Resting state on-demand sweating and high-stable universal flexible sensor and preparation method thereof

Abstract

A resting state on-demand sweating and high-stability universal flexible sensor and a preparation method thereof belong to the field of wearable biological sensing. An electrode array is integrated with iontophoresis electrodes, a current type three-electrode system and a voltage type two-electrode system. The surface of the working electrode of the current type three-electrode system is constructed with nickel cobalt hexacyanoferrate, biological enzymes and perfluorinated resin, and is used for universal detection of various metabolites; the voltage type two-electrode system realizes sweat pH monitoring by modifying polyaniline. Through the synergistic effect of iontophoresis and stimulation containing carbachol, the controllable induction and collection of resting state sweat are realized. The present application integrates sampling, transmission, identification, signal transduction and anti-interference, has good universality, and only needs to replace the oxidase to realize the detection of different metabolites; has high sensitivity, high stability and excellent anti-interference ability, and can realize the real-time and continuous monitoring of various biomarkers in human sweat under non-invasive conditions, and is suitable for wearable health monitoring and disease management fields.

Description

Technical Field

[0001] This invention relates to the field of wearable biosensing technology, and in particular to a resting-state on-demand sweating and highly stable universal flexible sensor and its preparation method, and especially to a flexible electrochemical biosensing platform suitable for constructing various oxidases and used for real-time detection of metabolites in sweat under resting conditions. Background Technology

[0002] Small molecule metabolites such as glucose, lactic acid, and uric acid are important biomarkers reflecting the body's energy metabolism, physiological stress state, and disease progression. Abnormal fluctuations in their concentrations are closely related to the occurrence and development of various chronic diseases, including diabetes, tissue hypoxia, gout, and cardiovascular disease. Therefore, sensitive, continuous, and real-time monitoring of these metabolites is of great significance for early disease screening, dynamic disease assessment, individualized intervention, and daily health management.

[0003] Currently, the detection of relevant metabolites still mainly relies on blood sample analysis, such as finger-prick or venous blood collection. Although blood testing methods are relatively mature and have high accuracy, they are invasive procedures, causing pain and psychological burden to subjects, affecting long-term monitoring compliance, and are difficult to meet the application requirements of high-frequency, continuous, and routine testing. In contrast, sweat contains a variety of biochemical components related to blood metabolic status, and its collection method is non-invasive, convenient, and suitable for continuous collection, thus becoming an important research direction in the field of wearable health monitoring.

[0004] However, existing sweat monitoring technologies still face significant limitations in practical applications. First, most sweat collection methods rely on exercise, high-temperature stimulation, or other external inducing conditions, which can easily cause fluctuations in the body's metabolic level and physiological state, thus affecting the accuracy and stability of the concentration of target analytes in sweat. At the same time, these methods are poorly applicable to the elderly, children, postoperative patients, patients with chronic diseases, and people in sedentary positions such as office workers, hindering the widespread application of sweat detection technology in clinical monitoring and daily health management.

[0005] Secondly, in terms of sensor construction, enzyme-based electrochemical sensors are widely used for sweat metabolite detection due to their advantages such as good molecular recognition ability, high sensitivity, low cost, and ease of miniaturization and integration. However, most existing enzyme-based sweat sensors are custom-designed around a single target molecule. Different analytes usually require different electrode materials, electron mediators, operating potentials, and enzyme immobilization strategies, resulting in a complex sensing interface construction process, poor device design versatility, difficulty in rapidly expanding to various metabolite detection scenarios, and hindering the formation of a standardized, modular, and large-scale manufacturing system. In other words, existing technologies generally lack a universal sensing platform that is compatible with multiple oxidases and allows for switching of detection targets through simple replacement of the recognition enzyme.

[0006] Furthermore, in terms of long-term continuous detection performance, existing enzyme-based sweat sensors still suffer from problems such as poor enzyme immobilization, insufficient interface stability, limited electron transmission efficiency, and weak anti-interference capabilities. On the one hand, oxidases are prone to inactivation, leakage, or detachment in flexible substrates or complex sweat environments, leading to rapid sensor response decay, poor repeatability, and short lifespan. On the other hand, sweat often contains ascorbic acid, urea, inorganic salts, and other electroactive or non-specific adsorbed components, which can easily cause background current drift, signal cross-response, or electrode contamination during detection, thereby affecting the accuracy and real-time performance of the detection results.

[0007] Therefore, there is an urgent need to develop a flexible electrochemical sensing technology solution for the detection of sweat metabolites. This solution should not only be able to achieve stable and controllable sweat acquisition in a resting state, but also have good compatibility with a variety of oxidases, excellent electron transport performance, reliable enzyme immobilization ability, and anti-interference ability in complex sweat environments, so as to meet the application requirements of multi-target, continuous, real-time and practical health monitoring. Summary of the Invention

[0008] The purpose of this invention is to address the problems in existing technologies, such as sweat sampling often relying on exercise or high temperature induction, enzyme-based wearable sensors mostly being single-target customized designs, insufficient interface stability and anti-interference capabilities, and the limitation of practical applications caused by the isolated design of devices and sampling methods. This invention provides a universal flexible sensor with broad-spectrum detection capabilities, high interface stability, and suitability for on-demand sweating in a resting state, along with its preparation method. This ensures high sensitivity, high accuracy, and long-term stable detection of multiple small-molecule metabolites in sweat under real physiological conditions, thereby improving the usability and applicability of clinical and daily health monitoring.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A resting-state, on-demand sweating and highly stable universal flexible sensor includes:

[0011] Flexible substrate;

[0012] An electrode array, disposed on a flexible substrate, includes an iontophoresis therapy electrode, a current-type three-electrode system, and a voltage-type two-electrode system. The iontophoresis therapy electrode is used to induce sweat on demand, the current-type three-electrode system is used for sweat metabolite concentration detection, and the voltage-type two-electrode system is used for sweat pH detection.

[0013] The working electrode surface of the current-type three-electrode system is sequentially provided with a bottom mediator layer, an intermediate enzymatic reaction layer and an outer encapsulation layer. The bottom mediator layer is nickel cobalt hexacyanoferrite, the intermediate enzymatic reaction layer contains oxidase, and the outer encapsulation layer is perfluorinated resin.

[0014] The working electrode surface of the voltage-type two-electrode system is provided with a pH-sensitive layer;

[0015] The iontophoresis therapy electrode includes an iontophoresis anode and an iontophoresis cathode. The iontophoresis anode is provided with an anodic stimulation gel containing carbacholine, and the iontophoresis cathode is provided with a cathodic stimulation gel containing sodium chloride.

[0016] A microfluidic channel layer is disposed on a flexible substrate. The microfluidic channel layer has a sensing chamber, an inlet channel, an outlet channel, a cathode gel through-hole aligned with the ion electroosmosis cathode, and an anode gel through-hole aligned with the ion electroosmosis anode.

[0017] The sample introduction layer is disposed above the microfluidic channel layer. The sample introduction layer has an array of sweat inlets aligned with the liquid inlet channel, a cathode gel contact window aligned with the cathode gel through-hole, and an anode gel contact window aligned with the anode gel through-hole.

[0018] The flexible substrate, microfluidic channel layer, and sample introduction layer are sequentially aligned and bonded from bottom to top, and the current-type three-electrode system and the voltage-type two-electrode system are both located in the sensing and detection chamber.

[0019] The current-type three-electrode system includes a first working electrode, a first reference electrode, and a first pair of electrodes; the voltage-type two-electrode system includes a second working electrode and a second reference electrode.

[0020] The bottom mediator layer is a cured layer formed by drying a nickel cobalt hexacyanoferrate solution; the intermediate enzymatic reaction layer is a biological enzyme membrane layer containing oxidase and stabilizer; and the outer encapsulation layer is a cured layer formed by drying a perfluorinated resin solution.

[0021] The area of ​​the ion electroosmosis anode is larger than the area of ​​the ion electroosmosis cathode.

[0022] The anodic stimulation gel is an agarose gel containing carbacholine, and the cathodic stimulation gel is an agarose gel containing sodium chloride.

[0023] The microfluidic channel layer is made of double-sided self-adhesive PET film or PET film laminated with medical double-sided adhesive tape; the sample injection layer is a flexible, skin-friendly film.

[0024] The sweat inlet array consists of several tiny sweat inlets arranged in an arc.

[0025] The method for fabricating a resting-state on-demand sweating and highly stable universal flexible sensor includes the following steps:

[0026] 1) An electrode array is fabricated on a flexible substrate, the electrode array comprising an iontophoresis therapy electrode, a current-type three-electrode system, and a voltage-type two-electrode system;

[0027] 2) A bottom mediator layer, an intermediate enzymatic reaction layer, and an outer encapsulation layer are sequentially modified on the working electrode surface of the current-type three-electrode system, and a pH-sensitive layer is modified on the working electrode surface of the voltage-type two-electrode system.

[0028] 3) Cast an anodic stimulation gel containing carbacholine onto the iontophoresis anode, and cast a cathodic stimulation gel containing sodium chloride onto the cathode of the iontophoresis therapy electrode.

[0029] 4) Preparation of microfluidic channel layer and sample introduction layer;

[0030] 5) Assemble the flexible substrate, microfluidic channel layer and sample injection layer from bottom to top, so that the current-type three-electrode system and voltage-type two-electrode system are located in the sensing and detection chamber of the microfluidic channel layer.

[0031] The bottom mediator layer is a nickel cobalt hexacyanoferrite solution, the intermediate enzymatic reaction layer is a biological enzyme membrane solution containing oxidase and stabilizer, the outer encapsulation layer is a perfluorinated resin solution, and the pH sensitive layer is a polyaniline sensitive layer.

[0032] The application of the sensor in the preparation of a non-invasive wearable device for monitoring metabolites.

[0033] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0034] 1. This invention possesses significant advantages as a universal platform, enabling rapid expansion of its detection capabilities for various metabolites. Using NiCoHCF as the core functional layer of the sensing interface, and leveraging its highly efficient electrocatalytic ability for hydrogen peroxide, a byproduct of various oxidase reactions, this invention constructs a universal flexible sensing platform suitable for multiple oxidases. Compared to existing technologies that design electrode materials, interface structures, and detection systems separately for different target analytes, this invention eliminates the need for repeated development of the underlying sensing architecture. Only the corresponding oxidase needs to be replaced to achieve the detection of different metabolites such as glucose, lactic acid, and uric acid. Therefore, this invention effectively overcomes the shortcomings of existing enzyme-based sweat sensors, such as poor versatility, long development cycles, complex manufacturing, and difficulty in large-scale promotion, possessing stronger platform expansion capabilities and industrial application potential.

[0035] 2. It possesses excellent detection performance, simultaneously achieving high sensitivity, high stability, and high anti-interference detection. In this invention, NiCoHCF not only provides a large specific surface area for effective immobilization of enzyme molecules but also promotes electron transport at the electrode interface, reducing electron transfer impedance, thereby significantly enhancing the sensor's current response and improving detection sensitivity. Simultaneously, the outer Nafion film provides stable coating and immobilization of enzyme molecules, reducing enzyme shedding and inactivation, and effectively inhibits the entry of electroactive interfering substances such as ascorbic acid from sweat into the reaction interface using its ion sieving properties. Therefore, this invention maintains good signal stability, detection accuracy, and selectivity in complex sweat environments, significantly improving the practical reliability of the flexible sweat sensor.

[0036] 3. Enables on-demand sweat collection and real-time monitoring even at rest, significantly expanding application scenarios. Addressing the issue that existing sweat detection methods generally rely on exercise or high-temperature induction, making them unsuitable for resting individuals, this invention further incorporates iontophoresis. Under an applied electric field, carbacholine promotes local sweat gland secretion, achieving controllable sweat collection even without exercise. Compared to traditional exercise-induced sweat collection methods, this approach reduces metabolic interference caused by exercise load and environmental changes, resulting in sweat samples that more accurately reflect the body's basic physiological state, thus improving the accuracy and stability of test results. This technology is particularly suitable for continuous health monitoring in non-exercise scenarios such as the elderly, children, patients with limited mobility, and daily office work, significantly enhancing the clinical and daily application value of this invention. Attached Figure Description

[0037] Figure 1 This is an exploded view of the hierarchical structure of the resting-state on-demand sweating and highly stable universal flexible sensor of the present invention.

[0038] Figure 2The results show the detection and analysis of the current-type three-electrode system, where: (A) is the response test graph of glucose solutions of different concentrations, (B) is the sensitivity of the test response of glucose solutions of different concentrations, and (C) is the anti-interference test graph.

[0039] Figure 3 The results show the detection and analysis of the voltage-type two-electrode system, where: (A) is the response test graph of standard solutions at different pH values, (B) is the sensitivity of the test response of standard solutions at different pH values, and (C) is the anti-interference test graph.

[0040] Figure 4 This invention provides an in vivo evaluation of the resting-state on-demand sweating and highly stable universal flexible sensor during daily activities.

[0041] Figure 5 The results show the detection and analysis of the current-type three-electrode system, where: (A) is the response test graph of lactic acid solution with different concentrations, (B) is the sensitivity of the test response of lactic acid solution with different concentrations, and (C) is the anti-interference test graph.

[0042] Reference numerals: 1. Flexible substrate; 101. Iontoosmosis therapy cathode; 102. Iontoosmosis therapy anode; 103. Counter electrode; 104. First working electrode; 105. First reference electrode; 106. Second working electrode; 107. Second reference electrode; 108. Sweat outlet; 109. Wire; 2. Microfluidic channel layer; 201. Cathode gel through-hole; 202. Anode gel through-hole; 203. Liquid inlet drainage channel; 204. Sensing chamber; 205. Liquid outlet drainage channel; 206. Sweat outlet; 3. Sample injection layer; 301. Cathode gel contact window; 302. Anode gel contact window; 303. Sweat outlet array. Detailed Implementation

[0043] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0044] This invention constructs a three-layer micro-nano interface (the bottom layer is a nickel cobalt hexacyanoferrite NiCoHCF mediator layer, the middle layer is an oxidase bio-enzyme membrane layer, and the outer layer is a perfluorinated resin encapsulation membrane such as Nafion) on a current-type three-electrode working electrode on a flexible substrate. This universal recognition-signal transduction interface is integrated with an iontophoresis on-demand sweat-inducing module (including an anode / cathode stimulation gel) and a microfluidic sampling / transmission structure to form a flexible sweat sensor system integrating "sampling-transport-recognition-transduction-anti-interference".

[0045] The specific implementation steps are as follows:

[0046] Step 1: Prepare an electrode array on a flexible substrate. The electrode array includes at least: an iontophoresis electrode (anode and cathode) for on-demand sweat induction, a current-type three-electrode system (first working electrode, first reference electrode and first pair of electrodes) for sweat metabolite concentration detection, and a voltage-type two-electrode system (second working electrode and second reference electrode) for sweat pH measurement.

[0047] Step 2: On the surface of the first working electrode of the current-type three-electrode system, the following layers are sequentially modified: a bottom mediator layer (NiCoHCF solution), an intermediate enzymatic reaction layer (a bio-enzyme membrane containing oxidase and stabilizer), and an outer encapsulation layer (a perfluorinated resin solution such as Nafion) to construct a three-layer micro-nano sensing interface; A pH-sensitive layer is modified on the surface of the working electrode of the voltage-type two-electrode system.

[0048] Step 3: Cast the anodic stimulation gel (agarose-based gel containing carbacholine) and the cathodic stimulation gel (agarose-based gel containing sodium chloride) onto the iontophoresis electrode, respectively.

[0049] Step 4: Prepare the microfluidic channel layer and the sample injection layer, and pre-set channel structures such as gel through-holes and sensing chambers aligned with the electrodes on the microfluidic layer;

[0050] Step 5: Assemble the flexible substrate, microfluidic channel layer and sample introduction layer from bottom to top, so that the current-type three-electrode system and voltage-type two-electrode system are located in the sensing chamber and form a sample collection and flow path.

[0051] The ratio of the area of ​​the ion electroosmosis anode to the area of ​​the cathode is 1.5 to 5:1 (the anode area is larger than the cathode) to improve the electroosmosis driving efficiency and reduce the intensity of local stimulation.

[0052] The preparation of the anodic stimulation gel includes: dissolving agarose in deionized water by heating and stirring, cooling to a suitable temperature, adding carbacholine and mixing to obtain an anodic gel solution; the preparation of the cathodic stimulation gel includes: dissolving agarose in deionized water by heating and stirring, cooling to a suitable temperature, adding sodium chloride and mixing to obtain a cathodic gel solution; the anodic gel solution and the cathodic gel solution are respectively cast onto the ion-electroosmosis anode and the ion-electroosmosis cathode and solidified at room temperature.

[0053] Furthermore, the agarose-based gel content is 0.5%–3% (w / v), the carbacholine concentration in the anolyte gel is 0.1–10 mg / mL, and the sodium chloride concentration in the cathode gel is 0.1–2 M, in order to achieve a controllable local sweat-inducing response.

[0054] The preparation of the nickel cobalt hexacyanoferrite solution includes: mixing sodium citrate, nickel chloride and cobalt chloride with dilute hydrochloric acid in a predetermined ratio, adding potassium ferrocyanide solution to the mixture at room temperature and stirring to react, aging under light-protected conditions, centrifuging to remove the supernatant and drying the precipitate, and finally preparing a NiCoHCF solution of the required concentration.

[0055] Furthermore, the NiCoHCF solution concentration is 10~30 mg / mL, and the Ni:Co molar ratio is 3:1~1:3, in order to balance catalytic activity and film formation properties.

[0056] The preparation of the bio-enzyme membrane solution containing oxidase and stabilizer includes: mixing glutaraldehyde solution with bovine serum albumin (BSA) to obtain a stabilizer, dissolving the target oxidase in PBS buffer to obtain an enzyme solution, and mixing the stabilizer and enzyme solution evenly and storing at low temperature to maintain enzyme activity.

[0057] Furthermore, the final concentration of glutaraldehyde in the bio-enzyme membrane solution is 0.01%~0.1%, and the cross-linking time is 5~60 minutes.

[0058] The preparation of the perfluorinated resin solution includes: diluting a high-concentration Nafion solution with anhydrous ethanol to the target mass fraction as needed to obtain the outer encapsulation solution.

[0059] Furthermore, the outer Nafion solution has a mass fraction of 0.1wt% to 5wt% to provide interference-resistant screening and enzyme encapsulation functions while maintaining throughput.

[0060] The pH-sensitive layer is a polyaniline-sensitive layer, and the polyaniline dispersion is drop-coated onto the surface of the second working electrode and then dried and cured.

[0061] The preparation of the polyaniline dispersion includes: dissolving aniline and ammonium persulfate in dilute hydrochloric acid, then mixing them, aging, centrifuging to remove the supernatant and drying the precipitate, and finally adding deionized water and sodium dodecyl sulfate to prepare a polyaniline nanowire dispersion.

[0062] Furthermore, the concentration of the polyaniline nanowire dispersion is preferably 5~10 mg / mL to ensure a good conductive network structure.

[0063] The microfluidic channel layer is made of double-sided self-adhesive PET film or PET film laminated with medical double-sided adhesive tape, and the microfluidic channel layer has the following: cathode gel through holes aligned with the ion electroosmosis cathode, anode gel through holes aligned with the ion electroosmosis anode, liquid inlet channel, sensing chamber, liquid outlet channel and sweat outlet.

[0064] The sample introduction layer is a flexible, skin-friendly film, and the sample introduction layer is provided with: a cathode gel contact window aligned with the cathode gel through hole, an anolyte gel contact window aligned with the anolyte gel through hole, and an array of sweat inlets aligned with the liquid inlet drainage channel.

[0065] Furthermore, the microfluidic channel and sweat inlet array structure can be defined as a channel layer composed of laminated PET and medical double-sided adhesive, and the sweat inlets can be several (e.g., seven) micro sweat inlets arranged in an arc to optimize sampling efficiency and local coverage.

[0066] Example 1: Fabrication of a resting-state on-demand sweating and highly stable universal glucose sweat flexible sensor, see [link to documentation]. Figure 1 .

[0067] Step 1: Printing of the flexible electrode array:

[0068] A working electrode and a counter electrode are printed on a flexible substrate 1 using conductive carbon paste, including a first working electrode 104, a second working electrode 106, and a counter electrode 103, and then dried in a vacuum drying oven at 80°C for 20 min. Next, a reference electrode and wires 109 are printed using Ag / AgCl ink. The reference electrode includes a first reference electrode 105 and a second reference electrode 107, and is also dried in a vacuum drying oven at 80°C for 20 min. The first working electrode 104 is used for enzyme-based current detection, and the second working electrode 106 and the second reference electrode 107 form a voltage-type two-electrode system for pH detection. The first working electrode 104 is a circle with a diameter of 4 mm, and the distance between it and the counter electrode 103 and the first reference electrode 105 is 1 mm. This compact design significantly reduces the uncompensated resistance of the solution.

[0069] On the flexible substrate 1, at the end position of the liquid drainage channel 205 of the subsequent microfluidic channel layer 2, a sweat outlet 108 is opened to drain excess sweat after it has flowed through the microfluidic channel.

[0070] Step 2: Preparation of the current-type three-electrode system for sweat glucose detection:

[0071] Accurately weigh 0.103 g sodium citrate, 0.024 g nickel chloride hexahydrate, and 0.024 g cobalt chloride hexahydrate, and add them sequentially to 10 mL of 0.1 M dilute hydrochloric acid. Stir until fully mixed to prepare solution 1. Accurately weigh 0.042 g potassium ferrocyanide and add it to 10 mL of deionized water to obtain solution 2. Add solution 2 dropwise to solution 1 using a constant pressure dropping funnel at 100 rpm at room temperature. After the addition is complete, stir vigorously at 700 rpm for 5 min at room temperature. Age the resulting solution at room temperature in the dark for 24 h. Centrifuge the reacted solution at 7000 rpm for 30 min, remove the supernatant, add deionized water, and repeat the washing and centrifugation twice. Dry the resulting precipitate under vacuum at 60 °C for 12 h. Prepare a 20 mg / mL NiCoHCF (Ni:Co = 1:2) solution, sonicate for 30 min, and store at 4 °C in the dark.

[0072] Take 250 mg of glutaraldehyde (50% in H2O), then add 4.75 g of PBS buffer and shake for 1 min to obtain a 2.5 wt% glutaraldehyde solution. Take 40 mg of BSA and add it to 4 mL of PBS buffer to obtain a 10 mg / mL BSA solution. Mix 40 μL of 2.5 wt% glutaraldehyde with 1.96 mL of 10 mg / mL BSA to obtain a BSA stabilizer. Take 40 mg of glucose oxidase (GOx) and add it to 0.5 mL of PBS buffer to obtain an 80 mg / mL GOx solution. Each time, take 25 μL of the 80 mg / mL GOx solution and mix it with 25 μL of BSA stabilizer (1:1, v / v) to obtain approximately 20 sets of 50 μL GOx solutions with a concentration of 40 mg / mL. These solutions need to be refrigerated at -20°C to ensure GOx activity.

[0073] A 0.1 wt% Nafion solution was prepared by diluting a 0.5 wt% Nafion solution with anhydrous ethanol.

[0074] A three-layer sensing system was modified on the surface of the first working electrode using a drop-coating method. The specific steps were as follows: 40 mg / mL glucose oxidase (GOx) bio-enzyme membrane solution was slowly thawed in a foam box containing ice. 20 mg / mL NiCoHCF solution was ultrasonically dispersed for 15 min. 4 μL of NiCoHCF solution was drop-coated onto the surface of the first working electrode (104), and dried in a 40℃ drying oven for 40 min, then removed and cooled to room temperature. 2.5 μL of GOx enzyme membrane solution was drop-coated onto the NiCoHCF layer and dried overnight at 4℃ in the dark. The electrode was then removed from the 4℃ freezer, allowed to return to room temperature, and 1.5 μL of 0.1 wt% Nafion solution was uniformly coated onto the outermost layer. The layer was then allowed to air dry and cure at room temperature for 30 min, completing the preparation of the current-type three-electrode system for sweat glucose detection.

[0075] Step 3: Preparation of a voltage-type two-electrode system for sweat pH detection:

[0076] Accurately weigh 372.5 mg of aniline and add it to 50 mL of 1M HCl solution. Sonicate for 5 min to dissolve completely, obtaining a 0.08 M aniline solution. Accurately weigh 228.2 mg of ammonium persulfate and add it to 50 mL of 1M HCl solution. Sonicate for 5 min to dissolve completely, obtaining a 0.02 M ammonium persulfate solution. Measure 10 mL of each of the two prepared solutions, mix them, and shake vigorously up and down about 30 times (approximately 10 seconds). Incubate the resulting solutions at 4℃ in the dark for 24 h to form polyaniline nanowires. Centrifuge the reacted polyaniline nanowires at 6000 rpm for 30 min, remove the supernatant, add 20 mL of deionized water, and repeat the centrifugation step 2-3 times under the same conditions until the pH of the supernatant is 7. Remove the supernatant. Add 10 mL of deionized water to the obtained precipitate, then add 0.08 g of sodium dodecyl sulfate and sonicate for 30 min to uniformly disperse the nanowires, thus obtaining polyaniline nanowires, which need to be stored in a refrigerator at 4°C in the dark.

[0077] Accurately weigh 237.3 mg of PVB and add it to 3 mL of methanol. Shake for 5 min to fully dissolve the PVB. Then add 150 mg of sodium chloride, sonicate for 30 min, and stir at 800 rpm for 6 h to obtain the PVB membrane solution.

[0078] The polyaniline sensitive layer on the surface of the second working electrode was modified using a drop-coating method. Specifically, the polyaniline nanowire dispersion was ultrasonically dispersed for 15 min. 3 μL of the polyaniline nanowire dispersion was then drop-coated onto the surface of the second working electrode. Similarly, the PVB film solution was modified on the surface of the second reference electrode 107 using the same drop-coating method. 3 μL of the PVB film solution was then drop-coated onto the surface of the second reference electrode 107. The electrode was then allowed to stand and dry at 4℃ for 12 h, thus completing the preparation of the voltage-type two-electrode system for sweat pH detection.

[0079] Step 4: Preparation and casting of the stimulation gel corresponding to the iontophoresis therapy electrode:

[0080] Accurately weigh 0.45 g of agarose and slowly add it to 14.55 g of deionized water. Heat the solution in an oil bath to 95°C and stir continuously at 500 rpm until the solution is clear and homogeneous, yielding 15 g of a 3% w / w agarose solution. After cooling to 70°C, add accurately weighed 0.15 g of carbachol and stir to mix, yielding a 1% w / w carbachol anolyte gel. Accurately weigh 0.45 g of agarose and slowly add it to 14.55 g of deionized water. Heat the solution in an oil bath to 95°C and stir continuously at 500 rpm until the solution is clear and homogeneous, yielding 15 g of a 3% w / w agarose solution. After cooling to 70°C, add accurately weighed 0.15 g of sodium chloride and stir to mix, yielding a 1% w / w sodium chloride cathode gel. Drop the anolyte gel and cathode gel onto the upper regions of the iontophoresis anode 102 and iontophoresis cathode 101, respectively, and allow them to solidify at room temperature.

[0081] Step 5: Fabrication and assembly of the microfluidic channel layer and the sample introduction layer:

[0082] Using an ultrafast laser three-dimensional micro / nano etching platform, cathode gel through-holes 201 and anolyte gel through-holes 202, a liquid inlet channel 203, a sensing chamber 204, a liquid outlet channel 205, and a sweat outlet 206 are cut into a PET film covered with 0.01 mm thick medical double-sided tape on both sides, forming a microfluidic channel layer 2. Cathode gel contact windows 301 and anolyte gel contact windows 302, and a sweat inlet array 303 containing seven 0.8 mm circular holes are cut into a skin-friendly film, forming a sample introduction layer 3. The three layers are precisely aligned and pressed together, so that the sweat outlet 206 of the microfluidic channel layer is vertically connected to the sweat outlet 108 of the flexible substrate, completing the assembly of a universal enzyme-based flexible sweat sensor.

[0083] A resting-state, on-demand sweating and highly stable universal flexible sensor has been successfully fabricated.

[0084] To verify the performance of the resting-state on-demand sweating and highly stable universal glucose sweat flexible sensor prepared in this invention, its sensing performance was explored using various electrochemical detection methods.

[0085] Figures 2 to 4 The performance test and in vivo evaluation results of the resting-state on-demand sweating and highly stable universal flexible sensor proposed in this invention are presented.

[0086] Figure 2 The results show the glucose sensing performance test results based on a current-mode three-electrode system. Figure 2 (A) shows the response curves of five glucose working electrodes prepared in the same batch, measured using the chronoamperometry method, in 20, 40, 80, and 160 μM glucose solutions. The results show that the electrodes exhibit good linear response over a wide concentration range, indicating that they have a wide linear detection range. Figure 2 (B) shows the sensitivity test results of the glucose working electrode in glucose solutions of different concentrations, indicating that it has good detection sensitivity; Figure 2 In the middle (C), the glucose working electrode was tested for anti-interference in a solution containing a variety of common interfering substances. The results showed that the electrode has high selectivity for glucose and good anti-interference performance.

[0087] Figure 3 The results are based on the pH sensing performance test of a voltage-type two-electrode system. Figure 3 Image (A) shows the response curves of the pH working electrode in PBS buffer solutions with pH values ​​of 3, 4, 5, 6, 7, and 8, tested using the open-circuit potential method. The results indicate that the electrode exhibits a good linear response over a wide pH range. Figure 3 (B) shows the sensitivity test results of the pH working electrode in buffer solutions of different pH values, indicating that it has good detection sensitivity; Figure 3 In the middle (C), the pH working electrode was tested for anti-interference in a solution containing a variety of common interfering substances. It still maintained a stable response signal, indicating that it has good anti-interference properties.

[0088] Figure 4 This paper demonstrates the in vivo assessment results of the sensor of this invention during daily activities. Sweat samples were collected at rest under stimulation by iontophoresis electrodes, and changes in glucose concentration and pH value in sweat before and after meals were detected and compared. The results show that the sensor can monitor fluctuations in physiological indicators in sweat in real time, making it suitable for non-invasive health monitoring during daily activities.

[0089] Example 2: Fabrication of a flexible sensor for resting-state on-demand sweating and highly stable universal lactic acid sweat

[0090] The preparation method is basically the same as in Example 1, except that in step three, GOx is replaced with an equal concentration of lactate oxidase (LOx) bio-enzyme membrane solution. The formulations and curing conditions of the bottom NiCoHCF and the outer Nafion remain unchanged. Test results show that the sensor also exhibits excellent catalytic oxidation performance for lactic acid, verifying the high efficiency and versatility of the underlying architecture of this invention.

[0091] Figure 5 The results are based on the lactic acid sensing performance test of the current-mode three-electrode system. Figure 5 (A) shows the response curves of five lactic acid working electrodes prepared in the same batch, measured using the chronoamperometry method, in 5, 10, 15, and 20 mM lactic acid solutions. The results show that the electrodes exhibit good linear response over a wide concentration range, indicating that they have a wide linear detection range. Figure 5 (B) shows the sensitivity test results of the lactic acid working electrode in lactic acid solutions of different concentrations, indicating that it has good detection sensitivity; Figure 5 In the middle (C), the anti-interference test of the lactic acid working electrode was carried out in a solution containing a variety of common interfering substances. The results showed that the electrode has high selectivity for lactic acid and good anti-interference performance.

Claims

1. A resting-state, on-demand sweating, highly stable, universal flexible sensor, characterized in that, include: Flexible substrate; An electrode array, disposed on a flexible substrate, includes an iontophoresis therapy electrode, a current-type three-electrode system, and a voltage-type two-electrode system. The iontophoresis therapy electrode is used to induce sweat on demand, the current-type three-electrode system is used for sweat metabolite concentration detection, and the voltage-type two-electrode system is used for sweat pH detection. The working electrode surface of the current-type three-electrode system is sequentially provided with a bottom mediator layer, an intermediate enzymatic reaction layer and an outer encapsulation layer. The bottom mediator layer is nickel cobalt hexacyanoferrite, the intermediate enzymatic reaction layer contains oxidase, and the outer encapsulation layer is perfluorinated resin. The working electrode surface of the voltage-type two-electrode system is provided with a pH-sensitive layer; The iontophoresis therapy electrode includes an iontophoresis anode and an iontophoresis cathode. The iontophoresis anode is provided with an anodic stimulation gel containing carbacholine, and the iontophoresis cathode is provided with a cathodic stimulation gel containing sodium chloride. A microfluidic channel layer is disposed on a flexible substrate. The microfluidic channel layer has a sensing chamber, an inlet channel, an outlet channel, a cathode gel through-hole aligned with the ion electroosmosis cathode, and an anode gel through-hole aligned with the ion electroosmosis anode. The sample introduction layer is disposed above the microfluidic channel layer. The sample introduction layer has an array of sweat inlets aligned with the liquid inlet channel, a cathode gel contact window aligned with the cathode gel through-hole, and an anode gel contact window aligned with the anode gel through-hole. The flexible substrate, microfluidic channel layer, and sample introduction layer are sequentially aligned and bonded from bottom to top, and the current-type three-electrode system and voltage-type two-electrode system are both located in the sensing and detection chamber.

2. The resting-state on-demand sweating and highly stable universal flexible sensor as described in claim 1, characterized in that: The current-type three-electrode system includes a first working electrode, a first reference electrode, and a first pair of electrodes; the voltage-type two-electrode system includes a second working electrode and a second reference electrode.

3. The resting-state on-demand sweating and highly stable universal flexible sensor as described in claim 1, characterized in that: The bottom mediator layer is a cured layer formed by drying a nickel cobalt hexacyanoferrate solution; the intermediate enzymatic reaction layer is a biological enzyme membrane layer containing oxidase and stabilizer; and the outer encapsulation layer is a cured layer formed by drying a perfluorinated resin solution.

4. The resting-state on-demand sweating and highly stable universal flexible sensor as described in claim 1, characterized in that: The area of ​​the ion electroosmosis anode is larger than the area of ​​the ion electroosmosis cathode.

5. The resting-state on-demand sweating and highly stable universal flexible sensor as described in claim 1, characterized in that: The anodic stimulation gel is an agarose gel containing carbacholine, and the cathodic stimulation gel is an agarose gel containing sodium chloride.

6. The resting-state on-demand sweating and highly stable universal flexible sensor as described in claim 1, characterized in that: The microfluidic channel layer is made of double-sided self-adhesive PET film or PET film laminated with medical double-sided adhesive tape; the sample injection layer is a flexible, skin-friendly film.

7. The resting-state on-demand sweating and highly stable universal flexible sensor as described in claim 1, characterized in that, The sweat inlet array consists of several tiny sweat inlets arranged in an arc.

8. A method for preparing a resting-state on-demand sweating and highly stable universal flexible sensor according to any one of claims 1 to 7, characterized in that, Includes the following steps: 1) An electrode array is fabricated on a flexible substrate, the electrode array comprising an iontophoresis therapy electrode, a current-type three-electrode system, and a voltage-type two-electrode system; 2) A bottom mediator layer, an intermediate enzymatic reaction layer, and an outer encapsulation layer are sequentially modified on the working electrode surface of the current-type three-electrode system, and a pH-sensitive layer is modified on the working electrode surface of the voltage-type two-electrode system. 3) Cast an anodic stimulation gel containing carbacholine onto the iontophoresis anode, and cast a cathodic stimulation gel containing sodium chloride onto the cathode of the iontophoresis therapy electrode. 4) Preparation of microfluidic channel layer and sample introduction layer; 5) Assemble the flexible substrate, microfluidic channel layer and sample injection layer from bottom to top, so that the current-type three-electrode system and voltage-type two-electrode system are located in the sensing and detection chamber of the microfluidic channel layer.

9. The preparation method according to claim 8, characterized in that: The bottom mediator layer is a nickel cobalt hexacyanoferrite solution, the intermediate enzymatic reaction layer is a biological enzyme membrane solution containing oxidase and stabilizer, the outer encapsulation layer is a perfluorinated resin solution, and the pH sensitive layer is a polyaniline sensitive layer.

10. The use of the sensor according to any one of claims 1 to 7 in the preparation of a non-invasive wearable device for monitoring metabolites.

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