MBAzy-based nanohybrid nanoszyme, electrode system, flexible sensor and detection method

Abstract

The application belongs to the technical field of electrochemical detection and analysis of stress biomarkers, and relates to an MBAzy heterojunction nanoscale enzyme, an electrode system, a flexible sensor and a detection method. 4 / 3 The MBAzy heterojunction nanoscale enzyme is a composite material of a single-atom nanoscale enzyme CuPC and MBene, the MBene is Mo B2MBene, and the mass ratio of the CuPC to the MBene is 10-20:1.The MBAzy heterojunction nanoscale enzyme can catalyze dopamine to generate electrons and cause an electrochemical reaction. The method based on the MBAzy heterojunction nanoscale enzyme can sensitively detect dopamine in a life flowing body.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemical detection and analysis of stress biomarkers, and specifically relates to a MBAzy heterojunction nanoscale enzyme, a modified electrode based on the MBAzy heterojunction nanoscale enzyme, an electrode system comprising the modified electrode, a flexible sensor formed by the electrode system, and a method for detecting the content of dopamine by using the flexible sensor. BACKGROUND

[0002] Dopamine (DA) is an important monoamine neurotransmitter, which is released by dopaminergic neurons, projecting throughout the central nervous system, controlling motor coordination, mood, cognition, and reward-driven behavior. In addition, dopamine, as an important immunoregulatory factor, plays an important role in diseases such as Parkinson's disease, neuropsychiatric diseases, nervous system HIV, inflammatory bowel disease, and rheumatoid arthritis. In order to explain the uncertainty of the physiological relevant concentration of dopamine, in vitro examination of dopamine should use a concentration close to the level of dopamine to which the studied cells may be exposed. In most tissues, this range is about 10 -5 M to 10 -11 M. Insufficient dopamine secretion can cause extracerebral vasodilation, attention deficit hyperactivity disorder (ADHD), Lewy body dementia, and Huntington's disease, while too high a level of dopamine is associated with brain cancer such as neuroblastoma. Therefore, the synthesis, release, structural changes, oxidation, and even single-molecule measurement of DA are of great significance to the study of its function.

[0003] Currently, techniques for effectively quantifying in vivo basal extracellular levels of dopamine can be divided into three types: (1) microdialysis, (2) imaging, and (3) electrochemical techniques. Microdialysis is usually used to measure neurochemicals, but it relies on non-real-time external analysis techniques and has a large probe that can easily cause inflammatory damage, affecting accurate sampling and being unsuitable for long-term human studies. The latest progress in dopamine detection has mainly focused on imaging and electrochemical techniques. Although functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) have been clinically applied and have the advantage of non-invasiveness, they currently cannot directly detect dopamine levels and have poor spatial resolution (greater than 1 millimeter), and the cost-effectiveness is much lower than that of electrochemical techniques. Therefore, many studies have focused on developing new electrochemical methods to detect dopamine in biological flow systems using their high spatial and temporal resolution. SUMMARY

[0004] The purpose of the present application is to provide a MBAzy heterojunction nanoscale enzyme, a flexible sensor based on a three-electrode system modified by the MBAzy heterojunction nanoscale enzyme, and a method for detecting dopamine by using the flexible sensor. The method can sensitively, non-invasively, and rapidly detect the content of dopamine in a life flow.

[0005] The first aspect of the present application provides an MBAzy heterojunction nanoscale enzyme, which is a composite material of single-atom nanoscale enzyme CuPC and MBene, wherein the MBene is Mo 4 / 3 B2 MBene, and the mass ratio of CuPC to MBene is 10-20:1.

[0006] The present application first uses single-atom nanoscale enzyme CuPC to synthesize MBAzy heterojunction nanoscale enzyme with MBene. The MBAzy heterojunction nanoscale enzyme can be prepared by conventional methods in the art.

[0007] According to one specific embodiment of the present application, the above-mentioned MBAzy heterojunction nanoscale enzyme can be prepared by the following method:

[0008] (1) mixing Mo, Y, Al, B powder mixture to perform solid phase sintering to obtain (Mo 2 / 3 Y 1 / 3 )2AlB2 powder; contacting (Mo 2 / 3Y 1 / 3 )2AlB2 powder with hydrofluoric acid aqueous solution to perform etching, and centrifuging, washing the obtained etching product until the supernatant is neutral, and then freeze-drying to obtain multi-layer Mo 4 / 3 B2 MBene powder, and further layering to obtain two-dimensional Mo 4 / 3 B2 MBene;

[0009] (2) slowly adding CuPC ethanol dispersion liquid into the solution of Mo 4 / 3 B2 MBene obtained in step (1), ultrasonic dispersion to mix uniformly, reacting under magnetic stirring, then centrifuging, removing supernatant, washing, collecting precipitate, and vacuum drying to obtain MBAzy heterojunction nanoscale enzyme powder.

[0010] Specifically, in step (1), the conditions of solid phase sintering include inert gas protection, temperature of 1000-1500℃, and time of 400-800min; the conditions of etching include temperature of 33-35℃, and time of 2-6 hours; the conditions of centrifugation include rotation speed of 3000-4000rpm, and time of 5-15min; in step (2), the conditions of reaction include temperature of 20-30℃, and time of 10-16 hours; the conditions of centrifugation include rotation speed of 6000-10000rpm, and time of 3-8min; and the conditions of vacuum drying include temperature of 40-80℃, and time of 2-6 hours.

[0011] The MBAzy heterojunction nanoscale enzyme of the present application comprises CuPC and MBene, because MBene has good electrocatalytic performance and a faster electron transfer rate, the combination of the single-atom nanoscale enzyme CuPC and MBene can simulate enzyme cascade reactions, so that the MBAzy heterojunction nanoscale enzyme has excellent electrochemical catalytic oxidation performance for dopamine, can oxidize dopamine to generate electrons, and then cause a change in current, and thus can be used as a modifier to further prepare an electrode.

[0012] The second aspect of the present application provides a modified electrode based on the MBAzy heterojunction nanoscale enzyme, wherein the electrode is modified with the MBAzy heterojunction nanoscale enzyme described above.

[0013] According to a preferred embodiment of the present application, the electrode material of the modified electrode is a graphene and TiO2 mixed ink, wherein the mass ratio of graphene to TiO2 is 1-5:1, preferably 2.5-3.5:1.

[0014] The method for modifying the MBAzy heterojunction nanoscale enzyme to the electrode can be, for example, drop coating the MBAzy heterojunction nanoscale enzyme solution on the electrode, and then placing it at room temperature for drying. This step can be performed multiple times to achieve the target modification amount of the MBAzy heterojunction nanoscale enzyme.

[0015] The third aspect of the present application provides an electrode system modified based on the MBAzy heterojunction nanoscale enzyme, wherein the electrode system is a three-electrode system or a two-electrode system, and the modified electrode described above is used as the working electrode.

[0016] The detection principle of the present application is that when the target substance is present, the CuPC in the MBAzy heterojunction nanoscale enzyme oxidizes the target substance to dopaminequinone, while generating electrons, thereby causing a change in current. The size of the generated current can be detected by connecting a portable electrochemical workstation. The size of the current value is positively correlated with the concentration of the target substance, and the target substance can be quantitatively detected to make a standard curve. The same method is used to determine the sample to be tested, and the measured current value is substituted into the standard curve to obtain the concentration of dopamine therein.

[0017] Based on the above principles of the present application, a flexible electrode with a two-electrode system or a three-electrode system can be designed. The ink of the electrode can be provided separately or printed on a TPU film through screen printing technology.

[0018] According to a preferred embodiment of the present application, the electrode system is a three-electrode system, which comprises a working electrode, a reference electrode and a counter electrode. In addition to the working electrode described above, the system further comprises:

[0019] (i) Reference electrode: Ag / AgCl;

[0020] (ii) the counter electrode: carbon and graphene oxide mixed electrode.

[0021] The present application does not particularly limit the amount of the two materials in the carbon and graphene oxide mixed electrode.

[0022] The fourth aspect of the present application provides a flexible sensor based on the MBAzy nanozyme modified three-electrode system, which comprises the working electrode, the reference electrode and the counter electrode described above in the form of a patch; and the material of the patch is TPU.

[0023] The method for preparing the sensor patch can be a conventional method in the art, such as screen printing technology, which prints the ink of the electrode onto the patch substrate.

[0024] The fifth aspect of the present application provides a sensor based on the MBAzy nanozyme modified three-electrode system, which comprises:

[0025] (1) the MBAzy nanozyme described above;

[0026] (2) the flexible electrode of the three-electrode system;

[0027] (3) the portable electrochemical workstation.

[0028] The MBAzy nanozyme can be packaged independently, and the solution of the MBAzy nanozyme is dropped and coated on the working electrode of the flexible electrode to achieve modification before use, or the MBAzy nanozyme can be pre-modified on the working electrode of the flexible electrode. When packaged independently, the MBAzy nanozyme can be in the form of powder, or the MBAzy nanozyme material can be prepared into a solution with a desired concentration with deionized water and stored at 4℃ for standby use. The flexible electrode of the three-electrode system in the sensor can be a conventional flexible electrode of the three-electrode system in the art, and the present application does not particularly limit it, and preferably, it can be the flexible sensor system described above, that is, it comprises:

[0029] (1) the working electrode: the graphene and TiO2 mixed ink GraphTiO-3 electrode;

[0030] (2) the reference electrode: Ag / AgCl;

[0031] (3) the counter electrode: carbon and graphene oxide mixed electrode.

[0032] The sixth aspect of the present application provides a method for detecting the content of dopamine based on the flexible sensor of the MBAzy nanozyme modified three-electrode system, which comprises:

[0033] (1) obtaining the MBAzy nanozyme described above;

[0034] (2) The flexible sensor is prepared by the following steps: preparing a three-electrode flexible electrode patch by a screen printing technology, and dropping and coating the MBAzy nano-enzyme solution obtained in step (1) on the working electrode of the flexible electrode, and placing and drying at room temperature;

[0035] (3) The electrode modified in step (2) is taken out, and a dopamine standard solution is added to the working area of the electrode, so that the solution completely wets the three electrodes;

[0036] (4) The current signal of the reaction product solution obtained in step (3) is measured, and a standard curve of the dopamine solution concentration and the current signal is established;

[0037] (5) The to-be-tested liquid containing dopamine is tested, and the obtained current signal value is substituted into the standard curve in step (4) to calculate the concentration of dopamine in the to-be-tested liquid.

[0038] According to the present application, the dopamine standard solution is a solution of a series of dopamine standard products with a concentration gradient, and the concentration range can be 512×10 -12 M-125×10 -3 M.

[0039] The conditions for measuring the current signal of the reaction product solution obtained in step (4) are related to the concentration of the measured solution, and a cyclic voltammetry method is adopted. The measurement conditions preferably include: the test range is-1V-1V, the scanning rate is 100mV / s, and the sensitivity is 10 -2 .

[0040] In the present application, the optimal volume of the MBAzy nano-enzyme used for modifying the working electrode can be determined by the following method:

[0041] (1) Take 5mg / mL MBAzy nano-enzyme suspension and 10nM dopamine standard solution out of the refrigerator and place at room temperature;

[0042] (2) Take 7 paper-based electrodes, numbered 1, 2, 3, 4, 5, 6, and 7, and modify 2, 3, 4, 5, 6, 7, and 8 of the MBAzy nano-enzyme suspension on the working electrode, respectively, and store in a 4℃ refrigerator;

[0043] (3) Turn on the electrochemical workstation, set the parameters, and preheat for 20min for standby.

[0044] (4) Respectively, 100 muL 10nM dopamine standard solution is added to the working area of the flexible electrode, so that it infiltrates the working electrode, the reference electrode and the counter electrode, then the green electrode is connected to the working electrode, the white electrode is connected to the reference electrode, the red electrode is connected to the counter electrode, after connection, click start directly, compare the current values of different volumes of MBAzy-based heterojunction nanometer enzyme, and determine that the optimal reaction volume is 5 muL.

[0045] According to the application, the optimal mixing ratio of graphene ink and TiO2 ink of the working electrode can be determined by the following method:

[0046] (1) The graphene ink and the TiO2 ink are prepared into mixed inks in five different ratios of 1:1, 2:1, 3:1, 4:1 and 5:1, and are respectively named as GraphTiO-1, GraphTiO-2, GraphTiO-3, GraphTiO-4 and GraphTiO-5;

[0047] (2) The sheet resistance and the resistance of the five different ratio inks are tested, and finally the optimal ratio is determined as GraphTiO-3.

[0048] According to the application, the optimal test temperature of the flexible sensor patch can be determined by the following method:

[0049] (1) The MBAzy-based heterojunction nanometer enzyme suspension and the 10nM dopamine standard solution are taken out from the refrigerator and placed at room temperature;

[0050] (2) Seven paper-based electrodes are taken, numbered as 1, 2, 3, 4, 5, 6 and 7, 5 muL of the MBAzy-based heterojunction nanometer enzyme suspension is modified on the working electrode, and is placed in a 4℃ refrigerator for storage;

[0051] (3) Seven 10nM dopamine standard solutions are subpackaged, and the temperature is adjusted to 5℃, 15℃, 25℃, 35℃, 45℃, 55℃ and 65℃ by means of water bath or refrigerator.

[0052] (4) The electrochemical workstation is turned on, the parameters are set, and it is preheated for 20 minutes for standby.

[0053] (5) 100 muL of dopamine standard solution at different temperatures is added to the working area of the flexible electrode, so that it infiltrates the working electrode, the reference electrode and the counter electrode, then the green electrode is connected to the working electrode, the white electrode is connected to the reference electrode, the red electrode is connected to the counter electrode, the yellow electrode is connected to another working electrode, after connection, click start directly, compare the current values of different temperatures of dopamine standard solution, from low temperature 5℃ to high temperature 65℃, the response signal of the MBAzy enzyme shows high stability and no significant change. This indicates that MBAzy has good thermal stability and wide temperature adaptability, and is suitable for application in various temperature environments.

[0054] According to the application, the optimal pH value of the dopamine content test experiment can be determined by the following method:

[0055] (1) Take the MBAzy heterojunction nanoscale enzyme suspension and 10 nM dopamine standard solution out of the refrigerator and place them at room temperature;

[0056] (2) Take 7 paper-based electrodes, numbered 1, 2, 3, 4, 5, 6, and 7, and modify 5 μL of the MBAzy heterojunction nanoscale enzyme suspension on the working electrode and store it in a 4°C refrigerator;

[0057] (3) Respectively, take 100 μL of 1×PBS buffer solution with pH=3.50; pH=4.50, pH=5.50; pH=6.50; pH=7.50; pH=8.50; and pH=9.50 and drop them onto the working area of the paper-based electrode, so that they can soak the working electrode, the reference electrode, and the counter electrode, then connect the working electrode with green, the reference electrode with white, the counter electrode with red, and the other working electrode with yellow, after the connection, directly click start, compare the current values of the 1×PBS buffer solutions with different pH values, and determine that pH=6.50 is the optimal test condition for dopamine.

[0058] The application utilizes the MBAzy heterojunction nanoscale enzyme to improve the speed of electron transfer and electrocatalytic kinetics, and the prepared sensor patch has high stability and high sensitivity. In addition, the application replaces the paper-based electrode with a flexible electrode, which does not need to distinguish between the hydrophilic region and the hydrophobic region through processing, reduces the experimental cost, and improves the portability and operability of the experiment. Therefore, the method of the application has the advantages of rapid response, non-invasive detection, non-toxicity, portability, and low cost, and has a wide application prospect and development potential in the fields of wearable sensors, clinical diagnosis, health monitoring, and personalized medical care, and can be used for non-invasive monitoring of human dopamine and stress state evaluation.

[0059] Other features and advantages of the application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0060] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0061] Figure 1 SEM image of MBene.

[0062] Figure 2 SEM image of MBAzy heterojunction nanoscale enzyme.

[0063] Figure 3TEM image of MBAzy-based nanoscale heterostructure nanoszyme.

[0064] Figure 4 A standard curve was plotted with the logarithm of the concentration of dopamine standard as the abscissa and the current value corresponding to each concentration as the ordinate.

[0065] Figure 5 A 3D graph of the standard curve.

[0066] Figure 6 A stability test of the flexible sensor patch based on the MBAzy-based nanoscale heterostructure nanoszyme modified three-electrode system for detecting dopamine content was performed, and the concentration was 10 nM. DETAILED DESCRIPTION

[0067] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0068] In the following examples, dopamine was provided by Alfa Aesar (China) Chemical Co., Ltd. (Shanghai, China). Artificial urine and artificial sweat were provided by Yuan Ye Sheng Wu (Shanghai) Co., Ltd. CuPC was provided by Macklin Biochemical Technology Co., Ltd. (Shanghai, China). TBAOH was purchased from Sigma. Commercial elemental powders Mo, B, Al, and Y were purchased from Sigma-Aldrich. Ascorbic acid (AA), uric acid (UA), urea, glucose (GLU), cysteine, glutathione (GSH), lactose, lactic acid (LA), glutamic acid (Glu), and potassium chloride (KCl) were purchased from Aladdin. The electrochemical workstation was purchased from Shanghai Huachen. Unless otherwise specified in the examples, the routine conditions or the conditions recommended by the manufacturer were used. Unless otherwise specified, the reagents or instruments used were conventional products that could be purchased on the market.

[0069] Example One

[0070] This example is used to illustrate the preparation of a flexible sensor patch based on the MBAzy-based nanoscale heterostructure nanoszyme modified three-electrode system and the establishment of a dopamine content detection method, including the following steps:

[0071] (1) MBene material synthesis method:

[0072] ① A Mo / Y / Al / B powder mixture was used to prepare (Mo 2 / 3 Y 1 / 3)2AlB2 powder: 127.92 mg Mo, 59.27 mg Y, 26.98 mg Al, 1.44 g B were weighed with stoichiometric molar ratio respectively and mixed thoroughly in a maroon mortar. The resulting homogeneous mixture was carefully transferred into a corundum crucible, which was placed in a tube furnace and then heated to 1200 °C for 600 min under Ar atmosphere. After cooling to room temperature, the loose sintered sample powder was sieved through a 200 mesh screen and stored in a 4 °C refrigerator for later use.

[0073] ② At room temperature, 1 g of the synthesized (Mo 2 / 3 Y 1 / 3 )2AlB2 i-MAB precursor was slowly added to 5 mL of 40 wt% aqueous hydrofluoric acid solution and then transferred to an oil bath for magnetic stirring at 33-35 °C for 4 h.

[0074] ③ The resulting etching product was transferred to a 50 mL centrifuge tube and centrifuged at 3500 rpm for 10 min, washed with deionized water several times until the supernatant pH was close to 7.

[0075] ④ Then, after freezing, it was placed in a freeze dryer for 48 h to obtain a gray-black multilayer Mo 4 / 3 B2 MBene powder. This powder was further layered to prepare two-dimensional Mo 4 / 3 B2 MBene, Figure 1 SEM image of the MBene.

[0076] (2) Synthesis method of MBAzy heterojunction nanoszyme:

[0077] ① 1.5000 g of CuPC was dispersed in 15 mL of anhydrous ethanol and ultrasonically dispersed for 30 min.

[0078] ② It was slowly added to 100 mL of Mo 4 / 3 B2 solution (1 mg / mL) and ultrasonically dispersed for another 30 min to mix well.

[0079] ③ The reaction was magnetically stirred at room temperature for 12 h.

[0080] ④ Then centrifuged at 8000 rpm for 5 min to remove the supernatant.

[0081] ④ Add ethanol in the test tube, centrifuge at 8000 rpm for 5 min at 4 °C, then pour out the supernatant, repeat at least 3 times.

[0082] ④ Collect the final precipitate and vacuum dry at 60 °C for 3 h to obtain MBAzy powder, which is stored in a 4 °C refrigerator for later use. Figure 2 SEM image of the MBAzy heterojunction nanoszyme. Figure 3Transmission electron microscopy of the MBAzy-based nanoheterostructure nanoszyme.

[0083] (3) Preparation of flexible electrode:

[0084] ① The working electrode, carbon and graphene oxide ink counter electrode, Ag / AgCl ink reference electrode were printed on the TPU flexible substrate, and placed in a 60°C oven for 30 min.

[0085] ② After room temperature, the GraphTiO-3 ink was printed on the working electrode, and placed in a 60°C oven for 30 min, and dried.

[0086] (4) Preparation of functionalized flexible electrode:

[0087] ① 5 mg / mL MBAzy-based nanoheterostructure nanoszyme suspension was placed at room temperature.

[0088] ② 5 μL of MBAzy-based nanoheterostructure nanosuspension was used to modify the working electrode by drop coating, 1 μL at a time, five times. After each modification, the electrode was placed at room temperature to dry for 2 h, and then repeated.

[0089] (5) The dopamine standard solution was taken out from the 4°C refrigerator, and 0.512 nM, 2.56 nM, 12.8 nM, 64 nM, 320 nM, 1600 nM, 8000 nM, 40000 nM, 200000 nM, 1000000 nM dopamine solutions were prepared with 1×PBS buffer.

[0090] (6) Open the electrochemical workstation, set the parameters, test range -1V~1V, scan rate 100 mV / s, sensitivity 10 -2 , preheat for 20 min.

[0091] (7) Take 100 μL of different concentrations of dopamine solution prepared in step (5) and drop it into the working area of the flexible electrode, so that the solution can soak the working electrode, reference electrode and counter electrode.

[0092] (8) Green for working electrode, white for reference electrode, red for counter electrode. After connection, click start to test.

[0093] (9) Detect different concentrations of dopamine (0.512 nM, 2.56 nM, 12.8 nM, 64 nM, 320 nM, 1600 nM, 8000 nM, 40000 nM, 200000 nM, 1000000 nM) and get the current value of each concentration. The standard curve is I[mA] = 1.6439 log(C DA )[×10 -9M]+0.6642, the detection linear range was 512x10 -12 M-125x10 -3 M, the detection limit was 4.44x10 -12 M (S / N=3). The standard curve was plotted with the current value as the ordinate and the dopamine standard concentration as the abscissa, as shown in Figure 4 . The 3D graph of the standard curve is shown in Figure 5 .

[0094] Example Two

[0095] The standard addition recovery experiment for detecting the content of dopamine based on the flexible sensor patch of the three-electrode system modified by the MBAzy nanoscale enzyme heterojunction includes the following steps:

[0096] (1) Add dopamine to artificial sweat and artificial urine at three different concentrations: 1 nM, 5 nM, and 10 nM. Mix the artificial sweat and dopamine in a test tube to obtain three different concentrations of the sample to be tested, and store in a 4°C refrigerator for later use.

[0097] (2) Take the MBAzy nanoscale enzyme heterojunction suspension out of the 4°C refrigerator and place it at room temperature.

[0098] (3) Measure 5 μL of the suspension and modify the working electrode by drop coating, measuring 1 μL at a time and modifying five times, and place the electrode in a room temperature drying oven for 2 h after each modification.

[0099] (4) Turn on the electrochemical workstation and set the parameter test range to -1 V-1 V, the scan rate to 100 mV / s, and the sensitivity to 10 -2 , and preheat for 20 min.

[0100] (5) Add 100 μL of the dopamine solution prepared in step (1) with different concentrations to the working area of the electrode modified in step (3) to allow the solution to soak the working electrode, the reference electrode, and the counter electrode.

[0101] (6) Connect the working electrode with the green terminal, the reference electrode with the white terminal, the counter electrode with the red terminal, and the other working electrode with the yellow terminal. After the connection is completed, click Start to perform the test.

[0102] (7) Calculate the detected concentration by the standard curve and compare it with the actual added concentration. The average recovery rate of DA in artificial sweat and urine was 92.65%-104.35% and 97.74%-100.37%, respectively, and the RSD of the three concentrations was less than 13%. The results show that the detection method can realize high-sensitivity detection of dopamine.

[0103] Example Three

[0104] The specificity of the dopamine content detection method was verified based on the flexible sensor patch of the three-electrode system modified by the MBAzy-based heterojunction nanosensor, including the following steps:

[0105] (1) Ascorbic acid, uric acid, glucose, I-cysteine, glutathione, lactose, lactic acid, glutamic acid and KCl were mixed to prepare three groups of solutions with concentrations of 40x10 -9 M, 200x10 -9 M and 1000x10 -9 M (the above concentrations are the concentrations of each component in the solution), which were placed in a 4°C refrigerator for storage.

[0106] (2) The MBAzy-based heterojunction nanosensor suspension was taken out from the 4°C refrigerator and placed at room temperature.

[0107] (3) 5 μL of the suspension was measured and modified on the working electrode by drop coating, 1 μL at a time, five times.

[0108] (4) The electrochemical workstation was turned on, and the parameters were set to a test range of -1V-1V, a scan rate of 100 mV / s, and a sensitivity of 10 -2 , and preheated for 20 min.

[0109] (5) 100 μL of ascorbic acid, uric acid, glucose, I-cysteine, glutathione, lactose, lactic acid, glutamic acid and KCl with different concentrations prepared in step (1) were added to the working area of the electrode modified in step (3), so that the solution infiltrated the working electrode, the reference electrode and the counter electrode.

[0110] (6) The working electrode was connected with green, the reference electrode was connected with white, the counter electrode was connected with red, and the other working electrode was connected with yellow. After the connection, the test was started directly by clicking.

[0111] (7) No obvious response was observed in the presence of a large number of these potential interfering species, but the sCMZ sensor showed a clear signal in the presence of dopamine, reflecting a high degree of specific dopamine recognition.

[0112] Example Four

[0113] The stability of the dopamine content detection method was verified based on the flexible sensor patch of the three-electrode system modified by the MBAzy-based heterojunction nanosensor, including the following steps:

[0114] (1) The dopamine standard solution was taken out from the 4°C refrigerator and configured into 10 groups of 10 nM standard solution with 1xPBS buffer.

[0115] (2) Take the MBAzy-based heterojunction nanoscale enzyme suspension out of the 4°C refrigerator and place it at room temperature.

[0116] (3) Measure 5 μL of the suspension and modify the working electrode by drop coating, measuring 1 μL at a time and modifying five times, and placing the electrode at room temperature to dry for 2 h after each modification.

[0117] (4) Turn on the electrochemical workstation and set the parameters: the test range is -1 V-1 V, the scan rate is 100 mV / s, the sensitivity is 10 -2 , and the preheating time is 20 min.

[0118] (5) Add 100 μL of the dopamine solution prepared in step (1) to the working area of the electrode modified in step (3) to soak the working electrode, the reference electrode and the counter electrode.

[0119] (6) Connect the working electrode with green, the reference electrode with white, the counter electrode with red, and the other working electrode with yellow, and then click Start to test.

[0120] (7) Select 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days and 100 days, test the current value of the dopamine solution prepared in step (1) according to the above method, and then obtain the column chart by software processing, as shown in Figure 6 , wherein the horizontal coordinate is the number of days and the vertical coordinate is the current value. Figure 6 It can be seen that the electrochemical response remains unchanged after 100 days of storage, proving that the detection method has good stability.

[0121] Example Five

[0122] The flexible sensor patch based on the MBAzy-based heterojunction nanoscale enzyme modified three-electrode system verifies the repeatability of the dopamine content detection method, including the following steps:

[0123] (1) Take the MBAzy-based heterojunction nanoscale enzyme suspension and 10 nM of dopamine standard solution out of the 4°C refrigerator and place it at room temperature.

[0124] (2) Measure 5 μL of the suspension and modify the working electrode by drop coating, measuring 1 μL at a time and modifying five times, and placing the electrode at room temperature to dry for 2 h after each modification.

[0125] (3) Turn on the electrochemical workstation and set the parameters: the test range is -1 V-1 V, the scan rate is 100 mV / s, the sensitivity is 10 -2 , and the preheating time is 20 min.

[0126] (4) To the working area of the electrode modified in step (2), 100 μL of the dopamine solution prepared in step (1) is added dropwise, so that the solution wets the working electrode, the reference electrode and the counter electrode.

[0127] (5) The working electrode is connected with the green color, the reference electrode is connected with the white color, the counter electrode is connected with the red color, and after the connection, the test can be directly started by clicking the start button.

[0128] (6) The measurement is repeated for 5 times, and the current value of each time is recorded to calculate the relative standard deviation. The results are shown in Table 1. It can be seen that the repeatability and reproducibility of the detection method of the application are good.

[0129] Table 1 Repeatability and reproducibility of dopamine

[0130]

[0131] Example Six

[0132] The flexible sensor patch based on the MBAzy-based heterojunction nanometer enzyme modified three-electrode system verifies the reversibility of the dopamine content detection method, including the following steps:

[0133] (1) The MBAzy-based heterojunction nanometer enzyme suspension and dopamine standard solutions with different concentrations are taken out from the 4℃ refrigerator and placed at room temperature.

[0134] (2) 5 μL of the suspension is measured and the working electrode is modified by drop coating, 1 μL at a time, and the modification is performed for five times. After each modification, the electrode is placed at room temperature for 2 h to dry.

[0135] (3) The electrochemical workstation is opened, and the parameters are set: the test range is-1V-1V, the scan rate is 100 mV / s, the sensitivity is 10 -2 , and the preheating time is 20 min.

[0136] (4) To the working area of the electrode modified in step (2), 100 μL of the dopamine solution prepared in step (1) is added dropwise, so that the solution wets the working electrode, the reference electrode and the counter electrode.

[0137] (5) The working electrode is connected with the green color, the reference electrode is connected with the white color, the counter electrode is connected with the red color, and after the connection, the test can be directly started by clicking the start button.

[0138] (6) The current value of each concentration is obtained by measuring from high concentration to low concentration and then washing the electrode with deionized water and measuring from low concentration to high concentration. The detection values of each time are close to the standard solution concentration, which proves that the detection method has excellent reversibility and small hysteresis.

[0139] Having described various embodiments of the application, it is to be understood that the above description is meant to be illustrative only, and that many modifications and variations of the embodiments are possible without departing from the scope and spirit of the described embodiments. Many modifications and variations of the described embodiments are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the described embodiments can be practiced otherwise than as specifically described.

Claims

1. A MBAzy heterojunction nanoszyme, which is a composite material of a single-atom nanoszyme CuPC and a MBene, the MBene being Mo 4 / 3 B2 MBene, and the mass ratio of the CuPC to the MBene is 10-20:

1. Mo 4 / 3 B2M Bene is made by a process comprising: solid state sintering of a Mo, Y, Al, B powder mixture to obtain (Mo 2 / 3 Y 1 / 3 )2AlB2 powder; etching of the (Mo 2 / 3 Y 1 / 3 )2AlB2 powder with an aqueous hydrofluoric acid solution, and centrifuging, washing until the supernatant is neutral, and freeze-drying of the resulting etched product to obtain a multilayer Mo 4 / 3 B2M Bene powder, further delaminated to obtain Mo 4 / 3 B2M Bene.

2. The preparation method of the MBAzy-based heterojunction nanoscale enzyme according to claim 1, comprising the following steps: (1) mixing Mo, Y, Al, B powder mixture to carry out solid phase sintering to obtain (Mo 2 / 3 Y 1 / 3 )2AlB2 powder; contacting (Mo 2 / 3 Y 1 / 3 )2AlB2 powder with hydrofluoric acid aqueous solution to carry out etching, and centrifuging, washing the obtained etching product until the supernatant is neutral, and then freeze-drying to obtain multi-layer Mo 4 / 3 B2 MBene powder, and further layering to obtain two-dimensional Mo 4 / 3 B2 MBene; (2) The CuPC ethanol dispersion solution is slowly added to the Mo prepared in step (1) 4 / 3 B2 MBene is uniformly mixed by ultrasonic dispersion, and the reaction is carried out under magnetic stirring, and then centrifuged, the supernatant is removed and washed, the precipitate is collected and vacuum dried to obtain MBAzy nano-enzyme powder.

3. The method of making according to claim 2, wherein, In step (1), the conditions for solid-phase sintering include: inert gas protection, temperature of 1000-1500°C, and time of 400-800 min; the conditions for etching include: temperature of 33-35°C, and time of 2-6 hours; and the conditions for centrifugation include: rotation speed of 3000-4000 rpm, and time of 5-15 min; In step (2), the conditions for reaction include: temperature of 20-30°C, and time of 10-16 hours; the conditions for centrifugation include: rotation speed of 6000-10000 rpm, and time of 3-8 min; and the conditions for vacuum drying include: temperature of 40-80°C, and time of 2-6 hours.

4. A modified electrode based on the MBAzy-based heterojunction nanoscale enzyme, wherein the electrode is modified with the MBAzy-based heterojunction nanoscale enzyme according to claim 1.

5. The modified electrode according to claim 4, wherein, The electrode material of the modified electrode is a graphene and TiO2 mixed ink, wherein the mass ratio of graphene to TiO2 is 1-5:

1. The modification amount of the MBAzy-based heterojunction nanoscale enzyme on the electrode is 0.0250-0.0300 mg, and the electrode is prepared by slow drop coating.

6. The modified electrode according to claim 5, wherein, The mass ratio of graphene to TiO2 is 2.5-3.5:

1.

7. An electrode system modified based on the MBAzy-based heterojunction nanoscale enzyme, wherein the electrode system is a three-electrode system or a two-electrode system, and the modified electrode according to any one of claims 4-6 is used as a working electrode.

8. The electrode system of claim 7, wherein, When the electrode system is a three-electrode system, it further comprises: (i) a reference electrode: Ag / AgCl; (ii) a counter electrode: a carbon and graphene oxide mixed electrode.

9. A flexible sensor based on a three-electrode system modified by the MBAzy-based heterojunction nanoscale enzyme, wherein the flexible sensor comprises the working electrode, the reference electrode and the counter electrode in the patch form according to claim 8, and the material of the patch is TPU.

10. A sensor based on a three-electrode system modified by the MBAzy-based heterojunction nanoscale enzyme, comprising: (1) the MBAzy-based heterojunction nanoscale enzyme according to claim 1; (2) a flexible electrode of a three-electrode system; (3) a portable electrochemical workstation.

11. A method for detecting the content of dopamine by a flexible sensor based on a three-electrode system modified by the MBAzy-based heterojunction nanoscale enzyme, comprising: (1) obtaining the MBAzy-based heterojunction nanoscale enzyme according to claim 1; (2) preparing the flexible sensor according to claim 9, comprising: preparing a three-electrode flexible electrode by screen printing technology, drop coating the MBAzy-based heterojunction nanoscale enzyme solution obtained in step (1) on the working electrode of the flexible electrode, and placing it at room temperature for drying; (3) taking out the modified electrode in step (2), and adding a dopamine standard solution to the working area of the electrode so that the solution completely wets the three electrodes; (4) measuring the current signal of the reaction product solution obtained in step (3), and establishing a standard curve of the concentration of the dopamine solution and the current signal. (5) The test liquid containing dopamine is tested according to the above method, and the current signal value obtained is substituted into the standard curve of step (4) to calculate the concentration of dopamine in the test liquid. (5) The test liquid containing dopamine is tested according to the above method, and the current signal value obtained is substituted into the standard curve of step (4) to calculate the concentration of dopamine in the test liquid.

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