Preparation method and application of indicator displacement sensor array for tea polyphenol identification

Abstract

The present invention discloses a preparation method and application of an indicator displacement sensor array for tea polyphenol identification. The array is prepared by using two arylboronic acids, 1-naphthaleneboronic acid (NapB) and 1-pyreneboronic acid (PyrB), as probes, and catechol-containing Alizarin Red S (ARS) and catechol violet (PV) as indicators. Based on the indicator displacement method (IDA) mechanism, a 2×2 colorimetric sensor array is designed and constructed for detecting and identifying different types of tea polyphenols, namely gallic acid, methyl gallate, epicatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate. The binding force between tea polyphenols and the boronic acid probe is stronger than that of the indicator. Different tea polyphenols have different binding abilities to the probe and different abilities to displace the indicator, resulting in different colorimetric responses of the IDA sensor array to tea polyphenols. In addition, the colorimetric sensing array was combined with principal component analysis (PCA) to successfully distinguish different types of tea polyphenols.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis and analytical chemistry, and in particular to a preparation method and application of an indicator displacement sensor array for identifying tea polyphenols. Background Art

[0002] Tea polyphenols (TPs), commonly known as tea tannins, are the most beneficial components extracted from tea leaves. Tea polyphenols are classified into four main chemical groups: catechins, flavonoids, anthocyanins, and phenolic acids. Catechins are the most abundant, accounting for 60% to 80% of the total tea polyphenols. The main catechins are gallic acid (GA), methyl gallate (MG), epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). Since the 1980s, tea polyphenols have been widely used in oils and fats, food, healthcare, and daily chemical products. Tea polyphenols have antioxidant, antibacterial, antiviral, free radical scavenging, anti-aging, fat-removing and weight-loss effects. Given the wide range of health benefits of tea polyphenols, effective detection and identification methods for different tea polyphenols are very demanding.

[0003] Methods for detecting various 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, require complex instrumentation, and require preliminary sample processing. Therefore, it is necessary to develop a rapid, economical, and sensitive method for the simultaneous identification of multiple tea polyphenols. With the continuous improvement of detection requirements and the development of instrument performance, there is a need to provide a technology for detecting tea polyphenols using colorimetric sensor arrays (CSAs). Summary of the Invention

[0004] The present invention is aimed at the deficiencies of the prior art.

[0005] The present invention is achieved through the following technical solutions: a preparation of an indicator displacement sensor array for identifying tea polyphenols, which realizes simple, rapid and effective identification of different tea polyphenols.

[0006] A method for preparing an indicator displacement sensor array for tea polyphenol identification is provided. The method uses 1-naphthaleneboronic acid NapB and 1-pyreneboronic acid PyrB as probes, and uses catechol-containing Alizarin Red S (ARS) and catechol purple PV as indicators to construct a 2×2 colorimetric sensor array for detecting and identifying tea polyphenols. The pH value is 8.2 and the concentration is 1×10 -2 mol / L HEPES buffer solution was used as the solvent; tea polyphenols were classified by combining principal component analysis (PCA); the specific steps are as follows:

[0007] (1) Design and synthesize the NapB probe, whose chemical structure is as follows:

[0008]

[0009] (2) Design and synthesize the PyrB probe, whose chemical structure is as follows:

[0010]

[0011] (3) Weigh a certain amount of NapB, PyrB, ARS, PV and six tea polyphenols and dissolve them in 1×10 -2 mol / L and pH=8.2 HEPES buffer solution for standby use; the six tea polyphenols are gallic acid GA, methyl gallate MG, epigallocatechin EGC, epicatechin EC, epigallocatechin gallate EGCG and epicatechin gallate ECG;

[0012] (4) Using NapB and PyrB as probes and ARS and PV as indicators, a 2×2 colorimetric sensor array was constructed to detect and identify six tea polyphenols: gallic acid GA, methyl gallate MG, epigallocatechin EGC, epicatechin EC, epigallocatechin gallate EGCG, and epicatechin gallate ECG, and the image of the colorimetric sensor array was obtained;

[0013] (5) Adobe Photoshop software was used to extract the RGB average value of all pixels in the central area of each reaction well, which had a diameter of 25 pixels, and all RGB values were classified and identified in combination with the PCA model.

[0014] Furthermore, the step (1) specifically comprises: at a temperature of -78°C, slowly mixing an anhydrous tetrahydrofuran solution containing 2g of 1-bromonaphthalene NapBr and a 2mol / L n-butyllithium solution in order by volume to obtain a mixed solution, then adding a triisopropyl borate solution within 1-10 minutes, and allowing the reaction mixture to naturally warm to room temperature after 1 hour; subsequently adding a hydrochloric acid solution, wherein the volume ratio of the 2mol / L n-butyllithium, the anhydrous tetrahydrofuran containing 2g of 1-bromonaphthalene NapBr, the triisopropyl borate and the hydrochloric acid solution is 5:30:3:2; and extracting with ethyl acetate, and washing the organic phase with water; drying the obtained solution with anhydrous magnesium sulfate, and evaporating the solvent to obtain the product 1-naphthalene boronic acid NapB as a white solid;

[0015] The NapBr structural formula is as follows:

[0016]

[0017] Furthermore, the step (2) specifically comprises: at an ambient temperature of -78°C, mixing anhydrous tetrahydrofuran containing 2g of 1-bromopyrene PyrBr with a 2mol / L n-butyllithium solution to obtain a mixed solution, and then adding triisopropyl borate within 1-10min. After 1 hour, the reaction mixture is naturally warmed to room temperature; subsequently, a hydrochloric acid solution is added, wherein the volume ratio of the 2mol / L n-butyllithium, the anhydrous tetrahydrofuran containing 2g of 1-bromopyrene PyrBr, triisopropyl borate and hydrochloric acid solution is 5:30:3:2; and extracting with ethyl acetate, and washing the organic phase with water; the obtained solution is dried over anhydrous magnesium sulfate, and the solvent is evaporated to obtain a light yellow solid product 1-pyrene borate PyrB;

[0018] The structure of PyrBr is as follows:

[0019]

[0020] Furthermore, the step (4) specifically includes: adding the buffer solution, boric acid probe, indicator and tea polyphenol solution into the wells of a 96-well plate, placing it on a micro-oscillator with a rotation speed of 300 rpm and oscillating for 10 minutes; placing the 96-well plate on an LED light and taking pictures to obtain an image.

[0021] An application of an indicator displacement sensor array for tea polyphenol identification is used to achieve the differentiated detection of six different tea polyphenols and the classification of different types and grades of green tea.

[0022] The beneficial effects of the present invention are as follows: the method for detecting and identifying six tea polyphenols based on the IDA colorimetric sensor array is rapid, simple, visual, and low-cost, and has application prospects in the detection of tea polyphenols in tea polyphenol products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the H NMR spectrum of the NapB probe;

[0024] Figure 2 is the carbon NMR spectrum of the NapB probe;

[0025] Figure 3 This is the high-resolution mass spectrum of the NapB probe;

[0026] Figure 4 is the H NMR spectrum of the PyrB probe;

[0027] Figure 5 is the carbon NMR spectrum of the PyrB probe;

[0028] Figure 6 This is the high-resolution mass spectrum of the PyrB probe;

[0029] Figure 7 is the chemical structure of tea polyphenols;

[0030] Figure 8 Figure (a) shows the colorimetric response of the sensor array to tea polyphenols; Figure (b) shows the PCA analysis results;

[0031] Figure 9 Figure (a) shows the colorimetric response of the sensor array to different concentrations of different types of tea polyphenols; Figure (b) shows the color difference.

[0032] Figure 10 The PCA analysis results of the sensor array's response to different concentrations of tea polyphenols;

[0033] Figure 11 Figure (a) shows the response of the colorimetric sensor array to different types of green tea; Figure (b) shows the PCA analysis results. DETAILED DESCRIPTION

[0034] In order to further illustrate the present invention, the following series of specific embodiments are given in conjunction with the accompanying drawings.

[0035] Example 1

[0036] Synthesis steps of NapB:

[0037] At -78°C, 2 mol / L n-butyl lithium (5 mL) was slowly added to anhydrous tetrahydrofuran (30 mL) containing 2 g of 1-bromonaphthalene (NapBr). Then, 3 mL of triisopropyl borate was added within 10 minutes. After 1 hour, the reaction mixture was naturally warmed to room temperature. Subsequently, 2 mL of hydrochloric acid was added, and the mixture was extracted with ethyl acetate, and the organic phase was washed with water. The resulting solution was dried over anhydrous magnesium sulfate and the solvent was evaporated to obtain the product 1-naphthaleneboronic acid (NapB) as a white solid (0.84 g, yield 50%). Figure 1 As shown, H NMR spectrum (DMSO-d6, 400 MHz) δ8.42-8.35 (m, 1H), 8.33 (s, 2H), 7.93-7.83 (m, 2H), 7.73 (d, J = 5.50 Hz, 1H), 7.53-7.42 (m, 3H). Figure 2 As shown, the nuclear magnetic resonance carbon spectrum (DMSO-d6, 100MHz) δ136.03, 133.27, 132.44, 129.55, 129.16, 128.62, 126.04, 125.77, 125.48. Figure 3 As shown, high resolution mass spectrum (m / s): measured value C 10 H8BO2 - [M] - =171.0610, calculated value is 171.0732.

[0038]

[0039] Example 2

[0040] Synthesis steps of PyrB:

[0041] At -78°C, PyrBr (2 g) was added to a 250 ml round-bottom flask filled with anhydrous tetrahydrofuran (30 mL), followed by the slow addition of 5 mL of n-butyl lithium (2 mol / L); 3 mL of triisopropyl borate was then added to the round-bottom flask over 10 min. After 1 hour, the mixture was naturally warmed to room temperature, 2 mL of HCl was added, and then extracted with ethyl acetate, and the organic phase was washed with water. The combined solution was dried over anhydrous MgSO4, and the solvent was evaporated to obtain the product 1-pyreneboronic acid (PyrB) as a light yellow solid (0.93 g, yield 53%). Figure 4 As shown, H NMR spectrum (DMSO-d6, 400 MHz) δ8.73 (d, J = 9.21 Hz, 1H), 8.56 (s, 2H), 8.32-8.24 (m, 4H), 8.21-8.15 (m, 3H), 8.06 (t, J = 7.63 Hz, 1H). Figure 5As shown, the nuclear magnetic resonance carbon spectrum (DMSO-d6, 100MHz) δ134.42,132.19,131.88,131.26,130.84,128.92,128.13,127.94,127.29,126.47,125.46,125.44,124.53,124.50,124.12. Figure 6 As shown, high resolution mass spectrum (m / s): measured value C 16 H 10 Bo2 - [M] - =245.0753, calculated value is 245.0852.

[0042]

[0043] Example 3

[0044] Qualitative and quantitative determination of six tea polyphenols:

[0045] (1) IDA colorimetric sensing array and acquisition of tea polyphenol colorimetric response images.

[0046] The reaction was carried out in HEPES (1×10 -2 50 μL of boronic acid probe (1×10 -3 mol / L) solution and 50 μL of indicator solution (8×10 -4 mol / L), and then add appropriate amount of tea polyphenols, different tea polyphenols structural formulas such as Figure 7 The final concentrations of boronic acid probe and indicator in each well were 2.5×10 - 4 mol / L and 2×10 -4 mol / L. Then place the plate on a micro-oscillator with a rotation speed of 300 rpm and take an image after oscillating for 10 minutes. The image is as follows Figure 8 Figure (a) and Figure 9 As shown in Figure (a), a 15W LED flatbed was covered with a black cloth, leaving an area in the center of the flatbed with the size of a 96-well plate, ensuring a uniform white background. The 96-well plate was placed in this area, and images were captured in darkness using a Canon EOS 750D digital camera. The camera was mounted on a tripod and captured at a shutter speed of 1 / 125s, an aperture setting of f / 11, and a film sensitivity of ISO 400. The camera parameters remained unchanged throughout the capture process.

[0047] (2) RGB acquisition of colorimetric images and PCA model analysis

[0048] To avoid uneven colors and edge shading, the RGB average of all pixels in the central area of each reaction well (25 pixels in diameter) was extracted using Adobe Photoshop. The colorimetric reaction was determined based on the color difference value (ΔRGB) based on the difference in R, G, and B values between the control and the test. For visualization purposes only, the color difference map was expanded by scaling the relevant color range (i.e., the RGB color range was expanded from 0 to 63 to 0 to 255). The resulting image is shown in Figure 2. Figure 8 Figure (a) and Figure 9 All the above colorimetric experiments were repeated five times. Then, MATLAB R2018b (Math Works, Natick, MA. USA) was used to run the PCA model to analyze the RGB data. The analysis results are shown in Figure (b). Figure 8 Figure (b) and Figure 10 shown.

[0049] Example 4

[0050] The present invention is applied to the differentiation of green tea:

[0051] The detailed information of four green tea samples (Liu'an Guapian, Mingqian Longjing, Duyun Maojian and Zhuyeqing) are shown in Table 1; i.e., the detailed information of the green tea samples and the sample number information;

[0052] Table 1

[0053]

[0054] The preparation process is as follows: 0.1 g of green tea sample was added to 10 mL of water, placed in a constant temperature water bath at 60°C for 15 min, and the filtered solution was used for further analysis. 50 μL of boronic acid probe (1×10 -3 mol / L) solution and 50 μL of indicator solution (8×10 -4 mol / L), and then 100 μL of green tea sample was added, and the photo was taken after 10 minutes. Adobe Photoshop was used to extract the RGB average value of all pixels in the central area of each reaction well (25 pixels in diameter). The colorimetric reaction was determined based on the color difference value (ΔRGB) according to the difference in R, G, and B values between the control and the test. For visualization purposes only, the color difference map was expanded by scaling the relevant color range (i.e., the RGB color range was expanded from 0 to 63 to 0 to 255), and the resulting image is shown in Figure 2. Figure 11 All the above colorimetric experiments were repeated five times. The PCA model was run using MATLAB R2018b (MathWorks, Natick, MA. USA) to analyze the RGB data. The analysis results are shown in Figure 1. Figure 11 As shown in Figure (b).

[0055] The contents described in the embodiments of this specification are merely an enumeration of the implementation forms of the inventive concept. The scope of protection of the present invention should not be regarded as limited to the specific forms described in the embodiments. The scope of protection of the present invention also includes equivalent technical means that can be thought of by those skilled in the art based on the inventive concept.

Claims

1. A method for preparing an indicator displacement sensor array for tea polyphenol identification, characterized in that: A 2×2 colorimetric sensor array was constructed to detect and identify tea polyphenols using 1-naphthaleneboronic acid NapB and 1-pyreneboronic acid PyrB as probes, and catechol-containing Alizarin Red S (ARS) and catechol purple PV as indicators. −2 mol / L HEPES buffer solution was used as the solvent; tea polyphenols were classified by combining principal component analysis (PCA); the specific steps are as follows: (1) Design and synthesize the NapB probe, whose chemical structure is as follows: ； (2) Design and synthesize the PyrB probe, whose chemical structure is as follows: ； (3) Weigh a certain amount of NapB, PyrB, ARS, PV and six tea polyphenols and dissolve them in 1×10 −2 mol / L and pH = 8.2 HEPES buffer solution for standby use; the six tea polyphenols are gallic acid GA, methyl gallate MG, epigallocatechin EGC, epicatechin EC, epigallocatechin gallate EGCG and epicatechin gallate ECG; (4) Using NapB and PyrB as probes and ARS and PV as indicators, a 2×2 colorimetric sensor array was constructed to detect and identify six tea polyphenols: gallic acid GA, methyl gallate MG, epigallocatechin EGC, epicatechin EC, epigallocatechin gallate EGCG, and epicatechin gallate ECG, and an image of the colorimetric sensor array was obtained; (5) Adobe Photoshop software was used to extract the RGB average value of all pixels in the central area of each reaction well, which has a diameter of 25 pixels, and the PCA model was used to classify and identify all RGB values.

2. The method for preparing the indicator displacement sensor array for tea polyphenol identification according to claim 1, characterized in that: The step (1) specifically comprises: at a temperature of −78 o At 400°C, an anhydrous tetrahydrofuran solution containing 2 g of 1-bromonaphthalene (NapBr) and a 2 mol / L n-butyllithium solution are slowly mixed in order by volume to obtain a mixed solution, and then a triisopropyl borate solution is added over 1-10 minutes. After 1 hour, the reaction mixture is naturally warmed to room temperature. Subsequently, a hydrochloric acid solution is added, wherein the volume ratio of the 2 mol / L n-butyllithium, the anhydrous tetrahydrofuran solution containing 2 g of 1-bromonaphthalene (NapBr), the triisopropyl borate, and the hydrochloric acid solution is 5:30:3:

2. The mixture is extracted with ethyl acetate, and the organic phase is washed with water. The resulting solution is dried over anhydrous magnesium sulfate, and the solvent is evaporated to obtain the product, 1-naphthaleneboronic acid (NapB), as a white solid. The NapBr structural formula is as follows: 。 3. The method for preparing the indicator displacement sensor array for tea polyphenol identification according to claim 1, characterized in that: The step (2) specifically includes: o At an ambient temperature of 1°C, anhydrous tetrahydrofuran containing 2 g of 1-bromopyrene PyrBr and a 2 mol / L n-butyl lithium solution are mixed to obtain a mixed solution, followed by the addition of triisopropyl borate within 1-10 minutes. After 1 hour, the reaction mixture is naturally warmed to room temperature; a hydrochloric acid solution is then added, wherein the volume ratio of the 2 mol / L n-butyl lithium, the anhydrous tetrahydrofuran containing 2 g of 1-bromopyrene PyrBr, triisopropyl borate, and hydrochloric acid solution is 5:30:3:2; extraction is performed with ethyl acetate, and the organic phase is washed with water; the resulting solution is dried over anhydrous magnesium sulfate, and the solvent is evaporated to obtain a light yellow solid product, 1-pyrene borate PyrB; The structure of PyrBr is as follows: 。 4. The method for preparing the indicator displacement sensor array for tea polyphenol identification according to claim 1, characterized in that: The step (4) specifically includes: adding the buffer solution, boric acid probe, indicator and tea polyphenol solution into the wells of a 96-well plate, placing it on a micro-oscillator with a rotation speed of 300 rpm and oscillating for 10 minutes; placing the 96-well plate on an LED light and taking pictures to obtain an image.

5. An application of an indicator displacement sensor array for tea polyphenol identification prepared according to the preparation method according to any one of claims 1 to 4, characterized in that: It is used to realize the differentiated detection of six different tea polyphenols and the classification of different types and grades of green tea.

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