Exocytosis monitoring electrochemical sensor, preparation method and application thereof

Abstract

The application relates to an electrochemical sensor for exocytosis monitoring and a preparation method and application thereof. A 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 porous carbonized wood as a substrate, and growing zinc oxide nanowires and organic framework compounds in situ on the substrate. The electrochemical sensor can monitor the exocytosis behavior of living cells by using the synergistic effect of nanometer materials while fixing single cells. The electrochemical sensor can be tested by cooperating with a conventional electrochemical workstation and a Faraday cage in a laboratory, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, and specifically relates to an electrochemical sensor for monitoring exocytosis, its preparation method, and its application. Background Technology

[0002] The release of neurotransmitters such as dopamine by nerve cells through exocytosis is a crucial physiological behavior in living organisms. It is a primary pathway for the nervous system to regulate bodily functions and a fundamental biological principle in various fields, including neurobiology, biochemistry, clinical medicine, pathology, and pharmacology. When exocytosis is tested using electrochemical amperometry, several discontinuous spike signals can be obtained. These spikes are transient current signals generated by the oxidation of neurotransmitters released during exocytosis. Analyzing the number and size of these spikes can provide information such as the frequency of exocytosis and the amount of dopamine molecules secreted, thus providing crucial data for communication between nerve cells or neurons. Cellular exocytosis may be altered under the influence of drugs. By studying the effects of drugs on cell exocytosis, compounds with biological activity, pharmacophores, or pharmacological effects can be efficiently screened from a vast array of natural products, providing crucial clues for drug discovery, from lead compounds to precursor compounds. This is particularly significant for the creation of new drugs from natural products. Currently, exocytosis research often relies on techniques such as cryo-electron microscopy, ultra-high resolution fluorescence microscopy, and carbon fiber electrodes (CFE). These techniques not only require expensive large-scale instruments and specialized operation, but also have low testing efficiency. New technologies are needed to provide efficient and powerful tools for screening bioactive natural products. Summary of the Invention

[0003] This invention provides an electrochemical sensor for monitoring exocytosis, its preparation method, and its application, which solves the problems of existing carbon fiber electrodes (CFE) that can only cover about 10% of the cell membrane, can only test adherent cells one by one, and cannot obtain multi-cell statistical data.

[0004] This invention provides an electrochemical sensor for monitoring exocytosis, wherein the working electrode of the electrochemical sensor is modified with a multidimensional nanomaterial; the multidimensional nanomaterial is obtained by in-situ growth of zinc oxide nanowires and organic framework compounds on a porous carbonized wood substrate.

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

[0006] This invention involves the in-situ growth of an array of ZnO nanowires on the natural pores of carbonized wood, with an outer layer of ZIF-8. The resulting synergistic effect facilitates the detection of real-time exocytosis in single cells: the pores of carbonized Finnish pine have a diameter of approximately 12 μm, matching the cell size, allowing cells to be immobilized within the pores without additional manipulation. Simultaneously, the excellent conductivity of carbonized wood facilitates the conduction of electrons generated by the electrochemical oxidation of dopamine to the working electrode. The in-situ grown array of ZnO nanowires maintains ample contact with the immobilized cells, receiving dopamine molecules released during exocytosis from multiple directions at close range. The ZIF-8 on the nanowire surface serves as a solid base nanocatalyst, deeply oxidizing dopamine molecules in solution. The electrical signals generated by electron transfer during the catalytic oxidation process can be used for real-time analysis of exocytosis events.

[0007] This invention also provides a method for preparing an electrochemical sensor for monitoring exocytosis, 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 them to obtain carbonized wood slices;

[0010] S3. Add an ethanol solution of zinc acetate dihydrate to the carbonized wood sheet, define the surface to which the solution is added as the front side, and then dry it.

[0011] S4. Calcine the carbonized wood sheets obtained in step S3, and remove them after cooling;

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

[0013] S6. The carbonized wood sheet obtained in step S4 is suspended face up on the surface of the precursor solution and heated;

[0014] S7. Take out the carbonized wood sheet, wash and dry it, calcine it a second time, cool it to room temperature and take it out. Repeat steps S5 and S6 to obtain carbonized wood-ZnO nanowires.

[0015] S8. Place 2-dimethylimidazole at the bottom of a glass container for preheating, then suspend the carbonized wood-ZnO nanowires above the 2-dimethylimidazole, heat at 100-120°C for 5-10 minutes, remove and cool to room temperature, wash and dry to obtain carbonized wood-ZnO nanowires-ZIF-8.

[0016] S9. A conductive carbon paste is coated on the working electrode area of ​​the screen-printed electrode, and the carbonized wood-ZnO nanowires-ZIF-8 are attached. After curing, the electrode is soaked in deionized water to obtain the electrochemical sensor for exocytosis monitoring.

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

[0018] Preferably, the carbonization process in step S1 is carried out at a temperature of 700–1000°C for 1–5 hours under an inert gas atmosphere.

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

[0020] Preferably, the calcination temperature in step S4 is 300–400°C, and the time is 10–30 min.

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

[0022] Preferably, in step S6, the heating temperature is 80–100°C and the time is 1–5 hours.

[0023] Preferably, the secondary calcination temperature in step S7 is 300–400°C, and the time is 10–30 min.

[0024] Preferably, in step S8, the preheating temperature is 100–120°C and the time is 20–40 min.

[0025] Existing methods for preparing electrochemical sensors for exocytosis monitoring require the synthetic materials to be thoroughly ground and ultrasonically processed into a slurry, which is then coated onto the surface of a screen-printed electrode. This method may damage the three-dimensional structure of the nanomaterials, affecting sensor performance. This invention uses carbonized wood as a substrate. A whole piece of carbonized wood is cut and then adhered to the working electrode using conductive carbon paste. This ensures conductivity while preserving the three-dimensional structure of the nanomaterials, enabling the sensor proposed in this invention to fix the target cells within the pores of the carbonized wood structure, achieving simple, rapid, and efficient real-time exocytosis studies.

[0026] The present invention also provides an application of an electrochemical sensor for monitoring exocytosis in real-time cell exocytosis.

[0027] Specifically, the following steps are included:

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

[0029] (2) Using a cell printer, the cells in the cell dispersion are printed onto the working electrode of the electrochemical sensor for exocytosis monitoring;

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

[0031] (4) Add the test liquid droplet to the working area of ​​the electrochemical sensor for exocytosis monitoring, fully covering the working electrode, reference electrode and counter electrode of the printed cell;

[0032] (5) Start monitoring using the amperometric method.

[0033] Preferably, in step (5), when monitoring is performed using the amperometric method, the monitoring potential is 0.34V, the sampling interval is 0.012 seconds, and the duration is 300 seconds.

[0034] In this invention, the term "dopaminergic cell" refers to a neuron that is capable of synthesizing and releasing dopamine.

[0035] Beneficial effects

[0036] (1) The electrochemical sensor for monitoring exocytosis of the present invention does not require a high-end current amplification and shielding system or complex and precise instrument operation. It can be used in conjunction with a conventional laboratory electrochemical workstation and a Faraday cage for testing.

[0037] (2) The electrochemical sensor for exocytosis monitoring of the present invention is simple to measure and requires no pretreatment process. The required number of dopaminergic cells can be placed on the electrochemical sensor for exocytosis monitoring for testing.

[0038] (3) The conventional electrode preparation method involves grinding the prepared nanomaterials into powder, mixing them with conductive resin and ultrasonically mixing them to form a slurry, which is then coated onto the working electrode area of ​​the screen-printed electrode. The grinding and ultrasonic processes may damage the three-dimensional structure of the nanomaterials. This invention uses carbonized wood as a substrate, which can be directly cut and fixed onto the working electrode of the screen-printed electrode using conductive slurry. This not only simplifies the operation steps but also preserves the three-dimensional structure of the nanomaterials to the greatest extent, which is used to fix cells on the sensing interface and improve monitoring efficiency.

[0039] (4) The electrochemical sensor electrode for exocytosis monitoring of the present invention can be used once, and the electrochemical sensor for exocytosis monitoring has low cost; fast detection time, sample preparation and testing time is less than 5 minutes; high research efficiency, can monitor single or multiple cells, and quickly obtain statistically significant data. Attached Figure Description

[0040] Figure 1 The images show SEM characterizations of the carbonized wood-ZnO nanowires-ZIF-8 prepared in Example 1 at different angles and magnifications.

[0041] Figure 2This is a schematic diagram of the live cell exocytosis monitoring device of the present invention.

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

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

[0044] Figure 5 To monitor the effect of different concentrations of the natural product hyperoside on the exocytosis of SH-SY5Y cells using the electrochemical sensor for exocytosis monitoring prepared in Example 1, (a) shows the real-time monitoring curves of SH-SY5Y cells treated with different concentrations of hyperoside, and (b) shows the test curves for each concentration of hyperoside 13 times. The effective exocytosis count was counted each time and the significance of the difference was analyzed by T detection (ns represents no significant difference, ** represents P < 0.01, **** represents P < 0.0001).

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

[0046] Figure 7 The electrochemical sensor for monitoring exocytosis prepared for Comparative Example 1(a) and Example 1(b) was used to test the real-time exocytosis of 10 SH-SY5Y cells. Detailed Implementation

[0047] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0048] An embodiment of the present invention provides an electrochemical sensor for monitoring exocytosis, wherein the working electrode of the electrochemical sensor is modified with a multidimensional nanomaterial hybrid; the multidimensional nanomaterial hybrid is obtained by in-situ growth of zinc oxide nanowires and an organic framework compound on a porous carbonized wood substrate. The organic framework compound is ZIF-8.

[0049] The advantage of the porous carbonized wood substrate of this invention lies in the fact that it utilizes the natural structure of wood to increase the specific surface area of ​​the material, making it more sensitive to the monitoring of dopamine molecules. The monitoring limit for dopamine on graphene substrate materials is 1 pM, while the monitoring limit of this invention is 0.5 pM. At the same time, the natural pores of the carbonized wood substrate can fix cells, simplify the testing method, and improve the monitoring efficiency.

[0050] The embodiments of the present invention also provide a method for preparing the above-mentioned electrochemical sensor for exocytosis monitoring. Using polyethyleneimine as a morphology directing agent, zinc nitrate hexahydrate as a zinc source, and hexamethylenetetramine as a base source and ligand, ZnO nanowires are directionally grown on carbonized wood sheets via a hydrothermal reaction to obtain carbonized wood-ZnO nanowires. Then, using 2-dimethylimidazole as a ligand, a metal-organic framework is grown in situ on the carbonized wood-ZnO nanowires to obtain carbonized wood-ZnO nanowires-ZIF-8. The carbonized wood-ZnO nanowires-ZIF-8 are then modified onto the working electrode to obtain the dopamine electrochemical sensor based on the natural structure of carbonized wood.

[0051] Unlike previous methods for preparing nanowires, this invention involves growing ZnO nanowires on a carbonized wood substrate using a two-stage hydrothermal method to ensure uniform ZnO distribution on the carbonized wood. This process guarantees that the carbonized wood is completely covered by the nanowires and that the nanowire length is significantly increased.

[0052] The electrochemical sensor for monitoring exocytosis provided in this embodiment of the invention can monitor the real-time exocytosis of dopaminergic cells.

[0053] In the following embodiments of the present invention, the steps for monitoring exocytosis using an electrochemical sensor are as follows:

[0054] S1. Cell dispersion was prepared by dispersing cells in sterile PBS buffer (pH=7.4) containing 2wt% bovine serum;

[0055] S2. Using the Shanghai Aorui cell printer, set the required number of cells and accurately print the cells in the cell dispersion onto the working electrode of the electrochemical sensor for exocytosis monitoring;

[0056] S3. Place the electrochemical sensor for exocytosis monitoring in a Faraday cage and connect it to the electrochemical workstation (connect the working area of ​​the electrochemical sensor for exocytosis monitoring to the electrochemical workstation);

[0057] S4. Using a micropipette, take 10 μL of the test solution and add it to the working area of ​​the electrochemical sensor for exocytosis monitoring, fully covering the working electrode, reference electrode, and counter electrode of the printed cell;

[0058] S5. Start monitoring using the amperometric method: Set the monitoring potential to 0.34V, the sampling interval to 0.012s, and continue for 300s.

[0059] S6. Terminate the experiment after 300 seconds of testing;

[0060] S7. Statistically analyze the number and area of ​​current spikes on the test curve, and use Faraday's formula to calculate the number of dopamine molecules in the exocytosis event.

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

[0062] This invention modifies a screen-printed electrode with carbonized wood-ZnO nanowires-ZIF-8 material to create an electrochemical sensor for exocytosis monitoring. Analysis can be performed by placing a specific number of cells in the working area of ​​the sensor. This exocytosis monitoring electrochemical sensor utilizes the synergistic effect of the natural structure of Finnish pine and multidimensional nanomaterials. When coupled with a standard laboratory electrochemical workstation and a small Faraday cage, it can achieve the recording and analysis of single-cell exocytosis events. The operation is simple, cost-effective, and suitable for efficient screening of natural product efficacy.

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

[0064] All raw materials used in the embodiments of this invention were obtained through commercial purchase.

[0065] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0066] The technical solution of the present invention will be further illustrated by the following embodiments.

[0067] Example 1

[0068] This embodiment provides a method for preparing an electrochemical sensor for exocytosis monitoring, the specific steps of which are as follows:

[0069] S1. Place a 3cm×1cm×1cm block of Finnish pine wood in a tube furnace and heat it at 800℃ for 2 hours while maintaining an Ar atmosphere (flow rate of 0.1L / min). The heating time is 4 hours to complete the carbonization process and obtain carbonized wood.

[0070] S2. Cut the carbonized wood into thin slices with a thickness of 700 μm, immerse them in ethanol for ultrasonic cleaning, and then dry them at 150°C.

[0071] S3. Weigh 22.22 mg of zinc acetate dihydrate and dissolve it in 20 mL of ethanol. Sonicate the solution until it is completely dissolved in the ethanol to obtain a solution. Then, use a 100 mL pipette to add 25 μL of the above solution dropwise onto a carbonized wood sheet (the side on which the solution is added should be the front). Dry the sheet at 150 °C and repeat four times.

[0072] S4. Place the carbonized wood veneer treated above in a tube furnace at 350°C and heat it at a constant temperature for 20 minutes, then cool it to room temperature and remove it.

[0073] S5. Weigh 0.387g of polyethyleneimine and 0.8g of zinc nitrate hexahydrate, add 10mL of deionized water and stir thoroughly to dissolve. Then add 0.35g of hexamethylenetetramine and dilute with deionized water to 100mL to obtain a well-mixed precursor solution.

[0074] S6. Pour the well-mixed precursor solution into a 100mL wide-mouth container, suspend the carbonized wood slab upside down on the surface of the precursor solution, and heat at 90℃ for 4h.

[0075] S7. Take out the carbonized wood sheet, wash it twice with deionized water, then wash it once with ethanol, dry it at 150°C, place it face up in a tube furnace at 350°C and heat it for 10 minutes, then wait for it to cool to room temperature and take it out; then repeat steps S5 and S6, take out the carbonized wood sheet, wash it twice with deionized water, then wash it once with ethanol, and dry it at 150°C to obtain carbonized wood-ZnO nanowires;

[0076] S8. Weigh 2g of 2-dimethylimidazole and place it at the bottom of a glass container. Preheat at 110℃ for 30min. Then, suspend the carbonized wood-ZnO nanowires obtained in step S7 8cm above the 2-dimethylimidazole and heat at 110℃ for 5min. After removing it and cooling it to room temperature, wash it once with ethanol and remove excess ethanol at 110℃ to obtain carbonized wood-ZnO nanowires-ZIF-8.

[0077] S9. Coat 1 μL of conductive carbon paste (Celanese BQ242) onto the working electrode area of ​​the screen-printed electrode (both the working electrode and the counter electrode are carbon electrodes, and the reference electrode is a silver / silver chloride electrode), and attach carbonized wood-ZnO nanowires-ZIF-8 divided into 1 mm × 1 mm to it.

[0078] S10. Place the material obtained in S9 in a 50℃ oven for 30 minutes to cure;

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

[0080] The SEM characterization images of the carbonized wood-ZnO nanowires-ZIF-8 prepared in this embodiment at different angles and magnifications are shown below. Figure 1As shown, an array of ZnO nanowires with an outer layer of ZIF-8 was grown in situ on the natural pores of carbonized wood. The resulting synergistic effect facilitates the detection of real-time exocytosis in single cells: the pores of carbonized Finnish pine are approximately 12 μm in diameter, matching the cell size, allowing cells to be immobilized within the pores without additional manipulation. Simultaneously, the excellent conductivity of carbonized wood facilitates the conduction of electrons generated by the electrochemical oxidation of dopamine to the working electrode. The in-situ grown array of ZnO nanowires maintains full contact with the immobilized cells, receiving dopamine molecules released during exocytosis from multiple directions at close range. The ZIF-8 on the nanowire surface serves as a solid base nanocatalyst, deeply oxidizing dopamine molecules in solution. The electrical signals generated by electron transfer during the catalytic oxidation process can be used for real-time analysis of exocytosis events.

[0081] Example 2

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

[0083] 1) Human neuroblastoma cells SH-SY5Y (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were used as the research model. They were cultured in SH-SY5Y special cell culture medium containing 1% penicillin-streptomycin double antibiotics. The environmental conditions were 37℃ and 5% CO2. The culture medium was changed every two days and passaged at a 1:1 ratio every four days.

[0084] 2) Disperse the cells in sterile PBS buffer containing 2 wt% bovine serum to prepare a cell dispersion;

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

[0086] 4) Place the electrochemical sensor for exocytosis monitoring in a Faraday cage and connect it to the electrochemical workstation;

[0087] 5) Using a micropipette, take 10 μL of PBS solution (pH = 7.4) containing 80 mM potassium chloride and 2 mM calcium chloride and add it to the working area of ​​the electrochemical sensor for exocytosis monitoring, fully covering the working electrode, reference electrode and counter electrode of the printed cells;

[0088] 6) Start monitoring using the amperometric method: Set the monitoring potential to 0.34V, the sampling interval to 0.012s, and continue for 300s;

[0089] 7) Terminate the experiment after 300 seconds of testing;

[0090] 8) The area Q obtained by integrating the peak curve is the total amount of charge transferred during the oxidation of DA molecules in an exocytosis event. According to the Faraday equation Q = nNF (Q is the peak area in coulombs; n is the number of electrons transferred by a DA molecule during oxidation; N is the number of DA molecules released in an exocytosis event), the average amount of DA released per exocytosis event in SH-SY5Y cells is approximately 122.46 2 mol.

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

[0092] Test results are as follows Figure 3 As shown in Figure a, current spike signals can be observed sequentially in the test curve, allowing for the observation of real-time exocytosis of a single cell. Furthermore, the current spike signals increase with the number of cells, verifying the monitoring capability of this electrochemical sensor for exocytosis monitoring of cell exocytosis events. Figure 3 As shown in b, the concentration of dopamine in the cell supernatant increases with the increase of cell number, and the trend is consistent with the trend detected by the electrochemical sensor for monitoring exocytosis of the present invention, verifying the feasibility of the electrochemical sensor for monitoring exocytosis of the present invention for observing live cell exocytosis.

[0093] Example 3

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

[0095] 1) Human neuroblastoma cells SH-SY5Y (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were used as the research model. They were cultured in SH-SY5Y special cell culture medium containing 1% penicillin-streptomycin double antibiotics. The environmental conditions were 37℃ and 5% CO2. The culture medium was changed every two days and passaged at a 1:1 ratio every four days.

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

[0097] 3) Using the Shanghai Aorui cell printer, set the number of cells to be printed to 10, and accurately print 10 SH-SY5Y cells onto the working electrode of the electrochemical sensor for exocytosis monitoring;

[0098] 4) Place the electrochemical sensor for cytotoxicity monitoring in a Faraday cage and connect it to the electrochemical workstation;

[0099] 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.

[0100] 6) Levodopa assay: Using a micropipette, 10 μL of PBS solution (pH = 7.4) containing 100 μM levodopa 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.

[0101] Test curves as follows Figure 4 As shown, it can be observed that, compared with cells treated with high concentrations of potassium chloride, cells treated with levodopa showed more current spikes in the test curve, indicating that levodopa promoted dopamine secretion in nerve cells, thus proving the practicality of the sensor in real-time monitoring of exocytosis.

[0102] Example 4

[0103] The effect of different concentrations of the natural product hyperoside on the exocytosis of SH-SY5Y cells was studied using the electrochemical sensor for exocytosis monitoring prepared in Example 1. The specific implementation procedures are as follows:

[0104] 1) Human neuroblastoma cells SH-SY5Y (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were used as the research model. They were cultured in SH-SY5Y special cell culture medium containing 1% penicillin-streptomycin double antibiotics. The environmental conditions were 37℃ and 5% CO2. The culture medium was changed every two days and passaged at a 1:1 ratio every four days.

[0105] 2) Disperse the cells in sterile PBS buffer containing 2 wt% bovine serum to prepare a cell dispersion;

[0106] 3) Using the Shanghai Aorui cell printer, set the number of cells to be printed to 10, and accurately print 10 SH-SY5Y cells onto the working electrode of the electrochemical sensor for exocytosis monitoring;

[0107] 4) Place the electrochemical sensor for cytotoxicity monitoring in a Faraday cage and connect it to the 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 b, statistical data revealed a concentration-dependent relationship between the number of bell-shaped exocytosis events. The peak count reached its maximum at 10 μM hyperoside and decreased at higher concentrations. The number of exocytosis events in SH-SY5Y cells increased with increasing hyperoside concentration, but decreased with further increases in hyperoside concentration above 10 μM. At 30 μM, there was no significant difference in peak count, and at 70 μM and 100 μM, the counts were lower than control levels. Sensor studies indicate that hyperoside has a significant concentration-dependent effect on the secretion of DA neurotransmitter in live SH-SY5Y cells.

[0114] Comparative Example 1

[0115] A method for preparing an electrochemical sensor for monitoring exocytosis, comprising the following specific steps:

[0116] S1. Place a 3cm×1cm×1cm block of Finnish pine wood in a tube furnace and heat it at 800℃ for 2 hours while maintaining an Ar atmosphere (flow rate 0.1L / min). The heating time is 4 hours to complete the carbonization process and obtain carbonized wood material.

[0117] S2. Cut the carbonized wood into thin slices with a thickness of 700 μm, immerse them in ethanol for ultrasonic cleaning, and then dry them at 150°C.

[0118] S3. Weigh 22.22 mg of zinc acetate dihydrate and dissolve it in 20 mL of ethanol. Sonicate the solution until it is completely dissolved in the ethanol to obtain a solution. Then, use a 100 mL pipette to add 25 μL of the above solution dropwise onto a carbonized wood sheet (the side on which the solution is added should be the front). Dry the sheet at 150 °C and repeat four times.

[0119] S4. Place the carbonized wood material in a tube furnace at 350℃ and heat it at a constant temperature for 20 minutes, then cool it to room temperature and remove it.

[0120] S5. Prepare an aqueous solution containing 25 mM zinc nitrate hexahydrate and 25 mM hexamethylenetetramine to obtain a precursor solution. Transfer the solution to a 100 mL wide-mouth container. Place the carbonized wood sheet upside down on the surface of the precursor solution and heat at 90 °C for 3 h to obtain carbonized wood-ZnO nanowires.

[0121] S6. Weigh 0.5g of 2-dimethylimidazole and place it at the bottom of a glass container. Preheat at 110℃ for 30min. Then, suspend the carbonized wood sheet 5cm above the 2-dimethylimidazole and heat at 110℃ for 5min. After removing it and cooling it to room temperature, wash it with ethanol and remove excess ethanol at 110℃ to obtain carbonized wood-ZnO nanowires-ZIF-8.

[0122] S7. Coat 1 μL of conductive carbon paste (Celanese BQ242) onto the working electrode area of ​​the screen-printed electrode (both the working electrode and the counter electrode are carbon electrodes, and the reference electrode is a silver / silver chloride electrode), and then attach carbonized wood-ZnO nanowires-ZIF-8 divided into 1 mm × 1 mm pieces onto it.

[0123] S8. Place in a 50℃ oven for 30 minutes to cure;

[0124] S9. To improve the hydrophilicity of the sensing material and ensure full contact between the electrolyte and electrode interface during electrochemical testing, the electrochemical sensor is soaked in deionized water for 24 hours.

[0125] S10. Prepare a cell dispersion by dispersing SH-SY5Y cells in sterile PBS buffer containing 2 wt% bovine serum;

[0126] S11. Using the Shanghai Aorui cell printer, set the number of cells to be printed to 10, and accurately print 10 SH-SY5Y cells onto the working electrode of the electrochemical sensor for exocytosis monitoring.

[0127] S12. Place the electrochemical sensor for exocytosis monitoring in a Faraday cage and connect it to the electrochemical workstation;

[0128] S13. Using a micropipette, take 10 μL of PBS solution containing 80 mM potassium chloride and 2 mM calcium chloride and add it to the working area of ​​the electrochemical sensor for exocytosis monitoring, fully covering the working electrode, reference electrode and counter electrode of the printed cells;

[0129] S14. Start monitoring using the amperometric method: Set the monitoring potential to 0.34V, the sampling interval to 0.012s, and continue for 300s; after 300s of testing, terminate the experiment.

[0130] Scanning electron microscope (SEM) images of the carbonized wood-ZnO nanowires-ZIF-8 prepared in Comparative Example 1 and Example 1 are shown below. Figure 6 As shown, the ZnO nanowires grown using the method of Comparative Example 1 cannot achieve complete coverage of carbonized wood. Using the method of Example 1, ZnO nanowires and ZIF-8 can be grown on the carbonized wood substrate. By performing two hydrothermal growth processes, ZnO nanowires can achieve complete coverage of the carbonized wood, and the length of the nanowires is significantly increased.

[0131] The electrochemical sensors prepared in Comparative Example 1 and Example 1 were used to monitor the real-time exocytosis of 10 SH-SY5Y cells. Figure 7 As shown, the material synthesized using existing methods (i.e., the material of Comparative Example 1) hardly exhibits any current spikes induced by exocytosis, while the material synthesized using the method of Example 1 of this invention exhibits multiple current spikes.

[0132] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the 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, growing zinc oxide nanowires and organic framework compounds on the substrate in situ; the organic framework compound is ZIF-8; the preparation method comprises the following steps: S1. Finnish pine blocks are subjected to carbonization treatment to obtain carbonized wood; S2. The carbonized wood is cut into thin slices, washed and dried to obtain carbonized wood slices; S3. Zinc acetate dihydrate ethanol solution is added dropwise on the carbonized wood slices, the dropwise surface is defined as the front surface, and the carbonized wood slices are dried in an oven; S4. The carbonized wood slices obtained in step S3 are calcined, and then taken out after cooling; S5. An aqueous solution containing polyethyleneimine, zinc nitrate hexahydrate and hexamethylenetetramine is prepared to obtain a precursor solution; S6. The front surface of the carbonized wood slices obtained in step S4 is inverted and suspended on the surface of the precursor solution, and heated; S7. The carbonized wood slices are taken out, washed, dried, subjected to secondary calcination, and then taken out after cooling to room temperature; after repeating steps S5 and S6, carbonized wood-zinc oxide nanowires are obtained; S8. 2-dimethylimidazole is preheated at the bottom of a glass container, then the carbonized wood-zinc oxide nanowires are hung above the 2-dimethylimidazole, heated at 100-120℃ for 5-10min, taken out and cooled to room temperature, washed and dried to obtain carbonized wood-zinc oxide nanowires-ZIF-8; S9. A conductive carbon paste is coated on the working electrode area of a screen-printed electrode, and the carbonized wood-zinc oxide nanowires-ZIF-8 is pasted, solidified, and then immersed in deionized water to obtain the electrochemical sensor for exocytosis monitoring.

2. The electrochemical sensor for exocytosis monitoring according to claim 1, wherein: In step S1, the carbonization treatment is carried out at a temperature of 700-1000℃ for 1-5h under inert gas protection.

3. The electrochemical sensor for exocytosis monitoring according to claim 1, wherein: In step S3, the concentration of zinc acetate dihydrate ethanol solution is 1.111mg / mL.

4. The electrochemical sensor for exocytosis monitoring according to claim 1, wherein: In step S4, the calcination temperature is 300-400℃, and the time is 10-30min.

5. The electrochemical sensor for exocytosis monitoring according to claim 1, wherein: In step S5, the concentration of polyethyleneimine in the precursor solution is 0.00387g / mL, the concentration of zinc nitrate hexahydrate is 0.008g / mL, and the concentration of hexamethylenetetramine is 0.0035g / mL.

6. Use of an electrochemical sensor for exocytosis monitoring according to claim 1 for monitoring real-time exocytosis of cells, characterized in that, The method comprises 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 exocytosis monitoring; (3) placing the electrochemical sensor for exocytosis monitoring in a Faraday cage and connecting it with an electrochemical workstation; (4) adding a to-be-tested liquid drop to the working area of the electrochemical sensor for exocytosis monitoring to fully cover the printed cell working electrode, reference electrode and counter electrode; (5) starting monitoring by amperometry: setting the monitoring potential to 0.34V, the sampling interval to 0.012s, and the duration to 300s; (6) after 300s of testing, terminating the experiment; (7) The number and area of current spikes on the test curve were counted, and the number of dopamine molecules released in the exocytosis event was calculated using the Faraday formula.

Images