A sweat sensor and a preparation method and application thereof

Abstract

The application relates to a sweat sensor and a preparation method and application thereof, relates to the technical field of biosensors, solves the blank of hybrid nanoflowers in the field of sweat analysis and the problem of long-term stability limitation of traditional biosensors. An enzyme solution is mixed with a copper sulfate solution and is uniformly oscillated, a mineralization reaction of enzyme molecules is generated in a water bath, and purified enzyme nanoflowers are obtained; a multi-electrode array is designed, a conductive silver paste is printed on a PET base, electrode wires and a silver electrode are formed, three working electrodes and one counter electrode are printed by using conductive carbon ink, a reference electrode is formed on the surface of the silver electrode, insulating ink is printed as a circuit protection layer, after electrode activation, multi-walled carbon nanotubes are dispersed in DMF, a PB / PPy film is formed on the surface of the electrode through electropolymerization after coating, enzyme nanoflower solution is added to the surface of the working electrode, a chitosan protection film is coated, and the sensor is obtained. The application has wide application prospects and important social value in the fields of medical detection and health monitoring.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, specifically to a sweat sensor, its preparation method, and its application. Background Technology

[0002] Human sweat, as an important bodily fluid, contains rich biological information, and its composition is closely related to health status and physiological metabolism. In recent years, sweat analysis has received widespread attention due to its non-invasive, real-time, and continuous monitoring characteristics, becoming an important research direction in the field of flexible electronics. By detecting metabolites (such as glucose, uric acid, lactic acid, and amino acids) and electrolytes (such as sodium ions, potassium ions, and chloride ions) in sweat, it is possible to assess a person's exercise status, nutritional status, and potential disease risks. In particular, the detection of biomarkers related to neurodegenerative diseases in sweat, such as levodopa, ascorbic acid, and glucose, is of great significance for the early diagnosis of diseases such as Parkinson's disease.

[0003] Biosensors, due to their high sensitivity and specificity, have been widely used in the detection of biomarkers. Traditional enzyme-based biosensors occupy an important position in the field of biosensors and are one of the earliest developed and most widely used types of biosensors. Enzyme-based biosensors utilize the high specificity and catalytic efficiency of enzymes to convert specific biochemical reactions into easily measurable electrical signals or other forms of signals, thereby achieving rapid and accurate detection of specific substances. However, traditional enzyme-based biosensors still face multiple challenges in practical applications, including: (1) the susceptibility of enzymes to inactivation, as enzymes are easily deactivated under conditions of high temperature, humidity changes, and long-term use, significantly reducing the reliability and lifespan of the sensor; (2) the influence of environmental interference, as sweat has a complex composition and is easily affected by factors such as pH value and ionic strength, leading to a decrease in the accuracy of detection results; and (3) insufficient long-term stability of the sensor, as existing sensors are prone to performance degradation under continuous monitoring conditions, making it difficult to meet the needs of real-time and dynamic monitoring. Therefore, improving the catalytic activity and stability of enzymes has become an important research direction in the field of biosensing.

[0004] In recent years, researchers have attempted to improve the stability of enzymes by encapsulating them with metal-organic frameworks (MOFs). However, while this method enhances stability, it inevitably leads to a decrease in the enzyme's catalytic activity, limiting its practical applications. Therefore, developing an innovative strategy that can simultaneously achieve both catalytic activity and stability remains a key problem that urgently needs to be solved in this field.

[0005] By combining organic materials (such as enzyme molecules) with inorganic materials (such as metal ions), the activity and stability of enzymes can be significantly improved, while enhancing the selectivity and anti-interference ability of sensors for target substances. Hybrid nanoflowers have the following advantages: (1) High specific surface area: which is conducive to the immobilization of enzyme molecules and the adsorption of target substances, thus improving detection sensitivity; (2) Enhanced catalytic performance: the inorganic components provide a stable microenvironment for enzyme molecules, significantly improving catalytic efficiency; (3) Excellent structural stability: the nanoflower structure can effectively protect enzymes from the influence of external environmental factors, improving long-term stability. However, although hybrid nanoflowers have shown great potential under laboratory conditions, their practical application in the field of sweat analysis is still in its early stages, especially real-time monitoring equipment for Parkinson's disease-related biomarkers is still scarce. In addition, the design of traditional flexible sensors usually makes it difficult to balance flexibility, stability and high sensitivity. Moreover, existing sensors mostly rely on complex structural designs and expensive materials, and the preparation process is cumbersome and costly, making it difficult to meet the needs of large-scale commercialization. Summary of the Invention

[0006] To address the current gaps in the application of hybrid nanoflowers in sweat analysis and the limited long-term stability of traditional biosensors, this invention proposes a sweat sensor based on organic-inorganic hybrid nanoflowers, its preparation method, and its applications.

[0007] The specific technical solution of the present invention is as follows:

[0008] A method for preparing a sweat sensor includes the following steps:

[0009] S1. Dissolve the enzyme in phosphate buffer solution to obtain an enzyme solution, then add copper sulfate solution and shake well; incubate the mixture in a water bath to allow the enzyme molecules to mineralize; collect the precipitate by centrifugation, wash with ultrapure water to remove unreacted enzyme, and obtain purified enzyme nanoflowers.

[0010] S2. Design a multi-electrode array and fabricate an external mask, which is then fixed onto a PET substrate;

[0011] Conductive silver paste is printed and coated to form electrode wires and a silver electrode, and then baked and cured.

[0012] Three working electrodes and one counter electrode are printed using conductive carbon ink.

[0013] FeCl3 solution was added dropwise to the surface of a silver electrode to form an Ag / AgCl reference electrode;

[0014] The printed insulating ink serves as a circuit protective layer and is cured under a UV lamp.

[0015] S3. Electrode activation was performed by cyclic voltammetry, followed by dispersing multi-walled carbon nanotubes in DMF solution, coating them onto the surface of the carbon working electrode, and drying them.

[0016] Pyrrole and Prussian blue are polymerized simultaneously on the surface of the working electrode to form a PB / PPy composite film.

[0017] The enzyme nanoflower solution obtained in step S1 was dropped onto the surface of the PB / PPy modified working electrode and dried overnight at room temperature.

[0018] A chitosan solution was drop-coated onto the surface of the working electrode and allowed to dry naturally to form a stable protective film, thus obtaining a sweat sensor.

[0019] Preferably, the phosphate buffer solution in step S1 has a concentration of 0.01 mol / L and a pH of 7.4; the copper sulfate solution has a concentration of 60 mmol / L to 150 mmol / L.

[0020] Specifically, the concentration of the copper sulfate solution can be 60 mmol / L, 80 mmol / L, 100 mmol / L, 120 mmol / L, or 150 mmol / L, with 120 mmol / L being the most preferred.

[0021] The concentration of the enzyme solution is 0.1 mg / mL to 1 mg / mL.

[0022] Preferably, the enzyme in step S1 is one of glucose oxidase, ascorbic acid oxidase, and tyrosinase; the concentration of the glucose oxidase solution is preferably 0.5 mg / mL, the concentration of the ascorbic acid oxidase solution is preferably 1 mg / mL, and the concentration of the tyrosinase solution is preferably 0.72 mg / mL.

[0023] Preferably, in step S1, the water bath temperature is 25°C, the culture time is 72 hours, the centrifugation speed is 3500 rpm, the centrifugation time is 10 minutes, and the washing is performed 3 times.

[0024] Preferably, the thickness of the PET substrate in step S2 is 0.1 mm; the size of the mask is 10 cm * 10 cm, and the thickness is 125 μm.

[0025] Preferably, the resistivity of the conductive carbon ink in step S2 is 10 Ω / cm. 2 .

[0026] Preferably, the baking and curing temperature in step S2 is 60°C, and the baking and curing time is 30 minutes.

[0027] Preferably, the concentration of the FeCl3 solution in step S2 is 0.05 mol / L, and the amount added is 5 mL.

[0028] Preferably, the amount of enzyme nanoflower solution added in step S3 is 5 μL, and the amount of chitosan solution added is 4 μL.

[0029] The present invention also provides a sweat sensor, which is prepared using the above-described preparation method.

[0030] The present invention also provides an application of the above-mentioned sweat sensor in a real-time monitoring device for Parkinson's disease-related biomarkers.

[0031] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0032] This approach utilizes a biomineralization method to prepare an organic-inorganic hybrid nanoflower structure from copper ions, glucose oxidase, ascorbic acid oxidase, and tyrosinase. Combined with a flexible substrate design, this results in a flexible wearable sweat sensor. By leveraging the unique biomineralization properties of the nanoflowers, the highly efficient catalytic properties of organic enzymes, and the stability of inorganic materials, the detection of Parkinson's disease-related biomarkers can be achieved.

[0033] 1. Improved sensitivity and stability:

[0034] The structure of organic-inorganic hybrid nanoflowers significantly enhances the catalytic activity of enzymes. This nanoflower structure provides enzymes with a larger specific surface area and a more suitable microenvironment, promoting enzyme-substrate contact and reaction, thereby enhancing the catalytic efficiency for Parkinson's disease-related biomarkers (such as levodopa, ascorbic acid, and glucose), enabling highly efficient detection.

[0035] Nanoflower structures effectively improve enzyme stability. Traditional enzymes are easily inactivated by external environments, while nanoflower structures can protect the active site of the enzyme, allowing it to maintain high activity over a long period. For example, in practical applications, even after prolonged use or under complex physiological conditions, the sensor can still function stably, ensuring the accuracy and reliability of the detection results.

[0036] 2. Flexibility and portability:

[0037] The sensor substrate is made of flexible PET material, which has excellent flexibility and mechanical adaptability. It can conform well to human skin, and its performance and wearing comfort will not be affected by bending, stretching or other movements of the body during human activity.

[0038] Due to its flexibility and thinness, the sensor can be easily integrated into wearable devices such as clothing and wristbands for extended wear. Users will hardly notice the sensor's presence in daily life, causing no disruption to their routines, making it ideal for real-time, continuous sweat monitoring.

[0039] 3. Multi-marker monitoring:

[0040] The sensor provided by this invention can simultaneously monitor multiple Parkinson's disease-related biomarkers, such as levodopa, ascorbic acid, and glucose. These biomarkers play an important indicative role in the occurrence and development of Parkinson's disease. By detecting them simultaneously, a more comprehensive understanding of the human physiological state can be achieved, providing richer information for the early diagnosis of the disease.

[0041] 4. The preparation process of this invention is relatively simple, requiring no complex equipment or harsh reaction conditions, thus reducing energy consumption and the use of chemical reagents, and is environmentally friendly. Furthermore, the materials used are low-cost and widely available, facilitating large-scale production and commercialization.

[0042] This invention provides new ideas for the design and development of wearable biosensors, and has broad application prospects and important social value in the fields of medical testing and health monitoring. Attached Figure Description

[0043] Figure 1 Microscopic morphology of the enzyme nanoflowers prepared in Example 1;

[0044] Figure 2 The elemental analysis results of the glucose oxidase nanoflowers prepared in Example 1 are shown in the figure.

[0045] Figure 3 The figure shows the results of comparing the long-term stability of three types of nanoflowers (GOx-NFs, AOx-NFs, and Tyr-NFs) with that of pure enzymes.

[0046] Figure 4 This is a schematic diagram of the fabrication process for screen-printed electrodes.

[0047] Figure 5 This is a schematic diagram of the structure of the biosensor provided by the present invention;

[0048] Figure 6 The electrochemical detection results of the biosensor provided by this invention;

[0049] Figure 7 The surface test results of the biosensor provided by the present invention;

[0050] Figure 8 This is a schematic diagram illustrating the detection principle of the biosensor provided by the present invention. Detailed Implementation

[0051] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0052] Example 1.

[0053] In this embodiment, glucose oxidase nanoflowers were prepared through the following steps:

[0054] Preparation and reaction of S1, CuSO4 solution and enzyme solution:

[0055] (1) Weigh 29.9 mg of copper sulfate pentahydrate powder and add it to 1 mL of ultrapure water. Dissolve it by sonication to form a clear blue solution, which is labeled as 120 mmol / L CuSO solution.

[0056] (2) Add 0.555 mL of 0.01 mol / L phosphate buffer solution (pH 7.4) to 10 KU of glucose oxidase (≥180 U / mg), shake well, and label as 100 mg / mL glucose oxidase solution. Then take 5 μL of the above glucose oxidase solution and add it to 995 μL of 0.01 mol / L phosphate buffer solution (pH 7.4), shake well, and label as 0.5 mg / mL glucose oxidase solution.

[0057] (3) Take 20 μL of the above CuSOD solution, add it to 1 mL of 0.5 mg / mL glucose oxidase solution, mix well, and place it in a 25℃ water bath for 72 h.

[0058] S2. After the reaction was completed, the precipitate was separated by centrifugation (3500 rpm, 10 min), and washed three times with ultrapure water to remove unbound enzyme and salt ions, yielding purified glucose oxidase nanoflower GO. x -NFs.

[0059] The microstructure of the enzyme nanoflower was scanned, and its microstructure is as follows: Figure 1 As shown. Elemental analysis of this enzyme nanoflower was performed, and the results are shown in the figure. Figure 2 This further proves that the enzyme nanoflower is composed of a copper phosphate backbone and glucose oxidase within it.

[0060] Example 2.

[0061] In this embodiment, ascorbic acid oxidase nanoflowers were prepared through the following steps:

[0062] Preparation and reaction of S1, CuSO4 solution and enzyme solution:

[0063] (1) Weigh 29.9 mg of copper sulfate pentahydrate powder and add it to 1 mL of ultrapure water. Dissolve it by sonication to form a clear blue solution, which is labeled as 120 mmol / L CuSO solution.

[0064] (2) Add 0.555 mL of 0.01 mol / L phosphate buffer solution (pH 7.4) to 10 KU of ascorbic acid oxidase (≥180 U / mg), shake well, and label as 100 mg / mL glucose oxidase solution. Then take 10 μL of the above ascorbic acid oxidase solution and add it to 990 μL of 0.01 mol / L phosphate buffer solution (pH 7.4), shake well, and label as 1 mg / mL ascorbic acid oxidase.

[0065] (3) Take 20 μL of the above CuSO solution, add 1 mL of 0.5 mg / mL ascorbic acid oxidase solution, mix well, and place in a 25℃ water bath for 72 h.

[0066] S2. After the reaction is complete, the precipitate is separated by centrifugation (3500 rpm, 10 min) and washed three times with ultrapure water to remove unbound enzymes and salt ions, thus obtaining purified ascorbic acid oxidase nanoflower AOx-NFs.

[0067] Example 3.

[0068] In this embodiment, ascorbic acid oxidase nanoflowers were prepared through the following steps:

[0069] Preparation and reaction of S1, CuSO4 solution and enzyme solution:

[0070] (1) Weigh 29.9 mg of copper sulfate pentahydrate powder and add it to 1 mL of ultrapure water. Dissolve it by sonication to form a clear blue solution, which is labeled as 120 mmol / L CuSO solution.

[0071] (2) Add 0.555 mL of 0.01 mol / L phosphate buffer solution (pH 7.4) to 10 KU of tyrosinase (≥180 U / mg), shake well, and label as 100 mg / mL tyrosinase solution. Then take 7.2 μL of the above tyrosinase solution and add it to 992.8 μL of 0.01 mol / L phosphate buffer solution (pH 7.4), shake well, and label as 0.72 mg / mL tyrosinase solution.

[0072] (3) Take 20 μL of the above CuSO solution, add it to 1 mL of 0.5 mg / mL tyrosinase solution, mix well, and place it in a 25℃ water bath for 72 h.

[0073] S2. After the reaction is complete, the precipitate is separated by centrifugation (3500 rpm, 10 min) and washed three times with ultrapure water to remove unbound enzymes and salt ions, thus obtaining purified tyrosinase nanoflowers Tyr-NFs.

[0074] Example 4.

[0075] This example tests the catalytic activity of the enzyme nanoflowers prepared in Examples 1-3:

[0076] S1, GO x and GO x -NFs catalytic activity test:

[0077] (1) Add 20 μL of GO to each container. x -NFs and GO x The solution (0.5 mg / mL) was added to 400 μL of PBS (pH = 7.4, 0.01 mol / L), followed by the addition of 600 μL of glucose solution at a concentration of 1–10 mmol / L.

[0078] (2) After incubating at 35°C for 20 min, add 100 μL TMB (20 mmol / L) and 20 μL HRP (0.1 mg / mL).

[0079] The absorbance was measured at 370 nm using a UV-Vis spectrometer.

[0080] Comparison of free GO x and GO x The relative activity of -NFs, with free GO x The activity is 100% for reference.

[0081] S2, AO x and AO x -NFs catalytic activity test:

[0082] (1) Add 70 μL of ascorbic acid (1-10 mmol / L), 150 μL of phen and 150 μL of NH4Fe(SO4)2·12H2O to 2150 μL of sodium acetate buffer (pH=5.6, 0.1M), incubate at 35°C for 10 min, and then use a UV-Vis spectrometer to measure the absorbance at 450 nm.

[0083] (2) Add 20 μL of AO to each 70 μL ascorbic acid (1-10 mmol / L) solution. x -NFs and AO x Incubate the solution (1 mg / mL) for 10 min. Then repeat (1).

[0084] (3) Calculate the difference in absorbance at 450 nm.

[0085] (4) With free AO x Using activity as a reference, evaluate immobilized AO x -NF activity.

[0086] S3, Tyr, and Tyr-NFs activity assays:

[0087] (1) Add 100 μL L-Dopa (1-5 mmol / L) and 100 μL Tyr-NFs (0.72 mg / mL) to 300 μL PBS (pH=7.4, 0.01 M) and incubate at 35 °C for 20 min.

[0088] (2) The absorbance was measured at 475 nm using a UV-Vis absorption spectrometer.

[0089] (3) Compare the relative activities of free Tyr and Tyr-NFs, with free Tyr activity as 100% as a reference.

[0090] Figure 3 Three types of nanoflowers (GO) x -NFs, AO x This diagram illustrates the long-term stability test results of the three purified enzymes (GO-NFs, Tyr-NFs, and GO-NFs) and the purified enzymes. The information in this diagram demonstrates that the three purified enzymes (GO-NFs, Tyr-NFs, and GO-NFs) exhibit stable long-term stability. x AO x The enzymes Tyr and Glycerol (GMOs) exhibited poor long-term stability, with their relative activities decreasing to varying degrees over time. Tyr showed the worst stability, almost completely losing its enzyme activity within a short period. However, the methods described in the above examples were used to prepare enzyme nanoflowers (GOs). x -NFs, AO x After incorporating NFs and Tyr-NFs, the stability was significantly improved. The relative activity of the enzyme nanoflowers remained at a high level over 60 days, especially for GO. x -NFs and AO x -NFs showed almost no significant decrease in relative activity.

[0091] All of the above tests were performed in parallel three times to ensure the reliability of the data.

[0092] Example 5.

[0093] This embodiment prepares a screen-printed electrode through the following steps, as illustrated in the schematic diagram of the preparation process. Figure 4 As shown.

[0094] S1. Electrode design and template preparation:

[0095] A multi-electrode array, consisting of three working electrodes (WE), one counter electrode (CE), and one reference electrode (RE), was designed using AutoCAD 2021. The array was fabricated as a 10cm*10cm mask with a thickness of 125μm and then fixed onto a PET substrate (0.1mm thick) using a semi-automatic screen printing machine.

[0096] S2, Silver Ink Printing:

[0097] Using an oil-based polyurethane squeegee, a layer of conductive silver paste is printed on the substrate at a speed of 5 cm per second to form the initial shape of conductive lines and electrodes. The printed substrate is then baked at 60°C for 30 minutes to cure the silver ink.

[0098] S3, Carbon Ink Printing:

[0099] Conductive carbon ink and acetone were mixed evenly at a volume ratio of 20:1. Then, conductive carbon ink (resistivity 10 Ωcm-2) was screen-printed using an oil-based polyurethane squeegee at a speed of 5 cm per second to prepare three working electrodes and a counter electrode, which were then baked at 60°C for 30 min.

[0100] S4. Preparation of reference electrode:

[0101] 5 mL of FeCl solution (0.05 mol / L) was dropped onto a silver electrode, reacted for 1 min, rinsed with ultrapure water, and dried for 30 min to prepare an Ag / AgCl reference electrode.

[0102] S5, Protective layer printing:

[0103] Using an oil-based polyurethane squeegee, insulating ink was screen-printed at a speed of 5 cm per second to cover the wire area connecting the working electrode and the interface, and then cured under 365 nm ultraviolet light for 2 hours.

[0104] S6. Conductivity verification:

[0105] Use a multimeter to test the resistance between the electrode and the interface of each conductive path to ensure the integrity and continuity of the electrode.

[0106] Example 6.

[0107] In this embodiment, the sensor was assembled and modified according to the following steps:

[0108] S1, Electrode activation:

[0109] One end of the screen-printed electrode is connected to an electrochemical workstation via an interface. The working electrode, reference electrode, and counter electrode at the other end are immersed in a 0.5 mol / L HSO solution. The electrodes are activated by cyclic voltammetry on the electrochemical workstation, with a potential range of -1.0 V to 1.5 V, a scan rate of 50 mV / s, and 30 cycles.

[0110] S2, MWCNT coating:

[0111] 10 mg MWCNT was dispersed in 1 mL DMF solution, ultrasonically dispersed for 1 h, and then 4 μL was dropped onto the surface of each carbon working electrode. The electrode was then placed on a 35 °C hot table to dry for 30 min.

[0112] S3, PB / PPy membrane electropolymerization:

[0113] Pyrrole (0.08 mol / L), KFe(CN) (2.5 mmol / L), FeCl (2.5 mmol / L), and HCl (0.1 mmol / L) were added sequentially to 5 mL of ultrapure water and stirred for 5 min. The electrode was connected to an electrochemical workstation, and CV was used to scan 20 times within the potential range of -0.2 V to 0.9 V to prepare the PB / PPy composite membrane. The electrode surface was then rinsed three times with ultrapure water.

[0114] S4, Nanoflower Modification:

[0115] 5 μL of the GO prepared in Examples 1-3 was drop-coated onto the PB / PPy modified working electrode. x -NFs, AO x -NFs and Tyr-NFs solutions were left at room temperature overnight and allowed to air dry.

[0116] S5, Chitosan protective film:

[0117] Add 0.05 g of chitosan powder to a clean beaker, add 50 mL of 0.1 mol / L acetic acid solution, sonicate for 5 min, and then stir with a magnetic stirrer for 2 h to obtain a clear and transparent solution. Drop 4 μL of the above chitosan solution onto the working electrode and allow it to dry at room temperature to form a protective film. The resulting sensor structure is shown below. Figure 5 As shown.

[0118] Example 7.

[0119] This embodiment performs performance testing on the sensor obtained in Embodiment 6:

[0120] S1. Calibration of biomarker concentration curves:

[0121] (1) Prepare 1-100 μmol / L levodopa solution, 1-500 μmol / L glucose solution and ascorbic acid solution using 0.01 mol / L phosphate buffer solution with pH 7.4.

[0122] (2) The chronoamperometry method was used to test the sensor's response to gradually changing concentrations of levodopa, glucose, and ascorbic acid. Different concentrations of the target substances were gradually added to phosphate buffer, and a standard curve of current density versus concentration was plotted. The test results are as follows: Figure 6 As shown in the figure, the overall electrochemical detection results indicate that the biosensor based on three enzyme nanoflowers showed good responses to glucose, ascorbic acid, and levodopa, three Parkinson's disease-related biomarkers. Within a certain concentration range, the concentration of the biomarkers and the current exhibited a good linear relationship, enabling quantitative detection of these biomarkers and providing valuable technical support for the diagnosis and monitoring of Parkinson's disease.

[0123] S2, Surface Tests:

[0124] Sensors were attached to the volunteers' skin, and sweating was induced by cycling under a constant exercise load. The concentration changes of markers in sweat were monitored in real time during exercise. The test results are as follows: Figure 7 As shown in the diagram, the test process was divided into three stages, labeled I, II, and III. In the first stage, since the volunteer had not yet sweated, the sensor did not show a significant signal. When the volunteer began to sweat and came into contact with the sensor surface, a strong signal change was generated instantaneously. Subsequently, due to the stabilization of the amount of sweat, the diffusion of the detectable substances in the sweat on the electrode surface also tended to stabilize, resulting in a stable and accurate test result. The test then ended, and the current signal disappeared after the sensor was removed. The test results demonstrate that the sensor can detect glucose, ascorbic acid, and levodopa in sweat in real time during surface testing, and exhibits a significant current response to changes in the concentration of these three substances. It can sensitively detect the dynamic changes in the concentration of these substances in sweat during sweating, indicating that the sensor has the ability to detect relevant substances in a real-world surface environment, providing a possibility for real-time monitoring of Parkinson's disease and related diseases.

[0125] Figure 8This diagram illustrates the working principle of the sensor provided by the present invention. The blue portion represents the enzyme nanoflower structure. Due to its large specific surface area, this structure can load a large amount of enzyme. Furthermore, by introducing additional metal ions to form a synergistic catalytic effect with the enzyme, it can improve catalytic efficiency and the stability of the sensing core. When a sample solution containing the substrate comes into contact with the sensor loaded with enzyme nanoflowers, the substrate molecules diffuse onto the surface of the enzyme nanoflowers. The enzyme specifically recognizes and catalyzes the substrate reaction, during which electrons are transferred between the enzyme and the substrate. The electrons generated by the enzyme-catalyzed reaction are transferred to the working electrode through the nanoflower structure, forming a redox current. The magnitude of the current is directly related to the analyte concentration. By measuring the change in the response current on the working electrode over time using chronoamperometry (as shown in the curve on the right), a current signal can be obtained. When the substrate concentration is high, the resulting redox current is large; conversely, when the substrate concentration is low, the redox current is small. Through analysis and calibration of the response current, a quantitative relationship between the substrate concentration and the change in current (ΔI) can be established, thereby enabling the detection and quantitative analysis of the substrate.

Claims

1. A method for preparing a sweat sensor, characterized in that, Includes the following steps: S1. Dissolve the enzyme in phosphate buffer solution to obtain an enzyme solution, then add copper sulfate solution and shake well; incubate the mixture in a water bath to allow the enzyme molecules to mineralize; collect the precipitate by centrifugation, wash with ultrapure water to remove unreacted enzyme, and obtain purified enzyme nanoflowers. S2. Design a multi-electrode array and fabricate an external mask, which is then fixed onto a PET substrate; Conductive silver paste is printed and coated to form electrode wires and a silver electrode, and then baked and cured. Three working electrodes and one counter electrode are printed using conductive carbon ink. FeCl3 solution was added dropwise to the surface of a silver electrode to form an Ag / AgCl reference electrode; The printed insulating ink serves as a circuit protective layer and is cured under a UV lamp. S3. Electrode activation was performed by cyclic voltammetry, followed by dispersing multi-walled carbon nanotubes in DMF solution, coating them onto the surface of the carbon working electrode, and drying them. Pyrrole and Prussian blue are polymerized simultaneously on the surface of the working electrode to form a PB / PPy composite film. The enzyme nanoflower solution obtained in step S1 was dropped onto the surface of the working electrode modified with PB / PPy composite film and dried overnight at room temperature. A chitosan solution was drop-coated onto the surface of the working electrode and allowed to dry naturally to form a stable protective film, thus obtaining a sweat sensor. The enzyme is one of glucose oxidase, ascorbic acid oxidase, or tyrosinase.

2. The method for preparing a sweat sensor according to claim 1, characterized in that, In step S1, the phosphate buffer solution has a concentration of 0.01 mol / L and a pH of 7.4; the copper sulfate solution has a concentration of 60 mmol / L to 150 mmol / L; and the enzyme solution has a concentration of 0.1 mg / mL to 1 mg / mL.

3. The method for preparing a sweat sensor according to claim 1, characterized in that, The concentration of the glucose oxidase solution was 0.5 mg / mL, the concentration of the ascorbic acid oxidase solution was 1 mg / mL, and the concentration of the tyrosinase solution was 0.72 mg / mL.

4. The method for preparing a sweat sensor according to claim 1, characterized in that, In step S1, the water bath temperature is 25℃ and the culture time is 72h; the centrifugation speed is 3500rpm and the centrifugation time is 10min; the washing is performed 3 times.

5. The method for preparing a sweat sensor according to claim 1, characterized in that, The PET substrate in step S2 has a thickness of 0.1 mm; the mask has a size of 10 cm * 10 cm and a thickness of 125 μm.

6. The method for preparing a sweat sensor according to claim 1, characterized in that, The resistivity of the conductive carbon ink mentioned in step S2 is 10 Ω / cm 2 .

7. The method for preparing a sweat sensor according to claim 1, characterized in that, The concentration of the FeCl3 solution in step S2 is 0.05 mol / L, and the amount added is 5 mL.

8. The method for preparing a sweat sensor according to claim 1, characterized in that, In step S3, the amount of enzyme nanoflower solution added is 5 μL, and the amount of chitosan solution added is 4 μL.

9. A sweat sensor, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the sweat sensor as described in claim 9 in a wearable device for real-time monitoring of Parkinson's disease-related biomarkers.

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