Novel method for constructing encoding microspheres by three-dimensionally assembling fluorescent dye labeled DNA molecules

Abstract

The invention discloses a novel method for constructing encoding microspheres by three-dimensionally assembling fluorescent dye labeled DNA molecules, which is characterized in that the fluorescent dye labeled DNA molecules are used for replacing organic fluorescent dyes and nano materials, and the fluorescent dye labeled DNA molecules and complementary DNA chains pre-anchored on the surfaces of the microspheres are assembled on the surfaces of the microspheres through DNA hybridization reaction. The method comprises the following steps: preparing a single-color or multi-color microsphere array with super-large coding capacity by taking the type and loading concentration of DNA molecules marked by a fluorescent dye as coding elements; streptavidin is modified to the surface of the coding microsphere through a biotin-streptavidin bridge, and the obtained functionalized coding microsphere can be efficiently combined with a biotinylated capture probe, so that simultaneous qualitative or quantitative detection of a plurality of target analytes is realized. The coding microsphere is large in coding capacity, low in decoding cost, environmentally friendly and high in universality, and has wide application prospects in the fields of clinical diagnosis, environment monitoring and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent microsphere encoding, and particularly relates to a preparation method of encoding microspheres constructed by three-dimensional assembly of dye molecules. Background Art

[0002] As our understanding of chemistry, biology, and medicine continues to deepen, the need for high-throughput and multiplex analysis of various molecules in a single sample is increasing. The emergence of high-throughput multiplex detection technologies has attracted wide attention in the fields of clinical diagnosis, gene expression analysis, and drug screening. The progress of biomedical science has enhanced the importance of these technologies, especially in early diagnosis and real-time tracking of disease progression. Liquid biochip technology represents a cutting-edge high-throughput detection platform for multi-component biological detection.

[0003] Fluorescent encoded microspheres (FEBs) have become an indispensable element in multiplex biomolecular analysis, driving the progress of our analytical capabilities. FEBs utilize fluorescent encoded microspheres as carriers for immune reactions and flow cytometry as an analytical technique, enabling the simultaneous quantitative analysis of hundreds or even more different biomolecules. This technology effectively addresses the limitations of solid-state array chips, such as poor repeatability, slow reaction kinetics, and low sensitivity, making it one of the most promising platforms for clinical multi-index detection. However, the encoding capacity achieved by existing methods has not reached the theoretical potential, especially when compared with planar biochip technology. To advance the development of this field, there is an urgent need to develop innovative fluorescent materials and novel preparation methods for fluorescent encoded microspheres, which are crucial for overcoming the limitations of low encoding capacity and potential bottlenecks in the preparation and application of current fluorescent materials.

[0004] Compared with alternative encoding technologies such as physical encoding, optical encoding, and chemical encoding, fluorescence-based optical encoding stands out due to its low production cost and simple analysis. The popularity of fluorescently encoded microspheres is attributed to their simple encoding process, cost-effectiveness, compatibility with various detection systems, and fast signal acquisition speed. Currently, the main method involves integrating dye molecules into polymer microspheres through the swelling method, and this technology is mainly owned by Luminex Corporation. Research shows that using fluorescent materials such as quantum dots (QDs), upconverting nanomaterials (UCNPs), aggregation-induced emission materials (AIEs), and surface-enhanced Raman scattering (SERS) spectroscopy, compared with organic dye microspheres, the encoding ability of dye molecules such as QDs and UCNPs has not been significantly improved. The main reasons are reabsorption phenomenon, aggregation effect, and Förster resonance energy transfer (FRET) effect. Aggregation and fluorescence quenching occur at high dye concentrations, which makes the encoding capacity unable to be further improved by increasing the concentration. Given the progress of DNA nanotechnology in recent years, dyes labeled with DNA molecules can form nanoparticles through self-assembly reactions (such as DNA barcode fluorescent probes and DNA origami). Therefore, we can effectively overcome the negative impacts brought by dye aggregation and fluorescence quenching using DNA nanotechnology, that is, by hybridizing dye-conjugated nucleic acid (DNA-dye) molecules, the dye molecules can be stereochemically assembled on magnetic microspheres, effectively improving their encoding ability. Summary of the Invention

[0005] The object of the present invention is to overcome the barriers existing in the prior art and provide a method for constructing encoded monochromatic or polychromatic microspheres by stereochemically assembling fluorescent dye-labeled DNA molecules and its preparation method.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] A method for constructing encoded microspheres by stereochemically assembling fluorescent dye-labeled DNA molecules, comprising the following steps:

[0008] (1) Hybridize the fluorescent dye-labeled DNA molecule with a biotin-labeled complementary DNA strand, the complementary DNA strand is modified with a functional group, and after the hybridization reaction, a fluorescent dye-labeled DNA molecule hybrid chain solution is formed;

[0009] (2) Activate the chemical group on the surface of the microsphere with an activator, and the chemical group is covalently coupled to the functional group modified on the DNA strand;

[0010] (3) Add the fluorescent dye-labeled DNA molecule hybrid chain solution to the activated microsphere, and the functional group on the complementary DNA strand is covalently coupled to the chemical group modified on the surface of the microsphere, thereby assembling the fluorescent dye-labeled DNA molecule onto the surface of the microsphere, and preparing monochromatic or polychromatic encoded microspheres with the types and loading concentrations of the fluorescent dye-labeled DNA molecules as encoding elements.

[0011] A fluorescent dye-labeled DNA molecule-encoded microsphere, comprising a microsphere, a fluorescent dye-labeled DNA molecule, and a DNA strand that hybridizes and complements the fluorescent dye-labeled DNA molecule. The surface of the microsphere is modified with a chemical group, and the DNA strand is modified with a functional group. The functional group on the DNA strand is covalently coupled to the chemical group modified on the surface of the microsphere. The fluorescent dye-labeled DNA molecule and the complementary DNA strand are assembled onto the surface of the microsphere through a hybridization reaction. Monochromatic or polychromatic encoded microspheres are prepared using the types and loading concentrations of the fluorescent dye-labeled DNA molecules as encoding elements.

[0012] Furthermore, streptavidin is modified onto the surface of the microsphere through a biotin-streptavidin bridge to obtain a functionalized encoded microsphere. The functionalized encoded microsphere can efficiently bind biotinylated capture probes (such as nucleic acids, antibodies, antigen proteins, receptor proteins, etc.), thereby enabling simultaneous qualitative and quantitative detection of multiple target analytes.

[0013] Furthermore, the fluorescent dye-labeled DNA molecule is one or a combination of DNA molecules labeled with FAM, Alexa Fluor, Cy3, Cy5, Cy5.5, Cy7, ROX, or Texas Red, and the loading concentration is 0 - 1000 nM. Monochromatic encoded microspheres are obtained by coupling the same type of fluorescent dye-labeled DNA molecules with different concentrations to the microspheres, resulting in a set of encoded microspheres with different monochromatic fluorescence intensities; polychromatic encoded microspheres are obtained by co-coupling two or more fluorescent dye-labeled DNA molecules to the same microsphere, resulting in a set of encoded microspheres with different polychromatic fluorescence intensities through different concentration combinations.

[0014] Furthermore, the microsphere is selected from one of polystyrene microspheres, silica microspheres, agarose gel microspheres, dextran gel microspheres, magnetic microspheres of magnetite or hematite, and the particle size of the microsphere is 1 - 30 μm.

[0015] Furthermore, the surface of the microsphere is modified with one of carboxyl, amino, maleimide, haloacetamide, gold atoms, alkynyl, or azide groups, and the functional group modified on the DNA strand that hybridizes and complements the fluorescent dye-labeled DNA molecule is one of amino, carboxyl, mercapto, thiol, aldehyde, epoxy, azide, alkynyl, hydroxyl, or cyano groups. The functional group on the DNA strand is covalently coupled to the chemical group modified on the surface of the microsphere.

[0016] The application of the encoded microsphere in the detection of one or more target substances includes the following steps:

[0017] (1) Specifically bind a biotin-labeled capture probe to the encoded microsphere modified with streptavidin;

[0018] (2) Capture the target analyte in the sample to be tested on the surface of the microspheres through the capture probes conjugated on the surface of the microspheres;

[0019] (3) The target analyte captured on the surface of the microspheres specifically binds to the fluorescently labeled reporter probe;

[0020] (4) Analyze the encoded microspheres with different fluorescence characteristics through a flow cytometer or a CCD imaging system, and detect the fluorescence signals of the reporter probes bound to the surface of each encoded microsphere.

[0021] Advantages of the present invention:

[0022] (1) Compared with other methods (swelling method, polymerization method or microengineering emulsification method), the present invention can directly use the chemical groups modified on the surface of the microspheres to covalently conjugate with the functional groups on the DNA strand for encoding, greatly simplifying the microsphere encoding process, ensuring the uniformity of the microsphere particle size, with a simple encoding process, good repeatability, low decoding cost, good detection compatibility, fast signal acquisition speed, and enabling batch preparation.

[0023] (2) The DNA molecule labeled with a fluorescent dye replaces the organic fluorescent dyes and inorganic nanomaterials used in the traditional method for preparing fluorescent microspheres. It has low cost, is easy to synthesize, has stable properties and no pollution, can effectively avoid the problems of aggregation-induced quenching of fluorescence and fluorescence leakage of the fluorescent dye, and is environmentally friendly, obtaining a microsphere matrix encoded with a large-capacity DNA molecule labeled with a fluorescent dye.

[0024] (3) Modify streptavidin on the surface of the encoded microspheres through a self-assembly method to form a fluorescent dye-labeled DNA molecule-streptavidin composite functional microsphere, avoiding the complex steps of excessive chemical group modification on the surface of the microspheres, and greatly reducing the microsphere preparation cost. This functional microsphere can efficiently bind to the biotinylated capture probe and achieve qualitative and quantitative detection of multiple target analytes simultaneously. Description of the drawings

[0025] Figure 1 : Signal detection diagram of the forward scatter angle (FSC) and side scatter angle (SSC) of the 10-μm (red) magnetic microspheres in Example 1 through a flow cytometer.

[0026] Figure 2 : Fluorescence microscope diagram of the monochromatic or bichromatic fluorescent dye-labeled DNA molecule-encoded microspheres (10-μm diameter) in Example 1. Figure a is the confocal microscope diagram of the magnetic microspheres in bright field; Figure b is the confocal microscope diagram of the DNA-Cy3 monochromatic encoded microspheres; Figure c is the confocal microscope diagram of the DNA-Cy5 monochromatic encoded microspheres; Figure d is the confocal microscope diagram of the DNA-Cy3 and DNA-Cy5 bichromatic encoded microspheres.

[0027] Figure 3 : Scatter plot of the monochromatic encoded microspheres decoded by flow cytometry in Example 1. The diameter of the microspheres is 10 μm. Figure a1 shows 8 suspension arrays of DNA-Cy3 monochromatic encoded microspheres; Figure b1 shows 13 suspension arrays of DNA-Cy5 monochromatic encoded microspheres.

[0028] Figure 4 : Analysis chart of the influence of the concentration of light dye-labeled DNA molecules on the encoding capacity in Example 1. Figure a1 shows the MFI of DNA-Cy3 monochromatic encoded microspheres; Figure a2 shows the linear relationship between the DNA-Cy3 concentration and MFI; Figure b1 shows the MFI chart of DNA-Cy5 monochromatic encoded microspheres; Figure b2 shows the linear relationship between the DNA-Cy5 concentration and MFI.

[0029] Figure 5 : Clustering chart of the dual-color encoded microspheres decoded by flow cytometry in Example 1. The diameter of the microspheres is 10 μm, and the DNA-Cy3 and DNA-Cy5 dual-color encoded microspheres form 104 suspension arrays.

[0030] Figure 6 : Specific fluorescence signal chart of DNA-Cy5 monochromatic encoded microspheres in Example 2 for detecting alpha-fetoprotein (AFP) by flow cytometry. Figures a1 and a2 show the FITC detection values of the negative group and their corresponding histograms, and Figures b1 and b2 show the FITC detection values of the positive group and their corresponding histograms.

[0031] Figure 7 : The dual-color encoded microspheres in Example 3 are used for the detection of multiple targets. Figure a shows the decoding of the dual-color encoded microspheres with the concentration combination of S4R5 and S4R7 in the FL2×FL5 detection channel; Figure b shows the specific determination results of tumor markers. Detailed implementation manners

[0032] The present invention will be further described in detail below in conjunction with the embodiments, so that those skilled in the art can implement it with reference to the embodiments. Unless otherwise specified, the reagents used in the embodiments of the present invention are all commercially available products.

[0033] The term "encoded microspheres" used herein refers to particles with recognizable characteristics that can be decoded by fluorescence microscopy and flow cytometry.

[0034] Example 1

[0035] This example provides a kind of encoded microspheres constructed by three-dimensional assembly of fluorescent dye-labeled DNA molecules, which are prepared by the following method, and the specific steps are as follows:

[0036] 1. Activate the groups on the surface of magnetic beads

[0037] Using commercially available carboxyl magnetic beads (Fe 3 O4 - Polymeric microspheres with a diameter of 10 μm, purchased from Beijing Bio-Tech Co., Ltd.) were used as substrate microspheres for encoding microspheres. Figure 1 It shows the forward scatter angle (FSC) and side scatter angle (SSC) signal detection diagrams of magnetic beads with a diameter of 10 μm (red) analyzed by flow cytometry. Activate the carboxyl groups on their surface using an activator:

[0038] 1) After mixing the carboxyl magnetic beads, pipette 1×10 10 microspheres into an EP tube, add 200 μL of 100 mM 2-(N-morpholino)ethanesulfonic acid sodium salt (MES, pH 5.0) solution to resuspend the magnetic beads, place the reaction tube on a magnetic separation rack and let it stand for 2 min, then aspirate the supernatant and wash twice.

[0039] 2) Quickly add 200 μL of freshly prepared 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC / NHS, with MES as the solvent) to the treated magnetic beads, and vortex to mix well to make the magnetic beads fully suspended and dispersed for standby.

[0040] 2. Loading fluorescent dye-labeled DNA molecules onto the surface of magnetic beads

[0041] 1) Dilute DNA-Cy3 (synthesized by Sangon Biotech, Shanghai, 5'-CGGTAATATCGGACTC-3'-Cy3) with MES Buffer to 8 concentrations of 0, 3.5, 13, 25, 45, 105, 260, 1000 nM (denoted as S0 - S7 respectively); dilute DNA-Cy5 (synthesized by Sangon Biotech, Shanghai, 5'-TAATGGAAACTGGATC-3'-Cy5) to 13 concentrations of 0, 0.02, 0.05, 0.1, 0.3, 0.8, 1.15, 3, 5, 12, 40, 230, 4500 nM (denoted as R0 - R12 respectively).

[0042] 2) Set the concentration of DNA-Cy3 in the microspheres encoded by monochromatic fluorescent dye-labeled DNA molecules to S0, S1, S2, S3, S4, S5, S6, S7, and the concentration of DNA-Cy5 to R0, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11, R12. Add 200 μL of different concentrations of DNA-Cy3 or DNA-Cy5 solution to each reaction tube and mix with biotinylated DNA strand (concentration 1 μM). The DNA strand hybridizes complementarily with DNA-Cy3 or DNA-Cy5, and incubate in a 37 °C metal bath for 40 min to form DNA-Cy3 or DNA-Cy5 hybrid chain solution.

[0043] 3) The DNA molecule-encoded microspheres labeled with dual-color fluorescent dyes are formed by conjugating a combination of two fluorescent dyes, DNA-Cy3 and DNA-Cy5, which are labeled with DNA molecules, to a single magnetic microsphere. The combination of DNA-Cy3 and DNA-Cy5 can be divided into 104 groups (S0R0, S0R1, S0R2, S0R3, S0R4, S0R5, S0R6, S0R7, S0R8, S0R9, S0R10, S0R11, S0R12, S1R0, S1R1, S1R2, S1R3, S1R4, S1R5, S1R6, S1R7, S1R8, S1R9, S1R10, S1R11, S1R12, S2R0, S2R1, S2R2, S2R3, S2R4, S2R5, S2R6, S2R7, S2R8, S2R9, S2R10, S2R11, S2R12, S3R0, S3R1, S3R2, S3R3, S3R4, S3R5, S3R6, S3R7, S3R8, S3R9, S3R10, S3R11, S3R12, S4R0, S4R1, S4R2, S4R3, S4R4, S4R5, S4R6, S4R7, S4R8, S4R9, S4R10, S4R11, S4R12, S5R0, S5R1, S5R2, S5R3, S5R4, S5R5, S5R6, S5R7, S5R8, S5R9, S5R10, S5R11, S5R12, S6R0, S6R1, S6R2, S6R3, S6R4, S6R5, S6R6, S6R7, S6R8, S6R9, S6R10, S6R11, S6R12, S7R0, S7R1, S7R2, S7R3, S7R4, S7R5, S7R6, S7R7, S7R8, S7R9, S7R10, S7R11, S7R12). 200 μL of solutions of DNA-Cy3 and DNA-Cy5 with different concentrations and biotinylated DNA strands (concentration 1 μM) are added to each reaction tube and mixed. The DNA strands are hybridized and complementary to DNA-Cy3 or DNA-Cy5 and labeled with amino groups, and incubated in a metal bath at 37 °C for 40 min to form a solution of DNA molecule hybrid strands labeled with dual-color fluorescent dyes.

[0044] 4) After adding 200 μL of the DNA molecule hybrid strand solution labeled with monochromatic or dual-color fluorescent dyes with different concentrations to the activated magnetic beads, mix well, and incubate with shaking in a constant temperature shaker at 140 rpm at 25 °C for 12 h. The amino groups labeled on the DNA molecule hybrid strands are coupled to the carboxyl groups modified on the surface of the magnetic beads, so that the DNA molecules labeled with fluorescent dyes are assembled onto the surface of the magnetic microspheres.

[0045] 5) After the reaction is completed, place the reaction tube on a magnetic separation rack and let it stand for 2 min. Aspirate the supernatant, add 500 μL of phosphate buffer (PBS, pH = 7.4) to fully suspend the magnetic beads. Place the reaction tube on the magnetic separation rack and let it stand for 2 min. Aspirate the supernatant and wash 3 times with PBS to obtain fluorescent dye-labeled DNA molecule-encoded microspheres, as shown in Figure 2 . The amount of magnetic beads used for each coding group is approximately 3×10 4 pieces.

[0046] 6) Analyze and decode the fluorescent dye-labeled DNA molecule-encoded microspheres using a flow cytometer. In the monochromatic fluorescent dye-labeled DNA molecule-encoded microspheres, the DNA-Cy3 monochromatic-encoded microspheres can be detected in the FL2-PE channel of the flow cytometer and can be divided into 8 suspension arrays (see a) in Figure 3 . The mean fluorescence intensity (MFI) of the DNA-Cy3 monochromatic-encoded microspheres slowly increases and tends to be stable with the increase of the DNA-Cy3 dye concentration, showing a good linear relationship in the range of 0 - 600 nM (see a1 and a2 in Figure 4 ); the DNA-Cy5 monochromatic-encoded microspheres can be detected in the FL5-APC channel of the flow cytometer and can be divided into 13 suspension arrays (see b) in Figure 3 . When the DNA-Cy5 dye concentration is higher than 700 nM, the MFI is stable, and it has good linearity at 0 - 700 nM (see b1 and b2 in Figure 4 ); in the dual-color fluorescent dye-labeled DNA molecule-encoded microspheres, the DNA-Cy3 and DNA-Cy5 dual-color-encoded microspheres can achieve 8×13 = 104 kinds of coding in the FL2-FL5 detection channel (see Figure 5 ).

[0047] 3. Introduce the biotin-streptavidin bridge system onto the surface of the fluorescent dye-labeled DNA molecule-encoded microspheres to form fluorescent dye-labeled DNA molecule-streptavidin composite functional microspheres

[0048] Add 20 μL of 1 mg / mL (final concentration 40 μg / mL) streptavidin solution to the fluorescent dye-labeled DNA molecule-encoded microspheres, and incubate with shaking in a constant temperature shaker at 25 °C and 140 rpm for 2 h. Wash 3 times with PBS to allow streptavidin to be linked to the microsphere surface through biotin to obtain functionalized encoded microspheres.

[0049] Example 2

[0050] Use the functionalized monochromatic fluorescent dye-labeled DNA molecule-encoded microspheres prepared in Example 1 as a carrier for the detection of tumor markers. The specific application steps are as follows:

[0051] (1) Take 5×10 55 functionalized DNA-Cy5 monochromatic coding microspheres (diameter 10 μm, concentration 230 nM) were added with 200 μL of biotin-labeled capture antibody RC6A (concentration 25 μg / mL, purchased from Nanjing Genscript Biotechnology Co., Ltd.) diluted with PBS. Incubated with shaking at 140 rpm at 37°C for 1 h, the biotin of the capture antibody label specifically binds to the streptavidin on the surface of the coding microspheres to immobilize the capture antibody on the microsphere surface. The reaction tube was placed on a magnetic separation rack and allowed to stand for 2 min, the supernatant was aspirated, and washed 3 times with PBS to wash away the unbound capture antibody.

[0052] (2) The antigen to be detected, alpha-fetoprotein (AFP), after dilution, was added to the coding microspheres immobilized with the capture antibody prepared in step (1). Incubated at 140 rpm at 37°C for 1 h, the reaction tube was placed on a magnetic separation rack and allowed to stand for 2 min, the supernatant was aspirated, and washed 3 times with PBS to wash away the unbound antigen. The concentration of the target AFP to be detected in the negative control group was 0 ng / mL.

[0053] (3) The green fluorescent protein StayGold (27 KDa, gene accession number: BCQ30682.1) was expressed and purified and used to label the detection antibody RC6B (purchased from Nanjing Genscript Biotechnology Co., Ltd.).

[0054] (4) The detection antibody RC6B labeled with the green fluorescent protein StayGold prepared in step (3) was added to the antigen-antibody complex microspheres prepared in step (2), and incubated at 140 rpm at 37°C for 1 h to form a "sandwich" type double antibody sandwich structure.

[0055] (5) After the reaction was completed, the fluorescence signal of StayGold on the microspheres was detected by flow cytometry. The detection channel of the fluorescence signal of StayGold in flow cytometry was FL1-FITC. The detection results of AFP are as Figure 6 shown, Figure 6 Figure a1 and Figure a2 in [reference] are the scatter plot and histogram of detecting AFP with the DNA-Cy5 monochromatic coding microspheres in the negative group; Figure 6 Figure b1 and Figure b2 in [reference] are the scatter plot and histogram of detecting AFP with the DNA-Cy5 monochromatic coding microspheres in the positive group.

[0056] Example 3

[0057] The functionalized DNA molecule coding microspheres labeled with dual-color fluorescent dyes prepared in Example 1 were used as carriers for the detection of multiple tumor markers, and the specific application steps are as follows:

[0058] (1) Take 5×10 5Functionalized Cy3 and Cy5 dual-color fluorescent dye-labeled DNA molecule-encoded microspheres (diameter 10 μm, select two suspension arrays S4R5 and S4R7). Add 200 μL of biotin-labeled capture antibody RC6A (concentration 25 μg / mL, purchased from Nanjing Genscript Biotech Co., Ltd.) diluted with PBS to the dual-color encoded microspheres in the S4R5 concentration combination. Add 200 μL of biotin-labeled capture antibody RC66 (concentration 25 μg / mL, purchased from Nanjing Genscript Biotech Co., Ltd.) diluted with PBS to the dual-color encoded microspheres in the S4R7 concentration combination. Incubate on a shaker at 37 °C and 140 rpm for 1 h. The biotin labeled on the capture antibody specifically binds to the streptavidin on the surface of the encoded microspheres, thereby immobilizing the capture antibody on the surface of the microspheres. Place the reaction tube on a magnetic separation rack and let it stand for 2 min. Aspirate the supernatant and wash 3 times with PBS to wash away the unbound capture antibody.

[0059] (2) Dilute the antigens to be detected, alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), and add them to the encoded microspheres immobilized with the capture antibody prepared in step (1). Incubate at 37 °C and 140 rpm for 1 h. Place the reaction tube on a magnetic separation rack and let it stand for 2 min. Aspirate the supernatant and wash 3 times with PBS to wash away the unbound antigen. The concentration of the target to be detected in the negative control group is 0 ng / mL.

[0060] (3) Add the detection antibodies RC6B and RC67 labeled with green fluorescent protein StayGold to the antigen-antibody complex microspheres prepared in step (2). Incubate at 37 °C and 140 rpm for 1 h to form a "sandwich" type double-antibody sandwich structure.

[0061] (4) After the reaction is completed, detect the fluorescence signal of StayGold on the encoded microspheres by flow cytometry. The detection channel of the fluorescence signal of StayGold in the flow cytometry is FL1-FITC. The results are as Figure 7 shown. No cross-reaction was found during the dual detection process, and the fluorescence signal can only be detected when the target specifically binds to the capture antibody.

Claims

1. A fluorescent dye-labeled DNA molecule encoding microsphere, characterized in that: The method comprises a microsphere, a DNA molecule labeled with a fluorescent dye, and a DNA chain hybridized and complementary to the DNA molecule labeled with the fluorescent dye. The surface of the microsphere is modified with a chemical group, the DNA chain is modified with a functional group, the functional group on the DNA chain is covalently coupled to the chemical group modified on the surface of the microsphere, the DNA molecule labeled with the fluorescent dye and the complementary DNA chain are assembled on the surface of the microsphere through a hybridization reaction, and a single-color or multi-color coded microsphere is prepared with the type and loading concentration of the DNA molecule labeled with the fluorescent dye as the coding element.

2. The coded microsphere according to claim 1, characterized in that: The surface of the fluorescent dye-labeled DNA molecule encoding microsphere is modified with streptavidin.

3. A method for constructing coding microspheres by three-dimensionally assembling fluorescent dye-labeled DNA molecules, characterized in that: The following steps are involved: (1) hybridizing a fluorescent dye-labeled DNA molecule with a biotin-labeled complementary DNA chain, wherein the complementary DNA chain is modified with a functional group, and forming a fluorescent dye-labeled DNA molecule hybrid chain solution after hybridization reaction; (2) activating the chemical groups on the surface of the microspheres with an activator, wherein the chemical groups are covalently coupled to the functional groups modified with the DNA chains; (3) Adding the fluorescent dye-labeled DNA molecule hybrid chain solution to the activated microspheres, the functional groups on the complementary DNA chains are covalently coupled with the chemical groups modified on the surface of the microspheres, thereby assembling the fluorescent dye-labeled DNA molecules onto the surface of the microspheres, and preparing single-color or multi-color coded microspheres with the type and loading concentration of the fluorescent dye-labeled DNA molecules as coding elements.

4. The method according to claim 3, characterized in that The streptavidin was modified onto the microsphere surface via a biotin-streptavidin bridge to obtain functionalized encoded microspheres.

5. The coded microsphere according to claim 1 or the method according to claim 3, characterized in that: The fluorescent dye-labeled DNA molecules are one or more combinations of DNA molecules labeled with FAM, Alexa Fluor, Cy3, Cy5, Cy5.5, Cy7, ROX or Texas Red.

6. The coded microsphere according to claim 1 or the method according to claim 3, characterized in that: The microspheres are selected from one of polystyrene microspheres, silica microspheres, agarose gel microspheres, dextran gel microspheres, ferroferric oxide or ferric oxide magnetic microspheres, and the particle size of the microspheres is 1 to 30 μm.

7. The coded microsphere according to claim 1 or the method according to claim 3, characterized in that: The surface of the microsphere is modified with one of carboxyl, amino, maleimide, haloacetamide, gold atom, alkynyl, and azido groups, and the functional group modified with the complementary DNA chain hybridized with the DNA molecule labeled with the fluorescent dye is one of amino, carboxyl, sulfhydryl, thiol, aldehyde, epoxy, azido, alkynyl, hydroxyl, and cyano groups.

8. The coded microsphere according to claim 1 or the method according to claim 3, characterized in that: The loading concentrations of the indicated fluorescent dye-labeled DNA molecules ranged from 0 to 1000 nM.

9. Use of the encoded microspheres according to claim 1 in detecting one or more targets, characterized in that: The following steps are involved: (1) The biotin-labeled capture probe is specifically bound to the encoded microspheres modified with streptavidin; (2) Capturing the target analyte in the sample to be tested onto the surface of the microspheres through the capture probes coupled to the surface of the microspheres; (3) The target analyte captured on the microsphere surface specifically binds to the fluorescently labeled reporter probe; (4) Identify the encoded microspheres with different fluorescence characteristics through flow cytometric analysis or CCD imaging system, and detect the fluorescence signal of the reporter probe bound to the surface of each encoded microsphere.

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