NbCat-NC-ZIF-8 electrochemical biosensor based on heterojunction design and preparation method of NbCat-NC-ZIF-8 electrochemical biosensor

Abstract

The invention provides a preparation method of an NbC-coated NC-ZIF-8 electrochemical biosensor based on heterojunction design, and belongs to the technical field of biological analysis and detection. The preparation method comprises the following steps: preparing an NbC-coated NC-ZIF-8 dispersion liquid, modifying the NbC-coated NC-ZIF-8 composite material on the surface of a GCE glassy carbon electrode by adopting a drop casting method, and fixing alcohol dehydrogenase ADH on the surface of the electrode by adopting a glutaraldehyde cross-linking method to obtain the NbC-coated NC-ZIF-8 / ADH / GCE modified electrode. The NbC-coated NC-ZIF-8 / ADH / GCE modified electrode is characterized in that the structural layers of the NbC-coated NC-ZIF-8 / ADH / GCE modified electrode are sequentially as follows from outside to inside: BSA (Bovine Serum Albumin), Glutaraldehyde, ADH, NbC-coated NC-ZIF-8 and Electrode surface. The NbC-coated NC-ZIF-8 / ADH / GCE modified electrode is used for NADH concentration detection, the preparation process is low in equipment requirement and simple, and the NADH / ADH / GCE modified electrode has the advantages of being high in sensitivity, high in response speed and not prone to being interfered by other components and is wide in application range.

Description

Technical Field

[0001] The present invention relates to the technical field of biological analysis and detection, and in particular to a NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design and a preparation method thereof. Background Art

[0002] In food science, nicotinamide adenine dinucleotide (NADH) is a key coenzyme, widely involved in cellular energy metabolism and redox reactions, such as glycolysis and the tricarboxylic acid cycle. NADH content serves not only as an important indicator for evaluating the nutritional value of food, but also as an effective marker for measuring food quality and freshness. Because changes in NADH concentration are closely related to biochemical processes such as oxidative stress in food, establishing a sensitive NADH detection method is of great theoretical and practical significance for accurately assessing food quality and ensuring food safety.

[0003] However, the concentration of NADH in food is usually low (μM level or even lower), and it is highly sensitive to light, heat and oxygen, and is easily degraded during the extraction and detection process, resulting in low measurement values. In addition, the complexity of the food matrix can significantly affect the extraction efficiency and detection accuracy of NADH, making selectivity a key challenge in the detection process. For example, food matrices such as beef contain a variety of interfering substances, such as polysaccharides, proteins, organic acids, etc., which may affect the detection signal of NADH and lead to inaccurate test results.

[0004] Currently, there are many methods for detecting NADH, including capillary electrophoresis, spectral analysis, high-performance liquid chromatography, etc. However, these methods generally have the following defects: insufficient sensitivity, the detection limit of existing methods is high, and it is difficult to detect low concentrations of NADH in food. For example, the detection limit of capillary electrophoresis and spectral analysis is usually at the μM level, and lower concentrations of NADH cannot be detected, resulting in the inability to meet the detection needs of low-concentration NADH in practical applications; slow response speed, the detection process of traditional methods is relatively cumbersome and time-consuming, and instant detection cannot be achieved. For example, high-performance liquid chromatography requires complex sample pretreatment and long analysis time, usually taking several hours or even longer to complete the detection, and cannot achieve rapid and real-time detection. ; Susceptibility to interference leads to limited scope of application. Other components in the food matrix may interfere with the detection signal of NADH and affect the accuracy of detection. For example, polysaccharides, proteins and other components in beef may interfere with the signal of spectral analysis, resulting in deviations in the detection results and affecting the reliability of detection; The equipment is complex and costly: Traditional methods usually require expensive instruments and professional technicians, which limits their application in actual detection. High-performance liquid chromatographs and spectrometers are expensive and require professional personnel for operation and maintenance, which increases the detection cost and technical threshold, making it difficult to promote and apply in small and medium-sized laboratories or food production companies.

[0005] In summary, the existing sensor technology has defects and shortcomings in the NADH detection process, such as insufficient sensitivity, slow response speed, limited scope of application, complex equipment and high cost. Summary of the Invention

[0006] In view of this, the present invention provides a NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design and a preparation method for biological NADH concentration detection. The preparation process has low equipment requirements and a simple process. It has the advantages of high sensitivity, fast response speed, and is not easily interfered by other components. It has a wide range of applications.

[0007] The technical solution adopted by the embodiment of the present invention to solve the technical problem is:

[0008] A method for preparing a NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design, comprising:

[0009] Step S1, preparing a NbC@NC-ZIF-8 dispersion, wherein the NC-ZIF-8 in the NbC@NC-ZIF-8 dispersion is prepared by pyrolysis of ZIF-8 powder;

[0010] In step S2, the NbC@NC-ZIF-8 composite material is modified on the surface of the GCE glassy carbon electrode by a drop casting method, and the alcohol dehydrogenase ADH is fixed on the electrode surface by a glutaraldehyde cross-linking method to obtain a NbC@NC-ZIF-8 / ADH / GCE modified electrode.

[0011] Preferably, the step S1 includes:

[0012] Step S11, preparing ZIF-8 powder by hydrothermal method:

[0013] Dissolve 7.3325 g of Zn(NO3)2·6H2O in 250 mL of methanol and continue stirring until completely dissolved. This is referred to as Solution A. At the same time, dissolve 16.25 g of 2-methylimidazole in 250 mL of methanol and continue stirring until completely dissolved. This is referred to as Solution B.

[0014] Slowly pour the B solution into the A solution while stirring, and continue stirring for 1 hour to ensure complete reaction;

[0015] After the reaction, the reaction product was washed with ethanol, centrifuged at 6000 r / min for 8 min, and washed three times. Finally, the product was dried in a vacuum oven at 60° C. for 15 h to obtain dry ZIF-8 powder.

[0016] Step S12, using ZIF-8 powder, to prepare NC-ZIF-8 by thermal decomposition method:

[0017] The ZIF-8 powder was transferred to a quartz boat, which was then placed in a tube furnace. Under a nitrogen atmosphere, the temperature was raised from an initial temperature of 20°C to a target temperature of 970°C at a rate of 5°C / min, and the temperature was maintained for 2 h after reaching the target temperature.

[0018] After the insulation is completed, the mixture is naturally cooled to room temperature to obtain the target product NC-ZIF-8;

[0019] Step S13, using NC-ZIF-8, a NbC@NC-ZIF-8 dispersion is prepared by thermal decomposition:

[0020] After the NbC particles and NC-ZIF-8 were fully mixed, they were placed in a quartz boat. The quartz boat was then moved into a tube furnace and heated from an initial temperature of 20°C to 1000°C at a rate of 5°C / min under a nitrogen atmosphere. After reaching the target temperature, the temperature was kept for 2 h.

[0021] After the heat preservation is completed, the mixture is naturally cooled to room temperature to obtain the target product NbC@NC-ZIF-8 heterojunction structure complex;

[0022] The NbC@NC-ZIF-8 heterojunction structure complex is further used to prepare a NbC@NC-ZIF-8 dispersion.

[0023] Preferably, the step S2 includes:

[0024] Step S21, pre-treating the bare electrode: polishing the surface of the GCE glassy carbon electrode with 0.3 μm and 0.05 μm alumina powder in sequence until the potential difference of the redox peak measured in a 0.1 M potassium chloride solution containing 5 mM K3[Fe(CN)6] is less than 90 mV; ultrasonically cleaning the pre-treated GCE glassy carbon electrode in ultrapure water and ethanol in sequence, drying it, and setting it aside;

[0025] Step S22, preparing NbC@NC-ZIF-8 / ADH / GCE modified electrode by drop casting method:

[0026] 7 μL of the NbC@NC-ZIF-8 dispersion was evenly drop-coated on the surface of the pretreated GCE glassy carbon electrode and dried under an infrared lamp. The concentration of the NbC@NC-ZIF-8 dispersion was 1.4 mg / mL.

[0027] After the temperature of the GCE glassy carbon electrode after coating was reduced to room temperature, 10 μL ADH, 5 μL glutaraldehyde solution and 5 μL BSA were added dropwise in sequence. Drying was performed after each addition of a reagent to finally obtain the NbC@NC-ZIF-8 / ADH / GCE modified electrode.

[0028] Preferably, the NbC@NC-ZIF-8 / ADH / GCE modified electrode is used to determine the NADH concentration. Under continuous stirring conditions, the NbC@NC-ZIF-8 / ADH / GCE modified electrode exhibits a significant current response to NADH, and the oxidation peak current Ipa is linearly related to the NADH concentration X. The fitting curve is expressed as:

[0029] Ipa(μA)=101.76+(5.08-101.76 / (1+(X / 589.54) ^1.04 )

[0030] In the formula, the fitting degree R 2 =0.999;

[0031] The low detection limit of the NbC@NC-ZIF-8 / ADH / GCE modified electrode was calculated according to the low detection limit LOD formula:

[0032] LOD=3σ / S

[0033] Where σ is the standard deviation of N blank measurements, S is the sensor signal, the signal-to-noise ratio S / N = 3, and N is the noise.

[0034] Preferably, the NbC@NC-ZIF-8 / ADH / GCE modified electrode has a wide linear detection range of 1 μM-1116 μM, and the lowest detection limit LOD is 0.075 μM.

[0035] Preferably, the structural hierarchy of the NbC@NC-ZIF-8 / ADH / GCE modified electrode is BSA, Glutaraldehyde, ADH, NbC@NC-ZIF-8, and Electrode surface from the outside to the inside.

[0036] The present invention provides a NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design, which is a NbC@NC-ZIF-8 / ADH / GCE modified electrode prepared by the aforementioned method, and is an electrode with a NbC@NC-ZIF-8 composite material on the surface of a GCE glassy carbon electrode.

[0037] Preferably, the NbC@NC-ZIF-8 / ADH / GCE modified electrode is used to determine the NADH concentration. Under continuous stirring conditions, the NbC@NC-ZIF-8 / ADH / GCE modified electrode exhibits a significant current response to NADH, and the oxidation peak current Ipa is linearly related to the NADH concentration X. The fitting curve is expressed as:

[0038] Ipa(μA)=101.76+(5.08-101.76 / (1+(X / 589.54) ^1.04 )

[0039] In the formula, the curve fitting degree R 2 =0.999;

[0040] The low detection limit of the NbC@NC-ZIF-8 / ADH / GCE modified electrode was calculated according to the low detection limit LOD formula:

[0041] LOD=3σ / S

[0042] Where σ is the standard deviation of N blank measurements, S is the sensor signal, the signal-to-noise ratio S / N = 3, and N is the noise.

[0043] Preferably, the NbC@NC-ZIF-8 / ADH / GCE modified electrode has a wide linear detection range of 1 μM-1116 μM, and the lowest detection limit LOD is 0.075 μM.

[0044] Preferably, the structural hierarchy of the NbC@NC-ZIF-8 / ADH / GCE modified electrode is BSA, Glutaraldehyde, ADH, NbC@NC-ZIF-8, and Electrode surface from the outside to the inside.

[0045] As can be seen from the above technical solution, the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design and preparation method provided in the embodiment of the present invention includes the following steps: first, preparing a NbC@NC-ZIF-8 dispersion, modifying the NbC@NC-ZIF-8 composite material on the surface of a GCE glassy carbon electrode by drop casting, and immobilizing alcohol dehydrogenase ADH on the electrode surface by glutaraldehyde cross-linking to obtain a NbC@NC-ZIF-8 / ADH / GCE modified electrode. The NbC@NC-ZIF-8 / ADH / GCE modified electrode of the present invention is used for biological NADH concentration detection. The preparation process has low equipment requirements and a simple process. It has the advantages of high sensitivity, fast response speed, and is not easily interfered with by other components, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a flow chart of the preparation method of the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design of the present invention.

[0047] Figure 2 Schematic diagram of the NbC@NC-ZIF-8 / ADH / GCE modified electrode structure of the present invention.

[0048] Figure 3Reference images for SEM images of NbC, ZIF-8, NbC@NC-ZIF-8, TEM image of NbC@NC-ZIF-8, and EDS spectrum of NbC@NC-ZIF-8.

[0049] Figure 4 This is the XPS spectrum of NbC@NC-ZIF-8.

[0050] Figure 5 CV response diagrams of different modified electrodes at 50 Mv / s.

[0051] Figure 6 Diagram of the catalytic oxidation mechanism of NADH on the NbC@NC-ZIF-8 / ADH / GCE modified electrode.

[0052] Figure 7 Figure 3 is the iT current response of the modified electrode to NADH and the linear relationship between Ipa and NADH concentration.

[0053] Figure 8 The data graph shows the current response, selectivity, repeatability and reproducibility of NbC@NC-ZIF-8 / ADH / GCE under different conditions.

[0054] Figure 9 EDS spectrum of NbC@NC-ZIF-8 and schematic diagram of the proportion of each element.

[0055] Figure 10 This is the XPS spectrum of Zn in the prepared NbC@NC-ZIF-8.

[0056] Figure 11 Comparison of the Cv curves of NbC@NC-ZIF-8 / ADH / GCE in the presence and absence of NADH. DETAILED DESCRIPTION

[0057] The technical solutions and technical effects of the present invention are further described in detail below with reference to the accompanying drawings of the present invention.

[0058] The present invention provides a NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design and its preparation method for high-sensitive detection of NADH in beef. The NbC@NC-ZIF-8 electrochemical biosensor is a NbC@NC-ZIF-8 / ADH / GCE modified electrode with a NbC@NC-ZIF-8 composite material on the surface of a GCE glassy carbon electrode. The preparation process is referenced to Figure 1 As shown:

[0059] Step S1, preparing NbC@NC-ZIF-8 dispersion:

[0060] First, ZIF-8 powder was prepared by hydrothermal method:

[0061] Dissolve 7.3325 g of Zn(NO3)2·6H2O in 250 mL of methanol and continue stirring until completely dissolved. This is referred to as Solution A. At the same time, dissolve 16.25 g of 2-methylimidazole in 250 mL of methanol and continue stirring until completely dissolved. This is referred to as Solution B.

[0062] Slowly pour solution B into solution A while stirring. Continue stirring for 1 hour to ensure complete reaction.

[0063] After the reaction, the reaction product was washed with ethanol, centrifuged at 6000 r / min for 8 min, and washed three times. Finally, the product was dried in a vacuum oven at 60 °C for 15 h to obtain dry ZIF-8 powder.

[0064] NC-ZIF-8 was prepared by thermal decomposition of ZIF-8 powder:

[0065] The ZIF-8 powder was transferred to a quartz boat, which was then placed in a tube furnace. Under a nitrogen atmosphere, the temperature was raised from an initial temperature of 20°C to a target temperature of 970°C at a rate of 5°C / min, and the temperature was maintained for 2 h after reaching the target temperature.

[0066] After the insulation is completed, the mixture is naturally cooled to room temperature to obtain the target product NC-ZIF-8;

[0067] Using NC-ZIF-8, NbC@NC-ZIF-8 dispersion was prepared by thermal decomposition method:

[0068] After the NbC particles and NC-ZIF-8 are fully mixed, they are placed in a quartz boat. The quartz boat is then moved into a tube furnace and heated from an initial temperature of 20°C to 1000°C at a rate of 5°C / min under a nitrogen atmosphere. After reaching the target temperature, the temperature is kept for 2 hours. NC-ZIF-8 is a nanoporous material. By heating, NbC is embedded in NC-ZIF-8.

[0069] After the heat preservation is completed, the mixture is naturally cooled to room temperature to obtain the target product NbC@NC-ZIF-8 heterojunction structure complex;

[0070] The NbC@NC-ZIF-8 heterojunction structure complex was further used to prepare the NbC@NC-ZIF-8 dispersion.

[0071] Step S2: The NbC@NC-ZIF-8 composite material is modified on the surface of the GCE glassy carbon electrode by drop casting, and the alcohol dehydrogenase ADH is immobilized on the electrode surface by glutaraldehyde cross-linking to obtain the NbC@NC-ZIF-8 / ADH / GCE modified electrode:

[0072] First, the bare electrode was pretreated: the surface of the GCE glassy carbon electrode was polished with 0.3 μm and then 0.05 μm alumina powder until the potential difference of the redox peak measured in a 0.1 M potassium chloride solution containing 5 mM K3[Fe(CN)6] was less than 90 mV. The pretreated GCE glassy carbon electrode was ultrasonically cleaned in ultrapure water and ethanol, respectively, and dried before use.

[0073] NbC@NC-ZIF-8 / ADH / GCE modified electrode was prepared by drop casting method:

[0074] 7 μL of NbC@NC-ZIF-8 dispersion was evenly dropped onto the surface of the pretreated GCE glassy carbon electrode and dried under an infrared lamp. The concentration of the NbC@NC-ZIF-8 dispersion was 1.4 mg mL -1 ;

[0075] After the temperature of the GCE glassy carbon electrode after coating dropped to room temperature, 10 μL ADH, 5 μL glutaraldehyde solution and 5 μL BSA were added dropwise in sequence. Drying was performed after each addition of a reagent to finally obtain the NbC@NC-ZIF-8 / ADH / GCE modified electrode.

[0076] As shown in the reference figure, the structural layers of the NbC@NC-ZIF-8 / ADH / GCE modified electrode are BSA, Glutaraldehyde, ADH, NbC@NC-ZIF-8, and Electrode surface from the outside to the inside.

[0077] This application fully considers the material properties when selecting the preparation materials:

[0078] NbC (niobium carbide): NbC is selected as the electrode material because of its high conductivity and excellent catalytic properties, which can effectively improve the current response and sensitivity of electrochemical sensors. NbC is a transition metal carbide with high hardness, high melting point and excellent electrical conductivity. It is widely used in cutting tools, wear-resistant coatings and electronic devices. Its unique electrical conductivity and catalytic properties make NbC show potential in some special electrochemical applications. NbC has good chemical stability, good surface conductivity, and can still maintain high thermal stability at high temperatures. It can work stably for a long time in extreme environments, especially under strong acid, strong alkali or high temperature conditions, which is crucial for the long-term stability and repeatability of electrochemical sensors.

[0079] ZIF-8 (metal-organic framework material): ZIF-8 was chosen as the carrier material because of its high specific surface area and porosity, which can provide more reaction sites and enhance the sensitivity and reaction speed of the sensor. ZIF-8 is a metal-organic framework material composed of zinc ions and imidazole ligands through coordination. It has unique structural characteristics, adjustable pore size, high chemical stability and excellent specific surface area. The high specific surface area and porosity of ZIF-8 give it a large surface area, which can provide more reaction sites. This is very important for improving the sensitivity of electrochemical sensors and accelerating the reaction process, especially when detecting trace substances, which can significantly enhance the performance of the sensor.

[0080] ADH (alcohol dehydrogenase): ADH was chosen as the biorecognition element due to its specific selectivity for NADH, which improves sensor selectivity. ADH is an enzyme that catalyzes the oxidation of ethanol and is widely present in the human body, offering excellent biocompatibility and environmental advantages. Unlike traditional chemical sensors, ADH is a naturally occurring enzyme. Electrochemical sensors using it as a catalyst offer environmental advantages, making it particularly suitable for applications with high environmental requirements.

[0081] Construction of heterojunction structure: NbC@NC-ZIF-8 composite materials were successfully prepared by hydrothermal and thermal decomposition methods to form a heterojunction structure. This structure not only significantly improves the electrical conductivity of the material, but also provides abundant active sites, thereby systematically improving the performance of the sensor. A heterojunction is a composite structure composed of two semiconductor materials with different band gaps and electronic properties. This unique structural design has a wide range of applications in the fields of electronic and optoelectronic devices. By constructing a heterojunction interface, not only is the electron transfer rate significantly improved, but it also provides abundant active sites, thereby systematically improving the performance of enzyme electrochemical sensors. Specifically, the heterojunction structure effectively overcomes the inherent defects of traditional enzyme electrodes, such as poor conductivity, slow electron transfer rate, and easy enzyme inactivation, ultimately achieving a comprehensive improvement in sensor sensitivity, response speed, and stability.

[0082] In order to explore the microstructure and assembly structure of the composite material, the present invention respectively carried out scanning electron microscopy (SEM) and transmission electron microscopy (TEM) characterization on NbC, ZIF-8 and NbC@NC-ZIF-8. Figure 3 As shown in region A of the graph, NbC is mainly distributed in irregular blocks with a particle size of about 1–4 μm. Figure 3 The ZIF-8 shown in the B area has a typical regular dodecahedron morphology, a smooth surface, high crystallinity, and a clear particle interface, showing a strong crystal orientation. Figure 3(SEM image of NbC@NC-ZIF-8 shown in region C of the image) The original structure of ZIF-8 partially collapses and reconstructs to form a nanoporous skeleton; at the same time, NbC particles are evenly embedded in it to obtain a composite structure. Figure 3 The D area is the TEM image of NbC@NC-ZIF-8. Figure 3 EDS spectrum of NbC@NC-ZIF-8 in the E region.

[0083] Figure 4 is the XPS spectrum of NbC@NC-ZIF-8, wherein, curve A is the total spectrum of the XPS spectrum of NbC@NC-ZIF-8, curve B is the XPS spectrum C1s of NbC@NC-ZIF-8, curve C is the XPS spectrum N1s of NbC@NC-ZIF-8, curve D is the XPS spectrum Nb 3d of NbC@NC-ZIF-8, curve E is the XRD spectrum of NbC, NC-ZIF-8 and NbC@NC-ZIF-8, and curve D is the FI-IR spectrum of NbC, NC-ZIF-8 and NbC@NC-ZIF-8.

[0084] Figure 5 Represents the CV response of different modified electrodes at 50Mv / s, where B represents EIS, C represents the CV response of NbC@NC-ZIF-8 / ADH / GCE at different scan rates of 20-200Mv / s, and the corresponding line graph of redox current and the square root of scan rate, and D represents the redox peak current response Ipa and I pc It is linearly correlated with the square root of the scan rate. The redox reaction kinetics of NbC@NC-ZIF-8 / GCE is a diffusion-controlled process. E represents the effect of different scan rates on the CV curves of NbC@NC-ZIF-8 / ADH / GCE. F represents the relationship between scan rate and Ipa.

[0085] Furthermore, the present invention conducted electrochemical testing on the prepared NbC@NC-ZIF-8 / ADH / GCE modified electrode to understand its properties. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), time-current amperometry (iT), and electrochemical impedance spectroscopy (EIS) were used to systematically study the structural characteristics and electrochemical behavior of the material. As a control, NbC / GCE, ZIF-8 / GCE, NbC@NC-ZIF-8 / GCE, and NbC@NC-ZIF-8 / ADH / GCE modified electrodes were prepared using the same method.

[0086] All electrochemical analysis methods were performed using a three-electrode system, in which the modified GCE was used as the working electrode, the platinum wire electrode and the Ag / AgCl electrode were used as the counter electrode and reference electrode, respectively. All electrochemical measurements were performed on a CHI660E electrochemical workstation. 3- / 4- CV, DPV, and EIS were performed in an electrolyte solution containing 0.1 M KCl and 0.1 M PBS (pH 7.4) containing the substrate NADH. NADH was detected by iT.

[0087] Sample preparation: Commercially available beef was used as an actual sample, and iT was used to detect NADH on the NbC@NC-ZIF-8 / ADH / GCE modified electrode. The specific sample pretreatment steps are as follows: Beef purchased from a supermarket was used as a sample to verify the practicality of the sensor. The above sample was simmered in a nutrient soup model for 2 hours. The soup was filtered through filter paper and then centrifuged at 11,000 rpm for 10 minutes at 4°C. The solution was then filtered three times using a 0.22μm microporous filter membrane and transferred to a centrifuge tube. NADH standard solutions of different concentrations were then added and tested using cyclic voltammetry.

[0088] NADH detection: In order to evaluate the performance and effectiveness of the electrochemical sensor based on NbC@NC-ZIF-8 / ADH / GCE, different concentrations of NADH were detected by iT (refer to Figure 6 shown). Figure 7 As shown in curve A (iT current response of the modified electrode to NADH (1-1116 μM) in 0.1 PBS (pH = 7.0)), under continuous stirring (1000 rpm), NbC@NC-ZIF-8 / ADH / GCE exhibits a significant current response to NADH. The results show that with the gradual increase in NADH concentration, the oxidation peak current (Ipa) shows a linear increasing trend. The corresponding equation is expressed as follows: Figure 7 As shown in curve B (linear relationship between Ipa and NADH concentration): Ipa (μA) = 101.76 + (5.08 - 101.76 / (1 + (X / 589.54) ^1.04 ), (R 2 =0.999). Under optimized experimental conditions, the peak current of NADH oxidation increased with increasing concentration within the range of 1 μM to 1116 μM, exhibiting a good linear relationship. Furthermore, based on LOD = 3σ / S (where σ is the standard deviation of 10 blank measurements and S is the slope of the calibration curve), the lower limit of detection (LOD) was calculated to be 0.075 μM (S / N = 3). This demonstrates that the constructed sensor exhibits high sensitivity and a wide linear detection range for NADH.

[0089] Table S1 lists NbC@NC-ZIF-8 / ADH / GCE and some reported NADH sensors for performance comparison:

[0090]

[0091] Table S1.Recoveries of NADH in Beef sample(n=5)

[0092] Reference analysis results Figure 8 , wherein, bar graph A represents the current response of NbC@NC-ZIF-8 / ADH / GCE to 1 μM NADH at different enzyme concentrations (4, 5, 6, 7, 8, 9 and 10 U / L), with a scan rate of 50 mV / s, bar graph B represents the current response of NbC@NC-ZIF-8 / ADH / GCE to 1 μM NADH at different temperatures (25, 30, 35, 40 and 45 °C), with a scan rate of 50 mV / s, bar graph C represents the current response of NbC@NC-ZIF-8 / ADH / GCE to 1 μM NADH at different pH (4, 5, 6, 7, 8 and 9), in the presence of 0.1 M KCl and 5 mM [Fe(CN)6] 3- / 4- Under the conditions of , histogram D shows experimental data for the selectivity of the modified electrode for 24 μM NADH, histogram E shows experimental data for the repeatability of the modified electrode for 24 μM NADH, and histogram F shows experimental data for the reproducibility of the modified electrode for 24 μM NADH. The experimental results show that the biosensor of the present invention exhibits excellent analytical performance: a wide linear detection range of 1-1116 μM, a minimum detection limit of 0.075 μM, and good selectivity, repeatability, and reproducibility.

[0093] Figure 9 EDS spectrum of NbC@NC-ZIF-8 and the ratio of each element. Figure 10 This is the XPS spectrum of Zn in the prepared NbC@NC-ZIF-8. Figure 11 Comparison of the Cv curves of NbC@NC-ZIF-8 / ADH / GCE in the presence and absence of NADH.

[0094] The NbC@NC-ZIF-8 / ADH / GCE modified electrode provided by the present invention has the following advantages:

[0095] High sensitivity: The sensor provided by the present invention has a detection limit as low as 0.075 μM, capable of detecting low concentrations of NADH in food. For example, in beef samples, the sensor provided by the present invention can accurately detect NADH concentrations as low as μM.

[0096] Fast response: The sensor has a wide linear detection range (1-1116 μM), enabling instant detection. For example, under continuous stirring conditions, the sensor exhibits a significant current response to NADH, with a short response time, enabling detection within seconds.

[0097] Excellent selectivity: The sensor's specific selectivity for ADH ensures the sensor's specific detection of NADH, avoiding interference from other components in the food matrix. For example, in beef, which contains multiple interfering substances, the sensor can accurately detect NADH without being affected by other components, significantly improving detection accuracy.

[0098] Low cost: The sensor is made from low-cost materials and is easy to operate, eliminating the need for expensive equipment and specialized technicians. For example, NbC and ZIF-8 are relatively inexpensive to prepare, and the sensor's simple fabrication process facilitates large-scale application, significantly reducing detection costs and technical barriers.

[0099] This invention, by constructing a heterojunction NbC@NC-ZIF-8 / ADH / GCE electrochemical biosensor, successfully overcomes the shortcomings of traditional NADH detection methods in terms of sensitivity, response speed, selectivity, and cost, providing a new solution for the rapid and accurate detection of NADH in food. The sensor boasts high sensitivity, rapid response, good selectivity, and low cost, making it suitable for rapid detection and quality control of NADH in food and promising broad application prospects.

[0100] The above disclosure is only a preferred embodiment of the present invention, and it is certainly not intended to limit the scope of the present invention. A person skilled in the art can understand that all or part of the processes of the above embodiment and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A method for preparing a NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design, characterized in that: include: Step S1, preparing a NbC@NC-ZIF-8 dispersion, wherein the NC-ZIF-8 in the NbC@NC-ZIF-8 dispersion is prepared by pyrolysis of ZIF-8 powder; In step S2, the NbC@NC-ZIF-8 composite material is modified on the surface of the GCE glassy carbon electrode by a drop casting method, and the alcohol dehydrogenase ADH is fixed on the electrode surface by a glutaraldehyde cross-linking method to obtain a NbC@NC-ZIF-8 / ADH / GCE modified electrode.

2. The method for preparing the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 1, characterized in that: The step S1 comprises: Step S11, preparing ZIF-8 powder by hydrothermal method: Dissolve 7.3325 g of Zn(NO3)2·6H2O in 250 mL of methanol and continue stirring until completely dissolved. This is referred to as Solution A. At the same time, dissolve 16.25 g of 2-methylimidazole in 250 mL of methanol and continue stirring until completely dissolved. This is referred to as Solution B. The B solution was slowly poured into the A solution while stirring, and the stirring was continued for 1 hour to ensure the complete reaction. After the reaction was completed, the reaction product was washed with ethanol, centrifuged at 6000 r / min for 8 minutes, and the washing was repeated three times. Finally, the product was placed in a vacuum oven at 60°C for 15 hours to obtain dry ZIF-8 powder. Step S12, using ZIF-8 powder, to prepare NC-ZIF-8 by thermal decomposition method: The ZIF-8 powder was transferred to a quartz boat, which was then placed in a tube furnace. Under a nitrogen atmosphere, the temperature was raised from an initial temperature of 20°C to a target temperature of 970°C at a rate of 5°C / min. After reaching the target temperature, the temperature was kept for 2 hours. After the temperature was kept, the mixture was naturally cooled to room temperature to obtain the target product NC-ZIF-8. Step S13, using NC-ZIF-8, a NbC@NC-ZIF-8 dispersion is prepared by thermal decomposition method: After the NbC particles and NC-ZIF-8 were fully mixed, they were placed in a quartz boat. The quartz boat was then moved into a tube furnace and heated from an initial temperature of 20°C to 1000°C at a rate of 5°C / min under a nitrogen atmosphere. After reaching the target temperature, the temperature was kept for 2 h. After the heat preservation is completed, the mixture is naturally cooled to room temperature to obtain the target product NbC@NC-ZIF-8 heterojunction structure complex; The NbC@NC-ZIF-8 heterojunction structure complex is further used to prepare a NbC@NC-ZIF-8 dispersion.

3. The method for preparing the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 2, characterized in that: The step S2 comprises: Step S21, pre-treating the bare electrode: polishing the surface of the GCE glassy carbon electrode with 0.3 μm and 0.05 μm alumina powder in sequence until the potential difference of the redox peak measured in a 0.1 M potassium chloride solution containing 5 mM K3[Fe(CN)6] is less than 90 mV; ultrasonically cleaning the pre-treated GCE glassy carbon electrode in ultrapure water and ethanol in sequence, drying it, and setting it aside; Step S22, preparing NbC@NC-ZIF-8 / ADH / GCE modified electrode by drop casting method: 7 μL of the NbC@NC-ZIF-8 dispersion was evenly drop-coated on the surface of the pretreated GCE glassy carbon electrode and dried under an infrared lamp. The concentration of the NbC@NC-ZIF-8 dispersion was 1.4 mg / mL. After the temperature of the GCE glassy carbon electrode after coating was reduced to room temperature, 10 μL ADH, 5 μL glutaraldehyde solution and 5 μL BSA were added dropwise in sequence. Drying was performed after each addition of a reagent to finally obtain the NbC@NC-ZIF-8 / ADH / GCE modified electrode.

4. The method for preparing the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 3, characterized in that: The NbC@NC-ZIF-8 / ADH / GCE modified electrode was applied to determine the NADH concentration. Under continuous stirring, the NbC@NC-ZIF-8 / ADH / GCE modified electrode exhibited a significant current response to NADH. The oxidation peak current Ipa was linearly related to the NADH concentration X, and the fitting curve was expressed as follows: Ipa(μA)=101.76+(5.08-101.76 / (1+(X / 589.54) ^1.04 ) In the formula, the fitting degree R 2 =0.999; The low detection limit of the NbC@NC-ZIF-8 / ADH / GCE modified electrode was calculated according to the low detection limit LOD formula: LOD=3σ / S Where σ is the standard deviation of N blank measurements, S is the sensor signal, the signal-to-noise ratio S / N = 3, and N is the noise.

5. The method for preparing the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 4, characterized in that: The NbC@NC-ZIF-8 / ADH / GCE modified electrode has a wide linear detection range of 1 μM-1116 μM and a minimum detection limit LOD of 0.075 μM.

6. The method for preparing the NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 5, characterized in that: The structural layers of the NbC@NC-ZIF-8 / ADH / GCE modified electrode are BSA, Glutaraldehyde, ADH, NbC@NC-ZIF-8, and Electrode surface from the outside to the inside.

7. A NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design, characterized in that: The NbC@NC-ZIF-8 / ADH / GCE modified electrode prepared according to any one of claims 1 to 6 is an electrode having a NbC@NC-ZIF-8 composite material on the surface of a GCE glassy carbon electrode.

8. The NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 7, characterized in that: The NbC@NC-ZIF-8 / ADH / GCE modified electrode was applied to determine the NADH concentration. Under continuous stirring, the NbC@NC-ZIF-8 / ADH / GCE modified electrode exhibited a significant current response to NADH. The oxidation peak current Ipa was linearly related to the NADH concentration X, and the fitting curve was expressed as follows: Ipa(μA)=101.76+(5.08-101.76 / (1+(X / 589.54) ^1.04 ) In the formula, the curve fitting degree R 2 =0.999; The low detection limit of the NbC@NC-ZIF-8 / ADH / GCE modified electrode was calculated according to the low detection limit LOD formula: LOD=3σ / S Where σ is the standard deviation of N blank measurements, S is the sensor signal, the signal-to-noise ratio S / N = 3, and N is the noise.

9. The NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 8, characterized in that: The NbC@NC-ZIF-8 / ADH / GCE modified electrode has a wide linear detection range of 1 μM-1116 μM and a minimum detection limit LOD of 0.075 μM.

10. The NbC@NC-ZIF-8 electrochemical biosensor based on heterojunction design according to claim 9, characterized in that: The structural layers of the NbC@NC-ZIF-8 / ADH / GCE modified electrode are BSA, Glutaraldehyde, ADH, NbC@NC-ZIF-8, and Electrode surface from the outside to the inside.

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