An electrochemical sensor and a preparation method and application thereof

Abstract

The application belongs to the technical field of electrochemical detection, and specifically provides an electrochemical sensor, which comprises a working electrode, the working electrode comprises a carbon fiber microelectrode and nanoparticles deposited on the surface of the carbon fiber microelectrode; the nanoparticles comprise nanoruthenium particles and nanogold particles. The electrochemical sensor utilizes the synergistic electrocatalysis of the bimetallic nanoparticles, and exhibits excellent sensitivity, stability and anti-interference performance for detecting ACOP, PAP and PNP. The electrochemical sensor can be successfully applied to the electrochemical detection of ACOP in human serum samples, and the recovery rate reaches 95.9-100.5%, which is expected to provide a solution for the quantitative analysis of ACOP and has great application potential in pharmacokinetics and pharmacology research. The application also provides a method for simultaneously or individually detecting ACOP, PAP and PNP, which exhibits sensitive voltammetric response to ACOP, PAP and PNP, wide linear range, and the detection limits are 6.192 nmol / L, 0.13 micromol / L and 0.05 micromol / L (S / N=3) respectively, and the method has high sensitivity, high selectivity and reliability.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical detection, and in particular relates to an electrochemical sensor and a preparation method and application thereof. Background Art

[0002] Acetaminophen (ACOP) has long been used as an antipyretic and analgesic, commonly used to treat fever and relieve mild to moderate pain caused by headaches, arthralgias, neuralgia, and myalgias. The synthesis of ACOP contains two major impurities: p-nitrophenol (PNP) and p-aminophenol (PAP). PNP, the raw material for its synthesis, is a highly hazardous phenolic compound that is known to cause cancer and mutagenesis. Even at very low concentrations, it is highly toxic to humans and the environment and can survive in the environment for extended periods without degradation. PAP, a synthetic intermediate or hydrolytic metabolite of ACOP, exhibits significant nephrotoxicity and teratogenicity. The Food and Drug Administration (FDA) has established an upper limit of 50 ppm for PAP in ACOP. In recent decades, various methods have been developed for the detection of ACOP, including spectrophotometry, chromatography, and chemiluminescence. However, most techniques are time-consuming and require complex pretreatment procedures. Electrochemical analysis, however, offers the advantages of simple preparation, ease of operation, rapid response, and significant sensitivity. However, so far, little research has been devoted to the simultaneous voltammetric determination of ACOPs and their synthetic impurities PAP and PNP in pharmaceutical preparations or biological samples. Therefore, establishing a low-cost and cost-effective electrochemical measurement for the simultaneous detection of multiple components remains a great challenge.

[0003] Carbon fiber microelectrodes (CFMEs) have the advantages of large specific surface area, good biocompatibility, excellent electron transfer performance, and extremely small size. They are widely used to detect neurotransmitters, metal ions, and monitor neurochemical substances in vivo. However, the electrochemical performance of CFMEs is extremely dependent on appropriate sensing surface modification. Gold nanoparticles have attracted much attention in the construction of new electrochemical detectors due to their advantages such as simple preparation, excellent electrocatalytic activity, large specific surface area, and excellent biocompatibility. Ruthenium nanoparticles have good conductivity and can serve as a medium for rapid electron transfer on the electrode surface, thus giving the sensor excellent detection sensitivity. Studies have shown that some possible synergistic effects of bimetallic nanoparticles provide new perspectives for improving electrocatalytic performance. For example, Au-Cu bimetallic nanoparticles show excellent anti-interference performance in arsenic (III) detection.

[0004] For this reason, Figure 1As shown, the present invention synthesizes ruthenium nanoparticles (RuNPs) and gold nanoparticles (AuNPs) and modifies them on the CFME surface to develop a potential electrochemical sensor. Thanks to the synergistic effect between RuNPs and AuNPs, the sensor has high selectivity and sensitivity for the simultaneous detection of ACOP, PAP, and PNP. Summary of the Invention

[0005] The object of the present invention is to provide an electrochemical sensor that can detect ACOP, PAP and PNP simultaneously or individually.

[0006] To this end, the present invention provides an electrochemical sensor, comprising a working electrode, wherein the working electrode comprises a carbon fiber microelectrode and nanoparticles deposited on the surface of the carbon fiber microelectrode; the nanoparticles comprise nanoruthenium particles and nanogold particles.

[0007] The present invention also provides a method for preparing the electrochemical sensor, comprising the following steps:

[0008] (1) preparing ruthenium nanoparticle colloid and gold nanoparticle colloid for later use;

[0009] (2) After washing the carbon fiber microelectrode, electrophoresis is performed in a colloid of nano-ruthenium particles to deposit nano-ruthenium particles on the electrode surface;

[0010] (3) The prepared ruthenium nanoparticles / carbon fiber microelectrode are thoroughly rinsed and electrophoretic deposition is continued in the gold nanoparticle colloid to obtain a gold nanoparticle / ruthenium nanoparticle / carbon fiber microelectrode (AuNPs / RuNPs / CFME) electrochemical sensor.

[0011] Specifically, in the above step (2), electrophoretic deposition is performed in the nano-ruthenium particle colloid at +1.4 to 1.8 V for 10 to 20 minutes.

[0012] Specifically, in the above step (3), electrophoretic deposition is performed in the nano-gold particle colloid at +1.4 to 1.8 V for 10 to 25 minutes.

[0013] The present invention also provides a method for simultaneously or individually detecting acetaminophen, p-aminophenol, and p-nitrophenol using the electrochemical sensor, comprising the following steps:

[0014] (1) Prepare standard solutions of acetaminophen, p-aminophenol, and p-nitrophenol at different concentrations using buffer;

[0015] (2) The working electrode and reference electrode of the electrochemical sensor constitute a two-electrode system. Using an electrochemical measurement method, the working electrode is immersed in each standard solution for incubation, and the corresponding current value is recorded. The concentration-current value standard curves of acetaminophen, p-aminophenol, and p-nitrophenol are drawn using the standard solution concentration and current value respectively;

[0016] (3) adding a buffer solution to the pre-treated sample to dilute it, detecting the current value of the sample using the electrochemical measurement method in step (2), and calculating the contents of acetaminophen, p-aminophenol, and p-nitrophenol in the sample according to a concentration-current value standard curve.

[0017] Specifically, the buffer solution is 0.05-0.2 mol / L PBS buffer solution with a pH of 4.0-7.0.

[0018] Specifically, the reference electrode is an Ag / AgCl electrode.

[0019] Specifically, the electrochemical measurement methods include cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry.

[0020] The cyclic voltammetry method recorded CV signals at a scan rate of 0.1 V / s between 0 and 1.0 V. The differential pulse voltammetry method was performed at a pulse amplitude of 0.1 V and a potential step of 0.005 V, using a potential range of -0.2 to 1.2 V. The electrochemical impedance spectroscopy method collected EIS measurements in a standard potassium ferricyanide solution at a frequency of 0.1 to 100 kHz. z , the open circuit potential is +0.20 V, and the voltage amplitude is 10 mV.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] The electrochemical sensor provided by the present invention utilizes the synergistic electrocatalytic effect of bimetallic nanoparticles to demonstrate excellent sensitivity, stability, and anti-interference performance for the detection of ACOP, PAP, and PNP. The electrochemical sensor can be successfully applied to the electrochemical detection of ACOP in human serum samples, with a recovery rate of 95.9-100.5%. It is expected to provide a solution for the quantitative analysis of ACOP and has great application potential in pharmacokinetic and pharmacological research. The method provided by the present invention exhibits a sensitive voltammetric response to ACOP, PAP, and PNP, with a wide linear range and detection limits of 6.192 nmol / L, 0.13 μmol / L, and 0.05 μmol / L (S / N = 3), respectively, demonstrating high sensitivity, high selectivity, and reliability.

[0023] The present invention will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of CFME (A), preparation of AuNPs and RuNPs (B), and representative scheme of electrochemical application (C).

[0025] Figure 2HRTEM micrographs of RuNPs (A, B) and AuNPs (C, D) at different magnifications; particle size distribution of RuNPs (E) and AuNPs (F); EDS spectra of RuNPs (G) and AuNPs (H) (the insets show the mass and atomic % distribution of various elements); elemental mapping of RuNPs (I) and AuNPs (J); SEM micrographs of CFME (K) and AuNPs / RuNPs / CFME (L).

[0026] Figure 3 The graph shows the DPV measurement results of 10 μmol / L ACOP solution containing 0.1 mol / L PBS, Tris-HCl and BR buffer using CFME and AuNPs / RuNPs / CFME electrodes, respectively.

[0027] Figure 4 (A) Four different electrodes in 10mmol / L[Fe(CN)6] 3- / 4- CV curves in solution; (B) CV curves of four different electrodes in 10mmol / L[Fe(CN)6] 3- / 4- The square root linear curve of peak current and scan rate in solution; (C) Four different electrodes in 10mmol / L[Fe(CN)6] 3- / 4- Nyquist plot in solution.

[0028] Figure 5 (A) CV curves of CFME, RuNPs / CFME, AuNPs / CFME and AuNPs / RuNPs / CFME in 10 μmol / LACOP; (B) DPV curves of four different electrodes in 10 μmol / LPAP, ACOP and PNP; (C) Effect of RuNPs and AuNPs deposition time on the oxidation peak current of 10 μmol / LACOP; (D) CV diagram of 10 μmol / LACOP on AuNPs / RuNPs / CFME; (E) Relationship curve between 10 μmol / LACOP current and square root of scan rate; (F) Calibration curve between logarithm of 10 μmol / LACOP current and logarithm of scan rate; (G) Linear relationship between potential and logarithm of scan rate for 10 μmol / LACOP; (H) CV curves of 10 μmol / LACOP in PBS solutions of different pH values; (I) Relationship between peak potential and peak current of 10 μmol / LACOP and pH of PBS solution.

[0029] Figure 6 Schematic diagram of the oxidation mechanism of ACOP.

[0030] Figure 7 (A) 15 consecutive DPV scans of AuNPs / RuNPs / CFME in 10 μmol / L ACOP (the inset is a local magnified view of the DPV curve); (B) 15 consecutive DPV scans of peak current of AuNPs / RuNPs / CFME in 10 μmol / L ACOP; (C) DPV response of AuNPs / RuNPs / CFME scanned continuously for 10 days in 10 μmol / L ACOP.

[0031] Figure 8 (AC) DPV curves of different concentrations of ACOP on AuNPs / RuNPs / CFME; (D) Linear relationship between peak current and ACOP concentration.

[0032] Figure 9 (AB) DPV curves of different concentrations of ACOP (PAP and PNP concentrations were fixed at 10 μmol / L); (C) Relationship between peak current and ACOP concentration; (D) DPV curves of different concentrations of PAP (containing 100 μmol / L ACOP and 10 μmol / L PNP); (E) DPV curves of different concentrations of PNP (containing 100 μmol / L ACOP and 10 μmol / L PAP); (F) DPV curves of different concentrations of ACOP, PAP, and PNP; Relationship between peak current and PAP (G) and PNP (H) concentrations; (I) Relationship between peak current and ACOP, PAP, and PNP concentrations. DETAILED DESCRIPTION

[0033] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0034] The electrochemical sensor of the present invention and the effects of its application are studied below through specific examples.

[0035] Example 1:

[0036] This embodiment provides an electrochemical sensor, which is prepared by the following method.

[0037] Acetaminophen, p-aminophenol, p-nitrophenol, and uric acid (UA) used in the examples of the present invention were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); ruthenium trichloride (RuCl3·3H2O), chloroauric acid (HAuCl4·3H2O), and glucose (GLU) were ordered from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); human serum was purchased from Shanghai Xinfan Biotechnology Co., Ltd. (Shanghai, China); and carbon fiber was purchased from Jilin Shenzhou Carbon Fiber Co., Ltd. (Jilin, China).

[0038] 1. Preparation of RuNPs by reducing RuCl3 with sodium triphosphate

[0039] Under vigorous stirring, 1% (w / v) RuCl3 solution (0.9 mL) was dissolved in 40 mL of boiling deionized water. Subsequently, 1% (w / v) trisodium citrate (1 mL) was quickly added and refluxed for 4 hours. The mixture was stirred continuously at 80°C ± 0.5°C until it turned light brown. After cooling, the resulting RuNPs colloid was stored in a refrigerator at 4°C.

[0040] 2. Synthesis of AuNPs by reducing HAuCl4 with sodium triphosphate

[0041] A 25.4 mmol / L HAuCl4 solution (0.5 mL) was brought to a boil with vigorous stirring, followed by the addition of 0.9 mL of 1% (w / v) trisodium citrate. The mixture was continuously stirred and boiled for 40 minutes, during which the colloid color was observed to change to wine red. Finally, the prepared AuNPs colloid was stored in a refrigerator at 4°C until use.

[0042] 3. Preparation of AuNPs / RuNPs / CFME

[0043] CFME was prepared using existing techniques. After washing the CFME with ethanol and then doubly distilled water, RuNPs were successfully deposited on the electrode surface by electrophoretic deposition (EPD) in a RuNPs colloid at +1.5V for 15 minutes. The prepared RuNPs / CFME was then thoroughly rinsed with doubly distilled water. After removal, electrophoretic deposition was continued in an AuNPs colloid at +1.5V for 10 minutes to produce an AuNPs / RuNPs / CFME electrochemical sensor.

[0044] 4. Characteristics of modified electrodes

[0045] Scanning electron microscopy (SEM) (SU8010, Hitachi, Japan) and high-resolution transmission electron microscopy (HRTEM) (Thermo Fisher Scientific, Czech Republic) were used to investigate the morphology of the modified materials and electrodes. Figure 2 TEM samples were prepared by dropping RuNPs or AuNPs colloids onto carbon-coated copper microscope grids.

[0046] Prepared RuNPs( Figure 2 A, B) and AuNPs( Figure 2 The morphology of (C, D) was characterized by HRTEM at different magnifications. As shown in the figure, RuNPs and AuNPs particles are evenly distributed and appear as uniform spheres. The particle size distribution histogram obtained from HRTEM ( Figure 2 E, F) The particle sizes of RuNPs and AuNPs were observed to be 2.05±0.3nm and 11.5±0.7nm, respectively. Figure 2 GJ) were subjected to elemental analysis, and the results showed that RuNPs and AuNPs were successfully synthesized.

[0047] The morphological characteristics of the CFME and AuNPs / RuNPs / CFME electrode surfaces were analyzed by SEM. Figure 2 As shown in Figure 4, the surface of CFME is smooth and flat, and the diameter of the carbon fibers is only about 7 μm. However, the surface of AuNPs / RuNPs / CFME is obviously different. Figure 2 As shown in Figure 5, many Ru and Au nanoparticles are bound on the surface of CFME with slight accumulation, indicating that the Ru and Au nanoparticles significantly increase the specific surface area of ​​the sensor.

[0048] Example 2:

[0049] In electrochemical analysis, the supporting electrolyte affects the thermodynamics, kinetics, and charge transfer rate of the electrode surface reaction. This example uses the electrochemical sensor provided in Example 1 to conduct experiments and study different buffer systems to determine the optimal buffer supporting electrolyte.

[0050] Electrochemical measurements were performed on a CHI660D electrochemical workstation (Chenhua, China). The two-electrode system consisted of two parts: AuNPs / RuNPs / CFME as the working electrode (WE) and Ag / AgCl electrode as the reference electrode (RE). The electrochemical measurement methods mainly included cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Among them, cyclic voltammetry recorded CV signals at a scan rate of 0.1 V / s in the range of 0-1.0 V. DPV experiments were performed at a pulse amplitude of 0.1 V and a potential step of 0.005 V, and the potential range used was -0.2-1.2 V. EIS measurements were collected in a standard potassium ferricyanide solution, and the frequency was set to 0.1-100 kHz. z , the open circuit potential is +0.20 V, and the voltage amplitude is 10 mV.

[0051] 0.1 mol / L phosphate buffer solution (PBS), Tris hydrochloride (Tris-HCl), and Britton-Robinson (BR) were used as supporting electrolytes, and each electrochemical experiment was repeated three times.

[0052] Several ACOP solutions with a concentration of 10 μmol / L were analyzed by CFME and AuNPs / RuNPs / CFME using DPV. The solutions were prepared with 0.1 mol / L PBS, Tris-HCl and BR buffer as supporting electrolytes. The results are shown in Figure 2. Figure 3 shown.

[0053] from Figure 3 It was observed that in the PBS buffer system, CFME and AuNPs / RuNPs / CFME had obvious DPV responses in ACOP solution, and the peak current of AuNPs / RNPs / CFME was 3.027 times that of bare CFME. Therefore, PBS buffer solution (0.1 mol / L) was preferred as the optimal electrolyte for electrochemical measurements.

[0054] Example 3:

[0055] Based on Example 2, this example further studies the electrochemical properties of the modified material, the electrochemical performance and condition optimization of the modified electrode, and the stability and anti-interference performance of the modified electrode.

[0056] 1. Study on the electrochemical properties of modified materials

[0057] At a concentration of 10 mmol / L[Fe(CN)6] 3- / 4- CV scans were performed on CFME modified with RuNPs, AuNPs and AuNPs / RuNPs in a solution (containing 0.1 mol / L KCl) to compare the electrochemical performance of CFME functionalized with different NPs ( Figure 4 A). As can be seen from the figure, the redox peak of the bare CFME electrode is weak, and the CV curve of the bare CFME shows a pair of redox peaks. The oxidation peak current (I pa ) is 2.193nA, and the peak potential difference (ΔE p ) is 0.38 V. Compared with bare CFME, the peak current generated by AuNPs / RuNPs / CFME is significantly increased, I pa Increased to 6.520nA, ΔE p The results show that the charge transfer efficiency of CFME is significantly improved after modification with Au-Ru nanoparticles. To examine the electroactive area of ​​the electrode before and after modification, the Figure 4 The oxidation peak current (I pa ) and the square root of the scan rate ( Figure 4 B). The electroactive surface area can be calculated using the Randles-Sevcik equation (3.1):

[0058] I pa =(2.69×10 5 )AD 1 / 2 n 3 / 2 ν 1 / 2 C(3.1)

[0059] Among them I pa is the oxidation peak current, n is the number of transferred electrons, and D is [Fe(CN)6] 3- / 4- Diffusion coefficient (cm 2 s -1 ), ν is the scan rate (Vs -1 ), C is [Fe(CN)6] 3- / 4- The results show that the electroactive surface areas of CFME, RuNPs / CFME, AuNPs / CFME and AuNPs / RuNPs / CFME are 0.315, 0.325, 0.418 and 0.431 cm, respectively. 2 .

[0060] EIS is a method to measure the electrochemical surface properties of the electrolyte interface of synthetic materials. Figure 4 C shows the Nyquist plots of CFME, RuNPs / CFME, AuNPs / CFME, and AuNPs / RuNPs / CFME in 10 mmol / L potassium ferrocyanide / potassium ferrocyanide containing 0.1 mol / L potassium chloride. The frequency in the experiment was 0.1-100 kHz. z , the voltage amplitude is 10mV. From the figure, we can see that there is an obvious semicircle in the Nyquist diagram of CFME, and the charge transfer resistance (R ct ) is approximately 8.46×10 4 Ω. After modification, the R ct are 7.078×10 4 Ω、5.105×10 4 Ω and 4.417×10 4 Ω. R ct The decrease of and the increase of A indicate that the electron transfer efficiency and electroactive area of ​​Ru-Au bimetallic nanoparticles are enhanced. ct The standard exchange current density (I0) and heterogeneous rate constant (K0) can be calculated using equations (3.2, 3.3).

[0061] I0=RT / nFR ct(3.2)

[0062] K0=RT / F 2 R ct AC(3.3)

[0063] Where I0, R, T, A, F, n, C and K0 represent the standard exchange current density, universal gas constant (8.314 Jmol -1 K -1 ), temperature (25°C), electroactive surface area, Faraday constant (96485Cmol -1 ), electron transfer number, [Fe(CN)6] 3- / 4- The results showed that the standard exchange current density I0 (58.14 nA cm) of AuNPs / RuNPs / CFME was 1.584 nm. -2 ) is significantly larger than the I0 of CFME (30.35nAcm -2 In addition, the K0 values ​​of CFME, RuNPs / CFME, AuNPs / CFME, and AuNPs / RuNPs / CFME were 3.146×10 -7 cms -1 , 3.760×10 -7 cms -1 , 5.213×10 -7 cms -1 and 6.025×10 -7 cms -1 This indicates that the Au-Ru nanoparticles modified on the CFME surface have good electrocatalytic activity.

[0064] 2. Electrochemical performance of modified electrodes and optimization of conditions

[0065] In PBS buffer solution with pH = 4.0, the CV curves of four different electrodes in 10 μmol / LACOP are shown in Figure 2. Figure 5 As shown in Figure 5A, a peak current around 0.4 V was obtained on the CFME electrode, which is attributed to the oxidation of ACOP (5A-b). The oxidation peak current responses on RuNPs / CFME (5A-c) and AuNPs / CFME (5A-d) increased compared to that of CFME. The composite modified electrode AuNPs / RuNPs / CFME exhibited the best current response to ACOP (5A-e) but showed no significant response to the supporting electrolyte (5A-a). Figure 5Figure B shows the DPV curves of 10 μmol / L ACOP at different electrodes in the presence of the same concentrations of PAP and PNP. The peak currents of ACOP, PAP, and PNP on CFME were approximately 1.036 nA, 1.857 nA, and 1.370 nA, respectively (5B-b). Compared with CFME, the peak currents on RuNPs / CFME increased to 3.877 nA, 6.176 nA, and 4.623 nA, respectively (5B-c). Compared with RuNPs / CFME (5B-d), AuNPs / RuNPs / CFME further improved the electrochemical responses of the three analytes, with significant increases in peak currents of 67.1%, 456.8%, and 111.7%, respectively (5B-e). These results demonstrate that the AuNPs / RuNPs composite nanomaterial possesses a large surface area and excellent electrical conductivity, significantly enhancing the enrichment effect of ACOP and the electron transfer rate.

[0066] The effect of electrodeposition time on the oxidation peak current of 10 μmol / L ACOP was investigated by DPV method in 0.1 mol / L PBS (pH 4.0) (n=3). Figure 5 As shown in Figure C, as the electrodeposition time of the RuNPs colloid increases, the DPV response begins to rise until the current reaches a peak, and then the current decreases. The oxidation peak current of the RuNPs-modified CFME reaches its peak when the electrodeposition time is 15 minutes. Furthermore, the maximum oxidation peak current is achieved when the AuNPs are deposited on the RuNPs / CFME for 10 minutes. Therefore, the optimal deposition time for RuNPs is 15 minutes, and for AuNPs is 10 minutes.

[0067] At the optimal deposition time, the peak current and scan rate of AuNPs / RuNPs / CFME in 10 μmol / L ACOP were investigated by CV method in 0.1 mol / L PBS (pH 4.0). Figure 5 As shown in D. As the scan rate increases, the oxidation peak current also increases accordingly, and the peak potential moves toward the positive potential direction. -1 In the range of Figure 5 E shows that the linear regression equation I pa =0.4096ν 1 / 2 +0.3154(R 2 =0.9901), indicating that the redox reaction of ACOP on the AuNPs / RuNPs / CFME surface is diffusion controlled. In addition, there is a good linear relationship between the logarithm of the scan rate and the logarithm of the peak current ( Figure 5F), the linear equation slope is 0.4702, close to the theoretical value of 0.5 for a pure diffusion process. Considering that a fast scan rate may lead to an increase in charge current and enhanced baseline noise, subsequent CV analysis will be performed at a scan rate of 0.1 V / s.

[0068] In addition, the logarithm of the scan rate and the peak potential showed a good linear relationship ( Figure 5 G), and the linear equation E pa (V) = 0.0507logν + 0.2384(R 2 =0.9912). The number of electrons transferred during ACOP oxidation is calculated according to the following Lavron equation (3.4, 3.5):

[0069] E pa =E θ '+(2.303RT / αnF)log(RTK0 / αnF)+(2.303RT / αnF)logν(3.4)

[0070] Slope=2.303RT / αnF(3.5)

[0071] Among them E θ ', R, T, α, F, K0, and n represent the standard electrode potential, gas constant, Kelvin temperature, electron transfer coefficient, Faraday constant, heterogeneous rate constant, and number of transferred electrons, respectively. Typically, α is assumed to be 0.5 in irreversible electrode processes. Since the slope of the linear plot is 0.0507, the value of αn is estimated to be 1.17, so n is approximately 2. The surface concentration or surface coverage of ACOP before and after electrode modification (Γ ACOP ) can be estimated by formula (3.6),

[0072] Γ ACOP =Q / nFA(3.6)

[0073] Where Q is the charge transferred by electrochemical reaction, and other symbols have conventional meanings. Calculate Γ for different electrodes ACOP The values ​​are 3.29×10 -5 molcm -2 (CFME), 3.19×10 -5 molcm -2 (RuNPs / CFME), 2.48×10 -5 molcm -2 (AuNPs / CFME) and 2.40×10 -5 molcm -2(AuNPs / RuNPs / CFME). This indicates that modifying the CFME surface with AuNPs and RuNPs increases the specific surface area and electron transfer rate of the electrode, thereby promoting the redox kinetics of ACOP.

[0074] In order to study the effect of the pH value of the background electrolyte (0.1 mol / L PBS) on the oxidation peak current of 10 μmol / L ACOP, 10 μmol / L ACOP solutions were prepared using PBS with different pH values, and the pH value of the buffer solution ranged from 4.0 to 7.0. p As the pH value increases, it decreases. The experimental results are as follows Figure 5 As shown in Figure 1, the oxidation peak current reaches its maximum at pH 4.0 and gradually decreases as the pH increases from 4.0 to 7.0. Therefore, a pH of 4.0 was selected as the optimal detection condition for subsequent experiments. Furthermore, the oxidation peak potential shifts toward negative potentials with increasing solution pH, indicating that protons participate in the redox reaction of ACOP. Figure 5 I shows the relationship between the oxidation peak potential and pH value, and the linear equation is E pa (mV)=672.9-55.0pH(R 2 =0.9995). The experimental results show that ACOP's E pa The slope of the linear equation is −55.0, which is almost equal to the theoretical Nernst value of −59.2, indicating that the number of protons and electrons involved in the redox process of ACOP is the same. Figure 6 The possible electrooxidation mechanism of ACOP was expounded, which was consistent with the mechanism reported in the literature.

[0075] 3. Stability and anti-interference of modified electrodes

[0076] In the supporting electrolyte (0.1 mol / L PBS pH 4.0), AuNPs / RuNPs / CFME was used to perform 15 consecutive differential pulse scans at 10 μmol / L ACOP. The results are shown in Figure 2. Figure 7 The relative standard deviation (RSD) of 15 replicate measurements of ACOP oxidation peak current was 0.20% ( Figure 7 B), indicating that the prepared modified electrode has good stability. Figure 7 Figure C shows an overlay of 10 consecutive differential pulse voltammetry curves of AuNPs / RuNPs / CFME in a 10 μmol / L ACOP solution. After the first measurement, the electrode was removed from the solution, rinsed with ultrapure water, and exposed to air for one day. The same experiment was repeated for 10 days. The results show a <5% decrease in ACOP peak current, demonstrating the stability and reproducibility of the modified electrode.

[0077] Under the optimized conditions, the DPV method was used to investigate the effects of some common interfering components on the ACOP determination of AuNPs / RuNPs / CFME. As shown in Table 1, the addition of 1 mmol / L inorganic ions (such as Na + , K + , Zn 2+ , Fe 2+ , Cl - , CO3 2- and SO4 2- ) and common organic compounds at a concentration of 100 μmol / L, such as glucose, dopamine, ascorbic acid, uric acid and levodopa, because these biomolecules are commonly present in complex biological samples. The experimental results show that the above substances have no significant effect on the detection of 10 μmol / L ACOP. pa The relative deviations were all less than ±5%, indicating that the electrochemical sensor prepared by the present invention had good anti-interference ability for the determination of ACOP.

[0078] Table 1 Effect of interfering components on 10 μmol / L ACOP detection (n=3)

[0079]

[0080]

[0081] Example 4:

[0082] This embodiment, based on the above embodiments, studies the detection effect of the electrochemical sensor on ACOP, PAP and PNP.

[0083] 1. AuNPs / RuNPs / CFME detection of ACOP

[0084] Figure 8 The DPV curves of ACOP with different concentrations on AuNPs / RuNPs / CFME under the optimal experimental conditions obtained in the above example are shown ( Figure 8 A-8C). In 0.1 mol / L PBS (pH 4.0) buffer solution, as the concentration of ACOP solution increases, the peak current value gradually increases, and in the concentration range of 0.04-100 μmol / L, the oxidation peak current of ACOP shows a good linear relationship with its concentration ( Figure 8 D), the linear equation is as follows: I pa1 (nA)=0.0717c(μmol / L)+0.1486(R 2 =0.9950,0.04-1μmol / L); I pa2 (nA)=0.1383c(μmol / L)+0.0803(R 2=0.9985, 1-10 μmol / L); I pa3 (nA)=0.0183c(μmol / L)+0.6452(R 2 =0.9974,10-100 μmol / L).

[0085] The calculated LOD and LOQ were 6.192 nmol / L and 20.6 nmol / L, respectively. In addition, compared with the results of the prior art (Table 2), the modified electrode has the characteristics of wide linear range and low sensitivity in the determination of ACOP.

[0086] Table 2 Comparison of analytical performance between AuNPs / RuNPs / CFME and existing ACOP detectors

[0087]

[0088]

[0089] 2. Robustness Analysis

[0090] The robustness of the analytical results was verified by varying the experimental parameters. At 10 μmol / L ACOP, some of the most critical procedural variables, such as the enrichment potential, pulse duration, and pH, were measured to observe their effects on recovery and standard deviation. As shown in Table 3, the recoveries for these procedural variables ranged from 97.30% to 104.29%, with standard deviations less than 5%, demonstrating that the proposed analytical method for determining ACOP using AuNPs / RuNPs / CFME is reliable and robust.

[0091] Table 3 Robustness results (n=3)

[0092]

[0093] 3. Simultaneous / independent determination of ACOP, PAP, and PNP using AuNPs / RuNPs / CFME

[0094] ACOP, PAP and PNP stock solutions were prepared into a series of gradient concentration solutions using 0.1 mol / L PBS (pH = 4.0) buffer solution, and then AuNPs / RuNPs / CFME was used for DPV determination. Figure 9 In AB, the concentrations of the other two components were fixed, and ACOP, PAP, and PNP were tested separately. When the concentrations of PAP and PNP were stabilized at 10 μmol / L, within the range of 0.5-10 μmol / L, the peak current of ACOP showed a good linear relationship with its concentration, and the linear equation was: I pa (nA)=0.0229c(μmol / L)+0.0503,R2 =0.9915( Figure 9 C), LOD is 0.34 μmol / L (S / N=3). Figure 9 As shown in G, in a solution containing 100 μmol / L ACOP and 10 μmol / L NP, the linear equation of PAP is: pa (nA)=0.2320c(μmol / L)+0.3017(R 2 =0.9927), its concentration was 1-10 μmol / L, and LOD was 0.19 μmol / L (S / N=3). In addition, in a solution containing 100 μmol / L ACOP and 10 μmol / L LPAP, the linear equation of PNP was obtained: pa (nA)=0.1117c(μmol / L)+1.8663(l-10μmol / L,R 2 =0.9972)( Figure 9 H), LOD was 0.015 μmol / L (S / N=3).

[0095] Figure 9 F is the DPV curve of AuNPs / RuNPs / CFME in mixtures of ACOP, PAP and PNP at different concentrations. ACOP, PAP and PNP show three well-separated oxidation peaks, and the peak current increases linearly with the increase of analyte concentration. Figure 9 As shown in I, the linear equations are: pa (nA)=0.0357c(μmol / L)+0.9823(10-100μmol / L,R 2 =0.9977,ACOP), I pa (nA)=0.3040c(μmol / L)+0.5360(1-10μmol / L,R 2 =0.9986, PAP) and I pa (nA)=0.0809c(μmol / L)+0.6511(1-10μmol / L,R 2 =0.9931, PNP). Therefore, the developed electrode shows a good linear range and low detection limit, and can be used for the simultaneous / independent determination of ACOP, PAP, and PNP.

[0096] 4. Actual sample analysis

[0097] Store the human serum sample in a refrigerator (4°C). First, treat the human serum sample with 1.5 mL of methanol as a protein precipitant. Then, centrifuge 10 mL of the human serum sample at 3000 rpm for 10 minutes to eliminate residual serum proteins. Filter the supernatant and dilute it with 0.1 mol / L PBS (pH 4.0) buffer. Then, add the appropriate amount of standard ACOP, PAP, and PNP solutions to the human serum sample and analyze it using the DPV method.

[0098] AuNPs / RuNPs / CFME was used to detect the spiked recovery of ACOP in human serum samples. The experimental results are shown in Table 4. The relative standard deviation of the DPV peak current of three parallel measurements was less than 2.39%, and the spiked recovery rate was between 95.9% and 101.9%, indicating that the electrochemical sensor provided by the present invention has potential application prospects in the determination of ACOP in clinical samples.

[0099] Table 4 DPV analysis of ACOP in human serum using AuNPs / RuNPs / CFME (n=3)

[0100]

[0101] The above examples are merely illustrative of the present invention and do not limit the scope of protection of the present invention. Any design that is identical or similar to the present invention falls within the scope of protection of the present invention.

Claims

1. An electrochemical sensor for the simultaneous or separate detection of acetaminophen, p-aminophenol, and p-nitrophenol, the electrochemical sensor comprising a working electrode, characterized in that: The working electrode comprises a carbon fiber microelectrode and nanoparticles deposited on the surface of the carbon fiber microelectrode; the nanoparticles comprise nanoruthenium particles and nanogold particles.

2. The use according to claim 1, characterized in that: The preparation method of the electrochemical sensor comprises the following steps: (1) preparing ruthenium nanoparticle colloid and gold nanoparticle colloid for use; (2) After washing the carbon fiber microelectrode, electrophoresis is performed in a colloid of nano-ruthenium particles to deposit nano-ruthenium particles on the electrode surface; (3) The prepared nano-ruthenium particles / carbon fiber microelectrode are thoroughly rinsed and electrophoretic deposition is continued in the nano-gold particle colloid to obtain a nano-gold particle / nano-ruthenium particle / carbon fiber microelectrode electrochemical sensor.

3. The use according to claim 2, characterized in that: In the step (2), electrophoretic deposition is performed in the nano-ruthenium particle colloid at a voltage of +1.4-1.8 V for 10-20 minutes.

4. The use according to claim 2, wherein: In the step (3), electrophoretic deposition is performed in the nano-gold particle colloid at a voltage of +1.4-1.8 V for 10-25 minutes.

5. A method for simultaneously or individually detecting acetaminophen, p-aminophenol and p-nitrophenol using an electrochemical sensor, characterized in that: The following steps are involved: (1) Prepare standard solutions of acetaminophen, p-aminophenol, and p-nitrophenol at different concentrations using buffer solution; (2) A working electrode and a reference electrode of the electrochemical sensor are formed into a two-electrode system, and the working electrode is immersed in each standard solution for incubation using an electrochemical measurement method, and the corresponding current value is recorded. The concentration-current value standard curves of acetaminophen, p-aminophenol and p-nitrophenol are drawn using the standard solution concentration and current value respectively; the working electrode of the electrochemical sensor includes a carbon fiber microelectrode and nanoparticles deposited on the surface of the carbon fiber microelectrode; the nanoparticles include ruthenium nanoparticles and gold nanoparticles; (3) Adding buffer solution to the pre-treated sample to dilute it, detecting the current value of the sample using the electrochemical measurement method in step (2), and calculating the content of acetaminophen, p-aminophenol and p-nitrophenol in the sample based on the concentration-current value standard curve.

6. The method according to claim 5, wherein: The buffer solution is 0.05-0.2 mol / L PBS buffer solution with a pH of 4.0-7.

0.

7. The method according to claim 5, wherein: The reference electrode is an Ag / AgCl electrode.

8. The method according to claim 5, wherein: The electrochemical measurement methods include cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry.

9. The method according to claim 8, wherein: The cyclic voltammetry method recorded CV signals at a scan rate of 0.1 V / s between 0 and 1.0 V. The differential pulse voltammetry method was performed with a pulse amplitude of 0.1 V and a potential step of 0.005 V, and the potential range used was -0.2 to 1.2 V. The electrochemical impedance spectroscopy method collected EIS measurements in a standard potassium ferricyanide solution with a frequency set between 0.1 and 100 kHz, an open circuit potential of +0.20 V, and a voltage amplitude of 10 mV.

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