An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid

Abstract

The application relates to the technical field of biosensing, and particularly discloses an implantable flexible microneedle sensor for continuously monitoring glucose and uric acid, which comprises a flexible substrate and a reference electrode, a counter electrode, a glucose detection working electrode and a uric acid detection working electrode integrated on the substrate; the two electrodes share the reference electrode and the counter electrode and are synchronously detected under a unified potential window of +0.1V to +0.2V. The former adopts a glucose oxidase (GOx) enzymatic detection scheme, is modified by a polypyrrole layer and a platinum nanoparticle layer, and is fixed with a GOx composite solution to realize specific recognition of glucose; the latter adopts a non-enzymatic scheme of a molecularly imprinted polymer (MIP), is constructed by a polyaniline / carbon nanotube composite layer and an MIP film to realize high-selectivity adsorption of uric acid. Through the triple innovation of recognition mechanism, potential design and integrated structure, the core problems of insufficient selectivity, signal crosstalk and serious interference are solved, in-situ continuous monitoring is realized in a minimally invasive manner, and the anti-interference performance and accuracy of synchronous detection are improved.

Description

Technical Field

[0001] This invention relates to the field of glucose and uric acid detection technology, and in particular to an implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid. Background Technology

[0002] Glucose is the body's primary energy source, participating in cellular metabolism. Its metabolism is regulated by hormones such as insulin and glucagon, maintaining a relatively stable concentration in the blood. Hyperglycemia caused by glucose imbalance is a typical marker of diabetes. Long-term hyperglycemia can lead to serious complications such as retinopathy, neuropathy, kidney damage, and cardiovascular and cerebrovascular diseases. Uric acid is the final product of purine nucleotide metabolism. In the body, it is oxidized from xanthine and excreted mainly through the kidneys. Uric acid has both antioxidant and pro-inflammatory effects; in appropriate amounts, it can scavenge free radicals, but excessive amounts can lead to the deposition of urate crystals, causing gout, and is closely related to metabolic syndrome, hypertension, cardiovascular disease, and kidney disease. Glucose and uric acid, as core biomarkers of energy metabolism and purine metabolism, respectively, are of great significance in assessing human metabolic status, diagnosing diseases, and managing interventions.

[0003] With the increasing prevalence of metabolic diseases and the growing demand for personalized management of chronic diseases, precise and simultaneous monitoring of multiple indicators has become an important development direction for biosensor technology. For example, Chinese patent application CN117554450A provides an electrochemical sensor based on screen-printed electrodes for the simultaneous detection of glucose and uric acid: glucose detection employs a non-enzymatic molecular imprinting strategy, utilizing the spatial structure of the imprinted channels to specifically identify glucose; uric acid detection employs a carbon nanomaterial catalytic strategy, directly oxidizing uric acid through the electrocatalytic activity of carbon nanomaterials, and using differential pulse voltammetry to detect the peak signal for quantification. However, neither involves biological enzymes. Glucose detection relies on the spatial structure recognition of the molecularly imprinted membrane, and its specificity is far lower than that of biological enzymes. It is prone to cross-reactivity with structurally similar substances such as fructose and maltose. Uric acid detection relies solely on the catalytic activity of carbon nanomaterials, lacking a specific recognition mechanism and exhibiting poor anti-interference capabilities, resulting in insufficient detection selectivity.

[0004] In addition, Chinese patent application CN119000820A discloses a flexible microneedle sensor with simultaneous detection of glucose and uric acid. Based on the catalytic properties of composite materials, glucose and uric acid undergo redox reactions at different potentials. The oxidation current signals of the two substances are collected separately using differential pulse voltammetry, and quantitative detection is achieved by combining linearity. However, the oxidation-dependent potential difference between glucose and uric acid is only 0.15V, which easily leads to overlapping oxidation peaks, resulting in insufficient specificity and poor accuracy of simultaneous detection.

[0005] Therefore, there is an urgent need for an implantable flexible microneedle sensor that can simultaneously detect glucose and uric acid while improving detection resistance and accuracy for continuous monitoring of glucose and uric acid. Summary of the Invention

[0006] This invention provides an implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid, which can simultaneously detect glucose and uric acid and improve detection resistance and accuracy.

[0007] To solve the above-mentioned technical problems, this application provides the following technical solution:

[0008] An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid includes a flexible substrate and a reference electrode, a counter electrode, a glucose detection working electrode, and a uric acid detection working electrode integrated into the flexible substrate. The glucose detection working electrode and the uric acid detection working electrode share the reference electrode and the counter electrode, and achieve synchronous detection within a unified potential window of +0.1V to +0.2V.

[0009] The preparation process of the glucose detection working electrode is as follows: the gold electrode substrate is ultrasonically cleaned with anhydrous ethanol and deionized water sequentially and then dried; cyclic voltammetry is performed for 8 cycles in a mixed solution of 0.2M pyrrole and 0.2M KCl at a potential range of 0~0.8V and a scan rate of 50mV / s to form a polypyrrole layer through in-situ electropolymerization; the electrode is immersed in a mixed solution of 1mM H2PtCl6 and 0.1M H2SO4 at a constant potential of –0.2V for 300s to form a platinum nanoparticle layer; a glucose oxidase composite solution is drop-coated and cured, wherein the composite solution is diluted with PBS buffer at pH 7.4 and contains 10mg / mL GOX, 1% w / v polyvinylpyrrolidone K30, 5mg / mL bovine serum albumin, 3% w / v trehalose and 0.05% v / v Tween 20;

[0010] The preparation process of the working electrode for uric acid detection is as follows: the gold electrode substrate is ultrasonically cleaned sequentially with anhydrous ethanol and deionized water, and then dried with nitrogen; a polyaniline / carbon nanotube composite conductive layer is deposited by cyclic voltammetry scanning for 5-10 cycles in a 1.0M HCl solution containing 0.2M aniline monomer and 1.0 mg / mL multi-walled carbon nanotubes within a potential range of −0.2V to +0.8V and a scan rate of 50mV / s; the electrode is then immersed in a pH 7.0 PBS electropolymerization solution containing 0.5mM uric acid, 10mM o-phenylenediamine, and 5mM Fe(CN)6³⁻ within a potential range of 0-0.8V for 20 cycles to construct a molecularly imprinted polymer film; after elution with PBS solution to remove uric acid template molecules, 1μL of 0.1wt% Nafion solution is drop-coated as a protective layer and then dried;

[0011] The reference electrode is a carbon electrode coated with Ag / AgCl mixed ink, and its surface is covered with a thin layer of Nafion ion exchange membrane; the counter electrode is a flexible carbon electrode that has been treated with oxygen plasma and then electrodeposited at a constant potential of +0.2V for 300s in a mixed solution of 0.5mM chloroplatinic acid and 0.5M sulfuric acid to form a Pt nanoparticle modified layer, and its surface is covered with a thin layer of Nafion ion exchange membrane.

[0012] The basic principles and beneficial effects of this solution are as follows: This solution employs a dual-path integration strategy, combining enzymatic detection of glucose oxidase with non-enzymatic detection of uric acid via a MIP membrane, precisely meeting the demands of continuous monitoring for device miniaturization and circuit simplification. Glucose detection leverages the specific recognition characteristics of glucose oxidase for β-D-glucose, while uric acid detection relies on the specific memory pores of the MIP membrane to adsorb and enrich target molecules. This dual specific recognition mechanism enhances anti-interference capabilities and effectively avoids interference from coexisting substances such as ascorbic acid and dopamine. Simultaneously, the two working electrodes share a reference electrode and a counter electrode, both operating within a unified potential window of +0.1V to +0.2V, eliminating the need for complex potential switching circuits and completely avoiding crosstalk between the two biomarkers, thus simplifying the solution design. Integrating dual-indicator detection into the same flexible microneedle electrode system enables in-situ minimally invasive sampling of subcutaneous tissue fluid and simultaneous continuous monitoring of multiple indicators. While ensuring detection accuracy, it meets the needs of simultaneous measurement of multiple body indicators in minimally invasive scenarios and is suitable for wearable continuous monitoring applications.

[0013] In this scheme, compared to the dual non-enzyme strategy, such as glucose MIP + uric acid carbon material, which relies on material / structure recognition, this scheme innovatively employs a hybrid strategy of enzyme method + MIP through recognition mechanism. Glucose detection uses glucose oxidase, which specifically recognizes β-D-glucose and is unresponsive to structurally similar sugars. Uric acid detection uses a molecularly imprinted polymer membrane, which specifically adsorbs uric acid through memory channels constructed from template molecules. This dual recognition mechanism fundamentally improves selectivity, effectively eliminating coexisting interfering substances. Furthermore, independent dual working electrodes are used, each matched with a dedicated recognition system, eliminating potential-dependent differences and reducing the likelihood of oxidation peak overlap, thus completely avoiding signal crosstalk. In addition, compared to the tendency for high-potential range in uric acid detection to amplify the influence of interfering substances and the introduction of stray signals in a wide potential range, this scheme's dual working electrodes operate within a low potential window of +0.1V to +0.2V, which is below the oxidation potential of interfering substances and avoids non-specific reactions at high potentials. It also eliminates the need for complex potential switching circuits, reducing signal resolution errors. Moreover, the low-potential design combined with the dedicated recognition mechanism improves signal purity and ensures detection accuracy.

[0014] In addition, compared to rigid screen-printed electrodes and microneedle array designs, the former cannot perform minimally invasive transdermal sampling and can only be used for single in vitro detection. It is also prone to loss and contamination during sample transmission, resulting in low accuracy. The latter has multiple microneedles dispersed in different areas of the skin, which can easily cause signal fluctuations due to differences in body fluid concentration and environmental conditions. This solution is based on a flexible polyimide substrate to construct a microneedle structure, which can minimally penetrate the stratum corneum of the skin and extract subcutaneous tissue fluid in situ, avoiding errors caused by samples leaving the body. It is suitable for continuous monitoring scenarios. At the same time, the dual working electrodes, reference electrodes, and counter electrodes are integrated into a single microneedle to ensure that each electrode contacts the same body fluid environment, reducing signal deviations caused by differences in skin impedance and conduction, and improving detection consistency and accuracy.

[0015] In summary, this solution, through triple innovation in identification mechanism, potential design, and integrated structure, not only solves the core problems of insufficient selectivity, signal crosstalk, and severe interference, but also achieves minimally invasive in-situ continuous monitoring, improving the anti-interference capability and accuracy of synchronous detection.

[0016] Furthermore, the flexible substrate is a polyimide film, and the glucose detection working electrode, uric acid detection working electrode, reference electrode and counter electrode are formed by pre-patterning by laser etching, followed by magnetron sputtering deposition of a 120nm thick gold film and mask peeling.

[0017] The beneficial effects are: the polyimide film has excellent flexibility and biocompatibility, the laser etching and magnetron sputtering process ensures the precision of the electrode pattern and the firm adhesion of the gold film, and the 120nm thickness can balance conductivity and minimal invasiveness.

[0018] Furthermore, the Ag / AgCl mixed ink of the reference electrode is coated onto the surface of the carbon electrode by screen printing, and after drying, a stable reference electrode is formed.

[0019] The beneficial effects are as follows: the screen printing process achieves uniform coating of Ag / AgCl mixed ink, and the reference electrode formed after drying is stable and reliable, which can provide accurate potential reference for dual working electrodes, reduce detection errors, and ensure the accuracy of quantitative detection of glucose and uric acid.

[0020] Furthermore, the pretreatment of the electrode includes: ultrasonic cleaning with ethanol and deionized water for 5 minutes each, drying with nitrogen, and then oxygen plasma treatment for 60 seconds.

[0021] The beneficial effects are: ultrasonic cleaning removes impurities from the electrode surface, oxygen plasma treatment enhances surface activity, strengthens the adhesion and conductivity of the subsequent Pt nanoparticle modification layer, ensures the electron transmission efficiency of the electrode, and improves the overall electrochemical stability of the sensor.

[0022] Furthermore, the GOx composite solution of the glucose detection working electrode is applied in a drop volume of 20 μL, and after standing at room temperature for 5 minutes, it is dried at 37°C for 15 minutes to complete the curing.

[0023] The beneficial effects are: precise control of the GOx composite liquid droplet application and curing process ensures uniform and firm adhesion of the enzyme layer; BSA, trehalose and other components work together to protect GOx activity, extend the service life of the enzyme layer, and ensure the sensitivity and long-term stability of glucose detection.

[0024] Furthermore, the PANI / CNT composite conductive layer of the uric acid detection working electrode is deposited in situ by cyclic voltammetry, wherein the scanning voltage range of the cyclic voltammetry is −0.2V to +0.8V, and the scanning rate is 50mV / s.

[0025] The beneficial effects are as follows: cyclic voltammetry with specific parameters enables in-situ uniform deposition of the PANI / CNT composite conductive layer, improves the electron transport efficiency and specific surface area of ​​the electrode, provides a good substrate for the construction of MIP membrane, enhances the ability of uric acid recognition and signal response, and ensures detection selectivity.

[0026] Furthermore, the Nafion ion exchange membranes on the surfaces of the reference electrode and the counter electrode are formed by drop-coating 1 μL of 0.5% Nafion solution and drying at room temperature.

[0027] The beneficial effects are as follows: the thin-layer Nafion ion exchange membrane can effectively block interfering ions and contaminants, protect the active sites on the surface of the reference electrode and the counter electrode, and at the same time, it does not affect ion transport, thereby improving the chemical stability and response consistency of the electrode in the physiological environment.

[0028] Furthermore, the glucose detection working electrode uses the amperometric method to record the current signal to achieve quantitative glucose detection, with a detection linear range covering 3.5~9.5 mmol / L.

[0029] The beneficial effects are: the amperometric method has a rapid response and stable signal, and the detection linear range accurately matches the clinical physiological glucose concentration range. It can accurately capture blood glucose fluctuations, meet the continuous monitoring needs of diabetic patients and other populations, and improve the clinical applicability of the test.

[0030] Furthermore, the uric acid detection working electrode uses cyclic voltammetry to record the change in current peak value to achieve quantitative detection of uric acid, with a detection linear range covering 150~450μmol / L.

[0031] The beneficial effects are as follows: cyclic voltammetry can accurately capture the peak signal of uric acid oxidation, and the detection linear range matches the normal and abnormal concentration range of uric acid in the human body. The high selectivity of the MIP membrane, combined with this method, effectively eliminates interference and ensures the accuracy and reliability of uric acid detection.

[0032] Furthermore, the sensor is a single flexible microneedle structure that integrates the reference electrode, counter electrode, glucose detection working electrode, and uric acid detection working electrode, for in-situ continuous sampling and synchronous detection of subcutaneous tissue fluid.

[0033] The beneficial effects are: the single microneedle structure significantly reduces puncture trauma and foreign body sensation, improving user compliance; the integrated multi-electrode enables in-situ sampling and simultaneous detection of tissue fluid, avoiding sample loss and contamination, reducing signal fluctuations, and ensuring the stability and convenience of continuous monitoring. Attached Figure Description

[0034] Figure 1 The front view of the electrode distribution diagram of the implantable flexible microneedle sensor;

[0035] Figure 2 The reverse side of the electrode distribution diagram of the implantable flexible microneedle sensor;

[0036] Figure 3 This is the result of a blood glucose signal test;

[0037] Figure 4 The uric acid results for test group 1;

[0038] Figure 5 The results for uric acid in test group 2.

[0039] Figure 6 The uric acid results for test group 3;

[0040] Figure 7 The uric acid results for test group 4;

[0041] Figure 8 The uric acid results for test group 5;

[0042] Figure 9 Calibrate the blood glucose results graph;

[0043] Figure 10 This is a calibration graph for uric acid results.

[0044] Figure labeling: Glucose detection working electrode 1, uric acid detection working electrode 2, reference electrode 3, counter electrode 4. Detailed Implementation

[0045] The following detailed description illustrates the specific implementation method:

[0046] This invention discloses an implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid, detailed as follows:

[0047] The implantable flexible microneedle sensor uses a polyimide film as a flexible substrate, on which a reference electrode 3 (RE), a counter electrode 4 (CE), a glucose detection working electrode 1 (WE1), and a uric acid detection working electrode 2 (WE2) are integrated. The electrode distribution is as follows: Figure 1 and Figure 2 As shown, Figure 1 The image shows the front view, with the glucose detection working electrode 1 in the upper left corner and the uric acid detection working electrode 2 in the lower right corner. Figure 2 On the reverse side, the reference electrode 3 is located in the lower left corner, and the counter electrode 4 is located in the upper right corner. WE1 and WE2 share the RE and CE electrodes, and all four work together to achieve simultaneous detection of glucose and uric acid within a unified potential window of +0.1V to +0.2V. The sensor as a whole is a single flexible microneedle structure, which can be minimally invasively implanted into subcutaneous tissue to avoid the trauma and signal deviation caused by multi-needle arrays.

[0048] First, the electrode fabrication process will be introduced.

[0049] Electrode patterns were pre-fabricated on a polyimide film surface using laser etching. Subsequently, a 120nm thick gold film was deposited by magnetron sputtering under high vacuum. After mask removal, a well-defined and firmly adhered gold electrode base structure was formed, providing a suitable substrate for subsequent functional modifications of each electrode and ensuring electrode conductivity and structural stability. A carbon electrode was selected as the substrate, and Ag / AgCl mixed ink was coated onto its surface using screen printing. After drying, a stable reference electrode 3 was formed. Then, 1μL of 0.5% Nafion solution was drop-coated, and after drying at room temperature, a thin Nafion ion exchange membrane was formed. This membrane prevents electrode surface contamination and limits the penetration of interfering ions, ensuring the stability of the reference potential. Using a flexible carbon electrode as a substrate, the electrode was ultrasonically cleaned with ethanol and deionized water for 5 minutes each, dried with nitrogen, and then subjected to oxygen plasma treatment for 60 seconds to improve surface activity. The treated CE was then immersed in a mixed solution of 0.5 mM chloroplatinic acid and 0.5 M sulfuric acid, and electrodeposited at a constant potential of +0.2 V for 300 seconds to form a dense Pt nanoparticle modification layer. After deposition, the electrode was rinsed and dried with deionized water, and 1 μL of 0.5% Nafion solution was drop-coated. After drying at room temperature, a counter electrode 4 with good conductivity and electrochemical stability was obtained, which can efficiently transport electrons and ensure the stability of the current response during detection.

[0050] The preparation of the glucose detection working electrode 1 is as follows: Electrode substrate pretreatment: The gold electrode substrate was ultrasonically cleaned for 5 minutes each in anhydrous ethanol and deionized water, and then dried at room temperature to ensure a clean and impurity-free surface, laying the foundation for subsequent modification layer adhesion. Polypyrrole (PPy) layer preparation: A mixed electropolymerization solution of 0.2M pyrrole and 0.2M KCl was prepared. The pretreated gold electrode was immersed in this solution, and a cyclic voltammetry scan was performed at a potential range of 0–0.8V and a scan rate of 50mV / s for 8 cycles to form a uniform PPy coating through in-situ electropolymerization. This layer imparts good mechanical flexibility and conductivity to the electrode, serving as a fixation layer and electron transport medium. Platinum nanoparticle (PtNP) layer preparation: A mixed deposition solution of 1M M H₂PtCl₆ and 0.1M M H₂SO₄ was prepared. The electrode was immersed in this solution and subjected to a constant potential of –0.2V. A uniformly distributed PtNP layer was formed by deposition for 300 s, significantly enhancing the electrocatalytic ability for H2O2 and improving the signal response for glucose detection. GOx enzyme composite solution was prepared and diluted to 10 mL with pH 7.4 PBS buffer. The composite solution contained 10 mg / mL glucose oxidase (GOx), 1% w / v polyvinylpyrrolidone K30, 5 mg / mL bovine serum albumin (BSA), 3% w / v trehalose, and 0.05% v / v Tween 20. 20 μL of this composite solution was dropped onto the surface of the PtNP layer, allowed to stand at room temperature for 5 minutes, and then dried at 37°C for 15 minutes to complete the curing. BSA and trehalose synergistically protected GOx activity and extended the enzyme layer's lifespan, while Tween 20 ensured uniform membrane spreading, ultimately yielding a highly specific glucose detection working electrode 1.

[0051] The preparation of the working electrode 2 for uric acid detection is as follows: Electrode substrate pretreatment: The gold electrode substrate was ultrasonically cleaned sequentially in anhydrous ethanol and deionized water, and then dried with nitrogen to ensure surface cleanliness. Preparation of the polyaniline / carbon nanotube (PANI / CNT) composite conductive layer: A 1.0M HCl solution containing 0.2M aniline monomer and 1.0 mg / mL multi-walled carbon nanotubes (MWCNTs) was prepared and ultrasonically treated for 30 minutes to form a homogeneous mixture. The pretreated gold electrode was immersed in this solution, and cyclic voltammetry was used to scan 5-10 times within a potential range of −0.2V to +0.8V at a scan rate of 50mV / s to deposit the PANI / CNT composite conductive layer in situ. This layer improves the electron transport efficiency and specific surface area of ​​the electrode, providing a good substrate for MIP film construction. Molecularly imprinted polymerase... MIP membrane preparation: An electropolymerization solution was prepared using PBS buffer (pH 7.0) as the solvent, containing 0.5 mM uric acid (template molecule), 10 mM o-phenylenediamine (functional monomer), and 5 mM MFe(CN)6³⁻ (auxiliary oxidant). The modified electrode was immersed in this solution, and 20 cyclic voltammetric scans were performed in the potential range of 0–0.8 V to construct the MIP membrane, forming "memory channels" in the membrane layer that match the molecular structure of uric acid. Template molecule elution and protective layer preparation: The electrode was repeatedly eluted in PBS solution to remove the uric acid template molecules embedded in the membrane layer, forming specific recognition holes. Subsequently, 1 μL of 0.1 wt% Nafion solution was dropped onto the electrode, and the electrode was allowed to dry naturally at room temperature to obtain uric acid detection working electrode 2. The Nafion protective layer further improved the membrane stability and anti-interference ability.

[0052] Next, the detection and verification of the sensor will be introduced.

[0053] Artificial sweat at pH 7.0 was prepared to simulate a physiological environment. Five test samples containing different concentrations of glucose and uric acid were prepared, with glucose concentrations of 3.5 mmol / L, 4.5 mmol / L, 6.1 mmol / L, 7.5 mmol / L, and 9.5 mmol / L, and uric acid concentrations of 150 μmol / L, 175 μmol / L, 200 μmol / L, 300 μmol / L, and 450 μmol / L, covering the physiological and common abnormal concentration ranges in the human body. The sensors were minimally invasively implanted into a simulated skin tissue model, allowing WE1 and WE2 to fully contact the subcutaneous tissue fluid for in-situ sampling. The detection potential was set to +0.2V (vsAg / AgCl). The response current of WE1 at different glucose concentrations was recorded using the amperometric method (i–t curve), and the peak current change of WE2 was recorded using the cyclic voltammetry (CV). After each sample was tested, the electrode surface was cleaned with PBS buffer to avoid cross-contamination. The five sample tests were completed sequentially.

[0054] glucose test results as follows Figure 3As shown in the blood glucose signal test results, the response current of WE1 exhibits a significant linear increasing trend with increasing glucose concentration. A standard curve was generated through data fitting. Figure 9 As shown in the figure (calibrated blood glucose results), the linear detection range covers 3.5~9.5 mmol / L, which conforms to the clinical physiological concentration range. It exhibits good sensitivity and stability, accurately reflecting changes in glucose concentration. The uric acid test results are as follows: Figures 4-8 (As shown in the uric acid results for test groups 1-5), the peak current of the cyclic voltammetry curves corresponding to different uric acid concentrations showed significant differences, and increased systematically with increasing uric acid concentration. The fitted standard curve is shown in the figure. Figure 10 (Uric acid result calibration) shows that the detection linear range covers 150~450μmol / L. The specific recognition function of the MIP membrane effectively eliminates interference, and the detection accuracy is excellent. Under the unified potential window, the detection signals of WE1 and WE2 have no obvious overlap and crosstalk. The synchronous detection results are highly consistent with the individual detection results, proving that the sensor can achieve accurate synchronous monitoring of two markers and meet the needs of multi-index detection.

[0055] The implantable flexible microneedle sensor prepared in this embodiment employs a hybrid recognition strategy of "GOx enzymatic + MIP non-enzymatic," combining a unified low-potential window with a single microneedle integrated structure. This approach leverages the specificity of GOx and the specific recognition capability of the MIP membrane to enhance detection resistance and accuracy, while the shared electrode and unified potential simplify circuit design, enabling device miniaturization. The minimally invasive design of the single microneedle improves user compliance, and in-situ detection avoids sample loss and contamination, meeting the needs of patients with metabolic diseases for continuous home monitoring. It possesses significant clinical application value and industrialization potential.

[0056] The combination of a GOx bio-enzyme-modified glucose electrode (specifically recognizing glucose) and a uric acid electrode constructed with a MIP molecularly imprinted membrane (specifically adsorbing uric acid) provides a dual recognition mechanism that ensures both selectivity and anti-interference capabilities, avoiding signal cross-interference. In contrast, in the "dual non-enzyme" strategy, the specificity of glucose MIPs is far lower than that of enzymes, and the single-material dual-catalysis strategy relies on potential differentiation, making glucose and uric acid detection signals prone to crosstalk. By using dual working electrodes and a unified low-potential window, crosstalk-free operation and circuit simplification are achieved, making it suitable for wearable devices. The electrode system operates at approximately +0.2V, eliminating the need for complex potential switching circuits and enabling simultaneous detection within a single potential window, thus improving detection efficiency. Based on in-situ tissue fluid detection using a single flexible microneedle, the single-needle integrated three-electrode design is more suitable for minimally invasive continuous monitoring scenarios, offering significant advantages in minimally invasiveness, signal stability, process integration, and wearable compatibility. It is suitable for scenarios such as long-term home monitoring, minimally invasive sampling, and simultaneous detection of two indicators, overcoming the inherent defects of microneedle array electrodes, such as large trauma, signal deviation, and complex manufacturing processes.

[0057] The above are merely embodiments of the present invention. The invention is not limited to the fields covered by these embodiments. Commonly known structures and characteristics in the solutions are not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the application date or priority date, are able to access all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical known structures or methods should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid, characterized in that, It includes a flexible substrate and a reference electrode, a counter electrode, a glucose detection working electrode, and a uric acid detection working electrode integrated on the flexible substrate. The glucose detection working electrode and the uric acid detection working electrode share the reference electrode and the counter electrode, and achieve synchronous detection within a unified potential window of +0.1V to +0.2V. The preparation process of the glucose detection working electrode is as follows: the gold electrode substrate is ultrasonically cleaned with anhydrous ethanol and deionized water sequentially and then dried; cyclic voltammetry is performed for 8 cycles in a mixed solution of 0.2M pyrrole and 0.2M KCl at a potential range of 0~0.8V and a scan rate of 50mV / s to form a polypyrrole layer through in-situ electropolymerization; the electrode is immersed in a mixed solution of 1mM H2PtCl6 and 0.1M H2SO4 at a constant potential of –0.2V for 300s to form a platinum nanoparticle layer; a glucose oxidase composite solution is drop-coated and cured, wherein the composite solution is diluted with PBS buffer at pH 7.4 and contains 10mg / mL GOX, 1% w / v polyvinylpyrrolidone K30, 5mg / mL bovine serum albumin, 3% w / v trehalose and 0.05% v / v Tween 20; The preparation process of the working electrode for uric acid detection is as follows: the gold electrode substrate is ultrasonically cleaned with anhydrous ethanol and deionized water in sequence and then dried with nitrogen; a polyaniline / carbon nanotube composite conductive layer is deposited by cyclic voltammetry scanning for 5 to 10 cycles in a 1.0M HCl solution containing 0.2M aniline monomer and 1.0 mg / mL multi-walled carbon nanotubes at a potential range of −0.2V to +0.8V and a scan rate of 50mV / s; the electrode is then immersed in a pH7.0 PBS electropolymerization solution containing 0.5mM uric acid, 10mM o-phenylenediamine and 5mM Fe(CN)6³⁻ for cyclic voltammetry scanning for 20 cycles to construct a molecularly imprinted polymer film. After eluting with PBS solution to remove uric acid template molecules, 1 μL of 0.1 wt% Nafion solution was dropped onto the substrate as a protective layer and then dried. The reference electrode is a carbon electrode coated with Ag / AgCl mixed ink, and its surface is covered with a thin layer of Nafion ion exchange membrane; the counter electrode is a flexible carbon electrode that has been treated with oxygen plasma and then electrodeposited at a constant potential of +0.2V for 300s in a mixed solution of 0.5mM chloroplatinic acid and 0.5M sulfuric acid to form a Pt nanoparticle modified layer, and its surface is covered with a thin layer of Nafion ion exchange membrane.

2. The implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 1, characterized in that, The flexible substrate is a polyimide film. The glucose detection working electrode, uric acid detection working electrode, reference electrode and counter electrode are formed by laser etching of pre-patterns, followed by magnetron sputtering deposition of a 120nm thick gold film and mask peeling.

3. The implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 2, characterized in that, The Ag / AgCl mixed ink of the reference electrode is coated onto the surface of the carbon electrode by screen printing, and after drying, a stable reference electrode is formed.

4. The implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 3, characterized in that, The pretreatment of the electrode includes: ultrasonic cleaning with ethanol and deionized water for 5 minutes each, drying with nitrogen, and then oxygen plasma treatment for 60 seconds.

5. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 4, characterized in that, The GOx composite solution of the glucose detection working electrode was applied in a drop volume of 20 μL. After standing at room temperature for 5 minutes, it was dried at 37°C for 15 minutes to complete the curing.

6. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 5, characterized in that, The PANI / CNT composite conductive layer of the uric acid detection working electrode is deposited in situ by cyclic voltammetry, wherein the scanning voltage range of the cyclic voltammetry is −0.2V to +0.8V and the scanning rate is 50mV / s.

7. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 6, characterized in that, The Nafion ion exchange membranes on the surfaces of the reference electrode and the counter electrode are formed by drop-coating 1 μL of 0.5% Nafion solution and drying at room temperature.

8. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 7, characterized in that, The glucose detection working electrode uses the amperometric method to record the current signal to achieve quantitative glucose detection, with a detection linear range covering 3.5~9.5 mmol / L.

9. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 8, characterized in that, The working electrode for uric acid detection uses cyclic voltammetry to record changes in current peak value to achieve quantitative detection of uric acid, with a detection linear range covering 150~450μmol / L.

10. An implantable flexible microneedle sensor for continuous monitoring of glucose and uric acid according to claim 9, characterized in that, The sensor is a single flexible microneedle structure that integrates the reference electrode, counter electrode, glucose detection working electrode, and uric acid detection working electrode, and is used for in-situ continuous sampling and synchronous detection of subcutaneous tissue fluid.

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