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CN-121978341-A - Single-molecule protein detection method based on microarray chip and chemiluminescent immunoassay technology

CN121978341ACN 121978341 ACN121978341 ACN 121978341ACN-121978341-A

Abstract

The invention relates to the technical field of detection, in particular to a single-molecule protein detection method based on a microarray chip and a chemiluminescent immunoassay technology. The invention uses 5-20 ten thousand micro array chips with 5-30um diameter to divide the antigen-antibody compound reaction liquid into several ten thousand micro array holes with single hole volume at pL level. Each microwell is used as an independent reaction unit for detection, wherein the microwell contains 1 or 0 antigen-antibody complexes, a chromogenic substrate or exciting light is added to collect signal data in all microwells, the number of all antigen-antibody complexes in a reaction system is directly and quantitatively detected according to a poisson distribution calculation formula, and then the concentration of the captured antigen in a microarray chip is calculated. The method has the advantages of small background signal interference, high sensitivity, high specificity, high accuracy and high precision, simple and quick operation, low cost and obvious advantages in the technical field of single-molecule detection, and the detection lower limit reaches fg/ml.

Inventors

  • GE GUANGJUN
  • HOU JUNHAO
  • WANG HUIMIN
  • XU FAN

Assignees

  • 臻准生物科技（上海）有限公司

Dates

Publication Date
20260505
Application Date
20241025

Claims (10)

  1. 1. A method for detecting single-molecule protein based on micro-array chip and chemiluminescent immunoassay technique is characterized in that antigen-antibody complex suitable for chemiluminescent immunoassay detection method is loaded into micro-array chip with 5-20 ten thousand micro-holes with diameter of 5-30um on the surface, micro-holes are identified and focused by chemiluminescent detection system, signal data in the micro-holes are collected, and the concentration of antigen in the micro-array chip is calculated and analyzed.
  2. 2. The method according to claim 1, wherein the chemiluminescent immunoassay is a direct chemiluminescent immunoassay, an enzymatic chemiluminescent immunoassay, an electrochemiluminescent immunoassay, or a photoexcitation chemiluminescent immunoassay.
  3. 3. The method for detecting single-molecule proteins according to claim 2, wherein the chemiluminescent immunoassay method is a direct chemiluminescent immunoassay method, an enzymatic chemiluminescent immunoassay method or a photoexcited chemiluminescent immunoassay method, and the antigen-antibody complex is a double antibody sandwich complex or an antigen-antibody complex.
  4. 4. The method for detecting single-molecule proteins according to any one of claims 1 to 3, wherein the microarray chip having 5 to 20 tens of thousands of micro-vias having a diameter of 5 to 30 μm on the surface thereof comprises a chip housing, a microwell chip and a substrate layer; the micro-pore chip is arranged above the bottom plate layer and provided with a gap to serve as a micro-channel layer, and the chip shell and the bottom plate layer are covered above the micro-pore chip in a sealing manner and provided with a gap; The chip shell is provided with a sample adding hole and a substrate hole, and the opposite sides of the sample adding hole and the substrate hole are arranged on the chip shell; injecting a reaction system to be detected into the micro-flow layer through the sample adding hole, so that the reaction system to be detected passes through the micro-through holes on the micro-hole chip, and sucking the reaction system to be detected into the micro-through holes through siphoning and hydrophilic action of hydrophilic groups in the micro-through holes; The micro-flow channel is arranged at the bottom of the micro-through hole and is suitable for the reaction system to be tested to pass through the bottom of the micro-through hole.
  5. 5. The method for detecting single-molecule proteins according to claim 4, wherein the microporous layer has 20 ten thousand micro-holes with a diameter of 10 μm.
  6. 6. The method for detecting single-molecule proteins according to claim 4, comprising the steps of: (1) Loading antigen-antibody complexes connected with chemiluminescent labels into a microarray chip through a sample loading hole, wherein each antigen-antibody complex contains a single protein to be tested; (2) Identifying the microarray chip by a chemiluminescent detection system, adding a substrate of the chemiluminescent label through the substrate wells, and combining the substrate with antigen-antibody complexes in all chip wells; (3) And after signal collection, carrying out threshold division, calibration and inspection by the chemiluminescent detection system, analyzing and counting the number of effective holes, the number of negative holes and the number of positive holes, removing the number of ineffective holes, and calculating the specific concentration of the protein in the sample to be detected.
  7. 7. The method for detecting single-molecule proteins according to claim 4, comprising the steps of: (1) Loading antigen-antibody complexes connected with the marker enzyme into a microarray chip through a sample loading hole, wherein each antigen-antibody complex contains a single protein to be detected; (2) Identifying the microarray chip by a chemiluminescent detection system, fixing and focusing a negative signal/background signal after the micro through holes detect the reloaded antigen-antibody complex; (3) Adding catalytic substrate into the substrate hole to combine the substrate with antigen-antibody complex in the chip hole, incubating for 5-15min, receiving positive signals generated in the micro through holes by a chemiluminescence detection system, performing threshold division, calibrating and detecting, analyzing and counting the number of effective holes, the number of negative holes and the number of positive holes, removing the number of ineffective holes, and calculating the concentration of single-molecule protein to be detected.
  8. 8. The method for detecting single-molecule proteins according to claim 4, comprising the steps of: (1) Loading the antigen-antibody complex into a microarray chip through a sample loading hole, wherein the antigen-antibody complex is directly coupled or indirectly coupled with a photosensitive microsphere and a luminescent microsphere; (2) The chemiluminescence detection system emits excitation light with specific wavelength to irradiate the microarray chip, and meanwhile, emitted light with different wave bands is collected to complete detection of the concentration of the single-molecule protein.
  9. 9. A single-molecule protein detection system comprises a microarray chip with 5-20 ten thousand micro through holes with diameters of 5-30um on the surface, a chemiluminescent detection system and a preparation for preparing antigen-antibody complexes; the microarray chip comprises a chip shell, a micropore chip and a bottom plate layer; the micro-pore chip is arranged above the bottom plate layer and provided with a gap to serve as a micro-channel layer, and the chip shell and the bottom plate layer are covered above the micro-pore chip in a sealing manner and provided with a gap; the chip shell is provided with a sample adding hole and a substrate hole, and the opposite sides of the sample adding hole and the substrate hole are arranged on the chip shell; injecting a reaction system to be detected into the micro-flow layer through the sample adding hole, so that the reaction system to be detected passes through the micro-through holes on the micro-hole chip, and the reaction system to be detected is sucked into the micro-through holes through siphoning action and hydrophilic action of hydrophilic groups in the micro-through holes; the micro-flow channel is arranged at the bottom of the micro-through hole and is suitable for the reaction system to be tested to pass through the bottom of the micro-through hole.
  10. 10. A microarray chip for use in any one of the following applications: (1) Loading the antigen-antibody complex onto the microarray chip, and quantitatively detecting single-molecule proteins by adopting a direct chemiluminescence immunoassay method; (2) Loading the antigen-antibody complex on the microarray chip, and quantitatively detecting single-molecule proteins by adopting an enzymatic chemiluminescence immunoassay method; (3) Loading the antigen-antibody complex onto the microarray chip, and quantitatively detecting single-molecule proteins by adopting a photoexcitation immunoassay method; the microarray chip comprises a chip shell, a micropore chip and a bottom plate layer; The microporous chip is arranged in the chip shell, 5-20 ten thousand micro through holes with the diameter of 5-30um are formed in the microporous layer, and a micro flow channel is formed in the chip shell; The chip shell is also provided with a sample adding hole, and the sample adding hole is suitable for injecting a reaction system to be tested into the micro-flow layer through the sample adding hole so that the reaction system to be tested passes through the micro-through holes on the micro-pore chip; The micro-flow channel is arranged between the micro-pore chip and the bottom plate layer and is suitable for the reaction system to be tested to pass through the bottom of the micro-through hole; and substrate holes are arranged on the opposite sides of the sample adding holes on the chip shell, and are suitable for injecting luminous substrates into the microporous chip through the substrate holes.

Description

Single-molecule protein detection method based on microarray chip and chemiluminescent immunoassay technology Technical Field The invention relates to the technical field of detection, in particular to a single-molecule protein detection method based on a microarray chip and a chemiluminescent immunoassay technology. Background Currently, the mainstream chemiluminescent immunoassay (Chemiluminescence immuno-assay, CLIA) mainly comprises a direct chemiluminescent method, an enzymatic chemiluminescent method, an electrochemiluminescent method, a photoexcitation chemiluminescent method and the like. The direct chemiluminescent immunoassay is a technology of directly emitting light with an oxidant (such as hydrogen peroxide) under alkaline conditions without a catalyst, which is represented by acridinium esters, has extremely high luminous efficiency, and reaches maximum luminous intensity about 0.4s after adding a luminous substrate. Enzymatic chemiluminescent immunoassay is a detection method combining enzymatic reaction with chemiluminescent technology, and common enzymes are horseradish peroxidase (Horseradish Peroxidase, HRP) and alkaline phosphatase (Alkaline phosphatase, ALP). Luminol and its derivatives are common enzymatic light-emitting photocatalytic substrates of HRP, under alkaline conditions, HRP catalyzes the oxidation-reduction process of oxidant (such as hydrogen peroxide) and luminol, the process emits light of about 425nm, AMPPD is the most common enzymatic light-emitting catalytic substrate of ALP, under alkaline conditions, ALP catalyzes the AMPPD to remove 1 molecule of phosphate group, unstable intermediate AMPD is formed, and the AMPD is cleaved to release light with the wavelength of about 470nm for tens of minutes. The photo-activated chemiluminescence is a homogeneous phase immunity technology, the process utilizes the specific combination of the photosensitive microsphere with the diameter of about 200nm and the luminescent microsphere through the antigen-antibody specific coupling directly or indirectly to enable the distance between the two microspheres to be smaller than 200nm, and when the photosensitive microsphere is irradiated by excitation light with the wavelength of about 680nm, the released singlet oxygen can act on the luminescent microsphere to emit light with the wavelength of about 610 nm. If the distance between the two microspheres is larger than 200nm, the singlet oxygen cannot act on the luminescent microspheres due to the short half-life period of the singlet oxygen, and no luminescent signal is generated. Under the continuous development of scientific research and application fields, the detection capability of the chemiluminescence method of each type is close to the theoretical limit, but the known protein ratio of the protein marker which can be stably detected in a human body is still 'iceberg angle', the quantitative detection level of single-molecule protein is difficult to achieve by the existing chemiluminescence immunoassay method, the concentration of target protein in a sample to be detected is calculated by the existing chemiluminescence detection instrument depending on the integral optical signal intensity in the solution and the standard substance, and the detection of the single-molecule protein cannot be realized in the detection principle. Therefore, some proteins with low expression abundance cannot be detected by chemiluminescence immunoassay, which provides a great challenge for the development of new biomarkers, the disease development and the treatment prognosis monitoring. The SimoA technology represented by Quanterix company at present shows extremely excellent performance in sensitivity, the lower detection limit of the technology reaches fg/ml, and the technology has extremely high application potential in detection of markers of tumors, neurological diseases and the like. However, simoA has high complexity, high instrument and consumable cost, so that the application and popularization of SimoA in clinical diagnosis markets are limited, and the SimoA cannot be better popularized and applied. The prior art also reports that a single-molecule protein detection chip based on electrochemiluminescence of an ultramicroelectrode array and a special chip containing an inclined micro-well array are used for detecting trace proteins to realize high-sensitivity qualitative detection, but the technology uses the inclined micro-well chip with complex preparation, complex and unstable reaction process operation, and the detection of trace proteins has high requirements (more than 40000) on the number and density of the electrode arrays, which is generally less than 1000 before. Therefore, there is an urgent need to provide a method for detecting single-molecule proteins, which is convenient to operate, high in sensitivity, high in specificity, rapid, accurate, low in cost, and free from expensive instruments and consumable reagents. Disclosure of In