Electrochemical sensor and electrochemical method for detecting fisetin

Abstract

The invention relates to the technical field of electrochemical analysis, in particular to an electrochemical sensor for detecting fisetin and an electrochemical method.The electrochemical sensor comprises a working electrode, the working electrode is Nafion-AuNPs / CFME, the Nafion-AuNPs / CFME comprises CFME, a Nafion layer and an AuNPs layer, and the Nafion layer and the AuNPs layer are sequentially modified on the surface of the CFME; the electrochemical sensor is adopted, a to-be-detected sample is detected through cyclic voltammetry or differential pulse voltammetry, response current is recorded, and then the concentration of fisetin in the to-be-detected sample is calculated according to a concentration-current linear regression equation. According to the electrochemical sensor, Nafion-AuNPs / CFME is adopted as a working electrode, the modification effect of AuNPs on the surface of CFME is enhanced through Nafion, and high-sensitivity, high-selectivity and wide-concentration-range detection of fisetin can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical analysis, and particularly relates to an electrochemical sensor and an electrochemical method for detecting fisetin. Background Art

[0002] Fisetin (FI) is a natural flavonoid polyphenol compound, also known as fisetinic acid, quercetin or butein, with the chemical name of 3,3',4',7-tetrahydroxyflavone, and its chemical structure is shown as follows:

[0003]

[0004] Fisetin widely exists in fruits, vegetables, nuts and wines, and is mainly extracted from plants of the Anacardiaceae family such as Cotinus coggygria and Toxicodendron vernicifluum, and has various biological activities such as anti-inflammatory and antioxidant, anti-cancer, nerve protection, anti-angiogenesis and anti-aging.

[0005] At present, the detection methods of fisetin mainly include ultraviolet spectrophotometry, high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), fluorescence analysis method, etc. However, these methods generally have high costs, high requirements for instruments, drug purity and operation techniques, and there are certain limitations in detection. Summary of the Invention

[0006] The purpose of the present invention is to provide an electrochemical sensor and an electrochemical method for detecting fisetin, which can realize rapid, stable and accurate quantitative detection of fisetin, and have the advantages of simple operation, high sensitivity and low cost.

[0007] To achieve the above purpose, the technical solution of the present invention is an electrochemical sensor for detecting fisetin, including a working electrode, and the working electrode is Nafion-AuNPs / CFME, and the Nafion-AuNPs / CFME includes CFME and a Nafion layer and an AuNPs layer sequentially modified on the surface of the CFME.

[0008] Further, the manufacturing method of the Nafion-AuNPs / CFME is: first immerse the CFME in a Nafion solution, take it out and dry it to obtain the CFME with a Nafion layer modified on the surface; then use the potentiostatic deposition method to modify the AuNPs layer on the surface of the CFME with a Nafion layer modified on the surface, wash and dry it to obtain Nafion-AuNPs / CFME.

[0009] Furthermore, the immersion time of the CFME in the Nafion solution is 4-6 min.

[0010] Furthermore, the potentiostatic deposition method is as follows: The CFME modified with a Nafion layer on the surface is immersed in the AuNPs solution together with a reference electrode, and electrodeposition is carried out at a voltage of +1.4 to 1.8 V for 10 to 25 minutes.

[0011] Furthermore, the electrochemical sensor further includes a reference electrode.

[0012] The present invention also provides an electrochemical method for detecting luteolin. Using the electrochemical sensor described in any one of the above, the sample to be tested is detected by cyclic voltammetry or differential pulse voltammetry, the response current is recorded, and then the concentration of luteolin in the sample to be tested is calculated according to the linear regression equation of concentration-current.

[0013] Furthermore, when the concentration of luteolin is in the range of 0.1 to 10 μmol / L, the linear regression equation of concentration-current is I p (nA) = 0.2664c (μmol / L) + 0.6500, R 2 = 0.996; when the concentration of luteolin is in the range of 0.01 to 0.1 μmol / L, the linear regression equation of concentration-current is I p (nA) = 1.3587c (μmol / L) + 0.4211, R 2 = 0.998; where c is the concentration of luteolin and I p is the current.

[0014] Furthermore, when using cyclic voltammetry, the potential range is -0.5 V to 0.7 V, and the scanning rate is 0.05 to 0.15 V / s; when using differential pulse voltammetry, the potential range is -0.1 to 0.7 V, the amplitude is 0.05 V, the pulse width is 0.1 s, the pulse time is 0.2 s, and the inhibition time is 2 s.

[0015] Furthermore, the preparation method of the sample to be tested is as follows: First, the actual sample is cleaned and dried, placed in a mortar and ground into fine powder, then anhydrous ethanol is added to the fine powder for dissolution, ultrasonic oscillation is carried out and then centrifuged, and then the supernatant is taken to obtain a sample solution, and then the sample solution is diluted with a buffer solution to obtain the sample to be tested.

[0016] Furthermore, the buffer solution is a PBS buffer solution with a pH value of 3.0 to 4.0.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The electrochemical sensor of the present invention uses Nafion-AuNPs / CFME as the working electrode. By enhancing the modification effect of AuNPs on the surface of CFME with Nafion, it can achieve the detection of luteolin with high sensitivity, high selectivity, and a wide concentration range;

[0019] (2) The linear range for detecting luteolin by the electrochemical sensor of the present invention is 0.01 - 0.10 μmol / L and 0.10 - 10 μmol / L. The detection limit (LOD, S / N = 3) is 0.078 μmol / L, and the quantification limit is 0.009 μmol / L;

[0020] (3) Compared with the ordinary carbon fiber electrode, the Nafion-AuNPs / CFME of the present invention has a larger response signal to luteolin, higher sensitivity, and excellent electrocatalytic performance;

[0021] (4) The electrochemical method of the present invention can also detect luteolin in the actual sample Cotinus coggygria Scop. var. pubescens Engl., which can provide reference and guiding value for the actual detection and application of luteolin. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0023] Figure 1 Among them: (a) is the TEM image of AuNPs; (b) is the SEM image of CFME; (c) is the SEM image of AuNPs / CFME; (d) is the SEM image of Nafion-AuNPs / CFME;

[0024] Figure 2 Among them: (a) is the EIS diagram of CFME, Nafion-AuNPs, and AuNPs / CFME; (b) is the DPV diagram of CFME, AuNPs / CFME, and Nafion-AuNPs / CFME in a 1×10 -6 mol / L luteolin solution; (c) is the CV diagram of CFME, AuNPs / CFME, and Nafion-AuNPs / CFME in a 1×10 -6 mol / L luteolin solution;

[0025] Figure 3 Among them: (a) is the Nafion-AuNPs / CFME electrode with different electrodeposition times in a 1×10 -6DPV graphs in luteolin solutions with a concentration of 1×10

[0026] Figure 4 -4 -6 mol / L; (b) is a line graph of the electrodeposition time of AuNPs and the oxidation peak current of luteolin; (a) is the CV graph of Nafion-AuNPs / CFME in 1×10

[0027] Figure 5 -4 -6 mol / L luteolin solution at different scan rates; (b) is the linear relationship between the oxidation-reduction peak current of luteolin and the scan rate; (c) is the linear relationship between the oxidation-reduction peak potential of luteolin and the logarithm of the scan rate; (a) is the DPV graph of Nafion-AuNPs / CFME in 1×10 -6 mol / L luteolin solution at different pH values; (b) is the linear relationship between the oxidation peak potential of Nafion-AuNPs / CFME in 1×10

[0028] Figure 6 -4

[0029] Figure 7 mol / L luteolin solution; (b) is the linear relationship between the concentration of luteolin and the peak current in the ranges of 0.01 - 0.1 μmol / L and 0.1 - 10 μmol / L; (a) is the DPV graph of different concentrations of luteolin on Nafion-AuNPs / CFME; (a) is the influence of interfering substances on the determination of luteolin; (b) is the DPV peak current values detected continuously 10 times in 1×10 - 6 -4 - 6 mol / L luteolin solution; (c) is the DPV peak current values detected continuously for 10 days in 1×10 Specific Embodiments

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] In this specification, unless otherwise specified, the technical terms used are common terms used by those of ordinary skill in the art; the experimental methods without specific conditions noted in this specification are conventional experimental methods; the test materials used in this specification are commercially available products without special instructions, and the compositions and preparation methods of various reagents can be referred to the operations in conventional experimental manuals.

[0032] This embodiment provides an electrochemical sensor for detecting fisetin, including a working electrode, and the working electrode is Nafion-AuNPs / CFME. The Nafion-AuNPs / CFME includes CFME and a Nafion layer and an AuNPs layer sequentially modified on the surface of the CFME. In this embodiment, Nafion-AuNPs / CFME is used as the working electrode of the electrochemical sensor, and the modification effect of AuNPs on the surface of CFME is enhanced through the Nafion layer, enabling the detection of fisetin with high sensitivity, high selectivity, and a wide concentration range.

[0033] Furthermore, the manufacturing method of the Nafion-AuNPs / CFME includes the following steps:

[0034] S1. Prepare CFME (carbon fiber microelectrode);

[0035] S2. Prepare an AuNPs (nano gold particles) solution;

[0036] S3. First, immerse the CFME in a Nafion (perfluorosulfonic acid cation exchanger) solution, take it out and dry it to obtain CFME with a Nafion layer modified on the surface; then use the potentiostatic deposition method to modify an AuNPs layer on the surface of the CFME with a Nafion layer modified on the surface, wash and dry it to obtain Nafion-AuNPs / CFME.

[0037] In this embodiment, CFME is used as the substrate, the polymer Nafion is used as the binder, and the AuNPs are modified on the surface of CFME by the potentiostatic deposition method to prepare Nafion-AuNPs / CFME with more AuNPs particles attached to the surface, improving the conductivity and catalytic activity of the electrochemical sensor.

[0038] Even further, the immersion time of the CFME in the Nafion solution is 4 - 6 min. Preferably, the immersion time of the CFME in the Nafion solution is 5 min.

[0039] Further, the potentiostatic deposition method is as follows: Immerse the CFME with a surface-modified Nafion layer and a reference electrode into the AuNPs solution together, and perform potentiostatic deposition at a voltage of +1.4 to 1.8 V for 10 to 25 minutes. Preferably, the reference electrode is an Ag / AgCl electrode, and potentiostatic deposition is performed at a voltage of 1.5 V for 15 minutes.

[0040] In step S1, the method for preparing CFME (carbon fiber microelectrode) is as follows: Place the glass capillary on the alcohol lamp flame and draw it until the inner diameter of its tip is reduced to about 20 - 30 μm; Use conductive glue to adhere the copper wire to the carbon fiber, and after natural drying, insert the carbon fiber into the thick end of the glass capillary and make it expose about 2 - 3 mm from the other end, and seal the end of the glass capillary with AB glue. After the glue is completely cured, place the tip of the glass capillary on the alcohol lamp flame for melting and sealing.

[0041] In step S2, the method for preparing the AuNPs (nano gold particles) solution is as follows: Heat the ultrapure water to boiling, slowly add the trisodium citrate solution and chloroauric acid solution under stirring, and continue heating and refluxing to obtain a purple-red AuNPs solution. Through the chemical reduction method, AuNPs with a negatively charged surface and a small particle size can be synthesized, and attaching them to the CFME can increase the specific surface area and conductivity.

[0042] Further, the electrochemical sensor further includes a reference electrode. Preferably, the reference electrode is an Ag / AgCl electrode.

[0043] This embodiment also provides an electrochemical method for detecting quercetin. Using the electrochemical sensor described in any one of the above, the sample to be tested is detected by cyclic voltammetry or differential pulse voltammetry, the response current is recorded, and then the concentration of quercetin in the sample to be tested is calculated according to the linear regression equation of concentration-current. This embodiment establishes an electrochemical analysis method for highly sensitive detection of quercetin through the above electrochemical sensor, and can achieve high-sensitivity, high-selectivity, and wide-concentration-range detection of quercetin.

[0044] Further, when using cyclic voltammetry, the potential range is -0.5 V to 0.7 V, and the scanning rate is 0.05 to 0.15 V / s; when using differential pulse voltammetry, the potential range is -0.1 to 0.7 V, the amplitude is 0.05 V, the pulse width is 0.1 s, the pulse time is 0.2 s, and the inhibition time is 2 s.

[0045] Further, when the concentration of quercetin is in the range of 0.1 to 10 μmol / L, the linear regression equation of concentration-current is I p (nA) = 0.2664c (μmol / L) + 0.6500, R 2= 0.996; When the concentration of fisetin is in the range of 0.01 - 0.1 μmol / L, the linear regression equation of concentration-current is I p (nA) = 1.3587c(μmol / L) + 0.4211, R 2 = 0.998; where c is the concentration of fisetin and I p is the current. By substituting the current detected by cyclic voltammetry or differential pulse voltammetry into the linear regression equation of concentration-current, the concentration of fisetin in the sample to be measured can be calculated.

[0046] Furthermore, the preparation method of the sample to be measured is as follows: First, clean and dry the actual sample, then place it in a mortar and grind it into fine powder. Then add anhydrous ethanol to the fine powder to dissolve it, perform ultrasonic oscillation and then centrifuge. After that, take the supernatant to obtain a sample solution, and then dilute the sample solution with a buffer solution to obtain the sample to be measured.

[0047] Even further, the buffer solution is PBS buffer solution with a pH value of 3.0 - 4.0, preferably 3.0.

[0048] Next, the electrochemical sensor and electrochemical method for detecting fisetin of the present invention are studied through specific examples.

[0049] The equipment used in this example includes CHI660D electrochemical workstation (Shanghai Chenhua), two-electrode system (working electrode: Nafion-AuNPs / CFME, reference electrode: Ag / AgCl), SU8010 cold field scanning electron microscope (Hitachi, Japan), Tecnai G220 S-TWIN transmission electron microscope (FEI, Czech Republic), AR224CN analytical balance (Shanghai Ohaus), PHSJ-6L pH meter (Shanghai Yidian), PJJ-1 precision magnetic stirrer (Guohua Changzhou), XD-RFL inverted microscope (Ningbo Shunyu), THW250ML electric heating mantle (Wuhan Ke'er).

[0050] The materials used in this example include carbon fiber (Jilin Shenzhou), methanol, ethanol, potassium ferricyanide, potassium ferrocyanide, ferrous sulfate, sodium citrate, calcium chloride, sodium chloride, ferric chloride, potassium sulfate, glucose (analytical pure, Sinopharm Group), chloroauric acid, fisetin (Shanghai Yuanye), AB glue (Deli), conductive glue (Hunan Baxiong New Materials), and PBS buffer solution (pH 7.0 Tris-HCl, self-made). All experimental water is ultrapure water. The actual sample of Cotinus coggygria leaves was collected from Wuhan, Hubei in November 2024, and it is the leaf of Cotinus coggygria var. pubescens Engl.

[0051] Example 1

[0052] The preparation method and electrochemical performance of Nafion-AuNPs / CFME will be described in detail below.

[0053] 1. Preparation of CFME (carbon fiber microelectrode)

[0054] A glass capillary tube (inner diameter 1 mm) is placed on the flame of an alcohol lamp and pulled until the inner diameter of its tip is reduced to about 25 μm. The copper wire is adhered to the carbon fiber using conductive glue. After natural drying, the carbon fiber is inserted into the thick end of the glass capillary tube and allowed to protrude about 2 - 3 mm from the other end. The end of the glass capillary tube is sealed with AB glue. After the glue is completely cured, the tip of the glass capillary tube is sealed by melting on the flame of an alcohol lamp. Before each use, the exposed carbon fiber is slightly ablated in the outer flame of the alcohol lamp, and the exposed length of the carbon fiber is measured to be 100 - 200 μm under an inverted microscope, thus obtaining CFME.

[0055] 2. Preparation of AuNPs (gold nanoparticles)

[0056] Add 40 mL of ultrapure water to a three-necked flask, heat it to boiling, and slowly add 900 μL of 0.1 mol / L trisodium citrate solution and 500 μL of 2.5×10 -4 mol / L chloroauric acid solution while stirring. Continue heating for 40 min and reflux for 20 min to obtain a purple-red AuNPs solution. After the reaction is completed, cool it to room temperature and store the solution in a refrigerator at 4 °C for later use.

[0057] 3. Preparation of Nafion-AuNPs / CFME and preparation of AuNPs / CFME

[0058] The tip of a newly prepared CFME is immersed in Nafion solution for 5 min, taken out and dried to obtain CFME with a surface-modified Nafion layer; the CFME with a surface-modified Nafion layer is used as the working electrode, and its tip and the tip of the reference electrode Ag / AgCl are immersed in the AuNPs solution together. Electrochemical deposition is carried out at a voltage of +1.5 V for 15 min using the potentiostatic deposition method to form an AuNPs layer on the surface of the CFME with a surface-modified Nafion layer. The electrode is washed successively with ethanol and distilled water and dried under an infrared lamp to obtain Nafion-AuNPs / CFME.

[0059] Another newly prepared CFME is directly immersed in the AuNPs solution together with the tip of the reference electrode Ag / AgCl. Electrochemical deposition is carried out at a voltage of +1.5 V for 15 min using the potentiostatic deposition method. The electrode is washed successively with ethanol and distilled water and dried under an infrared lamp to obtain AuNPs / CFME.

[0060] 4. Characterization of Nafion-AuNPs / CFME

[0061] The morphology of the prepared AuNPs was characterized by high-resolution transmission electron microscopy (TEM) to analyze the microscopic features of the modified electrode materials. Figure 1 (a) shows the TEM image of AuNPs. It can be seen that the AuNPs exhibit a relatively regular spherical morphology with a particle size of about 23 nm, indicating the successful preparation of gold nanoparticles.

[0062] Scanning electron microscopy (SEM) was used to analyze the surface morphologies of Nafion-AuNPs / CFME, AuNPs / CFME, and CFME. Figure 1 (b), (c), and (d) respectively show the SEM images of CFME, AuNPs / CFME, and Nafion-AuNPs / CFME. It can be seen that the surface of CFME is smooth and flat, while the surfaces of AuNPs / CFME and Nafion-AuNPs / CFME are rough and have spherical nanoparticles attached, indicating that AuNPs have been successfully modified onto the surface of CFME. Moreover, compared with AuNPs / CFME, the number of spherical nanoparticles on the surface of Nafion-AuNPs / CFME is more, and the electrode diameter also increases, indicating that Nafion can enhance the attachment of AuNPs on the surface of CFME, thereby improving the roughness and specific surface area of the electrode.

[0063] 5. Electrochemical performance of Nafion-AuNPs / CFME

[0064] A two-electrode system was constructed with the reference electrode Ag / AgCl and the working electrode Nafion-AuNPs / CFME of the electrochemical sensor, and the electrochemical performance of the electrode was evaluated by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). All experiments were carried out at room temperature.

[0065] The frequency range was set to 0.01 Hz to 100 kHz, and in 0.1 mol / L KCl and 5 mmol / L [Fe(CN)6] 3- / [Fe(CN)6] 4- redox probe solution, the three electrodes of CFME, AuNPs / CFME, and Nafion-AuNPs / CFME were characterized by EIS method. The open-circuit potential of the EIS method was -1.0 V, the voltage amplitude was 5.0 mV, and the frequency range was 0.1 to 10,000 Hz. Figure 2 (a) shows the EIS diagrams of the three electrodes. The transfer impedance of the electrode is proportional to the radius of the semicircle, which can reflect the conductivity of the electrode. It can be seen from the figure that the impedance values of Nafion-AuNPs / CFME and AuNPs / CFME are significantly lower than that of CFME, indicating that AuNPs can effectively reduce the interfacial impedance of CFME, thereby improving the electron transfer rate on the electrode surface.

[0066] Furthermore, the DPV method and CV method were further used to investigate the electrochemical properties of three electrodes, namely CFME, AuNPs / CFME, and Nafion-AuNPs / CFME, in a 1×10 -6 mol / L luteolin solution. The potential scanning range of the CV method was -0.5V to 0.7V, and the scanning rate was 0.1V / s; the potential range of the DPV method was -0.1 to 0.7V, the amplitude was 0.05V, the pulse width was 0.1s, the pulse time was 0.2s, and the inhibition time was 2s. Figure 2 (b) shows the DPV diagrams of the three electrodes. It can be seen that on the surface of CFME, the oxidation peak of luteolin appears at about 0.25V, and the peak current is 0.481nA; while on AuNPs / CFME, the oxidation peak potential is slightly negatively shifted, and the peak current increases to 2.800nA, about 5.8 times that of CFME; on Nafion-AuNPs / CFME, the peak current further increases to 3.380nA, about 7 times that of CFME. Figure 2 (c) shows the CV diagrams of the three electrodes. It can be seen that the reduction peak current of luteolin on different electrodes also shows the same increasing trend, indicating that Nafion-AuNPs / CFME has a larger electrode active area and stronger charge transfer ability, and has a more significant redox catalytic effect on luteolin than AuNPs / CFME and CFME. This result is consistent with the results of the electrochemical impedance spectroscopy (EIS) experiment.

[0067] Example 2

[0068] In this example, on the basis of Example 1, the effects of electrodeposition time, scanning rate, and pH value on Nafion-AuNPs / CFME in the detection of luteolin were further studied.

[0069] 1. Electrodeposition time

[0070] After immersing the tip of the clean CFME in the Nafion solution for 5 minutes, it was placed in 10 mL of AuNPs dispersion solution. At the initial potential of +1.5V, the CFME was electrodeposited by the potentiostatic deposition method for 5, 10, 15, 20, and 25 minutes (n = 3) to obtain Nafion-AuNPs / CFME with different AuNPs loadings. The oxidation peak current response of the Nafion-AuNPs / CFME electrode in a 1×10 -6 mol / L luteolin solution was tested by the DPV method to determine the optimal electrodeposition time. The results are as Figure 3As shown, within 0 - 15 min, the oxidation peak current of Nafion - AuNPs / CFME increases with the increase of the electrodeposition time. However, after exceeding 15 min, the oxidation peak current begins to decline. This is because the deposition amount of AuNPs on the surface of the carbon fiber microelectrode reaches saturation, resulting in the shedding of the surface modification layer and a decrease in the electrocatalytic activity. Thus, it can be seen that when the deposition time of AuNPs is 10 - 25 min, the oxidation peak current of Nafion - AuNPs / CFME remains relatively high. When the deposition time of AuNPs is 15 min, the oxidation peak current of Nafion - AuNPs / CFME is the best. Subsequent experiments were all carried out at the optimal electrodeposition time of 15 min.

[0071] 2. Scanning Rate

[0072] The CV method was used to study the electrochemical behavior of Nafion - AuNPs / CFME at different scanning rates (50 - 500 mV / s) to reveal the reaction mechanism of quercetin on the modified electrode. As Figure 4 (a) shows, the oxidation - reduction peak current (I p ) of quercetin shows an increasing trend with the increase of the scanning rate (v). At the same time, the oxidation peak potential (E pa ) shifts positively with the increase of the scanning rate, while the reduction peak potential (E pc ) gradually shifts negatively. This indicates that the oxidation - reduction reaction of quercetin occurring on the surface of Nafion - AuNPs / CFME is a quasi - reversible process. Figure 4 (b) shows that within the range of 50 - 500 mV / s, the oxidation peak current (I pa ) and reduction peak current (I pc ) of quercetin show a good linear relationship with the scanning rate (v). Their linear regression equations are respectively:

[0073] I pa (nA) = - 0.0123v(mV / s) - 1.1573, R 2 = 0.999;

[0074] I pc (nA) = 0.0109v(mV / s) + 0.3369, R 2 = 0.999;

[0075] This linear relationship indicates that the oxidation - reduction reaction of quercetin on the Nafion - AuNPs / CFME electrode is adsorption - controlled. In addition, Figure 4 (c) shows that there is also a linear relationship between the oxidation peak potential (E pa ) and the reduction peak potential (E pc ) and the logarithm of the scanning rate (lgv). The corresponding linear regression equations are respectively:

[0076] E pa (mV)=0.0595lgv + 0.2497, R 2 =0.997;

[0077] E pc (mV)= -0.0618lgv + 0.3738, R 2 =0.998;

[0078] Combined with the following Laviron's equation, the electron transfer coefficient (α) and the number of electron transfers (n) can be calculated:

[0079]

[0080] where E θ ' is the redox potential, the value of the ideal gas constant R is 8.314 J·mol -1 ·K -1 , the value of the Faraday constant F is 96485 C·mol -1 , and the value of the thermodynamic temperature T is 298 K. The calculated electron transfer coefficient α is 0.49, and the number of electron transfers n is 1.9 ≈ 2, indicating that the redox process of quercetin on the electrode involves the transfer of two electrons. It can be seen that due to the too fast scanning rate, the baseline noise will increase and the reversibility of the chemical reaction will decrease. The scanning rate can be selected from 50 to 150 mV / s, preferably 100 mV / s. The scanning rate of 100 mV / s is used in subsequent experiments.

[0081] 3. pH value

[0082] Prepare a PBS solution with a pH value of 2.0 - 7.0 containing 1×10 -6 mol / L quercetin, and use the DPV method to study the electrochemical response of quercetin on Nafion - AuNPs / CFME in PBS solutions with different pH values. As Figure 5 (a) shows, the oxidation peak potential gradually shifts negatively with the increase of the pH value, indicating that the reaction of quercetin on the Nafion - AuNPs / CFME electrode involves the participation of protons, and its oxidation process is a deprotonation reaction. At the same time, the oxidation - reduction peak current of quercetin gradually decreases with the increase of the pH value. Therefore, the pH value of the PBS solution used to prepare the quercetin sample solution to be measured can be 3.0 - 4.0, preferably 3.0. The PBS solution with a pH value of 3.0 is used in subsequent experiments.

[0083] Figure 5 (b) shows that there is a linear relationship between the oxidation peak potential (E p ) of quercetin and the pH value, and the regression equation is:

[0084] Ep (mV)=0.0610pH + 0.4408, R 2 =0.999。

[0085] This slope is close to the theoretical value of 0.0592 calculated by the Nernst equation, indicating that during the oxidation process of quercetin, the ratio of the number of electrons to the number of protons participating in the reaction is 1:1. According to the previously obtained electron transfer number n of quercetin as 2, it can be speculated that on the Nafion-AuNPs / CFME electrode, a two-proton two-electron transfer process occurs for quercetin, and its oxidation mechanism is as follows:

[0086]

[0087] Example 3

[0088] This example further studies the application of an electrochemical sensor using Nafion-AuNPs / CFME as the working electrode in the detection of quercetin.

[0089] Electrochemical detection was carried out on a CHI660D electrochemical workstation (Chenhua, China). The working electrode Nafion-AuNPs / CFME of the electrochemical sensor and the reference electrode Ag / AgCl were used to form a two-electrode system. Among them, for the CV method, the current was recorded at a scanning rate of 0.1 V / s in the range of -0.5 V to 0.7 V; for the DPV method, the current was recorded at an amplitude of 0.05 V, a pulse width of 0.1 s, and a pulse period of 0.2 s in the range of -0.1 V to 0.7 V.

[0090] 1. Linear range and detection limit of Nafion-AuNPs / CFME for detecting quercetin

[0091] The DPV method was used to investigate the electrochemical response of Nafion-AuNPs / CFME to quercetin at different concentrations under the optimal experimental conditions (electrodeposition time 15 min, pH 3.0, scan rate v = 100 mV / s). As Figure 6 (a) shows, as the concentration of quercetin increases, the peak current also gradually increases. And as can be seen from 6(b), the peak current shows a good linear relationship with the quercetin concentration in the ranges of 0.1 - 10 μmol / L and 0.01 - 0.1 μmol / L. By plotting a linear standard curve, a linear regression equation of concentration-current was established. When the quercetin concentration is in the range of 0.1 - 10 μmol / L, the linear regression equation of concentration-current is:

[0092] I p (nA)=0.2664c(μmol / L)+0.6500, R 2 =0.996;

[0093] When the luteolin concentration is in the range of 0.01 - 0.1 μmol / L, the linear regression equation of concentration - current is:

[0094] I p (nA) = 1.3587c(μmol / L) + 0.4211, R 2 = 0.998,

[0095] The detection limit (LOD, S / N = 3) is 0.078 μmol / L. According to the regression equation in the range of 0.01 - 0.1 μmol / L, the quantification limit is 0.009 μmol / L. Comparing the Nafion - AuNPs / CFME of this example with other electrodes, the results are shown in Table 1. It can be seen that the Nafion - AuNPs / CFME shows a lower detection limit (LOD) and a wider linear range (Linear range) compared with electrodes such as 2 - MES SAM / Au.

[0096] Table 1 Comparison of different modified electrodes for the detection of luteolin

[0097]

[0098]

[0099] 2. Stability and anti - interference analysis

[0100] To investigate the anti - interference ability of Nafion - AuNPs / CFME, glucose (GLU), Na -6 , K + , Fe + , Fe 2+ , Fe 3+ were added to a 1×10 -3 mol / L luteolin solution to make the concentration of interfering substances 1×10 Figure 7 (a) shows that the addition of interfering substances GLU, Na + , K + , Fe 2+ , Fe 3+ did not have a significant impact on the response signal of luteolin (RSD < 5.0%), indicating that the Nafion - AuNPs / CFME of the present invention has good anti - interference ability.

[0101] Take a newly prepared Nafion - AuNPs / CFME for electrode stability testing. Use the DPV method to detect a 1 μmol / L luteolin solution in parallel 10 times. The results are as shown in Figure 7 (b). The oxidation peak currents of luteolin are almost coincident. Use this modified electrode to conduct the same experiment continuously for 10 days. The results are asFigure 7 (c) As shown, the oxidation peak current of fisetin still did not change significantly, indicating that the Nafion-AuNPs / CFME of the present invention has good electrochemical stability.

[0102] 3. Detection of spike recovery of actual samples

[0103] To investigate the detection feasibility of Nafion-AuNPs / CFME for actual samples, the content of fisetin in actual samples was determined using Nafion-AuNPs / CFME, and spike recovery detection was carried out.

[0104] Pretreatment of actual sample Cotinus coggygria Scop. leaves: After washing and drying the Cotinus coggygria Scop. leaves, they were placed in a mortar and ground into fine powder. Exactly 1.000 g of fine powder was weighed, 10 mL of absolute ethanol was added for dissolution, and after ultrasonic oscillation for 20 min, centrifugation was carried out at a speed of 3000 r · min -1 for 15 minutes, and the supernatant was taken to obtain a sample solution for standby.

[0105] Preparation of sample to be measured: 10 μL of Cotinus coggygria Scop. leaf sample solution was diluted to 10 mL with PBS buffer solution at pH 3.0 to obtain a sample to be measured.

[0106] The concentration of fisetin in the sample to be measured was detected by DPV method using Nafion-AuNPs / CFME, the response current was recorded, and it was brought into the linear regression equation of concentration-current I p (nA)=0.2664c (μmol / L)+0.6500, R 2 =0.996, and the concentration of fisetin in the diluted Cotinus coggygria Scop. leaf solution was calculated to be 4.097 μmol / L. The content of fisetin detected by Nafion-AuNPs / CFME in Cotinus coggygria Scop. leaves was 1.173% (n = 3, RSD < 5%). Spike recovery of fisetin in the Cotinus coggygria Scop. leaf solution was carried out, and the recovery rate was 97.61% - 99.22%.

[0107] Table 2 Detection of the content of fisetin in Cotinus coggygria Scop. leaves

[0108]

[0109] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An electrochemical sensor for detecting fisetin, comprising a working electrode, characterized in that: The working electrode is Nafion-AuNPs / CFME, and the Nafion-AuNPs / CFME comprises CFME and a Nafion layer and an AuNPs layer sequentially modified on the surface of the CFME.

2. The electrochemical sensor according to claim 1, characterized in that The preparation method of Nafion-AuNPs / CFME is as follows: firstly immersing CFME in Nafion solution, taking it out and drying it to obtain CFME with a surface modified with Nafion layer; then modifying the AuNPs layer on the surface of the CFME with the surface modified with Nafion layer by constant potential deposition method, and obtaining Nafion-AuNPs / CFME after washing and drying.

3. The electrochemical sensor according to claim 2, characterized in that: The CFME is immersed in the Nafion solution for 4 to 6 minutes.

4. The electrochemical sensor according to claim 2, characterized in that The constant potential deposition method is as follows: CFME with a surface modified Nafion layer is used as a working electrode and immersed in an AuNPs solution together with a reference electrode, and the electrodeposition is performed at a voltage of +1.4 to 1.8 V for 10 to 25 minutes.

5. The electrochemical sensor according to claim 1, characterized in that: The electrochemical sensor also includes a reference electrode.

6. An electrochemical method for detecting fisetin, characterized in that: The electrochemical sensor according to any one of claims 1 to 5 is used to detect the sample to be tested by cyclic voltammetry or differential pulse voltammetry, the response current is recorded, and then the concentration of fisetin in the sample to be tested is calculated according to the concentration-current linear regression equation.

7. The electrochemical method according to claim 6, characterized in that: When the concentration of fisetin is in the range of 0.1 to 10 μmol / L, the linear regression equation of concentration-current is I p (nA)=0.2664c(μmol / L)+0.6500, R 2 =0.996; when the concentration of fisetin is in the range of 0.01-0.1 μmol / L, the linear regression equation of concentration-current is I p (nA)=1.3587c(μmol / L)+0.4211, R 2 =0.998; where c is the concentration of fisetin, I p For current.

8. The electrochemical method according to claim 6, characterized in that: When using cyclic voltammetry, the potential range is -0.5V~0.7V, and the scan rate is 0.05~0.15V / s; when using differential pulse voltammetry, the potential range is -0.1~0.7V, the amplitude is 0.05V, the pulse width is 0.1s, the pulse time is 0.2s, and the inhibition time is 2s.

9. The electrochemical method according to claim 6, characterized in that The preparation method of the sample to be tested is as follows: firstly, the actual sample is cleaned and dried, then ground into fine powder in a mortar, then anhydrous ethanol is added to the fine powder to dissolve, ultrasonically vibrated and centrifuged, then the supernatant is taken to obtain a sample solution, and then the sample solution is diluted with a buffer solution to obtain the sample to be tested.

10. The electrochemical method according to claim 9, characterized in that: The buffer solution is PBS buffer solution with a pH value of 3.0-4.0.

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