Bimetallic single-atom nanozyme electrochemical sensor and its preparation method and application

Abstract

The present invention discloses a bimetallic single-atom nanozyme electrochemical sensor and its preparation method and application. The bimetallic single-atom nanozyme electrochemical sensor is composed of a working electrode, a control electrode, a reference electrode and an electrical signal control device; the working electrode is formed by coating a gallium-copper bimetallic single-atom nanozyme and a conductive carbon slurry on a flexible carrier; the present invention constructs an electrochemical sensor by loading the catalytic material of the gallium-copper bimetallic single-atom nanozyme on an electrode substrate material, which can be used to detect the content of volatile amines in fresh meat, thereby realizing the detection of the freshness of fresh meat.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and more specifically, to a bimetallic single-atom nanoenzyme electrochemical sensor and a preparation method and application thereof. Background Art

[0002] During the processing, transportation, storage and sales of fresh meat, it is extremely susceptible to the influence of external and internal microorganisms, breeding a large number of bacteria. Under the action of specific microorganisms, proteins are further decomposed and volatile amines are produced, causing food to have peculiar smell, deteriorate or corrupt. Volatile amines can not only directly reflect the corruption and deterioration of meat products, but also cause harm to human health. Establishing a real-time and efficient detection method for volatile amines has become a necessary measure to ensure food quality and safety. Among them, traditional detection methods including gas chromatography, gas-mass spectrometry and high-performance liquid chromatography have certain defects, such as the need for professional operation and high cost, which cannot meet the current needs of rapid and portable detection.

[0003] In order to cope with the many challenges in meat freshness detection, some fast and portable sensing detection technologies have been applied to meat freshness detection. For example, colorimetric sensing detection method, fluorescence sensing detection method and electrochemical sensing detection method, but in the actual detection application process, the colorimetric sensing detection method has the defects of low detection sensitivity and strong subjectivity; the fluorescence sensing detection method mostly requires the use of toxic and harmful reagents in the preparation and synthesis process, and there are certain food safety risks. Therefore, compared with the above two sensing detection methods, the electrochemical sensing detection method has the advantages of high sensitivity, simple operation, high cost-effectiveness, green environmental protection, and no pollution, and the current application technology of electrochemical sensing detection method in meat freshness detection has not been reported.

[0004] As a new type of nanomaterial with high catalytic performance, single-atom nanozymes have been widely used in the field of food safety testing. However, in the application of rapid and portable detection of meat freshness, single-atom nanozymes have defects such as low catalytic activity, insufficient selectivity, and poor durability, which prevent them from being widely used in actual detection processes. Therefore, through reasonable design and structural optimization, bimetallic single-atom nanozyme catalytic materials are constructed. The catalytic activity, selectivity and durability can be significantly improved through the synergistic catalytic effect of bimetallic atoms, which is expected to be widely used in the field of meat freshness detection.

[0005] In summary, a fast, portable and low-cost electrochemical sensing detection method was constructed based on the excellent catalytic performance of bimetallic single-atom nanozymes, which has important research significance for the detection of volatile amines produced during the spoilage of meat. Summary of the invention

[0006] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0007] Another object of the present invention is to provide a bimetallic single-atom nanozyme electrochemical sensor and its preparation method and application. The present invention loads gallium-copper bimetallic single-atom nanozyme catalytic material on an electrode substrate material to construct an electrochemical sensor, which can be used to detect the content of volatile amines in fresh meat, and then evaluate the degree of meat spoilage, and finally detect the freshness of fresh meat. The detection method is simple and fast to operate, has high sensitivity, does not require the use of toxic and harmful reagents, and has the advantages of being green and environmentally friendly.

[0008] In order to achieve the purpose and other advantages of the present invention, a method for preparing a bimetallic single-atom nanozyme electrochemical sensor is provided, wherein the bimetallic single-atom nanozyme electrochemical sensor is composed of a working electrode, a control electrode, a reference electrode and an electrical signal control device; the working electrode is formed by coating a gallium-copper bimetallic single-atom nanozyme and a conductive carbon slurry on a flexible carrier;

[0009] The synthesis method of the gallium-copper bimetallic single-atom nanozyme is specifically as follows:

[0010] S1. Mix copper nitrate trihydrate, aluminum nitrate nonahydrate, anhydrous copper chloride, anhydrous gallium chloride and a solvent to obtain solution A; in solution A, the concentration of copper nitrate trihydrate is 12-18 mM, the concentration of aluminum nitrate nonahydrate is 4-7 mM, and the concentrations of anhydrous copper chloride and anhydrous gallium chloride are both 1.5-2.5 mg / mL;

[0011] S2, mixing sodium hydroxide with a solvent to obtain a solution B, wherein the concentration of sodium hydroxide in the solution B is 0.05-0.15 g / mL;

[0012] S3. Add solution A and solution B dropwise into the reaction solvent at the same time, maintain a stable pH during the dropping process, stir to allow sufficient reaction after the dropping is completed, filter and collect the residue after the reaction is completed, wash, dry and grind the residue into powder in sequence to obtain the gallium-copper bimetallic single-atom nanozyme; wherein the ratio of the solvent volume of solution A, the solvent volume of solution B and the reaction solvent volume is 1:1:2.

[0013] Preferably, the method for preparing the bimetallic single-atom nanozyme electrochemical sensor comprises the following steps:

[0014] Step 1: Print a conductive silver paste screen in the middle of the flexible carrier and cure it at 60° C. for 30 min to form a reference electrode after curing;

[0015] Step 2: Printing a conductive carbon paste screen on one side of the flexible carrier, curing at 60° C. for 30 min, and forming a control electrode after curing;

[0016] Step 3: Mix the gallium-copper bimetallic single-atom nanozyme and the conductive carbon paste in a mass ratio of 1:1 to obtain a composite; apply the composite to the other side of the flexible carrier, and form a working electrode after curing;

[0017] Step 4: Print a layer of insulating ink around the flexible carrier and cure it at 60°C for 20 min to obtain a bimetallic single-atom nanozyme electrochemical sensor chip;

[0018] Step 5: Connect the reference electrode, the control electrode and the working electrode to an electrical signal control device to construct the electrochemical sensor.

[0019] Preferably, in the method for preparing the bimetallic single-atom nanozyme electrochemical sensor, the flexible carrier is polyethyleneimine.

[0020] The present invention also provides a bimetallic single-atom nanozyme electrochemical sensor obtained based on the above preparation method.

[0021] The present invention also provides a method for detecting the freshness of meat based on the above-mentioned bimetallic single-atom nanozyme electrochemical sensor, which comprises:

[0022] Step A, preparing fresh meat sample solutions with different storage times, dripping the fresh meat sample solutions onto the bimetallic single-atom nanozyme electrochemical sensor chip, performing electrochemical tests using a chronoamperometry method, recording the corresponding steady-state current values, and drawing a regression curve between storage time and steady-state current values ​​to obtain regression equation 1;

[0023] Step B, determining the volatile basic nitrogen content in the fresh meat sample solution at different storage times, and drawing a regression curve between the storage time and the volatile basic nitrogen content to obtain regression equation 2;

[0024] Step C, based on regression equation 1 and regression equation 2, obtaining a regression curve of the steady-state current value and the volatile basic nitrogen content, and obtaining regression equation 3;

[0025] Step D, dripping the fresh meat sample solution to be tested onto the bimetallic single-atom nanozyme electrochemical sensor chip, obtaining the corresponding steady-state current value, and obtaining the corresponding volatile basic nitrogen content based on regression equation three;

[0026] Step E: evaluating the freshness of the raw meat to be tested based on the volatile basic nitrogen content.

[0027] The present invention has at least the following beneficial effects:

[0028] 1. During the catalytic reaction, gallium needs to overcome less energy than other metals, and the reaction is easier to proceed. The present invention combines gallium and copper to form a bimetallic nanozyme material, and utilizes the synergistic effect of the two metals, gallium and copper, to greatly improve the electron transmission performance during the catalytic reaction, thereby improving the detection sensitivity of the electrochemical sensor. The gallium-copper bimetallic single-atom nanozyme material synthesized by the eutectic precipitation method has a simple, easy-to-operate, economical and environmentally friendly synthesis method.

[0029] 2. The gallium-copper bimetallic single-atom nanozyme material is loaded on a flexible polyethyleneimine carrier using screen printing technology to construct a bimetallic single-atom nanozyme electrochemical sensor. This electrochemical sensor has the advantages of fast detection speed, wide linear range, low detection limit, strong anti-interference ability, high sensitivity, low cost and simple operation.

[0030] 3. The fresh meat freshness detection method constructed by the bimetallic single-atom nanozyme electrochemical sensor provided by the present invention can realize on-site rapid and portable, qualitative and quantitative detection of volatile amines produced during the spoilage of meat. Compared with large-scale instrument detection methods, it does not require complicated sample pretreatment, large-scale equipment and professional detection personnel, and has higher sensitivity.

[0031] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the structure of the bimetallic single-atom nanozyme electrochemical sensor prepared in Example 2 of the present invention;

[0033] Figure 2 Schematic diagram of the adsorption reaction relationship between gallium-copper bimetallic single-atom nanozyme and ammonia water under different temperature conditions in Experimental Example 1 of the present invention;

[0034] Figure 3 This is a schematic diagram of the current-time curve of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor to ammonia water in Experimental Example 2 of the present invention;

[0035] Figure 4 This is a schematic diagram of the response time of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor to ammonia water in Experimental Example 2 of the present invention;

[0036] Figure 5 This is a schematic diagram of the response signal results of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor to different ammonia concentrations in Experimental Example 2 of the present invention;

[0037] Figure 6It is a schematic diagram of the correlation results between different pH values ​​and the steady-state current value of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor responding to ammonia water in Experimental Example 2 of the present invention;

[0038] Figure 7 Schematic diagram of the correlation results between different potentials and the steady-state current value of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor responding to ammonia water in Experimental Example 2 of the present invention;

[0039] Figure 8 It is a schematic diagram of the response relationship between the ammonia concentration and the steady-state current value in Experimental Example 3 of the present invention;

[0040] Fig. 9 This is a schematic diagram of the linear relationship between the ammonia concentration and the steady-state current in Experimental Example 3 of the present invention;

[0041] Fig.10 This is a schematic diagram of the test results of repeated use of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor in Experimental Example 4 of the present invention;

[0042] Fig.11 This is a schematic diagram of the test results of different storage times of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor in Experimental Example 4 of the present invention;

[0043] Fig.12 This is a schematic diagram of the anti-interference test results of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor in Experimental Example 5 of the present invention;

[0044] Fig.13 This is a schematic diagram showing the statistical results of the steady-state current values ​​of pork, beef, mutton and chicken at different time periods under 25° C. storage conditions using the gallium-copper bimetallic single-atom nanozyme electrochemical sensor in Experimental Example 6 of the present invention;

[0045] Fig.14 This is a schematic diagram showing the statistical results of the steady-state current values ​​of pork, beef, mutton and chicken at different time periods under 4° C. storage conditions using the gallium-copper bimetallic single-atom nanozyme electrochemical sensor in Experimental Example 6 of the present invention;

[0046] Fig.15 This is a schematic diagram of the statistical results of the volatile basic nitrogen (TVB-N) values ​​of pork, beef, mutton and chicken at different time periods under the storage condition of 25° C. in Test Example 6 of the present invention;

[0047] Fig.16 This is a schematic diagram of the statistical results of the volatile basic nitrogen (TVB-N) values ​​of pork, beef, mutton and chicken at different time periods under the storage condition of 4°C in Experimental Example 6 of the present invention;

[0048] Fig.17It is a schematic diagram of the correlation results between the steady-state current value and the volatile basic nitrogen (TVB-N) of pork, beef, mutton and chicken under the storage condition of 25° C. in Test Example 6 of the present invention;

[0049] Fig.18 This is a schematic diagram of the correlation results between the steady-state current values ​​and volatile basic nitrogen (TVB-N) of pork, beef, mutton and chicken under 4°C storage conditions in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments so that those skilled in the art can implement the invention with reference to the description.

[0051] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0052] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0053] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used.

[0054] In the following examples, the instruments and equipment used without indicating the manufacturer are all conventional products that can be purchased through regular channels. The methods described are conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0055] The present invention provides a method for preparing a bimetallic single-atom nanozyme electrochemical sensor, wherein the bimetallic single-atom nanozyme electrochemical sensor is composed of a working electrode, a control electrode, a reference electrode and an electrical signal control device; the working electrode is formed by coating a gallium-copper bimetallic single-atom nanozyme and a conductive carbon slurry on a flexible carrier;

[0056] The synthesis method of the gallium-copper bimetallic single-atom nanozyme is specifically as follows:

[0057] S1. Mix copper nitrate trihydrate, aluminum nitrate nonahydrate, anhydrous copper chloride, anhydrous gallium chloride and a solvent to obtain solution A; in solution A, the concentration of copper nitrate trihydrate is 12-18 mM, the concentration of aluminum nitrate nonahydrate is 4-7 mM, and the concentrations of anhydrous copper chloride and anhydrous gallium chloride are both 1.5-2.5 mg / mL;

[0058] S2, mixing sodium hydroxide with a solvent to obtain a solution B, wherein the concentration of sodium hydroxide in the solution B is 0.05-0.15 g / mL;

[0059] S3. Add solution A and solution B dropwise into the reaction solvent at the same time, maintain a stable pH during the dropping process, react under stirring for 14 to 20 hours after the dropping is completed, filter and collect the residue after the reaction is completed, wash, dry and grind the residue into powder in turn to obtain the gallium-copper bimetallic single-atom nanozyme, wherein the ratio of the solvent volume of solution A, the solvent volume of solution B and the reaction solvent volume is 1:1:2.

[0060] The solvents used in step S1 and step S2, and the reaction solvent in step S3 are all ultrapure water.

[0061] In step S3, the stable pH value is 8.4-8.6; in step S3, the filter residue is washed by using anhydrous ethanol and ultrapure water for multiple times in sequence; the drying temperature of the washed filter residue is 50-70°C.

[0062] In another technical solution, the preparation method of the bimetallic single-atom nanozyme electrochemical sensor comprises the following steps:

[0063] Step 1: Print a conductive silver paste screen in the middle of the flexible carrier and cure it at 60°C for 30 min to form a reference electrode;

[0064] Step 2: Print a conductive carbon paste screen on one side of the flexible carrier and cure it at 60° C. for 30 min to form a control electrode;

[0065] Step 3: Mix the gallium-copper bimetallic single-atom nanozyme and the conductive carbon paste in a mass ratio of 1:1 to obtain a composite; apply the composite to the other side of the flexible carrier, and form a working electrode after curing;

[0066] Step 4: Print a layer of insulating ink around the flexible carrier and cure it at 60°C for 20 min to obtain a bimetallic single-atom nanozyme electrochemical sensor chip;

[0067] Step 5: Connect the reference electrode, the control electrode and the working electrode to an electrical signal control device to construct the electrochemical sensor.

[0068] In another technical solution, in the method for preparing the bimetallic single-atom nanozyme electrochemical sensor, the flexible carrier is polyethyleneimine.

[0069] The present invention also provides a bimetallic single-atom nanozyme electrochemical sensor obtained based on the above preparation method.

[0070] The present invention also provides a method for detecting the freshness of meat based on the above-mentioned bimetallic single-atom nanozyme electrochemical sensor, which comprises:

[0071] Step A, preparing fresh meat sample solutions with different storage times, dripping the fresh meat sample solutions onto the bimetallic single-atom nanozyme electrochemical sensor chip, performing electrochemical tests using a chronoamperometry method, recording the corresponding steady-state current values, and drawing a regression curve between storage time and steady-state current values ​​to obtain regression equation 1;

[0072] Step B, determining the volatile basic nitrogen content in the fresh meat sample solution at different storage times, and drawing a regression curve between the storage time and the volatile basic nitrogen content to obtain regression equation 2;

[0073] Step C, based on regression equation 1 and regression equation 2, obtaining a regression curve of the steady-state current value and the volatile basic nitrogen content, and obtaining regression equation 3;

[0074] Step D, dripping the fresh meat sample solution to be tested onto the bimetallic single-atom nanozyme electrochemical sensor chip, obtaining the corresponding steady-state current value, and obtaining the corresponding volatile basic nitrogen content based on regression equation three;

[0075] Step E: assessing the degree of spoilage of the fresh meat to be tested based on the volatile basic nitrogen content, thereby detecting the freshness of the meat.

[0076] In actual testing, meat usually has two storage conditions, room temperature (25°C) and refrigerator refrigeration (4°C). Therefore, the following uses 25°C and 4°C storage conditions as examples to further introduce this technology.

[0077] Construct a correlation model between the steady-state current value of meat and the concentration of volatile basic nitrogen in meat:

[0078] ① Construct the relationship between different storage times and corresponding steady-state current values: for example, at a storage temperature of 25°C, the meat sample solutions were stored for 0, 6, 12, 18, 24, 30, 36, 42, and 48 hours, respectively, and were added dropwise to the bimetallic single-atom nanozyme electrochemical sensor chip, respectively. The electrochemical test was performed by chronoamperometry, and the current was scanned to record the corresponding steady-state current value. The linear regression curve between time and steady-state current value was plotted with storage time as the horizontal coordinate and steady-state current value as the vertical coordinate, and the standard curve between storage time and steady-state current value was obtained. The relationship model formula of Y1=aX1 was obtained, which was recorded as regression equation 1, where Y1 is the steady-state current value and X1 is the storage time; at a storage temperature of 4°C, the meat sample solutions were stored for 1, 3, 5, 7, and 9 days, respectively, and the rest was the same as above. The linear regression curve between time and steady-state current value under the storage condition of 4°C was plotted, and the corresponding regression equation was obtained;

[0079] ② Draw the relationship between different storage time and volatile basic nitrogen content: At a storage temperature of 25°C, the meat sample solutions were stored for 0, 6, 12, 18, 24, 30, 36, 42, and 48 hours, and the volatile basic nitrogen content was determined in accordance with the national standard testing requirements GB5009.228-2016. The linear regression curve of storage time and volatile basic nitrogen content was drawn with storage time as the horizontal axis and volatile basic nitrogen content as the vertical axis. The regression curve of storage time and volatile basic nitrogen content was obtained, and the relationship model formula of Y2=aX2 was obtained, which was recorded as regression equation 2, where Y2 is the volatile basic nitrogen content and X2 is the storage time. As above, the regression curve of storage time and volatile basic nitrogen content under 4°C storage conditions was drawn and the corresponding regression equation was obtained.

[0080] ③ Establish the relationship between the steady-state current value and the volatile basic nitrogen content: Under the storage condition of 25°C, based on linear regression equation 1 and regression equation 2, with the steady-state current value as the horizontal coordinate and the volatile basic nitrogen content as the vertical coordinate, draw the linear regression curve of the steady-state current value and the volatile basic nitrogen content under the storage condition of 25°C, and obtain the regression curve of the steady-state current value and the volatile basic nitrogen content, and obtain the correlation model formula of Y=aX, recorded as regression equation 3, where Y is the volatile basic nitrogen content and X is the steady-state current value; as above, draw the regression curve of the steady-state current value and the volatile basic nitrogen content under the storage condition of 4°C and obtain the corresponding regression equation.

[0081] Weigh about 5 g of the meat sample to be tested stored at room temperature (25°C) or in a refrigerator (4°C), treat it with 25 mL of ultrapure water for 30 min, and collect the supernatant by centrifugation. Drop 50-150 μL of the supernatant on the gallium-copper bimetallic single-atom nanozyme electrochemical sensor chip (the method for preparing the above meat sample solution is the same as this method), measure the steady-state current value, and obtain the corresponding volatile basic nitrogen content of the meat to be tested based on the corresponding correlation model (under the same storage conditions).

[0082] In order to meet the needs of fast, efficient and portable food detection, the present invention provides a bimetallic single-atom nanozyme electrochemical sensor and its preparation method and application, which are used to detect volatile amines produced during the spoilage of fresh meat.

[0083] During the entire detection process, gallium-copper bimetallic single-atom nanozyme was first synthesized by the eutectic precipitation method, and then the gallium-copper bimetallic single-atom nanozyme electrochemical sensor chip was prepared and synthesized based on the screen printing technology; pork, beef, mutton and chicken sample solutions were dripped onto the electrochemical sensor chip, and an electrochemical test using the chronoamperometry method was carried out. By observing the changes in the steady-state current value, the changes in freshness were quantitatively identified, and at the same time, a correlation model between the steady-state current value and the volatile basic nitrogen content was constructed, thereby achieving reliable quantitative signal output detection.

[0084] 1. Experimental Materials

[0085] Anhydrous copper chloride (CuCl2, analytical grade, 98%), anhydrous gallium chloride (GaCl3, analytical grade, 98%), and ammonia (AA, analytical grade, 98%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Aluminum nitrate (Al(NO3)3, analytical grade, 98%) was purchased from Sinopharm Group Co., Ltd. Cadaverine (Cad, analytical grade, 98%) was obtained from Beijing Guangda Hengyi Technology Co., Ltd. Histamine (His, analytical grade, 98%), spermine (Spe, analytical grade, 98%), and putrescine (Put, analytical grade, 98%) were purchased from Beijing Baikai Biotechnology Co., Ltd. Tryptamine (Try, analytical grade, 98%) was purchased from Beijing Sola Biotechnology Co., Ltd. Phenylethylamine (Phe, analytical grade, 98%) was purchased from Beijing Nuochuang Chemical Technology Co., Ltd. Ultrapure water (18.2 MΩ, Millpore) was used to prepare solutions in all experiments. The portable electrochemical workstation (length × width × height = 45 mm × 21 mm × 4 mm, 19.1262 g) was purchased from Shenzhen Refresh Biosensor Technology Co., Ltd. The electrochemical sensor chip (length × width × height = 7 mm × 4 mm × 0.25 mm, 0.1461 g) was from Weihai Boteng Technology Co., Ltd. Pork, beef, mutton, and chicken were purchased from local supermarkets.

[0086] Example 1

[0087] Synthesis of Gallium-Copper Bimetallic Single-Atom Nanozymes

[0088] S1. Dissolve copper nitrate trihydrate (Cu(NO3)2•3H2O), aluminum nitrate nonahydrate (Al(NO3)3•9H2O), 40 mg anhydrous copper chloride (CuCl2), and 40 mg anhydrous gallium chloride (GaCl3) in 20 mL ultrapure water to obtain solution A; the concentration of copper nitrate trihydrate in solution A is 16 mM, and the concentration of aluminum nitrate nonahydrate is 5.33 mM;

[0089] S2. Dissolve 1.366 g of sodium hydroxide (NaOH) in 20 mL of ultrapure water to obtain solution B;

[0090] S3. Add solution A and solution B dropwise into 40 mL of reaction solvent at the same time, and maintain a stable pH of 8.6 during the dropping process (the pH is controlled by adjusting the dropping speed of solution A and solution B respectively). After the dropwise addition of one of the solutions (either solution A or solution B) is completed, stir the reaction at room temperature for 16 hours, stop the reaction, filter the reaction solution and collect the solid residue, wash the residue with anhydrous ethanol for multiple times, then wash the residue with ultrapure water for multiple times, dry at 60°C, and then grind into powder to obtain the gallium-copper bimetallic single-atom nanozyme.

[0091] Example 2

[0092] Preparation of Gallium-Copper Bimetallic Single-Atom Nanozyme Electrochemical Sensor

[0093] A. Print the conductive silver paste (silver / silver chloride, model: AG-7) on the middle of the flexible polyethyleneimine carrier by screen printing technology and cure it at 60°C for 30 min to form a reference electrode;

[0094] B. Print the conductive carbon paste (model: TC-205) on one side of the flexible polyethyleneimine carrier by screen printing technology and cure it at 60°C for 30 min to form a control electrode;

[0095] C. The synthesized gallium-copper bimetallic single-atom nanozyme solid powder was mixed with the conductive carbon paste in a mass ratio of 1:1 to prepare a composite; the composite was applied to the other side of the flexible polyethyleneimine carrier and cured at 60°C for 30 minutes to form a working electrode;

[0096] D. Then, a layer of insulating ink (model: LCN-D2) was printed around the polyethyleneimine carrier and cured at 60°C for 20 min to obtain a bimetallic single-atom nanozyme electrochemical sensor chip; the overall length, width and height of the chip are: 7 mm × 4 mm × 0.25 mm. The diameter of the circular area is 4 mm, the weight is 0.1461 g, and the composite loading is 2 mg;

[0097] E. Connect the reference electrode, the control electrode and the working electrode to an electrical signal control device (electrochemical workstation) to construct a bimetallic single-atom nanozyme electrochemical sensor; The present invention uses screen printing technology to construct an electrochemical sensor, specifically as follows Figure 1 As shown, the collaborative portable electrochemical workstation integrates an electrochemical detection platform.

[0098] In the following test examples, data analysis was performed using Origin software.

[0099] Test Example 1

[0100] Interaction between gallium-copper bimetallic single-atom nanozymes and volatile amines

[0101] In order to explore the interaction between gallium-copper bimetallic single-atom nanozymes and volatile amines, firstly, the present invention uses in situ Fourier transform infrared spectroscopy to explore the adsorption reaction relationship between gallium-copper bimetallic single-atom nanozymes and ammonia water under different temperature conditions, with ammonia water as a representative. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that with the increase of temperature, the GaCu bimetallic single-atom nanozyme in situ system has a peak at 2425 cm -1An obvious absorption peak was generated at 2425 cm-1, and as the temperature further increased to 325°C, the peak value reached the highest, indicating that the adsorption and binding ability between ammonia water and gallium-copper bimetallic single-atom nanozymes was the strongest. -1 The peaks at all showed a clear downward trend, indicating that a desorption reaction began to occur between ammonia water and the gallium-copper bimetallic single-atom nanozyme. It can be seen that there is an adsorption effect between the gallium-copper bimetallic single-atom nanozyme prepared by the present invention and the volatile amine.

[0102] Test Example 2

[0103] Optimization of detection parameters of bimetallic single-atom nanozyme electrochemical sensor

[0104] Taking ammonia water as the detection object, the sensing performance of the bimetallic single-atom nanozyme electrochemical sensor was optimized. Specifically, ammonia water was dropped onto the bimetallic single-atom nanozyme electrochemical sensor chip, and the electrochemical test was carried out using the chronoamperometry to record the steady-state current value.

[0105] Ammonia water with a concentration of 0.2 mM was added to the sensor chip of the bimetallic single-atom nanozyme electrochemical sensor, and the scanning current graph was shown in FIG. Figure 3~4 As shown by Figure 3~4 It can be seen that the current-time curve of the electrochemical sensor chip of the bimetallic single-atom nanozyme after contact with 0.2 mM ammonia solution has changed significantly compared with the control group (baseline) (the control group is to add an equal amount of ultrapure water on the chip) ( Figure 3 ), indicating that the bimetallic single-atom nanozyme electrochemical sensor chip has a good signal response to ammonia water, and the response time is about 4 s ( Figure 4 ).

[0106] Ammonia water with different concentrations (0.1 mM, 0.2 mM and 0.4 mM) was set up for the electrochemical test of the gallium-copper bimetallic single-atom nanozyme electrochemical sensor, and the corresponding steady-state current values ​​were recorded. Figure 5 As shown by Figure 5 It can be seen that as the concentration of ammonia water increases, the response signal also increases.

[0107] Ammonia water (0.2 mM) was used to optimize the pH value and potential of the detection conditions. Experiments with different pH values ​​(2.0, 4.0, 6.0, 8.0 and 12.0) and different potentials (300 mV, 400 mV, 500 mV, 600 mV and 700 mV) were set up and the corresponding steady-state current values ​​were recorded. The correlation results between different pH values ​​and steady-state current values ​​are shown in the figure. Figure 6 The correlation results between different potentials and steady-state current values ​​are shown in Figure 7 As shown by Figures 6 and 7It can be seen that the optimal response pH value of the bimetallic single-atom nanozyme electrochemical sensor to ammonia water is 4.0 and the potential is 600 mV.

[0108] Test Example 3

[0109] Detection sensitivity test of bimetallic single-atom nanozyme electrochemical sensor

[0110] Taking ammonia as an example, the sensitivity response test of the bimetallic single-atom nanozyme electrochemical sensor to volatile amines was carried out under the above-mentioned optimized detection conditions (pH=4.0, potential of 600 mV). In the linear range of ammonia concentration of 0.05 mM - 1 mM, the response relationship between ammonia concentration and steady-state current value was constructed. Figure 8 As shown; in the range of 0.05 mM -0.40 mM, the ammonia concentration and the steady-state current show a good linear relationship, as shown in Fig. 9 As shown, the decision coefficient R 2 =0.9906, correlation coefficient R=0.9953, and detection limit of 5.9 μM.

[0111] Test Example 4

[0112] Repeatability and stability test of bimetallic single-atom nanozyme electrochemical sensor

[0113] Under the detection conditions of 0.2 mM ammonia concentration, pH=4.0 and 600 mV potential, the detection activity of the repeated use of the bimetallic single-atom nanozyme electrochemical sensor was tested. After each scan, the chip of the bimetallic single-atom nanozyme electrochemical sensor was activated. The specific operation was to add 100 μL of 0.01 μM standard hydrochloric acid solution on the bimetallic single-atom nanozyme electrochemical sensor chip, let it stand for 2 min, then wipe it with absorbent paper, then add 100 μL of ultrapure water and let it stand for 2 min, wipe it with absorbent paper, repeat the operation twice and then perform the next test, record the current scan graph corresponding to each test, and calculate the relative activity [relative activity = (steady-state current value of the first scan-steady-state current value of the current scan) / steady-state current value of the first scan × 100%)]. The results are as follows Fig.10 (No.1 to No.7 are reused 1 to 7 times respectively, and the blank represents the reference of the control group) Fig.10 It can be seen that after being reused four times, the activity of the sensor chip can still be as high as 80%;

[0114] Similarly, under the detection conditions of 0.2 mM ammonia concentration, pH = 4.0 and 600 mV potential, the detection activity of the bimetallic single-atom nanozyme electrochemical sensor chip at different storage times (1, 3, 7, 14, 21 and 28 days) was tested. The results are as follows Fig.11As shown by Fig.11 It can be seen that after storage at room temperature for one week, the activity can still be maintained at 60% or above.

[0115] Test Example 5

[0116] Anti-interference test of gallium-copper bimetallic single-atom nanozyme electrochemical sensor

[0117] Ultrapure water was used as the solvent to prepare Na + (NaCl)、K + (KCl)、Cl - (NaCl)、NO 3- (NaNO3), SO4 2- (Na2SO4), urea, glucose, methionine, lysine, vitamin E, hydrogen sulfide, ammonia, cadaverine, histamine, putrescine, tryptamine, phenylethylamine solution, and a mixture of the above solutions (the concentration of each substance is 0.25mM);

[0118] Under the detection conditions of pH = 4.0 and potential of 600 mV, 100 μL of the above solution was added to the gallium-copper bimetallic single-atom nanozyme electrochemical sensor chip, and the steady-state current values ​​corresponding to each solution were recorded respectively. The specific results are as follows: Fig.12 As shown by Fig.12 It can be seen that the bimetallic single-atom nanozyme electrochemical sensor provided by the present invention has good anti-interference performance.

[0119] Test Example 6

[0120] Quantitative detection of livestock and poultry meat freshness using bimetallic single-atom nanozyme electrochemical sensor

[0121] Livestock (pork, beef and mutton) and poultry (chicken) were selected for actual sample testing. They were cut into small pieces of about 80 grams each and stored in commercially available fresh boxes at 25°C (durations of 0, 6, 12, 18, 24, 30, 36, 42 and 48 hours) and 4°C (durations of 0, 1, 3, 5, 7 and 9 days). Meat samples were taken at different time points, each weighing about 5 g, ultrasonically treated with 25 mL of ultrapure water for 30 min, and the supernatant was collected by centrifugation. 100 μL of the supernatant was dropped onto the gallium-copper bimetallic single-atom nanozyme electrochemical sensor chip for steady-state current value measurement. The results are shown in Figure 2. Fig.13 and 14 As shown in the figure, the experiment used the excellent sensing performance of the bimetallic single-atom nanozyme electrochemical sensor to detect the volatile amine content of pork, beef, mutton and chicken at different time points under storage conditions of 25℃ and 4℃, in order to evaluate the freshness of the meat. Figures 13 and 14It can be seen that as the storage time increases, the steady-state current value of the bimetallic single-atom nanozyme electrochemical sensor decreases;

[0122] In order to verify the reliability of the test results, the volatile basic nitrogen (TVB-N) values ​​of pork, beef, mutton and chicken samples at 25°C and 4°C at different storage times were tested according to the national standard method (GB 5009.228-2016, GB 2707-2016). The results are as follows: Figures 15 and 16 As shown in the figure, according to the GB5009.228-2016 standard, fresh meat can be judged to have spoiled if the TVB-N content exceeds 15 mg / 100g. Fig.15 It can be seen that at a storage temperature of 25°C, the spoilage time of pork, beef, mutton and chicken is 18, 24, 36 and 12 hours respectively. Fig.16 It can be seen that at a storage temperature of 4°C, the spoilage time of pork, beef, mutton and chicken is 5, 7, 7 and 3 days respectively;

[0123] Based on the above results, the response relationship between the steady-state current value and TVB-N value of each meat product was constructed. At a storage temperature of 25°C, there was a good linear relationship between the steady-state current value and TVB-N detected at different storage time points for pork, beef, mutton and chicken, such as Fig.17 As shown, the decision coefficient is R 2 =0.8521, correlation coefficient R=0.9231;

[0124] At 4°C storage temperature, there is also a good linear relationship between the steady-state current values ​​detected at different storage time points and TVB-N for pork, beef, mutton, and chicken, such as Fig.18 As shown, the decision coefficient is R 2 =0.9144, correlation coefficient R=0.9562;

[0125] In summary, the bimetallic single-atom nanozyme electrochemical sensor constructed in the present invention can realize reliable freshness detection of fresh livestock and poultry meat stored under room temperature (25°C) and refrigerated (4°C) conditions.

[0126] The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention will be obvious to those skilled in the art.

[0127] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a bimetallic single-atom nanozyme electrochemical sensor, characterized in that: The bimetallic single-atom nanozyme electrochemical sensor is composed of a working electrode, a control electrode, a reference electrode and an electrical signal control device; the working electrode is formed by coating a gallium-copper bimetallic single-atom nanozyme and a conductive carbon slurry on a flexible carrier; The synthesis method of the gallium-copper bimetallic single-atom nanozyme is specifically as follows: S1. Mix copper nitrate trihydrate, aluminum nitrate nonahydrate, anhydrous copper chloride, anhydrous gallium chloride and a solvent to obtain solution A; in solution A, the concentration of copper nitrate trihydrate is 12-18 mM, the concentration of aluminum nitrate nonahydrate is 4-7 mM, and the concentrations of anhydrous copper chloride and anhydrous gallium chloride are both 1.5-2.5 mg / mL; S2, mixing sodium hydroxide with a solvent to obtain a solution B, wherein the concentration of sodium hydroxide in the solution B is 0.05-0.15 g / mL; S3. Add solution A and solution B dropwise into the reaction solvent at the same time, maintain a stable pH during the dropping process, stir to allow sufficient reaction after the dropping is completed, filter and collect the residue after the reaction is completed, wash, dry and grind the residue into powder in sequence to obtain the gallium-copper bimetallic single-atom nanozyme; wherein the ratio of the solvent volume of solution A, the solvent volume of solution B and the reaction solvent volume is 1:1:

2.

2. The method for preparing the bimetallic single-atom nanozyme electrochemical sensor according to claim 1, characterized in that: The following steps are involved: Step 1: Print a conductive silver paste screen in the middle of the flexible carrier and cure it at 60° C. for 30 min to form a reference electrode after curing; Step 2: Printing a conductive carbon paste screen on one side of the flexible carrier, curing at 60° C. for 30 min, and forming a control electrode after curing; Step 3: Mix the gallium-copper bimetallic single-atom nanozyme and the conductive carbon paste in a mass ratio of 1:1 to obtain a composite; apply the composite to the other side of the flexible carrier, and form a working electrode after curing; Step 4: Print a layer of insulating ink around the flexible carrier and cure it at 60°C for 20 min to obtain a bimetallic single-atom nanozyme electrochemical sensor chip; Step 5: Connect the reference electrode, the control electrode and the working electrode to an electrical signal control device to construct the electrochemical sensor.

3. The method for preparing the bimetallic single-atom nanozyme electrochemical sensor according to claim 2, characterized in that: The flexible carrier is polyethyleneimine.

4. A bimetallic single-atom nanozyme electrochemical sensor obtained based on the preparation method described in claims 1-3.

5. The meat freshness detection method based on the bimetallic single-atom nanozyme electrochemical sensor according to claim 4 is characterized in that: include: Step A, preparing fresh meat sample solutions with different storage times, dripping the fresh meat sample solutions onto the bimetallic single-atom nanozyme electrochemical sensor chip, performing electrochemical tests using a chronoamperometry method, recording the corresponding steady-state current values, and drawing a regression curve between storage time and steady-state current values ​​to obtain regression equation 1; Step B, determining the volatile basic nitrogen content in the fresh meat sample solution at different storage times, and drawing a regression curve between the storage time and the volatile basic nitrogen content to obtain regression equation 2; Step C, based on regression equation 1 and regression equation 2, obtaining a regression curve of the steady-state current value and the volatile basic nitrogen content, and obtaining regression equation 3; Step D, dripping the fresh meat sample solution to be tested onto the bimetallic single-atom nanozyme electrochemical sensor chip, obtaining the corresponding steady-state current value, and obtaining the corresponding volatile basic nitrogen content based on regression equation three; Step E: evaluating the freshness of the raw meat to be tested based on the volatile basic nitrogen content.

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