Electrochemical biosensor, preparation method and application

Abstract

The invention discloses an electrochemical biosensor as well as a preparation method and application thereof, belongs to the technical field of detection, and aims to improve the detection sensitivity, specificity and detection accuracy of key biochemical substances of neurodegenerative diseases. The sensor comprises a substrate layer, a liquid inlet, a liquid outlet, a reference electrode and a counter electrode, the substrate layer comprises a PDMS flexible lower cover layer, a P3HT-Cu NWs conductive sensitive layer and a PDMS flexible upper cover layer, a micro-channel layer is arranged on the PDMS flexible upper cover layer, a channel is arranged in the micro-channel layer, and the P3HT-Cu NWs conductive sensitive layer is connected with a flexible flat cable. The preparation method comprises the following steps: preparing Cu NWs, preparing a P3HT-Cu NWs mixed solution, preparing a PDMS flexible lower cover layer, coating the PDMS flexible lower cover layer with the P3HT-Cu NWs mixed solution to obtain a P3HT-Cu NWs conductive sensitive layer, preparing a PDMS flexible upper cover layer, and bonding and sealing the PDMS flexible lower cover layer and the PDMS flexible upper cover layer to obtain the substrate layer.

Description

Technical Field

[0001] This invention belongs to the field of detection technology and relates to an electrochemical biosensor, particularly to an electrochemical biosensor for detecting biochemical substances in neurodegenerative diseases, its preparation method, and its application in the detection of biochemical substances in neurodegenerative diseases. Background Technology

[0002] In recent years, electrochemical biosensors have rapidly gained widespread attention due to their sensitivity and selectivity, offering advantages such as fast response, rapid result processing, user-friendliness, and low cost. An electrochemical biosensor typically consists of three parts: a biological receptor, a sensing electrode, and an electrochemical workstation. The electrochemical workstation converts the biological reaction signals occurring at the electrode into various forms of electrical signals (voltage, current, resistance), and by measuring these electrical signals, it determines the presence and concentration of the analyte.

[0003] In recent years, the application of this electrochemical (biological) sensor in the healthcare field has become increasingly frequent. For example, patent application number 2024115912884 discloses a biosensor of an organic electrochemical transistor and its fabrication method, which includes: a flexible substrate; a first gate; the first gate is placed above the flexible substrate; the first gate is a porous vertical gold nanowire gate, and multiple micron-sized conical micropores are disposed on the surface of the first gate, with the large opening end of the micron-sized conical micropores penetrating downward through the flexible substrate; a bio-oxidase and a biocatalyst are deposited above the first gate; the first gate has a Janus structure, with a hydrophilic top surface and a hydrophobic bottom surface of the flexible substrate; a second gate; the second gate is placed above the flexible substrate; the second gate is connected to the first gate; a stretchable source; a stretchable drain; the stretchable source and stretchable drain are placed side by side and spaced apart above the flexible substrate; a stretchable active layer; the stretchable active layer is placed above the stretchable source and stretchable drain; an electrolyte layer; the electrolyte layer is placed above the stretchable active layer and the second gate; and a flexible encapsulation layer; the flexible encapsulation layer covers the second gate, the stretchable source, the stretchable drain, the electrolyte, and the stretchable active layer. In this sensor, due to the capillary force of the conical micropores and the difference in hydrophilicity and hydrophobicity between the upper and lower surfaces of the device, sweat is spontaneously absorbed from the substrate side and transferred to the biofunctionalized vertical gold nanowire side, thus realizing the automated extraction and detection of sweat.

[0004] Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) severely affect muscle control, including muscle rigidity and tremors. These diseases can also lead to severe cognitive impairment, significantly impacting patients' physical and mental health. Taking Parkinson's disease (PD) as an example, its progression is closely related to the gradual degeneration of dopamine neurons in the brain. Therefore, real-time electrochemical monitoring of key biochemical substances (such as dopamine (DA) and ascorbic acid (AA)) is crucial for early diagnosis and disease surveillance.

[0005] Patent application No. 202511196769.X discloses a photoelectrochemical sensor for dopamine concentration detection, its preparation method, and its application. This photoelectrochemical sensor includes a working electrode, a counter electrode, and a reference electrode. The sensor is obtained by sequentially connecting the working electrode, counter electrode, and reference electrode to an electrochemical workstation. The working electrode is an indium tin oxide (ITO) electrode. A Bi4O5Br2 / g-C3N4 / Au composite material is deposited on the surface of the ITO electrode. The Bi4O5Br2 material exhibits photocatalysis, electrocatalysis, and ferroelectricity, and can produce a significant synergistic effect under visible light irradiation, providing a reliable new method for clinical dopamine (DA) detection / monitoring, and showing significant application potential in the early diagnosis of neurodegenerative diseases such as Parkinson's disease.

[0006] For neurodegenerative diseases such as Alzheimer's and Parkinson's, key biochemical substances include dopamine (DA) and ascorbic acid (AA). Simply detecting / monitoring dopamine (DA) as described above is insufficient to meet the application requirements. Therefore, it is necessary to provide an electrochemical biosensor with high selectivity for both dopamine (DA) and ascorbic acid (AA), offering an alternative solution for the detection of key biochemical substances in neurodegenerative diseases. Summary of the Invention

[0007] The purpose of this invention is to provide an electrochemical biosensor, its preparation method, and its application, thereby improving the detection sensitivity, specificity, and accuracy of key biochemical substances (such as dopamine DA and ascorbic acid AA) in neurodegenerative diseases.

[0008] To achieve the above objectives, the present invention specifically adopts the following technical solution: An electrochemical biosensor includes a substrate layer, an inlet and an outlet located on both sides of the substrate layer, a reference electrode and a counter electrode disposed on the top of the substrate layer, and the substrate layer includes a PDMS flexible lower cap layer, a P3HT-CuNWs conductive sensitive layer and a PDMS flexible upper cap layer disposed sequentially from bottom to top, a microchannel layer disposed on the side of the PDMS flexible upper cap layer near the P3HT-Cu NWs conductive sensitive layer, a flow channel disposed in the microchannel layer, and the two ends of the flow channel are connected to the inlet and the outlet, and a flexible ribbon cable is connected to one side of the P3HT-Cu NWs conductive sensitive layer.

[0009] Furthermore, the flow channels within the microchannel layer are serpentine.

[0010] Furthermore, the flow channels within the microchannel layer are tree-shaped, and a recessed cavity is provided in the middle of the PDMS flexible top cover layer. The lower ends of the reference electrode and the counter electrode both extend into the recessed cavity, and the bottom of the recessed cavity is connected to the flow channels of the microchannel layer.

[0011] A method for preparing an electrochemical biosensor includes the following steps: Step 1: Prepare Cu NWs; Step 2: Prepare a mixed solution of P3HT-Cu NWs; Step 3: Prepare a flexible PDMS capping layer; Step 4: Coat a mixed solution of P3HT-Cu NWs onto the PDMS flexible underlayer, and dry to obtain the P3HT-CuNWs conductive sensitive layer. Step 5: Prepare a flexible PDMS capping layer with a microfluidic channel layer; First, a positive mold is made on a silicon wafer using SU-8, and then a PDMS mold is made to obtain a flexible PDMS top cover layer with a microfluidic layer. Step 6: Bond and seal the PDMS flexible lower capping layer with the P3HT-Cu NWs conductive sensitive layer to the PDMS flexible upper capping layer with the microfluidic layer to obtain the substrate layer; Step 7: Open an inlet and an outlet on the substrate layer, and connect the inlet and outlet to the flow channels of the microfluidic layer; set a reference electrode and a counter electrode on the substrate layer.

[0012] Furthermore, in step 1, the specific steps for preparing Cu NWs are as follows: Step 1-1: Take 15-25 mL of a 15% sodium hydroxide (NaOH) solution and preheat it to 50-70°C. In steps 1-2, 0.5-2 mL of a 0.1% copper nitrate (Cu(NO3)2) solution, 0.1-0.2 mL of ethylenediamine (EDA), and 20-30 μL of a 35 wt% (mass percentage) hydrazine (N2H4) solution were added to the sodium hydroxide solution preheated in step 1-1 to obtain the reaction mixture. Steps 1-3: The reaction mixture from steps 1-2 is kept at 50-70°C for 1-3 hours to generate Cu NWs, resulting in a solution containing Cu NWs. Steps 1-4: If a faint red final product is observed during the reaction, it indicates that copper nanowires (Cu NWs) are present. The solution containing Cu NWs is centrifuged at a speed of 2000-3500 rpm to separate the copper nanowires (Cu NWs) from the solution. Steps 1-5: Wash the Cu NWs obtained from separation and collection in steps 1-4 to obtain Cu NWs.

[0013] Furthermore, the specific method for cleaning Cu NWs is as follows: Step 1-5-1, Initial cleaning; The Cu NWs obtained in steps 1-4 are redispersed in deionized water; then the deionized water containing the dispersed Cu NWs is placed in a centrifuge tube and centrifuged at a speed of 2000-3500 rpm. Repeat the above process of dispersing in deionized water and centrifuging 2-4 times to remove inorganic and soluble compounds; 1-5-2, then wash again; The initially cleaned Cu NWs were redispersed in ethanol and centrifuged (centrifugation parameters were the same as those for the initial cleaning) to obtain cleaned Cu NWs. During the subsequent cleaning, the above process of dispersing in ethanol and centrifuging can be repeated 2-4 times to remove residual organic solvents such as ethylenediamine (EDA) and hydrazine (N2H4).

[0014] Furthermore, the specific steps for preparing the P3HT-Cu NWs mixed solution are as follows: Step 2-1: Add 0.02-0.05 g of 2.8 wt% Cu NWs dispersion and 40-50 μL of 2825 μM 3HT solution to 0.5-1.5 mL of chloroform to obtain mixture one; Step 2-2: Add excess ferric chloride hexahydrate to 1.5-2 mL of chloroform to obtain mixture two; Steps 2-3: Perform ultrasonic treatment on mixture one and mixture two respectively, with an ultrasonic treatment time of 0.5-2 hours; Steps 2-4: Filter the ultrasonically treated mixture 2 to obtain a ferric chloride solution; mix and stir the ferric chloride solution with the ultrasonically treated mixture 1, and 3HT is oxidized and polymerized on the Cu NWs surface to obtain a P3HT-Cu NWs mixed solution.

[0015] Furthermore, the specific steps in preparing the flexible PDMS underlayer are as follows: Step 3-1: Mix PDMS and water at a weight ratio of 10:(0.5-2) to form a prepolymer; Step 3-2: Take 3-4g of the above prepolymer and drop it onto a single-sided polished silicon wafer with a diameter of two inches. Remove any remaining air bubbles in the prepolymer using vacuum. Step 3-3: Spin coat the prepolymer after removing bubbles from the silicon wafer onto the substrate at a speed of 400-600 rpm for 6-15 seconds. Steps 3-4: The substrate is thermocured at 60-85°C for 3-5 hours to obtain a flexible PDMS underlayer (thickness approximately 500 μm).

[0016] Furthermore, the specific steps for coating the P3HT-Cu NWs mixed solution are as follows: Step 4-1: The mixed solution of P3HT-Cu NWs obtained in step 2 is uniformly dispersed in a container containing 5-20 mL of deionized water to obtain a suspension; the concentration of the suspension is such that the total mass of P3HT-Cu NWs solids contained in 1 mL of the suspension is 1-4 mg.

[0017] Step 4-2: Take 2 mL of suspension and drop it evenly onto the PDMS flexible cover layer. After the suspension has covered the PDMS flexible cover layer, let it stand at room temperature for 1-2 min, rotate it at 300-500 rpm for 2-5 s, and dry it at 500-800°C for 2-5 h.

[0018] Application of electrochemical biosensors in instruments for detecting dopamine and / or ascorbic acid in neurodegenerative diseases.

[0019] The beneficial effects of this invention are as follows: 1. In this invention, the base layer of the sensor is made of flexible PDMS top and bottom capping layers and a P3HT-Cu NWs conductive sensitive layer. The microchannel layer of the PDMS flexible top capping layer is provided with channels. Target molecules (such as AA and DA) in the detected substance can diffuse to the P3HT-Cu NWs conductive sensitive layer when passing through the microchannel layer. The oxidation peaks of the target molecules under the detection conditions of this structure can reach -0.3V, -0.1V, and +0.16V. When the target molecules diffuse to the P3HT-Cu NWs conductive sensitive layer, the copper nanowires (Cu NWs) achieve high selectivity detection of AA and DA through fine crystals generated by constant potential electrochemical deposition, and generate current signals on the copper nanowires (Cu NWs). The P3HT-Cu NWs conductive sensitive layer has a fast electron transport speed, which improves the detection sensitivity and specificity of the sensor for key biochemical substances (dopamine DA, ascorbic acid AA, etc.) in neurodegenerative diseases, and improves the detection accuracy of the sensor (experimental detection data can be found in the experimental examples of the specific implementation). The entire sensor has good flexibility and stretchability, making it suitable for use as an implantable sensor. The overall mechanical modulus of the sensor is lower than that of brain tissue, making it less likely to induce inflammation. It is also less susceptible to protein adsorption and biofouling, resulting in high sensitivity and reliability.

[0020] 2. In this invention, a recessed cavity is formed in the middle of the PDMS flexible top cover layer, and the flow channels in the microchannel layer are tree-shaped. In this structure, cells are injected into the microchannel layer and adhere to and grow on the P3HT-Cu NWs conductive sensitive layer. Firstly, as the cells divide and grow, excess cells can enter the recessed cavity, which serves to accommodate the cells. Secondly, the syringe needle can be inserted into the recessed cavity at a 45° angle and drip the injected drug onto the cultured cells, stimulating the cells to react, causing the cells to release substances that are detected by the P3HT-Cu NWs conductive sensitive layer.

[0021] 3. In this invention, the P3HT-Cu NWs conductive sensitive layer is partially embedded 1-2 μm into the upper surface of the PDMS flexible capping layer. This reduces interface steps and micro-slits, decreases the probability of microfluidic leakage along the interface, and reduces noise caused by electrochemical baseline drift and flow / pressure changes. This significantly improves the adhesion and durability of the P3HT-Cu NWs conductive sensitive layer and the PDMS flexible capping layer, resulting in better sealing and baseline stability between the two. The surface is smoother with fewer dead corners, reducing non-specific protein / cell adhesion and improving anti-fouling and biocompatibility. The partial embedding can cover part of the copper nanolayer, making the sensitive layer more resistant to oxidation and damp heat, and the conductive network more stable, thus improving environmental stability. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 This is a cross-sectional view of the present invention; Figure 3 This is a schematic diagram of the flow channel structure of the microchannel layer in this invention; Figure 4 This is a schematic diagram of the flow channel structure of the microfluidic layer according to another embodiment of the present invention; The attached figures are labeled as follows: 1-reference electrode, 2-counter electrode, 3-outlet, 4-flexible cable, 5-PDMS flexible lower cover layer, 6-P3HT-Cu NWs conductive sensitive layer, 7-microchannel layer, 8-inlet, 9-recessed cavity, 10-flow channel, 11-PDMS flexible upper cover layer. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0024] Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0025] Example 1 This embodiment provides an electrochemical biosensor for detecting key biochemical substances such as dopamine (DA) and ascorbic acid (AA) in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0026] like Figure 1 , Figure 2 As shown, it includes a base layer, with an inlet 8 on the left side and an outlet 3 on the right side of the base layer, and a reference electrode 1 and a counter electrode 2 on the top of the base layer.

[0027] The substrate layer comprises, from bottom to top, a PDMS flexible lower cap layer 5, a P3HT-Cu NWs conductive sensing layer 6, and a PDMS flexible upper cap layer 11. Both the PDMS flexible lower cap layer 5 and the PDMS flexible upper cap layer 11 are made of PDMS (polydimethylsiloxane). The P3HT-Cu NWs conductive sensing layer 6 is disposed on the PDMS flexible lower cap layer 5. A flow channel 10 is provided on the side of the PDMS flexible upper cap layer 11 closest to the P3HT-Cu NWs conductive sensing layer 6. This layer on the PDMS flexible upper cap layer 11 corresponding to the area where the flow channel 10 is located forms a microchannel layer 7. The PDMS flexible lower cap layer 5 and the PDMS flexible upper cap layer 11 are stacked, the P3HT-Cu NWs conductive sensing layer 6 is bonded to the microchannel layer 7, and the PDMS flexible lower cap layer 5 and the PDMS flexible upper cap layer 11 are bonded and sealed to obtain the substrate layer.

[0028] The liquid inlet 8 on the left side and the liquid outlet 3 on the right side of the substrate layer are connected to the flow channel 10 of the microfluidic layer 7. A flexible cable 4 is connected to one side of the P3HT-Cu NWs conductive sensitive layer 6.

[0029] Preferably, the P3HT-Cu NWs conductive sensing layer 6 is partially embedded 1-2 μm into the upper surface of the PDMS flexible underlayer 5. This partial embedding reduces interface steps and micro-gaps, decreases the probability of microfluidic leakage along the interface, and reduces noise caused by electrochemical baseline drift and flow / pressure changes. It significantly improves the adhesion and durability of the P3HT-Cu NWs conductive sensing layer and the PDMS flexible underlayer, resulting in better sealing and baseline stability between the two. The surface is smoother, with fewer dead corners, reduced non-specific protein / cell adhesion, and better resistance to contamination and biocompatibility. The partial embedding can also cover part of the copper nanolayer, making the sensing layer more resistant to oxidation and damp heat, the conductive network more stable, and improving environmental stability.

[0030] This sensor allows the substance to be detected to enter the channel 10 of the microchannel layer 7, and also allows cells to be cultured within the channel 10 and / or the recessed cavity 9 of the microchannel layer 7. Therefore, the structure of the channel 10 differs for different applications. For allowing the substance to enter and be detected, the channel 10 is a serpentine channel (e.g., Figure 3 (As shown); if used for cell culture and targeting related molecules, the flow channel is a tree-shaped flow channel (e.g. Figure 4 As shown in the figure.

[0031] When the sensor is used to allow the detected substance to enter, the target molecules (DA, AA) enter the flow channel 10 of the microfluidic layer 7 from the inlet 8 and are finally discharged from the outlet 3; the flow channel 10 is preferably a serpentine flow channel (e.g., Figure 3 As shown in the diagram, the lower ends of the reference electrode 1 and the counter electrode 2 can extend into the flow channel 10, or be placed in the liquid outlet pool on one side of the liquid outlet 3 (to directly detect the flowing-out substance). When the target molecule passes through the microfluidic layer 7, it can diffuse to the surface of the P3HT-Cu NWs conductive sensitive layer 6, causing the P3HT-Cu NWs conductive sensitive layer 6 to generate a current signal, which is transmitted outward through the flexible ribbon cable 4 on one side.

[0032] When the sensor is used for cell culture, cells are injected into the microfluidic layer 7 through the inlet 8 and adhere to and grow on the P3HT-Cu NWs conductive sensitive layer 6. During cell culture, the culture medium enters through the inlet 8, perfuses through the microfluidic layer 7, and exits through the outlet 3. The channel 10 is preferably a tree-shaped channel, and the lower ends of the reference electrode 1 and the counter electrode 2 can extend into the channel 10 or be placed in the outlet pool on one side of the outlet 3 (to directly detect the outflowing substances). The syringe needle is inserted into the recessed cavity 9 at a 45° angle, and drugs are injected through the syringe needle and dripped onto the cultured cells to stimulate cell response. Molecules produced or consumed by the cells diffuse in the microfluidic layer 7 to the P3HT-Cu NWs conductive sensitive layer 6, which converts electrochemistry into a current signal, or the cells are covered with P3HT-Cu. The NWs conductive sensing layer 6 alters the interfacial impedance, and the current signal is transmitted outward via a flexible ribbon cable 4 on one side, used to characterize cell activity, metabolism, and drug response. During cell culture, cell division and growth occur, resulting in an increasing number and size of cells, potentially exceeding the space of the original flow channels for cell adhesion. Therefore, preferably, a recessed cavity 9 is formed in the middle of the PDMS flexible top cover layer 11, with its bottom connected to the tree-shaped flow channels 10 of the microfluidic layer 7. The lower ends of both the reference electrode 1 and the counter electrode 2 extend into the recessed cavity 9; during operation, the liquid in the flow channels 10 of the microfluidic layer 7 or the recessed cavity 9 submerges the lower ends of the reference electrode 1 and the counter electrode 2. By providing the recessed cavity 9, some cells can enter the recessed cavity 9 during cell growth, expanding the cell growth space.

[0033] The flexible PDMS underlayer 5 is one of the main innovations of the sensor in this embodiment. The specific steps of building the flexible PDMS underlayer 5 are as follows: Step 3-1: Mix PDMS and water at a ratio of 10:0.8 (by weight) to form a prepolymer.

[0034] Step 3-2: Take 3-4g of the prepolymer and drop it onto a single-sided polished silicon wafer with a diameter of two inches. Remove any remaining air bubbles from the prepolymer using vacuum.

[0035] Step 3-3: Spin coat the prepolymer from the silicon wafer after removing air bubbles onto the substrate at a speed of 400-600 rpm for 6-15 seconds.

[0036] Steps 3-4: The substrate is thermocured at 60-85°C for 3-5 hours to obtain the PDMS flexible underlayer 5 (thickness approximately 500μm).

[0037] The P3HT-CuNWs conductive sensing layer 6 is also one of the main innovations of the sensor in this embodiment. The specific steps for constructing the P3HT-CuNWs conductive sensing layer 6 are as follows: A mixed solution of P3HT-Cu NWs was coated onto the flexible PDMS capping layer 5 to obtain the P3HT-Cu NWs conductive sensing layer 6. The specific steps for coating the P3HT-Cu NWs mixed solution are as follows: Step 4-1: Disperse the mixed solution of P3HT-Cu NWs evenly in a container containing 5-20 mL of deionized water to obtain a suspension; the concentration of the suspension is such that the total mass of P3HT-Cu NWs solids contained in 1 mL of the suspension is 1-4 mg.

[0038] The specific preparation method of the P3HT-Cu NWs mixed solution is as follows: Step 2-1: Add 0.02-0.05 g of 2.8 wt% Cu NWs dispersion and 40-50 μL of 2825 μM 3HT solution to 0.5-1.5 mL of chloroform to obtain mixture one.

[0039] Step 2-2: Add excess ferric chloride hexahydrate to 1.5-2 mL of chloroform. The Fe in the solution... 3+ It can promote the formation of Cu NWs on the electrode surface to form 3HT polymer, resulting in mixed solution II.

[0040] Steps 2-3: Perform ultrasonic treatment on mixture one and mixture two respectively for 0.5-2 hours to promote dispersion and dissolution.

[0041] Steps 2-4: Filter the ultrasonically treated mixture 2 to obtain a ferric chloride solution; mix and stir the ferric chloride solution with the ultrasonically treated mixture 1, and 3HT is oxidized and polymerized on the Cu NWs surface to obtain a P3HT-Cu NWs mixed solution.

[0042] The preparation method of Cu NWs in the Cu NWs dispersion in step 2-1 is as follows: Step 1-1: Preheat the sodium hydroxide solution.

[0043] Take 15-25 mL of a 15% sodium hydroxide (NaOH) solution and preheat it to 50-70°C.

[0044] Step 1-2: Add copper nitrate solution, ethylenediamine and hydrazine solution to the sodium hydroxide solution preheated in step 1-1 to obtain a reaction mixture.

[0045] 0.5-2 mL of 0.1% copper nitrate (Cu(NO3)2) solution, 0.1-0.2 mL of ethylenediamine (EDA), and 20-30 μL of 35 wt% (35% by mass) hydrazine (N2H4) solution were added sequentially to the sodium hydroxide solution preheated in step 1-1 to obtain the reaction mixture.

[0046] In steps 1-3, the reaction mixture reacts to generate Cu NWs, resulting in a solution containing Cu NWs.

[0047] Incubate the reaction mixture from steps 1-2 at 50-70°C for 1-3 hours. During the reaction, ensure proper stirring to promote uniform reaction.

[0048] Steps 1-4 involve centrifuging the solution containing Cu NWs to separate the Cu NWs from the solution.

[0049] After the reaction is complete, if a faint red final product is observed, it indicates that copper nanowires (Cu NWs) have been present. Then, the reaction mixture is placed in a centrifuge tube and centrifuged at a speed of 2000-3500 rpm. Through centrifugation, the copper nanowires (Cu NWs) are separated from the solution.

[0050] Steps 1-5: Wash the Cu NWs obtained from separation and collection in steps 1-4 to obtain Cu NWs.

[0051] Step 4-2: Take 2 mL of suspension and drop it evenly onto the PDMS flexible cover layer 5. After the suspension has covered the PDMS flexible cover layer 5, let it stand at room temperature for 1 min, rotate at 400 rpm for 3 s, and dry at 700°C for 3 h.

[0052] The PDMS flexible capping layer 11 is also one of the main innovations of the sensor in this embodiment. During the fabrication of the PDMS flexible capping layer 11, a male mold is first made on a silicon wafer using an SU-8 mold. During the male mold making process, a structure adapted to the flow channels 10 of the microfluidic layer 7 is incorporated. Then, a PDMS casting process is performed to obtain the PDMS flexible capping layer 11 with the microfluidic layer 7. Based on the structure of the PDMS flexible capping layer 11, the microfluidic layer 7, and the flow channels 10 in this embodiment, those skilled in the art can easily implement the methods for making the male mold and performing the casting process without any creative effort.

[0053] Example 2 This embodiment provides a method for preparing an electrochemical biosensor, used to prepare the electrochemical biosensor described in Example 1. The preparation method specifically includes the following steps: Step 1: Prepare Cu NWs.

[0054] The specific steps for preparing Cu NWs are as follows: Step 1-1: Preheat the sodium hydroxide solution.

[0055] Take 16 mL of a 15% sodium hydroxide (NaOH) solution and preheat it to 52°C.

[0056] Step 1-2: Add copper nitrate solution, ethylenediamine and hydrazine solution to the sodium hydroxide solution preheated in step 1-1 to obtain a reaction mixture.

[0057] 0.7 mL of 0.1% copper nitrate (Cu(NO3)2) solution, 0.12 mL of ethylenediamine (EDA), and 21 μL of 35 wt% (35% by mass) hydrazine (N2H4) solution were added sequentially to the sodium hydroxide solution preheated in step 1-1 to obtain the reaction mixture.

[0058] In steps 1-3, the reaction mixture reacts to generate Cu NWs, resulting in a solution containing Cu NWs.

[0059] The reaction mixture from steps 1-2 was kept at 53°C for 1.5 hours. During the reaction, proper stirring was ensured to promote uniform reaction.

[0060] Steps 1-4 involve centrifuging the solution containing Cu NWs to separate the Cu NWs from the solution.

[0061] After the reaction is complete, if a faint red final product is observed, it indicates that copper nanowires (Cu NWs) have been present. Then, the reaction mixture is placed in a centrifuge tube and centrifuged at 2500 rpm. The copper nanowires (Cu NWs) are separated from the solution by centrifugation.

[0062] Steps 1-5: Wash the Cu NWs obtained from separation and collection in steps 1-4 to obtain Cu NWs.

[0063] The cleaning of Cu NWs involves two steps: initial cleaning and re-cleaning. The specific method is as follows: Step 1-5-1, Initial cleaning; The Cu NWs obtained in steps 1-4 were redispersed in deionized water; then the deionized water containing the dispersed Cu NWs was placed in a centrifuge tube and centrifuged at 2500 rpm.

[0064] Repeat the above process of dispersing in deionized water and centrifuging 2-4 times to remove inorganic and soluble compounds; 1-5-2, then wash again; The initially cleaned Cu NWs were redispersed in ethanol and centrifuged (using the same centrifugation parameters as the initial cleaning) to obtain cleaned Cu NWs.

[0065] During the subsequent cleaning, the above process of dispersing in ethanol and centrifuging can be repeated 2-4 times to remove residual organic solvents such as ethylenediamine (EDA) and hydrazine (N2H4).

[0066] Step 2: Prepare a mixed solution of P3HT-Cu NWs.

[0067] Using the Cu NWs obtained in step 1, a mixed solution of P3HT-Cu NWs was prepared. The specific steps are as follows: Step 2-1: Add 0.024 g of 2.8 wt% Cu NWs dispersion and 41 μL of 2825 μM 3HT solution to 0.6 mL of chloroform to obtain mixture one.

[0068] Step 2-2: Add excess ferric chloride hexahydrate to 1.6 mL of chloroform. The Fe in the solution... 3+ It can promote the formation of Cu NWs on the electrode surface to form 3HT polymer, resulting in mixed solution II.

[0069] Steps 2-3: Sonicate mixture one and mixture two separately for 0.8 hours to promote dispersion and dissolution.

[0070] Steps 2-4: Filter the ultrasonically treated mixture 2 to obtain a ferric chloride solution; mix and stir the ferric chloride solution with the ultrasonically treated mixture 1, and 3HT is oxidized and polymerized on the Cu NWs surface to obtain a P3HT-Cu NWs mixed solution.

[0071] Step 3: Prepare the PDMS flexible underlayer 5.

[0072] The specific steps for preparing the PDMS flexible underlayer 5 are as follows: Step 3-1: Mix PDMS and water in a ratio of 10:1 (by weight) to form a prepolymer.

[0073] Step 3-2: Take 3.335g of the prepolymer and drop it onto a single-sided polished silicon wafer with a diameter of two inches. Remove any remaining air bubbles from the prepolymer using vacuum.

[0074] Step 3-3: Spin coat the prepolymer from the silicon wafer after removing air bubbles onto the substrate at a speed of 440 rpm for 8 seconds.

[0075] Steps 3-4: The substrate is thermocured at 66°C for 3.5 hours to obtain the PDMS flexible underlayer 5 (thickness approximately 500 μm).

[0076] Step 4: Coat a mixed solution of P3HT-Cu NWs onto the PDMS flexible underlayer 5 to obtain the P3HT-Cu NWs conductive sensitive layer 6.

[0077] The specific steps for coating the P3HT-Cu NWs mixed solution are as follows: Step 4-1: The mixed solution of P3HT-Cu NWs obtained in step 2 is uniformly dispersed in a container containing 7 mL of deionized water to obtain a suspension; the concentration of the suspension is such that the total mass of P3HT-Cu NWs solids contained in 1 mL of the suspension is 1.3 mg.

[0078] Step 4-2: Take 2 mL of suspension and drop it evenly onto the PDMS flexible cover layer 5. After the suspension has covered the PDMS flexible cover layer 5, let it stand at room temperature for 1 min, rotate at 320 rpm for 2 s, and dry at 550°C for 2.5 h. Repeat the above steps of dropping, standing, spinning, and drying to spin-coat layer by layer and finally obtain the P3HT-CuNWs conductive sensitive layer 6. The number of repetitions can be determined by those skilled in the art based on the actual situation, without the need for creative effort (if the conductive network is not connected or the electrochemical sensitivity is insufficient, then spin-coating needs to be repeated).

[0079] Preferably, when preparing the P3HT-Cu NWs conductive sensing layer 6, the P3HT-Cu NWs conductive sensing layer 6 needs to be partially embedded 1-2 μm into the upper surface of the PDMS flexible lower capping layer 5. The specific method is as follows: First, following steps 4-1 to 4-2 above, the mixed solution of P3HT-Cu NWs is dried at room temperature on the PDMS flexible capping layer 5 to form a continuous film, thus forming a sample. Then place the sample in a chloroform vapor environment in a sealed container for 2-4 minutes to allow the PDMS surface (in the range of 3-5 micrometers) to swell and soften slightly. Remove the sample, cover the membrane with a PTFE sheet, and apply uniform light pressure (≈0.05–0.2 MPa, or equivalent to 0.5–2 kg / 10 cm²) for 1–3 min to encourage the composite membrane to embed into the surface of the upper layer of the PDMS flexible underlayer 5. Dry the sample at 60-80°C for 30-60 minutes to allow the solvent to be completely removed and the surface layer of the PDMS flexible cover layer 5 to shrink and set. at last Plasma is used to slightly remove the outermost organic "skin" of the P3HT-Cu NWs conductive sensitive layer 6 in 10-30 s (50-100 W), allowing the nanowires / composite film to be exposed, ensuring electrochemical contact with the aqueous phase, and also facilitating subsequent bonding with the top cap layer.

[0080] If the PDMS surface swells excessively, the degree of swelling can be reduced by appropriately shortening the steaming time or by using toluene instead.

[0081] Of the above steps, except for some steps which need to be carried out in a chloroform vapor environment, the remaining steps can be carried out in a slow-flowing nitrogen environment. After drying, the product should be packaged as soon as possible to avoid copper oxidation.

[0082] Step 5: Prepare the PDMS flexible top cover layer 11. First, make a positive mold on the silicon wafer using SU-8, and then use PDMS to make a mold to obtain the PDMS flexible top cover layer 11 with microchannel layer 7.

[0083] Step 6: Bond and seal the PDMS flexible lower cover layer 5 with P3HT-Cu NWs conductive sensitive layer 6 to the PDMS flexible upper cover layer 11 with microfluidic layer 7 to obtain the base layer.

[0084] Step 7: Open an inlet 8 and an outlet 3 on the substrate layer, which are connected to the flow channels 10 of the microfluidic layer 7; set a reference electrode 1 and a counter electrode 2 on the substrate layer. How to open the inlet 8 and outlet 3 and how to set the reference electrode 1 and counter electrode 2 are conventional methods in this field, and those skilled in the art can directly implement them based on existing technology.

[0085] Example 3 This embodiment provides a method for preparing an electrochemical biosensor, used to prepare the electrochemical biosensor described in Example 1. The preparation method specifically includes the following steps: Step 1: Prepare Cu NWs.

[0086] The specific steps for preparing Cu NWs are as follows: Step 1-1: Preheat the sodium hydroxide solution.

[0087] Take 20 mL of a 15% sodium hydroxide (NaOH) solution and preheat it to 60°C.

[0088] Step 1-2: Add copper nitrate solution, ethylenediamine and hydrazine solution to the sodium hydroxide solution preheated in step 1-1 to obtain a reaction mixture.

[0089] 1 mL of 0.1% copper nitrate (Cu(NO3)2) solution, 0.16 mL of ethylenediamine (EDA), and 25 μL of 35 wt% (mass percentage) hydrazine (N2H4) solution were added sequentially to the sodium hydroxide solution preheated in step 1-1 to obtain the reaction mixture.

[0090] In steps 1-3, the reaction mixture reacts to generate Cu NWs, resulting in a solution containing Cu NWs.

[0091] The reaction mixture from steps 1-2 was kept at 60°C for 2 hours. During the reaction, proper stirring was ensured to promote uniform reaction.

[0092] Steps 1-4 involve centrifuging the solution containing Cu NWs to separate the Cu NWs from the solution.

[0093] After the reaction is complete, if a faint red final product is observed, it indicates that copper nanowires (Cu NWs) have been present. Then, the reaction mixture is placed in a centrifuge tube and centrifuged at 3000 rpm. The copper nanowires (Cu NWs) are separated from the solution by centrifugation.

[0094] Steps 1-5: Wash the Cu NWs obtained from separation and collection in steps 1-4 to obtain Cu NWs.

[0095] The cleaning of Cu NWs involves two steps: initial cleaning and re-cleaning. The specific method is as follows: Step 1-5-1, Initial cleaning; The Cu NWs obtained in steps 1-4 were redispersed in deionized water; then the deionized water containing the dispersed Cu NWs was placed in a centrifuge tube and centrifuged at 3000 rpm.

[0096] Repeat the above process of dispersing in deionized water and centrifuging 2-4 times to remove inorganic and soluble compounds; 1-5-2, then wash again; The initially cleaned Cu NWs were redispersed in ethanol and centrifuged (using the same centrifugation parameters as the initial cleaning) to obtain cleaned Cu NWs.

[0097] During the subsequent cleaning, the above process of dispersing in ethanol and centrifuging can be repeated 2-4 times to remove residual organic solvents such as ethylenediamine (EDA) and hydrazine (N2H4).

[0098] Step 2: Prepare a mixed solution of P3HT-Cu NWs.

[0099] Using the Cu NWs obtained in step 1, a mixed solution of P3HT-Cu NWs was prepared. The specific steps are as follows: Step 2-1: Add 0.038 g of 2.8 wt% Cu NWs dispersion and 45 μL of 2825 μM 3HT solution to 0.9 mL of chloroform to obtain mixture one.

[0100] Step 2-2: Add excess ferric chloride hexahydrate to 1.5-2 mL of chloroform. The Fe in the solution... 3+ It can promote the formation of Cu NWs on the electrode surface to form 3HT polymer, resulting in mixed solution II.

[0101] Steps 2-3: Perform ultrasonic treatment on mixture one and mixture two respectively for 1 hour to promote dispersion and dissolution.

[0102] Steps 2-4: Filter the ultrasonically treated mixture 2 to obtain a ferric chloride solution; mix and stir the ferric chloride solution with the ultrasonically treated mixture 1, and 3HT is oxidized and polymerized on the Cu NWs surface to obtain a P3HT-Cu NWs mixed solution.

[0103] Step 3: Prepare the PDMS flexible underlayer 5.

[0104] The specific steps for preparing the PDMS flexible underlayer 5 are as follows: Step 3-1: Mix PDMS and water at a ratio of 10:1.5 (by weight) to form a prepolymer.

[0105] Step 3-2: Take 3.3250g of the prepolymer and drop it onto a single-sided polished silicon wafer with a diameter of two inches. Remove any remaining air bubbles from the prepolymer using vacuum.

[0106] Step 3-3: Spin coat the prepolymer from the silicon wafer after removing air bubbles onto the substrate at a speed of 500 rpm for 10 seconds.

[0107] Steps 3-4: The substrate is thermo-cured at 70°C for 4 hours to obtain the PDMS flexible underlayer 5 (thickness approximately 500 μm).

[0108] Step 4: Coat a mixed solution of P3HT-Cu NWs onto the PDMS flexible underlayer 5 to obtain the P3HT-Cu NWs conductive sensitive layer 6.

[0109] The specific steps for coating the P3HT-Cu NWs mixed solution are as follows: Step 4-1: The mixed solution of P3HT-Cu NWs obtained in step 2 is uniformly dispersed in a container containing 10 mL of deionized water to obtain a suspension; the concentration of the suspension is such that the total mass of P3HT-Cu NWs solids contained in 1 mL of the suspension is 2 mg.

[0110] Step 4-2: Take 2 mL of suspension and drop it evenly onto the PDMS flexible cover layer 5. After the suspension covers the PDMS flexible cover layer 5, let it stand at room temperature for 1 min, rotate at 400 rpm for 3 s, and dry at 700°C for 3 h. Repeat the above steps of dropping, standing, spinning, and drying to spin-coat layer by layer and finally obtain the P3HT-CuNWs conductive sensitive layer 6. The number of repetitions can be determined by those skilled in the art based on the actual situation, without the need for creative effort (if the conductive network is not connected or the electrochemical sensitivity is insufficient, then spin-coating needs to be repeated).

[0111] Step 5: Prepare the PDMS flexible top cover layer 11. First, make a positive mold on the silicon wafer using SU-8, and then use PDMS to make a mold to obtain the PDMS flexible top cover layer 11 with microchannel layer 7.

[0112] Step 6: Bond and seal the PDMS flexible lower cover layer 5 with P3HT-Cu NWs conductive sensitive layer 6 to the PDMS flexible upper cover layer 11 with microfluidic layer 7 to obtain the base layer.

[0113] Step 7: Open an inlet 8 and an outlet 3 on the substrate layer, which are connected to the flow channels 10 of the microfluidic layer 7; set a reference electrode 1 and a counter electrode 2 on the substrate layer. How to open the inlet 8 and outlet 3 and how to set the reference electrode 1 and counter electrode 2 are conventional methods in this field, and those skilled in the art can directly implement them based on existing technology.

[0114] Example 4 This embodiment provides a method for preparing an electrochemical biosensor, used to prepare the electrochemical biosensor described in Example 1. The preparation method specifically includes the following steps: Step 1: Prepare Cu NWs.

[0115] The specific steps for preparing Cu NWs are as follows: Step 1-1: Preheat the sodium hydroxide solution.

[0116] Take 23 mL of a 15% sodium hydroxide (NaOH) solution and preheat it to 66°C.

[0117] Step 1-2: Add copper nitrate solution, ethylenediamine and hydrazine solution to the sodium hydroxide solution preheated in step 1-1 to obtain a reaction mixture.

[0118] 1.6 mL of 0.1% copper nitrate (Cu(NO3)2) solution, 0.19 mL of ethylenediamine (EDA), and 28 μL of 35 wt% (35% by mass) hydrazine (N2H4) solution were added sequentially to the sodium hydroxide solution preheated in step 1-1 to obtain the reaction mixture.

[0119] In steps 1-3, the reaction mixture reacts to generate Cu NWs, resulting in a solution containing Cu NWs.

[0120] The reaction mixture from steps 1-2 was kept at 69°C for 2.6 hours. During the reaction, proper stirring was ensured to promote uniform reaction.

[0121] Steps 1-4 involve centrifuging the solution containing Cu NWs to separate the Cu NWs from the solution.

[0122] After the reaction is complete, if a faint red final product is observed, it indicates that copper nanowires (Cu NWs) have been present. Then, the reaction mixture is placed in a centrifuge tube and centrifuged at 3200 rpm. The copper nanowires (Cu NWs) are separated from the solution by centrifugation.

[0123] Steps 1-5: Wash the Cu NWs obtained from separation and collection in steps 1-4 to obtain Cu NWs.

[0124] The cleaning of Cu NWs involves two steps: initial cleaning and re-cleaning. The specific method is as follows: Step 1-5-1, Initial cleaning; The Cu NWs obtained in steps 1-4 were redispersed in deionized water; then the deionized water containing the dispersed Cu NWs was placed in a centrifuge tube and centrifuged at 3200 rpm.

[0125] Repeat the above process of dispersing in deionized water and centrifuging 2-4 times to remove inorganic and soluble compounds; 1-5-2, then wash again; The initially cleaned Cu NWs were redispersed in ethanol and centrifuged (using the same centrifugation parameters as the initial cleaning) to obtain cleaned Cu NWs.

[0126] During the subsequent cleaning, the above process of dispersing in ethanol and centrifuging can be repeated 2-4 times to remove residual organic solvents such as ethylenediamine (EDA) and hydrazine (N2H4).

[0127] Step 2: Prepare a mixed solution of P3HT-Cu NWs.

[0128] Using the Cu NWs obtained in step 1, a mixed solution of P3HT-Cu NWs was prepared. The specific steps are as follows: Step 2-1: Add 0.042 g of 2.8 wt% Cu NWs dispersion and 47 μL of 2825 μM 3HT solution to 1.3 mL of chloroform to obtain mixture one.

[0129] Step 2-2: Add excess ferric chloride hexahydrate to 1.9 mL of chloroform. The Fe in the solution... 3+It can promote the formation of Cu NWs on the electrode surface to form 3HT polymer, resulting in mixed solution II.

[0130] Steps 2-3: Perform ultrasonic treatment on mixture one and mixture two respectively for 1.5 hours to promote dispersion and dissolution.

[0131] Steps 2-4: Filter the ultrasonically treated mixture 2 to obtain a ferric chloride solution; mix and stir the ferric chloride solution with the ultrasonically treated mixture 1, and 3HT is oxidized and polymerized on the Cu NWs surface to obtain a P3HT-Cu NWs mixed solution.

[0132] Step 3: Prepare the PDMS flexible underlayer 5.

[0133] The specific steps for preparing the PDMS flexible underlayer 5 are as follows: Step 3-1: Mix PDMS and water at a ratio of 10:1.7 (by weight) to form a prepolymer.

[0134] Step 3-2: Take 3.385g of the prepolymer and drop it onto a single-sided polished silicon wafer with a diameter of two inches. Remove any remaining air bubbles from the prepolymer using vacuum.

[0135] Step 3-3: Spin coat the prepolymer from the silicon wafer after removing air bubbles onto the substrate at a speed of 560 rpm for 12 seconds.

[0136] Steps 3-4: The substrate is thermocured at 81°C for 4.8 hours to obtain the PDMS flexible underlayer 5 (thickness approximately 500 μm).

[0137] Step 4: Coat a mixed solution of P3HT-Cu NWs onto the PDMS flexible underlayer 5 to obtain the P3HT-Cu NWs conductive sensitive layer 6.

[0138] The specific steps for coating the P3HT-Cu NWs mixed solution are as follows: Step 4-1: The mixed solution of P3HT-Cu NWs obtained in Step 2 is uniformly dispersed in a container containing 18 mL of deionized water to obtain a suspension; the concentration of the suspension is such that the total mass of P3HT-Cu NWs solids contained in 1 mL of the suspension is 3.5 mg.

[0139] Step 4-2: Take 2 mL of suspension and drop it evenly onto the PDMS flexible cover layer 5. After the suspension has covered the PDMS flexible cover layer 5, let it stand at room temperature for 2 min, rotate at 460 rpm for 4 s, and dry at 750°C for 4.5 h. Repeat the above steps of dropping, standing, spinning, and drying to spin-coat layer by layer and finally obtain the P3HT-CuNWs conductive sensitive layer 6. The number of repetitions can be determined by those skilled in the art based on the actual situation, without the need for creative effort (if the conductive network is not connected or the electrochemical sensitivity is insufficient, then spin-coating needs to be repeated).

[0140] Step 5: Prepare the PDMS flexible top cover layer 11. First, make a positive mold on the silicon wafer using SU-8, and then use PDMS to make a mold to obtain the PDMS flexible top cover layer 11 with microchannel layer 7.

[0141] Step 6: Bond and seal the PDMS flexible lower cover layer 5 with P3HT-Cu NWs conductive sensitive layer 6 to the PDMS flexible upper cover layer 11 with microfluidic layer 7 to obtain the base layer.

[0142] Step 7: Open an inlet 8 and an outlet 3 on the substrate layer, which are connected to the flow channels 10 of the microfluidic layer 7; set a reference electrode 1 and a counter electrode 2 on the substrate layer. How to open the inlet 8 and outlet 3 and how to set the reference electrode 1 and counter electrode 2 are conventional methods in this field, and those skilled in the art can directly implement them based on existing technology.

[0143] Example 5 This embodiment provides a method for preparing an electrochemical biosensor, used to prepare the electrochemical biosensor described in Example 1. The preparation method specifically includes the following steps: Step 1: Prepare Cu NWs.

[0144] The specific steps for preparing Cu NWs are as follows: Step 1-1: Preheat the sodium hydroxide solution.

[0145] Take 19 mL of a 15% sodium hydroxide (NaOH) solution and preheat it to 64°C.

[0146] Step 1-2: Add copper nitrate solution, ethylenediamine and hydrazine solution to the sodium hydroxide solution preheated in step 1-1 to obtain a reaction mixture.

[0147] 0.7 mL of 0.1% copper nitrate (Cu(NO3)2) solution, 0.18 mL of ethylenediamine (EDA), and 22 μL of 35 wt% (35% by mass) hydrazine (N2H4) solution were added sequentially to the sodium hydroxide solution preheated in step 1-1 to obtain the reaction mixture.

[0148] In steps 1-3, the reaction mixture reacts to generate Cu NWs, resulting in a solution containing Cu NWs.

[0149] The reaction mixture from steps 1-2 was kept at 68°C for 1.5 hours. During the reaction, proper stirring was ensured to promote uniform reaction.

[0150] Steps 1-4 involve centrifuging the solution containing Cu NWs to separate the Cu NWs from the solution.

[0151] After the reaction is complete, if a faint red final product is observed, it indicates that copper nanowires (Cu NWs) have been present. Then, the reaction mixture is placed in a centrifuge tube and centrifuged at 3200 rpm. The copper nanowires (Cu NWs) are separated from the solution by centrifugation.

[0152] Steps 1-5: Wash the Cu NWs obtained from separation and collection in steps 1-4 to obtain Cu NWs.

[0153] The cleaning of Cu NWs involves two steps: initial cleaning and re-cleaning. The specific method is as follows: Step 1-5-1, Initial cleaning; The Cu NWs obtained in steps 1-4 were redispersed in deionized water; then the deionized water containing the dispersed Cu NWs was placed in a centrifuge tube and centrifuged at 2500 rpm.

[0154] Repeat the above process of dispersing in deionized water and centrifuging 2-4 times to remove inorganic and soluble compounds; 1-5-2, then wash again; The initially cleaned Cu NWs were redispersed in ethanol and centrifuged (using the same centrifugation parameters as the initial cleaning) to obtain cleaned Cu NWs.

[0155] During the subsequent cleaning, the above process of dispersing in ethanol and centrifuging can be repeated 2-4 times to remove residual organic solvents such as ethylenediamine (EDA) and hydrazine (N2H4).

[0156] Step 2: Prepare a mixed solution of P3HT-Cu NWs.

[0157] Using the Cu NWs obtained in step 1, a mixed solution of P3HT-Cu NWs was prepared. The specific steps are as follows: Step 2-1: Add 0.0255 g of 2.8 wt% Cu NWs dispersion and 48 μL of 2825 μM 3HT solution to 1.2 mL of chloroform to obtain mixture one.

[0158] Step 2-2: Add excess ferric chloride hexahydrate to 1.6 mL of chloroform. The Fe in the solution... 3+It can promote the formation of Cu NWs on the electrode surface to form 3HT polymer, resulting in mixed solution II.

[0159] Steps 2-3: Perform ultrasonic treatment on mixture one and mixture two respectively for 1.6 hours to promote dispersion and dissolution.

[0160] Steps 2-4: Filter the ultrasonically treated mixture 2 to obtain a ferric chloride solution; mix and stir the ferric chloride solution with the ultrasonically treated mixture 1, and 3HT is oxidized and polymerized on the Cu NWs surface to obtain a P3HT-Cu NWs mixed solution.

[0161] Step 3: Prepare the PDMS flexible underlayer 5.

[0162] The specific steps for preparing the PDMS flexible underlayer 5 are as follows: Step 3-1: Mix PDMS and water in a ratio of 10:1.3 (by weight) to form a prepolymer.

[0163] Step 3-2: Take 3.154g of the prepolymer and drop it onto a two-inch diameter single-sided polished silicon wafer. Remove any remaining air bubbles from the prepolymer using vacuum.

[0164] Step 3-3: Spin coat the prepolymer from the silicon wafer after removing air bubbles onto the substrate at a speed of 550 rpm for 8 seconds.

[0165] Steps 3-4: The substrate is thermo-cured at 80°C for 3.5 hours to obtain the PDMS flexible underlayer 5 (thickness approximately 500 μm).

[0166] Step 4: Coat a mixed solution of P3HT-Cu NWs onto the PDMS flexible underlayer 5 to obtain the P3HT-Cu NWs conductive sensitive layer 6.

[0167] The specific steps for coating the P3HT-Cu NWs mixed solution are as follows: Step 4-1: The mixed solution of P3HT-Cu NWs obtained in step 2 is uniformly dispersed in a container containing 8 mL of deionized water to obtain a suspension; the concentration of the suspension is such that the total mass of P3HT-Cu NWs solids contained in 1 mL of the suspension is 3.2 mg.

[0168] Step 4-2: Take 2 mL of suspension and drop it evenly onto the PDMS flexible cover layer 5. After the suspension has covered the PDMS flexible cover layer 5, let it stand at room temperature for 1 min, rotate at 450 rpm for 2 s, and dry at 650°C for 4 h. Repeat the above steps of dropping, standing, spinning, and drying to spin-coat layer by layer and finally obtain the P3HT-CuNWs conductive sensitive layer 6. The number of repetitions can be determined by those skilled in the art based on the actual situation, without the need for creative effort (if the conductive network is not connected or the electrochemical sensitivity is insufficient, then spin-coating needs to be repeated).

[0169] Step 5: Prepare the PDMS flexible top cover layer 11. First, make a positive mold on the silicon wafer using SU-8, and then use PDMS to make a mold to obtain the PDMS flexible top cover layer 11 with microchannel layer 7.

[0170] Step 6: Bond and seal the PDMS flexible lower cover layer 5 with P3HT-Cu NWs conductive sensitive layer 6 to the PDMS flexible upper cover layer 11 with microfluidic layer 7 to obtain the base layer.

[0171] Step 7: Open an inlet 8 and an outlet 3 on the substrate layer, which are connected to the flow channels 10 of the microfluidic layer 7; set a reference electrode 1 and a counter electrode 2 on the substrate layer. How to open the inlet 8 and outlet 3 and how to set the reference electrode 1 and counter electrode 2 are conventional methods in this field, and those skilled in the art can directly implement them based on existing technology.

[0172] Test case Experimental objective: To conduct detection tests on the sensitivity and specificity of dopamine (DA) and ascorbic acid (AA), key biochemical substances in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, using the P3HT-Cu NWs conductive sensitive layer 6 prepared in Example 5.

[0173] I. Experimental Materials: According to the method described in Example 5, the P3HT-Cu NWs conductive sensitive layer 6 obtained in the process (this sensitive layer is also the core innovation of this application) is used as the test material, and the P3HT-Cu NWs conductive sensitive layer 6 is detected by glassy carbon electrode (GCE).

[0174] II. Test Instruments: Electrochemical workstation: This workstation can measure current using cyclic voltammetry (CV) and differential pulse voltammetry (DPV).

[0175] III. Experimental Methods: Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed in 0.1 M phosphate-buffered saline (PBS) (pH 7.0) solution: 1. Cyclic voltammetry (CV): The P3HT-Cu NWs conductive sensitive layer 6 was tested using an electrochemical workstation. The scanning potential range was -1V to +1V, and the scanning rate was set to 100mV / s.

[0176] 2. Differential Pulse Voltammetry (DPV): DPV was used to test the electrical signals induced by ascorbic acid (AA) and dopamine (DA) at different concentrations in the P3HT-Cu NWs conductive sensitive layer 6. DPV parameters included an amplitude of 50 mV and a voltage range of -1 V to +1 V. The test concentration ranges were: AA from 1 μM to 60 μM, and DA from 1 μM to 100 μM.

[0177] IV. Test Results: 1. Cyclic voltammetry (CV): The test results are as follows: (a) 0 μM ascorbic acid (AA) and 0 μM dopamine (DA) showed no significant response to baseline current.

[0178] (b) When the concentration of AA is 10 μM, an oxidation peak of AA appears, with a peak potential of approximately -0.07 V.

[0179] (c) In the case of 10 μM AA and 50 μM DA, the oxidation peak of DA appears at +0.18 V.

[0180] In the absence of these three components, a distinct peak appeared at -0.07 V. This is because Cu NWs are readily oxidized to divalent copper ions near 0 V during the forward scan. Although this peak coincides with the oxidation peak potential of AA, the signal multiplication after the addition of AA does not affect the detection of AA. The results demonstrate the superiority of P3HT binding to Cu NWs, promoting the electro-oxidation of AA. When AA was added to PBS, an anodic peak at -0.07 V was observed. After the addition of DA, another peak appeared at +0.18 V.

[0181] 2. Differential Pulse Voltammetry (DPV): The test results are as follows: (a) 0 μM AA and 0 μM DA have no obvious current response.

[0182] (b) Under the conditions of 10 μM AA and 0 μM DA, AA showed a clear oxidation peak at -0.07 V.

[0183] (c) At 10 μM AA and 50 μM DA, the oxidation peak of AA remained at -0.07 V and the oxidation peak of DA was at +0.18 V.

[0184] As expected, Nafion / Cu NWs–P3HT / GCE simultaneously exhibited two well-defined oxidation peaks at potentials of -0.07 V and +0.18 V. These results indicate that Nafion / Cu NWs–P3HT / GCE exhibits simultaneously enhanced electrochemical activity for both AA and DA.

[0185] 3. Relationship between current and concentration: The experimental results show that the oxidation peak current of AA and DA exhibits a linear relationship with concentration at different concentrations (AA from 1 μM to 60 μM, and DA from 1 μM to 100 μM). Experimental data show that the current response of AA exhibits a linear relationship within the concentration range of 1 μM to 60 μM; and the current response of DA also shows good linearity within the concentration range of 1 μM to 100 μM.

[0186] AA showed a linear relationship in the concentration range of 1–60 μM, and DA showed a linear relationship in the concentration range of 1–100 μM, with detection limits of 0.06 μM and 0.43 μM, respectively (S / N=3). The corresponding fitting equations for AA and DA were calculated as follows: I(AA) = 2.5528 × C(AA) + 24.489 (R = 0.9943), I(DA) = 2.0439 × C(DA) + 7.9273 (R = 0.9968). The results indicate that it is feasible to simultaneously distinguish these two biomolecules in a mixed solution using Cu NWs–P3HT composite material via DPV.

[0187] 4. Relationship between pH and peak current and peak voltage: For the oxidation peak current variations of AA and DA at different pH values: For DA, the oxidation peak current signal does not change significantly between pH 3.0 and 5.0, remaining at 155–165 μA; the oxidation peak current gradually increases between pH 5.0 and 7.0, gradually increasing to 280–290 μA; the oxidation peak current does not change significantly between pH 7.0 and 8.0, remaining at 280–290 μA and tending to saturate. For AA, the oxidation peak current signal gradually decreases between pH 3.0 and 5.0, decreasing from around 140 μA to around 120 μA; the oxidation peak current signal gradually increases between pH 5.0 and 8.0, reaching its maximum at pH 8.0, increasing from around 120 μA to around 195–205 μA.

[0188] Regarding the changes in oxidation peak potentials of AA and DA at different pH values: as the pH value increases (from 3.0 to 8.0), the oxidation peak potentials of both AA and DA shift negatively. For DA, its oxidation peak potential gradually shifts negatively from around 0.5V to around 0.24~0.26V as the pH value increases from 3.0 to 8.0. For AA, its oxidation peak potential gradually shifts negatively from around 0.04V (close to 0V) to around -0.15~-0.17V as the pH value increases from 3.0 to 8.0. This indicates that even a lower anodic potential can distinguish between these two molecules.

Claims

1. An electrochemical biosensor, comprising a substrate layer, a liquid inlet (8) and a liquid outlet (3) located on both sides of the substrate layer, a reference electrode (1) and a counter electrode (2) arranged on the top of the substrate layer, characterized in that: The base layer comprises, from bottom to top, a PDMS flexible lower cover layer (5), a P3HT-Cu NWs conductive sensitive layer (6), and a PDMS flexible upper cover layer (11), the PDMS flexible upper cover layer (11) is provided with a micro-channel layer (7) on the side close to the P3HT-Cu NWs conductive sensitive layer (6), the micro-channel layer (7) is provided with a flow channel (10), the flow channel (10) is in communication with a liquid inlet (8) and a liquid outlet (3) at two ends, and the P3HT-Cu NWs conductive sensitive layer (6) is connected with a flexible flat cable (4) on one side.

2. An electrochemical biosensor as claimed in claim 1, wherein: The flow channel (10) in the micro-channel layer (7) is a serpentine flow channel.

3. An electrochemical biosensor as claimed in claim 1, wherein: The flow channel (10) in the micro-channel layer (7) is a tree-shaped flow channel, and the PDMS flexible upper cover layer (11) is provided with a recessed cavity (9) in the middle, the lower ends of the reference electrode (1) and the counter electrode (2) extend into the recessed cavity (9), and the bottom of the recessed cavity (9) is in communication with the flow channel (10) of the micro-channel layer (7).

4. A method of preparing an electrochemical biosensor, characterized by, The method comprises the following steps: Step 1, preparing Cu NWs; Step 2, preparing a mixed solution of P3HT-Cu NWs; Step 3, preparing a PDMS flexible lower cover layer (5); Step 4, coating the mixed solution of P3HT-Cu NWs on the PDMS flexible lower cover layer (5), and obtaining a P3HT-Cu NWs conductive sensitive layer (6) after drying; Step 5, preparing a PDMS flexible upper cover layer (11) with a micro-channel layer (7); Step 6, bonding and sealing the PDMS flexible lower cover layer (5) with the P3HT-Cu NWs conductive sensitive layer (6) and the PDMS flexible upper cover layer (11) with the micro-channel layer (7) to obtain a base layer; Step 7, opening a liquid inlet (8) and a liquid outlet (3) on the base layer, the liquid inlet (8) and the liquid outlet (3) being in communication with the flow channel (10) of the micro-channel layer (7); and arranging a reference electrode (1) and a counter electrode (2) on the base layer.

5. The method of claim 4, wherein the step of forming the enzyme layer is performed by applying a solution of the enzyme to the electrode surface. In step 1 of preparing Cu NWs, the specific steps are as follows: Step 1-1, preheating a sodium hydroxide solution; Step 1-2, adding a copper nitrate solution, an ethylenediamine solution and a hydrazine solution into the preheated sodium hydroxide solution to obtain a reaction mixture; Step 1-3, reacting the reaction mixture to generate Cu NWs, and obtaining a solution containing Cu NWs; Step 1-4, centrifuging the solution containing Cu NWs to separate Cu NWs from the solution; Step 1-5, cleaning the collected Cu NWs to obtain Cu NWs.

6. The method of claim 5, wherein the step of forming the biocompatible layer comprises forming a layer of a biocompatible polymer. In step 1-5 of cleaning Cu NWs, the specific method is as follows: Step 1-5-1, initial cleaning; The Cu NWs separated in step 1-4 are dispersed into deionized water again and centrifuged; Step 1-5-2, re-cleaning; The Cu NWs after initial cleaning are dispersed into ethanol again and centrifuged to obtain cleaned Cu NWs.

7. The method of claim 4, wherein the electrode is a gold electrode. 5 In step 2 of preparing a mixed solution of P3HT-Cu NWs, the specific steps are as follows: Step 2-1, adding a Cu NWs dispersion, a 3HT solution into chloroform to obtain a mixed solution one; Step 2-2, adding excess iron chloride hexahydrate in chloroform to obtain mixed solution two; Step 2-3, ultrasonic treatment of mixed solution one and mixed solution two respectively; Step 2-4, filtering mixed solution two after ultrasonic treatment to obtain iron chloride solution; mixing and stirring the iron chloride solution and mixed solution one after ultrasonic treatment, 3HT is oxidized and polymerized on the surface of Cu NWs to obtain a mixed solution of P3HT-Cu NWs.

8. The method of claim 4, wherein the electrode is a gold electrode. 5 Step 3, when preparing the PDMS flexible lower cover layer (5), the specific steps are as follows: Step 3-1, mixing PDMS and water in a ratio of 10:1 to form a prepolymer; Step 3-2, dropping the prepolymer on a silicon wafer to remove air bubbles in the prepolymer; Step 3-3, spin coating the prepolymer after removing air bubbles on the substrate; Step 3-4, heat curing the substrate to obtain the PDMS flexible lower cover layer (5).

9. The method of claim 4, wherein the electrode is a gold electrode. 0 Step 4, when coating the mixed solution of P3HT-Cu NWs, the specific steps are as follows: Step 4-1, uniformly dispersing the mixed solution of P3HT-Cu NWs prepared in step 2 in a container containing deionized water to obtain a suspension; Step 4-2, dropping the suspension on the PDMS flexible lower cover layer (5), and after the suspension covers the PDMS flexible lower cover layer (5), standing at room temperature, rotating, and drying to obtain a P3HT-Cu NWs conductive sensitive layer (6).

10. Use of the electrochemical biosensor of any one of claims 1-3 in the preparation of a dopamine and / or ascorbic acid detection instrument for neurodegenerative diseases.

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