Method for detecting antioxidant and polyphenol in food by constructing colorimetric sensing array based on monatomic iron nano-enzyme

Abstract

The invention discloses a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensing array based on monatomic iron nano-enzyme, and belongs to the field of nanotechnology and colorimetric sensing. The traditional monatomic nano enzyme shows limited enzyme-like activity and substrate specificity, and is difficult to meet the comprehensive requirements of detecting various antioxidants and polyphenols in food. In the research, a novel nano-enzyme system (sdsFeN (at) G) of monatomic iron nano-enzyme is developed. The SdsFeN (at) G shows excellent enzymatic activity, and a three-channel colorimetric sensing array is constructed by utilizing inhibition of different antioxidants and polyphenols on oxidation of three chromogenic substrates, so that simple, convenient and reliable identification and detection of five substances (tert-butylhydroquinone, butylated hydroxyanisole, propyl gallate, catechol and hydroquinone) in food are realized.

Description

Technical Field

[0001] The invention belongs to the field of nanotechnology and colorimetric sensing, and specifically relates to a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensing array based on iron-based nanoenzymes. Background Art

[0002] Food is easily oxidized and deteriorated during storage and preservation, especially some oily or oil-rich foods. Since antioxidants can effectively delay oxidation time, some antioxidants are added during the storage of food to extend the shelf life of food, thereby improving the stability of food and ensuring its nutrition and flavor. As a typical synthetic lipid antioxidant, it has significant chemical stability, strong antioxidant properties, good heat resistance, low cost and excellent anti-interference ability, and is widely used in the food industry. Antioxidants such as tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and propyl gallate (PG) have become food additives that are widely used in the field of oils and fats. However, it should be noted that excessive use of food additives can lead to food safety and human health problems. High doses of tert-butylhydroquinone and propyl gallate may cause toxicological effects and carcinogenic risks. Butylated hydroxyanisole (BHA) does not have mutagenic properties either in vivo or in vitro, but it may affect the metabolic process of some mutagenic compounds. And there is the possibility of it acting as an endocrine disruptor. Long-term exposure to such substances may cause health problems such as infertility and abnormal growth and development, which will have a negative impact on food quality and public health. Phenolic compounds are based on the benzene ring as the basic skeleton. The benzene ring is directly connected to the hydroxyl group. There is a special interaction between the hydroxyl group and the benzene ring. Phenolic substances have multiple isomers according to the different positions of the hydroxyl group on the benzene ring. Therefore, phenolic substances have unique physical and chemical properties.

[0003] Single-atom nanozymes have become a prominent focus in the field of nanozyme research due to their optimized atomic utilization, extremely high catalytic activity and selectivity. A large number of studies have shown that the unique physicochemical properties of single-atom nanozymes, such as the atomic size and the interaction between metals and carriers, not only give them excellent catalytic performance, but also significantly improve atomic utilization and reduce production costs, making them very promising in the monitoring and analysis of complex biochemical samples. In terms of food testing, single-atom nanozymes can catalyze rapid and visual detection of food substances, providing a new strategy for high-cost and technically cumbersome problems. With the continuous deepening of research, single-atom nanozymes are expected to play an important role in more fields and bring new development opportunities to many industries.

[0004] Colorimetric sensor array is a technology for detecting and analyzing chemical substances based on color changes. It consists of a series of sensor elements that have specific reactions to different chemical substances, and each sensor element changes color when it comes into contact with a specific substance. When applied to the detection process, the colorimetric sensor array can detect low concentrations of antioxidants and polyphenols, which is very important for evaluating the antioxidant and polyphenol content of food. And by designing specific sensor elements, the colorimetric sensor array can selectively identify and respond to different antioxidants and polyphenols, reducing the impact of interfering substances. In addition, the colorimetric sensor array can usually give results within a few minutes, which is very useful for quickly evaluating the content of antioxidants and polyphenols, especially on the production line or in field detection. Therefore, the use of colorimetric arrays has the advantages of high sensitivity, selectivity, and rapid response. The purpose of the present invention is to provide a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes, using the peroxidase activity of single-atom iron nanozymes to optimize enzyme activity, constructing a colorimetric sensor array for different antioxidants and polyphenols to detect and distinguish, forming a linear discriminant analysis diagram, and realizing simple, convenient, and reliable visual detection. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes, thereby achieving simple, convenient and reliable visual detection.

[0006] The purpose of the present invention is achieved through the following solutions:

[0007] A method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes, characterized in that it comprises the following steps:

[0008] A. Preparation of single-atom iron nanozymes;

[0009] B. Optimization of reaction conditions based on single-atom iron nanoperoxidase activity;

[0010] C. Constructing a colorimetric sensing array by changing the pH of the reaction system and adding the substance to be tested;

[0011] D. Use classic models to distinguish and identify them.

[0012] In step A, the preparation specifically includes: adding 50 mg of graphene oxide to 25 ml of distilled water and stirring, and then weighing 1.7 mg of FeCl 3 6H 2 O was added into a mixed solution of GO and distilled water and stirred, wherein Fe / GO = 0.7wt%, and placed into a rotary evaporator to evaporate the water completely. The calcination time was 2h under argon (Ar) to obtain the related product sdsFeN@G.

[0013] In step B, the parameters such as pH, substrate concentration, reaction time, etc. of the peroxidase activity system were optimized to obtain the optimal conditions. The optimized conditions were: 25°C, pH range 2.5-5, interval 0.5; reaction time range 1-7min, interval 1min; interval 0.02mM; TMB concentration range 5-50μM, interval 0.02mM. The absorbance value corresponding to the blue color was recorded using a UV-visible spectrometer.

[0014] In step C, three pH values ​​(pH=3.0, pH=3.5 and pH=4.0) were selected to construct a colorimetric sensing array, and the size of the colorimetric sensing array was three pH values*five test substances*five parallel groups. 2 O 2 , and the colorimetric substrate 3'3'5'5'-tetramethylbenzidine (TMB) and acetate buffers of different pH values ​​are added into a centrifuge tube, and then a certain amount of the substance to be tested is added into the tube. After reacting for 5 minutes, the colorimetric sensor detection array to be tested is obtained.

[0015] In step D, the classical model is linear discriminant analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 TEM image of the single-atom iron nanozyme proposed in Example 2

[0017] Figure 2 This is the high-resolution XPS full spectrum of the single-atom iron nanozyme proposed in Example 2

[0018] Figure 3 X-ray diffraction (XRD) pattern of the single-atom iron nanozyme proposed in Example 2

[0019] Figure 4 This is the optimization of the pH condition for the single-atom iron nanozyme in Example 3;

[0020] Figure 5 The optimization of the conditional substrate concentration TMB of the single-atom iron nanozyme in Example 3;

[0021] Figure 6 This is the optimization of the conditions and time for the single-atom iron nanozyme in Example 3;

[0022] Figure 7 The linear discriminant analysis diagram for different types of substances based on the colorimetric sensor array in Example 4 is implemented;

[0023] Figure 8 This is a linear discriminant analysis diagram of different concentrations of TBHQ based on the colorimetric sensor array in Example 4;

[0024] Fig. 9 This is a linear discriminant analysis diagram of different concentrations of BHA based on the colorimetric sensor array in Example 4.

[0025] Fig.10 The linear discriminant analysis diagram of different concentrations of PG based on the colorimetric sensor array in Example 4 is shown in FIG.

[0026] Fig.11 The linear discriminant analysis diagram of CC with different concentrations based on the colorimetric sensor array in Example 4 is shown in FIG.

[0027] Fig.12 This is a linear discriminant analysis diagram of different concentrations of HQ based on the colorimetric sensor array in Example 4.

[0028] Fig.13 The linear discriminant analysis diagram of the binary substance mixture system based on the colorimetric sensor array in Example 4 is implemented;

[0029] Fig.14 The linear discriminant analysis diagram of the ternary substance mixture system based on the colorimetric sensor array in Example 4 is realized; DETAILED DESCRIPTION

[0030] Embodiment 1:

[0031] The single-atom iron nanozyme was prepared by adding 50 mg of graphene oxide to 25 ml of distilled water and stirring, and then weighing 1.7 mg of FeCl 3 6H 2 O was added to a mixed solution of GO and distilled water and stirred for 15 minutes, wherein Fe / GO = 0.7wt%. The liquid was mixed evenly and then placed in a rotary evaporator until the water was completely evaporated. The solid was placed in a calcination furnace. In an argon (Ar) filled environment, the temperature was set to 750°C and the calcination time was 2h to obtain the relevant product sdsFeN@G, which was used for subsequent material preparation and related experimental exploration.

[0032] Embodiment 2:

[0033] In order to further explore the morphology and size of sdsFeN@G, Figure 1 The morphology of sdsFeN@G was carefully observed by transmission electron microscopy (TEM), and it was found that it exhibited a folded sheet structure. Figure 2 , X-ray photoelectron spectroscopy (XPS) was used to study the surface element composition and chemical state, and the results showed that sdsFeN@G contained Fe, C, N, and O elements. Figure 3, and use X-ray electron spectrometer to analyze the material, record the XPS spectrum through Al Kα radiation, so as to determine its chemical composition, element valence, chemical state and chemical bond, etc. X-ray electron diffraction (XRD) determines the phase of the material.

[0034] Embodiment 3:

[0035] The factors that can affect the catalytic effect of the peroxidase activity system, such as pH, TMB substrate concentration, and reaction time, were optimized in the hope of obtaining the optimal peroxidase catalytic activity of sdsFeN@G. The specific experimental conditions are as follows: 100 μL H 2 O 2 +100μL TMB+100μL sdsFeN@G (1mg / mL) were added to 700μL acetic acid (HAc-NaAc) buffer (pH=3.0), where the pH was 2, 3, 4, 5, 6, and 7, respectively, the TMB concentration was 0.8 to 2.2mmol / L, and the time was 1min-7min. Under the above conditions, the absorption spectrum was tested under the UV spectrophotometer and the relevant optimal conditions were obtained. Figure 4 The first step is to optimize the HAc-NaAc buffer with different pH values. After UV absorption spectrum measurement, the catalytic activity is at its highest value when the pH value of the HAc-NaAc buffer in the reaction system is 3. The HAc-NaAc buffer has the highest pH value at 3, and the activity on the left side gradually increases, while the activity on the right side gradually decreases. It can be concluded that pH 3 is the optimal pH value of the HAc-NaAc buffer, and the relevant buffer is configured for all subsequent experiments. Figure 5 , and then optimize the optimal concentration of the reaction substrate TMB. The optimization of the substrate concentration TMB selects a concentration range of 0.8-2.2mmol / L. The absorbance value curve corresponding to each concentration at 652nm first rises rapidly to the optimal point and then decreases, and the rapid oxidation of TMB to oxTMB changes to slow oxidation. The optimal point is 0.2mmol / L, and if the amount of TMB is too little, it will lack reactive substances, affecting subsequent experiments, and excessive concentration will lead to overreaction. Figure 6 When optimizing the reaction conditions with reference to other nanozymes, it was found that the reaction time of the nanozyme was also relatively important. The time was then optimized. Because the reaction was rapid, the UV absorption spectrum was tested every minute. When the concentration was 5-7 minutes, the reaction was already in a slow rising period. After comprehensive consideration, 5 minutes was selected as the optimal response time.

[0036] Embodiment 4:

[0037] In this paper, the peroxidase activity of sdsFeN@G is used as the basis to establish a three-channel colorimetric sensor array. Because the substrate TMB can be oxidized to oxTMB, showing a blue color, and the blue response is different under different pH conditions, a relatively stable colorimetric array is constructed for subsequent experiments. First, the colorimetric method is used to determine the standard curve of the five substances. The standard solutions of the five substances are prepared and 100 μL of H 2 O 2 (1mmol / L) + 100μLTMB (2mmol / L) + 100μLsdsFeN@G (1mg / mL) were added to 700μL (pH=3.0) HAc-NaAc buffer, and the mixture was reacted at room temperature for 5 minutes. Then, it was centrifuged at 1000rpm to obtain the supernatant. Then, 20μL of the substance to be tested was taken out and added to it. The reaction was continued for 5 minutes, and the absorbance spectrum was recorded at 652nm using an ultraviolet spectrophotometer. A0 and A were detected, and the difference between the two could change ΔA. A0 is the initial absorbance of the reaction, and A is the absorbance after adding the substance to be tested. The spectra before and after the reaction were detected to obtain the spectrum and absorbance value, and ΔA and substance concentration were taken as data. Origin software was used to plot each substance to obtain its calibration curve. Figure 7 As shown, our proposed visualization detection method can successfully distinguish five antioxidants at a concentration of 10 μM.

[0038] Secondly, the three-channel colorimetric sensor array is sensitive and simple, and it is used to distinguish and detect the five substances tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), propyl gallate (PG), catechol (CC), and hydroquinone (HQ). Prepare the standard solutions of the five substances, and add 100 μL H 2 O 2 , 100μLTMB, 100μLsdsFeN@G (1mg / mL), 700μL (pH=3.0, 4pH=3.5 and pH=4.0, HAc-NaAc) buffer solution were mixed and reacted at room temperature for 5 minutes, then centrifuged at 1000rpm to obtain the supernatant, 180μL was taken out from it and put into the well plate, then 20μL of the substance to be tested was taken out and added thereto, the reaction was continued for 5 minutes, and the change in absorbance value was recorded at 652nm using a multifunctional microplate reader. Using three different pH values, 5 parallel experiments were performed for each of the five substances to obtain a 3*5*5 array matrix, and finally, LDA analysis was performed using SPSS software, and a score chart was drawn. Figure 8 , 9As shown in Figures 10, 11, and 12, the order is TBHQ, BHA, PG, CC, and HQ. The colorimetric sensor array constructed in the present invention can successfully distinguish five antioxidants (10 μM-50 μM) of different concentrations.

[0039] The performance of the colorimetric sensor array was demonstrated using binary and ternary mixtures. Binary mixtures of PG and BHA at different molar ratios (PG:BHA=1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 8:2, 9:1, with a total concentration of 10 μmol / L) and ternary mixtures of TBHQ, BHA, and HQ at different molar ratios (TBHQ:BHA:HQ=1:2:7, 2:2:6, 1:5:4, 3:4:3, 2:3:5, with a total concentration of 10 μmol / L) were mixed with the supernatant of the reaction system according to the above method. After reacting for 5 minutes, the absorbance value was measured and the results were obtained by LDA analysis. Fig.13 , 14 As shown, the colorimetric sensor array constructed by the present invention can successfully identify binary and ternary mixed systems of antioxidants.

Claims

1. A method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes, characterized in that: The following steps are involved: A. Preparation and characterization of single-atom iron nanozymes; B. Optimize the reaction conditions based on the peroxidase activity of single-atom iron nanozymes; C. Constructing a colorimetric sensing array by changing the pH of the reaction system and adding the substance to be tested; D. Use classic models to distinguish and identify them.

2. According to claim 1, a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes, characterized in that: In step A, the preparation method includes: adding 50 mg of graphene oxide to 25 ml of distilled water for stirring, then weighing 1.7 mg of FeCl3·6H2O and adding it to a mixed solution of GO and distilled water for stirring, wherein Fe / GO=0.7wt%, and placing it in a rotary evaporator to completely evaporate the water, and calcining it under argon (Ar) for 2 hours to obtain the related product sdsFeN@G.

3. According to claim 1, a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes, characterized in that: In step B, the pH, substrate concentration, and reaction time parameters of the peroxidase activity system are optimized to obtain the optimal conditions; the optimized conditions are: 25° C., pH range 2.5-5, interval 0.5; reaction time range 1-7 min, interval 1 min; TMB concentration range 5-50 mM, interval 0.02 mM; and the absorbance value corresponding to the blue color is recorded using a UV-visible spectrometer.

4. A method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes according to claim 1, characterized in that: In step C, three pH values ​​(pH=3.0, pH=3.5 and pH=4.0) are selected to construct a colorimetric sensing detection array. The size of the colorimetric sensing detection array is three pH values* five test substances* five parallel groups.

5. A method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes according to claim 4, characterized in that: Step C specifically includes: adding iron nanozyme, H2O2, chromogenic substrate 3'3'5'5'-tetramethylbenzidine (TMB) and acetate buffer of different pH into a centrifuge tube, and then adding a certain amount of the substance to be tested into the tube, and after reacting for 5 minutes, a colorimetric sensing detection array of the substance to be tested is obtained.

6. The method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensor array based on single-atom iron nanozymes according to claim 1, characterized in that: In step D, the classical model is linear discriminant analysis.

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