Method for identifying tea polyphenol by using tetramethyl benzidine-hypochlorous acid colorimetric sensor array

Abstract

The invention discloses a method for identifying tea polyphenol by using a tetramethyl benzidine-hypochlorous acid colorimetric sensor array, which comprises the following steps: constructing a TMB-NaClO binary colorimetric sensor array, adding a NaClO mother solution and a tea polyphenol solution into deionized water, and finally adding a 3, 3 ', 5, 5'-tetramethyl benzidine mother solution to form a first TMB-NaClO sensing unit and a second TMB-NaClO sensing unit; the method is used for distinguishing and identifying nine different types of tea polyphenols. The TMB can be oxidized into compounds with different colors by NaClO with different concentrations, different tea polyphenols are different in hydroxyl number and position, so that the antioxidant activity is different, and the difference can amplify a product color signal by inhibiting the oxidation-reduction reaction of TMB-NaClO.

Description

Technical Field

[0001] The invention relates to the field of tea polyphenols identification, and in particular to a method for identifying tea polyphenols by utilizing a tetramethylbenzidine-hypochlorous acid colorimetric sensor array. Background Art

[0002] Tea polyphenols (TPs), commonly known as tea tannins, are the most beneficial ingredients extracted from tea leaves. Tea polyphenols are divided into four major categories according to their main chemical components: catechins, flavonoids, anthocyanins, and phenolic acids. Among them, catechins have the highest content, accounting for 60% to 80% of the total tea polyphenols. Among them, catechins are mainly gallic acid (Gallicacid-GA), catechin (Catechin-C), epicatechin (Epicatechin-EC), gallocatechin (Gallocatechin-GC), epigallocatechin (Epigallocatechin-EGC), catechin gallate (Catechin Gallate-CG), epicatechin gallate (Epicatechin Gallate-ECG), gallocatechin gallate (Gallocatechin Gallate-GCG) and epigallocatechin gallate (Epigallocatechin Gallate-EGCG). Since the 1980s, tea polyphenols have been widely used in oils, food, medical care, daily chemicals, etc. Tea polyphenols have antioxidant, antibacterial, antiviral, free radical scavenging, anti-aging, fat removal and weight loss effects. Given the wide range of health functions of tea polyphenols, effective detection and identification methods for different tea polyphenols are very demanding.

[0003] Various detection methods for tea polyphenols have been developed. Traditional instrumental detection often uses spectrophotometry, near-infrared spectroscopy, electrochemical analysis, and chromatography. However, these methods are costly, time-consuming, and require complex instrument operation and pre-processing of samples. Therefore, it is necessary to develop a rapid, economical, and sensitive method for simultaneously identifying multiple tea polyphenols. With the continuous improvement of detection requirements and the development of instrument performance, colorimetric sensor arrays have the advantages of high sensitivity, simple operation, and visualization, providing a new idea for the detection of tea polyphenols. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for identifying tea polyphenols by using a tetramethylbenzidine-hypochlorous acid colorimetric sensor array, so as to realize simple, rapid and effective identification of different tea polyphenols.

[0005] A method for identifying tea polyphenols using a tetramethylbenzidine-hypochlorous acid colorimetric sensor array comprises the following steps:

[0006] Step 1: Construction of colorimetric sensor array

[0007] Use dilute hydrochloric acid to prepare 3,3',5,5'-tetramethylbenzidine mother solution, use deionized water to prepare NaClO mother solution, and use deionized water to prepare tea polyphenol solution;

[0008] Take NaClO mother solution and tea polyphenol solution and add them into deionized water, and finally add 3,3',5,5'-tetramethylbenzidine mother solution to form the first sensing unit of TMB-NaClO;

[0009] Take NaClO mother solution and tea polyphenol solution and add them into deionized water, and finally add 3,3',5,5'-tetramethylbenzidine mother solution to form the second sensing unit of TMB-NaClO;

[0010] Step 2: Add the tea polyphenol sample to be tested into ethanol, perform ultrasonic extraction, filter the supernatant with a filter membrane, dilute the filtered solution, place the diluted solution in the first sensor unit and the second sensor unit respectively, and then place the test solution in the two sensor units into different storage holes of the reservoir, and then place the reservoir on an LED flat panel, cover the reservoir with a piece of black cloth to expose the storage hole, use a digital camera to take a colorimetric photo of the storage hole, obtain the photographed photo, select a range of 20 to 30 pixels in the center diameter of the storage hole in the photographed photo in the graphics processing software to calculate the RGB average value, and obtain the colorimetric data analysis result of the sample to be tested, so as to achieve the distinction and identification of different tea polyphenol compounds.

[0011] Step 3: Using TMB as a colorimetric developer and NaClO as a reducing agent, a 1×2 colorimetric sensor array TMB-NaClO was constructed to identify 9 different tea polyphenols (including four pairs of stereoisomers of tea polyphenol compounds—C and EC, GC and EGC, CG and ECG, GCG and EGCG). TMB can generate different forms of oxidized compounds under oxidation with different concentrations of NaClO, showing different colors. Due to the different number and position of hydroxyl groups of tea polyphenols, their antioxidant activities vary, which can inhibit the redox reaction of TMB and NaClO to different degrees, producing a specific colorimetric response. The unique color changes of the two sensor units of the colorimetric sensor array to different concentrations of tea polyphenols were converted into ED (ED = square root of the sum of squares of ΔRGB values) for quantitative analysis. After comparison with the quantitative results of ultraviolet absorption spectra, it was found that the quantitative effect of the colorimetric sensor array was better.

[0012] In step 1, the concentration of 3,3',5,5'-tetramethylbenzidine in the 3,3',5,5'-tetramethylbenzidine mother solution is (0.5-1.5)×10 -4 mol / L (most preferably 1×10 -4mol / L), the concentration of NaClO in the NaClO mother solution is (1.5-2.5)×10 -4 mol / L (most preferably 2×10 -4 mol / L), the concentration of tea polyphenols in the tea polyphenol solution is (2-3)×10 -4 mol / L (most preferably 2.5×10 -4 mol / L).

[0013] The concentration of 3,3',5,5'-tetramethylbenzidine in the first sensing unit of TMB-NaClO is 10-14 μmol / L (most preferably 12 μmol / L), and the concentration of NaClO in the first sensing unit of TMB-NaClO is 90-100 μmol / L (most preferably 95 μmol / L).

[0014] The concentration of 3,3',5,5'-tetramethylbenzidine in the second sensing unit of TMB-NaClO is 10-14 μmol / L (most preferably 12 μmol / L), and the concentration of NaClO in the second sensing unit of TMB-NaClO is 145-155 μmol / L (most preferably 150 μmol / L).

[0015] In step 1, the concentration ratio of NaClO in the first sensing unit of TMB-NaClO to the concentration ratio of NaClO in the second sensing unit of TMB-NaClO is 18-20:29-31 (most preferably 19:30).

[0016] Tea polyphenols (TPs), as a class of polyhydroxy compounds with biological activity in tea, have received extensive attention in health research in recent years and have gradually become one of the most promising health food molecules. However, tea polyphenols have similar polyhydroxy chemical structures, so it is not easy to develop a fast and simple differentiation detection method. The invention constructs a TMB-NaClO binary colorimetric sensor array for distinguishing and identifying 9 different types of tea polyphenols, including gallic acid (Gallic acid-GA), catechin (Catechin-C), epicatechin (Epicatechin-EC), gallocatechin (Gallocatechin-GC), epigallocatechin (Epigallocatechin-EGC), catechin gallate (Catechin Gallate-CG), epicatechin gallate (Epicatechin Gallate-ECG), gallocatechin gallate (Gallocatechin Gallate-GCG) and epigallocatechin gallate (Epigallocatechin Gallate-EGCG). TMB is a good color developer, while NaClO is a common reducing agent. Different concentrations of NaClO can oxidize TMB into compounds of different colors. Due to the different numbers and positions of hydroxyl groups of different tea polyphenols, there are certain differences in antioxidant activity. This difference can be amplified by inhibiting the redox reaction of TMB-NaClO to amplify the product color signal.

[0017] Compared with the prior art, the advantages and beneficial effects of the technical solution of the present invention are:

[0018] The invention is based on the construction of tetramethylbenzidine-hypochlorous acid colorimetric sensor array and a method for detecting and identifying nine kinds of tea polyphenols, which has the characteristics of rapidity, simplicity, visualization, low cost, etc. The detection of tea polyphenols in tea polyphenol products has application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The chemical structures of 9 types of tea polyphenols.

[0020] Figure 2 (a) Photographs of TMB solutions after adding different concentrations of NaClO; (b) UV absorption spectra of TMB after adding different concentrations of NaClO. (c) Oxidation products of TMB after being oxidized to different degrees by NaClO.

[0021] Figure 3 (a) Colorimetric response, (b) fingerprint, and (c) heat map of the TMB-NaClO sensor array to different tea polyphenols (8.5 μmol / L).

[0022] Figure 4 (a) is the colorimetric response of the first sensor unit of the TMB-NaClO sensor array to different concentrations and types of tea polyphenols, and (bj) is the linear relationship between the Euclidean distance (ED) of the first sensor unit of the TMB-NaClO sensor array and the tea polyphenol concentration.

[0023] Figure 5 (a) is the colorimetric response of the second sensor unit of the TMB-NaClO sensor array to different concentrations and types of tea polyphenols, and (bj) is the linear relationship between the Euclidean distance (ED) of the second sensor unit of the TMB-NaClO sensor array and the tea polyphenol concentration.

[0024] Figure 6 (a) Colorimetric response, (b) fingerprint, and (c) thermal map of the second sensor unit of the TMB-NaClO sensor array to green teas of different grades and types.

[0025] Figure 7 This is an evaluation of the antioxidant capacity of different tea polyphenols using the ABTS method.

[0026] Figure 8 Evaluation of antioxidant capacity of different types of green tea by ABTS method.

[0027] Table 1 shows the detailed information and sample number information of green tea samples. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with examples and drawings, but the present invention is not limited to the following embodiments.

[0029] A construction and application of a colorimetric sensor array for tea polyphenols identification comprises the following steps:

[0030] Step 1: Construction of colorimetric sensor array:

[0031] Prepare TMB stock solution (1×10 -4 mol / L), and NaClO mother solution (2×10 -4 mol / L) and tea polyphenols solution (2.5×10 -4mol / L). The reaction of the first sensing unit of TMB-NaClO is as follows: 2 mL of NaClO mother solution and an appropriate amount of tea polyphenol solution are added to deionized water in turn, reacted at room temperature of 25°C for 5 minutes, then 0.5 mL of TMB mother solution is added, and the reaction is continued for 5 minutes. In this detection system, the TMB concentration is 12 μmol / L and the NaClO concentration is 95 μmol / L. The reaction of the second sensing unit of TMB-NaClO is as follows: 3.15 mL of NaClO mother solution and an appropriate amount of tea polyphenol solution are added to deionized water in turn, reacted at room temperature of 25°C for 5 minutes, then 0.5 mL of TMB mother solution is added, and the reaction is continued for 5 minutes. In this detection system, the TMB concentration is 12 μmol / L and the NaClO concentration is 150 μmol / L. The two reaction solutions are placed in different storage holes of the reservoir, respectively, and then the reservoir is placed on an LED flat panel covered with a black cloth but exposing the reservoir area. The colorimetric photos were taken using an EOS 750D digital camera.

[0032] Step 2: Tea polyphenols colorimetric data analysis:

[0033] In Adobe Photoshop, a range of 25 pixels in diameter at the center of the reaction well was selected to calculate the RGB average value, and the colorimetric experiment was repeated six times.

[0034] Step 3: UV-visible spectrum response of colorimetric sensor array to tea polyphenols:

[0035] Prepare TMB stock solution (1×10 -4 mol / L), and NaClO mother solution (2×10 -4 mol / L) and tea polyphenols solution (2.5×10 -4 mol / L). The reaction of the first sensing unit of TMB-NaClO is as follows: 2 mL of NaClO mother solution and an appropriate amount of tea polyphenol solution are added to deionized water in turn, reacted at room temperature of 25°C for 5 minutes, then 0.5 mL of TMB mother solution is added, and the reaction is continued for 5 minutes. In this detection system, the TMB concentration is 12 μmol / L and the NaClO concentration is 95 μmol / L. The reaction of the second sensing unit of TMB-NaClO is as follows: 3.15 mL of NaClO mother solution and an appropriate amount of tea polyphenol solution are added to deionized water in turn, reacted at room temperature of 25°C for 5 minutes, then 0.5 mL of TMB mother solution is added, and the reaction is continued for 5 minutes. In this detection system, the TMB concentration is 12 μmol / L and the NaClO concentration is 150 μmol / L. The two reaction solutions are placed in cuvettes for UV-visible spectral analysis.

[0036] Step 4: Preparation of green tea samples:

[0037] The preparation process of green tea samples is as follows: 0.1g green tea sample is added to 10mL 70% ethanol, placed in a 70℃ constant temperature water bath for ultrasonic extraction for 45min, and the supernatant is filtered with a 0.22μm filter membrane. The filtered solution is diluted 10 times for further analysis and detection. The tea sample detection process is similar to the tea polyphenol detection process: TMB mother solution (1×10 -4 mol / L), and NaClO mother solution (2×10 -4 3.15 mL of NaClO stock solution and 100 μL of diluted green tea sample were added to deionized water in turn. After reacting at room temperature of 25°C for 5 min, 0.5 mL of TMB stock solution was added and the reaction was continued for 5 min. In this detection system, the TMB concentration was 12 μmol / L and the NaClO concentration was 150 μmol / L.

[0038] Step 5: ABTS free radical scavenging experiment:

[0039] Prepare ABTS stock solution (7mmol / L) and potassium persulfate solution (24.5mmol / L) with deionized water. Take 7mL ABTS stock solution and 1mL potassium persulfate solution and add them into 2mL deionized water. The concentration of potassium persulfate in the reaction solution is 2.45mmol / L and the concentration of ABTS is 4.9mmol / L. After reacting at room temperature at 25℃ in the dark for 16h, ABTS free radical cation (ABTS .+ ). ABTS .+ The solution was diluted with ethanol until the absorbance at 750 nm was 0.70 ± 0.02. 10 μL of the sample to be tested (tea polyphenol solution or green tea sample solution) was added to 1 mL of diluted ABTS .+ The absorbance of the solution was measured every 30 s and the reaction was monitored within 6 min using a UV-visible spectrometer.

[0040] The mechanism of action of the present invention is as follows: TMB can be oxidized by NaClO into different forms of oxidation products showing different colors. Figure 2As shown in the figure, low concentration of NaClO (5 μmol / L) can partially oxidize TMB into charge transfer complexes, with absorption peaks at 370, 450 and 650 nm, and the solution is blue-green. Continuing to increase the concentration of NaClO (15 μmol / L), TMB can be oxidized to protonated TMB diamine, with a strong absorption peak at 450 nm, and the solution color is yellow. Finally, TMB is completely oxidized to TMB diamine, with an absorption peak at 510 nm, and the solution is red. Different tea polyphenols have different numbers and positions of hydroxyl groups, which are closely related to antioxidant capacity. Generally speaking, the more hydroxyl groups there are, the stronger the antioxidant activity. Therefore, different tea polyphenols have different inhibitory effects on the redox reaction of TMB and NaClO, and eventually TMB becomes different forms of oxidized products, showing differential colorimetric responses.

[0041] Example 1

[0042] The present invention constructs a TMB-NaClO binary colorimetric sensor array for distinguishing and identifying 9 representative tea polyphenols including GA, C, EC, GC, EGC, CG, ECG, GCG and EGCG, among which four pairs of stereoisomers of tea polyphenol compounds are C and EC, GC and EGC, CG and ECG, GCG and EGCG, and the structures of the 9 tea polyphenols are as follows: Figure 1 shown.

[0043] Example 2

[0044] TMB can be oxidized by NaClO into different forms of oxidation products showing different colors. Figure 2 As shown in the figure, low concentration of NaClO (5 μmol / L) can partially oxidize TMB into a charge transfer complex with absorption peaks at 370, 450 and 650 nm, and the solution is blue-green. Continuing to increase the concentration of NaClO (15 μmol / L), TMB can be oxidized to protonated TMB diamine with a strong absorption peak at 450 nm and the solution color is yellow. Finally, TMB is completely oxidized to TMB diamine with an absorption peak at 510 nm and the solution is red.

[0045] Example 3

[0046] Under optimized conditions, the recognition performance of the TMB-NaClO sensor array for different tea polyphenols (GA, C, EC, GC, EGC, CG, ECG, GCG, and EGCG) was studied. Tea polyphenols have different degrees of inhibition on TMB oxidation, resulting in different colors and producing different colorimetric responses, such as Figure 3 The RGB values ​​of each sensing unit are extracted, and these values ​​form a unique fingerprint response for a single tea polyphenol, as shown in Figure 3As shown in Fig. b. To more clearly display the fingerprint response patterns of each type of tea polyphenol, the RGB values were converted into heatmaps and spider charts, as Figure 3 shown in Figs. c and 3d, which more clearly shows that the TMB sensing array can effectively identify 9 types of tea polyphenols.

[0047] Example 4

[0048] To explore the ability of the TMB-NaClO sensing array to quantitatively analyze tea polyphenols, a series of tea polyphenol concentration titration experiments were respectively carried out in two sensing units of the TMB-NaClO sensing array. As Figure 4 shown in Figs. a and 5a, for each type of tea polyphenol, an increase in the tea polyphenol content will cause an obvious rainbow-like color change (red, orange, yellow, green, cyan, blue), and this change is easily observable by the naked eye.

[0049] Example 5

[0050] When accurately determining the content of tea polyphenols, by converting the color changes caused by the change in tea polyphenol concentration in two sensing units of the sensing array into ED values (ED = the square root of the sum of the squares of the ΔRGB values), it was found that this value was positively correlated with the tea polyphenol concentration and showed a good linear relationship within a specific concentration range, as Figure 4 shown in Figs. b-j and Figure 5 b-j.

[0051] Example 6

[0052] To verify the reliability of this method, the commonly used ABTS method was adopted to evaluate the antioxidant capacity of different tea polyphenols and compare the results with those obtained by the colorimetric sensing array. In the ABTS method, a lower radical scavenging rate indicates a lower antioxidant activity. As Figure 7 shown, the order of antioxidant activities of 9 types of tea polyphenols is as follows: GA < C < EC < GC < EGC < CG < ECG < GCG < EGCG. It is basically consistent with the order of antioxidant activities obtained by the colorimetric sensing array, indicating that the constructed colorimetric sensing array can accurately distinguish and identify tea polyphenol compounds and green tea samples, and the method results are reliable and effective.

[0053] Application Example 1

[0054] Green tea is mainly produced in provinces such as Zhejiang, Guizhou, Henan, and Sichuan in China. Even the value difference between green teas with only slight differences in origin may be several times, but the appearance of these teas may be very similar. To distinguish these green teas, green teas of different grades from different production areas were selected, a total of 15 tea samples, as shown in Table 1.

[0055] Application Example 2

[0056] As Figure 6As shown in a), the colorimetric responses of a single sensing unit to these 15 green teas showed obvious differences. Among them, the Mingqian treasure of Xinyang Maojian (D1) showed strong antioxidant activity, while Huangshan Maofeng special grade second-class (C2), Duyun Maojian first grade (E3), and Liuan Guapian Meipian (F3) showed weak antioxidant activity. The fingerprint Figure 6 b) showed that these green teas had unique colorimetric responses. The heat map Figure 6 c) more clearly indicated that the sensing unit could effectively identify different types and grades of green teas. There were obvious differences in the colorimetric responses of different types of green teas. For green tea samples of the same type but different grades, their colorimetric responses also showed certain differences.

[0057] Application Example 3

[0058] To verify the reliability of this method, the commonly used ABTS method was adopted to evaluate the antioxidant capacity of different green tea samples and compare the results with those obtained by the colorimetric sensing array. In the ABTS method, a lower radical scavenging rate indicates lower antioxidant activity. As Figure 8 shown, the approximate order of antioxidant activity of representative tea samples among the 15 green tea samples was: E3 < F2 < C2 < A3 < B2 < D1. This was basically consistent with the order of antioxidant activity obtained by the colorimetric sensing array, indicating that the constructed colorimetric sensing array could accurately distinguish and identify tea polyphenol compounds and green tea samples, and the method results were reliable and effective.

[0059] Table 1

[0060]

Claims

1. A method for identifying tea polyphenols using a tetramethylbenzidine-hypochlorous acid colorimetric sensor array, characterized in that: The following steps are involved: Step 1: Construction of colorimetric sensor array Use dilute hydrochloric acid to prepare 3,3',5,5'-tetramethylbenzidine mother solution, use deionized water to prepare NaClO mother solution, and use deionized water to prepare tea polyphenol solution; Take NaClO mother solution and tea polyphenol solution and add them into deionized water, and finally add 3,3',5,5'-tetramethylbenzidine mother solution to form the first sensing unit of TMB-NaClO; Take NaClO mother solution and tea polyphenol solution and add them into deionized water, and finally add 3,3',5,5'-tetramethylbenzidine mother solution to form the second sensing unit of TMB-NaClO; Step 2: Add the tea polyphenol sample to be tested into ethanol, perform ultrasonic extraction, filter the supernatant with a filter membrane, dilute the filtered solution, place the diluted solution in the first sensor unit and the second sensor unit respectively, and then place the test solution in the two sensor units into different storage holes of the reservoir, and then place the reservoir on an LED flat panel, cover the reservoir with a piece of black cloth to expose the storage hole, use a digital camera to take a colorimetric photo of the storage hole, obtain the photographed photo, select a range of 20 to 30 pixels in the center diameter of the storage hole in the photographed photo in the graphics processing software to calculate the RGB average value, obtain the colorimetric data analysis result of the sample to be tested, and complete the distinction and identification of different tea polyphenol compounds.

2. The method for identifying tea polyphenols using tetramethylbenzidine-hypochlorous acid colorimetric sensor array according to claim 1, characterized in that: In step 1, the concentration of 3,3',5,5'-tetramethylbenzidine in the 3,3',5,5'-tetramethylbenzidine mother solution is (0.5-1.5)×10 -4 mol / L, the concentration of NaClO in the NaClO mother solution is (1.5~2.5)×10 - 4 mol / L, the concentration of tea polyphenols in the tea polyphenol solution is (2-3)×10 -4 mol / L.

3. The method for identifying tea polyphenols using tetramethylbenzidine-hypochlorous acid colorimetric sensor array according to claim 1, characterized in that: In step 1, the concentration of 3,3',5,5'-tetramethylbenzidine in the first sensing unit of TMB-NaClO is 10-14 μmol / L, and the concentration of NaClO in the first sensing unit of TMB-NaClO is 90-100 μmol / L.

4. The method for identifying tea polyphenols using tetramethylbenzidine-hypochlorous acid colorimetric sensor array according to claim 1, characterized in that: In step 1, the concentration of 3,3',5,5'-tetramethylbenzidine in the second sensing unit of TMB-NaClO is 10-14 μmol / L, and the concentration of NaClO in the second sensing unit of TMB-NaClO is 145-155 μmol / L.

5. The method for identifying tea polyphenols using tetramethylbenzidine-hypochlorous acid colorimetric sensor array according to claim 1, characterized in that: In step 1, the concentration ratio of NaClO in the first sensing unit of TMB-NaClO to NaClO in the second sensing unit of TMB-NaClO is 18-20:29-31.

6. The method for identifying tea polyphenols using tetramethylbenzidine-hypochlorous acid colorimetric sensor array according to claim 1, characterized in that: In step 2, the tea polyphenol sample to be tested refers to tea polyphenols containing one or more of the following tea structures:

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