An electrochemical imaging system and real-time monitoring method based on critical angle reflection

Abstract

The present invention discloses an electrochemical imaging system based on critical angle reflection and a real-time monitoring method for electrochemical reactions. The present invention utilizes critical angle reflection imaging to align the irradiation thickness of incident light on the working electrode with the diffusion layer thickness of the solution in the electrochemical cell on the surface of the working electrode. This maximally converts the changes in the diffusion layer of the redox reaction into the signal of reflected light, increasing the sensitivity. It can simultaneously observe the diffusion layer and the electrode reaction process without the use of luminescent probes or strict material conditions. This method aims to improve the time resolution, sensitivity, and compatibility with more types of materials, and can be integrated with other imaging modes such as fluorescence or ECL for use.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical imaging technology, and particularly relates to an electrochemical imaging system based on the principle of critical angle reflection and a method for real-time monitoring of electrochemical reactions. Background Art

[0002] Electrochemical imaging technology is essential for visualizing electrochemical processes, which greatly enhances the understanding of fields such as materials science, energy storage, and biosensing. Currently, several electrochemical imaging methods, including scanning electrochemical microscopy (SECM), electrochemiluminescence (ECL) imaging, surface plasmon resonance microscopy (SPRM), etc., each have some defects.

[0003] CN104502388A discloses a photoelectrochemical kinetics test system and method based on a scanning electrochemical microscope. The system includes a scanning electrochemical microscope device, a Pt ultramicroelectrode, a sample fixing device, a light source device, and a turntable control device. The scanning electrochemical microscope device includes a three-dimensional controller and an electrochemical workstation; the sample fixing device includes a polytetrafluoro chemical cell and a fixing member; the light source device includes a radiator, a DC power supply, and red, yellow, blue, and white LED light sources arranged in sequence on the edge of the radiator disc; the turntable control device includes a central processor, a disc with a light passing hole, a driver, a controller, and a stepping motor. Scanning electrochemical microscopy (SECM) is favored for its high spatial resolution and sensitivity, but its application is limited by slow mechanical scanning, resulting in insufficient time resolution and limited ability for high-throughput measurement.

[0004] CN117849135A discloses an electrochemiluminescence microscopy device and its application. The device includes an electrochemical reaction cell and an electrochemiluminescence microscope located above the electrochemical reaction cell for photographing a working electrode to be measured; the electrochemical reaction cell includes a plastic petri dish and an electrochemical workstation. The plastic petri dish is filled with an electrolyte, and a working electrode, an auxiliary electrode, and a reference electrode to be measured are inserted into the electrolyte. The working electrode, the auxiliary electrode, and the reference electrode are all electrically connected to the electrochemical workstation. The electrolyte includes a luminol reagent and a sodium hydroxide solution containing sodium carbonate. Using this device for early corrosion prediction of titanium and its alloys, the method is simple, easy to operate, economical, and environmentally friendly, and has a relatively high spatio-temporal resolution for the corrosion area on the surface of pure titanium and its alloys. It is an effective means for early corrosion prediction of pure titanium and its alloys in various complex environments, but it requires labeling samples and has a low photon emission efficiency.

[0005] Surface plasmon resonance microscopy (SPRM) can achieve label-free recording of fast electrochemical processes, but it requires strict conditions, such as a nanometer-thin gold film, specific wavelengths, and polarization, which limit its general applicability and compatibility with other technologies. Summary of the Invention

[0006] In view of the defects existing in electrochemical imaging technology, the present invention provides a label-free electrochemical imaging system, namely a Critical Angle Reflection based Electrochemical Imaging (CARE) system. This method has high time resolution and high sensitivity, and is applicable to real-time electrochemical imaging and monitoring.

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

[0008] An electrochemical imaging system based on critical angle reflection includes an electrochemical module, an imaging module, and a control module;

[0009] The electrochemical module includes an electrochemical workstation and an electrochemical cell; the working electrode, reference electrode, and counter electrode in the electrochemical cell are connected to the electrochemical workstation; the working electrode is a conductive glass sheet;

[0010] The imaging module includes a light source, a three-dimensional light source displacement stage, a focusing device, an imaging magnifying device, and an image acquisition device; the light source is used to emit incident light, the incident light is focused by the focusing device, and the incident angle of the incident light is controlled by the three-dimensional light source displacement stage, so that the incident light irradiates on the working electrode through the imaging magnifying device, and the image data in the electrochemical cell is collected by the image acquisition device;

[0011] The control module includes a control display device and a data collection device; the data collection device connects and collects the data of the electrochemical workstation and the image acquisition device, transmits it to the control display device, and the control display device synchronizes the data and controls the working parameters of the electrochemical workstation and the image acquisition device.

[0012] During the electrochemical process, the intensity of the current is proportional to the surface concentration gradient; from an optical perspective, the reflectivity is proportional to the surface refractive index gradient, that is, the concentration gradient, thereby linking the electrochemical and optical properties and establishing a quantitative relationship between the electrochemical current density and the reflectivity. Specifically, the light emitted by the light source passes through the focusing device, making the originally divergent light focus on the rear focal plane of the imaging magnifying device. By adjusting the displacement stage, the position of the focus on the rear focal plane is changed to achieve the adjustment of the incident angle. In the present invention, the incident angle is set to an angle slightly lower than the critical angle, and by receiving the reflected light signal, the light signal is converted into an electrical signal.

[0013] The incident angle of the incident light on the working electrode is slightly lower than the critical angle, and at this time, its irradiation thickness on the working electrode is aligned with the diffusion layer thickness of the solution in the electrochemical cell on the surface of the working electrode. Thus, the changes in the diffusion layer of the redox reaction are maximally converted into the signal of the reflected light, increasing the sensitivity.

[0014] The counter electrode is one or more of a platinum electrode, a graphite electrode, a stainless steel electrode, a gold electrode, and a carbon rod electrode; the reference electrode is one or more of a silver / silver chloride electrode, a saturated calomel electrode, and a standard hydrogen electrode.

[0015] The imaging magnification device is an optical microscope high numerical aperture objective lens, including oil immersion objective lenses with magnification multiples such as 40 times, 60 times, and 100 times.

[0016] The image acquisition device is a CMOS camera or a CCD camera.

[0017] The conductive glass sheet is a transparent substrate coated with a conductive material thin film, and the conductive material includes one or more of indium tin oxide, gold, graphene, copper, silver, and platinum.

[0018] The light source includes one or more of a superluminescent diode (SLD), a monochromatic LED, and a laser light source.

[0019] The present invention also provides a real-time monitoring method for electrochemical imaging based on critical angle reflection, which uses the electrochemical imaging system based on critical angle reflection to perform real-time monitoring on an electrochemical reaction, including the steps of:

[0020] Step 1, adding a material or solution to be analyzed into the electrochemical cell;

[0021] Step 2, connecting the three electrodes in the electrochemical cell to an electrochemical workstation, and the light source emits incident light to irradiate the working electrode. Adjust the incident angle of the incident light so that the irradiation thickness is consistent with the diffusion layer thickness, and apply a voltage to cause an oxidation-reduction reaction of the material or solution to be analyzed;

[0022] Step 3, obtaining an optical signal according to the control display device to synchronize the image acquisition device in real time, and realizing real-time monitoring of the electrochemical process in the electrochemical cell according to the relationship between the optical signal and the chemical signal;

[0023] t At a certain moment, the current density of the electrochemical cell j ( t ) and the reflectivity of the incident light on the conductive glass sheet R ( t ) The quantitative relationship is:

[0024] (1)

[0025] Wherein, L -1 Is the inverse Laplace transform, Is the change in the reflectivity of the conductive glass sheet The Laplace transform of, , Is tThe reflectivity of the conductive glass sheet at a moment, is the initial reflectivity of the conductive glass sheet; ; is the refractive index change of the reducing agent per unit concentration, is the diffusion coefficient of the reducing agent per unit concentration; is the refractive index change of the oxidizing agent per unit concentration, is the diffusion coefficient of the oxidizing agent per unit concentration; n represents the number of electrons transferred in the redox reaction of the material or solution to be analyzed; F is the Faraday constant, is the incident angle, is the initial concentration of the reactants in the solution;

[0026] When the initial concentration of the reactants is determined, , where k , b is a constant determined by the refractive index of the solution.

[0027] The constant k and b can be obtained according to the following steps. The specific steps include:

[0028] Through the Fresnel equation, calculate the relationship graph between the reflectivity θ and the refractive index of the solution at the known incident angle R ; linearly fit the refractive index intervals corresponding to the oxidizing agent and the reducing agent to obtain the slope β ;

[0029] Change θ to obtain a series of corresponding β values for plotting, and obtain that 1 / β has a linear relationship with θ to obtain the constants k and b .

[0030] The material to be analyzed in Step 1 includes any one of electrolyte solution, nanoparticles, and cells.

[0031] When the material to be analyzed is an electrolyte solution, the electrolyte solution contains reactants that can be oxidized or reduced, and the refractive index of the oxidation product or reduction product is different from that of the reactant;

[0032] When the material to be analyzed is nanoparticles, the particle diameter of the nanoparticles is between 40 and 500 nanometers. The nanoparticles are selected from metal nanoparticles such as silver, gold, and platinum. The nanoparticles must have electrocatalytic properties to cause the reactants in the solution to undergo a redox reaction, and the refractive index of the oxidation product or reduction product is different from that of the reactant;

[0033] When the material to be analyzed is a cell, the cell must have adhered and grown on the surface of a conductive glass slide and be placed in an electrolyte solution that contains reactants that can be oxidized or reduced, and the refractive index of the oxidation product or reduction product is different from that of the reactant.

[0034] In the present invention, the electrochemical imaging system based on critical angle reflection can be used for real-time optical imaging of homogeneous or heterogeneous surfaces of different types of electrolyte solutions, imaging analysis of ion concentration gradients at the surface and interface, catalytic activity analysis of nanomaterials at the single-particle level, high-resolution imaging of the electrochemical activity of single nanoparticles, or electrochemical imaging analysis of cells.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] In the electrochemical imaging system based on critical angle reflection of the present invention, by using critical angle reflection imaging, it is possible to simultaneously observe the diffusion layer and the electrode reaction process without the use of luminescent probes or strict material conditions. This method aims to improve the time resolution, sensitivity, and compatibility with more types of materials, and can be integrated with other imaging modes (such as fluorescence and ECL) for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic structural diagram of the electrochemical imaging system based on critical angle reflection in the present invention, where 11 is an electrochemical workstation, 21 is a working electrode, 22 is an electrochemical cell, 31 is a light source, 32 is a three-dimensional light source displacement stage, 33 is an imaging and magnification device, 34 is an image acquisition device, 35 is a focusing device, 41 is a control and display device, and 51 is a data collection device.

[0038] Figure 2 It is a diagram of electrochemical signals and optical signals in Example 1, where Figure 2 A is a graph of the voltage ( E ), recorded by the electrochemical workstation, varying with time ( t ); Figure 2 B is a graph of the current ( I ), recorded by the electrochemical workstation, varying with time ( t ); Figure 2 C is a graph of the optical signal (Int.) on the surface of the conductive glass electrode, collected by the image acquisition device, varying with time ( t ); Figure 2 D is a graph of the current density ( Figure 2 ), obtained by converting the optical signal in C of j CARE ), varying with time ( t ).

[0039] Figure 3 It is a diagram of electrochemical signals and optical signals in Example 2, where Figure 3A is the critical angle reflection imaging diagram of the area of the heterogeneous surface of the ITO electrode; Figure 3 B is the cyclic voltammogram ( I-E ) recorded by the electrochemical workstation before and after modifying with APTES; Figure 3 C is based on Figure 3 The current signal obtained by converting the optical signal in A of j CARE ) varying with voltage ( E ); Figure 3 D is the spatial distribution comparison diagram of the maximum current values of the bare ITO area and the APTES area during the cyclic voltammetry process.

[0040] Figure 4 are the electrochemical signal and optical signal diagrams during the catalytic hydrogen evolution process in Example 3, where Figure 4 A is the critical angle reflection imaging diagram of Pt nanoparticles at different voltages; Figure 4 B is the current signal obtained by converting the optical signals of Pt nanoparticles and the background ( j CARE ) varying with voltage ( E ); Figure 4 C is the statistical chart of the current peaks ( j peak ) converted from the optical signals of 151 Pt nanoparticles when the applied voltage is -0.7 V.

[0041] Figure 5 In A, it is the bright field and ECL imaging schematic diagrams of single cells (numbered 1, 2, 3) in Example 4, Figure 5 In B, it is the current signal diagram obtained by converting the optical signals of the same single cells (1, 2, 3) in Example 4 through critical angle reflection imaging; Figure 5 In C, it is the cyclic voltammogram of three cells and a background area in Example 4, and its current is obtained by critical angle reflection imaging. Detailed implementation manners

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the 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. Those skilled in the art make modifications or equivalent replacements on the basis of understanding the technical solutions of the present invention, and without departing from the spirit and scope of the technical solutions of the present invention, they should all be covered within the protection scope of the present invention.

[0043] As Figure 1 shown, an electrochemical imaging system based on critical angle reflection includes an electrochemical module, an imaging module and a control module;

[0044] The electrochemical module includes an electrochemical workstation 11 and an electrochemical cell 22; the working electrode 21, reference electrode, and counter electrode in the electrochemical cell 22 are connected to the electrochemical workstation. The electrochemical cell 22 is a reaction cell made of polydimethylsiloxane (PDMS). The counter electrode is a platinum electrode, and the reference electrode is a silver / silver chloride electrode. The working electrode 21 is a conductive glass sheet laid flat at the bottom of the electrochemical cell. The conductive glass sheet is a transparent substrate coated with an indium tin oxide thin film with a thickness of 99 nm, and the resistance is 10 - 15 Ω / square.

[0045] The imaging module includes a light source 31, a three-dimensional light source displacement stage 32, an imaging magnifying device 33, an image acquisition device 34, and a focusing device 35. The light source 31 is a superluminescent light-emitting diode (SLD) for generating incident light. The incident light passes through the focusing device 35 (lens group) and focuses the light on the rear focal plane of the imaging magnifying device 33. The position of the focal point on the rear focal plane is controlled by the three-dimensional light source displacement stage 32, so that the incident light irradiates the working electrode 21 through the imaging magnifying device 33 at an incident angle slightly lower than the critical angle (about 61.6°). The irradiation thickness of the incident light above the conductive glass sheet of the working electrode 21 is aligned with the diffusion layer of the electrolytic solution in the electrochemical cell on the surface of the conductive glass sheet, and the image data in the electrochemical cell is collected by the image acquisition device 34. The imaging magnifying device 33 is an optical microscope objective with a high numerical aperture, and the image acquisition device 34 is a CMOS camera.

[0046] The control module includes a control display device 41 and a data collection device 51. The data collection device 51 connects to and collects the data of the electrochemical workstation 11 and the image acquisition device 34, and transmits it to the control display device 41. The control display device 41 synchronizes the data and controls the working parameters of the electrochemical workstation 11 and the image acquisition device 34.

[0047] Example 1

[0048] Using the Figure 1 electrochemical imaging system based on critical angle reflection as shown to perform real-time imaging and monitoring of the electrochemical reaction. The specific method includes the steps:

[0049] Step 1, add 10 mM K 3 Fe(CN) 6 solution into the electrochemical cell, and control the angle of the incident light through the three-dimensional displacement stage so that it is slightly lower than the critical angle (61.64°), so that the illumination thickness of the incident light on the surface of the indium tin oxide (ITO) electrode is basically aligned with the diffusion layer during the Fe(CN) 6 3- redox reaction (about 50 microns);

[0050] Step 2: Connect the three electrodes in the electrochemical cell to the electrochemical workstation. Use cyclic voltammetry scanning (+0.5 V, -0.3 V, 100 mV / s). An oxidation-reduction reaction occurs in the electrochemical cell. , Record the optical signal by controlling the display device and the image acquisition device, and synchronously record the current signal for verification.

[0051] Step 3: Obtain the optical signal according to the control display device in real time to synchronize the image acquisition device. According to the relationship between the reflectivity and the current density in the optical signal, realize the real-time monitoring of the electrochemical reaction in the electrochemical cell;

[0052] After three cyclic voltammetry scans of the electrochemistry are completed, record the voltage signal E as Figure 2 shown in A; the current signal I as Figure 2 shown in B, Current I Reduction peaks and oxidation peaks start to appear at about +0.04 V and +0.25 V (relative to the Ag / AgCl reference), respectively, which is consistent with the oxidation-reduction potential of K 3 Fe(CN) 6 reported in the literature, confirming that an electrochemical oxidation-reduction reaction has occurred in the solution.

[0053] The optical signal recorded by the image acquisition device shows that when cyclic voltammetry is performed on the solution, the intensity of the optical signal Int. on the electrode surface changes with time as Figure 2 shown in C; Int. is the intensity of the reflected light and is proportional to the reflectivity . In the experiment of the change reflects the change of the concentration gradient of Fe(CN) 6 3- and Fe(CN) 6 4- on the ITO surface over time during this oxidation-reduction process.

[0054] The optical signal reflectivity can be converted into the current density j CARE of the electrical signal using formula (1) t , that is t at time j CARE the current density t of the electrochemical cell:

[0055] (1)

[0056] where L -1 is the inverse Laplace transform, The reflectivity change of the conductive glass sheet is the Laplace transform of , where t is the reflectivity of the conductive glass sheet at time , and

[0057] is the initial reflectivity of the conductive glass sheet; ; is the refractive index change of the reducing agent per unit concentration (in this example, Fe(CN) 6 4- ), which is 0.00006 RIU / (mol·m 3 ); is the diffusion coefficient of the reducing agent per unit concentration (in this example, Fe(CN) 6 4- ), which is 1×10 -9 m 2 / s; is the refractive index change of the oxidizing agent per unit concentration (in this example, Fe(CN) 6 3- ), which is 0.00005 RIU / (mol·m 3 ); is the diffusion coefficient of the oxidizing agent per unit concentration (in this example, Fe(CN) 6 3- ), which is 1×10 -9 m 2 / s; θ is the incident angle, which is 61.64° in this example; β is -72.0 RIU -1 ; n represents the number of electrons transferred in the redox reaction of the reactants (in this example n = 1); F is the Faraday constant, F = 96485 C / mol.

[0058] The graph of the electrical signal converted from the optical signal of C in Figure 2 versus time is shown in D of Figure 2 . It can be seen that it almost completely coincides with the current signal measured by the electrochemical workstation ( Figure 2 in B). It is confirmed that the present invention can achieve the conversion between optical signals and electrical signals and monitor the electrochemical process in real time.

[0059] Example 2

[0060] Using as Figure 1The electrochemical imaging system based on critical angle reflection shown performs real-time optical imaging and monitoring of the heterogeneous interface electrolyte. Taking the indium tin oxide (ITO) electrode modified with γ-aminopropyltriethoxysilane (APTES) as an example, the specific method includes:

[0061] Step 1: Dip a PDMS square of about 10×5 mm in an ethanol solution of 3% APTES, stick it on the right half of the ITO, after modification for 30 minutes, remove the PDMS, wash the ITO wafer with ethanol, and then dry it at 110 °C for 30 minutes to obtain an ITO electrode with half of its surface modified with APTES.

[0062] Step 2: Add PBS phosphate buffer containing 5 mM K 3 Fe(CN) 6 as the electrolyte into the electrochemical cell. Control the angle of the incident light at about 61.6° through a three-dimensional displacement stage, so that the illumination thickness of the incident light on the surface of the ITO electrode is aligned with the diffusion layer during the redox reaction of Fe(CN) 6 3- (about 50 μm);

[0063] Step 3: Connect the three electrodes in the electrochemical cell to an electrochemical workstation and use cyclic voltammetry scanning (+0.5 V, -0.3 V, 100 mV / s). Redox reactions occur in the electrochemical cell . Record the optical signal through a control display device and synchronously record the current signal for verification.

[0064] Step 4: Obtain the optical signal according to the control display device in real-time and synchronize it with the image acquisition device. According to the relationship between the reflectivity and current density in the optical signal, realize the real-time imaging monitoring of the redox reaction of Fe(CN) 6 3- on different regions of the ITO surface;

[0065] The critical angle reflection imaging diagram of the surface of the working electrode after half of its surface is modified with APTES is as shown in Figure 3 A (no voltage applied, scale bar 10 μm). The left side is the bare ITO area without modification, and the right side is the area modified with APTES. It can be seen that there are significant differences in the imaging of the bare ITO and APTES / ITO areas at this angle, and the latter shows a rough APTES pattern.

[0066] Figure 3In Figure B, the cyclic voltammograms recorded by the electrochemical workstation before and after modifying with APTES are used to verify the successful modification of APTES and the accuracy of critical angle reflection measurement. The peak values of the CV curve in the bare ITO region are located at +0.04 V and +0.25 V. After modifying with APTES, the peak values shift to higher potentials, located at +0.10 V and +0.30 V, and the current is larger, because the modification of APTES changes the electrochemical activity of ITO.

[0067] The optical signals in the bare ITO region ( Figure 3 region 1 in Figure A) and the APTES region ( Figure 3 region 2 in Figure A) are converted into current density according to the steps in Example 1 and formula (1). j CARE The graph of the change of current density with voltage (where the oxidant and reductant are Fe(CN) 6 3- and Fe(CN) 6 4- , and the method is the same as described in Example 1), and the results are shown in Figure 3 Figure C (region 1, 2 and the average of the whole image). The dotted line represents the average CV obtained from the whole field of view, including the bare ITO region and the APTES region, and its shape is basically the same as the signal obtained by the electrochemical workstation ( Figure 3 Figure B), which proves the measurement accuracy. At the same time, it also shows that the present invention can perform local and separate analysis on heterogeneous interfaces, revealing the differences in reaction efficiency and electron transfer kinetics in heterogeneous interfaces.

[0068] Figure 3 Figure D shows the redox current distribution maps in the bare ITO region and the APTES region. It can be seen that the surface current distribution of the APTES modification is significantly higher than that of the bare ITO region, and the spatial distribution is uneven, reflecting the application of this technology in imaging the current of uneven electrode surfaces.

[0069] Example 3

[0070] Using the critical angle reflection-based electrochemical imaging system shown in Figure 1 to perform high-throughput analysis on the catalytic activity of single nanoparticles. Taking the catalytic hydrogen evolution reaction of Pt nanoparticles (73 nm) in 1 M H 2 SO 4 solution as an example, the specific method includes the following steps:

[0071] Step 1: Disperse and coat Pt nanoparticles (PtNP) on the surface of the ITO electrode. After ultrasonic dispersing the PtNP solution for 3 minutes, take 100 μL and evenly distribute it on the ITO sheet, and let it dry naturally. At this time, PtNP is sparsely dispersed on the ITO surface in the form of single particles (the ITO area without PtNP coverage can be used as the background for the control experiment). Add 1 M H 2 SO 4 solution into the electrochemical cell; control the angle of the incident light to be about 61.6° through the three-dimensional displacement stage (the same as in Example 2);

[0072] Step 2: Connect the three electrodes in the electrochemical cell to the electrochemical workstation, and use cyclic voltammetry scanning (from 0 V to -0.7 V, 100 mV / s) to cause the catalytic reaction of PtNP in the electrochemical cell: hydrogen ions are reduced to hydrogen on the surface of PtNP, forming a concentration gradient near PtNP. Record the optical signal through the control display device, and synchronously record the current signal of the electrochemical workstation for verification.

[0073] Step 3: Obtain the optical signal according to the control display device by synchronously using the image acquisition device in real time. According to the relationship between the reflectivity and the current density in the optical signal, realize the real-time monitoring of hydrogen evolution on the surface of a single PtNP in the electrochemical cell;

[0074] Figure 4 In [Figure], A shows the critical angle reflection current diagrams (scale 1 μm) of three marked platinum nanoparticles PtNP (numbered 1 - 3) in a certain area on the working electrode surface at different potentials. It can be seen that the more negative the potential, the stronger the contrast between PtNP and the background, reaching the maximum value when the applied voltage is -0.7 V, indicating that the amount of hydrogen attached to the surface of PtNP reaches the peak.

[0075] Figure 4 In [Figure], B shows the CV curves obtained by converting the optical signals of three PtNP (numbered 1 - 3) and an ITO background area without PtNP according to formula (1) in Example 1. Among them, the oxidant and the reductant are H + and H 2 . The parameters used in the formula are: = 0.000005 RIU / (mol·m 3 ), = 9.3×10 -9 m 2 / s; =0, = 5.11×10 -9 m 2 / s, θ = 61.64°, β = -15.3 RIU -1 , n = 1,F = 96485 C / mol. It can be seen that the current in the background region is small and can be ignored. The three PtNPs show different current magnitudes in CV, indicating the heterogeneity of electrocatalytic activity among different nanoparticles.

[0076] Figure 4 In the C of, the current peak ( j peak ) distribution map of 151 PtNPs converted according to the optical signal at an applied voltage of -0.7 V is shown (the method is the same as that of B in Figure 4 ), further indicating the heterogeneity of the catalytic activity of individual nanoparticles. In summary, the high sensitivity and resolution of the present invention can achieve high-throughput imaging and analysis of electrochemical reactions at the single-particle scale.

[0077] Example 4

[0078] Using the electrochemical imaging system based on critical angle reflection as shown in Figure 1 and combining electrochemiluminescence (ECL) to image single cells. Among them, ECL uses the imaging amplification device 33 without adding other devices. Taking the cell ECL imaging in the Ru(bpy) 3 Cl 2 and TPrA solution system as an example, the specific method includes:

[0079] Step 1: Culture A549 cells on the surface of the ITO electrode overnight in a cell culture incubator to make the cells closely adhere to the electrode. Then wash the cell culture medium with PBS solution, and add 10 mM Ru(bpy) 3 Cl 2 and 20 mM TPrA (10 mM PBS) solution into the electrochemical working cell; control the angle of the incident light at 61.55° through a three-dimensional displacement stage;

[0080] Step 2: Connect the three electrodes in the electrochemical cell to an electrochemical workstation, and use cyclic voltammetry scanning (scanning range: 0 V to +1.4 V; scan rate: 100 mV / s). The redox reaction of Ru(bpy) 3 Cl 2 occurs in the electrochemical cell, and the reaction realizes luminescence by consuming the coreactant TPrA. By controlling the display device, record the ECL optical signal and the critical angle reflection optical signal successively, and synchronously record the current signal for verification.

[0081] Step 3: Obtain the optical signals according to the control display device to synchronously acquire the image acquisition device, including the ECL and critical angle reflection signals. Among them, the ECL signal is not further processed. For the critical angle reflection signal, calculate the current value using formula (1) according to the steps of Example 1.

[0082] Figure 5 In Figure A, the bright-field image and the corresponding ECL imaging of three cells labeled 1-3 in a certain area at a voltage of 1.0 V are shown (scale bar: 10 μm); in the ECL image, compared with the bright ITO background, the ECL images of cells 1-3 show dark stripes. This is because the cells adhere to the ITO surface, forming a closed space at the adhesion sites, and the ECL reaction process depletes the TPrA in the closed space, resulting in the inability to continuously emit light.

[0083] Figure 5 In Figure B, the critical angle reflection current signal map calculated by formula (1) for the three cells in the above area at an applied voltage of 1.2 V is shown, where the oxidant and reductant are Ru(bpy) 3 3+ and Ru(bpy) 3 2+ . The parameters used in the formula are: = 0.00027 RIU / (mol·m 3 ), = 5.9×10 -10 m 2 / s; = 0.00028 RIU / (mol·m 3 ), = 5.9×10 -10 m 2 / s; θ = 61.55°, β = - 26.3 RIU -1 , n =1, F = 96485 C / mol. It can be seen that, contrary to the ECL image, the cells all show bright stripes. This observation is attributed to the different detection mechanisms of ECL and the present invention: ECL depends on the reaction of Ru(bpy) 3 2+ and TPrA to emit light, and both need to coexist. Once the TPrA is depleted, the system will no longer emit light; while the signal of the present invention comes from the refractive index change caused by the redox reaction of Ru(bpy) 3 2+ (the contribution of TPrA to the refractive index is extremely small). Even if the TPrA is depleted, the redox reaction of Ru(bpy) 3 2+ will continue to occur, making the critical angle reflection signal still exist.

[0084] Figure 5 Figure C shows that during the cyclic voltammetry process, Figure 5The current as a function of voltage for the three cell regions shown in B, as well as the background region (where there are no cells). It can be seen that the current in the cell regions is stronger than that in the background region. This increase in current is due to the confinement effect of cell adhesion sites. At the adhesion sites, the cells form gaps ranging from dozens of nanometers to hundreds of nanometers, which greatly restricts the diffusion of Ru(bpy) 3 2+ and Ru(bpy) 3 2+ ions, resulting in an increase in the local ion concentration and thus an increase in current. This example shows that the present invention can directly image the change in ion concentration, that is, the sample itself does not need to have luminescent properties.

Claims

1. An electrochemical imaging system based on critical angle reflection, characterized in that: It includes an electrochemical module, an imaging module and a control module; The electrochemical module includes an electrochemical workstation and an electrochemical cell; the working electrode, reference electrode and counter electrode in the electrochemical cell are connected to the electrochemical workstation; the working electrode is a conductive glass sheet; The imaging module includes a light source, a three-dimensional light source displacement stage, a focusing device, an imaging magnifying device and an image acquisition device; the light source is used to emit incident light, the incident light is focused by the focusing device, the incident angle of the incident light is controlled by the three-dimensional light source displacement stage, the irradiation thickness of the incident light on the working electrode is aligned with the diffusion layer thickness of the solution in the electrochemical cell on the surface of the working electrode, the incident light is irradiated on the working electrode through the imaging magnifying device, and the image data in the electrochemical cell is acquired by the image acquisition device; The control module includes a control display device and a data collection device; the data collection device is connected to and collects data from the electrochemical workstation and the image acquisition device, and transmits the data to the control display device, which synchronizes the data and controls the working parameters of the electrochemical workstation and the image acquisition device; The control display device is synchronized with the image acquisition device in real time to obtain the optical signal, and the real-time monitoring of the electrochemical process in the electrochemical cell is realized according to the relationship between the optical signal and the chemical signal; t At this time, the current density of the electrochemical cell j ( t ) and the reflectivity of the incident light on the conductive glass sheet R ( t ) is: （1） in, L -1 is the inverse Laplace transform, is the reflectivity change of the conductive glass sheet The Laplace transform of , for t The reflectivity of the conductive glass sheet at this time, is the initial reflectivity of the conductive glass sheet; ; is the refractive index change per unit concentration of reducing agent, is the diffusion coefficient of the reducing agent per unit concentration; is the refractive index change per unit concentration of oxidant, is the diffusion coefficient of the oxidant per unit concentration; 𝑛 represents the number of electrons transferred by the redox reaction of the material or solution to be analyzed; F is the Faraday constant, is the incident angle, is the initial concentration of the reactant in the solution; When the initial concentration of the reactants is determined, in k , b is a constant determined by the refractive index of the solution, β is the slope.

2. The electrochemical imaging system based on critical angle reflection according to claim 1, characterized in that: The counter electrode is one or more of a platinum electrode, a graphite electrode, a stainless steel electrode, a gold electrode, and a carbon rod electrode; the reference electrode is one or more of a silver / silver chloride electrode, a saturated calomel electrode, and a standard hydrogen electrode.

3. The electrochemical imaging system based on critical angle reflection according to claim 1, characterized in that: The imaging magnification device is an optical microscope high numerical aperture objective lens.

4. The electrochemical imaging system based on critical angle reflection according to claim 1, characterized in that: The image acquisition device is a CMOS camera or a CCD camera.

5. The electrochemical imaging system based on critical angle reflection according to claim 1, characterized in that: The conductive glass sheet is a transparent substrate covered with a conductive material film, and the conductive material includes one or more of indium tin oxide, gold, graphene, copper, silver, and platinum.

6. The electrochemical imaging system based on critical angle reflection according to claim 1, characterized in that: The light source includes one or more of a superluminescent diode, a monochromatic LED or a laser light source.

7. A real-time monitoring method of electrochemical imaging based on critical angle reflection, characterized in that: Real-time monitoring of electrochemical reactions using the electrochemical imaging system based on critical angle reflection as described in any one of claims 1 to 6 comprises the following steps: Step 1, adding the material or solution to be analyzed into the electrochemical cell; Step 2, connecting the three electrodes in the electrochemical cell to the electrochemical workstation, irradiating the working electrode with incident light from a light source, adjusting the incident angle of the incident light so that the irradiation thickness is consistent with the diffusion layer thickness, and applying voltage to cause a redox reaction in the material or solution to be analyzed; Step 3, according to the control display device, the image acquisition device is synchronized in real time to obtain the optical signal, and according to the relationship between the optical signal and the chemical signal, the electrochemical process in the electrochemical cell is monitored in real time; t At this time, the current density of the electrochemical cell j ( t ) and the reflectivity of the incident light on the conductive glass sheet R ( t ) is: （1） in, L -1 is the inverse Laplace transform, is the reflectivity change of the conductive glass sheet The Laplace transform of , for t The reflectivity of the conductive glass sheet at this time, is the initial reflectivity of the conductive glass sheet; ; is the refractive index change per unit concentration of reducing agent, is the diffusion coefficient of the reducing agent per unit concentration; is the refractive index change per unit concentration of oxidant, is the diffusion coefficient of the oxidant per unit concentration; 𝑛 represents the number of electrons transferred by the redox reaction of the material or solution to be analyzed; F is the Faraday constant, is the incident angle, is the initial concentration of the reactant in the solution; When the initial concentration of the reactants is determined, in k , b is a constant determined by the refractive index of the solution, β is the slope.

8. The electrochemical imaging real-time monitoring method based on critical angle reflection according to claim 7, characterized in that: The material to be analyzed in step 1 includes any one of electrolyte, nanoparticles, and cells.

9. The electrochemical imaging real-time monitoring method based on critical angle reflection according to claim 7, characterized in that: When the material to be analyzed is an electrolyte, the electrolyte contains reactants that can be oxidized or reduced, and the refractive index of the oxidation product or the reduction product is different from the refractive index of the reactants; When the material to be analyzed is a nanoparticle, the nanoparticle has an electrocatalytic property, causing the reactants in the solution to undergo an oxidation-reduction reaction, and the refractive index of the oxidation product or the reduction product is different from the refractive index of the reactant; When the material to be analyzed is a cell, the cell has grown on the surface of the conductive glass sheet and is placed in an electrolyte, the electrolyte contains reactants that can be oxidized or reduced, and the refractive index of the oxidation product or the reduction product is different from the refractive index of the reactant.

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