Preparation method and application of self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin

Abstract

This invention relates to the fields of modified electrode preparation and drug analysis, specifically disclosing a method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin, and its application. The self-supporting flexible electrode is prepared by the following method: First, carbon fiber paper is immersed in an aqueous solution containing zinc salt, and then a zinc oxide seed-modified carbon fiber paper is obtained through a pyrolysis reaction; then, the zinc oxide seed-modified carbon fiber paper is subjected to a hydrothermal reaction to obtain a zinc oxide nanorod array-modified carbon fiber paper (ZnO@CFP); then, copper oxide nanosheets are deposited on ZnO@CFP by chemical deposition to obtain a copper oxide nanosheet-zinc oxide nanorod array composite material-modified carbon fiber paper (CuO-ZnO@CFP); finally, an aqueous solution of carbon nanotubes is sprayed onto CuO-ZnO@CFP to obtain a carbon nanotube-copper oxide nanosheet-zinc oxide nanorod array ternary composite material-modified carbon fiber paper (CNT-CuO-ZnO). The CuO-ZnO-CNT@CFP was cut and used as a self-supporting flexible working electrode to construct an electrochemical sensor that can simultaneously detect the content of baicalin and luteolin in traditional Chinese medicine samples. It has a wide detection range, high sensitivity and low detection limit, and has great potential for practical application.

Description

Technical Field

[0001] This invention relates to the fields of modified electrode preparation and drug analysis, and provides a method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin, as well as its application. Background Technology

[0002] Traditional Chinese medicine (TCM) has been practiced in China for thousands of years, and its efficacy has been historically verified, making it a treasure of Chinese traditional culture. However, TCM contains numerous and complex active ingredients. Currently, over 600 compounds, including flavonoids, alkaloids, terpenes, and polysaccharides, have been isolated and purified from TCM. Clarifying the relationship between the complex drug components and unique pharmacological effects of TCM is crucial for its rational and safe use and further development. Therefore, accurately detecting the content of various components in TCM has become an important topic in the field of pharmaceutical analysis. Baicalein and luteolin are two important flavonoid compounds, the main components of TCM such as Scutellaria baicalensis, Sophora flavescens, and Ginkgo biloba. These herbs typically possess anti-inflammatory, antibacterial, antitumor, and menopausal symptom-regulating effects. Currently, commonly used methods include high-performance liquid chromatography (HPLC), ultraviolet spectrophotometry (UV), flow injection chemiluminescence (FICL), and high-performance capillary electrophoresis (HPCE). Compared with these methods, electrochemical analysis (EC) has the advantages of being rapid, accurate, requiring simple instruments, and enabling continuous analysis. However, due to the similar structures of baicalin and luteolin, two flavonoids, the oxidation peaks of the two substances overlap on traditional electrodes, making their simultaneous determination more difficult. Currently, most electrochemical methods for detecting baicalin and luteolin focus on detecting single target compounds, while research on the simultaneous detection of baicalin and luteolin content is relatively limited.

[0003] p-type semiconductor ZnO is low-cost, non-toxic, and abundant; n-type semiconductor CuO possesses excellent electrocatalytic performance. Combining ZnO and CuO to construct pn heterostructures can provide more catalytically active sites for electrochemical reactions, stimulating synergistic effects between different metal oxides. Using these composites as electrode modifiers in electrochemical sensors can improve sensor sensitivity and selectivity, potentially enabling the simultaneous detection of baicalein and luteolin. Sahar Daemi et al. prepared ZnO-CuO composite nanofibers via electrospinning, using them as electrode modifiers to create a highly sensitive electrochemical sensor for hydrogen peroxide detection (Sahar Daemi, Shahram Ghasemi, Ali Akbar Ashkarran. Journal of Colloidand Interface Science, 2019, 550: 180-189). However, this method requires the addition of polyethylene polymer gel as a binder during electrospinning, which weakens the electrocatalytic activity of the ZnO-CuO composite material. Kajal Jindal et al. constructed a ZnO-CuO pn heterostructure by depositing CuO microcrystal clusters on a ZnO thin film using pulsed laser deposition technology. After loading uricase, they fabricated an electrochemical sensor for detecting uric acid (Kajal Jindal, Monika Tomar, Vinay Gupta. Sensors and Actuators B: Chemical, 2017, 253: 566-575). This method requires not only large-scale pulsed laser equipment but also the loading of uricase, limiting the practical application value of this material as an electrochemical sensor.

[0004] Furthermore, the poor conductivity of metal oxides limits their practical application as electrode modification materials. Carbon nanotubes (CNTs), on the other hand, possess advantages such as high conductivity, high stability, and good biocompatibility. Therefore, combining CNTs with metal oxides can create efficient electron transport channels in the composite material, enabling faster electron transfer of analytes at the electrode / electrolyte interface and improving the detection sensitivity as an electrochemical sensor.

[0005] With the development of nanomaterials and nanotechnology, modifying electrode surfaces with nanomaterials of different morphologies, sizes, and physicochemical properties can enable the development of ultrasensitive and highly selective electrochemical sensors. However, the immobilization of nanomaterials on electrode surfaces often requires the addition of binders. The encapsulation and dilution effects of binders can reduce the catalytic activity of nanomaterials, and prolonged immersion in electrolytes can cause the binders to degrade, leading to the detachment of the modifier from the electrode. Therefore, the immobilization of nanomaterials on electrode surfaces remains a challenging task.

[0006] Therefore, the invention of a simple and effective method without adding any binders to effectively combine nano-ZnO, nano-CuO and CNTs to prepare a highly sensitive and selective self-supporting flexible electrode that can simultaneously detect baicalein and luteolin has important research and application significance for the identification, content detection and drug safety of effective drug components in traditional Chinese medicine. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a self-supporting flexible electrode that is flexible, simple and effective, and capable of simultaneously detecting baicalein and luteolin without the need for adhesives, and its application.

[0008] The technical solution of this invention is: a method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin, and its application. The specific process is as follows:

[0009] (1) Immerse carbon fiber paper (CFP) in 20 mL of an aqueous solution containing zinc salt and hexamethylenetetramine for 20 seconds, remove it and place it directly in an oven, control the temperature at 50 ℃~100 ℃, and pyrolyze for 5~10 minutes; repeat the process 3 times to obtain carbon fiber paper modified with zinc oxide crystals.

[0010] (2) 3 mL of 5 mol / L ammonia water was slowly added dropwise to 25 mL of zinc salt aqueous solution. After stirring evenly, the solution was poured into a high-pressure reactor lined with polytetrafluoroethylene. The carbon fiber paper modified with zinc oxide seeds was also placed into the reactor. The temperature was controlled at 50 ℃~100 ℃. After hydrothermal reaction for 5~10 hours, the solution was cooled to room temperature. The carbon fiber paper was removed, rinsed with deionized water, and dried to obtain carbon fiber paper modified with zinc oxide nanorod array, denoted as ZnO@CFP.

[0011] (3) The carbon fiber paper modified with zinc oxide nanorod array was immersed in 10 mL of copper acetate aqueous solution and 10 mL of potassium hydroxide aqueous solution for 20 seconds each. After being taken out, it was air-dried at room temperature. The process was repeated 3 times. Then, it was placed on a heating plate and the temperature was controlled at 50 ℃~100 ℃ for chemical deposition reaction for 5~10 hours. The carbon fiber paper was removed, cooled to room temperature, rinsed with deionized water, and dried to obtain carbon fiber paper modified with copper oxide nanosheet-zinc oxide nanorod array composite material, denoted as CuO-ZnO@CFP.

[0012] (4) The carbon fiber paper modified with the above copper oxide nanosheet-zinc oxide nanorod array composite material was placed on a heating plate and the temperature was controlled at 50 ℃~100 ℃. The carbon nanotube (CNT) aqueous solution was sprayed onto CuO-ZnO@CFP. The spraying airflow rate was controlled at 90~150 mL / h and the spraying time was 10~30 seconds. The carbon fiber paper modified with the carbon nanotube-copper oxide nanosheet-zinc oxide nanorod array ternary composite material was obtained by thermal spraying. It was cut into a rectangle of 1 cm × 2 cm to obtain a self-supporting flexible electrode, which was denoted as CNT-CuO-ZnO@CFP electrode.

[0013] Furthermore, the zinc salt in process (1) is one of zinc acetate, zinc chloride, and zinc nitrate.

[0014] Furthermore, the molar concentration of the zinc salt aqueous solution in process (1) is 0.05 to 0.2 mol / L, and the molar ratio of zinc salt to hexamethylenetetramine is 1:1.

[0015] Furthermore, the zinc salt in process (2) is one of zinc acetate, zinc chloride, and zinc nitrate.

[0016] Furthermore, the molar ratio of zinc salt to ammonia in process (2) is 0.1 to 0.5:1.

[0017] Furthermore, the molar concentration of the copper acetate aqueous solution in process (3) is 0.05 mol / L to 2 mol / L, and the molar ratio of copper acetate to potassium hydroxide is 1:2.

[0018] Furthermore, the concentration of the carbon nanotube aqueous solution in process (4) is 1 to 3 mg / mL.

[0019] Furthermore, the self-supporting flexible electrode prepared by the method is used to simultaneously detect the content of baicalein and luteolin in traditional Chinese medicine. When the concentration of luteolin is fixed at 10 μM, the linear equation for the detection of baicalein by this self-supporting flexible electrode is as follows: I pba =11.92 C ba +49.93 ( C ba Regarding the concentration of baicalin, I pba (The peak current of baicalin); linear range: 0.03 μM~14 μM; sensitivity: 11.92 μA / μM·cm 2 The detection limit was 1.7 nM. When the baicalin concentration was fixed at 10 μM, the linear equation for the detection of luteolin by this self-supporting flexible electrode was: I plu = 21.85 Clu +28.45 ( C lu At the concentration of luteolin I plu (Peak current of luteolin); linear range: 0.03 μM~6 μM; sensitivity: 21.85 μA / μM·cm 2 The detection limit is 0.5 nM.

[0020] Compared with the prior art, the present invention has the following beneficial technical effects:

[0021] (1) This invention utilizes simple soaking pyrolysis, hydrothermal reaction, chemical deposition and thermal spraying methods to directly modify nano ZnO, nano CuO and CNT onto carbon fiber paper to obtain a self-supporting flexible electrode that does not require the addition of adhesives.

[0022] (2) The present invention forms an effective interface connection through the synergistic effect between the three components CNT-CuO-ZnO, which provides more catalytic active sites and electron transfer channels for the electrochemical reaction of the analytes, and realizes the simultaneous detection of baicalein and luteolin;

[0023] (3) The self-supporting flexible electrode obtained in this invention, when the luteolin concentration is fixed at 10 μM, has a linear range of 0.03 μM to 14 μM for detecting baicalein; and a sensitivity of 11.92 μA / μM·cm. 2 The detection limit reached 1.7 nM. When the baicalin concentration was fixed at 10 μM, the linear range for the detection of luteolin by this self-supporting flexible electrode was 0.03 μM to 6 μM; the sensitivity reached 21.85 μA / μM·cm. 2 The detection limit is 0.5 nM. Furthermore, the self-supporting flexible electrode obtained in this invention can be used for the detection of baicalin and luteolin in actual traditional Chinese medicine samples, showing broad application prospects. Attached Figure Description

[0024] Figure 1 This is a bending diagram of a self-supporting flexible electrode.

[0025] Figure 2 It is a scanning electron microscope image.

[0026] Figure 3 It is an XRD pattern.

[0027] Figure 4 It is a graph showing the linear relationship between peak current and concentration. Detailed Implementation

[0028] The invention will be further described below with reference to the accompanying drawings:

[0029] Implementation Case 1: A method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin, specifically including the following steps:

[0030] (1) Cut carbon fiber paper into 3 cm × 3 cm pieces, soak them in 20 mL of a mixed aqueous solution containing 0.05 mol / L zinc acetate and 0.05 mol / L hexamethylenetetramine for 20 seconds, take them out and put them directly into an oven, control the temperature at 100 ℃, and pyrolyze for 5 minutes; repeat the process 3 times to obtain carbon fiber paper modified with zinc oxide crystals.

[0031] (2) 3 mL of 5 mol / L ammonia water was slowly added dropwise to 25 mL of 0.2 mol / L zinc acetate aqueous solution. After stirring and mixing evenly, the solution was poured into a high-pressure reactor lined with polytetrafluoroethylene. The carbon fiber paper modified with zinc oxide seeds was also placed in the reactor. The temperature was controlled at 90℃. After hydrothermal reaction for 6 hours, the solution was cooled to room temperature. The carbon fiber paper was removed, rinsed with deionized water, and dried to obtain carbon fiber paper modified with zinc oxide nanorod array, which was denoted as ZnO@CFP-1.

[0032] (3) The carbon fiber paper modified with zinc oxide nanorod array was immersed in 10 mL of 0.05 mol / L copper acetate aqueous solution and 10 mL of 0.1 mol / L potassium hydroxide aqueous solution for 20 seconds each. After taking it out, it was dried at room temperature. The process was repeated 3 times. It was placed on a heating plate and the temperature was controlled at 90 °C for chemical deposition reaction for 5 hours. The carbon fiber paper was removed, cooled to room temperature, rinsed with deionized water, and dried to obtain carbon fiber paper modified with copper oxide nanosheet-zinc oxide nanorod array composite material, which was denoted as CuO-ZnO@CFP-1.

[0033] (4) Place the above CuO-ZnO@CFP-1 on a heating plate and control the temperature at 90 °C. Spray a 1 mg / mL CNT aqueous solution onto CuO-ZnO@CFP-1, control the spraying airflow rate at 120 mL / h, and the spraying time at 30 seconds. A carbon fiber paper modified with a ternary composite material of carbon nanotube-copper oxide nanosheet-zinc oxide nanorod array is prepared by thermal spraying. Cut it into a rectangle of 1 cm × 2 cm to prepare a self-supporting flexible electrode, which is denoted as CNT-CuO-ZnO@CFP-1 electrode.

[0034] Depend on Figure 1 As can be seen, the self-supporting flexible electrode that can simultaneously detect baicalein and luteolin can be bent, indicating that it has a flexible characteristic.

[0035] The inventors performed scanning electron microscopy on the ZnO@CFP-1, CuO-ZnO@CFP-1, and CNT-CuO-ZnO@CFP-1 samples obtained in the above process, and the results are as follows. Figure 2 As shown. Figure 2 The morphology of ZnO@CFP-1 is depicted, showing a dense array of vertically aligned ZnO nanorods covering the carbon fiber surface. This was achieved using a magnified scanning electron microscope. Figure 2 b. The ZnO nanorods are approximately 2-3 mm long. Scanning electron microscopy of CuO-ZnO@CFP-1 is shown below. Figure 2 As shown in Figure c, CuO nanosheets are uniformly coated on ZnO nanorods, forming a CuO-ZnO nanorod array resembling a sea cucumber. This is illustrated by a magnified scanning electron microscope. Figure 2 As can be seen, the CuO nanosheets are uniform in size, approximately 150 nm long and 50 nm wide. Figure 2 e and 2f are scanning electron microscope images of the CNT-CuO-ZnO@CFP-1 sample. As shown in the figure, CNTs are uniformly covered on CuO-ZnO nanorods, forming a composite electrode with a three-component structure and effective interfacial connection.

[0036] Depend on Figure 3 The XRD pattern shows that the prepared CNT-CuO-ZnO@CFP-1 self-supporting flexible electrode exhibits standard ZnO, CuO, and CNT characteristic diffraction peaks.

[0037] Implementation Case 2

[0038] In Implementation Case 1, the inventors prepared a self-supporting flexible electrode CNT-CuO-ZnO@CFP-1 and simultaneously detected the contents of baicalein and luteolin using differential pulse voltammetry to evaluate the electrochemical response capability of the CNT-CuO-ZnO@CFP-1 electrode. The prepared CNT-CuO-ZnO@CFP-1 self-supporting flexible electrode was used as the working electrode, a saturated calomel electrode as the reference electrode, and a platinum wire electrode as the counter electrode. Differential pulse voltammetry was performed in a BR buffer aqueous solution at pH 6.86. By changing the concentration of baicalein or luteolin added, the current response values ​​of the two electrodes under DPV technology were obtained, and the results are as follows:

[0039] Within a potential range of -0.2 V to 0.5 V, the concentrations of baicalein and luteolin were fixed at 10 μM, while the concentration of another analyte was varied. It was found that as the concentration of the analyte increased, the corresponding oxidation peak current increased accordingly. Figure 4 a, c). Differential pulse voltammetry showed a good linear relationship between the concentrations of baicalein and luteolin and the peak current within a certain range, yielding peak current-concentration linear relationship curves for either baicalein or luteolin. Figure 4(b, d). The optimal determination conditions for baicalein and luteolin were: BR buffer as the supporting electrolyte, with an optimal pH of 6.86.

[0040] When the concentration of luteolin was fixed at 10 μM, the linear range for detecting baicalein was 0.03 μM–14 μM; the linear equation was: I pba =11.92 C ba +49.93 ( C ba This refers to the concentration of baicalin. I pba (The peak current of baicalin); the sensitivity is 11.92 μA / μM·cm. 2 The detection limit is 1.7 nM.

[0041] When the concentration of baicalein was fixed at 10 μM, the linear range for detecting luteolin was 0.03 μM–6 μM; the linear equation was: I plu = 21.85 C lu +28.45 C lu This refers to the concentration of luteolin. I plu (The peak current of luteolin); sensitivity is 21.85 μA / μM·cm. 2 The detection limit is 0.5 nM.

[0042] Implementation Case 3: Simultaneous Determination of Baicalin and Luteolin in Shuanghuanglian Oral Liquid

[0043] Add 45 μL of Shuanghuanglian oral liquid to 10 mL of 0.1 mol / L BR buffer solution, sonicate for 30 minutes, and centrifuge at 12000 rpm for 10 minutes. Take the supernatant and dilute it with BR buffer solution to the detection concentration. Using the self-supported flexible electrode CNT-CuO-ZnO@CFP-1 prepared in Example 1 as the working electrode, the content of baicalein and luteolin in actual Shuanghuanglian oral liquid samples was detected by spiked recovery method. It was found that the electrode has a good recovery rate, reaching 97.0%–104.0%.

[0044] Table 1. Simultaneous determination of baicalin and luteolin content in Shuanghuanglian oral liquid samples using the spiking and recovery method.

[0045]

[0046] a. Five parallel measurements were performed.

[0047] Implementation Case 4: Simultaneous Determination of Baicalin and Luteolin in the Chinese Herb *Duyiwei*

[0048] Take 10 capsules of Duyiwei traditional Chinese medicine, grind the powder into a fine powder, accurately weigh 3.0 g, add 20 mL of ethanol, sonicate for 30 minutes, then reflux for 120 minutes, cool, and centrifuge at 12000 rpm for 10 minutes. Take the supernatant and dilute it to the detection concentration with BR buffer solution. Using the self-supporting flexible electrode CNT-CuO-ZnO@CFP-1 prepared in Example 1 as the working electrode, the content of baicalein and luteolin in actual Duyiwei capsule samples was detected by spiked recovery method. It was found that the electrode has a good recovery rate, reaching 96.0% to 104.0%, indicating that the present invention has good practical application prospects.

[0049] Table 2. Simultaneous determination of baicalin and luteolin content in Duyiwei capsules using the spiking recovery method.

[0050]

[0051] a. Five parallel measurements were performed.

[0052] The present invention has been disclosed above with reference to preferred embodiments only, but is not intended to limit the invention in any other way. Any modifications, equivalent substitutions, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the claims.

Claims

1. A method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin, characterized in that... Its preparation method includes the following process: Step (1) Immerse carbon fiber paper (CFP) in 20 mL of an aqueous solution containing zinc salt and hexamethylenetetramine for 20 seconds, remove it and place it directly in an oven, control the temperature at 50 ℃~100 ℃, and pyrolyze for 5~10 minutes; repeat this process 3 times to obtain carbon fiber paper modified with zinc oxide crystals. Step (2) Slowly add 3 mL of 5 mol / L ammonia water to 25 mL of zinc salt aqueous solution, stir evenly and pour into a high pressure vessel with a polytetrafluoroethylene liner. Put the carbon fiber paper modified with zinc oxide seeds into the vessel as well. Control the temperature at 50 ℃~100 ℃ and perform hydrothermal reaction for 5~10 hours. Cool to room temperature, take out the carbon fiber paper, rinse with deionized water and dry to obtain carbon fiber paper modified with zinc oxide nanorod array, denoted as ZnO@CFP; Step (3) The carbon fiber paper modified with zinc oxide nanorod array above was immersed in 10 mL of copper acetate aqueous solution and 10 mL of potassium hydroxide aqueous solution for 20 seconds each. After taking it out, it was air-dried at room temperature. This process was repeated 3 times. Then, it was placed on a heating plate and the temperature was controlled at 50 ℃~100 ℃ for chemical deposition reaction for 5~10 hours. The carbon fiber paper was removed and cooled to room temperature. After rinsing with deionized water, it was dried to obtain carbon fiber paper modified with copper oxide nanosheet-zinc oxide nanorod array composite material, denoted as CuO-ZnO@CFP. Step (4) Place the carbon fiber paper modified with the above copper oxide nanosheet-zinc oxide nanorod array composite material on a heating plate and control the temperature at 50 ℃~100 ℃. Spray the carbon nanotube (CNT) aqueous solution onto CuO-ZnO@CFP, control the spraying airflow rate at 90~150 mL / h, and the spraying time at 10~30 seconds. The carbon fiber paper modified with the carbon nanotube-copper oxide nanosheet-zinc oxide nanorod array ternary composite material is obtained by thermal spraying. Cut it into a rectangle of 1 cm × 2 cm to make a self-supporting flexible electrode, which is denoted as CNT-CuO-ZnO@CFP electrode. The self-supporting flexible electrode prepared by the method is used to simultaneously detect the content of baicalin and luteolin in traditional Chinese medicine. When the concentration of luteolin is fixed at 10 μM, the linear equation for the detection of baicalin by this self-supporting flexible electrode is as follows: I pba =11.92 C ba +49.93, C ba This refers to the concentration of baicalin. I pba The peak current of baicalin was measured; the linear range was 0.03 μM to 14 μM; and the sensitivity was 11.92 μA / μM·cm. 2 The detection limit is 1.7 nM. When the concentration of baicalin is fixed at 10 μM, the linear equation for the detection of luteolin by this self-supporting flexible electrode is as follows: I plu =21.85 C lu +28.45, C lu This refers to the concentration of luteolin. I plu The peak current of luteolin was measured; the linear range was 0.03 μM to 6 μM; and the sensitivity was 21.85 μA / μM·cm. 2 The detection limit is 0.5 nM.

2. The method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin according to claim 1, characterized in that... The zinc salt in step (1) is one of zinc acetate, zinc chloride, and zinc nitrate.

3. The method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin according to claim 1, characterized in that... The molar concentration of the zinc salt aqueous solution in step (1) is 0.05-0.2 mol / L, and the molar ratio of zinc salt to hexamethylenetetramine is 1:

1.

4. The method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin according to claim 1, characterized in that... The zinc salt in step (2) is one of zinc acetate, zinc chloride, and zinc nitrate.

5. A method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin according to claim 1, characterized in that... The molar ratio of zinc salt to ammonia in step (2) is 0.1 to 0.5:

1.

6. A method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin according to claim 1, characterized in that... The molar concentration of the copper acetate aqueous solution in step (3) is 0.05 mol / L to 2 mol / L, and the molar ratio of copper acetate to potassium hydroxide is 1:

2.

7. A method for preparing a self-supporting flexible electrode capable of simultaneously detecting baicalein and luteolin according to claim 1, characterized in that... The concentration of the carbon nanotube aqueous solution in step (4) is 1 to 3 mg / mL.

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