Preparation method of electrochemical biosensor for multi-mode sweat detection

Abstract

The invention belongs to the field of electrochemical biosensors, discloses a preparation method of an electrochemical biosensor capable of being used for multi-mode sweat detection, and particularly provides a single-electrode multifunctional arrangement method in a three-electrode system. A sensor material with high sensitivity and high selectivity is prepared, then the sensor material is printed through screen printing and assembled into a portable device to achieve simultaneous detection of multiple biomarkers in sweat, and the preparation method comprises the steps of selection and modification of an electrode material, sensor assembly and integration of a signal acquisition and analysis system. Through innovative material design and process optimization, the sensor not only can acquire information of various biomarkers in sweat in real time, but also can provide reliable data analysis, and shows excellent long-term stability and anti-interference capability in practical application. The technology provides an effective and reliable solution for real-time health monitoring.

Description

Technical Field

[0001] The invention relates to a method for preparing a biosensor, and in particular to the field of electrochemical biosensors. Background Art

[0002] Electrochemical biosensors are a sensor preparation method that combines electrochemical and biological systems. By modifying the relevant biological system on the electrode surface, the changes in the biological system are used to cause electrical changes on the electrode surface, and these changes are presented in the form of electrical signals. Electrochemical biosensors have been widely used in various biological analyses due to their high sensitivity, high resolution and portability. With the rapid development of modern electronic technology, the circuits of the electrical signal processing part have become increasingly miniaturized and sophisticated. Compared with optical analysis methods that require additional light detection structures, electrochemical biosensors have shown more prominent advantages in field deployment due to their simple and rapid electrical signal processing process.

[0003] In electrochemical analysis systems, the most common voltammetry-based analysis methods usually use a three-electrode system, including a counter electrode (CE), a working electrode (WE), and a reference electrode (RE). In this configuration, there is only a voltage relationship between the working electrode and the reference electrode, but no current relationship; there is only a current relationship between the working electrode and the counter electrode, but no voltage relationship. The reference electrode is used to control the voltage applied to the working electrode to ensure the accuracy of the voltage, and to obtain the current relationship under the relevant voltage through the current loop formed by the working electrode and the counter electrode. Through this three-electrode configuration, each electrode performs its own function and does not interfere with each other, which makes the traditional electrochemical biosensor have high stability and reliability.

[0004] However, with the increasing demand for multimodal detection, the limitations of the traditional three-electrode system are gradually becoming apparent. When using voltammetry to detect multiple analytes, the number of electrodes in the traditional system needs to be increased accordingly. For example, when detecting two analytes, at least four electrodes are required (one counter electrode, two working electrodes, and one reference electrode). When detecting n analytes, n+2 electrodes are required, where n is the number of working electrodes and the other two are the counter electrode and the reference electrode. This increase in the number of electrodes not only leads to an increase in the size of the sensor, but also limits it in certain application scenarios that require small-sized sensors, such as microvolume detection.

[0005] Especially in sweat analysis, since sweat contains a variety of physiological markers and there may be correlations between the markers, multimodal sensors are usually needed to obtain information on a variety of physiological markers in sweat to indirectly reflect the body's physiological state. However, the size of traditional multimodal sensors increases with the increase in the number of detected objects, which not only requires more sweat samples, but also limits the convenience of sensors in practical applications. Especially in the resting state, the amount of sweat produced by the human body is relatively small, which further highlights the urgency of reducing sensor size and sample requirements. Therefore, how to integrate multiple detection functions in a limited space has become a major challenge in the current field of electrochemical biosensors.

[0006] In response to this challenge, the present invention proposes an innovative electrode combination method, which integrates multiple electrochemical functions on a single electrode to achieve the simultaneous detection of multiple physiological indicators in a limited space. This method uses time switching on a single electrode to enable it to play different roles in different electrochemical analysis steps, avoiding the problem of size increase caused by multi-electrode arrangement. Through this innovative design, four electrodes can achieve four different electrochemical functions, including induced sweating, chloride ion detection, pH detection, and glucose detection in sweat analysis, greatly reducing the size of the sensor and the amount of sweat sample required. This technological breakthrough is of great significance in the development of miniaturized multimodal sensors. Summary of the invention

[0007] The present invention aims to solve the problem of low integration due to the large number of electrodes in the three-electrode system in existing electrochemical biosensors, and proposes a sensor preparation method that realizes multimodal sweat detection by switching multiple functions of a single electrode. This method realizes sweat induction, chloride ion detection, pH detection and glucose detection through four electrodes in an area of ​​about 8mm², greatly reducing the amount of sweat required and improving the integration and portability of the sensor.

[0008] A method for preparing an electrochemical biosensor that can be used for multimodal sweat detection, wherein the electrochemical biosensor consists of four electrodes, and the sensor is integrated by printing Ag / AgCl ink, conductive ink, conductive Prussian blue doped ink, and Ag / AgCl ink by a screen printing method, the screen photosensitive film thickness is 20-30 microns, the screen tension is 25-30 N, and the four electrodes can realize four functions of sweat induction, chloride ion detection, pH detection, and glucose detection.

[0009] In some of the embodiments, the preparation method of the electrochemical biosensor that can be used for multimodal sweat detection is characterized in that the ion release electrode for inducing sweat is prepared and connected by an electrochemical polymerization method; the specific steps are: immersing the entire electrode area from area 1 to area 4 in a mixed solution containing 3,4-ethylenedioxythiophene, sodium benzenesulfonate, heparin, and pilocarpine nitrate, and polymerizing at a constant potential of +1.3~1.5 V for 30~50 s to obtain an ion electrophoresis electrode containing pilocarpine nitrate for inducing sweat, which is recorded as SPCE-PEDOT@Hp electrode.

[0010] In some of the embodiments, the concentration of 3,4-ethylenedioxythiophene is 30-50 mmol / L, sodium benzenesulfonate is 3-5 mmol / L, heparin is 1-3 mmol / L, and pilocarpine nitrate is 20-60 mmol / L.

[0011] In some of the embodiments, 0.2-5 μL of dispersion containing glucose oxidase is drop-coated onto the electrode surface by a rapid enzyme dispenser. In particular, the preferred dispersion volume is 1-3 μL, and the preferred glucose enzyme concentration is 1-3 Unit / μL.

[0012] In some of the embodiments, when using an enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for drop coating, it is necessary to pre-treat the electrode in advance with a 0.1~0.2 mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives; the treatment conditions are: drop coating 4~6 μL of the above-mentioned mixed solution to the electrode surface, activating the electrode for 20~40 minutes, and after drop coating the enzyme dispersion, waiting for it to dry naturally to achieve the fixation of glucose oxidase, which is recorded as SPPE-GOx electrode.

[0013] In some of the embodiments, when the enzyme solution containing glutaraldehyde is drop-coated, acetic acid is used to prepare a solution with a pH value in the range of 5 to 7, and then the solution is used to dissolve a certain amount of glucose oxidase to form an enzyme solution of 1 to 2 Unit / μL, and then bovine serum albumin is added so that the mass ratio of bovine serum albumin to glucose oxidase is 3:7, and then glutaraldehyde solution is added so that the mass ratio of glutaraldehyde in the solution is 0.2% to 1.5%. After the enzyme dispersion is drop-coated, it is allowed to dry naturally to achieve the fixation of glucose oxidase.

[0014] In some of the embodiments, the preparation steps of the electrode with hydrogen ion selectivity and glucose detection ability are: after fixing the enzyme to the electrode surface, immersing the entire electrode area in a mixed solution containing o-aminobenzenesulfonic acid, aniline, and glucose. Preferably, the immersion time is 1 to 3 hours, and an electrode with both hydrogen ion selectivity and glucose detection ability is obtained, which is recorded as SPPE-GOx@SPAN electrode.

[0015] In some embodiments, the concentration of o-aminobenzenesulfonic acid is 1-3 mmol / L, the concentration of aniline is 40-60 mmol / L, and the concentration of glucose is 40-60 mmol / L.

[0016] (1) High integration: In an area of ​​8 mm², four electrodes are used to realize multimodal detection function in sweat analysis, which significantly improves the integration of the sensor.

[0017] (2) Miniaturization and micro-miniaturization: This method provides a new idea for the miniaturization of wearable devices. It can perform efficient detection under low sweat secretion and is suitable for practical applications.

[0018] (3) Strong practicality: By switching multifunctional electrodes, the number of electrodes and sample volume required for the sensor are reduced, making the sensor easier to integrate with other smart devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram of the application of multimodal detection electrochemical biosensors in the field of sweat analysis and its detection implementation principle; Figure 2 It is a physical picture of the four electrodes (areas 1 to 4 are printed using Ag / AgCl ink, conductive ink, conductive Prussian blue doped ink, and Ag / AgCl ink, respectively); Figure 3 is the relationship between the concentration and absorbance of pilocarpine nitrate; Figure 4 It is the realization of four sweat analysis functions of four electrodes, i-iv are the corresponding experimental steps and their corresponding experimental results. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the technical implementation method of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in combination with the technical process steps, specific implementation conditions and materials in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0021] Fabrication of electrochemical biosensor for multimodal sweat detection S1. The integrated electrode is printed by screen printing. The screen photosensitive film is 25 microns thick and the screen tension is 28N. Each electrode is made of Figure 2 Printed with the inks shown.

[0022] S2. Using the electrochemical polymerization method, the entire electrode was immersed in a mixture containing 40 mmol / L 3,4-ethylenedioxythiophene, 4 mmol / L sodium benzenesulfonate, 2 mmol / L heparin, and 30 mmol / L pilocarpine nitrate, and the printed electrodes were connected. Polymerization was performed at a constant potential of +1.4V for 40 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. It was recorded as SPCE-PEDOT@Hp electrode.

[0023] S3. A certain volume of dispersion containing glucose oxidase, bovine serum albumin, water, glutaraldehyde and acetic acid is drop-coated onto the electrode surface shown in region 3 by a rapid enzyme spotting machine. In particular, the preferred dispersion volume is 1 μL, and the preferred glucose enzyme concentration is 1 Unit / μL.

[0024] S4. When the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop coating, the electrode shown in area 3 needs to be pre-treated in advance with a 0.1mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives. The treatment conditions are: drop coating 4.5μL of the above mixed solution on the electrode surface and activating the electrode for 30 minutes. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase. It is recorded as SPPE-GOx electrode.

[0025] S5. When using an enzyme solution containing glutaraldehyde for drop coating, use acetic acid solution to configure the pH value to 5, and then use this solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 1 Unit / μL. Then add bovine serum albumin so that its mass ratio with glucose oxidase is 3:7. Then add glutaraldehyde solution so that its mass ratio in the solution is 0.25%. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase.

[0026] S6. After the enzyme was fixed to the electrode surface, the electrode was immersed in a mixture containing 2 mmol / L o-aminobenzenesulfonic acid, 50 mmol / L aniline, and 50 mmol / L glucose for 1 hour. An electrode with both hydrogen ion selectivity and glucose detection capabilities was obtained. It was recorded as SPPE-GOx@SPAN electrode.

[0027] S7. After preparing the modified screen-printed electrode, combine Figure 1 The three-electrode connection method and function realization sequence shown can realize the four functions of sweat induction, chloride ion detection, pH detection and glucose detection on the four electrodes. Example

[0028] Fabrication of electrochemical biosensor for multimodal sweat detection S1. The integrated electrode is printed by screen printing. The screen photosensitive film is 20 microns thick and the screen tension is 20N. Each electrode is made of Figure 2 Printed with the inks shown.

[0029] S2. Using the electrochemical polymerization method, the electrode was immersed in a mixture containing 30 mmol / L 3,4-ethylenedioxythiophene, 3 mmol / L sodium benzenesulfonate, 3 mmol / L heparin, and 20 mmol / L pilocarpine nitrate, and the printed electrodes were connected. Polymerization was performed at a constant potential of +1.3V for 30 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. It was recorded as SPCE-PEDOT@Hp electrode.

[0030] S3. A certain volume of dispersion containing glucose oxidase, bovine serum albumin, water, glutaraldehyde and acetic acid is drop-coated onto the electrode surface shown in region 3 by a rapid enzyme spotting machine. In particular, the preferred dispersion volume is 2 μL, and the preferred glucose enzyme concentration is 2 Unit / μL.

[0031] S4. When the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop coating, the electrode shown in region 3 needs to be pre-treated in advance with a 0.2mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives. The treatment conditions are: drop coating 4μL of the above-mentioned mixed solution on the electrode surface and activating the electrode for 35 minutes. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase. It is recorded as SPPE-GOx electrode.

[0032] S5. When using the enzyme solution containing glutaraldehyde for drop coating, use acetic acid solution to configure the pH value to 6, and then use the solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 1Unit / μL. Then add bovine serum albumin so that the mass ratio of it to glucose oxidase is 3:7. Then add glutaraldehyde solution so that its mass ratio in the solution is 0.2%. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase.

[0033] S6. After the enzyme was fixed to the electrode surface, the electrode was immersed in a mixture containing 1 mmol / L o-aminobenzenesulfonic acid, 40 mmol / L aniline, and 40 mmol / L glucose for 2 hours. An electrode with both hydrogen ion selectivity and glucose detection capabilities was obtained. It was recorded as SPPE-GOx@SPAN electrode.

[0034] S7. After preparing the modified screen-printed electrode, combine Figure 1 The three-electrode connection method and function realization sequence shown can realize the four functions of sweat induction, chloride ion detection, pH detection and glucose detection on the four electrodes. Example

[0035] Fabrication of electrochemical biosensor for multimodal sweat detection S1. The integrated electrode is printed by screen printing. The screen photosensitive film is 30 microns thick and the screen tension is 30N. Each electrode is made of Figure 2 Printed with the inks shown.

[0036] S2. Using the electrochemical polymerization method, the electrode was immersed in a mixture containing 50 mmol / L 3,4-ethylenedioxythiophene, 5 mmol / L sodium benzenesulfonate, 5 mmol / L heparin, and 40 mmol / L pilocarpine nitrate, and the printed electrodes were connected. Polymerization was performed at a constant potential of +1.5 V for 50 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. It was recorded as SPCE-PEDOT@Hp electrode.

[0037] S3. A certain volume of dispersion containing glucose oxidase, bovine serum albumin, water, glutaraldehyde and acetic acid is dropped onto the electrode surface shown in area 3 by a rapid enzyme dispenser in a drop coating manner. In particular, the preferred dispersion volume is 3 μL, and the preferred glucose enzyme concentration is 3 Unit / μL.

[0038] S4. When the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop coating, the electrode shown in area 3 needs to be pretreated in advance with a 0.15 mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives. The treatment conditions are: drop coating 6 μL of the above mixed solution on the electrode surface and activate the electrode for 20 minutes. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase. It is recorded as SPPE-GOx electrode.

[0039] S5. When using the enzyme solution containing glutaraldehyde for drop coating, use acetic acid solution to configure the pH value to 5.5, and then use this solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 3Unit / μL. Then add bovine serum albumin so that its mass ratio with glucose oxidase is 3:7. Then add glutaraldehyde solution so that its mass ratio in the solution is 0.3%. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase.

[0040] S6. After the enzyme was fixed to the electrode surface, the electrode was immersed in a mixture containing 3 mmol / L o-aminobenzenesulfonic acid, 60 mmol / L aniline, and 60 mmol / L glucose for 3 hours. An electrode with both hydrogen ion selectivity and glucose detection capabilities was obtained. It was recorded as SPPE-GOx@SPAN electrode.

[0041] S7. After preparing the modified screen-printed electrode, combine Figure 1 The three-electrode connection method and function realization sequence shown can realize the four functions of sweat induction, chloride ion detection, pH detection and glucose detection on the four electrodes. Example

[0042] Fabrication of electrochemical biosensor for multimodal sweat detection S1. The integrated electrode is printed by screen printing. The screen photosensitive film is 23 microns thick and the screen tension is 27N. Each electrode is made of Figure 2 Printed with inks shown.

[0043] S2. Using the electrochemical polymerization method, the electrode was immersed in a mixture containing 35 mmol / L 3,4-ethylenedioxythiophene, 4.5 mmol / L sodium benzenesulfonate, 4.5 mmol / L heparin, and 35 mmol / L pilocarpine nitrate, and the printed electrodes were connected. Polymerization was performed at a constant potential of +1.35 V for 45 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. It was recorded as SPCE-PEDOT@Hp electrode.

[0044] S3. A certain volume of dispersion containing glucose oxidase, bovine serum albumin, water, glutaraldehyde and acetic acid is dropped onto the electrode surface shown in area 3 by a rapid enzyme dispenser in a drop coating manner. In particular, the preferred dispersion volume is 2.5 μL, and the preferred glucose enzyme concentration is 3 Unit / μL.

[0045] S4. When the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop coating, the electrode shown in region 3 needs to be pre-treated in advance with a 0.12 mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives. The treatment conditions are: drop coating 5.5 μL of the above-mentioned mixed solution on the electrode surface and activating the electrode for 28 minutes. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase. It is recorded as SPPE-GOx electrode.

[0046] S5. When using an enzyme solution containing glutaraldehyde for drop coating, use acetic acid solution to configure the pH value to 5.3, and then use this solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 2.5Unit / μL. Then add bovine serum albumin so that its mass ratio with glucose oxidase is 3:7. Then add glutaraldehyde solution so that its mass ratio in the solution is 0.25%. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase.

[0047] S6. After the enzyme was fixed to the electrode surface, the electrode was immersed in a mixture containing 2.5 mmol / L o-aminobenzenesulfonic acid, 55 mmol / L aniline, and 55 mmol / L glucose for 2.5 hours. An electrode with both hydrogen ion selectivity and glucose detection capabilities was obtained. It was recorded as SPPE-GOx@SPAN electrode.

[0048] S7. After preparing the modified screen-printed electrode, combine Figure 1 The three-electrode connection method and function realization sequence shown can realize the four functions of sweat induction, chloride ion detection, pH detection and glucose detection on the four electrodes. Example

[0049] Fabrication of electrochemical biosensor for multimodal sweat detection S1. The integrated electrode is printed by screen printing. The screen photosensitive film is 27 microns thick and the screen tension is 27N. Each electrode is made of Figure 2 Printed with the inks shown.

[0050] S2. Using the electrochemical polymerization method, the entire electrode was immersed in a mixture containing 37 mmol / L 3,4-ethylenedioxythiophene, 4 mmol / L sodium benzenesulfonate, 4 mmol / L heparin, and 30 mmol / L pilocarpine nitrate, and the printed electrodes were connected. Polymerization was performed at a constant potential of +1.45 V for 45 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. It was recorded as SPCE-PEDOT@Hp electrode.

[0051] S3. A certain volume of dispersion containing glucose oxidase, bovine serum albumin, water, glutaraldehyde and acetic acid is dropped onto the electrode surface shown in area 3 by a rapid enzyme dispenser in a drop coating manner. In particular, the preferred dispersion volume is 2.3 μL, and the preferred glucose enzyme concentration is 2.3 Unit / μL.

[0052] S4. When the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop coating, the electrode shown in area 3 needs to be pre-treated in advance with a 0.12 mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives. The treatment conditions are: drop coating 5 μL of the above mixed solution on the electrode surface and activating the electrode for 26 minutes. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase. It is recorded as SPPE-GOx electrode.

[0053] S5. When using the enzyme solution containing glutaraldehyde for drop coating, use acetic acid solution to configure the pH value to 6.0, and then use the solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 2.5Unit / μL. Then add bovine serum albumin so that the mass ratio of it to glucose oxidase is 3:7. Then add glutaraldehyde solution so that its mass ratio in the solution is 1%. After drop coating the enzyme dispersion, wait for it to dry naturally to achieve the fixation of glucose oxidase.

[0054] S6. After the enzyme was fixed to the electrode surface, the electrode was immersed in a mixture containing 2.5 mmol / L o-aminobenzenesulfonic acid, 60 mmol / L aniline, and 60 mmol / L glucose for 2.7 hours. An electrode with both hydrogen ion selectivity and glucose detection capabilities was obtained. It was recorded as SPPE-GOx@SPAN electrode.

[0055] S7. After preparing the modified screen-printed electrode, combine Figure 1 The three-electrode connection method and function realization sequence shown can realize the four functions of sweat induction, chloride ion detection, pH detection and glucose detection on the four electrodes. Example

[0056] Fabrication and performance testing of electrochemical biosensors for multimodal sweat detection S1. Take the electrode prepared in Example 1 and perform electrochemical performance analysis in deionized water, McIlvaine buffer and artificial sweat to illustrate the sequential realization of the four functions of the electrode. Artificial sweat contains 85.56 mmol / L NaCl, 14.10 mmol / L NaH 2 PO 4and 2.385 mmol / L of L-histidine, and the final pH value of artificial sweat was 5.5.

[0057] S2. Use UV spectrophotometer to establish the relationship between the concentration of pilocarpine nitrate in deionized water solution and its corresponding absorbance. Figure 3 shown.

[0058] S3. The prepared electrode was subjected to the first to fourth experiments in the order of sweat induction, chloride ion detection, pH detection, and glucose detection to verify the realization of the multifunctionality of the single electrode and that the electrode role played by the single electrode in the previous electrochemical experiment would not affect its function in the subsequent experiments.

[0059] S4. The first step of the experiment was conducted in deionized water to verify the electrically controlled release of pilocarpine nitrate required for the sweat induction process. In order to eliminate the absorbance changes caused by the modified electrode itself under electrical stimulation and obtain the accurate release amount of pilocarpine nitrate, a relevant control experiment was designed. By normalizing the current response and absorbance changes of the control group SPCE-PEDOT@Hp electrode under long-term stimulation and subtracting them uniformly as the experimental background, the following results were obtained: Figure 4 The white part of the histogram shown in Figure i in Figure 2 represents the absorbance obtained after deducting the relevant background. Figure 3 The linear equation obtained can be used to obtain the accurate release amount of pilocarpine nitrate, and the relationship between the release amount and time is as follows: Figure 4 The red data points in Figure i are shown in Figure 5. Finally, within 10 minutes, the total amount of pilocarpine nitrate released was 37.2 μg / cm 2 , which indicates that the obtained electrode can achieve the release of pilocarpine nitrate in the first step for iontophoresis-based sweat induction.

[0060] S5. The second step of the experiment was carried out in a pH 7McIlvaine buffer to verify whether the electrode had an electrochemical response to the change in chloride ion concentration after the first step of the experiment. After the first step of pilocarpine nitrate release, the Ag / AgCl reference electrode in the pilocarpine nitrate release system was used as the chloride ion selective electrode in the chloride ion detection system, that is, the positive electrode of the corresponding primary battery system. The connection method of the relevant electrode alligator clips of the electrochemical workstation of the two-electrode system is the same as Figure 1 Therefore, the symbol of the primary cell used for chloride ion detection in this step is PEDOT@HpPN(s)|Cl-(aq)| Ag / AgCl(s)|. The detection of chloride ions in sweat is achieved by detecting the change of the open circuit voltage of the primary cell under different chloride ion concentrations. Figure 4As shown in Figure ii, as the chloride ion concentration in artificial sweat increases, the absolute value of the open circuit voltage of the two-electrode system gradually decreases. The quasi-logarithmic relationship between the open circuit voltage response and the chloride ion concentration shows that the relationship conforms to the Nernst equation, which verifies the proposed primary battery system. In addition, the two-electrode system has an obvious open circuit voltage response to chloride ions in the concentration range of 0-30mmol / L, indicating that the system can also clearly distinguish chloride ions in the concentration range above and below 30mmol / L, and can be actually used for the screening of cystic fibrosis. Therefore, after the first step of pilocarpine nitrate release, it was verified experimentally that the constructed electrochemical detection system can still realize the second step of chloride ion detection. Next, the pH detection in the third step of sequential detection was studied.

[0061] S6. The third step of the experiment was carried out in McIlvaine buffer with different pH values ​​to verify whether the electrode has an electrochemical response to the change of pH value after the previous experiments. Figure 1 It can be seen that PEDOT@HpPN for pH detection has been used twice, so when the electrode positions of the two-electrode detection system do not change, the symbol of the primary cell becomes SPAN(s)|H+(aq)|PEDOT@HpPN(s). The pH value in sweat can be detected by detecting the change in the open circuit voltage of the primary cell at different pH values. Figure 4 As shown in Figure iii, as the pH value increases, the open circuit voltage of the two-electrode system gradually increases. When the pH value range is 4-8, there is a good linear relationship between the open circuit voltage response and the pH value. This relationship also shows that the relationship between the change in open circuit voltage and the H+ concentration in the solution conforms to the Nernst equation, which also verifies the correctness of the proposed primary battery system. And this interval still covers the pH range of human sweat, indicating that the proposed pH detection system can still be used for pH detection of actual sweat after the first two steps of the sequential detection process, that is, the third step of the sequential detection is successfully achieved.

[0062] S7. Finally, after realizing the functions of the first three electrodes, a preliminary study was conducted on the most important fourth step glucose detection function. The Ag / AgCl electrode used as the reference electrode in the three-electrode system for glucose detection has been used twice in the previous detection, the Ag / AgCl electrode used as the counter electrode has been used once in the previous detection, and the SPPE-GOx@SPAN electrode used as the working electrode has been used once in pH detection. After these uses, the electrochemical response of the SPPE-GOx@SPAN electrode to glucose in the fourth step was experimentally studied. The results are shown in Figure 2. Figure 4As shown in the iV diagram. As can be seen from the figure, compared with the step response obtained under the same experimental conditions as the control (directly performing the fourth step test without the previous three steps), the step response of the SPPE-GOx@SPAN electrode to glucose in the fourth step has approximately the same step current change. The above sequential detection experiment verifies the multifunctionality of the proposed single electrode. Through careful electrode design and arrangement modification, the proposed electrode form and function can meet the needs of actual sweat detection. And the four functions of sweat induction, chloride ion detection, pH detection and glucose detection were successfully realized on the four electrodes.

[0063] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing an electrochemical biosensor for multimodal sweat detection, characterized in that The electrochemical biosensor is composed of four electrodes. The sensor is obtained by integrating four regions 1 to 4 obtained by printing Ag / AgCl ink, conductive ink, conductive Prussian blue doped ink, and Ag / AgCl ink by screen printing. The screen photosensitive film thickness is 20 to 30 microns, and the screen tension is 25 to 30 N. The four electrodes can realize four functions of sweat induction, chloride ion detection, pH detection, and glucose detection.

2. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 1, characterized in that The ion-releasing electrodes for inducing sweating were prepared and connected by an electrochemical polymerization method; the specific steps were: immersing the entire electrode area from area 1 to area 4 in a mixed solution containing 3,4-ethylenedioxythiophene, sodium benzenesulfonate, heparin, and pilocarpine nitrate, and polymerizing at a constant potential of +1.3~1.5 V for 30~50 s to obtain an ion electrophoresis electrode containing pilocarpine nitrate for inducing sweating, which was recorded as SPCE-PEDOT@Hp electrode.

3. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 2, characterized in that The concentration of the 3,4-ethylenedioxythiophene is 30-50 mmol / L, the concentration of sodium benzenesulfonate is 3-5 mmol / L, the concentration of heparin is 1-3 mmol / L, and the concentration of pilocarpine nitrate is 20-60 mmol / L.

4. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 1, characterized in that By drop coating, 0.2-5 μL of dispersion containing glucose oxidase is drop coated onto the electrode surface shown in region 3 by a rapid enzyme dispenser. In particular, the preferred dispersion volume is 1-3 μL, and the preferred glucose enzyme concentration is 1-3 Unit / μL.

5. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 1, characterized in that When using the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for drop coating, the electrode needs to be pretreated in advance with a 0.1~0.2 mmol / L morpholineethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives; the treatment conditions are: drop coating 4~6 μL of the above mixed solution to the electrode surface, activating the electrode for 20~40 minutes, and after drop coating the enzyme dispersion, waiting for it to dry naturally to achieve the fixation of glucose oxidase, which is recorded as SPPE-GOx electrode.

6. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 1, characterized in that When using an enzyme solution containing glutaraldehyde for drop coating, acetic acid is used to prepare a solution with a pH value in the range of 5 to 7, and then a certain amount of glucose oxidase is dissolved in the solution to form an enzyme solution of 1 to 2 Unit / μL. Bovine serum albumin is then added so that the mass ratio of bovine serum albumin to glucose oxidase is 3:7, and then glutaraldehyde solution is added so that the mass ratio in the solution is 0.2% to 1.5%. After drop coating of the enzyme dispersion, the glucose oxidase is fixed by allowing it to dry naturally.

7. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 1, characterized in that The preparation steps of the electrode with hydrogen ion selectivity and glucose detection ability are as follows: after fixing the enzyme to the electrode surface, the entire electrode area is immersed in a mixed solution containing o-aminobenzenesulfonic acid, aniline, and glucose. Preferably, the immersion time is 1 to 3 hours, and an electrode with both hydrogen ion selectivity and glucose detection ability is obtained, which is recorded as SPPE-GOx@SPAN electrode.

8. A method for preparing an electrochemical biosensor for multimodal sweat detection as claimed in claim 7, characterized in that The concentration of o-aminobenzenesulfonic acid is 1-3 mmol / L, the concentration of aniline is 40-60 mmol / L, and the concentration of glucose is 40-60 mmol / L.

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