An immune detection method and system

Abstract

An immunoassay method and system, the method comprising: a detection step, comprising providing a test solution containing a carrier immune complex, the carrier immune complex comprising a solid phase carrier, a luminescent material and a test object, under the action of a sheath flow, the test solution flows through a detection zone, and a scattering signal M of the solid phase carrier and a luminescent signal N of the luminescent material in the detection zone are collected; a calculation step, comprising calculating the concentration of the test object in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material. The present invention realizes digital segmentation from a physical principle through hydraulic focusing, does not require microplate consumables, and at the same time uses less solid phase carriers, is convenient and easy to obtain, and can achieve a dynamic detection range of 5 orders of magnitude.

Description

Technical Field

[0001] The present invention relates to the technical field of single molecule immunoassay, and in particular to an immunoassay method and system. Background Art

[0002] With the development of science and technology and the progress of human exploration, there is an urgent need for the detection of early biomarkers related to major diseases in the scientific research and clinical fields. These biomarkers are low in the early stages of the disease. For example, the neuromarkers that point to Alzheimer's disease are three or four orders of magnitude lower in plasma than in cerebrospinal fluid due to the existence of the human blood-brain barrier. How to detect trace markers is a great challenge to existing traditional methods.

[0003] Traditional detection methods, such as chemiluminescence and enzyme-linked immunosorbent assay for protein detection, are all performed in a reaction pool with a volume of several hundred microliters, detecting the light signal generated by the solution in the entire reaction pool. Generally speaking, there are hundreds of thousands or even millions of proteins to be tested in the reaction pool, and the signal can be easily detected under the detection of the photon counter.

[0004] When the concentration of the protein to be tested in the sample is as low as a certain level, such as thousands or tens of thousands, the light signal emitted by the protein to be tested in the reaction pool will be masked by background noise, such as photon loss caused by thermal motion of solution molecules, thermal noise of detection devices, and the influence of background stray light, resulting in failure to detect effective signals. Therefore, when traditional detection methods detect extremely low concentration samples, due to the existence of system background noise, the accuracy of detection is limited, and it is difficult to achieve high-sensitivity detection. For this reason, single-molecule detection technology came into being.

[0005] Single molecule detection is a new detection concept, originated from digital ELISA and digital PCR technology. Its core is to separate extremely low concentration magnetic bead immune complexes by physical means. In the reaction system of extremely low concentration of the tested protein, most magnetic beads do not capture antigens or capture one antigen, a small part of magnetic beads capture two or more antigens, and the proportion of magnetic beads that capture a specific number of antigens conforms to the Poisson distribution theory. The separated single magnetic bead immune complex is subjected to signal enhancement (digital PCR is nucleic acid amplification), thereby realizing the detection of individual tested proteins.

[0006] There are currently two mainstream single-molecule detection methods on the market. One is the femtoliter array method, which is to use gravity to settle the magnetic bead immune complex after the reaction to a microplate etched with hundreds of thousands of pits. The volume of each pit is at the femtoliter level, which is just enough to accommodate a single magnetic bead. When the magnetic bead enters the pit, the surface is treated with oil seals, so that the reactants are spatially divided into hundreds of thousands of parts through the pits spaced apart on the microplate. The enzyme bound to the magnetic bead in each pit excites the substrate in the pit to generate a light signal, and the microplate is photographed by an imaging device such as CCD or CMOS, and the fluorescence signal of the pit in the photographed image is analyzed. The shortcomings of this method include: 1) The manufacturing process of the femtoliter microplate is complicated and the cost of consumables is high, which is not conducive to popularization. The second is that the light signal emitted by a single pit is weak, and the exposure time of the CCD or CMOS needs to be increased; 2) The detection time is increased; 3) A refrigeration device needs to be added to avoid the increase in dark counts caused by high exposure time, which also increases the complexity and cost of the instrument to a certain extent.

[0007] In the prior art, some methods realize digital individual detection of the tested protein by focusing the tiny detection area of ​​the light spot. The disadvantage of this method is that the location of the fluorescent immune complex cannot be predicted, the randomness is large, and the scanning and detection optical path design is complex and requires high precision, which makes it difficult to promote.

[0008] In summary, the existing single-molecule detection technology has defects such as complex detection system, expensive detection equipment and consumables, such as the high precision requirements of femtoliter microplates, which leads to high costs and difficulty in large-scale promotion. These problems have greatly hindered the application of single-molecule detection technology in the scientific research and medical diagnosis market. Summary of the invention

[0009] According to the first aspect, in one embodiment, an immunoassay method is provided, comprising:

[0010] The detection step includes providing a test solution containing a carrier immune complex, wherein the carrier immune complex comprises a solid phase carrier, a luminescent material and a test object, and under the action of a sheath flow, the test solution flows through a detection area, and a scattering signal M of the solid phase carrier and a luminescent signal N of the luminescent material in the detection area are collected;

[0011] The calculation step includes calculating the concentration of the analyte in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material.

[0012] According to the second aspect, in one embodiment, there is provided an immunoassay system, comprising:

[0013] The detection module is used to make the test solution containing the carrier immune complex flow through the detection area under the action of the sheath flow, wherein the carrier immune complex contains a solid phase carrier, a luminescent material and a test object, and collect the scattering signal M of the solid phase carrier in the detection area and the luminescent signal N of the luminescent material;

[0014] The calculation step is used to calculate the concentration of the analyte in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material.

[0015] According to the third aspect, in one embodiment, an immunoassay device is provided, comprising:

[0016] Memory, used to store programs;

[0017] A processor is used to implement the immunoassay method as described in any one of the first aspects by executing the program stored in the memory.

[0018] According to the fourth aspect, in one embodiment, a computer-readable storage medium is provided, on which a program is stored, and the program can be executed by a processor to implement the immunoassay method as described in any one of the first aspects.

[0019] According to the fifth aspect, in one embodiment, an immunoassay system is provided, comprising:

[0020] A sheath flow forming module, used for forming a laminar flow, so that the carrier immune complex in the test solution passes through the detection area under the sheath flow, wherein the carrier immune complex comprises a solid phase carrier that can provide a scattering signal, a luminescent material that can provide a luminescent signal, and a test object;

[0021] A laser luminescence detection module is used to excite the carrier immune complex passing through the detection area to generate scattering signals and luminescence signals;

[0022] and an algorithm analysis module for analyzing a scatter diagram formed by the signal generated by the laser luminescence detection module, and calculating the concentration of the analyte in the test solution according to the scattered signals and luminescence signals in the scatter diagram.

[0023] According to the sixth aspect, in one embodiment, there is provided a carrier immune complex, comprising a solid phase carrier, a first binding substance, and a second binding substance directly or indirectly coupled to a luminescent material;

[0024] The first binding substance is bound to the solid support;

[0025] The first binding substance specifically binds to the first site of the analyte, and the second binding substance specifically binds to the second site of the analyte.

[0026] According to the seventh aspect, in one embodiment, a kit is provided, comprising the carrier immune complex of any one of the sixth aspect.

[0027] According to an immunoassay method and system of the above-mentioned embodiment, the present invention realizes digital segmentation based on physical principles through hydraulic focusing, does not require microplate consumables, and uses less solid phase carrier, which is convenient and easy to obtain.

[0028] In one embodiment, a dynamic detection range of 5 orders of magnitude can be achieved.

[0029] In one embodiment, high-throughput, multi-marker detection can be achieved by changing the characteristics of the magnetic beads, such as coating with multiple fluorescent dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram of a magnetic bead reaction according to an embodiment;

[0031] Figure 2 A schematic diagram of sheath flow detection according to an embodiment;

[0032] Figure 3 A schematic diagram of a detection optical path of an embodiment;

[0033] Figure 4.1 is a scatter plot of individual magnetic beads;

[0034] Figure 4.2 This is the scatter plot of the 0.02pg / mL sample;

[0035] Figure 4.3 It is the scatter plot when the antigen concentration is 0.1 pg / mL;

[0036] Figure 4.4 It is the scatter plot when the antigen concentration is 2 pg / mL;

[0037] Figure 4.5 It is a scatter plot when the antigen concentration is 200 pg / mL. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many detailed descriptions are intended to enable the present application to be better understood. However, those skilled in the art can effortlessly recognize that some of the features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, in order to avoid the core part of the present application being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

[0039] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0040] The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

[0041] As used herein, "antigen" (abbreviated as Ag) refers to a substance that can induce the production of antibodies, and is any substance that can induce an immune response.

[0042] As used herein, "sheath flow" refers to a laminar fluid state, that is, under laminar flow, there is no interaction between fluids and they move along predetermined streamlines. In the present invention, the laminar flow is composed of two liquids, one is the solution formed by the analyte, and the other is the sheath liquid, that is, the solution that encapsulates the analyte liquid. The sheath flow refers to the characteristics of this encapsulation. The advantage of the sheath flow encapsulation is that it can stabilize the streamline of the analyte solution, which is convenient for laser detection. At the same time, the analyte will not contact the detection pool, avoiding the contamination of the previous sample to the next sample.

[0043] To solve the problems existing in the prior art, in one embodiment, the present invention provides a single molecule detection method based on hydraulic focusing sheath flow technology, combined with laser fluorescence detection technology, which can achieve high-throughput and low-cost detection of extremely low concentration analytes such as proteins, nucleic acids, etc.

[0044] According to the first aspect, in one embodiment, an immunoassay method is provided, comprising:

[0045] The detection step includes providing a test solution containing a carrier immune complex, wherein the carrier immune complex comprises a solid phase carrier, a luminescent material and a test object, and under the action of a sheath flow, the test solution flows through a detection area, and a scattering signal M of the solid phase carrier and a luminescent signal N of the luminescent material in the detection area are collected;

[0046] The calculation step includes calculating the concentration of the analyte in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material.

[0047] The immunoassay method of the present invention can be either single molecule detection or conventional concentration immunoassay.

[0048] In one embodiment, the scattering signal M and the luminous signal N involved in the calculation may include signal amplitude, signal width or the number of signals.

[0049] In one embodiment, in the calculation step, based on the scatter plot formed by the luminescent signal N and the scattered signal M, the ratio R of the number of luminescent signals exceeding the luminescent signal threshold to the total number of all solid phase carriers is calculated. When R≤the preset threshold, the average number of luminescent materials bound to each solid phase carrier AFN (Average Fluorophore Number) is calculated. Based on AFN, the concentration of the analyte in the test solution is calculated.

[0050] In one embodiment, in the calculation step, according to the scatter plot formed by the luminescent signal N and the scattered signal M, the ratio R of the number of luminescent signals exceeding (i.e., ≥) the luminescent signal threshold to the total number of all solid phase carriers is calculated. When R≤the preset threshold, the average number of luminescent materials bound to each solid phase carrier AFN (Average Fluorophore Number) is calculated according to the following formula:

[0051] AFN = -ln(1-R) ​​(1).

[0052] The formulas here are only exemplary, and the specific algorithms are not limited. Other algorithms that can calculate the average number of luminescent materials bound to each solid phase carrier are within the scope of protection of the present invention. In one embodiment, the carrier immune complex includes a solid phase carrier, a first binder, and a second binder directly or indirectly coupled to a luminescent material; the luminescent material can be directly coupled to the second binder, that is, forming a luminescent material-second binder form, or it can be coupled to the second binder through at least one third binder, for example, it can be in the form of luminescent material-third binder 1-third binder 2-second binder, and the third binder 1 and the third binder 2 are different types of third binders;

[0053] The first binding substance is bound to the solid support;

[0054] The first binding substance specifically binds to the first site of the analyte, and the second binding substance specifically binds to the second site of the analyte.

[0055] In one embodiment, the analyte includes a protein or a small molecule, and the first binding substance includes a capture antibody for specifically binding to the first site of the analyte.

[0056] In one embodiment, proteins include but are not limited to antigens.

[0057] In one embodiment, the protein includes but is not limited to at least one of cardiac troponin I (cTnI), procalcitonin (PCT), cytokines, and the like.

[0058] In one embodiment, proteins include but are not limited to small molecules.

[0059] In one embodiment, small molecules may include molecules with a molecular weight lower than 1000 Daltons, such as vitamin D and total thyroxine TT4. Generally, small molecules are detected by competitive methods.

[0060] In one embodiment, the analyte includes a nucleic acid, and the first binding substance includes a probe for capturing the nucleic acid, that is, the probe includes a nucleotide sequence that can be reverse complementary to a target sequence in the nucleic acid.

[0061] In one embodiment, the nucleic acid includes but is not limited to DNA and RNA, and can be at least one of a single-stranded nucleic acid and a double-stranded nucleic acid.

[0062] In one embodiment, the second binding substance includes a detection antibody for specifically binding to the second site of the analyte.

[0063] In one embodiment, the luminescent material includes a fluorophore (also known as a fluorescent substance) or an up-conversion photoluminescent material.

[0064] In one embodiment, a fluorophore comprises a label having a spectral emission characteristic.

[0065] In one embodiment, the fluorophore is excitable by visible light.

[0066] In one embodiment, the quantum efficiency of the fluorophore is ≥ 40%.

[0067] In one embodiment, the fluorescence signal intensity values ​​of the fluorophore are distributed within 10 of the instrument measurement dynamic range. -2 ~10 -5 between.

[0068] In one embodiment, the fluorophore includes but is not limited to at least one of fluorescent dyes, fluorescent microspheres, and quantum dots.

[0069] In one embodiment, the solid phase carrier includes but is not limited to at least one of magnetic beads and microspheres.

[0070] In one embodiment, the particle size of the magnetic beads is ≤3 μm, including but not limited to 0.5 μm, 1 μm, 2 μm, and 3 μm. The particle size of the magnetic beads is equivalent to the hydraulic diameter, which is also equivalent to the average diameter.

[0071] In one embodiment, the reaction system may contain magnetic beads of only a single particle size, or may contain magnetic beads of multiple particle sizes.

[0072] In one embodiment, the reaction system may contain a single type of fluorescent dye or multiple types of fluorescent dyes.

[0073] In one embodiment, the particle size of the magnetic beads is 1-3 μm.

[0074] In one embodiment, during the detection step, the carrier immune complex passes through the detection zone under the sheath fluid.

[0075] In one embodiment, in the detection step, the carrier immune complexes pass through the detection zone one by one under the sheath flow.

[0076] In one embodiment, for immunoassay at a single molecule concentration level, in the detection step, the ratio of the number of solid phase carriers to the number of analytes in the reaction system is ≥ 5. That is, the number of solid phase carriers is in excess relative to the number of analytes.

[0077] In one embodiment, for conventional or high concentration samples, during the detection step, the number of analytes is greater than the number of solid phase carriers.

[0078] In one embodiment, in the detection step, when collecting the scattering signal M of the solid phase carrier in the detection area, the solid phase carrier includes the solid phase carrier in the carrier immune complex and the free solid phase carrier.

[0079] In one embodiment, in the detection step, when the carrier immune complex passes through the detection zone one by one under the sheath flow, in the calculation step, the number of solid phase carriers counted in a single measurement process is ≥ 10,000. In one embodiment, the preparation method of the carrier immune complex in the detection step includes:

[0080] The first complex preparation step includes solidifying the first binding substance onto a solid phase carrier, mixing the solid phase carrier solidified with the first binding substance with the sample to be tested, and the first binding substance specifically binds to the first site of the test substance in the test sample to obtain a first complex composed of the solid phase carrier, the first binding substance, and the test substance;

[0081] The first cleaning step includes fixing the solid phase carrier and cleaning to remove free substances in the reaction system; the free substances are mainly the first binding substance that is not bound to the solid phase carrier and the analyte that is not bound to the first binding substance;

[0082] A coupling step, comprising directly or indirectly coupling the luminescent material to the second binding substance;

[0083] The second complex preparation step includes mixing a second binding substance coupled with a luminescent material with the first complex, wherein the second binding substance specifically binds to a second site of the analyte, and obtaining a second complex of the first complex and the second binding substance coupled with a luminescent molecule, which is a carrier immune complex;

[0084] The second washing step includes fixing the solid phase carrier, washing and removing the free second binding substance and the luminescent material in the reaction system, and obtaining the washed carrier immune complex;

[0085] The test solution preparation step includes mixing the diluent with the carrier immune complex obtained in the second washing step to prepare a test solution that can be used in the detection step. The diluent is a liquid, specifically a suspension, which can prepare the washed carrier immune complex and the solid phase carrier (such as magnetic beads) into a uniformly distributed suspension liquid state.

[0086] In one embodiment, in the first washing step and the second washing step, the carrier immune complex and the solid phase carrier are fixed under the action of a magnetic field.

[0087] In one embodiment, in the first washing step and the second washing step, the carrier immune complex and the solid phase carrier are fixed to the container wall under the action of a magnetic field.

[0088] In one embodiment, in the calculation step, based on the scatter plot formed by the luminescent signal N and the scattered signal M, the ratio R of the number of luminescent signals exceeding the luminescent signal threshold to the total number of all solid phase carriers is calculated. When R>the preset threshold, the average number AFN of luminescent materials bound to each solid phase carrier is calculated. Based on AFN, the concentration of the analyte in the test solution is calculated.

[0089] In one embodiment, in the calculation step, according to the scatter plot formed by the luminescent signal N and the scattered signal M, the ratio R of the number of luminescent signals exceeding (i.e., ≥) the luminescent signal threshold to all carrier immune complex signals is calculated. When R> the preset threshold, the average number of luminescent materials bound to each solid phase carrier AFN (Average Fluorop hore Number) is calculated according to the following formula:

[0090]

[0091] In formula (2), I average It refers to the average luminescent signal pulse height or pulse area generated by the luminescent material in the carrier immune complex that exceeds (i.e., ≥) the luminescent signal threshold in the scatter plot;

[0092] I average single It refers to the pulse height or pulse area of ​​the luminescent signal generated by the luminescent material in the carrier immune complex when the solid phase carrier is combined with one luminescent material on average;

[0093] According to AFN, the concentration of the analyte in the test solution is calculated.

[0094] According to the second aspect, in one embodiment, a single molecule detection system is provided, comprising:

[0095] The detection module is used to make the test solution containing the carrier immune complex flow through the detection area under the action of the sheath flow, the carrier immune complex contains a solid phase carrier, a luminescent material and the test object, and collect the scattering signal M of the solid phase carrier in the detection area and the luminescent signal N of the luminescent material;

[0096] The calculation step is used to calculate the concentration of the analyte in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material.

[0097] According to the third aspect, in one embodiment, a single molecule detection device is provided, comprising:

[0098] Memory, used to store programs;

[0099] A processor, configured to implement any one of the methods of the first aspect by executing a program stored in a memory.

[0100] According to the fourth aspect, in one embodiment, a computer-readable storage medium is provided, on which a program is stored, and the program can be executed by a processor to implement the immunoassay method as described in any one of the first aspects.

[0101] According to the fifth aspect, in one embodiment, an immunoassay system is provided, comprising:

[0102] A sheath flow forming module, used for forming a laminar flow, so that a test solution containing a carrier immune complex passes through the detection area under the sheath flow, wherein the carrier immune complex contains a solid phase carrier that can provide a scattering signal, a luminescent material that can provide a luminescent signal, and a test object;

[0103] A laser luminescence detection module, used to excite substances in the solution to be tested passing through the detection zone to generate scattering signals and luminescence signals;

[0104] And an algorithm analysis module is used to analyze the scatter diagram formed by the signal generated by the laser luminescence detection module, and calculate the concentration of the analyte in the test solution according to the scattered signal and the luminescence signal in the scatter diagram.

[0105] In one embodiment, the algorithm molecule module is also used to draw a calibration curve, and calculate the concentration of the substance to be tested in the test solution based on the scattering signal and luminescence signal generated by the substance in the test solution and the calibration curve.

[0106] In one embodiment, the carrier immune complex comprises a solid phase carrier, a first binding substance, and a second binding substance directly or indirectly coupled to a luminescent material;

[0107] The first binding substance is bound to the solid support;

[0108] The first binding substance specifically binds to the first site of the analyte, and the second binding substance specifically binds to the second site of the analyte.

[0109] According to the sixth aspect, in one embodiment, there is provided a carrier immune complex, comprising a solid phase carrier, a first binding substance, and a second binding substance directly or indirectly coupled to a luminescent material;

[0110] The first binding substance is bound to the solid support;

[0111] The first binding substance is used to specifically bind to the first site of the object to be detected, and the second binding substance is used to specifically bind to the second site of the object to be detected. Since the time interval between preparation and detection is relatively short, the carrier immune complex is preferably prepared and used immediately.

[0112] In one embodiment, if the carrier immune complex can be preserved, it can be prepared in advance and taken out from the storage device when used.

[0113] In one embodiment, the method for preparing the carrier immune complex comprises:

[0114] The first complex preparation step includes solidifying the first binding substance onto a solid phase carrier, mixing the solid phase carrier solidified with the first binding substance with the sample to be tested, and the first binding substance specifically binds to the first site of the test substance in the test sample to obtain a first complex composed of the carrier, the first binding substance, and the test substance;

[0115] The first washing step includes fixing the solid phase carrier and washing away free substances in the reaction system, mainly free first binding substances;

[0116] A coupling step, comprising directly or indirectly coupling the luminescent material to the second binding substance;

[0117] The second complex preparation step includes mixing a second binding substance coupled with a luminescent material with the first complex, wherein the second binding substance specifically binds to a second site of the object to be detected, and obtaining a second complex of the first complex and the second binding substance coupled with the luminescent material, which is a carrier immune complex;

[0118] The second washing step includes fixing the solid phase carrier, washing and removing free substances in the reaction system, mainly free second binding substances and luminescent materials, to obtain a washed carrier immune complex;

[0119] The test solution preparation step includes mixing the diluent with the carrier immune complex obtained in the second washing step to prepare the test solution that can be used in the detection step.

[0120] In one embodiment, during the first cleaning step and the second cleaning step, the solid phase carrier is fixed under the action of a magnetic field.

[0121] In one embodiment, during the first cleaning step and the second cleaning step, the solid phase carrier is fixed to the container wall under the action of a magnetic field.

[0122] According to the seventh aspect, in one embodiment, a kit is provided, comprising the carrier immune complex of any one of the sixth aspect.

[0123] In one embodiment, magnetic beads are used as solid phase carriers, and the specific implementation steps are as follows:

[0124] 1. If Figure 1 The figure shows a schematic diagram of the magnetic bead reaction, in which a magnetic bead immune complex is formed according to the characteristics of the target protein.

[0125] 1.1 A capture antibody 100a that can specifically bind to an analyte (including but not limited to a protein to be tested) 101 is immobilized on a magnetic bead 100. The capture antibody 100a binds to the first site of the analyte 101, thereby capturing the analyte in the sample and forming a magnetic bead-capture antibody-protein to be tested complex.

[0126] 1.2 The magnetic beads 100 are fixed to the wall of the reaction cup under the action of a magnetic field, and unbound capture antibodies are washed away.

[0127] 1.3 The fluorescent microspheres 102a are coupled to the detection antibody to form the fluorescent secondary antibody 102.

[0128] 1.4 The coupled detection antibody is added to the reaction system, and the detection antibody binds to the second site of the analyte 101 to form a magnetic bead antigen antibody-fluorescent microsphere immune complex.

[0129] 1.5 Fix the magnetic beads to the wall of the reaction cup under the action of the magnetic field, and wash away the unbound detection antibodies and fluorescent microspheres.

[0130] 1.6 Add the suspension, which can be a cleaning solution, substrate, etc. Use a suspension that does not affect the reaction system, is transparent to light and has a refractive index that meets the requirements, to form a certain volume of the test solution, and mix it to form a monodisperse system of magnetic beads.

[0131] Figure 1 In the figure, 100 is a magnetic bead, and the particle size is generally between 0.5 μm and 5 μm. Magnetic beads with small particle size have better surface binding efficiency, but are not easy to clean, and large particle size has the problem of easy sedimentation. Therefore, the size of the magnetic beads in the present invention is required to be no more than 3 μm, preferably 1 to 3 μm.

[0132] 100a is a capture antibody immobilized on magnetic beads, which uses the principle of antigen-antibody specific binding to capture the analyte.

[0133] 101 is a test object, which can be an antigen, an antibody or a nucleic acid molecule.

[0134] 102 is a fluorescent secondary antibody, including a detection antibody and a fluorescent molecule 102a labeled on the detection antibody. The fluorescent molecule 102a can emit fluorescence under the irradiation of excitation light. The fluorescent molecule can be a labeling substance with spectral emission characteristics such as fluorescent microspheres, quantum dots or fluorescent dyes.

[0135] 103 is the immune complex after the reaction, which includes the magnetic beads 100 , the test substance 101 and the fluorescent secondary antibody 102 .

[0136] Figure 1 The schematic diagram shown is only a schematic diagram of the reaction process. The above reaction steps can be divided into a one-step method or a two-step method according to actual needs. The one-step method is to add the analyte, solid phase carrier, fluorescent marker, etc. to the reaction at the same time. The two-step method is to first add the analyte and solid phase carrier, wash them, then add the fluorescent marker reaction, and then wash them. The two methods have differences in the measurement range. It is preferred to use the two-step method for higher accuracy.

[0137] 2. Figure 2 The figure shows a schematic diagram of sheath flow detection, in which the solution after the reaction is wrapped in the sheath flow and transported to the detection area through the principle of hydraulic focusing.

[0138] 2.1 A certain amount of reaction liquid (ie, test solution) is drawn from the reaction cup 300 by using the first power device 301a, such as a syringe, a peristaltic pump, etc., and the reaction liquid is pushed into the detection area of ​​the flow chamber through the syringe and other components.

[0139] 2.2 At the same time, the second power device 301b is used to push the sheath liquid into the detection area.

[0140] 2.3 Using the principle of hydraulic focusing, laminar flow is formed, that is, the sheath liquid and the reaction liquid form two liquid flows, and the sheath liquid wraps the reaction liquid.

[0141] 2.4 As Figure 2 As shown in the method diagram on the upper right side, the magnetic bead immune complex 103 passes through the detection area one by one under the action of the sheath flow, forming a digital separation.

[0142] Figure 2 In the figure, 300 is a reaction cup, which is a place for reacting magnetic beads, test objects and fluorescent microspheres, and can be a disposable reaction cup or a fixed reaction container. The suspension after the reaction in the reaction cup 300 enters the sample needle of the sheath flow forming unit 202a under the action of the first power device 301a. 302 is a sheath liquid pool, which enters the sheath liquid channel of the sheath flow forming unit 202 under the action of the second power device 301b. The reaction liquid entering the sample needle forms a laminar flow 303 under the action of the sheath liquid, wherein the magnetic bead immune complex 103 in the laminar flow generates corresponding scattering signals and fluorescence signals under the irradiation of the optical system shaping light beam 201a.

[0143] 3. Detect the scattering signal and fluorescence signal of the magnetic bead immune complex passing through the detection area.

[0144] like Figure 3 The figure shows a schematic diagram of the detection light path, which detects the optical scattering signal of the magnetic bead immune complex, including at least one forward scattering signal and one side scattering signal, wherein the forward scattering signal angle is within 1° to 20°, and the side scattering angle is different from the forward scattering collection signal, and generally the collection center line is 90° perpendicular to the laser incident direction.

[0145] The fluorescence signal of the magnetic bead immune complex is detected, wherein the fluorescence wavelength is greater than the incident wavelength, and the detection signal includes the number of pulses, peak value, and area of ​​the pulses.

[0146] Figure 3 In the figure, 200 is a laser light source, which can be a semiconductor laser or a solid laser, and 201 is a beam shaping module, which is used to shape the light beam emitted by the laser light source into a size matching the magnetic beads. In the present invention, the size of the shaped beam does not exceed 300 μm in the direction parallel to the sample flow and does not exceed 20 μm in the direction perpendicular to the sample flow, preferably 5 μm*200 μm.

[0147] 202 is a sheath flow assembly, including a sheath flow forming unit 202a and an optical detection unit 202b. The optical detection unit is generally made of quartz material, which has a good spectral transmittance in the visible light band. Its inner hole is square or rectangular, and the side length is generally between 100 and 500 μm.

[0148] 203 is a forward scattered light collection module, 204 is a fluorescence detection module, and 205 is a side scattered light collection module.

[0149] 4. Algorithm Analysis

[0150] 4.1 Form at least two scatter plots, where the scatter plot dimensions include scattering signals and fluorescence signals.

[0151] 4.2 Based on the scatter plot information, the algorithm statistically obtains the scattering signal M and the fluorescence signal N. In this embodiment, the effective total number of solid phase carriers can be obtained through the scattering signal M, and the effective number of magnetic bead immune complexes can be obtained through the fluorescence signal N. "Effective" refers to the statistical value obtained after discarding the abnormal signal magnetic beads.

[0152] The calculation is described as follows:

[0153] In single-molecule level detection, the content of the analyte (i.e., the object to be detected) is very low. If an excess of solid phase carriers such as magnetic beads are added (in the present invention, the ratio of the number of solid phase carriers participating in the reaction to the number of analytes is not less than 5, that is, the number of solid phase carriers participating in the reaction is at least 5 times the number of analytes), after the reaction, most of the magnetic beads are not bound to the analyte, a small part is bound to one analyte, and a very small part is bound to two or three analytes. The distribution pattern of this binding conforms to the Poisson distribution.

[0154] Taking the test of a cardiac troponin I (cTn I) sample with a concentration of 10fg / mL as an example, the molecular weight of cTnI is about 24K. If the volume of serum involved in the reaction is 100μL, the total number of cTnI molecules contained in the sample is about 25,000. If the number of magnetic beads is selected to be 500,000, the ratio of the number of magnetic beads to the number of cTnI molecules is 20:1, that is, the average number of cTn I molecules and luminescent materials bound to one magnetic bead is 0.05.

[0155] According to Poisson distribution, it can be known that the proportion of magnetic beads binding to one cTnI molecule is about 4.76% of the total magnetic beads, and the proportion of magnetic beads binding to two cTnI molecules is about 0.12% of the total magnetic beads.

[0156] It can be seen that although the proportion of magnetic beads binding to two or more cTnI molecules is very low, the direct statistical proportion of magnetic beads binding to one cTnI molecule is 4.76% of the total magnetic beads, which is still somewhat deviated from the actual average binding number of 0.05.

[0157] According to the characteristics of Poisson distribution, we can use the proportion of unbound to convert. In this example, the theoretical proportion of bound quantity is (4.76% + 0.12%) = 4.88%. According to formula (1), we can get:

[0158] AFN = -ln(1-4.88%) = 0.05

[0159] Therefore, by calculating the effective luminescence signal N, the average luminescence binding number can be accurately calculated according to formula (1).

[0160] 4.4 Calculate the concentration of the analyte based on the pre-drawn standard curve of AFN and the analyte.

[0161] Example 1

[0162] This embodiment designs a method for detecting cTnI antigen molecules using a magnetic bead method.

[0163] The reagent system information is as follows:

[0164] The antigen to be tested was recombinant human cardiac troponin complex (IC) at a concentration of 0.6 mg / mL, and the buffer system was 10 mM PBS at a pH of 7.4.

[0165] The magnetic beads have a particle size of 1 μm, cTnI humanized monoclonal antibody HAb209, the magnetic beads have been pre-blocked to reduce nonspecific adsorption, the fluorescent secondary antibody is cTnI humanized monoclonal antibody HAb210 fluorescent microspheres, the particle size is 300 nm, the excitation wavelength is 480 nm, and the emission wavelength is 525 nm

[0166] As well as sample diluent and cleaning solution, the above test kits were purchased from Nanjing Huading Biotechnology.

[0167] The detection steps are as follows:

[0168] ① Sample dilution: dilute the antigen to 0pg / mL, 0.02pg / mL, 0.1pg / mL, 2pg / mL, and 200pg / mL with sample diluent;

[0169] ② Add 50 μL of each concentration gradient sample into the reaction cup;

[0170] ③ Add 20 μL of the mixed magnetic bead reagent;

[0171] ④ After mixing, incubate at 37°C for 15 minutes;

[0172] ⑤Wash 3 times with cleaning solution;

[0173] ⑥ Add 100 μL of diluted fluorescent microsphere antibody (by volume, the dilution ratio is 1:10);

[0174] ⑦ After mixing, incubate at 37°C for 15 minutes;

[0175] ⑧Wash 3 times with cleaning solution;

[0176] ⑨ Add 100 μL of diluent to prepare a suspended sample to be tested.

[0177] The above-mentioned samples to be tested were detected on a Beckman Cytoflex model, with FITC selected as the channel, medium speed 30 μL / min as the sample flow, and detection time of 20 s.

[0178] Figure 4.1 is a scatter plot of individual magnetic beads. Figure 4.2 This is the scatter plot of the 0.02pg / mL sample.

[0179] Comparing the two figures, it can be seen that the magnetic beads alone have basically no fluorescent signal in the FITC channel. After binding to the antigen to form a magnetic bead immune complex, a fluorescent signal is generated in the FITC channel after being irradiated by the laser.

[0180] The statistical results show that the ratio R of effective fluorescence signals is 0.8%.

[0181] When the antigen concentration increases gradually, the number of particles in the FITC channel increases gradually. Figure 4.3 and Figure 4.4 The scatter plots are for antigen concentrations of 0.1pg / mL and 2pg / mL, and the calculated ratios R are 1.5% and 4.5% respectively.

[0182] When the concentration increases to 200 pg / mL, most of the magnetic beads have bound to one or more cTnI antigens. Under laser irradiation, the fluorescence signal of the FITC channel is enhanced as a whole. Figure 4.5 As shown, the peak value of FITC is 6551. At 0.1 pg / mL, due to the low antigen concentration, most of the magnetic beads are not bound to the antigen, a small number of magnetic beads bind to one antigen, and the proportion of binding to more than two is very small. Therefore, the signal mean of the fluorescence channel at 0.1 pg / mL can be used as the average fluorescence intensity when binding to one fluorescent microsphere, and its value is 3184. At this time, according to formula (2), AFN=6551 / 3184=2.06 can be calculated.

[0183] Table 1

[0184] concentration AFN 0 0.004 0.02pg / mL 0.008 0.1pg / mL 0.015 2pg / mL 0.046 200pg / mL 2.06

[0185] The above AFN and antigen concentration are plotted into a curve, which shows that the present invention can detect the analyte at the single molecule level.

[0186] In one embodiment, the present invention solves the problems of complex design and high cost in the prior art solutions. The present invention realizes digital segmentation based on physical principles through hydraulic focusing, does not require microplate consumables, and uses less magnetic beads, which are convenient and easy to obtain. The entire solution can achieve a dynamic detection range of 5 orders of magnitude. At the same time, by changing the characteristics of the magnetic beads, such as coating them with a variety of fluorescent dyes, high-throughput, multi-marker detection can be achieved.

[0187] In one embodiment, a single molecule detection device is provided, comprising:

[0188] A reagent filling module, used to fill sample liquid and reaction reagents into the reaction module;

[0189] Reaction module, used for antigen-antibody immune reaction;

[0190] A magnetic bead washing module is used to wash the remaining reagent components that have not formed an immune reaction complex;

[0191] A sheath flow forming module, used for forming a laminar flow, wherein the magnetic beads pass through the detection area one by one under the sheath flow;

[0192] Laser fluorescence detection module, used to excite the magnetic bead immune complex passing through the detection area to produce scattering and fluorescence signals;

[0193] and an algorithm analysis module, which is used to analyze the scatter plot formed by the signal generated by the laser fluorescence detection module and the calibration curve, and calculate the concentration information of the protein to be tested.

[0194] In one embodiment, a single molecule detection system is provided, comprising:

[0195] Magnetic beads, the diameter of which does not exceed 3 μm, preferably 2 μm, and the ratio of the number of magnetic beads in the reaction system to the number of protein molecules to be measured is not less than 5, and the number of magnetic beads counted in a single measurement process is not less than 10,000;

[0196] Fluorescent dyes can be excited by visible light, with a quantum efficiency of not less than 40%, and the fluorescence signal intensity values ​​are distributed within 10% of the instrument's measurement dynamic range. -2 ~10 -5 between;

[0197] An optical detection module, comprising at least one visible light band laser, and the volume of the laser detection area does not exceed 1000 femtoliters;

[0198] At least two scattered signals and one fluorescent signal are collected. The scattered signal collection angle can be determined according to actual needs. The forward scattered signal collection angle can be 1° to 20°, and the side scattered signal collection angle can be 90°±28°.

[0199] A sheath flow forming module, wherein the sheath flow forming speed does not exceed 10 m / s and the sheath flow width does not exceed 50 μm;

[0200] The algorithm processing module performs processing based on the scatter plot information.

[0201] In one embodiment, regarding the light source excitation method, the laser can be replaced by an incoherent light source such as an LED or a xenon lamp, and used in conjunction with a filter.

[0202] In one embodiment, regarding the labeling method, the fluorescence mechanism can be replaced by an upconversion luminescence mechanism, that is, the excitation wavelength is shorter than the emission wavelength of the luminescence, which can reduce the background, but the excitation efficiency is lower.

[0203] In one embodiment, the fluorescent dye includes but is not limited to fluorescent microspheres, quantum dots, fluorescent proteins, and the like.

[0204] In one embodiment, the detection method includes but is not limited to the following methods:

[0205] The first is synchronous detection: for example, the existing single-element detection components such as photodiodes, photomultiplier tubes (PMTs), and avalanche photodiodes (APDs) are changed to multi-element photodetectors such as charge-coupled devices (CCDs) and silicon photomultipliers (SiPMs).

[0206] The second is asynchronous detection: if the labeled fluorescent dye has a long life span, it can be detected by time resolution, that is, after the laser is turned off, the excited fluorescent signal is detected within a certain period of time;

[0207] The third is single scattering + fluorescence detection, that is, the forward scattering and side scattering signals are not collected at the same time. A similar purpose can be achieved by increasing the number of fluorescence channels.

[0208] In one embodiment, the sheath flow can be achieved by microfluidics.

[0209] Those skilled in the art will appreciate that all or part of the functions of the various methods in the above-mentioned embodiments can be implemented by hardware or by computer programs. When all or part of the functions in the above-mentioned embodiments are implemented by computer programs, the program can be stored in a computer-readable storage medium, and the storage medium can include: read-only memory, random access memory, disk, optical disk, hard disk, etc., and the program is executed by a computer to implement the above-mentioned functions. For example, the program is stored in the memory of the device, and when the program in the memory is executed by the processor, all or part of the above-mentioned functions can be implemented. In addition, when all or part of the functions in the above-mentioned embodiments are implemented by computer programs, the program can also be stored in a storage medium such as a server, another computer, disk, optical disk, flash disk or mobile hard disk, and can be downloaded or copied and saved in the memory of the local device, or the system of the local device is updated, and when the program in the memory is executed by the processor, all or part of the functions in the above-mentioned embodiments can be implemented.

[0210] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. An immunoassay method, characterized in that: include: The detection step includes providing a test solution containing a carrier immune complex, allowing the test solution to flow through a detection zone under the action of a sheath flow, and collecting a scattering signal M of the solid phase carrier and a luminescent signal N of the luminescent material in the detection zone; The method for preparing the carrier immune complex in the detection step includes: The first complex preparation step includes solidifying the first binding substance onto a solid phase carrier, mixing the solid phase carrier solidified with the first binding substance with the sample to be tested, wherein the first binding substance specifically binds to the first site of the analyte in the sample to be tested, and obtaining a first complex composed of the solid phase carrier, the first binding substance, and the analyte; The first cleaning step includes fixing the solid phase carrier and cleaning to remove free substances in the reaction system; A coupling step, comprising directly or indirectly coupling the luminescent material to the second binding substance; The second complex preparation step includes mixing a second binding substance coupled with a luminescent material with the first complex, wherein the second binding substance specifically binds to a second site of the object to be detected, and obtaining a second complex of the first complex and the second binding substance coupled with a luminescent material, which is the carrier immune complex; The second washing step includes fixing the solid phase carrier, washing and removing the free second binding substance and the luminescent material in the reaction system, and obtaining the washed carrier immune complex; The step of preparing the test solution comprises mixing the diluent with the carrier immune complex obtained in the second washing step to prepare the test solution for the detection step; The calculation step comprises calculating the concentration of the analyte in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material; In the calculation step, according to the scatter plot formed by the luminescent signal N and the scattered signal M, the ratio R of the number of luminescent signals exceeding the luminescent signal threshold to the total number of all solid phase carriers is calculated. When R≤the preset threshold, the average solid phase carrier-bound luminescent material number AFN (Average Fluorophore Number) is calculated according to the following formula: AFN=-ln(1-R)(1); according to the AFN, the concentration of the analyte in the test solution is calculated; When R>preset threshold, the average number of luminescent materials bound to each solid phase carrier AFN is calculated according to the following formula, and the concentration of the analyte in the test solution is calculated according to the AFN; In formula (2), I average It refers to the average luminescent signal pulse height or pulse area generated by the luminescent material in the carrier immune complex that exceeds the luminescent signal threshold in the scatter plot; I averagesingle It refers to the average luminescent signal pulse height or pulse area generated by the luminescent material in the carrier immune complex when each solid phase carrier is bound to one luminescent material; the detection method is not for the purpose of disease diagnosis.

2. The immunoassay method according to claim 1, characterized in that In the detection step, the object to be detected includes a protein, and the first binding substance includes a capture antibody for specifically binding to a first site of the object to be detected.

3. The immunoassay method according to claim 1, characterized in that The object to be detected includes nucleic acid, and the first binding substance includes a probe for capturing the nucleic acid.

4. The immunoassay method according to claim 1, characterized in that The second binding substance includes a detection antibody, which is used for specifically binding to the second site of the substance to be detected.

5. The immunoassay method according to claim 1, characterized in that: The luminescent material includes a fluorophore or an up-conversion photoluminescent material.

6. The immunoassay method according to claim 5, characterized in that The fluorophore includes a label having a spectral emission characteristic.

7. The immunoassay method according to claim 6, characterized in that: The fluorophore includes at least one of fluorescent dyes, fluorescent microspheres, and quantum dots.

8. The immunoassay method according to claim 1, characterized in that In the detection step, the solid phase carrier includes at least one of magnetic beads and microparticles.

9. The immunoassay method according to claim 8, characterized in that The particle size of the magnetic beads is ≤3 μm.

10. The immunoassay method according to claim 8, characterized in that: In the detection step, the carrier immune complex passes through the detection zone under the sheath flow.

11. The immunoassay method according to claim 10, characterized in that: In the detection step, the carrier immune complexes pass through the detection zone one by one under the sheath flow.

12. The immunoassay method according to claim 1, characterized in that: In the detection step, the ratio of the number of solid phase carriers to the number of analytes in the reaction system is ≥5.

13. The immunoassay method according to claim 1, characterized in that: In the detection step, when collecting the scattering signal M of the solid phase carrier in the detection area, the solid phase carrier includes the solid phase carrier in the carrier immune complex and the free solid phase carrier.

14. The immunoassay method according to claim 1, characterized in that: In the first cleaning step and the second cleaning step, the solid phase carrier is fixed under the action of a magnetic field.

15. An immune detection system, characterized in that: include: The detection module is used to make the test solution containing the carrier immune complex flow through the detection area under the action of the sheath flow, and collect the scattering signal M of the solid phase carrier in the detection area and the luminescent signal N of the luminescent material; The method for preparing the carrier immune complex comprises: The first complex preparation step includes solidifying the first binding substance onto a solid phase carrier, mixing the solid phase carrier solidified with the first binding substance with the sample to be tested, wherein the first binding substance specifically binds to the first site of the analyte in the sample to be tested, and obtaining a first complex composed of the solid phase carrier, the first binding substance, and the analyte; The first cleaning step includes fixing the solid phase carrier and cleaning to remove free substances in the reaction system; A coupling step, comprising directly or indirectly coupling the luminescent material to the second binding substance; The second complex preparation step includes mixing a second binding substance coupled with a luminescent material with the first complex, wherein the second binding substance specifically binds to a second site of the object to be detected, and obtaining a second complex of the first complex and the second binding substance coupled with a luminescent material, which is the carrier immune complex; The second washing step includes fixing the solid phase carrier, washing and removing the free second binding substance and the luminescent material in the reaction system, and obtaining the washed carrier immune complex; The step of preparing the test solution comprises mixing the diluent with the carrier immune complex obtained in the second washing step to prepare the test solution for detection; A calculation module, used for calculating the concentration of the analyte in the test solution according to the scattering signal M collected from the solid phase carrier and the luminescent signal N collected from the luminescent material; In the calculation module, according to the scatter plot formed by the luminescent signal N and the scattered signal M, the ratio R of the number of luminescent signals exceeding the luminescent signal threshold to the total number of all solid phase carriers is calculated. When R≤the preset threshold, the average solid phase carrier-bound luminescent material number AFN (Average Fluorophore Number) is calculated according to the following formula: AFN=-ln(1-R)(1); according to the AFN, the concentration of the analyte in the test solution is calculated; When R>preset threshold, the average number of luminescent materials bound to each solid phase carrier AFN is calculated according to the following formula, and the concentration of the analyte in the test solution is calculated according to the AFN; In formula (2), I average It refers to the average luminescent signal pulse height or pulse area generated by the luminescent material in the carrier immune complex that exceeds the luminescent signal threshold in the scatter plot; I averagesingle It refers to the average luminescent signal pulse height or pulse area generated by the luminescent material in the carrier immune complex when each solid phase carrier is bound to one luminescent material.

16. An immune detection device, characterized in that: include: Memory, used to store programs; A processor, configured to implement the immunoassay method according to any one of claims 1 to 14 by executing the program stored in the memory.

17. A computer-readable storage medium, characterized in that: The medium stores a program, and the program can be executed by a processor to implement the immunoassay method according to any one of claims 1 to 14.

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