Wearable patch for detecting heat stress in animal and preparation method therefor

Abstract

Disclosed are a wearable patch for detecting heat stress in an animal and a preparation method therefor. The wearable patch comprises a flexible substrate and electrodes designed on the flexible substrate. The electrodes include a sweat-inducing electrode, a temperature-sensing electrode, a first working electrode, a second working electrode, and a reference electrode. The sweat-inducing electrode is configured for promoting sweating on the skin of the animal, and the temperature-sensing electrode, the first working electrode, and the second working electrode are configured for measuring the temperature, the pH, and the K+ concentration of sweat, respectively. In the present invention, on the basis of the principle of electrochemical wearable sensors, a wearable patch that can be configured for continuously monitoring various substances in animal sweat is constructed. The sensor is connected to an electrochemical measuring instrument by means of a conducting wire, and the sweat-inducing electrode is utilized to stimulate sweat secretion in the animal, thereby achieving in-situ, real-time, non-invasive, and continuous monitoring of metabolites and temperature.

Description

Wearable patch for animal heat stress detection and preparation method Technical Field

[0001] The present invention relates to the technical field of wearable electrochemical sensing analysis, and in particular to a wearable patch for animal heat stress detection and a preparation method thereof. Background Art

[0002] Heat stress is a nonspecific physiological response that frequently occurs in animals. Normally, animals have limited adaptability to ambient temperatures; when ambient temperatures exceed their tolerance range, they are susceptible to heat stress. There is a close relationship between animal health and heat stress. Under heat stress conditions, animals are susceptible to physiological disturbances, including elevated body temperature, increased respiratory rate, dehydration, and impaired immune function. These effects can lead to decreased endurance, impaired performance, and an increased risk of heat-related illnesses.

[0003] Traditional heat stress management typically utilizes a variety of methods, including physical environmental controls and nutritional management. While convenient, these traditional approaches often fail to meet the growing demand for precise management and continuous monitoring. Modern precision health management approaches for heat stress biomarkers, including traditional medical testing, often involve invasive and time-consuming procedures.

[0004] Among the numerous analytes, sweat contains a variety of chemical components and indicators, including electrolytes, glucose, lactate, etc., which have significant correlations with health and physiological status. However, most current sensors only involve the sensing of a single analyte and cannot achieve real-time monitoring (Zhao, et al., A flexible nonenzymatic sweat glucose sensor based on Au nanoflowers coated carbon cloth, Sensors and Actuators B: Chemical, VOL 388, August 2023, 133798). To date, there is an urgent need for a feasible, multi-faceted management method to achieve precise individual management of health problems caused by heat stress in animals.

[0005] Currently, there are no established devices on the market that utilize electrochemical wearable patches for multiplex analysis of animal sweat and precise health management. Among existing technologies for using electrochemical wearable sensors to detect sweat, the invention application with publication number CN116626117A, entitled "Method for preparing a flexible wearable electrochemical sensor for detecting cortisol in sweat," discloses a method for preparing a flexible wearable electrochemical sensor and a method for detecting cortisol in human sweat using the sensor. The flexible sensor obtained by this method has a large sensing surface area and excellent sensitivity. However, the sensor is complex to prepare and cannot achieve real-time analysis of multiple metabolites in sweat.

[0006] Summary of the Invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides a wearable patch for animal heat stress detection and a preparation method thereof.

[0008] A wearable patch for detecting heat stress in animals, comprising a flexible substrate and electrodes designed on the flexible substrate, each electrode comprising a sensing area as a working area and a conductive connection area as a non-working area, the electrodes comprising:

[0009] A sweat-inducing electrode for inducing sweat secretion from the skin of an animal in contact therewith, comprising an anode sweat-inducing electrode and a cathode sweat-inducing electrode. Under the action of an electric current, a current loop is formed between the anode sweat-inducing electrode and the cathode sweat-inducing electrode. The anode sweat-inducing electrode is modified with an anode gel containing a cholinergic substance. Under the action of electroosmosis, the cholinergic substance enters the skin to stimulate sweat secretion.

[0010] Temperature sensing electrodes for detecting sweat temperature;

[0011] The first working electrode is a pH detection electrode for detecting the pH of sweat;

[0012] The second working electrode is used to detect K in sweat + Concentration of K + Detection electrodes;

[0013] The reference electrode is a reference electrode used for the first working electrode and the second working electrode.

[0014] The sensing area of ​​each electrode is used to sense the corresponding indicator, and the tail end is used to connect to an external signal detection circuit, that is, to connect to an electrochemical workstation during detection. The portion between the sensing area and the area of ​​the tail end used for connecting to the electrochemical workstation is a conductive connection area serving as a non-working area. Preferably, the surface of the conductive connection area serving as the non-working area is insulated. The insulation treatment method can use conventional insulation treatment methods in the prior art, such as using polyimide tape for surface treatment to achieve insulation.

[0015] Preferably, the flexible substrate is made of polyimide, and the electrodes with laser-induced graphene are prepared on the surface of the polyimide flexible substrate by laser-induced cutting.

[0016] The principle of laser-induced graphene technology is mainly based on the photothermal effect and chemical reduction of laser radiation on the surface of the plastic substrate. Under the synergistic effect of photothermal effect and chemical reduction, graphite oxide decomposes into oxygen and other volatile molecules, while carbon atoms rearrange to form a two-dimensional graphene structure.

[0017] More preferably, the power, speed and dots per inch of the laser induced cutting are 5% to 10%, 10% to 20% and 500 to 800 respectively, wherein the ratio of the power and speed is a percentage of the respective maximum setting parameters.

[0018] The sweat inducing electrode is used to induce sweating on the animal skin, so any substance that promotes sweating on the animal skin can be used, for example, a cholinergic, which can be at least one of acetylcholine, methacholine, carbachol and bethanechol.

[0019] Preferably, the anodic gel modified on the sensing area of ​​the anodic sweat-inducing electrode is an agarose gel containing a choline-containing gel, wherein the mass percentage of the choline-containing gel is 0.1% to 10%. The cathodic gel modified on the sensing area of ​​the cathodic sweat-inducing electrode is also an agarose gel, but with the same mass percentage of NaCl instead of the choline-containing gel. During use, the anodic sweat-inducing electrode and the cathodic sweat-inducing electrode form a circuit. The anodic sweat-inducing electrode is modified with the anodic gel containing the choline-containing gel. Under electroosmotic action, the choline-containing gel enters the skin and stimulates sweat secretion.

[0020] The sensing area of ​​the first working electrode is modified with gold nanoparticles and a polyaniline film. A layer of gold nanoparticles is firstly electrodeposited on the sensing area of ​​the first working electrode, and then a layer of polyaniline film is modified in an aniline solution using cyclic voltammetry.

[0021] The sensing area of ​​the second working electrode is firstly electro-deposited with a layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) in a solution of 3,4-ethylenedioxythiophene and sodium polystyrene sulfonate, and then a layer of K + Selective membrane. + Valinomycin K + Selective membrane.

[0022] The reference electrode's sensing area is modified by first depositing a layer of silver, then adding a ferric chloride solution, followed by a methanol solution containing polyvinyl butyral and NaCl. The reference electrode provides a stable standard potential for the first and second working electrodes during detection, maintaining the stability of the detection system.

[0023] The present invention further provides a method for preparing the wearable patch for animal heat stress detection, comprising:

[0024] (1) Forming individual electrodes on a flexible substrate by laser-induced cutting;

[0025] (2) Functionally modify each electrode to form electrodes with different functions.

[0026] The anode gel modified on the sensing area of ​​the anode sweat induction electrode is an agarose gel added with a choline-like substance, wherein the mass percentage of the choline-like substance is 0.1% to 10%; the cathode gel modified on the sensing area of ​​the cathode sweat induction electrode is also an agarose gel, but the choline-like substance is replaced by NaCl at the same mass percentage;

[0027] The sensing area of ​​the first working electrode is modified with gold nanoparticles and a polyaniline film. A layer of gold nanoparticles is first electrodeposited on the sensing area of ​​the first working electrode, and then a layer of polyaniline film is modified using cyclic voltammetry in an aniline solution.

[0028] The sensing area of ​​the second working electrode is firstly electro-deposited with a layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) in a solution of 3,4-ethylenedioxythiophene and sodium polystyrene sulfonate, and then a layer of K + Selective membrane.

[0029] The present invention also provides a method for monitoring animal heat stress, comprising the following steps:

[0030] (1) attaching the wearable patch for animal heat stress detection to the skin of the animal to be detected, and connecting each electrode to a portable electrochemical workstation;

[0031] (2) Detect and record the pH and K of the sweat of the animal to be tested through each electrode + concentration and temperature;

[0032] (3) According to the pH and K + The concentration and temperature conditions are used to determine the heat stress state of the animals to be tested.

[0033] The animals to be tested need to be animals that can sweat through their skin, such as horses, pigs, sheep, cattle, etc.

[0034] During the test, the pH detection range is 4 to 8; K+ The concentration ranges from 1 to 32 mM. Temperature detection can be performed within the normal temperature range of animal sweat. The detection time can be short, such as a few minutes, or long, such as several hours.

[0035] For example, when testing horses, record the pH and K of horse sweat within 25 minutes. + The heat stress state of the horse can be judged by the changes in temperature and temperature. + When the concentration is greater than 3mM, there is no risk of heat stress in animals; when the temperature obtained by the test exceeds 40.5℃ and the pH is greater than 4 or K + When the concentration is greater than 3 mM, the animals are at risk of mild heat stress; when the monitored temperature exceeds 40.5 °C and the pH is less than 4 and K + When the concentration is less than 3mM, animals are at risk of severe heat stress and need to be cooled or supplemented with nutrients.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) Based on the principle of electrochemical wearable sensors, this invention constructs a wearable patch that can be used to continuously monitor multiple substances in animal sweat. The sensor is connected to an electrochemical measuring instrument via a wire, and sweat-inducing electrodes are used to stimulate animal sweat secretion, achieving in situ, real-time, non-invasive, and continuous metabolite and temperature detection.

[0038] (2) The sensor of the present invention integrates a laser-induced graphene sensing electrode array, sweat induction, and ion sensing modules. The integrated system can simultaneously detect pH, K, and + and temperature with high accuracy, stability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG1 is a schematic diagram of electrodes of a wearable patch for animal heat stress detection according to the present invention.

[0040] Figure 2 shows the open circuit potential response curve and standard curve of the sensor. Figure 2(a) shows the open circuit potential response curve of the sensor at pH 4 to 8; Figure 2(b) shows the open circuit potential response standard curve of the sensor at pH 4 to 8; Figure 2(c) shows the open circuit potential response standard curve of the sensor at K + The open circuit potential response curve of the sensor at a concentration of 1 to 32 mM; Figure 2(d) shows the open circuit potential response curve of the sensor at K + Figure 2(e) is the standard curve of the open circuit potential response at a concentration of 1 to 32 mM; Figure 2(f) is the standard curve of the resistance response of the sensor at a temperature of 25 to 40°C.

[0041] Figure 3 is a graph showing the pH and K levels of animal sweat. + Schematic diagram of the sensing system for temperature.

[0042] Figure numerals: 1. flexible substrate; 2. sweat inducing electrode; 3. temperature sensing electrode; 4. first working electrode; 5. reference electrode; 6. second working electrode. DETAILED DESCRIPTION

[0043] Example 1

[0044] Step 1: Design of a sensor (a wearable patch for animal heat stress detection according to the present invention):

[0045] The electrode shapes were designed using computer graphics software (CorelDRAW). The designed electrodes were evenly spaced and distributed on the surface of a polyimide (PI) flexible substrate 1. The electrodes included a sweat-inducing electrode 2, a temperature-sensing electrode 3, a first working electrode 4, a reference electrode 5, and a second working electrode 6. The electrode patch was designed to be square with a side length of 4 cm.

[0046] The sweat-inducing electrode 2 is used to induce sweat secretion from the skin of the animal in contact. It includes an anode sweat-inducing electrode and a cathode sweat-inducing electrode. Under the action of current, a current loop is formed between the anode sweat-inducing electrode and the cathode sweat-inducing electrode. The anode sweat-inducing electrode is modified with an anode gel containing acetylcholine. Under the action of electroosmosis, acetylcholine enters the skin to stimulate sweat secretion. The sweat-inducing electrode 2 needs to release acetylcholine to stimulate the animal's skin to sweat, so the entire sensing area is relatively large. The sensing areas of the anode sweat-inducing electrode and the cathode sweat-inducing electrode are on the periphery, enclosing the sensing areas of the other electrodes, making it easier for the other electrodes to detect sweat after stimulating the animal's skin to sweat.

[0047] The temperature sensing electrode is used to detect the temperature of sweat. The first working electrode is a pH detection electrode for detecting the pH of sweat. The second working electrode is used to detect the K in sweat. + Concentration of K + The reference electrode is a reference electrode for the first working electrode and the second working electrode.

[0048] The sensing area of ​​each electrode is used to sense the corresponding indicators, and the tail end is used to connect to the external signal detection circuit, that is, to connect to the electrochemical workstation during detection. The part between the sensing area and the area of ​​the tail end used to connect to the electrochemical workstation is the conductive connection area serving as the non-working area.

[0049] The sweat-inducing electrode 2, temperature-sensing electrode 3, first working electrode 4, reference electrode 5, and the other end (tail end) of the second working electrode 6 are evenly spaced along the length of the other side of the flexible substrate. The first working electrode 4, reference electrode 5, and second working electrode 6 maintain the same working area. The conductive connection area, which serves as the non-working area, i.e., the area outlined by the dotted line in Figure 1, is insulated. Insulating polyimide tape is used to insulate the conductive portion of the non-working area of ​​the electrodes.

[0050] Step 2: Production of flexible patch:

[0051] A flexible substrate 1 (PI film) was fixed to a copper plate and placed in a laser cutter. Laser-induced graphene (LEG)-based sweat-inducing electrode 2, temperature sensing electrode 3, first working electrode 4, reference electrode 5, and second working electrode 6 were cut on the PI film surface using a laser-induced cutting machine. The laser cutting power, speed, and dots per inch (DPI) were 8%, 15%, and 800, respectively. Insulating polyimide tape was then applied to the non-active areas of the sweat-inducing electrode 2, temperature sensing electrode 3, first working electrode 4, reference electrode 5, and second working electrode 6 for insulation.

[0052] Step 3: Functional modification of electrodes:

[0053] (1) The two sweat-inducing electrodes 2 prepared in step 2 have two sensing areas. The left side of Figure 1 is the anode sweat-inducing electrode, and the right side is the cathode sweat-inducing electrode. The electrodes are modified with anode gel and cathode gel, respectively. When in use, the anode sweat-inducing electrode and the cathode sweat-inducing electrode form a circuit. The anode sweat-inducing electrode is modified with anode gel containing cholinergics. Under the action of electroosmosis, cholinergics enter the skin to stimulate sweat secretion.

[0054] To prepare the anodic gel, a 1% (w / w) agarose solution is first prepared and heated to 250°C with continuous stirring. Once the mixture is completely dissolved, the solution is cooled to 150°C, and then 1% (w / w) acetylcholine is added to the above compounds. The cooled mixture is then slowly poured into a prefabricated mold until it completely solidifies at 4°C. The cathodic gel is prepared similarly to the anodic gel, but 1% (w / w) NaCl is used instead of acetylcholine to form the sweat-inducing electrode 2.

[0055] (2) On the sensing area of ​​the first working electrode 4 prepared in step 2, gold nanoparticles were first electrochemically deposited in a 10 mM chloroauric acid solution using linear sweep voltammetry on an electrochemical workstation. The deposition potential and time were set to -0.8 V and 60 s, respectively. Next, a polyaniline film was modified using cyclic voltammetry in a 0.1 M aniline solution. The cycle voltage and number of cycles were set to -0.2 to 1.0 V and 12 cycles, respectively, to form the first working electrode 4 for pH sensing. After modification of the gold nanoparticles and polyaniline film, the pH value of the detected system was determined. + It can pass through the polyaniline film to cause potential changes, thereby realizing pH measurement.

[0056] (3) Silver was deposited in a 0.5M AgNO3, 1M Na2S2O3, and 0.5M NaHSO3 solution on the surface of the sensing area of ​​the reference electrode 5 prepared in step 2, and then a 0.1M ferric chloride solution was added dropwise. Subsequently, 60mg of polyvinyl butyral and 60mg of NaCl were dissolved in 1mL of methanol with vigorous stirring. 6μL of the prepared reference solution was added dropwise to the electrode and dried overnight to obtain a homemade reference electrode 5. The reference electrode provides a stable standard potential, maintaining the stability of the detection system, and serves as the reference electrode for detection by the first working electrode and the second working electrode.

[0057] (4) In the sensing area of ​​the second working electrode 6 prepared in step 2, a constant current of 20 μA was applied and maintained for 10 min in a solution containing 0.05 M 3,4-ethylenedioxythiophene and 0.5 M sodium polystyrene sulfonate to thereby electrodeposit poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS). Then, 2% (w / w) valinomycin, 64.7% (w / w) dioctyl sebacate, 32.7% (w / w) polyvinyl chloride, and 0.6% (w / w) sodium tetraphenylborate were mixed to prepare K + Then dissolve the selective membrane in 400 μL cyclohexanone and drop 6 μL on the electrode to dry, thus forming a membrane for K + The second working electrode 6 for sensing.

[0058] In the modification of poly (3, 4-ethylenedioxythiophene) -poly (styrene sulfonic acid) and K + Selective membrane for K + Sensing. K in the detected system + Can pass through K + The selective membrane causes a potential change, thereby achieving K + Determination.

[0059] (5) The temperature sensing electrode 3 is a temperature electrode used to detect the temperature of sweat. The temperature sensing electrode can be used for temperature sensing without modification. When the temperature changes, the carbon atom arrangement on the graphene electrode changes, and the electrode resistance changes accordingly, thereby achieving temperature sensing.

[0060] Example 2

[0061] Example 2 of the present invention provides a method for detecting the performance of the sensor prepared in Example 1:

[0062] The electrodes obtained in Example 1 were connected to an electrochemical workstation. Specifically, the sweat induction electrode 2, the temperature sensing electrode 3 and the first working electrode 4 on the patch were connected to the working electrode clamp, and the reference electrode 5 was connected to the reference electrode clamp. The open circuit potential method was used to measure the pH and K at different levels. + Conduct analysis.

[0063] As shown in Figure 2(a) and Figure 2(b), a good linear relationship is shown in the pH range of 4 to 8. As shown in Figure 2(c) and Figure 2(d), in the K + The open circuit voltage of the sensor changes with the K + Figure 2(e) and Figure 2(f) show that the sensing shows a good linear relationship in the temperature range of 25 to 40°C.

[0064] Example 3

[0065] As shown in Figure 3, in an actual application scenario, horses that are prone to heat stress reactions were selected as an example for monitoring in an animal farm and connected to a portable electrochemical workstation. The pH and K in the horse sweat were recorded within 25 minutes. + The heat stress status of the horse can be judged by the changes in temperature and humidity. When the temperature is less than 40.5℃, the pH is greater than 4 and the K+ concentration is greater than 3mM, the animal is not at risk of heat stress; when the temperature is greater than 40.5℃, the pH is greater than 4 or the K+ concentration is greater than 3mM, the animal is not at risk of heat stress. + When the concentration is greater than 3 mM, the animals are at risk of mild heat stress; when the monitored temperature exceeds 40.5 °C and the pH is less than 4 and K + At concentrations below 3mM, animals are at risk of severe heat stress and require cooling or nutritional supplementation. Therefore, the resulting sensor can provide timely and personalized monitoring of animal heat stress, enabling precise animal health management.

Claims

1. A wearable patch for animal heat stress detection, comprising a flexible substrate and electrodes designed on the flexible substrate, each electrode comprising a sensing area as a working area and a conductive connection area as a non-working area, characterized in that: The electrode comprises: A sweat inducing electrode, used to induce sweat secretion from the skin of an animal in contact, comprising an anode sweat inducing electrode and a cathode sweat inducing electrode, wherein a current loop is formed between the anode sweat inducing electrode and the cathode sweat inducing electrode under the action of electric current; the anode sweat inducing electrode is modified with an anode gel containing choline, and under the action of electroosmosis, choline enters the skin to stimulate sweat secretion; Temperature sensing electrodes for detecting sweat temperature; The first working electrode is a pH detection electrode for detecting the pH of sweat; The second working electrode is used to detect K in sweat. + Concentration of K + Detection electrodes; The reference electrode is a reference electrode used for the first working electrode and the second working electrode.

2. The wearable patch for animal heat stress detection according to claim 1, characterized in that: The surface of the conductive connection area serving as a non-working area is insulated, and the end portion is used for an external signal detection circuit.

3. The wearable patch for animal heat stress detection according to claim 1, characterized in that: The flexible substrate is made of polyimide, and electrodes with laser-induced graphene are prepared on the surface of the polyimide flexible substrate by laser-induced cutting.

4. The wearable patch for animal heat stress detection according to claim 3, characterized in that: The power, speed and dots per inch of the laser induced cutting are 5% to 10%, 10% to 20% and 500 to 800 respectively.

5. The wearable patch for animal heat stress detection according to claim 1, characterized in that: The anode gel modified on the sensing area of ​​the anode sweat induction electrode is an agarose gel added with choline, wherein the mass percentage of choline is 0.1% to 10%; the cathode gel modified on the sensing area of ​​the cathode sweat induction electrode is also an agarose gel, but the same mass percentage of NaCl is used to replace the choline; The sensing region of the first working electrode is modified with gold nanoparticles and a polyaniline film, a layer of gold nanoparticles is firstly electrodeposited on the sensing region of the first working electrode, and then a layer of polyaniline film is modified by cyclic voltammetry in an aniline solution; The sensing area of ​​the second working electrode is firstly coated with 3,4-ethylenedioxythiophene and polystyrene sulfonic acid. A layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) was electrodeposited in sodium solution, and then a layer of K + Selective membrane.

6. The wearable patch for animal heat stress detection according to claim 5, characterized in that: The K + Valinomycin K + Selective membrane.

7. The wearable patch for animal heat stress detection according to claim 1, characterized in that: The cholinergic is at least one of acetylcholine, methacholine, carbachol and bethanechol.

8. The wearable patch for animal heat stress detection according to claim 1, characterized in that: When modifying the sensing area of ​​the reference electrode, a layer of silver is first deposited, then a ferric chloride solution is added dropwise, and then a methanol solution containing polyvinyl butyral and NaCl is added dropwise.

9. The method for preparing the wearable patch for animal heat stress detection according to any one of claims 1 to 8, characterized in that: The preparation method comprises: (1) Forming individual electrodes on a flexible substrate by laser-induced cutting; (2) Modify the functions of each electrode to form electrodes with different functions.

10. A method for monitoring animal heat stress, characterized in that: The following steps are involved: (1) Attaching the wearable patch for animal heat stress detection according to any one of claims 1 to 8 to the skin of an animal to be detected, and connecting each electrode to a portable electrochemical workstation; (2) Detect and record the pH and K of the sweat of the animal to be tested through each electrode + concentration and temperature; (3) According to the pH and K + The concentration and temperature conditions are used to determine the heat stress state of the animals being tested.

Images