A method for the preparation of an electrochemical biosensor usable for multi-modal sweat detection
By integrating multiple electrochemical functions on a single electrode, the problem of a large number of electrodes in traditional three-electrode systems is solved, achieving high integration and miniaturization of multimodal sweat detection, which is suitable for wearable devices.
Patent Information
- Application Number
- CN202411166076.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-23
- Publication Date
- 2026-01-13
- Estimated Expiration
- 2044-08-23
AI Technical Summary
Traditional three-electrode systems have a large number of electrodes in multimodal detection, which leads to an increase in sensor size and limits the convenience and sample requirements in small-size application scenarios, especially in sweat analysis.
By employing a single-electrode multi-functional switching approach, multiple electrochemical functions are integrated onto a single electrode. Time-based switching enables functions such as inducing sweating, chloride ion detection, pH detection, and glucose detection, thereby reducing the number of electrodes and increasing integration.
Four functions of detection are achieved within an 8mm² area, significantly reducing the size of the sensor and the amount of sweat samples, improving the sensor's integration and portability, and making it suitable for wearable devices.
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Figure CN119595727B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a method for preparing a biosensor, in particular to the field of electrochemical biosensor. BACKGROUND
[0002] An electrochemical biosensor is a sensor preparation method that combines electrochemistry and biological systems together. By modifying the relevant biological system on the electrode surface, the changes of the biological system can cause changes in the electrical properties of the electrode surface, and these changes can be 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 circuit for processing electrical signals has become increasingly miniaturized and sophisticated. Compared with optical analysis methods that require additional optical detection structures, electrochemical biosensors have more prominent advantages in field deployment due to their simple and fast electrical signal processing.
[0003] In an electrochemical analysis system, the most common analysis method based on voltammetry usually adopts 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, and no current relationship; there is only a current relationship between the working electrode and the counter electrode, and 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 through the current loop formed by the working electrode and the counter electrode, the current relationship under the relevant voltage is obtained. Through this three-electrode configuration, each electrode has its own function and does not interfere with each other, thereby making the traditional electrochemical biosensor have high stability and reliability.
[0004] However, under the background of increasing demand for multi-modal detection, the limitations of the traditional three-electrode system gradually appear. When using voltammetry to detect multiple analytes, the number of electrodes needs to be increased accordingly in the traditional system, for example, when detecting two analytes, at least four electrodes are needed (one counter electrode, two working electrodes, and one reference electrode). When detecting n analytes, n+2 electrodes are needed, 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 its use in some application scenarios that require small-sized sensors, such as micro-volume detection.
[0005] In particular, in sweat analysis, due to the presence of multiple physiological markers in sweat and the possible correlation between markers, it is usually necessary to use multi-modal sensors to obtain information on multiple physiological markers in sweat to indirectly reflect the physiological state of the body. However, the size of the traditional multi-modal sensor increases with the increase in the number of detected substances, which not only requires more sweat sample volume, but also limits the convenience of the sensor in practical application. Especially in the resting state, the amount of sweat produced by the human body is small, which further highlights the urgency of reducing the size of the sensor and the sample requirement. Therefore, how to integrate multiple detection functions in a limited space has become a major challenge in the field of electrochemical biosensors.
[0006] To address this challenge, the present application proposes an innovative electrode combination method, which realizes the simultaneous detection of multiple physiological indicators in a limited space by integrating multiple electrochemical functions on a single electrode. This method uses time switching on a single electrode to play different roles in different electrochemical analysis steps, avoiding the size increase problem caused by multi-electrode arrangement. Through this innovative design, four electrodes can realize four different electrochemical functions, including induced sweat, chloride detection, pH detection and glucose detection in sweat analysis, greatly reducing the size of the sensor and the required sweat sample volume. This technical breakthrough has important significance in the development of miniaturized multi-modal sensors. SUMMARY
[0007] The present application aims to solve the problem of low integration in existing electrochemical biosensors due to the large number of electrodes in a three-electrode system, and proposes a sensor preparation method for multi-modal sweat detection through single-electrode multi-function switching. This method realizes induced sweat, chloride detection, pH detection and glucose detection in an area of about 8 mm² using four electrodes, greatly reducing the required sweat volume and improving the integration and portability of the sensor.
[0008] A preparation method of an electrochemical biosensor for multi-modal sweat detection, wherein the electrochemical biosensor is composed of four electrodes, and the sensor is obtained by integrating four electrodes obtained by printing Ag / AgCl ink, conductive ink, conductive Prussian blue doped ink and Ag / AgCl ink using a screen printing method. The screen photosensitive film is 20-30 microns thick, and the screen tension is 25-30 N. The four electrodes can realize four functions of induced sweat, chloride detection, pH detection and glucose detection.
[0009] In some embodiments, the method for preparing the electrochemical biosensor for multi-modal sweat detection is characterized in that the ion release electrode for inducing sweat is prepared by an electrochemical polymerization method; the specific steps are: the electrode area from region 1 to region 4 is immersed in a mixed solution containing 3,4-ethylenedioxythiophene, sodium benzenesulfonate, heparin, and pilocarpine nitrate, and is polymerized at a constant potential of +1.3-1.5 V for 30-50 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweat, denoted as SPCE-PEDOT@Hp electrode.
[0010] In some embodiments, the concentration of 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.
[0011] In some embodiments, 0.2-5 μL of a dispersion liquid containing glucose oxidase is drop-coated onto the electrode surface by using a rapid point enzyme machine in a drop-coating manner; in particular, the preferred volume of the dispersion liquid is 1-3 μL, and the preferred concentration of glucose oxidase is 1-3 Unit / μL.
[0012] In some embodiments, when the enzyme liquid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop-coating, the electrode needs to be pretreated with 0.1-0.2 mmol / L of a morpholine ethanesulfonic acid aqueous solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and derivatives thereof; the treatment conditions are: 4-6 μL of the above-mentioned mixed solution is drop-coated onto the electrode surface, the electrode is activated for 20-40 minutes, and after the enzyme dispersion liquid is drop-coated, the glucose oxidase is fixed by naturally air-drying, denoted as SPPE-GOx electrode.
[0013] In some embodiments, when the enzyme liquid containing glutaraldehyde is used for drop-coating, an acetic acid solution with a pH value in the range of 5-7 is used, then a certain amount of glucose oxidase is dissolved in the solution to form an enzyme solution with a concentration of 1-2 Unit / μL, then bovine serum albumin is added so that the mass ratio of bovine serum albumin to glucose oxidase is 3:7, then a glutaraldehyde solution is added so that the mass ratio of glutaraldehyde in the solution is 0.2%-1.5%, and after the enzyme dispersion liquid is drop-coated, the glucose oxidase is fixed by naturally air-drying.
[0014] In some embodiments, the preparation steps of the electrode with hydrogen ion selectivity and glucose detection capability are as follows: after the enzyme is fixed to the electrode surface, the electrode region is immersed in a mixed solution containing o-aminobenzenesulfonic acid, aniline and glucose. Preferably, the immersion time is 1-3 hours. Thus, an electrode with hydrogen ion selectivity and glucose detection capability is obtained, which is denoted 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 realize multi-modal detection function in sweat analysis, significantly improving the integration of the sensor.
[0017] (2) Miniaturization and micromation: this method provides a new idea for the miniaturization of wearable devices, and can efficiently detect under low sweat secretion, suitable for practical application.
[0018] (3) Strong practicability: through the switching of multi-functional electrodes, the number of electrodes and the volume of samples required by the sensor are reduced, making the sensor more easily integrated with other intelligent devices. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 is a schematic diagram of the application of the multi-modal detection electrochemical biosensor in the field of sweat analysis and its detection implementation principle;
[0020] Figure 2 is a real object diagram of the four electrodes (region 1 to region 4 use Ag / AgCl ink printing, conductive ink printing, conductive Prussian blue doped ink printing, and Ag / AgCl ink printing, respectively);
[0021] Figure 3 is the relationship between the concentration of pilocarpine nitrate and the absorbance;
[0022] Figure 4 is the realization of four electrodes for four sweat analysis functions, i-iv are the corresponding experimental steps and their corresponding experimental results. DETAILED DESCRIPTION
[0023] In order to make the technical implementation method of the present application more convenient to understand, the technical process steps, specific implementation conditions and materials in the embodiments of the present application will be described below to make the technical solutions in the embodiments of the present application clear and complete. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. Embodiments
[0024] Fabrication of electrochemical biosensor for multi-modal sweat detection
[0025] S1. The integrated electrodes are printed by screen printing method. The screen photosensitive film is 25 microns thick, and the screen tension is 28 N. Each electrode is printed by ink as shown in Figure 2 .
[0026] S2. The electrodes are immersed in a mixed solution containing 40 mmol / L 3,4-ethylenedioxythiophene, 4 mmol / L sodium benzenesulfonate, 2 mmol / L heparin, and 30 mmol / L pilocarpine nitrate by electrochemical polymerization method, and each printed electrode is connected. Polymerize for 40 s at a constant potential of +1.4 V to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweat. Denoted as SPCE-PEDOT@Hp electrode.
[0027] S3. A certain volume of dispersion liquid containing glucose oxidase, bovine serum albumin, water, glutaraldehyde, and acetic acid is dropped and coated on the electrode surface in region 3 by a rapid point enzyme machine. In particular, the preferred volume of dispersion liquid is 1 μL, and the preferred concentration of glucose enzyme is 1 Unit / μL.
[0028] S4. When using enzyme liquid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for drop coating, the electrode in region 3 needs to be pretreated in advance with 0.1 mmol / L aqueous solution of morpholine ethanesulfonic acid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and its derivatives. The treatment conditions are: drop 4.5 μL of the above mixture on the electrode surface, and activate the electrode for 30 minutes. After drop coating the enzyme dispersion liquid, it is naturally air-dried to realize the immobilization of glucose oxidase. Denoted as SPPE-GOx electrode.
[0029] S5. When using the enzyme droplet coating containing glutaraldehyde, use acetic acid solution to configure the pH value to 5, then use the 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 the mass ratio of bovine serum albumin to glucose oxidase is 3:7. Then add a glutaraldehyde solution with a mass ratio of 0.25% in the solution. After the enzyme dispersion is coated, it is naturally dried to realize the fixation of glucose oxidase.
[0030] S6. After the enzyme is fixed to the surface of the electrode, the whole electrode is immersed in a mixed solution containing 2 mmol / L of o-aminobenzenesulfonic acid, 50 mmol / L of aniline, and 50 mmol / L of glucose for 1 hour. Both hydrogen ion selectivity and glucose detection capability are obtained. It is recorded as SPPE-GOx@SPAN electrode.
[0031] S7. After the modified screen-printed electrode is prepared, the three-electrode connection method and the function implementation sequence shown in Figure 1 can be used to realize the four functions of inducing sweat, chloride ion detection, pH detection, and glucose detection on the four electrodes. EMBODIMENT
[0032] Fabrication of electrochemical biosensor for multi-modal sweat detection
[0033] S1. The integrated electrode is printed by a screen printing method. The screen photosensitive film is 20 microns thick, and the screen tension is 20 N. Each electrode is printed by ink as shown in Figure 2 .
[0034] S2. The electrodes are immersed in a mixed solution containing 30 mmol / L of 3,4-ethylenedioxythiophene, 3 mmol / L of sodium benzenesulfonate, 3 mmol / L of heparin, and 20 mmol / L of pilocarpine nitrate by electrochemical polymerization, and the printed electrodes are connected and polymerized at a constant potential of +1.3 V for 30 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweat. It is recorded as SPCE-PEDOT@Hp electrode.
[0035] S3. A certain volume of dispersion liquid containing glucose oxidase, bovine serum albumin, water, glutaraldehyde, and acetic acid is dropped on the electrode surface in area 3 by a rapid enzyme point machine. In particular, the preferred dispersion liquid volume is 2 μL, and the preferred glucose enzyme concentration is 2 Unit / μL.
[0036] S4. When using an enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for drop coating, the electrode shown in region 3 needs to be pretreated with a 0.2 mmol / L aqueous solution of morpholine ethanesulfonic acid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and their derivatives. The treatment conditions are: drop 4 μL of the above mixture onto the electrode surface and activate the electrode for 35 minutes. After drop coating the enzyme dispersion, allow it to air dry naturally to achieve glucose oxidase immobilization. This is designated as the SPPE-GOx electrode.
[0037] S5. When using an enzyme solution containing glutaraldehyde for drop coating, prepare a pH of 6 using acetic acid solution, and then use this solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 1 Unit / μL. Next, add bovine serum albumin to achieve a mass ratio of 3:7 to glucose oxidase. Then, add glutaraldehyde solution to achieve a mass ratio of 0.2% in the solution. After drop coating the enzyme dispersion, allow it to air dry naturally to achieve glucose oxidase immobilization.
[0038] S6. After immobilizing the enzyme onto the electrode surface, immerse the entire electrode in a mixture containing 1 mmol / L o-aminobenzenesulfonic acid, 40 mmol / L aniline, and 40 mmol / L glucose for 2 hours. This yields an electrode with both hydrogen ion selectivity and glucose detection capability. This electrode is designated as the SPPE-GOx@SPAN electrode.
[0039] S7. After preparing the above-mentioned modified screen-printed electrode, combine it with... Figure 1 The three-electrode connection method and functional sequence shown can realize four functions: inducing sweating, chloride ion detection, pH detection, and glucose detection on four electrodes. Example
[0040] Fabrication of an electrochemical biosensor for multimodal sweat detection
[0041] S1. The integrated electrodes are printed using screen printing. The screen-printed photosensitive film is 30 micrometers thick, and the screen tension is 30N. Each electrode is manufactured using... Figure 2 It is printed with the ink shown.
[0042] S2. Using an electrochemical polymerization method, the entire electrode was immersed in a mixed solution containing 50 mmol / L 3,4-ethylenedioxythiophene, 5 mmol / L sodium benzenesulfonate, 5 mmol / L heparin, and 40 mmol / L pilocarpine nitrate. The printed electrodes were connected, and polymerization was carried out at a constant potential of +1.5V for 50 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. This electrode is denoted as SPCE-PEDOT@Hp electrode.
[0043] S3. The enzyme solution containing glucose oxidase, bovine serum albumin, water, glutaraldehyde, and acetic acid is dropped on the electrode surface in region 3 by using a rapid point enzyme machine. In particular, the preferred volume of the enzyme solution is 3 μL, and the preferred concentration of the glucose oxidase is 3 Unit / μL.
[0044] S4. When the enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for dropping, the electrode in region 3 is pretreated with a 0.15 mmol / L aqueous solution of morpholine ethanesulfonic acid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and derivatives thereof. The treatment conditions are as follows: 6 μL of the above mixture is dropped on the electrode surface, and the electrode is activated for 20 minutes. After the enzyme solution is dropped, the glucose oxidase is immobilized by naturally air-drying. This is denoted as an SPPE-GOx electrode.
[0045] S5. When the enzyme solution containing glutaraldehyde is used for dropping, an acetic acid solution is used to adjust the pH value to 5.5, and then a certain amount of glucose oxidase is dissolved in the solution to form an enzyme solution with a concentration of 3 Unit / μL. Then, bovine serum albumin is added to make the mass ratio of the bovine serum albumin to the glucose oxidase 3:7. Then, a glutaraldehyde solution is added to make the mass ratio of the glutaraldehyde in the solution 0.3%. After the enzyme solution is dropped, the glucose oxidase is immobilized by naturally air-drying.
[0046] S6. After the enzyme is immobilized on the electrode surface, the electrode is immersed in a mixed solution containing 3 mmol / L of o-aminobenzenesulfonic acid, 60 mmol / L of aniline, and 60 mmol / L of glucose for 3 hours. Thus, an electrode with both hydrogen ion selectivity and glucose detection capability is obtained. This is denoted as an SPPE-GOx@SPAN electrode.
[0047] S7. After the modified screen-printed electrode is prepared, the three-electrode connection method and the functional implementation sequence shown in Figure 1 are combined. Thus, the four functions of inducing sweat, chloride ion detection, pH detection, and glucose detection can be realized on the four electrodes. Embodiment
[0048] Fabrication of an electrochemical biosensor for multi-modal sweat detection
[0049] S1. The integrated electrode is printed by using a screen printing method. The screen film has a thickness of 23 microns, and the screen tension is 27 N. Each electrode is printed by using the ink shown in Figure 2 .
[0050] S2. Using an electrochemical polymerization method, the entire electrode was immersed in a mixed solution containing 35 mmol / L 3,4-ethylenedioxythiophene, 4.5 mmol / L sodium benzenesulfonate, 4.5 mmol / L heparin, and 35 mmol / L pilocarpine nitrate. The printed electrodes were connected, and polymerization was carried out at a constant potential of +1.35V for 45 s to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweating. This electrode is denoted as SPCE-PEDOT@Hp electrode.
[0051] S3. Using a drop-coating method, a certain volume of a dispersion containing glucose oxidase, bovine serum albumin, water, glutaraldehyde, and acetic acid is drop-coated onto the electrode surface shown in region 3 using a rapid enzyme dispensing machine. Specifically, the preferred dispersion volume is 2.5 μL, and the preferred glucose oxidase concentration is 3 Unit / μL.
[0052] S4. When using an enzyme solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide for drop coating, the electrode shown in region 3 needs to be pretreated with a 0.12 mmol / L aqueous solution of morpholine ethanesulfonic acid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and their derivatives. The treatment conditions are: drop 5.5 μL of the above mixture onto the electrode surface and activate the electrode for 28 minutes. After drop coating the enzyme dispersion, allow it to air dry naturally to achieve glucose oxidase immobilization. This is designated as the SPPE-GOx electrode.
[0053] S5. When using an enzyme solution containing glutaraldehyde for drop coating, prepare an acetic acid solution with a pH of 5.3, and then use this solution to dissolve a certain amount of glucose oxidase to form an enzyme solution of 2.5 Unit / μL. Next, add bovine serum albumin to achieve a mass ratio of 3:7 to glucose oxidase. Then, add glutaraldehyde solution to achieve a mass ratio of 0.25% in the solution. After drop coating the enzyme dispersion, allow it to air dry naturally to achieve glucose oxidase immobilization.
[0054] S6. After immobilizing the enzyme onto the electrode surface, immerse the entire electrode in a mixture containing 2.5 mmol / L o-aminobenzenesulfonic acid, 55 mmol / L aniline, and 55 mmol / L glucose for 2.5 hours. This yields an electrode with both hydrogen ion selectivity and glucose detection capability. This electrode is designated as the SPPE-GOx@SPAN electrode.
[0055] S7. After preparing the above-mentioned modified screen-printed electrode, combine it with... Figure 1 The three-electrode connection method and functional sequence shown can realize four functions: inducing sweating, chloride ion detection, pH detection, and glucose detection on four electrodes. Example
[0056] Electrochemical biosensor fabrication for multi-modal sweat detection
[0057] S1. The integrated electrodes were printed by screen printing method. The screen printing film thickness was 27 microns, and the screen tension was 27 N. Each electrode was printed by ink as shown in Figure 2 .
[0058] S2. The electrodes were immersed in a mixed solution containing 37 mmol / L of 3,4-ethylenedioxythiophene, 4 mmol / L of sodium benzenesulfonate, 4 mmol / L of heparin, and 30 mmol / L of pilocarpine nitrate, and the printed electrodes were connected and polymerized at a constant potential of +1.45 V for 45 s by electrochemical polymerization to obtain an iontophoresis electrode containing pilocarpine nitrate for inducing sweat. This electrode is denoted as SPCE-PEDOT@Hp electrode.
[0059] S3. A certain volume of enzyme dispersion liquid containing glucose oxidase, bovine serum albumin, water, glutaraldehyde, and acetic acid was dropped onto the electrode surface in region 3 by a rapid point enzyme machine in a drop coating manner. In particular, the preferred volume of the dispersion liquid is 2.3 μL, and the preferred concentration of glucose enzyme is 2.3 Unit / μL.
[0060] S4. When the enzyme liquid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is used for drop coating, the electrode shown in region 3 needs to be pretreated in advance with a 0.12 mmol / L aqueous solution of morpholine ethanesulfonic acid containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and derivatives thereof. The treatment conditions are as follows: 5 μL of the above-mentioned mixed solution is dropped onto the electrode surface to activate the electrode for 26 minutes. After the enzyme dispersion liquid is dropped, the glucose oxidase is fixed after the enzyme dispersion liquid is naturally air-dried. This electrode is denoted as SPPE-GOx electrode.
[0061] S5. When the enzyme liquid containing glutaraldehyde is used for drop coating, an acetic acid solution is used to configure a pH value of 6.0, and then a certain amount of glucose oxidase is dissolved in the solution to form an enzyme solution of 2.5 Unit / μL. Then bovine serum albumin is added so that the mass ratio of bovine serum albumin to glucose oxidase is 3:7. Then a glutaraldehyde solution is added so that the mass ratio of glutaraldehyde in the solution is 1%. After the enzyme dispersion liquid is dropped, the glucose oxidase is fixed after the enzyme dispersion liquid is naturally air-dried.
[0062] S6. After the enzyme is fixed to the electrode surface, the electrode is immersed in a mixed solution containing 2.5 mmol / L of o-aminobenzenesulfonic acid, 60 mmol / L of aniline, and 60 mmol / L of glucose for 2.7 hours to obtain an electrode with both hydrogen ion selectivity and glucose detection capability. This electrode is denoted as SPPE-GOx@SPAN electrode.
[0063] S7. After the modified screen-printed electrode is prepared, the three-electrode connection method and the functional implementation sequence shown in Figure 1 can be combined, so that the four functions of inducing sweat, chloride ion detection, pH detection and glucose detection can be realized on the four electrodes. Embodiment
[0064] Fabrication and performance test of electrochemical biosensor for multi-modal sweat detection
[0065] S1. Take the electrode prepared in Example 1, and analyze the electrochemical performance in deionized water, McIlvaine buffer and artificial sweat to illustrate the sequential implementation of the four functions of the electrode. The artificial sweat contains 85.56 mmol / L NaCl, 14.10 mmol / L NaH2PO4 and 2.385 mmol / L L-histidine, and the final pH value of the artificial sweat is 5.5.
[0066] S2. Establish the relationship between the concentration of pilocarpine nitrate in deionized water and the corresponding absorbance by ultraviolet spectrophotometry. The relationship is shown in Figure 3 .
[0067] S3. Perform the first to fourth step experiments on the prepared electrode in the order of inducing sweat, chloride ion detection, pH detection and glucose detection to verify the implementation of the multi-functionality of the single electrode and the electrode role playing of the single electrode in the previous electrochemical experiments does not affect its function in the subsequent experiments.
[0068] S4. The first step experiment is performed in deionized water to verify the electrically controlled release of pilocarpine nitrate required in the process of inducing sweat. To exclude the absorbance change caused by the modified electrode under electrical stimulation, thereby obtaining the accurate amount of pilocarpine nitrate released, a related control experiment is designed. By normalizing the current response and absorbance change of the control group SPCE-PEDOT@Hp electrode under long-term stimulation, and taking it as the experimental background for unified subtraction, the white part of the column chart shown in Figure 4 is obtained. This part represents the absorbance obtained after subtracting the relevant background. By bringing this absorbance into the linear equation shown in Figure 3 , the accurate amount of pilocarpine nitrate released can be obtained, and the relationship between the release amount and time is shown by the red data points in Figure 4 . Finally, the total amount of pilocarpine nitrate released in 10 min is 37.2 μg / cm 2 , which indicates that the electrode obtained can realize the release of pilocarpine nitrate in the first step for inducing sweat based on iontophoresis.
[0069] S5. The second step experiment was conducted in pH 7 McIlvaine buffer to verify whether the electrode after the first step experiment has electrochemical response to the change of chloride ion concentration. 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, i.e. the positive electrode of the corresponding primary cell system. The connection mode of the two-electrode system related electrode alligator clip was the same as Figure 1 . Therefore, the primary cell symbol for chloride ion detection in this step was PEDOT@HpPN(s) | Cl"(aq) | Ag / AgCl(s), and the detection of chloride ions in sweat was achieved by detecting the change of the open circuit voltage of the primary cell at different chloride ion concentrations. As shown in Fig. ii of Figure 4 , with the increase of the concentration of chloride ions in artificial sweat, the absolute value of the open circuit voltage of the two-electrode system gradually decreased. The logarithmic relationship between the open circuit voltage response and the concentration of chloride ions indicated that the relationship conformed to the Nernst equation, which verified the proposed primary cell system. Moreover, the two-electrode system had obvious open circuit voltage response to chloride ions in the concentration range of 0-30 mmol / L, indicating that the system could also obviously distinguish chloride ions in the concentration range above and below 30 mmol / L, and could be actually used for cystic fibrosis screening. Therefore, after the first step of pilocarpine nitrate release, it was verified by experiment that the constructed electrochemical detection system could still achieve the second step of chloride ion detection. Next, the third step of pH detection in the sequential detection was studied.
[0070] S6. The third step experiment was conducted in different pH McIlvaine buffer to verify whether the electrode after the previous experiments has electrochemical response to the change of pH value. After the second step of chloride ion detection, the Figure 1 It can be seen that PEDOT@HpPN used for pH value detection has been used twice, so under the condition that the electrode position of the two-electrode detection system does not change, the primary cell symbol becomes SPAN(s) | H+(aq) | PEDOT@HpPN(s). The detection of pH value in sweat was achieved by detecting the change of the open circuit voltage of the primary cell at different pH values. As shown in Fig. iii of Figure 4 , with the increase of pH value, the open circuit voltage of the two-electrode system gradually increased. In the pH value range of 4-8, the open circuit voltage response and the pH value had a good linear relationship. This relationship also indicated that the relationship between the change of open circuit voltage and the concentration of H+ in the solution conformed to the Nernst equation, which also verified the correctness of the proposed primary cell system. Moreover, this interval still covered the pH value range of human sweat, indicating that the proposed pH value detection system after the first two steps of the sequential detection process could still be used for actual pH detection of sweat, i.e. successfully achieved the third step of the sequential detection.
[0071] S7. Finally, after the first three electrode functions were realized, the most important fourth step glucose detection function was preliminarily studied. The Ag / AgCl electrode used as the reference electrode in the glucose detection three-electrode system 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 the pH detection. After these uses, the electrochemical response of the SPPE-GOx@SPAN electrode to glucose in the fourth step was tested and studied. The results are shown in FIG. 8. Figure 4 From the figure, it can be seen that the ladder current change of the SPPE-GOx@SPAN electrode in the fourth step to glucose is approximately the same as that obtained under the same test conditions as the control (without the previous three-step test, directly for the fourth step test). The above sequential detection experiment verifies the proposed multifunctionality of the single electrode. Through careful electrode design and arrangement modification, the proposed electrode form and function can meet the needs of actual sweat detection. And successfully realized the four functions of inducing sweat, chloride ion detection, pH detection and glucose detection on the four electrodes.
[0072] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.
Claims
1. A method of preparation of an electrochemical biosensor for multi-modal sweat detection, characterized by The electrochemical biosensor is composed of four electrodes, which can realize four different electrochemical functions, including sweat induction, chloride ion detection, pH detection and glucose detection in sweat analysis, and the sensor is printed by screen printing method to obtain four regions 1-4 integrated by Ag / AgCl ink, conductive ink, conductive Prussian blue doped ink and Ag / AgCl ink, the thickness of the screen photosensitive film is 20-30 microns, the screen tension is 25-30 N, and the four electrodes can realize the four functions of sweat induction, chloride ion detection, pH detection and glucose detection; the specific preparation method of the four electrodes is as follows: S1. The ion release electrode for inducing sweat is prepared by an electrochemical polymerization method; the specific steps are as follows: the electrode region is immersed in a mixed solution containing 3,4-ethylenedioxythiophene, sodium benzenesulfonate, heparin and pilocarpine nitrate from region 1 to region 4 as a whole, and is polymerized at a constant potential of +1.3-1.5 V for 30-50 s, so that the iontophoresis electrode containing pilocarpine nitrate for inducing sweat is obtained, which is recorded as SPCE-PEDOT@Hp electrode; S2. When using enzyme droplet coating containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the electrode needs to be pretreated in advance with 0.1-0.2 mmol / L aqueous morpholine ethanesulfonic acid solution containing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and its derivatives; the treatment conditions are as follows: 4-6 μL of the above-mentioned mixed solution is dropped on the electrode surface, and the electrode is activated for 20-40 minutes, after the enzyme dispersion solution is dropped, it is naturally dried to realize the fixation of glucose oxidase, which is recorded as SPPE-GOx electrode; S3. The preparation steps of the electrode with hydrogen ion selectivity and glucose detection capacity are as follows: after the enzyme is fixed to the electrode surface, the electrode region is immersed in a mixed solution containing o-aminobenzenesulfonic acid, aniline and glucose as a whole, and the immersion time is 1-3 hours, so that the electrode with hydrogen ion selectivity and glucose detection capacity is obtained, which is recorded as SPPE-GOx@SPAN electrode; S4. After the pilocarpine nitrate release in the first step, the Ag / AgCl reference electrode in the pilocarpine nitrate release system is used as the chloride ion selective electrode in the chloride ion detection system, that is, the positive electrode of the original battery system.
2. A method of preparing an electrochemical biosensor for multi-modal sweat detection as claimed in claim 1, characterized in that The concentration of 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.
3. A method of preparing an electrochemical biosensor for multi-modal sweat detection as claimed in claim 1, wherein 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.