Wearable microneedle ear tag for animal heat stress management

Abstract

The invention provides a wearable microneedle ear tag for animal heat stress management, the wearable microneedle ear tag comprises a female nail and a main body, the female nail comprises a detection disc, and the detection disc is provided with a microneedle electrode. The microneedle electrodes comprise a first working microneedle electrode, a second working microneedle electrode, a third working microneedle electrode, a reference microneedle electrode and a temperature sensing microneedle electrode. The temperature, pH, K + concentration and Ca < 2 + > concentration level of animal ear interstitial fluid can be monitored at the same time, and the physiological status of animals under the heat stress condition is comprehensively evaluated. According to the microneedle technology, the sensor can be more flexibly applied to animals of different types and sizes, wider application possibility is provided, the design of the microneedle is relatively small and light, invasive interference to the animals is reduced, stress to the animals is reduced, and the microneedle sensor is suitable for being worn by the animals for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of animal intelligent detection, and in particular to a wearable microneedle ear tag for animal heat stress management. Background Art

[0002] Animal heat stress refers to a series of physiological and psychological reactions caused by imbalanced temperature regulation in animals under high temperature conditions. This stress state has a negative impact on the health and production performance of animals, especially in the breeding industry. High temperatures can lead to decreased appetite, metabolic disorders, and reduced immunity in animals, thereby increasing the risk of disease and mortality. In addition, heat stress can also affect the reproductive capacity of animals, leading to decreased reproductive performance and even affecting the quality of products such as meat and milk. Effective management of heat stress is crucial, which not only helps to improve the quality of life of animals, but also significantly improves the efficiency of breeding. Therefore, paying attention to the management of animal heat stress is not only a responsibility for animal welfare, but also a necessary means to improve economic benefits and ensure food safety. Through scientific management and intervention measures, breeders can significantly reduce the occurrence of animal heat stress, improve production efficiency, and promote sustainable breeding development. In addition to body temperature, physiological and biochemical parameters of animal body fluids are also one of the indicators for achieving precise management of heat stress.

[0003] Animal ear tags play an indispensable role in modern animal husbandry, mainly used for identification, data management and health monitoring. With the expansion of breeding scale and the improvement of management requirements, traditional animal identification methods have gradually failed to meet the needs of modern breeding. The introduction of ear tags not only simplifies the animal management process, but also greatly improves breeding efficiency. Each ear tag usually contains a unique identification number or barcode, through which breeders can quickly obtain basic information about animals, including date of birth, health records, vaccination status and production performance.

[0004] Microneedle technology is a painless, minimally invasive sampling method that can extract body fluid samples, such as interstitial fluid, without causing significant harm to the animal. For most farmed animals, their sweat glands are underdeveloped, so sweat cannot be analyzed. As an important physiological body fluid, interstitial fluid can reflect the overall health status of animals, including water, electrolyte balance, metabolites and other indicators.

[0005] Currently, there is no equipment on the market that uses wearable microneedle ear tags for precise management of animal heat stress. In the existing use of animal ear tags to monitor animal body temperature, the invention application with publication number CN115428745A discloses a method of using thermochromic materials to monitor the body temperature of livestock, so as to detect and treat livestock plagues in the early stages. The color of the livestock thermochromic ear tag changes with the temperature of the animal to achieve body temperature monitoring. The ear tag is low in cost and can be used to monitor temperature using a low-cost camera. However, the sensor has low detection accuracy and cannot achieve real-time analysis of multiple biochemical indicators.

[0006] The invention application with the publication number CN108617533A discloses a livestock monitoring method, device and ear tag monitoring system based on ear tags, firstly obtaining the body temperature information collected by the temperature sensor and the movement information collected by the posture sensor; the temperature sensor and the posture sensor are arranged in the electronic ear tag; according to the above body temperature information and the movement information, the health analysis results corresponding to the corresponding livestock individuals are obtained; wherein the individual health information includes body temperature information, posture information, movement amount, movement amplitude and frequency. The temperature sensor and the posture sensor in the electronic ear tag detect the state of the livestock individuals from the two aspects of the physiology and movement of the livestock individuals, and the monitoring results are objective, quantitative and accurate, but the physiological and biochemical parameters of the livestock individuals are not detected. Summary of the invention

[0007] In order to solve the above technical problems existing in the prior art, the present invention provides a wearable microneedle ear tag for animal heat stress management, which can simultaneously monitor the temperature, pH, K of the interstitial fluid in the ear of the animal. + Concentration and Ca 2+ concentration levels to comprehensively assess the physiological status of animals under heat stress conditions.

[0008] The present invention provides a wearable microneedle ear tag for animal heat stress management, comprising a mother nail and a main body, wherein the main body comprises a label and a mounting hole arranged at the upper end of the label, the mother nail comprises a support column passing through the mounting hole when in use, a nail head with a pointed tip is provided at one end of the support column, and a detection disk is provided at the other end of the support column. When in use, the support column passes through the animal's ear, and the detection disk cooperates with the label to clamp the animal's ear with a side facing the main body.

[0009] The detection disk is provided with microneedle electrodes on one side facing the main body. The microneedle electrodes include a first working microneedle electrode, a second working microneedle electrode, a third working microneedle electrode, a reference microneedle electrode, and a temperature sensing microneedle electrode; the first working microneedle electrode is used to detect the pH of the interstitial fluid of the animal's ear, and the second working microneedle electrode is used to detect the K of the interstitial fluid of the animal's ear. +The third working microneedle electrode is used to detect Ca concentration in the interstitial fluid of the animal ear. 2+ concentration, the reference microneedle electrode is a reference electrode for the first working microneedle electrode, the second working microneedle electrode and the third working microneedle electrode, which maintains the potential of the first working electrode, the second working electrode and the third working electrode stable during sensing, thereby preventing interference from other ions; the temperature sensing microneedle electrode is used to detect temperature.

[0010] The support column is provided with a conductive column extending from each of the microneedle electrodes, and the interior of the mounting hole is provided with a positioning conductive groove corresponding to the conductive column one by one, and the side wall of the positioning conductive groove is provided with a conductive layer. When in use, the positioning conductive groove is matched and connected with the conductive column, so that the nameplate is stuck on the mother nail and will not rotate to cause the circuit between the nameplate and the mother nail to be disconnected.

[0011] The sign is provided with a circuit board for processing and transmitting detection data, and the circuit board is connected to the conductive layers of the positioning conductive grooves through wires. The circuit board includes a front-end analog circuit, a conversion circuit and a microcontroller circuit, and can realize real-time data transmission.

[0012] Preferably, each of the microneedle electrodes comprises a microneedle array composed of a plurality of microneedles, each microneedle is conical in shape and is used to penetrate the skin of an animal ear. The microneedle array works through multiple microneedles and can cover a larger area, while collecting signals at multiple points to achieve higher data collection efficiency and accuracy.

[0013] Further preferably, the bottom radius of the microneedle is 0.25-1 mm, the height is 1-2 mm, and the interval between adjacent microneedles is 0.5-2 mm.

[0014] Further preferably, the detection plate includes a connecting ring surrounding one end of the support column, and a plurality of connecting columns extending from the end of the support column to the connecting ring; each of the microneedle electrodes is arranged at a different position on the connecting column and / or the connecting ring.

[0015] Further preferably, the first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference microneedle electrode are respectively located on a connecting column; the temperature sensing microneedle electrode is located at the part where two connecting columns and the connecting ring are located between the two connecting columns.

[0016] Preferably, the side wall of the mounting hole extends toward the side of the sign facing away from the detection plate to form a column, and the mounting hole is arranged in the middle of the column. When in use, the column is used to fix the sign.

[0017] Preferably, one end of the support column is provided with a nail column for preventing the female nail from falling out of the mounting hole when in use, and the nail head is provided on the end surface of the nail column, and the diameter of the nail column is the same as the diameter of the base of the nail head. When in use, the female nail head and the nail column pass through one side of the animal's ear, and then the sign is inserted into the support column on the other side of the animal's ear. The material at the mounting hole of the sign can be slightly deformed to allow the nail head and the nail column to pass through. After the nail head and the nail column pass through the mounting hole, the material at the mounting hole of the sign resumes deformation. At this time, the diameter of the nail column is larger than the diameter of the mounting hole, so that the sign is stuck between the nail column and the animal's ear, and the female nail is not easy to fall out of the mounting hole of the sign.

[0018] Preferably, the support column is provided with an operation hole on the end surface of one end provided with the detection disk, and the operation hole extends axially inwardly of the support column. The provision of the operation hole facilitates the ear tag clamp to clamp the ear tag.

[0019] Preferably, when preparing the first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference microneedle electrode, a conductive metal layer is first sprayed on the surface of the corresponding area of ​​the mother nail, and then a layer of gold nanoparticles is deposited on the surface of the conductive metal layer, and the conductive metal layer is selected from gold or silver. Then, the areas where the microneedle electrodes are located are respectively subjected to the following treatments:

[0020] Depositing a layer of polyaniline film in the area where the first working microneedle electrode is located, and obtaining the first working microneedle electrode after drying;

[0021] A layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) (PEDOT:PSS, CAS: 155090-83-8) was deposited in the area where the second working microneedle electrode was located, and then a layer of K + selective membrane, to obtain the second working microneedle electrode;

[0022] A layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) was deposited in the area where the third working microneedle electrode was located, and then a layer of Ca 2+ selective membrane, to obtain the third working microneedle electrode;

[0023] A layer of Ag / AgCl slurry is coated on the area where the reference microneedle electrode is located, and then a methanol solution containing polyvinyl butyral and NaCl is added dropwise, and the reference microneedle electrode is obtained after drying;

[0024] The temperature sensing microneedle electrode is prepared by coating a layer of carbon nanotubes on the surface of the corresponding area of ​​the mother nail;

[0025] The conductive column is prepared by spraying a conductive metal layer on the surface of the corresponding area of ​​the mother nail;

[0026] A conductive film is pasted on the side wall of the positioning conductive groove as a conductive layer, and the conductive film is preferably a conductive copper tape.

[0027] Further preferably, when depositing gold nanoparticles, the electrodeposition of gold nanoparticles is carried out in a 5 mM chloroauric acid solution using linear sweep voltammetry on an electrochemical workstation, and the deposition potential and time are set to -0.8 V and 120 s, respectively.

[0028] When the polyaniline film is deposited, the polyaniline film is modified in the first working microneedle electrode region by cyclic voltammetry in a 0.1 M aniline solution, and the cycle voltage and the number of cycles are set to -0.2 to 1.0 V and 10 cycles, respectively.

[0029] When depositing poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), a constant current of 20 μA was applied and maintained for 10 minutes in a solution containing 0.05 M 3,4-ethylenedioxythiophene (CAS: 126213-50-1) and 0.5 M sodium polystyrenesulfonate (CAS: 25704-18-1), thereby electrodepositing poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid).

[0030] Coating K + When the selective membrane is used, 2 wt% of valinomycin, 64.7 wt% of diisooctyl sebacate (CAS: 122-62-3), 32.7 wt% of polyvinyl chloride and 0.6 wt% of sodium tetraphenylborate are mixed to prepare K + Selective membrane; prepare K + The selective membrane was dissolved in 400 μL of cyclohexanone to obtain K + Selective membrane solution, K + The selective membrane solution is coated on the surface of the second working microneedle electrode area and then dried.

[0031] Coating Ca 2+ When the selective membrane is used, 0.46 wt% of calcium ion carrier ETH129, 66.04 wt% of 2-nitrophenyl octyl ether, 33.02 wt% of polyvinyl chloride and 0.48 wt% of sodium tetraphenylborate are mixed to prepare Ca 2+ Selective membrane; prepare Ca 2+ The selective membrane was dissolved in 1 mL of tetrahydrofuran to obtain Ca 2+ Selective membrane solution, Ca 2+ The selective membrane solution is coated on the surface of the third working microneedle electrode area and then dried.

[0032] The microneedle ear tag of the present invention can be used for farmed animals such as pigs, cattle, and sheep.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Multi-parameter monitoring: The wearable microneedle ear tag for animal heat stress management of the present invention can simultaneously measure the temperature, pH, K of the interstitial fluid in the animal's ear. + Concentration and Ca 2+ The concentration level can comprehensively evaluate the physiological state of animals under heat stress conditions.

[0035] (2) Real-time data collection: The wearable microneedle ear tag for animal heat stress management of the present invention can collect detection data in real time, provide dynamic monitoring, help timely identify the heat stress state of animals and take corresponding intervention measures.

[0036] (3) Improving animal welfare: By timely monitoring and managing animal heat stress, the animal’s stress level can be effectively reduced, its welfare and production performance can be improved, and health problems caused by high temperatures can be reduced.

[0037] (4) Flexibility and adaptability: Microneedle technology allows sensors to be more flexibly applied to animals of different types and sizes, providing a wider range of application possibilities.

[0038] (5) Reduced invasiveness: Microneedles are designed to be relatively small and lightweight, which reduces invasive interference to animals and reduces stress on animals, making them suitable for long-term wear by animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a schematic diagram of the three-dimensional structure of the microneedle ear tag female nail of the present invention when it passes through the main body.

[0040] Figure 2 Schematic diagram of the exploded structure of the microneedle ear tag of the present invention.

[0041] Figure 3 It is a schematic diagram of the three-dimensional structure of the microneedle ear tag mother nail of the present invention.

[0042] Figure 4 It is a schematic diagram of the rear view structure of the microneedle ear tag female nail of the present invention.

[0043] Figure 5 It is a schematic diagram of the front view structure of the microneedle ear tag mother nail of the present invention.

[0044] Figure 6 for Figure 5 Cross-sectional view along AA direction.

[0045] Figure 7 It is a schematic diagram of the three-dimensional structure of the microneedle ear tag body of the present invention.

[0046] Figure 8 Schematic diagram of the three-dimensional structure of the microneedle ear tag body of the present invention from another viewing angle.

[0047] Fig. 9It is a schematic diagram of the structure of the circuit board in the microneedle ear tag of the present invention.

[0048] Fig.10 It is the open circuit potential response curve and standard curve of the sensor. Fig.10 (a) is the open circuit potential response curve of the sensor at pH 4 to 8; Fig.10 (b) is the standard curve of the open circuit potential response of the sensor at pH 4 to 8; Fig.10 (c) is the sensor at K + Open circuit potential response curves at concentrations of 1 to 32 mM; Fig.10 (d) is the sensor at K + Open circuit potential response standard curve graph with concentration ranging from 1 to 32 mM; Fig.10 (e) The sensor is in Ca 2+ Open circuit potential response curves for concentrations ranging from 0.5 to 16 mM; Fig.10 (f) The sensor is in Ca 2+ Standard curve of open circuit potential response with concentration ranging from 0.5 to 16 mM.

[0049] Fig.11 is the sensor's resistance response curve and standard curve. Fig.11 (a) is the resistance response curve of the sensor at a temperature of 25-40°C; Fig.11 (b) is the standard curve of the sensor's resistance response at temperatures between 25 and 40°C.

[0050] Reference numerals:

[0051] Mother nail 1, main body 2, nail head 3, nail column 4, support column 5, microneedle electrode 6, conductive column 7, connecting column 8, operation hole 9, first working microneedle electrode 10, second working microneedle electrode 11, third working microneedle electrode 12, reference microneedle electrode 13, temperature sensing microneedle electrode 14, column 15, sign 16, positioning conductive groove 17, wire 18, detection disk 19, connecting ring 20, mounting hole 21, circuit board 22. DETAILED DESCRIPTION

[0052] Example 1

[0053] like Figure 1-Figure 9 As shown, this embodiment provides a wearable microneedle ear tag for animal heat stress management, including a female nail 1 and a main body 2, the main body 2 includes a label 16 and a mounting hole 21 provided at the upper end of the label 16, the female nail 1 includes a support column 5 passing through the mounting hole 21 when in use, a nail head 3 with a sharp tip is provided at one end of the support column 5, and a detection disk 19 is provided at the other end of the support column 5. The side wall of the mounting hole 21 extends toward the side of the label 16 facing away from the detection disk 19 to form a column 15, and the middle of the column 15 is provided with a mounting hole 21.

[0054] The detection disk 19 is provided with a microneedle electrode 6 on the side facing the main body 2. The microneedle electrode 6 includes a first working microneedle electrode 10, a second working microneedle electrode 11, a third working microneedle electrode 12, a reference microneedle electrode 13, and a temperature sensing microneedle electrode 14; the first working microneedle electrode 10 is used to detect the pH of the interstitial fluid of the animal's ear, and the second working microneedle electrode 11 is used to detect the K of the interstitial fluid of the animal's ear. + The third working microneedle electrode 12 is used to detect the Ca concentration in the interstitial fluid of the animal ear. 2+ The reference microneedle electrode 13 is a reference electrode for the first working microneedle electrode 10, the second working microneedle electrode 11 and the third working microneedle electrode 12. During sensing, the potential of the first working microneedle electrode 10, the second working microneedle electrode 11 and the third working microneedle electrode 12 is maintained stable to prevent interference from other ions. The temperature sensing microneedle electrode 14 is used to detect temperature.

[0055] Each microneedle electrode 6 includes a microneedle array composed of a plurality of microneedles, each of which is conical in shape and is used to penetrate the skin of the animal's ear. The radius of the bottom surface of the microneedle is 0.25-1 mm, the height is 1-2 mm, and the interval between adjacent microneedles is 0.5-2 mm. The microneedle array can cover a larger area by working with multiple microneedles, and simultaneously collect signals at multiple points, thereby achieving higher data collection efficiency and accuracy.

[0056] The detection plate 19 further includes a connecting ring 20 surrounding one end of the support column 5, and a plurality of connecting columns 8 extending from the end of the support column 5 to the connecting ring 20. Each microneedle electrode 6 is arranged at a different position on the connecting column 8 and / or the connecting ring 20. The first working microneedle electrode 10, the second working microneedle electrode 11, the third working microneedle electrode 12 and the reference microneedle electrode 13 are respectively located on one connecting column 8; the temperature sensing microneedle electrode 14 is located at the two connecting columns 8 and the portion of the connecting ring 20 located between the two connecting columns 8.

[0057] The supporting column 5 is provided with a conductive column 7 extending from each microneedle electrode 6 , and the mounting hole 21 is provided with a positioning conductive groove 17 corresponding to the conductive column 7 , and the side wall of the positioning conductive groove 17 is provided with a conductive layer.

[0058] The support column 5 is provided with an operation hole 9 on the end surface of one end provided with the detection disk 19, which extends axially inwardly of the support column 5. The operation hole 9 is provided to facilitate the ear tag clamp to clamp the ear tag.

[0059] The support column 5 is provided with a nail column 4 at the end opposite to the detection plate 19 for preventing the female nail 1 from falling out of the mounting hole 21 during use. A nail head 3 is provided on the end face of the nail column 4, and the diameter of the nail column 4 is the same as the diameter of the base of the nail head 3.

[0060] The sign 16 is provided with a circuit board 22 for processing and transmitting detection data, and the circuit board 22 is connected to the conductive layer of each positioning conductive groove 17 through a wire 18. The circuit board 22 includes a front-end analog circuit, a conversion circuit and a microcontroller circuit, which can realize real-time data transmission.

[0061] The preparation of the conductive column, the first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference microneedle electrode comprises the following steps:

[0062] (1) An insulating polyimide tape is applied to the mother pin except for the conductive column, the first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference electrode, and a gold layer is sprayed on the mother pin after the mask processing by magnetron sputtering.

[0063] (2) The areas where the first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference electrode are located are subjected to electrodeposition of gold nanoparticles in a 5 mM chloroauric acid solution using linear sweep voltammetry on an electrochemical workstation, with the deposition potential and time being set to -0.8 V and 120 s, respectively.

[0064] (3) The polyaniline film was modified in the area where the first working microneedle electrode was located by cyclic voltammetry in a 0.1 M aniline solution. The cycle voltage and the number of cycles were set to -0.2 to 1.0 V and 10 cycles, respectively. After drying, the first working microneedle electrode for pH sensing was obtained. + The polyaniline film of the first working microneedle electrode can be passed through to cause a potential change, thereby realizing pH measurement.

[0065] (4) Electrodepositing poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid) (PEDOT:PSS, CAS: 155090-83-8) in the area where the second working microneedle electrode is located is achieved by applying a 20 μA constant current in a solution containing 0.05 M 3,4-ethylenedioxythiophene and 0.5 M sodium polystyrenesulfonate and maintaining it for 10 min. Then, 2 wt % of valinomycin, 64.7 wt % of diisooctyl sebacate, 32.7 wt % of polyvinyl chloride and 0.6 wt % of sodium tetraphenylborate are mixed to prepare K + Selective membrane. + The selective membrane was dissolved in 400 μL of cyclohexanone to obtain K + Selective membrane solution, K + The selective membrane solution is coated on the area where the second working microneedle electrode is located and then dried to obtain the second working microneedle electrode. + K that can pass through the second working microneedle electrode + Selective membrane, causing potential changes, thereby achieving K + Concentration determination.

[0066] (5) Electrodepositing poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) in the region where the third working microneedle electrode is located is achieved by applying a 20 μA constant current in a solution containing 0.05 M 3,4-ethylenedioxythiophene and 0.5 M sodium polystyrene sulfonate and maintaining it for 10 min. Then, 0.46 wt% of calcium ion carrier ETH129, 66.04 wt% of 2-nitrophenyl octyl ether, 33.02 wt% of polyvinyl chloride and 0.48 wt% of sodium tetraphenylborate were mixed to prepare Ca 2+ Selective membrane. Then the prepared Ca 2+ The selective membrane was dissolved in 1 mL of tetrahydrofuran to obtain Ca 2+ Selective membrane solution, Ca 2+ The selective membrane solution is coated on the area where the third working microneedle electrode is located and then dried to obtain the third working microneedle electrode. 2+ Can pass through Ca 2+ selective membrane, causing a potential change, thus achieving Ca 2+ Concentration determination.

[0067] (6) Ag / AgCl conductive silver paste was applied to the area where the reference microneedle electrode was located. Then, 60 mg of polyvinyl butyral and 60 mg of NaCl were vigorously stirred and dissolved in 1 mL of methanol to obtain a reference solution. The prepared reference solution was dripped onto the area where the reference microneedle electrode was located and dried overnight to obtain a reference microneedle electrode. The reference microneedle electrode provided a stable standard potential to maintain the stability of the detection system and was used as a reference electrode for the detection of the first working microneedle electrode, the second working microneedle electrode, and the third working microneedle electrode.

[0068] (7) Prepare a 1 mg / mL multi-walled carbon nanotube solution, drop it on the surface of the temperature sensing microneedle electrode, and after drying, obtain a temperature sensing microneedle electrode for temperature sensing. When the temperature changes, the number of electrons and phonons in the carbon nanotubes will change, resulting in a change in resistance.

[0069] In order to make the female pin of the ear tag and the main body conductively contact, a conductive copper tape is pasted on the side wall of the positioning conductive groove as a conductive layer.

[0070] When in use, the nail head and nail post of the female nail pass through one side of the animal's ear, the support post is inserted into the animal's ear, and then the sign is inserted into the support post on the other side of the animal's ear. The material at the mounting hole of the sign may be slightly deformed to allow the nail head and nail post to pass through. After the nail head and nail post pass through the mounting hole, the material at the mounting hole of the sign resumes its deformation. At this time, the diameter of the nail post is larger than the diameter of the mounting hole, so that the sign is stuck between the nail post and the animal's ear, and the female nail is not easy to fall out of the mounting hole of the sign. At this time, the side of the detection disk facing the sign cooperates with the sign to clamp the animal's ear. The positioning conductive grooves and conductive posts in the mounting hole are connected one by one, so that the sign is stuck on the female nail and will not rotate to cause the circuit between the sign and the female nail to be disconnected. The detection disk is attached to the animal's ear, and the microneedle electrodes on the detection disk pierce the skin of the animal's ear to detect the temperature of the animal's ear and the pH and K of the interstitial fluid in the animal's ear. + Concentration and Ca 2+ concentration, and transmit the detection data to the circuit board in real time for processing and output.

[0071] Test Example 1

[0072] The first working microneedle electrode, the second working microneedle electrode, and the third working microneedle electrode obtained in Example 1 were connected to a multi-channel electrochemical workstation to test the sensitivity and response characteristics of the first working microneedle electrode to pH changes and the second working microneedle electrode to K + The sensitivity and response characteristics of the third working microneedle electrode to Ca concentration changes 2+ Sensitivity and response characteristics to concentration changes.

[0073] When testing the sensitivity and response characteristics of the first working microneedle electrode to pH changes, a series of PBS buffer solutions with different pH values ​​(pH=4, pH=5, pH=6, pH=7, pH=8) were first prepared. Then, the first working microneedle electrode was immersed in these PBS buffer solutions with different pH values ​​to test its open circuit potential response for 20 seconds, and the instrument was stopped after 20 seconds. The test results are shown in Figure 2. Fig.10 (a) and Fig.10 As shown in (b), a good linear relationship is shown in the pH range of 4 to 8. Fig.10 In (b), the horizontal axis X is pH, the vertical axis Y is potential, and the fitting formula is Y = -0.0561X + 0.455, with a determination coefficient R 2 is 0.998.

[0074] When testing the second and third working microneedle electrodes, first immerse the microneedle electrodes in 10 mL of an aqueous solution containing 1 mM KCl and 0.5 mM CaCl2, and test the open circuit potential response of the second and third working microneedle electrodes for 20 seconds. After 20 seconds, stop the instrument. Add 10 μL of 1 M KCl aqueous solution and 5 μL of 1 M CaCl2 aqueous solution to the solution. At this time, K in the solution + and Ca 2+ The concentrations of KCl and CaCl2 were 2mM and 1mM respectively. The instrument was kept on for 20s and then stopped. 10μL of 1M KCl aqueous solution and 5μL of 1M CaCl2 aqueous solution were added to the solution. + and Ca 2+ The concentrations of K in the solution were 4mM and 2mM. The instrument was turned on for 20s and then stopped. 10μL of 1M KCl aqueous solution and 5μL of 1M CaCl2 aqueous solution were added to the solution. + and Ca 2+ The concentrations of KCl and CaCl2 were 8mM and 2mM respectively. The instrument was turned on for 20s and then stopped. 10μL of 1M KCl aqueous solution and 5μL of 1M CaCl2 aqueous solution were added to the solution. + and Ca 2+ The concentrations of K in the solution were 16mM and 8mM. The instrument was turned on for 20s and then stopped. 10μL of 1M KCl aqueous solution and 5μL of 1M CaCl2 aqueous solution were added to the solution. + and Ca 2+ The concentrations are 32 mM and 16 mM. The instrument is turned on and maintained for 20 seconds before being stopped.

[0075] The test results are as follows Fig.10 (c) and Fig.10 As shown in (d), in K + In the concentration range of 1 to 32 mM, the open circuit voltage of the sensor changes with K + The concentration increases with the increase, showing a good linear relationship. Fig.10 (d) The horizontal axis X is log 10 [K + ], the ordinate Y is the potential, the fitted formula is Y = 0.0578X + 0.0071, and the determination coefficient R 2 is 0.997. Fig.10 (e) and Fig.10 As shown in (f), in Ca 2+ The open circuit voltage of the sensor changes with the Ca concentration in the range of 0.5 to 16 mM during continuous detection. 2+The concentration increases with the increase, showing a good linear relationship. Fig.10 (f) The horizontal axis X is log 10 [Ca 2+ ], the ordinate Y is the potential, the fitting formula is Y = 0.0284X + 0.0042, the determination coefficient R 2 is 0.997.

[0076] Test Example 2

[0077] Connect the temperature sensing microneedle electrode obtained in Example 1 to a multimeter, immerse the temperature sensing microneedle electrode in a PBS buffer solution at a temperature of 25°C, observe and record the reading of the multimeter within 20 seconds. Then place the solution on a heater, heat the solution to 30°C, and continue to record with a multimeter for 20 seconds, and stop recording after 20 seconds. Continue to heat the solution to 35°C and record with a multimeter for 20 seconds, and stop recording after 20 seconds. Continue to heat the solution to 40°C and record with a multimeter for 20 seconds, and stop recording after 20 seconds. Continue to heat the solution to 45°C and record with a multimeter for 20 seconds, and stop recording after 20 seconds. The test results are as follows: Fig.11 (a) and Fig.11 As shown in (b), in the temperature range of 25-45°C, the temperature sensing microneedle electrode resistance and temperature show a good linear relationship. Fig.11 In (b), the horizontal axis X is temperature, the vertical axis Y is resistance, and the fitting formula is Y = -0.112X + 64.89, and the determination coefficient R 2 It is 0.993.

Claims

1. A wearable microneedle ear tag for animal heat stress management, comprising a female nail and a main body, wherein the main body comprises a label and a mounting hole arranged at the upper end of the label, the female nail comprises a support column passing through the mounting hole when in use, and one end of the support column is provided with a nail head with a sharp tip, characterized in that: The other end of the support column is provided with a detection plate. When in use, the support column passes through the animal's ear, and the detection plate faces the main body and cooperates with the label to clamp the animal's ear. The detection disk is provided with a microneedle electrode on a surface facing the main body; The microneedle electrode comprises a first working microneedle electrode, a second working microneedle electrode, a third working microneedle electrode, a reference microneedle electrode, and a temperature sensing microneedle electrode; the first working microneedle electrode is used to detect the pH of the interstitial fluid of the animal's ear, and the second working microneedle electrode is used to detect the K of the interstitial fluid of the animal's ear. + The third working microneedle electrode is used to detect Ca concentration in the interstitial fluid of the animal ear. 2+ concentration, the reference microneedle electrode is a reference electrode for the first working microneedle electrode, the second working microneedle electrode and the third working microneedle electrode; the temperature sensing microneedle electrode is used to detect temperature; The support column is provided with a conductive column led out from each of the microneedle electrodes, the mounting hole is provided with a positioning conductive groove corresponding to the conductive column one by one, the side wall of the positioning conductive groove is provided with a conductive layer, and when in use, the positioning conductive groove is matched and connected with the conductive column; The label is provided with a circuit board for processing and transmitting detection data, and the circuit board is connected to the conductive layers of the positioning conductive grooves through wires.

2. The wearable microneedle ear tag for animal heat stress management according to claim 1, characterized in that: Each of the microneedle electrodes comprises a microneedle array composed of a plurality of microneedles, and each microneedle is in a conical shape.

3. The wearable microneedle ear tag for animal heat stress management according to claim 2, characterized in that: The bottom radius of the microneedle is 0.25-1 mm, the height is 1-2 mm, and the interval between adjacent microneedles is 0.5-2 mm.

4. The wearable microneedle ear tag for animal heat stress management according to claim 2, characterized in that: The detection plate includes a connecting ring surrounding one end of the supporting column, and a plurality of connecting columns extending from the end of the supporting column to the connecting ring; Each of the microneedle electrodes is arranged at a different position on the connecting column and / or the connecting ring.

5. The wearable microneedle ear tag for animal heat stress management according to claim 4, characterized in that: The first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference microneedle electrode are respectively located on a connecting column; The temperature sensing microneedle electrode is located at the two connecting posts and the connecting ring is located at the part between the two connecting posts.

6. The wearable microneedle ear tag for animal heat stress management according to claim 1, characterized in that: The side wall of the mounting hole extends toward the side of the sign facing away from the detection plate to form a column, and the mounting hole is arranged in the middle of the column.

7. The wearable microneedle ear tag for animal heat stress management according to claim 1, characterized in that: One end of the support column is provided with a nail column for preventing the female nail from falling out of the mounting hole when in use, the nail head is provided on the end surface of the nail column, and the diameter of the nail column is the same as the diameter of the base of the nail head.

8. The wearable microneedle ear tag for animal heat stress management according to claim 1, characterized in that: The support column is provided with an operation hole extending axially inwardly of the support column on the end surface where one end of the detection disk is provided.

9. The wearable microneedle ear tag for animal heat stress management according to claim 1, characterized in that: When preparing the first working microneedle electrode, the second working microneedle electrode, the third working microneedle electrode and the reference microneedle electrode, a conductive metal layer is first sprayed on the surface of the corresponding area of ​​the mother nail, and then a layer of gold nanoparticles is deposited on the surface of the conductive metal layer; then the area where each of the microneedle electrodes is located is respectively subjected to the following treatments: Depositing a layer of polyaniline film in the area where the first working microneedle electrode is located, and obtaining the first working microneedle electrode after drying; A layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) was deposited in the area where the second working microneedle electrode was located, and then a layer of K + selective membrane, to obtain the second working microneedle electrode; A layer of poly(3,4-ethylenedioxythiophene)-poly(styrene sulfonic acid) was deposited in the area where the third working microneedle electrode was located, and then a layer of Ca 2+ selective membrane, to obtain the third working microneedle electrode; A layer of Ag / AgCl slurry is coated on the area where the reference microneedle electrode is located, and then a methanol solution containing polyvinyl butyral and NaCl is added dropwise, and the reference microneedle electrode is obtained after drying; The temperature sensing microneedle electrode is prepared by coating a layer of carbon nanotubes on the surface of the corresponding area of ​​the mother nail; The conductive column is prepared by spraying a conductive metal layer on the surface of the corresponding area of ​​the mother nail; A conductive film is pasted on the side wall of the positioning conductive groove as a conductive layer.

10. The wearable microneedle ear tag for animal heat stress management according to claim 8, characterized in that: When depositing gold nanoparticles, the electrodeposition of gold nanoparticles was carried out in 5 mM chloroauric acid solution using linear sweep voltammetry on an electrochemical workstation, and the deposition potential and time were set to -0.8 V and 120 s, respectively; When depositing the polyaniline film, the polyaniline film was modified on the first working microneedle electrode region by cyclic voltammetry in a 0.1 M aniline solution, and the cycle voltage and the number of cycles were set to -0.2 to 1.0 V and 10 cycles, respectively; When depositing poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid), 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 polystyrenesulfonate, thereby electrodepositing poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonic acid); Coating K + When the selective membrane is used, 2 wt% of valinomycin, 64.7 wt% of diisooctyl sebacate, 32.7 wt% of polyvinyl chloride and 0.6 wt% of sodium tetraphenylborate are mixed to prepare K + Selective membrane; prepare K + The selective membrane was dissolved in 400 μL of cyclohexanone to obtain K + Selective membrane solution, K + The selective membrane solution is coated on the surface of the second working microneedle electrode area and then dried; Coating Ca 2+ When the selective membrane is used, 0.46 wt% of calcium ion carrier ETH129, 66.04 wt% of 2-nitrophenyl octyl ether, 33.02 wt% of polyvinyl chloride and 0.48 wt% of sodium tetraphenylborate are mixed to prepare Ca 2+ Selective membrane; The prepared Ca 2+ The selective membrane was dissolved in 1 mL of tetrahydrofuran to obtain Ca 2+ Selective membrane solution, Ca 2+ The selective membrane solution is coated on the surface of the third working microneedle electrode area and then dried.

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