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

Abstract

The invention discloses an electrochemical imaging system based on critical angle reflection and an electrochemical reaction real-time monitoring method, which are characterized in that the irradiation thickness of incident light on a working electrode is aligned with the thickness of a diffusion layer of a solution in an electrochemical cell on the surface of the working electrode by utilizing critical angle reflection imaging. The present invention maximizes the conversion of changes in a diffusion layer of a redox reaction into a signal of reflected light, and increases sensitivity. The diffusion layer and the electrode reaction process can be observed at the same time, and a light-emitting probe or strict material conditions are not needed. The method aims to improve time resolution and sensitivity, is compatible with more types of materials, and can be integrated with other imaging modes such as fluorescence or ECL.

Description

Technical Field

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

[0002] Electrochemical imaging technology is essential for the visualization of electrochemical processes, and it has greatly improved the understanding of fields such as materials science, energy storage, and biosensing. Several current electrochemical imaging methods, including scanning electrochemical microscopy (SECM), electrochemiluminescence (ECL) imaging, surface plasmon resonance microscopy (SPRM), etc., 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 polytetrafluoroethylene 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 disk; the turntable control device includes a central processing unit, a disk with a light hole, a driver, a controller, and a stepper 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 high-throughput measurement capabilities.

[0004] CN117849135A discloses an electrochemical luminescence microscope device and its application, the device comprises an electrochemical reaction cell and an electrochemical luminescence microscope located above the electrochemical reaction cell for photographing the electrode to be tested; the electrochemical reaction cell comprises a plastic culture dish and an electrochemical workstation, the plastic culture dish is filled with an electrolyte, the electrode to be tested is inserted into the electrolyte as a working electrode, an auxiliary electrode and a reference electrode, the working electrode, the auxiliary electrode and the reference electrode are all electrically connected to the electrochemical workstation, and the electrolyte comprises a luminol reagent and a sodium hydroxide solution containing sodium carbonate. The device is used for early corrosion prediction of titanium and its alloys, the method is simple and easy to operate, economical and environmentally friendly, has a relatively high temporal and spatial resolution for the surface corrosion area of ​​pure titanium and its alloys, and is an effective means for early corrosion prediction of pure titanium and its alloys under various complex environments, but requires sample labeling and the efficiency of photon emission is low.

[0005] Surface plasmon resonance microscopy (SPRM) enables label-free recording of fast electrochemical processes, but it requires stringent conditions, such as nanometer-thin gold films, specific wavelengths, and polarization, which limit its general applicability and compatibility with other techniques. Summary of the invention

[0006] In view of the defects of electrochemical imaging technology, the present invention provides an electrochemical imaging system that does not require labeling, namely, an electrochemical imaging (CARE) system based on critical angle reflection. This method has high temporal resolution and high sensitivity and is suitable for real-time electrochemical imaging and monitoring.

[0007] To achieve the above object, the technical solution adopted by the present invention is: An electrochemical imaging system based on critical angle reflection comprises 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 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 connects 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.

[0008] In 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 a focusing device so that the originally divergent light is focused on the back focal plane of the imaging magnification device. By adjusting the displacement stage, the position of the focus on the back focal plane is changed to achieve the angle adjustment of the incident angle. In the present invention, the incident angle is set to an angle slightly lower than the critical angle, and the reflected light signal is received to convert the optical signal into an electrical signal.

[0009] The incident angle of the incident light on the working electrode is slightly lower than the critical angle, at which time the irradiation thickness on the working electrode is aligned with the thickness of the diffusion layer 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 converted into reflected light signals to the maximum extent, increasing sensitivity.

[0010] 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.

[0011] The imaging magnification device is an optical microscope high numerical aperture objective lens, including an oil immersion objective lens with magnifications of 40 times, 60 times, 100 times, etc.

[0012] The image acquisition device is a CMOS camera or a CCD camera.

[0013] 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.

[0014] The light source includes one or more of a superluminescent diode (SLD), a monochromatic LED or a laser light source.

[0015] The present invention also provides a real-time monitoring method of electrochemical imaging based on critical angle reflection, which uses the electrochemical imaging system based on critical angle reflection to monitor the electrochemical reaction in real time, comprising the steps of: 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 unit concentration of oxidant; nIt indicates 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.

[0016] constant k and b You can obtain it by following the steps below. The specific steps include: Using the Fresnel equations, calculate the known angle of incidence θ Below, reflectivity R The relationship diagram of the refractive index of the solution; linear fitting is performed on the refractive index intervals corresponding to the oxidant and the reductant to obtain the slope β ; Change θ Get a series of corresponding β Plot the values ​​and obtain 1 / β and θ is a linear relationship, and the constant k and b .

[0017] The material to be analyzed in step 1 includes any one of electrolyte, nanoparticles, and cells.

[0018] When the material to be analyzed is an electrolyte, the electrolyte contains a reactant 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; When the material to be analyzed is a nanoparticle, the particle diameter of the nanoparticle is between 40 and 500 nanometers, and the nanoparticle is selected from metal nanoparticles such as silver, gold, and platinum. The nanoparticle 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 the reduction product is different from the refractive index of the reactant; When the material to be analyzed is a cell, the cell must have grown on the surface of the conductive glass sheet and placed in an electrolyte containing 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.

[0019] The electrochemical imaging system based on critical angle reflection in the present invention can be used for real-time optical imaging of homogeneous or heterogeneous surfaces of different types of electrolytes, surface interface ion concentration gradient imaging analysis, 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.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The electrochemical imaging system based on critical angle reflection in the present invention can simultaneously observe the diffusion layer and electrode reaction process by using critical angle reflection imaging, without the need for luminescent probes or strict material conditions. This method aims to improve time resolution, sensitivity, and be compatible with a wider variety of materials, and can be integrated with other imaging modes (such as fluorescence and ECL). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the structure of the electrochemical imaging system based on critical angle reflection in the present invention, wherein 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 magnification device, 34 is an image acquisition device, 35 is a focusing device, 41 is a control display device, and 51 is a data collection device.

[0022] Figure 2 The electrochemical signal and optical signal diagram in Example 1, wherein Figure 2 A is the voltage recorded by the electrochemical workstation ( E ) over time ( t ) Change graph; Figure 2 B is the current recorded by the electrochemical workstation ( I ) over time ( t ) Change graph; Figure 2 C is the optical signal (Int.) of the conductive glass electrode surface collected by the image acquisition device over time ( t ) Change graph; Figure 2 D is based on Figure 2 The optical signal of C is converted into the current density ( j CARE ) over time ( t ) change graph.

[0023] Figure 3 The electrochemical signal and optical signal diagram in Example 2, wherein Figure 3 A is the regional critical angle reflection imaging image of the heterogeneous surface of the ITO electrode; Figure 3 B is the cyclic voltammetry recorded by the electrochemical workstation before and after modification of APTES ( IE )picture; Figure 3 C is based on Figure 3 The optical signal A is converted into the current signal ( j CARE ) with voltage ( E ) Change graph; Figure 3 D is a comparison of the spatial distribution of the maximum current in the bare ITO area and the APTES area during the cyclic voltammetry process.

[0024] Figure 4 Figure 3 shows the electrochemical and optical signals during the catalytic hydrogen evolution process. 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 signal of Pt nanoparticles and background ( j CARE ) with voltage ( E ) Change graph; Figure 4 C is the peak current value converted by 151 Pt nanoparticles according to the optical signal when the applied voltage is -0.7 V ( j peak ) statistical chart.

[0025] Figure 5 A is a schematic diagram of bright field and ECL imaging of single cells (numbered 1, 2, 3) in Example 4. Figure 5 B is a current signal diagram obtained by converting light signals through critical angle reflection imaging of the same single cell (1, 2, 3) in Example 4; Figure 5 C is a cyclic voltammogram of three cells and a background area in Example 4, and the current is obtained by critical angle reflection imaging. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0027] like Figure 1 As shown, an electrochemical imaging system based on critical angle reflection includes an electrochemical module, an imaging module and a control module; The electrochemical module includes an electrochemical workstation 11 and an electrochemical cell 22; the working electrode 21, the reference electrode and the counter electrode in the electrochemical cell 22 are connected to the electrochemical workstation, the electrochemical cell 22 is a polydimethylsiloxane (PDMS) material reaction cell, the counter electrode is a platinum electrode, and the reference electrode is silver / silver chloride; the working electrode 21 is a conductive glass sheet, which is laid flat on the bottom of the electrochemical cell, and the conductive glass sheet is a transparent substrate covered with a 99 nm thick indium tin oxide film, and the resistance is 10-15Ω / square.

[0028] 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 diode (SLD) for generating incident light; the incident light passes through the focusing device 35 (lens group) to focus the light on the back focal plane of the imaging magnifying device 33; the position of the focus on the back focal plane is controlled by the three-dimensional light source displacement stage 32, so that the incident light is irradiated on the working electrode 21 through the imaging magnifying device 33, the incident angle is 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 high numerical aperture objective lens, and the image acquisition device 34 is a CMOS camera.

[0029] The control module includes a control display device 41 and a data collection device 51; the data collection device 51 is connected to and collects data from the electrochemical workstation 11 and the image acquisition device 34, and transmits the data to the control display device 41, which synchronizes the data and controls the working parameters of the electrochemical workstation 11 and the image acquisition device 34.

[0030] Example 1 Use Figure 1 The electrochemical imaging system based on critical angle reflection is shown to perform real-time imaging and monitoring of electrochemical reactions. The specific method includes the following steps: Step 1: Add 10 mM K3Fe(CN)6 solution into the electrochemical cell, and use the three-dimensional translation stage to control the angle of the incident light to be 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 consistent with that of the Fe(CN)6 3- The diffusion layers during redox reactions are basically aligned (about 50 μm); Step 2: Connect the three electrodes in the electrochemical cell to the electrochemical workstation and use cyclic voltammetry (+0.5 V, -0.3 V, 100 mV / s) to generate a redox reaction in the electrochemical cell. , by controlling the display device and the image acquisition device to record the optical signal, and synchronously record the current signal for verification.

[0031] Step 3, according to the control display device, the image acquisition device is synchronized in real time to obtain an optical signal, and according to the relationship between the reflectivity and the current density in the optical signal, the electrochemical reaction in the electrochemical cell is monitored in real time; After completing three electrochemical cyclic voltammetry scans, the recorded voltage signal E like Figure 2 As shown in A; current signal I like Figure 2 As shown in B, Current I Reduction peaks and oxidation peaks began to appear at around +0.04 V and +0.25 V (relative to Ag / AgCl reference), respectively, which is consistent with the redox potential of K3Fe(CN)6 under this condition reported in the literature, confirming that an electrochemical redox reaction occurred in the solution.

[0032] The optical signal recorded by the image acquisition device shows that when the solution is subjected to cyclic voltammetry scanning, the optical signal intensity Int. on the electrode surface changes with time as shown in Figure 2 Int. is the reflected light intensity, which is related to the reflectivity. In the experiment The change in Fe(CN)6 3- and Fe(CN)6 4- Concentration gradient on the ITO surface as a function of time.

[0033] The optical signal reflectivity can be expressed as Converted into electrical signal current density j CARE ( t ),Right now t At this time, the current density of the electrochemical cell j CARE ( t ): (1)

[0034] 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; in, ; is the unit concentration of reducing agent (in this case, Fe(CN)6 4- ), which is 0.00006 RIU / (mol·m 3 ); is the unit concentration of reducing agent (in this case, Fe(CN)6 4- ) has a diffusion coefficient of 1×10 -9 m 2 / s; is the unit concentration of the oxidant (in this case, Fe(CN)6 3- ), which is 0.00005 RIU / (mol·m3 ); is the unit concentration of the oxidant (in this case, Fe(CN)6 3- ) has a diffusion coefficient of 1×10 -9 m 2 / s; θ is the angle of incidence, which is 61.64° in this example; β -72.0 RIU -1 ; n Indicates the number of electrons transferred by the reactants in the redox reaction (in this example n =1); F is the Faraday constant, F = 96485 C / mol.

[0035] through Figure 2 The electrical signal converted from the optical signal of C changes with time. Figure 2 As shown in D, it can be seen that the current signal measured by the electrochemical workstation ( Figure 2 The results of Figure 2 (A) and (B) are almost completely consistent. This proves that the present invention can realize the conversion of optical signals and electrical signals and monitor the electrochemical process in real time.

[0036] Example 2 Use Figure 1 The electrochemical imaging system based on critical angle reflection is shown to perform real-time optical imaging and monitoring of heterogeneous interface electrolytes. Taking the indium tin oxide (ITO) electrode modified with γ-aminopropyltriethoxysilane (APTES) as an example, the specific method includes: Step 1: Use a PDMS square of about 10×5 mm to dip into a 3% APTES ethanol solution and stick it to the right half of the ITO. After modification for 30 minutes, remove the PDMS, wash the ITO sheet with ethanol, and then dry it at 110°C for 30 minutes to obtain an ITO electrode with half of it modified with APTES.

[0037] Step 2: Add PBS phosphate buffer containing 5 mM K3Fe(CN)6 as the electrolyte in the electrochemical cell, and control the angle of the incident light to about 61.6° through the three-dimensional translation stage so that the illumination thickness of the incident light on the surface of the ITO electrode is consistent with that of the Fe(CN)6 3- The diffusion layer is aligned during redox reactions (about 50 μm); Step 3: Connect the three electrodes in the electrochemical cell to the electrochemical workstation and use cyclic voltammetry (+0.5 V, -0.3 V, 100 mV / s). Redox reaction occurs in the electrochemical cell. The optical signal is recorded by the control display device, and the current signal is recorded synchronously for verification.

[0038] Step 4, according to the control display device real-time synchronization image acquisition device to obtain optical signals, according to the relationship between reflectivity and current density in the optical signal, to achieve the Fe (CN) 6 3- Real-time imaging monitoring of redox reactions; The critical angle reflection imaging of the working electrode surface after half of the APTES is shown in the figure. Figure 3 As shown in A in the middle (no voltage applied, scale bar 10 μm), the left side is the unmodified bare ITO area, and the right side is the APTES-modified area. It can be seen that there is a significant difference in the imaging of the bare ITO and APTES / ITO areas at this angle, and the latter shows a rough APTES pattern.

[0039] Figure 3 Figure B is the cyclic voltammogram recorded by the electrochemical workstation before and after ATPES modification, which is used to verify the success of APTES modification and the accuracy of critical angle reflectance measurement; the peaks of the CV curve in the bare ITO area are located at +0.04 V and +0.25 V; after APTES modification, the peaks shift to higher potentials, located at +0.10 V and +0.30 V, and the current is larger, because APTES modification changes the electrochemical activity of ITO.

[0040] The bare ITO area ( Figure 3 Region 1 in A) and the APTES region ( Figure 3 The optical signal in region 2) of A is converted according to the steps in Example 1 according to formula (1) to obtain the current density j CARE The voltage variation diagram (where the oxidant and reductant are Fe(CN)6 3- and Fe(CN)6 4- , the method is the same as described in Example 1), and the results are as follows Figure 3 C (areas 1, 2, and the average of the entire image). The dashed line represents the average CV obtained from the entire field of view, including the bare ITO area and the APTES area, and its shape is similar to the signal obtained by the electrochemical workstation ( Figure 3 B) is also basically consistent, proving the accuracy of the measurement. At the same time, it also shows that the present invention can perform a local and separate analysis on the heterogeneous interface, revealing the differences in reaction efficiency and electron transfer kinetics in the heterogeneous interface.

[0041] Figure 3 Figure D in the middle shows the redox current distribution diagram in the bare ITO area and the APTES area. It can be seen that the surface current distribution of APTES-modified is significantly higher than that of the bare ITO area, and the spatial distribution is uneven, which reflects the application of this technology in imaging the surface current of uneven electrodes.

[0042] Example 3 Use Figure 1 The critical angle reflectance based electrochemical imaging system shown in the figure performs high-throughput analysis of the catalytic activity of a single nanoparticle. Taking the catalytic hydrogen evolution reaction of Pt nanoparticles (73 nm) in 1 M H2SO4 solution as an example, the specific method includes the following steps: Step 1, disperse and coat the Pt nanoparticles (PtNP) on the surface of the ITO electrode: After the PtNP solution is dispersed by ultrasound for 3 minutes, 100 microliters are evenly distributed on the ITO sheet and naturally air-dried. At this time, the PtNP is sparsely dispersed on the ITO surface in the form of single particles (the ITO area without PtNP coverage can be used as a background for control experiments). Add 1 M H2SO4 solution to the electrochemical cell; control the angle of the incident light to be about 61.6° through the three-dimensional translation stage (same as Example 2); Step 2: The three electrodes in the electrochemical cell are connected to an electrochemical workstation, and a cyclic voltammetry scan method (0 V to -0.7 V, 100 mV / s) is used to cause a catalytic reaction of the PtNP in the electrochemical cell: hydrogen ions are reduced to hydrogen gas on the surface of the PtNP, and a concentration gradient is formed near the PtNP. The optical signal is recorded by a control display device, and the current signal of the electrochemical workstation is synchronously recorded for verification.

[0043] Step 3, according to the control display device, the image acquisition device is synchronized in real time to obtain an optical signal, and according to the relationship between the reflectivity and the current density in the optical signal, the real-time monitoring of the hydrogen evolution on the surface of a single PtNP in the electrochemical cell is realized; Figure 4 A in the middle is the critical angle reflection current diagram of three labeled platinum nanoparticles PtNP (numbered 1-3) in a certain area on the working electrode surface at different potentials (scale 1 μm). 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 PtNP surface has reached its peak.

[0044] Figure 4 Figure B shows the CV curves obtained by converting the optical signals of three PtNPs (numbered 1-3) and an ITO background area without PtNPs according to formula (1) in Example 1. The oxidant and the reductant are H + and H2. 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 area is small and can be ignored. The three PtNPs show different current sizes in CV, indicating the heterogeneity of electrocatalytic activity among different nanoparticles.

[0045] Figure 4 The C of 151 PtNPs shows the peak current converted by the optical signal at an applied voltage of -0.7 V ( j peak ) distribution diagram (method and Figure 4 The results are shown in Figure 2 (same as in Figure B), 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.

[0046] Example 4 Use Figure 1 The electrochemical imaging system based on critical angle reflection is combined with electrochemiluminescence (ECL) to image single cells. Among them, ECL uses the imaging amplification device 33, and no other devices are added. Taking the cell ECL imaging in the Ru(bpy)3Cl2 and TPrA solution system as an example, the specific method includes: Step 1: Culture A549 cells on the surface of the ITO electrode in a cell culture incubator overnight to allow the cells to fit tightly to the electrode. Then, wash the cell culture medium with PBS solution, add 10 mM Ru(bpy)3Cl2 and 20 mM TPrA (10 mM PBS) solution to the electrochemical working cell; control the angle of the incident light at 61.55° using a three-dimensional displacement stage; Step 2: Connect the three electrodes in the electrochemical cell to the electrochemical workstation and use cyclic voltammetry (scanning range: 0 V to +1.4 V; scan rate: 100 mV / s) to generate redox reaction of Ru(bpy)3Cl2 in the electrochemical cell. The reaction consumes the co-reactant TPrA to generate light. The ECL optical signal and the critical angle reflection optical signal are recorded in sequence by controlling the display device, and the current signal is recorded synchronously for verification.

[0047] Step 3, according to the control display device, the image acquisition device is synchronized in real time to obtain an optical signal, including an ECL and a critical angle reflection signal. The ECL signal is not further processed. For the critical angle reflection signal, the current value is calculated using formula (1) according to the steps of Example 1.

[0048] Figure 5A in the middle shows the bright field image and corresponding ECL imaging image (scale 10 μm) of three cells marked 1-3 in a certain area at a voltage of 1.0 V; in the ECL image, compared with the bright ITO background, the ECL images of cells 1-3 appear as dark lines. This is because the cells are attached to the ITO surface, and closed spaces are formed at their adhesion sites. The ECL reaction process consumes the TPrA in the closed space, making it impossible to continue to emit light.

[0049] Figure 5 B shows the critical angle reflection current signal of the three cells in the above region when the applied voltage is 1.2 V, calculated by formula (1), where the oxidant and the 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, in contrast to the ECL image, the cells all appear as bright lines. This observation is attributed to the different detection mechanisms of ECL and the present invention: ECL relies on Ru(bpy)3 2+ The reaction between Ru(bpy) and TPrA requires the coexistence of the two. Once TPrA is exhausted, the system will no longer emit light. The signal comes from Ru(bpy)3 2+ The refractive index changes caused by the redox reaction (TPrA contributes very little to the refractive index), even if TPrA is depleted, Ru(bpy)3 2+ The redox reaction will continue to occur, so that the critical angle reflection signal still exists.

[0050] Figure 5 C shows that during the cyclic voltammetry process, Figure 5 The currents of the three cell regions shown in B and the background region (where there are no cells) vary with voltage. It can be seen that the current in the cell region is stronger than that in the background region. The increase in current is due to the confinement effect of the cell adhesion site. At the adhesion site, the cells form gaps ranging from tens to hundreds of nanometers, which greatly limits the Ru(bpy)3 2+and Ru(bpy)3 2+ The diffusion of ions leads to an increase in local ion concentration, thereby increasing the 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 luminescence 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 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 connects 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.

2. The electrochemical imaging system based on critical angle reflection according to claim 1, characterized in that: 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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 7 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 unit concentration of oxidant; n It indicates 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.

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

10. The electrochemical imaging real-time monitoring method based on critical angle reflection according to claim 8, characterized in that: When the material to be analyzed is an electrolyte, the electrolyte contains a reactant 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; 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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