Preparation and application of tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode

Abstract

The application belongs to the field of novel nano functional materials and electrochemical biosensing detection technology, and relates to a preparation method of a tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode and application thereof in detection of levodopa. The application aims to solve the problems of insufficient specific recognition performance, low electrocatalytic activity, low detection sensitivity and high detection limit in the detection of levodopa by existing electrochemical biosensors. The main preparation method is as follows: 1. preparation of CuNi@carbon nanosheet array / three-dimensional graphene composite material; 2. preparation of molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode; and 3. preparation of tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode. The electrode shows excellent specific recognition performance and high sensitivity (4.33 muA.mM ‑1 ·cm ‑2 ) when detecting 0-60 muM of levodopa, and the detection limit is 0.018 muM.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and electrochemical biosensor detection technology, and relates to a method for preparing a tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode and its application in the electrochemical detection of levodopa. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Treatment primarily relies on levodopa crossing the blood-brain barrier to replenish depleted dopamine levels. However, a key challenge in PD treatment is the accurate detection of levodopa (LD) concentrations. Too low an LD concentration may lead to treatment failure, while too high a concentration may damage the nervous system, ultimately exacerbating PD symptoms. Therefore, a reliable, accurate, and rapid method for monitoring LD concentrations is urgently needed. Electrochemical detection technology, with its advantages of fast detection speed, high sensitivity, and ease of operation, has rapidly developed and been widely applied. Among these advancements, developing novel electrode materials with high catalytic activity, high conductivity, and excellent interfacial mass transfer capabilities is a crucial approach to improving electrochemical detection performance.

[0003] In recent years, metal / carbon composites derived from metal-organic frameworks (MOFs) have attracted much attention in the field of electrochemical sensing due to their advantages such as low preparation cost, preservation of precursor morphology, high catalytic activity, and good conductivity. Bimetallic nanoparticle / carbon materials derived from MOFs can provide higher metal loading and abundant active sites. Among them, CuNi nanoparticles loaded with carbon, formed by the pyrolysis of CuNi-MOFs, exhibit excellent electrocatalytic activity and conductivity, and are expected to be applied to electrochemical detection in LD (Dielectric Discharge). Furthermore, the nanosheet array (NSA) morphology exposes more accessible active sites and promotes electrolyte penetration and rapid molecular diffusion, thereby significantly improving mass transfer efficiency. Using highly conductive three-dimensional graphene foam as a framework, CuNi@carbon nanosheet arrays can be generated in situ on its surface through high-temperature pyrolysis, forming a continuous conductive network; this structure is beneficial for the adsorption of high surface energy Cu and Ni nanoparticles onto LDs and promotes efficient charge transport.

[0004] Molecular imprinting technology can construct specific recognition cavities in polymer layers that are highly matched to template molecules in terms of size, shape, and functional group distribution, thereby endowing electrochemical sensors with excellent selectivity. Poly(3,4-ethylenedioxythiophene) (PEDOT) has excellent conductivity, electrochemical stability, and good film-forming properties, and is widely used to construct molecularly imprinted polymer sensing interfaces. By electropolymerizing a molecularly imprinted PEDOT layer on the surface of CuNi@carbon nanosheet array / 3D graphene, a specific recognition layer with high selectivity for LD can be formed, thereby establishing a signal response pathway based on a molecularly imprinted recognition mechanism. In addition, tyrosinase exhibits excellent specificity and catalytic activity for LD. Immobilizing tyrosinase on the surface of molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene can achieve efficient enzymatic catalytic signal amplification for LD, thereby constructing a second signal response pathway based on enzymatic catalysis. Therefore, the synergistic integration of molecularly imprinted recognition and enzymatic catalysis holds promise for achieving highly sensitive and selective dual-channel electrochemical detection of LD. In summary, this invention provides a method for preparing a tyrosinase-molecularly imprinted PEDOT / CuNi@CuNi@carbon nanosheet array / three-dimensional graphene composite electrode, and applies it to the electrochemical detection of an LD. Summary of the Invention

[0005] This invention aims to address the problems of insufficient selectivity, low sensitivity, and high detection limit in existing LD electrochemical detection processes, and provides a tyrosinase-modified molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode and its preparation method.

[0006] I. Preparation of CuNi@carbon nanosheet array / 3D graphene composite material Weigh 0.02-0.4 mM copper chloride, 0.02-0.4 mM nickel chloride, and 0.01-0.8 mM terephthalic acid and add them to a mixed solution consisting of 20-80 mL N,N-dimethylformamide (DMF) and 5-20 mL anhydrous ethanol. Stir at room temperature for 1-3 h. Immerse the three-dimensional graphene, which has been oxidized by a mixture of concentrated nitric acid and concentrated sulfuric acid (volume ratio 3:1), into the above precursor solution. Then, transfer the mixture to a 100 mL reaction vessel and keep it at 100-150°C for 6-12 h. After the reaction, wash the mixture three times each with deionized water and ethanol solution. After freeze-drying, calcine it at 500-900°C for 3 h under an argon protective atmosphere to obtain CuNi@carbon nanosheet array / three-dimensional graphene composite material. II. Fabrication of Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode The CuNi@carbon nanosheet array / 3D graphene composite material obtained in step one was loaded onto ITO conductive glass as the working electrode. Platinum wire and silver / silver chloride were used as the counter electrode and reference electrode, respectively. L-DOPA and 3,4-ethylenedioxythiophene (EDOT) were added to 10-40 mL of PBS buffer solution at a molar ratio of 1:1 to 1:5. The mixture was stirred at room temperature for 1-3 h. Cyclic voltammetry was used to scan the potential range of -0.2 to 0.8 V at a scan rate of 5-200 mV / s for 10-100 cycles to electropolymerize a molecularly imprinted PEDOT film on the surface of the working electrode, thus obtaining the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode. III. Fabrication of Tyrosinase-Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode Using the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode obtained in step two as a substrate, 4~16 μL of tyrosinase solution was added dropwise, and cross-linking and fixation were performed using 0.25~2.5% glutaraldehyde (GLU). The reaction was carried out at 4℃ or room temperature for 0.5~12 h. After the reaction, the electrode surface was washed with PBS buffer solution and naturally dried to obtain the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode.

[0007] Compared with existing technologies, the present invention has the following advantages: 1. This invention uses a one-step hydrothermal method combined with a high-temperature pyrolysis method to prepare CuNi@carbon nanosheet array / three-dimensional graphene composite material. By utilizing the excellent conductive network of three-dimensional graphene and the synergistic catalytic effect of CuNi bimetallic nanoparticles, the electron transport capability and electrocatalytic activity of the electrode material are improved. At the same time, the carbon nanosheet array structure increases the number of active sites on the electrode surface, which is beneficial to the rapid adsorption of LD molecules and interfacial electron transfer.

[0008] 2. This invention constructs a molecularly imprinted PEDOT recognition layer on the surface of CuNi@carbon nanosheet array / three-dimensional graphene composite material by electropolymerization. By utilizing the matching effect between the molecularly imprinted cavity and the LD in terms of spatial structure and functional group distribution, the specific recognition of the LD is achieved, effectively reducing the interference of coexisting substances in complex samples on the detection results and improving the detection selectivity.

[0009] 3. In this invention, tyrosinase is further immobilized on the surface of a molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode. By utilizing the specific catalytic effect of tyrosinase on LD, the detection signal is further amplified, thereby achieving high selectivity and high sensitivity detection of LD. It has a good linear response in the concentration range of 0~60 μM, a sensitivity of 4.33 μA·μM⁻¹·cm⁻², and a detection limit as low as 0.018 μM. Attached Figure Description

[0010] Figure 1 Morphological characterization of the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite material; Figure 2 Characterization of the structure, chemical bonding, valence state, and surface composition of the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite material; Figure 3 Electrochemical behavior of different electrodes; Figure 4 A schematic diagram of the DPV response and sensing mechanism of a tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode used for LD detection; Figure 5 Performance of tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode for LD detection in whole blood samples; Detailed Implementation

[0011] Specific Implementation Method 1: A method for preparing a tyrosinase-modified molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode, comprising the following steps: I. Preparation of CuNi@carbon nanosheet array / 3D graphene composite material Weigh 0.02-0.4 mM copper chloride, 0.02-0.4 mM nickel chloride, and 0.01-0.8 mM terephthalic acid and add them to a mixed solution consisting of 20-80 mL N,N-dimethylformamide (DMF) and 5-20 mL anhydrous ethanol. Stir at room temperature for 1-3 h. Immerse the three-dimensional graphene, which has been oxidized by a mixture of concentrated nitric acid and concentrated sulfuric acid (volume ratio 3:1), into the above precursor solution. Then, transfer the mixture to a 100 mL reaction vessel and keep it at 100-150°C for 6-12 h. After the reaction, wash the mixture three times each with deionized water and ethanol solution. After freeze-drying, calcine it at 500-900°C for 3 h under an argon protective atmosphere to obtain CuNi@carbon nanosheet array / three-dimensional graphene composite material. II. Fabrication of Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode The CuNi@carbon nanosheet array / 3D graphene composite material obtained in step one was loaded onto ITO conductive glass as the working electrode. Platinum wire and silver / silver chloride were used as the counter electrode and reference electrode, respectively. L-DOPA and 3,4-ethylenedioxythiophene (EDOT) were added to 10-40 mL of PBS buffer solution at a molar ratio of 1:1 to 1:5. The mixture was stirred at room temperature for 1-3 h. Cyclic voltammetry was used to scan the potential range of -0.2 to 0.8 V at a scan rate of 5-200 mV / s for 10-100 cycles to electropolymerize a molecularly imprinted PEDOT film on the surface of the working electrode, thus obtaining the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode. III. Fabrication of Tyrosinase-Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode Using the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode obtained in step two as a substrate, 4~16 μL of tyrosinase solution was added dropwise, and cross-linking and fixation were performed using 0.25~2.5% glutaraldehyde (GLU). The reaction was carried out at 4℃ or room temperature for 0.5~12 h. After the reaction, the electrode surface was washed with PBS buffer solution and naturally dried to obtain the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode.

[0012] Specific Implementation Method Two: A method for preparing a tyrosinase-modified molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode, comprising the following steps: The difference between this embodiment and embodiment one is that in step one, the CuNi@carbon nanosheet array / three-dimensional graphene composite material is obtained by calcination at 500~700℃ for 3 h under an argon protective atmosphere. Otherwise, it is the same as embodiment one. Specific Implementation Method 3: A method for preparing a tyrosinase-modified molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode, comprising the following steps: The difference between this embodiment and Embodiment 1 and Embodiment 2 is that in step 2, levodopa and 3,4-ethylenedioxythiophene (EDOT) are added to 15-30 mL of PBS solution at a molar ratio of 1:3 to 1:5, stirred for 1-3 hours, and cyclic voltammetry is used in the range of -0.2-0.6V at a scan rate of 30-60 mV / s for 10-60 cycles. The rest is the same as in Embodiment 1 to Embodiment 2. The effectiveness of the present invention was verified by the following experiments: This experiment presents a method for preparing a tyrosinase-modified molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode, comprising the following steps: I. Preparation of CuNi@carbon nanosheet array / 3D graphene composite material 0.2 mM copper chloride, 0.2 mM nickel chloride, and 0.4 mM terephthalic acid were weighed and added to a mixed solution consisting of 60 mL N,N-dimethylformamide (DMF) and 15 mL anhydrous ethanol. The mixture was stirred at room temperature for 1 h. The three-dimensional graphene oxidized by a mixture of concentrated nitric acid and concentrated sulfuric acid (volume ratio 3:1) was immersed in the above precursor solution. The mixture was then transferred to a 100 mL reactor and reacted at 125°C for 12 h. After the reaction, the mixture was washed three times each with deionized water and ethanol solution. After freeze-drying, the mixture was calcined at 600°C for 3 h under an argon atmosphere to obtain CuNi@carbon nanosheet array / three-dimensional graphene composite material. II. Fabrication of Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode The CuNi@carbon nanosheet array / 3D graphene composite material obtained in step one was loaded onto ITO conductive glass as the working electrode. Platinum wire and silver / silver chloride were used as the counter electrode and reference electrode, respectively. L-DOPA and 3,4-ethylenedioxythiophene (EDOT) were added to 20 mL of PBS buffer solution at a molar ratio of 1:4. The mixture was stirred at room temperature for 1 h. Cyclic voltammetry was used to scan the potential range of -0.2 to 0.6 V for 45 cycles at a scan rate of 50 mV / s to electropolymerize on the surface of the working electrode to form a molecularly imprinted PEDOT film, thus obtaining the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode. III. Fabrication of Tyrosinase-Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode Using the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode obtained in step two as a substrate, 12 μL of tyrosinase solution was added dropwise, and cross-linking and fixation were performed using 1% glutaraldehyde (GLU). The reaction was carried out at 4℃ or room temperature for 0.5 h. After the reaction, the electrode surface was washed with PBS buffer solution and allowed to dry naturally to obtain the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode.

[0013] This invention uses a three-electrode testing system to study the electrochemical properties of materials. The three electrodes consist of a platinum wire as the counter electrode, a silver / silver chloride electrode as the reference electrode, and a 0.1 M PBS solution as the electrolyte.

[0014] Figure 1 The morphology and structure of a tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite material (TYR / MI-PEDOT / CuNi@CNSA / 3D GF) are shown. CuNi@CNSA is uniformly grown on the surface of 3D GF, forming an interwoven nanosheet array structure, and the nanosheet surface is uniformly loaded with a large number of nanoparticles. Figure 1 After the MI-PEDOT layer is formed by electropolymerization on the CuNi@CNSA / 3D GF surface, the composite electrode still retains the original nanosheet array structure characteristics. Figure 1 d). After further fixation of TYR, the overall morphology of TYR / MI-PEDOT / CuNi@CNSA / 3D GF did not change significantly, but the size of the surface-loaded particles increased slightly, indicating that TYR was successfully modified onto the electrode surface. TEM images further showed that the nanosheet surface was uniformly covered with a large number of nanoparticles with a particle size of approximately 10–15 nm. Figure 1 High-resolution TEM images show that the nanoparticles have lattice fringe spacings of 0.208 nm and 0.205 nm, corresponding to the (111) crystal planes of Cu and Ni, respectively. Figure 1 Furthermore, the SAED pattern of CuNi@CNSA shows diffraction rings corresponding to the Cu and Ni (111) crystal planes, further confirming the formation of CuNi nanoparticles. Meanwhile, no obvious diffraction rings of carbon material crystals were observed, indicating that the MOF-derived carbon nanosheets mainly exhibit an amorphous structure. Figure 1 j). EDS elemental distribution results for CuNi@CNSA show that C, Ni, and Cu elements are uniformly distributed in the nanosheet structure. Figure 1 kn).

[0015] Figure 2 The crystal structure, chemical bonding, elemental valence states, and surface composition of TYR / MI-PEDOT / CuNi@CNSA / 3D GF are shown. Characteristic diffraction peaks of CuNi-MOFs appear at 2θ = 8.5°, 14.4°, 15.9°, 16.9°, and 29.9°. After high-temperature pyrolysis, the characteristic diffraction peaks of CuNi-MOFs disappear, while new diffraction peaks appear at 43.5°, 44.4°, 50.4°, and 51.5°, corresponding to the (111) and (200) crystal planes of Cu (JCPDS No. 04-0836) and Ni (JCPDS No. 04-0850), respectively. Furthermore, the intensity of the diffraction peak at 26.4° increases, and this peak is attributed to the (002) crystal plane of the carbon material. Figure 2a). The Raman spectrum of CuNi@CNSA / 3D GF shows three characteristic peaks at 1345, 1590, and 3045 cm⁻¹, corresponding to the D-band, G-band, and 2D-band of the carbon nanosheet array, respectively. Figure 2 b). Due to the lack of Raman active vibrational modes in metallic Cu and Ni, no obvious CuNi-related Raman peaks were observed. I0 of CuNi@CNSA / 3D GF D / I G A ratio of 1.08 indicates the presence of abundant defect structures and disordered carbon regions in the carbon framework, suggesting a low degree of graphitization. Meanwhile, the 2D peak near 3045 cm⁻¹ indicates the formation of a multilayered, stacked carbon structure.

[0016] XPS Full Spectrum ( Figure 2 c) The composite material exhibits characteristic peaks for C 1s, O 1s, Ni 2p, and Cu 2p, indicating the presence of C, O, Ni, and Cu elements. The C 1s spectrum can be fitted with three peaks at 284.8, 285.3, and 288.9 eV, corresponding to C-C, CO, and OC=O bonds, respectively. Figure 2 d). The O 1s spectrum can be decomposed into peaks at 529.5, 531.9, and 533.3 eV, which are attributed to metal-oxygen bonds (MO), surface-adsorbed oxygen, and hydroxyl groups (OH⁻), respectively. Figure 2 e). Ni 2p spectrum ( Figure 2 In f), the peaks at 853.2 and 870.9 eV correspond to metallic Ni. 0 The peaks at 855.7 and 873.9 eV are attributed to Ni²⁺, while Ni²⁺ satellite peaks appear at 861.4 and 879.9 eV, indicating that some Ni species have undergone oxidation on the surface. Cu 2p spectrum ( Figure 2 In g), the peaks at 932.6 and 952.1 eV correspond to Cu 2p3 / 2 and Cu 2p1 / 2, respectively, and mainly originate from Cu. 0 / Cu⁺ species; the peaks at 935.1 and 955.1 eV and the satellite peaks at 941.8 and 961.8 eV correspond to Cu²⁺ species, further indicating the presence of partially oxidized Cu species on the material surface. The coexistence of metallic and oxidized Cu and Ni species mainly stems from the surface-sensitive nature of XPS, whose detection depth is typically only a few nanometers, enabling the detection of oxide layers formed on the surface of metal nanoparticles exposed to air. FT-IR spectroscopy ( Figure 2The results showed that both CuNi@CNSA / 3D GF and TYR / MI-PEDOT / CuNi@CNSA / 3D GF exhibited a broad absorption peak at 3435 cm⁻¹, corresponding to the OH stretching vibration. Characteristic peaks at 1084, 980, and 855 cm⁻¹ corresponded to the COC and CSC vibrations in the PEDOT structure, respectively; the absorption peak at 539 cm⁻¹ was attributed to the MO bond stretching vibration. After fixing TYR, the absorption peak at 1640 cm⁻¹ was significantly enhanced, which is attributed to the C=O stretching vibration in the amide bond, indicating that TYR was successfully fixed on the MI-PEDOT / CuNi@CNSA / 3D GF electrode surface. Figure 2 i). In summary, the analytical results collectively demonstrate the successful construction of the TYR / MI-PEDOT / CuNi@CNSA / 3D GF composite electrode.

[0017] Figure 3 The electrochemical behavior and performance optimization results of electrodes prepared under different conditions are presented. Cyclic voltammetry (CV) curves are also shown. Figure 3 In section a), the CuNi@CNSA / 3D GF electrode exhibited a small CV curve area and a low redox peak current, indicating limited electrochemical activity. After the electropolymerization of the MI-PEDOT layer on the CuNi@CNSA / 3D GF surface to form a layer, the CV curve area and redox peak current of the electrode showed a trend of first increasing and then decreasing with the increase of the LD to EDOT molar ratio. When the LD to EDOT molar ratio was 1:4, the MI-PEDOT / CuNi@CNSA / 3D GF electrode exhibited the largest CV curve area and a high redox peak current, indicating that this ratio is conducive to the formation of an appropriate number of molecularly imprinted recognition sites and maintaining good electron transport capability. After further immobilization of TYR, the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode showed the largest CV curve area and the highest redox peak current, indicating that tyrosinase modification further enhanced the electrochemical response of the electrode.

[0018] Electrochemical impedance spectroscopy (EIS) was used to further investigate the effects of different TYR immobilization amounts (4, 8, 12, and 16 μL) on electrode performance. Figure 3 b). As the amount of TYR fixed increases, the semi-circular diameter in the high-frequency region gradually increases, i.e., the charge transfer resistance (Rct) increases from 8 Ω to 11 Ω, indicating that excessive TYR loading may hinder electron transport. Simultaneously, after TYR fixation, the slope in the low-frequency region gradually decreases with increasing TYR dosage, reflecting an increase in the diffusion impedance at the electrode interface. Based on the optimal TYR fixation amount (12 μL), the effect of different GLU mass fractions on tyrosinase fixation was further investigated. Figure 3c). As the mass fraction of GLU increases from 0.25% to 2.5%, Rct increases from 9 Ω to 18 Ω, and the slope in the low-frequency region gradually decreases, indicating that excessively high crosslinking agent concentration will increase interfacial mass transfer resistance. Figure 3 d represents the equivalent circuit model used to fit the EIS data, including solution resistance (Rs), constant phase angle element (CPE), charge transfer resistance (Rct), and Warburg impedance (W). Under optimized conditions (12 μL TYR and 0.5% GLU), the electrode exhibits a low Rct value (10 Ω). The electrolyte pH has a significant impact on the LD electrocatalytic oxidation process. Figure 3 e). As the pH value increased, the oxidation peak current first increased and then decreased, reaching its maximum at pH=7, indicating that neutral conditions are more favorable for the electrochemical oxidation reaction of LD on this electrode surface. Since the catalytic activity of TYR is affected by temperature, the catalytic stability of the electrode at different temperatures (15~55℃) was further investigated. Figure 3 f). Within the temperature range of 15–55 °C, the absorbance at 475 nm decreased by only about 0.1%, indicating that the electrode maintains good catalytic performance over a wide temperature range. Figure 3 g). Considering both stability and ease of operation, all subsequent experiments were conducted at room temperature (25℃). Furthermore, the effect of scan rate on the electrochemical behavior of the electrode was investigated. When the scan rate increased from 15 mV·s⁻¹ to 50 mV·s⁻¹, the redox peak current gradually increased, and the area of ​​the CV curve significantly increased ( Figure 3 The oxidation peak current showed a good linear relationship with the square root of the scan rate, and the fitting equation was: Ipa=(−0.201±0.013)+(0.103±0.003)v 1 / 2 (R²=0.9969) indicates that the electrochemical reaction process of LD on the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode is mainly diffusion-controlled. Figure 3 i).

[0019] The electrochemical detection performance of the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode for LD was studied using DPV, and the mechanism of this electrode's high-sensitivity recognition of LD was further explored through density functional theory (DFT) calculations. Figure 4 For the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode, its oxidation peak current gradually increases with increasing LD concentration (0~60 μM). Figure 4 a). A good linear relationship is observed between the oxidation peak current and the LD concentration, and the linear fitting equation is: I LD =(38.932±2.191)+(1.666±0.071)C LD(R²=0.9929) Figure 4 b). Under optimized conditions, the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode exhibited the highest detection sensitivity, reaching 4.33 μA·μM⁻¹·cm⁻². To further elucidate the sensing mechanism of this electrode for LD, a schematic diagram of the LD detection mechanism based on the synergistic effect of the dual channels was constructed ( Figure 4 c). TYR, immobilized on the surface of the MI-PEDOT layer, serves as a catalytic channel, catalyzing the oxidation of LD to dopaquinone, promoting electron generation, and achieving rapid electron transport through a highly conductive network constructed from MI-PEDOT and CuNi@CNSA / 3D GF, thereby enhancing the electrochemical response signal. Simultaneously, the MI-PEDOT layer acts as a molecular recognition channel; the molecularly imprinted cavity formed within it can achieve specific recognition of LD through spatial matching and functional group interactions, improving the sensor's selectivity. To further reveal the interaction between LD and the electrode interface, DFT calculations were used to construct adsorption models of LD on the MI-PEDOT / Cu(111) and MI-PEDOT / Ni(111) surfaces. The calculation results show that the adsorption energies of LD on the MI-PEDOT / Cu(111) and MI-PEDOT / Ni(111) surfaces are -5.59 eV and -0.48 eV, respectively, indicating that LD can form stable adsorption at the composite interface. Figure 4 After LD adsorption, both the MI-PEDOT / Cu(111) and MI-PEDOT / Ni(111) models showed significant charge redistribution, forming electron-rich regions (red) and electron-depleted regions (blue), and electrons were transferred from LD to the MI-PEDOT / Cu and MI-PEDOT / Ni interfaces. Figure 4 The above results indicate that the MI-PEDOT / CuNi@CNSA interface can promote the stable adsorption of LD molecules and interfacial electron transport. In summary, the efficient enzymatic catalysis of TYR combined with the specific molecular recognition ability of MI-PEDOT forms a synergistically enhanced dual-channel detection mechanism, achieving high sensitivity and high selectivity for LD detection.

[0020] To verify the self-filtration capability of polyacrylamide (PAM-H) hydrogel for blood samples, blood was first added to a glass bottle containing PAM-H for observation. During the 1-60 min test period, no blood penetration through the PAM-H structure was observed. Figure 5a). Further, when methylene blue (MB) stained blood was dropped onto the surface of PAM-H, a distinct blue color was observed inside PAM-H, while large molecular components in the blood did not show significant penetration. This indicates that large molecules such as blood cells, fibrinogen, and lipids can be effectively retained by PAM-H, while small aqueous components containing MB can pass through the PAM-H structure ( Figure 5 b). Subsequently, the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode was combined with a portable electrochemical sensor and further integrated with PAM-H hydrogel to evaluate its detection stability in complex blood environments. The current response of the unmodified PAM-H electrode decreased significantly after contact with blood, while the current signal of the electrode with PAM-H remained basically stable. Figure 5 c). The results showed that PAM-H can effectively block direct contact between macromolecular components in blood and the electrode interface, reducing the impact of biocontamination on electrode performance and enabling the electrode to maintain a stable electrochemical response in a blood environment. Further evaluation was conducted using IT to assess the electrode's ability to perform point-of-care testing (POCT) of LD in a blood environment. Within the 0–20 μM LD concentration range, the current response gradually increased with increasing LD concentration. Figure 5 d). A good linear relationship exists between LD concentration and current response, and the linear fitting equation is: I LD =(29.529±5.033)+(10.705±0.674)C LD (R²=0.9804) Figure 5 e). The above results indicate that the TYR / MI-PEDOT / CuNi@CNSA / 3D GF electrode with PAM-H self-filtration function can effectively reduce blood matrix interference and achieve rapid quantitative detection of LD in the blood environment.

Claims

1. The preparation and application of a tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode, characterized in that... The preparation method of this composite material electrode is carried out according to the following steps: I. Preparation of CuNi@carbon nanosheet array / 3D graphene composite material Weigh 0.02-0.4 mM copper chloride, 0.02-0.4 mM nickel chloride, and 0.01-0.8 mM terephthalic acid and add them to a mixed solution consisting of 20-80 mL N,N-dimethylformamide (DMF) and 5-20 mL anhydrous ethanol. Stir at room temperature for 1-3 h. Immerse the three-dimensional graphene, which has been oxidized by a mixture of concentrated nitric acid and concentrated sulfuric acid (volume ratio 3:1), into the above precursor solution. Then, transfer the mixture to a 100 mL reaction vessel and keep it at 100-150°C for 6-12 h. After the reaction, wash the mixture three times each with deionized water and ethanol solution. After freeze-drying, calcine it at 500-900°C for 3 h under an argon protective atmosphere to obtain CuNi@carbon nanosheet array / three-dimensional graphene composite material. II. Fabrication of Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode The CuNi@carbon nanosheet array / 3D graphene composite material obtained in step one was loaded onto ITO conductive glass as the working electrode. Platinum wire and silver / silver chloride were used as the counter electrode and reference electrode, respectively. L-DOPA and 3,4-ethylenedioxythiophene (EDOT) were added to 10-40 mL of PBS buffer solution at a molar ratio of 1:1 to 1:

5. The mixture was stirred at room temperature for 1-3 h. Cyclic voltammetry was used to scan the potential range of -0.2 to 0.8 V at a scan rate of 5-200 mV / s for 10-100 cycles to electropolymerize a molecularly imprinted PEDOT film on the surface of the working electrode, thus obtaining the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode. III. Fabrication of Tyrosinase-Molecularly Imprinted PEDOT / CuNi@Carbon Nanosheet Array / Three-Dimensional Graphene Composite Electrode Using the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode obtained in step two as a substrate, 4~16 μL of tyrosinase solution was added dropwise, and cross-linking and fixation were performed using 0.25~2.5% glutaraldehyde (GLU). The reaction was carried out at 4℃ or room temperature for 0.5~12 h. After the reaction, the electrode surface was washed with PBS buffer solution and naturally dried to obtain the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / 3D graphene composite electrode.

2. The method for preparing the CuNi@carbon nanosheet array / three-dimensional graphene composite material according to claim 1, characterized in that, In step one, 0.1-0.4 mM copper chloride, 0.1-0.4 mM nickel chloride and 0.2-0.8 mM terephthalic acid are weighed and added to a mixed solution consisting of 40-80 mL of N,N-dimethylformamide (DMF) and 10-20 mL of anhydrous ethanol.

3. The method for preparing the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode according to claim 1, characterized in that, In step two, levodopa and 3,4-ethylenedioxythiophene (EDOT) in a molar ratio of 1:2 to 1:4 were added to 10 to 30 mL of PBS buffer solution and stirred at room temperature for 1 to 3 h. Cyclic voltammetry was then used to scan the potential range of -0.2 to 0.6 V at a scan rate of 10 to 100 mV / s for 15 to 80 cycles.

4. The method for preparing the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode according to claim 1, characterized in that... In step three, glutaraldehyde (GLU) with a mass fraction of 0.25~1% is used as a crosslinking agent to immobilize 8~16 μL of tyrosinase on the surface of the molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode to form a tyrosinase modified layer.

5. The application of the tyrosinase-molecularly imprinted PEDOT / CuNi@carbon nanosheet array / three-dimensional graphene composite electrode prepared by the method according to any one of claims 1 to 4 in the electrochemical detection of levodopa.

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