A method for dual-mode quantitative detection of hypoxanthine to distinguish freshness of meat

Abstract

A dual-modal quantitative detection method for hypoxanthine to determine meat freshness includes the following steps: Step 1: Measuring the RGB values ​​of the color signal after the sample reaction is complete; Step 2: Measuring the temperature T of the reaction solution; Step 3: Constructing a linear equation between hypoxanthine concentration and color signal B / G or temperature T; Obtaining the color signal B / G based on a series of standard tests at different concentrations, and constructing the linear equation B / G = XC. Hx +y, where C Hx The concentration of the standard is given; based on the temperature T obtained from the testing of a series of standard concentrations, a linear equation is constructed: T = XC. Hx +y, where C Hx For the standard concentration; Step 4: Take the sample to be tested and repeat steps 1 and 2 to obtain the color signal B / G. n and temperature T n B / G n and T n Substituting the values ​​into the corresponding linear equation, the concentration of hypoxanthine in the sample is obtained. This invention employs a colorimetric-photothermal dual-modal detection method, which improves the reliability of the detection.

Description

Technical Field

[0001] This invention relates to a dual-modal quantitative detection method for hypoxanthine to determine the freshness of meat, belonging to the field of food testing technology. Background Technology

[0002] Meat freshness refers to the growth and decomposition process of microorganisms and enzymes in meat products. Its freshness level affects the quality and safety of meat products, directly impacting food hygiene and human health. Whether it's traditional meat products or the rapidly developing ready-to-eat meals, rapid, accurate, and effective methods for detecting meat product freshness remain crucial for ensuring food safety and health. Traditionally, meat freshness assessment is primarily based on human sensory characteristics, such as texture, odor, and color. However, this method suffers from subjectivity and reliability issues. In contrast, evaluating meat freshness by measuring the concentration of certain substances, such as volatile amines, histamine, trimethylamine, and degradation products of adenosine triphosphate (ATP), is more accurate and faster. Hypoxanthine (Hx), a product of ATP decomposition following a series of reactions during meat spoilage, accumulates in spoiled meat products. Therefore, hypoxanthine content is a key indicator of meat freshness, and achieving hypoxanthine measurement and freshness assessment has become a research hotspot in the meat processing industry.

[0003] Common methods for measuring hypoxanthine include high-performance liquid chromatography (HPLC), electrochemical methods, and spectral analysis-based methods. However, these methods often require specialized equipment and operators, resulting in high costs. To overcome these limitations, colorimetric detection is widely used by researchers due to its convenience, simplicity, low cost, and visualization advantages. Existing patent CN114113506A discloses a method for detecting hypoxanthine content in spoiled meat based on Fe-PDA nanozymes. This method uses a UV spectrophotometer to measure the absorbance intensity changes at 652 nm under different concentrations of hypoxanthine, establishing a calibration curve for hypoxanthine detection. The obtained LOD is 1.54 μmol / L. However, single colorimetric modes often lack sufficient accuracy. A visualized colorimetric and photothermal dual-modal strategy overcomes the limitations of single modes and demonstrates great potential for on-site quantitative detection of hypoxanthine. Summary of the Invention

[0004] The purpose of this invention is to provide a method for determining the freshness of meat by quantitative on-site detection of hypoxanthine based on bimetallic nanozymes in a dual-modal manner.

[0005] To achieve the above and other related objectives, the technical solution provided by this invention is: a method for quantitatively detecting hypoxanthine in a dual-modal manner to determine the freshness of meat, comprising the following steps:

[0006] Step 1: Measure the RGB values ​​of the color signal after the sample reaction is complete.

[0007] At room temperature, xanthine oxidase solution, 3,3,5,5-tetramethylbenzidine solution, Fe@CeO2 nanozyme and standard xanthine solutions of different concentrations were reacted in HAc-NaAc buffer. After the reaction was completed, the complete reaction solution was obtained, and color signals were collected using a smartphone with the Color Picker App installed.

[0008] Step 2: Measure the temperature T of the reaction solution:

[0009] After cooling the complete reaction solution in step 1 to room temperature, it was irradiated with a near-infrared 660nm laser. The solution after irradiation with the 660nm laser was photographed with a thermal imager and the temperature T of the reaction solution was recorded.

[0010] Step 3: Construct a linear equation between hypoxanthine concentration and color signal B / G or temperature T.

[0011] The color signal B / G was obtained from tests of a series of standard samples at varying concentrations. A linear equation was then constructed: B / G = XC. Hx +y, where C Hx For standard concentration;

[0012] Based on temperature differences T obtained from tests of a series of standard samples at varying concentrations, a linear equation is constructed: T = XC Hx +y, where C Hx For standard concentration;

[0013] Step 4: Take the sample to be tested and repeat steps 1 and 2 to obtain the color signal B / G. n and temperature T n B / G n and T n Substitute the values ​​into the corresponding linear equation to obtain the concentration of hypoxanthine in the sample to be tested.

[0014] The preferred technical solution is as follows: In step 1, the pH value of the HAc-NaAc buffer is 5.0; the concentration of Fe@CeO2 nanozyme is 50.0 μg / mL; the concentration of 3,3,5,5-tetramethylbenzidine solution is 1.0 mmol / L; the concentration of xanthine oxidase solution is 1.0 U / mL; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 32.0-37.0℃; and the reaction time is 40.0-50.0 min.

[0015] The preferred technical solution is that the laser irradiation time is 120.0-170.0s.

[0016] The preferred technical solution is as follows: In step 3, the B / G regression equation for the colorimetric signal is B / G = 0.00229C.Hx +0.97975; the photothermal temperature regression equation is T = 0.10087°C. Hx +28.64486.

[0017] The preferred technical solution is as follows: Meat freshness is judged using the following method: The freshness standard can be measured by the meat freshness K-value, which is the percentage of the sum of inosine adenosine and inosine (ATP) degradation products to the total amount of ATP-related compounds. The fresher the meat, the smaller the K-value, and vice versa. Taking common fish as an example, fish with a K-value of 20% are considered fresh, while those with a K-value higher than 70% are considered spoiled. For raw fish products or fresh meat products, a freshness K-value greater than 10% indicates spoilage. Specifically, when judging aquatic products, products with an Hx content ≤ 72.0 mg / kg are considered fresh; products with an Hx content of 72.0 mg / kg are considered fresh. <C Hx Products with a Hx content ≤118.0 mg / kg are considered less fresh; products with an Hx content ≥118.0 mg / kg are considered spoiled. Different types of meat and different storage conditions can lead to different Hx levels, therefore, the country needs to further develop a database or standard for Hx content assessment for different types of meat and storage conditions.

[0018] The preferred technical solution is as follows: the preparation method of the Fe@CeO2 nanozyme includes:

[0019] S1: Mix polyethylene glycol, ammonia, and ethanol in a brown bottle, stir until homogeneous, and then add to the tannic acid solution; react at room temperature for 3.0-8.0 min, then add formaldehyde solution and stir for 20.0-30.0 h;

[0020] S2: Add Ce(NO3)3 to the reaction system obtained in S1, and stir for 10.0-14.0 h; then, transfer the mixture to a reaction vessel, seal it, and place it in an oven to react at 95.0-105.0℃ for 10.0-14.0 h; after the reaction is completed, allow the reaction vessel to cool naturally to room temperature; finally, centrifuge the reactants, collect the precipitate after centrifugation, wash it with ethanol and centrifuge it again, and dry it; then heat the precipitate in a muffle furnace at 380.0-420.0℃ for 1.5-2.5 h to obtain CeO2 nanospheres;

[0021] S3: Disperse CeO2 nanospheres in deionized water, then add a DMSO solution containing ferrocene carboxylic acid, and stir for 5.0-7.0 h; subsequently, collect the Fe@CeO2 nanozyme by centrifugation, wash with ethanol and centrifuge again, then dry for later use. The purity of the ethanol, ammonia, tannic acid and formaldehyde solutions should be analytical grade.

[0022] Due to the application of the above technical solution, the advantages of this invention compared with the prior art are:

[0023] 1. The Fe@CeO2 nanozyme designed and synthesized in this invention has excellent peroxidase activity, and the method used does not require the addition of H2O2 and is simple to operate.

[0024] 2. The present invention has a short detection time, requiring only 45.0 minutes to observe the change in reagent color with the naked eye, thus realizing the detection of hypoxanthine.

[0025] 3. This invention employs a colorimetric-photothermal dual-modal detection method, which improves the reliability of the detection.

[0026] 4. This invention does not require complex instruments; it only requires a smartphone with the Color Picker App and a handheld thermal imager to collect colorimetric and photothermal signals.

[0027] 5. This invention enables the quantitative detection of hypoxanthine in fresh meat, aquatic products, processed meat products, and pre-cooked dishes, which has strong practical significance. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of a dual-mode sensing platform based on Fe@CeO2 nanozymes. This platform combines colorimetry and photothermal signal cascade catalytic enhancement technology for on-site detection of Hx to determine meat freshness.

[0029] Figure 2 Characterization of Fe@CeO2 nanozymes. (A) TEM image of Fe@CeO2 and (B) EDS spectrum; (C) TEM image; (D, E, F) elemental spectra of Fe, O and Ce, respectively.

[0030] Figure 3 Verification of the peroxidase activity of Fe@CeO2 bimetallic nanozymes (A) and feasibility of detecting Hx using the Fe@CeO2+XOD+TM B system (B).

[0031] Figure 4 The catalytic kinetics of Fe@CeO2 nanozymes were tested using double-reflection plots of H2O2 concentration versus reaction rate and TMB concentration versus reaction rate.

[0032] Figure 5 To optimize the optimal conditions for colorimetric mode detection in the sensor system based on the Fe@CeO2+XOD+TMB system.

[0033] Figure 6To optimize the optimal conditions for photothermal mode detection of the sensing system based on the Fe@CeO2+XOD+TMB system, and to assess the feasibility of detecting Hx, 1, 2, 3, and 4 represent (1) Fe@CeO2+XOD+TMB, (2) Fe@CeO2+XOD+TMB+660nm near-infrared laser irradiation (1.5W / cm²), respectively. 2 (3) Fe@CeO2+XOD+TMB+Hx (4) Fe@CeO2+XOD+TMB+Hx+660nm near-infrared laser irradiation (1.5W / cm) 2 ).

[0034] Figure 7 The standard curves show the color and temperature changes of hypoxanthine at concentrations ranging from 0.0 to 400.0 μmol / L, as well as the B / G values ​​of the colorimetric signals and the photothermal temperature.

[0035] Figure 8 This study evaluates the selectivity and anti-interference capabilities of the Fe@CeO2+XOD+TMB system. In 8A and 8B, 1-14 represent Hx, glucose, ascorbic acid, urea, lactic acid, bovine serum albumin, arginine, histidine, glycine, MgSO4, ZnCl2, Na2CO3, NaHCO3, and blank, respectively. In 8C and 8D, 1-13 represent blank, glucose, ascorbic acid, urea, lactic acid, bovine serum albumin, arginine, histidine, glycine, MgSO4, ZnCl2, Na2CO3, and NaHCO3, respectively. Detailed Implementation

[0036] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0037] Please see Figure 1-8 It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings are merely for illustrative purposes and to aid those skilled in the art in understanding and reading the invention. They are not intended to limit the scope of the invention and therefore have no substantial technical significance. Any modifications to the structure, changes in proportions, or adjustments to the size are not permitted. The following embodiments are provided to better understand the invention, but are not intended to limit it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0038] Unless otherwise specified, all reagents or materials described in the following examples are commercially available.

[0039] The technical solution of the present invention will be further described in detail below with reference to examples.

[0040] Example 1: Synthesis of Fe@CeO2 nanozymes

[0041] (1) Mix 0.1 g of polyethylene glycol with 0.35 mL of ammonia and water / ethanol (37.0 / 8.0 mL) in a brown bottle and stir with a magnetic stirrer for 0.5-1.5 h. Then, add the mixture to 8.0 mL of 25.0 mg / mL tannic acid solution. After reacting at room temperature for 3.0-8.0 min, add 0.38 mL of 37.0 wt% formaldehyde solution and stir with a magnetic stirrer for 20.0-30.0 h. (2) Add 2.0 mL of 35.0 mg / mL Ce(NO3)3 and stir with a magnetic stirrer for 10.0-14.0 h. Then, transfer the mixture to a reaction vessel, seal it, and place it in an oven to react at 100.0 °C for 10.0-14.0 h. After the reaction, allow the reaction vessel to cool naturally to room temperature. Finally, the reactants were centrifuged (4000.0 rpm, 10.0-15.0 min). After centrifugation, the precipitate was collected, washed three times with water / ethanol, centrifuged again, and dried. The precipitate was then heated in a muffle furnace at 380.0-420.0℃ for 1.5-2.5 h to obtain CeO2 nanospheres. (3) In this case, 20.0 mg CeO2 nanospheres were dispersed in 2.0 mL of deionized water, and then 200.0 μL of DMSO solution containing 20.0 mg ferrocene carboxylic acid was added. The mixture was stirred with a magnetic stirrer for 5.0-6.0 h. Afterward, Fe@CeO2 nanozymes were collected by centrifugation, washed three times with water / ethanol, centrifuged again, and dried for later use. The synthesized Fe@CeO2 nanozymes were characterized as follows: Figure 2 As shown.

[0042] Example 2: Validation of Fe@CeO2 nanozyme activity

[0043] H₂O₂ was used as the substrate, TMB as the chromogenic agent, and HAc-NaAc as the buffer solution. The following four reactions were performed: (1) Fe@CeO₂ + H₂O₂ + TMB, (2) CeO₂ + H₂O₂ + TMB, (3) Fe@CeO₂ + TMB, and (4) CeO₂ + TMB. After the reactions were complete, the samples were transferred to cuvettes, and the absorbance at 652 nm was measured using a UV spectrophotometer. The catalytic activity of different nanozymes was then compared. (See also...) Figure 3 A. Only the Fe@CeO2+TMB+H2O2 combination has a relatively large absorbance value, indicating that the Fe@CeO2 nanozyme prepared in this invention has high peroxidase activity.

[0044] In the above verification steps, the concentrations of CeO2 nanozyme and Fe@CeO2 nanozyme were 50.0 μg / mL; the TMB concentration was 1.0 mmol / L; the H2O2 solution concentration was 200.0 μmol / L; the pH of the added HAc-NaAc buffer was 4.5; the total mixed solution was 200.0 μL; the reaction temperature was 32.0-37.0℃; and the time for thorough mixing and reaction was 15.0 min.

[0045] Example 3: Feasibility verification of Hx detection using the Fe@CeO2+XOD+TMB system

[0046] TMB was used as the colorimetric reagent, and HAc-NaAc was used as the buffer solution. The following reactions were performed: (1) Hx + XOD + TMB, (2) TMB, (3) XOD + TMB, (4) Fe@CeO2 + XOD + TMB, (5) Fe@CeO2 + XOD + TMB + Hx. After the reactions were complete, the samples were transferred to cuvettes, and the absorbance at 652 nm was measured using a UV spectrophotometer. The values ​​were then compared. (See also...) Figure 3 B, only the combination of Fe@CeO2+XOD+TMB+Hx has a significant light absorption intensity, demonstrating the feasibility of the detection system proposed in this invention.

[0047] In the above verification steps, the concentration of Fe@CeO2 nanozyme was 50.0 μg / mL; the concentration of TMB was 1.0 mmol / L; the concentration of H2O2 solution was 200.0 μmol / L; the pH of the added HAc-NaAc buffer was 5.0; the total mixed solution was 200.0 μL; the reaction temperature was 35.0℃; and the mixing and reaction time was 45.0 min.

[0048] Example 4: Catalytic kinetics test of Fe@CeO2 nanozymes

[0049] Steady-state kinetics experiments were conducted in HAc-NaAc buffer containing Fe@CeO2 nanozymes by varying the concentrations of H2O2 (0.0–200.0 μmol / L) or TMB (0.0–1.5 mmol / L) while keeping the concentration of another substance constant. The bivariate plots of H2O2 concentration versus reaction rate and TMB concentration versus reaction rate were calculated using the following Michaelis-Menten formula:

[0050]

[0051] Where V represents the initial reaction rate, [S] is the substrate concentration, and K... m It is the Michelson constant, V max This indicates the maximum reaction rate. The results are as follows: Figure 4The Ki of Fe@CeO2 on H2O2 and TMB is shown. m The values ​​were 0.25 and 0.24, respectively, which are higher than the K values ​​of natural horseradish peroxidase HPR. m The values ​​are approximately 15.0 times and 2.0 times lower, indicating that the Fe@CeO2 nanozyme prepared in this invention has good catalytic activity.

[0052] In the above verification steps, the concentration of Fe@CeO2 nanozyme was 50.0 μg / mL; the pH of the added HAc-NaAc buffer was 4.5; the total mixed solution was 200.0 μL; the reaction temperature was 45.0℃; and the mixing and reaction time was 15.0 min.

[0053] Example 5: Optimization of optimal conditions for hypoxanthine detection in the Fe@CeO2+XOD+TMB system using colorimetric mode

[0054] To optimize the analytical performance of the Fe@CeO2+XOD+TMB system, the reaction time, pH value, temperature, and XOD concentration of the colorimetric detection system were studied in detail. The reaction time was first optimized, and the results are as follows: Figure 5 As shown in Figure A, the absorbance of the reaction system at 652 nm increased with increasing reaction time from 0.0 to 55.0 min, and remained essentially unchanged after 45.0 min. Therefore, 45.0 min was selected as the optimal reaction time for the system. The performance of the system was then investigated within the pH range of 4.5-7.0, and NaAc-HAc was selected as the reaction buffer. The results are as follows... Figure 5 As shown in Figure B, when the pH value is in the range of 4.5 to 5.0, the absorbance of the reaction system at 652 nm increases with increasing pH value of the buffer system. When the pH value exceeds 5.0, the absorbance gradually decreases with increasing pH; therefore, 5.0 was chosen as the optimal pH value for the system. Next, the performance of this system in the temperature range of 25.0℃–50.0℃ was investigated. The results are as follows... Figure 5 As shown in Figure C, when the temperature range is 25.0℃ to 35.0℃, the absorbance of the reaction system at 652nm increases with increasing reaction temperature. When the temperature exceeds 35.0℃ and increases to 50.0℃, the absorbance gradually decreases with further increases; therefore, 35.0℃ was chosen as the optimal temperature for the system. Finally, the XOD concentration has a significant effect on the reaction. Therefore, the XOD concentration was optimized, and the results are shown in Figure C. Figure 5 As shown in Figure D, the absorbance value at 652 nm after reaching 1.0 U / mL basically no longer increases. Therefore, 1.0 U / mL was chosen as the optimal concentration of XOD in the system.

[0055] Example 6: Optimal conditions for hypoxanthine detection in the Fe@CeO2+XOD+TMB system using photothermal mode.

[0056] To optimize the performance of the Fe@CeO2+XOD+TMB system in photothermal mode for detecting hypoxanthine, we optimized the near-infrared laser irradiation time. The results are as follows: Figure 6 As shown in A and 6B, the temperature of the solution increases with the increase of irradiation time, and the temperature increase approaches saturation at an irradiation time of 150.0 s. Therefore, an irradiation time of 150.0 s is selected as the optimal detection condition for the photothermal mode. Simultaneously, the following four groups were tested: (1) Fe@CeO2+XOD+TMB, (2) Fe@CeO2+XOD+TMB+660nm near-infrared laser irradiation (1.5W / cm²). 2 (3) Fe@CeO2+XOD+TMB+Hx (4) Fe@CeO2+XOD+TMB+Hx+660nm near-infrared laser irradiation (1.5W / cm) 2 The results showed that only group (4) had a significant temperature change, indicating the feasibility of this system for Hx photothermal mode detection.

[0057] Example 7: Colorimetric-Photothermal Dual-Mode Detection of Hypoxanthine Standards in Fe@CeO2+XOD+TMB System

[0058] First, different concentrations of hypoxanthine (including 0.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0, 120.0, 140.0, 160.0, 180.0, 200.0, 250.0, 300.0, and 400.0 μmol / L) were prepared. Then, 1.0 U / mL XOD solution, 50.0 μg / mL Fe@CeO2 nanozyme, and 1.0 mmol / L TMB solution were reacted with different concentrations of Hx in HAc-NaAc buffer (pH = 5.0) at 35.0 °C for 45.0 min.

[0059] After the reaction was complete, the reaction solution was collected, and color signals were collected using a smartphone with the Color Picker App installed. The B / G ratio of the color signal increased with increasing hypoxanthine concentration. Therefore, a linear equation was fitted using Origin software, with B / G as the ordinate and Hx concentration as the abscissa. Figure 7 As shown in Figure B, the concentration of Hx showed a linear relationship with the B / G ratio in the range of 4.51-200.0 μmol / L, with the regression equation B / G = 0.00229C. Hx The correlation coefficient was +0.97975, the detection limit was 1.35 μmol / L, and the correlation coefficient was 0.9963. Additionally, such as... Figure 7 As shown in Figure A, as the concentration of Hx increases, the solution exhibits a satisfactory color gradient, and the change in reagent color can be observed with the naked eye, thus enabling the detection of hypoxanthine.

[0060] Take the above reaction solution, and after it cools to room temperature, heat it at a power of 1.5 W / cm². 2 The reaction solution was irradiated with a 660nm near-infrared laser for 150.0s, and the temperature was recorded using a thermal imager. The highest temperature reached approximately 47.5℃ as the hypoxanthine concentration increased. Therefore, a linear equation was fitted using Origin software with temperature as the ordinate and Hx concentration as the abscissa. Figure 7 As shown in Figure C, the concentration of Hx is linearly related to temperature in the range of 2.77–200.0 μmol / L, with the regression equation being T = 0.10087C. Hx +28.64486, correlation coefficient 0.9955, detection limit 0.83 μmol / L. Furthermore, such as Figure 7 As shown in Figure A, as the concentration of Hx increases, a significant temperature gradient change appears on the thermal imaging analyzer, enabling the detection of hypoxanthine.

[0061] Example 8: Selection and anti-interference evaluation of the Fe@CeO2+XOD+TMB system

[0062] To evaluate the selectivity of a colorimetric and photothermal dual-modal visual sensing platform based on the Fe@CeO2+XOD+TMB system for Hx detection, representative substrates such as glucose, ascorbic acid, urea, lactic acid, bovine serum albumin, arginine, histidine, glycine, MgSO4, ZnCl2, Na2CO3, and NaHCO3 were used as proof-of-concept samples for both visual colorimetric and photothermal dual-modal quantitative detection. Figure 8 A and Figure 8 As shown in Figure B, only Hx can activate the Fe@CeO2+XOD+TMB system, achieving a cascaded catalytic enhancement of colorimetric and photothermal signals, while other reagents cannot initiate changes in colorimetric and photothermal signals. These findings demonstrate the good selectivity of the proposed dual-mode colorimetric and photothermal sensing platform based on the Fe@CeO2+XOD+TMB system in detecting Hx. Furthermore, the robustness of this proposed sensing platform to representative reagents on the aforementioned coexisting substrates was investigated. The results are as follows... Figure 8 C and 8D indicate that in the Fe@CeO2+XOD+TMB system, the effects of each coexisting reagent on the cascade enhancement of Hx-triggered colorimetric and photothermal signals are not significant. This suggests that the colorimetric and photothermal dual-mode sensing platform based on the Fe@CeO2+XOD+TMB system has strong anti-interference capabilities and can be used for the detection of Hx in actual meat.

[0063] Example 9: Colorimetric-Photothermal Dual-Modal Detection of Hypoxanthine in Fresh Meat

[0064] To verify the sensing performance of the Fe@CeO2+XOD+TMB system for hypoxanthine detection in fresh meat applications, we selected fresh chicken as the actual test sample. The chicken was purchased from Xingda City Shopping Center in Hefei, China; it was chilled chicken and stored at -2.0–6.0℃ in a fresh meat preservation cabinet. Approximately 5.0g of the purchased fresh chicken was taken as the test sample, washed, and cut into strips of approximately 2.0mm. It was then mixed with 10% trichloroacetic acid solution and sonicated for approximately 10.0min to homogenize. Next, it was centrifuged at 4000rpm / min for 5.0min, and the supernatant was filtered to obtain the sample solution. 1.0U / mL XOD solution, 50.0μg / mL Fe@CeO2 nanozyme, and 1.0mmol / L TMB solution were reacted with the sample solution in HAc-NaAc buffer (pH=5.0) at 35.0℃ for 45.0min. Take the above reaction solution, collect color signals using a smartphone with the ColorPicker App installed, and after cooling to room temperature, use a power of 1.5W / cm² to collect the color signals. 2 The solution was irradiated with a 660nm near-infrared laser for 150.0s, and the temperature was recorded by taking pictures with a thermal imager. Substituting the experimental results into the B / G and T regression equations in Example 7, the concentrations of hypoxanthine in fresh chicken meat under the two detection modes were found to be 20.02μmol / L and 19.72μmol / L, respectively.

[0065] Example 10: Colorimetric-Photothermal Dual-Modal Detection of Hypoxanthine in Fresh Aquatic Products

[0066] Compared to meats, aquatic products are more susceptible to microbial contamination and spoilage due to their aquatic environment, making the detection of aquatic product freshness crucial. To verify the sensing performance of the Fe@CeO2+XOD+TMB system for hypoxanthine detection in fresh aquatic products, we selected fresh squid as the actual test sample. The fresh squid was purchased from the Zhonghuan City Shopping Center in Hefei, China, and stored refrigerated on ice at -4.0 to 6.0℃. Approximately 5.0g of the purchased fresh squid was taken as the test sample, washed, and air-dried at approximately 25.0℃. It was then cut into strips approximately 2.0mm in diameter and mixed with a 10% trichloroacetic acid solution. The mixture was sonicated for approximately 10.0min to homogenize, followed by centrifugation at 4000rpm / min for 5.0min. The supernatant was filtered to obtain the sample solution. 1.0 U / mL XOD solution, 50.0 μg / mL Fe@CeO2 nanozyme, and 1.0 mmol / L TMB solution were reacted with the sample solution in HAc-NaAc buffer (pH = 5.0) at 35.0 °C for 45.0 min. The reaction solution was then used to collect color signals using a smartphone with the Color Picker App installed. After cooling to room temperature, the signals were collected at a power of 1.5 W / cm². 2 The solution was irradiated with a 660 nm near-infrared laser for 150.0 s, and the temperature of the reaction solution was recorded by taking pictures with a thermal imager. Substituting the experimental results into the B / G and T regression equations in Example 7, the concentrations of hypoxanthine in fresh squid were found to be 62.43 μmol / L and 65.80 μmol / L under the two detection modes.

[0067] Example 11: Colorimetric-Photothermal Dual-Modal Detection of Hypoxanthine in Processed Meat Products

[0068] Compared to traditional fresh meat, processed meat products require no further processing and can be consumed directly, making freshness testing even more crucial. To verify the sensing performance of Fe@CeO2 nanozyme for hypoxanthine detection in processed meat products, we selected pork ham sausage and herring sausage as actual test samples. The pork ham sausage and herring sausage were purchased from Zhonghuan City Shopping Center in Hefei, China, packaged in plastic film, stored at room temperature, and had a shelf life of 180 days. Approximately 5.0g of each of the purchased pork ham sausage and herring sausage were taken as test samples, cut into strips of approximately 2.0mm, mixed with 10% trichloroacetic acid solution, and sonicated for approximately 10.0min to homogenize. Then, the mixture was centrifuged at 4000rpm / min for 5.0min, filtered, and the supernatant was collected to obtain the sample solution. 1.0 U / mL XOD solution, 50.0 μg / mL Fe@CeO2 nanozyme, and 1.0 mmol / L TMB solution were reacted with the sample solution in HAc-NaAc buffer (pH = 5.0) at 35.0 °C for 45.0 min. The reaction solution was then used to collect color signals using a smartphone with the Color Picker App installed. After cooling to room temperature, the signals were collected at a power of 1.5 W / cm². 2 The reaction solution was irradiated with a 660 nm near-infrared laser for 150.0 s, and the temperature was recorded by taking pictures with a thermal imager. Substituting the experimental results into the B / G and T regression equations in Example 7, the hypoxanthine concentrations in pork ham sausage were found to be 14.54 μmol / L and 13.02 μmol / L under the two detection modes; the hypoxanthine concentrations in herring sausage were found to be 26.06 μmol / L and 27.48 μmol / L under the two detection modes.

[0069] Example 12: Colorimetric-Photothermal Dual-Modal Detection of Hypoxanthine in Pre-cooked Meat Dishes

[0070] Compared to the complex processing of ordinary foods, pre-cooked meals are much more convenient to prepare and consume. In recent years, the pre-cooked meal market has been booming, making timely detection of the freshness of pre-cooked meals crucial for ensuring their food safety. To verify the sensing performance of Fe@CeO2 nanozyme for hypoxanthine detection in pre-cooked meat dishes, we selected semi-cooked shrimp balls as the actual test sample. The semi-cooked shrimp balls in this study were purchased from the Zhonghuan City Shopping Center in Hefei, China, packaged in aluminum foil boxes, and stored frozen at -18.0℃. The main ingredients were lobster tails, drinking water, semi-solid compound seasoning, and solid seasoning. The shelf life was 18 months, and they could be directly heated in a pot for 5.0 minutes before consumption. One shrimp ball was selected from the purchased sample materials as the test sample. After washing, 5.0g of shrimp meat was taken and chopped, then mixed with 10% trichloroacetic acid solution and sonicated for about 10.0min to homogenize. The mixture was then centrifuged at 4000rpm / min for 5.0min, and the supernatant was filtered to obtain the sample solution. 1.0U / mL XOD solution, 50.0μg / mL Fe@CeO2 nanozyme, and 1.0mmol / L TMB solution were reacted with the sample solution in HAc-NaAc buffer (pH=5.0) at 35.0℃ for 45.0min. The reaction solution was then used to collect color signals using a smartphone with the Color Picker App installed. After cooling to room temperature, the color signal was collected at a power of 1.5W / cm². 2 The solution was irradiated with a 660nm near-infrared laser for 150.0s, and the temperature was recorded by taking pictures with a thermal imager. Substituting the experimental results into the B / G and T regression equations in Example 7, the hypoxanthine concentrations in the semi-cooked shrimp balls were found to be 34.63μmol / L and 35.75μmol / L under the two detection modes.

[0071] The data above demonstrates that, compared to other reported analytical methods, this colorimetric-photothermal dual-modal detection method for Hx, based on the Fe@CeO2+TMB+XOD enzyme cascade catalysis system, exhibits superior performance in terms of detection time, linear range, and sensitivity. In particular, compared to a method for detecting hypoxanthine content in spoiled meat based on Fe-PDA nanozymes disclosed in our team's previous authorized patent (CN114113506A), this method boasts higher sensitivity and a lower detection limit. Furthermore, it requires only a smartphone equipped with the Color Picker App and a handheld thermal imager to collect both colorimetric and photothermal signals, making it easy to operate. The combination of the two detection modes is also more accurate than a single detection mode.

[0072] Example 13: A method for quantitatively detecting hypoxanthine in a dual-modal manner to determine the freshness of meat.

[0073] A method for quantitatively detecting hypoxanthine in a dual-modal manner to determine the freshness of meat includes the following steps:

[0074] Step 1: Measure the RGB values ​​of the color signal after the sample reaction is complete.

[0075] At room temperature, xanthine oxidase solution (XOD), 3,3,5,5-tetramethylbenzidine solution (TMB), Fe@CeO2 nanozyme, and standard xanthine solutions (Hx) of different concentrations were reacted in HAc-NaAc buffer. After the reaction was completed, the complete reaction solution was obtained, and the color signal was collected using a smartphone with the Color Picker App installed (which can convert the colorimetric image into digital color RGB values).

[0076] Step 2: Measure the temperature T of the reaction solution:

[0077] After cooling the complete reaction solution in step 1 to room temperature of about 27.0℃, it was irradiated with a near-infrared 660nm laser. The solution after irradiation with the 660nm laser was photographed with a thermal imager and the temperature T of the reaction solution was recorded.

[0078] Step 3: Construct a linear equation between hypoxanthine concentration and color signal B / G or temperature T.

[0079] The color signal B / G was obtained from tests of a series of standard samples at varying concentrations. A linear equation was then constructed: B / G = XC. Hx +y, where C Hx For standard concentration;

[0080] Based on temperature differences T obtained from tests of a series of standard samples at varying concentrations, a linear equation is constructed: T = XC Hx +y, where C Hx For standard concentration;

[0081] Step 4: Take the sample to be tested and repeat steps 1 and 2 to obtain the color signal B / G. n and temperature T n B / G n and T n Substitute the values ​​into the corresponding linear equation to obtain the concentration of hypoxanthine in the sample to be tested.

[0082] The preferred embodiment is as follows: In step 1, the pH value of the HAc-NaAc buffer is 5.0; the concentration of Fe@CeO2 nanozyme is 50.0 μg / mL; the concentration of 3,3,5,5-tetramethylbenzidine solution is 1.0 mmol / L; the concentration of xanthine oxidase solution is 1.0 U / mL; the total volume of the mixed solution is 200.0 μL; the reaction temperature is 32.0-37.0℃; and the reaction time is 40.0-50.0 min.

[0083] The preferred embodiment is that the laser irradiation time is 120.0-170.0s.

[0084] The preferred implementation is as follows: In step 3, the B / G regression equation for the colorimetric signal is B / G = 0.00229C. Hx +0.97975; the photothermal temperature regression equation is T = 0.10087°C. Hx +28.64486.

[0085] A preferred implementation method is as follows: Meat freshness is judged using the following method: The freshness judgment standard can be based on the meat freshness K value, which is the percentage of the sum of inosine adenosine and inosine (ATP) degradation products to the total amount of ATP-related compounds. The fresher the meat, the smaller the K value, and vice versa. Taking common fish as an example, fish with a K value of 20% are considered fresh fish, while fish with a K value higher than 70% are considered spoiled. For raw fish products or fresh meat products, a freshness K value greater than 10% indicates spoilage. In particular, when judging the product as aquatic product, products with an Hx content ≤ 72.0 mg / kg are considered fresh products; products with an Hx content of 72.0 mg / kg are considered fresh products. <C Hx Products with a Hx content ≤118.0 mg / kg are considered less fresh; products with an Hx content ≥118.0 mg / kg are considered spoiled. Different types of meat and different storage conditions can lead to different Hx levels, therefore, the country needs to further develop a database or standard for Hx content assessment for different types of meat and storage conditions.

[0086] A preferred embodiment is as follows: The preparation method of the Fe@CeO2 nanozyme includes:

[0087] S1: Mix polyethylene glycol, ammonia, and ethanol in a brown bottle, stir until homogeneous, and then add to the tannic acid solution; react at room temperature for 3.0-8.0 min, then add formaldehyde solution and stir for 20.0-30.0 h;

[0088] S2: Add Ce(NO3)3 to the reaction system obtained in S1, and stir for 10.0-14.0 h; then, transfer the mixture to a reaction vessel, seal it, and place it in an oven to react at 95.0-105.0℃ for 10.0-14.0 h; after the reaction is completed, allow the reaction vessel to cool naturally to room temperature; finally, centrifuge the reactants, collect the precipitate after centrifugation, wash it with ethanol and centrifuge it again, and dry it; then heat the precipitate in a muffle furnace at 380.0-420.0℃ for 1.5-2.5 h to obtain CeO2 nanospheres;

[0089] S3: Disperse CeO2 nanospheres in deionized water, then add a DMSO solution containing ferrocene carboxylic acid, and stir for 5.0-7.0 h; subsequently, collect the Fe@CeO2 nanozyme by centrifugation, wash with ethanol and centrifuge again, then dry for later use. The purity of the ethanol, ammonia, tannic acid and formaldehyde solutions should be analytical grade.

[0090] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A method for quantitatively detecting hypoxanthine in a dual-modal manner to determine the freshness of meat, characterized in that: Includes the following steps: Step 1: Measure the RGB values ​​of the color signal after the sample reaction is complete. At room temperature, xanthine oxidase solution, 3,3,5,5-tetramethylbenzidine solution, Fe@CeO2 nanozyme and standard xanthine solutions of different concentrations were reacted in HAc-NaAc buffer. After the reaction was completed, the complete reaction solution was obtained, and color signals were collected using a smartphone with the Color Picker App installed. Step 2: Measure the temperature T of the reaction solution: After cooling the complete reaction solution in step 1 to room temperature, it was irradiated with a near-infrared 660 nm laser. The solution after irradiation with the 660 nm laser was photographed with a thermal imager and the temperature T of the reaction solution was recorded. Step 3: Construct a linear equation between hypoxanthine concentration and color signal B / G or temperature T. The color signal B / G was obtained from tests of a series of standard samples at varying concentrations. A linear equation was then constructed: B / G = XC Hx + y, where C Hx For standard concentration; Based on temperature differences T obtained from tests of a series of standard samples at varying concentrations, a linear equation is constructed: T = XC Hx + y, where C Hx For standard concentration; Step 4: Take the sample to be tested and repeat steps 1 and 2 to obtain the color signal B / G. n and temperature T n B / G n and T n Substitute the values ​​into the corresponding linear equation to obtain the concentration of hypoxanthine in the sample to be tested; The preparation method of the Fe@CeO2 nanozyme includes: S1: Mix polyethylene glycol, ammonia and ethanol in a brown bottle, stir until well mixed, and then add to the tannic acid solution; After reacting at room temperature for 3.0 - 8.0 min, formaldehyde solution is added, and the mixture is stirred for 20.0 - 30.0 h. S2: Add Ce(NO3)3 to the reaction system obtained in S1, and stir for 10.0 - 14.0 h; then, transfer the mixture to a reaction vessel, seal it, and place it in an oven to react at 95.0 - 105.0 °C for 10.0 - 14.0 h; after the reaction is completed, allow the reaction vessel to cool naturally to room temperature; finally, centrifuge the reactants, collect the precipitate after centrifugation, wash it with ethanol and centrifuge it again, and dry it; then heat the precipitate in a muffle furnace at 380.0 - 420.0 °C for 1.5 - 2.5 h to obtain CeO2 nanospheres; S3: Disperse CeO2 nanospheres in deionized water, then add DMSO solution containing ferrocene carboxylic acid, and stir for 5.0-7.0 h; then collect Fe@CeO2 nanozyme by centrifugation, wash with ethanol and centrifuge again, and then dry for later use.

2. The method for quantitative detection of hypoxanthine and determination of meat freshness using dual-modal methods according to claim 1, characterized in that: In step 1, the pH of the HAc-NaAc buffer was 5.0; the concentration of the Fe@CeO2 nanozyme was 50.0 μg / mL; the concentration of the 3,3,5,5-tetramethylbenzidine solution was 1.0 mmol / L; the concentration of the xanthine oxidase solution was 1.0 U / mL; the total volume of the mixed solution was 200.0 μL; the reaction temperature was 32.0 - 37.0 ℃; and the reaction time was 40.0 - 50.0 min.

3. The method for quantitative detection of hypoxanthine and determination of meat freshness using dual-modal methods according to claim 1, characterized in that: The laser irradiation time is 120.0 - 170.0 s.

4. The method for quantitative detection of hypoxanthine and determination of meat freshness using dual-modal methods according to claim 1, characterized in that: In step 3, the B / G regression equation for the colorimetric color signal is B / G = 0.00229 C. Hx + 0.97975; the photothermal temperature regression equation is T = 0.10087 C Hx + 28.64486.

5. The method for quantitative detection of hypoxanthine and determination of meat freshness using dual-modal methods according to claim 1, characterized in that: Meat freshness is judged using the following methods: The freshness standard can be measured by the meat freshness K-value, which is the percentage of the sum of inosine adenosine and hypoxanthine (the degradation products of adenosine triphosphate), to the total amount of adenosine triphosphate-related compounds. The fresher the meat, the lower the K-value, and vice versa. Taking common fish as an example, fish with a K-value of 20% are considered fresh, while those above 70% are spoiled. For raw fish or fresh meat products, a K-value greater than 10% indicates spoilage. When identifying aquatic products, products with an Hx content ≤ 72.0 mg / kg are considered fresh; an Hx content of 72.0 mg / kg is considered... <C Hx Products with ≤ 118.0 mg / kg are considered nearly fresh; products with Hx content ≥ 118.0 mg / kg are considered spoiled.

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