A multi-electrode subcutaneously implantable biosensor and a method of making

Abstract

This invention discloses a multi-electrode subcutaneous implantable biosensor, comprising a flexible composite substrate, a three-dimensional electrode system, and an in-situ self-maintaining system. The three-dimensional electrode system includes at least one first working electrode disposed on the front side of the flexible composite substrate, and a solid reference electrode and a counter electrode disposed on the back side. The working electrode, solid reference electrode, and counter electrode are electrically connected through inclined conductive vias penetrating the flexible composite substrate. The in-situ self-maintaining system is communicatively connected to a signal processing and control circuit, enabling active intervention on the surface microenvironment of the working electrode. By arranging the working electrode on the front side of the flexible composite substrate and the solid reference electrode and counter electrode on the back side, and utilizing inclined conductive vias to achieve three-dimensional electrical interconnection, the front working electrode is brought closer to the tissue fluid to be tested, optimizing mass transfer. The back electrode is located away from the implantation interface, which is heavily contaminated with biofouling, providing a more stable and cleaner electrochemical environment for the reference electrode.

Description

Technical Field

[0001] This invention relates to the field of sensor technology, and in particular to a multi-electrode subcutaneous implantable biosensor and its preparation method. Background Technology

[0002] In recent years, with the development of minimally invasive continuous monitoring technology, subcutaneous implantable biosensors have become a core technology in fields such as continuous glucose monitoring (CGM) and continuous lactate monitoring (CLM). These sensors are typically based on flexible printed circuits (FPC) or silicon-based micromachining technology, integrating a working electrode, a reference electrode, and a counter electrode on a single flexible substrate to form an electrochemical detection unit.

[0003] As shown in the patent "Improved Analyte Sensor and its Manufacturing and Use Method" (application number: 201080018303.6) published in China by Abbott Diabetes Care, its sensor adopts a typical "coplanar electrode structure," where all electrodes (working electrode, counter electrode, and reference electrode) are placed on the same substrate plane and electrically isolated through stacking and an insulating layer. This structure generates hydrogen peroxide through a glucose oxidase-catalyzed reaction, which then oxidizes the hydrogen peroxide to generate a weak current for detection. The technology is clear, the process is relatively mature, and it has become the mainstream architecture for current commercial CGM products (such as the FreestyleLibre series).

[0004] To further improve performance, subsequent technical solutions have optimized the electrode interface modification. For example, Chinese patent "Glucose Monitoring Probe and Its Working Electrode" (application number: 201822259382.6) discloses an electrode structure with a multilayer coating. The working electrode, from the inside out, includes a metal substrate, a transition layer, a noble metal catalytic layer, a coupling layer, an immobilized enzyme layer, and a polymer confinement membrane. The reference electrode uses an Ag / AgCl system and is covered with a polymer membrane. This approach aims to improve the electrode's biocompatibility, enzyme immobilization stability, and anti-interference capability through precise material stacking. Another similar silicon-based sensor structure (also seen in CN201822259382.6) further introduces a nanoparticle layer and a gradient film layer to enhance signal response and selectivity.

[0005] However, despite some improvements, the aforementioned existing technical solutions are still limited by their inherent "single-sided coplanar" or "simple stacking" electrode system architecture, leading to a series of interrelated fundamental challenges in long-term implantation applications: First, the coplanar electrode structure exposes all active interfaces to the same microenvironment susceptible to biofouling (such as nonspecific protein adsorption and fibrous membrane formation). The accumulation of fouling layers on the electrode surface forms a diffusion barrier, leading to progressively decreasing sensitivity and unpredictable baseline drift. This severely limits the sensor's effective lifespan (typically no more than 14 days) and forces patients to undergo frequent, painful external finger-prick blood calibrations.

[0006] Secondly, existing sensors rely entirely on the passive diffusion of analytes from interstitial fluid to the electrode surface. This process is slow, resulting in a 5-15 minute delay in physiological response. During periods of rapid blood glucose fluctuation (such as after a meal), the monitored values ​​lag significantly behind real-life changes, impacting the reliability and safety of real-time treatment decisions based on CGM data (such as closed-loop control of artificial pancreas).

[0007] Furthermore, with the increasing demand for comprehensive metabolic monitoring, integrating multiple working electrodes such as glucose, lactate, and ketone bodies onto the same device has become a trend. However, in a coplanar layout, significant electric field crosstalk and electrochemical cross-interference occur between densely arranged electrodes, severely affecting the specificity and accuracy of multi-index detection. Existing solutions using physical isolation or membrane screening have limited effectiveness.

[0008] Furthermore, existing sensors are essentially "passive sensing" elements, lacking the ability to perform in-situ real-time diagnosis of their own operating status (such as enzyme activity and degree of contamination), and even more so lacking mechanisms for proactive intervention (such as interface cleaning and calibration) when performance degrades. The reliability of the entire system is highly dependent on the initial manufacturing precision and the uncontrollable internal environment; once the interface fails, the system is essentially terminated. Summary of the Invention

[0009] The technical problem solved by this invention is to provide a multi-electrode subcutaneous implantable biosensor and its preparation method.

[0010] This application provides a multi-electrode subcutaneous implantable biosensor, comprising: A flexible composite substrate having a front and a back side that are positioned opposite to each other; A three-dimensional electrode system, integrating signal processing and control circuitry, includes at least one first working electrode disposed on the front side of the flexible composite substrate, a solid reference electrode disposed on the back side of the flexible composite substrate, and a counter electrode. The working electrode, the solid reference electrode, and the counter electrode are electrically connected through an inclined conductive via penetrating the flexible composite substrate, forming a spatially separated electrochemical detection circuit spanning the front and back sides of the flexible composite substrate. The in-situ self-maintenance system is communicatively connected to the signal processing and control circuit and is configured to actively intervene in the surface microenvironment of the working electrode under the control of the signal processing and control circuit.

[0011] Furthermore, the in-situ self-maintenance system includes: A flow field intervention unit is integrated into the flexible composite substrate and is used to generate controlled fluid motion in the working electrode surface area under the control of the signal processing and control circuit. An electric field intervention unit, which is electrically connected to the three-dimensional electrode system, is used to apply a controllable constraint electric field to the surface region of the working electrode under the control of the signal processing and control circuit. The signal processing and control circuit is configured to coordinate the control of the flow field intervention unit and the electric field intervention unit to execute a predetermined self-maintenance procedure.

[0012] Furthermore, the flow field intervention unit is a dual-coupling self-pressurizing microfluidic system, including a front microcavity and flow channel structure disposed on the front side of the flexible composite substrate and surrounding the working electrode, and a micro-airbag driving structure disposed on the back side of the flexible composite substrate; the micro-airbag driving structure is fluidly connected to the front microcavity and flow channel structure through inclined micropores, and is configured to generate compression-rebound by utilizing the periodic fluctuations of subcutaneous tissue fluid, so as to drive the generation of directional pulsating flow in the front microcavity under the monitoring or triggering of the signal processing and control circuit.

[0013] Furthermore, the flexible composite substrate is a time-gradient heterogeneous structure, including a rigid functional island array distributed at the implantation end and a flexible back layer constituting the main structure. The front microcavity and flow channel structure and the working electrode are disposed on the rigid functional island array. The rigid functional island array is embedded with a micro support frame that can be biodegraded within a predetermined time after implantation.

[0014] Preferably, the outer surface of the back flexible layer is provided with a passive mechanical anchoring structure; the material of the micro support frame is magnesium alloy or polyethylene glycol.

[0015] Furthermore, the electric field intervention unit includes a concentric ring microelectrode array and a nanofiber porous membrane; the concentric ring microelectrode array is disposed on the front side of the flexible composite substrate and is disposed around the working electrode and electrically isolated from it; the nanofiber porous membrane is disposed in the region between the working electrode and the concentric ring microelectrode array; the signal processing and control circuit is electrically connected to the concentric ring microelectrode array and is configured to apply a controllable potential to the concentric ring microelectrode array, which cooperates with the detection potential of the working electrode to form a gradient constraint electric field above the nanofiber porous membrane for repelling interfering substances.

[0016] Furthermore, the three-dimensional electrode system also includes a second working electrode disposed on the front side of the flexible composite substrate, and the surfaces of the first and second working electrodes are modified with different types of biosensitive membranes; the signal processing and control circuit is also configured to periodically measure the electrochemical impedance between the first and second working electrodes for self-diagnosis.

[0017] Furthermore, the solid reference electrode is a polymer electrolyte composite membrane electrode; the three-dimensional electrode system also includes a temperature-sensitive electrode and / or a pH-sensitive electrode disposed on the back side of the flexible composite substrate, and electrically connected to the signal processing and control circuit.

[0018] Furthermore, the signal processing and control circuit is configured to execute a self-maintenance program comprising at least one of the following modes: Periodic cleaning mode: The flow field intervention unit is activated in concert to generate a flushing flow, and the electric field intervention unit is controlled to apply desorption electrical pulses; Self-verification mode: Controls the three-dimensional electrode system to switch connection modes to perform cross-comparison of signals using different electrodes.

[0019] This application provides a method for preparing a multi-electrode subcutaneous implantable biosensor, comprising the following steps: Prepare a flexible composite substrate with tilted conductive vias; Electrodes of a three-dimensional electrode system are patterned on both sides of the flexible composite substrate, and three-dimensional electrical interconnection is achieved by filling the inclined conductive vias. The signal processing and control circuit is integrated and electrically connected to the three-dimensional electrode system; The flow field intervention unit and electric field intervention unit of the in-situ self-maintenance system are integrated in the flexible composite substrate, and a communication connection is established between them and the signal processing and control circuit. A biosensitive membrane and a functional layer are modified on the surface of the working electrode; Perform overall encapsulation.

[0020] Furthermore, the fabrication of the flow field intervention unit includes: forming a microcavity and flow channel on the front side of the flexible composite substrate, bonding a micro-airbag driving structure on the back side, and connecting the two by laser etching of tilted micro-holes; the fabrication of the electric field intervention unit includes: forming a concentric ring-shaped microelectrode array around the working electrode by photolithography, and depositing a nanofiber porous membrane on the device by electrospinning technology.

[0021] Compared with existing technologies, this invention arranges the working electrode on the front side (tissue contact side) of a flexible composite substrate, while arranging the solid reference electrode and counter electrode on the back side of the flexible composite substrate, and uses inclined conductive vias to achieve three-dimensional electrical interconnection. On the one hand, the front working electrode can be closer to the tissue fluid to be measured, optimizing mass transfer; on the other hand, the back electrode is far away from the implantation interface with heavy biofouling, providing a more stable and clean electrochemical environment for the reference electrode, significantly reducing the overall measurement error caused by reference potential drift. At the same time, the in-situ self-maintenance system actively drives the interstitial fluid to flow on the surface of the working electrode, greatly accelerating the delivery of analytes (such as glucose) to the electrode reaction center, while timely removing reaction products. It is expected to significantly shorten the physiological response delay of the sensor from 5-15 minutes in existing technologies, providing a more real-time and more realistic physiological fluctuation curve, and providing a more reliable data foundation for the refined management of diabetes and closed-loop insulin infusion systems. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention.

[0023] Figure 1 This is a schematic diagram of the structure of the multi-electrode subcutaneous implantable biosensor of the present invention; Figure 2 This is a cross-sectional view of the multi-electrode subcutaneous implantable biosensor of the present invention; Figure 3 This is a schematic diagram of the working principle of the dual-coupling self-pressurizing microfluidic system of the present invention; Figure 4 This is a schematic diagram of the planar layout and electric field distribution of the electric field interference unit of the present invention; Figure 5 This is a schematic diagram illustrating the evolution of the mechanical properties of the time-series gradient substrate according to the present invention; Figure 6 This is a flowchart of the preparation method of the electrode subcutaneous implantation biosensor of the present invention; Figure 7 This is a logical block diagram of the intelligent self-maintenance level of the present invention.

[0024] The reference numerals in the attached figures include: 1. Flexible composite substrate; 11. Rigid functional island array; 12. Flexible back layer; 13. Mechanical anchoring structure; 14. Micro-support frame; 15. Implantation end; 16. Internal intermediate layer; 2. Three-dimensional electrode system; 21. First working electrode; 22. Solid reference electrode; 23. Counter electrode; 24. Second working electrode; 25. Temperature-sensitive electrode; 26. pH-sensitive electrode; 27. Biosensitive membrane; 3. Tilted conductive via; 4. In-situ self-maintaining system; 41. Flow field intervention unit; 411. Ring microcavity; 412. Spiral gradient flow channel; 413. Micro-airbag; 414. Tilted micropore; 42. Electric field intervention unit; 421. Concentric ring microelectrode array; 422. Nanofiber porous membrane; 5. Signal processing and control chip. Detailed Implementation

[0025] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0026] like Figure 1 , Figure 2 As shown, the multi-electrode subcutaneous implantable biosensor of the present invention includes a flexible composite substrate 1, a three-dimensional electrode system 2, and an in-situ self-maintaining system 4; the specific structure is as follows: (1) Flexible composite substrate 1: A temporally gradient heterogeneous structure is employed. The specific structure and function are as follows: The front side (i.e., the implantation end 15, also the top layer) features three rigid functional islands made of polyimide (PI) with a diameter of approximately 1 mm, serving as rigid platforms to support the precision functional units (electrodes, microchannels). Their function is to provide sufficient local stiffness in the initial implantation stage, ensuring the sensor can accurately penetrate the subcutaneous tissue and maintain a stable posture. Simultaneously, they provide robust mechanical support for the fabrication and long-term operation of the front micro / nano structures, preventing functional unit failure due to deformation of the flexible material.

[0027] A biodegradable microsupport frame 14, made of magnesium alloy (AZ31) filament, is embedded in the center of the rigid functional island (i.e., the inner intermediate layer 16 of the substrate). It is hexagonal in shape with a filament diameter of 150 μm and provides additional axial and radial support during the critical initial stages after sensor implantation (e.g., the first 10-14 days), helping the rigid island maintain its shape. Its predetermined degradation cycle aims to achieve a smooth and controllable transition of the sensor's mechanical properties from a "rigid implantation state" to a "long-term flexible and biocompatible state." This design ensures the sensor possesses the necessary structural rigidity in the early stages of implantation to guarantee implantation accuracy and the stability of internal functional units. In the later stages of its service life, as the frame degrades, the sensor becomes more flexible, significantly reducing continuous mechanical stimulation to surrounding tissues, delaying fibrous capsule proliferation, and achieving superior tissue compatibility and signal stability throughout the sensor's entire design lifespan.

[0028] The flexible back layer 12 (i.e., the bottommost layer) is composed of medical-grade polydimethylsiloxane (PDMS) with a modulus of approximately 2 MPa, forming the main body of the sensor. Its function is to serve as a flexible carrier for the entire device, giving the sensor good overall flexibility and bendability, allowing it to adapt to the daily activities and deformations of subcutaneous tissue during long-term implantation, greatly reducing continuous mechanical stimulation to surrounding tissues. This is the basis for achieving good long-term tissue compatibility.

[0029] The passive mechanical anchoring structure 13 is a micron-sized barb array molded integrally onto the outer surface of the flexible back layer 12. Its function is to provide anti-migration anchoring force by combining with the surrounding newly formed tissue through mechanical interlocking effect after the micro support frame 14 degrades and the sensor becomes flexible as a whole, replacing the initial rigid support and ensuring that the sensor can remain stable in place during the long-term flexible phase, avoiding displacement caused by body movement.

[0030] This time-gradient substrate design systematically resolves the fundamental contradiction between the need for rigidity in short-term implantation and the need for flexibility in long-term compatibility. Through a time-sequential switch of a "degradable framework," it enables the intelligent evolution of the implant's mechanical properties, thereby ensuring implantation accuracy and initial stability throughout its entire lifespan while minimizing foreign body reactions and inhibiting excessive fibrous capsule proliferation, creating an excellent in vivo microenvironment for long-term stable monitoring.

[0031] (2) Three-dimensional electrode system 2 and signal processing control circuit: Front Electrode: Two circular electrodes are formed at the center of each rigid functional island using photolithography and sputtering processes. The first is a Φ300μm platinum-carbon first working electrode 21 for glucose monitoring; the second is a Φ300μm gold second working electrode 24 for lactate monitoring. They are positioned on the tissue contact side (front) to minimize analyte diffusion distance. This heterogeneous dual-working-electrode design supports simultaneous monitoring of multiple indicators. Its spatial separation from the back electrode is the core of this scheme's "spatially separated detection loop," effectively reducing the inherent electric field crosstalk of the coplanar layout.

[0032] Back electrode: Prepared by screen printing and magnetron sputtering, including a PEDOT:PSS / ionic liquid (poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate / ionic liquid composite material) composite film solid reference electrode 22, a platinum counter electrode 23, a boron nitride nanotube-doped carbon paste temperature-sensitive electrode 258, and an iridium oxide pH-sensitive electrode 26 (the platinum counter electrode 23, the temperature-sensitive electrode 258, and the pH-sensitive electrode 26 constitute the auxiliary electrode).

[0033] The solid-state reference electrode 22 provides a long-term stable potential reference that is independent of chloride ion concentration. Compared with traditional Ag / AgCl reference electrodes, this solid-state polymer electrolyte system fundamentally avoids potential drift caused by internal electrolyte leakage or dilution, and is the cornerstone of ensuring the long-term measurement accuracy of the entire electrochemical detection system. Positioning it on the back side, away from areas with severe biofouling on the front side, further enhances its operational stability.

[0034] The auxiliary electrode functions to form a current loop with the working electrode. The temperature and pH electrodes function to monitor the temperature and pH of the microenvironment in which the sensor is located in situ and synchronously. The acquired data is directly input into the signal processing circuit for real-time and dynamic temperature and pH compensation of the working electrode current signal, significantly improving the physiological accuracy and reliability of glucose and lactic acid concentration calculations, especially in pathological states such as fever and inflammation.

[0035] Three-dimensional interconnection: A femtosecond laser is used to drill inclined conductive vias 3 at a 45° angle to the normal and with a diameter of approximately 60 μm on the flexible composite substrate 1. These vias are then filled with electroplated copper, and the front working electrodes 21 are connected to the circuit nodes on the back side. The inclined conductive vias 3 are the key structure for realizing three-dimensional electrical interconnection. Their inclined angle design is beneficial for stress dispersion and improving interconnection reliability. They successfully connect the "sensing area" on the front side with the "reference area and control area" on the back side into a whole, forming a unique detection loop that spans both sides.

[0036] Circuit Integration: A customized mixed-signal processing and control chip 5 is mounted on the flexible back layer 12 using anisotropic conductive adhesive (ACF). This chip integrates a low-noise potential analyzer, a high-precision ADC, a programmable voltage output DAC, a microcontroller, and a wireless communication unit. The chip is electrically connected to all electrodes. The signal processing and control circuit is the "intelligent brain" of the system, integrating signal acquisition, amplification, analog-to-digital conversion, power management, wireless communication, and intelligent control algorithms. It not only processes sensor signals, but its core function is to coordinate and control the operation of the entire in-situ self-maintenance system 4, enabling intelligent diagnosis and on-demand maintenance.

[0037] (3) In-situ self-maintenance unit Flow field intervention unit 41—Dual-sided coupled self-pressurized microfluidic system: Frontal structure: On each rigid island, around the working electrode 21 region, an annular microcavity 411 with a depth of approximately 200 μm and a spiral gradient flow channel 412 connected thereto are formed through laser ablation and PDMS replication molding processes. A micro-weir with a height of 10 μm is provided at the end of the flow channel. A 30 μm thick medical silicone elastic film with uniform micropores (Φ20 μm) is bonded to the top as a cavity cover.

[0038] Back-side drive: Three independent silicone micro-airbags 413, each with a volume of approximately 1 μL, are pre-embedded and bonded within the corresponding flexible back layer 12.

[0039] Fluid connectivity: Inclined micropores 414 (Φ25μm) at a 30° angle to the substrate are etched using a femtosecond laser, connecting the inlet and outlet of each micro-airbag 413 to the starting end of the corresponding spiral flow channel on the front.

[0040] Working principle as follows Figure 3 As shown, the dual-coupling self-pressurizing microfluidic system exhibits two alternating operating states within a physiological pulsation cycle (such as tissue hydraulic fluctuations caused by heartbeat or exercise), thereby achieving directional fluid drive without external power. Compression phase (driving flushing flow): When the subcutaneous tissue hydraulic pressure increases, the pressure acts on the micro-airbag 413 driving structure located on the back of the flexible composite substrate 1, causing it to be compressed and deformed (e.g., Figure 3As shown in the left figure, the micro-airbag 413 is in a flattened state. The working fluid (such as pre-filled buffer solution or autologous interstitial fluid) inside the airbag is pumped under pressure through inclined micropores 414 at approximately a 30-degree angle to a spiral gradient flow channel 412 located on the front side of the substrate. The flow channel is designed with a depth and width gradient from the inlet to the outlet, and a micro-weir structure at the end. After being accelerated along the flow channel, the high-pressure fluid flows into the annular microcavity 411 surrounding the working electrode 21. As shown by the arrows and vortex lines in the figure, the fluid forms a vortex flow within the microcavity, effectively shearing and scouring the surface of the working electrode 21 located in the center of the microcavity, thereby actively removing biofouling and renewing the interstitial fluid at the interface.

[0041] Rebound period (preparation and anti-backflow): When tissue hydraulic pressure decreases, the micro-inflator 413 rebounds and re-inflates due to the elasticity of its material (e.g., Figure 3 As shown in the right sub-figure, the micro-airbag 413 returns to its full state, creating a brief negative pressure inside. At this time, a slight backflow tendency may occur at the connection between the inclined micro-orifice 414 and the front flow channel (as indicated by the thin dashed arrow in the figure). However, the micro-weir structure (a step about 10 μm high) at the end of the spiral gradient flow channel 412 utilizes the surface tension and gravity of the fluid itself, in conjunction with the angle design of the inclined micro-orifice 414, to jointly form a capillary valve effect, effectively preventing the liquid already carrying dirt in the micro-cavity from being drawn back, ensuring the unidirectionality of rinsing and cleaning efficiency.

[0042] This system pioneers a microfluidic design that utilizes physiological tissue hydraulic fluctuations (such as pulse) as a driving force. Its function is to actively generate controlled, directional pulsating flow on the surface of the working electrode 21. Specifically, tissue fluid pressure fluctuations drive the periodic deformation of the micro-bag 413, and this mechanical energy is converted into fluid kinetic energy through tilted micro-holes 414, forming a flushing flow within the frontal flow channel. The effects are threefold: 1) Active mass transfer: accelerating the transport of analytes and reaction products, reducing sensor response time from minutes to seconds; 2) Physical cleaning: periodically flushing the electrode surface using fluid shear force, delaying the accumulation of biofouling; 3) Energy self-sufficiency: eliminating the need for external pumps or batteries, achieving self-powered operation and significantly extending the sustainability of system maintenance.

[0043] Electric field intervention unit 42: like Figure 4 As shown, the planar layout and working principle of the electric field intervention unit 42 are as follows: On the top surface of the rigid functional island, the first working electrode 21 is located at the center as a circular electrode with a diameter of approximately 300 μm. Around it, three concentric ring-shaped microelectrodes are photolithographically fabricated in a strictly concentric manner, with a linewidth of approximately 15 μm and a spacing of approximately 30 μm between the rings. Each ring electrode is electrically isolated from the central working electrode by an insulating layer. Within the annular region between the central working electrode and the innermost ring electrode, a negatively charged nanofiber porous membrane 422, prepared by electrospinning, is covered.

[0044] The signal processing and control circuitry is configured to apply a precisely controllable potential to the electrode array. During normal detection, a detection potential (e.g., +0.6V) relative to the solid-state reference electrode 22 is applied to the central working electrode, while a lower auxiliary potential (e.g., +0.35V) is applied to the innermost annular electrode. Potentials that decrease sequentially or are grounded can be applied to the middle and outer rings. This creates a radial gradient electric field with a decreasing potential from the center outwards in the space above the nanofiber membrane.

[0045] This gradient electric field is effective against common electroactive interferences that are also negatively charged (such as ascorbate ions AA). - This generates a continuous electrostatic repulsive force, such as... Figure 4 The arrows symbolizing the repulsion of particles indicate that these particles are "driven away" from the sensitive reaction region of the working electrode. Simultaneously, the transport of electrically neutral target analytes (such as glucose) or reaction products (such as hydrogen peroxide) is not significantly affected by this electrostatic force. This charge-selective active repulsion mechanism, combined with the physical sieving effect of the nanofiber membrane, constitutes a dual, synergistic anti-interference barrier, significantly improving the selectivity and long-term stability of the detection. The effect is a leap from "passive blocking" to "active expulsion" in anti-interference mode, with high selectivity, stable and adjustable performance, solving the problem of anti-interference membrane performance degradation during long-term implantation. The ring electrode can also be used for electrochemical impedance spectroscopy measurements, enabling in-situ diagnosis of interface states.

[0046] (4) Biosensitive membrane 27 and functional layer: A platinum black nanocatalytic layer, a glucose oxidase (GOx) layer, and a polyurethane confinement membrane are sequentially modified on the surface of the first working electrode 21. A lactate oxidase (LOx) layer and a corresponding confinement membrane are modified on the surface of the second working electrode 2421. A PEDOT:PSS / p-benzoquinone / ionic liquid composite solid electrolyte membrane is coated on the surface of the solid reference electrode 22.

[0047] The biosensitive membrane 27 is the molecular recognition core of the sensor, functioning to convert specific biochemical reactions (such as glucose oxidation) into measurable electrical signals. The sophisticated coating design optimizes enzyme immobilization efficiency, enhances reaction kinetics, and controls the reaction region. Its effects directly determine the sensor's sensitivity, selectivity, and stability. In conjunction with the aforementioned self-maintenance system, it aims to maintain the high efficiency and cleanliness of this molecular recognition interface over the long term.

[0048] The intelligent workflow and self-maintenance procedures of the aforementioned biosensors are explained below, such as... Figure 7 As shown, the details are as follows; After the sensor is implanted under the skin, the chip is powered on and starts up, entering an intelligent working cycle.

[0049] Routine monitoring mode: The chip collects data cyclically in 1-minute cycles: a) glucose response current (I_G) of the first working electrode 21; b) lactic acid response current (I_L) of the second working electrode 2421; and c) readings of the temperature electrode and pH electrode (T, pH).

[0050] The signal processing and control circuit runs a built-in real-time multi-parameter compensation algorithm. This algorithm dynamically corrects the original response current I_G of the first working electrode 21 and the original response current I_L of the second working electrode 2421 based on real-time data synchronously measured by the temperature-sensitive electrode 25 and the pH-sensitive electrode 26. Specifically, the compensation algorithm is implemented through the following general model:

[0051] Here, F(T,pH) and F'(T,pH) are pre-calibrated binary compensation functions for temperature and pH, respectively, for glucose and lactate detection. These functions integrate the effects of temperature changes on enzyme reaction kinetics, diffusion coefficients, and electrochemical processes, as well as the effects of pH changes on enzyme activity and electrochemical equilibrium. The specific form of the compensation function can be polynomial, exponential, or an interpolation mapping table based on calibration data.

[0052] After compensation and correction, the currents I_G_corr and I_L_corr, combined with the sensor's calibration curve under standard conditions, are used to calculate the precise glucose and lactate concentrations. The microfluidic system continuously generates a low-frequency pulsating background flow driven by physiological pulsation, and the electric field intervention unit 42 continuously applies a gradient constraint electric field to actively resist interference.

[0053] Periodic self-diagnosis and self-maintenance procedures: A self-diagnostic process is performed every 6 hours: the chip switching circuit measures the electrochemical impedance spectroscopy (EIS, 10 mHz-100 kHz) between the first working electrode 21 and the second working electrode 2421. Characteristic parameters related to interface capacitance and charge transfer resistance in the impedance spectrum are analyzed. Simultaneously, the chip briefly switches connections, and signal cross-verification is performed using the theoretical response relationship of the two working electrodes 21 at the same glucose concentration.

[0054] Intelligent decision-making based on diagnostic results: If the EIS display shows that the dirt resistance at the interface increases by more than 20% of the threshold, or if there is a significant deviation in the signal verification, the "Enhanced Cleaning Mode" will be triggered immediately.

[0055] If all parameters are normal, the "normal maintenance mode" will be executed according to the preset cycle (e.g., every 24 hours).

[0056] Self-maintenance procedure execution: Cleaning Mode (Normal or Enhanced): The chip first controls the electric field intervention unit 42 to apply a -0.5V desorption electrical pulse lasting 100ms to the working electrode 21. Immediately afterwards, the flow field intervention unit 41 is triggered to initiate enhanced flushing (by micro-heating to trigger a stronger airbag pulse), generating a high-speed flushing flow lasting 5-10 seconds. This electro-current synergy effectively removes dirt.

[0057] Self-verification mode (every 12 hours): The chip temporarily reconfigures the electrode connections, for example, temporarily using the second working electrode 2421 as the counter electrode 23, forming a temporary detection loop with the first working electrode 21, measuring a "verification signal" and comparing it with the normal signal to verify the integrity of the circuit path.

[0058] Long-term evolution: like Figure 5 As shown, the mechanical properties of the flexible composite substrate 1 of this invention are not static, but rather evolve intelligently over time after implantation. This process can be divided into three stages, perfectly resolving the contradiction between the need for rigid support in the early stages of implantation and the long-term need for flexible compatibility: Phase I: Initial Post-Implantation Period (Approximately 0-10 Days) – Rigid Support Period like Figure 5As shown in the left sub-figure, the sensor exhibits a rigid overall shape during the initial implantation stage. At this stage, the rigid functional islands distributed at the implantation end 15 and the biodegradable micro-support frame 14 (such as magnesium alloy or PGA) embedded within them provide the primary mechanical strength. This frame exists as a complete mesh structure, ensuring that the sensor can penetrate the subcutaneous tissue with sufficient rigidity and maintain precise implantation depth and orientation, providing a stable working platform for the precise working electrode 21 on the front and the microfluidic structure. Meanwhile, the passive mechanical anchoring structure 13 (such as a barb array) integrated on the flexible layer 12 on the back is in a flat, latent state, closely adhering to the substrate surface, avoiding additional tissue trauma or difficulty in insertion during the implantation process.

[0059] Phase II: Post-implantation transition period (approximately 10-14 days) – Degradation and anchoring activation phase like Figure 5 As shown in the middle sub-figure, the biodegradable microsupport frame 14 begins to undergo controlled degradation in the in vivo environment over time (represented by the frame lines changing to dashed lines in the figure). Its degradation products (such as magnesium ions) have good biocompatibility. As the rigid support gradually weakens, the sensor as a whole begins to exhibit a certain degree of flexibility, enabling it to better conform to the natural contours of the tissue. At the same time, slight local environmental changes that may accompany the frame degradation process (such as slight alkalinity or temperature rise), or preset time triggers, will activate the shape memory polymer (SMP) on the back or cause deformation of the pre-embedded mechanical anchoring structure 13. As shown in the figure, some anchoring structures begin to pop out and lift from the surface, with their tips gradually piercing or hooking into the surrounding newly formed tissue fibers, thereby providing new anchoring forces derived from mechanical interlocking and beginning to assume the function of maintaining the sensor's stable position.

[0060] Phase III: Long-term post-implantation period (approximately 14 days or more) – Flexible stabilization period like Figure 5 As shown in the right sub-figure, once the biodegradable microsupport frame 14 has completely degraded and absorbed, the sensor's main mechanical properties are entirely dominated by the flexible back layer 12, transforming the entire sensor into a highly compliant and flexible state, capable of adapting to daily bodily activities without stress. At this point, the mechanical anchoring structure 13 on the flexible back layer 12 has fully popped out and is firmly embedded in the surrounding tissue (as shown by the interweaving of barbs and tissue fibers in the figure), forming a reliable long-term anti-migration anchor. This combination of "rigid-flexible transformation" and "active anchoring" greatly improves the long-term biocompatibility between the implant and the tissue.

[0061] like Figure 6 As shown, the preparation method of the multi-electrode subcutaneous implantable biosensor of the present invention mainly includes the following steps: S1: Fabrication of a time-gradient flexible composite substrate 1 with tilted conductive vias 3.

[0062] A technique combining laser-induced forward transfer (LIFT) and precision molding is employed. First, PI is selectively cured on a temporary carrier using a laser to form a prototype of a rigid functional island array 11 on the front side, with a pre-drilled groove in the center of each island. A pre-fabricated magnesium alloy microframe is then precisely placed into the groove. Subsequently, PDMS prepolymer is poured and cured to form a flexible back layer 12, while simultaneously molding the barbed structure on the surface. After demolding, a femtosecond laser is used to drill holes at a 45° angle from the back side of the substrate towards a predetermined position on the front side, forming a prototype of an inclined conductive via 3.

[0063] S2: Double-sided electrode patterning and three-dimensional electrical interconnection.

[0064] On the front side of the flexible composite substrate 1, the first and second working electrodes 24 and the concentric ring microelectrode array 421 are patterned through photolithography, electron beam evaporation (for Au electrodes), and lift-off processes. On the back side of the flexible composite substrate 1, the counter electrode 23, temperature electrode, and other patterns are formed by screen printing carbon paste and silver paste, combined with magnetron sputtering of Pt; the PEDOT:PSS composite pattern of the solid reference electrode 22 is formed through spraying and photolithography processes. Subsequently, copper is filled into the inclined vias through electroplating to achieve reliable interconnection of the front and back electrode circuits.

[0065] S3: Signal processing and control circuit integration.

[0066] Using a high-precision chip mounter, customized chips are flip-chip mounted onto designated pads on the back circuit using anisotropic conductive adhesive (ACF) and then thermo-bonded to achieve electrical connection and mechanical fixation.

[0067] S4: In-situ self-maintenance system 4 integration.

[0068] Flow field intervention unit 41: On the front side, a layer of photoresist is spin-coated, and a mold for microcavities and flow channels is defined using photolithography and reactive ion etching (RIE) processes. PDMS is poured in, cured, and then peeled off to obtain a front-side PDMS layer with microchannels, and a perforated silicone film is bonded thereon. On the corresponding position on the back side, a pre-formed silicone micro-airbag 413 is bonded. Finally, a femtosecond laser is used to precisely etch inclined micro-holes 414 connecting the airbags and the front-side flow channels from the side.

[0069] Electric field intervention unit 42: After completing the electrode preparation in step S2, PSS nanofibers are directionally deposited in the annular region between the working electrode 21 and the concentric ring electrode using an electrospinning device with a precision metal mask to form a nanofiber porous membrane 422.

[0070] S5: Biosensitive membrane 27 modification.

[0071] Using a microfluidic spotting system, glucose oxidase solution was precisely spotted onto the central region of the first working electrode 21, and lactate oxidase solution was spotted onto the second working electrode 2421, and cross-linked and cured under a temperature-controlled and humid environment. Subsequently, an ultrathin parylene layer was deposited on the surface of the working electrode 21 as a biocompatible confined encapsulation layer by chemical vapor deposition (CVD), exposing only the active center of the electrode.

[0072] S6: Overall packaging and testing.

[0073] The entire device was encapsulated by dipping it in medical-grade silicone, exposing only the active electrode area on the front, the microfluidic inlet and outlet, and the wireless communication antenna area on the back. After encapsulation, it underwent rigorous aging and sterilization processes. Finally, electrochemical performance testing, microfluidic function verification, and wireless communication testing were performed in a simulated interstitial fluid environment to ensure all functions met design specifications.

[0074] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0075] Furthermore, the use of terms such as "first" and "second" in this application is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed in this application.

[0076] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0077] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A multi-electrode subcutaneous implantable biosensor, characterized in that, include: A flexible composite substrate (1) having a front side and a back side arranged opposite to each other; The three-dimensional electrode system (2) integrates signal processing and control circuits. The three-dimensional electrode system (2) includes at least one first working electrode (21) disposed on the front side of the flexible composite substrate (1), a solid reference electrode (22) disposed on the back side of the flexible composite substrate (1), and a counter electrode (23). The working electrode (21), the solid reference electrode (22), and the counter electrode (23) are electrically connected through an inclined conductive via (3) penetrating the flexible composite substrate (1) to form a spatially separated electrochemical detection circuit spanning the front and back sides of the flexible composite substrate (1). The in-situ self-maintenance system (4) is communicatively connected to the signal processing and control circuit and is configured to actively intervene in the surface microenvironment of the working electrode (21) under the control of the signal processing and control circuit.

2. The multi-electrode subcutaneous implantable biosensor as described in claim 1, characterized in that, The in-situ self-maintenance system (4) includes: The flow field intervention unit (41) is integrated into the flexible composite substrate (1) and is used to generate controlled fluid motion in the surface area of ​​the working electrode (21) under the control of the signal processing and control circuit. An electric field intervention unit (42), which is electrically connected to the three-dimensional electrode system (2), is used to apply a controllable constraint electric field to the surface region of the working electrode (21) under the control of the signal processing and control circuit. The signal processing and control circuit is configured to coordinate the control of the flow field intervention unit (41) and the electric field intervention unit (42) to execute a predetermined self-maintenance procedure.

3. The multi-electrode subcutaneous implantable biosensor as described in claim 2, characterized in that, The flow field intervention unit (41) is a dual-coupling self-pressurizing microfluidic system, including a front microcavity and flow channel structure disposed on the front side of the flexible composite substrate (1) and surrounding the working electrode (21), and a micro-airbag driving structure disposed on the back side of the flexible composite substrate (1); the micro-airbag driving structure is fluidly connected to the front microcavity and flow channel structure through inclined micropores (414), and is configured to generate compression-rebound by utilizing the periodic fluctuation of subcutaneous tissue fluid, so as to drive the generation of directional pulsating flow in the front microcavity under the monitoring or triggering of the signal processing and control circuit.

4. The multi-electrode subcutaneous implantable biosensor as described in claim 3, characterized in that, The flexible composite substrate (1) is a time-gradient heterostructure, including a rigid functional island array (11) distributed at the implantation end (15) and a back flexible layer (12) constituting the main structure. The front microcavity and flow channel structure and the working electrode (21) are disposed on the rigid functional island array (11). The rigid functional island array (11) is embedded with a micro support frame (14) that can be biodegraded within a predetermined time after implantation. Preferably, the outer surface of the back flexible layer (12) is provided with a passive mechanical anchoring structure (13); the material of the micro support frame (14) is magnesium alloy or polyethylene glycol.

5. The multi-electrode subcutaneous implantable biosensor as described in claim 2, characterized in that, The electric field intervention unit (42) includes a concentric ring microelectrode array (421) and a nanofiber porous membrane (422). The concentric ring microelectrode array (421) is disposed on the front side of the flexible composite substrate (1) and is disposed around the working electrode (21) and electrically isolated from it. The nanofiber porous membrane (422) is disposed in the region between the working electrode (21) and the concentric ring microelectrode array (421). The signal processing and control circuit is electrically connected to the concentric ring microelectrode array (421) and is configured to apply a controllable potential to the concentric ring microelectrode array (421), which cooperates with the detection potential of the working electrode (21) to form a gradient constraint electric field above the nanofiber porous membrane (422) for repelling interfering substances.

6. The multi-electrode subcutaneous implantable biosensor as described in claim 5, characterized in that, The three-dimensional electrode system (2) further includes a second working electrode (24) disposed on the front side of the flexible composite substrate (1), and the surfaces of the first working electrode (21) and the second working electrode (24) are modified with different types of biosensitive membranes (27); the signal processing and control circuit is also configured to periodically measure the electrochemical impedance between the first working electrode (21) and the second working electrode (24) for self-diagnosis.

7. The multi-electrode subcutaneous implantable biosensor as described in claim 1, characterized in that, The solid reference electrode (22) is a polymer electrolyte composite membrane electrode; the three-dimensional electrode system (2) also includes a temperature-sensitive electrode (25) and / or a pH-sensitive electrode (26) disposed on the back side of the flexible composite substrate (1) and electrically connected to the signal processing and control circuit.

8. The multi-electrode subcutaneous implantable biosensor as described in claim 3, characterized in that, The signal processing and control circuitry is configured to execute a self-maintenance program comprising at least one of the following modes: Periodic cleaning mode: The flow field intervention unit (41) is activated in concert to generate a flushing flow, and the electric field intervention unit (42) is controlled to apply a desorption electric pulse; Self-verification mode: Control the three-dimensional electrode system (2) to switch the connection mode so as to use different electrodes to perform signal cross-comparison.

9. The method for preparing a multi-electrode subcutaneous implantable biosensor as described in any one of claims 1 to 8, characterized in that, Includes the following steps: A flexible composite substrate (1) with tilted conductive vias (3) was prepared. Electrodes of a three-dimensional electrode system (2) are patterned on both sides of the flexible composite substrate (1), and three-dimensional electrical interconnection is achieved by filling the inclined conductive vias (3). The signal processing and control circuit is integrated and electrically connected to the three-dimensional electrode system (2); The flow field intervention unit (41) and electric field intervention unit (42) of the in-situ self-maintenance system (4) are integrated in the flexible composite substrate (1), and a communication connection is established between them and the signal processing and control circuit. A biosensitive membrane (27) and a functional layer are modified on the surface of the working electrode (21); Perform overall encapsulation.

10. The method for preparing a multi-electrode subcutaneous implantable biosensor as described in claim 9, characterized in that, The fabrication of the flow field intervention unit (41) includes: forming a microcavity and flow channel on the front side of the flexible composite substrate (1), bonding a micro airbag driving structure on the back side, and connecting the two by laser etching of tilted micro-holes (414); the fabrication of the electric field intervention unit (42) includes: forming a concentric ring microelectrode array (421) around the working electrode (21) by photolithography, and depositing a nanofiber porous membrane (422) on the device by electrospinning.

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