Histamine electrochemical biosensor based on g-C3N4 / TiO2 heterojunction as well as preparation method and application of histamine electrochemical biosensor

Abstract

The invention discloses a high-performance biosensor based on a g-C3N4 / TiO2 heterojunction as well as a preparation method and application of the high-performance biosensor, and belongs to the technical field of biosensing and food safety detection. The sensor is constructed by taking a glassy carbon electrode as a substrate and sequentially modifying a g-C3N4 / TiO2 heterojunction composite material and diamine oxidase (DAO). The g-C3N4 / TiO2 heterojunction is prepared through a one-step calcination method, a type II p-n junction structure is formed, photo-generated charge separation and interface electron transfer are effectively promoted, and the conductivity, catalytic activity and enzyme immobilization capacity of the electrode are remarkably improved. The invention discloses a synergistic mechanism of heterojunction interface enhanced histamine selective adsorption and catalytic reaction through density functional theory calculation. The sensor shows excellent detection performance on histamine: the linearity is good in a range of 0.05-10 mM, and the detection limit is as low as 0.075 M; the selectivity is still higher than 93% in the presence of an interferent with ten times of concentration; when the method is applied to detection of actual samples of beef, mutton, fish and chicken, the adding standard recovery rate is 93.60%-101.8%, and the relative standard deviation is smaller than 5%. The sensor has the advantages of high sensitivity, good selectivity, strong anti-interference capability, good reproducibility and the like, and a reliable new method is provided for rapid and accurate screening of histamine in food.

Description

Technical Field

[0001] This invention relates to the field of biosensing and food safety detection technology, specifically to a high-performance biosensor based on g-C3N4 / TiO2 heterojunction, its preparation method, and its application in detecting histamine content in meat products. Background Technology

[0002] Histamine (HIS) is a major biogenic amine spoilage metabolite produced by the degradation of histidine during the storage and transportation of aquatic and livestock products. Excessive accumulation of histamine can directly trigger allergic reactions, headaches, vomiting, and other symptoms of poisoning. It is also a key indicator for evaluating food freshness and safety. Therefore, domestic and international regulatory agencies have established strict limits on the histamine content in food.

[0003] Currently, while methods such as high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) can be used for detection, they suffer from problems such as high instrument costs, complex operation, and long detection cycles. Electrochemical sensors show potential due to their advantages of rapid response, low cost, and ease of miniaturization, but they still face challenges such as insufficient conductivity and stability of traditional electrode materials, low efficiency of biological enzyme immobilization, and poor resistance to interference in complex food matrices. Based on this, this invention proposes a high-performance biosensor based on a g-C3N4 / TiO2 heterojunction, aiming to provide a rapid, sensitive, highly selective, and novel method for histamine detection in complex meat matrices. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a high-performance biosensor based on g-C3N4 / TiO2 heterojunction and its preparation method, thereby solving the technical problems of existing histamine detection methods, such as cumbersome operation, high cost, and insufficient sensitivity and anti-interference ability in complex food matrices.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A high-performance biosensor based on a g-C3N4 / TiO2 heterojunction is fabricated through the following steps: 1) Preparation of g-C3N4 / TiO2 heterojunction composite material: g-C3N4 / TiO2 powder was ultrasonically dispersed in 50% ethanol at a mass ratio of 2:1 for 30 minutes. After vacuum drying for 24 hours, it was heat-treated at 550 ℃ for 2.0 hours under argon protection by heating at 5 ℃ / min. After cooling, it was ground to obtain the composite material.

[0006] 2) Construction of DAO / g-C3N4 / TiO2 / GCE biosensor: The bare glassy carbon electrode (GCE) was polished, cleaned, and air-dried. 8.0 μL of g-C3N4 / TiO2 dispersion was drop-coated onto the electrode surface and dried at 45°C. Subsequently, 5.0 μL of BSA solution (1.0 mM), 8.0 μL of DAO solution (1.0 U / mL), and 5.0 μL of glutaraldehyde solution (5.0 mM) were added drop-coated sequentially. After each drop-coating step, the electrode was dried at a constant temperature of 45°C to obtain the biosensor.

[0007] Due to the adoption of the above technical solution, the beneficial effects of this application are: 1. This invention effectively promotes the separation and transfer of interfacial charges by constructing g-C3N4 / TiO2 heterojunctions, significantly improving the conductivity and electron transport efficiency of the substrate, and providing a high specific surface area platform for enzyme immobilization.

[0008] 2. Combining density functional theory (DFT) calculations, the mechanism of the specific adsorption enhancement effect of the heterojunction interface on histamine was revealed, thereby realizing the efficient and highly selective conversion of biorecognition signals into electrochemical signals.

[0009] 3. The prepared sensor exhibits excellent performance: it has a wide linear detection range (0.05-10 mM) and a low detection limit (0.075 μM) for histamine; its selectivity is higher than 93% in the presence of ten times the concentration of interfering substances; when applied to the detection of actual meat samples, the spiked recovery rate reaches 93.60%-101.8%, and the relative standard deviation (RSD) is less than 5%, demonstrating excellent accuracy, stability and practicality.

[0010] 4. The preparation method described in this invention is simple and low in cost, and the constructed sensor provides a reliable new tool for rapid and on-site screening of histamine in food. Attached Figure Description

[0011] Figure 1 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of different materials (TiO2g-C3N4 and g-C3N4 / TiO2 composite materials); Figure 2 X-ray diffraction patterns and Fourier transform infrared spectra of different materials (g-C3N4, TiO2 and g-C3N4 / TiO2); Figure 3 The image shows the X-ray photoelectron spectroscopy (XPS) analysis of the g-C3N4 / TiO2 composite material. Figure 4 Cyclic voltammetric response curves and electrochemical impedance spectroscopy for different modified electrodes (bare GCE, g-C3N4 / GCE, TiO2 / GCE, g-C3N4 / TiO2 / GCE and DAO / g-C3N4 / TiO2 / GCE); Figure 5 A schematic diagram of the electrochemical oxidation mechanism of histamine catalyzed by diamine oxidase; Figure 6 The adsorption model and differential charge density plot of histamine on the surface of g-C3N4 / TiO2 heterojunction are calculated based on density functional theory. Figure 7 The figure shows the optimization results of key preparation and detection parameters of the sensor (g-C3N4 / TiO2 coating amount, detection pH value, DAO enzyme concentration); Figure 8 The differential pulse voltammetry response curve for histamine detection by the sensor and its calibration curve; Figure 9 The bar chart shows the reproducibility test results of the sensor and its selective response under multiple coexisting interfering substances. Figure 10 Histamine detection graph in a real sample. Detailed Implementation

[0012] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all of them.

[0013] Example 1: Preparation and characterization of g-C3N4 / TiO2 heterojunction composite material 15 g of melamine was weighed and placed in a quartz boat. The mixture was calcined at 550 °C for 2.0 hours under an argon atmosphere at a rate of 5 °C / min. After cooling, it was ground to obtain a light yellow g-C3N4 powder.

[0014] Weigh the above g-C3N4 powder and commercial nano TiO2 powder at a mass ratio of 2:1, place them in a 50% ethanol aqueous solution, and ultrasonically disperse for 30 minutes to mix them evenly.

[0015] The mixed suspension was vacuum dried for 24 hours to remove the solvent.

[0016] The dried solid mixture was heated to 550 °C at a rate of 5 °C / min under argon protection and held at this temperature for 2.0 hours.

[0017] After heat treatment, the product is naturally cooled to room temperature and then ground into fine powder to obtain g-C3N4 / TiO2 heterojunction composite material.

[0018] The material was characterized using scanning electron microscopy, transmission electron microscopy, X-ray diffraction, infrared spectroscopy, and X-ray photoelectron spectroscopy. The results confirmed the successful synthesis of a heterojunction structure, with TiO2 nanoparticles uniformly loaded on g-C3N4 sheets (see...). Figures 1-3 ).

[0019] Example 2: Construction of DAO / g-C3N4 / TiO2 / GCE biosensor Pretreatment of glassy carbon electrode: The glassy carbon electrode with a diameter of 3 mm was polished with 0.3 μm and 0.05 μm alumina powder in sequence, and then ultrasonically cleaned with ultrapure water and anhydrous ethanol for 1 minute each, and then dried at room temperature for later use.

[0020] Preparation of heterojunction modification layer: Take 5 mg of the g-C3N4 / TiO2 composite material prepared in Example 1, disperse it in 1 mL of ultrapure water to prepare a dispersion. Take 8.0 μL of this dispersion and drop it onto the surface of the pretreated glassy carbon electrode, and place it in an oven at 45 ℃ until completely dry.

[0021] Enzyme immobilization: 5.0 μL of bovine serum albumin solution (1.0 mM), 8.0 μL of diamine oxidase solution (1.0 U / mL), and 5.0 μL of glutaraldehyde solution (5.0 mM) were sequentially added to the surface of the electrode modified with the composite material. After each addition of a solution, the electrode was dried at 45 °C to finally obtain the DAO / g-C3N4 / TiO2 / GCE biosensor.

[0022] Example 3: Electrochemical performance characterization and parameter optimization of the sensor A three-electrode system was constructed using a CHI 660E electrochemical workstation, with an Ag / AgCl electrode as the reference electrode, a platinum wire electrode as the counter electrode, and the prepared biosensor as the working electrode.

[0023] In a solution containing 5 mM [Fe(CN)6] 3- / 4- Cyclic voltammetry and electrochemical impedance spectroscopy were performed on different modified electrodes in a 0.1 M KCl solution containing a redox couple. The results showed that g-C3N4 / TiO2 / GCE exhibited the lowest charge transfer resistance and the highest response current, demonstrating that the heterojunction effectively improved conductivity (see...). Figure 4 ).

[0024] The sensor's response to histamine was studied using differential pulse voltammetry in 0.1 M PBS buffer. The optimal drop volume of the g-C3N4 / TiO2 dispersion (optimized to 8.0 μL), the pH of the detection system (optimized to 7.0), and the fixed concentration of the DAO enzyme (optimized to 0.6 U / mL) were optimized (see [link to relevant documentation]). Figure 7 ).

[0025] Example 4: Sensor Analysis Performance Test Under optimal conditions, the DPV method was used to determine histamine standard solutions of different concentrations. The results showed that the histamine concentration exhibited a good linear relationship with the oxidation peak current in the range of 0.05 mM to 10 mM, with the linear equation I(μA) = 5.211 C(mM) + 4.70 and a correlation coefficient R² = 0.999. Based on a signal-to-noise ratio (S / N) of 3, the limit of detection was 0.075 μM (see [link to relevant documentation]). Figure 8 ).

[0026] Using six independently prepared sensors to detect a 2.0 mM histamine solution, the relative standard deviation of the current response was 1.52%, indicating good reproducibility of the sensor preparation.

[0027] Adding ten times the concentration of common interfering substances (such as tyramine, putrescine, cadaverine, and phenylethylamine) to a 2.0 mM histamine solution resulted in a sensor response change of less than 7%, demonstrating its excellent anti-interference capability (see [link to original text]). Figure 9 ).

[0028] Example 5: Testing of actual meat samples Sample pretreatment: Fresh beef, mutton, carp, and chicken samples from the market were chopped and homogenized. Appropriate amounts of sample were weighed, and histamine standards at low, medium, and high concentrations were added respectively. Extraction and purification were performed according to literature methods. Finally, the extract was redissolved in 0.1 M PBS for analysis.

[0029] Detection and recovery calculation: The sensor of this invention was used to detect the DPV of the spiked sample extract, and the histamine content was calculated using a standard curve. Simultaneously, high-performance liquid chromatography (HPLC) was used for comparative verification.

[0030] The results are as follows Figure 10 As shown, the spiked recoveries detected by the sensor ranged from 93.60% to 101.8%, which were in good agreement with the results obtained by the HPLC method. The relative standard deviations were all less than 5%, which verified the accuracy and reliability of the method in the application of real complex samples.

Claims

1. An electrochemical biosensor for detecting histamine, characterized in that: It includes a working electrode and a heterojunction composite material layer and a biometric layer sequentially modified on the working electrode; the heterojunction composite material layer comprises g-C3N formed by combining graphitic carbon nitride and titanium dioxide. 4 / TiO2 heterojunction; the biorecognition layer contains diamine oxidase.

2. The biosensor based on g-C3N4 / TiO2 heterojunction according to claim 1, characterized in that: In the g-C3N4 / TiO2 heterojunction, the mass ratio of graphitic carbon nitride to titanium dioxide is 2:

1.

3. The biosensor based on g-C3N4 / TiO2 heterojunction according to claim 1, characterized in that: The working electrode is a glassy carbon electrode.

4. A method for fabricating a biosensor based on a g-C3N4 / TiO2 heterojunction as described in any one of claims 1-3, characterized in that: Includes the following steps: 1) Preparation of g-C3N4 / TiO2 heterojunction composite material; 2) The g-C3N4 / TiO2 heterojunction composite material dispersion is coated on the surface of the working electrode to form a heterojunction composite material layer; 3) Immobilize diamine oxidase on the heterojunction composite material layer to form a biorecognition layer, thereby obtaining the biosensor.

5. The preparation method according to claim 4, characterized in that: In step 1), graphitic carbon nitride and titanium dioxide powder are ultrasonically dispersed in 50% ethanol at a mass ratio of 2:

1. After vacuum drying, the mixture is heat-treated at 550℃ for 2.0 hours under argon protection by heating at 5℃ / min. After cooling, it is ground to obtain g-C3N4 / TiO2 heterojunction composite material.

6. The preparation method according to claim 4, characterized in that: In step 3), BSA solution, DAO solution and glutaraldehyde solution are added dropwise to the surface of the heterojunction composite material layer, and the diamine oxidase is fixed after drying.

7. A method for detecting histamine using a biosensor based on a g-C3N4 / TiO2 heterojunction as described in any one of claims 1-3, characterized in that: Using the biosensor as the working electrode, electrochemical detection technology was employed to quantitatively analyze histamine in the sample.

8. The method according to claim 7, characterized in that: The electrochemical detection technique is differential pulse voltammetry; the detection is carried out in a phosphate buffer solution with a pH of 7.

0.

9. The application of the biosensor based on g-C3N4 / TiO2 heterojunction as described in any one of claims 1-3 in detecting histamine content in food.

Images