Continuous glucose monitoring sensor based on one-dimensional titanium dioxide material and preparation method thereof

Abstract

The invention belongs to the technical field of biosensors, and particularly relates to a one-dimensional titanium dioxide material-based continuous glucose monitoring sensor and a preparation method thereof, the preparation method comprises the following steps: S1, preparation of a one-dimensional titanium dioxide material: dispersing multi-walled carbon nanotubes in isopropanol, adding diethylenetriamine and titanium tetraisopropoxide for a reaction, filtering, washing, and drying to obtain the one-dimensional titanium dioxide material; then centrifuging, drying and calcining to obtain a one-dimensional titanium dioxide material; s2, GOx immobilization: dispersing the one-dimensional titanium dioxide material in a GOx solution, and oscillating to form a Gox-TiO2 material; s3, electrode preparation: preparing a microneedle array three-electrode system from the microneedle array electrode base material through 3D printing and magnetron sputtering processes; and S4, performing functional layer modification on the working electrode, wherein the functional layer modification comprises cysteine modification, glutaraldehyde modification and glucose oxidase immobilization. Through the sensor disclosed by the invention, the sensing performance of the existing CGMS can be improved, the medium loss of the sensor is prevented, the stability is improved, and the detection range is expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material and a preparation method thereof. Background Art

[0002] Continuous glucose monitoring (CGM), often referred to as "dynamic glucose monitoring," is a technology that continuously monitors blood glucose concentrations. It uses a glucose sensor to measure glucose concentrations in the interstitial fluid beneath the skin, indirectly reflecting blood glucose levels. CGM is designed to provide continuous, comprehensive, and reliable blood glucose information throughout the day, detect asymptomatic hypoglycemia and hyperglycemia, and help understand the trends and patterns of blood glucose fluctuations.

[0003] The CGM system consists of a sensor, a transmitter, and a receiver. The sensor continuously monitors glucose levels in the interstitial fluid of the subcutaneous tissue via a microprobe implanted in the subcutaneous tissue. The transmitter then wirelessly transmits the blood glucose data to an information receiver, such as a system-specific mobile app.

[0004] Existing second-generation glucose sensors rely on artificial mediators (such as ferrocene derivatives) to transfer electrons, which have problems such as easy loss of mediators, poor stability and insufficient detection range. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention provides a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material and a preparation method thereof, so as to improve the sensing performance of the existing CGMS, prevent the loss of sensor medium, improve stability, and expand the detection range.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The present invention aims to provide a method for preparing a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material, comprising the following steps:

[0008] S1. Preparing a one-dimensional titanium dioxide material: dispersing multi-walled carbon nanotubes in isopropyl alcohol, adding diethylenetriamine and tetraisopropoxytitanium to react, and then centrifuging, drying, and calcining to obtain a one-dimensional titanium dioxide material;

[0009] S2, GOx immobilization: dispersing one-dimensional titanium dioxide material in GOx solution and oscillating to form Gox-TiO2 material;

[0010] S3. Electrode preparation: The microneedle array electrode substrate is prepared by 3D printing and magnetron sputtering to form a microneedle array three-electrode system;

[0011] S4. Perform functional layer modification on the working electrode, including cysteine modification, glutaraldehyde modification, and glucose oxidase immobilization.

[0012] Furthermore, the method for preparing a one-dimensional titanium dioxide material includes: dispersing acid-treated multi-walled carbon nanotubes in isopropanol, adding diethylenetriamine and tetraisopropoxytitanium, and reacting at 170-190°C for 20-40 hours; after the reaction solution is cooled to room temperature, it is transferred to a centrifuge tube, centrifuged at 11000-13000rpm for 8-12 minutes, and the supernatant is discarded; 15-25mL of deionized water is added, ultrasonicated for 3-7 minutes, centrifuged at 11000-13000rpm for 8-12 minutes, the supernatant is discarded, and the water washing is repeated twice; 15-25mL of anhydrous ethanol is added, stirred for 8-12 minutes, centrifuged at 11000-13000rpm for 8-12 minutes, the supernatant is discarded, and the ethanol washing is repeated once, the precipitate is transferred to a culture dish, and vacuum dried at 50-70°C for 20-28 hours to obtain a black powder; and then calcined at 500-600°C for 1-3 hours to obtain a one-dimensional titanium dioxide material.

[0013] Furthermore, the GOx immobilization method includes: dispersing 8-12 mg of one-dimensional titanium dioxide material in 1-2 mL of GOx solution, shaking at room temperature for 0.5-1 hour, and letting the solution stand at 3-5°C overnight to form a Gox-TiO2 material.

[0014] Furthermore, the electrode preparation method includes: using polylactic acid as a raw material using a 3D printer, setting the nozzle temperature to 200-220°C, the platform temperature to 50-70°C, the layer height to 0.03-0.07mm and the printing speed to 15-25mm / s to prepare a microneedle array, ultrasonically cleaning with isopropyl alcohol and treating with ultraviolet ozone, spin coating a 3-7% PMMA chlorobenzene solution at 2000-4000rpm using a sizing machine to form an insulating layer, and curing on a hot plate at 70-90°C; then using a magnetron sputtering instrument to form a 5×10 -6 Three thin films of Cr, Pt, and Ag were deposited sequentially under a Torr background vacuum. After the electrode pattern was defined by photolithography, the Ag layer in the working electrode and counter electrode areas was removed by wet etching with a 1:10 nitric acid solution for 20-40 seconds, exposing the Pt counter electrode. A 90-110 nm thick Au layer was then deposited as the working electrode under mask protection by magnetron sputtering. Finally, the photoresist was ultrasonically removed using acetone, and electrolysis was performed in a 0.1-0.2 M HCl solution at a voltage of +0.4-0.6 V for 20-40 seconds to convert the Ag on the reference electrode surface into Ag / AgCl.

[0015] Furthermore, the microneedle array has a base width of 100-300 μm, a height of 500-700 μm, a tip diameter of 10-30 μm, a base spacing of 100-300 μm, and a base array of 6×6.

[0016] Furthermore, the photolithography process includes: spin coating at 2000-4000 rpm, pre-baking at 80-100°C for 50-70 seconds, and 365nm UV light at 90-110 mJ / cm 2 Exposure, and processing with AZ 300MIF developer.

[0017] Furthermore, the method for modifying the functional layer of the working electrode includes:

[0018] 1) Cysteine modification: The gold electrode region was site-specifically modified with a 20-40 mM cysteine aqueous solution and incubated at room temperature for 20-28 hours. The electrode was rinsed with deionized water to remove loosely attached cysteine.

[0019] 2) Glutaraldehyde modification: Modify the Au-Cys region with 2-3% glutaraldehyde in phosphate buffer, incubate at room temperature for 0.5-1.5 hours, and rinse the electrode with the same phosphate buffer;

[0020] 3) Immobilization of glucose oxidase: The Au-Cys-GA modified area was modified with Gox-TiO2 solution, incubated at 3-5°C for 5-7 hours, and the electrode was rinsed with PBS buffer to obtain the final Au-Cys-GA-Gox-TiO2 working electrode.

[0021] A continuous glucose monitoring sensor based on one-dimensional titanium dioxide material is prepared by the above preparation method.

[0022] Compared with the prior art, the beneficial technical effects of the present invention are:

[0023] 1. The present invention prepares a novel one-dimensional titanium dioxide material by a solvothermal method, and covalently bonds the one-dimensional titanium dioxide immobilized with glucose oxidase (Gox) to the working electrode to form a sensor. The porous structure and high specific surface area of the one-dimensional titanium dioxide provide a good immobilization environment for the protein and promote direct electron transfer. Glucose is oxidized to produce gluconic acid under the catalysis of GOx, and the reduced state of the enzyme (FADH2) is directly transferred to the gold electrode through the surface of the one-dimensional titanium dioxide. No dissolved oxygen or artificial medium is required, successfully realizing high-performance glucose sensing without a medium.

[0024] 2. The microneedle array electrode sensor of the present invention is applied to glucose monitoring in interstitial fluid, which can realize wide range glucose detection (2-25mM for commercial products and 0.1-30mM for the present invention) and achieve stable continuous glucose monitoring for 19 days (14 days for commercial products).

[0025] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above contents of the present invention and its objectives, features and advantages more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a standard curve diagram of glucose concentration and response current in the test example of the present invention.

[0027] Figure 2 This is a graph showing the stability monitoring results of the sensor of the present invention obtained by continuous monitoring for 19 days. DETAILED DESCRIPTION

[0028] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0029] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] This paper constructs a third-generation microneedle array electrode sensor for interstitial fluid glucose monitoring. A one-dimensional titanium dioxide material, used for immobilizing glucose oxidase (Gox) and promoting direct electron transfer, is prepared using a hot solvent method. Polylactic acid (PLA) is used as the substrate for the microneedle array electrodes. The three-electrode microneedle array system is fabricated through 3D printing and magnetron sputtering. Finally, the working electrode is modified with a functional layer to form the microneedle array electrode sensor.

[0031] Example 1

[0032] A method for preparing a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material comprises the following steps:

[0033] 1. Material Preparation and GOx Immobilization

[0034] ① Material preparation: Acid-treated multi-walled carbon nanotubes (MWCNTs) were dispersed in isopropanol, diethylenetriamine (DETA) and tetraisopropoxytitanium were added, and the reaction was carried out at 180°C for 30 hours; after the reaction solution was cooled to room temperature, it was transferred to a centrifuge tube, centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded; 20 mL of deionized water was added, ultrasonicated for 5 minutes (power 100 W), centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The water washing was repeated twice; 20 mL of anhydrous ethanol was added, stirred for 10 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded. The ethanol washing was repeated once;

[0035] The precipitate was transferred to a culture dish and vacuum dried at 60°C for 24 hours to obtain a loose black powder (CNT-TiO2 composite material); the composite material was calcined at 550°C for 2 hours to remove the MWCNTs template and obtain one-dimensional TiO2.

[0036] ②GOx immobilization: 10 mg of the synthesized one-dimensional titanium dioxide was dispersed in 1 mL of GOx solution (10 mg / mL, pH 7.4 in PBS), and the mixture was vigorously shaken at room temperature for 0.5 h. The solution was allowed to stand at 4°C overnight to allow protein adsorption to form Gox-TiO2 material.

[0037] 2. Electrode Preparation and Functionalization

[0038] ① Electrode preparation: A high-precision FDM 3D printer (Ultimaker S5) was used to prepare microneedle arrays (base width 200 μm, height 600 μm, tip diameter 20 μm, base spacing 200 μm, base array 6×6) using polylactic acid (PLA) as the raw material. The nozzle temperature was set at 210°C, the platform temperature was set at 60°C, the layer height was set at 0.05 mm, and the printing speed was set at 20 mm / s. After ultrasonic cleaning with isopropyl alcohol and UV ozone treatment, a 5% PMMA chlorobenzene solution was spin-coated at 3000 rpm using a spin coater to form an insulating layer and cured on an 80°C hot plate. Subsequently, a magnetron sputtering device (Kurt J. Lesker PVD 75) was used to deposit the microneedle arrays at 5×10 -6 Three thin films of Cr (200W / 5min / 20nm), Pt (200W / 10min / 100nm) and Ag (150W / 15min / 200nm) were deposited in sequence under a Torr background vacuum. The three thin films were deposited by photolithography (AZ 5214 photoresist, 3000rpm spin coating, 90℃ pre-bake for 60 seconds, 365nm ultraviolet 100mJ / cm 2 After defining the electrode pattern (exposure, AZ300MIF developer treatment), the Ag layer in the working electrode and counter electrode areas was removed by wet etching with a 1:10 nitric acid solution for 30 seconds, exposing the Pt counter electrode. A 100nm thick Au layer was then deposited as the working electrode under mask protection by magnetron sputtering. Finally, the photoresist was removed by ultrasonic acetone, and electrolysis was performed in a 0.1M HCl solution with a +0.5V voltage (vs. SCE) for 30 seconds to convert the Ag on the reference electrode surface into Ag / AgCl. Key process controls include 3D printed microneedle accuracy (height error <5%), sputtered film thickness uniformity (fluctuation <5%), and AgCl electrode stability (potential drift <2mV / h).

[0039] ② Working electrode fixed-point modification

[0040] 1) Cysteine (Cys) modification: The gold electrode region was site-specifically modified with 30 mM cysteine aqueous solution and incubated at room temperature for 24 h. The electrode was rinsed with deionized water to remove loosely attached cysteine.

[0041] 2) Glutaraldehyde (GA) modification: 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 8.0) was added to the Au-Cys region for site-specific modification, incubated at room temperature for 1 hour, and the electrode was rinsed with the same phosphate buffer.

[0042] 3) Glucose oxidase (GOx) immobilization: A Gox-TiO2 solution was site-specifically modified in the Au-Cys-GA modified area. The electrode was incubated at 4°C for 6 hours. The electrode was rinsed with PBS buffer (pH 7.4) to obtain the final Au-Cys-GA-Gox-TiO2 working electrode. The electrode was dried at room temperature and set aside for use.

[0043] 3. Sensor assembly:

[0044] The three patterned electrodes were connected to silver wires, and the other ends of the silver wires were connected to the interfaces of the electrochemical workstation.

[0045] Example 2

[0046] A method for preparing a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material comprises the following steps:

[0047] 1. Material Preparation and GOx Immobilization

[0048] ① Material preparation: Acid-treated multi-walled carbon nanotubes (MWCNTs) were dispersed in isopropanol, diethylenetriamine (DETA) and tetraisopropoxytitanium were added, and the reaction was carried out at 170°C for 20 hours; after the reaction solution was cooled to room temperature, it was transferred to a centrifuge tube, centrifuged at 11000 rpm for 8 minutes, and the supernatant was discarded; 15 mL of deionized water was added, ultrasonicated for 3 minutes (power 100 W), centrifuged at 11000 rpm for 8 minutes, and the supernatant was discarded. The water washing was repeated twice; 15 mL of anhydrous ethanol was added, stirred for 8 minutes, centrifuged at 11000 rpm for 8 minutes, and the supernatant was discarded. The ethanol washing was repeated once;

[0049] The precipitate was transferred to a culture dish and vacuum dried at 50°C for 20 hours to obtain a loose black powder (CNT-TiO2 composite material); the composite material was calcined at 500°C for 1 hour to remove the MWCNTs template and obtain one-dimensional TiO2.

[0050] ②GOx immobilization: 8 mg of the synthesized one-dimensional titanium dioxide was dispersed in 2 mL of GOx solution (10 mg / mL, pH 7.4 in PBS), and the mixture was vigorously shaken at room temperature for 1 hour. The solution was left to stand at 3°C overnight to allow protein adsorption to form Gox-TiO2 material.

[0051] 2. Electrode Preparation and Functionalization

[0052] ① Electrode preparation: A high-precision FDM 3D printer (Ultimaker S5) was used to prepare microneedle arrays (base width 100 μm, height 500 μm, tip diameter 10 μm, base spacing 100 μm, base array 6×6) using polylactic acid (PLA) as the raw material. The nozzle temperature was set at 200°C, the platform temperature was set at 50°C, the layer height was set at 0.03 mm, and the printing speed was set at 15 mm / s. After ultrasonic cleaning with isopropyl alcohol and UV ozone treatment, a 3% PMMA chlorobenzene solution was spin-coated at 2000 rpm using a spin coater to form an insulating layer and cured on a hot plate at 70°C. Subsequently, a magnetron sputtering device (Kurt J. Lesker PVD 75) was used to deposit the microneedles on a 5×10 -6 Three thin films of Cr (200W / 5min / 20nm), Pt (200W / 10min / 100nm) and Ag (150W / 15min / 200nm) were deposited in sequence under a Torr background vacuum. The three thin films were deposited by photolithography (AZ 5214 photoresist, 2000rpm spin coating, 80℃ pre-bake for 50 seconds, 365nm ultraviolet ray 90mJ / cm 2 After defining the electrode pattern (exposure, AZ 300MIF developer treatment), the Ag layer in the working electrode and counter electrode areas was removed by wet etching with a 1:10 nitric acid solution for 20 seconds, exposing the Pt counter electrode. A 90nm thick Au layer was then deposited as the working electrode under mask protection by magnetron sputtering. Finally, the photoresist was removed by ultrasonication with acetone, and electrolysis was performed in a 0.2M HCl solution with a voltage of +0.4V (vs. SCE) for 20 seconds to convert the Ag on the reference electrode surface into Ag / AgCl. Key process controls include 3D printed microneedle accuracy (height error <5%), sputtered film thickness uniformity (fluctuation <5%), and AgCl electrode stability (potential drift <2mV / h).

[0053] ② Working electrode fixed-point modification

[0054] 1) Cysteine (Cys) modification: The gold electrode region was site-specifically modified with 20 mM cysteine aqueous solution and incubated at room temperature for 20 h. The electrode was rinsed with deionized water to remove loosely attached cysteine.

[0055] 2) Glutaraldehyde (GA) modification: 2% glutaraldehyde phosphate buffer (0.1 M, pH 8.0) was added to the Au-Cys region for site-specific modification, incubated at room temperature for 0.5 h, and the electrode was rinsed with the same phosphate buffer.

[0056] 3) Glucose oxidase (GOx) immobilization: A Gox-TiO2 solution was site-specifically modified in the Au-Cys-GA modified area. The solution was incubated at 3°C for 5 h. The electrode was rinsed with PBS buffer (pH 7.4) to obtain the final Au-Cys-GA-Gox-TiO2 working electrode. The electrode was dried at room temperature and then used.

[0057] 3. Sensor assembly:

[0058] The three patterned electrodes were connected to silver wires, and the other ends of the silver wires were connected to the interfaces of the electrochemical workstation.

[0059] Example 3

[0060] A method for preparing a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material comprises the following steps:

[0061] 1. Material Preparation and GOx Immobilization

[0062] ① Material preparation: Acid-treated multi-walled carbon nanotubes (MWCNTs) were dispersed in isopropanol, diethylenetriamine (DETA) and tetraisopropoxytitanium were added, and the reaction was carried out at 190°C for 40 hours; after the reaction solution was cooled to room temperature, it was transferred to a centrifuge tube, centrifuged at 13000rpm for 12 minutes, and the supernatant was discarded; 25mL of deionized water was added, ultrasonicated for 7 minutes (power 100W), centrifuged at 13000rpm for 12 minutes, and the supernatant was discarded. The water washing was repeated twice; 25mL of anhydrous ethanol was added, stirred for 12 minutes, centrifuged at 13000rpm for 12 minutes, and the supernatant was discarded. The ethanol washing was repeated once;

[0063] The precipitate was transferred to a culture dish and vacuum dried at 70°C for 28 hours to obtain a loose black powder (CNT-TiO2 composite material); the composite material was calcined at 600°C for 3 hours to remove the MWCNTs template and obtain one-dimensional TiO2.

[0064] ②GOx immobilization: 12 mg of the synthesized one-dimensional titanium dioxide was dispersed in 1 mL of GOx solution (10 mg / mL, pH 7.4 in PBS), and the mixture was vigorously shaken at room temperature for 0.5 h. The solution was allowed to stand at 5°C overnight to allow protein adsorption to form Gox-TiO2 material.

[0065] 2. Electrode Preparation and Functionalization

[0066] ① Electrode preparation: A high-precision FDM 3D printer (Ultimaker S5) was used to prepare microneedle arrays (base width 300 μm, height 700 μm, tip diameter 30 μm, base spacing 300 μm, base array 6×6) using polylactic acid (PLA) as the raw material. The nozzle temperature was set at 220°C, the platform temperature was set at 70°C, the layer height was set at 0.07 mm, and the printing speed was set at 25 mm / s. After ultrasonic cleaning with isopropyl alcohol and UV ozone treatment, a 7% PMMA chlorobenzene solution was spin-coated at 4000 rpm using a spin coater to form an insulating layer and cured on a hot plate at 90°C. Subsequently, a magnetron sputtering device (Kurt J. Lesker PVD 75) was used to deposit the microneedle arrays at 5×10 -6 Three thin films of Cr (200W / 5min / 20nm), Pt (200W / 10min / 100nm) and Ag (150W / 15min / 200nm) were deposited in sequence under a Torr background vacuum. The three thin films were deposited by photolithography (AZ 5214 photoresist, 4000rpm spin coating, 100℃ pre-bake for 70 seconds, 365nm ultraviolet 110mJ / cm 2 After defining the electrode pattern (exposure, AZ300MIF developer treatment), the Ag layer in the working electrode and counter electrode areas was removed by wet etching with a 1:10 nitric acid solution for 40 seconds, exposing the Pt counter electrode. A 110nm thick Au layer was then deposited as the working electrode under mask protection by magnetron sputtering. Finally, the photoresist was removed by ultrasonic acetone, and electrolysis was performed in a 0.1M HCl solution at a voltage of +0.6V (vs.SCE) for 40 seconds to convert the Ag on the reference electrode surface into Ag / AgCl. Key process controls include 3D printed microneedle accuracy (height error <5%), sputtered film thickness uniformity (fluctuation <5%), and AgCl electrode stability (potential drift <2mV / h).

[0067] ② Working electrode fixed-point modification

[0068] 1) Cysteine (Cys) modification: The gold electrode region was site-specifically modified in a 40 mM cysteine aqueous solution and incubated at room temperature for 28 h. The electrode was rinsed with deionized water to remove loosely attached cysteine.

[0069] 2) Glutaraldehyde (GA) modification: 3% glutaraldehyde phosphate buffer (0.1 M, pH 8.0) was added to the Au-Cys region for site-specific modification, incubated at room temperature for 1.5 hours, and the electrode was rinsed with the same phosphate buffer.

[0070] 3) Glucose oxidase (GOx) immobilization: A Gox-TiO2 solution was site-specifically modified in the Au-Cys-GA modified area. The electrode was incubated at 5°C for 7 hours. The electrode was rinsed with PBS buffer (pH 7.4) to obtain the final Au-Cys-GA-Gox-TiO2 working electrode. The electrode was dried at room temperature and set aside for use.

[0071] 3. Sensor assembly:

[0072] The three patterned electrodes were connected to silver wires, and the other ends of the silver wires were connected to the interfaces of the electrochemical workstation.

[0073] Test example

[0074] The following tests were performed using the continuous glucose monitoring sensor prepared in Example 1:

[0075] 1) Test system: Hydrogels were prepared to simulate skin; glucose solutions of different concentrations (0.1, 0.5, 1, 4, 5, 10, 15, 25, and 30 mM) were prepared using artificial interstitial fluid at pH 7.0.

[0076] 2) Determination of glucose detection range: Connect the continuous glucose monitoring sensor to the electrochemical workstation, turn on the electrochemical workstation, and after the current stabilizes, add equal amounts of 0.1mM, 0.5mM, 1.5mM, 4mM, 5mM, 10mM, 15mM, 25mM, and 30mM glucose solutions into the hydrogel every 100s to obtain the relationship between the change in response current and the change in glucose concentration. Draw a standard curve between glucose concentration and response current to determine the detection range of glucose concentration, such as Figure 1 The results showed that the formula for obtaining the standard curve of glucose concentration and response current was: I = 2.795C + 6.4415, R 2 = 0.9829, where I is the response current (μA) and C is the glucose concentration (mM). The results show that the detection range of the array microneedle system obtained by the present invention can be 0.1-30 mM.

[0077] 3) Sensor stability test: Connect the continuous glucose monitoring sensor to the electrochemical workstation, turn on the electrochemical workstation, and after the current stabilizes, add 5mM glucose solution (pH 7.0) prepared from artificial interstitial fluid into the hydrogel. Repeat the measurement three times and record the average current response value. The stability monitoring results are obtained after continuous monitoring for 19 days. Figure 2 The results showed that after 19 days of continuous monitoring, the sensor performance remained basically stable.

[0078] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0079] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A method for preparing a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material, characterized in that: The following steps are involved: S1. Preparing a one-dimensional titanium dioxide material: dispersing multi-walled carbon nanotubes in isopropyl alcohol, adding diethylenetriamine and tetraisopropoxytitanium to react, and then centrifuging, drying, and calcining to obtain a one-dimensional titanium dioxide material; S2, GOx immobilization: dispersing one-dimensional titanium dioxide material in GOx solution and oscillating to form Gox-TiO2 material; S3. Electrode preparation: The microneedle array electrode substrate is prepared by 3D printing and magnetron sputtering to form a microneedle array three-electrode system; S4. Perform functional layer modification on the working electrode, including cysteine modification, glutaraldehyde modification, and glucose oxidase immobilization.

2. The method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to claim 1, wherein: The method for preparing a one-dimensional titanium dioxide material comprises: dispersing acid-treated multi-walled carbon nanotubes in isopropanol, adding diethylenetriamine and tetraisopropoxytitanium, and reacting at 170-190°C for 20-40 hours; after the reaction solution is cooled to room temperature, transferring it to a centrifuge tube, centrifuging it at 11000-13000 rpm for 8-12 minutes, and discarding the supernatant; adding 15-25 mL of deionized water, ultrasonicating it for 3-7 minutes, centrifuging it at 11000-13000 rpm for 8-12 minutes, discarding the supernatant, and repeating water washing twice; adding 15-25 mL of anhydrous ethanol, stirring it for 8-12 minutes, centrifuging it at 11000-13000 rpm for 8-12 minutes, discarding the supernatant, and repeating ethanol washing once, transferring the precipitate to a culture dish, and vacuum drying it at 50-70°C for 20-28 hours to obtain a black powder; and then calcining it at 500-600°C for 1-3 hours to obtain the one-dimensional titanium dioxide material.

3. The method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to claim 1, characterized in that: The GOx immobilization method includes: dispersing 8-12 mg of one-dimensional titanium dioxide material in 1-2 mL of GOx solution, shaking at room temperature for 0.5-1 hour, and letting the solution stand at 3-5°C overnight to form a Gox-TiO2 material.

4. The method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to claim 1, wherein: The electrode preparation method includes: printing the microneedle array electrode substrate using a 3D printer, setting the nozzle temperature to 200-220°C, the platform temperature to 50-70°C, the layer height to 0.03-0.07mm, and the printing speed to 15-25mm / s to prepare the microneedle array, ultrasonically cleaning with isopropyl alcohol and treating with ultraviolet ozone, spin coating a 3-7% PMMA chlorobenzene solution at 2000-4000 rpm using a spunbond to form an insulating layer, and curing on a hot plate at 70-90°C; then using a magnetron sputtering apparatus to form a 5×10 -6 Three thin films of Cr, Pt, and Ag were deposited sequentially under a Torr background vacuum. After the electrode pattern was defined by photolithography, the Ag layer in the working electrode and counter electrode areas was removed by wet etching with a 1:10 nitric acid solution for 20-40 seconds, exposing the Pt counter electrode. A 90-110 nm thick Au layer was then deposited as the working electrode under mask protection by magnetron sputtering. Finally, the photoresist was ultrasonically removed using acetone, and electrolysis was performed in a 0.1-0.2 M HCl solution at a voltage of +0.4-0.6 V for 20-40 seconds to convert the Ag on the reference electrode surface into Ag / AgCl.

5. The method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to claim 4, characterized in that: Polylactic acid is used as the substrate for the microneedle array electrode.

6. The method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to claim 4, characterized in that: The base width of the microneedle array is 100-300 μm, the height is 500-700 μm, the tip diameter is 10-30 μm, the base spacing is 100-300 μm, and the base array is 6×6.

7. The method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to claim 6, characterized in that: The photolithography process includes: 2000-4000rpm spin coating, 80-100℃ pre-bake for 50-70 seconds, 365nm UV light 90-110mJ / cm 2 Exposure, and processing with AZ 300MIF developer.

8. The method for preparing a continuous glucose monitoring sensor based on one-dimensional titanium dioxide material according to claim 1, characterized in that: Methods for modifying the working electrode with a functional layer include: 1) Cysteine modification: The gold electrode region was site-specifically modified with a 20-40 mM cysteine aqueous solution and incubated at room temperature for 20-28 hours. The electrode was rinsed with deionized water to remove loosely attached cysteine. 2) Glutaraldehyde modification: Modify the Au-Cys region with 2-3% glutaraldehyde in phosphate buffer, incubate at room temperature for 0.5-1.5 hours, and rinse the electrode with the same phosphate buffer; 3) Immobilization of glucose oxidase: The Au-Cys-GA modified area was modified with Gox-TiO2 solution, incubated at 3-5°C for 5-7 hours, and the electrode was rinsed with PBS buffer to obtain the final Au-Cys-GA-Gox-TiO2 working electrode.

9. A continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material, prepared by the method for preparing a continuous glucose monitoring sensor based on a one-dimensional titanium dioxide material according to any one of claims 1 to 8.

Images