A method for detecting tannic acid based on prussian blue nanoscale enzyme and application thereof

Abstract

The application relates to a tannic acid detection method based on Prussian blue nanoscale enzyme and application thereof, and the method comprises the following steps: adding HCl in K3[Fe(CN)6] * 3H2O, heating reaction, centrifugal collection of the precipitate, washing and drying to obtain Prussian blue nanoscale enzyme; mixing the aqueous solution of the Prussian blue nanoscale enzyme, a chromogenic substrate, peroxide and tannic acid standard working solutions with different concentrations, water bath reaction, measuring the absorbance at 650-655 nm, and drawing a standard curve; mixing the aqueous solution of the Prussian blue nanoscale enzyme, the chromogenic substrate, the peroxide and a sample to be measured, water bath reaction, measuring the absorbance at 650-655 nm, and calculating the tannic acid concentration in the sample to be measured according to the standard curve. The detection method constructed by the application is simple in operation, high in sensitivity and good in selectivity, can be accurately and quickly used for quantitative analysis of tannic acid in tobacco, and has a good application prospect in tobacco quality evaluation and related fields.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a method for detecting tannic acid based on Prussian blue nanozymes and its application. Background Technology

[0002] Tannic acid, also known as tannin, is a natural polyphenolic compound widely found in the leaves, peels, and wood of various plants. It gradually turns black upon exposure to air and has a pronounced astringent taste with no particular odor. Chemically, tannic acid is a hydrolyzable tannin, capable of hydrolyzing under heating conditions to produce simple phenolic substances such as gallic acid. Furthermore, tannic acid has the property of forming polynuclear complexes with metal ions. Tannic acid is a tannic acid naturally present in tobacco and is one of the main sources of astringency, significantly impacting the sensory quality of tobacco. On the one hand, excessively high tannic acid content needs to be controlled through techniques such as microbial degradation to improve the bitterness and unpleasant odor of tobacco smoke; on the other hand, the unique chemical properties of tannic acid make it promising for applications in nicotine release regulation, free radical scavenging, and process performance improvement.

[0003] The main methods for detecting tannins include spectrophotometry, chromatography and coupled techniques, and traditional chemical analysis methods. Currently, multiple detection methods have been established for tannin content in various fields, covering different sample types. For example, GB / T 15686-2008 "Determination of Tannin Content in Sorghum", GB / T 27985-2011 "Determination of Tannins in Feed - Spectrophotometric Method", and GB 1886.303-2021 "National Food Safety Standard for Food Additives - Edible Tannins" clearly stipulate the technical requirements and test methods for tannins used as food additives. However, there are currently few reported methods for detecting tannins in tobacco, and a rapid detection method needs to be established.

[0004] Nanozymes are a class of nanomaterials that possess catalytic activity similar to natural enzymes. First discovered in 2007, they bridged the gap between inorganic and organic life. Compared to traditional natural enzymes (mainly proteins), nanozymes not only have similar catalytic mechanisms but also offer significant advantages such as high stability (heat and acid / alkali resistance), low cost, and ease of large-scale preparation. Currently, research on constructing colorimetric sensors based on nanozymes for the detection of tannic acid is limited.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting tannic acid based on Prussian blue nanozymes and its application, which can be used for accurate and rapid quantitative analysis of tannic acid in tobacco.

[0007] In a first aspect, the present invention provides a method for detecting tannic acid based on Prussian blue nanozymes, comprising the following steps: S1. HCl was added to K3[Fe(CN)6]·3H2O and stirred to obtain a clear solution. The reaction was heated and then cooled to room temperature after the reaction was completed. The precipitate was collected by centrifugation, washed several times with water and ethanol, and dried to obtain Prussian blue nanozyme. S2. Mix the aqueous solution of Prussian blue nanozyme, chromogenic substrate, peroxide and standard working solutions of tannic acid of different concentrations. After mixing evenly, react in a water bath, cool to room temperature, and measure the absorbance at 650-655 nm (preferably 652 nm). Plot a standard curve based on the relationship between tannic acid concentration and absorbance. S3. Mix the aqueous solution of Prussian blue nanozyme, chromogenic substrate, peroxide and the sample to be tested. After mixing evenly, react in a water bath, cool to room temperature, and measure the absorbance at 650-655 nm (preferably 652 nm). Calculate the tannic acid concentration in the sample to be tested based on the standard curve.

[0008] Preferably, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0009] Preferably, the peroxide is hydrogen peroxide (H2O2).

[0010] Preferably, the pH of the water bath reaction is controlled at 3-5, more preferably 4, and the Prussian blue nanozyme has good enzyme catalytic activity.

[0011] Preferably, the temperature of the water bath reaction is controlled at 37-57°C, more preferably 37°C, and the Prussian blue nanozyme has good enzyme catalytic activity.

[0012] Preferably, the water bath reaction time is controlled at 10-60 min, more preferably 10 min, and Prussian blue nanozyme has good enzyme catalytic activity.

[0013] Preferably, in the water bath reaction, the concentration of Prussian blue nanozyme is controlled at 10-50 μg / mL, more preferably 10 μg / mL, as Prussian blue nanozyme has good enzyme catalytic activity.

[0014] In this invention, using 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide as substrates, it was found that Prussian blue nanozymes have peroxidase activity. The addition of tannic acid can significantly affect the absorbance of the system, indicating that tannic acid has an inhibitory effect on the catalytic activity of Prussian blue nanozymes. This system can be well used for the detection of tannic acid.

[0015] Preferably, when the tannic acid concentration is in the range of 0.5-15 μM, the absorbance exhibits a good linear relationship with the concentration, and the linear equation is y=0.038x+0.9479, R 2 =0.9955.

[0016] Preferably, the limit of detection for tannic acid is 0.12 μM and the limit of quantification is 0.37 μM.

[0017] Preferably, the sample to be tested is a tobacco sample, and its preparation includes the following steps: adding an ethanol solution to the tobacco, vortexing at room temperature and centrifuging, and then transferring the supernatant.

[0018] Preferably, the preparation of the tannic acid standard working solutions of different concentrations includes the following steps: The tannic acid standard was dissolved in water to obtain a tannic acid stock solution; The tannic acid stock solution was diluted to obtain a tannic acid solution. Different volumes of tannic acid solution were transferred and diluted with buffer solution to obtain standard working solutions of tannic acid with different concentrations.

[0019] In a second aspect, the present invention provides a tannic acid detection sensing platform based on Prussian blue nanozymes, wherein the tannic acid detection method based on Prussian blue nanozymes is used for detection.

[0020] A third aspect of the present invention provides the application of the aforementioned Prussian blue nanozyme-based tannic acid detection sensing platform in the detection of tannic acid in tobacco.

[0021] The present invention has at least the following beneficial effects: This invention successfully prepared Prussian blue nanozymes (PB NPs) at room temperature and conducted a systematic study on their enzyme-mimicking properties. The optimal catalytic efficiency of PB NPs was determined through optimization of catalytic conditions and enzymatic performance evaluation. Simultaneously, the study found that tannic acid can effectively inhibit the enzyme-mimicking activity of PB NPs. Based on this unique inhibitory effect, this invention constructed a tannic acid detection method based on Prussian blue nanozymes. By plotting a standard curve of absorbance versus tannic acid concentration, the method was successfully applied to the quantitative detection of tannic acid in tobacco samples. Furthermore, this method exhibits good anti-interference ability against potentially interfering substances in tobacco samples. This method is simple to operate, highly sensitive, highly resistant to interference, and has good selectivity, showing promising application prospects in tobacco quality assessment and related fields. Therefore, this invention not only deepens the exploration of the regulatory conditions of PB NPs enzyme-mimicking but also provides a new approach for the rapid screening of tannic acid in food / agricultural product safety testing. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 The UV-vis spectra of the TMB-PB NPs, TMB-H2O2 and TMB-H2O2-PB NPs solutions provided by the present invention are shown in the inset at the top left corner. In the inset at the top left corner, a, b and c are photographs of the TMB-PB NPs, TMB-H2O2 and TMB-H2O2-PB NPs solutions, respectively.

[0024] Figure 2 The graph shows the effects of pH, PB NP concentration, temperature, and reaction time on the activity of PB NPs-mimicking enzymes provided by this invention.

[0025] Figure 3 The graph shows the detection results of tannic acid provided by the present invention; where a is the absorption spectrum of tannic acid at different concentrations added to the TMB-H2O2-PB NPs system, and b is the standard curve of tannic acid concentration versus absorbance.

[0026] Figure 4 The figure shows the stability verification results of the PB NPs provided in this invention.

[0027] Figure 5 The absorbance results of different interfering substances in the TMB-H2O2-PB NPs-TA system provided by this invention are shown in the figure. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Preparation of Prussian Blue Nanozymes (PB NPs) Accurately weigh 168.9 mg of K3[Fe(CN)6]·3H2O into a beaker, add 40 mL of 0.1 M HCl solution, and stir magnetically for 5 min to obtain a clear solution. Place the beaker in an oven at 80 °C for 20 h, cool to room temperature, centrifuge to collect the precipitate, and wash several times with distilled water and ethanol. After drying at room temperature for 12 h, collect the PB NPs crystals.

[0032] Example 2: Activity Evaluation of Prussian Blue Nanozymes (PB NPs) The peroxidase activity of PB NPs was studied colorimetrically using TMB as the chromogenic substrate. 90 μL of an aqueous solution of PB NPs (1 mg / mL), 90 μL of 10 mM TMB solution, and 30 μL of 100 mM H₂O₂ solution were sequentially added to 2790 μL of HAc-NaAc (pH 4.0, 0.1 M) buffer solution. After thorough mixing, the mixture was reacted in a 37°C water bath for 10 min. After cooling to room temperature, the UV absorption spectrum was measured using UV-vis. A blank experiment was performed using HAc-NaAc buffer solution instead of PB NPs.

[0033] from Figure 1 As shown in illustration b, the solution has no obvious color when TMB and H2O2 are mixed. The addition of PB NPs can significantly accelerate the colorimetric reaction rate, causing the solution to turn blue rapidly and exhibit characteristic absorption. Figure 1 Illustration c). Conversely, from Figure 1 As shown in illustration a, TMB cannot be oxidized to oxTMB in the absence of H2O2, indicating that PB NPs have peroxidase activity.

[0034] Example 3: Optimization of Prussian Blue Nanozymes (PB NPs) Activity To evaluate the catalytic activity of PB NPs nanozymes, this example optimized the pH of the HAc-NaAc buffer solution, the concentration of PB NPs, the enzyme-catalyzed reaction temperature, and the reaction time, and determined the optimal experimental conditions, as follows: (1) pH optimization of HAc-NaAc buffer solution: HAc-NaAc buffer solutions with different pH (3.0-7.0) were prepared to replace 2790 μL of HAc-NaAc (pH 4.0, 0.1 M) buffer solution in Example 2. Other conditions remained unchanged, and the absorbance corresponding to different pH was measured to study the effect of different pH on the catalytic activity of PB NPs.

[0035] like Figure 2 As shown in a, at 0.1 mol L -1 In the HAc-NaAc buffer system, the catalytic activity of PB NPs gradually increased with increasing pH from 3 to 4. As the pH continued to increase, the enzyme-mimicking activity of PB NPs gradually decreased. Based on this, subsequent colorimetric experiments were conducted in an HAc-NaAc buffer solution at pH 4.0.

[0036] (2) Optimization of PB NPs concentration: Different volumes of 1 mg / mL PB NPs solution were transferred to make the concentration range of PB NPs 10~50 μg / mL. Other conditions were the same as in Example 2. The effect of PB NPs concentration on PB NPs catalytic activity was studied.

[0037] from Figure 2 As shown in b, the absorbance gradually increases with the increase of PB NPs concentration. Considering the cost of the experiment, 10 μg / mL of PB NPs was chosen for subsequent experiments.

[0038] (3) Optimization of reaction temperature: The temperature of the water bath reaction was changed (17~67℃), while other conditions were the same as in Example 2. The absorbance of the system at 652 nm was measured at different temperatures to study the effect of temperature on the catalytic activity of PB NPs.

[0039] from Figure 2 As shown in section c, within the temperature range of 17℃-37℃, the absorbance increases significantly with increasing temperature. However, when the temperature exceeds 37℃, the absorbance change is minimal, indicating that PB NPs have a wider temperature adaptability range compared to natural enzymes. Based on this, 37℃ was chosen as the optimal temperature for subsequent experiments.

[0040] (4) Optimization of reaction time: The reaction time in the water bath was changed (10~60 min), and other conditions were the same as in Example 2. The absorbance of the system at 652 nm was measured at different reaction times to study the effect of reaction time on the catalytic activity of PB NPs.

[0041] At optimal temperature and pH, the effect of reaction time on absorbance is as follows: Figure 2As shown in d in the figure. Under 37℃ water bath conditions, the enzyme activity of PB NPs increased with time. Considering time cost, 10 min was selected as the reaction time for subsequent experiments.

[0042] Example 4: A colorimetric method for the detection of tannic acid based on Prussian blue nanozymes (PB NPs) Accurately weigh 0.1701 g of tannic acid standard and dissolve it in 10 mL of water to prepare a 10 mM tannic acid stock solution. Dilute the 10 mM tannic acid stock solution to 10 mL. -4 The tannic acid solution of M was used to transfer 15 μL, 30 μL, 90 μL, 150 μL, 240 μL, 300 μL and 450 μL of 10 μL of the solution. -4 A tannic acid solution of M was mixed with 90 μL of PB NPs (1 mg / mL) aqueous solution, 90 μL of 10 mM TMB solution, and 30 μL of 100 mM H2O2 solution. The mixture was then brought to a final volume of 3 mL with HAc-NaAc (0.1 M) buffer solution at pH 4.0 to obtain 0.5 μM–15 μM tannic acid standard working solutions. After thoroughly mixing the standard working solutions, the mixture was reacted in a 37°C water bath for 10 min. After cooling to room temperature, the absorbance at 652 nm was measured, and a standard curve was plotted based on the relationship between concentration and absorbance.

[0043] Depend on Figure 3 As shown in Figure a, when different concentrations of tannic acid are added, the absorbance of the TMB-H2O2-PB NPs system decreases to varying degrees, and the catalytic activity of PB NPs is significantly inhibited. Figure 3 As shown in b, within the range of 0.5-15 μM, the absorbance of the system at 652 nm exhibits a good linear relationship with the concentration of tannic acid, with the linear equation being y = 0.038x + 0.9479, R0. 2 =0.9955. The limit of detection of the method is 0.12 μM (S / N=3), and the limit of quantitation is 0.37 μM (S / N=10).

[0044] Example 5: Reproducibility and Stability Study The reproducibility of PB NPs preparation was verified. Five batches of PB NPs were prepared according to the preparation method in Example 1. Solutions of the same concentration were prepared for each batch and added to TMB-H2O2-TA to study their reproducibility. The results are as follows: Figure 4As shown in Table 1, under the same reaction conditions, the absorbance of the five batches of PB NPs added to TMB-H2O2-TA remained essentially consistent (deviation less than 3%). Furthermore, the storage stability of the PB NPs solution at room temperature over time was investigated. The results are shown in Table 1; after 30 days of storage, the absorbance of the system showed no significant change, indicating that the catalytic activity of PB NPs remained stable. These results demonstrate that PB NPs, as a mimic enzyme, exhibit excellent stability at room temperature.

[0045] Table 1. Changes in absorbance of the TMB-H2O2-PB NPs-TA system at different storage times.

[0046] Example 6: Study on the anti-interference ability of the method Tobacco leaf samples are complex systems containing numerous compounds, which may influence tannic acid detection. To evaluate the performance of this method in detecting tannic acid in tobacco leaves, common coexisting substances in tobacco leaves were added to the TMB-H2O2-PBNPs-TA system to study the effect of interfering substances on the detection of tannic acid content. Different concentrations of interfering substances were added to a 3 μM tannic acid solution, and the concentrations of each interfering substance are shown in Table 2. The absorbance of different interfering substances after adding them to the TMB-H2O2-PBNPs-TA system is shown in Table 2. Figure 5 As shown, the absorbance of the system did not change by more than 5% before and after the addition of different interfering substances, indicating that the coexisting substances in the tobacco leaf sample would not interfere with the detection of tannic acid. This method can be used to detect the tannic acid content in actual tobacco leaf samples.

[0047] Table 2 Concentration of Coexisting Substances

[0048] Example 7: Analysis of actual samples (1) Sample pretreatment: Accurately weigh about 0.04 g of flue-cured tobacco powder into a 10 mL centrifuge tube, add 4 mL of 20% ethanol solution, vortex at room temperature for 40 min, and then centrifuge at 7000 rmp for 5 min to obtain the supernatant.

[0049] (2) Detection steps: Transfer 150 μL of the supernatant and mix it with 90 μL of PB NPs (1 mg / mL) aqueous solution, 90 μL of 10 mM TMB solution, and 30 μL of 100 mM H2O2 solution. Adjust the volume to 3 mL with HAc-NaAc (0.1 M) buffer solution at pH 4.0. After mixing thoroughly, react in a water bath at 37℃ for 10 min. After the reaction, measure the absorbance. Calculate the tannic acid concentration in the sample based on the standard curve plotted in Example 4. Calculate the tannic acid content in the tobacco sample using the following formula (…). ):

[0050] c - concentration, μM; m - sample weight, g; M - relative molecular mass of tannic acid.

[0051] The results are shown in Table 3. Each sample was measured three times, and the relative standard deviation (RSD) was less than 5%, indicating that the method has good repeatability.

[0052] Table 3. Determination results of tannic acid content in flue-cured tobacco leaf samples

[0053] (3) Evaluation of method accuracy: The accuracy of the method was evaluated by the standard addition method (Table 4). The recovery rate of tannic acid was between 98% and 100%, indicating that the method has good accuracy and high precision in the spiked recovery experiment of tobacco leaf samples. The method can be used for the accurate determination of tannic acid content in tobacco leaves.

[0054] Table 4. Spiked recoveries of tannins in flue-cured tobacco leaf samples

[0055] In summary, this invention prepared Prussian blue nanozymes (PB NPs) using a room-temperature method and systematically investigated their catalytic performance. The peroxidase-like activity of PB NPs was verified using TMB as a chromogenic substrate. The results showed that PB NPs exhibited optimal catalytic activity in an acetate buffer solution at pH 4.0. Compared with natural peroxidases, PB NPs maintained high catalytic efficiency over a wider temperature range, demonstrating excellent temperature tolerance. Further research revealed that tannic acid, a typical polyphenol compound, effectively inhibited the enzymatic activity of PB NPs, leading to a significant decrease in the absorbance of the TMB-H₂O₂-PB NPs chromogenic system. Under optimal experimental conditions, the tannic acid concentration showed a good linear relationship with the system absorbance change, with a correlation coefficient (R²) greater than 0.99. Interference assessment of common coexisting substances in tobacco confirmed that this method has good selectivity for the detection of tannic acid. The accuracy of this method in actual samples was evaluated using the spiked recovery method. The spiked recovery rate was between 98% and 100%, indicating that this method can be accurately and rapidly used to determine the tannic acid content in tobacco samples. The tannic acid detection method based on Prussian blue nanozyme constructed in this invention has good application prospects in tobacco quality assessment and related fields.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting tannic acid based on Prussian blue nanozymes, characterized in that, Includes the following steps: S1. HCl was added to K3[Fe(CN)6]·3H2O and stirred to obtain a clear solution. The reaction was heated and then cooled to room temperature after the reaction was completed. The precipitate was collected by centrifugation, washed several times with water and ethanol, and dried to obtain Prussian blue nanozyme. S2. Mix the aqueous solution of Prussian blue nanozyme, chromogenic substrate, peroxide and standard working solutions of tannic acid of different concentrations. After mixing evenly, react in a water bath, cool to room temperature, and measure the absorbance at 650-655 nm. Plot a standard curve based on the relationship between tannic acid concentration and absorbance. S3. Mix the aqueous solution of Prussian blue nanozyme, chromogenic substrate, peroxide and the sample to be tested. After mixing evenly, react in a water bath, cool to room temperature, and measure the absorbance at 650-655 nm. Calculate the tannic acid concentration in the sample to be tested based on the standard curve.

2. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine, and the peroxide is hydrogen peroxide.

3. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, The pH of the water bath reaction was controlled at 4.0, the reaction temperature at 37°C, and the reaction time at 10 min.

4. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, In the water bath reaction, the concentration of Prussian blue nanozyme was controlled at 10 μg / mL.

5. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, The Prussian blue nanozyme has peroxidase activity, and the tannic acid inhibits the catalytic activity of the Prussian blue nanozyme.

6. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, When the concentration of tannic acid is in the range of 0.5-15 μM, the absorbance shows a good linear relationship with the concentration.

7. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, The limit of detection for tannic acid was 0.12 μM, and the limit of quantitation was 0.37 μM.

8. The method for detecting tannic acid based on Prussian blue nanozymes according to claim 1, characterized in that, The sample to be tested is a tobacco sample, and its preparation includes the following steps: adding ethanol solution to tobacco, vortexing at room temperature and centrifuging, and then transferring the supernatant.

9. A tannic acid detection sensing platform based on Prussian blue nanozymes, characterized in that, The tannic acid detection method based on Prussian blue nanozyme as described in any one of claims 1-8 was used for detection.

10. The application of the tannic acid detection sensing platform based on Prussian blue nanozyme as described in claim 9 in the detection of tannic acid in tobacco.

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