Electronic patch for automatically sampling and detecting sweat lactic acid and preparation method thereof

Abstract

The invention relates to the technical field of biosensors, in particular to an electronic patch for autonomously sampling and detecting sweat lactic acid and a preparation method thereof, and the method comprises the following steps: step 1, preparing an electrode on a single side of a substrate to obtain a substrate-electrode structure; 2, arranging a graphene film on the surface of an electrode in the substrate-electrode structure to obtain a substrate-electrode-graphene film structure; step 3, soaking the substrate-electrode-graphene film structure in a reaction solution, and sequentially modifying PAZE, lactic dehydrogenase, NAD + enzyme and Tween 80 on the surface of the graphene film to form a sensing layer; and 4, connecting an integrated circuit layer above the sensing layer to obtain the sweat lactic acid autonomous sampling electronic patch which can realize sweat autonomous collection and filtration and long-term continuous real-time monitoring of sweat lactic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and more specifically to an electronic patch for autonomous sampling and detection of sweat lactic acid and a preparation method thereof. Background Art

[0002] In the medical field, changes in lactic acid concentration can be used to evaluate certain disease states, such as diabetic ketoacidosis, lactic acidosis, etc. In sports science, lactic acid testing can help evaluate an individual's exercise endurance and training status to determine the training intensity and recovery effect.

[0003] Detecting lactic acid through blood is currently the most widely used method. However, blood testing is an invasive detection method, and at the same time, it is necessary to send the blood to the laboratory for pretreatment, which is not conducive to the real-time monitoring of lactic acid. Research shows that there is a positive correlation between the lactic acid concentration in sweat and that in blood. Compared with blood testing, sweat testing has greater advantages in terms of convenience, non-invasiveness, real-time performance, etc.

[0004] Existing sweat lactic acid sensors have the following limitations: 1. The collection and detection of sweat are carried out separately, which is not conducive to the real-time monitoring of sweat lactic acid, and sweat is prone to evaporation and contamination, affecting accuracy; 2. The lactate oxidase or dehydrogenase used for detecting lactic acid is only attached to the sensitive element of the sensor by drop coating. In this physical attachment method, lactate oxidase or dehydrogenase cannot be tightly combined with the sensitive element of the sensor, which is not conducive to the stability of the detection signal; 3. It is necessary to process and analyze the detection signal on an external device such as a smartphone or a computer, which is not convenient to carry and affects the real-time performance of the signal; 4. The reversibility, reusability and anti-interference ability are not analyzed, and it cannot be ensured that the sensor can achieve continuous and long-term detection, and it cannot be determined whether the sensor has the ability to resist interference such as temperature change, pH change, and mechanical deformation, so it cannot be widely applied. Summary of the Invention

[0005] The present invention provides an electronic patch for autonomous sampling and detection of sweat lactic acid and a preparation method thereof, aiming to achieve autonomous collection and filtration of sweat and long-term continuous real-time monitoring of sweat lactic acid.

[0006] The above object is achieved by the following technical solutions:

[0007] An electronic patch for autonomous sampling and detection of sweat lactic acid includes a sweat autonomous sampling layer, a sensing layer, and an integrated circuit layer fixedly connected in sequence from bottom to top;

[0008] The sweat self - sampling layer includes microchannels for transporting liquid to the sensing layer. The sensing layer includes a graphene film, a linking molecule with an aromatic ring structure, lactate dehydrogenase, nicotinamide adenine dinucleotide (NAD+ enzyme), and Tween 80, which are sequentially modified through an electrode layer. The sensing layer provides a detection signal to the integrated circuit layer, and the integrated circuit layer realizes the acquisition of the detection signal.

[0009] The method for preparing the electronic patch for self - sampling and detection of sweat lactate includes the following steps:

[0010] Step 1: Prepare electrodes on one side of the substrate to obtain a substrate - electrode structure;

[0011] Step 2: Set a graphene film on the surface of the electrode in the substrate - electrode structure to obtain a substrate - electrode - graphene film structure;

[0012] Step 3: Immerse the substrate - electrode - graphene film structure into a reaction solution, and sequentially modify PAZE, lactate dehydrogenase, NAD+ enzyme, and Tween 80 on the surface of the graphene film to form a sensing layer;

[0013] Step 4: Connect an integrated circuit layer above the sensing layer to obtain an electronic patch for self - sampling and detection of sweat lactate.

[0014] The beneficial effects of the electronic patch and its preparation method for self - sampling and detection of sweat lactate in the present invention are as follows:

[0015] A micro - wearable electronic patch for self - sampling and detection of sweat lactate that can achieve self - sampling and online intelligent analysis of sweat lactate has high stability and accuracy, can realize self - collection and filtration of sweat and long - term continuous real - time monitoring of sweat lactate, and can give a warning prompt when the lactate level exceeds a reasonable range.

[0016] 1. The present invention realizes the functions of automatic collection and filtration of sweat through a sweat self - sampling patch and a bilateral asymmetric structure fiber membrane, and integrates the functions of sweat collection and detection in the same device, realizing real - time and continuous detection of sweat lactate;

[0017] 2. The present invention uses a graphene film as a sensitive element, modifies lactate dehydrogenase on the surface of the graphene film through PAZE as a linking molecule, and forms a passivation layer on the graphene surface without modified enzyme through Tween 80. Compared with the traditional drop - coating method, this modification method makes the combination of lactate dehydrogenase and the sensitive element more firm, greatly improving the accuracy and stability of sensing;

[0018] 3. The present invention realizes the acquisition and conversion of detection signals through an integrated circuit connected above the sensing layer, and wirelessly transmits the signals to an Android smartwatch through a Bluetooth module, getting rid of the traditional operation mode of a smartphone or computer, and realizing the wearable of the sweat lactate sensor;

[0019] 4. The present invention has comprehensively experimentally verified the sensing performance of the prepared electronic patch for autonomous sampling and detection of sweat lactate. The experiments show that the electronic patch for autonomous sampling and detection of sweat lactate provided by the present invention can continuously monitor the sweat lactate level for a long time, and at the same time has a certain ability to resist temperature changes, pH changes, and mechanical deformations, and has good environmental adaptability;

[0020] 5. The present invention can detect different types of biomolecules in sweat by replacing lactate dehydrogenase with a specific recognition molecule corresponding to other target molecules, and has a wider practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flow chart of the preparation method of an electronic patch for autonomous sampling and detection of sweat lactate according to the present invention;

[0022] Figure 2 Two-dimensional schematic diagram of an electronic patch for autonomous sampling and detection of sweat lactate according to the present invention;

[0023] Figure 3 Three-dimensional exploded view of an electronic patch for autonomous sampling and detection of sweat lactate according to the present invention;

[0024] Figure 4 Specific diagram of the gold / chromium composite electrode prepared in the example;

[0025] Figure 5 and 6 Atomic force microscope (AFM) height characterization diagrams of the graphene surface before and after modifying lactate dehydrogenase, NAD+, and Tween 80;

[0026] Figure 7 X-ray energy spectrometer (EDS) characterization diagrams of carbon, nitrogen, and phosphorus elements on the graphene surface before and after modifying lactate dehydrogenase, NAD+, and Tween 80;

[0027] Figure 8 Physical diagram of the integrated circuit customized in the example;

[0028] Figure 9 Graphene transfer characteristic curve diagrams of the electronic patch for autonomous sampling and detection of sweat lactate prepared in the example for different concentrations of lactate;

[0029] Figure 10Hill equation fitting curve graphs of the detection signals obtained after the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples were placed at room temperature for different times;

[0030] Figure 11 Response signal graphs of the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples under different temperature and pH conditions;

[0031] Figure 12 Electrical property graphs of the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples under different types of mechanical deformations;

[0032] Figure 13 Response signal graphs of different concentrations during the forward - reverse - forward detection process of the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples;

[0033] Figure 14 Integrated circuit system block diagram designed for the examples;

[0034] Figures 15 to 18 Integrated circuit diagram designed for the examples.

[0035] In the figure: sweat autonomous sampling layer 1, filtration layer 2, sensing layer 3, and integrated circuit layer 4. Detailed implementation manners

[0036] An electronic patch for autonomous sampling and detection of sweat lactic acid, comprising a sweat autonomous sampling layer 1, a filtration layer 2, a sensing layer 3, and an integrated circuit layer 4 fixedly connected in sequence from bottom to top;

[0037] Among them, the material of the sweat autonomous sampling layer 1 is polydimethylsiloxane (PDMS). The sweat autonomous sampling layer 1 includes microchannels for directional liquid transportation. The microchannels include a liquid collection chamber in the center, a liquid outlet disposed at the center of the liquid collection chamber, a flow channel communicated with the liquid collection chamber, and a liquid inlet communicated with the flow channel. The cross - sections of the center of the liquid collection chamber and the liquid inlet are circular, and the radii decrease in sequence, forming a low - surface - energy area at the center of the microchannel and a high - surface - energy area away from the center of the microchannel. The liquid inlets can be circumferentially distributed around the liquid collection chamber, and the liquid inlets can also continue to communicate with new liquid inlets, so that the liquid is driven from the high - surface - energy area on the outer periphery to the low - surface - energy area at the liquid collection chamber; the sweat autonomous sampling layer 1 concentrates the surrounding sweat to its own center and discharges it to the filtration layer 2 for filtration through the liquid outlet;

[0038] Among them, the filtration layer 2 is a double - sided asymmetric structure filtration fiber membrane, having a three - dimensional reticular fiber structure, and having different hydrophilic / hydrophobic properties on both sides. Due to the surface energy difference on both sides of the fiber membrane, sweat can be directionally transported from the hydrophobic side to the hydrophilic side under the drive of the Laplace pressure, and impurities such as sebum in the sweat can be blocked outside the fiber membrane;

[0039] Among them, the sensing layer 3 includes a graphene film, a linking molecule with an aromatic ring structure, lactate dehydrogenase, nicotinamide adenine dinucleotide (NAD+ enzyme), and Tween 80, which are sequentially modified through an electrode layer;

[0040] The linking molecule with an aromatic ring structure is 1-pyrenebutanoic acid succinimidyl ester (hereinafter simply referred to as PASE);

[0041] Furthermore, the electrode layer includes a substrate and four electrodes diagonally arranged on one side surface of the substrate. The substrate is preferably polyethylene naphthalate (PEN) with a thickness of 6 μm. The number of electrodes is four and they are diagonally arranged.

[0042] Preferably, the electrodes are metal electrodes, more preferably gold / chromium composite electrodes. The gold / chromium composite electrodes specifically include a chromium layer and a gold layer sequentially stacked on the surface of the substrate. The thickness of the chromium layer is preferably 2 nm, and the thickness of the gold layer is preferably 43 nm.

[0043] Furthermore, the graphene film is stacked in the center of the surface of the electrode layer and on the surface of a partially exposed substrate. The graphene film is preferably single-layer graphene. PAZE, lactate dehydrogenase, NAD+ enzyme, and Tween 80 are sequentially modified on the surface of the graphene film.

[0044] Among them, the integrated circuit layer 4 includes an integrated circuit which is a customized flexible printed circuit board, and a signal conversion circuit, a signal processing circuit, an amplification and voltage division circuit, a battery, and a Bluetooth module arranged on the printed circuit board. For details, please refer to Figures 15 to 18 . Among them, Figure 15 is the power supply and signal processing circuit, Figure 16 is the -5V reverse voltage conversion circuit, Figure 17 is the +2.5V reference potential circuit, Figure 18 is the power supply and signal processing circuit for the main control chip.

[0045] In the present invention, unless otherwise specified, the preparation raw materials used are all commercially available products well-known to those skilled in the art.

[0046] A preparation method for an electronic patch for self-sampling and detection of sweat lactic acid as described above. Example: The method includes the following steps:

[0047] Step 1: Prepare electrodes on one side of the substrate, ensuring that the electrodes are completely set on the substrate and meet the thickness requirements. Preferably, use electron beam evaporation to prepare gold / chromium composite electrodes on one side of a PEN substrate with a size of 1.5×2 cm 2 , with a chromium layer thickness of 2 nm and a gold layer thickness of 43 nm, to obtain a substrate-electrode structure;

[0048] Step 2: Using commercially available CVD graphene as the substrate, such as CVD graphene grown on copper foil, and covering a layer of polymethyl methacrylate (PMMA) protective layer on one side of the graphene surface (marked as the front side), use the wet transfer method to set a graphene film on the surface of the electrode in the substrate-electrode structure, and pattern the graphene film through photolithography and oxygen ion etching techniques. The size of the graphene is 40×100 μm 2 , to obtain a substrate-electrode-graphene film structure;

[0049] Step 3: Immerse the substrate-electrode-graphene film structure into the PASE solution. The substrate is polyethylene naphthalate (PEN) with a thickness of 6 μm, as Figure 2 shown. The electrode includes four gold / chromium composite electrodes arranged diagonally. The solvent of the PAZE solution is preferably N,N-dimethylformamide (DMF), the concentration is preferably 5 mM, and the immersion time is preferably 2 hours. Then wash the surface of the substrate-electrode-graphene film structure with DMF, ethanol solution, and deionized water respectively, and then dry it with nitrogen, that is, complete the PASE modification on the surface of the graphene film. Drop the prepared lactate dehydrogenase solution onto the surface of the graphene film modified with PAZE. The solvent of the lactate dehydrogenase solution is preferably 1×PBS (phosphate buffer solution), the concentration is preferably 0.5 mg / ml, and the dosage is preferably 100 μL. Place the graphene film structure in an environment of 4°C for 3 h to complete the modification of lactate dehydrogenase. After the modification is completed, take out the device and rinse the surface of the graphene film with PBS, and then dry it with nitrogen. Then immerse the substrate-electrode-graphene film structure modified with lactate dehydrogenase into the prepared NAD+ solution. The solvent of the NAD+ solution is preferably 1×PBS, and the concentration is preferably 10 mM. Place the graphene film structure in an environment of 4°C for 3 h for NAD+ modification. After the modification is completed, rinse the surface of the graphene film with PBS solution again, and finally dry it with nitrogen, that is, complete the modification of lactate dehydrogenase and NAD+. Finally, immerse the modified structure into 0.1% Tween 80 reagent and then take it out to obtain the sensing layer 3;

[0050] Among them, Figure 3 is the atomic force microscope (AFM) height characterization of the graphene surface before and after the modification of lactate dehydrogenase, NAD+, and Tween 80. Dense height protrusions appear on the modified graphene surface, Figure 4 is the elemental characterization of the graphene surface before and after the modification. The nitrogen and phosphorus elements on the surface of the modified graphene are significantly increased, indicating that lactate dehydrogenase and NAD+ are successfully modified on the surface of the graphene film.

[0051] Step 4. The self-sampling layer 1 can be prepared by various methods such as molding, laser processing, photolithography, etc. Preferably, the sweat self-sampling patch is prepared by molding. The mold for the sweat self-sampling patch is prepared using 3D printing technology. PDMS and its curing agent are fully mixed in a mass ratio of 10:1 and then injected into the mold. After being placed in a vacuum for 30 min to remove air bubbles, it is taken out and placed in an environment at 60 °C and left to stand for 20 h for complete curing. After demolding and taking out, the sweat self-sampling layer 1 can be obtained. The sweat self-sampling layer 1 is fixedly connected to the lower end face of the filter layer 2.

[0052] Step 5. The modified graphene film surfaces of the filter layer 2 and the sensing layer 3 are bonded and connected; an integrated circuit layer 4 is connected above the sensing layer 3 to obtain an electronic patch for sweat lactic acid self-sampling and detection.

[0053] Among them, preferably, a commercially available polyethylene terephthalate-polyvinylidene fluoride (PET-PVDF) fiber membrane is used as the substrate. The PVDF side of the fiber membrane is placed naked in an oxygen plasma cleaner for plasma treatment. The treatment time is preferably 100 - 140 s, more preferably 120 s. After treatment and taking out, the surface of the naked side of the fiber membrane is modified to be hydrophilic, and the hydrophilic side is bonded to the graphene film.

[0054] Among them, preferably, the wet transfer method is used to transfer the graphene film to the surface of the electrode layer. The specific operation steps of the wet transfer method are as follows:

[0055] The CVD graphene is cut into a square with a size preferably of 3×3 mm 2 . The graphene on the back of the copper foil is removed using oxygen ion etching technology. Then, the side (front side) of the copper foil with the PMMA protective layer is facing up and suspended in a 4 wt% concentration of APS-100 copper etching solution for 3 h to completely dissolve the copper foil, obtaining a graphene film-PMMA protective layer structure. The graphene film-PMMA protective layer structure is fished out using a cover glass and placed in a petri dish filled with deionized water to make the graphene film contact the deionized water and left to stand for 15 min. This process is repeated 2 - 3 times to ensure complete removal of the residual APS-100 copper etching solution on the surface of the graphene film. The substrate-electrode structure is immersed in the petri dish, and the graphene film suspended in deionized water is aligned with the center of the electrode. The graphene film-PMMA protective layer structure is fished out and placed in a vacuum drying oven at 25 °C for vacuum standing for 5 h, and then placed on a hot plate at 180 °C for heating for 1 h to soften the PMMA protective layer and strengthen the connection between the graphene and the substrate. After taking out, it is soaked in acetone for 1 h to remove the PMMA protective layer from the surface of the graphene film. Then, it is rinsed successively with isopropyl alcohol (IPA) and deionized water and dried with nitrogen, and the graphene film can be transferred to the surface of the electrode layer.

[0056] Pattern the graphene film on the electrode layer after transferring graphene. The method for patterning graphene in the present invention is not particularly limited, as long as a graphene film with a suitable position and meeting the size requirements can be obtained. The present invention preferably uses photolithography and oxygen ion etching techniques to complete the patterning of graphene, obtaining a substrate - electrode - graphene film structure. The size of the graphene film is preferably 40×100μm 2 。

[0057] Step six: Customize the integrated circuit layer 4 fixed above the sensing layer 3. The specific structure of the integrated circuit layer 4 is as Figure 5 shown. The entire electronic patch for autonomous sampling and detection of sweat lactic acid is powered by a micro - lithium - ion battery. The digital - to - analog converter DAC8830 is connected to the planar gate of the electrode layer to give a constant gate voltage V g , and a precise and stable low - noise drain - source voltage V ds is provided through a self - designed voltage - dividing circuit. The original signal is converted into a voltage signal using an amplifier circuit. Then, the amplified signal is collected by the analog - to - digital converter ADS1271 and processed by the built - in ultra - low - power microcontroller STM32L4. Finally, the information can be transmitted to an Android smartwatch through a Bluetooth module and an on - board antenna, and the lactic acid concentration information can be monitored through a customized Android application on the smartwatch.

[0058] In the present invention, the graphene film is used as a conductive sensitive element. The arsenic group structure of PASE molecules can combine with the hexagonal lattice of graphene in the form of π - π bonds, so as to modify PASE on the surface of the graphene film; the ester group of PASE will combine with the amino groups of lactate dehydrogenase and NAD+ enzyme to undergo a Schiff base condensation reaction, and NAD+ enzyme and lactate dehydrogenase are connected and modified on the surface of the graphene film through PASE. After wearing the electronic patch for autonomous sampling and detection of sweat lactic acid on the human body, sweat is collected through the sweat autonomous sampling patch and transported to the sensing layer through a bilateral asymmetric structure filter fiber membrane. Lactic acid in the sweat is catalyzed by lactate dehydrogenase to generate pyruvate, while NAD+ is reduced to NADH and hydrogen ions are generated, introducing new positive charges into the sweat. This will cause an equal amount of negative charges to be generated inside the graphene film, changing the carrier density of graphene, thereby changing the conductivity of graphene. Finally, the graphene transfer characteristic curve and the Dirac point will shift with the change of the concentration of the target molecule, thus generating a detection signal corresponding to the concentration of the target molecule. At the same time, Tween 80 has a low affinity with the complex components in sweat and can form a passivation layer on the surface of some graphene that is not modified by enzymes, further improving the stability of sensing.

[0059] A drain-source voltage and a gate voltage are provided to the sensing layer 3 through the integrated circuit layer 4 fixedly connected above the sensing layer 3, and the acquisition and conversion of detection signals are realized. The signals are transmitted to the Android smart watch through the Bluetooth module. The electronic patch for autonomous sampling and detection of sweat lactic acid is operated through a customized Android smart watch built-in App, and the real-time lactic acid level data is read. When the lactic acid level exceeds the reasonable range, the green sign representing "normal" will be converted into a dark pink "warning" sign, and a warning signal is conveyed through vibration.

[0060] Since the bilateral asymmetric structure filtering fiber membrane in the present invention has a three-dimensional network fiber structure and different hydrophilic / hydrophobic properties on both sides, due to the surface energy difference on both sides of the fiber membrane, sweat can be directionally transported from the hydrophobic side to the hydrophilic side under the drive of the Laplace pressure, and impurities such as sebum in the sweat can be blocked outside the fiber membrane, realizing the isolation of non-target molecules in the solution to be measured from the graphene film, preventing the surface of the graphene film from being contaminated by impurities and affecting its electrical properties, which is beneficial to improving the stability and accuracy of the sensor.

[0061] In the present invention, lactic acid in sweat is converted into pyruvic acid under the catalytic action of lactate dehydrogenase. This reaction is a reversible reaction, that is, when the lactic acid concentration decreases, the reaction will proceed in the reverse direction, and at the same time, the hydrogen ion concentration in the sweat to be measured is reduced, thereby reversely changing the conductivity of graphene, ensuring the continuity of the sensing signal and the reusable of the electronic patch for autonomous sampling and detection of sweat lactic acid, and finally realizing the continuous monitoring of the sweat lactic acid concentration. The results of the examples show that in the forward-reverse-forward detection process, the response signal values corresponding to the same concentration have high consistency, indicating that the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention can realize the reversible detection and continuous detection of lactic acid; at the same time, the present invention conducts a long-term placement test and anti-interference test on the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples. The tests show that the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention has little influence on the sensing performance after being placed for 30 days, and a certain degree of temperature change, pH change and mechanical deformation have little influence on the sensing performance of the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention. The maximum detection limit of the lactic acid concentration of the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples reaches 400 mM, and the minimum detection limit is 0.5 mM, and the theoretical minimum detection limit is 0.31 mM.

[0062] Test example:

[0063] 1. Use PBS buffer with a pH of 7.4 as a solvent to prepare lactic acid solutions with different concentrations, namely 0.5 mM, 1.5 mM, 3 mM, 4.5 mM, 6 mM, 12.5 mM, 50 mM, and 400 mM. Store them in an environment of 2 - 4 °C for later use. Use a pipette to separately add 30 μL of lactic acid solutions with different concentrations to the sensing layer modified with lactate dehydrogenase and NAD⁺, and use PBS buffer without added lactic acid as a control group. Use a dual-channel power supply to provide the drain-source voltage and the gate voltage respectively. Set the drain-source voltage to 5 mV, and the range of the gate voltage is -0.1 - 0.3 V, and measure the graphene transfer characteristic curves respectively.

[0064] The measurement results are as Figure 6 shown. As the lactic acid concentration increases from 0 to 400 mM, the Dirac point moves 84 mV in the negative x-axis direction. The results show that the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention can identify different lactic acid molecules in the solution and generate corresponding response signals. The relationship between the response signal and the lactic acid concentration can be expressed by the Hill equation:

[0065]

[0066] where, ΔV Dirac is the change in the Dirac point V Dirac , c is the lactic acid concentration, and the equilibrium dissociation coefficient k D ≈23.098 mM and the Hill coefficient A≈1.606 are estimated by fitting the curve.

[0067] 2. Place the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the example in an environment of 4 °C for 3 days, 7 days, and 30 days respectively, and then detect the signals. Figure 7 The Hill equation fitting curves obtained from detecting the signals of the electronic patch for autonomous sampling and detection of sweat lactic acid after being placed in a room temperature environment for different times are shown. The results show that the fitting curves obtained from the detection signals of the electronic patch for autonomous sampling and detection of sweat lactic acid after being placed in a room temperature environment for 3 days, 7 days, and 30 days are basically the same, and the deviation is less than 6.86% within 30 days. This shows that the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention has good stability and will not have an obvious attenuation effect on the sensing stability of the electronic patch for autonomous sampling and detection of sweat lactic acid when placed in a room temperature environment for a long time.

[0068] 3. Place the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the example at 25 °C, 35 °C, and 40 °C for 1 h, and conduct the same experiment as in step 1. And for the case of 25 °C, use PBS buffer with pH values of 5.5 and 6.5 as solvents to prepare lactic acid solutions with different concentrations for comparative experiments. The results are as Figure 8As shown, the results show that under different temperature and pH conditions, the influence of the same lactic acid concentration on the response signal ΔV Dirac is very small, indicating that the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention is hardly affected by temperature and pH in a general measurement environment.

[0069] 4. Test the ability of the sensing layer of the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples to resist mechanical deformation. In the bending test, the sensing layer was wound around a cylindrical tube with a radius of 1 mm, peeled off after 1 minute, and repeated 100 times in a cycle. In the torsion test, the sensing layer was twisted at an angle from -180° to +180°, and repeated 100 times in a cycle. In the folding test, the sensing layer was folded in half and opened, and repeated 100 times in a cycle. In addition, the sensing layer was shrunk from its original size into a small ball and then flattened again, and repeated 100 times in a cycle. After various mechanical deformation tests, lactic acid detections with concentrations increasing from 0.5 to 400 mM and decreasing from 200 to 0.37 mM were carried out respectively. The experimental results are as Figure 9 shown. At each tested lactic acid concentration, the response signals measured after different mechanical deformation tests are basically the same, and the maximum deviation is less than 4.2%.

[0070] 5. Conduct reversibility and continuity experimental verification on the electronic patch for autonomous sampling and detection of sweat lactic acid prepared in the examples. The experimental results are as Figure 10 shown. First, the concentration of the lactic acid solution to be tested was gradually increased from 0 to 400 mM, and the corresponding response signal ΔV Dirac gradually increased from 2 mV to 85.3 mV. Secondly, the lactic acid concentration was gradually decreased from 400 mM to 0, and the above detections were repeated. Then, the concentration of the lactic acid solution to be tested was gradually increased from 0 to 400 mM to complete the forward-reverse-forward detection process. Moreover, during the forward-reverse-forward detection process, the response signals ΔV Dirac corresponding to the same concentration basically overlapped, and the maximum deviation was less than 5.6%, proving that the electronic patch for autonomous sampling and detection of sweat lactic acid provided by the present invention can achieve reversible and continuous detection of lactic acid.

Claims

1. An electronic patch for autonomous sampling and detection of sweat lactic acid, characterized in that, It includes a sweat self - sampling layer (1), a sensing layer (3) and an integrated circuit layer (4) fixedly connected in sequence from bottom to top; The sweat self - sampling layer (1) includes microchannels for transporting liquid to the sensing layer (3). The sensing layer (3) includes a graphene film, a linking molecule with an aromatic ring structure, lactate dehydrogenase, NAD+ enzyme, and Tween 80 modified successively through an electrode layer. The sensing layer (3) provides a detection signal to the integrated circuit layer (4), and the integrated circuit layer (4) realizes the acquisition of the detection signal.

2. The electronic patch for self - sampling and detection of sweat lactate according to claim 1, wherein the sweat self - sampling layer (1) includes microchannels for directing the transport of liquid to the sensing layer (3).

3. The electronic patch for self - sampling and detection of sweat lactate according to claim 1, wherein a filter layer (2) is provided between the sweat self - sampling layer (1) and the sensing layer (3), and the filter layer (2) is fixedly connected to the self - sampling layer (1) and the sensing layer (3) respectively.

4. The electronic patch for self - sampling and detection of sweat lactate according to claim 1, wherein the integrated circuit layer (4) includes a circuit board, and a signal conversion circuit, a signal processing circuit, an amplification and voltage - division circuit, and a battery electrically connected to the printed circuit board.

5. The electronic patch for self - sampling and detection of sweat lactate according to claim 4, wherein the integrated circuit layer (4) further includes a wireless module electrically connected to the circuit board.

6. A method for preparing an electronic patch for autonomous sampling and detection of sweat lactic acid, characterized in that, It includes the following steps: Step 1: Prepare an electrode on one side of a substrate to obtain a substrate - electrode structure; Step 2: Set a graphene film on the surface of the electrode in the substrate - electrode structure to obtain a substrate - electrode - graphene film structure; Step 3: Immerse the substrate - electrode - graphene film structure into a reaction solution, and successively modify PAZE, lactate dehydrogenase, NAD+ enzyme, and Tween 80 on the surface of the graphene film to form the sensing layer (3); Step 4: Connect the integrated circuit layer (4) above the sensing layer (3) to obtain an electronic patch for self - sampling and detection of sweat lactate.

7. The method for preparing an electronic patch for self - sampling and detection of sweat lactate according to claim 6 further includes Step 5: Fix the sweat self - sampling layer (1) below the sensing layer (3).

8. The method for preparing an electronic patch for self - sampling and detection of sweat lactate according to claim 7 further includes Step 6: Fix the filter layer (2) between the sweat self - sampling layer (1) and the sensing layer (3).

9. The method for preparing an electronic patch for autonomous sampling and detection of sweat lactic acid according to claim 6, in step three: Immerse the substrate-electrode-graphene film structure into the PASE solution, then wash the surface of the substrate-electrode-graphene film structure with DMF, ethanol solution and deionized water respectively, and then dry it with nitrogen, thus completing the PASE modification on the surface of the graphene film; Drop the prepared lactic dehydrogenase solution onto the surface of the graphene film after PAZE modification, and place the graphene film structure in an environment of 1 to 8 °C for 1 to 5 h to complete the modification of lactic dehydrogenase; After the modification is completed, take out the device and rinse the surface of the graphene film with PBS, then dry it with nitrogen; then immerse the substrate - electrode - graphene film structure modified with lactate dehydrogenase into the prepared NAD+ solution, place it in an environment of 1 to 8 °C for 1 to 5 h for NAD+ modification; After the modification is completed, rinse the surface of the graphene film with PBS solution again, and finally dry it with nitrogen, that is, complete the modification of lactate dehydrogenase and NAD+; finally, immerse the modified structure into the Tween 80 reagent and then take it out to obtain the sensing layer (3).

10. The method for preparing an electronic patch for self - sampling and detection of sweat lactate according to claim 8 uses a wet transfer method to transfer the graphene film to the surface of the electrode layer.

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