A single molecule detection method based on electrochemiluminescence

Abstract

This invention discloses a single-molecule detection method based on electrochemiluminescence, comprising an analyte capture and labeling system, an electrochemiluminescence reaction system, a photon signal acquisition system, and a single-molecule data processing system. The electrochemiluminescence reaction system triggers an electrochemical reaction on probe molecules of the analyte to generate a detectable light signal. The photon signal acquisition system collects single photon signals or isolated, minimal photon signals released by the electrochemiluminescence reaction. The single-molecule data processing system correlates isolated photon signals with the number of single molecules. Utilizing the above-mentioned technical solutions included in the photon signal acquisition system and isolated photon signal processing system of this invention, single-molecule detection, localization, and quantification can be achieved. This invention overcomes the sensitivity limitations of traditional electrochemiluminescence immunoassay techniques, achieving detection at the single-molecule level, with a detection limit for carcinoembryonic antigen below 10 fg / mL.

Description

Technical Field

[0001] This invention relates to the field of electrochemiluminescence analysis, and more particularly to a single-molecule detection method based on electrochemiluminescence. This method is applicable to single-molecule immunoassay, single-molecule nucleic acid analysis, and other similar single-molecule analysis fields. Background Technology

[0002] Detecting the levels of disease biomarkers in serum is of great significance for clinical disease diagnosis and monitoring. Currently, the main clinical method for detecting serum disease biomarkers utilizes immunoassays, which rely on the specific recognition and binding of the analyte antigen and labeled antibody to detect a single biomarker. While current immunoassays can detect most proteins in serum, they cannot accurately measure low-abundance proteins that appear in the early stages of disease development. Studies have shown that in the early stages of most cancers and infections, the levels of disease biomarkers in serum are below 10%. -16 -10 -12 The concentration is between mol / L, however, most immunoassay methods can only detect concentrations above 10. -12 The presence of low-expression cancer biomarkers (in mol / L) is a significant concern. Therefore, there is an urgent need to develop a novel immunoassay method to detect the presence of these biomarkers.

[0003] Single-molecule detection technology is an effective way to solve the above problems. Reducing the detection sensitivity to the single-molecule level can effectively lower the detection limit, thereby enabling the detection of low-expressed proteins. Currently, the most mature single-molecule detection technology in the field of immunoassay is the single-molecule fluorescence method. This method separates immunoassay complexes labeled with fluorescent probes in a very small reaction array, excites the complex with a laser, and statistically analyzes the distribution of its fluorescence intensity to achieve single-molecule-level detection. However, using lasers can lead to photobleaching and interference from background signals, thus affecting the accuracy of the detection.

[0004] Electrochemiluminescence (ECL) is favored in immunoassays due to its immunity to background interference, ease of voltage regulation, and high sensitivity. Currently, ECL methods have achieved the detection of low concentrations of proteins in serum; for example, the detection limit can be as low as 28.75 aM by specifically binding the analyte protein to a nucleotide-labeled probe molecule. Another method to lower the detection limit is to increase the concentration of the probe molecule conjugated to the detection antibody, for example, by doping Ru(bpy)3. 2+ SiO2 / Au nanoparticles, used as luminescent probes, can effectively enhance electrochemiluminescence signals, thereby enabling the detection of low-concentration analytes. However, the aforementioned electrochemiluminescence methods all introduce numerous coupling steps, increasing detection complexity, and none of them achieve protein detection at the single-molecule level. Furthermore, their absolute sensitivity is low, and they lack spatial resolution.

[0005] To date, few studies have utilized electrochemiluminescence (ECL) technology for the detection and quantification of biomolecules at the single-molecule level. We have developed a single-molecule ECL method and investigated its application in immunoassay. This method is simple to operate, requiring no additional coupling steps, and achieves precise localization and quantification of individual analyte molecules by analyzing the isolated photon signals generated during ECL. A major advantage of this method is the combination of isolated photon signals with single-molecule information, fundamentally elevating the sensitivity of immunoassay to the single-molecule level. This facilitates our understanding of certain life processes and their mechanisms of action at the single-protein level. Summary of the Invention

[0006] The purpose of this invention is to provide an analytical method based on electrochemical analysis and explore its application in biomolecular detection. Taking a common carcinoembryonic antigen (CEA) immunoassay system as an example, this method can overcome the sensitivity limitations of traditional electrochemiluminescence immunoassay techniques, achieving detection at the single-molecule level, with a detection limit for CEA below 10 fg / mL.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention discloses a single-molecule detection method based on electrochemiluminescence, comprising a analyte capture and labeling system, an electrochemiluminescence reaction system, a photon signal acquisition system, and a single-molecule data processing system. The electrochemiluminescence reaction system is used to trigger an electrochemical reaction on the probe molecules of the analyte to generate a detectable light signal. The photon signal acquisition system is used to acquire single photon signals or isolated, very small amounts of photon signals released by the electrochemiluminescence reaction. The single-molecule data processing system is used to correlate isolated photon signals with the number of single molecules.

[0009] As a further improvement, the electrochemiluminescence reaction system of the present invention is realized through an electrochemiluminescence reaction unit and a host computer; the electrochemiluminescence reaction unit includes a sample cell containing electrochemical reactants, a working electrode, a reference electrode, a counter electrode, a data acquisition card or an electrochemical workstation or other triggering device that can apply voltage, the working electrode, the reference electrode, and the counter electrode are set in the sample cell, and the data acquisition card is connected to the reference electrode, the counter electrode, the working electrode and the host computer respectively, and the electrochemical reactants include an immunoassay complex and a co-reactant.

[0010] As a further improvement, the acquisition card of the present invention sets a preset voltage through the host and applies voltage to the working electrode and the counter electrode, acquires the voltage across the reference electrode and the working electrode, and sends the acquired voltage value to the host. The host compares the voltage value acquired by the acquisition card with the preset voltage value, and controls the acquisition card to adjust the voltage signal sent to the working electrode and the counter electrode according to the comparison result.

[0011] As a further improvement, the electrochemical reactants of this invention also include free probe molecules to participate in the catalytic pathway of the electrochemiluminescence reaction, thereby enhancing the electrochemical signal generated by the co-reactant pathway. This solves the problem of weak luminescence signal in traditional electrochemical immunoassay systems.

[0012] As a further improvement, the photon signal acquisition system described in this invention is realized through a photon information acquisition unit and a host. The photon information acquisition unit includes a microscopic imaging system and a photon detector. The microscopic imaging system is located directly below the sample cell and is connected to the photon detector. The other end of the photon detector is connected to the host.

[0013] As a further improvement, the photon detector described in this invention is used to continuously acquire isolated photon signals released in the sample cell and send them sequentially to the host computer. The host computer generates a grayscale image for analysis and processing based on the isolated photon signals sent by the photon detector.

[0014] As a further improvement, this invention obtains isolated photon signals, which are relatively isolated in both time and space, by controlling the reactant concentration and the exposure time. The co-reactant concentration ranges from 1 picomolar per liter to 200 millimoles per liter; the immunoassay complex concentration ranges from 50 micrograms to 5 milligrams per liter; and the exposure time ranges from 10 microseconds to 50 milliseconds. Its emphasis on acquisition parameters means that only after acquiring isolated photon signals can subsequent molecular localization be performed.

[0015] As a further improvement, the isolated photon signal of the present invention includes the pixel position of the photon at the current moment and the grayscale value information of multiple adjacent pixels.

[0016] As a further improvement, the microscopic imaging system of the present invention is equipped with an oil immersion microscope objective with a high numerical aperture.

[0017] As a further improvement, the photon detector described in this invention is an electron multiplier camera, a complementary oxide semiconductor camera, a photomultiplier diode, an avalanche photodiode, or a photodetector and its array with high sensitivity.

[0018] As a further improvement, the single-molecule data processing system of this invention correlates isolated photon signals with single-molecule position information. The host analyzes the grayscale values ​​of the pixels of the single photon or a very small number of isolated photons sent by the photon detector, as well as the grayscale values ​​of their neighboring pixels. It then uses a two-dimensional Gaussian or other function with spatial positioning capabilities to fit the spatial position of the single photon or a very small number of isolated photons to obtain the positioning information and standard deviation of the single photon or isolated photon. This positioning method can accurately determine the position of the luminescent molecule.

[0019] As a further improvement, the spatial positions of multiple single photons or isolated, extremely small numbers of photons located over multiple acquisition time periods can be accumulated to obtain a positioning image that breaks the optical diffraction limit. After noise reduction processing of the above positioning points and merging of identical signals, the number of labeled molecules can be determined from the corrected single-molecule positioning information, thus improving the accuracy of determining the number of molecules from positioning.

[0020] As a further improvement, the merging of identical signals can be achieved by post-processing the positioning signals by setting a time threshold or a spatial threshold, thereby reducing over-counting caused by repeated positioning.

[0021] As a further improvement, the detection method is used for single-molecule immunoassay based on electrochemiluminescence, single-molecule nucleic acid analysis based on electrochemiluminescence, and other single-molecule detection and analysis applications based on electrochemiluminescence in principle, and is used to achieve highly sensitive quantitative detection of common antigens; for example, it achieves quantitative analysis of carcinoembryonic antigen, with a detection limit of less than 10 fg / mL for carcinoembryonic antigen.

[0022] The beneficial effects of this invention are as follows:

[0023] By utilizing the photon signal acquisition system and isolated photon signal processing system of this invention, the detection, localization, and quantification of single molecules can be achieved. This invention can be applied to immunoassay techniques based on this method, providing a protein detection method with ultra-high sensitivity.

[0024] This invention overcomes the sensitivity limitations of traditional electrochemiluminescence immunoassay technology, achieving detection at the single-molecule level, with a detection limit of less than 10 fg / mL for carcinoembryonic antigen.

[0025] Adding a small amount of electrochemiluminescence probe molecules to the electrochemiluminescence reaction system can enhance the electrochemiluminescence signal, especially for working electrodes that are not prone to co-reactant mechanisms, such as transparent tin oxide (ITO) electrodes.

[0026] In single-molecule data processing methods, a localization image that breaks the optical diffraction limit can be obtained by accumulating the spatial positions of multiple individual photons or isolated, very small numbers of photons located over multiple acquisition time periods. Since the single-response time of most single molecules is longer than the single-frame sampling time in this invention, individual molecules may be counted repeatedly, leading to the localization of molecular clusters at relatively close locations. Therefore, it is impossible to determine the total number of molecules based on the accumulation of localized molecules, and correction is needed for repeatedly localized molecules. Over-localization caused by continuous flickering from the same molecule can be merged by setting a reasonable time threshold.

[0027] In single-molecule data processing methods, spatial thresholds are set to incorporate localization biases caused by multiple reactions of the same molecule. Two-dimensional Gaussian fitting or other localization fitting methods introduce uncertainty, leading to discrepancies in the localization information of the same molecule at different times. Setting a spatial threshold can reasonably eliminate this spatial uncertainty. Attached Figure Description

[0028] Figure 1 This is a flowchart of a single-molecule electrochemical immunoassay method;

[0029] Figure 2 This is a schematic diagram of the electrochemiluminescence immunoassay detection steps on magnetic beads;

[0030] Figure 3 This is a schematic diagram of the structure of the system of the present invention;

[0031] Figure 4 This is a diagram showing the optimization effect of electrochemical reaction system conditions;

[0032] Figure 5 This is a comparison chart of single-molecule localization methods;

[0033] Figure 6 This is a graph showing the relationship between the concentration and the number of molecules of carcinoembryonic antigen (CEA). Detailed Implementation

[0034] This invention discloses a single-molecule detection method based on electrochemiluminescence, including a analyte capture and labeling system, an electrochemiluminescence reaction system, a photon signal acquisition system, and an isolated photon signal processing system;

[0035] The analyte capture and labeling system is used to immobilize the antigen or analyte at a specific location and connect it to a probe molecule for subsequent collection of the corresponding luminescent signal; the electrochemiluminescence reaction system is used to trigger an electrochemical reaction on the probe molecule of the analyte to generate a detectable light signal; the photon signal acquisition system is used to collect single photon signals or isolated, very small amounts of photon signals generated by the electrochemiluminescence reaction and display them on a computer in grayscale form; the single-molecule data processing system is used to analyze and process the collected isolated photon signals, correlate the isolated photon signals with molecular position information, and determine the position and number of analyte molecules.

[0036] The capture method in the analyte capture and labeling system can be: an antibody-antigen-antibody sandwich structure based on magnetic beads; a hybridization structure based on DNA modified on a glass slide array; or a direct antigen-antibody specific recognition structure. The labeling probe in the analyte capture and labeling system can be ruthenium terpyridine, iridium terpyridine, or other transition metal complexes; it can be an enzyme molecule with electrochemiluminescence catalytic activity; or it can be nanoparticles with electrochemiluminescence catalytic activity, such as gold nanoparticles, platinum nanoparticles, carbon quantum dots, and nanoparticles loaded with the aforementioned probe molecules.

[0037] Figure 1 This is a flowchart of a single-molecule electrochemical immunoassay method. The electrochemiluminescence reaction system triggers an electrochemical reaction on the probe molecules of the analyte, generating a detectable light signal. This is achieved through an electrochemiluminescence reaction unit and a host computer. The electrochemiluminescence reaction unit includes a sample cell containing electrochemical reactants, a working electrode, a reference electrode, a counter electrode, a data acquisition card or electrochemical workstation, or other voltage-applying triggering device. The working electrode, reference electrode, and counter electrode are located in the sample cell. The data acquisition card is connected to the reference electrode, counter electrode, working electrode, and host computer, respectively. The electrochemical reactants include an immunoassay complex and a co-reactant. The reactants can undergo an electrochemical reaction within the sample cell and release a light signal. The data acquisition card is set with a preset voltage by the host computer and applies a voltage to the working electrode and the counter electrode. The data acquisition card simultaneously acquires the voltage across the reference electrode and the working electrode and sends the acquired voltage value to the host computer. The host computer compares the voltage value acquired by the data acquisition card with the preset voltage value and controls the data acquisition card to adjust the voltage signal sent to the working electrode and the counter electrode based on the comparison result.

[0038] The sample cell also includes free probe molecules to participate in the catalytic pathway of the electrochemiluminescence reaction, thereby enhancing the electrochemical signal of the co-reactant pathway. For some working electrodes that do not readily undergo co-reactant mechanisms, such as transparent tin oxide (ITO) electrodes, adding small amounts of luminescent probe molecules, such as ruthenium terpyridine, iridium terpyridine, or other transition metal complexes, to the above reaction system can allow them to participate in the catalytic pathway of electrochemiluminescence, thereby enhancing the electrochemiluminescence signal. The co-reactants in the electrochemiluminescence reaction system can be reducing agents, such as tripropylamine, or oxidizing agents, such as hydrogen peroxide.

[0039] The photon signal acquisition system is implemented through a photon information acquisition unit and a host computer. The photon information acquisition unit includes a microscopic imaging system and a photon detector. The microscopic imaging system is positioned directly below the sample cell and connected to the photon detector, the other end of which is connected to the host computer. The photon detector continuously acquires single-photon information emitted from the sample cell and sequentially transmits it to the host computer. The host computer generates a grayscale image for analysis and processing based on the single-photon information transmitted by the photon detector.

[0040] The photon signal acquisition system uses a high numerical aperture objective lens in a microscopic imaging system to collect single photon signals or isolated, extremely small amounts of photon signals generated by single-molecule electrochemical reactions. These signals are then captured by a photon detector with high quantum efficiency and low readout noise. The photon detector sequentially sends the collected single photon information to the host computer and generates an image.

[0041] Isolated photon signals, which are relatively isolated in both time and space, are obtained by controlling the concentration of reactants and the exposure time of acquisition. The concentration range of the co-reactant is 1 picomolar per liter to 200 millimoles per liter; the concentration range of the immunoassay complex is 50 micrograms to 5 milligrams per liter; and the exposure time ranges from 10 microseconds to 50 milliseconds.

[0042] An isolated photon signal contains the pixel position of the photon at the current moment and the grayscale information of multiple neighboring pixels.

[0043] The microscopic imaging system is equipped with an oil immersion microscope objective with a high numerical aperture, and the photon detector is an electron multiplier camera, a complementary oxide semiconductor camera, a photomultiplier diode, an avalanche photodiode, or a photodetector and its array with high sensitivity.

[0044] The single-molecule data processing system correlates isolated photon signals with single-molecule position information. The host analyzes the pixel information of the single photon or isolated very few photons sent by the photon detector and the gray value information of multiple adjacent pixels. The spatial position is fitted using a two-dimensional Gaussian or other functions with spatial positioning capabilities to obtain the positioning information and standard deviation of the single photon or isolated very few photons.

[0045] By accumulating the spatial positions of multiple single photons or isolated, extremely small numbers of photons located over multiple acquisition time periods, a positioning image that breaks the optical diffraction limit can be obtained. After noise reduction processing of these positioning points and merging of identical signals, the number of labeled molecules can be determined from the corrected single-molecule positioning information. Merging of identical signals can be achieved by post-processing the positioning signals by setting time or spatial thresholds.

[0046] The method of this invention enables the quantitative analysis of carcinoembryonic antigen (CEA), with a detection limit of less than 10 fg / mL and a detection sensitivity at the single-molecule level.

[0047] To explain the purpose and technical approach of this invention in detail, the single-molecule detection method based on electrochemiluminescence proposed in this invention will be further described below through specific embodiments and in conjunction with the accompanying drawings.

[0048] Example 1: Electrochemical Immunoassay for Acquisition and Processing of Isolated Photon Signals

[0049] An electrochemiluminescence method for detecting carcinoembryonic antigen (CEA) comprises the following steps: capturing and labeling CEA, triggering an electrochemiluminescence reaction, acquiring isolated photon signals generated by the single-molecule reaction, and post-processing the isolated photon signals. Figure 1 Flowchart of single-molecule electrochemical immunoassay method, the specific flowchart is as follows: Figure 1 :

[0050] I. Capture and labeling of carcinoembryonic antigen on magnetic beads:

[0051] Figure 2 This is a schematic diagram of the electrochemiluminescence immunoassay detection steps on magnetic beads; the first step in performing immunoassay on magnetic beads is to prepare an immunoassay complex with a sandwich structure, and the preparation process is as follows:

[0052] ① Wash the streptavidin-coated magnetic beads three times with PBS buffer containing 0.1% BSA to prepare a magnetic bead solution with a final concentration of 1 mg / mL. Take 20 μL of the magnetic bead solution and add 50 μL of 3 mg / mL biotinylated capture antibody solution to it. Vortex the solution at room temperature for 1 h.

[0053] ② After the reaction, wash the magnetic beads three times with PBS buffer containing 0.1% BSA. Then add 10 μL of 100 ng / mL carcinoembryonic antigen solution and react at 37°C for 1 h.

[0054] ③ After the reaction, wash the magnetic beads three times with PBS buffer containing 0.1% BSA. Add 40 μL of 4 mg / mL ruthenium tripyridine-labeled detection antibody solution. Incubate at 37°C for 1 h. Finally, wash the magnetic beads three times with PBS buffer containing 0.1% BSA to obtain the immunolabeled complex that can be used for electrochemiluminescence detection.

[0055] II. Triggering the electrochemiluminescence reaction

[0056] The electrochemiluminescence reaction system should include: the above-labeled immunoassay complex, co-reactant, working electrode, counter electrode, reference electrode, and acquisition card, or an electrochemical workstation or other device that can apply voltage to trigger the electrochemical reaction.

[0057] The co-reactant in the electrochemiluminescence reaction system is 10 mL of 50 μM / L tripropylamine. The working electrode is a transparent ITO electrode, the reference electrode is Ag / AgCl, and the counter electrode is a Pt electrode. The required voltage is applied through a data acquisition card. Figure 3 This is a schematic diagram of the system of the present invention. The acquisition card is connected to the working electrode, reference electrode, counter electrode, and host. The acquisition card is set with a preset voltage by the host and applies voltage to the working electrode and the counter electrode. The acquisition card simultaneously acquires the voltage across the reference electrode and the working electrode and sends the acquired voltage value to the host. The host compares the voltage value acquired by the acquisition card with the preset voltage value and controls the acquisition card to adjust the voltage signal sent to the working electrode and the counter electrode according to the comparison result. The probe molecules marked on the magnetic beads undergo an electrochemiluminescence reaction and release photons under voltage regulation.

[0058] Since the signal intensity of the electrochemiluminescence reaction is affected by various factors such as reactant concentration, applied voltage, and solution pH, the above electrochemical reaction conditions were optimized. The specific steps are as follows:

[0059] ① Take 20 μL of the 0.2 mg / mL immunolabeled complex from step 1, ③, and add it to the above electrochemiluminescence reaction system. The concentrations of free ruthenium terpyridine in the reaction system are 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 μM / L, respectively.

[0060] ② Apply DC voltages of 0.9, 0.95, 1, 1.05, 1.1, 1.15, and 1.2 V (vs. Ag / AgCl) to the ITO electrode using a data acquisition card, and measure the electrochemiluminescence image contrast of the magnetic beads at different voltages for each free terpyridine ruthenium concentration gradient in step ②①. The acquisition area is 512*512 pixels (81.92*81.92μm), and the exposure time is 5s.

[0061] ③ To reduce the interference of background fluctuations on the electrochemiluminescence signal, voltage and concentration conditions with the highest image contrast should be selected. The electrochemiluminescence image acquired in step ② is read using MATLAB, and the signal intensity value on the magnetic bead and the background intensity value are analyzed respectively. The contrast on the magnetic bead is defined as (signal intensity - background intensity) / background intensity. Figure 4 This is a diagram showing the optimized conditions of the electrochemical reaction system; the optimal concentration of free terpyridine ruthenium was determined to be 1.5 μM / L, and the optimal voltage condition was determined to be 1.2 V.

[0062] III. Acquisition of Isolated Photon Signals

[0063] The single-photon information acquisition unit includes a photon detector and a microscopic imaging system. The microscopic imaging system is positioned directly below the electrochemiluminescence reaction system and is fixed together with the photon detector and connected to the host. The photon detector is used to continuously acquire single-photon information released by the electrochemiluminescence reaction and send it to the host in sequence. The host generates a grayscale image for analysis and processing based on the single-photon information sent by the photon detector.

[0064] The steps for acquiring isolated photon signals from a single magnetic bead using the above-mentioned isolated photon signal acquisition system under the optimized electrochemiluminescence reaction conditions in step two ③ are as follows:

[0065] ① Add the immunoassay complex marked in step 1, ③ above to the sample cell of the electrochemiluminescence reaction system. Apply voltage to the electrochemiluminescence reaction system according to step 2.

[0066] ② The microscopic imaging system below the sample cell is equipped with a high numerical aperture oil immersion objective lens to collect isolated photon signals. After the photons are absorbed by the objective lens, they are detected by an electron-multiplying photon detector and transmitted to the host. By adjusting the exposure time and reactant concentration, isolated photon signals can be acquired. After accumulating more than 10,000 images, single-molecule localization analysis can be performed. In this step, the acquisition area is 128*128 pixels (20.48*20.48μm), the single-frame exposure time is 0.005s, and a total of 15,000 frames are acquired for each magnetic bead.

[0067] IV. Post-processing of isolated photon signals

[0068] Single-molecule localization is mainly achieved by analyzing the pixel position information of a single photon or a very small number of isolated photons received by the host computer, along with the grayscale information of multiple neighboring pixels. A two-dimensional Gaussian or other function with spatial localization capabilities is used to fit the photon's position information at that moment. By repeatedly fitting the single-photon position information from multiple consecutively acquired images, the spatial position information of the single molecule represented by the single photon or the very small number of isolated photons can be obtained. The steps for locating a single molecule using isolated photon signals are as follows:

[0069] ① Analyze the pixel position information of a single photon or a very small number of isolated photons in a single frame image and the gray value information of multiple adjacent pixels. Use a two-dimensional Gaussian to fit the position information of the photon at that moment. This step will introduce a certain positioning uncertainty.

[0070] ② By accumulating the single-molecule data located in each frame in space, a molecular positioning image with a resolution higher than the diffraction limit can be obtained. In order to correct the positioning uncertainty and over-counting problems caused by the above method, the positioning points in step 4① above are corrected by setting a time threshold. The time threshold is defined as a multiple of the exposure time. Photon signals that continuously flash in the same pixel within the time threshold are merged into the same molecule.

[0071] ③ Because the two-dimensional Gaussian fitting method introduces a certain degree of uncertainty, the positioning information of the same molecule at different times will deviate. Therefore, a spatial threshold is set to eliminate the spatial uncertainty of positioning. This spatial threshold can be a fixed spatial distance, or a parameter describing the similarity between two positioning points, such as the Hellinger distance.

[0072] After setting and filtering thresholds twice, the location of a single molecule can be correlated with the number of single molecules, that is, the number of location points is equal to the number of labeled carcinoembryonic antigen molecules. Figure 5 This is a comparison chart of single-molecule localization methods; the difference in the number of molecules localized before and after correction is shown below. Figure 5 As shown.

[0073] Example 2: Quantitative Analysis of Carcinoembryonic Antigen

[0074] The single-molecule electrochemical immunoassay method was used for the quantitative analysis of carcinoembryonic antigen (CEA). The electrochemiluminescence method described in Example 1 was used, including the capture and labeling of CEA, triggering of the electrochemiluminescence reaction, acquisition of isolated photon signals, and post-processing of isolated photon signals.

[0075] I. Capture and labeling of carcinoembryonic antigen on magnetic beads:

[0076] The difference from step one of Example 1 is that the concentration of carcinoembryonic antigen in step one ② of Example 1 is changed to a series of concentration gradients (0-10). 3 (ng / mL).

[0077] II. Triggering the electrochemiluminescence reaction

[0078] According to the optimized electrochemiluminescence conditions in step two of Example 1, the electrochemical reaction system under different carcinoembryonic antigen concentration gradients in step one above is triggered by the acquisition card to produce a single-molecule electrochemiluminescence reaction.

[0079] III. Acquisition of Isolated Photon Signals

[0080] Following the acquisition steps and conditions in Step 3 of Example 1, isolated photon signals were acquired from the magnetic beads labeled with different carcinoembryonic antigen concentrations in Step 2 above, with five parallel acquisitions at each concentration gradient.

[0081] IV. Post-processing of isolated photon signals

[0082] The data processing method described in step four of Example 1 above is used to analyze each set of single-photon data collected in step three above, to obtain the number of localizing molecules at different carcinoembryonic antigen (CEA) labeling concentrations, thereby obtaining a standard curve for quantitative analysis. Figure 6 This is a graph showing the relationship between the concentration and molecular number of carcinoembryonic antigen (CEA). (0-10) -3 Five gradients between ng / mL were used to determine the method's detection limit, 10 -2 -10 3 The detection range is ng / mL, and the quantification range is 0.5-50 ng / mL. It can be seen that there is a good linear correlation between the concentration of carcinoembryonic antigen and the number of molecules located, which verifies the feasibility and effectiveness of the above single-molecule detection method based on electrochemiluminescence. It can be used for the accurate quantification of antigens and other analytes.

[0083] The concentration gradients in step one are 0 and 10, respectively. -6 10 -5 10 -4 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 ,0.5,5,20,30,40,50ng / mL.

[0084] The above examples are merely preferred embodiments of the present invention. The present invention is not limited to the above embodiments. Other improvements and variations that can be directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A single-molecule detection method based on electrochemiluminescence, characterized in that, The system includes a analyte capture and labeling system, an electrochemiluminescence reaction system, a photon signal acquisition system, and a single-molecule data processing system. The electrochemiluminescence reaction system triggers an electrochemical reaction on the probe molecules of the analyte to generate a detectable light signal. The photon signal acquisition system collects single photon signals or isolated, minimal photon signals released by the electrochemiluminescence reaction. The single-molecule data processing system correlates the photon signals with the number of single molecules. The photon signal acquisition system is implemented through a photon information acquisition unit and a host computer. The photon information acquisition unit includes a microscopic imaging system and a photon detector. The single-molecule data processing system correlates isolated photon signals with single-molecule position information. The host computer analyzes the pixel information of the single photon or isolated, minimal photon sent by the photon detector and the grayscale value information of multiple adjacent pixels. It uses a two-dimensional Gaussian or other function with spatial positioning capabilities to fit the spatial position to obtain the positioning information and standard deviation of the single photon or isolated, minimal photon.

2. The electrochemiluminescence single-molecule detection method according to claim 1, characterized in that, The electrochemiluminescence reaction system is realized through an electrochemiluminescence reaction unit and a host computer. The electrochemiluminescence reaction unit includes a sample cell containing electrochemical reactants, a working electrode, a reference electrode, a counter electrode, a data acquisition card or an electrochemical workstation or other triggering device that can apply voltage. The working electrode, reference electrode, and counter electrode are set in the sample cell. The data acquisition card is connected to the reference electrode, counter electrode, working electrode and host computer respectively. The electrochemical reactants include a combination of electrochemiluminescence probe molecules and target analytes.

3. The single-molecule detection method based on electrochemiluminescence according to claim 2, characterized in that, The acquisition card sets a preset voltage through the host and applies the voltage to the working electrode and the counter electrode, acquires the voltage across the reference electrode and the working electrode, and sends the acquired voltage value to the host. The host compares the voltage value acquired by the acquisition card with the preset voltage value, and controls the acquisition card to adjust the voltage signal sent to the working electrode and the counter electrode according to the comparison result.

4. The single-molecule detection method based on electrochemiluminescence according to claim 2 or 3, characterized in that, The electrochemical reactants also include free probe molecules that participate in the catalytic pathway of the electrochemiluminescence reaction to enhance the electrochemical signal generated by the co-reactant pathway.

5. The single-molecule detection method based on electrochemiluminescence according to claim 2 or 3, characterized in that, The microscopic imaging system is positioned directly below the sample cell and connected to a photon detector, the other end of which is connected to the host computer.

6. The single-molecule detection method based on electrochemiluminescence according to claim 5, characterized in that, The photon detector is used to continuously acquire isolated photon signals emitted in the sample cell and send them sequentially to the host computer. The host computer generates a grayscale image for analysis and processing based on the isolated photon signals sent by the photon detector.

7. The single-molecule detection method based on electrochemiluminescence according to claim 6, characterized in that, The isolated photon signal, which is relatively isolated in both time and space, is obtained by controlling the concentration of reactants and the exposure time of the acquisition. The combination of the electrochemiluminescent probe molecule and the target analyte includes an immunoassay complex. The concentration range of the immunoassay complex is 50 micrograms to 5 milligrams per liter; the exposure time range is 10 microseconds to 50 milliseconds; when the electrochemical reactants also include a co-reactant, the concentration range of the co-reactant is 1 picomolar per liter to 200 millimoles per liter.

8. The single-molecule detection method based on electrochemiluminescence according to claim 6 or 7, characterized in that... An isolated photon signal contains the pixel position of the photon at the current moment and the grayscale information of multiple adjacent pixels.

9. The single-molecule detection method based on electrochemiluminescence according to claim 5, characterized in that, The microscopic imaging system is equipped with a high numerical aperture oil immersion microscope objective.

10. The single-molecule detection method based on electrochemiluminescence according to claim 5, characterized in that, The photon detector is an electron multiplier camera, a complementary oxide semiconductor camera, a photomultiplier diode, an avalanche photodiode, or a photodetector and its array with high sensitivity.

11. The single-molecule detection method based on electrochemiluminescence according to claim 1, 2, 3, 6, 7, 9, or 10, characterized in that, By accumulating the spatial positions of multiple single photons or isolated photons located within multiple acquisition time periods, a positioning image that breaks the optical diffraction limit can be obtained. After noise reduction processing and merging of identical signals, the number of labeled molecules can be determined from the corrected single-molecule positioning information.

12. The single-molecule detection method based on electrochemiluminescence according to claim 10, characterized in that, Merging identical signals involves post-processing the positioning signals by setting time or spatial thresholds.

13. The single-molecule detection method based on electrochemiluminescence according to claim 1, 2, 3, 6, 7, 9, 10, or 12, characterized in that, The detection method described herein is used for single-molecule immunoassay based on electrochemiluminescence, single-molecule nucleic acid analysis based on electrochemiluminescence, and other single-molecule detection and analysis applications based on electrochemiluminescence in principle, and is used to achieve highly sensitive quantitative detection of common antigens.

Images