Wearable microneedle sensor for animal ketogenic health assessment and preparation method

Abstract

The invention discloses a wearable microneedle sensor for animal ketogenic health assessment and a preparation method thereof. A sucker array is arranged on the front end face of the PDMS flexible patch, the rear end face of the PDMS flexible patch is fixedly connected to the surface of one side of the fixing support, the soft silica gel shell is fixedly connected to the surface of the other side of the fixing support, a hollowed-out groove is formed in the middle of the fixing support, a microneedle electrode is installed in the hollowed-out groove, a cavity is formed in the middle of the soft silica gel shell, and an electric assembly is arranged in the cavity. The PDMS flexible patch and the sucker array on the front end surface of the PDMS flexible patch are integrally made of a PDMS material; the inner wall of the suction cup is coated with a tannic acid-polyethylene glycol composite coating. The compliance to the skin is improved, dynamic balance is achieved through the synergistic effect of physical negative pressure adsorption and chemical adhesion, rebounding of microneedles in the skin is inhibited, a combined needle locking structure is constructed, high-fidelity signal collection in a motion scene is achieved, beta-hydroxybutyric acid, glucose and the pH value in interstitial fluid can be synchronously detected, and then ketogenic risk assessment is achieved.

Description

Technical Field

[0001] This invention relates to the field of intelligent sensing technology for animal information, specifically to a wearable microneedle sensor for assessing the ketogenic health of animals and its preparation method. Background Technology

[0002] The ketogenic diet has garnered significant attention due to its remarkable efficacy in treating obesity and other conditions. Under ketogenic diet intervention, ketone body synthesis in the hepatic mitochondrial matrix is ​​significantly enhanced, with the main products including acetoacetic acid, β-hydroxybutyrate, and acetone. Acetone is metabolized via the respiratory pathway, while acetoacetic acid and β-hydroxybutyrate serve as alternative energy substrates for high-energy-consuming tissues such as myocardium, skeletal muscle, and brain tissue. However, abnormally elevated concentrations may lead to pathological risks such as hypoglycemia, myocardial fibrosis, and arrhythmias. Therefore, the clinical safety of this therapy is highly dependent on the dynamic balance of key metabolic markers, requiring synergistic monitoring of β-hydroxybutyrate and blood glucose, which poses challenges to the real-time and continuous nature of detection methods. Traditional metabolic monitoring mainly relies on laboratory biochemical analysis, requiring periodic venous blood sampling and detection of biomarker concentrations using enzymatic colorimetry or gas chromatography-mass spectrometry. While this method offers high accuracy, it suffers from long sampling intervals, poor timeliness, insufficient continuity, and inability to capture rapid fluctuations in metabolic markers. Furthermore, the invasive nature of the procedure can cause pain and discomfort.

[0003] Wearable biosensors based on electrochemical detection principles have demonstrated unique advantages in the detection of biofluids such as sweat and interstitial fluid, providing emerging tools for real-time, in-situ monitoring to achieve personalized health management. Among them, microneedle biosensors, with their micrometer-scale diameter, excellent mechanical strength, and high aspect ratio, can achieve minimally invasive, painless epidermal penetration and target the detection of physiologically relevant biomarkers from deeper layers of skin tissue. However, the skin has a hairy, wrinkled, and curved surface morphology, and undergoes complex dynamic deformations during movement. These characteristics pose significant challenges to the bio-electronic interface. Skin curvature causes microneedles to shift and be partially extruded from the dermis. Hair and wrinkles on the epidermis hinder the stable adhesion of microneedle devices, resulting in voids at the contact interface and significantly increasing contact impedance. Shear stress caused by motion can lead to interface slippage, producing motion artifacts and accelerating device failure. Traditional microneedle biosensors, limited by planar manufacturing processes and rigid material systems, struggle to achieve conformal contact with biological tissues at the microscale, thus limiting signal fidelity.

[0004] Current technologies face a contradiction between the need for high-precision real-time clinical monitoring and insufficient equipment capabilities, and also encounter the challenge of dynamic interface instability. There is an urgent need for an integrated system capable of high-fidelity simultaneous monitoring of β-hydroxybutyrate, glucose, and pH in exercise scenarios, with localized ketogenic risk warning functions, to achieve precise management of health problems caused by excessive ketogenic diets. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides a wearable microneedle sensor for assessing the ketogenic health of animals and its preparation method.

[0006] This invention enables high-fidelity simultaneous monitoring of β-hydroxybutyrate, glucose, and pH in sports scenarios, and has a localized ketogenic risk warning function, which can be used to accurately assess health problems caused by excessive ketogenic diet.

[0007] The technical solution adopted in this invention is as follows:

[0008] I. A wearable microneedle sensor for assessing ketogenic health in animals:

[0009] The wearable microneedle sensor includes a PDMS flexible patch with a suction cup array, a fixing bracket, and a soft silicone shell. The front end of the PDMS flexible patch is provided with a suction cup array, the rear end of the PDMS flexible patch is fixedly connected to one side of the fixing bracket, the soft silicone shell is fixedly connected to the other side of the fixing bracket, the fixing bracket has a hollow groove in the middle, and microneedle electrodes are installed in the hollow groove. The soft silicone shell has a cavity in the middle, and electrical components are arranged in the cavity.

[0010] The PDMS flexible patch, the fixing bracket, and the soft silicone shell are connected together to form a five-pointed star shape.

[0011] The suction cup array includes multiple suction cups arranged on the front surface of the fixed bracket. The multiple suction cups are arranged in an array to form a suction cup array, and the PDMS flexible patch and the suction cup array on its front end are made of the same material as PDMS.

[0012] The inner wall of each suction cup in the suction cup array is coated with a tannic acid-polyethylene glycol composite coating. Specifically, the tannic acid-polyethylene glycol composite coating is composed of a 50% wt tannic acid aqueous solution and a 50% wt polyethylene glycol aqueous solution in a volume ratio of 671:329.

[0013] The fixed bracket has five hollow slots in the middle. Each hollow slot is diamond-shaped and arranged in the shape of a five-pointed star. Each hollow slot has an electrode installed in it. Each electrode has a microneedle sensing array consisting of no more than six microneedle structures near the center. The microneedles of different electrodes are arranged at intervals and do not contact each other, thus forming a microneedle electrode.

[0014] Each microneedle of the microneedle sensing array has a chromium / gold bilayer metal material deposited on it.

[0015] The electrodes in the five hollowed-out slots are respectively the first working electrode, the reference electrode, the second working electrode, the third working electrode, and the counter electrode.

[0016] The first working electrode, the second working electrode, and the third working electrode operate independently in electrochemical detection, sharing the same reference electrode and the same pair of electrodes. Their specific configuration is as follows:

[0017] β-hydroxybutyrate sensor: consists of a three-electrode system comprising a first working electrode, a reference electrode, and a counter electrode;

[0018] Glucose sensor: A three-electrode system consisting of a second working electrode, a reference electrode, and a counter electrode;

[0019] pH sensor: It consists of a two-electrode system consisting of a third working electrode and a reference electrode.

[0020] The electrical components include a connector, a button battery, and a printed circuit board. Both the connector and the button battery are mounted on the printed circuit board. The button battery powers the printed circuit board. One end of the connector is electrically connected to each microneedle of the microneedle sensing array of each electrode, and the other end is electrically connected to the printed circuit board.

[0021] The wearable microneedle sensor is worn on an animal. The PDMS flexible patch is adsorbed onto the animal's surface via a suction cup array, and at the same time, the microneedle sensor array is inserted into the animal's body. The reaction interface of the microneedle sensor contacts the interstitial fluid in the animal's body, generating an electrochemical response signal caused by dynamic changes in β-hydroxybutyrate, glucose, and pH. The electrochemical response signal is input to a printed circuit board for signal analysis to obtain the dynamic changes in blood metabolic state. The metabolic state is analyzed by a lightweight classification model built into the printed circuit board, thereby realizing the ketogenic health assessment of the animal.

[0022] The innovation of this invention lies in the design of a biomimetic starfish-inspired five-armed star-shaped flexible patch and a suction cup-microneedle combined locking structure. Through biomimetic design, the adhesion of the sensor to the body surface is improved, thereby enhancing the stability of animal ketogenic health detection and assessment.

[0023] II. A method for fabricating a wearable microneedle sensor:

[0024] This invention further provides a method for preparing the wearable microneedle sensor for animal ketogenic health assessment, the method comprising:

[0025] Step 1: Fabrication of microneedle array

[0026] Each microneedle of the microneedle electrode is machined into a conical structure using a CNC machine tool, with a bottom diameter of 300 μm and a height of 1000 μm. A bilayer metal layer of chromium and gold is sequentially deposited on the surface of the microneedle by magnetron sputtering to generate conductivity. The five microneedle electrodes, excluding the counter electrode, are functionalized as a β-hydroxybutyrate electrode (modified with a polythione layer and hydroxybutyrate dehydrogenase), a glucose electrode (modified with a Prussian blue layer and glucose oxidase), a pH electrode (modified with a polyaniline film), and a reference electrode (Ag / AgCl layer and polyvinyl butyral protective layer), while the counter electrode (no additional modification required).

[0027] Step 2: Preparation of suction cup patch

[0028] An integral structure consisting of a PDMS flexible patch and microneedles was prepared by integrally curing and molding each microneedle electrode with a PDMS precursor solution.

[0029] Step 3: Overall Assembly

[0030] The PDMS substrate of the combined pin module is cut into a five-armed star shape using laser cutting, and the printed circuit boards of the microneedle electrodes and electrical components in the soft silicone shell are connected by connectors.

[0031] More specifically, the solder joints of the electrode microneedles are connected to the pins via conductive silver paste and aligned with the printed circuit board connector.

[0032] In step one: the β-hydroxybutyrate electrode is formed by modifying a polythione layer on a chromium and gold bilayer metal layer on the microneedle surface and then depositing hydroxybutyrate dehydrogenase, serving as the first working electrode; the glucose electrode is formed by modifying a Prussian blue layer on a chromium and gold bilayer metal layer on the microneedle surface and then depositing glucose oxidase, serving as the second working electrode; the pH electrode is formed by modifying a polyaniline film on a chromium and gold bilayer metal layer on the microneedle surface, serving as the third working electrode; the reference electrode is formed by first depositing an Ag / AgCl layer and then a polyvinyl butyral protective layer on a chromium and gold bilayer metal layer on the microneedle surface, serving as the reference electrode.

[0033] Step two specifically involves:

[0034] First, a suction cup array structure on the PDMS flexible patch is formed using a polyurethane mold. Then, a PDMS precursor solution is injected and degassed under vacuum to eliminate air bubbles.

[0035] Then, each microneedle electrode and its microneedle sensing array are placed in the uncured PDMS precursor solution for preparing the patch according to the pre-set positioning points. After curing at 70°C for 3 hours, the patch is demolded to form a combined locking needle module consisting of a PDMS flexible patch and microneedle electrodes.

[0036] Finally, a tannic acid-polyethylene glycol composite coating is applied to the inner wall of each suction cup in the suction cup array.

[0037] The tannic acid-polyethylene glycol composite coating is prepared using the following process and conditions:

[0038] A 50% wt aqueous solution of tannic acid dissolved in water and a 50% wt aqueous solution of polyethylene glycol dissolved in water were mixed at a volume ratio of 671:329. After centrifugation at 13,500 rpm for 3 minutes, the supernatant was removed to obtain a tannic acid-polyethylene glycol composite coating material. This tannic acid-polyethylene glycol composite coating material was uniformly coated onto the inner wall of a PDMS suction cup to form a tannic acid-polyethylene glycol composite coating, serving as a biomimetic adhesion enhancement layer. The combined needle-locking module suppresses microneedle displacement through the negative pressure adsorption mechanism of the suction cup array. By combining a pentagonal-shaped bending motion to adhere to the surface, it improves adsorption and adhesion, thereby enhancing the stability of the microneedles penetrating the body surface. During the adhesion compression stage, the normal adhesion pressure reduces the volume of the suction cup chamber; during the rebound stage, the suction cup generates an anti-peel pressure difference, increasing the adhesion strength to 16.2 kPa.

[0039] In practice, sensitivity results are obtained through electrochemical performance testing.

[0040] Electrochemical performance verification showed that the β-hydroxybutyrate sensor exhibited a bilinear response in the 0-4.5 mM range, with a sensitivity of 0.02058 μA / mM (R² = 0.9971) in the 0-3 mM range and 0.04306 μA / mM (R² = 0.9997) in the 3-4.5 mM range. The glucose sensor showed a sensitivity of 0.4112 μA / mM (R² = 0.9974) in the 0-1.5 mM glucose concentration range and 0.06032 μA / mM (R² = 0.9928) in the 1.5-5 mM range. The pH sensor showed a sensitivity of 42.63 mV / pH (R² = 0.9969) in the pH 4-9 range.

[0041] This invention also provides a method for assessing the ketogenic health of animals, comprising the following steps:

[0042] In practical applications, microneedle sensors are attached to the skin of the neck of Bama pigs. A short-term metabolic intervention involves injecting a 50% glucose solution into the animal's ear vein to induce rapid changes in blood glucose levels. Sensor readings reflect the dynamic changes in metabolites in the blood, effectively capturing analyte fluctuations.

[0043] Furthermore, a long-term dietary intervention was conducted on Bama miniature pigs to induce ketosis. Sensor readings showed a high degree of consistency with blood reference values ​​during metabolic transitions, validating its ability to dynamically monitor ketotic health status.

[0044] The overall structure of this invention adopts a five-armed star-shaped structure inspired by a starfish, enhancing its conformity to the skin. A biomimetic suction cup surrounding the microneedle electrode array forms a locking structure, achieving a dynamic balance through the synergistic effect of physical negative pressure adsorption and chemical adhesion, thus inhibiting microneedle rebound in the skin.

[0045] The microneedle sensor described in this invention mimics the predatory behavior of starfish, constructing a five-armed star-shaped flexible patch and a suction cup-microneedle combined locking structure to achieve high-fidelity signal acquisition in motion scenarios. The sensor integrates a three-channel microneedle sensing array to simultaneously detect β-hydroxybutyrate, glucose, and pH in interstitial fluid, and triggers a local optical alarm through an edge computing model, thereby being used for ketosis risk assessment.

[0046] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0047] (1) Bionic interlocking interface: The sensor described in this invention integrates a suction cup-microneedle joint interlocking structure in a five-armed star-shaped flexible patch, which can conformally adhere to the skin surface and suppress microneedle rebound under skin movement deformation to ensure the stability of the bioelectronic interface and achieve high-fidelity signal acquisition.

[0048] (2) Real-time monitoring of multiple parameters: The microneedle sensing array of the present invention can measure metabolic biomarkers such as β-hydroxybutyrate, glucose and pH in interstitial fluid in a minimally invasive, synchronous and continuous manner, providing laboratory-grade accuracy for real-time assessment of ketosis and metabolic risk, and eliminating the need for repeated invasive sampling.

[0049] (3) On-chip intelligent risk warning: The lightweight model deployed on a single microcontroller described in this invention can realize localized, low-power ketogenic risk prediction and LED optical warning, realizing the on-chip architecture of "biochemical sensing-edge computing-closed-loop feedback", providing a closed-loop paradigm with clinical translation potential for wearable health monitoring.

[0050] In summary, this invention can simultaneously monitor the concentrations of β-hydroxybutyrate, glucose, and pH in animal interstitial fluid, enabling precise assessment of ketogenic risk. This invention utilizes a biomimetic locking needle structure and on-chip intelligent technology to achieve in-situ, continuous, high-fidelity, and intelligent metabolic health monitoring. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the three-dimensional structure of the microneedle sensor. Among them, Figure 1 (a) is a schematic diagram of the sensor assembly; Figure 1 (b) is a schematic diagram of the exploded structure of the sensor.

[0052] Figure 2 This is a schematic diagram of the three-dimensional structure of the microneedle sensor array.

[0053] Figure 3The graphs show the electrochemical response curve and standard curve of the sensor. Figure 3 (a) is the amperometric response curve of the sensor at β-hydroxybutyric acid concentrations of 0-4.5 mM; Figure 3 (b) is a standard curve of the sensor at β-hydroxybutyric acid concentrations of 0-4.5 mM; Figure 3 (c) is the amperometric response curve of the sensor at glucose concentrations of 0-5 mM; Figure 3 (d) is the standard curve of the sensor at glucose concentrations of 0-5 mM;

[0054] Figure 3 (e) is the open-circuit potential response curve of the sensor at pH 4-9; Figure 3 (f) is the standard curve of the sensor at pH 4-9.

[0055] Figure 4 The figures show the finite element simulation analysis of the suction cup's mechanical behavior. Figure 4 (a) Simulation of the suction cup compression state; Figure 4 (b) is a simulation of the suction cup under tension.

[0056] Figure 5 This is a schematic diagram illustrating the biomechanical interaction between the patch and the skin. Among them, Figure 5 (a) is a schematic diagram of the force analysis of the mechanical interaction between the patch and the skin; Figure 5 (b) shows the simulation analysis results of the mechanical interaction between the patch and the skin.

[0057] Figure 6 The results of dynamic β-hydroxybutyrate, glucose, and pH measurements using sensors and standard methods are presented in a graphical format.

[0058] Figure 7 This is a graph showing the sensor's in-situ detection error analysis.

[0059] Figure 8 This is a schematic diagram of a lightweight binary classification model for edge computing. Figure 8 (a) is the process of random hyperparameter optimization; Figure 8 (b) is the confusion matrix of the lightweight model.

[0060] Figure 9 A comparison chart of in-situ signal stability of sensor patches.

[0061] Reference numerals: 1. PDMS flexible patch with suction cup array; 2. Microneedle sensor array; 3. Fixing bracket; 4. Connector; 5. Button battery; 6. Printed circuit board; 7. Soft silicone shell; 8. First working electrode; 9. Reference electrode; 10. Second working electrode; 11. Third working electrode; 12. Counter electrode. Detailed Implementation

[0062] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0063] The embodiments of the present invention are as follows:

[0064] Example 1

[0065] like Figure 1 As shown, the structure of the embodiment includes a PDMS flexible patch 1 with a suction cup array, a fixing bracket 3, and a soft silicone shell 7; the PDMS flexible patch 1, the fixing bracket 3, and the soft silicone shell 7 are connected together to form a pentagram shape.

[0066] The front end of the PDMS flexible patch 1 is provided with a suction cup array. The rear end of the PDMS flexible patch 1 is fixedly connected to one side of the fixed bracket 3. The soft silicone shell 7 is fixedly connected to the other side of the fixed bracket 3. The fixed bracket 3 has a hollow groove in the middle, in which microneedle electrodes are installed. The soft silicone shell 7 has a cavity in the middle, in which electrical components are arranged.

[0067] The suction cup array includes multiple suction cups arranged on the front surface of the fixed bracket 3. The multiple suction cups are arranged in an array to form a radial suction cup array. The PDMS flexible patch 1 and the suction cup array on its front end are made of the same material PDMS. The inner wall of each suction cup in the suction cup array is coated with a tannic acid-polyethylene glycol composite coating. Specifically, the tannic acid-polyethylene glycol composite coating is composed of tannic acid (50% wt aqueous solution) and polyethylene glycol (50% wt aqueous solution) in a volume ratio of 671:329.

[0068] The fixed bracket 3 has five hollow slots in the middle, each hollow slot is diamond-shaped, and the five hollow slots are arranged in the shape of a five-pointed star. An electrode is installed in each hollow slot. Each electrode has a microneedle sensing array 2 composed of multiple microneedle structures near the center. The microneedles of different electrodes are arranged at intervals and do not contact each other, thus forming a microneedle electrode. Chromium / gold double-layer metal material is deposited on each microneedle of the microneedle sensing array 2.

[0069] The electrodes in the five hollow slots are the first working electrode 8, the reference electrode 9, the second working electrode 10, the third working electrode 11, and the counter electrode 12. The first working electrode 8, the second working electrode 10, and the third working electrode 11 operate independently in electrochemical detection, sharing the same reference electrode 9 and the same counter electrode 12. The specific configuration is as follows: the first working electrode 8, the reference electrode 9, and the counter electrode 12 form a three-electrode system for the β-hydroxybutyrate sensor; the second working electrode 10, the reference electrode 9, and the counter electrode 12 form a three-electrode system for the glucose sensor; and the third working electrode 11 and the reference electrode 9 form a two-electrode system for the pH sensor.

[0070] The electrical assembly includes a connector 4, a button battery 5, and a printed circuit board 6 integrating an LED warning module. Both the connector 4 and the button battery 5 are mounted on the printed circuit board 6. The button battery 5 powers the printed circuit board 6. One end of the connector 4 is electrically connected to each microneedle of the microneedle sensing array 2, and the other end is electrically connected to the printed circuit board 6 for alignment between the microneedle electrodes and the printed circuit board. The multi-layered structure of the connector 4, button battery 5, and printed circuit board 6 is encapsulated in a silicone shell, with a total weight of 4.5g.

[0071] The microneedle electrode contains five independent microneedle modules, each with six conical microneedles arranged in a row. A chromium / gold layer is sputtered onto the surface of the microneedles as a conductive layer. The mounting bracket 3 is used to integrate the microneedle array and the suction cup patch to form a combined locking structure. The connector 4 enables the alignment connection between the microneedle array and the printed circuit board. The button battery 5 is used for system power supply. The printed circuit board 6 integrates signal acquisition, Bluetooth transmission, STM32 microcontroller and LED alarm module. The soft silicone shell 7 is an encapsulation layer with high transparency to accommodate LED visualization function.

[0072] In practice, wearable microneedle sensors are worn on the animal body, for example, on the back of the animal's neck. The PDMS flexible patch 1 is adsorbed onto the animal's surface through a suction cup array, and at the same time, the microneedle sensor array 2 is inserted into the animal's body. The reaction interface of the microneedle sensor contacts the interstitial fluid in the animal's body, generating an electrochemical response signal caused by the dynamic changes of β-hydroxybutyrate, glucose, and pH. The signal is input into the printed circuit board 6, and the metabolic state is analyzed by the lightweight classification model built into the printed circuit board 6, thereby realizing the ketogenic health assessment of the animal.

[0073] The manufacturing process of the wearable microneedle sensor for animal ketogenic health assessment described in Example 1 includes the following steps:

[0074] Step 1: Fabrication of microneedle electrodes.

[0075] The shape of the microneedle electrode was designed using the computer graphics software SolidWorks. For example... Figure 2 The electrode block includes a first working electrode 8, a reference electrode 9, a second working electrode 10, a third working electrode 11, and a counter electrode 12. The electrode block is designed in a rhomboid shape and has a thickness of 1 mm. The first working electrode is a β-hydroxybutyrate detection electrode for detecting the concentration of β-hydroxybutyrate in the interstitial fluid. The second working electrode is a glucose detection electrode for detecting the concentration of glucose in the interstitial fluid. The third working electrode is a pH detection electrode for detecting the pH value of the interstitial fluid. The reference electrode is a shared reference electrode used for the first, second, and third working electrodes. The counter electrode is a shared counter electrode used for the first and second working electrodes.

[0076] The sensing areas of each electrode are used to sense and detect the corresponding indicators, the tail solder joints are used for external signal detection circuits, and the part between the sensing areas and the tail external pin area is a conductive connection area that serves as a non-working area.

[0077] First, five electrode blocks were fabricated on a polymethyl methacrylate substrate using micromachining technology. The sensing area of ​​each electrode contained six conical microneedles with a height of 1000 μm, a bottom diameter of 300 μm, and a center-to-center spacing of 700 μm between adjacent microneedles. A 15 nm chromium bonding layer and a 200 nm gold conductive layer were deposited on the surface of the microneedle array by magnetron sputtering.

[0078] Step 2: Functionalization of microneedle electrodes.

[0079] Electrodeposition modification was performed using an electrochemical workstation with a microneedle electrode as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. After activation by 40 cyclic voltammetric scans in 0.1M sulfuric acid solution at a scan rate of 1V / s, a gold nanoparticle layer was deposited for 200s at a constant potential of -0.22V in a solution of 1.2 mg / mL chloroauric acid, 0.1M sodium chloride, and 1.5 wt% hydrochloric acid.

[0080] β-Hydroxybutyrate detection electrode: Cyclic voltammetric deposition was performed in a 1 mM thionine solution dissolved in PBS buffer (potential range -0.2 V to 0.8 V, scan rate 50 mV / s, 20 cycles). Subsequently, 4 μL of enzyme mixture (containing 0.25 M nicotinamide adenine dinucleotide and 500 U / mL hydroxybutyrate dehydrogenase) was dropped onto the electrode. 1 μL of glutaraldehyde was added for cross-linking. Finally, 4 μL of polyvinyl chloride solution was dropped onto the electrode as an anti-interference layer.

[0081] Glucose detection electrode: A Prussian blue layer was deposited by cyclic voltammetry in a solution containing 2.5 mM ferric chloride, 2.5 mM potassium ferricyanide, and 100 mM hydrochloric acid (potential range -0.15 V to 0.3 V, scan rate 20 mV / s, 20 cycles). This layer was then stabilized by cyclic voltammetry in a solution containing 100 M potassium chloride and hydrochloric acid (potential range -0.2 V to 0.5 V, scan rate 50 mV / s, 5 cycles). A NiHCF protective layer was then deposited by cyclic voltammetry in a solution containing 0.5 mM nickel chloride, 0.5 mM potassium ferricyanide, 100 mM potassium chloride, and 100 mM hydrochloric acid (potential range 0 V to 0.8 V, scan rate 100 mV / s, 5 cycles). 5 μL of glucose oxidase solution (5 KU / mL) was dropped onto the electrode and immobilized by glutaraldehyde crosslinking. Finally, 4 μL of Nafion was dropped onto the electrode as an anti-interference layer.

[0082] pH detection electrode: Polyaniline film was cyclically polymerized in a 1M hydrochloric acid solution containing 0.1M aniline (potential range -0.2V to 1.0V, scan rate 100mV / s, 30 cycles).

[0083] Reference electrode: After coating with conductive silver paste, it was cured at 60°C for 1 hour, and then chlorinated for 30 seconds with 0.1M ferric chloride solution to form an Ag / AgCl layer, followed by modification with a polyvinyl butyral protective layer. The polyvinyl butyral protective layer was prepared by adding 4 μL of a methanol mixed solution containing 79.1 mg of polyvinyl butyral and 50 mg of sodium chloride.

[0084] A biodegradable protective coating was uniformly deposited on the microneedle electrodes by drop-coating a mixture of 5 wt% water-soluble silk fibroin and 5 wt% hyaluronic acid (1:1 volume ratio). All microneedle electrodes were then dried and stored at 4°C.

[0085] Step 3: Preparation of suction cup patch.

[0086] A polyurethane mold with a suction cup chamber structure having an inner diameter of 2 mm, a height of 1 mm, and a wall thickness of 0.2 mm was used. PDMS precursor and curing agent were mixed at a mass ratio of 20:1, vacuum degassed for 15 minutes to eliminate air bubbles, and then injected into the mold. The five electrode blocks described in step two were embedded and subjected to a second vacuum treatment. After curing at 70°C for 3 hours, the mixture was demolded to form a flexible patch with embedded microneedle electrodes, i.e., a combined locking needle structure. A tannic acid aqueous solution (50 wt%) and a polyethylene glycol aqueous solution (50 wt%) were mixed at a volume ratio of 671:329. After centrifugation at 13,500 rpm for 3 minutes, the supernatant was removed and coated onto the inner wall of the suction cup to form an adhesive layer. Finally, a five-armed star-shaped flexible substrate was formed by laser cutting.

[0087] Step 4: System Integration and Assembly. Using conductive silver paste, the electrode tail solder points are connected to the pin connectors, and then integrated with the printed circuit board via connectors. Finally, all components are encapsulated in a transparent soft silicone protective shell.

[0088] A) Sensor performance test in Example 1:

[0089] β-hydroxybutyrate sensor: such as Figure 3 (a) and Figure 3 (b) Ampere detection of β-hydroxybutyrate PBS solutions with concentration gradients of 0–4.5 mM was performed at a constant potential of 0.3 V. The response current showed a linear relationship with concentration in the 0–3 mM range, with a sensitivity of 0.02058 μA / mM (R² = 0.9971). The sensitivity in the 3–4.5 mM range was 0.04306 μA / mM (R² = 0.9971). 2 =0.9997).

[0090] glucose sensor: such as Figure 3(c) and Figure 3 (d) Ampere detection was performed on glucose PBS solutions with concentration gradients of 0–5 mM at a constant potential of 0.6 V. The sensitivity was 0.4112 μA / mM (R² = 0.9974) in the 0–1.5 mM glucose concentration range and 0.06032 μA / mM (R² = 0.9974) in the 1.5–5 mM range. 2 =0.9928).

[0091] pH sensor: such as Figure 3 (e) and Figure 3 (f) The open-circuit potential method was used to detect phosphate buffer solutions with pH values ​​of 4–9. The potential response showed a linear negative correlation with pH (R0). 2 =0.9969).

[0092] The mechanical behavior of the suction cup is analyzed through finite element simulation. For example... Figure 4 (a) During the compression phase, the normal pressure causes the suction cup chamber volume to decrease; after the external force is removed, the chamber volume rebound is limited by the inclined sealing edge, forming a negative pressure adsorption steady state. For example... Figure 4 (b) During the stretching process, the expansion of the chamber generates an anti-peel pressure differential. Peel tests show that the maximum adhesion strength of the suction cup patch reaches 16.2 kPa. Furthermore, theoretical analysis and simulation were conducted on the stress conditions after microneedles were inserted into the skin. Figure 5 (a) In the first stage, during the insertion of the microneedle under pressure, the microneedle experiences resistance and friction from the skin. In the second stage, after insertion is complete, the external pressure is released, and the needle body is partially extruded due to the skin's elastic deformation. Figure 5 (b) In the final state, the microneedle patch with suction cup remains deeper in the skin, demonstrating the needle-locking effect. During the rebound phase, the needle tip stress in the planar patch fluctuates violently, indicating that the needle is rapidly extruded; while the needle tip stress in the suction cup patch decreases gradually and stabilizes, indicating that the force is balanced under the suction cup's adsorption. During the downward compression, the volume of the suction cup cavity compresses, and the tensile stress on the inner wall increases; during the rebound, the volume of the cavity expands, and the tensile stress on the inner wall decreases slightly, generating local negative pressure to resist the skin's extrusion force, ultimately achieving force balance. Compared to the planar patch, in the combined needle-locking structure integrated with the suction cup, the strong adhesion of the suction cup can resist the skin's extrusion force, providing a stable microneedle-skin interface for subsequent monitoring.

[0093] B) Animal testing of Example 1:

[0094] The continuous monitoring capability of the microneedle sensor prepared in Example 1 was verified in the Bama miniature pig model.

[0095] Bama pigs weighing 30±2kg were selected. They were fasted for 16 hours before the procedure, and their necks were shaved. The device was attached to the skin surface of their necks, and pressure was applied for 10 seconds to achieve stable sensor attachment. Throughout the experiment, a multi-parameter physiological monitor continuously tracked the Bama pigs' respiratory rate, body temperature, blood oxygen saturation, and heart rate.

[0096] Short-term metabolic intervention: Injecting a 50% glucose solution into the ear vein of an animal to induce rapid changes in blood glucose levels. For example... Figure 6 The sensor readings reflected the dynamic changes in metabolites in the blood. Due to prolonged fasting, the initial glucose concentration was low. After injection, glucose reached a peak concentration of 27.15 mM. β-hydroxybutyrate concentration decreased synchronously due to inhibition of lipolysis, and pH showed a slow decreasing trend. These results indicate that the microneedle sensor prepared in Example 1 can effectively capture analyte fluctuations, has dynamic response capabilities, and is suitable for real-time physiological monitoring.

[0097] Long-term ketogenic intervention: Bama miniature pigs underwent a 14-day ketogenic diet intervention to induce ketosis, validating the ability of the microneedle sensor prepared in Example 1 to dynamically assess ketotic health status during metabolic transition. Figure 7 The sensor readings are highly consistent with blood reference values: the correlation coefficient R of β-hydroxybutyrate is high. 2 =0.9809, glucose R 2 =0.9582, pH value R 2 =0.9448, verifying the reliability of the system in a dynamic biological environment.

[0098] C) Performance testing of the ketogenic risk assessment model in Example 1:

[0099] Based on in vivo data obtained from long-term ketogenic intervention in animal testing in Example 1, a dataset was constructed and a ketogenic health risk assessment was conducted.

[0100] Based on the long-term ketogenic intervention experiment, classification labels were assigned: "healthy" before the ketogenic diet intervention and "risky" after the intervention. The sensor operated at a sampling frequency of T = 0.1 s, collecting 27,000 s of data for model training. The three-channel signals from the sensor were used as input, and the average signal value was calculated every 10 s. The dataset was divided into training and validation sets in an 8:2 ratio.

[0101] To achieve real-time edge computing, a fully connected neural network binary classification model was developed and deployed on an STM32 microcontroller. For example... Figure 8As shown, after hyperparameter optimization (hidden layer nodes = 4, batch size = 8, training epochs = 60), the model size is compressed to within 4KB, and the classification accuracy exceeds 95%. During deployment, the trained model is rewritten in C language and burned into the STM32 microcontroller. The system reads sensor signals at 10-second intervals and inputs them into the edge model mounted on the microcontroller. An LED warning module is integrated into the printed circuit board; when the classification model outputs "risk," an LED flashing signal is triggered, with a response delay of less than 10ms, forming a real-time "sensing-analysis-warning" feedback system.

[0102] Comparative Example 1:

[0103] This comparative example provides a microneedle device integrated into a planar patch, serving as a control device. The control device includes a PDMS flexible planar patch without a suction cup array, a fixing bracket, and a soft silicone shell; the front face of the PDMS flexible planar patch does not have a suction cup array, and the remaining configuration is the same as in Example 1. Specifically, the rear face of the PDMS flexible patch is fixedly connected to one side of the fixing bracket, and the soft silicone shell is fixedly connected to the other side of the fixing bracket. A hollow groove is formed in the middle of the fixing bracket, in which microneedle electrodes are installed and arranged. A cavity is formed in the middle of the soft silicone shell, in which electrical components are arranged.

[0104] Then, in vivo monitoring of Bama miniature pigs was conducted using the sensors of Example 1 and Comparative Example 1, and comparative tests were performed on planar, concave, and convex pig skin areas, respectively. Compared with planar devices, the suction cup patch microneedle device of Example 1 of the present invention showed significantly reduced signal fluctuations.

[0105] like Figure 9 For voltage-type pH sensors, the pH sensing signal fluctuation of the planar device in Comparative Example 1 exceeded 300mV, while the fluctuation of the suction cup patch microneedle in Example 1 was controlled within 7.9mV. For current-type sensors (β-hydroxybutyrate and glucose sensors), due to the enhanced contact between the microneedle and interstitial fluid by the combined locking needle structure of Example 1, the response of the β-hydroxybutyrate sensor was 5.49 times that of the planar device in Comparative Example 1, and the response of the glucose sensor was 1.42 times that of the planar device in Comparative Example 1. Skin rebound caused partial extrusion of the microneedles from the planar device, resulting in a significant increase in contact resistance.

[0106] Therefore, the combined locking needle structure in the suction cup patch microneedle device of the present invention suppresses microneedle displacement through suction cup negative pressure, maintains the stability of the electrode-tissue interface, and improves sensitivity and reliability in dynamic biological environments.

[0107] In summary, the wearable microneedle sensor of this invention integrates multi-parameter biosensing and edge intelligent analysis systems, enabling high-fidelity dynamic monitoring of β-hydroxybutyrate, glucose, and pH in interstitial fluid, and providing real-time early warning of health risks associated with ketosis metabolism, which is of great significance for ketosis health management.

[0108] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A wearable microneedle sensor for assessing ketogenic health in animals, characterized in that: It includes a PDMS flexible patch (1) with a suction cup array, a fixing bracket (3) and a soft silicone shell (7); the front end of the PDMS flexible patch (1) is provided with a suction cup array, the rear end of the PDMS flexible patch (1) is fixedly connected to one side of the fixing bracket (3), the soft silicone shell (7) is fixedly connected to the other side of the fixing bracket (3), the fixing bracket (3) has a hollow groove in the middle, and microneedle electrodes are installed in the hollow groove. The soft silicone shell (7) has a cavity in the middle, and electrical components are arranged in the cavity.

2. The wearable microneedle sensor for assessing ketogenic health in animals according to claim 1, characterized in that: The PDMS flexible patch (1), the fixing bracket (3) and the soft silicone shell (7) are connected together to form a five-pointed star shape.

3. The wearable microneedle sensor for assessing ketogenic health in animals according to claim 1, characterized in that: The suction cup array includes multiple suction cups arranged on the front surface of the fixed bracket (3), the multiple suction cups are arranged in an array to form a suction cup array, and the PDMS flexible patch (1) and the suction cup array on its front end are made of the same material as PDMS. The inner wall of each suction cup in the suction cup array is coated with a tannic acid-polyethylene glycol composite coating. Specifically, the tannic acid-polyethylene glycol composite coating is composed of a 50% wt tannic acid aqueous solution and a 50% wt polyethylene glycol aqueous solution in a volume ratio of 671:

329.

4. A wearable microneedle sensor for assessing ketogenic health in animals according to claim 1, characterized in that: The fixed bracket (3) has five hollow slots in the middle. The five hollow slots are arranged in the shape of a five-pointed star. Each hollow slot has an electrode installed in it. Each electrode has a microneedle sensing array (2) composed of 6 microneedles near the center, thus forming a microneedle electrode. The electrodes in the five hollowed-out slots are the first working electrode (8), the reference electrode (9), the second working electrode (10), the third working electrode (11), and the counter electrode (12).

5. A wearable microneedle sensor for assessing ketogenic health in animals according to claim 1, characterized in that: The electrical components include a connector (4), a button battery (5), and a printed circuit board (6). Both the connector (4) and the button battery (5) are mounted on the printed circuit board (6). The button battery (5) is used to power the printed circuit board (6). One end of the connector (4) is electrically connected to each microneedle of the microneedle sensing array (2) of each electrode, and the other end is electrically connected to the printed circuit board (6).

6. A wearable microneedle sensor for assessing ketogenic health in animals according to claim 1, characterized in that: The wearable microneedle sensor is worn on the animal body. The PDMS flexible patch (1) is adsorbed onto the animal body surface through a suction cup array, and at the same time as adsorption, the microneedle sensor array (2) is inserted into the animal body. The reaction interface of the microneedle sensor contacts the interstitial fluid in the animal body, generating an electrochemical response signal caused by the dynamic changes of β-hydroxybutyric acid, glucose and pH value. The electrochemical response signal is input into the printed circuit board (6) for signal analysis to obtain the metabolic state, thereby realizing the ketogenic health assessment of the animal.

7. A method for preparing the wearable microneedle sensor according to any one of claims 1-6, characterized in that: The method includes: Step 1: Fabrication of microneedle array Each microneedle of the microneedle electrode is processed into a conical structure. A double metal layer of chromium and gold is sequentially deposited on the surface of the microneedle by magnetron sputtering. The five microneedle electrodes, excluding the counter electrode, are functionalized as β-hydroxybutyrate electrode, glucose electrode, pH electrode and reference electrode, respectively. Step 2: Preparation of suction cup patch An integral structure consisting of a PDMS flexible patch (1) and microneedles was prepared by integrally curing and molding each microneedle electrode with a PDMS precursor solution; Step 3: Overall Assembly The combined locking pin module is cut into a five-armed star shape using laser cutting, and the microneedle electrode is connected to the printed circuit board in the soft silicone shell (7).

8. The preparation method according to claim 7, characterized in that: In step one: The β-hydroxybutyrate electrode is formed by modifying a polythione layer on a chromium and gold bilayer metal layer on the surface of the microneedle with hydroxybutyrate dehydrogenase, and serves as the first working electrode (8). The glucose electrode is formed by modifying a Prussian blue layer on a chromium and gold bilayer metal layer on the surface of the microneedle with glucose oxidase, and serves as the second working electrode (10). The pH electrode is formed by modifying a polyaniline film on a chromium and gold bilayer metal layer on the surface of the microneedle, and serves as the third working electrode (11). The reference electrode is formed by first depositing an Ag / AgCl layer on the surface of the microneedle and then depositing a polyvinyl butyral protective layer, and then serving as the reference electrode (9).

9. The preparation method according to claim 7, characterized in that: Step two specifically involves: First, the suction cup array structure on the PDMS flexible patch (1) is formed by molding, and then the PDMS precursor solution is injected and degassed under vacuum to eliminate air bubbles. Then, each microneedle electrode was placed in an uncured PDMS precursor solution, cured at 70°C for 3 hours, and then demolded to form a combined locking needle module consisting of a PDMS flexible patch (1) and microneedle electrodes. Finally, a tannic acid-polyethylene glycol composite coating is applied to the inner wall of each suction cup in the suction cup array.

10. The preparation method according to claim 9, characterized in that: The tannic acid-polyethylene glycol composite coating is prepared using the following process and conditions: A 50% wt aqueous solution of tannic acid dissolved in water and a 50% wt aqueous solution of polyethylene glycol dissolved in water were mixed at a volume ratio of 671:

329. After centrifugation at 13,500 rpm for 3 min, the supernatant was removed to obtain a tannic acid-polyethylene glycol composite coating material. The tannic acid-polyethylene glycol composite coating material was uniformly coated on the inner wall of a PDMS chuck to form a tannic acid-polyethylene glycol composite coating.

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