Aspartic acid molecularly imprinted electrochemical sensor and its preparation and detection method

Abstract

This paper presents a molecularly imprinted electrochemical sensor for aspartic acid (Asp) and its preparation and detection methods. Belonging to the field of electrochemical analysis and detection, the method involves physically mixing polyethyleneimine (PEI) with multi-walled carbon nanotubes (MXenes) to form a single-layer tubular fiber structure. The MXenes are then bridged by polyethyleneimine to disperse and load the MXenes onto the surface and between the pores of layered MXenes, resulting in a highly conductive MWCNTs-PEI-MXene nanocomposite material that serves as the substrate, enhancing the signal response of the molecularly imprinted sensor. Using o-phenylenediamine as the functional monomer and aspartic acid as the template molecule, a molecularly imprinted electrochemical sensor is constructed, improving the specificity of the electrochemical sensor for Aspartic acid. This preparation method is simple, and the resulting molecularly imprinted electrochemical sensor exhibits good selectivity, reproducibility, and long-term stability, maintaining 92.8% of its initial value even after 7 days of continuous monitoring. This achieves sensitive and specific analysis of Aspartic acid in real samples.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical analysis and detection, specifically relating to an aspartic acid molecularly imprinted electrochemical sensor and its preparation and detection method. Background Technology

[0002] Aspartic acid (Asp) is a natural amino acid widely found in plants such as legumes, beets, and tea. It possesses various physiological functions, including promoting plant growth, enhancing stress resistance, participating in human energy metabolism, and protecting the cardiovascular system and liver. Under normal dietary intake, aspartic acid is non-toxic and harmless to both humans and plants. However, long-term or excessive intake of high doses of aspartic acid may cause mild discomfort such as headaches, dizziness, and nausea. Currently, aspartic acid can be detected using high-performance liquid chromatography-ultraviolet (HPLC-UV) and HPLC-mass spectrometry (HPLC-MS), but these methods require complex pretreatment steps, skilled technicians, and expensive equipment. Therefore, finding an efficient and sensitive method for aspartic acid detection is both necessary and urgent.

[0003] In contrast, electrochemical methods are simple to operate, have a fast response time, and are low in cost. Based on the electroactivity of Asp, detection can be achieved using the electrochemical signals generated by its oxidation on the electrode surface. To improve the specificity of detection, introducing molecularly imprinted polymers (MIPs) as recognition elements is beneficial for enhancing sensor selectivity. Currently, electropolymerization has become an important method for preparing Asp molecularly imprinted electrochemical sensors due to its advantages such as precise control of imprint film thickness, thorough template elution, uniform and robust film layers, and environmental friendliness. However, current molecularly imprinted electrochemical sensors still face challenges such as limited mass transfer efficiency, weak electrochemical signal response, and the need to improve sensitivity. Therefore, there is an urgent need to develop novel sensing interfaces and materials to overcome these difficulties. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of insufficient selectivity and sensitivity of the currently developed Asp electrochemical sensors, and to provide an aspartic acid molecularly imprinted electrochemical sensor and its preparation and detection method.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is: a method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid, comprising the following steps: (1) Multi-walled carbon nanotubes and polyethyleneimine were dispersed in ultrapure water and mixed using a cell disruptor to allow polyethyleneimine to be physically adsorbed onto the surface of multi-walled carbon nanotubes. The treated suspension was washed, centrifuged, and then vacuum dried to obtain MWCNTs-PEI solid powder.

[0006] MWCNTs-PEI solid powder and MXene solid were added to ultrapure water in a certain proportion, and after ultrasonic dispersion, MWCNTs-PEI-MXene solid powder was obtained by freeze drying.

[0007] (2) The glassy carbon electrode is treated with aluminum oxide powder, then cleaned and dried to obtain the treated glassy carbon electrode.

[0008] (3) The prepared MWCNTs-PEI-MXene solid powder was ultrasonically dispersed in ultrapure water, and the resulting dispersion was added to the surface of the treated glassy carbon electrode. After drying, an electrode coated with a sensitive material substrate layer was obtained.

[0009] (4) Immerse the electrode coated with the sensitive material substrate in an acetate buffer solution containing o-phenylenediamine and aspartic acid, and perform cyclic voltammetry scans within a fixed voltage range; after the scan is completed, remove the electrode and dry it at room temperature. (5) After immersing the dried electrode in the elution solution for a period of time, rinse it with ultrapure water and dry it to obtain the molecularly imprinted electrochemical sensor for detecting aspartic acid.

[0010] In step (1), the mass concentration ratio of multi-walled carbon nanotubes to polyethyleneimine in the suspension is 1:1; multi-walled carbon nanotubes and polyethyleneimine are dispersed in ultrapure water and mixed using a cell disruptor with an ultrasonic power of 100W. During the process, the ultrasonic operation lasts for 2 seconds and the interval is 2 seconds; after the process, the mixture is centrifuged at 10,000 rpm and then vacuum dried at 60 ℃ for 12 h.

[0011] In step (1), the mass ratio of MWCNTs-PEI solid powder to MXene solid is 2:1; the ultrasonic dispersion time is 30 min; the freeze-drying time is 6 h, the freezing temperature is -20 ℃, and the drying time is 24 h.

[0012] In step (2), the diameter of the glassy carbon electrode is 3 mm; the particle size of the aluminum oxide powder used is 0.05 μm.

[0013] In step (3), the concentration of MWCNTs-PEI-MXene in the dispersion is 0.1~2 mg / mL; the drying temperature is 37 ℃ and the drying time is 1 h.

[0014] In step (4), the mass ratio of aspartic acid to o-phenylenediamine is 1:1~4, the concentration of acetate buffer solution is 0.1 mol / L, and the pH value is 4.0~6.0; the potential range of cyclic voltammetry scan is -0.2~1.0 V, the scan rate is 50 mV / s, and the number of cycles is 5~20.

[0015] In step (5), the elution solution is a mixture of ethanol and acetic acid in a ratio of V. 乙醇 :V 乙酸 =9:1, soaking time is 3 min.

[0016] The present invention also provides a molecularly imprinted electrochemical sensor for detecting aspartic acid, comprising a sensitive material substrate layer coated on an electrode, and a molecularly imprinted polymer layer constructed on the sensitive material substrate layer using o-phenylenediamine as a functional monomer and aspartic acid as a template molecule; wherein the sensitive material substrate layer is obtained by bridging multi-walled carbon nanotubes and layered MXene with polyethyleneimine, wherein the polyethyleneimine and multi-walled carbon nanotubes are physically mixed and adsorbed together, so that the multi-walled carbon nanotubes form a single-layer tubular fiber structure and are dispersed and loaded on the surface of the layered MXene and between the pores of each layer.

[0017] A method for detecting aspartic acid using a molecularly imprinted electrochemical sensor includes the following steps: (1) Immerse the molecularly imprinted electrochemical sensor in a standard solution containing different concentrations of aspartic acid. Each concentration of aspartic acid standard solution corresponds to one molecularly imprinted electrochemical sensor. After soaking and incubating for a period of time, take out the molecularly imprinted electrochemical sensor and rinse it with ultrapure water to remove unbound aspartic acid.

[0018] (2) The incubated molecularly imprinted electrochemical sensor was used as the working electrode. Differential pulse voltammetry was used to detect and record the electrochemical signals generated by different concentrations of aspartic acid in the buffer solution. The concentration of the aspartic acid standard solution was used as the abscissa and the generated electrical signal was used as the ordinate to establish a standard curve.

[0019] (3) When it is necessary to detect aspartic acid in a sample, obtain the sample solution to be tested, immerse the molecularly imprinted electrochemical sensor in the sample solution for a period of time, and then rinse it with ultrapure water; use the processed molecularly imprinted electrochemical sensor as the working electrode for detection, and substitute the measured current signal into the standard curve established in step (2) to obtain the concentration of aspartic acid in the sample to be tested.

[0020] The concentration of the aspartic acid standard solution was 0.1~20 mmol / L, the soaking and incubation time was 10~20 min, the buffer solution used for detection was 40 mmol / L BR buffer solution with pH 2.0, and the scanning voltage range for detection was 0.5~1.0 V.

[0021] The beneficial effects of this invention are as follows: First, polyethyleneimine and multi-walled carbon nanotubes are physically mixed to form a single-layer tubular fiber structure. The multi-walled carbon nanotubes are then dispersed and loaded onto the surface of layered MXene and between the pores of each layer through polyethyleneimine bridging, resulting in a MWCNTs-PEI-MXene nanocomposite material with excellent conductivity. Using this nanocomposite material as a substrate can improve the signal response of molecularly imprinted sensors. Second, o-phenylenediamine (OPD), which has high affinity, is selected as the electropolymerizable functional monomer to prepare a molecularly imprinted polymer (MIP), which is beneficial for improving the specificity of electrochemical sensors for Asp analysis.

[0022] This invention utilizes polyethyleneimine-linked multi-walled carbon nanotubes (MWCNTs) and layered MXene. Polyethyleneimine can be physically mixed and adsorbed onto the MIPs, dispersing them to form a single-layer tubular fiber structure. The abundant amino groups on the surface of polyethyleneimine facilitate electrostatic binding with layered MXene, and the excess amino groups also contribute to the formation of electropolymerized MIPs. This method offers advantages such as simple preparation, high selectivity, good reproducibility, and high reusability, achieving sensitive and specific analysis of Asp in real samples. The prepared molecularly imprinted electrochemical sensor for Asp detection exhibits a linear range of 0.1–20 mmol / L and a detection limit as low as 0.1 mmol / L.

[0023] This invention employs electrochemical polymerization to prepare molecularly imprinted polymers with controllable morphology and excellent selectivity. The resulting imprinted cavities specifically bind Asp via hydrogen bonds and π-π conjugation, thereby improving the selectivity of the sensor. The constructed molecularly imprinted electrochemical sensor exhibits good reproducibility and long-term stability, maintaining 92.8% of its initial value even after 7 days of continuous monitoring. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the fabrication process of the molecularly imprinted electrochemical sensor of the present invention; wherein (A) is the fabrication process of the MWCNTs-PEI-MXene composite material; and (B) is the process of fabricating a molecularly imprinted polymer layer on the electrode GCE.

[0025] Figure 2The following are transmission electron microscope (TEM) images of the materials: (A) is MWCNTs; (B) is MWCNTs-PEI; (C) is layered MXene; (D) is a MWCNTs-PEI-MXene composite material.

[0026] Figure 3 These are cyclic voltammetry plots for MIP / MWCNTs-PEI-MXene / GCE and NIP / MWCNTs-PEI-MXene / GCE.

[0027] Figure 4 These are impedance diagrams for MIP / MWCNTs-PEI-MXene / GCE and NIP / MWCNTs-PEI-MXene / GCE.

[0028] Figure 5 This is a graph showing the current signals corresponding to standard solutions of aspartic acid at different concentrations.

[0029] Figure 6 This is a graph showing the linear relationship between different concentrations of aspartic acid standard solution and current signals.

[0030] Figure 7 This is a selectivity diagram of the constructed molecularly imprinted electrochemical sensor.

[0031] Figure 8 The chart shows the reproducibility between batches (top) and the stability over 7 consecutive days (bottom). Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. The specific contents listed in the following embodiments are not limited to the technical features necessary to solve the technical problem of the present invention. Furthermore, the listed embodiments are merely a part of the present invention, and not all embodiments.

[0033] This invention first prepares a polyethyleneimine (PEI)-bridged multi-walled carbon nanotube and layered MXene nanocomposite material (MWCNTs-PEI-MXene) with excellent conductivity, which can be used as a substrate material to improve the signal response of the molecularly imprinted sensor. Secondly, o-phenylenediamine (OPD) with high affinity is selected as an electropolymerization functional monomer to prepare a molecularly imprinted polymer (MIP), which is beneficial to improve the specificity of the electrochemical sensor for Asp analysis.

[0034] Specific preparation methods are as follows: Figure 1 As shown, it includes the following steps: 1. Preparation of MWCNTs-PEI-MXene nanocomposites: First, multi-walled carbon nanotubes (MWCNTs) and polyethyleneimine (PEI) were dispersed in ultrapure water and treated with a cell disruptor for a period of time. The resulting suspension was then washed and centrifuged three times, and then vacuum dried to obtain a solid powder. The obtained product was defined as MWCNTs-PEI and stored at room temperature for further use.

[0035] The preferred mass concentration ratio of MWCNTs to PEI is 1:1. The ultrasonic power of the cell disruptor is 100 W, the total time is 20 min, including 2 s of ultrasonic operation and 2 s interval, the centrifugation speed is 10000 rpm, the drying temperature is 60℃, and the drying time is 12 h.

[0036] A certain amount of MWCNTs-PEI and layered MXene solids were weighed and added to ultrapure water. After being ultrasonically dispersed for a period of time, they were freeze-dried to obtain solid powder. The obtained product was defined as MWCNTs-PEI-MXene and stored at room temperature for further use.

[0037] The preferred mass ratio of MWCNTs-PEI to MXene is 2:1, the volume of ultrapure water is 10 mL, the ultrasonic power is 99%, the ultrasonic time is 30 min, the freezing time is 6 h, the freezing temperature is -20 ℃, and the drying time is 24 h.

[0038] The MWCNTs-PEI-MXene composite material prepared by the above method has a loose accordion-like two-dimensional layered structure, and its surface and channels are loaded with nanotubes, which is beneficial to electron transport.

[0039] 2. Fabrication of molecularly imprinted electrochemical sensors: A method for fabricating a molecularly imprinted electrochemical sensor includes modifying a substrate material by coating the MWCNTs-PEI-MXene composite material onto an electrode surface and drying it to obtain a sensitive material layer; further designing a molecularly imprinted layer on the sensitive material layer substrate by polymerizing functional monomers through electrochemical polymerization to form a polymer layer containing template molecules; and removing the template molecules from the polymer layer to form imprinted cavities. The construction process of this molecularly imprinted electrochemical sensor includes the following steps: (1) The glassy carbon electrode (GCE) is treated with aluminum oxide powder, and then ultrasonically cleaned in water, ethanol, acetone and water in sequence and dried to obtain the treated glassy carbon electrode.

[0040] (2) The prepared MWCNTs-PEI-MXene composite material was ultrasonically dispersed in ultrapure water. The prepared MWCNTs-PEI-MXene dispersion was dropped onto the surface of the treated glassy carbon electrode and dried to obtain MWCNTs-PEI-MXene / GCE.

[0041] (3) Immerse the obtained MWCNTs-PEI-MXene / GCE in an acetate buffer solution containing o-phenylenediamine (OPD, as a functional monomer) and aspartic acid (Asp, as a template molecule) and perform cyclic voltammetry scans within a fixed voltage range; after the scan is completed, remove the electrode and dry it at room temperature. The resulting electrode is denoted as OPD / MWCNTs-PEI-MXene / GCE.

[0042] (4) After soaking the obtained electrode OPD / MWCNTs-PEI-MXene / GCE in the elution solution for a period of time, it can be rinsed with ultrapure water and dried to obtain the MWCNTs-PEI-MXene composite material-based molecularly imprinted electrochemical sensor, denoted as MIP / MWCNTs-PEI-MXene / GCE.

[0043] In one embodiment, the glassy carbon electrode has a diameter of 3 mm, and the alumina powder used to treat the glassy carbon electrode has a particle size of 0.05 μm. A MWCNTs-PEI-MXene dispersion with a concentration of 0.1–2 mg / mL is added dropwise to the surface of the glassy carbon electrode in a volume of 6 μL. The drying temperature at the end of the dropwise addition is 37 °C, and the drying time is 1 h.

[0044] The preferred ratio of Asp (template molecule) to OPD (functional monomer) is 1:1 to 4; the concentration of the acetate buffer solution is 0.1 mol / L; the pH value is 4.0 to 6.0; the potential range of the cyclic voltammetry scan is -0.2 to 1.0 V; the scan rate is 50 mV / s; and the number of cycles is 5 to 20.

[0045] The elution solution is a mixture of ethanol and acetic acid in a ratio of V. 乙醇 :V 乙酸 =9:1, soaking time is 3 min. Example 1:

[0046] (1) Weigh 20 mg of multi-walled carbon nanotubes (MWCNTs) and 20 mg of polyethyleneimine (PEI) and disperse them in 20 mL of ultrapure water. Use a cell disruptor to ultrasonically disrupt the suspension for 20 min at an ultrasonic power of 100 W and an ultrasonic working time of 2 s and an interval of 2 s. Then, centrifuge and wash the resulting suspension three times at 10,000 rpm. Then, vacuum dry it at 60 °C for 12 h to obtain a solid powder. The obtained product is defined as MWCNTs-PEI and stored at room temperature for further use.

[0047] Weigh 20 mg of MWCNTs-PEI solid and 10 mg of MXene solid and add them to 10 mL of ultrapure water. Set the ultrasonic power to 99% and ultrasonically disperse the sample for 30 min. Then freeze the sample in a -20 ℃ freezer for 6 h. After freezing, freeze-dry the sample for 24 h to obtain a black powder sample, which is defined as MWCNTs-PEI-MXene. Store the sample at room temperature for further use.

[0048] Figure 2 (A) shows that MWCNTs exhibit an aggregated multilayer tubular structure, which is due to their poor water solubility leading to material aggregation; Figure 2 (B) is MWCNTs treated with PEI-PEI, which exhibits a single-layer tubular fiber structure that is intertwined and entangled, indicating that PEI-treated MWCNTs are more loosely distributed. Figure 2 (C) is a two-dimensional layered structure MXene that exhibits an accordion-like appearance; Figure 2 (D) shows that the MWCNTs-PEI-MXene composite material prepared by both methods has a looser multilayer structure, with MWCNTs-PEI loaded on both the surface and the pores, which increases the distance between MXene layers and is beneficial for electron transport.

[0049] (2) The glassy carbon electrode (GCE, 3 mm) was treated with 0.05 μm aluminum oxide powder, and then ultrasonically cleaned in water, ethanol, acetone and water in sequence and dried to obtain the treated glassy carbon electrode.

[0050] (3) The MWCNTs-PEI-MXene composite material prepared in step (1) is ultrasonically dispersed in ultrapure water to prepare a 1 mg / mL MWCNTs-PEI-MXene dispersion; 6 μL of MWCNTs-PEI-MXene dispersion is added dropwise to the glassy carbon electrode surface treated in step (2) using a pipette, and MWCNTs-PEI-MXene / GCE is obtained after drying for 1 h.

[0051] (4) Immerse the MWCNTs-PEI-MXene / GCE obtained in step (3) in a 0.1 mol / L acetate buffer solution (pH 4.0~6.0) containing o-phenylenediamine (OPD, as a functional monomer) and aspartic acid (Asp, as a template molecule), wherein the ratio of Asp (template molecule) to OPD (functional monomer) is 1:1~4. Then, by electrochemical polymerization, perform 5~20 consecutive cyclic voltammetric scans at 50 mV / s in the voltage range of -0.2~1.0 V. After the scan is completed, remove the electrode, dry it at room temperature, and obtain the modified electrode, which is denoted as OPD / MWCNTs-PEI-MXene / GCE.

[0052] (5) Immerse the modified electrode OPD / MWCNTs-PEI-MXene / GCE obtained in step (4) in an elution solution of ethanol and acetic acid (V 乙醇 :V 乙酸 After 3 min in a mixture of 9:1, the molecularly imprinted electrochemical sensor based on MWCNTs-PEI-MXene can be obtained by rinsing with ultrapure water and drying, denoted as MIP / MWCNTs-PEI-MXene / GCE.

[0053] Comparative Example 1: The non-imprinted polymer (NIP) was prepared according to the same steps as in Example 1, except that the template molecule aspartic acid was not added during the polymerization process in step (4). The resulting sensor was denoted as NIP / MWCNTs-PEI-MXene / GCE.

[0054] The sensor MIP / MWCNTs-PEI-MXene / GCE provided in Example 1 and the NIP / MWCNTs-PEI-MXene / GCE in Comparative Example 1 were immersed in a 0.1 mol / L potassium chloride aqueous solution containing 5 mmol / L potassium ferricyanide. The electrochemical properties of the sensor were investigated by cyclic voltammetry and electrochemical impedance spectroscopy.

[0055] Due to the lack of effective imprinting sites, NIP / MWCNTs-PEI-MXene / GCE exhibits a lower oxidation peak current ( Figure 3 and larger impedance values ​​( Figure 4 ).

[0056] The molecularly imprinted electrochemical sensor based on MWCNTs-PEI-MXene prepared by the aforementioned method includes a sensitive material substrate layer coated on the electrode, and a molecularly imprinted polymer layer constructed on the sensitive material substrate layer using o-phenylenediamine as a functional monomer and aspartic acid as a template molecule. The sensitive material substrate layer is obtained by bridging multi-walled carbon nanotubes and layered MXene with polyethyleneimine, wherein polyethyleneimine and multi-walled carbon nanotubes are physically mixed and adsorbed together, so that the multi-walled carbon nanotubes form a single-layer tubular fiber structure and are dispersed and loaded on the surface of the layered MXene and between the pores of each layer.

[0057] A method for detecting aspartic acid using a molecularly imprinted electrochemical sensor prepared in this manner includes the following steps: (1) Immerse the sensor prepared in the above steps in a standard solution containing aspartic acid (Asp) of different concentrations, for example, select multiple Asp standard solutions with concentrations in the range of 0.1~20 mmol / L. One concentration of Asp solution corresponds to one sensor, and the concentration and sensor have a one-to-one correspondence. After incubation for 10~20 min, take it out and rinse the electrode with ultrapure water to remove unbound Asp, and obtain the incubated electrode, which is denoted as Asp / MIP / MWCNTs-PEI-MXene / GCE; (2) Using the Asp / MIP / MWCNTs-PEI-MXene / GCE incubated in step (1) as the working electrode, the Ag / AgCl (saturated KCl) electrode as the reference electrode, and the platinum wire electrode as the counter electrode, the electrochemical signals generated by different concentrations of Asp were detected and recorded in the buffer solution using differential pulse voltammetry (DPV) on an electrochemical workstation of model CHI660E. A standard curve was established with the concentration of the Asp standard solution as the abscissa and the generated electrical signal as the ordinate. (3) Detection of Asp in actual samples: First, obtain the sample solution, place the MIP / MWCNTs-PEI-MXene / GCE sensor in the sample solution and incubate for 10~20 min, then rinse the sensor with ultrapure water and then perform detection. Obtain the corresponding current signal through electrochemical testing. Substitute the generated current signal into the standard curve constructed in step (2) to obtain the concentration of Asp in the actual sample, thus realizing the purpose of detecting Asp in unknown samples.

[0058] The electrochemical tests in steps (2) and (3) were performed in a 40 mmol / L BR (pH=2.0) buffer solution with a scanning voltage range of 0.5~1.0 V. Example 2:

[0059] The MIP / MWCNTs-PEI-MXene / GCE provided in Example 1 was immersed in standard solutions containing 0.1, 1, 5, 10, and 20 mM aspartic acid (Asp), respectively. After incubation for 10-15 min, the electrodes were removed and rinsed with ultrapure water to remove unbound Asp, resulting in the incubated electrodes, denoted as Asp / MIP / MWCNTs-PEI-MXene / GCE. The aforementioned product (Asp / MIP / MWCNTs-PEI-MXene / GCE) was used as the working electrode, an Ag / AgCl (saturated KCl) electrode as the reference electrode, and a platinum wire electrode as the counter electrode. The electrochemical signal generated by this product was detected and recorded using differential pulse voltammetry (DPV) in a 40 mmol / L BR (pH=2.0) buffer solution on a CHI660E electrochemical workstation. The DPV scan range was 0.5–1.0 V. A standard curve was established by plotting the Asp current signal on the ordinate and the corresponding concentration of the standard solution on the abscissa for detecting the Asp concentration in actual samples.

[0060] from Figure 5 and Figure 6 As can be seen, the peak current signal gradually increases with increasing Asp concentration, exhibiting a good linear relationship within the concentration range of 0.1–20 mM. Its standard curve is I. Asp =67.47C col +288.01 (R) 2 =0.992), and the detection limit is 0.1 mM. Example 3:

[0061] The analytical performance of the prepared molecularly imprinted electrochemical sensor was investigated. The MIP / MWCNTs-PEI-MXene / GCE provided in Example 1 was immersed in a standard solution containing 10 mM aspartic acid (Asp) and incubated for 10–15 min. After incubation, the electrode was removed and rinsed with ultrapure water to remove unbound Asp, yielding the incubated electrode, denoted as MIP / MWCNTs-PEI-MXene / GCE. The selectivity, reproducibility, and long-term stability of the molecularly imprinted sensor were investigated.

[0062] Figure 7The selectivity of the prepared molecularly imprinted electrochemical sensor is shown, with interfering substances including L-glutamate (L-GA), thyroxine (T4), cysteine ​​(Cys), D-glutamate (D-GA), phenylalanine (PHE), glycine (Gly), proline (Pro), malic acid (MA), histidine (His), aspartic acid (Asp), and a mixture of aspartic acid and other substances (Asp+Mix). Blank serves as a blank control. The figure shows a significant current response in the presence of Asp, indicating good specificity of the developed product.

[0063] Figure 8 The results showed that the RSD for batch-to-batch reproducibility was 4.42%, indicating excellent reproducibility of the developed product. Furthermore, after 7 consecutive days of testing, the signal of the constructed imprinted electrochemical sensor maintained 92.8% of its initial value on the 7th day. Figure 8 (See the figure below), which shows good long-term stability. Example 4:

[0064] The constructed molecularly imprinted electrochemical sensor based on MWCNTs-PEI-MXene was used to analyze the actual sample. The steps are as follows: (1) Purchase a sample of leafy greens from a local supermarket, weigh 2 g of leafy greens, crush them, add 6 mL of ultrapure water for extraction, shake for 30 min, centrifuge at 1000 rpm for 10 min, and extract the supernatant. Further, filter using a 0.22 μm filter membrane to obtain the final extract. The molecularly imprinted electrochemical sensing method constructed in this invention was used for testing.

[0065] (2) Extracts containing 5 and 10 mM Asp were prepared by the standard addition method and tested using the constructed analytical method to verify the practicality of the constructed method.

[0066] The results of this invention, using molecularly imprinted electrochemical sensing and HPLC-MS methods, for detecting Asp content in leafy green vegetables are shown in the table below: In the blank unspiked sample, Asp was not detected. After spiking, the average recovery rate of the molecularly imprinted electrochemical sensor constructed by this invention was 106.2%. At the same time, the detection result was basically consistent with the result of the national standard method (HPLC-MS), indicating that the method of this invention has reliability and practicality; and realizes the application of col detection in unknown samples.

[0067] The above description of specific embodiments is only for the purpose of helping to understand the technical concept and core idea of ​​the present invention. Although specific preferred embodiments have been used to describe and illustrate the technical solutions, they should not be construed as limiting the present invention itself. Those skilled in the art can make various changes in form and detail without departing from the technical concept of the present invention. These easily conceived changes or substitutions should all be covered within the protection scope of the present invention.

Claims

1. A method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid, characterized in that: Includes the following steps: (1) Multi-walled carbon nanotubes and polyethyleneimine were dispersed in ultrapure water and mixed using a cell disruptor to allow polyethyleneimine to be physically adsorbed onto the surface of multi-walled carbon nanotubes. The treated suspension was washed, centrifuged, and then vacuum dried to obtain MWCNTs-PEI solid powder. MWCNTs-PEI solid powder and MXene solid were added to ultrapure water in a certain proportion, and after ultrasonic dispersion, MWCNTs-PEI-MXene solid powder was obtained by freeze drying. (2) The glassy carbon electrode is treated with aluminum oxide powder, then cleaned and dried to obtain the treated glassy carbon electrode; (3) The prepared MWCNTs-PEI-MXene solid powder was ultrasonically dispersed in ultrapure water, and the resulting dispersion was added to the surface of the treated glassy carbon electrode. After drying, an electrode coated with a sensitive material substrate layer was obtained. (4) Immerse the electrode coated with the sensitive material substrate in an acetate buffer solution containing o-phenylenediamine and aspartic acid, and perform cyclic voltammetry scans within a fixed voltage range; after the scan is completed, remove the electrode and dry it at room temperature. (5) After immersing the dried electrode in the elution solution for a period of time, rinse it with ultrapure water and dry it to obtain the molecularly imprinted electrochemical sensor for detecting aspartic acid.

2. The method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid as described in claim 1, characterized in that: In step (1), the mass concentration ratio of multi-walled carbon nanotubes to polyethyleneimine in the suspension is 1:1; multi-walled carbon nanotubes and polyethyleneimine are dispersed in ultrapure water and mixed using a cell disruptor with an ultrasonic power of 100W. During the process, the ultrasonic operation lasts for 2 seconds and the interval is 2 seconds; after the process, the mixture is centrifuged at 10,000 rpm and then vacuum dried at 60 ℃ for 12 h.

3. The method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid as described in claim 1, characterized in that: In step (1), the mass ratio of MWCNTs-PEI solid powder to MXene solid is 2:1; the ultrasonic dispersion time is 30 min; the freeze-drying time is 6 h, the freezing temperature is -20 ℃, and the drying time is 24 h.

4. The method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid as described in claim 1, characterized in that: In step (2), the diameter of the glassy carbon electrode is 3 mm; the particle size of the aluminum oxide powder used is 0.05 μm.

5. The method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid as described in claim 1, characterized in that: In step (3), the concentration of MWCNTs-PEI-MXene in the dispersion is 0.1~2 mg / mL; the drying temperature is 37 ℃ and the drying time is 1 h.

6. The method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid as described in claim 1, characterized in that: In step (4), the mass ratio of aspartic acid to o-phenylenediamine is 1:1~4, the concentration of acetate buffer solution is 0.1 mol / L, and the pH value is 4.0~6.0; the potential range of cyclic voltammetry scan is -0.2~1.0 V, the scan rate is 50 mV / s, and the number of cycles is 5~20.

7. The method for preparing a molecularly imprinted electrochemical sensor for detecting aspartic acid as described in claim 1, characterized in that: In step (5), the elution solution is a mixture of ethanol and acetic acid in a ratio of V. 乙醇 :V 乙酸 =9:1, soaking time is 3 min.

8. A molecularly imprinted electrochemical sensor for detecting aspartic acid, characterized in that: The material includes a sensitive material substrate layer coated on the electrode, and a molecularly imprinted polymer layer constructed on the sensitive material substrate layer using o-phenylenediamine as a functional monomer and aspartic acid as a template molecule. The sensitive material substrate layer is obtained by bridging multi-walled carbon nanotubes and layered MXene with polyethyleneimine, wherein the polyethyleneimine and multi-walled carbon nanotubes are physically mixed and adsorbed together, so that the multi-walled carbon nanotubes form a single-layer tubular fiber structure and are dispersed and loaded on the surface of the layered MXene and between the pores of each layer.

9. A method for detecting aspartic acid using the molecularly imprinted electrochemical sensor according to claim 8, characterized in that: Includes the following steps: (1) Immerse the molecularly imprinted electrochemical sensor in a standard solution containing different concentrations of aspartic acid. Each concentration of aspartic acid standard solution corresponds to one molecularly imprinted electrochemical sensor. After soaking and incubating for a period of time, take out the molecularly imprinted electrochemical sensor and rinse it with ultrapure water to remove unbound aspartic acid. (2) The incubated molecularly imprinted electrochemical sensor was used as the working electrode. Differential pulse voltammetry was used to detect and record the electrochemical signals generated by different concentrations of aspartic acid in the buffer solution. The concentration of the aspartic acid standard solution was used as the abscissa and the generated electrical signal was used as the ordinate to establish a standard curve. (3) When it is necessary to detect aspartic acid in a sample, obtain the sample solution to be tested, immerse the molecularly imprinted electrochemical sensor in the sample solution for a period of time, and then rinse it with ultrapure water; use the processed molecularly imprinted electrochemical sensor as the working electrode for detection, and substitute the measured current signal into the standard curve established in step (2) to obtain the concentration of aspartic acid in the sample to be tested.

10. The method as described in claim 9, characterized in that: The concentration of the standard solution of aspartic acid is 0.1~20 mmol / L, the soaking and incubation time is 10~20 min, the buffer solution used for detection is 40 mmol / L BR buffer solution with pH 2.0, and the scanning voltage range for detection is 0.5~1.0 V.

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