Electrochemical sensor for monitoring exocytosis as well as preparation method and application of electrochemical sensor

Abstract

The invention relates to an electrochemical sensor for monitoring exocytosis as well as a preparation method and application of the electrochemical sensor. A working electrode of the electrochemical sensor is modified with a multi-dimensional nano mixed material; the multi-dimensional nano mixed material is prepared by taking porous carbonized wood as a substrate and growing a zinc oxide nanowire and an organic framework compound on the substrate in situ. The electrochemical sensor disclosed by the invention can be used for efficiently monitoring the exocytosis behavior of living cells by utilizing the synergistic effect of the nano material while fixing single cells, can be used for testing by matching with a conventional electrochemical workstation and a Faraday cage in a laboratory, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biosensing, and particularly relates to an electrochemical sensor for monitoring exocytosis and a preparation method and application thereof. BACKGROUND

[0002] Exocytosis of neurotransmitters such as dopamine by nerve cells is one of the important physiological behaviors of organisms, is the main way of the nervous system to regulate the body, and is a basic biological principle in the fields of neurobiology, biochemistry, clinical medicine, pathology and pharmacy. When the exocytosis is tested by using an electrochemical amperometric method, a plurality of discontinuous spike signals can be obtained, and the spikes are transient current signals generated by oxidation of neurotransmitters released by exocytosis. By analyzing the number and size of the spikes, information such as the frequency of exocytosis and the number of secreted dopamine molecules can be obtained, thereby providing key data for information exchange between nerve cells or neurons. Under the action of drugs, the exocytosis behavior of cells may change. By studying the influence of drugs on cell exocytosis, compounds with biological activity, drug efficacy groups or drug effects can be efficiently screened from a large number of natural products, thereby providing important clues for drug discovery from lead compounds to promising compounds. Especially, it is of great significance for creating new drugs from natural products. At present, exocytosis research often relies on technologies such as cryo-electron microscopy, ultra-high resolution fluorescence microscopy and carbon fiber electrode (CFE), which not only need expensive large-scale instruments and professional operation, but also have low testing efficiency. Therefore, new technologies are needed to provide efficient tools for active natural product screening. SUMMARY

[0003] The application provides an electrochemical sensor for monitoring exocytosis and a preparation method and application thereof, and solves the problems that the existing carbon fiber electrode (CFE) can only cover about 10% of the cell membrane, can only test adherent cells one by one, and cannot obtain statistical data of multiple cells.

[0004] The application provides an electrochemical sensor for monitoring exocytosis, wherein a working electrode of the electrochemical sensor is modified with a multi-dimensional nanometer hybrid material; and the multi-dimensional nanometer hybrid material is obtained by using porous carbonized wood as a substrate, and growing zinc oxide nanowires and organic framework compounds in situ on the substrate.

[0005] Preferably, the organic framework compound is ZIF-8.

[0006] The application in-situ grows arrayed ZnO nanowires on natural pores of carbonized wood, and the outer layer is ZIF-8, and the synergistic effect helps to detect the real-time exocytosis behavior of single cells: the pore diameter of the carbonized Finnish pine is about 12 μm, which matches the cell size, and the cells can be fixed in the pores without additional operation steps, and the carbonized wood has good conductivity, which helps the electron generated by the electrochemical oxidation of dopamine to be conducted to the working electrode. The in-situ grown arrayed ZnO nanowires are in full contact with the fixed cells and receive the dopamine molecules released by exocytosis from multiple directions at close range. The ZIF-8 on the surface of the nanowires is a solid base nanocatalyst, which deeply oxidizes the dopamine molecules in the solution, and the electric signal generated by the electron transfer in the catalytic oxidation process can be used for real-time analysis of the exocytosis event.

[0007] The application also provides a preparation method of an electrochemical sensor for exocytosis monitoring, comprising the following steps:

[0008] S1. Carbonize Finnish pine blocks to obtain carbonized wood;

[0009] S2. Cut the carbonized wood into thin slices, wash and dry to obtain carbonized wood slices;

[0010] S3. Drop zinc acetate dihydrate ethanol solution on the carbonized wood slices, define the dropping surface as the front surface, and dry;

[0011] S4. Calcine the carbonized wood slices obtained in step S3, and take out after cooling;

[0012] S5. Prepare an aqueous solution containing polyethyleneimine, zinc nitrate hexahydrate and hexamethylenetetramine to obtain a precursor solution;

[0013] S6. Invert the front surface of the carbonized wood slices obtained in step S4 and suspend them on the surface of the precursor solution, and heat;

[0014] S7. Take out the carbonized wood slices, wash and dry, perform secondary calcination, cool to room temperature and take out, repeat steps S5 and S6 to obtain carbonized wood-ZnO nanowires;

[0015] S8. Preheat 2-dimethylimidazole at the bottom of a glass container, then suspend the carbonized wood-ZnO nanowires above the 2-dimethylimidazole, heat at 100-120℃ for 5-10 min, take out and cool to room temperature, wash and dry to obtain carbonized wood-ZnO nanowires-ZIF-8;

[0016] S9. Apply conductive carbon paste to the working electrode area of the screen-printed electrode, and paste the carbonized wood-ZnO nanowires-ZIF-8, solidify, and soak in deionized water to obtain the electrochemical sensor for exocytosis monitoring.

[0017] Preferably, the size of the pine wood block in step S1 is 3cm*1cm*1cm.

[0018] Preferably, the carbonization treatment in step S1 is performed at a temperature of 700-1000 DEG C for 1-5h under inert gas protection.

[0019] Preferably, the concentration of the zinc acetate dihydrate ethanol solution in step S3 is 1.111mg / mL.

[0020] Preferably, the calcination temperature in step S4 is 300-400 DEG C for 10-30min.

[0021] Preferably, the concentration of polyethyleneimine in the precursor solution in step S5 is 0.00387g / mL, the concentration of zinc nitrate hexahydrate is 0.008g / mL, and the concentration of hexamethylenetetramine is 0.0035g / mL.

[0022] Preferably, the heating temperature in step S6 is 80-100 DEG C for 1-5h.

[0023] Preferably, the secondary calcination temperature in step S7 is 300-400 DEG C for 10-30min.

[0024] Preferably, the preheating temperature in step S8 is 100-120 DEG C for 20-40min.

[0025] The existing preparation method of the electrochemical sensor for monitoring exocytosis needs to fully grind and ultrasonically prepare a slurry of a synthetic material, and coat the slurry on the surface of a screen-printed electrode, which may damage the three-dimensional structure of the nanomaterial and affect the performance of the sensor. The present application uses carbonized wood as a substrate, cuts the carbonized wood into a whole piece, and then pastes the carbonized wood on the working electrode using conductive carbon paste, so as to ensure the conductivity and also retain the three-dimensional structure of the nanomaterial from being damaged, so that the sensor proposed in the present application can fix the cells to be measured in the holes of the carbonized wood structure, and realize simple, rapid and efficient real-time exocytosis research.

[0026] The present application also provides a use of the electrochemical sensor for monitoring exocytosis in monitoring real-time exocytosis of cells.

[0027] Specifically, the following steps are included:

[0028] (1) dispersing cells in a sterile PBS buffer containing bovine serum to prepare a cell dispersion liquid;

[0029] (2) using a cell printer to print the cells in the cell dispersion liquid onto the working electrode of the electrochemical sensor for monitoring exocytosis;

[0030] (3) Place the exocytosis monitoring electrochemical sensor in a Faraday cage and connect it to an electrochemical workstation;

[0031] (4) Add the liquid sample to be tested to the working area of the exocytosis monitoring electrochemical sensor, so as to fully cover the printed cell working electrode, reference electrode and counter electrode;

[0032] (5) Start monitoring by amperometry.

[0033] Preferably, in step (5), the monitoring potential is 0.34 V, the sampling interval is 0.012 s, and the duration is 300 s.

[0034] In the present application, the term "dopaminergic cell" refers to a neuron capable of synthesizing and releasing dopamine.

[0035] Advantages

[0036] (1) The exocytosis monitoring electrochemical sensor of the present application does not require high-end current amplification and shielding system, complex and precise instrument operation, and can be tested in combination with a laboratory conventional electrochemical workstation and a Faraday cage.

[0037] (2) The exocytosis monitoring electrochemical sensor determination method of the present application is simple and does not require a pretreatment process. Only the required number of dopaminergic cells need to be placed on the exocytosis monitoring electrochemical sensor to test.

[0038] (3) The electrode preparation method grinds the prepared nanomaterial into powder, mixes the nanomaterial with conductive resin, and ultrasonically mixes the slurry to coat the working electrode area of the screen-printed electrode. The grinding and ultrasonic process may damage the three-dimensional structure of the nanomaterial. The present application uses carbonized wood as a substrate, which can be directly cut and fixed on the working electrode of the screen-printed electrode through conductive paste. Not only is the operation step simple, but also the three-dimensional structure of the nanomaterial can be preserved to the greatest extent, which is used for cell fixation on the sensing interface and improves the monitoring efficiency.

[0039] (4) The exocytosis monitoring electrochemical sensor electrode of the present application can be used once, and the exocytosis monitoring electrochemical sensor has low cost; the detection time is fast, the sample preparation and testing time is less than 5 minutes; the research efficiency is high, and single or multiple cells can be monitored to quickly obtain statistically significant data. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 SEM characterization images of carbonized wood-ZnO nanowire-ZIF-8 prepared in Example 1 at different angles and different magnifications.

[0041] Figure 2A schematic diagram of the live cell exocytosis monitoring device of the present application.

[0042] Figure 3 (a) is the real-time exocytosis of different numbers of live cells tested using the exocytosis monitoring electrochemical sensor of Example 1, and (b) is the concentration of dopamine in the supernatant of different numbers of cells monitored using ELISA.

[0043] Figure 4 Results of the exocytosis of SH-SY5Y cells after treatment with levodopa, a drug that promotes dopamine secretion, monitored using the exocytosis monitoring electrochemical sensor prepared in Example 1.

[0044] Figure 5 Results of the effect of different concentrations of the natural product hyperoside on the exocytosis of SH-SY5Y cells, monitored using the exocytosis monitoring electrochemical sensor prepared in Example 1, in which (a) is the real-time monitoring curve of SH-SY5Y cells treated with different concentrations of hyperoside, and (b) is the curve of 13 tests for each concentration of hyperoside, the number of effective exocytosis for each test is counted and analyzed for significant difference using T-test (ns represents no significant difference, ** represents P<0.01, **** represents P<0.0001).

[0045] Figure 6 Scanning electron microscope images of the carbonized wood-ZnO nanowire-ZIF-8 prepared in Comparative Example 1 (a) and Example 1 (b).

[0046] Figure 7 Real-time exocytosis of 10 SH-SY5Y cells tested using the exocytosis monitoring electrochemical sensor prepared in Comparative Example 1 (a) and Example 1 (b). DETAILED DESCRIPTION

[0047] The present application will be further described with reference to the following specific examples. It should be understood that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content of the present application, and these equivalent forms also fall within the scope of the appended claims.

[0048] The embodiment of the present application provides an exocytosis monitoring electrochemical sensor, wherein the working electrode of the electrochemical sensor is modified with a multi-dimensional nanohybrid material; the multi-dimensional nanohybrid material is obtained by using porous carbonized wood as a substrate, and growing zinc oxide nanowires and an organic framework compound on the substrate in situ. The organic framework compound is ZIF-8.

[0049] The porous carbonized wood substrate has the advantages of increasing the specific surface area of the material by utilizing the natural structure of wood, and being more sensitive to the monitoring of dopamine molecules, and the monitoring limit of the graphene substrate material for dopamine is 1 pM, and the monitoring limit of the application is 0.5 pM; meanwhile, the natural pores of the carbonized wood substrate can fix cells, simplify the test method, and improve the monitoring efficiency.

[0050] The embodiment of the application also provides a preparation method of the above-mentioned exocytosis monitoring electrochemical sensor, in which polyethyleneimine is used as a morphology directing agent, zinc nitrate hexahydrate is used as a zinc source, hexamethylenetetramine is used as an alkali source and a complexing agent, ZnO nanowires are directionally grown on carbonized wood flakes through a hydrothermal reaction, and carbonized wood-ZnO nanowires are obtained; then 2-dimethylimidazole is used as a ligand to in-situ grow metal organic frameworks on the carbonized wood-ZnO nanowires, and carbonized wood-ZnO nanowire-ZIF-8 is obtained; and the carbonized wood-ZnO nanowire-ZIF-8 is modified on the working electrode to obtain the dopamine electrochemical sensor based on the natural structure of carbonized wood.

[0051] Different from the previous method for preparing nanowires, the ZnO nanowires are grown twice on the carbonized wood substrate in the application in order to uniformly distribute the ZnO on the carbonized wood, so that the carbonized wood is completely covered by the nanowires and the length of the nanowires is significantly increased.

[0052] The exocytosis monitoring electrochemical sensor provided by the embodiment of the application can monitor the real-time exocytosis of dopaminergic cells.

[0053] In the following embodiments of the application, the steps of monitoring exocytosis by the exocytosis monitoring electrochemical sensor are as follows:

[0054] S1. dispersing cells in sterile PBS buffer (pH=7.4) containing 2wt% bovine serum to prepare a cell dispersion liquid;

[0055] S2. using a Shanghai Aorui cell printer, setting the required number of cells, and accurately printing the cells in the cell dispersion liquid to the working electrode of the exocytosis monitoring electrochemical sensor;

[0056] S3. placing the exocytosis monitoring electrochemical sensor in a Faraday cage and connecting it with an electrochemical workstation (connecting the working area of the exocytosis monitoring electrochemical sensor with the electrochemical workstation);

[0057] S4. using a micropipette to take 10 μL of a to-be-tested liquid and drop it on the working area of the exocytosis monitoring electrochemical sensor, so as to fully cover the working electrode, the reference electrode and the counter electrode on which the cells are printed;

[0058] S5. starting monitoring by amperometry: setting the monitoring potential to 0.34 V, the sampling interval to 0.012 s, and the duration to 300 s.

[0059] S6. After 300s of testing, terminate the experiment;

[0060] S7. Count the number and area of current spikes on the test curve, and calculate the number of dopamine molecules in the exocytosis release event using the Faraday formula.

[0061] In the embodiment of the present application, the test solution is a potassium chloride / calcium chloride solution, a levodopa solution or a hyperoside solution.

[0062] The carbonized wood-ZnO nanowire-ZIF-8 material is modified on the screen-printed electrode to prepare the exocytosis monitoring electrochemical sensor, and a specific number of cells are placed in the working area of the exocytosis monitoring electrochemical sensor for analysis. The exocytosis monitoring electrochemical sensor utilizes the synergistic effect of the natural structure of the Finnish pine and the multi-dimensional nanomaterial, and is combined with a laboratory general electrochemical workstation and a small Faraday cage to realize the recording and analysis of single exocytosis events of single cells, which is simple to operate, low in cost and can be used for efficient screening of natural product efficacy.

[0063] Unless otherwise specified, the room temperature in the present application is 25±2℃.

[0064] The raw materials used in the embodiment of the present application are all commercially available.

[0065] It should be noted that the details not described in the present application are all conventional operation means in the art and are not the focus of the present application.

[0066] The technical solutions of the present application are further described below through examples.

[0067] Example 1

[0068] The present embodiment provides a preparation method of an exocytosis monitoring electrochemical sensor, and the specific steps are as follows:

[0069] S1. A 3cm×1cm×1cm Finnish pine block is placed in a tube furnace and heated at 800℃ for 2h under an Ar atmosphere (flow rate of 0.1L / min), and the temperature is raised for 4h to complete the carbonization treatment to obtain carbonized wood;

[0070] S2. The carbonized wood is cut into a 700μm thick slice, ultrasonically cleaned in ethanol, and then dried at 150℃;

[0071] S3. 22.22mg of zinc acetate dihydrate is dissolved in 20mL of ethanol, ultrasonically dissolved in ethanol to obtain a solution; then 25μL of the above solution is added on the carbonized wood slice at one time using a 100mL pipette (at this time, the solution is added on the front surface), and dried at 150℃, repeated four times;

[0072] S4. The carbonized wood flake after the above treatment was placed in a tubular furnace at 350℃ for 20 min, and then cooled to room temperature and taken out;

[0073] S5. 0.387 g of polyethyleneimine and 0.8 g of zinc nitrate hexahydrate were weighed, respectively, and then added to 10 mL of deionized water and stirred to dissolve, followed by adding 0.35 g of hexamethylenetetramine, and then deionized water was added to make up to 100 mL to obtain a mixed precursor solution;

[0074] S6. The mixed precursor solution was poured into a 100 mL wide-mouth container, and the carbonized wood flake was inverted and suspended on the surface of the precursor solution, and then heated at 90℃ for 4 h;

[0075] S7. The carbonized wood flake was taken out, washed twice with deionized water, and then washed once with ethanol, and then dried at 150℃, and then placed with the front face upward in a tubular furnace at 350℃ for 10 min, and then cooled to room temperature and taken out; then steps S5 and S6 were repeated, and then the carbonized wood flake was taken out, washed twice with deionized water, and then washed once with ethanol, and then dried at 150℃ to obtain carbonized wood-ZnO nanowires;

[0076] S8. 2 g of 2-dimethylimidazole was weighed and placed at the bottom of a glass container, and then preheated at 110℃ for 30 min, and then the carbonized wood-ZnO nanowires obtained in step S7 were hung 8 cm above the 2-dimethylimidazole, and then heated at 110℃ for 5 min, and then taken out and cooled to room temperature, and then washed once with ethanol, and then excess ethanol was removed at 110℃ to obtain carbonized wood-ZnO nanowires-ZIF-8;

[0077] S9. 1 μL of conductive carbon paste (Celanese BQ242) was coated on the working electrode area of a screen-printed electrode (the working electrode and the counter electrode were both carbon electrodes, and the reference electrode was a silver / silver chloride electrode), and then the carbonized wood-ZnO nanowires-ZIF-8 divided into 1 mm x 1 mm was pasted thereon;

[0078] S10. The material obtained in S9 was placed in a 50℃ oven for 30 min for curing;

[0079] S11. In order to improve the hydrophilicity of the sensing material and ensure sufficient contact between the electrolyte and the electrode interface in the electrochemical test, the cured electrochemical sensor was soaked in deionized water for 24 h to obtain an exocytosis monitoring electrochemical sensor.

[0080] The SEM characterization graph of the carbonized wood-ZnO nanowires-ZIF-8 prepared in this example under different angles and different magnifications is as follows: Figure 1As shown, the arrayed ZnO nanowires are in-situ grown on the natural pores of the carbonized wood, and the outer layer is ZIF-8, which produces a synergistic effect to help detect the real-time exocytosis behavior of single cells: the pore diameter of the carbonized pine wood is about 12 μm, which matches the size of the cells, and the cells can be fixed in the pores without additional operation steps. Meanwhile, the carbonized wood has good electrical conductivity, which helps the electrons generated by the electrochemical oxidation of dopamine to be conducted to the working electrode. The in-situ grown arrayed ZnO nanowires are in full contact with the fixed cells and receive the dopamine molecules released by exocytosis from multiple directions at close range. The ZIF-8 on the surface of the nanowires is a solid base nanocatalyst, which deeply oxidizes the dopamine molecules in the solution, and the electrical signal generated by the electron transfer in the catalytic oxidation process can be used for real-time analysis of the exocytosis event.

[0081] Example 2

[0082] The exocytosis monitoring electrochemical sensor prepared in Example 1 was used to monitor the exocytosis of different numbers of living cells under the stimulation of high concentration of potassium ions, and the schematic diagram of the living cell exocytosis monitoring device is as shown in Figure 2 As shown, the specific operation is as follows:

[0083] 1) Human neuroblastoma cells SH-SY5Y (purchased from Wuhan Punsai Life Science and Technology Co., Ltd.) were used as the research model, and the cells were cultured in SH-SY5Y special cell culture medium containing 1% penicillin-streptomycin double antibody, and the environmental conditions were 37°C, 5% CO2, the culture medium was replaced every two days, and the cells were subcultured at a ratio of 1:1 every four days;

[0084] 2) The cells were dispersed in sterile PBS buffer containing 2wt% bovine serum to prepare a cell dispersion solution;

[0085] 3) The Shanghai Aorui cell printer was used, and the required number of cells was set to accurately print the cells onto the working electrode of the exocytosis monitoring electrochemical sensor;

[0086] 4) The exocytosis monitoring electrochemical sensor was placed in a Faraday cage and connected to an electrochemical workstation;

[0087] 5) A micropipette was used to take 10 μL of PBS solution (pH = 7.4) containing 80 mM potassium chloride and 2 mM calcium chloride and drop it on the working area of the exocytosis monitoring electrochemical sensor, fully covering the working electrode, reference electrode and counter electrode printed with cells;

[0088] 6) Start monitoring by amperometry: set the monitoring potential to 0.34 V, the sampling interval to 0.012 s, and the duration to 300 s;

[0089] 7) After 300 s of testing, the experiment was terminated;

[0090] 8) The area Q of the peak curve is the total transferred electric quantity in the process of oxidation of DA molecules in an exocytosis event. According to Faraday equation Q = nNF (Q is the area of the peak, unit: coulomb; n is the number of electrons transferred in the oxidation of one DA molecule; N is the number of DA molecules released in one exocytosis event), the number of DA molecules released by one exocytosis event of the average SH-SY5Y cell is about 122.46zmol;

[0091] 9) ELISA verification: 10, 100 and 1000 SH-SY5Y cells were respectively transferred into 100 μL PBS solution containing 80 mM potassium chloride and 2 mM calcium chloride (pH = 7.4), and the cell supernatant was taken after 300 s, and the dopamine concentration in the supernatant was tested using an ELISA kit.

[0092] The test results are shown in Figure 3 a. In the test curve, the current peak signal can be observed in turn, the real-time exocytosis of one cell can be observed, and the current peak signal increases with the increase of the number of cells, verifying the monitoring ability of the exocytosis monitoring electrochemical sensor for cell exocytosis events. As shown in Figure 3 b, the concentration of dopamine in the cell supernatant increases with the increase of the number of cells, which is consistent with the trend tested by the exocytosis monitoring electrochemical sensor of the application, verifying the feasibility of the exocytosis monitoring electrochemical sensor of the application to observe the exocytosis of living cells.

[0093] Example 3

[0094] Using the exocytosis monitoring electrochemical sensor prepared in Example 1, the exocytosis of SH-SY5Y cells after treatment with levodopa, a drug that promotes dopamine secretion, was studied. The specific implementation operation is as follows:

[0095] 1) Human neuroblastoma cells SH-SY5Y (purchased from Wuhan Punsai Life Science and Technology Co., Ltd.) were used as the research model, and were cultured using SH-SY5Y special cell culture medium containing 1% penicillin-streptomycin double antibody, and the environmental conditions were 37℃, 5% CO2, the culture medium was replaced every two days, and the cells were subcultured at a ratio of 1:1 every four days;

[0096] 2) The cells were dispersed in sterile PBS buffer solution (pH = 7.4) containing 2wt% bovine serum to prepare a cell dispersion liquid;

[0097] 3) The Shanghai Aorui cell printer was used, and the number of printed cells was set to 10, and 10 SH-SY5Y cells were accurately printed on the working electrode of the exocytosis monitoring electrochemical sensor;

[0098] 4) The exocytosis monitoring electrochemical sensor was placed in a Faraday cage and connected to an electrochemical workstation;

[0099] 5) Control experiment: 10 μL of PBS solution containing 80 mM potassium chloride and 2 mM calcium chloride was added to the working area of the exocytosis monitoring electrochemical sensor using a micropipette, fully covering the printed cell working electrode, reference electrode and counter electrode; start monitoring using amperometry: set the monitoring potential to 0.34 V, sampling interval to 0.012 s, and duration to 300 s; after 300 s of testing, terminate the experiment;

[0100] 6) Levodopa experiment: 10 μL of PBS solution (pH = 7.4) containing 100 μM levodopa was added to the working area of the exocytosis monitoring electrochemical sensor using a micropipette, fully covering the printed cell working electrode, reference electrode and counter electrode; start monitoring using amperometry: set the monitoring potential to 0.34 V, sampling interval to 0.012 s, and duration to 300 s; after 300 s of testing, terminate the experiment.

[0101] The test curve is shown in Figure 4 It can be observed that, compared with the cells treated with high-concentration potassium chloride, more current spike signals appear in the test curve of the cells treated with levodopa, the promotion of levodopa on dopamine secretion of nerve cells is observed, and the practicability of the sensor in real-time monitoring of exocytosis is proved.

[0102] Example 4

[0103] The exocytosis monitoring electrochemical sensor prepared in Example 1 was used to study the effect of different concentrations of natural product hyperoside on the exocytosis of SH-SY5Y cells. The specific implementation operation is as follows:

[0104] 1) Human neuroblastoma cells SH-SY5Y (purchased from Wuhan Punsai Life Science and Technology Co., Ltd.) were used as the research model, and were cultured in SH-SY5Y special cell culture medium containing 1% penicillin-streptomycin double antibody, and the environmental conditions were 37°C, 5% CO2, the culture medium was replaced every two days, and the cells were subcultured at a ratio of 1:1 every four days;

[0105] 2) The cells were dispersed in sterile PBS buffer containing 2wt% bovine serum to prepare a cell dispersion liquid;

[0106] 3) A Shanghai Aorui cell printer was used, and the number of printed cells was set to 10, and 10 SH-SY5Y cells were accurately printed on the working electrode of the exocytosis monitoring electrochemical sensor;

[0107] 4) The exocytosis monitoring electrochemical sensor was placed in a Faraday cage and connected to an electrochemical workstation;

[0108] 5) Control experiment: Using a micropipette, 10 μL of PBS solution containing 80 mM potassium chloride and 2 mM calcium chloride was added to the working area of ​​the electrochemical sensor for exocytosis monitoring, fully covering the working electrode, reference electrode, and counter electrode that had been printed with cells; monitoring was started using the amperometric method: the monitoring potential was set to 0.34 V, the sampling interval was 0.012 s, and the duration was 300 s; the experiment was terminated after 300 s of testing.

[0109] 6) Hyperoside Assay: Hyperoside PBS solutions with concentrations of 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 70 μM, and 100 μM were prepared using PBS solution (pH = 7.4). 10 μL of each solution was added to the working area of ​​the electrochemical sensor used for exocytosis monitoring, fully covering the working electrode, reference electrode, and counter electrode of the printed cell. Monitoring was initiated using the amperometric method: the monitoring potential was set to 0.34 V, the sampling interval was 0.012 s, and the duration was 300 s. The experiment was terminated after 300 s of testing.

[0110] 7) To obtain statistically valid data, each hyperoside concentration was tested 13 times;

[0111] 8) Data analysis: Peak analysis was performed using Origin software. The criteria for determining a valid exocytosis event were: the signal-to-noise ratio (SNR) of the peak signal was more than three times. Data with a half-width (t1 / 2, defined as the width at 50% of the maximum peak value) greater than 20ms were removed to eliminate interference from multiple events.

[0112] 9) Statistical analysis: The results were evaluated using a two-tailed unpaired t-test. A p-value < 0.05 was considered statistically significant.

[0113] The effects of different concentrations of the natural product hyperoside on the exocytosis of SH-SY5Y cells were monitored using the electrochemical sensor for exocytosis monitoring prepared in Example 1. The results are as follows: Figure 5 As shown, by Figure 5 The amperometric curve of a showed that the number of exocytosis events in SH-SY5Y cells increased with increasing hyperoside concentration, reaching a peak at a hyperoside concentration of 10 μM. Figure 5As shown in Figure b, the statistical data shows a bell-shaped number of exocytosis events-concentration dependence, with the peak count reaching a maximum at 10 mM hyperoside and decreasing at higher hyperoside concentrations. The number of exocytosis events in SH-SY5Y cells increased with the increase of the concentration of hyperoside, but when the concentration of hyperoside was higher than 10 mM, the number of exocytosis events decreased with the increase of the concentration of hyperoside. There was no significant difference in the peak count at 30 mM, and it was lower than the control level at 70 mM and 100 mM. The sensor study results show that hyperoside has a significant concentration-dependent effect on the secretion of DA neurotransmitters in living SH-SY5Y cells.

[0114] Comparative Example 1

[0115] A method for preparing an electrochemical sensor for monitoring exocytosis, the specific steps are as follows:

[0116] S1. A 3 cm x 1 cm x 1 cm piece of Finnish pine was placed in a tube furnace and heated at 800°C for 2 h under an Ar atmosphere (flow rate 0.1 L / min) for 4 h to complete the carbonization treatment, obtaining a carbonized wood material;

[0117] S2. The carbonized wood was cut into 700 μm thick slices and ultrasonically cleaned in ethanol, then dried at 150°C;

[0118] S3. 22.22 mg of zinc acetate dihydrate was weighed into 20 mL of ethanol and ultrasonically dissolved in ethanol to obtain a solution; then 25 μL of the above solution was added to the carbonized wood slices with a 100 mL pipette (at this time the solution was added to the front surface), and dried at 150°C, repeated four times;

[0119] S4. The carbonized wood material was placed in a tube furnace at 350°C for 20 min, then cooled to room temperature and removed;

[0120] S5. An aqueous solution containing 25 mM zinc nitrate hexahydrate and 25 mM methenamine was prepared to obtain a precursor solution, which was transferred to a 100 mL wide-mouth container, and the carbonized wood slices were inverted and suspended on the surface of the precursor solution, heated at 90°C for 3 h to obtain carbonized wood-ZnO nanowires;

[0121] S6. 0.5 g of 2-dimethylimidazole was weighed into a glass container and preheated at 110°C for 30 min, then the carbonized wood slices were hung 5 cm above the 2-dimethylimidazole and heated at 110°C for 5 min, then cooled to room temperature, washed with ethanol, and the excess ethanol was removed at 110°C to obtain carbonized wood-ZnO nanowires-ZIF-8;

[0122] S7. 1 μL of conductive carbon paste (Celanese BQ242) was coated on the working electrode area of the screen-printed electrode (both working electrode and counter electrode were carbon electrodes, and the reference electrode was a silver / silver chloride electrode), and the carbonized wood-ZnO nanowire-ZIF-8 divided into 1 mm x 1 mm was pasted on it;

[0123] S8. Curing was performed by placing in a 50°C oven for 30 min;

[0124] S9. In order to improve the hydrophilicity of the sensing material and ensure sufficient contact between the electrolyte and the electrode interface in the electrochemical test, the electrochemical sensor was immersed in deionized water for 24 h;

[0125] S10. The SH-SY5Y cells were dispersed in sterile PBS buffer containing 2 wt% bovine serum to prepare a cell dispersion liquid;

[0126] S11. The Shanghai Aolai cell printer was used, and the number of printed cells was set to 10. Precisely 10 SH-SY5Y cells were printed on the working electrode of the exocytosis monitoring electrochemical sensor;

[0127] S12. The exocytosis monitoring electrochemical sensor was placed in a Faraday cage and connected to an electrochemical workstation;

[0128] S13. 10 μL of PBS solution containing 80 mM potassium chloride and 2 mM calcium chloride was added dropwise to the working area of the exocytosis monitoring electrochemical sensor using a micropipette, so as to fully cover the printed cell working electrode, reference electrode and counter electrode;

[0129] S14. Start monitoring by amperometry: set the monitoring potential to 0.34 V, the sampling interval to 0.012 s, and the duration to 300 s; after 300 s of testing, terminate the experiment.

[0130] The scanning electron microscope images of the carbonized wood-ZnO nanowire-ZIF-8 prepared in Comparative Example 1 and Example 1 are shown in FIGS. 1 and 2, respectively. Figure 6 It can be seen that the ZnO nanowires grown by the method of Comparative Example 1 cannot completely cover the carbonized wood; the method of Example 1 can grow ZnO nanowires and ZIF-8 on the carbonized wood substrate, and the two times of hydrothermal growth of ZnO nanowires can completely cover the ZnO nanowires on the carbonized wood, and the length of the nanowires is significantly increased.

[0131] The real-time exocytosis of 10 SH-SY5Y cells was tested by the exocytosis monitoring electrochemical sensor prepared in Comparative Example 1 and Example 1, as shown in FIGS. 3 and 4, respectively. Figure 7 It can be seen that the material synthesized by the existing method (i.e. the material of Comparative Example 1) can hardly monitor the current spike caused by exocytosis, while the material synthesized by the method of Example 1 of the present application can monitor multiple current spikes.

[0132] The above merely provides the preferred embodiment of the present application, and the protection scope of the present application is not limited thereto. Any modification or replacement within the technical scope disclosed by the present application can be easily conceived by those skilled in the art, and should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. An electrochemical sensor for exocytosis monitoring, characterized by: The working electrode of the electrochemical sensor is modified with a multi-dimensional nanometer hybrid material; The multi-dimensional nanometer hybrid material is obtained by taking carbonized wood as a substrate, and growing zinc oxide nanowires and organic framework compounds on the substrate in situ.

2. The electrochemical sensor for exocytosis monitoring according to claim 1, characterized in that: The organic framework compound is ZIF-8.

3. A preparation method of an electrochemical sensor for monitoring exocytosis, comprising the following steps: S1. Carbonizing pine wood blocks to obtain carbonized wood; S2. Cutting the carbonized wood into thin slices, washing and drying to obtain carbonized wood slices; S3. Dropping zinc acetate dihydrate ethanol solution on the carbonized wood slices, defining the dropping surface as the front surface, and drying; S4. Calcining the carbonized wood slices obtained in step S3, and taking them out after cooling; S5. Preparing an aqueous solution containing polyethyleneimine, zinc nitrate hexahydrate and hexamethylenetetramine to obtain a precursor solution; S6. Inverting the front surface of the carbonized wood slices obtained in step S4 and suspending them on the surface of the precursor solution, and heating; S7. Taking out the carbonized wood slices, washing and drying, and performing secondary calcination, and taking them out after cooling to room temperature, and repeating steps S5 and S6 to obtain carbonized wood-zinc oxide nanowires; S8. Preheating 2-dimethylimidazole at the bottom of a glass container, then suspending the carbonized wood-zinc oxide nanowires above the 2-dimethylimidazole, heating at 100-120℃ for 5-10 min, taking out and cooling to room temperature, washing and drying to obtain carbonized wood-zinc oxide nanowires-ZIF-8; S9. Coating conductive carbon paste on the working electrode area of a screen-printed electrode, and pasting the carbonized wood-zinc oxide nanowires-ZIF-8, curing, and immersing in deionized water to obtain the electrochemical sensor for monitoring exocytosis.

4. The method of claim 3, wherein: The carbonization temperature in step S1 is 700-1000℃, and the time is 1-5h, and the process is carried out under inert gas protection.

5. The method of claim 3, wherein: The concentration of zinc acetate dihydrate ethanol solution in step S3 is 1.111mg / mL.

6. The method of claim 3, wherein: The calcination temperature in step S4 is 300-400℃, and the time is 10-30min.

7. The method of claim 3, wherein: The concentration of polyethyleneimine in the precursor solution in step S5 is 0.00387g / mL, the concentration of zinc nitrate hexahydrate is 0.008g / mL, and the concentration of hexamethylenetetramine is 0.0035g / mL.

8. Use of the electrochemical sensor for monitoring exocytosis according to claim 1 in monitoring real-time exocytosis of cells.

9. Use according to claim 8, characterized in that: Comprising the following steps: (1) dispersing cells in sterile PBS buffer containing bovine serum to prepare a cell dispersion liquid; (2) using a cell printer to print cells in the cell dispersion liquid onto the working electrode of the electrochemical sensor for monitoring exocytosis; (3) placing the electrochemical sensor for monitoring exocytosis in a Faraday cage and connecting it with an electrochemical workstation; (4) dropping the test liquid onto the working area of the electrochemical sensor for monitoring exocytosis, and fully covering the printed cell working electrode, reference electrode and counter electrode; (5) starting monitoring by amperometry.

10. Use according to claim 9, characterized in that: When the amperometric method is used in the step (5) for monitoring, the monitoring potential is 0.34 V, the sampling interval is 0.012 seconds, and the duration is 300 seconds.

Images