Microneedle biosensor and wearable monitoring device for glucagon-like peptide-1 detection

Abstract

The application provides a microneedle biosensor and a wearable monitoring device for detection of glucagon-like peptide-1. The microneedle biosensor comprises a three-electrode detection system formed by functionalization of conductive microneedles and a flexible conductive circuit connected thereto. The three-electrode detection system comprises a working electrode, a reference electrode and a counter electrode. The working electrode is constructed by depositing a layer of gold nanoparticles on the surface of the conductive microneedle and anchoring glucagon-like peptide-1 antibodies; the reference electrode is constructed by coating the surface of the conductive microneedle with Ag / AgCl material; and the conductive microneedle itself serves as the counter electrode. The microneedle biosensor for GLP-1 monitoring can be integrated into a wearable device and can directly monitor the changes in GLP-1 concentration in the interstitial fluid. Compared with the traditional sweat monitoring, the accuracy of monitoring can be improved, and efficient, convenient and real-time GLP-1 level monitoring can be realized, which has a wide application space in the field of obesity treatment and health management.

Description

Technical Field

[0001] This invention belongs to the field of biomedical sensing and relates to the intelligent detection of glucagon-like peptide-1 (GLP-1), specifically to a microneedle biosensor and wearable monitoring device for the detection of GLP-1. Background Technology

[0002] Obesity is a chronic disease with a high relapse rate, characterized by excessive accumulation of adipose tissue. Clinically, a body mass index (BMI) ≥ 30 kg / m² is defined as obesity. 2 The obese population is defined as individuals with obesity. Obesity is recognized as a multifactorial disease caused by a systemic energy imbalance, often leading to various complications, including diabetes, cardiovascular disease, respiratory diseases, and malignant tumors. The global obesity rate has been gradually increasing in recent years, with over 1 billion obese individuals currently living there, and more than 2.8 million people dying annually from obesity and its complications. In response to the rising obesity rate in my country, the National Health Commission has launched the "Weight Management Year" campaign, incorporating "nationwide weight loss" into the Healthy China initiative. Therefore, precise management of obesity has become a key research direction in the medical field.

[0003] Glucagon-like peptide-1 (GLP-1) is a human hormone closely related to blood glucose regulation and energy metabolism. GLP-1 can control the secretion of insulin and glucagon, and is of great value in the treatment of obesity and complications such as diabetes. The hormone level of GLP-1 is of guiding significance for the analysis and modification of drug use and treatment plans. At present, the detection and analysis of GLP-1 levels in vivo mainly rely on laboratory immunoassay and molecular biology methods. The mainstream technologies include enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical staining (IHC). The above methods are all offline, ex vivo, and invasive detection methods, and have common limitations such as complex sample pretreatment, long detection cycle, and cumbersome operation procedures. The overall detection time is usually 1 to 2 working days.

[0004] In terms of sample type and testing process: ELISA requires the collection of human venous blood, separation and preparation of serum or plasma samples, and quantitative detection based on antigen-antibody specific binding reaction. It is currently the routine method for assessing human GLP-1 levels in clinical practice. In clinical testing, blood samples need to be collected from patients before and after fasting meals at designated locations. The entire testing process and report generation take about 24 hours. Western blot is only applicable to in vitro cell or ex vivo tissue samples and is only used for semi-quantitative analysis of GLP-1 protein in basic research. It cannot be applied to clinical in vivo testing. Immunohistochemistry requires obtaining ex vivo tissue specimens for immunostaining and microscopic observation. It can only achieve tissue localization analysis and cannot achieve in vivo, real-time, and quantitative detection.

[0005] In summary, existing GLP-1 detection technologies all have drawbacks such as invasive sampling, detection lag, complex operation, and inability to dynamically monitor, making it difficult to meet the clinical demand for rapid, real-time, minimally invasive, and continuous monitoring of GLP-1 levels.

[0006] Wearable detection devices are highly efficient and convenient miniaturized devices that are gaining increasing attention in fields such as disease diagnosis and biomarker detection. Microneedles are patches composed of an array of micron-sized needles. When piercing the skin, the microneedles only penetrate the epidermis without stimulating the dermis, which contains pain receptors. Furthermore, the micropores they create allow for trans-skin transport of substances, effectively improving the efficiency of disease marker diagnosis and drug delivery. For example, Chinese invention patent CN119414022A provides a wearable C-peptide detection device and its detection method and application. Its detection chip includes a microneedle array, a reaction chamber, and an injection unit connected sequentially through a liquid outlet channel. The microneedles are dual-hole microneedles, with holes penetrating the entire microneedle but without a pointed tip. The reaction chamber, microneedle array, and injection unit are connected. The reaction chamber contains a pre-embedded, fixed trap and receives the reaction liquid (including detection antibodies, washing solution, chromogenic solution, and eluent) injected by the syringe, reacting with the solution. After the reaction is complete, detection is performed via RGB readings. However, almost all steps in this detection process, such as sampling, reaction solution injection, elution, and reading, require manual operation, making the detection process cumbersome and difficult to meet the needs of real-time and continuous monitoring.

[0007] Wearable biosensors can be used to monitor GLP-1, enabling accurate, efficient, and convenient real-time acquisition of hormone levels. This is a highly effective measure for regulating GLP-1 levels in real time to control blood glucose and energy metabolism, thus meeting the needs of precision treatment for obesity. However, wearable biosensors based on sweat monitoring suffer from limitations such as difficulty in obtaining sweat and the extremely low concentration of large-molecule GLP-1 in sweat, resulting in insufficient sensitivity or inaccurate monitoring results in practical applications. Therefore, there is an urgent need to develop a precise and convenient device for GLP-1 level monitoring to achieve real-time monitoring of GLP-1.

[0008] Therefore, based on the above, the present invention aims to design a GLP-1 wearable monitoring device based on a microneedle biosensor. Summary of the Invention

[0009] This invention addresses the bottleneck of continuous monitoring of GLP-1 hormone levels in precision obesity treatment by constructing a microneedle biosensor for GLP-1 monitoring and integrating it into a wearable device. This allows for direct monitoring of GLP-1 concentration changes in interstitial fluid. Compared to traditional sweat monitoring, which may improve accuracy, this invention promises to achieve efficient, convenient, and real-time GLP-1 level monitoring, with broad application prospects in obesity treatment and health management.

[0010] In a first aspect, the present invention provides a microneedle biosensor for detecting glucagon-like peptide-1, comprising a three-electrode detection system formed by functionalizing conductive microneedles and a flexible conductive circuit connected thereto.

[0011] The three-electrode detection system includes a working electrode, a reference electrode, and a counter electrode.

[0012] The working electrode was constructed by depositing a layer of gold nanoparticles on the surface of conductive microneedles and anchoring glucagon-like peptide-1 antibody; the reference electrode was constructed by coating the surface of conductive microneedles with Ag / AgCl material.

[0013] The conductive microneedle itself serves as the counter electrode.

[0014] The preferred conditions for each component of the present invention are as follows:

[0015] (1) Regarding conductive microneedles

[0016] The conductive microneedle is obtained by conductive treatment of the prepared microneedle.

[0017] Preferably, the microneedle includes a substrate and a microneedle array integrally formed with the substrate. The microneedle substrate adopts an n×n conical microneedle array, in which each microneedle has a length of 500~1000μm and a tip spacing of 500~700μm.

[0018] In this invention, the shape of the microneedle array is not limited to a cone shape, but also includes a pyramid shape, etc.; in the array specification, n is selected from any integer from 3 to 10, preferably any integer from 3 to 6.

[0019] Furthermore, the microneedle array and the substrate are made of polystyrene (PS) or AS resin and are prepared by template centrifugation.

[0020] In a preferred embodiment of the present invention, the microneedles are prepared as follows: 200 μL to 500 μL of PS solution is added to a polydimethylsiloxane (PDMS) microneedle template, and the template is centrifuged at 3000 to 4000 rpm for 5 to 10 minutes. After the PS solution in the microneedle template is completely dried, the template is demolded to obtain PS microneedles.

[0021] Preferably, the conductive processing method for the microneedle array includes depositing a metal conductive layer on the surface of the microneedle using physical sputtering, vacuum evaporation, or chemical deposition.

[0022] Furthermore, the conductive metal layer is selected from gold, platinum, palladium, rhodium, iridium, iron, or chromium; preferably gold or platinum.

[0023] In one preferred embodiment of the present invention, the method for setting a metal conductive layer by physical sputtering is as follows: the microneedle is placed with the tip facing upward in the chamber of a vacuum sputtering instrument, and the target conductive layer metal target is used as the metal source for metal deposition. The vacuum degree of the deposition environment is 1.0 Pa to 3.0 Pa, the deposition process current is 20 mA to 30 mA, and the deposition time is 200 s to 100 s.

[0024] (2) Working electrode

[0025] When constructing the working electrode, gold nanoparticles were deposited on the surface of conductive microneedles as antibody anchoring sites by electrodeposition. Specifically, the conductive microneedles were used as the working electrode, forming a three-electrode electrochemical system with a saturated calomel reference electrode and a platinum wire counter electrode. Electrochemical deposition was carried out in a 1 mM~5 mM chloroauric acid solution, with a deposition voltage of -0.25 V~-0.5 V and a deposition time of 30 s~300 s.

[0026] Subsequently, a glucagon-like peptide-1 antibody was anchored onto the gold nanoparticles using a self-assembled monolayer covalent modification method. The self-assembled monolayers used included, but were not limited to, aminopropyltriethoxysilane (APTES) and mercaptoundecanoic acid.

[0027] In this process, aminopropyltriethoxysilane is linked to gold nanoparticles via 6-mercapto-1-hexanol, and mercaptoundecanoic acid is directly linked to gold nanoparticles via Au-S bonds. Subsequently, the specific antibody is anchored via amide bonds in a 0.1~0.5 mg / mL glucagon-like peptide-1 antibody solution to complete the antibody modification.

[0028] In one preferred embodiment of the present invention, the method for modifying GLP-1 specific antibodies using the APTES self-assembled monolayer covalent modification method is as follows: (1) The conductive microneedle electrode with gold nanoparticles attached to its surface is immersed in an anhydrous ethanol solution of 50 mM~150 mM 6-mercapto-1-hexanol for 1 h~3 h to perform hydroxylation of the gold nanoparticle surface; (2) After hydroxylation, the microneedle surface is repeatedly washed three times with anhydrous ethanol and deionized water. After washing, the microneedle electrode is immersed in 95% ethanol solvent containing 5%~10% APTES for 1 h~3 h to perform surface amination. After amination, the microneedle surface is repeatedly washed three times with anhydrous ethanol and deionized water; (3) After washing, the microneedle electrode is immersed in 2.5% glutaraldehyde solution with PBS as solvent for 1 h~3 h and repeatedly washed three times with deionized water; (4) After washing, the microneedle electrode is immersed in GLP-1 antibody solution of 200 μg / mL~800 μg / mL for 1 h~3 h. h was used for antibody modification, wherein the pH of the GLP-1 antibody solution was 8 and Tris-HCl buffer was used as the solvent; (5) After antibody modification, the microneedle electrode was immersed in 50 mM~100 mM ethanolamine solution for blocking. After blocking, it was washed three times with deionized water to obtain the working electrode of the GLP-1 antibody-modified microneedle biosensor.

[0029] (3) Regarding the reference electrode and the counter electrode

[0030] Reference electrode functionalization methods include, but are not limited to, Ag / AgCl slurry drop coating, spin coating, or preparation by FeCl3 oxidation after Ag metal sputtering. In a preferred embodiment of the present invention, the reference electrode is functionalized using the Ag / AgCl slurry drop coating method, with the amount of Ag / AgCl slurry dropped being 50-200 μL.

[0031] (4) Regarding sensor assembly

[0032] The reference electrode, counter electrode, and working electrode are connected to an external circuit through flexible conductive paths and assembled to complete the fabrication of the microneedle sensor, and its performance is then tested.

[0033] Preferably, the core modules of the external circuit system include, but are not limited to, the STM32F0 processing module and the RC6621A Bluetooth transmission module.

[0034] Based on the above description, a second aspect of the present invention provides a preferred assembly method for a microneedle biosensor, comprising the following steps:

[0035] S1, the microneedle substrate is made of polystyrene (PS) material and is prepared by template centrifugation.

[0036] S2, the obtained polystyrene microneedle substrate is conductively treated by metal sputtering, and a platinum metal conductive layer is physically deposited on the surface of the microneedle to complete the conductive treatment of the microneedle.

[0037] S3, the microneedle biosensor comprises three characteristic parts: working electrode, reference electrode and counter electrode. The working electrode is prepared by antibody modification. First, a layer of gold nanoparticles is deposited on the surface of the conductive microneedle described in S2 by electrodeposition. Then, a single-molecule self-assembled layer is used to anchor the glucagon-like peptide-1 antibody to complete the construction of the working electrode.

[0038] S4, the reference electrode is prepared by coating the surface of the conductive microneedles described in S2 with Ag / AgCl material to complete the functionalization of the reference electrode, and the counter electrode is the platinum-plated conductive microneedles described in S2.

[0039] S5. The reference electrode, counter electrode, and working electrode are connected to the external circuit through flexible conductive paths, and the assembly is completed to complete the fabrication of the microneedle sensor and test its performance.

[0040] In a third aspect, the present invention provides a wearable monitoring device for detecting glucagon-like peptide-1, comprising the microneedle biosensor described above, and further supplemented by an integrated circuit and a software module.

[0041] Furthermore, the device is wirelessly connected to a visualized smart terminal. The microneedle biosensor is used to monitor glucagon-like peptide-1 (GLP-1) levels in real time, and the changes in current detected by the sensor are visualized to the mobile smart terminal via wireless transmission, thus completing real-time monitoring of GLP-1 hormone levels.

[0042] Compared with the prior art, the technical solution provided by the present invention has the following significant advantages and beneficial effects:

[0043] Compared to existing GLP-1 detection technologies, this invention, based on minimally invasive sensing microneedles for real-time GLP-1 detection, achieves key breakthroughs in detection principle, implementation method, response speed, and clinical applicability. Its core advantages are as follows:

[0044] (1) Ultra-fast response: The detection system of the present invention has efficient and stable signal transmission. It can complete specific identification and output a stable and reliable GLP-1 detection signal within 30 seconds, without waiting for sample processing and complex reaction process, thus realizing instant detection;

[0045] (2) No need for ex vivo samples: The entire testing process does not require venous blood collection, serum / plasma preparation, or extraction of cells and ex vivo tissue samples, completely avoiding the invasive sampling process of traditional methods, greatly reducing the difficulty of clinical operation and patient discomfort;

[0046] (3) Minimally invasive and painless: Flexible sensing microneedles are used as detection carriers. The microneedles are small in size and the insertion depth is controllable, resulting in minimal damage to the skin and body tissues, achieving truly minimally invasive and painless detection with higher safety.

[0047] (4) Real-time dynamic monitoring: It can realize the real-time and continuous monitoring of GLP-1 level changes in vivo, breaking through the limitations of traditional methods such as single-point, offline and lagging detection. It can dynamically capture the GLP-1 fluctuation pattern in key time windows such as before and after meals and before and after drug administration, and has good continuity and long-term monitoring feasibility. Attached Figure Description

[0048] Figure 1 A schematic diagram (a) and a sensing mechanism diagram (b) of the GLP-1 microneedle biosensor are shown.

[0049] Figure 2 The image shows a scanning electron microscope image of the GLP-1 microneedle biosensor;

[0050] Figure 3 The mechanical properties of the GLP-1 microneedle biosensor are shown.

[0051] Figure 4 The biosafety results of the GLP-1 microneedle biosensor are shown;

[0052] Figure 5 The electrochemical impedance spectroscopy of the GLP-1 microneedle biosensor is shown.

[0053] Figure 6 The current-time curve of the GLP-1 microneedle biosensor in response to GLP-1 is shown.

[0054] Figure 7 The sensitivity fitting curve of the GLP-1 microneedle biosensor is shown;

[0055] Figure 8 A block diagram of the GLP-1 microneedle biosensor wearable device design is shown. Detailed Implementation

[0056] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0057] Example 1: Structure and fabrication of GLP-1 microneedle biosensor

[0058] This invention constructs a microneedle biosensor for GLP-1 monitoring and integrates it into a wearable device, which can directly monitor changes in GLP-1 concentration in interstitial fluid.

[0059] As a core component of the GLP-1 wearable monitoring device, the fabrication diagram and sensing mechanism diagram of the GLP-1 microneedle biosensor are shown below. Figure 1 As shown, GLP-1-specific antibodies were covalently modified onto the surface of microneedle electrodes, and the changes in electrode characteristics caused by the specific binding of the antibodies to GLP-1 were analyzed, thereby achieving GLP-1 sensing and monitoring.

[0060] See the schematic diagram of the fabrication process of the GLP-1 microneedle biosensor. Figure 1 a. The specific process is as follows:

[0061] (1) Substrate microneedles were prepared by template centrifugation method.

[0062] PS was used as the preparation material. 10-30 g of polystyrene (PS) was weighed and added to a 500 mL Erlenmeyer flask, and 50 mL-150 mL of N,N-dimethylformamide (DMF) was added. The mixture was heated to 120 °C until completely dissolved to obtain a PS solution.

[0063] 200 μL–500 μL of PS solution was added to a polydimethylsiloxane (PDMS) microneedle template, and the template was centrifuged at 3000–4000 rpm for 5–10 minutes. After the PS solution inside the microneedle template was completely dried, the template was demolded to obtain the PS microneedle substrate. The microneedle biosensor array parameters adopted a 3×3 conical microneedle array with a tip length of 500 μm–1000 μm and a tip spacing of 500 μm–700 μm.

[0064] In this example, the tip shape of the substrate microneedles is conical, and the array size is 3×3. However, the tip shape is not limited to conical; it can also be pyramidal, etc., and the array size is not limited to 3×3. It can be set according to actual needs.

[0065] (2) Polystyrene-based microneedles are conductively treated by metal sputtering, and a platinum metal conductive layer is physically deposited on the surface of the microneedles to complete the conductive treatment of the microneedles.

[0066] After obtaining the PS microneedle substrate, the PS microneedles are subjected to conductive treatment. The polarization methods include, but are not limited to, magnetron sputtering, vacuum evaporation, chemical deposition, etc., and the polarization metal materials include, but are not limited to, gold, platinum, etc.

[0067] In this embodiment, platinum metal deposition is used to make the microneedles conductive. Specifically, the prepared PS microneedles are placed with the tip facing upward in the chamber of a vacuum sputtering instrument, and metal deposition is performed using a platinum target as the metal source. The vacuum level of the deposition environment is 1.0 Pa to 3.0 Pa, the deposition current is 20 mA to 30 mA, and the deposition time is 200 s to 100 s. After the platinum metal deposition is completed, conductive microneedles are obtained.

[0068] (3) Construction of the three-electrode system

[0069] The microneedle biosensor comprises three components: a working electrode, a reference electrode, and a counter electrode. The working electrode is fabricated using an antibody-modified method. First, a layer of gold nanoparticles is deposited on the surface of a conductive microneedle. Then, a single-molecule self-assembled layer is used to anchor a glucagon-like peptide-1 antibody, completing the construction of the working electrode. Details are as follows:

[0070] Gold nanoparticles were deposited on the surface of conductive microneedles as antibody anchoring sites via electrodeposition. Specifically, the conductive microneedles were used as the working electrode, forming a three-electrode electrochemical system with a saturated calomel reference electrode and a platinum wire counter electrode. Electrochemical deposition was carried out in a 1 mM to 5 mM chloroauric acid solution, with a deposition voltage of -0.25 V to -0.5 V and a deposition time of 30 s to 300 s, resulting in a microneedle electrode with gold nanoparticles attached to its surface.

[0071] GLP-1 specific antibodies were modified onto gold nanoparticles on the surface of a microneedle electrode using a self-assembled monolayer covalent modification method. The self-assembled monolayers used included, but were not limited to, aminopropyltriethoxysilane (APTES) and mercaptoundecanoic acid. In this embodiment, APTES self-assembled monolayer covalent modification was used for GLP-1 specific antibody modification. Microneedle electrodes with gold nanoparticles attached to their surface were immersed in 50 mM–150 mM 6-mercapto-1-hexanol (anhydrous ethanol as solvent) for 1–3 h to hydroxylate the gold nanoparticles. After hydroxylation modification, the microneedle surface was repeatedly washed three times with anhydrous ethanol and deionized water. Following washing, the microneedle electrodes were immersed in 5%–10% APTES (95% ethanol solvent) for 1–3 h to amination. After amination, the microneedle surface was repeatedly washed three times with anhydrous ethanol and deionized water. After washing, the microneedle electrodes were immersed in 2.5% glutaraldehyde solution (PBS solvent) for 1–3 h and repeatedly washed three times with deionized water. After washing, the microneedle electrodes were immersed in 200 μg / mL–800 μg / mL GLP-1 antibody solution (Tris-HCl buffer solvent, pH 8) for 1–3 h to modify the antibody. After antibody modification, the microneedle electrodes were immersed in 50 mM–100 mM 6-mercapto-1-hexanol for 1–3 h to hydroxylate the gold nanoparticles. The microneedle sensor was blocked in a mM ethanolamine solution, and after blocking, it was washed three times with deionized water to obtain the working electrode of the GLP-1 antibody-modified microneedle sensor.

[0072] The reference electrode of the microneedle biosensor is an Ag / AgCl microneedle reference electrode. The preparation methods of Ag / AgCl material include, but are not limited to, drop coating, spin coating, sputtering, and chemical synthesis. In this embodiment, the drop coating Ag / AgCl slurry preparation method is adopted, in which 50 μL~200 μL of Ag / AgCl slurry is drop-coated onto the surface of conductive microneedles. After the slurry is completely dried, the Ag / AgCl microneedle reference electrode is obtained.

[0073] The counter electrode of the microneedle biosensor uses the aforementioned conductive microneedles deposited with metallic platinum.

[0074] (4) Assembly of microneedle biosensors

[0075] The reference electrode, counter electrode, and working electrode are connected to an external circuit through flexible conductive paths and assembled to complete the fabrication of the microneedle sensor, and its performance is then tested.

[0076] For the sensing mechanism of microneedle biosensors, please refer to [link / reference]. Figure 1b. The working electrode of the GLP-1 microneedle biosensor includes a substrate microneedle, a platinum conductive layer, a gold nanoparticle layer, a self-assembled monolayer, and a GLP-1 specific antibody layer. The self-assembled monolayer is fixed to the surface of the gold nanoparticles by gold-sulfur bonds, and the self-assembled monolayer is anchored to the GLP-1 antibody by amide bonds. With the help of the GLP-1 antibody sensor, GLP-1 can be specifically captured. After GLP-1 specifically binds to the antibody on the sensor surface, it will affect the conductivity of the electrode surface and cause a change in current. Current information can be obtained by current-time monitoring to complete GLP-1 sensing.

[0077] Example 2: Morphology of GLP-1 microneedle biosensing electrode

[0078] Sample: The working electrode of the GLP-1 microneedle biosensor prepared in Example 1.

[0079] Experimental Methods: The working electrode of the prepared GLP-1 microneedle biosensor was fixed to the sample holder of a scanning electron microscope using conductive tape. The morphology of the microneedle sensor was observed using a field emission scanning electron microscope (FEI, Apero Hivoc). Results are shown below. Figure 2 The study showed that a metal layer appeared on the smooth PS microneedle surface after platinum sputtering, followed by the appearance of obvious granular gold nanoparticles on the surface during electrodeposition, which provided a good microneedle platform for subsequent antibody modification and GLP-1 detection.

[0080] Example 3 Mechanical Performance Testing of GLP-1 Microneedle Biosensing Electrode

[0081] Sample: The working electrode of the GLP-1 microneedle biosensor prepared in Example 1.

[0082] Experimental method: The working electrode of the prepared GLP-1 microneedle biosensor was attached and fixed to the center of the base below the material mechanical property tester with the needle tip facing upward. The microneedle was compressed by the platform above the tester, and the displacement and pressure curve of the pressure stage were recorded.

[0083] Test results are as follows Figure 3 The results show that the mechanical properties of the GLP-1 microneedle biosensor exhibit a fracture hardness of 0.3 N / needle in the pressure test, which is much greater than the mechanical performance requirement of 0.058 N / needle required for skin puncture. This indicates that the microneedle can effectively puncture the skin to monitor and sense GLP-1 in the interstitial fluid.

[0084] Example 4: Biosafety Test of GLP-1 Microneedle Biosensing Electrode

[0085] Sample: The working electrode of the GLP-1 microneedle biosensor prepared in Example 1.

[0086] Experimental Methods: The biosafety of the prepared GLP-1 microneedle biosensor working electrode was assessed by cytotoxicity evaluation through co-culture with normal cells. Mouse epithelial-like fibroblasts (L929) were used as the cell line. After sterilization, the GLP-1 microneedle biosensor was placed in sterile culture dishes and immersed in high-glucose DMEM medium containing 10% fetal bovine serum under a 5% CO2 atmosphere. L929 cells were seeded at a density of 20,000 cells / well in 96-well cell culture plates and cultured for 24 h. The medium used to immerse the GLP-1 microneedle biosensor was then added to the wells, replacing the original medium. After 24 h of culture, live and dead cells were stained using Calcein-AM / PI, and cell viability was observed using fluorescence microscopy to assess the biosafety of the GLP-1 microneedle biosensor.

[0087] The results are as follows Figure 4 This indicates that the GLP-1 microneedle biosensor has good biosafety and no obvious cytotoxicity.

[0088] Example 5: Electrochemical Impedance Spectroscopy of GLP-1 Microneedle Biosensing Electrode

[0089] Sample: The working electrode of the GLP-1 microneedle biosensor prepared in Example 1.

[0090] Experimental Methods: During the fabrication of the GLP-1 microneedle biosensor, the adhesion of different materials to the conductive microneedles affects the charge migration characteristics of the electrode surface. The charge migration state of the electrode surface was measured by electrochemical impedance spectroscopy. The GLP-1 microneedle biosensor was used as the working electrode, platinum conductive microneedles as the counter electrode, and Ag / AgCl microneedles as the reference electrode. Electrochemical impedance spectra were collected from 100 Hz to 10000 Hz in K3[Fe(CN)6] / K4[Fe(CN)6] electrolyte at a test voltage of 0.1 V and an amplitude of 0.05 V.

[0091] Test results are as follows Figure 5 The results show that the surface impedance of the GLP-1 microneedle biosensor changes during the microneedle processing. The resistance decreases slightly after electrodeposition of Au nanoparticles, but increases significantly after antibody modification due to the effect of the non-conductive protein layer on charge transfer, proving that the GLP-1 antibody modification was successful.

[0092] Example 6: Response of GLP-1 microneedle biosensor to GLP-1

[0093] Sample: The working electrode of the GLP-1 microneedle biosensor prepared in Example 1.

[0094] Experimental methods: GLP-1 microneedle biosensor was used as the working electrode, platinum conductive microneedles as the counter electrode, and Ag / AgCl microneedles as the reference electrode. The current-time curves of the working electrode were recorded in PBS solutions containing different concentrations of GLP-1.

[0095] result Figure 6 The results show that the GLP-1 microneedle biosensor exhibits a good current response to different concentrations of GLP-1, with the current decreasing as the GLP-1 concentration increases. This decrease in current response is due to the fact that GLP-1 specifically binds to the antibody, hindering charge transfer and thus reducing the charge mobility on the electrode surface. The sensitivity fitting curve of the GLP-1 microneedle biosensor is shown in the figure. Figure 7 As shown, the results indicate that the sensor has a linear response to GLP-1.

[0096] Example 7: GLP-1 Microneedle Biosensor Wearable System Design

[0097] Sample: GLP-1 microneedle biosensor prepared in Example 1.

[0098] Experimental Methods: A wearable GLP-1 level monitoring sensor was fabricated using a GLP-1 microneedle biosensor as the working electrode, along with a silver / silver chloride microneedle reference electrode and a platinum counter electrode, forming a three-electrode detection system. The system was developed through microcircuit design and flexible circuit fabrication. The circuitry includes current detection and signal amplification circuitry for the three-electrode system, an analog-to-digital converter, and a Bluetooth transmission module. A block diagram of the main components on the flexible circuit board is shown below. Figure 8 As shown.

[0099] The core modules of the circuit system used include, but are not limited to, the STM32F0 processing module and the RC6621A Bluetooth transmission module.

[0100] At the same time, the wearable system connects to a smart visual terminal, receiving analysis data from the wearable system to display and summarize the detection data in real time.

[0101] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above specific embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A microneedle biosensor for detecting glucagon-like peptide-1, characterized in that, This includes a three-electrode detection system formed by functionalizing conductive microneedles and a flexible conductive circuit connected to it. The three-electrode detection system consists of a working electrode, a reference electrode, and a counter electrode. The working electrode was constructed by depositing a layer of gold nanoparticles on the surface of conductive microneedles and anchoring glucagon-like peptide-1 antibody. The reference electrode was constructed by coating the surface of conductive microneedles with Ag / AgCl material. The conductive microneedle itself serves as the counter electrode.

2. The microneedle biosensor for glucagon-like peptide-1 detection according to claim 1, characterized in that: in, Microneedles include a substrate and an array of microneedles integrally formed with the substrate, wherein each microneedle in the array is of length... The diameter is 500~1000μm, and the needle tip spacing is 500~700μm; The conductive processing methods for microneedle arrays include depositing a metal conductive layer on the surface of the microneedles using physical sputtering, vacuum evaporation, or chemical deposition.

3. The microneedle biosensor for glucagon-like peptide-1 detection according to claim 2, characterized in that: in, The microneedle array and the substrate are made of polystyrene (PS) or AS resin; The conductive metal layer is selected from gold, platinum, palladium, rhodium, iridium, iron, or chromium; The method for setting a metal conductive layer using physical sputtering is as follows: the microneedle is placed with the tip facing upward in the vacuum sputtering chamber, and the target conductive layer metal target is used as the metal source for metal deposition. The vacuum degree of the deposition environment is 1.0 Pa to 3.0 Pa, the deposition process current is 20 mA to 30 mA, and the deposition time is 200 s to 100 s.

4. The microneedle biosensor for glucagon-like peptide-1 detection according to claim 1, characterized in that: in, When constructing the working electrode, gold nanoparticles were deposited on the surface of conductive microneedles as antibody anchoring sites by electrodeposition. Specifically, the conductive microneedles were used as the working electrode, forming a three-electrode electrochemical system with a saturated calomel reference electrode and a platinum wire counter electrode. Electrochemical deposition was carried out in a 1 mM~5 mM chloroauric acid solution, with a deposition voltage of -0.25 V~-0.5 V and a deposition time of 30 s~300 s.

5. The application according to claim 1, characterized in that: in, A self-assembled monolayer covalent modification method was used to anchor glucagon-like peptide-1 antibody onto gold nanoparticles. The self-assembled monolayers used included, but were not limited to, aminopropyltriethoxysilane (APTES) and mercaptoundecanoic acid. In this process, aminopropyltriethoxysilane is linked to gold nanoparticles via 6-mercapto-1-hexanol, and mercaptoundecanoic acid is directly linked to gold nanoparticles via Au-S bonds. Subsequently, the specific antibody is anchored via amide bonds in a 0.1~0.5 mg / mL glucagon-like peptide-1 antibody solution to complete the antibody modification.

6. The microneedle biosensor for glucagon-like peptide-1 detection according to claim 5, characterized in that: in, The method for modifying GLP-1 specific antibodies using the APTES self-assembled monolayer covalent modification method is as follows: (1) Immerse the conductive microneedle electrode with gold nanoparticles attached to its surface in anhydrous ethanol solution of 50 mM~150 mM 6-mercapto-1-hexanol for 1 h~3 h to perform hydroxylation of the gold nanoparticle surface; (2) After completing the hydroxylation modification, wash the microneedle surface three times with anhydrous ethanol and deionized water. After washing, immerse the microneedle electrode in 95% ethanol solvent containing 5%~10% APTES for 1 h~3 h to perform surface amination. After amination, wash the microneedle surface three times with anhydrous ethanol and deionized water; (3) After cleaning, immerse the microneedle electrode in 2.5% glutaraldehyde solution with PBS as solvent for 1 h~3 h and wash it three times with deionized water; (4) After cleaning, immerse the microneedle electrode in GLP-1 antibody solution of 200 μg / mL~800 μg / mL for 1 h~3 h. h was used for antibody modification, wherein the pH of the GLP-1 antibody solution was 8 and Tris-HCl buffer was used as the solvent; (5) After antibody modification, the microneedle electrode was immersed in 50mM~100mM ethanolamine solution for blocking. After blocking, it was washed three times with deionized water to obtain the working electrode of the GLP-1 antibody-modified microneedle biosensor.

7. The microneedle biosensor for glucagon-like peptide-1 detection according to claim 1, characterized in that: in, The reference electrode, counter electrode, and working electrode are each connected to an external circuit via a flexible conductive path.

8. A wearable monitoring device for detecting glucagon-like peptide-1, characterized in that, Includes the microneedle biosensor, integrated circuit, and software module as described in any one of claims 1 to 6.

9. The wearable monitoring device for glucagon-like peptide-1 detection according to claim 8, characterized in that, The device connects wirelessly to a visual smart terminal. The microneedle biosensor is used to monitor glucagon-like peptide-1 levels in real time, and the changes in current detected by the sensor are visualized to a mobile smart terminal via wireless transmission, thus completing the real-time monitoring of glucagon-like peptide-1 hormone levels.

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