A method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes.
By constructing a colorimetric sensor array based on single-atom iron nanozymes, the problem of detecting antioxidants and polyphenols in food in existing technologies has been solved, enabling rapid and accurate visual detection and differentiation, and reducing detection costs and complexity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2026-04-03
AI Technical Summary
Existing technologies are insufficient for quickly and accurately detecting and distinguishing the content of antioxidants and polyphenols in food, especially in on-line or field testing, where high testing costs and cumbersome techniques exist.
A colorimetric sensor array based on single-atom iron nanozymes was constructed. By optimizing reaction conditions such as pH, substrate concentration and reaction time, the peroxidase activity of single-atom iron nanozymes was utilized to construct the colorimetric sensor array for detection and differentiation, forming a linear discriminant analysis diagram.
It achieves simple, fast, and reliable visual detection, and can accurately distinguish and detect different types and concentrations of antioxidants and polyphenols in food within minutes, reducing detection costs and complexity.
Smart Images
Figure CN120102556B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the fields of nanotechnology and colorimetric sensing, specifically relating to a method for detecting antioxidants and polyphenols in food by constructing a colorimetric sensing array based on iron-based nanozymes. Background Technology
[0002] Foods are easily oxidized and spoiled during storage, especially oily or oil-rich foods. Antioxidants, which effectively slow down oxidation, are often added to extend the shelf life of stored foods, thereby improving their stability and preserving their nutritional value and flavor. As a typical synthetic lipid antioxidant, it possesses significant chemical stability, strong antioxidant properties, good heat resistance, low cost, and excellent anti-interference ability, and is widely used in the food industry. Tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and propyl gallate (PG) are among the most widely used food additives in the oil and fat industry. However, it is important to note 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. While butylated hydroxyanisole (BHA) does not possess mutagenic properties in either in vivo or in vitro environments, it may affect the metabolic processes of some mutagenic compounds. Furthermore, these substances have the potential to act as endocrine disruptors. Long-term exposure may lead to health problems such as infertility and abnormal growth and development, negatively impacting food quality and public health. Phenolic compounds, with a benzene ring as their basic skeleton and hydroxyl groups directly attached to the benzene ring, exhibit unique interactions with the benzene ring. Depending on the position of the hydroxyl group on the benzene ring, phenolic substances have various isomers, thus possessing unique physical and chemical properties.
[0003] Single-atom nanozymes have become a prominent focus in the field of nanozyme research due to their optimized atom utilization, extremely high catalytic activity, and selectivity. Numerous studies have shown that the unique physicochemical properties of single-atom nanozymes, such as their atomic size and the interaction between the metal and the support, not only endow them with excellent catalytic performance but also significantly improve atom utilization and reduce production costs, making them highly promising for the monitoring and analysis of complex biochemical samples. In food detection, single-atom nanozymes can catalyze rapid and visual detection of food substances, providing a new strategy to address the problems of high costs and cumbersome techniques. With continued research, single-atom nanozymes are expected to play an important role in more fields, bringing new development opportunities to numerous industries.
[0004] Colorimetric sensor arrays are a technique for detecting and analyzing chemical substances based on color changes. They consist of a series of sensing elements that specifically react to different chemical substances, each changing color upon contact with a particular substance. When applied to detection processes, colorimetric sensor arrays can detect low concentrations of antioxidants and polyphenols, which is crucial for assessing their content in food. Furthermore, by designing specific sensing elements, colorimetric sensor arrays can selectively identify and respond to different antioxidants and polyphenols, reducing the influence of interfering substances. In addition, colorimetric sensor arrays typically provide results within minutes, which is extremely useful for rapidly assessing antioxidant and polyphenol content, especially in production line or on-site testing. Therefore, colorimetric arrays offer advantages such as high sensitivity, selectivity, and rapid response. The purpose of this invention is to provide a method for detecting antioxidants and polyphenols in food using a colorimetric sensor array constructed based on single-atom iron nanozymes. Utilizing the peroxidase activity of single-atom iron nanozymes, enzyme activity is optimized to construct a colorimetric sensor array for the detection and differentiation of different antioxidants and polyphenols, forming a linear discriminant analysis graph. This achieves simple, convenient, and reliable visual detection. Summary of the Invention
[0005] The purpose of this 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, which achieves simple, convenient and reliable visual detection.
[0006] The objective of this invention is achieved through the following solution:
[0007] A method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes, characterized by comprising the following steps:
[0008] A. Preparation of single-atom iron nanozymes;
[0009] B. Optimize reaction conditions based on the activity of single-atom iron nanoparticles peroxidase;
[0010] C. Construct a colorimetric sensor array by changing the pH of the reaction system and adding the analyte;
[0011] D. Use the classic model to distinguish and judge them.
[0012] In step A, the preparation specifically includes: adding 50 mg of graphene oxide to 25 ml of distilled water and stirring, then weighing 1.7 mg of FeCl3·6H2O and adding it to a mixed solution of GO and distilled water and stirring, wherein Fe / GO = 0.7 wt%, placing it in a rotary evaporator to evaporate the water completely, and calcining it under argon (Ar) for 2 h to obtain the relevant product sdsFeN@G.
[0013] In step B, the parameters of the peroxidase activity system, including pH, substrate concentration, and reaction time, were optimized to obtain the optimal conditions. The optimized conditions were: 25℃, pH range 2.5 ~ 5, interval 0.5; reaction time range 1 ~ 7 min, interval 1 min, interval 0.02 mM; TMB concentration range 0.8-2.2 μM, interval 0.02 mM. The absorbance values corresponding to blue were recorded using a UV-Vis spectrometer.
[0014] In step C, three pH values (pH=3.0, pH=3.5, and pH=4.0) are selected to construct a colorimetric sensor array. The size of the colorimetric sensor array is three pH values * five analytes * five parallel sets. Iron nanozyme, H₂O₂, the chromogenic substrate 3'3'5'5'-tetramethylbenzidine (TMB), and acetate buffer solutions at different pH values are added to centrifuge tubes. Then, a certain amount of the analytes is added to the tubes. After reacting for 5 minutes, the colorimetric sensor array is obtained.
[0015] In step D, the classic mode is linear discriminant analysis. Attached Figure Description
[0016] Figure 1 TEM image of the single-atom iron nanozyme proposed in Example 2.
[0017] Figure 2 The high-resolution XPS full spectrum of the single-atom iron nanozyme proposed in Example 2.
[0018] Figure 3 The X-ray diffraction (XRD) pattern of the single-atom iron nanozyme proposed in Example 2.
[0019] Figure 4 This is an optimization of the pH conditions for the single-atom iron nanozyme in Example 3;
[0020] Figure 5 This is an optimization of the conditional substrate concentration TMB for the single-atom iron nanozyme in Example 3;
[0021] Figure 6 This is an optimization of the conditions and time for the single-atom iron nanozyme in Example 3;
[0022] Figure 7 This is the linear discriminant analysis diagram of different types of substances based on a colorimetric sensor array in Example 4;
[0023] Figure 8 This is a linear discriminant analysis diagram of different concentrations of TBHQ based on a colorimetric sensor array, as shown in Example 4.
[0024] Figure 9This is the linear discriminant analysis diagram for different concentrations of BHA based on a colorimetric sensor array, as shown in Example 4.
[0025] Figure 10 This is a linear discriminant analysis diagram of different concentrations of PG based on a colorimetric sensor array, as shown in Example 4.
[0026] Figure 11 This is a linear discriminant analysis diagram of different concentrations of CC based on a colorimetric sensor array, as shown in Example 4.
[0027] Figure 12 This is the linear discriminant analysis diagram for different concentrations of HQ based on a colorimetric sensor array in Example 4.
[0028] Figure 13 This is a linear discriminant analysis diagram of a binary substance mixture system based on a colorimetric sensor array, as shown in Example 4.
[0029] Figure 14 This is a linear discriminant analysis diagram of a ternary substance mixture system based on a colorimetric sensor array, as shown in Example 4. Detailed Implementation
[0030] Example 1:
[0031] The preparation of single-atom iron nanozymes specifically included: adding 50 mg of graphene oxide to 25 ml of distilled water and stirring; then weighing 1.7 mg of FeCl3·6H2O and adding it to a mixed solution of GO and distilled water and stirring for 15 min, where Fe / GO = 0.7 wt%. After the liquid was mixed evenly, it was placed in a rotary evaporator until the water was completely evaporated. The resulting solid was placed in a calcination furnace and calcined at 750℃ for 2 h in an argon (Ar) atmosphere to obtain the product sdsFeN@G, which was used for subsequent material preparation and related experimental investigations.
[0032] Example 2:
[0033] In order to further explore the morphology and size of sdsFeN@G, such as Figure 1 Detailed observation of the morphology of sdsFeN@G using transmission electron microscopy (TEM) revealed that it exhibits a folded, sheet-like structure. Figure 2 The surface elemental composition and chemical state were studied using X-ray photoelectron spectroscopy (XPS), and the results showed that sdsFeN@G contains Fe, C, N, and O elements. Figure 3Furthermore, X-ray electron spectroscopy (XPS) was used to analyze the materials, recording XPS spectra using Al Kα radiation to determine their chemical composition, elemental valence states, chemical states, and chemical bonds. X-ray electron diffraction (XRD) was used to determine the phases of the materials.
[0034] Example 3:
[0035] The factors influencing the catalytic activity of the peroxidase activity system, including pH, TMB substrate concentration, and reaction time, were optimized to obtain the optimal peroxidase catalytic activity of sdsFeN@G. Specific experimental conditions were as follows: 100 μL H₂O₂ + 100 μL TMB + 100 μL sdsFeN@G (1 mg / mL) were added to 700 μL acetic acid (HAc-NaAc) buffer (pH = 3.0), with pH values of 2, 3, 4, 5, 6, and 7 respectively. TMB concentrations ranged from 0.8 to 2.2 mmol / L, and reaction times ranged from 1 min to 7 min. The absorbance spectra were measured under these conditions using a UV spectrophotometer to obtain the optimal conditions. Figure 4 The first step was to optimize the HAc-NaAc buffer solutions at different pH values. Through UV absorption spectroscopy, it was found that the catalytic activity was highest at pH 3 in the reaction system. Furthermore, the activity of the HAc-NaAc buffer solution peaked at pH 3, with the left side showing a gradual increase and the right side showing a gradual decrease. Therefore, pH 3 was determined to be the optimal pH value for the HAc-NaAc buffer solution, and buffer solutions were prepared accordingly for all subsequent experiments. Figure 5 Secondly, the optimal concentration of the substrate TMB was optimized. The TMB concentration range was selected as 0.8-2.2 mmol / L. The absorbance curves at 652 nm for each concentration showed a rapid increase to the optimum before decreasing, indicating a change from rapid oxidation of TMB to oxTMB to slow oxidation. The optimum TMB substrate concentration was found to be 0.2 mmol / L. Insufficient TMB would result in a lack of reactants, affecting subsequent experiments, while excessive concentration would lead to over-reaction. Figure 6 When optimizing reaction conditions by referring to other nanozymes, it was found that the reaction time of nanozymes is also quite important. Therefore, the time was also optimized. Because the reaction is rapid, the UV absorption spectrum was tested every minute. When the concentration reached 5-7 minutes, the reaction was already in the slow rise period. Taking all factors into consideration, 5 minutes was selected as the optimal response time.
[0036] Example 4:
[0037] This paper uses the peroxidase activity of sdsFeN@G as a basis to establish a three-channel colorimetric sensing array. Because the substrate TMB can be oxidized to oxTMB, exhibiting a blue color, and the blue response varies under different pH conditions, a relatively stable colorimetric array was constructed for subsequent experiments. First, a standard curve was determined for five substances using a colorimetric method. Standard solutions for the five substances were prepared by adding 100 μL H2O2 (1 mmol / L) + 100 μL TMB (2 mmol / L) + 100 μL sdsFeN@G (1 mg / mL) to 700 μL (pH = 3.0) HAc-NaAc buffer solution. The mixture was reacted at room temperature for 5 min, then centrifuged at 1000 rpm to obtain the supernatant. 20 μL of the analyte was then added to the supernatant, and the reaction was continued for another 5 min. The absorbance spectrum was recorded at 652 nm using a UV spectrophotometer. The absorbance of A0 and A is measured, and the difference between them is ΔA, where A0 is the initial absorbance of the reaction and A is the absorbance after the addition of the analyte. The spectra before and after the reaction are measured to obtain spectra and absorbance values. ΔA and the substance concentration are then used as data to plot the absorbance of each substance using Origin software, resulting in a standardization curve. For example... 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 was used to distinguish and detect these five substances: tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), propyl gallate (PG), catechol (CC), and hydroquinone (HQ). Standard solutions for the five substances were prepared, and 100 μL of H₂O₂, 100 μL of TMB, 100 μL of sdsFeN@G (1 mg / mL), and 700 μL of buffer solution (pH = 3.0, pH = 3.5, and pH = 4.0, HAc-NaAc) were mixed and reacted at room temperature for 5 min. Afterward, the mixture was centrifuged at 1000 rpm to obtain the supernatant. 180 μL of the supernatant was transferred to a well plate, and 20 μL of the analyte was added. The reaction was continued for 5 min, and the absorbance change was recorded at 652 nm using a multi-mode microplate reader. Using three different pH values and five parallel experiments for each of the five substances, a 3*5*5 array matrix was obtained. Finally, LDA analysis was performed using SPSS software, and a scoring plot was generated. 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 this invention can successfully distinguish five antioxidants (10 μM-50 μM) at different concentrations.
[0039] Performance was demonstrated using binary and ternary mixtures of contrast-enhanced colorimetric sensors. Binary mixtures of PG and BHA with different molar ratios (PG:BHA = 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 8:2, 9:1, total concentration 10 μmol / L) and ternary mixtures of TBHQ, BHA, and HQ with different molar ratios (TBHQ:BHA:HQ = 1:2:7, 2:2:6, 1:5:4, 3:4:3, 2:3:5, total concentration 10 μmol / L) were mixed with the supernatant of the reaction system according to the above method. After reacting for 5 min, the absorbance was measured, and the results were obtained by LDA analysis. Figure 13 , 14 As shown, the colorimetric sensor array constructed in this invention can successfully identify binary and ternary mixed systems of antioxidants.
Claims
1. A method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes, characterized in that, Includes the following steps: A. Preparation and characterization of single-atom iron nanozymes; In step A, the preparation method includes: adding 50 mg of graphene oxide to 25 ml of distilled water and stirring, then weighing 1.7 mg of FeCl3·6H2O and adding it to a mixed solution of GO and distilled water and stirring, wherein Fe / GO = 0.7 wt%, placing it in a rotary evaporator to evaporate the water completely, and calcining it at 750 °C for 2 h under argon (Ar) to obtain the relevant product sdsFeN@G; B. Optimize reaction conditions based on the peroxidase activity of single-atom iron nanozymes; C. Construct a colorimetric sensor array by changing the pH of the reaction system and adding the analyte; D. Use the classic model to distinguish and judge them.
2. The method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes according to claim 1, characterized in that, In step B, the pH, substrate concentration, and reaction time parameters of the peroxidase activity system were optimized to obtain the optimal conditions. The optimized conditions were: 25℃, pH range 2.5–5, with an interval of 0.5; reaction time range 1–7 min, with an interval of 1 min; TMB concentration range 0.8–2.2 mM, with an interval of 0.02 mM; and the absorbance value corresponding to blue was recorded using a UV-Vis spectrometer.
3. The method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes according to claim 1, characterized in that, In step C, three pH values of 3.0, 3.5, and 4.0 are selected to construct a colorimetric sensor array. The size of the colorimetric sensor array is three pH values * five analytes * five parallel sets.
4. The method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes according to claim 3, characterized in that, Step C specifically includes: adding iron nanozyme, H2O2, chromogenic substrate 3'3'5'5'-tetramethylbenzidine (TMB) and acetate buffer at different pH values into a centrifuge tube, then adding a certain amount of the analyte into the tube, and after reacting for 5 minutes, the colorimetric sensing array of the analyte is obtained.
5. The method for detecting antioxidants and polyphenols in food based on a colorimetric sensor array constructed using single-atom iron nanozymes according to claim 1, characterized in that, In step D, the classic mode is linear discriminant analysis.