Electrochemical microscopic imaging analysis method based on fluorescence signal

Abstract

The invention discloses an electrochemical microscopic imaging analysis method based on a fluorescence signal, which comprises the following steps of: constructing a fluorescence response electrochemical signal instrument by combining electrochemistry with a fluorescence microscope, and observing the redox potential of K3 [Fe (CN) 6] by taking a terpyridyl ruthenium (Ru (bpy) 32 +) / potassium ferricyanide (K3 [Fe (CN) 6]) fluorescence system as a research object. Influences of electrochemical behaviors on fluorescence signals are deeply explored, and the relationship between fluorescence signal changes and electric signal changes is revealed. According to the present invention, the combination of the electrochemical technology and the fluorescence signal is achieved, the disadvantage of the poor spatial-temporal resolution of the electrochemiluminescence is made up, the new dimension capable of being regulated and controlled by the electrical parameter is provided for the fluorescence imaging, and the Faraday current and the non-Faraday current can be completely reflected by the fluorescence signal. Therefore, the method has wide application value in the aspects of exploring the conductivity of the nano material and the like.

Description

Technical Field

[0001] The present invention is an electrochemical microscopic imaging analysis method based on fluorescence signals. The technical essence is photoluminescence, but it is regulated by electrochemical technology, belonging to both the field of electrochemical imaging technology and the field of optical imaging technology. Background Art

[0002] In the field of electrochemical analysis, although traditional electrochemical methods can provide rich quantitative information, they have limitations in spatial resolution and real-time imaging (see Yu X-Y. Imaging mass spectrometry tackles interfacial challenges in electrochemistry[J]. Current Opinion in Electrochemistry, 2017, 6(1): 53-9.). In recent years, with the development of optical imaging technology, the combination of fluorescence microscopy and electrochemical technology has brought new breakthroughs to electrochemical analysis. This combination not only retains the high sensitivity and selectivity of electrochemical methods but also realizes real-time and in-situ imaging of the electrochemical reaction process by virtue of the spatial resolution advantage of optical imaging technology (see Mei Y, Zhong C, Li L, et al. Single-layer graphene-coated gold chip for electrochemical surface plasmon resonance study[J]. Anal Bioanal Chem, 2019, 411(19): 4577-85.).

[0003] Meanwhile, fluorescence technology occupies an important position in the field of optical imaging due to its advantages such as high sensitivity, high selectivity, and non-invasiveness. Fluorescence microscopes can provide intuitive visual information to help researchers better understand the dynamic processes of chemical reactions. However, fluorescence technology also has its drawbacks. For example, fluorescence signals are easily affected by background fluorescence and photobleaching, resulting in a decrease in signal intensity and affecting the stability and accuracy of imaging (Morgana S, Casentini B, Tirelli V, Grasso F, Amalfitano S. Fluorescence-based detection: A review of current and emerging techniques to unveil micro / nanoplastics in environmental samples. Trac-Trends in Analytical Chemistry, 2024, 172: 117559.). In addition, fluorescence technology usually requires an external excitation light source, which may introduce additional background noise and reduce the signal-to-noise ratio of imaging.

[0004] In view of this, it is particularly necessary to combine electrochemical technology with fluorescence technology. Researchers are developing enhanced spectroelectrochemical techniques for the indirect detection of redox events, studying photoelectrochemical reactions, the heterogeneity of electrode surfaces, and the modification of electrode surfaces. In existing fluorescence electrochemistry-related research, electrical signals are mostly obtained based on scanning electrochemical microscopy (SECM), which cannot effectively balance spatial and temporal resolution; in addition, the systems of most fluorescence electrochemistry-related research limit the synchronous study of Faraday current and non-Faraday current, resulting in insufficient exploration of the relationship between changes in fluorescence signals and changes in electrical signals. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide an electrochemical microscopic imaging analysis method based on fluorescence signals, which improves the problem of poor spatial and temporal resolution of electrochemical technology imaging and also supplements the regulation methods of fluorescence imaging, broadening its fluorescence analysis methods for electrochemical reactions.

[0006] Technical Solution: The electrochemical microscopic imaging analysis method based on fluorescence signals described in the present invention includes the following steps:

[0007] Place the electrolyte containing the fluorescent molecule Ru(bpy)3 2+ and K3[Fe(CN)6] in an electrochemical reaction cell, use a fluorescence microscope to image the surface of the working electrode, apply a voltage to the working electrode through an electrochemical workstation, and through Ru(bpy)3 2+By determining the change of the fluorescence signal and the relationship between the fluorescence signal and the electrical signal, the imaging analysis of the local electrochemical activity on the electrode surface can be realized.

[0008] Furthermore, the fluorescence microscope includes a white light excitation light source, an excitation filter, an emission filter, a semi-transmissive and semi-reflective mirror, a water immersion objective lens, and a camera for collecting fluorescence signals.

[0009] Furthermore, a 570nm long-pass filter is selected as the long-pass emission end filter, and a (450±20)nm filter is selected as the excitation end filter.

[0010] Furthermore, the electrochemical reaction cell consists of an electrochemical workstation and its three-electrode system. Among them, the three-electrode system uses an ITO glass electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, and a Pt electrode as the counter electrode.

[0011] Furthermore, a silicone rubber gasket is pasted on the conductive surface of the ITO glass electrode, and the silicone rubber gasket and the ITO glass electrode form an electrochemical reaction cell.

[0012] Furthermore, K3[Fe(CN)6] is used as the system redox molecule, which has good electrochemical behavior and represents the electrochemical technology part; Ru(bpy)3 2+ can emit fluorescence and is a typical fluorescent molecule, representing the fluorescence part. Therefore, the two need to be combined to form Ru(bpy)3 2+ / K3[Fe(CN)6] fluorescence electrochemical system.

[0013] Furthermore, the electrolyte is prepared with phosphate buffer solution as the solvent. The concentration of the phosphate buffer solution is 0.05 - 0.15mol / L, preferably 0.1mol / L. The concentrations of Ru(bpy)3 2+ and K3[Fe(CN)6] in the electrolyte are both 300 - 700μmol / L, preferably 500μmol / L, which can achieve the best effect for subsequent experiments.

[0014] Furthermore, the working mode of the electrochemical workstation is cyclic voltammetry (CV). The cyclic voltammetry scanning parameters are: the initial potential is below -0.5V, preferably -0.6V; the highest potential is 0.5 - 1.0V, preferably 1.0V; the lowest potential is below -0.5V, preferably -0.6V; the termination potential is below -0.6V, preferably -0.6V; the scanning speed is 0.05 - 0.15V / s, preferably 0.1V / s.

[0015] Further, when determining the relationship between the fluorescence signal and the electrical signal, the fluorescence signal is first preprocessed, including smoothing the fluorescence signal into a smooth curve, and then normalizing the fluorescence signal and taking the negative derivative with respect to time. The specific data processing formula is as follows:

[0016]

[0017] where I n ’ is the processed value, I n is the fluorescence intensity value corresponding to the nth image, I1 is the fluorescence intensity value corresponding to the first image, and t is the imaging time of the corresponding image.

[0018] Furthermore, the obtained data I n ’ is used as Y2, and is fitted with the cyclic voltammetry voltage value X1, the cyclic voltammetry current value Y1, and the voltage value X2 corresponding to the imaging time of each image on a double Y-axis ordinate graph to determine the relationship between the fluorescence signal and the electrical signal.

[0019] Furthermore, Origin software is used for fitting.

[0020] Further, during the experiment, ensure that the fluorescence signal is acquired simultaneously at the moment when the electrochemical workstation applies the voltage. To reduce operation errors, the electrochemical workstation is set to sweep three cycles, and the second cycle is selected when processing the data. Since they start and end simultaneously, each fluorescence intensity obtained can calculate a uniquely corresponding voltage through the relationship between the imaging time and the sweep rate, thus establishing the relationship between the fluorescence intensity and the voltage, that is, the relationship between the fluorescence signal and the electrical signal.

[0021] Furthermore, the voltage at the imaging time is calculated using the following formula:

[0022] The voltage value at the imaging time (V x ) = initial voltage (V0) + imaging time (T) × sweep rate (u)

[0023] At this time, the voltage signal is used as X1, the current signal is used as Y1, the voltage value V x at the imaging time is used as X2, and the processed fluorescence signal I n ’ after normalization and taking the negative derivative with respect to time is used as Y2. A double Y-axis coordinate graph can be made for fitting to determine the relationship between the fluorescence signal and the electrical signal.

[0024] Further, the variation relationship of the fluorescence signal with the voltage signal is: when the oxidation reaction occurs (when the oxidation peak starts), the fluorescence intensity decreases; when the reduction reaction occurs (when the reduction peak starts), the fluorescence intensity increases.

[0025] Further, according to different specific experimental purposes, the working electrode surface can be covered with substances for treatment.

[0026] Further, the covering is an ink, a chlorella or a chitosan suspension.

[0027] The present invention can change the change of fluorescence intensity by quantitatively adjusting electrochemical parameters, so as to establish a synchronous acquisition method for electrical signals and fluorescence signals.

[0028] Regulation mechanism: The present invention constructs a fluorescence electrochemical microscopy imaging technology capable of responding to changes in electrochemical signals by using Ru(bpy)3 2+ as a fluorescent molecule. Cyclic voltammetry can well control the redox reaction of K3[Fe(CN)6] occurring on the electrode surface. The electron transfer in this process causes a change in the local electron density, ultimately resulting in the change of the fluorescence signal of Ru(bpy)3 2+ When a reduction reaction occurs, the fluorescence intensity increases; when an oxidation reaction occurs, the fluorescence intensity decreases, thus showing periodic change peaks in cyclic voltammetry scans.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0030] The method of the present invention utilizes wide-field imaging technology, and the advantage is that it has higher spatio-temporal resolution; the method of the present invention can effectively capture the electron transfer during the charge-discharge process and the redox process, and these processes are presented in the form of fluorescence, so as to comprehensively reflect the Faraday current and the non-Faraday current. The Faraday current refers to the current formed by the redox reaction on the electrode and follows Faraday's law; while the non-Faraday current is related to the charging process of the electric double layer on the electrode surface. Traditional electrochemical techniques mainly focus on the Faraday current, while the fluorescence signal detected by the inventive method can reflect both the Faraday current and the non-Faraday current, providing more comprehensive and in-depth information for electrochemical analysis. Description of the drawings

[0031] Figure 1 Cyclic voltammogram of the Ru(bpy)3 2+ solution in Example 1, where the scanning range is -0.6 to 1.5 V;

[0032] Figure 2 Cyclic voltammogram after adding K3[Fe(CN)6] in Example 1, where the scanning range is -0.6 to 1.0 V;

[0033] Figure 3 Fluorescence emission spectra of Ru(bpy)3 2+ and K3[Fe(CN)6] in Example 1;

[0034] Figure 4 Bright-field image of the ITO glass electrode surface in Example 2;

[0035] Figure 5 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in Example 2;

[0036] Figure 6 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in the ink-free covered area in Example 3;

[0037] Figure 7 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in the ink-covered area in Example 3;

[0038] Figure 8 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in the chitosan-free covered area in Example 3;

[0039] Figure 9 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in the chitosan-covered area in Example 3;

[0040] Figure 10 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in the Chlorella vulgaris-free covered area in Example 3;

[0041] Figure 11 It is the fitting diagram of the fluorescence signal and the cyclic voltammetry signal in the Chlorella vulgaris-covered area in Example 3;

[0042] Figure 12 It is the bright-field imaging diagram of the ITO glass electrode surface etching in Example 4;

[0043] Figure 13 It is the diagram of the fluorescence signal and the cyclic voltammetry signal in the unetched area in Example 4;

[0044] Figure 14 It is the diagram of the fluorescence signal and the cyclic voltammetry signal in the etched area in Example 4;

[0045] Figure 15 It is the fluorescence data diagram collected in Example 5 using the data in Example 3;

[0046] Figure 16 It is the diagram of the fluorescence intensity and the cyclic voltammetry signal (including three cycles) collected in Example 5 using the data in Example 3;

[0047] Figure 17 It is the fluorescence data diagram collected in Example 5 using the etched data in Example 4;

[0048] Figure 18 It is the diagram of the fluorescence intensity and the cyclic voltammetry signal (including three cycles) collected in Example 5 using the data in Example 4. Detailed implementation manners

[0049] In the fluorescence system of Example 1, determination of different molecular functions

[0050] In this fluorescence system, there are two main components, namely Ru(bpy)3Cl2 and K3[Fe(CN)6] molecules. Among them, the solvent is 0.1 mol / L phosphate buffer solution (pH = 7.4) (PBS).

[0051] 1. Determination of redox molecules

[0052] (1) Preparation of electrolyte: Prepare the electrolyte with 0.1 mol / L phosphate buffer solution (pH = 7.4) (PBS) and Ru(bpy)3Cl2 so that the concentration of Ru(bpy)3Cl2 in the electrolyte is 500 μmol / L.

[0053] Preparation of ITO glass electrode:

[0054] (1) Pretreatment of ITO glass electrode: Cut the ITO glass into small pieces of 25 mm × 25 mm, ultrasonicate them in acetone, ethanol, and secondary water for 5 - 10 min respectively, then wash with secondary water, and finally dry the ITO glass with nitrogen to obtain the ITO glass electrode.

[0055] (2) Paste a silicone rubber gasket with a diameter of 20 mm and a height of 2 mm on the conductive surface of the ITO glass electrode. The silicone rubber gasket and the ITO glass electrode form an electrochemical reaction cell, and the electrolyte is injected into it.

[0056] Place the prepared electrolyte into the electrochemical reaction cell. Use the ITO glass electrode as the working electrode, immerse the Ag / AgCl reference electrode and the platinum counter electrode into the electrolyte to form a three - electrode system; turn on the electrochemical workstation and perform cyclic voltammetry scanning on it, where the initial voltage is -0.6 V, the highest voltage is 1.5 V, the lowest voltage is -0.6 V, the termination voltage is -0.6 V, and the scan rate is 0.1 V / s. Obtain the cyclic voltammogram as Figure 1 shown. According to Figure 1 the cyclic voltammogram, both the oxidation peak and the reduction peak of Ru(bpy)3Cl2 are greater than 1.0 V.

[0057] (2) Prepare the fluorescence system solution: Prepare the electrolyte with 0.1 mol / L phosphate buffer solution (pH = 7.4) (PBS), Ru(bpy)3Cl2, and K3[Fe(CN)6] so that the concentrations of Ru(bpy)3Cl2 and K3[Fe(CN)6] in the electrolyte are both 500 μmol / L. Arrange the three - electrode system in the same method as in step (1), set the same electrochemical parameters, but change the highest voltage from 1.5 V to 1.0 V. Obtain the cyclic voltammogram as Figure 2 shown. It can be seen from Figure 2 that there are oxidation - reduction peaks at less than 1.0 V.

[0058] Since Ru(bpy)3Cl2 does not show a peak at less than 1.0 V, this peak must come from K3[Fe(CN)6], that is, K3[Fe(CN)6] is the only redox molecule in this system under the experimental conditions.

[0059] 2. Determination of the fluorescent molecule

[0060] Prepare a 500 μmol / L Ru(bpy)3Cl2 solution and a 500 μmol / L K3[Fe(CN)6] solution respectively according to Step 1, and the solvent is 0.1 mol / L PBS solution. Measure their fluorescence spectra respectively. From Figure 3 it can be seen that under the experimental conditions, selecting a 570 nm long-pass filter at the emission end can ensure that the emission light of Ru(bpy)3Cl2 can be collected best. Therefore, it can be considered that Ru(bpy)3Cl2 is the only fluorescent molecule in this system under the experimental conditions. Among them, the experimental conditions refer to selecting a 450 nm filter at the excitation end and a 570 nm long-pass filter at the emission end. Since Ru(bpy)3Cl2 exists in the form of Ru(bpy)3 2+ in the electrolyte, the following are all Ru(bpy)3 2+ .

[0061] Example 2 clarifies the relationship between the fluorescence signal and the electrical signal

[0062] Draw a thick line on the surface of the ITO glass electrode with an ink pen for marking, aiming to locate the microscope to focus on the electrode surface. Since charge transfer occurs directly on the electrode surface, only by focusing on the electrode surface can the change law of the fluorescence signal with the electrical signal be better obtained.

[0063] Prepare a fluorescent system solution containing 500 μmol / L Ru(bpy)3 2+ and 500 μmol / L K3[Fe(CN)6] with 0.1 mol / L PBS. Take about 1 mL of it and place it in an electrochemical reaction cell, and set up a three-electrode system. Observe the electrode surface by bright-field imaging, move the electrode position and adjust the focus point so that the edge of the thick ink pen line can be clearly shown in the bright-field imaging, as Figure 4As shown. At this time, fluorescence imaging is performed, and the exposure time is not limited (set to 100 ms in this experiment). The working mode of the electrochemical workstation is set to cyclic voltammetry, with the initial voltage of -0.6 V, the highest voltage of 1.0 V, the lowest voltage of -0.6 V, the termination voltage of -0.6 V, the scanning rate of 0.1 V / s, a 2-s delay in power-on, and a total of 3 scans. Fluorescence imaging is started at the moment when the voltage is applied and ended at the end moment, and the electrical signal and optical signal are exported (ensure that the fluorescence signal is obtained simultaneously at the moment when the electrochemical workstation applies the voltage. In order to reduce operation errors, the electrochemical workstation is set to scan three cycles, and the second cycle is selected when processing data).

[0064] Take the data corresponding to the second cycle and perform normalization on the fluorescence intensity (if it is not smooth enough, smoothing and noise reduction are required; if it is smooth enough, it can be ignored), and then take the derivative with respect to the imaging time. Among them, the imaging time refers to the time value at the time of imaging corresponding to each photo. After taking the derivative, take the opposite number as the final fluorescence intensity result. The specific data processing formula is as follows:

[0065]

[0066] Among them, I n ’ is the processed value, I n is the fluorescence intensity value corresponding to the nth image, I1 is the fluorescence intensity value corresponding to the first image, and t is the imaging time corresponding to the image.

[0067] The electrical signal includes voltage and current signals. Similarly, take the data of the second cycle and calculate the voltage value at the imaging time corresponding to each fluorescence signal. The common formula is as follows:

[0068] The voltage value at the imaging time (V x ) = initial voltage (V0) + imaging time (T) × scanning rate (u)

[0069] At this time, take the voltage signal as X1, the current signal as Y1, the voltage value V x at the imaging time as X2, and the processed fluorescence signal after normalization and taking the derivative with respect to time and adding a negative sign as Y2. A double Y-axis coordinate graph can be made, as Figure 5 shown. It can be seen that there is a fitting effect between the two to a certain extent.

[0070] Example 3 explores the influence of the covering on the electrode surface on the fitting effect of the two.

[0071] To explore the influence of the covering on the electrode surface on the fitting effect of the two, draw a thick line on the surface of the ITO glass electrode with an ink pen for marking, aiming to position the microscope to focus on the electrode surface. Because charge transfer occurs directly on the electrode surface.

[0072] Prepare a solution containing 500 μmol / L Ru(bpy)3 with 0.1 mol / L PBS2+ and a fluorescent system solution of 500 μmol / L K3[Fe(CN)6]. Take about 1 mL of it and place it in an electrochemical reaction cell, and set up a three-electrode system. Observe the electrode surface using bright-field imaging, move the electrode position and adjust the focus point so that the thick edge of the ink pen can be clearly shown in the bright-field imaging, as Figure 4 shown. At this time, perform fluorescence imaging, and the exposure time is not limited (set to 100 ms in this experiment). The working mode of the electrochemical workstation is set to cyclic voltammetry, with an initial voltage of -0.6 V, a maximum voltage of 1.0 V, a minimum voltage of -0.6 V, a termination voltage of -0.6 V, a scan rate of 0.1 V / s, a delay in power-on of 2 s, and a total of 3 scans. Turn on fluorescence imaging at the moment of applying voltage and turn it off at the end moment, and export the electrical signal and optical signal. The results are as Figures 6 - 7 shown. From Figure 6 and Figure 7 , it can be seen that the fitting degree of the ink-free area is significantly better than that of the ink-covered area (note the differences in the peak shape and the right ordinate).

[0073] Set up the experiment in the same way, replace the ink with Chlorella or chitosan, and the results are as Figure 8 , Figure 9 , Figure 10 , Figure 11 shown. From Figures 8 - 10 , the same conclusion can be obtained, that is, the fitting degree of the uncovered area is significantly better than that of the covered area. Therefore, it can be concluded that if the surface coverings are such as ink, Chlorella, and chitosan, etc., the fitting effect of the two will be reduced. It should be noted that such coverings are usually poor conductors of electricity, and the specific reasons are shown in Example 4.

[0074] Example 4 explores the regulation mechanism of electrochemical technology on the fluorescent system

[0075] Etch off part of the conductive layer on the surface of the ITO glass electrode so that part of the area is conductive and part of the area is non-conductive. Prepare a fluorescent system solution containing 500 μmol / L Ru(bpy)3 2+ and 500 μmol / L K3[Fe(CN)6] with 0.1 mol / L PBS. Take about 1 mL of it and place it in an electrochemical reaction cell, and set up a three-electrode system. Observe the electrode surface using bright-field imaging, move the electrode position and adjust the focus point so that the edge of the etched area can be clearly shown in the bright-field imaging, as Figure 12 shown. At this time, perform fluorescence imaging, and the exposure time is not limited (set to 100 ms in this experiment). The working mode of the electrochemical workstation is set to cyclic voltammetry, with an initial voltage of -0.6 V, a maximum voltage of 1.0 V, a minimum voltage of -0.6 V, a termination voltage of -0.6 V, a scan rate of 0.1 V / s, a delay in power-on of 2 s, and a total of 3 scans. Turn on fluorescence imaging at the moment of applying voltage and turn it off at the end moment, and export the electrical signal and optical signal.

[0076] The fluorescence intensity data and electrical signal data of the unetched area and the etched area are respectively selected for analysis. At this time, the voltage signal is taken as X1, the current signal is taken as Y1, and the voltage value V at the imaging moment x is taken as X2, and the corresponding normalized fluorescence signal is Y2. A double Y-axis coordinate graph can be made to obtain the final data as Figure 13 、 Figure 14 shown. From Figure 13 、 14 analysis, it can be seen that there is almost no fitting effect between the fluorescence signal and the cyclic voltammogram in the etched part, nor is there a regular change (note that the right vertical coordinates of the two figures are different); while in the unetched part, the fluorescence signal and the cyclic voltammogram follow a certain fitting effect and change rule. Therefore, it can be known that the etched part loses control, and the direct reason is that the local charge density has not changed significantly. Therefore, the regulation mechanism is to change the local charge density of the solution through electrochemical technology, thereby changing the intensity of the fluorescence emitted by it.

[0077] Example 5 explores the relationship between the fluorescence signal and the voltage signal

[0078] According to Examples 1 to 4, it can be determined that the fluorescence signal can change with the change of the voltage signal. For Example 3, based on the experiment with chitosan as the covering material, the fluorescence data of its uncovered part are listed (such as Figure 15 ), and then with the fluorescence intensity as the Y1 axis, the voltage value V at the imaging moment x as the X1 axis, the derived normalized current signal as the Y2 axis, and the derived voltage signal V at the imaging moment x as the X2 axis, a double Y-axis graph is made Figure 16 shown. According to Figure 15 , the fluorescence intensity generally shows a downward trend from the beginning to the end, which means that Figure 16 the black line in should be interpreted clockwise, and the decrease is based on the principle of fluorescence photobleaching. According to Figure 16 , when V x ≈0.1V, the oxidation peak starts, and at this time the fluorescence intensity just shows a downward trend; when V x ≈0.3V, the reduction peak starts, and at this time the fluorescence intensity just shows an upward trend. Similarly, according to the data of the unetched part in Example 4, Figure 17 、 Figure 18 are made, and by the same analysis, it can be known that when V x ≈0.1V, the oxidation peak starts, and at this time the fluorescence intensity just shows a downward trend; when V x ≈0.3V, the reduction peak starts, and at this time the fluorescence intensity just shows an upward trend. Therefore, the conclusion is drawn that the relationship between the fluorescence signal and the voltage signal is: when the oxidation reaction occurs (when the oxidation peak starts), the fluorescence intensity decreases; when the reduction reaction occurs (when the reduction peak starts), the fluorescence intensity increases.

Claims

1. An electrochemical microscopic imaging analysis method based on fluorescence signals, characterized in that: The following steps are involved: The fluorescent molecule Ru(bpy)3 2+ The electrolyte of K3[Fe(CN)6] and K3[Fe(CN)6] was placed in an electrochemical reaction cell, the surface of the working electrode was imaged using a fluorescence microscope, and a voltage was applied to the working electrode through an electrochemical workstation. 2+ The change of fluorescence signal determines the relationship between fluorescence signal and electrical signal, thus realizing imaging analysis of local electrochemical activity on the electrode surface.

2. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: A fluorescence microscope consists of a white light excitation source, an excitation filter, an emission filter, a half-transparent and half-reflective mirror, a water immersion objective, and a camera for collecting fluorescence signals.

3. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: The electrochemical reaction cell comprises an electrochemical workstation and a three-electrode system thereof, wherein the three-electrode system comprises an ITO glass electrode as a working electrode, an Ag / AgCl electrode as a reference electrode, and a Pt electrode as a counter electrode.

4. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: The silicone rubber gasket is adhered to the conductive surface of the ITO glass electrode, and the silicone rubber gasket and the ITO glass electrode constitute an electrochemical reaction cell.

5. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: The electrolyte is prepared with phosphate buffer solution, the concentration of phosphate buffer solution is 0.05-0.15 mol / L, and Ru(bpy)3 2+ The concentrations of K3[Fe(CN)6] and K3[Fe(CN)6] were both 300-700 μmol / L.

6. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: The electrochemical workstation works in cyclic voltammetry (CV) mode, and the cyclic voltammetry scanning parameters are: initial potential below -0.5V, maximum potential 0.5-1.0V, minimum point below -0.5V, end point below -0.6V, and scanning speed 0.05-0.15V / s.

7. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: When determining the relationship between the fluorescence signal and the electrical signal, the fluorescence signal is preprocessed, including smoothing the fluorescence signal to a smooth curve, and then normalizing the fluorescence signal and taking the derivative with respect to time and then taking the negative number. The specific data processing formula is as follows: Among them, I n ' is the value obtained by processing, I n is the fluorescence intensity value corresponding to the nth image, I1 is the fluorescence intensity value corresponding to the first image, and t is the imaging time of the corresponding image.

8. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 7, characterized in that: The data I n ' As Y2, the cyclic voltammetry voltage value X1, the cyclic voltammetry current value Y1, and the current value X2 corresponding to each image imaging are fitted on the double Y vertical coordinate graph to determine the relationship between the fluorescence signal and the electrical signal.

9. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 8, characterized in that: The fitting was performed using Origin software.

10. The electrochemical microscopic imaging analysis method based on fluorescence signals according to claim 1, characterized in that: Depending on the specific experimental purpose, the surface of the working electrode can be treated by covering it with substances.

Images