A molecularly imprinted electrochemical sensor for simultaneous detection of glutamic acid and serotonin and a construction method thereof

Abstract

The application discloses a kind of molecularly imprinted electrochemical sensor capable of detecting glutamic acid and serotonin simultaneously.The molecularly imprinted electrochemical sensor includes glassy carbon electrode, and (1) amino-functionalized reduced graphene oxide and (2) double-template molecularly imprinted polymer with glutamic acid and serotonin as template molecules and o-phenylenediamine as monomer successively modified on the surface of glassy carbon electrode.Compared with prior art, the molecularly imprinted electrochemical sensor of the application can realize simultaneous detection of non-electrically active glutamic acid and electrically active 5-hydroxytryptamine, thereby saving detection operation and cost, showing good linear relationship and low detection limit for Glu and 5-HT in the concentration range of 1-100 μM, and having good precision, accuracy, selectivity and stability.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection and analysis, specifically relating to a molecularly imprinted electrochemical sensor capable of simultaneously detecting glutamate and serotonin. Background Technology

[0002] In depression research, glutamate (Glu) and serotonin (5-HT) have attracted much attention as potential biomarkers. Glutamate (Glu), as an important excitatory neurotransmitter, is relied upon by more than 50% of neurons in brain tissue for signal transduction. Under the action of glutamate decarboxylase, Glu is decarboxylated to produce GABA, and Glu and GABA are reuptaken by glial cells and converted back into glutamine and glutamate, thus forming the glutamate metabolic loop. Disorders of the glutamate metabolic loop are associated with the occurrence and development of depression, and patients have elevated Glu levels. Serotonin (5-HT), on the other hand, is a messenger that produces pleasurable mood and participates in the regulation of human emotions. 5-HT deficiency or relative deficiency can participate in the occurrence and development of depression through gene polymorphism, neural networks, neurotransmitter metabolism and transmission. The monoamine hypothesis suggests that decreased levels of monoamine neurotransmitters such as 5-HT in the synaptic cleft are an important cause of depression and also provide a theoretical basis for the development of antidepressants.

[0003] Various methods exist for the detection of Glu and 5-HT, among which electrochemical methods have attracted widespread attention due to their unique advantages. For example, Alam et al. (…) Journal of Luminescence A zinc oxide / nickel oxide / aluminum oxide nanoparticle film was deposited on a glassy carbon electrode to prepare an enzyme-free Glu sensor. This sensor exhibited high sensitivity, fast response, good reproducibility and stability in phosphate buffer, and was not affected by electrolytes in biological samples. Li et al. (Electroanal, 2022, 34(6): 1048-1053) prepared a label-free electrochemical aptamer by utilizing the specific binding between 5-HT and the aptamer, and directly measured the oxidation of 5-HT. The measurement of 5-HT in the concentration range of 1~100 μM was achieved, with a detection limit of 0.3 μM. In addition, there are many other reports on the detection of Glu or 5-HT by electrochemical sensors. However, there is currently no electrochemical sensor that can simultaneously detect Glu and 5-HT. This is because, under certain conditions, Glu is less likely to generate an electrochemical signal and can be considered non-electroactive, while 5-HT is more likely to generate an electrochemical signal and can be considered electroactive. Summary of the Invention

[0004] In view of the shortcomings of existing electrochemical sensors for the detection of Glu and 5-HT, the purpose of this invention is to provide a molecularly imprinted electrochemical sensor that can simultaneously detect glutamate and serotonin.

[0005] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0006] A molecularly imprinted electrochemical sensor capable of simultaneously detecting glutamic acid and serotonin, the molecularly imprinted electrochemical sensor comprising an electrode, and (1) amino-functionalized reduced graphene oxide and (2) a dual-template molecularly imprinted polymer with glutamic acid and serotonin as template molecules and o-phenylenediamine as monomer, sequentially modified on the electrode surface.

[0007] Preferably, the amino-functionalized reduced graphene oxide is prepared by a method comprising the following steps:

[0008] (a) Graphene oxide and 3-aminopropyltriethoxysilane were reacted by heating in a solvent to obtain amino-functionalized graphene oxide;

[0009] (b) The amino-functionalized graphene oxide and the reducing agent are heated in a solvent to obtain amino-functionalized reduced graphene oxide.

[0010] More preferably, the reducing agent is hydrazine hydrate.

[0011] More preferably, the reaction temperature in step (a) is 60~80℃ and the reaction time is 4~7h, and the reaction temperature in step (b) is 90~100℃ and the reaction time is 80~100min.

[0012] In step (a), 3-aminopropyltriethoxysilane is kept in excess relative to graphene oxide. After the reaction, the excess 3-aminopropyltriethoxysilane is washed away. Preferably, the mass ratio of graphene oxide to 3-aminopropyltriethoxysilane is 1:4~5, and the reaction is carried out in an ethanol solvent.

[0013] In step (b), the reducing agent is kept in excess relative to the amino-functionalized graphene oxide. When the reducing agent is hydrazine hydrate, the mass ratio of amino-functionalized graphene oxide to hydrazine hydrate is 5:4.

[0014] Preferably, the molar ratio of glutamic acid, serotonin, and o-phenylenediamine is 1:1:2 to 10; more preferably, the molar ratio of glutamic acid, serotonin, and o-phenylenediamine is 1:1:4 to 8; and most preferably, the molar ratio of glutamic acid, serotonin, and o-phenylenediamine is 1:1:6.

[0015] The method for constructing the above-mentioned molecularly imprinted electrochemical sensor includes the following steps:

[0016] (1) Amino-functionalized reduced graphene oxide was modified onto the electrode surface using a drop-coating method;

[0017] (2) Dissolve glutamic acid and o-phenylenediamine in PBS buffer and let stand for more than 30 minutes to obtain solution A. Dissolve serotonin and o-phenylenediamine in PBS buffer and let stand for more than 30 minutes to obtain solution B. Then combine solution A and solution B to obtain the assembly solution.

[0018] (3) The electrode after step (1) is immersed in the assembly solution and cyclic voltammetry is performed. After the scan is completed, the template molecules are removed to obtain the molecularly imprinted electrochemical sensor.

[0019] Preferably, the amount of amino-functionalized reduced graphene oxide used is 2~10 μg, more preferably, the amount of amino-functionalized reduced graphene oxide used is 4 μg.

[0020] Preferably, the concentration of glutamic acid in the assembly solution is 1 mmol / L. Equal volumes of solutions A and B are combined.

[0021] Preferably, cyclic voltammetry scanning is performed within a potential range of 0–0.8 V at a scan rate of 0.05 V / s. -1 The number of scans is 10 to 25; more preferably, the number of scans is 15.

[0022] Preferably, after scanning, the electrode is first rinsed with water and then placed in hydrochloric acid solution for constant potential elution for 200-800 s. More preferably, the concentration of hydrochloric acid solution is 0.5 mol / L, and constant potential elution is performed at -0.4 V for 400 s.

[0023] The above-mentioned molecularly imprinted electrochemical sensor is applied in the detection of glutamate and serotonin using differential pulse voltammetry. The detection steps include:

[0024] (1) After incubating the molecularly imprinted electrochemical sensor in a standard solution containing glutamic acid and serotonin for a certain period of time, the electrochemical response of the molecularly imprinted electrochemical sensor in K3[Fe(CN)6] / K4[Fe(CN)6] solution was detected by differential pulse voltammetry, and the linear equations of glutamic acid concentration and serotonin concentration with electrochemical response value were obtained.

[0025] (2) After incubating the molecularly imprinted electrochemical sensor in the sample solution for a certain period of time, the electrochemical response of the molecularly imprinted electrochemical sensor in the K3[Fe(CN)6] / K4[Fe(CN)6] solution is detected by differential pulse voltammetry. Based on the corresponding electrochemical response value obtained by detection and the linear equation obtained in step (1), the concentrations of glutamic acid and serotonin are obtained.

[0026] Preferably, the incubation time is 20-25 minutes. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the construction process of the molecularly imprinted electrochemical sensor of the present invention.

[0028] Figure 2 Scanning electron microscope images of GO (a), NRGO (b), MIP / NRGO / GCE (c), and NIP / NRGO / GCE (d).

[0029] Figure 3 Infrared curves for GO, RGO, and NRGO.

[0030] Figure 4 Effective surface area diagrams (a) and Qt for bare GCE, GO / GCE, and NRGO / GCE 1 / 2 Fitting relationship diagram (b).

[0031] Figure 5 The CV (a), EIS (b), and DPV (c) plots of bare GCE, GO / GCE, and NRGO / GCE in 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution are shown.

[0032] Figure 6 The CV (a) and EIS (b) plots are for bare GCE, NRGO / GCE, MIP / NRGO / GCE, and NIP / NRGO / GCE in 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution.

[0033] Figure 7 The DPV response diagram of MIP / NRGO / GCE in 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution after incubation with different substances.

[0034] Figure 8 The current response of MIP / NRGO / GCE and the amount of NRGO modification (a), the ratio of template to monomer (b), are given by ΔI. DPV-Glu (As an evaluation index), the ratio of template to monomer (c, with ΔI) DPV-5-HT The graph shows the relationship between the number of electropolymerization cycles (d), elution time (e), and incubation time (f), which are used as evaluation indicators.

[0035] Figure 9 The DPV diagram (a) of MIP / NRGO / GCE in 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution after incubation with different concentrations of Glu, and the corresponding probe current (ΔI) [Fe(CN)6]3- / 4- Linear relationship between 5-HT and Glu concentration (b); DPV diagram of MIP / NRGO / GCE in 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution after incubation with different concentrations of 5-HT (c), corresponding linear relationship between 5-HT peak current and 5-HT concentration (d), probe current (ΔI) [Fe(CN)6] 3- / 4- The linear relationship between 5-HT concentration and 5-HT concentration (e).

[0036] Figure 10 DPV response after incubating MIP / NRGO / GCE with different analytes. Detailed Implementation

[0037] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0038] Unless otherwise specified, the concentration units mM in this invention represent mmol / L and M represents mol / L.

[0039] The molecularly imprinted electrochemical sensor constructed in this invention, capable of simultaneously detecting glutamic acid and serotonin, includes an electrode, and (1) amino-functionalized reduced graphene oxide and (2) a dual-template molecularly imprinted polymer with glutamic acid and serotonin as template molecules and o-phenylenediamine as a monomer, which are sequentially modified on the electrode surface.

[0040] Amino-functionalized reduced graphene oxide was modified using a drop-coating method.

[0041] The dual-template molecularly imprinted polymer first self-assembles with o-phenylenediamine via template molecules, and then modifies the electrode surface through electropolymerization.

[0042] The principle of this invention's molecularly imprinted electrochemical sensor for simultaneous detection of Glu and 5-HT is as follows: First, the molecularly imprinted electrochemical sensor is incubated in the test solution. Then, the sensor is placed in a K3[Fe(CN)6] / K4[Fe(CN)6] solution, and the generated electrochemical response signal is detected using differential pulse voltammetry. On the one hand, due to the recombination of Glu and 5-HT with the pores of the dual-template molecularly imprinted polymer, the active probe [Fe(CN)6]... 3- / 4- The reduced pathways for electrode transfer and electrochemical reactions decrease the current signal I. [Fe(CN)6] 3- / 4- On the one hand, it decreases; on the other hand, 5-HT will generate its own oxidation peak current signal I. 5-HT Based on the changes in these two signals, simultaneous detection of Glu and 5-HT can be achieved. Example

[0043] The construction process of the molecularly imprinted electrochemical sensor of this invention is as follows: Figure 1 As shown.

[0044] 1. Experimental Section

[0045] 1.1 Experimental Instruments and Reagents

[0046] Three-electrode detection system: glassy carbon electrode (GCE) as working electrode, saturated calomel electrode as reference electrode, and platinum wire electrode as auxiliary electrode; MetrohmAutolab® Nova electrochemical workstation (Metrohm GmbH, Switzerland); FTIR-650 infrared spectrometer (Tianjin Gangdong Technology Co., Ltd.); JSM-6701F cold field emission scanning electron microscope (Nippon Electron Optics Co., Ltd.); Z36HK centrifuge (Hermle GmbH, Germany); DF-101S magnetic stirrer (Zhengzhou Changcheng Science & Industry Trade Co., Ltd.); KQ-50 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), etc.

[0047] Graphene oxide (GO) was purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd.; reagents such as Glu and o-phenylenediamine (OPD) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; 5-HT was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; other chemicals were of analytical grade; 0.1 M PBS buffer (pH=7.4); unless otherwise specified, the water used in the experiment was double-distilled water.

[0048] 1.2 Laboratory Animals

[0049] Kunming mice weighing 18-22 g were purchased from the Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (License No. SYXK(GAN)2019-0002). All operations involving animal experiments strictly complied with the relevant regulations of the school and college and were in accordance with animal ethics.

[0050] 1.3 Preparation of amino-functionalized reduced graphene oxide (NRGO)

[0051] The first step involved functionalizing GO with 3-aminopropyltriethoxysilane (APTES) to obtain amino-functionalized GO (NGO). The specific experimental steps were as follows: 0.1 g of GO was dispersed in 10 mL of anhydrous ethanol and sonicated in a sealed conical flask for 1 h. Then, 0.5 mL of APTES was added to initiate the reaction, and the mixture was magnetically stirred at 60°C for 6 h. The mixture was then washed and filtered several times with anhydrous ethanol to remove unreacted APTES, yielding NGO.

[0052] The second step involves reducing NGO with a reducing agent to obtain NRGO. The specific experimental steps are as follows: Under oil bath conditions, at 5 mg / mL... -1N₂H₄·H₂O was added to the NGO suspension, with a mass ratio of NGO to N₂H₄·H₂O of 5:4. The mixture was heated to 95°C and refluxed for 80–100 min. After the reaction solution cooled to room temperature, it was centrifuged at 13,000 rpm for 6 min, washed three times, the supernatant was discarded, and the precipitate was freeze-dried to obtain NRGO.

[0053] 1.4 Construction of Molecularly Imprinted Electrochemical Sensors

[0054] NRGO was redispersed with ultrapure water to obtain 1 mg·mL⁻¹ -1 NRGO suspension.

[0055] The GCE electrode was polished with 0.30 mm and 0.05 mm alumina powder, then rinsed with ethanol and water. The electrode was then placed in 0.1 M sulfuric acid solution and cyclically scanned using CV at a potential range of -0.4 to 0.8 V until stable. Afterward, it was removed, washed, and dried for later use. 4 μL of 1 mg·mL⁻¹... -1 An NRGO suspension was dropped onto the GCE surface and dried under an infrared lamp to obtain an NRGO / GCE electrode.

[0056] Freshly prepared 0.1 M PBS buffer (pH=7.4) containing 2 mM Glu and 6 mM OPD and 0.1 M PBS buffer (pH=7.4) containing 2 mM 5-HT and 6 mM OPD were incubated for 30 min each. This facilitated the self-assembly of the two template molecules and OPD in a single matrix via hydrogen bonding. Then, equal volumes of the two solutions were mixed to obtain an assembly solution containing 1 mM Glu, 1 mM 5-HT, and 6 mM OPD. The NRGO / GCE electrode was then immersed in the assembly solution at a concentration of 0.05 V / s. -1 The electropolymerization of the dual-template molecularly imprinted polymer (MIP) was completed by cyclic voltammetry for 15 consecutive scans within a potential range of 0–0.8 V. Afterward, the electrode was rinsed with double-distilled water to remove unbound and weakly bound monomers and template molecules. Finally, the electrode was eluted in 0.5 M HCl solution at a constant potential of -0.4 V for 400 s to remove the template molecules coated on the MIP layer. After thorough drying, the molecularly imprinted electrochemical sensor (MIP / NRGO / GCE) was obtained. As a comparison, a non-imprinted electrode (NIP / NRGO / GCE) was constructed using the same method, except that no template molecules were present.

[0057] 1.5 Electrochemical Detection

[0058] The electrochemical responses of different modified electrodes and sensors in 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution in 0.1 M KCl were determined using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV).

[0059] Detection of Glu and 5-HT by the sensor: The constructed sensor was incubated in a sample solution containing Glu and 5-HT for a certain period of time, and then the electrochemical response of the sensor in a 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution of 0.1 M KCl was determined by DPV method.

[0060] CV detection conditions: potential range of −0.4~0.8 V, scan frequency of 0.1 V / s. −1 EIS testing conditions: Frequency range 10 -1 ~10 5 Hz, sinusoidal signal amplitude 10 mV, voltage 0.20V; DPV detection conditions: potential range is 0~0.7 V, pulse width 50 ms, pulse amplitude 25 mV, pulse period 0.5 s.

[0061] 1.7 Processing of actual samples

[0062] Normal mouse serum was collected as the actual sample for the detection of Glu and 5-HT. Blood was collected from the eyeball and placed in a 1.5 mL EP tube. It was allowed to coagulate at room temperature for 2 h. After the blood cells settled, it was centrifuged at 3000 rpm for 30 min. The supernatant was collected and aliquoted into 1.5 mL centrifuge tubes and stored at -80℃ for later use.

[0063] 2 Results

[0064] 2.1 Physical characterization of materials and electrodes

[0065] The surface morphology of the material and electrodes was observed using a scanning electron microscope (SEM). Figure 2 'a' is unmodified GO, with a wrinkled surface that is relatively smooth and has a small specific surface area. After amino-functionalization and reductive treatment, NRGO is obtained. Figure 2 b. Its surface becomes rougher due to the emergence of richer wrinkles, and its specific surface area increases significantly. This change can improve the sensitivity of electrode detection. Figure 2 c and Figure 2Images d show the morphological characteristics of MIP / NRGO / GCE and NIP / NRGO / GCE after elution. A comparison clearly shows that MIP / NRGO / GCE exhibits varying degrees of cavity structures, while NIP / NRGO / GCE presents a dense monomer film. This is likely because the template molecules in MIP / NRGO / GCE are involved, leading to their removal after electrochemical elution and the creation of imprinted cavities. This facilitates subsequent specific re-adsorption of target molecules. In contrast, NIP / NRGO / GCE, lacking template molecules, has its electrode surface covered by a dense, non-imprinted polymer film. Even after electrochemical elution, it cannot form effective imprinted pores, resulting in poor conductivity.

[0066] Fourier transform infrared spectroscopy was used to perform infrared spectral analysis on GO, NGO, and NRGO, such as... Figure 3 For GO, 1620 cm -1 Corresponding to C=C stretching vibration, 1398 cm -1 Corresponding to the CO stretching vibration peak of the carboxyl group, these are characteristic absorption peaks of GO. After APTES functionalization, amino-functionalized NGO was obtained. It can be seen that, compared to GO, NGO significantly increased by 1195 cm⁻¹. -1 1074 cm -1 and 793 cm -1 These three characteristic absorption peaks, among which the one at 1230 cm⁻¹ is the most prominent. -1 ~1030 cm -1 The peaks within this range belong to the stretching vibration peaks of the CN bonds in primary and secondary amines, at 793 cm⁻¹. -1 The peaks belong to the stretching vibrations of silicon-oxygen bonds, indicating that APTES has been bound to the GO sheets, achieving amino functionalization of GO. For NRGO, the original NGO peak was at 1398 cm⁻¹. -1 The CO stretching vibration peak of the corresponding carboxyl group disappeared, indicating that the amino-functionalized NGO was reduced after treatment with hydrazine hydrate, which shows that NRGO was successfully prepared.

[0067] 2.2 Effective surface area of ​​different modified electrodes

[0068] Using K3[Fe(CN)6] as a probe molecule, the electrochemical effective surface area of ​​three electrodes—bare GCE, GO / GCE, and NRGO / GCE—was characterized by chronocoulometric analysis. Figure 4 a. According to Anson's equation (1):

[0069]

[0070] n is the electron transfer number, F is the Faraday constant, A is the electrochemical effective surface area of ​​the electrode, c represents the concentration of the substrate in the electrolyte, D is the diffusion coefficient, and Q... dl Q is the charge of the double-layer electric field. ads For induced charge. From Figure 4 b shows that Q and t 1 / 2 A linear relationship exists, and the effective surface areas of bare GCE, GO / GCE, and NRGO / GCE can be calculated from the slope to be 1.33, 1.15, and 3.87 cm², respectively. 2 It is believed that the electrochemical effective surface area of ​​GCE is significantly increased by nearly two times after modification with NRGO, which can greatly promote the enrichment of target molecules Glu and 5-HT on the electrode surface, thereby enhancing the electrochemical response signal of Glu and 5-HT. Subsequent CV, EIS and DPV results also confirmed this.

[0071] 2.3 Electrochemical characterization of electrodes with different modifications

[0072] Bare GCE, GO / GCE, and NRGO / GCE were characterized using CV, EIS, and DPV in a 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution of 0.1 M KCl. Figure 5 Figure a shows a comparison of the CV responses of different modified electrodes. It can be seen that when GCE is modified with NRGO (NRGO / GCE), the peak current value is significantly larger than that of unmodified GCE and graphene oxide-modified GCE (GO / GCE). This is because NRGO, after undergoing amino functionalization and reduction, possesses a wrinkled structure of varying degrees and a faster electron transfer rate, thereby increasing the specific surface area and conductivity of the electrode. Electrochemical indexing (EIS) is an effective method for studying the surface and interfacial characteristics of modified electrodes, such as... Figure 5 b is a comparison of the EIS responses of different modified electrodes. Relatively regular semicircles can be observed in the high-frequency region, and the diameter of these semicircles is related to the electron transfer resistance (R0). ct This is consistent with the conductivity characteristics of the electrode-solution interface. It can be seen that the semicircle of NRGO / GCE has a smaller diameter compared to other electrodes. ct The smaller size indicates that NRGO modification reduces electron transport resistance and effectively promotes electron transport on the electrode surface. The DPV results also confirm this. Figure 5 c. NRGO / GCE exhibits the largest DPV response. In other words, the results from CV, EIS, and DPV are consistent, indicating that NRGO / GCE has superior conductivity under the same conditions.

[0073] 2. Electrochemical characterization of the 4-molecule imprinted electrochemical sensor

[0074] The construction process of molecularly imprinted electrochemical sensors was characterized using colorimetry (CV). For example... Figure 6 a. The bare GCE exhibits a pair of reversible peaks. The peak current increases after adding NRGO to the electrode surface, indicating that NRGO has good conductivity and current amplification, enhancing the response of the base electrode. After constructing a MIP on the NRGO / GCE surface, the peak current decreases, indicating that the MIP prepared under these conditions has poor conductivity. Even if template molecules are eluted and form imprinted cavities, this partially hinders the active probe from reaching the electrode surface for electron transfer, thus reducing the peak current. For NIP / NRGO / GCE, the decrease in peak current is more pronounced, with almost no observable response. This suggests that MIP / NRGO / GCE with template molecules forms imprinted cavities after elution, while the polymer layer on the surface of NIP / NRGO / GCE without template molecules is dense and has poor conductivity. Even after elution, imprinted cavities cannot be formed, preventing the active probe from successfully penetrating the polymer film to reach the electrode surface.

[0075] To further illustrate the formation of molecularly imprinted polymers, the sensor construction process was also characterized using EIS, such as... Figure 6 b (The inset shows an enlarged view of GCE, NRGO / GCE, and NIP / NRGO / GCE). Compared to GCE, NRGO / GCE has a smaller diameter, R... ct Smaller. When MIPs are constructed on the NRGO / GCE surface, their semicircles become larger, R ct The increased size indicates the formation of a poorly conductive polymer on the electrode surface, hindering electron transfer. For NIP / NRGO / GCE, the semicircle has a larger diameter, R... ct Larger. This is because even after elution, no imprinted cavities can be formed on the surface of the NIP / NRGO / GCE electrode. The dense film with low conductivity hinders electron transfer. The EIS analysis results are consistent with the CV analysis results mentioned above.

[0076] 2.5 DPV Response of Molecularly Imprinted Electrochemical Sensor (MIP Electrode) After Incubation with Different Substances

[0077] To illustrate the peak behavior of Glu and 5-HT, the DPV response of the MIP electrode in 1 mL of K3[Fe(CN)6] / K4[Fe(CN)6] solution was investigated using K3[Fe(CN)6] / K4[Fe(CN)6] as the active probe. The results were as follows: Figure 7The MIP electrode incubated with blank PBS and PBS solution containing Glu showed only one peak of the active probe, while the MIP electrode incubated with PBS solution containing 5-HT also showed an additional 5-HT oxidation peak. Therefore, it can be concluded that when an active probe is introduced for detection, I... [Fe(CN)6] 3- / 4- The changes are attributed to the simultaneous adsorption of Glu and 5-HT on the dual-template imprinted electrode, while I 5-HT The changes are only related to 5-HT itself, and based on this, it is possible to detect both simultaneously.

[0078] 2.6 Condition Optimization

[0079] 2.6.1 Optimization of NRGO Modification Dosage

[0080] 1 mg·mL⁻¹ was sequentially dropped onto the surface of the treated bare GCE electrode. -1 NRGO suspensions of 2, 4, 6, 8, and 10 μL were dried and then subjected to CV scanning. The results are as follows: Figure 8 a. As the amount of modification increases, the current response value first increases and then decreases. The response value is the largest when the amount of modification is 4 μL, which is the optimal amount of modification for NRGO.

[0081] 2.6.2 Optimization of the template molecule to monomer ratio

[0082] Following the construction conditions described in section 1.4 above, the concentration of OPD was adjusted to 2, 4, 6, 8, and 10 mM, respectively, to construct molecularly imprinted electrochemical sensors with molar ratios of Glu:5-HT:OPD of 1:1:2, 1:1:4, 1:1:6, 1:1:8, and 1:1:10. The DPV response of Glu and 5-HT were used as evaluation indicators for optimization, and the results are as follows. Figure 8 b and 8c. When the ratio of the three is 1:1:6, regardless of △I DPV-Glu Or △I DPV-5-HT As an evaluation metric, the difference in DPV response reached its maximum value. Therefore, the optimal ratio of Glu, 5-HT, and OPD was determined to be 1:1:6. It should be noted that through ratio optimization, it can be found that regardless of ΔI... DPV-Glu As an evaluation indicator or ΔI DPV-5-HT As an evaluation indicator, the results are consistent. Combined with the fact that the ratios of Glu and 5-HT are roughly equal, it can be assumed that their competitive abilities to enter the imprint cavities are roughly equal. Therefore, in the subsequent optimization of the number of electropolymerization cycles, elution time, and incubation time, only the response signal of Glu was used as the evaluation indicator, and no further optimization studies were conducted using the response signal of 5-HT as the evaluation indicator.

[0083] 2.6.3 Optimization of the number of scan cycles during electropolymerization

[0084] To investigate the effect of the number of electropolymerization cycles on the sensor response, the electrochemical responses were examined for 5, 10, 15, 20, and 25 electropolymerization scan cycles. Electrodes with different numbers of polymerization cycles were eluted, and imprinted pores matching Glu and 5-HT were left on the surface of the polymer film. The more pores, the more Glu and 5-HT reach the electrode surface, and the greater the corresponding response difference. The number of electropolymerization cycles directly affects the thickness of the imprinted film, which in turn affects the number of imprinted pores and the response difference. The results are as follows: Figure 8 d, The maximum ΔI was obtained when the number of electropolymerization cycles was 15. DPV Increasing the number of electropolymerization cycles further reduced the difference, indicating that too few polymerization cycles cannot form sufficiently effective imprint cavities, which will affect the recognition and detection of target molecules; while too many polymerization cycles will result in an overly dense polymer layer, making it difficult to elute template molecules, thus reducing detection performance.

[0085] 3.6.4 Optimization of elution method and elution time

[0086] A comparison of eluents using methanol:glacial acetic acid (V:V=9:1) and 0.5 M HCl revealed that the former was almost ineffective for elution; therefore, 0.5 M HCl was chosen as the final eluent. Using 0.5 M HCl as the eluent, the effects of stirring elution and elution at a constant potential of -0.4 V were investigated. The results showed that stirring elution was not ideal, thus the final elution method was determined to be elution at a constant potential of -0.4 V. This may be because the double-imprinted polymer film is denser than the single-imprinted polymer film, making it difficult to elute the template molecules using only the physical method of stirring elution; a constant potential electrochemical method is required for successful elution.

[0087] The effect of potentiostatic elution time on the sensor response was then investigated. The results are as follows: Figure 8 e. When the elution time is less than 400 s, the current response difference increases with the increase of elution time, indicating that imprint cavities are constantly forming; however, when the elution time is greater than 400 s, the current difference decreases instead, indicating that excessively long elution time is not conducive to the stable maintenance of imprint cavities. Therefore, the final elution time is determined to be 400 s.

[0088] 2.6.5 Optimization of incubation time

[0089] Too short an incubation time makes it difficult to achieve complete binding of the target molecule to the pores of the imprinted polymer, while too long an incubation time consumes detection time. Therefore, the incubation time of the sensor was also optimized. The results are as follows: Figure 8f. As the incubation time increases, the sensor response difference gradually increases. When it reaches 20 min, the sensor response difference tends to stabilize, indicating that the re-adsorption of target molecules in the sample solution into the pores of the imprinted polymer has reached saturation, which is the optimal incubation time.

[0090] 2.7 Linear range and detection limit for Glu and 5-HT

[0091] Under the optimized conditions described above, the constructed molecularly imprinted electrochemical sensor was incubated in different concentrations of Glu and 5-HT standard solutions, respectively. The electrochemical response of the sensor in a 1 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution of 0.1 M KCl was then measured using the DPV method. For Glu, the results are as follows: Figure 9 a and Figure 9 b, I [Fe(CN)6] 3- / 4- The linear detection range is 1–100 μM, and the linear equation is ΔI. [Fe(CN)6] 3- / 4- =0.92 lnC Glu +0.86 (R) 2 =0.9944), the detection limit is 0.067 μM (S / N=3), where ΔI [Fe(CN)6] 3- / 4- This refers to the peak current difference corresponding to the active probe after the template molecule is removed and re-bound. For 5-HT, the result is as follows: Figure 9 c, with the increase of 5-HT concentration, I [Fe(CN)6] 3- / 4- Decrease, while I 5-HT Increase. I [Fe(CN)6] 3- / 4- The signal drop is due to 5-HT occupying the imprint cavities, while I 5-HT The signal increase is due to the rise in 5-HT concentration. These two signal changes can be combined into a dual signal for 5-HT detection. A linear fit was performed on the 5-HT concentration and its corresponding signal change, and the results are as follows: Figure 9 d and 9e, in the range of 1~100 μM, I 5-HT The change in 5-HT concentration is linearly related to the linear equation: I 5-HT =0.035 lnC 5-HT +0.20 (R) 2 =0.9921); while △I [Fe(CN)6] 3- / 4- The change in 5-HT concentration also showed a linear relationship, with the linear equation being ΔI. [Fe(CN)6] 3- / 4- =0.036 C 5-HT +0.68 (R)2 =0.9903), and the detection limit of this method is 0.047 μM (S / N=3). These results further validate that Glu and 5-HT can be measured using the same sensor.

[0092] 2.8 Selective Investigation

[0093] To avoid interference from structural analogs and potential coexisting components during subsequent testing of actual samples, this invention selected glycine (Gly), tyrosine (Tyr), dopamine (DA), ascorbic acid (AA), and glucose as interfering agents, with [Fe(CN)6] as the target. 3- / 4- The response value was used as the evaluation index. The ΔI values ​​of the molecularly imprinted electrochemical sensor of this invention were measured before and after incubation in PBS solutions containing 10 μM Glu, 10 μM 5-HT, and 20 μM interfering substances. DPV To verify the selectivity and anti-interference capabilities of MIP / NRGO / GCE, the results are as follows: Figure 10 The sensor showed a significantly greater difference in current response before and after incubation of Glu and 5-HT than other interfering substances, indicating that the sensor has a good ability to identify Glu and 5-HT and can selectively identify Glu and 5-HT.

[0094] 2.9 Reproducibility, repeatability, and stability

[0095] Five MIP / NRGO / GCE electrodes were constructed using the same method, and DPV measurements were performed on 10 μM Glu and 10 μM 5-HT, respectively. The calculated RSDs for Glu and 5-HT detection results were 3.08% and 4.28%, respectively, indicating that MIP / NRGO / GCE has good reproducibility.

[0096] Repeatability can be assessed by continuously detecting, eluting, and re-detecting the same MIP / NRGO / GCE electrode. The results showed that the RSDs of the sensor for the first three detections of Glu and 5-HT were 3.55% and 4.07%, respectively, indicating that the electrode can still exhibit good electrochemical response within three repeated uses.

[0097] The constructed MIP / NRGO / GCE electrode was stored in a 4°C refrigerator for 10 days, and then the same concentration of Glu and 5-HT solution was measured again. The results showed that the sensor still showed no less than 95.69% of the initial performance, which can be considered as having good stability.

[0098] 2.10 Actual Sample Testing

[0099] In actual sample testing, we can obtain I 5-HT and ,in yes and The sum of these values, combined with the linear equation obtained above, allows us to calculate the concentrations of Glu and 5-HT in the actual sample.

[0100] To evaluate the accuracy and practicality of this method, the standard addition method was used to spike and recover Glu and 5-HT from mouse serum. The same concentrations of Glu and 5-HT were simultaneously spiked into mouse serum, and the results are shown in Table 1. The recoveries ranged from 97.15% to 102.05%, with RSD < 4.05%, indicating that the MIP / NRGO / GCE electrode has good analytical performance and can achieve the detection of Glu and 5-HT in real samples.

[0101] Table 1 Results of spiked recovery tests (n=3)

[0102]

[0103] a is the average of three consecutive measurements.

[0104] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A molecularly imprinted electrochemical sensor capable of simultaneously detecting glutamate and serotonin, characterized in that: The molecularly imprinted electrochemical sensor includes a glassy carbon electrode and (1) amino-functionalized reduced graphene oxide and (2) a dual-template molecularly imprinted polymer with glutamic acid and serotonin as template molecules and o-phenylenediamine as monomer, which are sequentially modified on the surface of the glassy carbon electrode. The amino-functionalized reduced graphene oxide was prepared by a method comprising the following steps: (a) Graphene oxide and 3-aminopropyltriethoxysilane were reacted by heating in a solvent to obtain amino-functionalized graphene oxide; (b) The amino-functionalized graphene oxide and the reducing agent are heated in a solvent to obtain amino-functionalized reduced graphene oxide.

2. The molecularly imprinted electrochemical sensor according to claim 1, characterized in that: The reducing agent is hydrazine hydrate.

3. The molecularly imprinted electrochemical sensor according to claim 1, characterized in that: The reaction temperature for step (a) is 60~80℃ and the reaction time is 4~7h. The reaction temperature for step (b) is 90~100℃ and the reaction time is 80~100min.

4. The molecularly imprinted electrochemical sensor according to claim 1, characterized in that: The molar ratio of glutamic acid, serotonin, and o-phenylenediamine is 1:1:2~10.

5. The molecularly imprinted electrochemical sensor according to claim 1, characterized in that: The molar ratio of glutamic acid, serotonin, and o-phenylenediamine is 1:1:

6.

6. A method for constructing the molecularly imprinted electrochemical sensor according to any one of claims 1 to 5, comprising the following steps: (1) Amino-functionalized reduced graphene oxide was modified onto the electrode surface using a drop-coating method; (2) Dissolve glutamic acid and o-phenylenediamine in PBS buffer and let stand for more than 30 minutes to obtain solution A. Dissolve serotonin and o-phenylenediamine in PBS buffer and let stand for more than 30 minutes to obtain solution B. Then combine solution A and solution B to obtain the assembly solution. (3) The electrode after step (1) is immersed in the assembly solution and cyclic voltammetry is performed. After the scan is completed, the electrode is rinsed with water and then placed in hydrochloric acid solution. The template molecules are removed by constant potential elution to obtain the molecularly imprinted electrochemical sensor.

7. The construction method according to claim 6, characterized in that: The amount of amino-functionalized reduced graphene oxide used is 2~10 μg.

8. The construction method according to claim 7, characterized in that: The amount of amino-functionalized reduced graphene oxide used was 4 μg.

9. The construction method according to claim 6, characterized in that: The concentration of glutamic acid in the assembly solution was 1 mmol / L, and solutions A and B were combined in equal volumes.

10. The construction method according to claim 6, characterized in that: Cyclic voltammetry scans were performed within the potential range of 0–0.8 V at a scan rate of 0.05 V / s. -1 The number of scans is 10-25.

11. The construction method according to claim 10, characterized in that: The number of scans is 15.

12. The construction method according to claim 6, characterized in that: Elute at a constant potential for 200~800 s.

13. The construction method according to claim 12, characterized in that: The hydrochloric acid solution had a concentration of 0.5 mol / L, and elution was performed at a constant potential of -0.4 V for 400 s.

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