A FeCe / NC-TMB colorimetric sensor and its preparation method and application

Abstract

The present invention discloses an FeCe / NC enzyme and a preparation method thereof. The present invention also discloses an FeCe / NC-TMB colorimetric sensor. The present invention also discloses the application of the FeCe / NC or the FeCe / NC-TMB colorimetric sensor in detecting tannic acid. The present invention also discloses a method for detecting tannic acid. The present invention constructs an FeCe / NC-TMB colorimetric sensor, realizing highly sensitive and highly specific detection of the antioxidant TA, with a minimum detection limit of 0.26 μM. Moreover, the colorimetric sensor constructed by the present invention has high selectivity for TA.

Description

Technical Field

[0001] The present invention belongs to the technical field of sensor applications, and particularly relates to an FeCe / NC-TMB colorimetric sensor, a preparation method thereof, and an application thereof. Background Art

[0002] Long-term addition of antibiotics to livestock and poultry feed will cause a large amount of antibiotic residues in animals, posing a hazard to human health. Antioxidants were originally often added to feed to prevent or delay the oxidation and deterioration of active ingredients in the feed. In recent years, it has been found that natural antioxidants such as tannic acid also have antibacterial and anti-inflammatory effects, and have gradually become popular alternatives after the ban on antibiotics. Among them, tannic acid (TA) is a natural polyphenolic substance widely present in plants. It has multiple ortho-phenolic hydroxyl groups and is easily oxidized to release H + ⁺, which can compete with free radicals for binding and is a very strong antioxidant. At the same time, tannic acid can also use phenolic hydroxyl groups to form hydrogen bonds with the polypeptide carbonyl groups on proteins in virus microorganisms, thereby inhibiting enzyme activity and achieving antibacterial effects. In addition, it can also extend the residence time of chyme in the small intestine, reduce the phenomenon of overfeeding in the small intestine, prevent animal diarrhea, and improve animal survival rate. However, excessive use of antioxidants will also cause the accumulation of antioxidants in animals and damage animal health.

[0003] At present, many analytical methods have been developed for the detection of antioxidants in feed, such as high-performance liquid chromatography, gas chromatography, electrochemistry, etc. Shang Jun et al. optimized the chromatographic detection wavelength, mobile phase, and flow rate, and then used high-performance liquid chromatography to simultaneously measure five synthetic antioxidants, namely propyl gallate (PG), tert-butylhydroquinone (TBHQ), ethoxyquinoline (EQ), butylated hydroxyanisole (BHA), and dibutylhydroxytoluene (BHT). The lowest detection limit of PG was 1.8 mg / kg. Chen Jiyuan used gas chromatography to detect five commonly used synthetic antioxidants. After the target sample was extracted with ethanol, it was separated by a capillary chromatographic column, and had a good linear range within 2 - 40 μg / mL, with the lowest detection limit of 5 mg / kg. Chan-Eam et al. studied the detection of antioxidant BHA by differential pulse voltammetry in micellar solutions and emulsified solutions. The lowest detection limit in the emulsified solution was as low as 8.5×10 -7 ⁻⁶ mol / L. Although these methods have the advantages of high accuracy, good reproducibility, and low detection limit, most of them require large instruments, have relatively high requirements for the skills of operators, and cannot meet the requirements of on-site real-time detection. Therefore, establishing a simpler and more portable antioxidant detection method is of great significance in the field of feed monitoring.

[0004] In recent years, due to the high catalytic activity of single-atom nanozymes, there have been an increasing number of reports on constructing biosensors for detecting antioxidants. Zhang et al. synthesized SA-Pd / NPC with peroxidase-like activity and constructed a Pd / NPC-H2O2-TMB detection system, achieving colorimetric detection of thiol-containing antioxidant glutathione (GSH) and thiol-free ascorbic acid (AA). The detection ranges of GSH and AA were 0.01 - 0.1 mM and 1 - 13 μM, respectively, and the lowest detection limits were 3 μM and 0.3 μM, respectively. Jing et al. synthesized FeN4 with oxidase-like activity and constructed a three-channel colorimetric sensing platform using its property of catalyzing TMB, OPD, and ABTS, which could simultaneously identify GSH, ascorbic acid (AA), L-cysteine (L-Cys), uric acid (UA), and melatonin (MT). The detection limits for these five antioxidants were as low as 100 nM. Taking GSH as a model, spiked recovery was carried out in human serum, and the recovery rate was 93.3 - 103.5%, providing a strategy for Fe-N / C single-atom nanozymes in constructing sensor arrays. Shen et al. prepared Fe-N / C synthesized by three different methods and based on these three enzymes prepared a colorimetric sensing array, using machine learning for color discrimination to achieve simultaneous detection of six antioxidants, namely AA, GSH, L-Cys, UA, dopamine (DA), and dithiothreitol (DTT). From these studies, it was concluded that biosensors constructed using SANs had broad application prospects in the field of antioxidant detection.

[0005] In recent years, metal-organic framework (MOF) materials, which are highly porous crystalline coordination compounds composed of inorganic metal nodes and organic linkers, have been considered as the preferred materials for constructing carbon-based SANs due to their advantages such as large specific surface area, adjustable pore structure, and diverse compositions. In particular, zinc-based zeolitic imidazolate framework-8 (ZIF-8) has attracted much attention due to its sodalite-like structure, abundant nitrogen-containing organic linkers, and high thermal stability. For example, Chen et al. prepared single-atom nanozymes with Fe as the active center by high-temperature pyrolysis of ZIF-8, which exhibited oxidase-like activity and was successfully used for the detection of alkaline phosphatase activity. Zhao et al. prepared FeCo / SANs with a bimetallic non-alloy structure by carbonizing the FeCo-ZIF-8 precursor. It had both oxidase activity and peroxidase-like activity and could directly catalyze H2O2 and O2 to form reactive oxygen species (ROS) simultaneously. The generated ROS could significantly induce apoptosis and inhibit tumor growth.

[0006] However, single-atom nanozymes constructed from a single element are gradually unable to meet the needs of practical detection. To further improve the activity of SANs, a large number of research reports have found that by changing single metal atoms into dual-metal atoms or multi-metal atoms, the availability of active surface sites can be altered through the interaction between atoms, thereby further enhancing the catalytic activity of SANs. For example, Wu et al. prepared SANs co-doped with three elements, Fe, Co, and Zn. Through experimental comparison, it was found that the SANs co-doped with Fe, Co, and Zn had higher enzyme activity than those prepared from a single element. Ji et al. found that replacing N on FeN4 with a P to form FeN3P could also improve the catalytic effect of SANs.

[0007] Cerium (Ce) is one of the most abundant elements on Earth and is very inexpensive compared to other rare earth metals. In addition, due to its special outer electron structure [Xe]4f15d16s2, Ce has a significant redox pair (Ce 3+ / Ce 4+ ). Therefore, nanozymes composed of Ce elements have been widely studied at present. In 2010, Pirmohamed first discovered that CeO2 has peroxidase-like activity. Hang et al. prepared three typical nanostructures, namely nanorods, nanopolyhedra, and nanocubes, by adjusting the scale of CeO2 nanozymes, and found that they have oxidase-like activity. However, most of these studies have focused on CeO2, and there are relatively few studies on Ce / NC alone. Therefore, further experimental verification is still needed to develop Ce / NC with high catalytic activity. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide a new highly active FeCe / NC enzyme with high catalytic activity and its preparation method.

[0009] Another technical problem to be solved by the present invention is to provide a highly sensitive and highly specific FeCe / NC-TMB colorimetric sensor.

[0010] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned FeCe / NC or FeCe / NC-TMB colorimetric sensor in the detection of tannic acid.

[0011] The last technical problem to be solved by the present invention is to provide a highly sensitive and highly specific detection method for TA.

[0012] Technical Solution: To solve the above technical problems, the present invention provides a preparation method for FeCe / NC, including the following steps:

[0013] 1) Fe(NO3)3·9H2O, gZn(NO3)2·6H2O and Ce(NO3)3·6H2O are dissolved in a methanol solution, and then added to a methanol solution containing 2-methylimidazole (MIM). After vigorous stirring at room temperature, the mixture is allowed to stand for aging, the precipitate is collected by centrifugation, washed with a methanol solution, and dried in a vacuum oven to obtain FeCe-ZIF-8;

[0014] 2) The dried FeCe-ZIF-8 is thoroughly ground into powder, then placed in a ceramic boat and put into a tubular furnace. Under the protection of high-purity nitrogen, it is heated to 600 - 1100 °C, held at a constant temperature, and then cooled to finally obtain FeCe / NC.

[0015] Among them, the molar ratio of Fe(NO3)3·9H2O, gZn(NO3)2·6H2O and Ce(NO3)3·6H2O in step 1) is: 5∶201.6∶12 - 5∶201.6∶144.

[0016] Among them, the mass-volume ratio of MIM and methanol is 6 - 13 mg / mL.

[0017] Among them, the heating rate in step 2) is 5 - 10 °C / min.

[0018] Preferably, the holding time in step 2) is 2 - 3 hours; preferably, the heating temperature in step 2) is 1000 °C;

[0019] Preferably, the cooling step in step 2) is: cooling to 500 °C at a rate of 10 °C / min, and then cooling to room temperature.

[0020] The present invention also includes FeCe / NC obtained by the above preparation method.

[0021] The present invention also includes a FeCe / NC-TMB colorimetric sensor, which includes the above FeCe / NC and TMB. Preferably, the concentration of FeCe / NC is 3.0 - 7.0 mg / L, the concentration of TMB is 2 - 4 mM, and it also includes a 200 - 300 mM acetic acid-sodium acetate buffer solution with a pH of 2 - 6.

[0022] The present invention also includes the application of the above FeCe / NC-TMB colorimetric sensor in detecting tannic acid.

[0023] The present invention also includes a method for detecting tannic acid, which includes the following steps: mixing the FeCe / NC-TMB colorimetric sensor with different concentrations of TA to obtain a standard curve of the relationship between the absorbance value and the concentration of TA, and then determining the concentration of tannic acid in the sample to be tested according to this standard curve.

[0024] Among them, the concentration of FeCe / NC in the FeCe / NC-TMB colorimetric sensor is 3.0 - 4.0 mg / L. Preferably, the concentration of FeCe / NC is 4.0 mg / L.

[0025] Among them, the pH of the acetic acid-sodium acetate buffer solution in the FeCe / NC-TMB colorimetric sensor is 3.5.

[0026] Mechanism of the present invention: A novel FeCe / NC with high catalytic activity is prepared in the present invention. First, a FeCe-ZIF-8 precursor is prepared by self-assembly, and the carbonization temperature is optimized to obtain a highly active FeCe / NC enzyme. By comparing the enzyme reaction kinetics of FeCe / NC and Fe / NC, it is proved that the prepared novel FeCe / NC has higher catalytic activity. Then, a colorimetric biosensing platform is constructed to detect the antioxidant TA. The detection principle is that FeCe / NC has peroxidase-like activity and can catalyze O2 to generate free radicals, oxidizing the colorless substrate TMB into blue oxTMB, which has an absorption peak at 652 nm. When TA is added, the antioxidant can scavenge free radicals, thereby inhibiting the generation of blue oxTMB and reducing the absorbance value of the solution at 652 nm. The concentration of TA is proportional to the decrease in the absorbance value at 652 nm. Therefore, colorimetric detection of TA can be achieved based on this principle. Further, in order to improve the sensitivity of the detection, the present invention optimized the reaction pH, reaction temperature, reaction time, and FeCe / NC enzyme concentration of the FeCe / NC-TMB detection system, and obtained that the optimal experimental conditions are at pH 3.5, reaction temperature 30 °C, reaction time 12 minutes, and FeCe / NC enzyme concentration of 4 mg / L, and the system has the highest sensitivity.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention constructs a FeCe / NC-TMB colorimetric sensor, realizing highly sensitive and highly specific detection of the antioxidant TA. The decrease in the absorbance value at 652 nm has a good linear range with the concentration of added TA. The detection range of TA is 1.0 - 6.0 μM, and R 2 is 0.995, and the lowest detection limit is 0.26 μM. In addition, the selectivity of the constructed colorimetric sensor for TA was verified. The results show that only TA can cause a significant decrease in absorbance, and the influence of other common interfering ions on absorbance can be ignored. The sensor has good selectivity for TA. Finally, by detecting TA in the actual sample feed and comparing it with the national standard method, there is no difference between the two, proving that this method can be applied to the detection of actual samples. Description of the Drawings

[0028] Figure 1Schematic diagram of the principle of the FeCe / NC-TMB colorimetric sensor for detecting the antioxidant tannic acid;

[0029] Figure 2 Effect of synthesis temperature on the activity of FeCe / NC;

[0030] Figure 3 Investigation of the peroxidase-like activity of FeCe / NC, (A) Absorption spectra of FeCe / NC catalyzing the oxidation of different substrates; (B) UV-visible spectra of the FeCe / NC-TMB system;

[0031] Figure 4 Steady-state kinetics of FeCe / NC and Fe / NC towards TMB, (A) Steady-state kinetic analysis of FeCe / NC; (B) Lineweaver-Burk plot corresponding to FeCe / NC; (C) Steady-state kinetic analysis of Fe / NC; (D) Lineweaver-Burk plot corresponding to Fe / NC;

[0032] Figure 5 Optimization of the pH of the FeCe / NC-TMB detection system;

[0033] Figure 6 Optimization of the reaction temperature of the FeCe / NC-TMB detection system;

[0034] Figure 7 Optimization of the reaction time of the FeCe / NC-TMB detection system;

[0035] Figure 8 Optimization of the FeCe / NC concentration in the FeCe / NC-TMB detection system;

[0036] Figure 9 Linear regression curve of the FeCe / NC-TMB for detecting TA;

[0037] Figure 10 Specificity evaluation of the FeCe / NC-TMB detection system. Detailed implementation method

[0038] Example 1 Preparation of FeCe / NC

[0039] First, 0.1 g of Fe(NO3)3·9H2O, 2.975 g of Zn(NO3)2·6H2O, and 3.126 g of Ce(NO3)3·6H2O were dissolved in 265 mL of methanol solution, and then added to a solution containing 3.456 g of 2-methylimidazole (MIM) and 265 mL of methanol. After vigorously stirring at room temperature for 2 hours, it was left to age for 24 hours. Finally, the precipitate was collected by centrifugation at 8000 rpm for 8 minutes, then washed 3 times with methanol solution, and dried in a vacuum oven at 60 °C to obtain FeCe-ZIF-8.

[0040] The dried FeCe-ZIF-8 was thoroughly ground into powder using a mortar, then placed in a ceramic boat, and finally the ceramic boat containing the powder was put into a tube furnace. Under the protection of high-purity nitrogen, the temperature was raised at a rate of 5 °C / min. To explore the effect of synthesis temperature on enzyme activity, it was heated to 600, 700, 800, 900, 1000, and 1100 °C respectively, then held at the target temperature for 2 hours, cooled to 500 °C at a rate of 10 °C / min, and then naturally cooled to room temperature. Finally, FeCe / NC was obtained. The preparation of Fe / NC was carried out without adding Ce(NO3)3·6H2O, and other steps remained unchanged.

[0041] First, Fe in Fe(NO3)3 and Ce in Ce(NO3)3 were encapsulated by ZIF-8 to form trimetal-doped FeCe-ZIF-8. The peroxidase-like activity of FeCe / NC is mainly related to its specific surface area, active site density, and electron conductivity. Changing the carbonization temperature of FeCe / NC can directly affect these three factors. Therefore, we investigated six carbonization temperatures of 600, 700, 800, 900, 1000, and 1100 °C. As Figure 2 shown, due to the low carbonization temperature, highly active sites could not be formed at 600 °C and 700 °C, so FeCe / NC had low catalytic activity. When the calcination temperature was continuously increased, the peroxidase-like activity of FeCe / NC was greatly improved. When the carbonization temperature continued to rise to 1100 °C, the peroxidase-like activity decreased. This may be because the best balance of these three factors was achieved at 1000 °C. Therefore, 1000 °C was selected as the optimal reaction temperature.

[0042] Example 2 Determination of peroxidase-like activity of FeCe / NC

[0043] To evaluate its peroxidase-like properties, first, we used different chromogenic substrates including 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and o-phenylenediamine (OPD). As Figure 3As shown in , FeCe / NC oxidizes TMB to blue oxTMB, which has an obvious absorption peak at 652 nm; it oxidizes OPD to yellow oxOPD, which has an obvious absorption peak at 450 nm; it oxidizes ABTS to green oxABTS, which has an obvious absorption peak at 412 nm. Compared with the other two substrates, TMB is non-carcinogenic, more sensitive, and has a faster oxidation rate, resulting in faster color development. Therefore, TMB was selected as the substrate for subsequent experiments.

[0044] TMB was selected as the colorimetric substrate. FeCe / NC catalyzes the formation of blue oxTMB from colorless TMB, and its absorption peak is located at 652 nm. Add FeCe / NC (100 μL, 500 μg / mL) to 1.8 mL of acetate buffer (200 mM, pH 3.5), and then add TMB (100 μL, 4 mM). Then incubate the mixture at 30 °C and measure the change rate of absorbance (λ652 nm) for 12 minutes. As Figure 3 As shown in , in acetic acid-sodium acetate buffer (200 mM, pH 3.5), TMB, FeCe / NC, and TMB + FeCe / NC were added respectively. The colorimetric cuvettes with TMB and FeCe / NC added alone showed no color change. Only when TMB and FeCe / NC were added simultaneously did the solution turn blue. Therefore, it was proved that the color change of the substrate was caused by the peroxidase-like property of FeCe / NC.

[0045] Example 3 Steady-state kinetic analysis experiment of FeCe / NC

[0046] TMB was selected as the substrate molecule to evaluate the steady-state kinetics of FeCe / NC. The specific steps are as follows: First, add 100 μL of 500 μg / mL FeCe / NC to 1.8 mL of 200 mM acetic acid-sodium acetate buffer (pH 3.5). Finally, add 0.1 mL of TMB with different concentrations (0.01, 0.02, 0.03, 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 1 mM) respectively. React at 30 °C, and record the absorbance value (λ652 nm) once every 30 seconds using a spectrophotometer after reacting for 30 seconds. Then, convert the absorbance OD value at 652 nm to the concentration of oxTMB substance through the Beer-Lambert law (A = kbC). Among them, A represents the absorbance OD value, k represents the molar extinction coefficient, and the molar extinction coefficient of oxTMB here is 39000 M -1 cm -1 , b represents the optical path cm, and C represents the concentration of the substance. Finally, the kinetic parameters were calculated through the Lineweaver-Burk equation:

[0047]

[0048] Among them, Vmax represents the maximum reaction rate, Km represents the Michaelis constant, V0 is the initial rate, and [S] is the TMB concentration. The enzyme reaction kinetics of Fe / NC was measured under the same conditions.

[0049] The enzyme kinetic results of FeCe / NC and Fe / NC are summarized in Figure 4 , which shows a typical Michaelis-Menten curve. The Vmax and Km were derived using the double-reciprocal curve Lineweaver-Burk equation.

[0050]

[0051]

[0052] The equations for FeCe / NC and Fe / NC are y = 0.019x + 0.146, R 2 = 0.998 and y = 0.042x + 0.179, R 2 = 0.993, where y is the reciprocal of the initial reaction rate, x is the reciprocal of the substrate concentration, and R 2 is the correlation coefficient. The Vmax and Km of FeCe / NC are 7.68×10 -7 Ms -1 and 0.15 mM, respectively, and the Vmax and Km of Fe / NC are 5.59×10 -7 Ms -1 and 0.237 mM, respectively. A lower Km value represents a stronger interaction between the enzyme and its substrate. The Km value of FeCe / NC is lower than that of Fe / NC, indicating that FeCe / NC exhibits higher catalytic performance.

[0053] Compared with other reported Fe / NC in the literature, the enhanced peroxidase-like activity of FeCe / NC may be attributed to the following reasons. (1) The synergistic effect of Fe and Ce bimetallic active centers. (2) The FeCe / NC prepared in this example has a higher BET specific surface area, exposing more active sites, increasing the contact probability of O2 with FeCe / NC, and thus improving the catalytic efficiency.

[0054] Example 4 Construction and optimization of the FeCe / NC-TMB system, establishment of the standard curve, and sensitivity detection experiment of TA

[0055] As an antioxidant, TA can scavenge free radicals and inhibit the oxidation of TMB. Therefore, we constructed a FeCe / NC-TMB biosensing platform for the detection of TA.

[0056] The FeCe / NC-TMB colorimetric biosensor includes: FeCe / NC with a concentration of 4.0 - 7.0 mg / L, TMB with a concentration of 2 - 4 mM, and 200 - 300 mM acetic acid - sodium acetate buffer with a pH of 2 - 6.

[0057] To improve the sensitivity of the FeCe / NC-TMB system for detecting TA, several experimental parameters were optimized, including the pH of the reaction, reaction time, reaction temperature, and the concentration of FeCe / NC.

[0058] pH optimization: 0.1 mL of 25 mg / L FeCe / NC was added to 1.8 mL of 200 mM acetic acid - sodium acetate buffer (pH 2, 3, 3.5, 4, 5, 6). Finally, 0.1 mL of 4 mM TMB was added, and the reaction was carried out at 30 °C for 12 minutes, and then the absorbance value was recorded. As Figure 5 shown, when FeCe / NC is in the acetic acid buffer with a pH range of 2 - 3.5, as the acidity weakens, the pseudo - enzyme activity of FeCe / NC gradually increases. The reason is that under more acidic conditions, FeCe / NC agglomerates in the solution to form large black particles, which affects the catalytic activity of the enzyme. And when the pH is less than 2, TMB can convert the blue cationic radical into a yellow quinone - type compound, resulting in no absorbance at 652 nm. When the pH continues to increase in the range of 3.5 - 6, as the acidity weakens, the solubility of TMB gradually decreases, resulting in a decrease in the absorbance of the TMB product at 652 nm. Therefore, the optimal reaction pH of FeCe / NC is 3.5.

[0059] Secondly, the pseudo - oxidase of FeCe / NC is also sensitive to the reaction temperature. Therefore, the reaction temperature was further optimized: 0.1 mL of 25 mg / L FeCe / NC was added to 1.8 mL of 200 mM acetic acid - sodium acetate buffer (pH 3.5). Finally, 0.1 mL of 4 mM TMB was added, and the reaction was carried out at 20, 25, 30, 35, 40, 50, 60 °C for 12 minutes, and then the absorbance value was recorded. As Figure 6 shown, when the temperature is in the range of 20 - 30 °C, as the temperature increases, the pseudo - oxidase activity remains almost unchanged; then when the temperature continues to increase, the pseudo - oxidase activity gradually decreases. Therefore, 30 °C is the optimal reaction temperature.

[0060] Then, the optimization of the reaction time of the FeCe / NC pseudo - oxidase was investigated: 0.1 mL of 25 mg / L FeCe / NC was added to 1.8 mL of 200 mM acetic acid - sodium acetate buffer (pH 3.5). Finally, 0.1 mL of 4 mM TMB was added, and the reaction was carried out at 30 °C for 12 - 25 minutes, and then the absorbance value was recorded. As Figure 7As shown, the absorbance gradually increases at 0 = 12 minutes and levels off at 12 - 25 minutes. Therefore, the optimal reaction time was selected as 12 minutes.

[0061] Finally, when constructing the detection system, the concentration of FeCe / NC has a great influence on the sensitivity of the detection system and can greatly improve the detection sensitivity. Therefore, under the condition that other reaction conditions remain unchanged, the enzyme concentration of FeCe / NC in the detection system was further optimized: 0.1 mL (3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L) of FeCe / NC was added to 1.7 mL of 200 mM acetic acid - sodium acetate buffer (pH 3.5), and then 0.1 mL of 5 μM TA was added. Finally, 0.1 mL of 4 mM TMB was added, and the reaction was carried out at 30 °C for 12 minutes, and then the absorption value was recorded. As Figure 8 shown, when the concentration of FeCe / NC is between 3.0 - 4.0 mg / L, the sensitivity of the FeCe / NC - TMB detection system gradually increases. When the enzyme concentration continues to increase from 4.0 - 7.0 mg / L, the sensitivity of the detection system gradually decreases. Therefore, the concentration of FeCe / NC was selected as 4.0 mg / L in the experiment.

[0062] Under the optimal conditions (pH 3.5 and temperature 30 °C), the absorbance (652 nm) of different concentrations of TA was measured after 12 - minute reaction. As Figure 9 shown, the concentration of TA is linearly related to the change in absorbance at A652 in the range of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 μM, that is, in the range of 1.0 - 6.0 μM. The equation is y = 0.0446x + 0.0394. Here, y and x represent the decrease in absorbance at A652 nm and the concentration of TA respectively, and the correlation coefficient (R 2 ) is 0.995, indicating a good linear relationship between them. The lowest detection limit (LOD) of TA was calculated to be 0.26 μM (S / N = 3), proving that the FeCe / NC - TMB colorimetric sensor has high sensitivity for TA detection.

[0063] Example 5 Selectivity Experiment of FeCe / NC - TMB

[0064] To verify the anti - interference ability of the constructed FeCe / NC - TMB system for antioxidant detection, some common interfering ions such as Ca 2+ , K + , Na +, Arg, Gly, Glu, and Met were added to the developed system. Here, the concentration of the added anti-interference ions was much higher than the maximum detection concentration of TA, which is 6 μM. The other experimental steps were the same as those for detecting TA. 0.1 mL of 4 mg / L FeCe / NC was added to 1.7 mL of 200 mM acetic acid-sodium acetate buffer (pH 3.5), and then 0.1 mL of 6 μM TA and 0.1 mL of 100 μM Ca 2+ , K+, and Na + , interference ions such as arginine (Arg), glycine (Gly), glutamic acid (Glu), and methionine (Met) were added. Finally, 0.1 mL of 4 mM TMB solution was added, and the reaction was carried out at 30 °C for 12 minutes, and then the absorbance value was recorded. The absorbance value at the maximum concentration of 6 μM within the standard curve range of TA was selected as the ordinate and compared with the absorbance values of other anti-interference ions. As Figure 10 shown, when 100 μM of high-concentration interference ions were added, the absorbance value of the system remained almost unchanged compared with the blank absorbance value, indicating that the changes caused by these interference ions were negligible. Only when TA was added did the absorbance value at 652 nm decrease significantly, demonstrating that the constructed FeCe / NC-TMB colorimetric biosensor has high specificity for the antioxidant TA.

[0065] Example 6 Repeatability and reproducibility experiments of the FeCe / NC-TMB colorimetric sensor

[0066] To study the repeatability of the constructed FeCe / NC-TMB colorimetric sensor, the sample containing 5.0 μM TA was measured in parallel three times. The results showed that the relative standard deviation (RSD) was 3.22%, indicating that the biosensor has good repeatability for the antioxidant TA. The stability of the constructed colorimetric sensor mainly depends on the stability of FeCe / NC stored at room temperature. After storing the synthesized FeCe / NC at room temperature for 30 days, the sensor was used to detect the 5 μM TA sample. The test result was 4.54 + 0.09 μM, the absolute error was 0.46 μM, and the RSD was 1.67% (<5%), so the method has good reproducibility for the detection of TA.

[0067] Example 7 Detection of actual samples

[0068] To further verify the reliability of the constructed sensor for the detection of antioxidants in actual samples, TA in feed was tested. The FeCe / NC-TMB sensor was used to detect the content of TA in actual samples, and the content of TA in feed was detected by the national standard method (NYSL-1006-2022, China) for comparison, and Folin-Ciocalteu method was selected as the reference method. The feasibility of this method in practical applications was evaluated. First, the feed was crushed into powder using a grinder, 2 g was accurately weighed and placed in a conical flask, 50 mL of acetone solution was added, and it was stirred in a water bath at 55 °C for 1 hour. After cooling sufficiently, it was centrifuged at 10000 r / min for 10 minutes, and the supernatant was taken for standby. The feed extract was filtered through a syringe filter filtered, then the extract was diluted, and finally an appropriate amount of TA was added for detection. Three parallel tests were carried out for each sample.

[0069] Table 1 Comparison of the determination results of tannic acid in feed by the national standard method and the developed method

[0070]

[0071] The results are shown in Table 1. TA was added to three feed samples at 0.5 mmol / kg, 1 mmol / kg, and 2.5 mmol / kg, respectively. The recoveries were 97.0%, 101.9%, and 98.6%, respectively, and the RSDs were 5.8%, 2.3%, and 4.3%, respectively. There was no significant statistical difference between the Folin-Ciocalteu method and the FeCe / NC-TMB method (p > 0.05), which verified again the reliability of the constructed method for detecting antioxidants. The results showed that the biosensing system constructed with FeCe / NC had broad application prospects in analytical detection.

Claims

1. A preparation method of FeCe / NC, characterized in that, It includes the following steps: 1) Fe(NO3)3•9H2O, Zn(NO3)2•6H2O and Ce(NO3)3•6H2O are dissolved in a methanol solution, and then added to a methanol solution containing 2-methylimidazole. After vigorous stirring at room temperature, it is left to age, the precipitate is collected by centrifugation, then washed with a methanol solution, and dried in a vacuum oven to obtain FeCe-ZIF-8; 2) The dried FeCe-ZIF-8 is thoroughly ground into powder, then placed in a ceramic boat and put into a tube furnace. Under the protection of high-purity nitrogen, it is heated to 800-1100 °C, held at a constant temperature, and then cooled to finally obtain FeCe / NC. The molar ratio of Fe(NO3)3•9H2O, Zn(NO3)2•6H2O and Ce(NO3)3•6H2O in step 1) is 5:201.6: (12-144).

2. The preparation method of FeCe / NC according to claim 1, characterized in that, The heating rate in step 2) is 5-10 °C / min.

3. The preparation method of FeCe / NC according to claim 1, wherein, The holding time in step 2) is 2-3 hours.

4. The preparation method of FeCe / NC according to claim 1, characterized in that, The heating temperature in step 2) is 1000 °C.

5. The preparation method of FeCe / NC according to claim 1, characterized in that, The cooling step in step 2) is: cooling to 500 °C at 10 °C / min, and then cooling to room temperature.

6. FeCe / NC obtained by the preparation method according to any one of claims 1-5.

7. A FeCe / NC-TMB colorimetric sensor, characterized in that, The FeCe / NC-TMB colorimetric sensor includes FeCe / NC according to claim 5 and TMB.

8. The FeCe / NC-TMB colorimetric sensor according to claim 7, wherein The concentration of the FeCe / NC is 3.0-7.0 mg / L, the concentration of TMB is 2-4 mM, and it also includes a 200-300 mM acetic acid-sodium acetate buffer solution with a pH of 2-6.

9. The application of FeCe / NC according to claim 6 or the FeCe / NC-TMB colorimetric sensor according to claim 7 or 8 in detecting tannic acid.

10. A method for detecting tannic acid, characterized in that, It includes the following steps: mixing the FeCe / NC-TMB colorimetric sensor according to claim 7 with tannic acid TA at different concentrations to obtain a standard curve of the relationship between the absorbance value and the concentration of tannic acid TA, and then measuring the concentration of tannic acid in the sample to be measured according to this standard curve.

11. The method according to claim 10, wherein The concentration of FeCe / NC in the FeCe / NC-TMB colorimetric sensor is 3.0-4.0 mg / L.

12. The method according to claim 11, wherein The concentration of the FeCe / NC is 4.0 mg / L.

13. The method according to claim 10, characterized in that The pH of the acetic acid-sodium acetate buffer solution in the FeCe / NC-TMB colorimetric sensor is 3.5.

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