CGM electrode sensor and preparation method thereof

Abstract

The invention belongs to the technical field of CGM sensors, and particularly relates to a CGM electrode sensor and a preparation method thereof.The preparation method comprises the following steps that three sets of stainless steel microneedle arrays are prepared from a stainless steel substrate and serve as a counter electrode, a reference electrode and a working electrode respectively; mechanically polishing the stainless steel microneedle array, and performing composite pickling activation; after surface pretreatment, a sol-gel deposition aluminum oxide layer is adopted; preparing a gold plating solution, and plating gold on the stainless steel microneedle array deposited with the aluminum oxide layer by adopting two-stage pulse electroplating; the working electrode is prepared by PMMA coating, adhesion layer construction, glucose oxidase immobilization and limiting layer construction; the counter electrode, the reference electrode and the working electrode are respectively embedded into the substrate to form the microneedle array electrode sensor with a three-electrode system. Based on a nickel-free direct gold plating process, good uniformity, strong binding force and lower cost after gold plating can be realized, so that the stability of the continuous glucose sensor in use is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of CGM sensors, and particularly relates to a CGM electrode sensor and a preparation method thereof. Background Art

[0002] Continuous glucose monitoring (CGM) is a new minimally invasive blood glucose monitoring technology that records the electrical signals generated by the glucose oxidation reaction in interstitial fluid through microelectrodes implanted in subcutaneous tissue, indirectly reflecting the measured blood glucose. The hardware system of CGM blood glucose monitoring products mainly includes three core components: a sensor, a transmitter, and a receiver. Among them, the sensor, as the key component inserted into subcutaneous tissue and continuously measuring glucose levels, is responsible for sensing glucose in tissue fluid and converting it into an electrical signal, which is the part with the highest technical barrier and the most core in CGM products.

[0003] In the prior art, the gold plating process of traditional stainless steel CGM sensors needs to perform pretreatment, nickel plating (2 - 5μm intermediate layer), and gold plating in sequence. The nickel layer is used to enhance the bonding force of the gold layer and block the migration of matrix metal ions; however, this process has significant defects: nickel, as a strong sensitizer, is prone to cause allergic or inflammatory reactions after implantation. The high brittleness of the nickel layer leads to cracking of the coating and peeling of the gold layer under dynamic load, and uneven current density distribution results in poor coating uniformity; in addition, the nickel plating process requires additional processes (such as electroplating solution maintenance, activation treatment), resulting in a 30% increase in production cost and limiting miniaturized design (the thickness of the nickel layer cannot adapt to microneedles with a diameter < 30μm). Summary of the Invention

[0004] In order to solve the problems in the prior art, the present invention provides a CGM electrode sensor and a preparation method thereof, achieving the purpose of good uniformity, strong bonding force, and lower cost after gold plating based on a nickel - free direct gold plating process, thereby improving the stability of continuous glucose sensors during use.

[0005] The present invention solves its technical problems by adopting the following technical solutions: The present invention aims to provide a preparation method of a CGM electrode sensor, including the following steps: S1. Preparation of stainless steel microneedle arrays: Three groups of stainless steel microneedle arrays are prepared using a stainless steel substrate as the counter electrode, reference electrode, and working electrode respectively; S2. Surface pretreatment: The stainless steel microneedle arrays are first mechanically polished and then subjected to composite pickling activation; S3. Preparation of a nano - transition layer: The stainless steel microneedle arrays after surface pretreatment are deposited with an alumina layer by sol - gel method; S4. Direct gold plating: Prepare a gold plating solution and perform two-stage pulse electroplating to plate the stainless steel micro-needle array after depositing the alumina layer in S3 with gold; S5. Preparation of the electrode: The working electrode is further prepared by PMMA coating, adhesion layer construction, glucose oxidase immobilization, and construction of a limiting layer; S6. Sensor assembly: The counter electrode, reference electrode, and working electrode are respectively embedded in the substrate to form a micro-needle array electrode sensor with a three-electrode system.

[0006] Furthermore, the method for preparing three groups of stainless steel micro-needle arrays using a stainless steel substrate includes: immersing the stainless steel substrate in a deionized water medium, setting the discharge voltage at 30 - 50V, the cutting feed rate at 5 - 6 mm / min, the electrode gap at 0.04 - 0.08 mm, and controlling the ambient temperature at 20 ± 1°C to complete the processing.

[0007] Furthermore, the three groups of prepared stainless steel micro-needle arrays have the same geometric characteristics: the height of the pyramid-shaped needle body structure is maintained at 1 - 3 mm, the base width is 700 - 900 μm, the center distance between adjacent needles is 0.5 - 1.5 mm, among which the working electrode and the reference electrode are arranged in a 1×3 array, and the counter electrode is configured in a 2×3 array layout.

[0008] Furthermore, mechanical polishing uses sandpaper with particle sizes from coarse to fine: #800 - #2000 to polish the surface of the stainless steel micro-needle array until the surface roughness Ra is 0.2 - 0.4 μm.

[0009] Furthermore, the method for composite pickling activation includes: soaking the mechanically polished stainless steel micro-needle array in a mixed solution of HCl, HF, and an inhibitor at 20 - 40°C for 3 - 7 minutes to remove the passivation film; then performing pulsed current treatment in an electrolyte containing thiourea and citric acid for 8 - 12 minutes to form a sulfide interface layer.

[0010] Furthermore, the method for preparing the nano-transition layer includes: immersing the surface-pretreated stainless steel micro-needle array in a mixed sol of aluminum isopropoxide and nitric acid, and performing heat treatment at 50 - 70°C for 20 - 40 minutes to form a 40 - 60 nm dense Al 2 O 3 layer.

[0011] Further, the preparation method of the gold plating solution includes: adding 1-2 g of potassium chloroaurate dihydrate into 500 mL of deionized water, and then successively adding 4-6 g of disodium ethylenediaminetetraacetate, 23-27 g of buffer, 7-8 g of potassium dihydrogen phosphate, 30-50 mL of formic acid, 1-2 mL of nonionic fluorocarbon compound, and 4-6 g of cobalt sulfate heptahydrate, and stirring with a magnetic stirrer; wherein, the preparation method of the buffer is: mixing citric acid and potassium citrate in a molar ratio of 1:1, weighing 23-27 g of the mixture, adding it into 500 mL of deionized water, and stirring until completely dissolved to prepare a buffer solution for maintaining the pH value of the gold plating solution.

[0012] Further, the method for gold plating the stainless steel microneedle array after depositing an alumina layer on S3 by two-stage pulse electroplating includes: immersing the stainless steel microneedle array after depositing the alumina layer into the prepared gold plating solution, controlling the temperature of the gold plating solution at 40-60 °C, and the electroplating is divided into two stages: in the initial stage, the current density is set at 7-9 A / dm², the voltage is 5-9 V, and the duration is 8-12 seconds. Subsequently, it enters the steady state stage, the current density drops to 1-2 A / dm², and the voltage is adjusted to 3-5 V, and the duration is 45-55 seconds.

[0013] Further, the preparation method of the working electrode includes: a. PMMA coating: Coating the microneedles with a PMMA solution with a mass ratio of 1%-3%, and drying at 70-90 °C for 15-17 h; b. Adhesion layer construction: Mixing a single-walled carbon nanotube and an ethanol solution of Nafion in a volume ratio of 1:3-5, electro-depositing at a potential of 0.5-0.7 V for 50-70 seconds, and washing with ethanol and drying after electro-deposition; c. Glucose oxidase immobilization: Using a PBS solution, preparing a mixed solution according to 40-60 mg / mL of glucose oxidase, 8-12 mg / mL of mediator, and 2-3 mg / mL of glutaraldehyde. Dipping the microneedles treated with Nafion into the mixed solution, and then lifting and drying; d. Construction of the limiting layer: Coating the outer surface of the microneedles with a dimethylformamide solution containing polyurethane to form a limiting layer.

[0014] A CGM electrode sensor is prepared by the above preparation method.

[0015] Compared with the prior art, the beneficial technical effects of the present invention are as follows: 1) Enhanced biocompatibility: The nickel-free gold plating process avoids the harm of nickel release to the human body, is suitable for medical devices in contact with the human body, ensures the safety and reliability of the product, and reduces the risk of adverse reactions caused by nickel allergy in patients; 2) Long-term stability: After soaking in artificial interstitial fluid simulating the human body (pH 7.4, 37 °C) for 30 days, there is no obvious corrosion or peeling of the coating; 3) Cost saving: It reduces the cumbersome steps such as the use and maintenance of electroplating solution and activation treatment during the nickel plating process, achieving the effects of cost saving and reducing complex process steps; 4) Miniaturization adaptability: The coating evenly covers the tip of the microneedle (diameter < 30 μm), ensuring the complete function of the complex micro-structure; 5) Firm bonding force: After using the nano-transition layer and then plating with gold, a dense inorganic layer is constructed on the clean stainless steel substrate in advance, providing an adhesive and forceful substrate for the subsequent gold layer and blocking the migration of other ions, ensuring the stability and reliability of the product during long-term use.

[0016] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above content, objects, features and advantages of the present invention more obvious and understandable, the following specific embodiments of the present invention are given. Description of the Drawings

[0017] Figure 1 It is the standard curve graph of glucose concentration and response current in the test example of the present invention.

[0018] Figure 2 It is the monitoring result graph of the sensor stability in the test example of the present invention. Detailed Embodiments

[0019] The following further details the technical solution of the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative and explanatory purposes of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0020] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or prepared by existing methods. Example 1

[0021] The preparation method of a continuous glucose sensor based on a nickel-free direct gold plating process is as follows: ① Preparation of stainless steel microneedle array: A Swiss Charmilles CUT20P slow wire cutting machine tool is used to prepare the microneedle array, and 0.20 mm molybdenum wire is used as the cutting electrode. During the processing, the 316L stainless steel substrate is immersed in deionized water medium, the discharge voltage is set at 40 V, the cutting feed rate is 5.5 mm / min, the electrode gap is 0.06 mm, and the processing is completed under the condition that the ambient temperature is controlled at 20 °C; Three groups of prepared microneedle arrays have the same geometric features: the height of the pyramid-shaped needle body structure is kept at 2 mm, the width of the base is 800 μm, and the center distance between adjacent needles is 1 mm. Among them, the working electrode and the reference electrode are arranged in a 1×3 array, and the counter electrode is configured in a 2×3 array layout; ② Surface pretreatment: 1) Mechanical polishing: The surface of the microneedle array is polished with sandpapers of different grits (from coarse to fine: #800 - #2000) until the surface roughness Ra is 0.3 μm to remove burrs and large scratches on the surface; 2) Composite pickling activation: a. Pickling solution: The polished microneedle array is immersed in HCl (10 wt%) + HF (2 wt%) + corrosion inhibitor (sodium dodecylbenzenesulfonate 0.1 wt%) at 30 °C for 5 minutes to remove the passivation film; b. Electrolytic activation: The electrolyte containing thiourea (5 g / L) + citric acid (20 g / L) is treated with a pulsed current (peak value 8 A / dm², duty cycle 20%) for 10 minutes to form a sulfide interface layer; ③ Nano-transition layer (nano-aluminum oxide replaces nickel): Sol-gel deposition of aluminum oxide layer: The surface-pretreated microneedles are immersed in a mixed sol of aluminum isopropoxide (0.1 M) and nitric acid (0.05 M), and heat-treated at 60 °C for 30 minutes to form a 50-nm dense Al 2 O 3 layer; ④ Nickel-free direct gold plating 1) Preparation of gold plating solution: 1 g of potassium chloroaurate dihydrate (KAuCl 4 ·2H 2 O) is added to 500 mL of deionized water to provide gold ions as the main salt; then 5 g of disodium ethylenediaminetetraacetate (Na 2 EDTA) is added to stabilize the gold ions in the plating solution. The buffer is a mixture of citric acid and potassium citrate in a 1:1 molar ratio, and the addition amount is 25 g to maintain the pH value of the plating solution. 7.5 g of potassium dihydrogen phosphate (KH 2 PO 4 ) is added as a conductive salt to enhance the conductivity of the plating solution. Formic acid (HCOOH) is used as an acidic regulator, and the addition amount is 40 mL to adjust the acidic environment of the plating solution. 1 mL of non-ionic fluorocarbon compound (e.g., FCF-204 non-ionic fluorocarbon surface leveling agent) is added as a surfactant to improve the uniformity of the coating; finally, 5 g of cobalt sulfate heptahydrate (CoSO 4 ·7H 2 O) is added as a hardening agent to increase the hardness of the coating. All reagents are added in sequence and stirred with a magnetic stirrer; 2) Nickel-free direct gold plating process: The gold plating process uses a two-stage pulse electroplating technique. The instruments used include a pulse electroplating power supply and a constant temperature water bath. The stainless steel micro-needle array with a nano-transition layer is immersed in the prepared gold plating solution. The temperature of the plating solution is controlled at 50 °C. The electroplating is divided into two stages: In the initial stage, the current density is set to 8 A / dm², the voltage is 7 V, and the duration is 10 seconds. This stage aims to achieve rapid nucleation of the gold layer. Subsequently, it enters the steady state stage, where the current density drops to 1.5 A / dm², the voltage is adjusted to 4 V, and the duration is 50 seconds. This stage is used to form a dense gold layer with a thickness of 0.3 ± 0.05 μm; By adjusting the pulsed current, it is ensured that the coating uniformly covers the tips of the micro-needles (diameter < 30 μm). At the same time, combined with the application of a nano-transition layer (such as an alumina layer), the bonding force between the gold layer and the stainless steel substrate is significantly improved, ensuring the stability and reliability of the coating during long-term use. After electroplating, the micro-needle array is taken out, rinsed with deionized water and dried for standby; ⑤ Preparation and modification of electrodes: 1) Counter electrode (CE), reference electrode (RE): The nickel-free gold-plated micro-needle electrodes arranged in 2×3 and 1×3 arrays are used as the counter electrode (CE) and reference electrode (RE) respectively; 2) Preparation of working electrode (WE): a. PMMA coating: The micro-needles are coated with a 2% (by mass) PMMA (polymerization degree 8500) solution and dried at 80 °C for 16 h; b. Construction of the adhesion layer: An ethanol solution of single-walled carbon nanotubes (SWCNT) and Nafion (perfluorosulfonic acid polymer solution) is mixed in a volume ratio of 1:4 and electro-deposited at a potential of 0.6 V for 60 seconds. The electro-deposited micro-needle electrode is cleaned with ethanol and naturally dried at room temperature overnight; c. Immobilization of glucose oxidase (GOD): A mixed solution is prepared using a PBS solution with a pH of 7.0 at a concentration of glucose oxidase (GOx): 50 mg / mL, mediator (Osmium mediator): about 10 mg / mL, and glutaric dialdehyde: 2.5 mg / mL. The micro-needles treated with Nafion are dipped into the enzyme solution and then lifted and dried at room temperature to immobilize the enzyme on the surface of the micro-needles; d. Construction of the confinement layer: The outer surface of the micro-needles is coated with an N,N-dimethylformamide (DMF) solution containing polyurethane (PU, polymerization degree 1200) to form a confinement layer to prevent enzyme leakage and improve the biocompatibility of the electrode; ⑥ Sensor assembly: Integration of three - electrode system: Polyimide (PI, degree of polymerization is 2000) material is used as the substrate, and electron etching technology is used to drill holes in the substrate. The WE, CE, and RE are respectively embedded in the substrate, so that the microneedles pass through the substrate and protrude 1 mm, forming a three - electrode system with a microneedle array electrode. The WE, CE, and RE are respectively connected to silver wires, and the other ends of the silver wires are respectively connected to the interfaces of the electrochemical workstation. Example 2

[0022] Preparation method of a nickel - free direct gold - plated continuous glucose sensor, the specific steps are as follows: ① Preparation of stainless - steel microneedle array: A Swiss Charmilles CUT20P slow - wire electrical discharge machining machine is used to prepare the microneedle array, and 0.20 mm molybdenum wire is used as the cutting electrode. During the processing, the 316L stainless - steel substrate is immersed in deionized water medium. The discharge voltage is set at 30 V, the cutting feed rate is 5 mm / min, the electrode gap is 0.04 mm, and the processing is completed under the condition that the ambient temperature is controlled at 19℃. The three groups of prepared microneedle arrays have the same geometric characteristics: the height of the pyramid - shaped needle body structure is 1 mm, the base width is 700 μm, and the center distance between adjacent needle bodies is 0.5 mm. Among them, the working electrode and the reference electrode are arranged in a 1×3 array, and the counter electrode is configured in a 2×3 array layout; ② Surface pretreatment: 1) Mechanical polishing: Sandpapers with different grits (from coarse to fine: #800 - #2000) are used to polish the surface of the microneedle array until the surface roughness Ra is 0.2 μm to remove surface burrs and large scratches; 2) Composite pickling activation: a. Pickling solution: The polished microneedle array is immersed in HCl (10 wt%) + HF (2 wt%) + inhibitor (sodium dodecylbenzenesulfonate 0.1 wt%) at 20℃ for 3 minutes to remove the passivation film; b. Electrolytic activation: The electrolyte containing thiourea (5 g / L) + citric acid (20 g / L) is used for 8 minutes with a pulsed current (peak value 8 A / dm², duty cycle 20%) to form a sulfide interface layer; ③ Nano - transition layer (nano - alumina replaces nickel): Sol - gel deposition of alumina layer: The surface - pretreated microneedles are immersed in a mixed sol of aluminum isopropoxide (0.1 M) and nitric acid (0.05 M), and heat - treated at 50℃ for 20 minutes to form a 40 - nm dense Al 2 O 3 layer; ④ Nickel - free direct gold - plating 1) Preparation of gold - plating solution: Potassium chloroaurate dihydrate (KAuCl 4 ·2H2 O) 1 g is added to 500 mL of deionized water to provide gold ions as the main salt; then 4 g of disodium ethylenediaminetetraacetate (Na 2 EDTA) is added to stabilize the gold ions in the plating solution. The buffer is a mixture of citric acid and potassium citrate in a 1:1 molar ratio, and the addition amount is 23 g to maintain the pH value of the plating solution. 7 g of potassium dihydrogen phosphate (KH 2 PO 4 ) is added as a conductive salt to enhance the conductivity of the plating solution. Formic acid (HCOOH) is used as an acidic regulator, and the addition amount is 30 mL to adjust the acidic environment of the plating solution. 1 mL of a non-ionic fluorocarbon compound (e.g., FCF-204 non-ionic fluorocarbon surface leveling agent) is added as a surfactant to improve the uniformity of the coating. Finally, 4 g of cobalt sulfate heptahydrate (CoSO 4 ·7H 2 O) is added as a hardening agent to increase the hardness of the coating. All reagents are added in sequence and stirred using a magnetic stirrer; 2) Process of nickel-free direct gold plating: The gold plating process uses a two-stage pulse electroplating technique, and the instruments used include a pulse electroplating power supply and a constant temperature water bath. The stainless steel micro-needle array with a nano-transition layer is immersed in the prepared gold plating solution. The temperature of the plating solution is controlled at 40 °C. The electroplating is divided into two stages: in the initial stage, the current density is set to 7 A / dm², the voltage is 5 V, and the duration is 8 seconds. This stage aims to achieve rapid nucleation of the gold layer. Subsequently, it enters the steady state stage, where the current density drops to 1 A / dm², the voltage is adjusted to 3 V, and the duration is 45 seconds. This stage is used to form a dense gold layer with a thickness of 0.3 ± 0.05 μm; By adjusting the pulsed current, it is ensured that the coating uniformly covers the tips of the micro-needles (diameter < 30 μm). At the same time, by combining the application of a nano-transition layer (such as an alumina layer), the bonding force between the gold layer and the stainless steel substrate is significantly improved, ensuring the stability and reliability of the coating during long-term use. After electroplating, the micro-needle array is taken out, rinsed with deionized water, and dried for standby; ⑤ Preparation and modification of electrodes: 1) Counter electrode (CE), reference electrode (RE): The nickel-free gold-plated micro-needle electrodes arranged in 2×3 and 1×3 arrays are used as the counter electrode (CE) and reference electrode (RE) respectively; 2) Preparation of working electrode (WE): a. PMMA coating: The micro-needles are coated with a 1% (by mass) PMMA (polymerization degree 8500) solution and dried at 70 °C for 15 h; b. Adhesion layer construction: An ethanol solution of single-walled carbon nanotubes (SWCNT) and Nafion (perfluorosulfonic acid polymer solution) is mixed at a volume ratio of 1:3, and electro-deposited for 50 seconds at a potential of 0.5V. After electro-deposition, the microneedle electrode is cleaned with ethanol and naturally air-dried overnight at room temperature; c. Glucose oxidase (GOD) immobilization: A mixed solution is prepared using PBS solution with a pH of 7.0, at concentrations of glucose oxidase (GOx): 40 mg / mL, mediator (Osmium mediator): approximately 8 mg / mL, and glutaric dialdehyde: 2 mg / mL. The Nafion-treated microneedles are dipped into the enzyme solution, then lifted and air-dried at room temperature to immobilize the enzyme on the surface of the microneedles; d. Restriction layer construction: The outer surface of the microneedles is coated with an N,N-dimethylformamide (DMF) solution containing polyurethane (PU, degree of polymerization 1200) to form a restriction layer to prevent enzyme leakage and improve the biocompatibility of the electrode; ⑥ Sensor assembly: Integration of three-electrode system: A polyimide (PI, degree of polymerization 2000) material is used as the substrate, and the substrate is drilled using electron etching technology. The working electrode (WE), counter electrode (CE), and reference electrode (RE) are respectively embedded in the substrate, and the microneedles pass through the substrate and protrude 1 mm to form a three-electrode system with a microneedle array electrode. The WE, CE, and RE are respectively connected to silver wires, and the other ends of the silver wires are respectively connected to the interfaces of the electrochemical workstation. Example 3

[0023] Preparation method of a continuous glucose sensor based on a nickel-free direct gold plating process, the specific steps are as follows: ① Preparation of stainless steel microneedle array: A Swiss Charmilles CUT20P slow wire electrical discharge machining machine is used to prepare the microneedle array, and a 0.20 mm molybdenum wire is used as the cutting electrode. During the processing, the 316L stainless steel substrate is immersed in deionized water medium, the discharge voltage is set at 50V, the cutting feed rate is 6 mm / min, the electrode gap is 0.08 mm, and the processing is completed under the condition that the ambient temperature is controlled at 21℃; The three groups of prepared microneedle arrays have the same geometric characteristics: the height of the pyramid-shaped needle body structure is maintained at 3 mm, the base width is 900 μm, and the center distance between adjacent needle bodies is 1.5 mm. Among them, the working electrode and the reference electrode are arranged in a 1×3 array, and the counter electrode is configured in a 2×3 array layout; ② Surface pretreatment: 1) Mechanical polishing: The surface of the microneedle array is polished using sandpapers with different grits (from coarse to fine: #800 - #2000) until the surface roughness Ra is 0.4 μm to remove surface burrs and large scratches; 2) Composite pickling activation: a. Pickling solution: Immerse the polished microneedle array in HCl (10 wt%) + HF (2 wt%) + corrosion inhibitor (sodium dodecylbenzenesulfonate 0.1 wt%) at 40 °C for 7 minutes to remove the passivation film; b. Electrolytic activation: Treat with an electrolyte containing thiourea (5 g / L) + citric acid (20 g / L) under a pulsed current (peak value 8 A / dm², duty cycle 20%) for 12 minutes to form a sulfide interface layer; ③ Nano transition layer (nano alumina replaces nickel): Sol-gel deposition of alumina layer: Immerse the surface-pretreated microneedles in a mixed sol of aluminum isopropoxide (0.1 M) and nitric acid (0.05 M), and heat-treat at 70 °C for 40 minutes to form a 60-nm dense Al 2 O 3 layer; ④ Nickel-free direct gold plating 1) Preparation of gold plating solution: Add 2 g of potassium chloroaurate dihydrate (KAuCl 4 ·2H 2 O) to 500 mL of deionized water to provide gold ions as the main salt; then add 6 g of disodium ethylenediaminetetraacetate (Na 2 EDTA) to stabilize the gold ions in the plating solution. The buffer is a 1:1 molar ratio mixture of citric acid and potassium citrate, and the addition amount is 27 g to maintain the pH value of the plating solution. Add 8 g of potassium dihydrogen phosphate (KH 2 PO 4 ) as a conductive salt to enhance the conductivity of the plating solution. Formic acid (HCOOH) is used as an acidic regulator, and the addition amount is 50 mL to adjust the acidic environment of the plating solution. Add 2 mL of a non-ionic fluorocarbon compound (e.g., FCF-204 non-ionic fluorocarbon surface leveling agent) as a surfactant to improve the uniformity of the coating. Finally, add 6 g of cobalt sulfate heptahydrate (CoSO 4 ·7H 2 O) as a hardening agent to increase the hardness of the coating. Add all the reagents in sequence and stir with a magnetic stirrer; 2) Nickel-free direct gold plating process: The gold plating process uses a two-stage pulse electroplating technique, and the instruments used include a pulse electroplating power supply and a constant temperature water bath. The stainless steel micro-needle array with a nano-transition layer is immersed in the prepared gold plating solution. The temperature of the plating solution is controlled at 60 °C. The electroplating is divided into two stages: In the initial stage, the current density is set to 9 A / dm², the voltage is 9 V, and the duration is 12 seconds. This stage aims to achieve rapid nucleation of the gold layer. Subsequently, it enters the steady state stage, where the current density drops to 2 A / dm², the voltage is adjusted to 5 V, and the duration is 55 seconds. This stage is used to form a dense gold layer with a thickness of 0.3 ± 0.05 μm; By adjusting the pulse current, it is ensured that the coating uniformly covers the tips of the micro-needles (diameter < 30 μm). At the same time, by combining the application of a nano-transition layer (such as an alumina layer), the bonding force between the gold layer and the stainless steel substrate is significantly improved, ensuring the stability and reliability of the coating during long-term use. After electroplating, the micro-needle array is taken out, rinsed with deionized water and dried for standby; ⑤ Preparation and modification of the electrode: 1) Counter electrode (CE), reference electrode (RE): The nickel-free gold-plated micro-needle electrodes arranged in 2×3 and 1×3 arrays are used as the counter electrode (CE) and reference electrode (RE) respectively; 2) Preparation of the working electrode (WE): a. PMMA coating: The micro-needles are coated with a 3% (by mass ratio) PMMA (polymerization degree of 8500) solution and dried at 90 °C for 17 h; b. Construction of the adhesion layer: An ethanol solution of single-walled carbon nanotubes (SWCNT) and Nafion (perfluorosulfonic acid polymer solution) is mixed in a volume ratio of 1:5, and electro-deposited at a potential of 0.7 V for 70 seconds. After electro-deposition, the micro-needle electrode is cleaned with ethanol and air-dried at room temperature overnight; c. Immobilization of glucose oxidase (GOD): A mixed solution is prepared using a PBS solution with a pH of 7.0 at concentrations of glucose oxidase (GOx): 60 mg / mL, mediator (Osmium mediator): approximately 12 mg / mL, and glutaric dialdehyde: 3 mg / mL. The micro-needles treated with Nafion are dipped into the enzyme solution, then lifted and air-dried at room temperature to immobilize the enzyme on the surface of the micro-needles; d. Construction of the limiting layer: The outer surface of the micro-needles is coated with an N,N-dimethylformamide (DMF) solution containing polyurethane (PU, polymerization degree of 1200) to form a limiting layer to prevent enzyme leakage and improve the biocompatibility of the electrode; ⑥ Sensor assembly: Integration of three - electrode system: Polyimide (PI, degree of polymerization is 2000) material is used as the substrate, and electron etching technology is used to drill holes in the substrate. The WE, CE, and RE are respectively embedded in the substrate, so that the microneedles penetrate through the substrate and expose 2 mm, forming a three - electrode system with a microneedle array electrode. The WE, CE, and RE are respectively connected to silver wires, and the other ends of the silver wires are respectively connected to the interfaces of the electrochemical workstation. Test Examples

[0024] Electrochemical tests: a. Test conditions and system: Chronoamperometry (i - t method), three - electrode system, 0.25 mM potassium ferricyanide solution with pH = 6.8, constant potential (300 mV), scan time (1000 s), glucose solutions with different concentrations (0, 1, 2, 5, 10, 15, 25, 30 mM); b. Detection range test: Connect the gold - plated microneedle array electrode sensor prepared in Example 1 to the electrochemical workstation. Turn on the electrochemical workstation. After the current tends to be stable, add 3 mL of 0.25 mM potassium ferricyanide solution with pH = 6.8 to the three - electrode system, and conduct electrochemical tests on glucose solutions with concentrations of (0, 1, 2, 5, 10, 15, 25, 30 mM) in sequence. Obtain the change relationship between the response current value and the glucose concentration, draw the standard curve between the glucose concentration and the response current, and determine the detection range of the glucose concentration; As Figure 1 The results show that the formula of the standard curve between the glucose concentration and the response current is: I = 0.0668C+0.57, R 2 = 0.9868, where I is the response current (unit: μA) and C is the glucose concentration (unit: mM). The results indicate that the array microneedle system of the present invention has a good linear response in the glucose concentration range of 1 - 30 mM, and the detection range is 1 - 30 mM; c. Long - term stability test: Within 20 days, connect the gold - plated microneedle array electrode sensor prepared in Example 1 to the electrochemical workstation every day. After the current baseline is stable, add different glucose concentrations to 3 mL of 0.25 mM potassium ferricyanide solution with pH = 6.8 for measurement, repeat three times, and record the average current response value. Continuously monitor for 20 days to obtain the stability monitoring results.

[0025] As Figure 2 The results show that after 20 - day continuous monitoring, the performance of the sensor remains basically stable.

[0026] The present invention realizes nickel-free direct gold plating on a stainless-steel substrate through a composite pickling activation + nano-transition layer + pulse electroplating technique, covalently modifies the micro needles after gold plating with glucose oxidase (GOD), and finally assembles a micro needle array electrode sensor with a three-electrode system. It solves the problems of nickel sensitization, low coating adhesion, and complex process. By measuring the detection range of glucose concentration and monitoring the stability of the sensor, it is proved that the sensor has stable and reliable performance and meets the requirements of miniaturization and long-term stability of CGM sensors.

[0027] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0028] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. A method for preparing a CGM electrode sensor, characterized in that: The following steps are involved: S1. Preparation of stainless steel microneedle arrays: Three groups of stainless steel microneedle arrays were prepared using stainless steel substrates, which served as counter electrode, reference electrode and working electrode respectively; S2, surface pretreatment: the stainless steel microneedle array is first mechanically polished, and then composite acid washing and activation is performed; S3, preparation of nano transition layer: using sol-gel deposition of aluminum oxide layer on the stainless steel microneedle array after surface pretreatment; S4, direct gold plating: prepare a gold plating solution, and use two-stage pulse electroplating to gold plate the stainless steel microneedle array after the aluminum oxide layer is deposited in S3; S5. Preparation of electrode: the working electrode is then prepared by PMMA coating, adhesion layer construction, glucose oxidase immobilization and restriction layer construction; S6. Sensor assembly: The counter electrode, reference electrode and working electrode are respectively embedded in the substrate to form a microneedle array electrode sensor with a three-electrode system.

2. A method for preparing a CGM electrode sensor as claimed in claim 1, characterized in that: The method for preparing three groups of stainless steel microneedle arrays using a stainless steel substrate includes: immersing the stainless steel substrate in a deionized water medium, setting a discharge voltage of 30-50V, a cutting feed rate of 5-6mm / min, an electrode gap of 0.04-0.08mm, and completing the processing under the condition of controlling the ambient temperature at 20±1℃.

3. A method for preparing a CGM electrode sensor as claimed in claim 2, characterized in that: The three groups of stainless steel microneedle arrays prepared have the same geometric characteristics: the height of the pyramid-shaped needle structure is maintained at 1-3mm, the base width is 700-900μm, the center distance between adjacent needles is 0.5-1.5mm, the working electrode and the reference electrode are arranged in a 1×3 array, and the counter electrode is configured in a 2×3 array layout.

4. A method for preparing a CGM electrode sensor as claimed in claim 1, characterized in that: Mechanical polishing uses sandpaper with a particle size ranging from coarse to fine: #800-#2000 to polish the surface of the stainless steel microneedle array to a surface roughness Ra of 0.2-0.4 μm.

5. A method for preparing a CGM electrode sensor as claimed in claim 4, characterized in that: The composite pickling activation method includes: immersing the mechanically polished stainless steel microneedle array in a mixture of HCl, HF and corrosion inhibitor at 20-40°C for 3-7 minutes to remove the passivation film; and then immersing it in an electrolyte containing thiourea and citric acid with a pulse current for 8-12 minutes to form a sulfide interface layer.

6. A method for preparing a CGM electrode sensor as claimed in claim 1, characterized in that: The method for preparing the nano transition layer comprises: immersing the stainless steel microneedle array after surface pretreatment in a mixed sol of aluminum isopropoxide and nitric acid, and heat treating at 50-70°C for 20-40 minutes to form a 40-60nm dense Al2O3 layer.

7. A method for preparing a CGM electrode sensor as claimed in claim 1, characterized in that: The preparation method of the gold plating solution comprises: adding 1-2 g of potassium chloroaurate dihydrate into 500 mL of deionized water, then sequentially adding 4-6 g of disodium ethylenediaminetetraacetate, 23-27 g of a buffer, 7-8 g of potassium dihydrogen phosphate, 30-50 mL of formic acid, 1-2 mL of a non-ionic fluorocarbon compound and 4-6 g of cobalt sulfate heptahydrate, and stirring with a magnetic stirrer; wherein the preparation method of the buffer comprises: mixing citric acid and potassium citrate in a 1:1 molar ratio, weighing 23-27 g of the mixture, adding it into 500 mL of deionized water, stirring until it is completely dissolved, and preparing a buffer solution.

8. A method for preparing a CGM electrode sensor as claimed in claim 7, characterized in that: The method of gold-plating a stainless steel microneedle array after S3 deposition of an aluminum oxide layer using two-stage pulse electroplating includes: immersing the stainless steel microneedle array after the aluminum oxide layer is deposited in a configured gold plating solution, the temperature of the gold plating solution is controlled at 40-60°C, and the electroplating is divided into two stages: an initial stage, the current density is set to 7-9A / dm², the voltage is 5-9V, and the duration is 8-12 seconds, and then enters a steady-state stage, the current density drops to 1-2A / dm², the voltage is adjusted to 3-5V, and the duration is 45-55 seconds.

9. A method for preparing a CGM electrode sensor as claimed in claim 1, characterized in that: The preparation method of the working electrode comprises: a. PMMA coating: Use 1%-3% PMMA solution to coat the microneedles and dry at 70-90°C for 15-17h; b. Adhesion layer construction: Mix single-walled carbon nanotubes and Nafion ethanol solution in a volume ratio of 1:3-5, electrodeposit at a potential of 0.5-0.7V for 50-70 seconds, wash with ethanol after electrodeposition, and dry; c. Immobilization of glucose oxidase: Using PBS solution, prepare a mixed solution of 40-60 mg / mL glucose oxidase, 8-12 mg / mL mediator, and 2-3 mg / mL glutaraldehyde, dip the Nafion-treated microneedles into the mixed solution, and then lift them up to dry; d. Construction of restriction layer: A dimethylformamide solution containing polyurethane is applied to the outer surface of the microneedle to form a restriction layer.

10. A CGM electrode sensor obtained by the method for preparing a CGM electrode sensor according to any one of claims 1 to 9.

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