA) is a linear polymer synthesized from acrylic acid monomers. Its side chains contain numerous carboxylic acid groups (-COOH). The carboxyl groups on the PAA chain are nucleophilic and can undergo ring-opening reactions with epoxy groups. Under heating conditions, the carboxyl groups of PAA attack the methylene carbon atoms of unreacted epoxy groups on the SU-8 surface, leading to ring-opening of the epoxy groups and the formation of ester bonds. Each epoxy group site can react with PAA, extending the reaction to more sites. The reaction principle is shown in formula (II).

[0065] Formula (II).

[0066] In an optional embodiment, the epoxy groups are provided by at least one epoxy resin.

[0067] In optional embodiments, the epoxy resin includes, but is not limited to, multifunctional epoxy resins containing glycidyl ether structures, and may further optionally include, at least one of phenolic epoxy resins, bisphenol type epoxy resins, and alicyclic epoxy resins.

[0068] In optional embodiments, the epoxy resin includes, but is not limited to, one or more of SU-8 photoresist, ECN-type o-cresol epoxy resin, E-51 / E-44 bisphenol A epoxy resin, 3,4-epoxycyclohexylmethyl-3,4-epoxycyclohexylcarboxylate and glycidyl methacrylate (GMA).

[0069] In an optional embodiment, the epoxy resin includes SU-8 photoresist.

[0070] In an optional embodiment, the polycarboxylated polymer includes at least one of polyacrylic acid, polymethacrylic acid, polymaleic anhydride, polyaspartic acid, polyglutamic acid, carboxymethyl cellulose, and carboxymethyl dextran.

[0071] In optional embodiments, the polycarboxylated polymer is polyacrylic acid, and the viscosity-average molecular weight of polyacrylic acid is 1,000 to 5,000,000, for example, but not limited to 1,000, 2,000, 5,000, 8,000, 10,000, 20,000, 50,000, 80,000, 100,000, 200,000, 500,000, 800,000, 100,000, 200,000, or 5,000,000; further optionally, the viscosity-average molecular weight of polyacrylic acid is 20,000 to 1,000,000; further optionally, the viscosity-average molecular weight of polyacrylic acid is 450,000.

[0072] The epoxy groups on the surface of the solid support can be provided by any part of the solid support, such as a solid support prepared from a polymer containing epoxy groups, or a coating of a substance containing epoxy groups on the surface of the solid support; the solid support may optionally have epoxy groups on part or all of its surface.

[0073] In optional embodiments, the solid support includes microparticles, nanoparticles, membranes, sheets, microporous plates, columns, and microfluidic chips.

[0074] In an optional implementation, the particles include magnetic particles.

[0075] In an optional embodiment, the solid carrier includes a magnetic barcode; more preferably, it includes a nickel barcode.

[0076] In an optional embodiment, the surface of the solid support contains SU-8 photoresist.

[0077] In an optional embodiment, the reaction system in which the solid support with epoxy groups on its surface reacts with the polycarboxylated polymer solution also contains a surfactant. The surfactant's role is to improve the dispersibility of the solid support during the modification process and reduce the adsorption and aggregation of the solid support on the inner wall of the reaction vessel.

[0078] In optional embodiments, the surfactant includes, but is not limited to, Tween-20.

[0079] In an optional embodiment, the working concentration of Tween-20 in the reaction system is 0.002 wt% to 0.05 wt%, for example, but not limited to 0.002 wt%, 0.005 wt%, 0.008 wt%, 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt% or 0.05 wt%, preferably 0.01 wt%.

[0080] In an optional embodiment, the reaction conditions for the reaction between the solid support with epoxy groups on its surface and the polycarboxylated polymer solution satisfy at least one of (a) to (d):

[0081] (a) The concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.9 wt% to 1.1 wt%, preferably 1 wt%;

[0082] (b) The reaction temperature is 55℃~65℃, preferably 60℃;

[0083] (c) The reaction time is 3 h to 4 h, preferably 3.5 h.

[0084] (d) When the polycarboxylated polymer is polyacrylic acid, the reaction conditions for the reaction between the solid support with epoxy groups on the surface and the polycarboxylated polymer solution include: the concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.1 wt%~1 wt%, the reaction temperature is 60℃~90℃, and the reaction time is 1 h~6 h.

[0085] Secondly, some embodiments provide a carboxylated solid support prepared by the carboxylation modification method of the first aspect, wherein a polymer brush is formed on the surface of the carboxylated solid support and the carboxyl grafting density is high.

[0086] Thirdly, some embodiments provide a functional molecule-modified solid support, which is formed by linking a carboxylated solid support from the second aspect to a functional molecule. The functional molecule can be directly linked through carboxyl groups on the surface of the solid support, or it can be linked through linkers. The functional molecule can also be linked to other reactive groups on the surface of the solid support besides carboxyl groups.

[0087] In optional embodiments, the functional molecules include nucleic acids, peptides, sugars, lipids, metals, and small molecule compounds.

[0088] In optional implementations, the functional molecules include probes, signaling molecules, drug molecules, and antibodies.

[0089] Fourthly, a liquid phase-array kit is provided, comprising combinations of carboxylated solid-phase supports with different encodings from the first aspect, and capture probes for different target analytes. The combinations of carboxylated solid-phase supports and capture probes in this liquid phase-array kit can be packaged separately, or they can directly contain carboxylated solid-phase supports modified with capture probes, i.e., solid-phase supports modified with functional molecules as described in the second aspect, wherein the functional molecules are capture probes.

[0090] In an optional embodiment, the solid support in the liquid phase chip kit includes microparticles, nanoparticles, and polymer chains. Microparticles may optionally include, but are not limited to, microspheres, microsheets, and microrods.

[0091] In an alternative embodiment, the solid-phase support obtains magnetic feature encoding by controlling the magnetic material.

[0092] In optional embodiments, the capture probes include, but are not limited to, nucleic acid probes, such as DNA probes and RNA probes; peptide probes, such as antibody probes, antigen probes and other peptides with affinity; and polysaccharide probes, lipid probes and small molecule compound probes.

[0093] In an optional embodiment, the liquid phase chip kit further includes one or more of the following: buffer reagents, primers, hybridization reagents, washing reagents, enzyme preparations, chromogenic substances, positive controls, and negative controls.

[0094] Fifthly, some embodiments also provide the application of the carboxylated solid-phase carrier of the second aspect, the functionally modified solid-phase carrier of the third aspect, or the liquid-phase chip kit of the fourth aspect in the preparation of in vitro diagnostic products. This in vitro diagnostic product utilizes a carboxylated solid-phase carrier with a high surface carboxyl grafting density as a probe carrier, which can be used for the detection of various indicators, such as proteins and nucleic acids. Applying the above-mentioned carboxylated solid-phase carrier, functionally modified solid-phase carrier, and liquid-phase chip kit to in vitro diagnostics helps to develop novel liquid-phase chip systems that integrate high-precision magnetic encoding, magneto-optical synergistic signal reading, and universal surface modification. While breaking through the throughput limit of multi-target detection, it also promotes the evolution of equipment towards portability and low cost, providing a next-generation technology platform for applications such as pathogen multi-detection and tumor molecular subtyping.

[0095] The following are some examples.

[0096] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0097] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0098] The BMB used in the following examples and comparative examples is model C004-B01B05 (purchased from Wuhan Dexin Biotechnology Co., Ltd.). This BMB is prepared by SU8 photoresist and contains epoxy groups on its surface.

[0099] Examples 1-9

[0100] Examples 1-9 each provide a method for preparing a carboxylated magnetic barcode, comprising the following steps:

[0101] (1) Weigh polyacrylic acid (Aladdin, P104272, Mv: 450,000) powder, and prepare a reaction solution with a certain mass / volume concentration (w / v) using an aqueous solution containing 0.01wt% Tween-20 according to the required concentration in Table 1.

[0102] (2) Take BMB (500K, i.e. 500,000 PCS) and put it into a 2mL centrifuge tube. Remove the storage solution after magnetic suction. Wash twice with ultrapure water and add 2mL of the prepared polyacrylic acid solution. Place it in a constant temperature shaking metal bath and adjust the speed to 800rpm. React according to the temperature and time in Table 1.

[0103] (3) After the reaction is complete, remove the reaction solution by magnetic attraction, add 2 mL of 1×PBS-T buffer and sonicate to wash 3 times; finally, store in 2 mL of 1×PBS-T buffer at 4℃.

[0104] Table 1 Experimental parameters

[0105]

[0106] Examples 10-13

[0107] Using polypropylene polymers with molecular weights in Table 2, BMB was carboxylated under the conditions of 0.5% polyacrylic acid concentration, 60°C reaction temperature, and 3.5h reaction time.

[0108] (1) Weigh polyacrylic acid powders of different molecular weights and prepare a polymer concentration of 0.5%wt, containing 0.01%wt Tween 20.

[0109] (2) Take BMB (500K, i.e. 500,000 PCS) and put it into a 2mL centrifuge tube. Remove the storage solution after magnetic suction. Wash twice with ultrapure water and add 2mL of the prepared polyacrylic acid solution. Place it in a constant temperature shaking metal bath and adjust the speed to 800rpm. React at 60℃ for 3.5h.

[0110] (3) After the reaction is complete, remove the reaction solution by magnetic attraction, add 2 mL of 1×PBS-T buffer and sonicate to wash 3 times; finally, store in 2 mL of 1×PBS-T buffer at 4℃.

[0111] Table 2

[0112]

[0113] Comparative Example 1

[0114] Comparative Example 1 served as a blank control. Following the preparation steps of Example 1 and the parameters in Table 1, BMB chips were placed in a reaction at 90°C for 6 hours as a blank control group. BMB was reacted with a reaction solution that did not contain polyacrylic acid.

[0115] Comparative Example 2

[0116] Comparative Example 2 involved carboxylation modification of the BMB surface with a SU-8 surface using glycine. Under alkaline conditions, the epoxy groups on the SU-8 surface formed a ring-opening link with the amino terminus of glycine, generating a carboxyl group at each epoxy site. Unlike Examples 1-9, Example 1 allowed for the linking of a polyacrylic acid polymer chain at each epoxy site, resulting in hundreds or thousands of carboxyl groups.

[0117] The modification method is as follows:

[0118] Weigh 15 mg of glycine and add it to 2 mL of pH 10 sodium carbonate buffer to prepare a 0.1 M glycine solution. Take BMB particles (50 K~1000 K) and add them to a 2 mL centrifuge tube. After removing the storage solution with a magnetic aspirator, wash the tube three times with pH 10 sodium carbonate buffer. Add 2 mL of 0.1 M glycine solution. Place the tube in a constant temperature shaker at 45 °C and adjust the speed to 800 rpm for 24 h. After the reaction, wash the tube three times with 2 mL of PBS-T using sonication. Store the tube in 2 mL of PBS-T buffer at 4 °C.

[0119] Example 1

[0120] Grafting density was evaluated by coupling with biomolecules: carboxylated BMB was activated and coupled with NH2-PEG3-Biotin for biotinylation modification, and then combined with SA-Alexa Fluor 532 Dye. The degree of carboxylation modification was characterized by fluorescence intensity.

[0121] 1. Experimental Procedure:

[0122] (1) Solution preparation:

[0123] 25mM MES (2-(N-Morpholino)ethanesulfonic acid 2-morpholinoethanesulfonic acid) buffer: Dissolve 0.53g MES in 90mL of water and adjust the pH to 6.0;

[0124] 20 mg / mL EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride) solution: Weigh 20 mg of EDC and dissolve it in 1 mL of 25 mM MES;

[0125] 20 mg / mL NHS (N-Hydroxysuccinimide) solution: Weigh 20 mg of Sulfo-NHS and dissolve it in 1 mL of 25 mM MES;

[0126] 100mM NH2-PEG3-Biotin (amino-polyethylene glycol-biotin) solution: Weigh 8mg NH2-PEG3-Biotin and dissolve it in 0.2mL 1:× PBS;

[0127] 1 mg / mL SA-Alexa Fluor 532 Dye (Streptavidin-modified Alexa Fluor™ 532 fluorescent dye) solution: Dissolve 1 mg of SA-Alexa Fluor 532 in 1 mL of PBS;

[0128] Take 20 μL of the modified BMB from the examples and comparative examples respectively and add it to a 2 mL centrifuge tube. Wash twice with 200 μL of 25 mM MES buffer using magnetic suction. After washing, add 25 μL of 20 mg / mL EDC solution and 25 μL of 20 mg / mL NHS solution to 100 μL of MES buffer. Place the tube in a constant temperature shaker, adjust the temperature to 25°C, and the rotation speed to 800 rpm for 30 min. After the reaction, the reaction solution was removed by magnetic suction, and the sample was washed three times with PBS using magnetic suction. Then, 190 μL of PBS and 10 μL of 100 mM NH2-PEG3-Biotin solution were added. The mixture was shaken at 25 °C for 2 h. After the reaction, the sample was washed three times with PBS-T solution using magnetic suction. 95 μL of PBS buffer was added to a centrifuge tube, followed by 5 μL of 1 mg / mL SA-Alexa Fluor 532 Dye solution. The mixture was reacted at 25 °C for 15 min. After the reaction, the sample was washed three times with PBS using magnetic suction. An appropriate amount of sample was placed under an Olympus fluorescence microscope for observation. The parameters were set as follows: excitation light 530 nm, power 10 W, exposure 50 ms, and gain 500%. The captured images were processed using ImageJ, and the grayscale value in the red channel was used to evaluate the degree of binding to SA-Alexa Fluor 532 Dye and characterize the degree of surface carboxylation.

[0129] Using Example 1 and Comparative Example 2 as examples, images of carboxylated BMB modified with streptavidin-Alexa Fluor 532Dye under natural light and under 530nm excitation are shown: The image of the carboxylated BMB prepared in Example 1 under natural light is shown below. Figure 1 As shown, the SA-Alexa Fluor532 Dye fluorescence spectra of the carboxyl-modified BMB in Example 1 and Comparative Example 2 under 530 nm excitation light are as follows: Figure 2 and Figure 3 As shown, from Figure 2 and Figure 3 It can be seen that the fluorescence intensity of polypropylene-modified BMB is significantly higher than that of glycine-modified BMB, indicating that the BMB modified in Example 1 can provide more carboxyl sites and has a higher degree of carboxylation modification, thus resulting in a high grafting density.

[0130] The characterization results of the modified BMB combined with SA-Alexa Fluor 532 Dye in Examples 1-9 and Comparative Examples 1-2 are shown in Table 3, and the statistical results are shown in the figure below. Figure 4 As shown.

[0131] Table 3 Characterization results of SA-Alexa Fluor 532 Dye

[0132]

[0133] As shown in Table 3, the fluorescence intensity of Examples 1-9 was higher than that of Comparative Examples 1-2, generally exhibiting a higher grafting density. Concentration was the key factor affecting fluorescence value (marginally significant), and increasing it (0.1%~1%) significantly improved the fluorescence value. Temperature and time had no significant effect, but the trends were better at 60℃ and 3.5h. These experimental results show that, compared with the unmodified solid support, the carboxyl group density was significantly increased, improving the immobilization ability of coupled biomolecules.

[0134] The fluorescence characterization results of Examples 10-13 are shown in Table 4.

[0135] Table 4

[0136]

[0137] Table 4 shows that polyacrylic acid with different molecular weights can be successfully modified by carboxylation. Among them, the molecular weight of around 450,000 has a higher fluorescence value, while the molecular weight of more than 1,250,000 is prone to aggregation.

[0138] Example 2

[0139] To detect anti-jo-1 antibodies, carboxylated modified BMB prepared according to the method in Example 2 was used to coat jo-1 antigen, and clinical samples of different concentrations were tested. Then, secondary antibodies labeled with PE were used for fluorescence signal detection. The fluorescence signal test results are as follows.

[0140] Table 5. Detection results of anti-jo-1 antibodies

[0141]

[0142] Conclusion: Negative samples (blood from physical examination) and positive samples can be clearly distinguished, and strong positive, moderate positive and weak positive samples can be successfully distinguished.

[0143] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for carboxylation modification of a solid-phase support, characterized in that, include: A solid support with epoxy groups on its surface is reacted with a solution of a polycarboxylic acid polymer, so that the polycarboxylic acid polymer is grafted onto the surface of the solid support through epoxy groups. The concentration of the polycarboxylated polymer in the solution is 0.01 wt% to 10 wt%, the reaction temperature is 40℃ to 100℃, and the reaction time is 1 h to 8 h.

2. The method for carboxylation modification of a solid support according to claim 1, characterized in that, The epoxy groups are provided by at least one epoxy resin; Optionally, the epoxy resin includes a multifunctional epoxy resin containing a glycidyl ether structure; Optionally, the epoxy resin includes at least one of phenolic epoxy resin, bisphenol type epoxy resin, and alicyclic epoxy resin; Optionally, the epoxy resin includes one or more of SU-8 photoresist, ECN-type o-cresol epoxy resin, E-51 / E-44 bisphenol A epoxy resin, 3,4-epoxycyclohexylmethyl-3,4-epoxycyclohexylcarboxylate, and glycidyl methacrylate (GMA).

3. The method for carboxylation modification of a solid support according to claim 1, characterized in that, The polycarboxylated polymer includes at least one of polyacrylic acid, polymethacrylic acid, polymaleic anhydride, polyaspartic acid, polyglutamic acid, carboxymethyl cellulose, and carboxymethyl dextran; Optionally, the polycarboxylated polymer is polyacrylic acid, and the viscosity-average molecular weight of the polyacrylic acid is 1,000 to 5,000,000. Optionally, the viscosity-average molecular weight of the polyacrylic acid is 20,000 to 1,000,000; Further optionally, the polyacrylic acid has a viscosity-average molecular weight of 450,000.

4. The method for carboxylation modification of a solid support according to claim 1, characterized in that, The solid support includes microparticles, nanoparticles, membranes, sheets, microporous plates, columns, polymer chains, and microfluidic chips; Optionally, the particles include magnetic particles; Optionally, the solid carrier includes a magnetic barcode; more optionally, it includes a nickel barcode. Optionally, the surface of the solid support contains SU-8 photoresist.

5. The carboxylation modification method according to any one of claims 1 to 4, characterized in that, The reaction system also contains surfactants; Optionally, the surfactant includes Tween-20; Optionally, the working concentration of Tween-20 in the reaction system is 0.002wt%~0.05wt%.

6. The carboxylation modification method according to any one of claims 1 to 5, characterized in that, The reaction conditions satisfy at least one of (a) to (d): (a) The concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.9 wt% to 1.1 wt%; (b) The reaction temperature is 55~65℃; (c) The reaction time is 3 h to 4 h; (d) The polycarboxylated polymer is polyacrylic acid, the concentration of the polycarboxylated polymer in the polycarboxylated polymer solution is 0.1 wt%~1 wt%, the reaction temperature is 60℃~90℃, and the reaction time is 1 h~6 h.

7. A carboxylated solid support prepared by the carboxylation modification method according to any one of claims 1 to 6.

8. A solid-phase support modified with functional molecules, characterized in that, It is formed by linking the carboxylated solid support as described in claim 7 with functional molecules; Optionally, the functional molecules include nucleic acids, peptides, sugars, lipids, metals, and small molecule compounds; Optionally, the functional molecules include probes, signaling molecules, drug molecules, and antibodies.

9. A liquid phase chip reagent kit, characterized in that, Combinations of carboxylated solid supports as described in claim 7 with different codes, and capture probes for different target analytes; Optionally, the solid support includes microparticles, nanoparticles, and polymer chains; Optionally, the solid-phase support obtains magnetic feature encoding by controlling the magnetic material; Optionally, a solid-phase support modified with the functional molecule of claim 8, with different encodings, wherein the functional molecule is the capture probe.

10. The application of the carboxylated solid-phase support of claim 7, the functionally modified solid-phase support of claim 8, or the liquid-phase chip kit of claim 9 in the preparation of in vitro diagnostic products.

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