Microneedle sensor for continuously monitoring glucose in interstitial fluid and manufacturing method

Abstract

The invention provides a microneedle sensor for continuously monitoring glucose in interstitial fluid and a manufacturing method of the microneedle sensor. The microneedle sensor comprises a substrate material and a microneedle array electrode system, the microneedle array electrode system is embedded into the substrate material and is 1mm higher than the substrate material; wherein the microneedle array electrode system is made of stainless steel, is pyramid-shaped and comprises a working electrode, a counter electrode and a reference electrode. Direct electron transfer between glucose oxidase and the electrode is realized through a third-generation sensing technology, and the high biocompatibility of the stainless steel microneedle array and a stable coating process are combined, so that the detection range is remarkably widened, and the continuous monitoring period is prolonged. The six-layer GOD / GNPs composite membrane is covalently modified, so that the enzyme is effectively prevented from falling off, the stability of the sensor is improved, the problems of potential interference and enzyme inactivation in the prior art are solved, and the GOD / GNPs composite membrane is suitable for long-term and accurate blood glucose monitoring requirements of diabetic patients.

Description

Technical Field

[0001] The present invention relates to the field of biochemical sensing technology, and in particular to a microneedle sensor for continuous monitoring of glucose in interstitial fluid and a manufacturing method thereof. Background Art

[0002] Existing continuous glucose monitoring systems (CGMS) mostly use first- or second-generation sensing technologies. First-generation technologies rely on oxygen as an electron acceptor, resulting in high potential and low electron transfer efficiency. Second-generation technologies introduce redox mediators, but are susceptible to interference from oxygen competition, and glucose oxidase (GOD) is easily detached due to physical adsorption or cross-linking, leading to sensor failure. The present invention utilizes third-generation sensing technology, utilizing direct electron transfer between the enzyme active center and the electrode, significantly improving sensing performance and stability. Summary of the Invention

[0003] Based on this, it is necessary to provide a microneedle sensor and a manufacturing method for continuous monitoring of glucose in interstitial fluid to address the above technical problems.

[0004] A microneedle sensor for continuous monitoring of glucose in interstitial fluid comprises: a substrate material and a microneedle array electrode system; the microneedle array electrode system is embedded in the substrate material and is 1 mm higher than the substrate material; wherein the microneedle array electrode system is made of stainless steel and is pyramid-shaped and comprises: a working electrode, a counter electrode, and a reference electrode.

[0005] In one embodiment, the microneedle array electrode system is prepared by:

[0006] Three pyramidal microneedle arrays with a height of 1.8-2 mm, a base of 800-1000 μm, and a microneedle spacing of 0.8-1 mm were prepared from a stainless steel sheet using a wire-cut cutting machine.

[0007] The surface of the microneedle array is subjected to degreasing, acid washing and activation treatment in sequence to obtain a treated microneedle array;

[0008] Platinum is coated on any of the treated microneedle arrays by magnetron sputtering to obtain a counter electrode;

[0009] Gold is plated on another treated microneedle array by magnetron sputtering to obtain an initial working electrode; glucose oxidase and gold nanoparticles are immobilized on the surface of the initial working electrode by covalent modification to obtain a working electrode;

[0010] A reference electrode was prepared by electrodeposition for the remaining treated microneedle array.

[0011] In one embodiment, a stainless steel sheet is processed by a wire-cutting machine to prepare three pyramid-shaped microneedle arrays with a height of 1.8-2 mm, a base of 800-1000 μm, and a microneedle spacing of 0.8-1 mm, including:

[0012] The processing parameters include a copper wire electrode diameter of 0.15-0.3 mm, a voltage of 35-50 V, a cutting speed of 6.3-10 mm / min, an electrode gap of 0.08-0.2 mm, and the stainless steel sheet is immersed in pure water during the processing.

[0013] In one embodiment, the surface of the microneedle array is subjected to degreasing, acid washing and activation treatments in sequence to obtain the treated microneedle array, comprising:

[0014] The microneedle array is ultrasonically cleaned with acetone or ethanol for 10-20 minutes to obtain a degreased array;

[0015] Soaking the ultrasonically treated array in a 10% HCl solution for 10-20 minutes and cleaning it to obtain an acid-washed array;

[0016] The acid-washed array was immersed in a 10% H2SO4 solution for 1-2 minutes, cleaned, and dried to obtain a treated microneedle array.

[0017] In one embodiment, the preparation parameters of the platinum coating and the gold coating are: magnetron sputtering time 60-120 seconds, vacuum degree 0.04-0.06 MPa, and current 10-15 mA.

[0018] In one embodiment, the process of immobilizing glucose oxidase and gold nanoparticles on the surface of the initial working electrode by covalent modification to obtain the working electrode comprises:

[0019] The surface of the initial working electrode is modified by cysteamine, the carbohydrate groups of GOD are oxidized by sodium periodate to form Schiff base bonding, and the adsorption of gold nanoparticles is repeated for more than five times to form a multilayer film to obtain a working electrode.

[0020] In one embodiment, gold nanoparticles are prepared by reducing chloroauric acid with sodium citrate, and the solution is boiled until the color changes to deep red.

[0021] In one embodiment, preparing a reference electrode by electrodeposition on the remaining treated microneedle array comprises:

[0022] The remaining treated microneedle array is electroplated with a silver nitrate solution and then converted with a saturated NaCl solution to obtain a reference electrode.

[0023] A method for preparing a microneedle sensor for continuous monitoring of glucose in interstitial fluid, the preparation method comprising:

[0024] A pyramid-shaped microneedle array was prepared by processing a stainless steel sheet using a wire-cut cutting machine.

[0025] The surface of the microneedle array is subjected to degreasing, acid washing and activation treatment in sequence to obtain a treated microneedle array;

[0026] Platinum is plated on any one of the treated microneedle arrays by magnetron sputtering to obtain a counter electrode; gold is plated on another of the treated microneedle arrays by magnetron sputtering to obtain an initial working electrode; glucose oxidase and gold nanoparticles are immobilized on the surface of the initial working electrode by covalent modification to obtain a working electrode; a reference electrode is prepared on the remaining treated microneedle array by electrodeposition; the counter electrode, the working electrode, and the reference electrode constitute the microneedle array electrode system;

[0027] The microneedle array electrode system is embedded in a prefabricated substrate material to obtain a microneedle sensor.

[0028] Compared to existing technologies, the advantages and beneficial effects of this invention are as follows: It utilizes third-generation sensing technology to achieve direct electron transfer between glucose oxidase and electrodes. Combined with the high biocompatibility and stable coating process of the stainless steel microneedle array, it significantly broadens the detection range and extends the continuous monitoring period. Furthermore, by covalently modifying the GOD / GNPs composite membrane with six or more layers, it effectively prevents enzyme shedding and improves sensor stability, resolving the potential interference and enzyme inactivation issues encountered in existing technologies. This makes it suitable for the long-term, accurate blood glucose monitoring needs of diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 1 is a schematic flow chart of a method for preparing a microneedle sensor for continuous monitoring of glucose in interstitial fluid in one embodiment;

[0030] Figure 2 A schematic diagram of a standard curve of glucose concentration and response current in one embodiment;

[0031] Figure 3 Schematic diagram of sensor stability monitoring results in one embodiment. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0033] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in one or more embodiments of this specification should have the usual meanings understood by people with ordinary skills in the field to which the invention belongs. The words "first", "second" and similar terms used in one or more implementations of this specification do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "include" or "comprise" mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Words such as "connect" or "connected" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may also change accordingly.

[0034] In one embodiment, a microneedle sensor for continuous monitoring of glucose in interstitial fluid is provided, comprising a substrate material and a microneedle array electrode system. The microneedle array electrode system is embedded in the substrate material and extends 1 mm above the substrate material. The microneedle array electrode system is made of stainless steel and has a pyramidal shape, including a working electrode, a counter electrode, and a reference electrode.

[0035] The microneedle array electrode system was prepared as follows:

[0036] Three pyramidal microneedle arrays with a height of 1.8-2 mm, a base of 800-1000 μm, and a microneedle spacing of 0.8-1 mm were prepared from a stainless steel sheet using a wire-cut cutting machine.

[0037] The surface of the microneedle array is subjected to degreasing, acid washing and activation treatment in sequence to obtain a treated microneedle array;

[0038] Platinum is coated on any of the treated microneedle arrays by magnetron sputtering to obtain a counter electrode;

[0039] Gold is plated on another treated microneedle array by magnetron sputtering to obtain an initial working electrode; glucose oxidase and gold nanoparticles are immobilized on the surface of the initial working electrode by covalent modification to obtain a working electrode;

[0040] A reference electrode was prepared by electrodeposition for the remaining treated microneedle array.

[0041] Specifically, 316L stainless steel, known for its excellent biocompatibility and mechanical properties, was used as the microneedle array substrate. This stainless steel was processed using wire-cut electrocuting. The processed stainless steel microneedle arrays were then surface treated, plated, and covalently modified with glucose oxidase. Finally, a three-electrode microneedle array sensor was assembled. The sensor's stable and reliable performance was demonstrated through measurements of the glucose concentration detection range and sensor stability monitoring.

[0042] Three pyramidal microneedle arrays with a height of 1.8-2 mm, a base of 800-1000 μm, and a microneedle spacing of 0.8-1 mm were prepared from a stainless steel sheet using a wire-cutting machine. The arrays include:

[0043] The processing parameters include a copper wire electrode diameter of 0.15-0.3 mm, a voltage of 35-50 V, a cutting speed of 6.3-10 mm / min, an electrode gap of 0.08-0.2 mm, and the stainless steel sheet is immersed in pure water during the processing.

[0044] The surface of the microneedle array is sequentially subjected to degreasing, pickling and activation treatments to obtain a treated microneedle array comprising:

[0045] The microneedle array is ultrasonically cleaned with acetone or ethanol for 10-20 minutes to obtain a degreased array;

[0046] Soaking the ultrasonically treated array in a 10% HCl solution for 10-20 minutes and cleaning it to obtain an acid-washed array;

[0047] The acid-washed array was immersed in a 10% H2SO4 solution for 1-2 minutes, cleaned, and dried to obtain a treated microneedle array.

[0048] The preparation parameters of the platinum coating and the gold coating are: magnetron sputtering time 60-120 seconds, vacuum degree 0.04-0.06MPa, and current 10-15mA.

[0049] The surface of the initial working electrode is fixed with glucose oxidase and gold nanoparticles by covalent modification, and the obtained working electrode includes:

[0050] The surface of the initial working electrode was modified by cysteamine, the carbohydrate groups of GOD were oxidized by sodium periodate to form Schiff base bonding, and the adsorption of gold nanoparticles was repeated more than five times to form a multilayer film to obtain a working electrode.

[0051] Gold nanoparticles were prepared by reducing chloroauric acid with sodium citrate, and the solution was boiled until the color turned deep red.

[0052] The reference electrode for the remaining processed microneedle array is prepared by electrodeposition, including:

[0053] The remaining processed microneedle array was electroplated with silver nitrate solution and then converted with saturated NaCl solution to obtain a reference electrode.

[0054] Based on the same inventive concept, corresponding to any of the above embodiments of the microneedle sensor for continuous monitoring of glucose in interstitial fluid, the present invention also provides a method for preparing a microneedle sensor for continuous monitoring of glucose in interstitial fluid.

[0055] like Figure 1 As shown, the preparation method includes:

[0056] Step S1, processing a stainless steel sheet by a wire-cut cutting machine to prepare a pyramid-shaped microneedle array;

[0057] Step S2, sequentially subjecting the surface of the microneedle array to degreasing, acid washing, and activation treatments to obtain a treated microneedle array;

[0058] Step S3, platinum coating is applied to any one of the treated microneedle arrays by magnetron sputtering to obtain a counter electrode; gold coating is applied to another of the treated microneedle arrays by magnetron sputtering to obtain an initial working electrode; glucose oxidase and gold nanoparticles are immobilized on the surface of the initial working electrode by covalent modification to obtain a working electrode; a reference electrode is prepared by electrodeposition on the remaining treated microneedle array; the counter electrode, the working electrode, and the reference electrode constitute the microneedle array electrode system;

[0059] Step S4: embedding the microneedle array electrode system into a prefabricated substrate material to obtain a microneedle sensor.

[0060] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0061] Example 1

[0062] ① Preparation of stainless steel microneedle array:

[0063] Microneedle arrays were fabricated using a Mitsubishi MV1200R wire-cut electro-cutting machine in Japan, using 0.15mm copper wire electrodes. During processing, 316L stainless steel sheets were immersed in pure water. The process was conducted at a voltage of 35V, a cutting speed of 6.3mm / min, and an electrode gap of 0.08mm at room temperature of 22°C. Three microneedle arrays were prepared, serving as the unmodified working, counter, and reference electrodes, respectively. The microneedles in each of the three microneedle arrays were pyramidal in shape, 2mm in height, 800μm in base width, and 1mm in spacing. The unmodified working and reference electrodes had a 1x3 matrix, while the unmodified counter electrode matrix had a 2x3 matrix.

[0064] ②Microneedle array surface treatment:

[0065] 1) Degreasing: Immerse the microneedle array in acetone or ethanol, ultrasonically clean for 10 minutes, and then rinse with deionized water;

[0066] 2) Acid washing: Immerse the degreased microneedle array in 10% HCl (mass fraction) for 10 minutes, and then rinse with deionized water;

[0067] 3) Activation: Immerse the acid-washed microneedle array in a 10% H2SO4 solution for 1-2 minutes, then rinse with deionized water, and finally rinse with deionized water and ethanol, and blow dry for later use.

[0068] ③ Preparation of microneedle array coating:

[0069] 1) Gold / platinum sputtering was performed using a Cressington 108 sputtering apparatus with the following sputtering parameters: time 60 s, vacuum 0.04 MPa, current 10 mA. A gold-sputtered stainless steel microneedle array was used as the unmodified working electrode, and a platinum-sputtered stainless steel microneedle array was used as the counter electrode (CE).

[0070] 2) Prepare 0.2 mol / L silver nitrate solution, adjust the pH to 4-6 with dilute nitric acid or sodium hydroxide, and test at 3 mA / cm 2 Ag was deposited on the stainless steel microneedle array at a current density of 10 min. The stainless steel microneedle array after Ag deposition was immersed in a saturated NaCl solution at room temperature, and a voltage of +0.3 V was applied for 20 min to promote the conversion of Ag to AgCl, forming an Ag / AgCl reference electrode (RE).

[0071] ④ Preparation of working electrode (WE):

[0072] 1) Preparation of gold nanoparticles: Using the sodium citrate reduction method, a 1 mM (w / v) aqueous solution of chloroauric acid (HAuCl4) was used as the gold source. 100 mL of 1 mM HAuCl4 was heated to boiling, and 33 mL of a pre-prepared 38.8 mM sodium citrate solution was quickly added. Heating was continued for 10 minutes until the solution turned deep red, and the solution was allowed to cool naturally.

[0073] 2) Glucose enzyme modification: The initial working electrode was first polished with 1.0, 0.3 and 0.05 μm alumina slurries and then thoroughly rinsed with double distilled water; sonicated in a 1:1 mixture of nitric acid, acetone and double distilled water and dried in a highly purified N2 flow; the clean initial working electrode was immersed in an aqueous solution of cysteamine (20 mM) for 2 hours in the dark; 20 μM glucose oxidase (GOD) solution (5 mL, in 0.1 M PBS, pH 6.8) was reacted with 30 mg of sodium metaiodate at 4°C in the dark for 1 hour, and the reaction was stopped with 25 mM ethylene glycol at room temperature for 30 minutes, so that the carbohydrate groups on the peripheral surface of the glucose oxidase molecules were oxidized to carbon aldehydes together with periodate; the initial working electrode modified with amino groups was immersed in IO4-oxidized GOD solution for 1 hour to form a Schiff base between the -CHO group of GOD and the -NH2 group of cysteamine; the initial working electrode was again placed in the cysteamine solution for 1 hour to introduce -SH groups on the periphery of the electrode. Finally, the gold nanoparticles were covalently attached to the -SH groups by immersing the initial working electrode modified with thiol groups in the above-mentioned gold nanoparticle solution for 1 hour. The above steps were repeated 5 times to form a six-layer GOD / GNPs multilayer film, which was then washed with deionized water and set aside for use.

[0074] ⑤Assembly test:

[0075] 1) Assembly: A resin substrate was used as the substrate, and holes were drilled into the substrate using laser drilling technology to obtain a prefabricated substrate. WE, CE, and RE were embedded into the substrate, respectively. The microneedles were allowed to pass through the substrate and protrude 1 mm, forming a three-electrode system with a microneedle array electrode. The WE, CE, and RE were connected to silver wires, and the other ends of the silver wires were connected to the interfaces of the electrochemical workstation.

[0076] 2) Testing:

[0077] Test system: Hydrogels were prepared to mimic skin; glucose solutions of varying concentrations (0.5, 1.5, 5, 10, 15, 25, and 30 mM) were prepared using artificial interstitial fluid at pH 7.0.

[0078] Determination of the detection range of glucose concentration: Connect the microneedle array electrode system to the electrochemical workstation, turn on the electrochemical workstation, and add equal amounts of 0.5mM, 1.5mM, 5mM, 10mM, 15mM, 25mM, and 30mM glucose solutions into the hydrogel every 80s after the current stabilizes. 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, and determine the detection range of glucose concentration, such as Figure 2 The results showed that the formula for obtaining the standard curve of glucose concentration and response current was: I = 1.7339C + 1.5927, R 2 =0.9896, where I is the response current (μA) and C is the glucose concentration (mM). The detection range of the array microneedle system obtained by the present invention can be 0.5-30 mM.

[0079] Sensor stability test: Connect the microneedle array electrode system 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. Continuous monitoring for 20 days to obtain stability monitoring results, such as Figure 3 ,The results show that after 20 days of continuous monitoring, ,the sensor performance remains basically stable.

[0080] This invention utilizes third-generation sensing technology to achieve direct electron transfer between glucose oxidase and electrodes. Combined with the high biocompatibility and stable coating process of a 316L stainless steel microneedle array, this significantly broadens the detection range (0.5-30mM) and extends the continuous monitoring period to 20 days. Furthermore, by covalently modifying the six-layer GOD / GNPs composite membrane, enzyme shedding is effectively prevented, improving sensor stability and addressing potential interference and enzyme inactivation issues encountered in existing technologies. This device is suitable for the long-term, accurate blood glucose monitoring needs of diabetic patients.

[0081] It should be noted that the above description is limited to some embodiments of the present invention. Other embodiments are within the scope of the appended claims. In some cases, the actions or steps recited in the claims may be performed in an order different from that described in the above embodiments and still achieve the desired results. Furthermore, the processes depicted in the accompanying drawings do not necessarily require the specific order or sequential order shown to achieve the desired results. In certain embodiments, multitasking and parallel processing are also possible or may be advantageous.

[0082] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0083] While specific details have been set forth to describe exemplary embodiments of the present invention, it will be apparent to those skilled in the art that embodiments of the present invention may be practiced without or with variations in these specific details. Therefore, these descriptions should be considered illustrative rather than restrictive. Although the present invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0084] The embodiments of the present invention are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present invention should be included in the scope of protection of the present invention.

Claims

1. A microneedle sensor for continuous monitoring of glucose in interstitial fluid, characterized in that: include: Substrate materials and microneedle array electrode systems; The microneedle array electrode system is embedded in the substrate material and is 1 mm higher than the substrate material. The microneedle array electrode system is made of stainless steel and is pyramid-shaped and includes: a working electrode, a counter electrode, and a reference electrode.

2. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 1, characterized in that: The microneedle array electrode system is prepared by the following method: Three pyramidal microneedle arrays with a height of 1.8-2 mm, a base of 800-1000 μm, and a microneedle spacing of 0.8-1 mm were prepared from a stainless steel sheet using a wire-cut cutting machine. The surface of the microneedle array is subjected to degreasing, acid washing and activation treatment in sequence to obtain a treated microneedle array; Platinum is coated on any of the treated microneedle arrays by magnetron sputtering to obtain a counter electrode; Gold is plated on another treated microneedle array by magnetron sputtering to obtain an initial working electrode; glucose oxidase and gold nanoparticles are immobilized on the surface of the initial working electrode by covalent modification to obtain a working electrode; A reference electrode was prepared by electrodeposition for the remaining treated microneedle array.

3. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 2, characterized in that: The method of processing a stainless steel sheet by a wire cutting machine to prepare three pyramid-shaped microneedle arrays with a height of 1.8-2 mm, a base of 800-1000 μm, and a microneedle spacing of 0.8-1 mm comprises: The processing parameters include a copper wire electrode diameter of 0.15-0.3 mm, a voltage of 35-50 V, a cutting speed of 6.3-10 mm / min, an electrode gap of 0.08-0.2 mm, and the stainless steel sheet is immersed in pure water during the processing.

4. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 2, characterized in that: The surface of the microneedle array is subjected to degreasing, pickling and activation treatments in sequence to obtain a treated microneedle array, comprising: The microneedle array is ultrasonically cleaned with acetone or ethanol for 10-20 minutes to obtain a degreased array; Soaking the ultrasonically treated array in a 10% HCl solution for 10-20 minutes and cleaning it to obtain an acid-washed array; The acid-washed array was immersed in a 10% H2SO4 solution for 1-2 minutes, cleaned, and dried to obtain a treated microneedle array.

5. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 2, characterized in that: The preparation parameters of the platinum coating and the gold coating are: magnetron sputtering time 60-120 seconds, vacuum degree 0.04-0.06 MPa, and current 10-15 mA.

6. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 2, characterized in that: The fixing of glucose oxidase and gold nanoparticles on the surface of the initial working electrode by a covalent modification method to obtain the working electrode comprises: The surface of the initial working electrode is modified by cysteamine, the carbohydrate groups of GOD are oxidized by sodium periodate to form Schiff base bonding, and the adsorption of gold nanoparticles is repeated for more than five times to form a multilayer film to obtain a working electrode.

7. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 6, characterized in that: The gold nanoparticles are prepared by reducing chloroauric acid with sodium citrate, and the solution is boiled and reacted until the color changes to deep red.

8. The microneedle sensor for continuous monitoring of glucose in interstitial fluid according to claim 2, characterized in that: The step of preparing a reference electrode by electrodeposition on the remaining processed microneedle array comprises: The remaining treated microneedle array is electroplated with a silver nitrate solution and then converted with a saturated NaCl solution to obtain a reference electrode.

9. The method for preparing a microneedle sensor for continuous monitoring of glucose in interstitial fluid according to any one of claims 1 to 8, wherein: The preparation method comprises: A pyramid-shaped microneedle array was prepared by processing a stainless steel sheet using a wire-cut cutting machine. The surface of the microneedle array is subjected to degreasing, acid washing and activation treatment in sequence to obtain a treated microneedle array; Platinum is plated on any one of the treated microneedle arrays by magnetron sputtering to obtain a counter electrode; gold is plated on another of the treated microneedle arrays by magnetron sputtering to obtain an initial working electrode; glucose oxidase and gold nanoparticles are immobilized on the surface of the initial working electrode by covalent modification to obtain a working electrode; a reference electrode is prepared on the remaining treated microneedle array by electrodeposition; the counter electrode, the working electrode, and the reference electrode constitute the microneedle array electrode system; The microneedle array electrode system is embedded in a prefabricated substrate material to obtain a microneedle sensor.

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