WO2021258618A1 - 一种生物样品检测方法及检测试剂盒 - Google Patents

一种生物样品检测方法及检测试剂盒 Download PDF

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WO2021258618A1
WO2021258618A1 PCT/CN2020/128738 CN2020128738W WO2021258618A1 WO 2021258618 A1 WO2021258618 A1 WO 2021258618A1 CN 2020128738 W CN2020128738 W CN 2020128738W WO 2021258618 A1 WO2021258618 A1 WO 2021258618A1
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binding substance
analyte
binding
marker
magnetic
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French (fr)
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王哲
柳可
熊贵
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瑞芯智造(深圳)科技有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/5375—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by changing the physical or chemical properties of the medium or immunochemicals, e.g. temperature, density, pH, partitioning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4737—C-reactive protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/585—Calcitonins

Definitions

  • the invention belongs to the technical field of biomolecule detection, and specifically relates to a method for quantitative detection of biological samples.
  • enzyme-linked immunoassay is currently the most widely used immunoassay method.
  • the method combines the specificity of the antigen-antibody reaction with the action of the enzyme-catalyzed substrate by labeling the secondary antibody with the enzyme, and judging the test result according to the color change of the substrate after the enzyme acts on the substrate, and the sensitivity can reach the level of ng.
  • Commonly used enzymes for labeling include horseradish peroxidase (HRP), alkaline phosphatase (AP) and so on. Because enzyme-linked immunosorbent assay does not require special equipment and is simple to detect, it is widely used in disease detection. Commonly used methods are indirect method, sandwich method and BAS-ELISA.
  • the indirect method is to hold the protein to be tested in the well plate, then add the primary antibody, the enzyme-labeled secondary antibody, and the substrate to develop color, and then quantitatively detect the antigen through an instrument (such as a microplate reader).
  • This method is simple to operate but has poor specificity due to high background. It has been gradually replaced by the sandwich method.
  • the sandwich method uses two primary antibodies to capture and fix the target antigen, which greatly improves the specificity of the reaction while ensuring sensitivity.
  • the current general sandwich method requires one end to be connected to a group that can directly or indirectly emit light, which is limited by the solution environment and the sensitivity of the detector.
  • Quanterix has developed a method that can simultaneously detect thousands of individual protein molecules. Using the same reagents as conventional ELISA, this method has been used to measure proteins in various matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) in femtomolar (fg/mL) concentrations, thereby reducing The sensitivity has been increased by approximately 1000 times.
  • This method utilizes soaring-sized reaction chamber arrays, which are called single-molecule arrays (Simoa TM ), which can isolate and detect individual enzyme molecules. Because the volume of the array is about 2 billion times smaller than that of the conventional ELISA, if the labeled protein is present, the fluorescent product will be quickly generated.
  • the purpose of the present invention is to provide a quantitative detection method for biological samples.
  • Another object of the present invention is to provide a quantitative detection kit for biological samples.
  • a biological sample detection method which is characterized in that it comprises the following steps:
  • first binding substance and the second binding substance respectively specifically bind to the first binding site and the second binding site of the analyte in the sample.
  • the method of step (2) is: under the control of a magnetic field, the magnetic beads in the mixture are adsorbed, and the unbound label modified with the second binding substance in the mixture is removed.
  • the intensity of the magnetic field is not less than 0.0001T, preferably 0.0001T-0.1T;
  • the separation of the label and the magnetic beads can be to disconnect the magnetic beads from the first binding substance, or to disconnect the first binding substance from the analyte, or to disconnect the second binding substance.
  • the specific method can be:
  • the dissociating agent can be a citric acid buffer, a strong acid buffer, or a strong alkaline buffer. Liquid, or other chemical dissociation agent;
  • the specific decomposition enzyme decomposes the first conjugate, the analyte, and the second conjugate;
  • step (4) uses magnetic field adsorption to remove magnetic beads in the sample
  • the counting of markers in step (4) is detected by nanopore counting method, or NTA detection method, or nanoflow counting method, or digital fluorescence counting method.
  • the marker is a particle with a diameter of 10 nm to 1000 nm.
  • the label is polyethylene microspheres, silica microspheres, nano-gold or fluorescent microspheres.
  • the first binding substance or the second binding substance is an antigen, an antibody, a receptor, or an aptamer.
  • the modification of the first conjugate and the magnetic bead, or the modification of the second conjugate and the label is the bonding of the nitrobenzyl group to the DNA molecular chain, or the modification of the dithioethylamino group and the carboxyl group
  • the bond is either the bond between sulfonate and oligonucleotide, or the bond between 6C or 12C organic molecular chain and biotin.
  • the 6C or 12C organic molecular chain may be a hexaalkyl chain or a dodecyl chain.
  • the analyte is one or more than one
  • the first binding substance is one or more than one, and each first binding substance specifically binds to the first binding site of the corresponding analyte, and the first binding substance is modified on the surface of the magnetic bead ;
  • the second binding substances are one or more than one kind, each second binding substance specifically binds to the second binding site of the corresponding analyte, and each second binding substance is modified to different The surface of the markers, each marker has a measurable difference in size and/or surface potential;
  • the types of different markers are distinguished and counted, so as to obtain the type and content of the analyte in the sample.
  • the difference in diameter of each marker is more than 5%, or the difference in surface potential is more than 10%.
  • this technical solution detects the size and/or surface potential of the markers in the sample through nanopores with different pore diameters to distinguish and count the types of different markers, so as to obtain the type and concentration of the analyte in the sample.
  • the analyte is one or more than one
  • the first binding substance is one or more than one, each first binding substance specifically binds to the first binding site of the corresponding analyte, and each first binding substance is modified in a different On the surface of the magnetic beads, each magnetic bead has a measurable difference in size and/or magnetic induction;
  • the second binding substances are one or more than one kind, each second binding substance specifically binds to the second binding site of the corresponding analyte, and each second binding substance is modified to different The surface of the markers, each marker has a measurable difference in size and/or surface potential;
  • step (3) by magnetic field gradient, magnetic field deflection or centrifugal method, magnetic beads of different sizes or different magnetic induction intensities are grouped;
  • the types of different markers are distinguished and counted according to the size and/or surface potential of the markers, so as to obtain the type and content of the analyte in the sample, so as to realize the simultaneous detection of different analytes to a greater extent.
  • the diameter difference of each type of magnetic beads is more than 5%, or the magnetic induction intensity difference is more than 5%;
  • the difference in diameter of each marker is more than 5%, or the difference in surface potential is more than 10%.
  • a biological sample detection kit which includes:
  • the magnetic beads modified with the first binding substance, the first binding substance can specifically bind to the first binding site of the analyte
  • a label modified with a second binder which can specifically bind to the second binding site of the analyte.
  • the marker is a particle with a diameter of 10 nm to 1 um.
  • the label is polyethylene microspheres, silica microspheres, nano-gold or fluorescent microspheres.
  • the first binding substance or the second binding substance is an antigen, an antibody, a receptor, or an aptamer.
  • the modification of the first conjugate and the magnetic bead, or the modification of the second conjugate and the label is the bonding of the nitrobenzyl group to the DNA molecular chain, or the modification of the dithioethylamino group and the carboxyl group
  • the bond is either the bond between sulfonate and oligonucleotide, or the bond between 6C or 12C organic molecular chain and biotin.
  • the 6C or 12C organic molecular chain may be a hexaalkyl chain or a dodecyl chain.
  • the analyte is one or more than one
  • the first binding substance is one or more than one, and each first binding substance specifically binds to the first binding site of the corresponding analyte, and the first binding substance is modified on the surface of the magnetic bead ;
  • the second binding substances are one or more than one kind, each second binding substance specifically binds to the second binding site of the corresponding analyte, and each second binding substance is modified to different The surface of the markers, each marker has a measurable difference in size and/or surface potential;
  • the difference in diameter of each marker is more than 5%, or the difference in surface potential is more than 10%.
  • the analyte is one or more than one
  • the first binding substance is one or more than one, each first binding substance specifically binds to the first binding site of the corresponding analyte, and each first binding substance is modified in a different On the surface of the magnetic beads, each magnetic bead has a measurable difference in size and/or magnetic induction;
  • the second binding substances are one or more than one kind, each second binding substance specifically binds to the second binding site of the corresponding analyte, and each second binding substance is modified to different The surface of the markers, each marker has a measurable difference in size and/or surface potential;
  • the diameter difference of each type of magnetic beads is more than 5%, or the magnetic induction intensity difference is more than 5%.
  • the difference in diameter of each marker is more than 5%, or the difference in surface potential is more than 10%.
  • the kit further includes a buffer, a dissociating agent, and a small magnet device, or a coil device for generating a magnetic field.
  • the present invention forms a ternary complex (magnetic bead-antigen-marker) by immunospecifically binding magnetic beads.
  • the ternary complex is collected under the action of a magnetic field.
  • the markers in the ternary immune complex are eluted, the markers and magnetic beads are separated under the action of a magnetic field, the eluate containing the markers is collected and the wash of the markers is redispersed with a strong electrolyte. Deliquation is then passed through a nanoparticle counter to achieve absolute quantification, as shown in Figure 2.
  • the advantage of the connection between the first binding substance and the magnetic beads is that the magnetic beads can be uniformly dispersed in the mixed solution, so that the first binding substance can fully bind to the analyte in the sample to be tested, and the capture rate is improved.
  • the ternary complex with magnetic beads can be enriched under the action of a magnetic field, which is convenient for subsequent steps to use for detection and counting; in addition, the "magnetic bead-antigen-marker" ternary complex is grouped in the magnetic field, The number of types of analytes that can be detected simultaneously can be further increased.
  • the present invention can detect the types and contents of multiple different analytes by using different markers in combination with different analytes, thereby realizing simultaneous detection of multiple targets.
  • the method of the present invention uses a nanoparticle counting method instead of an optical reading method, which can greatly increase the lower limit of detection. Counting particles can be read stably in a solution that is conducive to improving the signal-to-noise ratio of nanopore counting. This method can be used for counting in an environment where antigen and antibody are unstable.
  • the method of the present invention can detect trace proteins below the detection limit of conventional immunoassays, and can be widely used in immunological detection, microbial detection, cell separation and other fields.
  • Figure 1 is a schematic diagram of the structure of the "magnetic bead-antigen-marker" ternary complex of the present invention.
  • Figure 2 is a schematic diagram of the detection principle of the detection method of the present invention: 1 is the mixing of the magnetic beads modified with the first conjugate and the sample containing the analyte, and 2 is the first combination of the magnetic beads modified with the first conjugate and the analyte.
  • the binding site specifically binds
  • 3 is the addition of the label modified with the second binding substance to specifically bind to the second binding site of the analyte, and the unbound label modified with the second binding substance is removed under a magnetic field.
  • 4 To separate the label from the magnetic beads, collect the eluted label from the supernatant.
  • Figure 3 is a graph of the count curve obtained with different initial concentrations of troponin cTnI: the data dot (o) is the count value of the standard concentration sample, and the count value (x) of the measured sample can correspond to the corresponding concentration on the standard curve.
  • Figure 4 is a schematic diagram of the simultaneous detection of troponin cTnI and cTnT in Example 2: Different markers 1 and 2 are separated from magnetic beads, and the eluted markers 1 and 2 from the supernatant are collected. Marker 2 has a larger size than marker 1 and corresponds to a larger pulse signal, so that markers 1 and 2 can be distinguished.
  • Figure 5 shows the simultaneous detection and counting results of troponin cTnI and cTnT in Example 2: the pulse data of the mixture of markers 1 and 2, and a single pulse corresponds to a single marker particle.
  • Marker 2 has a larger size than marker 1 and corresponds to a larger pulse signal. Markers 1 and 2 can be distinguished by pulse value, so that markers 1 and 2 can be counted.
  • Figure 6 is a schematic diagram of the magnetic field separation of different magnetic beads: the direction of the magnetic field is orthogonal to the initial direction of the fluid.
  • the ternary complex is deflected under the action of a magnetic field.
  • the ternary complex of small magnetic beads flows out from the first outlet, and the ternary complex of large magnetic beads flows out from the second outlet because of its larger size and smaller deflection.
  • the detection of troponin cTnI is taken as an example to further illustrate the detection method of the present invention.
  • the immunomagnetic beads (Ademtech) were replaced with MES buffer three times, and the pH of the solution was 4-5 and the ionic strength was 0.1M.
  • 1mg nanoparticles polyethylene PS microspheres, diameter 200nm
  • a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, until EDC and NHS are in the solution
  • the final concentration is 0.2mg/mL.
  • the supernatant is discarded by centrifugation.
  • the precipitate after discarding the supernatant was reconstituted with the PBS solution, antibody 2 (4tc2-20c6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • a nanopore single particle counting device (model: qNano, Izon Science Ltd.) based on the Coulter principle (U.S. Patent 2,656, 508.1953) was used to count the nanoparticles dispersed in the KCl buffer in step (6).
  • samples to be tested with different concentrations of troponin cTnI (10 fg/ml, 100 fg/ml, 1000 fg/ml) can be eluted to obtain nanoparticle supernatants with corresponding concentrations.
  • the supernatant was counted by the method described above.
  • Figure 3 there are count data obtained with different initial concentrations of troponin cTnI at 10 fg/ml, 100 fg/ml, and 1000 fg/ml (Table 1).
  • the standard curve of the concentration and the number of counts can be obtained by counting.
  • the data dot (o) is the count value of the standard concentration sample, and the count value (x) of the measured sample can correspond to the corresponding concentration of 500fg/ml on the standard curve.
  • concentration constant A is obtained from the standard curve and is 0.42 ⁇ 0.02ml/pg.
  • troponin cTnI at a concentration of 500 fg/ml. After testing, the number of counts is 212, the concentration is 498fg/ml after conversion by the calculation formula, and the accuracy rate is more than 99%.
  • the simultaneous detection of troponin cTnI and troponin cTnT is taken as an example to further illustrate the detection method of the present invention.
  • the immunomagnetic beads (Ademtech) were replaced with MES buffer three times, and the pH of the solution was 4-5 and the ionic strength was 0.1M.
  • the precipitate after the supernatant was reconstituted with the MES buffer, and the capture troponin cTnI antibody 1 (a34600, biospacific) and the capture troponin cTnT antibody 1 (1C11, HyTest) were added, mixed and coated for 1 hour, then Add the terminator BSA (Bovine Serum Albumin) to a concentration of 1% to stop the reaction.
  • BSA Bovine Serum Albumin
  • 1mg nanoparticles polyethylene PS microspheres, 200 nanometers
  • a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, until the EDC and NHS are in the solution
  • the final concentration is 0.2mg/mL, after 30 minutes of activation, the supernatant is discarded by centrifugation.
  • the precipitate after discarding the supernatant was reconstituted with the PBS solution, antibody 2 (4tc2-20c6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • 1mg nanoparticles (silica microspheres, 100 nanometers) are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, until the EDC and NHS are in the solution
  • the final concentration is 0.2mg/mL, after 30 minutes of activation, the supernatant is discarded by centrifugation.
  • the precipitate after the supernatant was discarded was reconstituted with the PBS solution, antibody 2 (9G6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • the simultaneous detection of troponin cTnI, troponin cTnT, C-reactive protein (CRP) and procalcitonin (PCT) is taken as an example to further illustrate the detection method of the present invention.
  • the immunomagnetic beads (150 nm, Ademtech) were replaced with MES buffer three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • the immunomagnetic beads (150 nm, Ademtech) were replaced with MES buffer three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • the immunomagnetic beads (800 nm, Thermo Fisher, magnetic induction intensity equivalent to 150 nm Ademtech magnetic beads) were replaced with MES buffer solution three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • Add 1 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) dissolved in DMSO to the balanced solution. , After 20 minutes of reaction, centrifuge and discard the supernatant;
  • the immunomagnetic beads (800 nm, Thermo Fisher, magnetic induction intensity equivalent to 150 nm Ademtech magnetic beads) were replaced with MES buffer solution three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • Add 1 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) dissolved in DMSO to the balanced solution. , After 20 minutes of reaction, centrifuge and discard the supernatant;
  • 1mg nanoparticles polyethylene PS microspheres, 1000 nanometers
  • a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, until EDC and NHS are in the solution
  • the final concentration is 0.2mg/mL, after 30 minutes of activation, the supernatant is discarded by centrifugation.
  • the precipitate after discarding the supernatant was reconstituted with the PBS solution, cTnI antibody 2 (4tc2-20c6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • 1mg nanoparticles polyethylene PS microspheres, 1000 nanometers
  • a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, until EDC and NHS are in the solution
  • the final concentration is 0.2mg/mL, after 30 minutes of activation, the supernatant is discarded by centrifugation.
  • the precipitate after discarding the supernatant was reconstituted with the PBS solution, CRP antibody 2 (K1017, Okaybio) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • 1mg nanoparticles (nano-gold, 10 nanometers) are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then EDC and NHS are added separately until the final concentrations of EDC and NHS in the solution are both 0.2mg/mL, after 30 minutes of activation, centrifuge to discard the supernatant.
  • the precipitate after discarding the supernatant was reconstituted with the PBS solution, cTnT antibody 2 (9G6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • 1mg nanoparticles (nano-gold, 10 nanometers) are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then EDC and NHS are added separately until the final concentrations of EDC and NHS in the solution are both 0.2mg/mL, after 30 minutes of activation, centrifuge to discard the supernatant.
  • the precipitate after discarding the supernatant was reconstituted with the PBS solution, PCT antibody 2 (K58w3, Okaybio) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • the simultaneous detection of troponin cTnI, troponin cTnT, C-reactive protein (CRP) and procalcitonin (PCT) is taken as an example to further illustrate the detection method of the present invention.
  • the immunomagnetic beads (150 nm, Ademtech) were replaced with MES buffer three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • the immunomagnetic beads (150 nm, Ademtech) were replaced with MES buffer three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • the immunomagnetic beads (800 nm, Thermo Fisher, magnetic induction intensity equivalent to 150 nm Ademtech magnetic beads) were replaced with MES buffer solution three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • Add 1 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) dissolved in DMSO to the balanced solution. , After 20 minutes of reaction, centrifuge and discard the supernatant;
  • the immunomagnetic beads (800 nm, Thermo Fisher, magnetic induction intensity equivalent to 150 nm Ademtech magnetic beads) were replaced with MES buffer solution three times, and the solution was balanced to pH 4-5 and ionic strength 0.1M.
  • Add 1 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) dissolved in DMSO to the balanced solution. , After 20 minutes of reaction, centrifuge and discard the supernatant;
  • 1mg nanoparticles polyethylene PS microspheres, 200nm, surface potential -50mV are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, to EDC and The final concentration of NHS in the solution was 0.2 mg/mL. After 30 minutes of activation, the supernatant was discarded by centrifugation. The precipitate after discarding the supernatant was reconstituted with the PBS solution, cTnI antibody 2 (4tc2-20c6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • cTnI antibody 2 (4tc2-20c6, HyTest
  • 1mg nanoparticles (polyethylene PS microspheres, 200 nanometers, surface potential -20mV) are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, to EDC and The final concentration of NHS in the solution was 0.2 mg/mL. After 30 minutes of activation, the supernatant was discarded by centrifugation. The precipitate after discarding the supernatant was reconstituted with the PBS solution, CRP antibody 2 (K1017, Okaybio) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • CRP antibody 2 K1017, Okaybio
  • 1mg nanoparticles (polyethylene PS microspheres, 200nm, surface potential -50mV) are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, to EDC and The final concentration of NHS in the solution was 0.2 mg/mL. After 30 minutes of activation, the supernatant was discarded by centrifugation. The precipitate after discarding the supernatant was reconstituted with the PBS solution, cTnT antibody 2 (9G6, HyTest) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • cTnT antibody 2 (9G6, HyTest
  • 1mg nanoparticles polyethylene PS microspheres, 200 nanometers, surface potential -20mV are diluted in a 0.1M PBS solution to a final concentration of 0.1mg/mL of nanoparticles; then add EDC and NHS, respectively, to EDC and The final concentration of NHS in the solution was 0.2 mg/mL. After 30 minutes of activation, the supernatant was discarded by centrifugation. The precipitate after discarding the supernatant was reconstituted with the PBS solution, PCT antibody 2 (K58w3, Okaybio) was added, labeled for 25 minutes, and BSA was added to the concentration of 0.5% to stop the reaction.
  • PCT antibody 2 K58w3, Okaybio
  • the above mixing passes through the bifurcated pipeline as shown in the figure below, enters from the left nozzle, and flows out from the three nozzles on the right and upper side.
  • a constant magnetic field of 0.001T which is generated by a winding coil with a constant current, and the direction of the magnetic field is upward.
  • the ternary composite composed of magnetic beads is deflected under the action of a magnetic field.
  • the ternary composite of 150 nanometer magnetic beads flows out from the first exit, and the ternary composite of 800 nanometer magnetic beads is relatively small due to its larger size. Flow out from the second outlet. So as to realize the grouping according to the size of the magnetic beads.
  • Dissociate after separation The dissociated marker nanoparticles are counted, and the charge measurement is used for statistics of different markers.
  • the specific method is to use a single particle surface potential meter for single particle potential measurement (model: qNano, Izon Science Ltd.).
  • the method of the present invention can detect trace proteins below the detection limit of conventional immunoassays, and can be widely used in immunological detection, microbial detection, cell separation and other fields.
  • the present invention can detect the types and contents of multiple different test substances by using different markers in combination with different test substances, and realize the simultaneous detection of multiple targets.

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Abstract

一种生物样品检测方法及检测试剂盒，该方法通过免疫特异性结合磁珠的方式，形成三元免疫复合物——磁珠-抗原-标记物，在磁场作用下收集三元免疫复合物。在特定化学试剂下，将三元免疫复合物中的标记物洗脱下来，在磁场作用下将标记物和磁珠分离，收集包含标记物的洗脱液并用强电解质重新分散。检测标记物的洗脱液进而通过纳米颗粒计数器从而实现绝对定量。该方法可以检出常规免疫检测检出下限以下的痕量蛋白，在免疫学检测、微生物检测、细胞分离等领域可广泛应用。

Description

一种生物样品检测方法及检测试剂盒 技术领域
本发明属于生物分子检测技术领域，具体涉及一种生物样品的定量检测方法。
背景技术
疾病的发生、发展与蛋白质的异常表达或特定蛋白表达密切相关。准确测定疾病相关蛋白的含量在传染病防控、癌症筛查和精准诊断等方面具有重要意义。
几种常用的免疫学技术，酶联免疫检测是目前应用最广泛的免疫检测方法。该方法是将二抗标记上酶，抗原抗体反应的特异性与酶催化底物的作用结合起来，根据酶作用底物后的显色颜色变化来判断试验结果，其敏感度可达ng水平。常见用于标记的酶有辣根过氧化物酶(HRP)、碱性磷酸酶(AP)等。由于酶联免疫法无需特殊的仪器，检测简单，因此被广泛应用于疾病检测。常用的方法有间接法、夹心法以及BAS－ELISA。间接法是先将待测的蛋白抱被在孔板内，然后依次加入一抗、标记了酶的二抗和底物显色，通过仪器(例如酶标仪)定量检测抗原。这种方法操作简单但由于高背景而特异性较差。目前已逐渐被夹心法取代。夹心法利用二种一抗对目标抗原进行捕获和固定，在确保灵敏度的同时大大提高了反应的特异性。目前通用的夹心法需要一端连接可以直接或者间接发光的基团，会受到溶液环境和检测器灵敏度的限制。
最近，Quanterix开发了一种可同时检测数千个单个蛋白质分子的方法。使用与常规ELISA相同的试剂，该方法已用于以飞摩尔(fg/mL)浓度测量各种不同基质(血清，血浆，脑脊髓液，尿液，细胞提取物等)中的蛋白质，从而将灵敏度大约提高了1000倍。这种方法利用了飞升大小的反应室阵列，这些阵列称为单分子阵列(Simoa TM)，可以隔离和检测单个酶分子。因为阵列体积比常规ELISA小约20亿倍，所以如果存在标记的蛋白质，则会快速生成荧光产物。随着扩散的消除，可以容易地观察到这种高的局部产物浓度。只需一个分子即可达到检测极限。该方法也被定义为数字ELISA。但是，Simoa方法还是依赖于计算每个反应室的光学信号强度通过柏松公式推算浓度，还不能实现完全的绝对定量。
发明内容
针对现有技术中所存在的不足，本发明的目的在于提供一种生物样品的定量检测方法。
本发明的另一个目的是提供一种生物样品的定量检测试剂盒。
本发明所采取的技术方案是：
一种生物样品检测方法，其特征在于：包括如下步骤：
(1)将修饰有第一结合物的磁珠与含有待分析物的样品、及修饰有第二结合物的标记物混合，得混合液；
(2)除去混合液中未结合的修饰有第二结合物的标记物；
(3)将混合液中的标记物与磁珠分离；
(4)去除样品中的磁珠，通过对标记物进行计数和/或电荷、粒径测量得出样品中待分析物的含量以及种类；
其中，第一结合物和第二结合物分别与所述样品中的待分析物的第一结合位点、第二结合位点特异性结合。
作为优选的：步骤(2)的方法为：在磁场的控制下，吸附住混合物中的磁珠，除去混合液中未结合的修饰有第二结合物的标记物。其中，磁场的强度不低于0.0001T，优选为0.0001T-0.1T；
作为优选的：步骤(3)中，标记物和磁珠的分离可以是断开磁珠与第一结合物的连接，或者断开第一结合物与待分析物的结合，或者断开第二结合物与待分析物的结合，或者断开第二结合物与标记物的连接；
具体的方法可以为：
1)利用解离剂，解离磁珠与第一结合物的连接或第二结合物与标记物的连接，所述解离剂可以是柠檬酸缓冲液、强酸性缓冲液、强碱性缓冲液，或者是其他的化学解离剂；
或者2)使用第一结合物的竞争分子、待分析物的竞争分子、或第二结合物的竞争分子使得标记物被从磁珠上替换下来；
或者3)特异性分解酶分解第一结合物、待分析物、第二结合物；
或者4)采用以上方法的组合。
作为优选的：步骤(4)使用磁场吸附，去除样品中的磁珠；
作为优选的，步骤(4)中标记物的计数采用纳米孔计数法、或者NTA检测法、或者纳米流式计数法、或者数字荧光计数法检测。
作为优选的：所述标记物为直径在10nm～1000nm的颗粒。
进一步优选的：所述的标记物为聚乙烯微球，二氧化硅微球，纳米金或者荧光微球。
作为优选的：第一结合物或第二结合物为抗原、抗体、受体、或者是适配体。
作为优选的：第一结合物与磁珠的修饰方式，或者第二结合物与标记物的修饰方式为硝基苄基与DNA分子链的键合，或者是二硫代乙基氨基与羧基的键合，或者是磺酸盐与寡核 苷酸的键合，或者是6C或12C有机分子链与生物素的键合。
作为优选的：所述6C或12C有机分子链可以是六烷基链或者十二烷基链。
本发明的另一个技术方案为：
所述待分析物为一种或一种以上；
对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，第一结合物修饰在磁珠的表面；
对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
根据标记物的尺寸和/或表面电位，来区分不同标记物的种类并计数，从而得到样品中待分析物的种类和含量。
作为优选的：每种标记物的直径差异5％以上，或者表面电位差异10％以上。
作为优选的：本技术方案通过不同孔径的纳米孔检测样品中标记物的尺寸和/或表面电位，来区分不同标记物的种类并计数，从而得到样品中待分析物的种类和浓度。
本发明的另一个技术方案为：
所述待分析物为一种或一种以上；
对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，每种第一结合物分别修饰在不同的磁珠表面，每种磁珠在尺寸和/或磁感应强度上有可测量的差异；
对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
在步骤(3)之前，通过磁场梯度、磁场偏转或者离心的方法，将不同尺寸或者不同磁感应强度的磁珠分组；
在不同分组中根据标记物的尺寸和/或表面电位，来区分不同标记物的种类并计数，从而得到样品中待分析物的种类和含量，从而更大限度实现不同待测物的同时检测。
作为优选的：每种磁珠的直径差异在5％以上，或者磁感应强度差异在5％以上；
作为优选的：每种标记物的直径差异在5％以上，或者表面电位差异10％以上。
一种生物样品检测试剂盒，其包括：
a.修饰有第一结合物的磁珠，第一结合物能够与待分析物的第一结合位点特异性结合；
b.修饰有第二结合物的标记物，第二结合物能够与待分析物的第二结合位点特异性结合。
作为优选的：所述标记物为直径在10nm～1um的颗粒。
进一步优选的：所述的标记物为聚乙烯微球，二氧化硅微球，纳米金或者荧光微球。
作为优选的：第一结合物或第二结合物为抗原、抗体、受体、或者是适配体。
作为优选的：第一结合物与磁珠的修饰方式，或者第二结合物与标记物的修饰方式为硝基苄基与DNA分子链的键合，或者是二硫代乙基氨基与羧基的键合，或者是磺酸盐与寡核苷酸的键合，或者是6C或12C有机分子链与生物素的键合。
作为优选的：所述6C或12C有机分子链可以是六烷基链或者十二烷基链。
本发明试剂盒的另一个技术方案为：
所述待分析物为一种或一种以上；
对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，第一结合物修饰在磁珠的表面；
对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
作为优选的：每种标记物的直径差异5％以上，或者表面电位差异10％以上。
本发明试剂盒的另一个技术方案为：
所述待分析物为一种或一种以上；
对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，每种第一结合物分别修饰在不同的磁珠表面，每种磁珠在尺寸和/或磁感应强度上有可测量的差异；
对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
作为优选的：每种磁珠的直径差异在5％以上，或者磁感应强度差异在5％以上。
作为优选的：每种标记物的直径差异在5％以上，或者表面电位差异在10％以上。
进一步优选的：所述试剂盒还包括缓冲液、解离剂以及小型磁铁装置，或者用于产生磁场的线圈装置。
本发明的有益效果是：
1、本发明通过免疫特异性结合磁珠的方式，形成三元复合物(磁珠-抗原-标记物)， 如图1所示，在磁场作用下收集三元复合物。在特定化学试剂下，将三元免疫复合物中的标记物洗脱下来，在磁场作用下将标记物和磁珠分离，收集包含标记物的洗脱液并用强电解质重新分散检测标记物的洗脱液进而通过纳米颗粒计数器从而实现绝对定量，如图2所示。
2、本发明方法中，第一结合物与磁珠连接的优势在于：磁珠可以均匀分散在混合液中，能够使第一结合物充分结合待测样品中的待测物，提高捕获率，提高检测下限；同时带磁珠的三元复合物能够在磁场的作用下进行富集，便于后续步骤使用检测计数；此外“磁珠-抗原-标记物”三元复合物在磁场中的分组，可以更进一步增加同时检测的待分析物的种类数。
3、标记物与磁珠的多种解离方式的组合运用的优势在于：可以最大限度地提高解离率。
4、本发明通过使用不同的标记物结合不同的待测物的方法，可以检测多个不同待测物的种类和含量，实现多靶标的同时检测。
5、本发明方法使用纳米颗粒计数法，不使用光学读取的方法，可以极大的提高检出下限。计数颗粒可以在有利于提高纳米孔计数信噪比的溶液中稳定读取，该方法可以在抗原抗体不稳定的环境中进行计数。
6、本发明方法可以检出常规免疫检测检出下限以下的痕量蛋白，在免疫学检测、微生物检测、细胞分离等领域可广泛应用。
附图说明
图1为本发明“磁珠-抗原-标记物”三元复合物结构示意图。
图2为本发明检测方法的检测原理示意图：1为修饰有第一结合物的磁珠与含有待分析物的样品混合，2为修饰有第一结合物的磁珠与待分析物的第一结合位点特异性结合，3为加入修饰有第二结合物的标记物与待分析物的第二结合位点特异性结合，在磁场下去除未结合的修饰有第二结合物的标记物4为标记物与磁珠分离，收集上清中洗脱下来的标记物。
图3为不同初始浓度肌钙蛋白cTnI得到的计数曲线图：其中数据圆点(o)为标准浓度样本计数值，实测样本的计数值(x)可在标准曲线上对应相应浓度。
图4为实施例2肌钙蛋白cTnI和cTnT同时检测示意图：不同标记物1和2与磁珠分离，收集上清中洗脱下来的标不同标记物1和2。标记物2较标记物1有较大的尺寸，对应较大的脉冲信号，以此来区分标记物1和2。
图5为实施例2肌钙蛋白cTnI和cTnT同时检测计数结果：标记物1和2混合物的脉冲数据，单个脉冲对应单个标记物颗粒。标记物2较标记物1有较大的尺寸，对应较大的脉冲信号，通过脉冲值可以区分标记物1和2，从而实现标记物1和2的计数。
图6为不同磁珠的磁场分离示意图：磁场方向和流体初始方向正交。三元复合物在磁场 作用下发生偏转，小磁珠的三元复合物从第一个出口流出，大磁珠的三元复合物因为尺寸较大，偏转比较小，从第二个出口流出。
具体实施方式
为了能够更清楚地理解本发明的技术内容，特举以下实施例结合附图详细说明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
本实施例以肌钙蛋白cTnI的检测为例，对本发明的检测方法作进一步的说明。
1、制备抗体1标记的免疫磁珠
免疫磁珠(Ademtech)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获肌钙蛋白抗体1(a34600，biospacific)，混合包被1小时，然后加入终止剂BSA(牛血清白蛋白)至BSA的浓度为1％，终止反应。
2、制备抗体2标记的纳米颗粒
lmg纳米颗粒(聚乙烯PS微球，直径200纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入抗体2(4tc2-20c6，HyTest)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
3、肌钙蛋白cTnI检测方法
(1)将不同浓度的肌钙蛋白cTnI待检测样本(10fg/ml，100fg/ml,1pg/ml)与抗体1标记的免疫磁珠混合，在37℃孵育1h；
(2)在铷磁铁的磁场(磁场强度为0.0001T)吸引下，采用含0.02％tween20的PBS缓冲液(pH＝7.4)通过移液枪吸取去除上清液，反复3次；
(3)向其中加入抗体2标记的纳米颗粒，在37℃孵育1h；
(4)除去未结合的纳米颗粒：在磁场下，采用含0.02％tween20的PBS缓冲液(pH＝7.4)清洗3次去除上清液；
(5)向免疫三元复合物中先后加入解离液柠檬酸缓冲液(pH＝3)和NaOH溶液(pH＝13)洗脱纳米颗粒；
(6)磁场下吸取上清液重新分散在1M KCl缓冲液中。
(7)利用基于库尔特原理(U.S.Patent 2,656,508.1953)的纳米孔单颗粒计数装置(型号：qNano,Izon science Ltd.)对步骤(6)分散于KCl缓冲液中的纳米颗粒进行计数。
本实施例中，不同浓度的肌钙蛋白cTnI待检测样本(10fg/ml，100fg/ml，1000fg/ml)可以洗脱得到相应浓度的纳米颗粒上清液。将该上清液用上述方法进行计数。在图3中有10fg/ml，100fg/ml，1000fg/ml不同初始浓度肌钙蛋白cTnI得到的计数数据(表1)，通过计数可以得到浓度和计数数目的标准曲线。其中数据圆点(o)为标准浓度样本计数值，实测样本的计数值(x)可在标准曲线上对应相应浓度为500fg/ml。
表1 不同初始浓度肌钙蛋白cTnI得到的计数数据
肌钙蛋白cTnI浓度 计数数目
10fg/ml 4
100fg/ml 43
1000pg/ml 425
浓度计算公式：单位时间计数(N)＝浓度常数(A)*浓度(C)。这里浓度常数A由标准曲线得到，为0.42±0.02ml/pg。
进一步以浓度为500fg/ml的肌钙蛋白cTnI进行验证。经检测，计数数目为212，经计算公式换算后浓度为498fg/ml，准确率达99％以上。
实施例2
本实施例以肌钙蛋白cTnI和肌钙蛋白cTnT同时检测为例，对本发明的检测方法作进一步的说明。
1、制备多种抗体1标记的免疫磁珠
免疫磁珠(Ademtech)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获肌钙蛋白cTnI抗体1(a34600，biospacific)和捕获肌钙蛋白cTnT抗体1(1C11，HyTest)，混合包被1小时，然后加入终止剂BSA(牛血清白蛋白)至BSA的浓度为1％，终止反应。
2、制备抗体2标记的纳米颗粒
lmg纳米颗粒(聚乙烯PS微球，200纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗 粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入抗体2(4tc2-20c6，HyTest)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
lmg纳米颗粒(二氧化硅微球，100纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入抗体2(9G6，HyTest)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
3、肌钙蛋白cTnI和cTnT同时检测方法
洗脱方法在实施例1中一样的步骤之前，还通过添加肌钙蛋白分解酶的方法进行洗脱，得到两种标记物纳米颗粒的混合上清液，用于纳米孔计数。检测原理如图4所示，计数结果如图5所示，不同颗粒大小得到不同幅值的脉冲。检测结果如表2所示。
表2 不同初始浓度肌钙蛋白的检测数据
蛋白 初始浓度 标记物计数数目 换算浓度 准确率
cTnI 75fg/ml 31 72fg/ml 98％
cTnT 500fg/ml 210 485fg/ml 97％
实施例3
本实施例以肌钙蛋白cTnI，肌钙蛋白cTnT，C反应蛋白(CRP)和降钙素原(PCT)同时检测为例，对本发明的检测方法作进一步的说明。
1、制备多种抗体1标记的免疫磁珠
免疫磁珠(150纳米，Ademtech)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获肌钙蛋白cTnI抗体1(a34600，biospacific)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
免疫磁珠(150纳米，Ademtech)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获CRP抗体1(K1016，Okaybio)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
免疫磁珠(800纳米，赛默飞，磁感应强度与150纳米Ademtech磁珠相当)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获肌钙蛋白cTnT抗体1(1C11，HyTest)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
免疫磁珠(800纳米，赛默飞，磁感应强度与150纳米Ademtech磁珠相当)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获PCT抗体1(K3b8，Okaybio)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
2、制备抗体2标记的纳米颗粒
lmg纳米颗粒(聚乙烯PS微球，1000纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入cTnI抗体2(4tc2-20c6，HyTest)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
lmg纳米颗粒(聚乙烯PS微球，1000纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入CRP抗体2(K1017，Okaybio)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
lmg纳米颗粒(纳米金，10纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入cTnT抗体2(9G6，HyTest)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
lmg纳米颗粒(纳米金，10纳米)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入PCT抗体2(K58w3，Okaybio)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
3.四种蛋白的同时检测
(1)将四种蛋白样本与四种抗体1标记的免疫磁珠混合，在37℃孵育1h；
(2)在磁场(磁场强度0.0001T)吸引下，采用含0.02％tween20的PBS缓冲液(pH＝7.4)通过移液枪吸取去除上清液，反复3次；
(3)向其中加入四种抗体2标记的纳米颗粒，在37℃孵育1h；
(4)除去未结合的纳米颗粒：在磁场下，采用含0.02％tween20的PBS缓冲液(pH＝7.4)清洗3次去除上清液；
(5)磁场作用下的分离：上述混合物经过如图6所示的分叉管路，从左边管口进入，从右侧和上侧的三个管口流出。在管路路径上有由通上恒定电流的绕线线圈产生的大小为0.0001T的恒定磁场，磁场方向向上。在磁珠组成的三元复合物在磁场作用下发生偏转，150纳米磁珠的三元复合物从第一个出口流出，800纳米的磁珠三元复合物因为尺寸较大，偏转比较小，从第二个出口流出。从而实现了根据磁珠尺寸实现分组。
(6)分离后进行解离。解离的标记物纳米颗粒进行计数，根据粒径大小的测量用于不同标记物的统计。
检测结果如表3所示。
表3 不同初始浓度待检蛋白的检测数据
蛋白 初始浓度 标记物计数数目 换算浓度 准确率
cTnI 75fg/ml 31 72fg/ml 98％
cTnT 500fg/ml 210 485fg/ml 97％
CRP 100fg/ml 42 98fg/ml 98％
PCT 50fg/ml 22 52fg/ml 99％
实施例4
本实施例以肌钙蛋白cTnI，肌钙蛋白cTnT，C反应蛋白(CRP)和降钙素原(PCT)同时检测为例，对本发明的检测方法作进一步的说明。
1、制备多种抗体1标记的免疫磁珠
免疫磁珠(150纳米，Ademtech)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获肌钙蛋白cTnI抗体1(a34600，biospacific)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
免疫磁珠(150纳米，Ademtech)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获CRP抗体1(K1016，Okaybio)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
免疫磁珠(800纳米，赛默飞，磁感应强度与150纳米Ademtech磁珠相当)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获肌钙蛋白cTnT抗体1(1C11，HyTest)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
免疫磁珠(800纳米，赛默飞，磁感应强度与150纳米Ademtech磁珠相当)经MES缓冲液置换三次，平衡至溶液pH为4-5、离子强度0.1M。平衡后的溶液中分别加入DMSO溶解的EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)与NHS(N-羟基丁二酰亚胺)各1mg，反应20分钟后离心弃上清；
以所述MES缓冲液复溶弃上清后的沉淀物，加入捕获PCT抗体1(K3b8，Okaybio)，混合包被1小时，然后加入终止剂BSA(牛血清蛋白)至BSA的浓度为1％，终止反应。
2、制备抗体2标记的纳米颗粒
lmg纳米颗粒(聚乙烯PS微球，200纳米，表面电位-50mV)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入cTnI抗体2(4tc2-20c6，HyTest)，标记25分钟，加入BSA至其 浓度为0.5％终止反应。
lmg纳米颗粒(聚乙烯PS微球，200纳米，表面电位-20mV)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入CRP抗体2(K1017，Okaybio)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
lmg纳米颗粒(聚乙烯PS微球，200纳米，表面电位-50mV)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入cTnT抗体2(9G6，HyTest)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
lmg纳米颗粒(聚乙烯PS微球，200纳米，表面电位-20mV)在浓度为0.1M的PBS溶液中稀释至纳米颗粒的终浓度为0.1mg/mL；然后分别加入EDC与NHS，至EDC与NHS在溶液中的终浓度均为0.2mg/mL，活化30分钟后，离心弃上清。将弃上清之后的沉淀物以所述PBS溶液复溶，加入PCT抗体2(K58w3，Okaybio)，标记25分钟，加入BSA至其浓度为0.5％终止反应。
3.四种蛋白的同时检测
(1)将四种蛋白样本与四种抗体1标记的免疫磁珠混合，在37℃孵育1h；
(2)在磁场(磁场强度0.1T)吸引下，采用含0.02％tween20的PBS缓冲液(pH＝7.4)通过移液枪吸取去除上清液，反复3次；
(3)向其中加入四种抗体2标记的纳米颗粒，在37℃孵育1h；
(4)除去未结合的纳米颗粒：在磁场下，采用含0.02％tween20的PBS缓冲液(pH＝7.4)清洗3次去除上清液；
(5)磁场作用下的分离
上述混合经过如下图所示的分叉管路，从左边管口进入，从右侧和上侧的三个管口流出。在管路路径上有由通上恒定电流的绕线线圈产生的大小为0.001T的恒定磁场，磁场方向向上。在磁珠组成的三元复合物在磁场作用下发生偏转，150纳米磁珠的三元复合物从第一个出口流出，800纳米的磁珠三元复合物因为尺寸较大，偏转比较小，从第二个出口流出。从而实现了根据磁珠尺寸实现分组。
分离后进行解离。解离的标记物纳米颗粒进行计数，根据电荷的测量用于不同标记物的 统计。具体方式为用单粒子表面电位仪进行单粒子电位测量(型号：qNano,Izon science Ltd.)。
表4 不同初始浓度待检蛋白的检测数据
蛋白 初始浓度 标记物计数数目 换算浓度 准确率
cTnI 15fg/ml 7 18fg/ml 96％
cTnT 50fg/ml 23 54fg/ml 97％
CRP 10fg/ml 5 12fg/ml 96％
PCT 50fg/ml 22 52fg/ml 98％
由以上实施例可以看出：本发明方法可以检出常规免疫检测检出下限以下的痕量蛋白，在免疫学检测、微生物检测、细胞分离等领域可广泛应用。本发明通过使用不同的标记物结合不同的待测物的方法，可以检测多个不同待测物的种类和含量，实现多靶标的同时检测。
本领域的技术人员可以理解：在不脱离本发明的原理和宗旨的情况下，对这些实施例所进行的同等效果的修改和替换，均落入本发明权利要求的保护范围。

Claims (20)

  1. 一种生物样品检测方法，其特征在于：包括如下步骤：
    (1)将修饰有第一结合物的磁珠与含有待分析物的样品、修饰有第二结合物的标记物混合，得混合液；
    (2)除去混合液中未结合的修饰有第二结合物的标记物；
    (3)将步骤(2)处理后的混合液中的标记物与磁珠分离；
    (4)去除样品中的磁珠，通过对标记物进行计数和/或电荷、粒径测量得出样品中待分析物的含量以及种类；
    其中，第一结合物和第二结合物分别依次与所述待分析物的第一结合位点、第二结合位点特异性结合。
  2. 根据权利要求1的检测方法，其特征在于：步骤(2)的方法为：在磁场的控制下，吸附混合物中的磁珠，除去混合液中未结合的修饰有第二结合物的标记物，磁场的强度不低于0.0001T。
  3. 根据权利要求1所述的检测方法，其特征在于：步骤(3)中，标记物和磁珠的分离方法为：
    1)利用解离剂，解离磁珠与第一结合物的连接或第二结合物与标记物的连接；
    或者2)使用第一结合物的竞争分子、待分析物的竞争分子、或第二结合物的竞争分子使得标记物被从磁珠上替换下来；
    或者3)特异性分解酶分解第一结合物、待分析物或第二结合物；
    或者4)采用以上方法的组合。
  4. 根据权利要求1所述的检测方法，其特征在于：步骤(4)是通过磁场吸附，去除样品中的磁珠；标记物的计数采用纳米孔计数法、或者NTA检测法、或者纳米流式计数法、或者数字荧光计数法检测。
  5. 根据权利要求1所述的检测方法，其特征在于：所述标记物为直径在10nm～1000nm的颗粒。
  6. 根据权利要求5所述的检测方法，其特征在于：所述的标记物为聚乙烯微球，二氧化硅微球，纳米金或者荧光微球。
  7. 根据权利要求1所述的检测方法，其特征在于：第一结合物或第二结合物为抗原、抗体、受体、或者是适配体。
  8. 根据权利要求7所述的检测方法，其特征在于：第一结合物与磁珠的修饰方式，或者第二结合物与标记物的修饰方式为硝基苄基与DNA分子链的键合，或者是二硫代乙基氨基与羧基的键合，或者是磺酸盐与寡核苷酸的键合，或者是6C或12C有机分子链与生物素 的键合。
  9. 根据权利要求1-8任一项所述的检测方法，其特征在于：
    所述待分析物为一种或一种以上；
    对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，第一结合物修饰在磁珠的表面；
    对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
    根据标记物的尺寸和/或表面电位，来区分不同标记物的种类并计数，从而得到样品中待分析物的种类和含量。
  10. 根据权利要求9所述的检测方法，其特征在于，每种标记物的直径差异在5％以上，或者表面电位差异在10％以上。
  11. 根据权利要求1-8任一项所述的检测方法，其特征在于：
    所述待分析物为一种或一种以上；
    对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，每种第一结合物分别修饰在不同的磁珠表面，每种磁珠在尺寸和/或磁感应强度上有可测量的差异；
    对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸或者表面电位上有可测量的差异；
    在步骤(3)之前，通过磁场梯度、磁场偏转或者离心的方法，将不同尺寸或者不同磁感应强度的磁珠分组；
    在不同分组中根据标记物的尺寸和/或表面电位，来区分不同标记物的种类并计数，从而得到样品中待分析物的种类和含量，更大限度实现不同待测物的同时检测。
  12. 根据权利要求11所述的检测方法，其特征在于，每种磁珠的直径差异在5％以上，或者磁感应强度差异在5％以上；每种标记物的直径差异在5％以上，或者表面电位差异在10％以上。
  13. 一种生物样品检测试剂盒，其包括：
    a.修饰有第一结合物的磁珠，第一结合物能够与待分析物的第一结合位点特异性结合；
    b.修饰有第二结合物的标记物，第二结合物能够与待分析物的第二结合位点特异性结合。
  14. 根据权利要求13所述的生物样品检测试剂盒，其特征在于：所述标记物为直径在10nm～ 1000nm的颗粒。
  15. 根据权利要求14所述的生物样品检测试剂盒，其特征在于：所述的标记物为聚乙烯微球，二氧化硅微球，纳米金或者荧光微球。
  16. 根据权利要求13的生物样品检测试剂盒，其特征在于：第一结合物或第二结合物为抗原、抗体、受体、或者是适配体。
  17. 根据权利要求16所述的生物样品检测试剂盒，其特征在于：第一结合物与磁珠的修饰方式，或者第二结合物与标记物的修饰方式为硝基苄基与DNA分子链的键合，或者是二硫代乙基氨基与羧基的键合，或者是磺酸盐与寡核苷酸的键合，或者是6C或12C有机分子链与生物素的键合。
  18. 根据权利要求13-17任一项所述的生物样品检测试剂盒，其特征在于：
    所述待分析物为一种或一种以上；
    对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，第一结合物修饰在磁珠的表面；
    对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
    作为优选的，每种标记物的直径差异在5％以上，或者表面电位差异在10％以上。
  19. 根据权利要求13-17任一项所述的生物样品检测试剂盒，其特征在于：
    所述待分析物为一种或一种以上；
    对应地，所述第一结合物为一种或一种以上，每种第一结合物分别与对应的待分析物的第一结合位点特异性结合，每种第一结合物分别修饰在不同的磁珠表面，每种磁珠在尺寸和/或磁感应强度上有可测量的差异；
    对应地，所述第二结合物为一种或一种以上，每种第二结合物分别与对应的待分析物的第二结合位点特异性结合，每种第二结合物分别修饰到不同的标记物的表面，每种标记物在尺寸和/或表面电位上有可测量的差异；
    作为优选的，每种磁珠的直径差异在5％以上，或者磁感应强度差异在5％以上；
    作为优选的：每种标记物的直径差异在5％以上，或者表面电位差异在10％以上。
  20. 根据权利要求13-17任一项所述的生物样品检测试剂盒，其特征在于：所述试剂盒还包括缓冲液、解离剂以及小型磁铁装置，或者用于产生磁场的线圈装置。
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