Biological sample detection method and detection kit
阅读说明：本技术 一种生物样品检测方法及检测试剂盒 (Biological sample detection method and detection kit ) 是由 王哲 柳可 熊贵 于 2020-06-24 设计创作，主要内容包括：本发明公开了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,形成三元复合物(磁珠-抗原-标记物),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。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。(The invention discloses a biological sample detection method and a detection kit, wherein a ternary complex (magnetic bead-antigen-marker) is formed by combining immunospecificity with a magnetic bead, and the ternary complex is collected under the action of a magnetic field. Eluting the label in the ternary immune complex under the action of a specific chemical reagent, separating the label from the magnetic beads under the action of a magnetic field, collecting the eluate containing the label and re-dispersing the eluate by using a strong electrolyte. The eluate of the detection marker is then passed through a nanoparticle counter to achieve absolute quantitation. The method can detect trace protein below the lower limit of the conventional immunoassay, and can be widely applied to the fields of immunoassay, microbial detection, cell separation and the like.)
1. A method for detecting a biological sample, comprising: the method comprises the following steps:
(1) mixing the magnetic beads modified with the first conjugate with a sample containing an analyte and a label modified with a second conjugate to obtain a mixed solution;
(2) removing the unbound label modified with the second binding substance from the mixture;
(3) separating the label from the magnetic beads in the mixed solution treated in the step (2);
(4) removing magnetic beads in the sample, and obtaining the content and the type of an analyte in the sample by counting the markers and/or measuring the charges and the particle sizes;
wherein the first and second binding substances are specifically bound to the first and second binding sites of the analyte, respectively.
2. The detection method according to claim 1, characterized in that: the method in the step (2) comprises the following steps: and adsorbing the magnetic beads in the mixture under the control of a magnetic field, and removing the unbound label modified with the second conjugate from the mixed solution, wherein the strength of the magnetic field is not lower than 0.0001T.
3. The detection method according to claim 1, characterized in that: in the step (3), the separation method of the label and the magnetic bead comprises the following steps:
1) dissociating the connection between the magnetic beads and the first binding substance or the connection between the second binding substance and the label by using a dissociating agent;
or 2) displacing the label from the magnetic bead using the competitor molecule of the first binder, the competitor molecule of the analyte, or the competitor molecule of the second binder;
or 3) a specific lytic enzyme that cleaves the first binding substance, the analyte or the second binding substance;
or 4) a combination of the above methods.
4. The detection method according to claim 1, characterized in that: removing magnetic beads in the sample through magnetic field adsorption; counting of the markers is detected by nanopore counting, or NTA detection, or nano flow counting, or digital fluorescence counting.
5. The detection method according to claim 1, characterized in that: the marker is a particle with the diameter of 10 nm-1000 nm.
6. The detection method according to claim 5, characterized in that: the marker is polyethylene microsphere, silicon dioxide microsphere, nanogold or fluorescent microsphere.
7. The detection method according to claim 1, characterized in that: the first or second binding agent is an antigen, an antibody, a receptor, or an aptamer.
8. The detection method according to claim 7, characterized in that: the modification mode of the first combination and the magnetic beads or the modification mode of the second combination and the label is the bonding of nitrobenzyl and a DNA molecular chain, or the bonding of dithioethylamino and carboxyl, or the bonding of sulfonate and oligonucleotide, or the bonding of a 6C or 12C organic molecular chain and biotin.
9. The detection method according to any one of claims 1 to 8, characterized in that:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, and the first binding substances are modified on the surface of the magnetic beads;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
the different labels are distinguished and counted according to their size and/or surface potential, thereby obtaining the type and amount of analyte in the sample.
10. The detection method according to claim 9, wherein the difference in diameter of each of the markers is 5% or more, or the difference in surface potential is 10% or more.
11. The detection method according to any one of claims 1 to 8, characterized in that:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, each of the first binding substances is modified on the surface of a different magnetic bead, and each of the magnetic beads has a measurable difference in size and/or magnetic induction intensity;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size or surface potential;
before the step (3), grouping magnetic beads with different sizes or different magnetic induction intensities by a magnetic field gradient, magnetic field deflection or centrifugation method;
in different groups, the types of different markers are distinguished and counted according to the sizes and/or surface potentials of the markers, so that the types and the content of the analytes in the sample are obtained, and the simultaneous detection of different analytes is realized to a greater extent.
12. The detection method according to claim 11, wherein the difference in diameter of each magnetic bead is 5% or more, or the difference in magnetic induction is 5% or more; the difference in diameter of each marker is above 5%, or the difference in surface potential is above 10%.
13. A biological sample detection kit, comprising:
a. magnetic beads modified with a first binding compound capable of specifically binding to a first binding site of an analyte;
b. a label modified with a second binding substance capable of specifically binding to a second binding site of the analyte.
14. The biological sample detection kit according to claim 13, characterized in that: the marker is a particle with the diameter of 10 nm-1000 nm.
15. The biological sample detection kit according to claim 14, characterized in that: the marker is polyethylene microsphere, silicon dioxide microsphere, nanogold or fluorescent microsphere.
16. The biological sample detection kit according to claim 13, characterized in that: the first or second binding agent is an antigen, an antibody, a receptor, or an aptamer.
17. The biological sample detection kit according to claim 16, characterized in that: the modification mode of the first combination and the magnetic beads or the modification mode of the second combination and the label is the bonding of nitrobenzyl and a DNA molecular chain, or the bonding of dithioethylamino and carboxyl, or the bonding of sulfonate and oligonucleotide, or the bonding of a 6C or 12C organic molecular chain and biotin.
18. The biological sample detection kit according to any one of claims 13 to 17, characterized in that:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, and the first binding substances are modified on the surface of the magnetic beads;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
preferably, the difference in diameter of each marker is 5% or more, or the difference in surface potential is 10% or more.
19. The biological sample detection kit according to any one of claims 13 to 17, characterized in that:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, each of the first binding substances is modified on the surface of a different magnetic bead, and each of the magnetic beads has a measurable difference in size and/or magnetic induction intensity;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
preferably, the diameter difference of each magnetic bead is more than 5%, or the magnetic induction difference is more than 5%;
preferably, the method comprises the following steps: the difference in diameter of each marker is above 5%, or the difference in surface potential is above 10%.
20. The biological sample detection kit according to any one of claims 13 to 17, characterized in that: the kit also comprises a buffer solution, a dissociation agent and a small magnet device or a coil device for generating a magnetic field.
Technical Field
The invention belongs to the technical field of biomolecule detection, and particularly relates to a quantitative detection method for a biological sample.
Background
The occurrence and development of diseases are closely related to the abnormal expression of proteins or the expression of specific proteins. The accurate determination of the content of the disease-related protein has important significance in the aspects of infectious disease prevention and control, cancer screening, accurate diagnosis and the like.
Several commonly used immunological techniques, enzyme-linked immunoassay, are currently the most widely used immunological detection methods. The method combines the specificity of antigen-antibody reaction and the action of enzyme catalysis substrate, and judges the test result according to the color change of the enzyme after acting on the substrate, and the sensitivity can reach ng level. Common enzymes for labeling are horseradish peroxidase (HRP), Alkaline Phosphatase (AP), and the like. The enzyme-linked immunosorbent assay is widely applied to disease detection because special instruments are not needed and the detection is simple. Commonly used methods are indirect, sandwich and BAS-ELISA. The indirect method is to wrap the protein to be detected in a pore plate, then add primary antibody, secondary antibody marked with enzyme and substrate in sequence for color development, and quantitatively detect the antigen through an instrument (such as an enzyme-labeling instrument). This method is simple to operate but is less specific due to the high background. Has been gradually replaced by the sandwich method. The sandwich method utilizes two kinds of primary antibodies to capture and fix the target antigen, thereby greatly improving the specificity of the reaction while ensuring the sensitivity. Current sandwich methods require a group attached at one end that can emit light either directly or indirectly, and are limited by the solution environment and sensitivity of the detector.
Recently, Quanterix developed a method that can detect thousands of individual protein molecules simultaneously. This method has been used to measure proteins in various matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.) at femtomolar (fg/mL) concentrations using the same reagents as in conventional ELISA, thereby increasing the sensitivity by approximately 1000-fold. This method utilizes arrays of reaction chambers of the fly-up size, known as single molecule arrays (Simoa)TM) Single enzyme molecules can be isolated and detected. Because the array volume is about 20 hundred million times smaller than conventional ELISA, if labeled proteins are present, it will be fasterThe fluorescent product is rapidly generated. This high local product concentration can be easily observed with elimination of diffusion. Only one molecule is needed to reach the detection limit. This method is also defined as digital ELISA. However, the Simoa method still relies on calculating the optical signal intensity of each reaction chamber to estimate the concentration by the poisson formula, and complete absolute quantification cannot be achieved.
Disclosure of Invention
In view of the shortcomings of the prior art, the present invention aims to provide a method for quantitative detection of a biological sample.
Another object of the present invention is to provide a quantitative detection kit for biological samples.
The technical scheme adopted by the invention is as follows:
a method for detecting a biological sample, comprising: the method comprises the following steps:
(1) mixing the magnetic beads modified with the first conjugate, a sample containing an analyte and a marker modified with the second conjugate to obtain a mixed solution;
(2) removing the unbound label modified with the second binding substance from the mixture;
(3) separating the label from the magnetic beads in the mixed solution;
(4) removing magnetic beads in the sample, and obtaining the content and the type of an analyte in the sample by counting the markers and/or measuring the charges and the particle sizes;
wherein the first and second binding substances specifically bind to the first and second binding sites, respectively, of the analyte in the sample.
Preferably, the method comprises the following steps: the method in the step (2) comprises the following steps: and adsorbing the magnetic beads in the mixture under the control of a magnetic field, and removing the unbound label modified with the second binding substance in the mixed solution. Wherein the intensity of the magnetic field is not less than 0.0001T, preferably 0.0001T-0.1T;
preferably, the method comprises the following steps: in the step (3), the separation of the label and the magnetic bead may be to disconnect the magnetic bead from the first binder, or disconnect the first binder from the analyte, or disconnect the second binder from the label;
the specific method can be as follows:
1) dissociating the connection between the magnetic beads and the first binding substance or the connection between the second binding substance and the label by using a dissociating agent, wherein the dissociating agent can be a citric acid buffer solution, a strong alkaline buffer solution or other chemical dissociating agents;
or 2) displacing the label from the magnetic bead using the competitor molecule of the first binder, the competitor molecule of the analyte, or the competitor molecule of the second binder;
or 3) a specific decomposition enzyme decomposes the first binding substance, the analyte, and the second binding substance;
or 4) a combination of the above methods.
Preferably, the method comprises the following steps: removing magnetic beads in the sample by using magnetic field adsorption;
preferably, the counting of the labels in step (4) is performed by nanopore counting, NTA detection, nanoflow counting, or digital fluorescence counting.
Preferably, the method comprises the following steps: the marker is a particle with the diameter of 10 nm-1000 nm.
Further preferred is: the marker is polyethylene microsphere, silicon dioxide microsphere, nanogold or fluorescent microsphere.
Preferably, the method comprises the following steps: the first or second binding agent is an antigen, an antibody, a receptor, or an aptamer.
Preferably, the method comprises the following steps: the modification mode of the first combination and the magnetic beads or the modification mode of the second combination and the label is the bonding of nitrobenzyl and a DNA molecular chain, or the bonding of dithioethylamino and carboxyl, or the bonding of sulfonate and oligonucleotide, or the bonding of a 6C or 12C organic molecular chain and biotin.
Preferably, the method comprises the following steps: the 6C or 12C organic molecular chain may be a hexaalkyl chain or a dodecaalkyl chain.
The other technical scheme of the invention is as follows:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, and the first binding substances are modified on the surface of the magnetic beads;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
the different labels are distinguished and counted according to their size and/or surface potential, thereby obtaining the type and amount of analyte in the sample.
Preferably, the method comprises the following steps: the difference in diameter of each marker is more than 5%, or the difference in surface potential is more than 10%.
Preferably, the method comprises the following steps: according to the technical scheme, the sizes and/or surface potentials of the markers in the sample are detected through the nano-pores with different pore diameters, so that the types of different markers are distinguished and counted, and the type and the concentration of the analyte in the sample are obtained.
The other technical scheme of the invention is as follows:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, each of the first binding substances is modified on the surface of a different magnetic bead, and each of the magnetic beads has a measurable difference in size and/or magnetic induction intensity;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
before the step (3), grouping magnetic beads with different sizes or different magnetic induction intensities by a magnetic field gradient, magnetic field deflection or centrifugation method;
in different groups, the types of different markers are distinguished and counted according to the sizes and/or surface potentials of the markers, so that the types and the content of the analytes in the sample are obtained, and the simultaneous detection of different analytes is realized to a greater extent.
Preferably, the method comprises the following steps: the diameter difference of each magnetic bead is more than 5%, or the magnetic induction difference is more than 5%;
preferably, the method comprises the following steps: 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, comprising:
a. magnetic beads modified with a first binding compound capable of specifically binding to a first binding site of an analyte;
b. a label modified with a second binding substance capable of specifically binding to a second binding site of the analyte.
Preferably, the method comprises the following steps: the marker is a particle with the diameter of 10 nm-1 um.
Further preferred is: the marker is polyethylene microsphere, silicon dioxide microsphere, nanogold or fluorescent microsphere.
Preferably, the method comprises the following steps: the first or second binding agent is an antigen, an antibody, a receptor, or an aptamer.
Preferably, the method comprises the following steps: the modification mode of the first combination and the magnetic beads or the modification mode of the second combination and the label is the bonding of nitrobenzyl and a DNA molecular chain, or the bonding of dithioethylamino and carboxyl, or the bonding of sulfonate and oligonucleotide, or the bonding of a 6C or 12C organic molecular chain and biotin.
Preferably, the method comprises the following steps: the 6C or 12C organic molecular chain may be a hexaalkyl chain or a dodecaalkyl chain.
The other technical scheme of the kit is as follows:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, and the first binding substances are modified on the surface of the magnetic beads;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
preferably, the method comprises the following steps: the difference in diameter of each marker is more than 5%, or the difference in surface potential is more than 10%.
The other technical scheme of the kit is as follows:
the analytes are one or more than one;
correspondingly, the first binding substances are one or more, each of the first binding substances is specifically bound with the first binding site of the corresponding analyte, each of the first binding substances is modified on the surface of a different magnetic bead, and each of the magnetic beads has a measurable difference in size and/or magnetic induction intensity;
correspondingly, the second binding substance is one or more than one, each second binding substance is respectively and specifically combined with the second binding site of the corresponding analyte, each second binding substance is respectively modified to the surface of different labels, and each label has measurable difference in size and/or surface potential;
preferably, the method comprises the following steps: the diameter difference of each magnetic bead is more than 5%, or the magnetic induction difference is more than 5%.
Preferably, the method comprises the following steps: the difference in diameter of each marker is above 5%, or the difference in surface potential is above 10%.
Further preferred is: the kit also comprises a buffer solution, a dissociation agent and a small magnet device or a coil device for generating a magnetic field.
The invention has the beneficial effects that:
1. according to the invention, a ternary complex (magnetic bead-antigen-marker) is formed by immunospecifically binding to the magnetic bead, and the ternary complex is collected under the action of a magnetic field as shown in figure 1. Under a specific chemical reagent, the label in the ternary immune complex is eluted, the label is separated from the magnetic beads under the action of a magnetic field, the eluent containing the label is collected and is redispersed by a strong electrolyte to detect the eluent of the label, and absolute quantification is realized by a nanoparticle counter, as shown in fig. 2.
2. In the method of the present invention, the advantages of the first binding substance linked to the magnetic beads are: the magnetic beads can be uniformly dispersed in the mixed solution, so that the first binding substance can be fully combined with a substance to be detected in a sample to be detected, the capture rate is improved, and the lower limit of detection is improved; meanwhile, the ternary complex with the magnetic beads can be enriched under the action of a magnetic field, so that detection and counting in the subsequent steps are facilitated; furthermore, the grouping of the "magnetic bead-antigen-label" ternary complexes in the magnetic field can further increase the number of species of analytes to be detected simultaneously.
3. The combined use of multiple dissociation modes of the label and the magnetic beads has the advantages that: the dissociation rate can be improved to the maximum.
4. The invention can detect the types and the contents of a plurality of different objects to be detected by using the method of combining different markers with different objects to be detected, thereby realizing the simultaneous detection of multiple targets.
5. The method of the invention uses a nanoparticle counting method and does not use an optical reading method, thereby greatly improving the lower limit of detection. Counting particles can be stably read in a solution which is beneficial to improving the signal-to-noise ratio of the nanopore count, and the method can count in an environment where antigen and antibody are unstable.
6. The method can detect trace protein below the lower limit of the conventional immunoassay, and can be widely applied to the fields of immunoassay, microbial detection, cell separation and the like.
Drawings
FIG. 1 is a schematic structural diagram of a "magnetic bead-antigen-label" ternary complex of the present invention.
FIG. 2 is a schematic diagram of the detection principle of the detection method of the present invention: mixing the magnetic beads modified with the first binding substances with a sample containing an analyte, specifically binding the magnetic beads modified with the first binding substances with a first binding site of the analyte, specifically binding the magnetic beads modified with the second binding substances with a second binding site of the analyte, removing the unbound labels modified with the second binding substances under a magnetic field, separating the labels from the magnetic beads, and collecting the labels eluted from the supernatant.
Fig. 3 is a graph of counts obtained for different initial concentrations of troponin cTnI: wherein the data dots (o) are standard concentration sample counts and the measured sample counts (x) can correspond to the corresponding concentrations on the standard curve.
Fig. 4 is a schematic diagram of the simultaneous detection of the troponins cTnI and cTnT of example 2: the
Fig. 5 shows the results of simultaneous detection and counting of the troponins cTnI and cTnT in example 2: pulse data for mixtures of
FIG. 6 is a schematic diagram of magnetic field separation of different magnetic beads: the magnetic field direction is orthogonal to the fluid initial direction. The ternary complex is deflected under the action of a magnetic field, the ternary complex of the small magnetic beads flows out from the first outlet, and the ternary complex of the large magnetic beads flows out from the second outlet because the size of the ternary complex is larger and the deflection is smaller.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.