A single-photon signal acquisition method for electrochemiluminescence, imaging system and application thereof

Abstract

A method for collecting single photon signals or isolated extremely small photon signals of electrochemiluminescence, comprising an electrochemiluminescence reaction system and a photon signal collection system; an electrochemiluminescence reaction is triggered in the electrochemiluminescence reaction system; the photon signal collection system is used to collect single photon signals or isolated extremely small photon signals released by the electrochemiluminescence reaction. The single photon signal or isolated extremely small photon signal is generated by a single-molecule electrochemical reaction. An electrochemiluminescence imaging system and its application can improve the resolution of imaging, break through the optical diffraction limit to achieve super-resolution, and the image resolution obtained by imaging using this method can be as low as within the Abbe diffraction limit.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemiluminescence detection, and in particular to an electrochemiluminescence single-photon signal acquisition method, an imaging system and applications thereof. Background Art

[0002] Electrochemiluminescence (ECL) is a specific chemiluminescent reaction initiated by electrochemistry on the electrode surface. It actually includes two processes: electrochemistry and chemiluminescence. This analytical method is a highly sensitive detection method developed in the early 1990s. Its working principle is as follows: a certain potential is applied to the working electrode, and terpyridine ruthenium [Ru(bpy)3 2+ The divalent ruthenium in the electrode undergoes oxidation reaction on the electrode surface and loses one electron to become trivalent [Ru(bpy)3 3+ At the same time, tripropylamine (TPrA) in the reaction system is also oxidized on the electrode surface to become a cationic free radical TPA. +· , and rapidly and spontaneously removes a proton to form the free radical TPA · . Due to Ru(bpy)3 3+ It is a strong oxidizing agent, and TPA · It is a strong reducing agent, and the two will then undergo a redox reaction, resulting in Ru(bpy)3 3+ Reduced to excited state Ru(bpy)3 2+* . Excited state of Ru(bpy)3 2+* It decays by radiative transition mechanism, releasing a photon with a central wavelength of 610-620nm, and returns to the ground state Ru(bpy)3 2+ Continue to participate in the subsequent electrochemiluminescence reaction on the electrode surface, so that the oxidation-reduction reaction on the electrode surface is cyclical, and the process continuously releases photons. As the counting time of the photon counter increases, the measured signal continues to increase, improving the signal-to-noise ratio and greatly improving the detection sensitivity. ECL also has the following advantages: no radioactive hazard, it is a specific chemiluminescence reaction triggered by electrochemistry on the electrode surface, the detection speed is fast, compared with other chemiluminescence systems, the marker terpyridine ruthenium is relatively stable, the luminescence intensity changes relatively stably over time, and the luminescence process is more controllable. The detection method in the prior art usually adopts an electrochemiluminescence reaction sample cell with a magnetic enrichment electrode, a constant potentiostat, a single photon counting module, and a signal acquisition and processing system. The working electrode and the counter electrode in the sample cell are respectively made of platinum sheet and annular platinum wire structure, and the photon collection and transmission adopts a quartz fiber bundle (light-through section 5mm 2). The single-photon counting module is integrated with a high-sensitivity channel photomultiplier tube, a high-voltage power supply, a signal preamplifier, an amplifier discriminator, etc. It converts the single photon signal into a standard TTL pulse, and then the counting module collects the digital pulse signal and inputs it into the computer. However, the above-mentioned electrochemiluminescence technology based on photomultiplier tubes does not have spatial resolution and cannot be used for direct imaging; it has not achieved super-resolution imaging that breaks through the optical diffraction limit of electrochemiluminescence.

[0003] In summary, in the detection process of electrochemiluminescence reaction in the prior art, the detected signal is generated by repeatedly performing the electrochemiluminescence reaction process on the electrode surface to generate many photons to enhance the light signal, and then further amplify the signal before it can be collected. In addition, the imaging resolution after processing by the prior art is not good. The values ​​usually reported can only achieve sub-micron imaging, and it is difficult to image smaller objects. (Resolution is usually understood as the ability to distinguish the smallest object. In actual operation, there are point resolution (the minimum distance to distinguish two points), line resolution (the minimum spacing to distinguish two lines), and resolution calculated by Fourier ring (FRC) related methods.) The resolution of imaging is often limited by two factors. One is the sampling resolution, that is, the size of the smallest object that can be distinguished is twice the pixel size. The other is the optical diffraction limit. The worst of these two resolutions is the resolution of the imaging system. Among them, the sampling resolution can be improved to a certain extent by using a lens with a higher magnification and a photon detector with a lower pixel size; while the resolution bottleneck caused by the Abbe diffraction limit is difficult to eliminate by conventional imaging methods. It is necessary to use an imaging method that can break through the optical diffraction limit, namely electrochemiluminescence imaging super-resolution. The optical diffraction limit refers to the Abbe diffraction limit. The Abbe diffraction limit is defined as: S = 0.61×λ / NA, λ is the imaging wavelength, and NA is the numerical aperture of the high-power oil-immersion objective. The Abbe diffraction limit corresponds to 200-400 nanometers in common electrochemiluminescence systems. That is, traditional electrochemiluminescence imaging cannot achieve this limit of spatial resolution (Zhang et al, Annu. Rev. Anal. Chem. 2019.12: 275–95). Therefore, in the field of electrochemiluminescence, how to break through the Abbe diffraction limit of imaging methods to achieve a breakthrough improvement in resolution is a technical problem that needs to be solved urgently. Summary of the invention

[0004] Based on the aforementioned technical defects, the purpose of the present invention is to provide an electrochemiluminescence single-photon signal acquisition method, an imaging system and its application, which can improve the resolution of imaging, break through the optical diffraction limit and achieve super-resolution. The image resolution obtained by imaging using the method of the present invention can be as low as within the Abbe diffraction limit.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] The present invention provides a method for collecting a single photon signal or an isolated very small amount of photon signals of electrochemiluminescence, comprising an electrochemiluminescence reaction system and a photon signal collection system;

[0007] triggering an electrochemiluminescent reaction in the electrochemiluminescent system;

[0008] The photon signal collection system is used to collect a single photon signal or an isolated very small amount of photon signal released by the electrochemical luminescence reaction, wherein the single photon signal or the isolated very small amount of photon signal is generated by a single molecule electrochemical reaction.

[0009] Preferably, the luminescent probe in the electrochemiluminescent reaction system can be: a transition metal coordination compound, such as terpyridine ruthenium, terpyridine iridium, etc., or a derivative thereof, such as terpyridine ruthenium modified with a carboxyl group; it can be a carbon material such as carbon quantum dots, polymer carbon dots, graphene quantum dots, etc.; it can be a substance that can be used as a chemiluminescent probe itself, such as luminol and its derivatives, acridinium esters and their derivatives, 9,10-diphenylanthracene as a representative polycyclic compound and its derivatives; it can also be nanoparticles such as gold nanoparticles, quantum dots, metal organic porous compounds, covalent porous compounds, and the aforementioned luminescent materials loaded on nanoparticles;

[0010] Preferably, the co-reactant in the electrochemiluminescence system can be: a reducing substance represented by tripropylamine; an oxidizing substance represented by hydrogen peroxide.

[0011] In particular, for some reactions in the annihilation mechanism, that is, the same ion undergoes redox reactions at the anode and cathode, and the oxidation product and the reduction product after the reaction can continue to react chemically and emit light. When such molecules are used as luminescent probes, no additional co-reactants need to be added. For example, terpyridine ruthenium, 9,10-diphenylanthracene, etc.

[0012] In particular, for some small molecules (molecular weight less than 1000) such as luminol and its derivatives, there is an experimental possibility to reduce the applied voltage to 0V by rationally designing the system.

[0013] Preferably, the single-photon signal acquisition method collects the single photon signal or isolated extremely small amount of photon signals through a high numerical aperture objective lens arranged in the photon signal acquisition system, and then captures them by a photon detector with high quantum efficiency and low readout noise.

[0014] For a system using terpyridine ruthenium as a luminescent probe, the high quantum efficiency is achieved by selecting a photodetector with a higher quantum efficiency at the central wavelength of the luminescence.

[0015] Further preferably, the low readout noise means that the readout noise of the photodetector selected during the experiment is low.

[0016] In order to provide the aforementioned acquisition method, the present invention further provides an electrochemiluminescence imaging system, comprising: an electrochemiluminescence unit, a photon information acquisition unit and a host;

[0017] The electrochemical luminescence unit includes a sample pool containing reactants, wherein the reactants can undergo an electrochemical reaction in the sample pool and release a single photon signal or an isolated very small amount of photon signal;

[0018] The photon information acquisition unit is arranged at a position corresponding to the sample pool and is electrically connected to the host. The photon information acquisition unit continuously acquires the single photon signal or the isolated extremely small photon signal and sends it to the host in sequence. The host generates an image based on the information of the single photon sent in sequence by the photon signal or the isolated extremely small photon signal acquisition unit. The image is a super-resolution image of electrochemical luminescence, and the resolution of the image exceeds the Abbe optical diffraction limit.

[0019] Preferably, the photon information acquisition unit includes a photon detector and a microscopic imaging system;

[0020] The sample pool is placed on the microscopic imaging system, the photon detector is fixed to the microscopic imaging system and electrically connected to the host, and the photon detector collects the single photon signal or isolated extremely small amount of photon signal in the sample pool through the microscopic imaging system and sends the information to the host.

[0021] Preferably, the microscopic imaging system is equipped with a microscopic imaging objective lens.

[0022] Preferably, the photon detector is an electron multiplying camera or a complementary oxide semiconductor camera or a photomultiplier tube or an avalanche photodiode or a high-sensitivity photodetector with similar functions and an array thereof.

[0023] Preferably, the electrochemical luminescence unit further comprises: an acquisition card, a reference electrode, a counter electrode and a working electrode; or an electrochemical workstation or other similar device capable of applying voltage to trigger an electrochemical reaction that realizes equivalent functions;

[0024] Preferably, the acquisition card is electrically connected to the reference electrode, the counter electrode, the working electrode and the host respectively, and the reference electrode, the counter electrode and the working electrode are arranged on the sample pool;

[0025] Preferably, the acquisition card sends voltage signals to the working electrode and the counter electrode, and the acquisition card collects current information of the electrochemical luminescence unit through the counter electrode, and sends the current information to the host for display.

[0026] Preferably, the electrochemical luminescence unit further comprises a current amplifier;

[0027] The counter electrode is electrically connected to the acquisition card through the current amplifier to send the current information of the electrochemical luminescence unit to the acquisition card.

[0028] Preferably, a voltage value is preset in the host;

[0029] The acquisition card 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 sent 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.

[0030] The present invention further provides an electrochemiluminescence imaging method implemented based on the above-mentioned single-photon signal acquisition method, using an electrochemiluminescence imaging system with any of the above technical features, comprising the imaging steps of:

[0031] S100, the photon information acquisition unit sequentially and continuously acquires the spatial position information of a single photon or a very small amount of isolated photons generated at the first moment, the spatial position information of a single photon or a very small amount of isolated photons generated at the second moment, ..., the spatial position information of a single photon or a very small amount of isolated photons generated at the Nth moment in the sample pool, and sequentially and continuously sends the spatial position information to the host;

[0032] S200, the host receives the spatial position information of the single photon or the isolated very small amount of photons generated at the first moment, the spatial position information of the single photon or the isolated very small amount of photons generated at the second moment, ..., the spatial position information of the single photon or the isolated very small amount of photons generated at the Nth moment, and then processes the data to generate an image, wherein the image is a super-resolution image of electrochemical luminescence, and the resolution of the image exceeds the Abbe optical diffraction limit;

[0033] Wherein N is an integer greater than 1.

[0034] Preferably, the imaging step comprises the steps of:

[0035] S100, the photon information acquisition unit sequentially and continuously acquires the spatial position information of the single photon or isolated very small amount of photons generated at the first moment in the sample pool, including the grayscale value of the pixel of the single photon or isolated very small amount of photons at the first moment and the grayscale value of the multiple pixels adjacent to it, the spatial position information of the single photon or isolated very small amount of photons generated at the second moment includes the grayscale value of the pixel of the single photon or isolated very small amount of photons at the second moment and the grayscale value of the multiple pixels adjacent to it, ..., the spatial position information of the single photon or isolated very small amount of photons generated at the Nth moment includes the grayscale value of the pixel of the single photon or isolated very small amount of photons at the Nth moment and the grayscale value of the multiple pixels adjacent to it, and sends it sequentially and continuously to the host;

[0036] Preferably, the step S200 includes the steps of:

[0037] S210, the host receives the spatial position information of the single photon or the isolated very small number of photons generated at the first moment, including the grayscale values ​​of the pixel of the single photon or the isolated very small number of photons at the first moment and the multiple pixels adjacent to it, and performs two-dimensional Gaussian or similar function fitting with a spatial positioning function to obtain the spatial position information of the single photon or the isolated very small number of photons generated at the first moment, performs two-dimensional Gaussian or similar function fitting with a spatial positioning function to the spatial position information of the single photon or the isolated very small number of photons generated at the first moment, including the grayscale values ​​of the pixel of the single photon or the isolated very small number of photons at the first moment and the multiple pixels adjacent to it, and obtains the second position coordinates of the single photon at the second moment, ..., performs two-dimensional Gaussian or similar function fitting with a spatial positioning function to the grayscale values ​​of the pixel of the single photon at the Nth moment and the multiple pixels adjacent to it, and obtains the Nth position coordinates of the single photon at the Nth moment. * The position coordinates are fitted into an image and displayed, wherein the image is a super-resolution image of electrochemical luminescence, and the resolution of the image exceeds the Abbe optical diffraction limit;

[0038] Wherein N is an integer greater than 1.

[0039] Preferably, in step S100, the spatial position information of the single photon or isolated very small number of photons generated at the first moment collected includes the grayscale value of the pixel of the single photon at the first moment and the multiple pixels adjacent to it, the spatial position information of the single photon or isolated very small number of photons generated at the second moment includes the grayscale value of the pixel of the single photon at the second moment and the multiple pixels adjacent to it, ..., the spatial position information of the single photon or isolated very small number of photons generated at the Nth moment includes the grayscale value of the pixel of the single photon at the Nth moment and the multiple pixels adjacent to it;

[0040] Preferably, step S200 includes the steps of:

[0041] S210, performing two-dimensional Gaussian or similar function fitting with a spatial positioning function on the spatial position information of the single photon or the isolated very small number of photons generated at the first moment, including the grayscale values ​​of the pixel of the single photon or the isolated very small number of photons at the first moment and the multiple pixels adjacent to it, to obtain the spatial position information of the single photon or the isolated very small number of photons generated at the first moment, performing two-dimensional Gaussian or similar function fitting with a spatial positioning function on the spatial position information of the single photon or the isolated very small number of photons generated at the first moment, including the grayscale values ​​of the pixel of the single photon or the isolated very small number of photons at the first moment and the multiple pixels adjacent to it, to obtain the second position coordinates of the single photon at the second moment,…, performing two-dimensional Gaussian or similar function fitting with a spatial positioning function on the grayscale values ​​of the pixel of the single photon at the Nth moment and the multiple pixels adjacent to it, to obtain the Nth position coordinates of the single photon at the Nth moment. * Location coordinates;

[0042] Preferably, S220, an image is generated according to the first position coordinate, the second position coordinate, ... and the Nth position coordinate, so that the time-space position combination of the single photon or the isolated very small number of photons is superimposed to generate a super-resolution image of electrochemical luminescence, so that the resolution of the image exceeds the Abbe optical diffraction limit, wherein the single photon corresponds to the first position coordinate at the first moment, corresponds to the second position coordinate at the second moment, ... corresponds to the Nth position coordinate at the Nth moment.

[0043] Preferably, step S210 also includes analyzing the standard deviation corresponding to the position coordinates of a single photon or an isolated very small number of photons at each moment or part of the moment, accumulating the fitted signals, fusing the same signals and performing noise reduction processing, and determining the position coordinates of a single photon at different times and generating a super-resolution image of electrochemiluminescence.

[0044] Preferably, a resolution determination step is included after the imaging step;

[0045] The resolution determination step comprises:

[0046] S100', dividing the first position coordinates, the second position coordinates, ... and the Nth position coordinates into two sets in a statistical sense,

[0047]

[0048] in, and are the statistical correlations after Fourier transformation of two statistically significant sets in a constant circular space of size q, where

[0049] S200', calculate the resolution FRC(q) curve and take the abscissa value corresponding to the curve value at 1 / e as the calculated spatial frequency. The reciprocal of the abscissa is the resolution (Nieuwenhuizen et al, Nature Methods, 2013, 10, 557-562).

[0050] Preferably, the photon information acquisition unit includes a photon detector and an inverted microscope;

[0051] The sample pool is placed on the inverted microscope, the photon detector is fixed to the inverted microscope and electrically connected to the host, the photon detector collects the single photon information generated in the sample pool through the inverted microscope and sends the information to the host, and the inverted microscope is equipped with a high-power oil-immersion objective lens;

[0052] In the resolution determination step, the step S200′ includes the following steps:

[0053] S300' compares the resolution FRC(q) calculated in step S200' with the Abbe diffraction limit S, and if FRC(q) < S, it is super-resolution;

[0054] Where S = 0.61 × λ / NA, λ is the imaging wavelength, and NA is the numerical aperture of the high-power oil-immersion objective.

[0055] Preferably, an electrochemical detection step is included before the imaging step;

[0056] The electrochemical detection step comprises the steps of

[0057] S100”, the electrochemiluminescence unit applies voltage to the sample cell, so that the reactants in the sample cell undergo electrochemical reactions and release a single photon or a very small number of isolated photons;

[0058] The S200" electrochemical luminescence unit collects the current information in the sample cell and sends the information to the host.

[0059] Preferably, the electrochemical luminescence unit further comprises: a collection card, a reference electrode, a counter electrode and a working electrode;

[0060] The acquisition card is electrically connected to the reference electrode, the counter electrode, the working electrode and the host respectively, and the reference electrode, the counter electrode and the working electrode are arranged on the sample pool;

[0061] In step S100", the acquisition card outputs analog voltage signals to both ends of the working electrode and the counter electrode;

[0062] In step S200", the acquisition card acquires the current information of the electrochemical luminescence unit through the pair of electrodes, and sends the current information to the host for display.

[0063] Preferably, the electrochemical luminescence unit further comprises a current amplifier, and the counter electrode is electrically connected to the acquisition card via the current amplifier;

[0064] In step S200", the current information of the electrochemical luminescence unit is amplified by the current amplifier and then sent to the acquisition card.

[0065] Preferably, a voltage value is preset in the host.

[0066] In step S100", the acquisition card acquires the voltages at both ends of the reference electrode and the working electrode, and sends the acquired voltage values ​​to the host. The host compares the voltage value sent by the acquisition card with the preset voltage value, and controls the acquisition card to adjust the analog voltage signal sent to the working electrode and the counter electrode according to the comparison result.

[0067] The electrochemiluminescence imaging system and imaging method of the present invention continuously collects single photon information by using the photon information acquisition unit and sends it to the host in sequence. The host generates images according to the information of the single photons sent in sequence by the photon information acquisition unit. The technical solution can perform single photon detection, thereby improving the resolution of imaging, breaking through the optical diffraction limit to achieve super-resolution, and the image is an electrochemiluminescence super-resolution image. The technology of the present invention can be applied to micro-nanoscale spatial resolution electrochemical measurement, micro-nanostructure imaging, catalyst characterization, surface chemical structure characterization, biological imaging, immune detection, etc., and provides an electrochemical super-resolution image with ultra-high sensitivity and ultra-high spatiotemporal resolution achieved by the present invention.

[0068] The present invention also provides a micro-nano scale spatially resolved electrochemical measurement method, the electrochemical measurement method comprising: using the electrochemical luminescence single photon signal or isolated extremely small photon signal acquisition method as described above to achieve; or using any form of electrochemical luminescence imaging system as described above to achieve; or using any form of electrochemical luminescence imaging method as described above to achieve; preferably, the electrochemical measurement method utilizes the single photon acquisition signal and the electrochemical luminescence imaging method to perform optical signal reading of information such as current and potential in electrochemical measurements.

[0069] The present invention also provides an imaging method for a micro-nano structure, the imaging method comprising: using the electrochemiluminescence single photon signal or isolated very small photon signal acquisition method as described above to achieve; or using any form of electrochemiluminescence imaging system as described above to achieve; or using any form of electrochemiluminescence imaging method as described above to achieve; preferably, the micro-nano structure can be used as an electrochemiluminescence electrode material or a load material on an electrode for the acquisition of a single photon signal or isolated very small photon signal of electrochemiluminescence.

[0070] The present invention also provides a catalyst characterization method, which comprises: using the single photon signal of electrochemiluminescence or the isolated extremely small photon signal acquisition method as described above to achieve; or using any form of electrochemiluminescence imaging system as described above to achieve; or using any form of electrochemiluminescence imaging method as described above to achieve.

[0071] The present invention also provides a surface chemical structure characterization method, which comprises: using the single photon signal of electrochemiluminescence or the isolated extremely small photon signal acquisition method as described above to achieve; or using any form of electrochemiluminescence imaging system as described above; or using any form of electrochemiluminescence imaging method as described above to achieve.

[0072] The present invention also provides a biological imaging method, which includes: using the single photon signal or isolated extremely small photon signal acquisition method of electrochemiluminescence as described above to achieve it; or using any form of electrochemiluminescence imaging system as described above to achieve it; or using any form of electrochemiluminescence imaging method as described above to achieve it.

[0073] The present invention also provides an immunoassay method, which comprises: adopting the electrochemiluminescence single photon signal or isolated extremely small photon signal acquisition method as described above to achieve; or adopting any form of electrochemiluminescence imaging system as described above to achieve; or adopting any form of electrochemiluminescence imaging method as described above to achieve.

[0074] The present invention also provides a single-photon source or quantum light source based on a chemiluminescent reaction, wherein the single-photon source or quantum light source comprises a chemical reaction luminescent system, wherein the chemical reaction luminescent system can only emit a single photon signal during the spontaneous radiation lifetime, wherein a chemiluminescent reaction occurs in the chemical reaction luminescent system, and the emission of a single photon signal during the spontaneous radiation lifetime is achieved by controlling reaction condition parameters such as reactant concentration and / or temperature in the chemical reaction; preferably, the chemiluminescent reaction is an electrochemiluminescent reaction, and the emission of a single photon signal during the spontaneous radiation lifetime is achieved by controlling at least one of reaction condition parameters such as reactant concentration, temperature, electrode activity, voltage, and voltage application method in the electrochemical reaction; preferably, the reactant in the electrochemical reaction is a terpyridine ruthenium-tripropylamine system, and the concentration range of the terpyridine ruthenium is: 1 picomoles per liter to 2 millimoles per liter; the concentration range of the tripropylamine is: 1 picomoles per liter to 200 millimoles per liter.

[0075] The beneficial effect of the present invention is that for the first time, single photon signals or isolated extremely small amounts of photon signals generated by single-molecule electrochemiluminescence reactions are used for imaging with time resolution and spatial resolution, achieving electrochemiluminescence super-resolution imaging that breaks through the Abbe diffraction limit. Breaking through the important bottleneck of existing electrochemiluminescence technology, the technology of the present invention can be applied to micro-nanoscale spatially resolved electrochemical measurements, micro-nanostructure imaging, catalyst characterization, surface chemical structure characterization, biological imaging, immune detection, etc., providing ultra-high sensitivity and ultra-high temporal and spatial resolution electrochemical super-resolution images achieved by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Schematic diagram of the structure of the electrochemiluminescence imaging system in Example 1.

[0077] Figure 2 This is a comparison chart of the signal detection sensitivity limit of the present invention and the fluorescence technology.

[0078] Figure 3 This is a graph of single-photon signal results with high time resolution collected by the present invention.

[0079] Figure 4 This is a flow chart of the electrochemiluminescence imaging method in Example 2.

[0080] Figure 5 It is a schematic diagram comparing the electrochemiluminescence detection imaging in the prior art and the single photon collection imaging method of the present invention.

[0081] Figure 6 The imaging experiment results of the micro-nano structure described in Example 3.

[0082] Figure 7Schematic diagram of the principle of label-free electrochemiluminescence imaging of cell adhesion in Example 4.

[0083] Figure 8 The figure shows the imaging result of the method of the present invention applied to living cells.

[0084] Fig. 9 It is the bright field optical imaging of fixed cells and the super-resolution electrochemiluminescence imaging of the present invention.

[0085] Fig.10 It is a comparison diagram of the bright field optical image, the super-resolution electrochemiluminescence imaging image of the present invention and the super-resolution fluorescence imaging image of the same cell.

[0086] Fig.11 Result diagram of micro-nanoscale electrochemical measurement in Example 5.

[0087] Fig.12 This is a diagram showing the evaluation results of the catalyst activity in Example 6.

[0088] Fig.13 This is a diagram of the ultra-high sensitivity single molecule detection results in Example 7.

[0089] Fig.14 Generates a single-photon result plot for a chemiluminescent reaction.

[0090] Among them: 1-electrochemical luminescence unit; 11-sample cell; 12-acquisition card; 13-reference electrode; 14-counter electrode; 15-working electrode; 16-current amplifier; 2-photon information acquisition unit; 21-photon detector; 22-inverted microscope; 3-host. DETAILED DESCRIPTION

[0091] In order to make the purpose, technical solution and advantages of the present invention more clear, the electrochemiluminescence imaging system and imaging method of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0092] Example 1: Single photon collection from single-molecule electrochemiluminescence reaction

[0093] like Figure 1As shown, the present invention provides an electrochemiluminescence imaging system, including: an electrochemiluminescence unit 1, a photon information acquisition unit 2 and a host 3. The electrochemiluminescence unit 1 includes a sample pool 11 containing reactants, and the reactants can undergo electrochemical reactions in the sample pool 11 and release photons. The photon information acquisition unit 2 is arranged at a position corresponding to the row of the sample pool 11, and is electrically connected to the host 3. The photon information acquisition unit 2 continuously acquires single photon information and sends it to the host 3 in sequence. The host 3 generates an image based on the information of the single photons sent in sequence by the photon information acquisition unit 2. The reactant mentioned here is a terpyridine ruthenium-tripropylamine system, wherein the concentration range of terpyridine ruthenium is: 1 picomoles per liter to 2 millimoles per liter; the concentration range of tripropylamine is: 1 picomoles per liter to 200 millimoles per liter; the exposure time range is: 10 microseconds to 50 milliseconds. Its redox reaction mechanism is:

[0094] Ru(bpy)3 2+ -e - →Ru(bpy)3 3+

[0095] hv refers to the photons produced.

[0096] Among them, a single terpyridine ruthenium molecule is first oxidized on the surface of the working electrode indium tin oxide (ITO) through a heterogeneous electrochemical reaction, and then reduced back to a divalent excited state by a tripropylamine radical and emits a photon with an energy of about 2 electron volts in the solution.

[0097] By using the photon information acquisition unit 2 to continuously acquire single photon information and send it to the host 3 in sequence, the host 3 generates an image based on the information of the single photons sent in sequence by the photon information acquisition unit 2, which can improve the resolution of the imaging and break through the optical diffraction limit to achieve super-resolution.

[0098] Specifically, Figure 1 As shown, the photon information acquisition unit 2 includes a photon detector 21 and an inverted microscope 22. The sample pool 11 is placed on the inverted microscope 22, wherein the inverted microscope 22 is equipped with a high-power oil-immersion objective lens. The photon detector 21 is fixed to the inverted microscope 22 and electrically connected to the host 3. The photon detector 21 collects the single photon information generated in the sample pool 11 through the inverted microscope 22, and sends the information to the host 3. In this way, the photons emitted by the electrochemical reaction are efficiently absorbed by the high-power oil-immersion objective lens and then efficiently detected by the electron multiplying photon detector. By controlling the reaction concentration and exposure time, single photon signals that are relatively isolated in space and time are collected. Usually, the next step of reconstruction can be carried out after more than 10,000 acquisitions. In actual work, the photon detector 21 can be an electron multiplying photon detector or a complementary oxide semiconductor photon detector, but is not limited to this.

[0099] As an implementation method, Figure 1 As shown, the electrochemical luminescence unit 1 also includes: an acquisition card 12, a reference electrode 13, a counter electrode 14 and a working electrode 15. The acquisition card 12 is electrically connected to the reference electrode 13, the counter electrode 14, the working electrode 15 and the host 3 respectively, and the reference electrode 13, the counter electrode 14 and the working electrode 15 are arranged on the sample cell 11. In this way, the electrochemical luminescence unit 1 forms a three-electrode trigger control system. Its working process is that the acquisition card 12 sends a voltage signal to the working electrode 15 and the counter electrode 14, so that the reactants can undergo an electrochemical reaction in the sample cell 11 and generate a single photon, and the acquisition card 12 collects the current information of the electrochemical luminescence unit 1 through the counter electrode 14, and sends the current information to the host 3 for display. In this way, electrochemical detection in the electrochemical luminescence process is realized.

[0100] Preferably, if Figure 1 As shown, the electrochemical luminescent unit 1 also includes a current amplifier 16, and the electrode 14 is electrically connected to the acquisition card 12 through the current amplifier 16, so as to amplify the current information of the electrochemical luminescent unit 1 and send it to the acquisition card 12. In actual work, a suitable amplification gain is selected according to the current size. The principle of selecting the gain is to select a large gain as much as possible without overloading the current amplifier 16. The value collected by the acquisition card 12 after being amplified by the current amplifier 16 and converted into a voltage and the selected gain is the measured current value.

[0101] Preferably, the voltage is preset in the host 3; the acquisition card 12 acquires the voltage at both ends of the reference electrode 13 and the working electrode 15, and sends the acquired voltage value to the host 3. The host 3 compares the voltage value sent by the acquisition card 12 with the preset voltage value, and controls the acquisition card 12 to adjust the voltage signal sent to the working electrode 15 and the counter electrode 14 according to the comparison result. The acquisition card 12 outputs the analog voltage signal to both ends of the working electrode 15 and the counter electrode 13, and measures and inputs the voltage at both ends of the reference electrode 13 and the working electrode 15 to the acquisition card 12. The difference between the measured voltage and the preset voltage is calculated, and the host 3 controls the acquisition card 12 to adjust the voltage signal sent to the working electrode 15 and the counter electrode 14 according to the difference, so that the difference is less than 0.001V.

[0102] Example 2: Super-resolution electrochemiluminescence imaging

[0103] The present invention provides an electrochemiluminescence imaging method, which uses the electrochemiluminescence imaging system described in Example 1, including voltage application, imaging steps, and analysis results to determine resolution.

[0104] The imaging step includes the following steps: S100, the photon information acquisition unit sequentially and continuously acquires the first spatiotemporal information, the second spatiotemporal information, ..., the Nth spatiotemporal information of a single photon generated in the sample pool, and sequentially and continuously sends them to the host; S200, the host fits the received first spatiotemporal information, the second spatiotemporal information, ..., the Nth spatiotemporal information into an image and displays it; wherein N is an integer greater than 1.

[0105] Compared with traditional fluorescence imaging methods, the background can be reduced to nearly 0 by voltage-induced luminescence and then detected by photodetectors, because the interference of excitation light on the imaging background is avoided, and the signal-to-noise ratio of single-photon detection can be greater than 5. Figure 2 , where a is the background value of different imaging methods, b is the background noise value, imaging noise is defined as the standard deviation of its background value, and c is the signal-to-noise ratio of different imaging methods for a single photon. Therefore, the present invention can achieve high signal-to-noise ratio detection of single photons.

[0106] The detected single photon signal is shown as Figure 3 in, Figure 3 a Background value when the exposure time is 0.507ms, Figure 3 bd is the ECL imaging signal when the exposure time is 0.507ms, 4ms and 15ms. Figure 3 eh is the counting diagram of the number of photons detected by the selected pixels corresponding to ad within 400 frames. Figure 3 i is the average intensity in the entire field of view at different exposure times. Figure 3 j is the percentage of pixels with detected signals in the next frame at different exposure times to the total pixels. Figure 3 k is the relationship between the statistical distribution of the number of detected photons and the exposure time.

[0107] The flow chart of the imaging process can be obtained by Figure 4 express

[0108] Figure 5 Schematic diagram of the positioning processing of single-photon signal imaging information and the reconstruction method of super-resolution images.

[0109] Specifically, in step S100, the collected first spatiotemporal information includes the grayscale values ​​of the pixel of a single photon at the first moment and its eight adjacent pixels, the second spatiotemporal information includes the grayscale values ​​of the pixel of a single photon at the second moment and its eight adjacent pixels, ..., the Nth spatiotemporal information ( Figure 5 a) Includes the grayscale values ​​of the pixel of a single photon at the Nth moment and its 8 adjacent pixels ( Figure 5b). Step S200 includes the steps of: S210, performing two-dimensional Gaussian fitting on the grayscale values ​​of the pixel of the single photon at the first moment and the eight adjacent pixels, obtaining the first position coordinates of the single photon at the first moment, performing two-dimensional Gaussian fitting on the grayscale values ​​of the pixel of the single photon at the second moment and the eight adjacent pixels, obtaining the second position coordinates of the single photon at the second moment, ..., performing two-dimensional Gaussian fitting on the grayscale values ​​of the pixel of the single photon at the Nth moment and the eight adjacent pixels ( Figure 5 c), obtaining the Nth position coordinates of a single photon at the Nth moment; S220, generating an image according to the first position coordinates, the second position coordinates, ... and the Nth position coordinates ( Figure 5 d). Preferably, in step S210, the method further includes analyzing the standard deviation of the position coordinates of a single photon or a very small number of isolated photons at each moment or part of the moment, and accumulating the fitted signals, fusing the same signals, and performing noise reduction processing. By determining the position coordinates of a single photon at different times and generating a super-resolution image of electrochemical luminescence, the resolution can be improved, the image can be clearer, and super-resolution can be achieved by breaking through the optical diffraction limit ( Figure 5 e).

[0110] Further, a resolution determination step is included after the imaging step.

[0111] The resolution determination step includes: S100', dividing the first position coordinate, the second position coordinate, ... and the Nth position coordinate into two sets in a statistical sense, namely and

[0112] S200', calculation resolution FRC(q), This enables the calculation of the resolution of the generated image.

[0113] In actual work, the photon information acquisition unit includes a photon detector and an inverted microscope. The sample pool is placed on the inverted microscope. The photon detector is fixed to the inverted microscope and electrically connected to the host. The photon detector collects the single photon information generated in the sample pool through the inverted microscope and sends the information to the host. The inverted microscope is equipped with a high-power oil-immersion objective. In the resolution determination step, step S200' includes the following steps: S300' compares the resolution FRC(q) calculated in step S200' with the optical diffraction limit S. If FRC(q) < S, it is super-resolution, where S = 0.61 × λ / NA, λ is the imaging wavelength, and NA is the numerical aperture of the high-power oil-immersion objective. The use of such a technical solution can accurately determine whether the imaging is super-resolution.

[0114] As an implementation method, Figure 4As shown, an electrochemical detection step is included prior to the imaging step.

[0115] The electrochemical detection step includes step S100", the electrochemical luminescence unit inputs voltage to the sample cell to make the reactants in the sample cell generate a single photon; S200", the electrochemical luminescence unit collects current information in the sample cell and sends the information to the host for display.

[0116] Specifically, the electrochemical luminescence unit also includes: an acquisition card, a reference electrode, a counter electrode and a working electrode. The acquisition card is electrically connected to the reference electrode, the counter electrode, the working electrode and the host respectively, and the reference electrode, the counter electrode and the working electrode are arranged on the sample cell. At this time, step S100" includes that the acquisition card outputs an analog voltage signal to both ends of the working electrode and the counter electrode to input a voltage to the sample cell so that the reactants in the sample cell emit photons. This method is to form a three-electrode trigger control system for the electrochemical luminescence unit, and control the reactants in the sample cell to emit photons through the system. At the same time, step S200" includes that the acquisition card collects the current information of the electrochemical luminescence unit through the counter electrode, and sends the current information to the host for display, so as to realize electrochemical detection during the electrochemical luminescence process. It should be noted that the specific method of controlling the reactants in the sample cell to emit photons is not limited to the above technical solution. The potential of the reference electrode can also be defined as the potential zero point through the custom reference function of the acquisition card, and the voltage can be directly applied to the working electrode and the counter electrode through the analog output interface to realize the voltage application of the three-electrode system. Electrochemical measurements can also be achieved using an electrochemical workstation (potentiostat, bipotentiostat) or the like.

[0117] Furthermore, the electrochemical luminescence unit also includes a current amplifier, and the electrode is electrically connected to the acquisition card through the current amplifier. In step S200", the current information of the electrochemical luminescence unit is sent to the acquisition card after being reflected by the current amplifier. The specific working principle of this step can be referred to the description in Example 1.

[0118] As an implementation method, a voltage value is preset in the host. In step S100", the acquisition card 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 sent by the acquisition card with the preset voltage value, and controls the acquisition card to adjust the analog voltage signal sent to the working electrode and the counter electrode according to the comparison result. The specific working principle of this step can be referred to the description in Example 1.

[0119] Example 3: Micro-nanostructure imaging

[0120] This is the result diagram of the micro-nano structure imaging described in the present invention. In order to demonstrate the imaging capability, the present invention adopts a micro-nano processing method of focused ion beam etching, pre-processes a template of a known structure for the ITO electrode, and performs super-resolution electrochemiluminescence imaging on it.

[0121] from Figure 6 It is possible to clearly see the single-molecule ECL imaging and the collection of single photon or isolated very small amount of photon signals on the micro-nanostructured indium tin oxide (ITO) electrode of the present invention, wherein the surface of the micro-nanostructured indium tin oxide (ITO) electrode is first finely characterized using a scanning electron microscope.

[0122] Figure 6 .Single-molecule ECL imaging of ITO structures. Figure 6 a, ITO imaging template with known structure collides with a single ECL probe molecule. The ruthenium complex probe is free in the solution and emits light in a limited area on the electrode surface after collision and ECL reaction on the ITO surface. Figure 6 b, SEM image of FIB-patterned ITO. Scale bar: 3 μm. Figure 6 c, Figure 6 Intensity profile of the indicated line in b. Figure 6 d, Figure 6 Super-resolved single-molecule ECL image of the same FIB-patterned ITO shown in b. Figure 6 e, Figure 6 Intensity distribution of the lines indicated in d. Figure 6 f, Representative clusters showing three reaction sites. Figure 6 g, FRC quantification of image resolution from three randomly selected regions of interest (ROIs). Figure 6 h, Quantitative localization analysis of selected ROIs (300 nm × 300 nm, indicated by white arrows) on the FIB-patterned ITO coverslip. ROI 1 is located on the ITO surface, and ROI 2 is located on the insulating part of the glass layer.

[0123] The above results demonstrate a new type of super-resolution microscopy that does not require laser excitation. Although the current imaging object is still confined to the electrode surface, in the future it may be possible to achieve three-dimensional imaging using strategies such as split beam paths or point spread function engineering. Since the use of lasers can be avoided, the ECL-based method can minimize the background to the level of the camera bias background, which provides ultra-high sensitivity for resolving single photons (such as Figure 2 ). In addition, single-molecule ECL can be spatiotemporally controlled by the magnitude of the applied voltage, which is suitable for solving dynamic imaging needs.

[0124] Example 4: Biological Imaging

[0125] The present invention further uses single-molecule ECL to image cells. Cell adhesion to the cell growth matrix is ​​an important process in many basic biological processes (such as cell migration and proliferation). Imaging the adhesion of living cells requires a technology that takes into account both temporal and spatial resolution. Some super-resolution fluorescence microscopy techniques have been shown to meet these requirements for live cell imaging. Although super-resolution fluorescence microscopy is compatible with physiological operations, the use of fluorescent labels for adhesion proteins is cumbersome and is not conducive to the physiological processes of living cells. ECL microscopy has been shown to avoid such direct labeling problems for single-cell imaging (see Figure 7 , Schematic diagram illustrating negative contrast ECL imaging of cell adhesion. Figure 7 a, Partial bonding. Figure 7 b. Complete adhesion. ), but the insufficient resolution of conventional ECL limits the use of this method to study more detailed adhesion structures. Imaging cell adhesion by applying single-molecule ECL. Single cell Figure 8 a Direct imaging on the ITO surface ( Figure 8 b). Compared to the exposed ITO surface, the cell adhesion area hinders the diffusion of ECL reactants into the ITO and leads to dark contrast, thereby imaging the adhesion site without directly labeling the adhesion protein. Compared with the specific fluorescence imaging of labeled protein clusters, single-molecule ECL microscopy can image the overall cell adhesion rather than the specific labeled protein. Due to the advantages of sensitivity and time resolution, the present invention can effectively use ECL signals for image reconstruction at a relatively fast imaging speed. Super-resolution ECL images of living HEK293 cells are shown in Figure 2. Figure 8 b and 8d, Figure 8 a and Figure 8 c reveals specific information in contrast to bright field images and traditional ECL images. The present invention further explored the dynamic process of cell adhesion and observed the movement rate of the adhesion area with a time resolution of 12s and a spatial resolution of 150nm, breaking through the Abbe diffraction limit. This resolution is comparable to the optimized super-resolution fluorescence microscopy method for imaging adhesion sites. Based on the analysis of the dynamic process, the present invention obtained the average velocity of the selected adhesive area, where Figure 8 The motion of the region in e is 1.37 μm 2 min -1 , Figure 8 The area in f is 1.02 μm 2 min -1 .

[0126] Fig. 9 DIC and single-molecule ECL imaging of fixed PC12 cells. Fig. 9 a, DIC image. Fig. 9b, Single-molecule ECL image. The image was reconstructed from 15,000 frames with an exposure time of 10 milliseconds. Scale bar: 5 μm.

[0127] By comparing super-resolution fluorescence imaging of fixed cells (see Fig.10 ) and single-molecule ECL imaging confirmed the feasibility of single-molecule ECL in super-resolution imaging of cell adhesion, see Fig.10 shown.

[0128] Fig.10 Comparison of DIC, single-molecule ECL, and super-resolution fluorescence microscopy STORM images of the same PC 12 cells. Fig.10 ab, DIC images of PC12 cells. Fig.10 ef, STORM image. Fig.10 gh, Merged single-molecule ECL and STORM images. Fig.10 a. Fig.10 c. Fig.10 e and Fig.10 The scale bar in g is 5 μm. Fig.10 b. Fig.10 d. Fig.10 f and Fig.10 The scale bar in h is 2 μm. Comparative imaging experiments on the same PC 12 cells were performed with the following procedures: cell fixation, DIC / BF imaging, single-molecule ECL imaging, membrane lysis of fixed cells, fluorescent labeling of adhesion proteins, and STORM imaging. Fig.10 The results in show the overall correlation between the different imaging techniques. Adhesion includes many different types (>100) of protein complexes, while the STORM imaging adhesion of the present invention is only one type of protein complex. Therefore, STORM imaging only shows one type of possible adhesion site. On the other hand, single molecule ECL imaging reveals the overall adhesion structure.

[0129] One of the experimental schemes for cell imaging using the scheme of the present invention is as follows. Please note that this example is for illustrating the implementation of this patent and does not limit its specific implementation conditions:

[0130] Chemicals: Tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate, tri-n-propylamine, sodium dihydrogen phosphate dihydrate, and disodium hydrogen phosphate dodecahydrate were prepared with deionized (DI) water.

[0131] The device used in the present invention is carried out on an inverted optical microscope. The photons emitted by the electrochemical reaction are collected by an objective lens with a numerical aperture of 1.49 and detected by a water-cooled EMCCD camera with an EM gain of 500.

[0132] A three-electrode electrochemical system was used to record the electrochemiluminescence reaction. Ag / AgCl and Pt plates (15 mm × 15 mm × 0.1 mm) were used as reference and counter electrodes; ITO was used as a working electrode. A voltage of 1.4 V (vs. Ag / AgCl) was applied to trigger the ECL reaction. Phosphate buffer (0.15 M, pH 7.4) containing 50 mM and different concentrations was used as the electrolyte.

[0133] A data acquisition card is used to control the feedback voltage and acquire the associated data from a low-noise current amplifier with a custom programmed control interface. The electrochemiluminescence reaction current recording and image data acquisition (EMCCD camera) are synchronized together by using TTL (+5V) signals.

[0134] The imaging template was fabricated on a 240 nm thick ITO coverslip by using a focused ion beam operating at 120 pA ion beam at 30 kV. The patterned ITO coverslip was placed in an electrochemical cell containing 50 mM of . Due to the influence of the ion beam during FIB processing and the uneven surface activity of different ITO samples, the voltage (1.2 V-1.6 V) and different concentrations (10 μM-210 μM, depending on the different FIB samples, due to the interaction of the ion beam with ITO, the ion beam can deplete the active ITO layer) need to be adjusted according to the imaging feedback from the experimental event rate to obtain optimized random single-molecule observation. Note that the present invention generally requires the use of a higher concentration of for FIB-processed ITO samples to obtain event rates comparable to those of sputtered ITO samples. For super-resolution electrochemiluminescence imaging, a TIRF objective with a numerical aperture of 1.45 was used. The exposure time of the EMCCD was 5.6 ms per frame, and the field of view was 27.3 μm × 27.3 μm. The imaging data were fitted with an integrated Gaussian function to localize the luminescent molecules, and then super-resolution images were reconstructed by overlapping single molecules. Custom filters were applied to the super-resolution images to remove artifacts.

[0135] Human embryonic kidney cells (HEK293) were cultured in high glucose DEME medium (4.5 g / L glucose, without phenol red) at 37°C and 5% CO2. The medium was mixed with 10% normal fetal bovine serum and 1% penicillin-streptomycin. For live cell experiments, HEK293 cells were digested with trypsin-EDTA solution for 5 min and then transferred to ITO coverslips that had been sterilized in 75% alcohol for 10 min and volatilized in fire. The transferred cells were then cultured in DEME medium (without phenol red) at 37°C and 5% CO2 for 24 h. All cell culture reagents were provided by Sangon Biotech Co., Ltd. A 60× objective lens (NA=1.49) was used for super-resolution ECL imaging of live cells. Before the ECL imaging experiment, the ITO coverslips with cells were rinsed 3 times with PBS buffer to remove the culture medium residues. The ECL imaging experiments were performed at a pulse voltage of 1.2 V (frequency: 1 Hz) with 100 μM and 50 mM 1× PBS buffer. The exposure time for each frame was 3.013 milliseconds. After recording for 129 seconds, a total of 40,000 frames were collected, of which 20,000 frames were without voltage and used as background reference for data analysis. For fixed cell imaging, cells were first fixed with 4% paraformaldehyde (PFA) in PBS at 4°C, followed by immunolabeling and associated super-resolution optical fluorescence STORM imaging.

[0136] Single-molecule ECL microscopy can potentially be developed to visualize cell structures by labeling probe molecules with ECL. As with fluorescence, amplification of the emission photons of probe molecules labeled in ECL through multiple excitation processes can further improve imaging capabilities and specificity. Although ECL avoids laser-induced problems (such as photobleaching and autofluorescence), the use of voltage and ECL probes may also affect cells. Nevertheless, the present invention believes that single-molecule ECL has advantages in its sensitivity and can be used as an alternative and complementary technology to fluorescence methods for single-molecule imaging. Based on the spatial and temporal isolation of single reaction events, the present invention seeks to directly detect single ECL reactions by utilizing the single-photon emission generated, demonstrating significant improvements in observing electrochemical reactions in aqueous solutions. Finally, super-resolution ECL imaging is applied in bioassays and cell imaging.

[0137] Example 5: Micro-nanoscale electrochemical measurements

[0138] Since the ECL reaction mechanism determines the conversion relationship between one electron and one photon emission, the frequency detected by an isolated photon can be associated with the electron transfer rate, that is, the current. The relationship between the applied voltage and the corresponding frequency of photon detection is explored, and the Butler-Volmer equation in electrochemistry is used to fit the area of ​​the spatial super-resolution scale. The intrinsic kinetic constant of the electrochemical reaction can be obtained relative to the value of the substrate and the equilibrium potential of the reaction. Effect reference Fig.11 . Fig.11 AI is the distribution diagram of photon detection frequency at different spatial positions under different voltages. Fig.11 J is the relationship between the photon detection frequency and voltage when selecting different positions, and the corresponding BV equation fitting. Fig.11 K is the value of the fitted intrinsic kinetic constants in different regions relative to the substrate. Fig.11 L is the equilibrium potential when the fitted chemical reaction occurs.

[0139] Example 6: Catalyst characterization method

[0140] In the process of catalyst characterization, turnover frequency is often used as an indicator of the catalytic activity of the catalyst. That is, the number of substrate molecules catalyzed by the catalyst per unit time at a single site. For the experiment of the present invention, the present invention can obtain the turnover frequency of different positions by calculating the number of photons per unit time through the one-to-one correspondence between the number of photons in the reaction mechanism and the reaction cycle, thereby realizing the imaging of the active sites on the catalyst surface and the evaluation of the catalytic activity. For the effect, please refer to Fig.12 . Fig.12 a is the conventional ECL diagram, Fig.12 b is the super-resolution ECL imaging result. Fig.12 c is the associated scanning electron microscope image, Fig.12 d is the FRC resolution result, with a resolution of <31nm, breaking the Abbe diffraction limit. Fig.12 e is the comparison between traditional ECL imaging and super-resolution ECL imaging. Fig.12 f is the turnover frequency measurement diagram at 1.3V corresponding to e.

[0141] Example 6: Highly sensitive immunoassay

[0142] Based on the existing magnetic bead immunoelectrochemiluminescence detection scheme, combined with single molecule reaction signal detection, the detection and quantification of extremely low concentration substances in the solution can be achieved. Combined with the positioning information, the number of molecules on a single magnetic bead can be quantified through the number of positioning points. Through long-term signal accumulation, the detection signal-to-noise ratio of a single positioning point, that is, a single molecule on a magnetic bead, can exceed 1500 (SNR>1500) within 100 seconds, thereby achieving ultra-high sensitivity single molecule detection. Please refer to the effect. Fig.13 .in Fig.13 a is the principle diagram, Fig.13 b is the imaging image of ECL, Fig.13 c shows the relatively high signal-to-noise ratio after 100 seconds of accumulation of a single molecule.

[0143] Based on this method, it is possible to use existing electrochemiluminescence commercial kits to achieve single-molecule level detection, greatly expanding the detection limit of existing technologies. Therefore, it can be used for early diagnosis of diseases; trace detection of biological samples such as viruses; pregnancy detection and analysis of fetal gender and genetic diseases, as well as detection, quantification and imaging of explosives or biological traces in judicial processes, achieving extremely high detection sensitivity.

[0144] Similarly, the electrochemiluminescence technology described in the present invention can also be used to characterize and image surface chemical structures or similar biological imaging.

[0145] Example 7: Single-photon source or quantum light source based on chemiluminescent reaction

[0146] Single photon source or quantum light source requires that the luminescent system can only emit one photon during the lifetime of spontaneous radiation. The main ways to achieve single photon source in the existing technology are to use photoluminescence and electroluminescence. This application proposes to use chemiluminescence reaction as a way to generate single photon source. Specifically, take electrochemiluminescence reaction as an example. Fig.14 a Schematic diagram of the electrochemiluminescence reaction detection device generated by single photons, and Fig.14 The electrochemiluminescence measurement results shown in bg. By controlling the reaction parameters such as reaction concentration and applied voltage similar to the above, for example, for the electrochemiluminescence reaction, by controlling at least one of the reaction condition parameters such as reactant concentration, temperature, electrode activity, voltage, and voltage application method in the electrochemical reaction, a single photon signal is emitted during the spontaneous radiation lifetime. This embodiment takes the electrochemiluminescence reaction system of terpyridine ruthenium-tripropylamine as an example, the terpyridine ruthenium concentration range is: 1 pmole per liter to 2 mmol per liter; the tripropylamine concentration range is: 1 pmole per liter to 200 mmol per liter, so that the electrochemiluminescence reaction generates a single photon signal that is relatively isolated in space and time, which can be used as a single photon source. Fig.14 f shows the single photon signal of a single-molecule electrochemiluminescence reaction collected by the present application after time and space control. Although the present application takes the electrochemiluminescence reaction as an example, other chemiluminescence systems can also be used as single-photon sources or quantum light sources to achieve similar effects as long as they can produce a reaction system that satisfies the emission of a single photon each time.

[0147] Although the present application takes the terpyridine ruthenium-tripropylamine electrochemiluminescent system as an example, it is not limited to this electrochemiluminescent probe system. Similarly, the luminescent system in the electrochemiluminescent reaction system can be realized by the following combination. The luminescent agent includes a transition metal coordination compound, such as terpyridine ruthenium, terpyridine iridium, etc., or a derivative thereof, such as terpyridine ruthenium modified with a carboxyl group; it can be a carbon material such as carbon quantum dots, polymer carbon dots, graphene quantum dots, etc.; it can be a substance that can be used as a chemiluminescent probe itself, such as luminol and its derivatives, acridinium esters and their derivatives, 9,10-diphenylanthracene as a representative of condensed ring compounds and their derivatives; it can also be nanoparticles such as gold nanoparticles, quantum dots, metal organic porous compounds, covalent porous compounds, and the aforementioned luminescent materials loaded on nanoparticles; the co-anti-agent in the electrochemiluminescent system can be: a reducing substance represented by tripropylamine; an oxidizing substance represented by hydrogen peroxide. In particular, for some reactions in the annihilation mechanism, that is, the same ion undergoes redox reactions at the anode and cathode respectively, and the oxidation product and the reduction product after the reaction can continue to react chemically and emit light. When such molecules are used as luminescent probes, no additional co-reactants can be added. Such as terpyridine ruthenium, 9,10-diphenylanthracene, etc. In particular, for some small molecules (molecular weight less than 1000) such as luminol and its derivatives, there is an experimental possibility to reduce the applied voltage to 0V by rationally designing the system.

[0148] The electrochemiluminescence imaging method described in the present invention only requires the luminescence system to provide a photon signal, so it does not depend on the specific electrochemiluminescence system itself. As long as the photon signal of the electrochemiluminescence reaction can be generated, it can achieve an effect similar to the terpyridine ruthenium-tripropylamine electrochemiluminescence system exemplified in the embodiment of the present invention.

[0149] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An electrochemiluminescence imaging method, using an electrochemiluminescence imaging system, the electrochemiluminescence imaging system comprising: An electrochemical luminescence unit (1), a photon information acquisition unit (2) and a host (3); the electrochemical luminescence unit (1) comprises a sample pool (11) containing a reactant; the reactant can undergo an electrochemical luminescence reaction in the sample pool (11) and release a single photon signal or an isolated very small amount of photon signal; the photon information acquisition unit (2) is arranged at a position corresponding to the sample pool (11) and is electrically connected to the host (3); the photon information acquisition unit (2) continuously acquires the single photon signal or the isolated very small amount of photon signal and sends it to the host (3) in sequence; the host (3) generates an image according to the single photon signal or the isolated very small amount of photon signal sent in sequence by the photon information acquisition unit (2); the imaging step is characterized in that it comprises the following steps: S100, the photon information acquisition unit sequentially and continuously acquires the spatial position information of a single photon or a very small amount of isolated photons generated at the first moment, the spatial position information of a single photon or a very small amount of isolated photons generated at the second moment, ..., the spatial position information of a single photon or a very small amount of isolated photons generated at the Nth moment in the sample pool, and sequentially and continuously sends the spatial position information to the host; S200. The host receives the spatial position information of the single photon or the isolated very small amount of photons generated at the first moment, the spatial position information of the single photon or the isolated very small amount of photons generated at the second moment, ..., the spatial position information of the single photon or the isolated very small amount of photons generated at the Nth moment, and processes the received data to combine and superimpose the time and space positions of the single photons or the isolated very small amount of photons to generate a super-resolution image of electrochemical luminescence, the resolution of which exceeds the Abbe optical diffraction limit; wherein N is an integer greater than 1.

2. The electrochemiluminescence imaging method according to claim 1, characterized in that: In step S100, the spatial position information of the single photon or isolated very small amount of photons generated at the first moment collected includes the grayscale values ​​of the pixel of the single photon or isolated very small amount of photons at the first moment and multiple pixels adjacent to it, the spatial position information of the single photon or isolated very small amount of photons generated at the second moment includes the grayscale values ​​of the pixel of the single photon or isolated very small amount of photons at the second moment and multiple pixels adjacent to it, ..., the spatial position information of the single photon or isolated very small amount of photons generated at the Nth moment includes the grayscale values ​​of the pixel of the single photon or isolated very small amount of photons at the Nth moment and multiple pixels adjacent to it; Step S200 includes the steps of: S210, performing two-dimensional Gaussian or similar function fitting with spatial positioning function on the spatial position information of the single photon or isolated very small number of photons generated at the first moment, including the grayscale values ​​of the pixel of the single photon or isolated very small number of photons at the first moment and the multiple pixels adjacent thereto, to obtain the first position coordinates of the single photon or isolated very small number of photons generated at the first moment, performing two-dimensional Gaussian or similar function fitting with spatial positioning function on the spatial position information of the single photon or isolated very small number of photons generated at the second moment, including the grayscale values ​​of the pixel of the single photon or isolated very small number of photons at the second moment and the multiple pixels adjacent thereto, to obtain the second position coordinates of the single photon or isolated very small number of photons generated at the second moment, ..., performing two-dimensional Gaussian or similar function fitting with spatial positioning function on the spatial position information of the single photon or isolated very small number of photons generated at the Nth moment, including the grayscale values ​​of the pixel of the single photon or isolated very small number of photons at the Nth moment and the multiple pixels adjacent thereto, to obtain the Nth position coordinates of the single photon or isolated very small number of photons generated at the Nth moment; S220, generating an image according to the first position coordinates, the second position coordinates, ... and the Nth position coordinates, so that the time-space position combination of the single photon or the isolated very small number of photons is superimposed to generate a super-resolution image of electrochemical luminescence, so that the time-space resolution of the image exceeds the Abbe optical diffraction limit.

3. The electrochemiluminescence imaging method according to claim 2, characterized in that: In step S210, it also includes analyzing the standard deviation corresponding to the position coordinates of a single photon or an isolated very small number of photons at each moment or part of the first moment, the second moment, ..., and the Nth moment, accumulating the fitted signals, fusing the same signals, and performing noise reduction processing, and determining the position coordinates of single photons at different times and generating a super-resolution image of electrochemiluminescence.

4. The electrochemiluminescence imaging method according to any one of claims 1 to 3, characterized in that: An electrochemical detection step is included before the imaging step; the electrochemical detection step includes: S110, the electrochemical luminescence unit inputs a voltage to the sample cell, so that the reactants in the sample cell undergo an electrochemical reaction and release a single photon or a very small amount of isolated photons; S120, the electrochemical luminescence unit collects current information in the sample cell and sends the information to the host.

5. The electrochemiluminescence imaging method according to claim 4, characterized in that: The electrochemical luminescence unit also includes: a collection card, a reference electrode, a counter electrode and a working electrode; The acquisition card is electrically connected to the reference electrode, the counter electrode, the working electrode and the host respectively, and the reference electrode, the counter electrode and the working electrode are arranged on the sample pool; In step S110, the acquisition card outputs an analog voltage signal to both ends of the working electrode and the counter electrode; In step S120, the acquisition card acquires the current information of the electrochemical luminescence unit through the pair of electrodes, and sends the current information to the host.

6. The electrochemiluminescence imaging method according to claim 5, characterized in that: The electrochemical luminescence unit further comprises a current amplifier, and the counter electrode is electrically connected to the acquisition card via the current amplifier; In step S120, the current information of the electrochemical luminescence unit is amplified by the current amplifier and then sent to the acquisition card.

7. The electrochemiluminescence imaging method according to claim 6, characterized in that: Presetting a voltage waveform in the host; In step S110, the acquisition card 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 sent by the acquisition card with a preset voltage value, and controls the acquisition card to adjust the analog voltage signal sent to the working electrode and the counter electrode according to the comparison result.

8. An electrochemiluminescence imaging system, characterized in that: include: An electrochemical luminescence unit (1), a photon information collection unit (2) and a host (3); the electrochemical luminescence unit (1) comprises a sample pool (11) containing a reactant; the reactant can undergo an electrochemical luminescence reaction in the sample pool (11) and release a single photon signal or an isolated very small amount of photon signal; the photon information collection unit (2) is arranged at a position corresponding to the sample pool (11) and is electrically connected to the host (3); the photon information collection unit (2) continuously collects the single photon signal or the isolated very small amount of photon signal and sends it to the host (3) in sequence; the host (3) generates an image according to the single photon signal or the isolated very small amount of photon signal sent in sequence by the photon information collection unit (2), comprising the following imaging steps: S100, the photon information acquisition unit sequentially and continuously acquires the spatial position information of a single photon or a very small amount of isolated photons generated at the first moment, the spatial position information of a single photon or a very small amount of isolated photons generated at the second moment, ..., the spatial position information of a single photon or a very small amount of isolated photons generated at the Nth moment in the sample pool, and sequentially and continuously sends the spatial position information to the host; S200. The host receives the spatial position information of the single photon or the isolated very small amount of photons generated at the first moment, the spatial position information of the single photon or the isolated very small amount of photons generated at the second moment, ..., the spatial position information of the single photon or the isolated very small amount of photons generated at the Nth moment, and processes the received data to combine and superimpose the time and space positions of the single photons or the isolated very small amount of photons to generate a super-resolution image of electrochemical luminescence, the resolution of which exceeds the Abbe optical diffraction limit; wherein N is an integer greater than 1.

9. The electrochemiluminescence imaging system according to claim 8, characterized in that: The photon information collection unit (2) comprises a photon detector (21) and a microscopic imaging system (22); The sample pool (11) is placed on the microscopic imaging system (22), the photon detector (21) is fixed to the microscopic imaging system (22) and is interconnected with the host (3), and the photon detector (21) collects the single photon signal or isolated extremely small amount of photon signal generated in the sample pool (11) through the microscopic imaging system (22), and sends the information to the host (3).

10. The electrochemiluminescence imaging system according to claim 9, characterized in that: The microscopic imaging system (22) is equipped with a microscopic objective lens.

11. The electrochemiluminescence imaging system according to claim 9, characterized in that: The photon detector (21) is an electron multiplying camera or a complementary oxide semiconductor camera or a photomultiplier tube or an avalanche photodiode or a high-sensitivity photodetector with similar functions and an array thereof.

12. The electrochemiluminescence imaging system according to any one of claims 8 to 11, characterized in that: The electrochemical luminescence unit (1) further comprises: a data acquisition card (12), a reference electrode (13), a counter electrode (14) and a working electrode (15); The data acquisition card (12) is respectively connected to the reference electrode (13), the counter electrode (14), the working electrode (15) and the host (3); the reference electrode (13), the counter electrode (14) and the working electrode (15) are arranged on the sample pool (11); The acquisition card (12) sends a voltage signal to the working electrode (15) and the counter electrode (14), and the acquisition card (12) collects current information of the electrochemical luminescence unit (1) through the counter electrode (14), and sends the current information to the host (3).

13. The electrochemiluminescence imaging system according to claim 12, characterized in that: The data acquisition card is replaced by an electrochemical workstation or a device with a voltage application function capable of triggering an electrochemical luminescence reaction; the counter electrode (14) and the reference electrode (13) are replaced by the same counter electrode to achieve the same effect.

14. The electrochemiluminescence imaging system according to claim 12, characterized in that: The electrochemical luminescence unit (1) further comprises a current amplifier (16); The counter electrode (14) is electrically connected to the data acquisition card (12) via the current amplifier (16) so as to send the current information of the electrochemical luminescence unit (1) to the acquisition card (12).

15. The electrochemiluminescence imaging system according to claim 14, characterized in that: The current collection function realized by the current amplifier is replaced by an electrochemical workstation or a device capable of realizing the current collection function.

16. A method for collecting single photon signals or isolated extremely small photon signals of electrochemiluminescence, implemented by the electrochemiluminescence imaging system according to any one of claims 8 to 15, characterized in that: It includes an electrochemiluminescence reaction system and a photon signal collection system; triggering an electrochemiluminescent reaction in the electrochemiluminescent reaction system; It is characterized in that the photon signal collection system is used to collect a single photon signal or an isolated very small amount of photon signal released by the electrochemiluminescence reaction, wherein the single photon signal or the isolated very small amount of photon signal is generated by a single molecule electrochemical reaction.

17. A micro-nanoscale spatially resolved electrochemical measurement method, characterized in that: The micro-nanoscale spatially resolved electrochemical measurement method includes: being implemented by the electrochemiluminescence imaging method described in any one of claims 1 to 7; or being implemented by the electrochemiluminescence imaging system described in any one of claims 8 to 15; or being implemented by the electrochemiluminescence single photon signal or isolated extremely small amount of photon signal acquisition method described in claim 16.

18. The micro-nanoscale spatially resolved electrochemical measurement method according to claim 17, characterized in that: The micro-nanoscale spatially resolved electrochemical measurement method utilizes the electrochemiluminescence single photon signal or isolated extremely small photon signal acquisition method and the electrochemiluminescence imaging method to perform optical signal reading of current and potential information in electrochemical measurement.

19. A method for imaging a micro-nano structure, characterized in that: The imaging method of the micro-nano structure includes: using the electrochemiluminescence imaging method described in any one of claims 1-7; or using the electrochemiluminescence imaging system described in any one of claims 8-15; or using the electrochemiluminescence single photon signal or isolated extremely small amount of photon signal acquisition method described in claim 16.

20. The imaging method of micro-nanostructure according to claim 19, characterized in that: The micro-nano structure can be used as an electrode material for electrochemiluminescence or a material loaded on an electrode for collecting a single photon signal or a solitary extremely small amount of photon signals of electrochemiluminescence.

21. A catalyst characterization method, characterized in that: The catalyst characterization method comprises: implementing it by using the electrochemiluminescence imaging method described in any one of claims 1 to 7; or implementing it by using the electrochemiluminescence imaging system described in any one of claims 8 to 15; or implementing it by using the electrochemiluminescence single photon signal or isolated extremely small amount of photon signal acquisition method described in claim 16.

22. A method for characterizing a surface chemical structure, characterized in that: The surface chemical structure characterization method includes: using the electrochemiluminescence imaging method described in any one of claims 1 to 7; or using the electrochemiluminescence imaging system described in any one of claims 8 to 15; or using the electrochemiluminescence single photon signal or isolated extremely small photon signal acquisition method described in claim 16.

23. A biological imaging method, characterized in that: The biological imaging method includes: using the electrochemiluminescence imaging method described in any one of claims 1-7; or using the electrochemiluminescence imaging system described in any one of claims 8-15; or using the electrochemiluminescence single photon signal or isolated extremely small photon signal acquisition method described in claim 16.

24. An immunoassay method, characterized in that: The immunoassay method comprises: being implemented by the electrochemiluminescence imaging method described in any one of claims 1 to 7; or being implemented by the electrochemiluminescence imaging system described in any one of claims 8 to 15; or being implemented by the electrochemiluminescence single photon signal or isolated extremely small amount of photon signal acquisition method described in claim 16.

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