WO2018214822A1 - Microparticle chrominance clustering analysis method and reagent kit - Google Patents

Abstract

A microparticle chrominance clustering analysis method and a reagent kit. The method comprises: S1, constructing biological target detection reagents, coating microparticles of different chrominances by using different biological probes I, so as to form the biological target detection reagents, the biological target detection reagents capable of simultaneously having specific binding reactions with M different types of biological targets in a sample to be measured; S2, carrying out biological detection reactions to form a combination of color microparticles, biological probes I, biological targets to be tested and isolating reagents; S3, isolating biological detection reaction products, and separating out polymerized substances of the color particles and the biological probes I that do not react with the detection reagents; and S4, determining the detection result, collecting statistics about the types and the contents of the biological targets to be detected in the detected sample according to a one-to-one correspondence between the chrominances of color microparticles and the types of the biological targets. In the present invention, multiple detection of a single sample and rapid multiple detection of multiple samples are implemented.

Description

Microparticle chroma clustering analysis method and kit Technical field

The invention belongs to the field of biomedical technology, and particularly relates to a microparticle chroma clustering analysis method and a kit.

Background technique

Biomacromolecules, including proteins, nucleic acids, etc., embody or record information about life and disease. They are extremely important analytical objects in biological research and disease diagnosis. Testing their existence and content is the basic work of scientific research and disease diagnosis. In addition to biomacromolecule detection, in some cases in disease diagnosis and biological research, it is also necessary to detect viruses and cells. These biomolecules, virus particles and cells are called biological targets to be tested in detection experiments. Biotargets, DBTs).

If DBTs are biological macromolecules, they are also called target molecules (TMs). There are generally two technical solutions for the detection of a single biomolecule (TM) to be tested:

One is the sandwich method, which uses a known biomolecule (probe I) that specifically binds to TM to bind TM, and then adds another known, labeled detectable signal that can be combined with TM or The probe I-TM complex specifically binds to the biomolecule (Probe II), forms the probe I-TM-Probe II-detectable signal complex, and separates and measures the strong detectable signal in this complex. Weak, according to the calibration curve between the detectable signal intensity determined by the experiment and the content of the TM standard, the content of the TM in the sample to be tested is found. To implement this method, the TM needs to have more than two probe recognition sites that do not affect each other, or the TM has only one probe recognition site but another probe that recognizes the TM-Probe I complex exists;

The second is the competition method, which is generally used when the TM has only one probe recognition site, and the probe I is labeled with a detectable signal, respectively reacted with the TM standard, reacted with the sample to be tested, and then with the TM standard. The reaction is used to estimate the TM content of the sample to be tested by the difference in the detectable signal intensity of the TM standard-probe I-detectable signal complex formed in the two reactions.

If the DBTs are virus particles or cells, the detection of one or more marker molecules can be used to determine which virus is to be tested and which cells are to be tested. At this point, each marker molecule only needs to have a probe I labeled with a detectable signal.

In the above technical solution, the detectable signal for labeling the bioprobe is known as an enzyme, a quantum dot, a chemiluminescent material, a colloidal gold, a colloidal selenium, a radioactive isotope, a fluorescein, etc. due to different kinds of detection signals. Different detection methods are generally not mixed in one detection reaction, and the same type of detection signal often has only one or several identifiable signal abundances, so that one detection reaction can only detect one or several biological targets to be tested (DBTs). ).

The occurrence, development and outcome of diseases are often influenced and regulated by multiple factors. Therefore, it is generally necessary to simultaneously detect multiple DBTs in biological research and disease diagnosis. In order to improve detection efficiency, save samples and save detection costs, and provide more analytical indicators for research and diagnosis, people have invented multiple detection techniques that can simultaneously detect multiple DBTs in one detection reaction, mainly including:

1. Microarray technology, also known as biochip technology. This technology has evolved from membrane hybridization technology, and its basic technical idea is to simultaneously carry out detection reactions for different TMs at different planar positions of the same detection reaction system. The basic technical realization is that the probe I which can specifically bind to different TMs is sequentially fixed at different positions of the solid phase substrate or the base film, that is, the biochip; the sample to be tested is reacted with the biochip, and the sample is in the sample. The existing TMs are captured by the probe I at the corresponding array site in the biochip, and the biochip site of the TM captures the fluorescently labeled probe II due to the bridging action of the TM; by detecting different arrays of the biochip Whether the micro-point has a fluorescent signal can determine whether there is a corresponding target molecule in the sample. Because the high-density biochip spotter can point out a matrix of more than 400 dots per square centimeter on the substrate, this technology can simultaneously detect tens of thousands of target molecules, providing genomics, proteomics and other research. High-throughput research methods and tools. However, in this technology, the reaction between the sample and the biochip belongs to a solid-liquid phase reaction, and the steric hindrance is large, and the binding reaction is slow, which often takes several hours, and it is difficult to meet the medical test with high time limit.

2.xMAP technology, also known as liquid chip technology. The technology is developed from flow cytometry. The basic idea is to liquefy the lattice of microarray technology to reduce steric hindrance and accelerate the detection reaction. The basic technology is realized in plastic microspheres with different fluorescence characteristics. The different plastic probes are labeled with different probes I, and the probes which specifically bind to different target molecules are labeled with fluorescein which has different fluorescence characteristics from the microspheres, and the different plastic microspheres-probe I, probe II-fluorescence The molecules are mixed together to form a liquid phase detection reagent. After binding reaction with the sample, the target molecule in the sample will be combined with the corresponding probe in the reagent to form a plastic microsphere-probe I-TM-probe II-fluorescein. a complex, the complex is based on a flow analyzer with a double excitation (one laser for exciting the fluorescence of the microsphere and the other for exciting the fluorescent molecule on the probe II) The fluorescence characteristics of the ball and the forward scattered light characteristics distinguish different microspheres. According to whether the fluorescence characteristics of the probe II are simultaneously present on different microspheres, whether the surface of the microspheres has a binding reaction is determined, thereby pushing back the sample. What are the detection target molecules? Although the number of target molecules that can be simultaneously detected by xMAP technology is far less than that of microarray technology, it not only significantly improves the efficiency of the binding reaction, but also enables semi-quantitative detection, which is closer to the requirements of medical detection. However, although this technology can simultaneously analyze multiple detection molecules, due to its flow detection technology, the instrument detection unit analyzes thousands to millions of microspheres in the system one by one, although The analysis of a single specimen is completed in a few minutes, but it is difficult to complete the detection of hundreds of specimens in a short period of time, and it is difficult to meet the detection requirements of a large number of clinical specimens.

3. πcode technology, which uses microfabrication technology to engrave different patterns on elliptical discs with diameters from tens of microns to hundreds of microns to distinguish different discs and then probe them on different discs. Needle I, coating the probe II on a magnetic material (such as a superparamagnetic magnetic bead), and combining different disc-probe I, magnetic material-probe II into a detection reagent, and reacting with the sample, in the sample The target molecule will bind to the corresponding probe in the reagent to form a complex of the disc-probe I-target molecule-probe II-magnetic material, and the above complex is separated from the disc which has not undergone the binding reaction by magnetic force. Then, through microscopic imaging and image recognition technology, the type and number of discs participating in or not participating in the reaction are determined, thereby estimating the type and content of the target molecules contained in the sample. This technology can detect different specimens in different micropores of a microplate, not only can simultaneously detect multiple target molecules in one detection system, but also complete the detection of multiple specimens in a short time. xMAP technology is difficult to detect hundreds of specimens in a short period of time. However, the disc of this technology adopts micro-machining technology, the cost is difficult to reduce, and the disc is easy to form a stack in the detection system, forming a large interference.

It can be seen that people have been exploring multiple detection technologies, and multiple detection techniques have been progressing. However, there is still no reliable, cost-effective technology that can simultaneously achieve single sample multiple detection and multi-sample rapid multiple detection.

Summary of the invention

In view of the above-mentioned deficiencies of the prior art, the present invention provides a microparticle chromaticity clustering analysis method, which aims to realize single sample multiplex detection and multi-sample rapid multiplex detection; and correspondingly provide a kit thereof.

In order to achieve the above object, the technical solution adopted by the present invention is as follows:

A particle chromaticity clustering analysis method, the technical idea is to use colored microparticles as a bioreactive marker, and the color spectroscopy is clustered by chromaticity through large field microscopic imaging and image processing, according to different before and after biological reaction. The change in the number of color particles, calculate the concentration of different biomolecules or biological particles in the same detection system, including the following steps:

S1: Construction of biological target detection reagents

Color granules of different chromaticity are coated with different biological probes I to form colored microparticle-bioprobe I polymer, and different colored microparticle-bioprobe I polymer is used for different biological targets to be tested. Specific binding reaction; mixing M kinds of colored microparticle-bioprobe I polymer to form a biological target detection reagent, which can simultaneously specifically bind to M different biological targets in the specimen to be tested Reaction; wherein M is a natural number ≥1.

S2: performing a biological detection reaction

The biological target detection reagent, the sample to be tested, and the N separation reagents are added to the reaction cup I to form a reaction suspension, and the biological detection reaction is performed to form a colored microparticle-bioprobe I-subject biological target-separating reagent combination ;N is a natural number ≥1.

S3: separation of biological detection reaction products

All of the reaction products generated in the step S2 were separated from the colored microparticle-bioprobe I polymer in which the detection reagent did not undergo a binding reaction.

S4: Determination of test results

The color microparticle-bioprobe I polymer involved in the biological reaction separated in step S3 is clustered and counted according to the color particle chromaticity thereof, and the statistical detection is performed according to the one-to-one correspondence between the color particle chromaticity and the biological target species. The type and content of the biological target to be tested contained in the specimen.

Alternatively, the color microparticle-bioprobe I polymer remaining after separating the reaction product in the step S3 is clustered and counted according to the color particle chromaticity thereof, and the type of all the colored microparticle-bioprobe I polymer in the reagent Calculating the clustering count of the color microparticle-bioprobe I polymer involved in the reaction in step S3, and then calculating the specimen to be tested according to the one-to-one correspondence between the color particle chromaticity and the type of the biological target to be tested. The type and content of the biological target to be tested.

Further, the colored fine particles have a diameter of not more than 100 μm.

Further, the specific separation method of the step S3 is: adsorbing all the separation reagents and the colored microparticle-bioprobe I-tested biological target-separating reagent conjugate with a magnetic needle, and placing it in the reaction cup II, and then adding the biological probe-to-be Measuring the biological target depolymerization agent, and then using the magnetic needle to adsorb and discard all the magnetic components, leaving the colored microparticle-bioprobe I polymer in the reaction cup II, and then proceeding to step S4;

Alternatively, an external magnetic field is used to adsorb all the separation reagents as well as the color microparticle-bioprobe I-test biological target-separating reagent conjugate in the reaction cup I, and the color microparticle-bioprobe I polymer not combined with the separation reagent Transfer to the cuvette III with a pipette, and proceed to step S4.

Wherein, the biological probe-tested biological target depolymerizing agent functions to separate the separation reagent from the colored microparticle-bioprobe I-subject biological target-separating reagent for subsequent detection. Commonly used biological probes - biological target depolymerizing agents to be tested include, but are not limited to, the following types:

Acid dissociation agent: pH value of 1.5 to 3, such as glycine solution.

Alkaline dissociator: pH value of 10 ~ 12.5, such as triethylamine solution.

High-salt dissociation agent: such as a MgCl ₂ solution having a concentration of 3 to 5 mol/L, a LiCl solution having a concentration of 5 to 10 mol/L, or the like.

Ionic detergent: such as a SDS solution having a concentration of 0.5 to 2% by weight.

The cracking agent: for example, urea having a concentration of 2 to 8 mol/L, a concentration of 2 to 5 mol/L of guanidine hydrochloride, and a concentration of 5 to 20% by weight of thiocyanuric acid.

Organic solvent: such as a concentration of 25 to 50% ethylene glycol, such as a concentration of 5 to 20% of dioxane.

Further, the clustering count is such that the color microparticle-bioprobe I polymer is dispersed in a single layer, and then subjected to large field microscopic color photographing, and the obtained photograph is subjected to image processing to extract the chromaticity of all the microparticles, and according to the color. Degrees are counted.

The preferred clustering counting method is: using the filtering technique, the colored microparticle-bioprobe I polymer obtained in step S3 is deposited on the filter screen to be arranged in a single layer; and then the large field of view microscopic color photographing is performed, and the obtained photograph is obtained. Through the image processing, the chromaticity of all the microparticles is extracted, and the clustering is performed according to the chromaticity; the number of the colored microparticle-bioprobe I polymer participating in the reaction reflects the number of species of the biological target to be tested in the specimen to be tested, and the color The chromaticity of the colored microparticles in the microparticle-bioprobe I polymer corresponds to the species of the biological target to be tested and the number of microparticles per chromaticity corresponds to the content of the biological target to be tested.

Further, the biological target is at least one of an antibody, an antigen, a ligand, a receptor, an oligonucleotide fragment, a cell, a virus particle, and an immune complex.

The separating reagent is a magnetic material coated with a biological probe II capable of specifically binding to a biological target to be tested, and has a biological probe III capable of specifically recognizing all the biological targets-bioprobe I-color microparticle complexes described above. At least one of the coated magnetic material and the magnetic material coated with the M biological targets to be tested.

The biological probe I, the biological probe II and the biological probe III are at least one of an antibody, an antigen, a ligand, a receptor, an oligonucleotide fragment, and a peptide nucleic acid.

The biological probe II is a substance capable of specifically reacting with a target to be tested, for example, in the double antibody sandwich method, the target to be detected is an antigen, the biological probe I is an antibody of a target, and the biological probe II is Another antibody to the target. In the double antigen sandwich method, the target to be tested is an antibody, the biological probe I is an antigen for detecting a target, and the biological probe II is also a target antigen, which is the same as the biological probe I.

Bioprobe III is a substance that can react with all bio-probe I-target polymer to be tested, for example, complement c1q, which can bind to immune complexes; for example, bio-probe I is an antigen, and the target is to be treated. Since the detection antibody is a human antibody, the Fc segment of the antibody is the same, so an animal anti-human antibody can be used as the biological probe III.

Correspondingly, the color microparticle-bioprobe I-tested biological target-separating reagent conjugate formed by the reaction is a color microparticle-biological probe I-tested biological target-biological probe II-magnetic material, colored microparticle-bioprobe Needle I - biological target to be tested - bioprobe III - magnetic material and / or color microparticle - bioprobe I - biological target to be tested - magnetic material combination.

A kit for particle size cluster analysis, comprising the biological target detection reagent constructed by the above S1.

Further, the above separation reagent may also be included.

Further, a biological probe - a biological target depolymerizing agent to be tested is also included.

In the kit, the biological target detection reagent, the separation reagent, and/or the biological probe - the biological target depolymerization agent to be tested are independently packaged.

The colored microparticles are on the order of micrometers and have a diameter of not more than 100 μm. Colored microparticles refer to microspheres dyed with distinguishable colors. The colors can be different in color or fluorescence, or they can be different in depth of the same color. The microspheres may be natural polymer microspheres, such as starch microspheres, albumin microspheres, gelatin microspheres, chitosan microspheres, etc.; or synthetic polymer microspheres, such as polystyrene microspheres, polyacrylic acid microspheres. Balls, silica microspheres, and the like.

The dyeing of the colored microparticles may be carried out by adding a dye or by dyeing during the preparation. For example, the Chinese patent with the application number of 0213936.5 and the Chinese patent with the application number of 20041003508.3.

Compared with the prior art, the present invention has the following beneficial effects:

1. Up to tens of thousands of biological targets can be detected simultaneously in one detection reaction. The detection technique of the present invention is directed to the formation of a target biomolecule complex, and since the chroma has a chroma of up to 36,000, it can almost meet all the requirements of the existing biomedical detection.

2, can quickly complete the above detection of up to hundreds of specimens. The detection technology of the invention can simultaneously perform multi-index detection reaction of 384 specimens on a 384-well microplate, and the result analysis uses an automatic clustering analysis system of microparticle chromaticity, which can automatically complete the analysis of the detection reaction. While achieving high-throughput analysis of single-sample detection indicators, high-throughput testing of multiple specimens can be performed.

3. It can perform qualitative analysis and quantitative detection. Since each target biomolecule is combined with a chromatic spherical microparticle and a detection probe molecule to form a target biomolecule complex, the number of microparticles involved in the reaction is positively correlated with the content of the biological target to be tested, and may be based on the microspheres participating in the reaction. The concentration of the corresponding detection target is calculated by counting.

4. The detection sensitivity is good. In theory, as long as there is a target to be tested in the specimen to be tested, a corresponding colored particle is separated, so the theoretical value of the analytical sensitivity is a single molecule level.

5. Low cost. Since the color microparticles used in the present invention are different in chromaticity, fluorescein is not necessary, so the cost of the dye is lower than that of the conventional technique. At the same time, without the use of single-molecule channels, laser emitters and micro-machining technology, not only can save a lot of cost, but also easier to achieve mass production.

detailed description

The present invention will be further described in detail below in conjunction with specific embodiments. In the following Examples 1 to 4, the diameter of the colored microparticles was 8 μm, and the optical microscopy system for microscopic imaging had a field of view of 6.36 mm in diameter, an optical resolution of 3 μm, and an effective magnification of 4.44 times.

Embodiment 1

Microparticle chromaticity clustering analysis for identification of common accidental antibodies in human blood type

Human blood type accidental antibody refers to the immune antibody produced by the body after blood transfusion and pregnancy, which does not have blood group antigens or antigen subtypes. It not only causes difficulty in blood transfusion when retransfusion, but also produces unexpected antibodies in women of childbearing age. May cause fetal hemolytic disease in the fetus. Therefore, accidental antibody screening and accidental antibody identification are important tests for blood transfusion and pregnancy testing. At present, accidental antibody screening and accidental antibody identification are used as two test items. First, the antibody screening reagent cells and the sample to be tested are used for reaction, and agglutination indicates that there is an unexpected antibody, and no agglutination indicates that there is no unexpected antibody; In other cases, it is necessary to use a set of antibody identification reagent cells to react with the sample to be tested, and according to the agglutination reaction of the different reagent cells and the blood group antigen composition on the reagent cells, the solution of the unexpected antibody is to be an antibody against the blood group antigen. Since the reagent cell group is composed of human red blood cells containing different rare blood type antigens, the resources are scarce, the price is expensive, the detection operation is cumbersome, the result analysis is complicated, and the detection efficiency is extremely low, and a simple and efficient method is needed to improve the detection efficiency and the detection quality. . In this embodiment, the multi-analyte detection capability of the microparticle chroma clustering analysis method and the high-throughput detection capability of the specimen are utilized to realize high-efficiency detection of common accidental antibody identification of human blood type. Since there are hundreds of human erythrocyte blood group antigens, for the sake of convenience, this example only uses the highest incidence of anti-A2, anti-B2, anti-D, anti-E, anti-e, anti-C, anti-c and other 7 kinds of unexpected antibodies. Identification is an example. The specific implementation steps are as follows:

S1: Preparation of microparticle color clustering analysis human blood type accidental antibody identification reagent

7 kinds of colored microspheres (the hues of the chromaticity are 10°, 20°, 30°, 40°, 50°, 60°, 70°, respectively), and the coated A2 antigen with a hue of 10° has a hue of 20°. Coated B2 antigen, coated D antigen with a color tone of 30°, coated E antigen with a color tone of 40°, coated e antigen with a color tone of 50°, coated C antigen with a color tone of 60°, and a color tone of 70° The coating is c antigen and blocked with polyethylene glycol to obtain microspheres (10)-A2, microspheres (20)-B2, microspheres (30)-D, microspheres (40)-E, microspheres (50 )-e, microspheres (60)-C, microspheres (70)-c, suspended in PBS buffer of pH 7.4 in a ratio of 1:1:1:1:1:1:1, each The concentration of the microspheres is about 100 / microliter, and the main reagent (reagent I) for detecting the human blood type accidental antibody is prepared by microparticle color cluster analysis, and placed at 4 ° C for use;

100 nm magnetic beads were coated with mouse anti-human IgG, blocked with polyethylene glycol, and suspended in PBS buffer at pH 7.4. The concentration of magnetic beads-anti-human IgG particles was about 10,000 / microliter. Microparticle color cluster analysis method for human blood type accidental antibody identification and separation reagent (Reagent II), placed at 4 ° C for use;

Glycine buffer with a pH of 2.5 was prepared as a dissociation reagent (Reagent III)

The microspheres were coated with antigens according to the methods reported by Chen Qilong, Liu Jiaxuan, Wang Yujia, etc. (Journal of Beijing University of Chemical Technology - Natural Science Edition, Vol. 41, No. 3). Magnetic beads coated with anti-human IgG were reported by Guo Huifang, Zhang Wenhong, Wen Dongqing, etc. The method (Journal of Immunology, Vol. 22, No. 5) was carried out.

S2: detection reaction

Take conventional 384-well plastic microplates (120 μl per well), add 30 μl of different test specimens to different micropores, and add 30 μl of reagent I obtained in S1 step, 40 μl of reagent II, at 37 The binding reaction was carried out by allowing to stand at °C for 10 minutes.

S3: separation reaction product

All the substances in the holes that can be adsorbed by the magnetic field are transferred to the corresponding holes of the other 384-well microplate by the electromagnetic needle. The reaction holes and the transfer holes are in one-to-one correspondence, and the diameter of the filter holes is 7 at the bottom of each hole. Micron filter membrane; add 100 μl of reagent III in the transfer hole, dissociate the reaction under shaking at 37 ° C for 5 minutes; then use the electromagnetic needle to absorb the substances in each well that can be adsorbed by the magnetic field. . The transfer holes were subjected to pressure filtration, and the colored microspheres in the holes were embedded in the sieve to be arranged in a single layer.

S4: Detection and result determination

Using a color particle sub-cluster analysis system, the filter membrane of each transfer hole was subjected to microsphere focusing photographing, and the chromaticity of each microsphere in the photograph was analyzed, and classified and counted according to chromaticity. If the count of the microspheres (10) is greater than zero, it indicates that there is an anti-A2 antibody in the specimen, and the number of microspheres (10) is positively correlated with the concentration of the anti-A2 antibody; if the count of the microspheres (20) is greater than zero, it indicates There is anti-B2 antibody in the specimen, and the number of microspheres (20) is positively correlated with the concentration of anti-B2 antibody; if the count of microspheres (30) is greater than zero, it indicates that there are anti-D antibodies in the specimen, and the microspheres ( The number of 30) is positively correlated with the concentration of anti-D antibody; if the count of microspheres (40) is greater than zero, it indicates that there is anti-E antibody in the specimen, and the number of microspheres (40) and the concentration of anti-E antibody are positive. Correlation; if the count of the microspheres (50) is greater than zero, indicating that there is an anti-e antibody in the specimen, and the number of microspheres (50) is positively correlated with the concentration of the anti-e antibody; if the count of the microspheres (60) is greater than zero , indicating that there is anti-C antibody in the specimen, and the number of microspheres (60) is positively correlated with the concentration of anti-C antibody; if the count of microspheres (70) is greater than zero, it indicates that there is anti-c antibody in the specimen, and micro The number of spheres (70) is positively correlated with the concentration of anti-c antibody. The dose-effect relationship between the detection target and the microsphere count is determined by the calibration curve.

The color particle clustering analysis system can perform automated hole-by-hole analysis on the transfer holes of the above microplates, and quickly obtain the results of accidental blood group antibody identification of one to 384 specimens to be tested.

It should be understood that the present embodiment is only used to describe the implementation process of the microparticle chroma clustering analysis for the identification of human blood type accidental antibodies, and the human blood group antibodies that can be detected are not limited to the above seven kinds.

Embodiment 2

Microparticle chromaticity clustering analysis for tumor marker detection

Tumors are common diseases and frequently-occurring diseases, which seriously endanger the life safety of patients. Laboratory diagnosis is an important method for early detection of tumors and for the treatment of patients. Due to the diversity of tumors, it is often necessary to simultaneously detect dozens of tumor markers in order to find tumors on a broad spectrum. In this embodiment, the multi-analyte detection capability of the microparticle chroma clustering analysis method and the high-throughput detection capability of the specimen are utilized to achieve high-efficiency detection of tumor markers. Since there are dozens of tumor markers that have been discovered, for the sake of convenience, this example only uses the most common alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and neuron-specific enolase (NSE). Detection of seven tumor markers including squamous cell carcinoma associated antigen (SCC-Ag), prostate specific antigen (PSA), cytokeratin fragment antigen 21-1 (CYFRA21-1), and carbohydrate antigen 242 (CA242) For example, the specific implementation steps are as follows:

S1: Preparation of microparticle color clustering analysis tumor marker detection reagent

7 kinds of colored microspheres having a particle diameter of 8 μm (the hue components of chromaticity are 10°, 20°, 30°, 40°, 50°, 60°, 70°, respectively), and the color of the coating is 10°. AFP antibody, coated mouse anti-human CEA antibody with a color tone of 20°, coated mouse anti-human NSE antibody with a color tone of 30°, coated mouse anti-human SCC-Ag antibody with a color tone of 40°, package with a color tone of 50° Mouse anti-human PSA antibody, coated mouse anti-human CYFRA-1 antibody with a color tone of 60°, coated mouse anti-human CA242 antibody with a color tone of 70°, and blocked with polyethylene glycol to obtain microsphere (10)-anti- AFP, microspheres (20)-anti-CEA, microspheres (30)-anti-NSE, microspheres (40)-anti-SCC-Ag, microspheres (50)-anti-PSA, microspheres (60)-anti-CYFRA21-1 Microspheres (70)-CA242 were suspended in PBS buffer of pH 7.4 at a ratio of 1:1:1:1:1:1:1, and the concentration of each microsphere was about 100/μl. The main reagent (Reagent I) for the identification of human blood type accidental antibodies was prepared by microparticle chroma cluster analysis, and placed at 4 ° C for use;

100 nm magnetic beads were coated with rabbit anti-mouse IgG, blocked with polyethylene glycol, and suspended in PBS buffer at pH 7.4. The concentration of magnetic beads-anti-mouse IgG particles was about 10,000/μl. Microparticle color clustering analysis method for tumor marker detection and separation reagent (Reagent II), placed at 4 ° C for use;

Glycine buffer with a pH of 2.0 was prepared as a dissociation reagent (Reagent III)

The microspheres were coated with antigens according to the methods reported by Chen Qilong, Liu Jiaxuan, Wang Yujia, etc. (Journal of Beijing University of Chemical Technology - Natural Science Edition, Vol. 41, No. 3). Magnetic beads coated with anti-mouse IgG were reported by Guo Huifang, Zhang Wenhong, Wen Dongqing, etc. The method (Journal of Immunology, Vol. 22, No. 5) was carried out.

S2: detection reaction

Take conventional 384-well plastic microplates (120 μl per well), add 30 μl of different test specimens to different micropores, and add 30 μl of reagent I obtained in S1 step, 40 μl of reagent II, at 37 The binding reaction was carried out by allowing to stand at °C for 10 minutes.

S3: separation reaction product

All the substances in the holes that can be adsorbed by the magnetic field are transferred to the corresponding holes of the other 384-well microplate by the electromagnetic needle. The reaction holes and the transfer holes are in one-to-one correspondence, and the diameter of the filter holes is 7 at the bottom of each hole. Micron filter membrane; add 100 μl of reagent III in the transfer hole, dissociate the reaction under shaking at 37 ° C for 5 minutes; then use the electromagnetic needle to absorb the substances in each well that can be adsorbed by the magnetic field. . The transfer holes were subjected to pressure filtration so that the colored microspheres in the holes were embedded in the sieve and arranged in a single layer.

S4: Detection and result determination

Using a color particle sub-cluster analysis system, the filter membrane of each transfer hole was subjected to microsphere focusing photographing, and the chromaticity of each microsphere in the photograph was analyzed, and classified and counted according to chromaticity. If the count of the microspheres (10) is greater than zero, it indicates that there is AFP in the specimen, and the number of microspheres (10) is positively correlated with the concentration of AFP; if the count of the microspheres (20) is greater than zero, it indicates that there is CEA, and the number of microspheres (20) is positively correlated with the concentration of CEA; if the count of microspheres (30) is greater than zero, it indicates that there is NSE in the specimen, and the number of microspheres (30) and the concentration of NSE are positive. Correlation; if the count of the microspheres (40) is greater than zero, indicating that there is SCC-Ag in the specimen, and the number of microspheres (40) is positively correlated with the concentration of SCC-Ag; if the count of the microspheres (50) is greater than zero , indicating that there is PSA in the specimen, and the number of microspheres (50) is positively correlated with the concentration of PSA; if the count of microspheres (60) is greater than zero, it indicates that there is CYFRA21-1 in the specimen, and the microspheres (60) The number is positively correlated with the concentration of CYFRA21-1; if the count of the microspheres (70) is greater than zero, it indicates that there is CA242 in the specimen, and the number of microspheres (70) is positively correlated with the concentration of CA242. The dose-effect relationship between the detection target and the microsphere count is determined by the calibration curve.

The color particle clustering analysis system can automatically and vertically analyze the transfer holes of the above microplates, and quickly obtain the detection results of multiple tumor markers from 1 to 384 samples to be tested.

It should be understood that the present embodiment is only used to describe the implementation process of the microparticle chromaticity clustering analysis method for tumor markers, and the tumor markers that can be detected are not limited to the above seven kinds.

Embodiment 3

Microparticle chromaticity clustering analysis for blood donor infection screening

In order to avoid transfusion-transmitted diseases, the screening of blood-borne pathogens in blood donor specimens is an international practice in the blood supply industry. The mandatory screening programs in China's regulations include HIV-1 antibodies, HIV-p24, HBsAg, HCV antibodies and TP antibodies. For six kinds of detection indicators, blood stations often need to repeat the above screening for dozens or even hundreds of blood donors at the same time. In this embodiment, the multi-analyte detection capability of the microparticle chroma clustering analysis method and the high-throughput detection capability of the specimen are utilized to realize blood donor infection screening. For convenience of expression, in this example, HIV-GP41 antibody is used as a representative of HIV-1 antibody, HCV-NS3 antibody is representative of HCV antibody, and TP-47 antibody is representative of syphilis antibody, and simultaneous detection with HBsAg and HIV-p24 is For example, the specific implementation steps are as follows:

S1: Preparation of microparticle color clustering analysis for blood donor infection screening

Five kinds of colored microspheres having a particle diameter of 8 μm (the color components of chromaticity are 10°, 20°, 30°, 40°, and 50°, respectively), and the color of the coating is 10° coated with HIV-GP41 antigen, and the color tone is 20°. The coated anti-HBsAg antibody is coated with HCV-NS3 antigen, coated with TP-47 antigen with a color tone of 30°, coated with human anti-HIV-p24 antibody with a color tone of 40°, and coated with human anti-HBsAg antibody. The alcohol is blocked to obtain microspheres (10)-HIV-GP41, microspheres (20)-HCV-NS3, microspheres (30)-TP-47, microspheres (40)-anti-HIV-p24, microspheres (50) - Anti-HBsAg, suspended in PBS buffer solution of pH 7.4 in a ratio of 1:1:1:1:1, the concentration of each microsphere is about 100 /μl, and made into a particle size cluster analysis method. Blood type accidental antibody identification main reagent (Reagent I), placed at 4 ° C for use;

100 nm magnetic beads were coated with complement C1q subunit, polyethylene glycol was blocked, and the suspension was suspended in PBS buffer of pH 7.4. The concentration of magnetic beads-C1q particles was about 10000/μl to prepare microparticles. Chromatic clustering analysis method for human blood type accidental antibody identification and separation reagent (Reagent II), placed at 4 ° C for use;

Glycine buffer with a pH of 2.0 was prepared as a dissociation reagent (Reagent III)

The microspheres were coated with antigens according to the methods reported by Chen Qilong, Liu Jiaxuan, Wang Yujia, etc. (Journal of Beijing University of Chemical Technology - Natural Science Edition, Vol. 41, No. 3). The magnetic beads coated complement C1q was reported by Guo Huifang, Zhang Wenhong, Wen Dongqing, etc. Method (Journal of Immunology, Vol. 22, No. 5).

S2: detection reaction

Take conventional 384-well plastic microplates (120 μl per well), add 30 μl of different test specimens to different micropores, and add 30 μl of reagent I obtained in S1 step, 40 μl of reagent II, at 37 The binding reaction was carried out by allowing to stand at °C for 10 minutes.

S3: separation reaction product

All the substances in the holes that can be adsorbed by the magnetic field are transferred to the corresponding holes of the other 384-well microplate by the electromagnetic needle. The reaction holes and the transfer holes are in one-to-one correspondence, and the diameter of the filter holes is 7 at the bottom of each hole. Micron filter membrane; add 100 μl of reagent III in the transfer hole, dissociate the reaction under shaking at 37 ° C for 5 minutes; then use the electromagnetic needle to absorb the substances in each well that can be adsorbed by the magnetic field. . The transfer holes were subjected to pressure filtration, and the colored microspheres in the holes were embedded in the sieve to be arranged in a single layer.

S4: Detection and result determination

Using a color particle sub-cluster analysis system, the filter membrane of each transfer hole was subjected to microsphere focusing photographing, and the chromaticity of each microsphere in the photograph was analyzed, and classified and counted according to chromaticity. If the count of the microspheres (10) is greater than zero, it indicates that there is an anti-HIV-GP41 antibody in the specimen, and the number of microspheres (10) is positively correlated with the concentration of the anti-HIV-GP41 antibody; if the microspheres (20) are counted Greater than zero, indicating that the sample has anti-HCV-NS3 antibody, and the number of microspheres (20) is positively correlated with the concentration of anti-HCV-NS3 antibody; if the count of the microsphere (30) is greater than zero, it indicates that there is Anti-TP-47 antibody, and the number of microspheres (30) is positively correlated with the concentration of anti-TP-47 antibody; if the count of microspheres (40) is greater than zero, it indicates that there is HIV-p24 in the specimen, and the microspheres ( The number of 40) is positively correlated with the concentration of HIV-p24; if the count of the microspheres (50) is greater than zero, it indicates that there is HBsAg in the specimen, and the number of microspheres (50) is positively correlated with the concentration of HBsAg. The dose-effect relationship between the detection target and the microsphere count is determined by the calibration curve.

The color particle clustering analysis system can perform automated hole-by-hole analysis on the transfer holes of the above microplates, and quickly obtain the infection screening results of one to 384 samples to be tested.

It should be understood that the present embodiment is only used to describe the implementation process of the particle chromaticity clustering analysis method for blood donor infection screening, and the infectious markers that can be detected are not limited to the above five kinds.

Embodiment 4

Microparticle particle clustering analysis of human erythrocyte blood group detection

The human erythrocyte blood type is an allogeneic antigen on the surface of human red blood cells. The blood that is incompatible with blood type may cause hemolysis and accidental antibody production, and the life and fertility safety of the critically ill patients. Therefore, both the blood donor and the recipient must perform the human red blood cell blood type. Detection. As many as hundreds of red blood cell types have been discovered, this embodiment utilizes the multi-analyte detection capability of the microparticle color cluster analysis method and the high-throughput detection capability of the specimen to realize the detection of various blood types on human red blood cells for convenience. In the present embodiment, the simultaneous detection of A antigen, B antigen, D antigen, E antigen, e antigen, C antigen, and c antigen is taken as an example, and the specific implementation steps are as follows:

S1: Preparation of microparticle color clustering analysis method for human red blood cell blood type detection reagent

7 kinds of colored microspheres having a particle diameter of 8 μm (the hue components of chromaticity are 10°, 20°, 30°, 40°, 50°, 60°, 70°, respectively), and the coating is anti-A with a color tone of 10°. Antibody, coated anti-B antibody with a color tone of 20°, coated anti-D antibody with a color tone of 30°, coated anti-E antibody with a color tone of 40°, coated anti-e antibody with a color tone of 50°, Coated anti-C antibody with a color tone of 60°, coated anti-e antibody with a color tone of 70°, and blocked with polyethylene glycol to obtain microspheres (10)-anti-A, microspheres (20)-anti-B, Microspheres (30) - anti-D, microspheres (40) - anti-E, microspheres (50) - anti-e, microspheres (60) - anti-C, microspheres (70) - anti-c, according to 1:1: The ratio of 1:1:1:1:1 was suspended in PBS buffer of pH 7.4, and the concentration of each microsphere was about 100/μl, which was made into the microparticle color cluster analysis method. Reagent (Reagent I), placed at 4 ° C for use;

100 nm magnetic beads were coated with phytohemagglutinin, blocked with polyethylene glycol, and suspended in PBS buffer at pH 7.4. The concentration of magnetic beads-hemagglutinin particles was about 10,000/μl. Microparticle color cluster analysis method for human blood type accidental antibody identification and separation reagent (Reagent II), placed at 4 ° C for use;

Use deionized water as dissociation reagent (Reagent III)

The microspheres were coated with antigens according to the methods reported by Chen Qilong, Liu Jiaxuan, Wang Yujia, etc. (Journal of Beijing University of Chemical Technology - Natural Science Edition, Vol. 41, No. 3). Magnetic beads coated with hemagglutinin were reported by Guo Huifang, Zhang Wenhong, Wen Dongqing, etc. The method (Journal of Immunology, Vol. 22, No. 5) was carried out.

S2: detection reaction

Take conventional 384-well plastic microplates (120 μl per well), add different detection specimens to different micropores, add 2 μL of erythrocyte physiological saline suspension, and add the reagent I30 microliters and reagents prepared in S1 step. II40 μl, and allowed to stand at 37 ° C for 10 minutes for the binding reaction.

S3: separation reaction product

All the substances in each hole that can be adsorbed by the magnetic field are transferred to the corresponding holes of another 384-well microplate by an electromagnetic needle. The reaction holes and the transfer holes are in one-to-one correspondence, and the bottom of each hole of the transfer hole has a hole diameter of 7 μm. Filter membrane; 100 μl of reagent III was added to the transfer well, and the dissociation reaction was carried out under shaking at 37 ° C for 5 minutes; then the magnetic field was used to absorb the substances adsorbed by the magnetic field. The transfer holes were pressure-filtered so that the colored microspheres in the holes were embedded in the sieve and arranged in a single layer.

S4: Detection and result determination

Using a color particle sub-cluster analysis system, the filter membrane of each transfer hole was subjected to microsphere focusing photographing, and the chromaticity of each microsphere in the photograph was analyzed, and classified and counted according to chromaticity. If the count of the microspheres (10) is greater than zero, it indicates that there is an A antigen on the red blood cells of the specimen; if the count of the microspheres (20) is greater than zero, it indicates that there is a B antigen on the red blood cells of the specimen; if the count of the microspheres (30) is greater than zero , indicating that the sample has D antigen on the red blood cells; if the count of the microspheres (40) is greater than zero, it indicates that the sample has E antigen on the red blood cells; if the count of the microspheres (50) is greater than zero, it indicates that the sample has red antigen on the red blood cells; If the count of the microspheres (60) is greater than zero, it indicates that there is a C antigen on the red blood cells of the specimen; if the count of the microspheres (70) is greater than zero, it indicates that there is a c antigen on the red blood cells of the specimen.

The color particle clustering analysis system can perform automated hole-by-hole analysis on the transfer holes of the above microplates, and quickly obtain the blood cell type test results of one to 384 samples to be tested.

It should be understood that the present embodiment is only used to describe the implementation process of the microparticle chroma clustering analysis method for human red blood cell blood type, and the human red blood cell blood type that can be detected is not limited to the above seven kinds.

Embodiment 5

Detection of human myocardial injury markers by microparticle chroma clustering analysis

In the present embodiment, the color microparticles have a diameter of 3 μm and are superparamagnetic, and the optical microscopy system for microscopic imaging has a field of view of 6.36 mm in diameter, an optical resolution of 3 μm, and an effective magnification of 4.44 times.

Acute myocardial infarction (AMI), acute coronary syndrome (ACS), unstable angina and myocarditis often lead to acute myocardial damage, which is a serious threat to the lives of patients. Early diagnosis and early treatment are the key factors affecting the success rate of acute myocardial injury. In order to obtain early diagnosis and a long diagnostic window as much as possible, it is often necessary to simultaneously detect creatine kinase isoenzyme hybridization (CK-MB), troponin I subunit (cTnI), and myoglobin (Myo). Using the multi-analyte detection capability of the microparticle chroma clustering method and the high-throughput detection capability of the specimen, the serum levels of CK-MB, cTnI and Myo can be detected simultaneously in only one test, and can also be detected simultaneously. For multiple patients, the specific implementation steps are as follows:

S1: Preparation of microparticle color clustering analysis method for human myocardial injury marker detection reagent

Three types of colored microspheres having a particle diameter of 3 μm and having superparamagnetic properties (tone components of chromaticity are 10°, 20°, and 30°, respectively), and a CK-MB primary antibody having a color tone of 10° and a color tone of 20 ° coated with cTnI primary antibody, coated with Myo primary antibody at a color tone of 30°, and blocked with polyethylene glycol to obtain microspheres (10)-anti-CK-MB, microspheres (20)-anti-cTnI, microspheres ( 30)-anti-Myo, suspended in PBS buffer of pH 7.4 in a ratio of 1:1:1, the concentration of each microsphere is about 100 / microliter, made into microparticle color cluster analysis human myocardial damage Marker detection reagent I, placed at 4 ° C for use;

CK-MB secondary antibody, cTnI secondary antibody and Myo secondary antibody were combined to coat 384-well microplates at a concentration ratio of 1:1:1 to prepare microparticle color cluster analysis method for human myocardial injury marker detection reagent II, 4 °C Drain, seal with a parafilm, and store at 4 °C. The microspheres were carried out by the secondary antibodies with reference to the methods reported by Chen Qilong, Liu Jiaxuan, Wang Yujia, etc. (Journal of Beijing University of Chemical Technology - Natural Science Edition, Vol. 41, No. 3).

S2: detection reaction

Take a 384-well microplate with CK-MB secondary antibody, cTnI secondary antibody, and Myo secondary antibody (microparticle color cluster analysis method for human myocardial injury marker detection reagent II), and tear the N-hole sealing film for use. N is equal to the number of specimens to be tested.

Different specimens correspond to different wells, and 30 μl of the specimen to be tested (patient serum or plasma) was added, and then 30 μl of the detection reagent I was added to each well, and the reaction was shaken at 37 ° C for 10 minutes, and allowed to stand for 5 minutes.

S3: Detection and result determination

Using the color particle clustering analysis system to focus on the microspheres at the bottom of each hole of the microplate, analyze the chromaticity of each microsphere in the photo, and classify and count according to the chromaticity, and obtain the color microparticles in each detection system. The starting quantity. The iron bismuth boron magnet was placed on top of the microplate. After 2 minutes, the microspheres at the bottom of each hole of the microplate were photographed by a color microparticle clustering analysis system, and the chromaticity of each microsphere in the photo was analyzed, and the chromaticity was performed according to the chromaticity. The number of the microparticles involved in the binding reaction in each detection system was obtained by classification.

The ratio of the number of microparticles (10) involved in the binding reaction or the amount thereof to the initial amount is positively correlated with the concentration of CK-MB in the patient's serum; the number of microparticles (20) participating in the binding reaction or the ratio of the number of microparticles (20) involved in the binding reaction, It is positively correlated with the concentration of cTnI in the patient's serum; the ratio of the number of microparticles (30) involved in the binding reaction or its initial number is positively correlated with the concentration of Myo in the patient's serum. For different batches of detection reagents, a correction curve can be prepared, and the myocardial damage markers in the test specimens are quantitatively detected. By using the one-to-one correspondence between the number of the specific hole on the orifice plate and the patient specimen number, the detection result of each hole can be correlated with the patient.

It should be understood that the present embodiment is only used to describe the implementation process of the microparticle chromaticity clustering analysis for detecting human myocardial injury markers, and the myocardial damage markers that can be detected are not limited to the above three types. An electromagnet or other magnetic field can also be used in place of the iron-boron boron magnet in the step S3.

Embodiment 6

Detection of human antibiotic antibodies by microparticle chroma clustering analysis

In the present embodiment, the diameter of the colored microparticles is 100 μm, and the optical microscope system for microscopic imaging has a field of view of 6.36 mm in diameter, an optical resolution of 3 μm, and an effective magnification of 4.44 times.

Antibiotics are powerful tools for humans to fight against microbial infections such as bacteria. They are not only good medicines for treating infectious diseases, but also often used to prevent wound infections before and after surgery. With the widespread use of antibiotics in the clinic, it has been found that certain antibiotics or their degradation products stimulate the body's immune response, producing antibodies against antibiotics. Even in the case of cross-antigen reactions, some patients who have not been exposed to antibiotics have antibodies that specifically bind to antibiotics. Injecting or taking the antibiotic with a patient who has produced an antibiotic antibody may reduce the efficacy of the antibiotic due to the neutralization of the antibody, and may cause an adverse reaction such as immune hemolytic anemia or nephritis. Therefore, antibiotic antibody testing before patients use antibiotics is an important measure to prevent related adverse reactions and ensure efficacy. In this example, a method for detecting human antibiotic antibodies by microparticle color cluster analysis is described by taking penicillin antibody, cephalosporin antibody and sulfa antibody as examples. The specific implementation steps are as follows:

S1: Preparation of microparticle color clustering analysis method for human myocardial injury marker detection reagent

Three kinds of colored microspheres having a particle diameter of 100 μm (tone components of chromaticity are 10°, 20°, and 30°, respectively), and penicillin-conjugated bovine serum albumin (BSA-P) having a color tone of 10°, color tone a 20° coated cephalosporin-conjugated bovine serum albumin (BSA-C), a 30° toned sulfonamide-conjugated bovine serum albumin (BSA-S), and blocked with polyethylene glycol. The microspheres (10)-BSA-C, microspheres (20)-BSA-C, and microspheres (30)-BSA-S were obtained and suspended in a 1:1 buffer of pH 7.4 at a ratio of 1:1:1. , the concentration of each microsphere is about 100 / microliter, made into a microparticle color cluster analysis human antibiotic antibody detection reagent I, placed at 4 ° C;

BSA-P, BSA-C, and BSA-S were combined to coat superparamagnetic beads in a concentration ratio of 1:1:1 to prepare a microparticle color cluster analysis method for human antibiotic antibody detection reagent II (magnetic bead concentration 100) / microliter), placed at 4 ° C for use. Magnetic beads and colored microspheres were carried out by BSA-P, BSA-C, and BSA-S according to the methods reported by Chen Qilong, Liu Jiaxuan, and Wang Yujia (Journal of Beijing University of Chemical Technology - Natural Science Edition, Vol. 41, No. 3).

S2: detection reaction

Take 384-well microplates, add 30 microliters of specimens to be tested (patient serum or plasma) according to different specimens, and add 30 μl of detection reagent to each well, 30 μl of detection reagent II, and rotate horizontally at 37 °C. The reaction was shaken for 10 minutes and allowed to stand for 2 minutes.

S3: Detection and result determination

Using the color particle clustering analysis system to focus on the microspheres at the bottom of each hole of the microplate, analyze the chromaticity of each microsphere in the photo, and classify and count according to the chromaticity, and obtain the color microparticles in each detection system. The starting quantity. The iron bismuth boron magnet was placed on top of the microplate. After 2 minutes, the microspheres at the bottom of each hole of the microplate were photographed by a color microparticle clustering analysis system, and the chromaticity of each microsphere in the photo was analyzed, and the chromaticity was performed according to the chromaticity. The number of the microparticles involved in the binding reaction in each detection system was obtained by classification.

The ratio of the number of microparticles (10) involved in the binding reaction or the amount thereof to the initial amount is positively correlated with the concentration of penicillin antibody in the patient's serum; the number of microparticles (20) involved in the binding reaction or the ratio of the number of microparticles involved in the binding reaction, The concentration of cephalosporin antibody in the patient's serum was positively correlated; the ratio of the number of microparticles (30) involved in the binding reaction or the ratio of its initial number was positively correlated with the concentration of sulfa antibody in the patient's serum. For different batches of detection reagents, a correction curve can be prepared, and the antibiotic antibodies in the specimens to be tested are quantitatively detected. By using the one-to-one correspondence between the number of the specific hole on the orifice plate and the patient specimen number, the detection result of each hole can be correlated with the patient.

It should be understood that the present embodiment is only used to describe the implementation process of the microparticle chroma clustering analysis method for human antibiotic antibody detection, and the antibiotic antibodies that can be detected are not limited to the above three kinds. An electromagnet or other magnetic field can also be used in place of the iron-boron boron magnet in the step S3.

Comparative example one

In order to verify the test efficiency of the present invention, the method disclosed in the present invention and the method disclosed in "Study on Magnetic Ball and Fluorescent Encoding Microsphere Labeled Microarray Blood Type Identification Technique", pages 1-66, respectively, detect 5, 100, and 300 indicators. Comparative experiments were carried out, and the results are shown in Table 1. The invention adopts the color microparticles with a diameter of 8 μm, and the optical microscope system for microscopic imaging has a field of view of 6.36 mm in diameter, an optical resolution of 3 μm, and an effective magnification of 4.44 times.

Table 1 Comparison of the length of time required to complete the number of different indicators for a single specimen

Technical solutions	D3 time required (seconds)	Time required for the invention (seconds)
Detect 5 indicators	61	1
Detect 100 indicators	80	1
Detect 300 indicators	120	1

Note: (1) In D3 and the detection system of the present invention, the index microsphere ratio (the number of each color microsphere in the detection system) is the same, both are 1000; (2) D3 needs to perform 60 seconds after each test. The instrument is clean and is not required by the present invention.

Comparative example two

In order to verify the test efficiency of the present invention, the method disclosed in the present invention and the method disclosed in "Study on Magnetic Ball and Fluorescent Encoding Microsphere Labeled Microarray Blood Type Identification Technique", pages 1-66, respectively, are detected. 100 and 300 microspheres were compared and the results are shown in Table 2. The color microparticles of the present invention have a diameter of 8 μm, and the optical microscope system for microscopic imaging has a field of view of 6.36 mm in diameter, an optical resolution of 3 μm, and an effective magnification of 4.44 times.

Table 2 Comparison of the time required to complete the 20 indicators of different quantitative specimens

Technical solutions	D3 time required (seconds)	Time required for the invention (seconds)
Test 1 specimen	170	3
Testing 100 specimens	9090	300
Testing 300 specimens	27090	900

Note: (1) In D3 and the detection system of the present invention, the index microsphere ratio (the number of each color microsphere in the detection system) is the same, both are 1000; (2) using D3 technology, each sample is analyzed for 20 seconds, The instrument is cleaned for 60 seconds. It takes an average of 10 seconds to manually replace the next test sample, and 1 control per batch. (3) Using the technique of the present invention, each sample is measured once before and after each reaction. Each measurement is 1 second, and the next test is automatically replaced by the instrument, which takes only 1 second, without the need for an additional control test.

The above-described embodiments of the present invention are merely illustrative of the invention, and are not intended to limit the embodiments of the invention. Other variations and modifications of the various forms can be made by those skilled in the art in light of the above description. It is not possible to exhaust all implementations here. Obvious changes or variations that come within the scope of the present invention are still within the scope of the invention.

Claims (11)

1. A particle chromaticity clustering analysis method, comprising the steps of:

   S1: Construction of biological target detection reagents

   Color granules of different chromaticity are coated with different biological probes I to form colored microparticle-bioprobe I polymer, and different colored microparticle-bioprobe I polymer is used for different biological targets to be tested. Specific binding reaction; mixing M kinds of colored microparticle-bioprobe I polymer to form a biological target detection reagent, which can simultaneously specifically bind to M different biological targets in the specimen to be tested a reaction; wherein, M is a natural number ≥1;

   S2: performing a biological detection reaction

   The biological target detection reagent, the sample to be tested, and the N separation reagents are added to the reaction cup I to form a reaction suspension, and the biological detection reaction is performed to form a colored microparticle-bioprobe I-subject biological target-separating reagent combination ;N is a natural number ≥1;

   S3: separation of biological detection reaction products

   Separating all the reaction products generated in the step S2 from the colored microparticle-bioprobe I polymer in which no binding reaction occurs in the detection reagent;

   S4: Determination of test results

   The color microparticle-bioprobe I polymer involved in the biological reaction separated in step S3 is clustered and counted according to the color particle chromaticity thereof, and the statistical detection is performed according to the one-to-one correspondence between the color particle chromaticity and the biological target species. The type and content of the biological target to be tested contained in the specimen;

   And/or, the color microparticle-bioprobe I polymer remaining after separating the reaction product in step S3 is clustered and counted according to the color particle chromaticity thereof, and all the colored microparticle-bioprobe I polymer in the reagent The difference between the type and the quantity is obtained, and the clustering count of the colored microparticle-bioprobe I polymer participating in the reaction in step S3 is obtained, and then the one-to-one correspondence between the color particle chromaticity and the type of the biological target to be tested is determined. The type and content of the biological target to be tested contained in the specimen.
2. The microparticle color clustering analysis method according to claim 1, wherein the specific separation method of step S3 is: adsorption of all separation reagents by magnetic needles and color microparticle-bioprobe I-testing biological target-separating reagent combination And placed in the reaction cup II, then add the bio-probe - the biological target depolymerization agent to be tested, and then use the magnetic needle to adsorb and discard all the magnetic components, leaving the colored microparticle-bioprobe I polymer in the reaction cup II In step S4;

   Alternatively, an external magnetic field is used to adsorb all the separation reagents as well as the color microparticle-bioprobe I-test biological target-separating reagent conjugate in the reaction cup I, and the color microparticle-bioprobe I polymer not combined with the separation reagent Transfer to the cuvette III with a pipette, and proceed to step S4.
3. The microparticle chroma clustering analysis method according to claim 1, wherein the clustering count is to disperse the color microparticle-bioprobe I polymer in a single layer, and then perform large-field microscopic color photographing. The resulting photograph is image-processed, the chromaticity of all the micro-particles is extracted, and the clustering is performed according to the chromaticity.
4. The microparticle chroma clustering analysis method according to claim 3, wherein the color particle sub-cluster counting method used in step S4 is: depositing the color microparticle-bioprobe I polymer obtained in step S3 by using a filtering technique. On the filter screen, it is arranged in a single layer; then the large field of view microscopic color photographing, the obtained photograph is processed by image processing, the chromaticity of all the microparticles is extracted, and the clustering is performed according to the chromaticity; the colored microparticles participating in the reaction - The number of species of the bio-probe I polymer reflects the number of species of the biological target to be tested in the specimen to be tested, the chromaticity of the colored microparticles in the color microparticle-bioprobe I polymer corresponds to the species of the biological target to be tested, and each chromaticity The number of microparticles corresponds to the content of the biological target to be tested.
5. The microparticle color clustering analysis method according to claim 1, wherein the biological target is an antibody, an antigen, a ligand, a receptor, an oligonucleotide fragment, a cell, a virus particle, and an immune complex. At least one.
6. The microparticle color clustering analysis method according to claim 1, wherein the separation reagent is a magnetic material coated with a bioprobe II capable of specifically binding to a biological target to be tested, and has specific recognition. At least one of the biological probe III coated magnetic material of the foregoing biological target-bioprobe I-color microparticle complex and the magnetic material coated with the M biological target to be tested.
7. The microparticle chroma clustering analysis method according to claim 6, wherein the bioprobe I, the bioprobe II and the bioprobe III are antibodies, antigens, ligands, receptors, and oligonucleosides. At least one of an acid fragment and a peptide nucleic acid.
8. The fine particle color clustering analysis method according to claim 1, wherein the color fine particles have a diameter of micro-order and ≤ 100 μm.
9. A microparticle kit for use in a method of particle size cluster analysis, characterized in that the microparticle kit comprises the biological target detection reagent constructed in S1 of claim 1.
10. The use according to claim 8, further comprising the separation reagent of claim 5.
11. The use according to claim 8, further comprising a biological probe - the biological target depolymerizing agent to be tested.