Ketone body biosensor and preparation method thereof

Abstract

The invention discloses a ketone body biosensor and a preparation method thereof.The ketone body biosensor comprises a substrate, a working electrode, a reference electrode, a counter electrode, an active enzyme layer and an outer membrane adjusting layer, the working electrode and the reference electrode are distributed on one face of the substrate, the counter electrode is distributed on the other opposite face of the substrate, the active enzyme layer is arranged on the surface of the working electrode, and the outer membrane adjusting layer is arranged on the surface of the working electrode. And the working electrode, the reference electrode and the counter electrode are completely wrapped by the outer membrane adjusting layer. The ketone body biosensor provided by the invention is stable in performance, safe, reliable and high in sensitivity, can continuously monitor the change of the beta-hydroxybutyric acid level in the body, is stable and mature in preparation process, and is beneficial to improving the production efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biosensors, and particularly relates to a ketone body biosensor and a preparation method thereof. BACKGROUND

[0002] As an important branch of electrochemical sensors, biosensors can identify the chemical-related signals generated by a certain substance and convert them into electrochemical signals, and through further signal amplification, specific, highly selective, and highly sensitive detection and analysis of the specific substance can be achieved. Biosensor detection technology greatly shortens the detection time, has the characteristics of simple operation and low cost, and has been widely used in clinical testing, food safety, drug analysis, environmental monitoring, and military and other important fields.

[0003] Diabetes is a common disease caused by the interaction of genetic and environmental factors, and its main clinical manifestation is high blood sugar. Common symptoms include polydipsia, polyuria, polyphagia, and weight loss. Diabetic patients have the possibility of developing hyperglycemic crisis at any time, and the most common acute conditions include diabetic ketosis and diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic syndrome (HHS), especially DKA, which is fast-acting and severe, and seriously endangers the health of diabetic patients. The main reason for the occurrence of DKA is insulin deficiency, accompanied by inappropriate elevation of various hyperglycemic hormones such as glucagon, corticosteroids, catecholamines, and growth hormone. If not treated in time, it may cause brain edema, permanent nerve damage, and even death. In recent years, studies have shown that blood ketone determination can effectively reflect the severity of the disease in diabetic patients, and has important significance for early and rapid diagnosis of diabetic patients with ketoacidosis, prevention of disease progression, disease monitoring, and reduction of the incidence and mortality of DKA.

[0004] Monitoring the content of ketone bodies in the human body requires a reliable, fast, sensitive, and accurate method. Ketone bodies are composed of β-hydroxybutyric acid (β-HBA), acetoacetic acid (AcAc), and acetone, with proportions of 78%, 20%, and 2%, respectively. β-HBA is the main substance that causes DKA. Therefore, ketone body detection is mainly to detect the β-HBA component. β-HBA is dehydrogenated to AcAc under the catalysis of β-hydroxybutyric acid dehydrogenase, and then metabolized to the outside through a series of pathways.

[0005] The existing ketone body detection method has the following problems: 1. Today, the traditional nitroprusside method is commonly used in clinical practice to monitor urine ketones, which is a semi-quantitative method and cannot accurately reflect the specific content of ketones. Moreover, it can only detect the acetoacetic acid component in ketones. 2. At present, blood ketone monitoring can significantly improve the positive rate of DKA patients, and has important guiding significance in the treatment of DKA, but all are in vitro detection, which does not have timeliness and continuity; 3. The ketone body continuous monitoring biosensor has no successful commercial application, which is mainly limited by low sensitivity, and the stability in continuous monitoring application needs to be further improved.

[0006] Based on this, the application discloses a ketone body biosensor and a preparation method thereof. SUMMARY

[0007] In order to solve the problems in the prior art, the purpose of the present application is to provide a ketone body biosensor and a preparation method thereof.

[0008] In order to achieve the above-mentioned purposes and achieve the above-mentioned technical effects, the technical scheme adopted by the present application is as follows: A ketone body biosensor, comprising a substrate, a working electrode, a reference electrode, a counter electrode, an active enzyme layer and an outer membrane regulating layer, the working electrode and the reference electrode are distributed on one side of the substrate, the counter electrode is distributed on the other side of the substrate opposite to the one side, the active enzyme layer is arranged on the surface of the working electrode, and the working electrode, the reference electrode and the counter electrode are completely wrapped by the outer membrane regulating layer.

[0009] Further, the substrate is made of flexible high polymer inert material, and the thickness is 0.075-0.20 mm.

[0010] Further, the working electrode is a gold electrode with a surface electroplated platinum black layer.

[0011] Further, the reference electrode is an Ag / AgCl reference electrode.

[0012] Further, the counter electrode is a gold electrode.

[0013] Further, the active enzyme layer is prepared by the following steps: Tris-HCI buffer solutions of beta-hydroxybutyric acid dehydrogenase and oxidized coenzyme I are respectively prepared, and the solutions are fully mixed in a certain proportion to obtain a solution S1; An osmium complex with an amino group is dissolved in anhydrous ethanol to obtain a solution S2; An ethanol solution of polymethacrylate and vinylpyridine copolymer is configured as a solution S3; A Tris-HCI buffer solution of 1,10-phenanthroline-5,6-dione (PD) is configured to obtain a solution S4; Mixing solution S1, solution S2, solution S3 and solution S4 uniformly according to a certain proportion, then adding polyethylene glycol diglycidyl ether, mixing and oscillating fully, through the method of brush coating, drop coating or dip coating, repeating operation multiple times, crosslinking at 30-40℃ for a period of time and drying, the active enzyme layer can be obtained.

[0014] Further, the outer membrane regulating layer comprises a diffusion layer and a hydrophilic surface layer arranged in order from inside to outside.

[0015] Further, the diffusion layer is prepared by polyester, polymethylsiloxane, polyphenol, o-phenylenediamine, polyaniline, polypyrrole, o-aminophenol, perfluorosulfonic acid type polymer or copolymer with hydrophilic component.

[0016] Further, the hydrophilic surface layer adopts a copolymer of methacrylic acid sulfobetaine and vinylpyridine, and a hydrophilic polymer film is formed by chemical crosslinking.

[0017] The application further discloses a preparation method of the ketone body biosensor. 1) preparing a substrate, a working electrode, a reference electrode and a counter electrode; 2) distributing the working electrode and the reference electrode on one side of the substrate, and distributing the counter electrode on the other side of the substrate; 3) manufacturing an active enzyme layer on the surface of the working electrode; 4) manufacturing a diffusion layer and a hydrophilic surface layer on the surfaces of the working electrode, the reference electrode and the counter electrode in sequence to form an outer membrane regulating layer.

[0018] Compared with the prior art, the application has the following beneficial effects: The application discloses a ketone body biosensor and a preparation method thereof. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Fig. 1 is a structural schematic diagram of the application; wherein, Figure 1 a is a position schematic diagram of the working electrode and the reference electrode, Figure 1 b is a position schematic diagram of the counter electrode; Figure 2 Fig. 2 is a β-HBA continuous response signal diagram of the application; Figure 3 Fig. 3 is a ketone body biosensor linear response diagram obtained through data processing. Figure 2 Fig. 3 is a ketone body biosensor linear response diagram obtained through data processing. DETAILED DESCRIPTION

[0020] The present application will be described in detail below so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the scope of protection of the present application can be more clearly defined.

[0021] The following presents a simplified summary of one or more aspects in order to provide a basic understanding of such aspects. This summary is not an extensive overview of all contemplated aspects, and is intended to neither identify key or critical elements of all aspects nor delineate the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that is presented later.

[0022] As shown in Figures 1-3 The present application discloses a kind of ketone body biosensor, it mainly includes substrate, working electrode 1, reference electrode 2, counter electrode 3, active enzyme layer and outer membrane adjustment layer, wherein, working electrode 1 and reference electrode 2 are distributed in one side of substrate, counter electrode 3 is distributed in the opposite side of substrate, working electrode 1 is gold electrode with surface electroplating platinum black layer, reference electrode 2 is Ag / AgCl reference electrode, counter electrode 3 is gold electrode, active enzyme layer is arranged on the surface of working electrode 1, outer membrane adjustment layer completely wraps three electrodes (working electrode 1, reference electrode 2, counter electrode 3).

[0023] In some embodiments, the substrate is made of flexible high polymer inert material, including at least one of polyvinylidene fluoride (PVDF), polypropylene (PP), polyimide resin (PI), polyethylene terephthalate (PET) and polyester fiber (PES), which can maintain a certain degree of shape without twisting too much like fabric, without affecting the fixing effect of the sensor. The thickness of the substrate is 0.075-0.20mm.

[0024] In some embodiments, the electrode is made by screen printing process, which has the advantages of flexible design, simple processing method, low cost, high sensitivity and easy miniaturization. The electrode is made by laser cutting process, which has the advantages of fast cutting speed, high processing precision, no damage to the surface of the material, smooth cutting edge and no shear burr.

[0025] In some embodiments, the working electrode 1 is made of gold nanoparticle slurry, and the platinum black layer is plated on the surface of the working electrode 1 by magnetron sputtering method. The high conductivity of gold electrode can provide stable current transmission and ensure the stability and reliability of the sensor performance; the platinum black layer is a kind of porous material with high surface area and high catalytic performance, which can improve the catalytic activity of the electrode surface and promote the biochemical reaction, thereby improving the sensitivity of the sensor. By increasing the surface area of the electrode, the platinum black layer can reduce the current density, thereby reducing the polarization effect and capacitance interference, forming a stable electrochemical interface on the electrode surface, and effectively improving the stability and reproducibility of the electrode.

[0026] In some embodiments, the reference electrode 2 is an Ag / AgCl reference electrode prepared by chloridizing an Ag-plated electrode immersed in a 2-6 mol / L KCl solution by chronopotentiometry.

[0027] In some embodiments, the counter electrode 3 is a gold electrode prepared using a gold nanoparticle slurry.

[0028] In some embodiments, the preparation steps of the active enzyme layer include: A Tris-HCI buffer solution of beta-hydroxybutyric acid dehydrogenase and oxidized coenzyme I (NAD+, 0-5 mg / mL) is prepared respectively, and uniformly mixed in a certain proportion to obtain a solution S1; An osmium complex with an amino group is dissolved in anhydrous ethanol to obtain a solution S2; An ethanol solution of polymethacrylate and vinylpyridine copolymer is prepared as a solution S3; A Tris-HCI buffer solution of 1,10-phenanthroline-5,6-dione (PD) is prepared to obtain a solution S4; The solution S1, the solution S2, the solution S3, and the solution S4 are uniformly mixed in a certain proportion, and polyethylene glycol diglycidyl ether is added, mixed and shaken, and then cross-linked and dried at 30-40℃ by repeated operations such as brushing, dripping or dipping, to obtain the active enzyme layer.

[0029] In the present application, the osmium complex is a highly efficient electron mediator that can oxidize the enzyme and be reduced itself, and the reduced electron mediator is oxidized on the electrode in the cycle process of electron transfer, which can reduce the dependence on the oxygen concentration in the solution and improve the detection sensitivity. The polymer POEGMA-co-PVP has good chemical inertness and is relatively stable. After cross-linking reaction with PEGDGE and the osmium complex, a hydrophilic microporous membrane layer is formed, and the covalent bonding method and the embedding method are used to achieve good fixation effect of beta-hydroxybutyric acid dehydrogenase, coenzyme and electron mediator molecules, which does not affect the activity of the enzyme and does not cause the loss of the electron mediator, ensuring the service life of the sensor.

[0030] In some embodiments, the outer membrane regulating layer includes a diffusion layer and a hydrophilic surface layer arranged in order from inside to outside.

[0031] In some embodiments, the diffusion layer is prepared by polyester, polymethylsiloxane, polyphenol, o-phenylenediamine, polyaniline, polypyrrole, o-aminophenol, perfluorosulfonic acid type polymer or copolymer with hydrophilic components.

[0032] In some embodiments, the diffusion layer is a blend of polyurethane and polydimethylsiloxane (PDMS). The modified polyurethane has a certain degree of hydrophilicity, which can regulate the penetration rate of the analyte molecules by adjusting the coating thickness, and a small amount of hydrophobic PDMS can promote oxygenation ability while increasing the adhesion of the membrane layer, both of which work together to regulate the penetration rate of β-hydroxybutyric acid molecules.

[0033] In some more specific embodiments, the diffusion layer is a blend of polyurethane and polydimethylsiloxane (PDMS). A tetrahydrofuran film solution of polyurethane and PDMS is configured. By means of brush coating, drop coating or dip coating, the operation is repeated 2-3 times, and the electrode surface is uniformly coated, and dried at 32-37°C for 30-60 min.

[0034] In some embodiments, the hydrophilic surface layer uses a methacrylic acid sulfobetaine (SBMA) and vinylpyridine (PVP) copolymer (PSBMA-co-PVP) to form a hydrophilic polymer film through chemical cross-linking. SBMA is a zwitterionic compound with excellent biocompatibility, which has good solubility in aqueous solution, salt solution and alcohol solution; PVP has a large number of pyridine groups as active sites, which can react with cross-linking agents such as polyethylene glycol diglycidyl ether (PEGDGE) to form covalent bonds. The presence of the two components not only ensures the biological properties of the sensor, but also enhances the firmness of the membrane layer, which helps to improve the service life of the sensor.

[0035] In some more specific embodiments, the hydrophilic surface layer uses PSBMA-co-PVP, and a 0.2-1.0 wt% ethanol solution is configured, and 5 mg / mL polyethylene glycol diglycidyl ether (PEGDGE) is added, mixed and shaken, and the electrode surface is uniformly coated by means of brush coating, drop coating or dip coating, and dried at 28-37°C for 30-60 min.

[0036] The application also discloses a preparation method of a ketone body biosensor, comprising the following steps: 1) preparing a substrate, a working electrode 1, a reference electrode 2 and a counter electrode 3; 2) distributing the working electrode 1 and the reference electrode 2 on one side of the substrate, and distributing the counter electrode 3 on the other side of the substrate; 3) preparing an active enzyme layer on the surface of the working electrode 1; 4) sequentially preparing a diffusion layer and a hydrophilic surface layer on the surfaces of the working electrode 1, the reference electrode 2 and the counter electrode to form an outer membrane regulating layer.

[0037] Example 1 As Figures 1-3As shown, a ketone body biosensor mainly comprises a substrate, a working electrode 1, a reference electrode 2, a counter electrode 3, an active enzyme layer and an outer membrane regulating layer, wherein the working electrode 1 and the reference electrode 2 are distributed on one side of the substrate, the counter electrode 3 is distributed on the opposite side of the substrate, the active enzyme layer is arranged on the surface of the working electrode 1, and the outer membrane regulating layer completely wraps the three electrodes (the working electrode 1, the reference electrode 2 and the counter electrode 3).

[0038] In this embodiment, the substrate is made of polyimide resin (PI) with a thickness of 0.20 mm.

[0039] The electrode is made by screen printing process, which has the advantages of flexible design, simple processing method, low cost, high sensitivity and easy miniaturization. The electrode is made by laser cutting process, which has the advantages of fast cutting speed, high processing precision, no damage to the surface of the material, smooth cutting edge and no shear burr.

[0040] The working electrode 1 is made of gold nanoparticle slurry, and a platinum black layer is plated on the surface of the working electrode 1 by magnetron sputtering. The high conductivity of the gold electrode can provide stable current transmission and ensure the stability and reliability of the sensor performance. The platinum black layer is a material with high porosity, high surface area and high catalytic performance, which can improve the catalytic activity of the electrode surface and promote the biochemical reaction, thereby improving the sensitivity of the sensor. By increasing the surface area of the electrode, the platinum black layer can reduce the current density, thereby reducing the polarization effect and capacitive interference, forming a stable electrochemical interface on the electrode surface, and effectively improving the stability and reproducibility of the electrode.

[0041] The reference electrode 2 is an Ag / AgCl reference electrode prepared by immersing an Ag-plated electrode in a 3 mol / L KCl solution for chlorination by chronopotentiometry.

[0042] The counter electrode 3 is a gold electrode prepared by using gold nanoparticle slurry.

[0043] The preparation steps of the active enzyme layer include: Tris-HCI buffer solutions of β-hydroxybutyric acid dehydrogenase and oxidized coenzyme I are prepared respectively, and the two solutions are mixed in a volume ratio of 1:2 to obtain a solution S1, so that the active enzyme molecules are stable in the solution; An osmium complex with an amino group is dissolved in anhydrous ethanol to obtain a dark brown solution S2; An ethanol solution of poly(ethylene glycol) methacrylate and vinylpyridine (POEGMA-co-PVP) copolymer is prepared as solution S3; A Tris-HCI buffer solution of 1,10-phenanthroline-5,6-dione (PD) is prepared to obtain solution S4; Mix solutions S1, S2, S3, and S4 in a volume ratio of 2:2:1:2, then add 100 mg / mL polyethylene glycol diglycidyl ether (PEGDGE), mix thoroughly and shake, and repeat the process twice by brushing. Crosslink the mixture at 37°C and dry it to obtain the active enzyme layer.

[0044] The outer membrane conditioning layer includes a diffusion layer and a hydrophilic surface layer arranged sequentially from the inside to the outside.

[0045] The diffusion layer is a blend of polyurethane and polydimethylsiloxane (PDMS). A tetrahydrofuran film solution of polyurethane and PDMS was prepared. The solution was applied evenly to the electrode surface by brushing, dripping, or dipping, repeating the process twice, and then dried at 37°C for 30 minutes.

[0046] The hydrophilic surface layer uses PSBMA-co-PVP. A 0.5wt% ethanol solution of PSBMA is prepared, and then 5mg / mL polyethylene glycol diglycidyl ether (PEGDGE) is added. The mixture is thoroughly mixed and shaken. The process is repeated twice by brushing, dripping, or dipping to evenly coat the electrode surface. The electrode is then dried at 37℃ for 30 minutes.

[0047] This embodiment also discloses a method for preparing a ketone body biosensor, including the following steps: 1) Prepare the substrate, working electrode 1, reference electrode 2, and counter electrode 3; 2) A working electrode 1 and a reference electrode 2 are distributed on one side of the substrate, and a counter electrode 3 is distributed on the opposite side of the substrate; 3) An active enzyme layer is fabricated on the surface of working electrode 1; 4) A diffusion layer and a hydrophilic surface layer are sequentially fabricated on the surfaces of the working electrode 1, the reference electrode 2, and the counter electrode to form an outer film conditioning layer.

[0048] The sensitivity of the ketone body sensor prepared in the examples was tested using an electrochemical workstation. The test results are shown in [link to relevant documentation]. Figures 2-3 . Figure 2 This indicates that the current signal gradually increases with the increase of β-HBA concentration in the test solution. Figure 3 This indicates that the ketone body sensor exhibits a good linear response within the concentration range of 0–5 mmol / L.

[0049] Any parts or structures not specifically described in this invention can be made using existing technologies or products, and will not be elaborated upon here.

[0050] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A ketone body biosensor characterized by, The application relates to a novel enzyme electrode, which comprises a substrate, a working electrode, a reference electrode, a counter electrode, an active enzyme layer and an outer membrane regulating layer.

2. The ketone body biosensor according to claim 1, wherein The substrate is made of flexible high polymer inert material, and the thickness is 0.075-0.20 mm.

3. The ketone body biosensor according to claim 1, wherein The working electrode is a gold electrode with a surface-plated platinum black layer.

4. The ketone body biosensor according to claim 1, wherein The reference electrode is an Ag / AgCl reference electrode.

5. The ketone body biosensor according to claim 1, wherein The counter electrode is a gold electrode.

6. The ketone body biosensor according to claim 1, wherein The active enzyme layer is prepared by the following steps: Tris-HCl buffer solutions of beta-hydroxybutyric acid dehydrogenase and oxidized coenzyme I are respectively prepared, and the solutions are uniformly mixed according to a certain proportion to obtain solution S1. An osmium complex with an amino group is dissolved in anhydrous ethanol to obtain solution S2. An ethanol solution of polymethacrylate and vinylpyridine copolymer is prepared as solution S3. A Tris-HCl buffer solution of 1,10-phenanthroline-5,6-dione is prepared to obtain solution S4. Solution S1, solution S2, solution S3 and solution S4 are uniformly mixed according to a certain proportion, polyethylene glycol diglycidyl ether is added, and the mixture is fully mixed and shaken, and then the active enzyme layer is obtained by repeatedly operating through brushing, dropping or dipping coating methods, cross-linking at 30-40 DEG C for a period of time and drying.

7. The ketone body biosensor according to claim 1, wherein The outer membrane regulating layer comprises a diffusion layer and a hydrophilic surface layer arranged in sequence from inside to outside.

8. The ketone body biosensor according to claim 7, wherein The diffusion layer is prepared by polyester, polymethylsiloxane, polyphenol, o-phenylenediamine, polyaniline, polypyrrole, o-aminophenol, perfluorosulfonic acid type polymer or copolymer with hydrophilic components.

9. The ketone body biosensor according to claim 7, wherein The hydrophilic surface layer is formed by chemical cross-linking of a sulfobetaine methacrylic acid and vinylpyridine copolymer into a hydrophilic polymer film.

10. The method of claim 1-9, wherein the ketone body biosensor is prepared by the steps of: The application further discloses a preparation method of the enzyme electrode, which comprises the following steps: 1) preparing a substrate, a working electrode, a reference electrode and a counter electrode; 2) distributing the working electrode and the reference electrode on one side of the substrate, and distributing the counter electrode on the other side of the substrate; 3) manufacturing an active enzyme layer on the surface of the working electrode; 4) sequentially manufacturing a diffusion layer and a hydrophilic surface layer on the surfaces of the working electrode, the reference electrode and the counter electrode to form an outer membrane regulating layer.

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