Flexible electrochemical detection circuit and preparation method thereof

Abstract

The invention relates to the field of electrochemical sensors, and particularly discloses a flexible electrochemical detection circuit and a preparation method thereof.The flexible electrochemical detection circuit comprises a flexible substrate, a three-electrode system fixed to the flexible substrate and a hydrogel layer used for packaging the three-electrode system; a working electrode in the three-electrode system comprises a conductive circuit and a porous conductive carrier covering the conductive circuit, the porous conductive carrier is polyglycolide porous particles adsorbed with carbon nanotubes, and a biological enzyme is fixed on the porous conductive carrier. Based on the flexible circuit, the porous enzyme carrier with high specific surface area is compounded, so that the electrochemical detection efficiency, sensitivity and accuracy are effectively improved.

Description

Technical Field

[0001] The present application relates to the field of electrochemical sensors, and in particular to a flexible electrochemical detection circuit and a preparation method thereof. Background Art

[0002] Metabolite indicators are an important basis for evaluating human health status, physiological functions and possible disease states. As people pay more attention to and demand for health monitoring, how to achieve real-time, portable, easy-to-operate and low-cost metabolite detection is of great significance for health management and medical diagnosis.

[0003] Traditional metabolite detection usually uses invasive blood testing methods, which can cause pain and the risk of wound infection to patients. In addition, blood testing is usually cumbersome and cannot achieve real-time monitoring. Therefore, wearable detection devices based on flexible biosensors have received increasing attention. One of the core components of flexible biosensors is the flexible electrochemical detection circuit, which needs to meet the requirements of achieving high-precision detection under the efficient catalysis of enzymes. Summary of the invention

[0004] In order to improve the detection efficiency, sensitivity and accuracy of a flexible electrochemical detection circuit, the present application provides a flexible electrochemical detection circuit and a preparation method thereof.

[0005] The present application provides a method for preparing a flexible electrochemical detection circuit using the following technical solution: A method for preparing a flexible electrochemical detection circuit comprises the following steps: preparing a flexible substrate; Inkjet printing of nanosilver conductive circuits on flexible substrates; preparing a counter electrode and a reference electrode on the nanosilver conductive circuit; Preparation of polyglycolide porous particles: preparing porous polyglycolide by a solvent replacement method, and crushing the porous polyglycolide to obtain micrometer-sized polyglycolide porous particles; Preparation of working electrode: fix the biological enzyme on polyglycolide porous particles, mix with carbon nanotube dispersion, and drip the obtained mixture on the corresponding area on the nanosilver conductive line to obtain the working electrode; the working electrode, counter electrode and reference electrode form a three-electrode system; The three-electrode system was encapsulated using hydrogel.

[0006] Furthermore, the method for preparing the polyglycolide porous particles comprises: preparing a hexafluoroisopropanol solution of polyglycolide; Under stirring conditions, deionized water is added to the hexafluoroisopropanol solution of polyglycolide to precipitate porous polyglycolide; The stirring speed is increased to break the porous polyglycolide into micron-sized porous polyglycolide particles.

[0007] Further, the method for preparing the hexafluoroisopropanol solution of polyglycolide comprises: The polyglycolide particles are subjected to heat pressing and air quenching to obtain a low-crystallinity polyglycolide film; The low crystallinity polyglycolide film is dissolved in hexafluoroisopropanol to obtain a hexafluoroisopropanol solution of polyglycolide.

[0008] First, polyglycolide particles with a diameter of several millimeters are pressed into a film hundreds of microns thick by hot pressing, which greatly increases the heat exchange area and can be quickly cooled in room temperature air. At the same time, the high thermal conductivity of aluminum foil is used to quench and cool the polyglycolide film in the air, blocking the recrystallization process of polyglycolide, thereby reducing the crystallinity of polyglycolide and making it easily soluble in organic solvents such as hexafluoroisopropanol. Compared with the common process of reducing crystallinity by high-temperature heating followed by liquid nitrogen quenching, the hot pressing-air quenching process provided by this application is simple and can effectively reduce crystallinity.

[0009] Furthermore, in the step of adding deionized water to the hexafluoroisopropanol solution of polyglycolide, a micro-injection pump is used to add the deionized water at an injection speed of 80-120 μL / min.

[0010] Furthermore, in the step of breaking the porous polyglycolide into micrometer-sized porous polyglycolide particles, the stirring speed is 600-800 rpm and the stirring time is 36-60 hours.

[0011] In the process of adding deionized water to the hexafluoroisopropanol solution of polyglycolide, the stronger affinity between water molecules and hexafluoroisopropanol molecules leads to a decrease in the degree of binding between polyglycolide molecular chains and hexafluoroisopropanol molecules, and polyglycolide and water molecules cannot form hydrogen bonds; polyglycolide molecular chains re-aggregate and precipitate to form crystal nuclei, and then more polyglycolide precipitates and grows around the crystal nuclei. When sufficient deionized water is added to the solution, hexafluoroisopropanol molecules and water molecules are completely combined, polyglycolide is completely precipitated, and the irregular precipitation growth makes polyglycolide present a porous flocculent structure.

[0012] The mixed liquid is then heated and stirred at high speed to completely evaporate the hexafluoroisopropanol from the solution, and the polyglycolide porous floccules are broken to obtain polyglycolide porous particles; as the stirring time increases, the size of the polyglycolide porous particles gradually decreases. As a biological enzyme carrier, polyglycolide porous particles need to meet the structural requirements of a large specific surface area, and at the same time, the appropriate stirring time should be selected by comprehensively considering factors such as time cost.

[0013] A flexible electrochemical detection circuit prepared by the above method includes: Flexible substrate; A three-electrode system, fixed on the flexible substrate, comprising a counter electrode, a reference electrode and a working electrode; wherein the working electrode comprises a conductive circuit in a cross-comb structure and a porous conductive carrier covering the conductive circuit, wherein the porous conductive carrier is a polyglycolide porous particle adsorbed with carbon nanotubes; a biological enzyme is fixed on the porous conductive carrier, and the biological enzyme can undergo a redox reaction with the analyte; The hydrogel layer is used to encapsulate the three-electrode system.

[0014] Furthermore, the biological enzyme is glucose oxidase, and the analyte is glucose.

[0015] The flexible electrochemical detection circuit provided in this application can be used for wearable detection, such as for detecting glucose in sweat, or for detecting glucose in skin interstitial fluid in combination with a microneedle array. When in use, the body fluid to be tested is absorbed by the hydrogel layer and diffused to the three-electrode system. The glucose in the body fluid to be tested reacts with the glucose oxidase in the working electrode to produce hydrogen peroxide. When a certain potential is applied externally, the hydrogen peroxide oxidizes and decomposes to produce electrons. The higher the glucose concentration, the more hydrogen peroxide is produced, that is, the more electrons, the greater the current generated. Therefore, the glucose concentration can be analyzed by detecting the magnitude of the current.

[0016] The conductive circuits in the working electrode area present a cross-comb structure, which exposes part of the PDMS substrate in the electrode area, thus having good hydrophilicity after oxygen plasma surface modification, allowing the porous conductive carrier with fixed biological enzymes to be evenly distributed in the electrode area, optimizing the contact area between the working electrode and the solution to be tested, and improving the detection sensitivity and signal-to-noise ratio.

[0017] Furthermore, the polyglycolide porous particles have a micrometer-scale spatial network structure.

[0018] The polyglycolide porous particles have a high specific surface area, providing ample space for the attachment of enzymes and sufficient reaction domains for subsequent enzymatic reactions. Compared with the polyglycolide porous film, the polyglycolide porous particles have a shorter electron transfer path when used as enzyme carriers for electrochemical detection, which is conducive to improving detection efficiency and sensitivity. At the same time, the polyglycolide porous particles have excellent hydrophilicity and biocompatibility. As enzyme carriers, they can effectively maintain the activity of enzymes, thereby ensuring the accuracy and stability of detection.

[0019] Furthermore, the size of the polyglycolide porous particles is 10-20 μm.

[0020] Larger polyglycolide porous particles have a smaller specific surface area, while smaller polyglycolide porous particles have a lower porosity and a relatively weaker ability to immobilize enzymes. Polyglycolide porous particles with a size of 10-20 μm have a rich pore structure, which meets the demand for high specific surface area carriers for enzyme immobilization.

[0021] Furthermore, the flexible substrate comprises a polyimide layer and a dimethylsiloxane layer, and the three-electrode system is fixed on the dimethylsiloxane layer.

[0022] Polyimide film is flexible but not ductile, and is easily scratched during transportation and storage. Therefore, printing circuits directly on polyimide film is likely to affect the quality of circuit formation. Dimethylsiloxane is prepared in real time and has good surface flatness. It can produce high-quality circuits through inkjet printing after surface modification by oxygen plasma. The two materials are compounded, and the inextensibility of polyimide is used to ensure that the circuit will not be broken due to extension. At the same time, the flatness of the dimethylsiloxane surface is used to improve the processing quality, and ultimately the prepared flexible circuit has good bendability and electrical stability.

[0023] In summary, the present application includes at least one of the following beneficial technical effects: 1. This application is based on a flexible circuit and a porous enzyme carrier with a high specific surface area, which helps to improve the efficiency, sensitivity, accuracy and stability of detection; 2. The micron-sized polyglycolide porous particles prepared by the solvent replacement method have high specific surface area, excellent hydrophilicity and biocompatibility, can effectively fix biological enzymes and maintain enzyme activity, and meet the needs of electrochemical detection; the preparation method is simple and easy to achieve large-scale preparation; 3. The polyimide-dimethylsiloxane composite flexible substrate is used to make the prepared flexible circuit have good bendability and electrical stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : is a schematic diagram of the structure of the flexible electrochemical detection circuit in the embodiment of the present application, wherein (a) is a top view; (b) is a front view; Figure 2 is a process flow chart for preparing polyglycolide porous particles in an embodiment of the present application; Figure 3 is a resistivity variation curve of the inkjet-printed circuit of the flexible substrate at different bending angles in the embodiment of the present application; Figure 4 1 is a microscopic morphology of enzyme immobilized on polyglycolide porous particles in the embodiment of the present application, wherein (a) is the enzyme solution after drying on the silicon wafer substrate; (b) is the polyglycolide porous particles without enzyme immobilized; (c) is the polyglycolide porous particles with enzyme immobilized; Figure 5 The results of the flexible electrochemical detection circuit in the embodiment of the present application for glucose detection are shown in FIG. 1 , wherein (a) is the current-time response curve under different glucose concentrations; and (b) is the fitting line of the relationship between the steady-state current and the glucose concentration.

[0025] Figure numerals: 1. flexible substrate; 2. nanosilver conductive circuit; 3. reference electrode; 4. working electrode; 5. counter electrode; 6. lower substrate; 7. upper substrate; 8. porous conductive carrier with fixed biological enzymes; 9. hydrogel layer. DETAILED DESCRIPTION

[0026] The English abbreviations in the text are as follows: PGA(Polyglycolide acid):polyglycolide; PDMS (Polydimethylsiloxane): dimethylsiloxane; HFIP (Hexafluoroisopropanol): Hexafluoroisopropanol; PI(Polyimide) The following is combined with Figure 1-5 This application is described in further detail.

[0027] The present application discloses a method for preparing a flexible electrochemical detection circuit, comprising the following steps: Step 1: Prepare a flexible substrate, the specific method is as follows: The PMDS precursor main liquid and the auxiliary liquid were mixed at a mass ratio of 10:1 to obtain a PDMS prepolymer solution, which was placed in a degassing mixer for degassing; Take 2 mL of degassed PDMS prepolymer solution on a 40 mm × 40 mm × 50 μm PI film, and transfer it to a 50 mm × 50 mm × 1 mm glass slide. Then place it in the center of the glue spreader and turn on the vacuum pump for adsorption and fixation. Set the speed to 1500 r / min and the time to 30 s before starting glue spread. After the PDMS sample was coated, it was placed flat in an incubator and the temperature was adjusted to 85°C for 30 min to completely cure it, thereby obtaining a PI-PDMS composite substrate, in which the thickness of the PI layer was 50 μm and the thickness of the PDMS layer was 70 μm. The PI-PDMS composite substrate was placed in a plasma treatment chamber for plasma treatment at a power of 30 W for 30 s to obtain a modified PI-PDMS composite substrate to enhance the hydrophilicity of the PDMS layer.

[0028] Step 2: inkjet print the nanosilver conductive circuit on the PDMS layer. After printing, place the nanosilver conductive circuit and the substrate in a vacuum drying oven for sintering at a sintering temperature of 150° C. and a sintering time of 45 min.

[0029] Step 3: Prepare a counter electrode and a reference electrode on the nanosilver conductive circuit. The specific method is as follows: The nanosilver conductive circuit and substrate were immersed in a 1M FeCl3 solution for 1 min to chlorinate the circuit material into Ag / AgCl, and then the circuit surface was rinsed with deionized water to remove the residual solution. After drying, the substrate surface was treated with air plasma for 30 s to make the substrate surface hydrophilic. The counter electrode and reference electrode obtained were both Ag / AgCl electrodes.

[0030] Step 4, preparing porous polyglycolide particles: preparing porous polyglycolide by solvent replacement method, crushing the porous polyglycolide to obtain micrometer-sized porous polyglycolide particles, referring to Figure 2 , the specific method is: Step 4-1, preparing a hexafluoroisopropanol solution of polyglycolide: The polyglycolide particles were wrapped in aluminum foil, and then placed on a hot press bottom plate preheated at 230°C, and heated for 100 seconds to completely melt the polyglycolide particles; then the temperature was maintained and the hot press was operated to press for 100 seconds at the set pressure; after the hot pressing treatment, the aluminum foil wrapped with polyglycolide was taken out of the hot press, cooled at room temperature for 15 seconds, and peeled off from the aluminum foil to obtain a low crystallinity polyglycolide film; The low crystallinity polyglycolide film is dissolved in hexafluoroisopropanol to obtain a hexafluoroisopropanol solution of polyglycolide.

[0031] Step 4-2, preparing porous polyglycolide by solvent replacement method: Take a 20 mL screw-cap bottle and put it into a magnetic rotor, add 2 mL of 50 mg / mL polyglycolide hexafluoroisopropanol solution (PGA-HFIP solution) into the bottle, and place the screw-cap bottle on a magnetic stirrer; Start the magnetic stirrer and set the speed to 300 rpm. Use a micro-injection pump to add deionized water to the PGA-HFIP solution. During the injection process, the needle is inserted into the screw-mouth bottle and close to the wall. The injection speed is 100 μL / min, and the feed is constant for 20 minutes. The total injection volume of deionized water is 2 mL. A large amount of white floccules are precipitated in the screw-mouth bottle, which is porous polyglycolide.

[0032] Step 4-3, crushing the porous polyglycolide to obtain micron-sized porous polyglycolide particles: The floccules in the screw-cap bottle were transferred to a beaker containing 40 mL of deionized water, and a large magnetic rotor was replaced and placed on a magnetic stirrer for stirring. The speed was set to 700 rpm to break the porous polyglycolide; After stirring for 48 hours, polyglycolide porous particles with a size of 10-20 μm were obtained. After standing, the supernatant was removed and 2 mL of milky white polyglycolide porous particle suspension was retained. At this time, the theoretical value of the ratio of the mass of polyglycolide porous particles to the volume of the suspension was 50 mg / mL.

[0033] Step 5: Preparation of working electrode: Glucose oxidase is mixed with the polyglycolide porous particles obtained in step 4-3, and then mixed with a carbon nanotube dispersion with a concentration of 13% and a particle size range of 5-15 nm. The obtained mixed solution is dripped onto the corresponding area on the conductive circuit, and a working electrode is obtained after drying; the working electrode, the counter electrode and the reference electrode constitute a three-electrode system.

[0034] Step 6: Encapsulate the three-electrode system using hydrogel.

[0035] A flexible electrochemical detection circuit was prepared by the above method. Figure 1 The flexible electrochemical detection circuit includes a flexible substrate 1, a three-electrode system fixed on the flexible substrate 1, and a hydrogel layer 9 for encapsulating the three-electrode system. The three-electrode system includes a counter electrode 5, a reference electrode 3, and a working electrode 4.

[0036] Reference Figure 1 The flexible substrate 1 includes a lower substrate 6 (polyimide layer) and an upper substrate 7 (dimethylsiloxane layer), the upper substrate 7 is printed with a nanosilver conductive circuit 2 with a line width of 100 μm, and the counter electrode 5 and the reference electrode 3 are Ag / AgCl electrodes obtained by chlorination treatment of the nanosilver conductive circuit 2.

[0037] Since electrochemical sensors rely on flexible circuits to conduct electrical signals, considering the elasticity of human skin and the curvature of the body, changes in the resistivity of the circuit will affect the final detection accuracy and stability of the device. Since the bending angle of the flexible circuit in most application scenarios is less than 90°, the bending measurement range of the circuit is set to 10-90°, and the measurement interval is 10°. Figure 3 As shown in the figure, when the bending angle is less than 40°, the line resistivity remains between 2.3-3.0Ω / mm. As the bending angle increases further, the resistivity begins to increase rapidly. When the line is bent to 90°, the average resistivity of the line rises to 3.5Ω / mm, but it is still at a relatively low value level. The results show that the flexible circuit has good bendability and electrical stability using a polyimide-dimethylsiloxane composite flexible substrate.

[0038] Reference Figure 1 The nanosilver conductive line 2 in the working electrode 4 region is in a cross-comb structure with a tooth spacing of 150 μm, and the region is covered with a porous conductive carrier 8 with a biological enzyme fixed thereon to obtain the working electrode 4. The porous conductive carrier is a polyglycolide porous particle adsorbed with carbon nanotubes, and the biological enzyme fixed thereon is glucose oxidase. The size of the polyglycolide porous particle is 10-20 μm, and has a micron-scale spatial network structure.

[0039] Due to its high specific surface area, excellent hydrophilicity and biocompatibility, polyglycolide porous particles can effectively immobilize biological enzymes. Figure 4 As shown in (a), after the enzyme solution is dried on the silicon wafer, it presents a flat film-like structure with obvious surface texture and a small amount of micropores. Figure 4 As shown in (b), the spatial network structure of the polyglycolide porous carrier without enzyme immobilization is clear and distinct, with distinct boundaries and good pore distribution. Figure 4 As shown in (c), the surface of the polyglycolide porous particles with fixed enzymes is covered with an enzyme film, showing morphological characteristics such as thickened spatial network structure and reduced porosity. In addition, no thick enzyme layer was observed on the silicon wafer substrate, which indicates that the enzyme solution is gradually adsorbed on the polyglycolide porous particles during the drying process, and finally the enzyme is tightly fixed in the micrometer-level spatial network structure of the carrier.

[0040] The flexible electrochemical detection circuit provided in this embodiment is used for glucose detection: 1 µL of glucose solution is dripped onto a PGA porous film with a diameter of 5 mm, and then the PGA porous film is covered on the electrode reaction area of ​​the flexible electrochemical detection circuit for chronoamperometry detection; different concentrations of glucose solutions are used for detection and the data is recorded.

[0041] like Figure 5 As shown in (a), the current response gradually stabilizes as the detection proceeds, and the steady-state current value gradually increases as the glucose concentration in the solution increases, proving that the flexible electrochemical detection circuit provided in this embodiment can better distinguish the glucose concentration. The response current of glucose solutions of different concentrations has reached a stable state at 10s, so the response current at 10s is selected as the steady-state current of electrochemical detection for subsequent linear fitting.

[0042] The fitting line of the relationship between steady-state current and glucose concentration is as follows Figure 5 As shown in (b), the results show that in the range of 1-10 mM glucose concentration, there is a good linear relationship between the steady-state current value and the glucose concentration, with a sensitivity of 2.63 μA / mM and R 2=0.984. In addition, in order to evaluate the detection limit of the flexible electrochemical detection circuit, the blank control group PBS solution was tested 10 times using the same test method, and the standard deviation of the 10 test results was calculated to be 0.12. The detection limit of the flexible electrochemical detection circuit was calculated to be 0.05mM according to the signal-to-noise ratio method (3N / S).

[0043] In summary, the flexible electrochemical detection circuit provided in this embodiment has good glucose concentration detection capability and can achieve linear high-resolution rapid detection in the glucose concentration range of 1-10mM, laying a good foundation for the subsequent integration of flexible glucose sensors with other components such as porous microneedle arrays.

[0044] The above are all preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Therefore, any equivalent changes made according to the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a flexible electrochemical detection circuit, characterized in that: The following steps are involved: preparing a flexible substrate; Inkjet printing of nanosilver conductive circuits on flexible substrates; preparing a counter electrode and a reference electrode on the nanosilver conductive circuit; Preparation of polyglycolide porous particles: preparing porous polyglycolide by a solvent replacement method, and crushing the porous polyglycolide to obtain micrometer-sized polyglycolide porous particles; Preparation of working electrode: fix the biological enzyme on polyglycolide porous particles, mix with carbon nanotube dispersion, and drip the obtained mixture on the corresponding area on the nanosilver conductive line to obtain the working electrode; the working electrode, counter electrode and reference electrode form a three-electrode system; The three-electrode system was encapsulated using hydrogel.

2. The method for preparing a flexible electrochemical detection circuit according to claim 1, characterized in that: The method for preparing polyglycolide porous particles comprises: preparing a hexafluoroisopropanol solution of polyglycolide; Under stirring conditions, deionized water is added to the hexafluoroisopropanol solution of polyglycolide to precipitate porous polyglycolide; The stirring speed is increased to break the porous polyglycolide into micron-sized porous polyglycolide particles.

3. The method for preparing a flexible electrochemical detection circuit according to claim 2, characterized in that: In the step of adding deionized water to the hexafluoroisopropanol solution of polyglycolide, a micro-injection pump is used to add the deionized water at an injection speed of 80-120 μL / min.

4. The method for preparing a flexible electrochemical detection circuit according to claim 2, characterized in that: In the step of breaking the porous polyglycolide into micrometer-sized porous polyglycolide particles, the stirring speed is 600-800 rpm and the stirring time is 36-60 hours.

5. The method for preparing a flexible electrochemical detection circuit according to claim 2, characterized in that: The method for preparing a hexafluoroisopropanol solution of polyglycolide comprises: The polyglycolide particles are subjected to heat pressing and air quenching to obtain a low-crystallinity polyglycolide film; The low crystallinity polyglycolide film is dissolved in hexafluoroisopropanol to obtain a hexafluoroisopropanol solution of polyglycolide.

6. A flexible electrochemical detection circuit, prepared by the method for preparing a flexible electrochemical detection circuit according to any one of claims 1 to 5, characterized in that: include: Flexible substrate; A three-electrode system, fixed on the flexible substrate, comprising a counter electrode, a reference electrode and a working electrode; wherein the working electrode comprises a conductive circuit in a cross-comb structure and a porous conductive carrier covering the conductive circuit, wherein the porous conductive carrier is a polyglycolide porous particle adsorbed with carbon nanotubes; a biological enzyme is fixed on the porous conductive carrier, and the biological enzyme can undergo a redox reaction with the analyte; The hydrogel layer is used to encapsulate the three-electrode system.

7. A flexible electrochemical detection circuit according to claim 6, characterized in that: The polyglycolide porous particles have a micron-scale spatial network structure.

8. The flexible electrochemical detection circuit according to claim 6, characterized in that: The size of the polyglycolide porous particles is 10-20 μm.

9. A flexible electrochemical detection circuit according to claim 6, characterized in that: The biological enzyme is glucose oxidase, and the analyte is glucose.

10. The flexible electrochemical detection circuit according to claim 6, characterized in that: The flexible substrate comprises a polyimide layer and a dimethylsiloxane layer, and the three-electrode system is fixed on the dimethylsiloxane layer.

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