A CGM electrode sensor and its preparation method

Abstract

The present invention belongs to the technical field of CGM sensors, and specifically relates to a CGM electrode sensor and a preparation method thereof. The preparation method includes the following steps: preparing three groups of stainless steel microneedle arrays using a stainless steel substrate, which are used as a counter electrode, a reference electrode, and a working electrode respectively; mechanically grinding the stainless steel microneedle arrays and then subjecting them to composite pickling activation; after surface pretreatment, depositing an alumina layer by sol-gel; preparing a gold plating solution and plating the stainless steel microneedle arrays after depositing the alumina layer by two-stage pulse electroplating; the working electrode is further prepared by PMMA coating, adhesion layer construction, glucose oxidase immobilization, and construction of a limiting layer; the counter electrode, the reference electrode, and the working electrode are respectively embedded in a substrate to form a microneedle array electrode sensor with a three-electrode system. Based on a nickel-free direct gold plating process, the present invention can achieve good uniformity, strong adhesion, and lower cost after gold plating, thereby improving the stability of the continuous glucose sensor during use.

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 to continuously measure glucose levels, is responsible for sensing glucose in tissue fluid and converting it into electrical signals, 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 requires pretreatment, nickel plating (2 - 5μm intermediate layer), and gold plating in sequence. The nickel layer is used to enhance the adhesion 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 the uneven current density distribution results in poor coating uniformity; in addition, the nickel plating process requires additional processes (such as electroplating solution maintenance and 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 adhesion, 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:

[0006] The present invention aims to provide a preparation method of a CGM electrode sensor, comprising the following steps:

[0007] 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;

[0008] S2. Surface pretreatment: The stainless steel microneedle arrays are first mechanically polished and then subjected to composite pickling activation;

[0009] S3. Preparation of a nano-transition layer: The surface-pretreated stainless-steel microneedle array is coated with an alumina layer by sol-gel deposition;

[0010] S4. Direct gold plating: Prepare a gold plating solution, and use two-stage pulse electroplating to plate the stainless-steel microneedle array after depositing the alumina layer in S3;

[0011] S5. Preparation of the electrode: The working electrode is prepared by further coating with PMMA, constructing an adhesion layer, immobilizing glucose oxidase, and constructing a limiting layer;

[0012] 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.

[0013] Furthermore, 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 the discharge voltage at 30 - 50 V, 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.

[0014] Furthermore, the three groups of prepared stainless-steel microneedle 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 needle bodies is 0.5 - 1.5 mm, where 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.

[0015] Furthermore, for mechanical polishing, sandpapers with particle sizes ranging from coarse to fine: #800 - #2000 are used to polish the surface of the stainless-steel microneedle array until the surface roughness Ra is 0.2 - 0.4 μm.

[0016] Furthermore, the method for composite pickling activation includes: immersing the mechanically polished stainless-steel microneedle array in a mixed solution of HCl, HF, and an inhibitor, soaking at 20 - 40 °C for 3 - 7 minutes to remove the passivation film; then, in an electrolyte containing thiourea and citric acid, performing pulsed current treatment for 8 - 12 minutes to form a sulfide interface layer.

[0017] Furthermore, the method for preparing the nano-transition layer includes: immersing the surface-pretreated stainless-steel microneedle array in a mixed sol of aluminum isopropoxide and nitric acid, performing heat treatment at 50 - 70 °C for 20 - 40 minutes to form a 40 - 60 nm dense Al2O3 layer.

[0018] Furthermore, the preparation method of the gold plating solution includes: adding 1-2 g of potassium chloroaurate dihydrate to 500 mL of deionized water, and then sequentially 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 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 is: mixing citric acid and potassium citrate in a molar ratio of 1:1, weighing 23-27 g of the mixture, adding it to 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.

[0019] Furthermore, 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 in 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, the voltage is adjusted to 3-5 V, and the duration is 45-55 seconds.

[0020] Furthermore, the preparation method of the working electrode includes:

[0021] 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;

[0022] 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;

[0023] 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 in the mixed solution, and then lifting and drying;

[0024] d. Construction of the limiting layer: Coating the outer surface of the microneedles with a dimethylformamide solution containing polyurethane to form a limiting layer.

[0025] A CGM electrode sensor is prepared by the above preparation method.

[0026] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0027] 1) Enhanced biocompatibility: The nickel-free gold plating process avoids the harm of nickel release to the human body, is applicable to medical devices in contact with the human body, ensures the safety and reliability of the product, and reduces the risk of adverse reactions in patients due to nickel allergy;

[0028] 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;

[0029] 3) Cost savings: It reduces the cumbersome steps such as the use and maintenance of electroplating solution and activation treatment during the nickel plating process, achieving the effect of cost savings and reducing complex process steps;

[0030] 4) Miniaturization adaptability: The coating evenly covers the tip of the microneedle (diameter < 30 μm), ensuring the complete function of complex microstructures;

[0031] 5) Firm adhesion: After using a nano-transition layer for gold plating, a dense inorganic layer is pre-constructed on the clean stainless steel substrate, 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.

[0032] The above description is only an overview of the technical solution of the present invention. In order to be able to more clearly understand the technical means of the present invention, it can be implemented in accordance with the content of the specification. And in order to make the above content, its purpose, features and advantages of the present invention more obvious and understandable, the following specific embodiments of the present invention are specifically given. Description of the Drawings

[0033] Figure 1 It is the standard curve graph of glucose concentration and response current in the test example of the present invention.

[0034] Figure 2 It is the monitoring result graph of the sensor stability in the test example of the present invention. Detailed Embodiments

[0035] 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.

[0036] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchases or prepared by existing methods. Example 1

[0037] A preparation method of a continuous glucose sensor based on a nickel-free direct gold plating process is as follows:

[0038] ① Preparation of stainless steel microneedle arrays:

[0039] A Swiss Charmilles CUT20P slow wire electrical discharge machining (EDM) machine is used to prepare the microneedle arrays, with a 0.20 mm molybdenum wire as the cutting electrode. During the machining process, 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 machining is completed under the condition that the ambient temperature is controlled at 20 °C.

[0040] The three groups of prepared microneedle arrays have the same geometric features: the height of the pyramid-shaped needle body structure is 2 mm, the base width 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.

[0041] ② Surface pretreatment:

[0042] 1) Mechanical polishing: The surface of the microneedle arrays is polished with sandpapers of different grits (from coarse to fine: #800 - #2000) until the surface roughness Ra is 0.3 μm to remove surface burrs and large scratches.

[0043] 2) Composite pickling and activation:

[0044] a. Pickling solution: The polished microneedle arrays are 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.

[0045] b. Electrolytic activation: The electrolyte containing thiourea (5 g / L) + citric acid (20 g / L) is used for 10 minutes with a pulsed current (peak value 8 A / dm², duty cycle 20%) to form a sulfide interface layer.

[0046] ③ Nano-transition layer (nano-aluminum oxide replacing nickel):

[0047] 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 Al2O3 layer.

[0048] ④ Nickel-free direct gold plating

[0049] 1) Preparation of gold plating solution:

[0050] 1 g of potassium tetrachloroaurate dihydrate (KAuCl4·2H2O) was added to 500 mL of deionized water to provide gold ions as the main salt; 5 g of disodium ethylenediaminetetraacetate (Na2EDTA) was further added to stabilize the gold ions in the plating solution. The buffer was a mixture of citric acid and potassium citrate in a 1:1 molar ratio, with an addition amount of 25 g, used to maintain the pH value of the plating solution. 7.5 g of potassium dihydrogen phosphate (KH2PO4) was added as a conductive salt to enhance the conductivity of the plating solution. Formic acid (HCOOH) was used as an acidic regulator, with an addition amount of 40 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 surfactant) was added as a surfactant to improve the uniformity of the coating; finally, 5 g of cobalt sulfate heptahydrate (CoSO4·7H2O) was added as a hardening agent to increase the hardness of the coating. All reagents were added in sequence and stirred using a magnetic stirrer;

[0051] 2) Nickel-free direct gold plating process:

[0052] The gold plating process used a two-stage pulse electroplating technique, and the instruments used included a pulse electroplating power supply and a constant temperature water bath. The stainless steel micro-needle array with a nano-transition layer was immersed in the prepared gold plating solution. The temperature of the plating solution was controlled at 50 °C. The electroplating was divided into two stages: in the initial stage, the current density was set at 8 A / dm², the voltage was 7 V, and the duration was 10 s. This stage aimed to achieve rapid nucleation of the gold layer. Subsequently, it entered the steady state stage, where the current density dropped to 1.5 A / dm², the voltage was adjusted to 4 V, and the duration was 50 s. This stage was used to form a dense gold layer with a thickness of 0.3 ± 0.05 μm;

[0053] By adjusting the pulsed current, it was ensured that the coating uniformly covered 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 was significantly improved, ensuring the stability and reliability of the coating during long-term use. After electroplating, the micro-needle array was taken out, rinsed with deionized water, and dried for standby;

[0054] ⑤ Preparation and modification of electrodes:

[0055] 1) Counter electrode (CE), reference electrode (RE): The nickel-free gold-plated micro-needle electrodes arranged in 2×3 and 1×3 arrays were used as the counter electrode (CE) and reference electrode (RE), respectively;

[0056] 2) Preparation of working electrode (WE):

[0057] a. PMMA coating: The micro-needles were coated with a 2% (by mass) PMMA (polymerization degree of 8500) solution and dried at 80 °C for 16 h;

[0058] b. Adhesion layer construction: Mix an ethanol solution of single-walled carbon nanotubes (SWCNT) and Nafion (perfluorosulfonic acid polymer solution) at a volume ratio of 1:4, and electro-deposit for 60 seconds at a potential of 0.6V. After electro-deposition, the microneedle electrode is cleaned with ethanol and air-dried overnight at room temperature;

[0059] c. Glucose oxidase (GOD) immobilization: Prepare a mixed solution using PBS solution with a pH of 7.0, at concentrations of glucose oxidase (GOx): 50mg / mL, mediator (Osmium mediator): approximately 10mg / mL, and glutaric dialdehyde: 2.5mg / mL. Dip the Nafion-treated microneedles into the enzyme solution, then lift and air-dry at room temperature to immobilize the enzyme on the surface of the microneedles;

[0060] d. Restriction layer construction: Coat the outer surface of the microneedles with an N,N-dimethylformamide (DMF) solution containing polyurethane (PU, polymerization degree of 1200) to form a restriction layer to prevent enzyme leakage and improve the biocompatibility of the electrode;

[0061] ⑥ Sensor assembly:

[0062] Integration of the three-electrode system: Use polyimide (PI, polymerization degree of 2000) material as the substrate, and use electron etching technology to drill holes in the substrate. Embed the WE, CE, and RE into the substrate respectively, so that the microneedles pass through the substrate and protrude 1mm, 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

[0063] A preparation method of a continuous glucose sensor based on a nickel-free direct gold plating process is as follows:

[0064] ① Preparation of a stainless steel microneedle array:

[0065] Use a Swiss Charmilles CUT20P slow wire cutting machine to prepare the microneedle array, and use 0.20mm molybdenum wire as the cutting electrode. During the processing, immerse the 316L stainless steel substrate in deionized water medium, set the discharge voltage at 30V, the cutting feed rate at 5 mm / min, the electrode gap at 0.04mm, and complete the processing 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 pyramidal needle body structure is maintained at 1mm, the base width is 700μm, and the center distance between adjacent needle bodies is 0.5mm. 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;

[0066] ② Surface pretreatment:

[0067] 1) Mechanical polishing: Use sandpapers with different grits (from coarse to fine: #800 - #2000) to polish the surface of the microneedle array until the surface roughness Ra reaches 0.2 μm, so as to remove burrs and large scratches on the surface;

[0068] 2) Composite pickling activation:

[0069] a. Pickling solution: Immerse the polished microneedle array in HCl (10wt%) + HF (2wt%) + corrosion inhibitor (sodium dodecylbenzenesulfonate 0.1wt%) at 20 °C for 3 minutes to remove the passivation film;

[0070] b. Electrolytic activation: Use an electrolyte containing thiourea (5 g / L) + citric acid (20 g / L), and treat it with a pulsed current (peak value 8 A / dm², duty cycle 20%) for 8 minutes to form a sulfide interface layer;

[0071] ③ Nano-transition layer (nano-aluminum oxide replaces nickel):

[0072] Sol-gel deposition of aluminum oxide layer: Immerse the surface-pretreated microneedles into a mixed sol of aluminum isopropoxide (0.1M) and nitric acid (0.05M), and perform heat treatment at 50 °C for 20 minutes to form a 40-nm dense Al2O3 layer;

[0073] ④ Nickel-free direct gold plating

[0074] 1) Preparation of gold plating solution:

[0075] Add 1 g of potassium tetrachloroaurate dihydrate (KAuCl4·2H2O) to 500 mL of deionized water to provide gold ions as the main salt; then add 4 g of disodium ethylenediaminetetraacetate (Na2EDTA) 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. Add 7 g of potassium dihydrogen phosphate (KH2PO4) 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. Add 1 mL of 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 4 g of cobalt sulfate heptahydrate (CoSO4·7H2O) as a hardening agent to increase the hardness of the coating. Add all the reagents in sequence and stir with a magnetic stirrer;

[0076] 2) Nickel-free direct gold plating process:

[0077] The gold plating process adopts 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 at 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;

[0078] By adjusting the pulsed current, it is ensured that the coating evenly 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 is completed, the micro-needle array is taken out, rinsed with deionized water and dried for standby;

[0079] ⑤ Preparation and modification of electrodes:

[0080] 1) Counter electrode (CE), reference electrode (RE): The nickel-free gold-plated micro-needle electrodes arranged in arrays of 2×3 and 1×3 are used as the counter electrode (CE) and reference electrode (RE) respectively;

[0081] 2) Preparation of working electrode (WE):

[0082] a. PMMA coating: The micro-needles are coated with a 1% (by mass ratio) PMMA (polymerization degree of 8500) solution and dried at 70 °C for 15 h;

[0083] 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 1:3, and electro-deposited at a potential of 0.5 V for 50 seconds. After electro-deposition, the micro-needle electrode is cleaned with ethanol and naturally dried at room temperature overnight;

[0084] 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): 40 mg / mL, mediator (Osmium mediator): approximately 8 mg / mL, and glutaric dialdehyde: 2 mg / mL. The micro-needles treated with Nafion are dipped into the enzyme solution, then lifted and dried at room temperature to immobilize the enzyme on the surface of the micro-needles;

[0085] d. Construction of the confinement layer: Coat the outer surface of the microneedles with an N,N-dimethylformamide (DMF) solution containing polyurethane (PU, degree of polymerization 1200) to form a confinement layer to prevent the leakage of enzymes and improve the biocompatibility of the electrodes;

[0086] ⑥ Sensor assembly:

[0087] Integration of the three-electrode system: Use polyimide (PI, degree of polymerization 2000) material as the substrate, and use electron etching technology to drill holes in the substrate. Embed the WE, CE, and RE into the substrate respectively, 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 3

[0088] A preparation method of a continuous glucose sensor based on a nickel-free direct gold plating process, the specific steps are as follows:

[0089] ① Preparation of the stainless steel microneedle array:

[0090] Use a Swiss Charmilles CUT20P slow wire cutting machine to prepare the microneedle array, and use 0.20 mm molybdenum wire as the cutting electrode. During the processing, immerse the 316L stainless steel substrate in deionized water medium, set the discharge voltage at 50V, the cutting feed rate at 6 mm / min, the electrode gap at 0.08 mm, and complete the processing under the condition that the ambient temperature is controlled at 21°C;

[0091] The three groups of prepared microneedle arrays have the same geometric characteristics: the height of the pyramid-shaped needle body structure is kept 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;

[0092] ② Surface pretreatment:

[0093] 1) Mechanical polishing: Polish the surface of the microneedle array with sandpapers of different grits (from coarse to fine: #800 - #2000) until the surface roughness Ra is 0.4 μm to remove burrs and large scratches on the surface;

[0094] 2) Composite pickling activation:

[0095] a. Pickling solution: Immerse the polished microneedle array in HCl (10 wt%) + HF (2 wt%) + inhibitor (sodium dodecylbenzenesulfonate 0.1 wt%) and soak at 40°C for 7 minutes to remove the passivation film;

[0096] b. Electrolytic activation: The electrolyte containing thiourea (5 g / L) + citric acid (20 g / L) is treated with pulsed current (peak value 8 A / dm², duty cycle 20%) for 12 minutes to form a sulfide interfacial layer;

[0097] ③ Nano-transition layer (nano-aluminum oxide replaces nickel):

[0098] Sol-gel deposition of aluminum oxide layer: The pretreated microneedles are immersed in a mixed sol of aluminum isopropoxide (0.1 M) and nitric acid (0.05 M), and heat-treated at 70 °C for 40 minutes to form a 60-nm dense Al2O3 layer;

[0099] ④ Nickel-free direct gold plating

[0100] 1) Preparation of gold plating solution:

[0101] 2 g of potassium tetrachloroaurate dihydrate (KAuCl4·2H2O) is added to 500 mL of deionized water to provide gold ions as the main salt; then 6 g of disodium ethylenediaminetetraacetate (Na2EDTA) 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 27 g to maintain the pH value of the plating solution. 8 g of potassium dihydrogen phosphate (KH2PO4) 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 50 mL to adjust the acidic environment of the plating solution. 2 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, 6 g of cobalt sulfate heptahydrate (CoSO4·7H2O) 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;

[0102] 2) Nickel-free direct gold plating process:

[0103] The gold plating process uses two-stage pulse electroplating technology, and the instruments used include a pulse electroplating power supply and a constant temperature water bath. The stainless steel microneedle 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, 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;

[0104] By adjusting the pulsed current, ensure that the coating evenly covers the tips of the microneedles (diameter < 30 μm). Meanwhile, combined with the application of a nano-transition layer (such as an alumina layer), significantly improve the bonding force between the gold layer and the stainless-steel substrate, ensuring the stability and reliability of the coating during long-term use. After electroplating, take out the microneedle array, rinse it with deionized water and dry it for standby;

[0105] ⑤ Preparation and modification of the electrodes:

[0106] 1) Counter electrode (CE), reference electrode (RE): Use nickel-free gold-plated microneedle electrodes arranged in 2×3 and 1×3 arrays respectively as the counter electrode (CE) and the reference electrode (RE);

[0107] 2) Preparation of the working electrode (WE):

[0108] a. PMMA coating: Coat the microneedles with a 3% (by mass) PMMA (degree of polymerization 8500) solution and dry at 90 °C for 17 h;

[0109] b. Construction of the adhesion layer: Mix an ethanol solution of single-walled carbon nanotubes (SWCNT) and Nafion (perfluorosulfonic acid polymer solution) in a volume ratio of 1:5 and electro-deposit at a potential of 0.7 V for 70 s. After electro-deposition, clean the microneedle electrode with ethanol and air-dry it at room temperature overnight;

[0110] c. Immobilization of glucose oxidase (GOD): Prepare a mixed solution with a pH of 7.0 PBS solution at concentrations of glucose oxidase (GOx): 60 mg / mL, mediator (Osmium mediator): about 12 mg / mL, and glutaric dialdehyde: 3 mg / mL. Dip the Nafion-treated microneedles into the enzyme solution, then lift them and air-dry at room temperature to immobilize the enzyme on the surface of the microneedles;

[0111] d. Construction of the confinement layer: Coat the outer surface of the microneedles with an N,N-dimethylformamide (DMF) solution containing polyurethane (PU, degree of polymerization 1200) to form a confinement layer to prevent enzyme leakage and improve the biocompatibility of the electrode;

[0112] ⑥ Sensor assembly:

[0113] Integration of the three-electrode system: Use polyimide (PI, degree of polymerization 2000) material as the substrate and use electron etching technology to drill holes in the substrate. Embed the WE, CE, and RE into the substrate respectively, so that the microneedles pass through the substrate and protrude 2 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.

[0114] Test Example

[0115] Electrochemical Test:

[0116] 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);

[0117] b. Detection Range Test: Connect the gold-plated microneedle array electrode sensor prepared in Example 1 to an electrochemical workstation. Turn on the electrochemical workstation. After the current stabilizes, 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, plot the standard curve between the glucose concentration and the response current, and determine the detection range of the glucose concentration;

[0118] As Figure 1 The results show that the formula for the standard curve of glucose concentration and 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;

[0119] 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 stabilizes, 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.

[0120] As Figure 2 The results show that after 20 days of continuous monitoring, the performance of the sensor remains basically stable.

[0121] The present invention realizes direct gold plating on a stainless steel substrate without nickel through a composite pickling activation + nano-transition layer + pulse electroplating technique, covalently modifies the gold-plated microneedles with glucose oxidase (GOD), and finally assembles a microneedle array electrode sensor with a three-electrode system. Solve the problems of nickel sensitization, low coating adhesion, and complex process. Prove that the performance of the sensor is stable and reliable by measuring the detection range of glucose concentration and monitoring the stability of the sensor, meeting the requirements of miniaturization and long-term stability of CGM sensors.

[0122] The serial numbers of the embodiments of the present invention above are only for description and do not represent the superiority or inferiority of the embodiments.

[0123] 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 rather than restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these fall within the protection scope of the present invention.

Claims

1. A preparation method of a CGM electrode sensor, characterized in that, It includes the following steps: S1. Preparation of stainless steel micro-needle arrays: Three groups of stainless steel micro-needle arrays are prepared using a stainless steel substrate, serving as the counter electrode, reference electrode, and working electrode respectively. S2. Surface pretreatment: The stainless steel micro-needle arrays are first mechanically polished and then subjected to composite pickling activation. S3. Preparation of nano-transition layer: The stainless steel micro-needle arrays after surface pretreatment are deposited with an alumina layer by sol-gel method. S4. Direct gold plating: A gold plating solution is prepared, and the stainless steel micro-needle arrays after depositing the alumina layer in S3 are plated with gold using two-stage pulse electroplating. S5. Preparation of the electrode: The working electrode is further prepared by PMMA coating, construction of the adhesion layer, immobilization of glucose oxidase, and construction of the 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.

2. The preparation method of a CGM electrode sensor according to claim 1, wherein: 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 - 6mm / min, the electrode gap at 0.04 - 0.08mm, and controlling the ambient temperature at 20 ± 1°C to complete the processing.

3. The preparation method of a CGM electrode sensor according to claim 2, characterized in that: The three groups of stainless steel micro-needle arrays prepared have the same geometric features: the height of the pyramid-shaped needle body structure is maintained at 1 - 3mm, the base width is 700 - 900μm, the center distance between adjacent needles is 0.5 - 1.5mm. 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.

4. The preparation method of a CGM electrode sensor according to claim 1, wherein: For mechanical polishing, sandpapers with particle sizes from coarse to fine: #800 - #2000 are used to polish the surface of the stainless steel micro-needle arrays until the surface roughness Ra is 0.2 - 0.4μm.

5. The preparation method of a CGM electrode sensor according to claim 4, wherein: The method for composite pickling activation includes: immersing the mechanically polished stainless steel micro-needle arrays in a mixed solution of HCl, HF, and inhibitor, soaking at 20 - 40°C for 3 - 7 minutes to remove the passivation film; then, in an electrolyte containing thiourea and citric acid, performing pulsed current treatment for 8 - 12 minutes to form a sulfide interface layer.

6. The preparation method of a CGM electrode sensor according to claim 1, wherein: The method for preparing the nano-transition layer includes: immersing the stainless steel micro-needle arrays after surface pretreatment in a mixed sol of aluminum isopropoxide and nitric acid, performing heat treatment at 50 - 70°C for 20 - 40 minutes to form a 40 - 60nm dense Al2O3 layer.

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

8. The preparation method of a CGM electrode sensor according to claim 7, characterized in that: The method for gold plating the stainless steel micro-needle array after depositing an alumina layer on S3 by two-stage pulse electroplating includes: immersing the stainless steel micro-needle 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, the voltage is adjusted to 3 - 5 V, and the duration is 45 - 55 seconds.

9. The preparation method of a CGM electrode sensor according to claim 1, characterized in that: The preparation method of the working electrode includes: a. PMMA coating: Coating the micro-needles with a PMMA solution with a mass ratio of 1% - 3%, drying at 70 - 90 °C for 15 - 17 h; b. Adhesion layer construction: Mixing single-walled carbon nanotubes 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, washing with ethanol after electro-deposition, and drying in air; c. Glucose oxidase immobilization: Using a PBS solution, preparing a mixed solution according to 40 - 60 mg / mL glucose oxidase, 8 - 12 mg / mL mediator, and 2 - 3 mg / mL glutaraldehyde. Dipping the micro-needles treated with Nafion into the mixed solution, and then lifting and drying; d. Construction of the limiting layer: Coating the outer surface of the micro-needles with a dimethylformamide solution containing polyurethane to form a limiting layer.

10. A CGM electrode sensor prepared by the preparation method of a CGM electrode sensor according to any one of claims 1 - 9.

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