Electrochemical sensor for monitoring beta cell insulin secretion in real time as well as preparation method and application of electrochemical sensor

Abstract

The invention relates to an electrochemical sensor for monitoring beta cell insulin secretion in real time and a preparation method and application thereof, the sensor is an electrochemical chip modified by a multi-dimensional nano material, and the multi-dimensional nano material on the electrochemical chip is used for capturing dopamine released by beta cells; and determining the release quantity of dopamine according to the electrochemical signal. According to the method, the insulin secretion condition of the beta cells is monitored in real time by taking dopamine as a tracer agent, and the exocytosis process of the beta cells can be researched under natural and normal conditions. According to the invention, only a common Faraday cage and a basic electrochemical workstation are needed for detection, one or more beta cells can be detected, the operation is simple, and the research efficiency of insulin secretion is greatly improved. In addition, the medicine for regulating beta cell insulin secretion can be efficiently screened, and a foundation is laid for research of diabetes treatment medicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to an electrochemical sensor for real-time monitoring of insulin secretion in beta cells, and a preparation method and application thereof. Background Art

[0002] Insulin is a pancreatic hormone secreted through exocytosis by pancreatic beta cells. Its function is to regulate blood sugar levels and maintain normal metabolism. Abnormal pancreatic beta cell function leads to decreased insulin secretion, which can lead to diabetes and other diseases, seriously affecting human health. Therefore, methods for the real-time quantitative observation of insulin in living cells will play a key role in diabetes drug screening and efficacy evaluation.

[0003] Currently, the main methods for detecting insulin secretion by β cells are: (1) enzyme-linked immunosorbent assay (ELISA), which uses insulin-specific antibodies for sandwich detection and quantification through enzyme-labeled signals; (2) electrochemiluminescence immunoassay (ECLIA), which combines electrochemiluminescence labeling with immune reaction to detect luminescent signals. However, the above two methods can only detect the concentration of insulin secreted by cells into the supernatant and cannot observe the process and behavior of pancreatic islet cells secreting insulin through exocytosis in real time; (3) electrochemical methods, which are mainly based on the interaction between insulin and electroactive substances or the construction of specific recognition elements, and indicate the presence and concentration of insulin by measuring changes in electrical signals on the electrode surface. For example, carbon fiber electrodes (CFE) can study the real-time secretion of insulin by β cells by directly oxidizing insulin. However, using CFE to directly oxidize insulin requires complex electrode modification and has limited sensitivity. CFE operation also requires advanced equipment, a dedicated electromagnetic shielding room, and professional operators. Only one cell can be detected at a time, and efficiency needs to be improved. (4) Serotonin (5-HT) is used as a tracer to monitor insulin release from β cells. However, the tracer 5-HT is present in relatively low concentrations in β cells, requiring the cells to absorb a large amount of 5-HT in advance. This process may inhibit the normal secretion of insulin, leading to inaccurate results.

[0004] In addition, current detection methods still have technical bottlenecks that make it difficult to achieve real-time and dynamic monitoring of insulin secretion in beta cells. For these reasons, there is an urgent need for a method that is simple to operate, real-time, rapid and sensitive to monitor insulin secretion. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an electrochemical sensor for real-time monitoring of insulin secretion from β cells, as well as its preparation method and application. The electrochemical sensor uses multidimensional nanomaterials with synergistic effects to highly sensitively capture dopamine released by β cells and indicate the secretion of insulin in real time.

[0006] The present invention provides an electrochemical sensor for real-time monitoring of insulin secretion from β cells. The sensor is an electrochemical chip modified with multidimensional nanomaterials. The multidimensional nanomaterials on the electrochemical chip are used to capture dopamine released by β cells, and the amount of dopamine released is determined based on the electrochemical signal.

[0007] The β cells release dopamine and insulin synchronously, and the amount of insulin secreted by the β cells is indicated according to the amount of dopamine released, so as to achieve real-time monitoring of the secretion of insulin by the β cells using dopamine as a tracer.

[0008] Preferably, the multidimensional nanomaterial is MNG-1 material; the MNG-1 material is obtained based on a solvent-free chemical vapor deposition method, wherein 2-methylimidazole vapor and ZnO-on-rGO nanomaterial are heated to react at 100-110°C for 1-10 minutes to form a zeolite imidazolate skeleton-8 on the surface of the ZnO-on-rGO nanomaterial.

[0009] Preferably, the preparation method of the ZnO-on-rGO nanomaterial comprises:

[0010] (a) A single layer of graphene oxide was loaded on the surface of a silicon wafer to prepare rGO nanosheets;

[0011] (b) loading zinc oxide nanodots on the rGO nanosheets by calcination to prepare a silicon wafer;

[0012] (c) The silicon wafer is placed in a mixed solution of zinc nitrate and hexamethylenetetramine for hydrothermal reaction to obtain ZnO-on-rGO nanomaterials.

[0013] Preferably, the calcination in step (b) is carried out at 350-400° C. for 20-40 min.

[0014] Preferably, the molar ratio of zinc nitrate to hexamethylenetetramine in step (c) is 1:1.

[0015] Preferably, the hydrothermal reaction temperature in step (c) is 80-100° C. and the reaction time is 3-6 h.

[0016] The present invention also provides a method for preparing an electrochemical sensor for real-time monitoring of insulin secretion in β cells, comprising the following steps:

[0017] The multidimensional nanomaterial is dispersed in a mixed solvent system to form a homogeneous dispersion; the dispersion is evenly coated on the working electrode area of ​​the electrochemical sensor chip, and the chip is allowed to stand at room temperature until the solvent evaporates naturally.

[0018] Preferably, the mass-to-volume ratio of the multidimensional nanomaterial and the mixed solvent system is 1 mg:100-120 μL.

[0019] Preferably, the mixed solvent system is formed by mixing ethanol and Nafion solution in a volume ratio of 5-10:1.

[0020] Preferably, the concentration of the Nafion solution is 1 wt%-10 wt%.

[0021] The present invention also provides an application of an electrochemical sensor for real-time monitoring of insulin secretion from beta cells in screening drugs for regulating insulin secretion from beta cells.

[0022] The present invention also provides an application of an electrochemical sensor for real-time monitoring of insulin secretion by beta cells in screening drugs for treating diabetes.

[0023] The present invention also provides an application of an electrochemical sensor for real-time monitoring of insulin secretion in beta cells in the preparation of a product for evaluating the regulatory effect of a drug on the secretory function of beta cells.

[0024] The present invention uses an electrochemical sensor based on MNG-1 multidimensional nanomaterial (abbreviated as MNG-1 material) to monitor insulin secretion by β cells in real time. Figure 1 Although insulin is not electrochemically active, its release occurs simultaneously with the secretion of dopamine molecules, which are easily oxidized. The MNG-1 material is composed of zeolitic imidazolate framework-8 (ZIF-8), which can efficiently catalyze dopamine, ZnO nanowires that are in close contact with cells in multiple directions, and reduced graphene oxide with excellent conductive properties. The synergistic effect of this material can efficiently capture the release of exocytotic dopamine and indirectly reflect the secretion of insulin based on the electrochemical signal generated by dopamine oxidation.

[0025] Beneficial effects

[0026] (1) The multidimensional nanomaterial used in the present invention, namely the MNG-1 material, is composed of ZIF-8, ZnO nanowires, and reduced graphene oxide nanosheets. The multidimensional nanostructure has a synergistic effect on the detection of dopamine secreted by living cells in real time: ① The ZIF-8 shell acts as a solid base catalyst, which can effectively oxidize the neurotransmitter dopamine released during cell exocytosis, promoting electron transfer and thus achieving electrochemical sensing. ② The arrayed ZnO nanowires are conducive to comprehensively capturing the occurrence of exocytosis events from multiple directions, and the ZnO nanowires also provide a zinc source for the growth of ZIF-8. ③ The reduced graphene oxide nanosheets have excellent electrical conductivity, which is conducive to the transmission of electrical signals.

[0027] (2) The present invention utilizes a single-cell printing system to precisely load the desired number of cells onto the MNG-1 material interface. After printing, the cells spontaneously adhere to the MNG-1 material surface, with the cell membrane forming close contact with the tentacle-like structures of the ZnO nanowires. This method, which enables the loading of cells onto the chip and their close contact with the catalytic material without requiring a sophisticated micromanipulation system, achieves efficient and rapid sample preparation, resolving the cumbersome and time-consuming nature of single-cell analysis.

[0028] (3) β cells release catecholamine neurotransmitters such as dopamine while secreting insulin through exocytosis. Insulin is difficult to detect directly through electrochemical oxidation, while these catecholamine neurotransmitters are easily oxidized to produce electrochemical signals due to their catechol structure. Therefore, the present invention uses dopamine as a tracer to overcome the drawbacks of using 5-HT as a tracer to detect insulin secretion through β cell exocytosis. This eliminates the need for cells to take up additional insulin in advance, and allows the study of β cell exocytosis under natural, normal conditions.

[0029] (4) The present invention combines an MNG-1-modified sensor chip with a multidimensional nanomaterial having a synergistic effect to highly sensitively capture dopamine released by β cells, thereby indicating insulin secretion in real time. The method for preparing the MNG-1-modified sensor chip is simple and easy to implement. The method based on the chip, which uses dopamine as a tracer to monitor the exocytosis and secretion of insulin by β cells in real time, only requires an ordinary Faraday cage and a basic electrochemical workstation for detection. It can detect single or multiple cells, is simple to operate, and greatly improves the efficiency of research on the insulin secretion behavior of β cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the working principle of the sensor of the present invention for real-time monitoring of insulin secretion by β cells.

[0031] Figure 2 ad are scanning electron microscope (SEM) images of MNG-1 material at different magnifications.

[0032] Figure 3 are the elemental analysis results of MNG-1 materials; ab are SEM images of ZIF-8 coated ZnO nanowires at different magnifications, and c is the EDS element mapping result.

[0033] Figure 4 Schematic diagram of the sensor of the present invention.

[0034] Figure 5 The working electrode of the screen-printed chip was modified with MNG-1 material, and an electrochemical sensor was constructed after loading a precise number of β cells. Its microstructure was observed through SEM images; a shows four β cells loaded on the working electrode, and bd are both single β cells loaded on the working electrode.

[0035] Figure 6 The results of the behavioral events of dopamine released by living β cells are measured; a is the amperometric test curve of different β cell numbers; b is an amplification of a peak in graph a; c is the ELISA test result; d is the feasibility and effectiveness of the method of the present invention verified using the insulin secretion-promoting drugs gliclazide and tolbutamide.

[0036] Figure 7 Figure 3. Real-time electrochemical monitoring of dopamine exocytosis in β-cells after treatment with different concentrations of melatonin. a is the amperometric curve, b is the number of exocytotic events, and c is the average number of dopamine molecules released during exocytotic events. n = 13 independent measurements. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns indicates no significant difference. DETAILED DESCRIPTION

[0037] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0038] Example 1

[0039] 1. Synthesis of MNG-1 material

[0040] 1.1 Preparation of ZnO-on-rGO

[0041] (1) A 1 cm × 1 cm silicon wafer was used as the substrate, and then 25 μL of a single-layer reduced graphene oxide (rGO) dispersion (2 mg / mL) was evenly drop-coated on the surface of the silicon wafer. After drying, the process was repeated three times to obtain rGO nanosheets.

[0042] (2) To grow ZnO nanowire arrays on the surface of rGO nanosheets, a 5 mM ZnO nanodot dispersion was used as a seed. Zn(CH3COO)2·2H2O was mixed with an ethanol solution to prepare a 5 mM solution. Ultrasonic treatment was performed for 5 minutes to obtain a uniformly dispersed solution, namely the ZnO nanodot dispersion. The ZnO nanodot dispersion was then drop-coated on the rGO nanosheets prepared in step (1) and placed in an oven until the solvent was fully evaporated. This was repeated four times.

[0043] (3) Subsequently, the silicon wafer was placed in a tube furnace and treated at 350 °C for 20 min, and the ZnO seeds could be firmly fixed on the surface of the rGO nanosheets.

[0044] (4) The silicon wafer was suspended face down in a stock solution containing 25 mM Zn(NO3)2 and 25 mM hexamethylenetetramine (molar ratio 1:1) and subjected to a hydrothermal reaction in a constant temperature vacuum drying oven at 90 °C for 3-6 h to obtain the ZnO-on-rGO sample.

[0045] 1.2 Synthesis of MNG-1 Material

[0046] 0.5 mg of 2-methylimidazole was placed in a 50 mL polypropylene reaction bottle and preheated at 110 ° C for 30 minutes. Subsequently, the ZnO-on-rGO sample was suspended in the above polypropylene bottle, ensuring that the position of the ZnO-on-rGO sample was 5 cm higher than the upper surface of the 2-methylimidazole reagent, and then heated at 110 ° C for 5 minutes. In a sealed reaction bottle, 2-methylimidazole vapor was reacted with ZnO by solvent-free chemical vapor deposition (CVD) to form zeolitic imidazolate framework-8 (ZIF-8) on the surface. After the reaction is complete, the sample is removed from the reaction bottle and rinsed with ethanol to remove residual reactants.

[0047] 1.3 Characterization results

[0048] The vertical growth of ZnO nanowires on both sides of graphene nanosheets was successfully achieved by hydrothermal method. Figure 2 As shown, SEM images show that these nanowires exhibit excellent order and orientation, forming a uniform and dense vertical array with an average diameter of about 50 nm and an average length of about 1.3 μm. Figure 2 Figures ad show the microstructural features of the MNG-1 material at different magnifications. The blanket-like MNG-1 material is a complex composite structure consisting of one-dimensional ZnO nanowire arrays, two-dimensional graphene oxide (rGO) nanosheets, and three-dimensional ZIF-8 nanoshells. This arrangement ensures effective exposure of the nanowires on the substrate and provides space for interconnection between the nanowires. At higher magnifications ( Figure 2d), the morphological characteristics of a single ZnO nanowire were observed. At this time, the nanowire had a certain regularity and the surface energy could be preliminarily observed to be wrapped by a layer of ZIF-8 nanoshell.

[0049] The SEM images and corresponding EDS element mapping results of the in situ growth of ZIF-8 metal organic framework (MOFs) coating on the surface of ZnO nanowires by CVD are shown in Figure 3 .like Figure 3 As shown in ab, SEM images reveal the changes in the surface morphology of ZIF-8 after coating ZnO nanowires. Compared with unmodified ZnO nanowires, it can be observed that some colloidal substances are wrapped on the surface of the nanowires. ZIF-8 tends to attach to the ZnO nanowires in the form of thin layers or particles, significantly increasing the specific surface area of ​​the material and providing more active sites for the subsequent oxidation reaction of dopamine. Figure 3 EDS elemental mapping results, shown in Figure c, further confirm the uniform distribution of ZIF-8 on the ZnO nanowire surface. The Zn and O elements in the figure correspond to the distribution of ZnO, while the C and N elements reflect the presence of ZIF-8. Although the N content is low, its uniform distribution indicates that 2-methylimidazole has been successfully introduced and the ZIF-8 structure has been effectively formed on the nanowire surface. This provides strong support for subsequent electrochemical sensing of dopamine.

[0050] 2. Cell culture

[0051] INS-1E cells are a cell line derived from rat islet cell tumors and are widely used in the study of pancreatic beta cell function. The INS-1E cells used in the present invention were purchased from the Cell Bank / Stem Cell of the Chinese Academy of Sciences (derived from ATCC). Cell culture was performed using Roswell Park Memorial Institute 1640 culture medium supplemented with 10% FBS, 100mM sodium pyruvate, 5mM 2-mercaptoethanol, 200mM Glutamine, 1M HEPES buffer, and 1% penicillin-streptomycin mixture. Cell culture was performed in a 37°C constant temperature environment with 95% air humidity and 5% CO2, and passaged at a ratio of 1:2. Fresh culture medium was replaced 2-3 times a week, and cell viability (>95%) was regularly monitored by trypan blue staining.

[0052] 3. Preparation of MNG-1 modified electrochemical sensor

[0053] Screen-printed electrodes (SPE) typically utilize a three-electrode system consisting of a working electrode, a counter electrode, and a reference electrode. First, the reference electrode is printed using silver / silver chloride ink. Next, the working and counter electrodes are printed sequentially using carbon paste. Then, silver paste is used to create conductive circuits to ensure efficient transmission of electrical signals. Finally, an insulating paste is applied to form a protective layer, isolating the electrode material from air, thereby reducing the risk of oxidation and improving detection reliability.

[0054] Subsequently, the MNG-1 material was peeled off from the silicon wafer and accurately weighed. Then, 1.0 mg of sample was taken after accurate weighing (accuracy ± 0.1 mg); it was then dispersed in 112.5 μL of the mixed solvent system (V 乙醇 :V Nafion溶液 (5wt%) = 8:1) and ultrasonically treated (40kHz, 30min) to form a homogeneous dispersion. Subsequently, 5μL of the dispersion was accurately taken with a micropipette (accuracy ±0.1μL) and evenly coated on the working electrode area of ​​the sensor chip. After standing at room temperature for the solvent to evaporate naturally, the functionalized electrode (i.e., the MNG-1 modified electrochemical sensor, see Figure 4 ) can be directly used for electrochemical detection.

[0055] 4. Use a single-cell printer to load cells onto the sensor

[0056] Using Orei Technologies' single-cell printing system (SCP4000), equipped with a biojet printing chip B150 manufactured using a standard CMOS-MEMS process, the β cells were centrifuged (1200 rpm, 5 min) and the supernatant removed. The cells were then resuspended in 1 mL of PBS buffer containing less than 2% bovine serum to prepare a cell suspension (density approximately 1×10 4 cells / mL). Take 100 μL of suspension and place it in the liquid reservoir of the bioprinter head, let it stand for 60 seconds and wait for the cells to settle. Then, fix the MNG-1 modified sensor chip on the three-dimensional positioning platform to ensure that the working electrode is aligned with the cell landing point. After setting the single cell sedimentation parameters to determine the required number of target cells, the target cells are screened in real time by intelligent image recognition technology, and air pressure pulses are used to achieve precise deposition of single cells or a specific number. After printing, the cells can spontaneously attach to the surface of the MNG-1 material, and the cell membrane forms a close contact with the tentacle-like structure of the ZnO nanowires (see Figure 5 ), thereby achieving efficient detection of exocytosis events.

[0057] 5. Measuring dopamine / insulin release from living beta cells

[0058] First, 10 μL of the test solution was slowly dripped onto the surface of the MNG-1-modified sensor chip with an integrated three-electrode system, onto the working electrode containing the precise number of β cells, ensuring that the test solution completely covered the effective detection area formed by the reference electrode, counter electrode, and working electrode. Subsequently, a CHI660E electrochemical workstation (equipped with a CHI200B Faraday shield system) was used for signal acquisition. This configuration and setup can accurately measure the output current in the pA to nA range, and the current detection range of this system covers 1 pA-10 nA. Under constant potential of 0.38 V, the real-time current signal was continuously recorded for 200 s at a sampling interval of 12 ms. According to Faraday's law, as shown in formula (1), the current peak area is proportional to the amount of dopamine released, which can quantitatively characterize the exocytosis dynamics of dopamine released from β cell vesicles.

[0059] Q=nNF (1)

[0060] In this experiment, Q represents the amount of charge transferred during each exocytosis, N represents the number of dopamine molecules, F is the Faraday constant (96485 C / mol), and n is the number of electrons transferred per oxidized dopamine molecule. Because the MNG-1 material can deeply oxidize dopamine molecules, n is 4.

[0061] 6. Determination of small molecule regulatory effects

[0062] The small molecule solution system required for the test contains 80mM K + with 2 mM Ca 2+ PBS buffer (pH = 7.4) was used as the base medium. Subsequently, the target number of β cells was screened using image recognition technology and accurately printed onto the sensing electrode, and 10 μL of the test solution was slowly added dropwise. The amperometric current-time curve was recorded at a constant potential of 0.38 V using a CHI660E electrochemical workstation, with a sampling interval of 12 ms and a continuous acquisition time of 200 s. To ensure sufficient statistical evaluation of the stimulatory effect of the test substance, the test was repeated 13 times for each test substance concentration.

[0063] 7. Statistical analysis

[0064] Origin software was used for signal processing and visualization analysis. The criterion for determining an effective exocytosis event was that the signal-to-noise ratio (SNR) of the peak signal exceeded three times, where the baseline noise was calculated from the pre-acquisition period (the first 20 seconds). To eliminate the interference of multiple events, the half-height width (t 1 / 2 , defined as I maxNonspecific spike data greater than 20 ms (width at 50% of the mean) were included. Data are presented using a Tukey boxplot format: boxes represent the 25%–75% interquartile range, the centerline marks the median, dashed lines extend to 1.5 times the interquartile range (IQR), and discrete points represent outliers. Statistical analysis was performed using GraphPad Prism and assessed by a two-tailed unpaired t-test. Differences were considered significant when P < 0.05.

[0065] 8. Experimental Results

[0066] 8.1 As Figure 6 As shown in Figure a, it shows the results of real-time electrochemical monitoring of exocytosis events of different numbers of β cells (1, 10, and 100). In order to induce β cell exocytosis, the present invention added 80mM K to the MNG-1 modified sensor chip during the electrochemical test. + and 2 mM Ca 2+ The solution is used to induce cell membrane depolarization, activate ion channels, and stimulate the fusion of vesicles containing dopamine / insulin with the cell membrane. This method utilizes the electrophysiological properties of the cell membrane when stimulated and can efficiently capture the cell exocytosis process. As the number of β cells increases, the number of spikes in the electrochemical signal also increases significantly. This confirms that the MNG-1 modified sensor chip designed by the present invention can be used to study β cell exocytosis events. The electrochemical signal of one β cell is relatively stable, with only sporadic spikes. The number of spikes in the electrochemical signal of 10 β cells increases significantly, and the electrochemical signal of 100 β cells shows more spikes.

[0067] like Figure 6 As shown in b, it is Figure 6 A magnified image of a single peak recorded in (a) can be used to calculate the amount of dopamine released during a single exocytosis event. By integrating a single peak, the charge (Q) proportional to the amount of dopamine released can be obtained. The number of dopamine molecules released during a single exocytosis event can then be calculated using Equation (1), allowing for quantitative analysis of exocytosis events. The gray peak area represents the number of released dopamine molecules, a crucial parameter for studying exocytosis events.

[0068] 8.2 Enzyme-linked immunosorbent assay (ELISA) is a classic quantitative analysis method that is often used to detect small molecules secreted into the supernatant by living cells. The present invention uses a commercial ELISA kit (Wuhan Huamei Bioengineering Co., Ltd.) to detect + and 2 mM Ca 2+After solution treatment, the levels of dopamine and insulin secreted by different numbers of cells (references for insulin detection methods: doi.org / 10.1016 / j.toxrep.2024.101742, doi.org / 10.1159 / 000443896).

[0069] The results are as follows Figure 6 As shown in Figure c, ELISA can be used to measure dopamine and insulin secretion levels at different β-cell numbers. Due to the limited sensitivity of ELISA, it is difficult to detect secretion from a single cell; at least 10 cells are required to reach the detection threshold. With increasing β-cell number (the horizontal axis represents the log10 value of the cell number), the secretion of both insulin and dopamine increases synchronously, with consistent trends. This trend is consistent with the electrochemical detection results, further confirming the co-release of dopamine and insulin.

[0070] Although ELISA is a commonly used method for insulin detection, it lacks single-cell testing capabilities and requires offline analysis of cell culture supernatants, making it impossible to monitor exocytosis events in real time. To overcome these limitations, the present invention constructs a sensor chip modified with MNG-1 material. This chip is expected to simultaneously monitor dopamine and insulin exocytosis events in real time through electrochemical methods, thereby addressing the shortcomings of ELISA and providing a new tool for drug effect research. By analyzing changes in β-cell exocytosis events before and after drug treatment, the effects of drugs on β-cell secretory function can be assessed, providing a new approach for drug development for diseases such as diabetes.

[0071] 8.3 To validate the effectiveness of the method of the present invention, changes in exocytosis events were observed by real-time electrochemical monitoring after treating β cells with gliclazide and tolbutamide, two widely used insulin secretagogues in clinical practice. This study observed exocytosis behavior after treating β cells with these two drugs. The specific procedures were:

[0072] The drug solution system required for the test contains 80mM K + with 2 mM Ca 2+ PBS buffer (pH = 7.4) was used as the base medium. Gliclazide and tolbutamide standard solutions were prepared into 10 μM test solutions using the same solvent system (because they are difficult to dissolve in water, DMSO was added for solubilization, and the final DMSO concentration was ≤0.8%). β-cells containing the target number were screened using image recognition technology and accurately printed onto the sensor electrode, and 10 μL of the test solution (gliclazide or tolbutamide) was slowly added dropwise. The current-time curve was recorded at a constant potential of 0.38 V using a CHI660E electrochemical workstation, with a sampling interval of 12 ms and continuous acquisition for 200 s.

[0073] result Figure 6 As shown in Figure d, compared with the control group (no drug added), the exocytosis frequency of β cells treated with gliclazide and tolbutamide was significantly increased. At the clinically recommended dose, both gliclazide and tolbutamide significantly increased the exocytosis frequency of β cells, indicating that both drugs can effectively promote insulin release from β cells. The number of spikes in the electrochemical signal of β cells treated with gliclazide and tolbutamide increased significantly. The experimental results confirmed that both drugs studied can significantly enhance insulin secretion activity. This experiment also further verified the effectiveness and feasibility of using dopamine as a "tracer" to explore the insulin release process of β cells.

[0074] Example 2

[0075] After verifying the effectiveness and feasibility of the sensor chip, this study further utilized it to investigate the regulatory effects of different concentrations of melatonin on β-cell insulin secretion. In today's fast-paced modern lifestyle, public awareness of health and wellness is increasing. This trend has fueled the booming health food industry, with melatonin, a widely sought-after ingredient, gaining widespread recognition and acceptance. Insomnia and circadian rhythm disruption are common health issues worldwide, primarily due to decreased endogenous melatonin levels. Diabetes often presents with severe insomnia, leading to widespread use of melatonin in this population. Further investigation of its potential insulin-regulating effects is of clinical importance. Therefore, this study focused on melatonin, investigating its dose-dependent effects on β-cell insulin secretion. Melatonin is an indoleamine hormone secreted by the pineal gland, whose primary physiological function is to regulate the sleep-wake cycle and circadian rhythm. As a dietary supplement, melatonin is widely used to improve sleep quality and alleviate insomnia, and its market size continues to grow. Studies have shown that melatonin receptors exist in pancreatic β-cells, suggesting that melatonin has a potential regulatory effect on insulin secretion. Clinical epidemiological studies have shown that decreased melatonin secretion is significantly associated with the risk of developing type 2 diabetes. Furthermore, experimental studies have confirmed that melatonin has a protective effect on β-cells and may have potential benefits in the treatment of metabolic syndrome. Taken together, these findings suggest that melatonin's regulatory effects on insulin may provide a new strategy for the treatment of diabetes. However, existing studies are largely based on indirect evidence, such as molecular biology or epidemiology, and there is a lack of direct studies examining the regulatory effects of melatonin dose on β-cell insulin secretion to quantitatively reveal its direct effects on insulin secretion.

[0076] Methods: After treating β cells with different concentrations of melatonin, dopamine exocytosis was monitored by real-time electrochemical monitoring. The specific procedures were as follows:

[0077] Using 80mM K + with 2 mM Ca 2+ Phosphate-buffered saline (PBS, pH = 7.4) was used as the base medium for preparing the drug solution system required for testing. Melatonin solution: Given the low solubility of melatonin in water (<2 mg / mL), 0.8% (v / v) dimethyl sulfoxide (DMSO) was initially used as a cosolvent when preparing the melatonin solution. Subsequently, melatonin was dissolved in PBS buffer containing DMSO to prepare a series of concentration gradients: 10 nM, 1 nM, 1 μM, 10 μM, 100 μM, and 1 mM. Beta cells containing the target number were screened using image recognition technology and accurately printed onto the sensing electrode. 10 μL of the test solution (melatonin solutions of varying concentrations) was then slowly added dropwise. Current-time curves were recorded at a constant potential of 0.38 V using a CHI660E electrochemical workstation, with a sampling interval of 12 ms and a continuous acquisition time of 200 s. To ensure statistically adequate evaluation of the stimulatory effect of the test substance, the test was repeated 13 times for each test concentration.

[0078] Results: As Figure 7 As shown in Figure a, beta cells treated with different concentrations of melatonin showed significant differences in spike frequency compared to the control group (no melatonin). Low-concentration melatonin (10nM, 100nM, 1μM): Within the concentration range of 10nM to 1μM, melatonin showed a moderate promoting effect on the spike frequency of beta cells. In particular, at concentrations of 100nM and 1μM, a significant increase in spike frequency was observed. High-concentration melatonin (10μM, 100μM, 1mM): As the melatonin concentration increased, its promoting effect on the exocytosis frequency of beta cells gradually weakened, ultimately reaching a level comparable to that of the control group. Figure 7 b shows the statistical results of the frequency of exocytosis events of 10 β cells under different concentrations of melatonin treatment within 200 seconds in the form of a box plot. Figure 7 Further analysis and summary of the real-time electrochemical monitoring data shown in (a) aim to more clearly demonstrate the quantitative effects of different melatonin concentrations on β-cell exocytosis frequency. The boxplot clearly shows the distribution of exocytosis events in the different melatonin treatment groups, including the median, upper and lower quartiles, maximum, minimum, and outliers.

[0079] Statistical analysis results showed that there were significant differences in the frequency of exocytosis events among groups treated with melatonin at different concentrations. Compared with the control group, the 10nM (P<0.01), 100nM (P<0.001), and 1μM (P<0.0001) melatonin-treated groups all showed a significant increase in exocytosis frequency, indicating that melatonin can effectively promote insulin release from β cells within a lower concentration range. In particular, the 1μM group showed a very significant promoting effect. When the melatonin concentration increased to 10μM, 100μM, and 1mM, its promoting effect on the frequency of β cell exocytosis gradually weakened until there was no significant difference compared with the control group (ns), indicating that higher concentrations of melatonin neither promoted nor inhibited insulin secretion.

[0080] like Figure 7 As shown in Figure c, melatonin can increase the amount of dopamine released by exocytosis within a specific concentration range. Compared with the control group, the amount of dopamine released in the 0.1μM and 1μM melatonin treatment groups was significantly increased, indicating that melatonin promotes greater neurotransmitter release from β cells within this concentration range. However, at an excessively high concentration (1mM), the average number of dopamine molecules released by β cell exocytosis was not significantly different from that in the control group (ns), indicating that the promoting effect of melatonin disappears.

[0081] The above results indicate that the method of the present invention, which uses dopamine as a tracer to monitor insulin secretion by β-cell exocytosis, not only achieves real-time monitoring of insulin secretion but also can detect single to multiple cells, is simple to operate, and greatly improves the efficiency of insulin secretion research. Furthermore, it can also be used to evaluate the effects of drugs on insulin secretion by β-cell exocytosis and to screen new drugs that regulate insulin secretion by β-cell exocytosis, thereby providing a new means for the research and screening of diabetes treatment drugs.

[0082] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An electrochemical sensor for real-time monitoring of insulin secretion in β cells, characterized by: The sensor is an electrochemical chip modified with multidimensional nanomaterials. The multidimensional nanomaterials on the electrochemical chip are used to capture dopamine released by β cells, and the amount of dopamine released is determined based on electrochemical signals.

2. The electrochemical sensor for real-time monitoring of insulin secretion in β cells according to claim 1, characterized in that: The multidimensional nanomaterial is an MNG-1 material; the MNG-1 material is obtained based on a solvent-free chemical vapor deposition method, wherein 2-methylimidazole vapor and ZnO-on-rGO nanomaterial are heated to react at 100-110° C. for 1-10 minutes to generate a zeolite imidazolate skeleton-8 on the surface of the ZnO-on-rGO nanomaterial.

3. The electrochemical sensor for real-time monitoring of insulin secretion in β cells according to claim 1, characterized in that: The preparation method of the ZnO-on-rGO nanomaterial comprises: (a) A single layer of graphene oxide was loaded on the surface of a silicon wafer to prepare rGO nanosheets; (b) loading zinc oxide nanodots on the rGO nanosheets by calcination to prepare a silicon wafer; (c) The silicon wafer is placed in a mixed solution of zinc nitrate and hexamethylenetetramine for hydrothermal reaction to obtain ZnO-on-rGO nanomaterials.

4. A method for preparing an electrochemical sensor for real-time monitoring of insulin secretion in β cells according to any one of claims 1 to 3, comprising the following steps: The multidimensional nanomaterial is dispersed in a mixed solvent system to form a homogeneous dispersion; the dispersion is evenly coated on the working electrode area of ​​the electrochemical sensor chip, and the chip is allowed to stand at room temperature until the solvent evaporates naturally.

5. The preparation method according to claim 4, characterized in that: The mass-to-volume ratio of the multidimensional nanomaterial and the mixed solvent system is 1 mg:100-120 μL.

6. The preparation method according to claim 4 or 5, characterized in that: The mixed solvent system is formed by mixing ethanol and Nafion solution in a volume ratio of 5-10:

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7. The preparation method according to claim 6, characterized in that: The concentration of the Nafion solution is 1 wt%-10 wt%.

8. Use of the electrochemical sensor for real-time monitoring of insulin secretion from β cells according to any one of claims 1 to 3 in screening drugs that regulate insulin secretion from β cells.

9. Use of the electrochemical sensor for real-time monitoring of insulin secretion from β cells according to any one of claims 1 to 3 in screening drugs for treating diabetes.

10. Use of the electrochemical sensor for real-time monitoring of insulin secretion in β cells according to any one of claims 1 to 3 in the preparation of a product for evaluating the regulatory effect of a drug on the secretory function of β cells.

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