A subcutaneously implantable multi-metabolic index continuous monitoring sensor and a preparation method and application thereof

Abstract

This invention provides a subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators, its preparation method, and its applications, belonging to the field of biosensor technology. The subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators provides this invention uniformly distributes the electrodes in an electrode system on two surfaces at one end of a flexible substrate. An insulating layer is used to cover the electrodes to prevent signal crosstalk between different electrodes. Different enzyme-modified layers are composed of different components in the working electrodes, enabling the detection of one metabolite on each working electrode, preventing mutual interference between different enzymes, and allowing for expansion to monitor multiple indicators simultaneously as needed. By setting a vinyl homopolymer protective layer on the enzyme-modified layer, this invention reduces interference from the detection environment on the detection results, while improving the stability of the enzyme-modified layer, thereby improving long-term monitoring accuracy.

Description

Technical Field

[0001] This invention relates to the field of biosensor technology, and in particular to a subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators, its preparation method, and its application. Background Technology

[0002] A biosensor is a device that uses bioactive units as sensing elements, combined with chemical and physical conversion elements, to achieve high selectivity for the analyte. It boasts advantages such as high sensitivity, fast detection speed, ease of operation, low cost, and the ability to perform continuous dynamic monitoring, making it an indispensable advanced detection and monitoring device for the development of biotechnology. With the advancement of the sensor field, biosensors are being used in more and more aspects of daily life, with subcutaneous implantable sensors being a key area of ​​development.

[0003] Subcutaneous implantable sensors have attracted much attention due to their advantages such as being minimally invasive, convenient, continuous, reliable, and providing accurate data. Currently, the most common subcutaneous implantable sensors are the traditional three-electrode structure. The traditional three-electrode structure can usually only achieve continuous monitoring of glucose, and it is difficult to monitor multiple analytes such as glucose, lactate, and β-hydroxybutyrate simultaneously. When detecting multiple indicators, multiple sensors need to be implanted, which is highly invasive.

[0004] To provide continuous monitoring of multiple indicators, some studies have stacked and concentrated multiple working electrodes in the same local area. However, when multiple working electrodes are stacked and concentrated in the same local area, the excessive concentration of multiple functional areas may lead to low local area utilization, increased interlayer interference, and decreased flexibility and stability, resulting in lower monitoring accuracy. Distributing multiple working electrodes at different depths or in different tissue layers, although all within the interstitial fluid environment, can affect the comparability and stability between multiple analyte channels due to differences in local perfusion, oxygen concentration, metabolic state, and tissue response; this also leads to lower monitoring accuracy. Furthermore, integrating different enzyme systems is challenging. Glucose detection often uses glucose oxidase (GOx), lactate detection often uses lactate oxidase (LOx), while β-hydroxybutyrate detection often uses β-hydroxybutyrate dehydrogenase (β-HBDH), and uric acid detection uses uricase oxidase (UOx). Cholesterol detection uses a dual-enzyme system combining cholesterol esterase and cholesterol oxidase (COD+CEH), and triglycerides use a triple-enzyme system combining lipase, glycerol kinase, and glycerol-3-phosphate oxidase (LPL+GK+GPO). These enzyme systems differ in reaction mechanisms, stability, and membrane requirements, making integration difficult. When multiple working electrodes coexist, problems such as local oxygen competition, diffusion of reaction byproducts, background drift, and long-term degradation of biocompatibility may occur.

[0005] Furthermore, the needs for metabolite monitoring vary across different scenarios. For example, high-risk diabetic patients require simultaneous monitoring of blood glucose, lactate, and blood ketones to comprehensively predict the risk of ketoacidosis and tissue hypoxia; individuals with metabolic syndrome require combined monitoring of blood glucose, uric acid, and triglycerides to dynamically assess the degree of glucose, lipid, and purine metabolism disorders; and patients with cardiovascular chronic diseases rely on combined monitoring of uric acid, cholesterol, and triglycerides to assess the risk of atherosclerosis and gout. Existing sensors mostly have fixed indicator configurations, and a single substrate can only achieve a single detection combination. Developing different functional models requires re-molding and adjusting the entire printing and production process, resulting in long development cycles and high mass production costs.

[0006] Therefore, there is an urgent need to provide a subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators that can be freely combined for simultaneous detection of multiple indicators, has high detection accuracy, and strong anti-interference ability. Summary of the Invention

[0007] The purpose of this invention is to provide a subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators with simultaneous detection of multiple indicators, high detection accuracy and strong anti-interference ability, as well as its preparation method and application.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators, including a flexible substrate and an electrode system and a connection system disposed on the surface of the flexible substrate; The electrode system includes a working electrode, a counter electrode, and a reference electrode; the number of working electrodes is 1 to 3; each electrode in the electrode system is uniformly distributed on two surfaces at one end of the flexible substrate; The working electrode comprises a working electrode layer, an enzyme-modified layer, and a protective layer sequentially stacked on the surface of a flexible substrate; the enzyme-modified layer in different working electrodes has a different composition; the protective layer covers the surfaces of the enzyme-modified layer, the counter electrode, and the reference electrode; The connection system includes working electrode wires and working electrode contacts that are sequentially connected to each working electrode; the working electrode wires are wires with an insulating layer on their surface; the working electrode contacts are located on the surface of the flexible substrate opposite to the electrode system; and the working electrode wires are located on the surface of the flexible substrate between the working electrodes and the working electrode contacts.

[0009] Preferably, the flexible substrate is L-shaped and includes an implant arm and a connecting arm connected at an angle; the working electrode is disposed at the non-connecting end of the implant arm; and the working electrode contact is disposed at the non-connecting end of the connecting arm.

[0010] Preferably, the included angle is 45° to 135°.

[0011] Preferably, the implant arm has a width of 0.5-2 mm, a length of 5-20 mm, and a rounded or semi-circular end; the connecting arm has a width of 2-5 mm and a length of 3-15 mm.

[0012] Preferably, the enzyme in the enzyme-modified layer is selected from one of the following: glucose oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, uricase, cholesterol esterase combined with cholesterol oxidase dual enzyme system, and glycerol phosphate oxidase-peroxidase coupled enzyme system.

[0013] Preferably, the protective layer is a vinyl homopolymer protective layer or a Nafion film.

[0014] Preferably, the vinyl homopolymer protective layer is formed by cross-linking and curing a vinyl polymer solution; the vinyl polymer solution includes: medical-grade poly(hydroxyethyl methacrylate)-methyl methacrylate copolymer, vitamin E polyethylene glycol succinate, vinyl prepolymer, and solvent.

[0015] Preferably, the thickness of the enzyme-modified layer is 5~10 μm.

[0016] This invention also provides a method for preparing the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor described in the above technical solution, comprising the following steps: (1) Screen printing is performed on the front and back surfaces of the flexible substrate to obtain the working electrode layer, wires, working electrode contacts, counter electrode and reference electrode; then insulating paste is covered on the surface of the working electrode wires and dried to obtain the primary electrode; (2) After coating the surface of each working electrode layer of the initial electrode obtained in step (1) with an enzyme-modified solution, a first cross-linking and curing reaction is performed to form an enzyme-modified layer on the surface of the working electrode layer; after coating the surface of the enzyme-modified layer, the counter electrode and the reference electrode with a protective layer solution, a second cross-linking and curing reaction is performed to obtain a subcutaneous implantable multi-metabolic indicator continuous monitoring sensor.

[0017] The present invention also provides the application of the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor described in the above technical solution or the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor prepared by the preparation method described in the above technical solution in the simultaneous detection of multiple metabolic indicators.

[0018] This invention provides a subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators. The sensor includes a flexible substrate and an electrode system and a connection system disposed on the surface of the flexible substrate. The electrode system comprises a working electrode, a counter electrode, and a reference electrode. The number of working electrodes is 1 to 3, and each electrode is uniformly distributed on two surfaces at one end of the flexible substrate. This electrode arrangement prevents signal crosstalk between different electrodes, thereby improving monitoring accuracy. Each working electrode comprises a working electrode layer, an enzyme-modified layer, and a protective layer sequentially stacked on the surface of the flexible substrate. The enzyme-modified layer in different working electrodes has a different composition. This allows for the detection of one metabolite on each working electrode, preventing mutual interference between different enzymes, and can be expanded to monitor multiple indicators simultaneously. By setting a protective layer, this invention can inhibit bioaccumulation and corrosion, reduce interference from the detection environment on the detection results, improve biocompatibility, and enhance the stability of the enzyme-modified layer, thereby improving long-term monitoring accuracy and stability. The connection system provided by this invention includes working electrode wires and working electrode contacts sequentially connected to each working electrode; the working electrode wires are wires with an insulating layer on their surface; the working electrode contacts are disposed on the surface of the flexible substrate opposite to the electrode system; the working electrode wires are disposed on the surface of the flexible substrate between the working electrodes and the working electrode contacts. The results of the embodiments show that the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor provided by this invention can simultaneously monitor multiple metabolic indicators and has excellent anti-interference ability and monitoring accuracy. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor of the present invention; In this diagram, 100 represents the front of the sensor; 101 represents contact 1; 102 represents contact 2; 103 represents contact 3; 104 represents working electrode 1; 105 represents working electrode 2; 106 represents the reference electrode; 200 represents the back of the sensor; 201 represents contact 4; 202 represents contact 5; 203 represents working electrode 4; and 204 represents counter electrode 5. Figure 2 The image shows the response of the subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in Example 1 of this invention to a gradient glucose solution. Detailed Implementation

[0020] The present invention provides a subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators, including a flexible substrate and an electrode system and a connection system disposed on the surface of the flexible substrate; The electrode system includes a working electrode, a counter electrode, and a reference electrode; the number of the working electrodes is 1 to 3. The working electrode comprises a working electrode layer, an enzyme-modified layer, and a protective layer sequentially stacked on the surface of a flexible substrate; the enzyme-modified layer in different working electrodes has a different composition; the protective layer covers the surfaces of the enzyme-modified layer, the counter electrode, and the reference electrode; The connection system includes working electrode wires and working electrode contacts that are sequentially connected to each working electrode; the working electrode wires are wires with an insulating layer on their surface; the working electrode contacts are located on the surface of the flexible substrate opposite to the electrode system; and the working electrode wires are located on the surface of the flexible substrate between the working electrodes and the working electrode contacts.

[0021] The subcutaneous implantable multi-metabolic continuous monitoring sensor provided by the present invention includes a flexible substrate.

[0022] In this invention, the flexible substrate is preferably made of a polymer, more preferably of polyimide (PI) or polyester (PET). The use of a polymer-based flexible substrate in this invention enables the substrate to be flexible and reduces the cost of the sensor.

[0023] In this invention, the flexible substrate is preferably L-shaped, comprising an implant arm and a connecting arm connected at an angle. In this invention, the working electrode is preferably disposed at the non-connecting end of the implant arm; the working electrode contact is preferably disposed at the non-connecting end of the connecting arm.

[0024] In this invention, the width of the implant arm is 0.5~2mm, and the length of the implant arm is preferably 5~20mm. As an embodiment of this invention, the width of the implant arm can be 0.5mm, 1mm, 1.5mm, or 2mm; and the length of the implant arm can be 5mm, 6mm, 7mm, 8mm, 9mm, 10mm, 11mm, 12mm, 13mm, 14mm, 15mm, 16mm, 17mm, 18mm, 19mm, or 20mm. This invention controls the width and length of the implant arm within the above ranges, which is beneficial for electrode integration and also reduces the wound area when the sensor is implanted subcutaneously.

[0025] In this invention, the end of the implant arm is preferably rounded or semi-circular. Setting the end of the implant arm to rounded or semi-circular makes subcutaneous implantation more convenient.

[0026] In this invention, the width of the connecting arm is preferably 2-5 mm, and the length of the connecting arm is preferably 3-15 mm. As an embodiment of this invention, the width of the connecting arm can be 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, or 5 mm; and the length of the connecting arm can be 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, or 15 mm. This invention controls the width and length of the connecting arm within the above ranges, which is beneficial for connecting an external receiver.

[0027] In this invention, the included angle is preferably 45° to 135°. As an embodiment of this invention, the included angle can be 45°, 50°, 60°, 70°, 80°, 90°, 100°, 110°, 120°, 130°, or 135°, with 90° being the most preferred. In this invention, the connection point of the included angle has a smooth transition, which avoids stress concentration.

[0028] The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor provided by the present invention includes an electrode system disposed on the surface of the flexible substrate.

[0029] In this invention, the electrode system includes a working electrode, a counter electrode, and a reference electrode.

[0030] In this invention, the number of working electrodes is 1 to 3. As an embodiment of this invention, the number of working electrodes can be 2 or 3. In this invention, when the number of working electrodes is 2 or 3, the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor is a 4-electrode system or a 5-electrode system, capable of continuously monitoring 2 or 3 metabolic indicators, respectively.

[0031] In this invention, the electrodes in the electrode system are uniformly distributed on two surfaces at one end of the flexible substrate. By uniformly distributing the electrodes on two surfaces at one end of the flexible substrate, this invention integrates the working electrode, counter electrode, and reference electrode at the front end of the flexible substrate, facilitating subcutaneous implantation and simultaneous measurement of multiple subcutaneous metabolic indicators.

[0032] In this invention, the working electrode comprises a working electrode layer, an enzyme modification layer, and a protective layer sequentially stacked on the surface of a flexible substrate.

[0033] In this invention, the working electrode layer is preferably made of pure carbon; the thickness of the working electrode layer is preferably 1~20μm, more preferably 5~15μm.

[0034] In this invention, the surface roughness of the working electrode layer is preferably Ra = 0.5~2 μm. By controlling the surface roughness of the working electrode layer within the above range, this invention further enhances the adhesion of the enzyme-modified layer.

[0035] In this invention, the enzyme-modified layer in different working electrodes has a different composition.

[0036] In this invention, the enzyme in the enzyme-modified layer is selected from one of the following: glucose oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, uricase, a cholesterol esterase combined with cholesterol oxidase dual-enzyme system, and a glycerol phosphate oxidase-peroxidase coupled enzyme system. In this invention, glucose oxidase (GOx) is used for glucose detection, lactate oxidase (LOx) for lactate detection, β-hydroxybutyrate dehydrogenase (β-HBDH) for β-hydroxybutyrate detection, uricase (UOX) for uric acid detection, the cholesterol esterase combined with cholesterol oxidase dual-enzyme system (COD+CEH) for cholesterol detection, and the glycerol phosphate oxidase-peroxidase coupled enzyme system (LPL+GK+GPO) for triglyceride detection. This invention, by controlling the types of enzymes in the enzyme-modified layer, can be used to monitor the corresponding metabolites of the enzymes. This invention does not have specific limitations on the source of the enzymes in the enzyme-modified layer; conventional commercially available products can be used.

[0037] In this invention, the thickness of the enzyme-modified layer is preferably 5-10 μm, more preferably 6-8 μm.

[0038] In this invention, the enzyme-modified layer is preferably obtained by cross-linking and curing an enzyme-modified solution.

[0039] In this invention, when the enzyme is glucose oxidase, the reaction principle is: glucose + O2 → gluconic acid + H2O2, generating a current that is positively correlated with the glucose concentration.

[0040] In one embodiment of the present invention, the enzyme-modified solution preferably comprises: glucose oxidase (GOx), human serum albumin, carbon nanotubes, and a solvent. In this invention, the solvent is preferably N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES solution), and the pH value of the HEPES solution is preferably 7.3-7.5, more preferably 7.4. In this invention, the concentration of GOx in the enzyme-modified solution is preferably 5-50 mg / mL; the concentration of human serum albumin is preferably 5-50 mg / mL; and the concentration of carbon nanotubes is preferably 1-20 mg / mL. As an embodiment of the present invention, the concentration of GOx in the enzyme-modified solution can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the concentration of human serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; and the concentration of carbon nanotubes can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, or 20 mg / mL.

[0041] In this invention, when the enzyme is glucose oxidase, as one embodiment of the invention, the composition of the enzyme-modified solution preferably includes: GOx, human serum albumin, polyethylene glycol diglycidyl ether (PEGDGE), polypyridine osmium complex, and a solvent. In this invention, the solvent is preferably a HEPES solution, and the pH value of the HEPES solution is preferably 7.0-7.6, more preferably 7.4. In this invention, the concentration of GOx in the enzyme-modified solution is preferably 5-50 mg / mL; the concentration of human serum albumin is preferably 5-50 mg / mL; the concentration of PEGDGE is preferably 1-30 mg / mL; and the concentration of the polypyridine osmium complex is preferably 5-50 mg / mL. As an embodiment of the present invention, the concentration of GOx in the enzyme-modified solution can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the concentration of human serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or 50 mg / mL. The concentrations of PEGDGE can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL; the concentrations of the polypyridine osmium complex can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL.

[0042] In this invention, when the enzyme is lactate oxidase, the reaction principle is: lactic acid + O2 → pyruvate + H2O2; H2O2 electrochemically oxidizes to generate current, and pure carbon electrode catalyzes and improves reaction efficiency.

[0043] In this invention, the enzyme-modified solution preferably comprises: lactate oxidase (LOx), bovine serum albumin, glutaraldehyde, graphene, and PBS solution. In this invention, the concentration of LOx in the enzyme-modified solution is preferably 5-50 mg / mL; the concentration of bovine serum albumin is preferably 5-50 mg / mL; the mass-volume concentration of glutaraldehyde is preferably 0.1-5%; and the concentration of graphene is preferably 3-50 mg / mL. As an embodiment of this invention, the concentration of LOx can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the concentration of bovine serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, etc. The concentration of glutaraldehyde is 45 mg / mL or 50 mg / mL; the mass-volume concentration of glutaraldehyde can be 0.1%, 1%, 2%, 3%, 4%, or 5%; the concentration of graphene can be 3 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the pH of the PBS solution is preferably 7.1~7.3, more preferably 7.2. In this invention, the mass-volume concentration of glutaraldehyde refers to the mass / volume percentage concentration, that is, the number of grams of solute contained in 100 mL of solution. 100%.

[0044] In this invention, when the enzyme is β-hydroxybutyrate dehydrogenase, the reaction principle is: β-hydroxybutyrate + NAD+ + → Acetoacetic acid + NADH; NADH is oxidized on the surface of a pure carbon electrode to generate current, and the electron mediator accelerates electron transfer.

[0045] In this invention, the enzyme-modified solution preferably comprises: β-hydroxybutyrate dehydrogenase (β-HBDH), human serum albumin, PEGDGE, iron oxide, and a solvent. In this invention, the concentration of β-HBDH in the enzyme-modified solution is preferably 5-50 mg / mL; the concentration of human serum albumin is preferably 5-50 mg / mL; the concentration of PEGDGE is preferably 1-50 mg / mL; and the concentration of iron oxide is preferably 1-50 mg / mL. As an embodiment of the present invention, the concentration of β-HBDH can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the concentration of human serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; and the concentration of PEGDGE can be 1 mg / mL, 5 mg / mL, or 1 mg / mL. The concentrations of the iron oxide can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the solvent is preferably a HEPES solution, and the pH value of the HEPES solution is preferably 7.0~7.6, more preferably 7.4.

[0046] In this invention, when the enzyme is uricase, as one embodiment of the invention, the composition of the enzyme-modified solution preferably includes: UOx, bovine serum albumin, glutaraldehyde, graphene, and a solvent. In this invention, the solvent is preferably a PBS solution, and the pH value of the PBS solution is preferably 7.0-7.6, more preferably 7.2. In this invention, the concentration of UOx in the enzyme-modified solution is preferably 5-40 mg / mL; the concentration of bovine serum albumin is preferably 5-40 mg / mL; the volume concentration of glutaraldehyde is preferably 0.1%-1.0%; and the concentration of graphene is preferably 1-8 mg / mL. As an embodiment of the present invention, the concentration of UOx in the enzyme-modified solution can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, or 40 mg / mL; the concentration of bovine serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, or 40 mg / mL; the volume concentration of glutaraldehyde can be 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, or 1.0%; and the concentration of graphene can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, or 8 mg / mL.

[0047] In this invention, when the enzyme is a combination system of cholesterol esterase and cholesterol oxidase, as one embodiment of the invention, the composition of the enzyme modification solution preferably includes: CEH, COD, human serum albumin, PEGDGE, ferrocene derivative mediator, and solvent. In this invention, the solvent is preferably a HEPES solution, and the pH value of the HEPES solution is preferably 7.0-7.6, more preferably 7.4. In this invention, the concentration of CEH in the enzyme modification solution is preferably 3-30 mg / mL; the concentration of COD is preferably 3-30 mg / mL; the concentration of human serum albumin is preferably 5-50 mg / mL; the concentration of PEGDGE is preferably 1-30 mg / mL; and the concentration of the ferrocene derivative mediator is preferably 5-40 mg / mL. As an embodiment of the present invention, the concentration of CEH in the enzyme-modified solution can be 3 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL; the concentration of COD can be 3 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL; the concentration of human serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL; The concentrations of the ferrocene derivative mediator can be 5 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL; the concentration of the PEGDGE can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL; the concentration of the ferrocene derivative mediator can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, or 40 mg / mL.

[0048] In this invention, when the enzyme is a triple enzyme system consisting of lipase, glycerol kinase, and glycerol-3-phosphate oxidase, as one embodiment of the invention, the composition of the enzyme modification solution preferably includes: LPL, GK, GPO, bovine serum albumin, PEGDGE, carbon nanotubes, and a solvent. In this invention, the solvent is preferably a HEPES solution, and the pH value of the HEPES solution is preferably 7.0-7.6, more preferably 7.3. In this invention, the concentration of LPL in the enzyme modification solution is preferably 2-25 mg / mL; the concentration of GK is preferably 2-25 mg / mL; the concentration of GPO is preferably 2-25 mg / mL; the concentration of bovine serum albumin is preferably 5-40 mg / mL; the concentration of PEGDGE is preferably 1-25 mg / mL; and the concentration of carbon nanotubes is preferably 1-6 mg / mL. As an embodiment of the present invention, the concentration of LPL in the enzyme-modified solution can be 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL; the concentration of GK can be 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL; the concentration of GPO can be 2 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL; The concentration of bovine serum albumin can be 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, or 40 mg / mL; the concentration of PEGDGE can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or 25 mg / mL; and the concentration of carbon nanotubes can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, or 6 mg / mL.

[0049] In this invention, the protective layer is preferably a vinyl homopolymer protective layer or a Nafion film.

[0050] In this invention, the vinyl homopolymer protective layer is preferably formed by cross-linking and curing a vinyl polymer solution; the vinyl polymer solution includes: medical-grade poly(hydroxyethyl methacrylate)-methyl methacrylate copolymer, vitamin E polyethylene glycol succinate, vinyl prepolymer, and solvent. The vinyl homopolymer protective layer used in this invention can prevent the adsorption and deposition of large molecular proteins and lipids in human tissue fluid on the electrode surface, isolate corrosive ions from eroding conductive circuits and enzyme layers, effectively delay enzyme activity decay, avoid electrode corrosion, and significantly improve the long-term stability of the sensor; simultaneously, it can weaken the local microenvironment fluctuations caused by the accumulation of large molecular metabolic impurities, reduce additional signal deviations, and indirectly optimize anti-interference capabilities, but it does not replace a dedicated anti-interference film to block small molecule interfering substances such as ascorbic acid and uric acid.

[0051] In this invention, the thickness of the vinyl homopolymer protective layer is preferably 2~1000μm, more preferably 100~500μm.

[0052] In this invention, the concentration of the medicinal poly(hydroxyethyl methacrylate)-methyl methacrylate copolymer in the vinyl polymer solution is preferably 15-30 wt%, more preferably 20-25 wt%.

[0053] In this invention, the concentration of vitamin E polyethylene glycol succinate in the vinyl polymer solution is preferably 0.3-0.6 wt%. As an embodiment of this invention, the concentration of vitamin E polyethylene glycol succinate in the vinyl polymer solution can be 0.3 wt%, 0.4 wt%, 0.5 wt%, or 0.6 wt%. The addition of vitamin E polyethylene glycol succinate in this invention improves biocompatibility.

[0054] In this invention, the concentration of vinyl prepolymer in the vinyl polymer solution is preferably 1.0-2.0%. As an embodiment of this invention, the concentration of vinyl prepolymer in the vinyl polymer solution can be 1.0 wt%, 1.5 wt%, or 2.0 wt%. This invention adds vinyl prepolymer to enhance the density of the protective film. In this invention, the molecular weight of the vinyl prepolymer is preferably 800-3000 Da. This invention uses a low molecular weight vinyl prepolymer, which is more conducive to enhancing the density of the protective film.

[0055] In this invention, the solvent is preferably a mixture of anhydrous ethanol and deionized water. In an embodiment of this invention, the volume ratio of anhydrous ethanol to deionized water can be 7:3.

[0056] In this invention, the electrode system includes a counter electrode. Preferably, the counter electrode is an Ag / AgCl composite electrode; the thickness of the counter electrode is preferably 10-200 μm, more preferably 100-150 μm. This invention utilizes the counter electrode to provide a stable reference potential (drift within ±5 mV).

[0057] In this invention, the electrode system includes a reference electrode.

[0058] In this invention, the reference electrode is preferably a pure carbon electrode; the thickness of the reference electrode is preferably 1~20μm, more preferably 5~15μm. The reference electrode in this invention ensures the stability of the current loop.

[0059] The subcutaneous implantable multi-metabolic continuous monitoring sensor provided by the present invention includes a connection system disposed on the surface of the flexible substrate.

[0060] In this invention, the connection system includes working electrode wires and working electrode contacts that are sequentially connected to each working electrode.

[0061] In this invention, the working electrode wire is a wire with an insulating layer on its surface. The insulating layer is preferably made of polyimide (PI), and its thickness is preferably 10-500 μm. By covering the working electrode wire with an insulating layer, this invention achieves electrical isolation between electrodes, shortening the signal transmission path, improving detection accuracy, and enabling simultaneous detection of multiple indicators by expanding the number of working electrodes at the implantation end, while also considering process scalability and cost advantages.

[0062] In this invention, the working electrode contact is disposed on the other end of the flexible substrate surface opposite to the electrode system; the working electrode wire is disposed on the surface of the flexible substrate between the working electrode and the working electrode contact.

[0063] A preferred schematic diagram of the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor provided by the present invention is shown below. Figure 1 As shown. In Figure 1 In the diagram, 100 represents one surface of the subcutaneous implantable multi-metabolic continuous monitoring sensor, referred to as the front of the sensor; 200 represents the other surface, referred to as the back of the sensor.

[0064] This invention also provides a method for preparing the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor described in the above technical solution, comprising the following steps: (1) Screen printing is performed on the front and back surfaces of the flexible substrate to obtain the working electrode layer, wires, working electrode contacts, counter electrode and reference electrode; then insulating paste is covered on the surface of the working electrode wires and dried to obtain the primary electrode; (2) After coating the surface of each working electrode layer of the initial electrode obtained in step (1) with an enzyme-modified solution, a first cross-linking and curing reaction is performed to form an enzyme-modified layer on the surface of the working electrode layer; after coating the surface of the enzyme-modified layer, the counter electrode and the reference electrode with a protective layer solution, a second cross-linking and curing reaction is performed to obtain a subcutaneous implantable multi-metabolic indicator continuous monitoring sensor.

[0065] The present invention obtains a working electrode layer, a wire, a working electrode contact, a counter electrode, and a reference electrode by screen printing on the front and back surfaces of a flexible substrate; then, an insulating paste is covered on the surface of the working electrode wire and dried to obtain the initial electrode.

[0066] In this invention, the working electrode, wires, working electrode contacts, and counter electrode are preferably made of pure carbon. In embodiments of this invention, the carbon paste used for screen printing can be 300-mesh carbon paste. Preferably, the carbon paste is dried at 120°C after screen printing to obtain the working electrode, wires, working electrode contacts, and counter electrode. This invention adopts a one-piece, direct-connection structure with screen printing on the same side for the working electrode, working electrode wires, and working electrode contacts. The circuits on the front and back sides are independent of each other, and each circuit is led out separately. The connection between the wires and the electrodes / contacts is rounded and an insulating layer is used to achieve electrical isolation. The external contacts are connected to the transmitter via elastic spring clips.

[0067] In this invention, the reference electrode is preferably an Ag / AgCl composite electrode, and the thickness of the reference electrode is preferably 10-200 μm. In this invention, the screen printing includes sequentially printing the reference electrode paste and curing. In an embodiment of this invention, the preferred composition of the reference electrode paste is: 50 wt% flake silver powder, 20 wt% ultrafine silver chloride powder, 4 wt% medical polyurethane, 2 wt% polyvinyl butyral (PVB), 15 wt% diethylene glycol butyl ether, 8 wt% isophorone, 0.8 wt% silicone leveling agent, and 0.2 wt% polyamide thixotropic agent. In this invention, the curing temperature is preferably 110-150°C, more preferably 120°C, and the curing time is preferably 30 min.

[0068] In this invention, the insulating slurry is preferably a PI slurry. This invention does not have a particular limitation on the composition of the PI slurry; any conventional PI slurry can be used. This invention forms an insulating layer by adding PI slurry and then drying it.

[0069] After obtaining the initial electrode, the present invention coats the surface of each working electrode layer of the initial electrode with an enzyme-modified solution and then performs a first cross-linking and curing reaction to form an enzyme-modified layer on the surface of the working electrode layer; after coating the surface of the enzyme-modified layer, the counter electrode and the reference electrode with a protective layer solution, a second cross-linking and curing reaction is performed to obtain a subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators.

[0070] In this invention, the composition of the enzyme-modified solution is the same as that of the enzyme-modified solution described in the above technical solution, and will not be repeated here.

[0071] In this invention, the coating method is preferably spot coating. In this invention, an enzyme-modified solution is coated onto the surface of each working electrode layer.

[0072] In this invention, the temperature of the first crosslinking curing reaction is preferably 20~40℃, more preferably 35~37℃; the time of the first crosslinking curing reaction is preferably 1~48h. As an embodiment of the present invention, the time for the first crosslinking curing reaction can be 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, or 48h.

[0073] In this invention, the protective layer solution is preferably a vinyl homopolymer solution, and the composition of the vinyl homopolymer solution is the same as that of the above-described technical solution, and will not be repeated here.

[0074] In this invention, the temperature of the second crosslinking curing reaction is preferably 30~55°C, more preferably 40°C. This invention does not have a particular limitation on the time of the second crosslinking curing reaction, as long as it is sufficient to completely dry the vinyl homopolymer solution.

[0075] The preparation method provided by this invention is simple to operate. By rationally arranging the electrodes, a subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators with simultaneous detection of multiple indicators, high detection accuracy, and strong anti-interference can be obtained.

[0076] The present invention also provides the application of the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor described in the above technical solution or the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor prepared by the preparation method described in the above technical solution in the simultaneous detection of multiple metabolic indicators.

[0077] The present invention does not have any special limitations on the application method of the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor; conventional application methods for subcutaneous implantable sensors can be used.

[0078] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0079] Example 1 A subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators comprises a flexible substrate and an electrode system and a connection system disposed on the surface of the flexible substrate; the flexible substrate is L-shaped and consists of an implant arm and a connecting arm connected at an angle; the implant arm is 2 mm wide, 8 mm long, and has a semi-circular end; the connecting arm is 4 mm wide, 10 mm long, and has an angle of 90°. The electrode system consists of a pure carbon working electrode (WE), a pure carbon counter electrode (CE), and a reference electrode Ag / AgCl (RE). There are two working electrodes, named WE1 and WE2 respectively. WE1, WE2, and RE are distributed on the same surface of the implant arm of the flexible substrate, and from the end of the implant arm to the connection part, they are WE2, WE1, and RE in sequence. CE is disposed on the other surface of the implant arm of the flexible substrate. The working electrode consists of a working electrode layer, an enzyme-modified layer, and a protective layer sequentially stacked on the surface of a flexible substrate; the enzyme in the WE1 enzyme-modified layer is glucose oxidase, and the enzyme in the WE2 enzyme-modified layer is β-hydroxybutyrate dehydrogenase; wherein, the thickness of the WE1 enzyme-modified layer is 8 μm, the thickness of the WE2 enzyme-modified layer is 7 μm, and the thickness of the vinyl homopolymer protective layer is 12 μm. The connection system consists of working electrode wires and working electrode contacts that are sequentially connected to each working electrode; the working electrode wires are wires with a surface covered by a 1μm thick PI insulating layer; the working electrode contacts are disposed at one end of the connecting arm on the surface of the flexible substrate; and the working electrode wires are disposed on the surface of the flexible substrate between the working electrode and the working electrode contacts.

[0080] The preparation method of the subcutaneous implantable multi-metabolic continuous monitoring sensor described in this embodiment is as follows: (1) Provide an L-shaped PI flexible substrate, and prepare two working electrode layers (300 mesh carbon paste) with a thickness of 10 μm, a wire with a thickness of 10 μm, a working electrode contact with a thickness of 10 μm and a counter electrode with a thickness of 10 μm on the surface of the flexible substrate by screen printing process. After drying at 120°C, prepare a reference electrode with a thickness of 10 μm by screen printing process; then cover the surface of the working electrode wire with PI paste, and obtain the initial electrode after drying. (3) A glucose oxidase modification solution was spot-coated onto the surface of one working electrode layer of the initial electrode obtained in step (2). The composition of the glucose oxidase modification solution was (GOx 30 mg / mL, human serum albumin 30 mg / mL, carbon nanotubes 5 mg / mL, HEPES solution pH 7.4 as solvent). After thermal cross-linking and curing at 37°C for 2.5 h, WE1 was obtained. A β-hydroxybutyrate dehydrogenase modification solution was spot-coated onto the surface of the second working electrode layer. The composition of the glucose oxidase modification solution was (β-HBDH 20 mg / mL, human serum albumin 30 mg / mL, PEGDGE 8 mg / mL, iron oxide (electron mediator) 4 mg / mL, HEPES solution pH 7.5 as solvent). After thermal cross-linking and curing at 40°C, WE2 was obtained. Then, a protective film layer was coated on top to obtain a subcutaneous implantable multi-metabolic continuous monitoring sensor.

[0081] Test Example 1 The subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in Example 1 was calibrated, and a concentration-current standard curve was established in simulated tissue fluid to ensure detection accuracy.

[0082] The experimental steps are as follows: 1. Prepare a 37℃ constant-temperature human simulated tissue solution, and prepare standard concentration solutions of glucose, lactic acid, and β-hydroxybutyric acid in gradients; 2. Immerse the sensor to be calibrated completely in the simulated tissue solution, connect it to the electrochemical workstation, and let it stand for 5 minutes to allow the system to reach equilibrium; 3. Prepare glucose standard solutions covering the range of 2.2 mmol / L to 22 mmol / L with at least 5 gradients using phosphate buffer. The solutions contain the lower limit of measurement of 2.2 mmol / L, the manufacturer-specified characteristic concentrations in the range of 4 mmol / L to 11.1 mmol / L, and the upper limit of measurement of 22 mmol / L. The test environment is controlled at 23℃±5℃. After the temperature of each concentration solution stabilizes and the sensor output current reaches a steady state, the steady-state current values ​​corresponding to each working electrode at different standard concentrations are measured sequentially. Each concentration group is tested three times and the average value is taken; 4. Perform a univariate linear regression to fit the standard curve with glucose concentration as the abscissa and the mean steady-state current of the corresponding electrode as the ordinate, solve the linear regression equation to obtain the detection sensitivity, and calculate the linear correlation coefficient r. 5. Substitute the current at each concentration into the regression equation to back-calculate the glucose concentration, and obtain the absolute deviation and relative deviation respectively; then check according to the standard judgment index: the linear correlation coefficient of the curve r≥0.99, the linear deviation of all concentration points does not exceed ±0.83mmol / L or ±15% (take the larger of the two limits), and the linearity and error of all electrode curves meet the above indicators, then the calibration is deemed qualified, and this linearity test procedure is completed; if it is necessary to verify the temperature response performance at the same time, the entire set of concentration gradient tests should be repeated under constant temperature conditions of 34℃, 38℃, and 42℃ (±0.5℃), and the correlation coefficient of the fitted curve at each temperature should still meet r≥0.99.

[0083] The response of the subcutaneously implanted multi-metabolic continuous monitoring sensor prepared in this embodiment to gradient glucose solution is shown in the figure below. Figure 2 As shown.

[0084] The test results of the subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in this embodiment are as follows: (a) Anti-interference performance: In the mixed interfering substance system, the glucose signal deviation is ≤2.6%, the β-hydroxybutyric acid signal deviation is ≤2.4%, and there is no cross-response between the two components; (b) Long-term stability: After continuous immersion in simulated body fluid at constant temperature for 21 days, the GOx enzyme activity was 91% and the β-HBDH activity was 89% retained on day 14; the GOx activity was 83% and the β-HBDH activity was 80% on day 21; the total drift of the Ag / AgCl reference potential was ≤±4.5mV, and there was no membrane peeling or circuit breakage.

[0085] Example 2 A subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators, which differs from Example 1 in that the enzyme in the WE2 enzyme-modified layer is lactate oxidase, the thickness of the enzyme-modified layer is 6 μm, and the rest of the structure is the same as that in Example 1. The preparation method of the subcutaneous implantable multi-metabolic continuous monitoring sensor differs from that in Example 1 in step (3) by cross-linking and curing the enzyme-modified solution (composition: LOx 25mg / mL, bovine serum albumin 25mg / mL, 0.5% glutaraldehyde, graphene 3mg / mL, PBS pH7.2) on one electrode at 37°C for 2.5h to obtain WE2, and coating the surface of the enzyme-modified layer with a Nafion anti-interference membrane; the remaining steps and parameters are the same as in Example 1.

[0086] This embodiment prepares a four-electrode sensor for simultaneous detection of glucose and lactic acid.

[0087] Test Example 2 The anti-interference performance of the subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in Example 2 was tested: after adding the same concentration of interfering substances (0.1mM ascorbic acid, 0.1mM uric acid, 1μM dopamine), the glucose signal deviation was ≤2.5%, the lactate signal deviation was ≤1.8%, and the Nafion membrane effectively blocked the interfering substances; the system response time was stable at ≤35s. Long-term stability test: Continuous monitoring in an in vitro simulated body fluid environment for 21 days showed that GOx activity was 90% and LOx activity was 90% on day 14; GOx activity was 82% and LOx activity was 81% on day 21; there was no electrochemical crosstalk between the two working electrodes, and the dynamic metabolic signal was continuously and stably output.

[0088] Example 3 A subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators, which differs from Example 1 in that the enzyme in the WE1 enzyme-modified layer is lactate oxidase and the thickness of the enzyme-modified layer is 6 μm, while the rest of the structure is the same as in Example 1. The preparation method of the subcutaneous implantable multi-metabolic continuous monitoring sensor differs from that of Example 1 in that the enzyme modification solution (composition: LOx 25mg / mL, 0.5% glutaraldehyde, PBS pH7.2) on one electrode is cross-linked and cured at 37°C for 2.5h to obtain WE1. The remaining steps and parameters are the same as those in Example 1.

[0089] This embodiment prepares a four-electrode sensor for simultaneous detection of lactic acid and ketone bodies.

[0090] Test Example 3 Performance tests were conducted on the subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in Example 3: Interference resistance and detection accuracy: Simulated subcutaneous tissue interference fluid was prepared using pH 7.4 and 0.01 mol / L HEPES buffer, containing 0.1 mM ascorbic acid, 0.1 mM uric acid, and lactic acid. Signal deviation was ≤1.7%, and β-hydroxybutyrate signal deviation was ≤2.3%. There was no glucose cross-interference, and the full-range detection error was ≤5%. Long-term stability: After 21 days of continuous immersion in simulated body fluid, the LOx activity was 91% and the β-HBDH activity was 88% on day 14; and the LOx activity was 82% and the β-HBDH activity was 79% on day 21. The reference electrode potential fluctuation was ≤±4mV, making it suitable for long-term subcutaneous metabolic monitoring and blood disease risk warning.

[0091] Example 4 A subcutaneous implantable sensor for continuous monitoring of multiple metabolic indicators comprises a flexible substrate and an electrode system and a connection system disposed on the surface of the flexible substrate; the flexible substrate is L-shaped and consists of an implant arm and a connecting arm connected at an angle; the implant arm is 2 mm wide, 8 mm long, and has a semi-circular end; the connecting arm is 4 mm wide, 10 mm long, and has an angle of 90°. The electrode system consists of a pure carbon working electrode (WE), a pure carbon counter electrode (CE), and a reference electrode Ag / AgCl (RE). There are three working electrodes, named WE1, WE2, and WE3. WE1, WE2, and RE are distributed on the same surface of the implant arm of the flexible substrate, and are arranged sequentially from the end of the implant arm to the connection. WE3 and CE are disposed on the other surface of the implant arm of the flexible substrate, and are arranged sequentially from the end of the implant arm to the connection. The working electrode consists of a working electrode layer, an enzyme-modified layer, and a protective layer sequentially stacked on the surface of a flexible substrate; the enzyme in the WE1 enzyme-modified layer is glucose oxidase, the enzyme in the WE2 enzyme-modified layer is β-hydroxybutyrate dehydrogenase, and the enzyme in the WE3 enzyme-modified layer is lactate oxidase; wherein, the thickness of the WE1 enzyme-modified layer is 8 μm, the thickness of the WE2 enzyme-modified layer is 7 μm, and the thickness of the WE3 enzyme-modified layer is 8 μm; the thickness of the vinyl homopolymer protective layer is 12 μm for each layer. The connection system consists of working electrode wires and working electrode contacts that are sequentially connected to each working electrode; the working electrode wires are wires with a surface covered by a 1μm thick PI insulating layer; the working electrode contacts are disposed at one end of the connecting arm on the surface of the flexible substrate; and the working electrode wires are disposed on the surface of the flexible substrate between the working electrode and the working electrode contacts.

[0092] The preparation method of the subcutaneous implantable multi-metabolic continuous monitoring sensor described in this embodiment is as follows: (1) Provide an L-shaped PI flexible substrate, and prepare three working electrode layers (300 mesh carbon paste) with a thickness of 10 μm, a wire with a thickness of 10 μm, a working electrode contact with a thickness of 10 μm and a counter electrode with a thickness of 10 μm on the surface of the flexible substrate by screen printing process. After drying at 120℃, prepare a reference electrode with a thickness of 10 μm by screen printing process; then cover the surface of the working electrode wire with PI paste, and obtain the initial electrode after drying. (3) A glucose oxidase modification solution was applied to the surface of a working electrode layer of the initial electrode obtained in step (2). The composition of the glucose oxidase modification solution was (GOx 30mg / mL, human serum albumin 30mg / mL, carbon nanotubes 5mg / mL, HEPES solution pH 7.4 as solvent). After thermal crosslinking and curing at 37°C for 2.5h, WE1 was obtained. A β-hydroxybutyrate dehydrogenase modification solution was spot-coated onto the surface of the second working electrode layer. The glucose oxidase modification solution consisted of β-HBDH 20 mg / mL, human serum albumin 30 mg / mL, PEGDGE 8 mg / mL, iron oxide (electron mediator) 4 mg / mL, and HEPES solution at pH 7.5 as the solvent. After thermal cross-linking and curing at 40°C, WE2 was obtained. A third enzyme modification solution (consisting of LOx 25 mg / mL, bovine serum albumin 25 mg / mL, graphene 3 mg / mL, 0.5% glutaraldehyde, and PBS at pH 7.2) was spot-coated onto the surface of the third working electrode layer. After thermal cross-linking and curing at 45°C, WE3 was obtained. A protective film was then coated onto the surface to obtain a subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators.

[0093] This embodiment prepares a five-electrode sensor for the simultaneous detection of glucose, lactic acid, and ketone bodies.

[0094] Comparative Example 1 A subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators differs from Example 1 in that it does not have a vinyl homopolymer protective layer, but the rest of the structure is the same as that of Example 1.

[0095] The preparation method of the subcutaneous implantable multi-metabolic continuous monitoring sensor differs from that in Example 1 in that step (3) is as follows: an enzyme-modified solution (composition: GOx 30mg / mL, osmium complex 30mg / mL, HEPES pH 7.4) is applied to the surface of one working electrode layer of the initial electrode obtained in step (2), and PEGDGE is cross-linked and cured at 36℃ for 2.5h to obtain WE1. An enzyme-modified solution (composition: β-HBDH 20mg / mL, iron oxide mediator 4mg / mL, HEPES pH 7.5) is applied to the surface of the other working electrode layer, and cross-linked and cured at 36℃ for 2.5h to obtain WE2. The enzyme-modified layer is obtained, and the subcutaneous implantable multi-metabolic continuous monitoring sensor is obtained. The remaining preparation steps are the same as in Example 1.

[0096] Test Example 4 The subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in Comparative Example 1 was tested in simulated tissue fluid containing 0.2 mmol / L ascorbic acid and 0.4 mmol / L uric acid. The overall average signal deviation reached 8.6%, and the anti-interference performance was far worse than that of the subcutaneous implantable multi-metabolic continuous monitoring sensor prepared in Example 1.

[0097] As can be seen from the above structure, the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor obtained by the present invention, through the rational layout of the sensor structure, can achieve simultaneous detection of multiple indicators and has the advantages of high detection accuracy and strong anti-interference ability. In addition, the present invention separates the different functional working electrodes on the front and back of the flexible substrate, and sets an insulating layer to physically separate adjacent electrodes on the same side, thus spatially isolating the diffusion of by-products of different enzyme modification layers and local oxygen competition; and each working electrode is independently coated with the corresponding special enzyme modification solution, each enzyme system forms a film independently and does not come into contact with each other, eliminating the need for co-curing and mixing multiple enzymes, and completely avoiding the problems of different enzyme reaction mechanisms and film layer adaptation conflicts; at the same time, the entire substrate hardware is universal, and only the enzyme modification layer of a single electrode needs to be replaced to freely combine any monitoring combination such as blood glucose, lactate, blood ketones, etc., which greatly reduces the difficulty of multi-indicator sensor integration process.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A subcutaneously implantable sensor for continuous monitoring of multiple metabolic indicators, characterized in that, It includes a flexible substrate and an electrode system and a connection system disposed on the surface of the flexible substrate; The electrode system includes a working electrode, a counter electrode, and a reference electrode; the number of working electrodes is 1 to 3; each electrode in the electrode system is uniformly distributed on two surfaces at one end of the flexible substrate; The working electrode comprises a working electrode layer, an enzyme-modified layer, and a protective layer sequentially stacked on the surface of a flexible substrate; the enzyme-modified layer in different working electrodes has a different composition; the protective layer covers the surfaces of the enzyme-modified layer, the counter electrode, and the reference electrode; The connection system includes working electrode wires and working electrode contacts that are sequentially connected to each working electrode; the working electrode wires are wires with an insulating layer on their surface; the working electrode contacts are located on the surface of the flexible substrate opposite to the electrode system; and the working electrode wires are located on the surface of the flexible substrate between the working electrodes and the working electrode contacts.

2. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 1, characterized in that, The flexible substrate is L-shaped and includes an implant arm and a connecting arm connected at an angle; the working electrode is disposed at the non-connecting end of the implant arm; the working electrode contact is disposed at the non-connecting end of the connecting arm.

3. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 2, characterized in that, The included angle is 45°~135°.

4. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 2, characterized in that, The implant arm has a width of 0.5~2mm and a length of 5~20mm, and the end of the implant arm is rounded or semi-circular; the connecting arm has a width of 2~5mm and a length of 3~15mm.

5. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 1, characterized in that, The enzyme in the enzyme-modified layer is selected from one of the following: glucose oxidase, lactate oxidase, β-hydroxybutyrate dehydrogenase, uricase, cholesterol esterase combined with cholesterol oxidase dual enzyme system, and glycerol phosphate oxidase-peroxidase coupled enzyme system.

6. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 1, characterized in that, The protective layer is a vinyl homopolymer protective layer or a Nafion film.

7. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 6, characterized in that, The vinyl homopolymer protective layer is formed by cross-linking and curing a vinyl polymer solution; the vinyl polymer solution includes: medical-grade poly(hydroxyethyl methacrylate)-methyl methacrylate copolymer, vitamin E polyethylene glycol succinate, vinyl prepolymer and solvent.

8. The subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to claim 1, characterized in that, The thickness of the enzyme-modified layer is 5~10 μm.

9. A method for preparing the subcutaneous implantable multi-metabolic indicator continuous monitoring sensor according to any one of claims 1 to 8, characterized in that, Includes the following steps: (1) Screen printing is performed on the front and back surfaces of the flexible substrate to obtain the working electrode layer, wires, working electrode contacts, counter electrode and reference electrode; then insulating paste is covered on the surface of the working electrode wires and dried to obtain the primary electrode; (2) After coating the surface of each working electrode layer of the initial electrode obtained in step (1) with an enzyme-modified solution, a first cross-linking and curing reaction is performed to form an enzyme-modified layer on the surface of the working electrode layer; after coating the surface of the enzyme-modified layer, the counter electrode and the reference electrode with a protective layer solution, a second cross-linking and curing reaction is performed to obtain a subcutaneous implantable multi-metabolic indicator continuous monitoring sensor.

10. The application of the subcutaneous implantable multi-metabolic continuous monitoring sensor according to any one of claims 1 to 8 or the subcutaneous implantable multi-metabolic continuous monitoring sensor prepared by the preparation method according to claim 9 in the simultaneous detection of multiple metabolic indicators.

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