A method for preparing and application of flexible colorimetric-electrochemical dual-mode sensor for sweat marker monitoring

Abstract

This invention discloses a method for preparing and applying a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers. A FeSN-MXene single-atom nanozyme with excellent H2O2 enzyme activity is synthesized using MXene material, FeSO4, and urea as reactants. This nanozyme is then incorporated into a hydrogel stock solution to form a functionalized nanocomposite hydrogel. A three-dimensional honeycomb porous graphene flexible electrode is prepared on a flexible polyimide film using laser-induced graphene technology as the electrochemical sensor substrate. A colorimetric probe TMB and a bio-enzyme are sequentially added and immersed in the highly sensitive and specific catalytic nanocomposite hydrogel to prepare a color-changing hydrogel patch, i.e., a flexible colorimetric sensor. The nanocomposite hydrogel and the bio-enzyme are then sequentially modified onto the working electrode of the electrochemical sensor substrate to prepare a flexible electrochemical sensor. The color-changing hydrogel patch and the flexible electrochemical sensor can achieve highly sensitive and specific colorimetric-electrochemical dual-mode monitoring of sweat biomarkers.

Description

Technical Field

[0001] This invention belongs to the field of flexible wearable biosensor technology, specifically relating to a method for preparing and applying a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers. Background Technology

[0002] Traditional medical testing is like a "snapshot," capturing only the physiological state at the moment of sampling. For example, diabetic patients need to have their blood drawn multiple times a day to test their blood sugar, which not only increases their physical discomfort but also easily misses crucial blood sugar fluctuations during non-sampling periods such as meal intervals and nighttime sleep, failing to provide a complete picture of the patient's 24-hour blood sugar variation. In recent years, wearable sensing technology has developed rapidly. These devices can achieve long-term, continuous, and dynamic monitoring of physiological markers, effectively compensating for the shortcomings of traditional monitoring methods.

[0003] Studies have shown a correlation between glucose levels in surface sweat and blood glucose concentration. Sweat, as the most readily available bodily fluid, is an excellent medium for non-invasive biomarker analysis. Furthermore, surface sweat is easier to obtain quickly and continuously, and is less prone to degradation, avoiding the pollution, irritation, and inconvenience associated with collecting other non-invasive bodily fluids (such as saliva, tears, and urine), making it an ideal carrier for in-situ dynamic analysis of biomarkers. With the rapid development of artificial intelligence and the Internet of Things, the combination of wearable device detection and smartphone displays has made point-of-care testing (POCT) of biomarkers in sweat increasingly convenient for the public, especially based on simple, visually perceptible methods and highly sensitive, easily miniaturized, and integrated electrochemical sensing technologies. Therefore, constructing colorimetric-electrochemical multimodal flexible wearable sweat sensors based on smartphones has the potential for portable, real-time, sensitive, and non-invasive sweat analysis and metabolic function feedback, becoming an important research direction and hot topic for realizing biomarker molecular-level mobile health management and medical monitoring.

[0004] Existing technology, patent document CN201910283778.0 discloses a fabrication technique for a coplanar flexible electrochemical sensor electrode and array. It employs a flexible substrate and fabricates an electrode array on the substrate, consisting of two silver electrodes as the working electrode and a counter electrode, and a silver chloride electrode as the reference electrode—a three-electrode electrochemical sensor integrating the three electrodes. Furthermore, the fabricated electrodes are coplanar and flexible, allowing them to be attached to any curved surface, thus overcoming the limitations of traditional working, counter, and reference electrodes in terms of integration, coplanarity, and bending. Patent document CN202510530139.5 discloses a microstructured electrode flexible electrochemical sensor, comprising a flexible electrode substrate with a hemispherical array and three electrodes on the surface: a working electrode, a counter electrode, and a reference electrode. The working electrode includes a raised metal array and a through-hole array. The raised metal corresponds to the hemispherical array on the substrate, and the through-hole array penetrates the substrate. This sensor increases the reaction area and promotes electron transfer through its three-dimensional structure, while the through-hole array ensures oxygen supply. The flexible substrate and structural design enhance mechanical stability, making it suitable for wearable devices. Patent document CN202511874247.0 discloses a flexible electrochemical sensor, its preparation method, and its application. The flexible electrochemical sensor includes a flexible substrate, three electrodes, a conductive strip, an external wire, and a multi-cavity polypyrrole film. The three electrodes consist of a working electrode, a reference electrode, and an auxiliary electrode, which are printed on the flexible substrate. The conductive strip is connected to the three electrodes and is sealed with insulating tape, exposing only the working area of ​​the three electrodes. The external wire is attached to the conductive strip with conductive silver paste. The multi-cavity polypyrrole film is modified on the surface of the working electrode. This invention addresses the shortcomings of existing devices in terms of portability and their inability to meet the needs of rapid on-site screening and immediate analysis of large batches of samples. None of the aforementioned patent documents contain any information regarding the preparation of flexible electrochemical sensors by co-modifying working electrodes with FeSN-MXene single-atom nanozymes and bioenzymes, or the preparation of colorimetric patch sensors by co-modifying FeSN-MXene single-atom nanozymes, colorimetric probes (TMB), and bioenzymes. Furthermore, there is no mention of any technical inspiration related to the formation of nanozyme-bioenzyme complexes using FeSN-MXene single-atom nanozymes with excellent H2O2 enzyme activity and bioenzymes to promote substrate-H2O2 catalytic cascade reactions, thereby achieving highly sensitive and specific electrochemical and colorimetric dual-mode detection of sweat markers. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers and its application. The sensor can be used for highly sensitive, specific and real-time colorimetric-electrochemical dual-mode monitoring of sweat biomarkers.

[0006] This invention focuses on biomarkers in sweat used for disease and exercise monitoring, such as glucose and lactic acid. A highly conductive, highly adhesive, and well-permeable hydrogel is prepared using a dual cross-linking strategy, incorporating biocompatible N,N'-methylenebisacrylamide, chitosan, glucose, and acrylamide. This hydrogel serves as a color-changing substrate and functionalization carrier. Guided by research into nanozyme libraries and theoretical calculations, FeSN-MXene single-atom nanozymes (FeSN-MXene SAzymes) with excellent H2O2 enzyme activity are synthesized using MXene, FeSO4, and urea as raw materials through low-temperature annealing. These FeSN-MXene single-atom nanozymes are then introduced into the synthesized hydrogel for functionalization, and their biomarker responsiveness, freeze resistance, and self-healing properties are adjusted to enhance their intelligent capabilities, resulting in PAAM-CS / FeSN-MXene. SAzymes nanocomposite hydrogel; a three-dimensional honeycomb porous graphene flexible electrode is prepared in one step on a flexible polyimide (PI) film using laser-induced graphene technology (LIG) as the substrate for an electrochemical sensor. At the same time, a colorimetric gel patch is prepared by introducing a low-toxicity and high-sensitivity colorimetric probe TMB. The colorimetric probe TMB and bioenzymes are sequentially added and immersed in the highly sensitive and specific catalytic nanocomposite hydrogel to form a colorimetric hydrogel patch, i.e., a flexible colorimetric sensor. The nanocomposite hydrogel and bioenzymes are sequentially modified on the LIG working electrode to obtain a flexible electrochemical sensor. This electrochemical sensor is combined with a control panel and power system that integrate data conversion and wireless communication functions. The detection data is output in real time via a smartphone, realizing real-time, in-situ, dynamic, and multimodal digital monitoring of multiple markers in sweat.

[0007] The core technology of this invention is the design and synthesis of a self-adhesive hydrogel with tissue-like softness, excellent ionic conductivity, and good biocompatibility as a color-changing substrate and electrochemical catalytic functionalization carrier. This hydrogel is then combined with a novel FeSN-MXene single-atom nanozyme and a bioenzyme synthesized at low temperature to form a nanozyme-bioenzyme complex, promoting a cascade reaction. By using a smartphone camera and Bluetooth connection to a mini-program, a colorimetric-electrochemical dual-mode sensor is constructed, significantly improving the sensitivity, specificity, stability, and portability of the flexible sensor. The shape-controllable semi-solid color-changing hydrogel patch and LIG technology enable large-scale direct writing of electrodes with arbitrarily complex patterns, which can be further expanded to simultaneous monitoring of multimodal, multi-channel sweat markers.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers. The specific preparation process is as follows: A highly conductive, strongly adhesive, and well-permeable hydrogel is prepared by cross-linking biocompatible N,N'-methylenebisacrylamide, chitosan, glucose, and acrylamide using a dual cross-linking strategy. This hydrogel serves as the color-changing substrate and electrochemical catalytic functionalization carrier. A FeSN-MXene single-atom nanozyme with excellent H2O2 enzyme activity is synthesized by low-temperature annealing using MXene material, FeSO4, and urea as reactants. The FeSN-MXene single-atom nanozyme is then introduced into the synthesized hydrogel for functionalization, and its responsiveness to sweat biomarkers, antifreeze properties, and self-healing properties are adjusted to obtain PAAM-CS / FeSN-MXene. SAzymes nanocomposite hydrogel; a three-dimensional honeycomb porous graphene flexible electrode was prepared on a flexible polyimide film using laser-induced graphene technology as an electrochemical sensor substrate; a color-changing hydrogel patch, i.e., a flexible colorimetric sensor, was prepared by sequentially adding and immersing a colorimetric probe TMB and a bio-enzyme into the nanocomposite hydrogel with high sensitivity and specific catalytic effect; the nanocomposite hydrogel and bio-enzyme were sequentially modified on the working electrode of the electrochemical sensor substrate to prepare a flexible electrochemical sensor; the color-changing hydrogel patch and the flexible electrochemical sensor can realize high-sensitivity and specific colorimetric-electrochemical dual-mode monitoring of sweat markers.

[0009] Further specifying, the sweat markers are glucose and / or lactic acid.

[0010] A method for preparing a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers, the specific preparation steps of which are as follows: Step S1, Preparation of FeSN-MXene single-atom nanozyme: LiF was dissolved in concentrated hydrochloric acid, and titanium aluminum carbide was added and stirred at 30~40℃ to obtain a black suspension. The suspension was centrifuged and washed repeatedly with deionized water until the supernatant was neutral to obtain the exfoliated MXene suspension. After freeze-drying, the suspension was placed in a tube furnace and annealed at 500~600℃ under a nitrogen atmosphere with a heating rate of 4~6℃ / min to obtain a layered MXene structure. The layered MXene structure was used as a template and ultrasonically dispersed in deionized water to obtain dispersion A. Urea was dissolved in deionized water to obtain solution B, and ferrous sulfate was dissolved in deionized water to obtain solution C. Under stirring conditions, solutions B and C were added to dispersion A in batches. After mixing evenly, the mixture was stirred at room temperature. The resulting suspension was then freeze-dried and annealed at 500~600℃ under a nitrogen atmosphere with a heating rate of 4~6℃ / min to obtain FeSN-MXene single-atom nanozyme. Step S2, Preparation of PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel: Acrylamide (AAm) was added to PBS buffer solution and stirred at 40-60℃ until completely dissolved to obtain an acrylamide solution. Then, glucose, N,N'-methylenebisacrylamide (MBA) and chitosan (CS) were added sequentially and stirred at 40-60℃ until completely dissolved. After cooling to room temperature, potassium persulfate and lithium chloride (LiCl) were added and stirred to dissolve. High-purity nitrogen gas was introduced to remove oxygen, and then ultrasonication was used to remove bubbles to obtain the hydrogel stock solution. FeSN-MXene single-atom nanozyme was added and stirred continuously to mix evenly to obtain the hydrogel / nanozyme stock solution. The hydrogel / nanozyme stock solution was poured into a mold and reacted at 60-80℃. After cooling to 2-5℃, it was removed from the mold to obtain PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel. Step S3, Construction of TMB colorimetric sensor patch PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel was made into a composite hydrogel patch. Then, the colorimetric probe TMB and bio-enzyme were sequentially added and immersed into the composite hydrogel patch with high sensitivity and specific catalytic effect to obtain a TMB-based colorimetric sensor patch, i.e., a flexible colorimetric sensor. Step S4, Electrode preparation: A three-dimensional honeycomb porous graphene electrode is printed on a flexible polyimide film using laser-induced graphene technology as a sensing substrate. The three-dimensional honeycomb porous graphene electrode includes a reference electrode, a working electrode, and a counter electrode. The working electrode is located between the reference electrode and the counter electrode. Silver paste is applied to the lower ends of the counter electrode and the working electrode to serve as the graphene counter electrode and the graphene working electrode. Silver paste is applied to the lower end of the reference electrode and silver chloride paste is applied above the reference electrode to serve as the silver chloride reference electrode. The electrode is then dried at 50~70℃ for later use. Step S6, Construction of PAAM-CS / FeSN-MXene SAzymes / GOx / LIG flexible electrochemical sensor: The hydrogel / nanozyme stock solution was modified on the surface of the graphene working electrode, and then the bio-enzyme was modified and air-dried at room temperature to obtain the PAAM-CS / FeSN-MXene SAzymes / GOx / LIG flexible electrochemical sensor.

[0011] Furthermore, the bioenzyme mentioned in step S3 is one or more of glucose oxidase (GOx) or lactate oxidase (LOx).

[0012] Furthermore, in step S4, the reaction area of ​​the three-electrode system printed on the polyimide film is connected to the sweat microfluidic collection-export channel. The specific preparation process of the sweat microfluidic collection-export channel is as follows: the size of the microfluidic channel is drawn using CAD to customize the channel mold. The crosslinking agent and curing agent of PDMS are mixed evenly in a mass ratio of 10:1 and injected into the mold. Vacuum is drawn to remove the air bubbles in the mixture. Then, it is placed in an oven at 80°C for curing for 1 hour. After demolding, a sweat microfluidic collection-export channel with uniform texture is obtained. Finally, the interior and surface of the channel are modified by hydrophilicity and hydrophobicity using plasma cleaning equipment to obtain the sweat microfluidic collection-export channel.

[0013] The flexible colorimetric-electrochemical dual-mode sensor for sweat biomarker monitoring described in this invention is used in highly sensitive and selective colorimetric-electrochemical dual-mode monitoring of glucose and / or lactic acid biomarkers in sweat.

[0014] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention introduces a two-dimensional MXene material with good conductivity as a template, and synthesizes a novel FeSN-MXene SAzymes with excellent H2O2 enzyme activity by low temperature annealing. Using H2O2 as a reaction intermediate, it promotes the catalytic cascade reaction of substrate-H2O2 with biological enzymes such as glucose oxidase (GOx) and lactate oxidase (LOx). The nanoenzyme is dispersed in PAAM-CS hydrogel stock solution to prepare PAAM-CS / FeSN-MXene SAzymes / GOx complex with good catalytic activity and mechanical stability. It is then directly gelled in situ on LIG working electrode with large specific surface area, realizing the ultra-high sensitivity, excellent specificity and good mechanical stability of flexible electrochemical sensor, and further broadening the application range of flexible sensor.

[0015] (2) This invention is based on direct imaging of semi-solid, self-adhesive, functionalized hydrogel patches with smartphones to quantitatively monitor the color changes of biomarkers such as glucose and lactic acid in sweat, realizing the portability of visual rough measurement and colorimetric quantification. Moreover, the patch array can realize the simultaneous monitoring of multiple biomarkers. Attached Figure Description

[0016] Figure 1 The schematic diagram of the flexible colorimetric-electrochemical dual-mode sensor in this invention is as follows: (A) Synthesis of composite hydrogel; (B) Synthesis of FeSN-MXene Sazymes; (C) Schematic diagram of colorimetric-electrochemical dual-mode monitoring.

[0017] Figure 2Mechanical properties of PAAM-CS hydrogel were characterized as follows: (A) tensile and adhesive properties; (B) stress-strain tensile properties; (C) stress-strain compressibility; (D) rheological frequency; (E) rheological amplitude.

[0018] Figure 3 Characterization of the conductivity of PAAM-CS hydrogel: (A) Light bulb experiment and multimeter measurement of the resistance of gels of different lengths, (B) Conductivity of gels with different doping.

[0019] Figure 4 Water retention and freeze resistance tests for PAAM-CS hydrogel: (A) LiCl concentration optimization; (B) water retention stability; (C) freeze resistance.

[0020] Figure 5 SEM images for morphological characterization of the materials: (A) Mxene; (B) Fe single-atom nanozyme; (C) Polyacrylamide-chitosan hydrogel; (D) Hydrogel and Fe single-atom doped nanogel and local magnified image.

[0021] Figure 6 Images of LIG single-channel, dual-channel, and triple-channel electrodes.

[0022] Figure 7 (A) Glucose gradient graph and linear fitting graph determined by colorimetric method; (B) Selectivity test graph.

[0023] Figure 8 For H2O2 testing of flexible electrochemical sensor: (A) it gradient curve; (B) linear fitting curve.

[0024] Figure 9 For glucose testing using a flexible electrochemical sensor: (A) it gradient curve; (B) linear fitting curve.

[0025] Figure 10 (A) Schematic diagram and mini-program interface for electrochemical testing of glucose in sweat; (B) Electrochemical test diagram of glucose before and after meals; (C) Colorimetric test of glucose in sweat. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified. Example 1

[0027] Using glucose as the detection target model, a flexible colorimetric-electrochemical dual-mode sensor based on a smartphone for highly sensitive, specific, and real-time monitoring of sweat biomarkers is provided, along with its preparation method and application, including the following steps: Step S1: Preparation and characterization of polyacrylamide-chitosan (PAAM-CS) hydrogel A certain amount of acrylamide (AAm) was weighed and added to 10 mM PBS buffer solution. The solution was stirred in a 50°C water bath until completely dissolved to obtain a 30 wt% AAm solution. Then, 6 wt% glucose, 0.045 wt% N,N'-methylenebisacrylamide (MBA), and 1 wt% chitosan (CS) were added sequentially and stirred in a 50°C water bath until completely dissolved. After cooling to room temperature, 0.13 wt% potassium persulfate and 20 wt% lithium chloride (LiCl) were added and stirred until dissolved. High-purity nitrogen gas was purged for 10 min to remove oxygen, and then sonicated for 5 min to remove air bubbles to prepare a hydrogel stock solution. The prepared hydrogel stock solution was poured into a mold and reacted in a 70°C oven for 2 h. After cooling to 4°C, the hydrogel was removed from the mold.

[0028] The morphology, structure, adhesion, tensile and compressive mechanical properties, water retention, freeze resistance and electrical conductivity of the synthesized hydrogel were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, rotational rheometer and multimeter.

[0029] Step S2: Synthesis and Characterization of FeSN-MXene SAzymes Nanozymes 0.5 g LiF was dissolved in 10 mL of concentrated hydrochloric acid with a concentration of 9 mol / L, and then 0.5 g titanium aluminum carbide was slowly added. The mixture was stirred in a water bath at 35 °C for 24 h to obtain a black suspension. The suspension was centrifuged and washed repeatedly with deionized water (centrifuged at 7000 rpm for 10 min) until the supernatant was neutral to obtain the exfoliated MXene suspension. After freeze-drying, the suspension was placed in a tube furnace and annealed at 550 °C for 5 h under a nitrogen atmosphere at a heating rate of 5 °C / min to obtain a layered MXene structure.

[0030] 100 mg of the exfoliated MXene sheet structure was weighed as a template and ultrasonically dispersed in 15 mL of deionized water for 1 h to obtain dispersion A. 0.3 g of urea was dissolved in 5 mL of deionized water to obtain solution B, and 6.8 mg of ferrous sulfate was dissolved in 5 mL of deionized water to obtain solution C. During stirring, solutions B and C were added to dispersion A in small amounts several times. After mixing evenly, the mixture was stirred at room temperature for 24 h. The resulting suspension was freeze-dried and then annealed at 550 °C for 5 h under a nitrogen atmosphere at a heating rate of 5 °C / min to obtain FeSN-MXene SAzymes.

[0031] Its morphology, crystal form and elemental distribution were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), spherical aberration electron microscopy, elemental synchrotron radiation, X-ray photoelectron spectroscopy (XPS), and X-ray powder diffraction (XRD).

[0032] Step S3: Preparation and Synthesis of PAAM-CS / FeSN-MXene SAzymes Nanocomposite Hydrogel A certain amount of acrylamide (AAm) was weighed and added to 10 mM PBS buffer solution. The solution was stirred in a 50°C water bath until completely dissolved to obtain a 30 wt% AAm solution. Then, 6 wt% glucose, 0.045 wt% N,N'-methylenebisacrylamide (MBA), and 1 wt% chitosan (CS) were added sequentially, and the mixture was stirred in a 50°C water bath until completely dissolved. After cooling to room temperature, 0.13 wt% potassium persulfate and 20 wt% lithium chloride (LiCl) were added and stirred until dissolved. High-purity nitrogen gas was purged for 10 min to remove oxygen, and then the mixture was sonicated for 5 min to remove air bubbles, thus preparing a hydrogel stock solution. Finally, 0.03 mg / mL FeSN-MXene was added. Sazymes nanozymes were continuously stirred for 1.0 h to obtain a hydrogel / nanozyme stock solution. The prepared hydrogel / nanozyme stock solution was poured into a mold and reacted in an oven at 70°C for 2 h. After cooling to 4°C, it was removed from the mold to obtain PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel.

[0033] Step S4: Construction of TMB-based colorimetric sensor patch A circular patch with a diameter of 1 cm and a thickness of 0.5 mm was fabricated from PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel. The prepared hydrogel patch was immersed in 100 µL of a solution containing 5 mM TMB. After the TMB solution was completely absorbed into the hydrogel patch, mixtures containing 10 mg / mL glucose oxidase (COx) and different concentrations of glucose were added dropwise. The color change of the hydrogel patch was observed. The grayscale value of the hydrogel color was analyzed using ImageJ software after taking photos under LED light with a mobile phone.

[0034] Step S5: Preparation of LIG electrode The PI film was cleaned with ethanol and deionized water for 5 minutes each, and then dried in an oven at 60°C for later use. Electrode patterns were designed using AutoCAD software and imported into a computer. A computer-controlled laser was used to batch print three-dimensional honeycomb porous graphene electrode structures on biocompatible PI films. These three-dimensional honeycomb porous graphene electrodes included a reference electrode, a working electrode, and a counter electrode. The working electrode was located between the reference and counter electrodes. The laser power, scanning frequency, number of scans, and focal length of the engraving machine were optimized to control and improve the microstructure of the three electrodes according to experimental requirements. Silver paste was applied to the lower ends of the counter and working electrodes to serve as the graphene counter electrode and graphene working electrode, respectively. Silver paste was applied to the lower end of the reference electrode, and silver chloride paste was applied above the reference electrode to serve as the silver chloride reference electrode. The electrodes were then dried at 60°C for later use.

[0035] Step S6: Measure H2O2 using PAAM-CS / FeSN-MXene SAzymes / LIG flexible electrochemical sensor. 3 μL of PAAM-CS hydrogel containing 0.1 mg / mL FeSN-MXene Sazymes nanozyme was modified onto the surface of the LIG working electrode. After gelation, a PAAM-CS / FeSN-MXene Sazymes / LIG flexible electrochemical sensor was constructed. Chronoamperometry (It) was performed on different concentrations of H2O2 to verify the sensor's response to H2O2.

[0036] Step S7: Construction of the PAAM-CS / FeSN-MXene SAzymes / GOx / LIG flexible electrochemical sensor 3 μL of PAAM-CS hydrogel stock solution containing 0.1 mg / mL FeSN-MXene Sazymes nanozyme was modified onto the surface of the LIG working electrode, and then 3 μL of 10 mg / mL GOx was added. After air drying at room temperature, a PAAM-CS / FeSN-MXeneSAzymes / GOx / LIG flexible electrochemical sensor was obtained.

[0037] Step S8: Glucose test in actual sweat Colorimetric hydrogel patches soaked in TMB and GOx in sequence, along with a microfluidic sweat collection-export channel and a PAAM-CS / FeSN-MXene SAzymes / GOx / LIG flexible electrochemical sensor, were attached to the arm and connected to a circuit signal processing unit. Changes in glucose in sweat before and after meals were continuously monitored via smartphone photography and a Bluetooth mini-program.

[0038] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for preparing a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers, characterized in that... The specific preparation process is as follows: A highly conductive, highly adhesive, and well-permeable hydrogel is prepared by cross-linking biocompatible N,N'-methylenebisacrylamide, chitosan, glucose, and acrylamide using a dual cross-linking strategy. This hydrogel serves as the color-changing substrate and electrochemical catalytic functionalization carrier. Using MXene, FeSO4, and urea as reactants, FeSN-MXene single-atom nanozymes with excellent H2O2 enzyme activity are synthesized through low-temperature annealing. The FeSN-MXene single-atom nanozymes are then introduced into the hydrogel stock solution for gelation and functionalization, and their responsiveness to sweat markers, antifreeze properties, and self-healing properties are adjusted to obtain PAAM-CS / FeSN-MXene. SAzymes nanocomposite hydrogel; a three-dimensional honeycomb porous graphene flexible electrode was prepared on a flexible polyimide film using laser-induced graphene technology as an electrochemical sensor substrate; a color-changing hydrogel patch, i.e., a flexible colorimetric sensor, was prepared by sequentially adding and immersing a colorimetric probe TMB and a bio-enzyme into the nanocomposite hydrogel with high sensitivity and specific catalytic effect; the nanocomposite hydrogel and bio-enzyme were sequentially modified on the working electrode of the electrochemical sensor substrate to prepare a flexible electrochemical sensor; the color-changing hydrogel patch and the flexible electrochemical sensor can realize high-sensitivity and specific colorimetric-electrochemical dual-mode monitoring of sweat markers.

2. The method for preparing the flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers according to claim 1, characterized in that: The sweat markers are glucose and / or lactic acid.

3. The method for preparing the flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers according to claim 1, characterized in that... The specific preparation steps are as follows: Step S1, Preparation of FeSN-MXene single-atom nanozyme: LiF was dissolved in concentrated hydrochloric acid, and titanium aluminum carbide was added and stirred at 30~40℃ to obtain a black suspension. The suspension was centrifuged and washed repeatedly with deionized water until the supernatant was neutral to obtain the exfoliated MXene suspension. After freeze-drying, the suspension was placed in a tube furnace and annealed at 500~600℃ under a nitrogen atmosphere with a heating rate of 4~6℃ / min to obtain a layered MXene structure. The layered MXene structure was used as a template and ultrasonically dispersed in deionized water to obtain dispersion A. Urea was dissolved in deionized water to obtain solution B, and ferrous sulfate was dissolved in deionized water to obtain solution C. Under stirring conditions, solutions B and C were added to dispersion A in batches. After mixing evenly, the mixture was stirred at room temperature. The resulting suspension was then freeze-dried and annealed at 500~600℃ under a nitrogen atmosphere with a heating rate of 4~6℃ / min to obtain FeSN-MXene single-atom nanozyme. Step S2, Preparation of PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel: Acrylamide was added to PBS buffer solution and stirred at 40~60℃ until completely dissolved to obtain an acrylamide solution. Then, glucose, N,N'-methylenebisacrylamide and chitosan were added in sequence and stirred at 40~60℃ until completely dissolved. After cooling to room temperature, potassium persulfate and lithium chloride were added and stirred to dissolve. High-purity nitrogen was introduced to remove oxygen, and then the bubbles were removed by sonication to obtain the hydrogel stock solution. FeSN-MXene single-atom nanozyme was added and stirred continuously to mix evenly to obtain the hydrogel / nanozyme stock solution. The hydrogel / nanozyme stock solution was poured into a mold and reacted at 60~80℃. After cooling to 2~5℃, it was removed from the mold to obtain PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel. Step S3, Construction of TMB colorimetric sensor patch PAAM-CS / FeSN-MXene SAzymes nanocomposite hydrogel was made into a composite hydrogel patch. Then, the colorimetric probe TMB and bio-enzyme were sequentially added and immersed into the composite hydrogel patch with high sensitivity and specific catalytic effect to obtain a TMB-based colorimetric sensor patch, i.e., a flexible colorimetric sensor. Step S4, Electrode preparation: A three-dimensional honeycomb porous graphene electrode is printed on a flexible polyimide film using laser-induced graphene technology as a sensing substrate. The three-dimensional honeycomb porous graphene electrode includes a reference electrode, a working electrode, and a counter electrode. The working electrode is located between the reference electrode and the counter electrode. Silver paste is applied to the lower ends of the counter electrode and the working electrode to serve as the graphene counter electrode and the graphene working electrode. Silver paste is applied to the lower end of the reference electrode and silver chloride paste is applied above the reference electrode to serve as the silver chloride reference electrode. The electrode is then dried at 50~70℃ for later use. Step S6, Construction of PAAM-CS / FeSN-MXene SAzymes / GOx / LIG flexible electrochemical sensor: The hydrogel / nanozyme stock solution was modified on the surface of the graphene working electrode, and then the bio-enzyme was modified and air-dried at room temperature to obtain the PAAM-CS / FeSN-MXene SAzymes / GOx / LIG flexible electrochemical sensor.

4. The method for preparing the flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers according to claim 3, characterized in that... The bioenzyme mentioned in step S3 is one or more of glucose oxidase (GOx) or lactate oxidase (LOx).

5. The method for preparing a flexible colorimetric-electrochemical dual-mode sensor for monitoring sweat biomarkers according to claim 3, characterized in that... In step S4, the reaction area of ​​the three-electrode system printed on the polyimide film is connected to the sweat microfluidic collection-export channel. The specific preparation process of the sweat microfluidic collection-export channel is as follows: the size of the microfluidic channel is drawn using CAD to customize the channel mold. The crosslinking agent and curing agent of PDMS are mixed evenly in a mass ratio of 10:1 and injected into the mold. Vacuum is drawn to remove the air bubbles in the mixture. Then, it is placed in an oven at 80°C for curing for 1 hour. After demolding, a sweat microfluidic collection-export channel with uniform texture is obtained. Finally, the interior and surface of the channel are modified by hydrophilicity and hydrophobicity using plasma cleaning equipment to obtain the sweat microfluidic collection-export channel.

6. The application of the flexible colorimetric-electrochemical dual-mode sensor for sweat biomarker monitoring prepared by the method according to any one of claims 1 to 5 in highly sensitive and selective colorimetric-electrochemical dual-mode monitoring of glucose and / or lactic acid biomarkers in sweat.

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