A glucose microneedle sensing electrode and a preparation method and application thereof

Abstract

The application provides a glucose microneedle sensing electrode and a preparation method and application thereof, and belongs to the technical field of nanomaterial preparation and electrochemical sensing. The glucose microneedle sensing electrode comprises a conductive microneedle base and a Prussian blue layer, a self-assembled monolayer, a glucose oxidase layer and a protective layer arranged on the surface of the conductive microneedle base. The Prussian blue layer serves as an electron transfer mediator, the self-assembled monolayer is used for assisting the fixation of the glucose oxidase, and the glucose oxidase layer is used for catalyzing the glucose reaction. The Prussian blue layer, the self-assembled monolayer and the glucose oxidase immobilization layer are sequentially constructed on the surface of the acupuncture needle, and the glutaraldehyde cross-linking and the Nafion protection are combined, so that the stable fixation and signal transmission of the enzyme are realized. The obtained electrode is used for the real-time detection of glucose in interstitial fluid, and has good stability and application potential.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation and electrochemical sensing technology, specifically relating to a glucose microneedle sensing electrode, its preparation method, and its application. Background Technology

[0002] Direct detection of subcutaneous interstitial fluid offers a pathway for measuring biomarkers such as glucose in relation to blood levels. However, existing systems typically rely on invasive electrodes of millimeter length, which can cause pain, infection, or tissue inflammation, and also lack long-term stability. Microneedle technology, as a minimally invasive platform, is small enough to penetrate the stratum corneum and directly contact the interstitial fluid, making it a promising candidate for wearable chemisensing.

[0003] However, existing glucose microneedle sensors have the following problems: Firstly, there is the issue of enzyme immobilization and stability. Glucose oxidase (GOx) is prone to diffusion and leakage in aqueous solutions. Physically adsorbed GOx can gradually detach from the electrode surface in complex and dynamic physiological environments due to mechanical erosion and protein competitive adsorption, leading to a continuous decrease in sensor sensitivity over time and a limited lifespan. Furthermore, GOx is susceptible to conformational changes in high-concentration cross-linking agents or organic solvents, resulting in loss of enzyme activity. Maintaining high catalytic activity of GOx while achieving stable interfacial immobilization is the core issue limiting the long-term stability of the sensor.

[0004] Secondly, there is the issue of electron transfer efficiency. The active site of GOx is deeply embedded within the three-dimensional structure of the protein, making direct electron transfer between it and the electrode surface extremely difficult. Typically, an electron transfer mediator is needed to achieve a cascade of electron transfer from the electrode to the mediator to the enzyme's active site in order to obtain a measurable current signal. The choice of mediator and the design of its interface configuration with the enzyme and electrode directly determine the sensor's sensitivity and linear range.

[0005] Third, there is the issue of eliminating interfering substances. Electroactive substances coexisting in the interstitial fluid have certain electrochemical activity near the working potential of Prussian blue. It is necessary to effectively shield the interference signals through reasonable interface design to ensure the selectivity of glucose detection.

[0006] In summary, how to construct a stable glucose oxidase immobilization interface on the surface of microneedles and combine it with wearable wireless monitoring systems is a technical problem that needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a glucose microneedle sensing electrode, its preparation method, and its application, and to improve upon the problems of insufficient enzyme immobilization stability, limited linear detection range, and poor long-term monitoring signal stability in existing glucose microneedle sensors.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, the present invention provides a glucose sensing interface, which comprises, from the inside out, a Prussian blue layer, a self-assembled monolayer, and a glucose oxidase immobilization layer. The Prussian blue layer serves as an electron transport mediator. The self-assembled monolayer is disposed on the surface of the Prussian blue layer. The glucose oxidase immobilization layer is disposed on the surface of the self-assembled monolayer.

[0009] Self-assembled monolayers are formed by the combination of mercaptocarboxylic acids and mercaptoalcohols.

[0010] Preferably, the mercaptocarboxylic acid is mercaptoundecanoic acid.

[0011] Preferably, the mercapto alcohol is mercaptoethanol.

[0012] More preferably, the self-assembled monolayer is formed by mercaptoundecanoic acid and mercaptoethanol.

[0013] Preferably, the glucose oxidase immobilization layer includes glucose oxidase and film-forming aids.

[0014] Preferably, the film-forming aid material includes chitosan.

[0015] Preferably, the glucose oxidase immobilization layer is cross-linked with a cross-linking agent.

[0016] Preferably, the crosslinking agent is glutaraldehyde.

[0017] Preferably, the glucose sensing interface further includes a protective layer.

[0018] More preferably, the protective layer is a Nafion film.

[0019] On the other hand, the present invention provides an electrode.

[0020] The electrode includes a conductive microneedle substrate and the aforementioned glucose sensing interface covering the conductive microneedle substrate.

[0021] In another aspect, the present invention provides a method for preparing the above-mentioned electrode, the preparation of which includes the following steps: S1. A Prussian blue layer is formed on the surface of a conductive microneedle substrate to obtain Prussian blue modified microneedles. S2. A self-assembled monolayer is formed on the surface of the Prussian blue-modified microneedles to obtain self-assembled monolayer-modified microneedles. S3, activates the carboxyl groups in the self-assembled monolayer; S4. Immobilize glucose oxidase on the surface of a self-assembled monolayer after carboxyl activation to obtain glucose oxidase-modified microneedles. S5. Cross-linking treatment of glucose oxidase-modified microneedles; S6. A protective layer is formed on the surface of the cross-linked microneedles to obtain a glucose-sensing microneedle electrode.

[0022] Preferably, in S1, the conductive microneedle substrate is an acupuncture needle.

[0023] Preferably, in S1, a Prussian blue layer is formed on the surface of the acupuncture needle by electrodeposition.

[0024] Preferably, the electrodeposition method uses an electrodeposition solution containing ferric salts, ferricyanide, potassium chloride, and hydrochloric acid.

[0025] Preferably, the ferric salt is ferric chloride.

[0026] Preferably, the ferricyanide is potassium ferricyanide.

[0027] Preferably, electrodeposition is performed under constant potential conditions.

[0028] Preferably, the electrodeposition solution contains 2.5 mM ferric chloride, 100 mM potassium chloride, 2.5 mM potassium ferricyanide, and 100 mM hydrochloric acid.

[0029] More preferably, the constant potential is 0.4V relative to the Ag / AgCl reference electrode.

[0030] More preferably, the electrodeposition time is 200 s.

[0031] Preferably, in step S2, the Prussian blue-modified microneedles are immersed in an ethanol solution containing mercaptoundecanoic acid and mercaptool, so that a self-assembled monolayer is formed on their surface.

[0032] More preferably, the concentration of mercaptoundecanoic acid is 0.5 mM and the concentration of mercaptoethanol is 1 mM.

[0033] Preferably, in S3, sodium salts of ethyl dimethylaminopropyl carbodiimide and N-hydroxysulfosuccinimide are used to activate the carboxyl groups in the self-assembled monolayer.

[0034] Preferably, the carboxyl activation is carried out in a 2-(N-morpholino)ethanesulfonic acid buffer solution.

[0035] Preferably, glucose oxidase in S4 is immobilized in the form of a glucose oxidase-chitosan mixed solution.

[0036] More preferably, the chitosan solution concentration is 1 wt%.

[0037] More preferably, the concentration of the glucose oxidase solution is 20 mg / mL.

[0038] More preferably, the volume ratio of glucose oxidase solution to chitosan solution is 1:2.

[0039] Preferably, in S5, the crosslinking treatment is carried out using a glutaraldehyde solution.

[0040] Preferably, in S6, the protective layer is formed by immersing the cross-linked microneedles in a Nafion solution.

[0041] The present invention also provides a glucose detection device, comprising the above-mentioned electrode having a glucose sensing interface, a reference electrode, and a counter electrode.

[0042] Preferably, the glucose detection device further includes a housing, a circuit board, and a wireless communication module.

[0043] Preferably, the shell is a polytetrafluoroethylene shell.

[0044] Preferably, the circuit board is electrically connected to an electrode, a reference electrode, and a counter electrode having a glucose sensing interface, for acquiring glucose detection signals.

[0045] Preferably, the wireless communication module is used to transmit the glucose detection signal to the mobile terminal.

[0046] Preferably, the wireless communication module is a Bluetooth communication module.

[0047] The present invention also provides the application of the above-mentioned glucose sensing interface, electrode or glucose detection device in glucose detection.

[0048] The technical solution of this invention solves the key problems of the prior art through the following technical means: Regarding enzyme immobilization and stability, this invention introduces a two-component SAM strategy of mercaptoundecanoic acid / mercaptoethanol: mercaptoundecanoic acid provides a terminal carboxyl functional group, which, upon activation, forms an amide covalent bond with the amino group of lysine residues on the surface of glucose oxidase, achieving directional covalent anchoring of glucose oxidase; mercaptoethanol acts as a diluent to regulate the functional group density of the SAM layer, while simultaneously blocking the non-specific adsorption of proteins at exposed electrode sites, ensuring the bioactive conformation of glucose oxidase. Furthermore, glutaraldehyde crosslinking further forms a crosslink network between adjacent glucose oxidase molecules, significantly enhancing the mechanical integrity of the enzyme layer, effectively inhibiting enzyme leakage and inactivation, thereby greatly extending the sensor's lifespan.

[0049] Regarding electron transfer efficiency, this invention employs electrodeposition to grow a uniform and dense Prussian blue film in situ on the surface of acupuncture needles. Prussian blue has excellent electrocatalytic activity and can efficiently catalyze the reduction of H2O produced by glucose oxidase reaction at low working potentials. This avoids the co-oxidation of interfering substances such as ascorbic acid and uric acid when detecting H2O2 at high potentials, thereby improving the selectivity of the sensor and reducing power consumption.

[0050] In terms of interference removal, the Nafion outer coating, as an anion exchange membrane, can effectively repel negatively charged interference, further improving the selectivity of glucose detection.

[0051] In the field of microneedle-scale interface engineering, electrodeposition, SAM self-assembly, and enzyme impregnation are all liquid-phase processes that are gentle to operate and suitable for the micron-scale electrode of acupuncture needles. They can achieve uniform and repeatable functional interface construction in a limited area.

[0052] Compared with the prior art, the present invention has the following beneficial effects: 1. In this invention, Prussian blue is electrodeposited on the surface of acupuncture needles. Prussian blue, as an electron transport mediator, is beneficial for constructing an electrochemical detection interface for glucose.

[0053] 2. This invention utilizes mercaptoundecanoic acid and mercaptoethanol to form a self-assembled monolayer, which is then activated to immobilize glucose oxidase. This self-assembled monolayer-assisted immobilization strategy enables the formation of a more regular and dense enzyme layer, thereby improving the interfacial robustness of the glucose working electrode.

[0054] 3. The present invention uses glucose oxidase mixed with chitosan to form an enzyme layer, which is then cross-linked with glutaraldehyde and treated with Nafion, which helps to improve the immobilization stability of glucose oxidase on the surface of the microneedle electrode.

[0055] 4. Compared with electrodes not modified with self-assembled monolayers, the glucose working electrode of the present invention containing self-assembled monolayers can extend the linear response range and improve operational stability. Electrochemical tests in the original disclosure showed that although the enzyme electrode without self-assembled monolayers had a slightly higher initial current response, it had a narrower linear detection range and poorer operational stability; the electrode containing self-assembled monolayers can form a more regular and dense enzyme layer, enhancing enzyme retention and interfacial robustness.

[0056] 5. This invention uses acupuncture needles as the substrate for microneedle electrodes and integrates them with a reference electrode, a counter electrode, a polytetrafluoroethylene shell, a wireless Bluetooth circuit board, and a mobile data acquisition system, enabling real-time online monitoring of interstitial fluid glucose.

[0057] 6. In the human wear verification, the present invention can transmit glucose data in interstitial fluid to the user interface in real time via Bluetooth, and compare and verify it with commercial finger-prick blood testing equipment, proving that it has the potential for wearable glucose monitoring applications. Attached Figure Description

[0058] Figure 1 In the image, (a) is a SEM image of the GOx / SAM / PB / AN electrode in Example 1, and (b) is a SEM image of the GOx / PB / AN electrode. Figure 2 In Example 1 of this invention, (a) is the chronoamperometry curve of the GOx / SAM / PB / AN electrode with glucose continuously added to 0.1M PBS solution, and (b) is the linear curve of its current versus concentration. Figure 3 In Example 1 of this invention, (a) is the chronoamperometry curve of the GOx / PB / AN electrode in 0.1M PBS solution with glucose added continuously, and (b) is the linear curve of its current versus concentration. Figure 4 In Example 2 of this invention, (a) represents the relative current response of the electrode containing the SAM layer in 0.1M PBS containing 2mM, and (b) represents the relative current response of the electrode without the SAM layer in 0.1M PBS containing 2mM. Figure 5 In Example 3 of the present invention, (a) is the relative current response value of the electrode containing the SAM layer after one month of testing in 0.1M PBS containing 2 mM, and (b) is the relative current response value of the electrode without the SAM layer after one month of testing in 0.1M PBS containing 2 mM. Figure 6 This is the current response diagram of the GOx / SAM / PB / AN microelectrode in Test Example 4 of the present invention during the anti-interference experiment when detecting glucose; Figure 7 This is a schematic diagram of the wearable sensor in Embodiment 3 of the present invention, wherein (a) is a photograph of the actual sensor and (b) is a photograph of the front of the electrode. Figure 8 The wearable sensor in Test Example 5 was used to monitor the glucose concentration curve in the interstitial fluid in real time. Detailed Implementation

[0059] To enable those skilled in the art to better understand the present application, the technical solutions in specific embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by those skilled in the art.

[0060] Example 1 This embodiment provides a method for preparing an electrode with a glucose sensing interface.

[0061] The specific steps are as follows: Conductive acupuncture needles were selected as the microneedle substrate. Before use, the acupuncture needles were cleaned to remove surface oil, particles, and other impurities. Cleaning methods may include ultrasonic cleaning with ethanol and deionized water in sequence, or other metal microelectrode cleaning methods commonly used in the field.

[0062] After cleaning, the acupuncture needles are dried in a nitrogen stream or air for later use. The pretreated acupuncture needles possess conductivity and mechanical strength, making them suitable as a substrate for subsequent electrodeposition of Prussian blue and the construction of glucose-sensing interfaces.

[0063] Prussian blue was electrodeposited on the surface of acupuncture needle electrodes using a potentiostatic method in a freshly prepared solution containing 2.5 mM FeCl3, 100 mM KCl, 2.5 mM K3[Fe(CN)6], and 100 mM HCl. In the potentiostatic method, the potential was 0.4 V relative to the Ag / AgCl reference electrode, and the electrodeposition time was 200 s. The deposited Prussian blue layer served as an electron transport mediator, resulting in the PB / AN electrode.

[0064] The PB / AN electrode was immersed overnight in ethanol containing 0.5 mM mercaptoundecanoic acid (MUA) and 1 mM mercaptoethanol (MCH) to form a self-assembled monolayer. Afterward, it was rinsed with ethanol, then with deionized water, and dried in an airflow to obtain the SAM / PB / AN electrode.

[0065] The electrode was placed in a humid chamber at room temperature and incubated for 35 min with 10 μL of 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing 0.4 M ethyl dimethylaminopropyl carbodiimide (EDC) and 0.1 M N-hydroxysulfosuccinimide sodium salt (sulfo-NHS). The pH of the MES buffer was 5.0.

[0066] A 1 wt% chitosan solution was prepared by dissolving chitosan in a 2% acetic acid solution and stirring for 1 hour. A glucose oxidase solution was then mixed with the chitosan solution at a volume ratio of 1:2, wherein the glucose oxidase solution concentration was 20 mg / mL.

[0067] The aforementioned electrode was immersed in the enzyme solution for 5 minutes and then dried at 4°C. Subsequently, it was immersed in a 50% glutaraldehyde solution for 1 minute for cross-linking. Finally, it was immersed in a 0.05% Nafion solution for 30 seconds (Nafion is a perfluorosulfonic acid ion exchange resin membrane developed and commercialized by DuPont), and then dried at 4°C to obtain the GOx / SAM / PB / AN glucose working electrode, the SEM image of which is shown below. Figure 1 As shown in (a) in the figure.

[0068] In this context, GOx represents glucose oxidase, SAM represents self-assembled monolayer, PB represents Prussian blue, and AN represents acupuncture needle.

[0069] As shown in the figure, the electrode surface modified with GOx is clearly covered with a film, which is the enzyme, indicating that the enzyme has been successfully immobilized on the electrode surface. In contrast, the electrode surface without the modified SAM layer has a significantly smaller coverage, suggesting that modifying the SAM layer facilitates enzyme attachment and immobilization.

[0070] Comparative Example 1 This comparative example provides a glucose sensing control electrode without a self-assembled monolayer, for comparison with the glucose working electrode containing a self-assembled monolayer in Example 1.

[0071] First, a Prussian blue layer was electrodeposited on the surface of the acupuncture needle according to the method in Example 1 to obtain a PB / AN electrode.

[0072] Subsequently, without performing MUA and MCH self-assembly treatment, a glucose sensing control electrode without a self-assembled monolayer was prepared directly following the steps in Example 1: glucose oxidase immobilization, drying at 4°C, 50% glutaraldehyde cross-linking, and 0.05% Nafion treatment. Its SEM image is shown below. Figure 1 As shown in (b), this control electrode was used to evaluate the effect of self-assembled monolayers on glucose oxidase immobilization and electrode detection performance.

[0073] Test Example 1 The GOx / SAM / PB / AN electrode prepared in Example 1 was used as the working electrode. The reference electrode and the counter electrode together formed the electrochemical testing system, which was placed in a 0.1M PBS solution containing glucose for chronoamperometry testing. The current response signal was recorded by injecting different concentrations of glucose into the 0.1M PBS solution.

[0074] Meanwhile, the control electrode without a self-assembled monolayer prepared in Comparative Example 1 was tested under the same conditions.

[0075] like Figure 2 and Figure 3 As shown, the test results indicate that although the enzyme electrode without the SAM layer exhibits a slightly higher initial current response, its linear detection range is significantly narrower and its operational stability is poor. Specifically, the GOx / SAM / PB / AN electrode achieved a wide linear detection range of 1–100 mM, with correlation coefficients of 0.992 and 0.994 for the two linear intervals, respectively. In contrast, the GOx / PB / AN electrode has a linear detection range of only 1–20 mM and a correlation coefficient of only 0.858, indicating a poor linear response.

[0076] This difference may stem from the relatively disordered enzyme interface structure formed by direct immobilization, which easily leads to enzyme molecule leakage and gradual decline in activity. In contrast, the SAM-assisted immobilization strategy is beneficial for constructing more regular and dense enzyme layers. Although this interface structure may slow down electron transfer kinetics to some extent, it significantly improves enzyme retention and interface robustness, thereby broadening the linear response range and improving the sensor's operational stability.

[0077] Test Example 2 Enzyme electrodes with and without SAM layers were prepared in different batches using the same preparation method. They were then placed in 0.1M PBS solution containing 2mM glucose for chronoamperometry testing to evaluate the preparation repeatability and signal consistency between different electrodes.

[0078] like Figure 4 As shown in the results, the batch-to-batch reproducibility of the prepared enzyme electrodes was significantly improved after the introduction of the SAM layer, with the relative response generally remaining above 90%. In contrast, the electrodes without the modified SAM layer showed poor reproducibility, with the relative response of some electrodes even as low as 62%, indicating insufficient consistency in preparation and signal reproducibility.

[0079] These results demonstrate that the SAM layer can improve the consistency of enzyme immobilization and interface structure between different electrodes, thereby reducing batch-to-batch response differences and improving the reproducibility of electrode preparation.

[0080] Test Example 3 To evaluate the long-term stability of the electrodes, enzyme electrodes with and without SAM layers were prepared using the same preparation method, and chronoamperometry was performed in 0.1M PBS solution containing 2mM glucose. Current responses were recorded on days 1, 2, 5, 8, 10, 20, and 30 to compare the signal retention capabilities of the two electrodes during long-term detection.

[0081] like Figure 5 As shown, the test results indicate that the enzyme electrode loaded with the SAM layer maintained a relatively stable current response within one month, demonstrating its good long-term operational stability. In contrast, the electrode without the modified SAM layer showed a significant decrease in current response, with the lowest relative response value dropping to 58%, indicating poor interface stability and signal retention.

[0082] These results indicate that the introduction of the SAM layer can effectively reduce signal attenuation during long-term detection and improve the working stability of the enzyme electrode under continuous or long-term use conditions.

[0083] Test Example 4 This test example was used to evaluate the effect of the GOx enzyme layer on the anti-interference performance of the glucose-sensing microneedle electrode. The GOx / SAM / PB / AN electrode prepared in Example 1 was placed in 0.1M PBS solutions containing glucose and different interfering substances for electrochemical response testing, and its chronocurrent response was recorded.

[0084] like Figure 6 As shown, the test results demonstrate that the GOx / SAM / PB / AN electrode maintains a stable glucose response even in the presence of common interfering substances, exhibiting good anti-interference ability. This result indicates that the prepared electrode has high selectivity for glucose detection and possesses potential for glucose detection in complex sample environments.

[0085] The test results above demonstrate that the SAM-assisted immobilization strategy proposed in this invention plays a crucial role in improving the detection performance, repeatability, and long-term stability of the glucose sensing electrode. In this invention, the self-assembled monolayer is not merely a surface modification layer, but rather a key interface for achieving stable immobilization of glucose oxidase.

[0086] Specifically, the self-assembled monolayer constructed using MUA and MCH provides relatively ordered and stable carboxyl sites on the surface of Prussian blue-modified acupuncture needles. After activation with EDC / sulfo-NHS, these carboxyl sites can effectively couple with glucose oxidase, thereby promoting the stable immobilization of GOx on the electrode surface. Compared to electrodes without a SAM layer, electrodes containing a SAM layer can form a more regular, dense, and stable enzyme immobilization interface, reducing enzyme molecule desorption, leakage, and activity decay during preparation, soaking, and detection.

[0087] Therefore, the introduction of the SAM layer not only improves the consistency of enzyme loading and interface structure and enhances the repeatability between different electrodes, but also effectively reduces signal drift during long-term detection, thereby broadening the linear range of glucose detection and significantly improving the long-term working stability and practical application reliability of the electrode.

[0088] Example 2 This embodiment provides a method for preparing a reference electrode and a counter electrode. The reference electrode is prepared by electrodeposition and construction of a polymer film protective layer to create a silver / silver chloride-modified acupuncture needle electrode. Specifically, electrodeposition is carried out in a mixed solution of AgNO3, Na2S2O3, and NaHSO3 under the conditions of -0.01 mA for 300 to 600 s. Subsequently, the electrodeposited acupuncture needle is first immersed in FeCl3 solution, and then immersed in a mixed solution of PVB and NaCl for 10 to 60 s to obtain the reference electrode. The counter electrode is prepared by cleaning and modifying the acupuncture needle with silane, followed by impregnation to modify the acupuncture needle with hollow mesoporous carbon spheres to obtain the counter electrode. The electrode prepared in Example 1, the above-mentioned reference electrode, and the counter electrode can constitute an electrode system for glucose detection.

[0089] Example 3 This embodiment provides an application of the glucose working electrode prepared in Example 1, specifically a wearable glucose sensor.

[0090] like Figure 7 As shown, the glucose working electrode prepared in Example 1, the reference electrode prepared in Example 2, and the counter electrode are embedded in a polytetrafluoroethylene (PTFE) shell. A circuit board with Bluetooth functionality is connected to the tail end of the acupuncture needle inside the shell and then encapsulated. Data is collected in real time via a mobile phone, completing the construction of a wearable wireless monitoring system. The PTFE shell is fabricated through 3D modeling and 3D printing. The circuit board with Bluetooth data transmission capability is a custom-designed circuit board. This wearable glucose sensor can collect glucose detection signals through the electrode system and transmit the data to a mobile phone via Bluetooth, achieving real-time data acquisition.

[0091] Test Example 5 This test case validates the wearable glucose sensor in vivo. Healthy subjects aged 20 to 30 years were continuously monitored. After fasting overnight, a wearable sensor based on an acupuncture needle array was inserted into the dermis of the upper arm, transmitting glucose data from the interstitial fluid to the user interface in real time via Bluetooth. During monitoring, finger-prick blood samples were collected periodically and compared with a commercially available Sinocare finger-prick blood glucose meter for validation.

[0092] like Figure 8 As shown in the figure, the test results indicate that the wearable glucose sensor of the present invention can monitor the changes in glucose concentration in interstitial fluid in real time. The test results are basically consistent with those of commercial blood glucose meters, indicating that the sensor has the application potential for real-time monitoring of glucose in interstitial fluid.

[0093] Industrial Applicability: The present invention provides a clear method for preparing a wearable glucose microneedle sensor based on acupuncture needles. The acupuncture needles, Prussian blue electrodeposition system, self-assembled monolayer, glucose oxidase, chitosan, glutaraldehyde, and Nafion used can all be used to construct the glucose working electrode. This glucose working electrode can be integrated with a reference electrode, a counter electrode, a polytetrafluoroethylene shell, a wireless Bluetooth circuit board, and a mobile data acquisition system to form a wearable wireless monitoring system for real-time online monitoring of glucose content in interstitial fluid, demonstrating practical application value.

[0094] Finally, it should be noted that the described embodiments are merely some, not all, of the embodiments of the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents; that is, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A glucose microneedle sensing electrode, characterized in that, The device includes a conductive microneedle substrate and, from the inside out, a Prussian blue layer, a self-assembled monolayer, a glucose oxidase layer, and a protective layer disposed on the surface of the conductive microneedle substrate. The self-assembled monolayer is formed by mercaptoundecanoic acid and mercaptoethanol and disposed on the surface of the Prussian blue layer to assist in the immobilization of glucose oxidase. The glucose oxidase layer includes glucose oxidase and chitosan and is cross-linked with glutaraldehyde. The glucose oxidase layer is used to catalyze the glucose reaction. The protective layer is a Nafion layer.

2. A method for preparing the glucose microneedle sensing electrode according to claim 1, characterized in that, Includes the following steps: S1. A Prussian blue layer is electrodeposited on the surface of a conductive microneedle substrate to obtain Prussian blue-modified conductive microneedles. S2. Prussian blue-modified conductive microneedles are immersed in an ethanol solution containing mercaptoundecanoic acid and mercaptoethanol to form a self-assembled monolayer. The concentration of mercaptoundecanoic acid in the ethanol solution containing mercaptoundecanoic acid and mercaptoethanol is 0.5 mM and the concentration of mercaptoethanol is 1 mM. S3. Conductive microneedles modified with self-assembled monolayers were activated in a 2-(N-morpholino)ethanesulfonic acid buffer containing sodium 2-(N-morpholino)ethanesulfonic acid at a pH of 5.0 and a concentration of 0.4 M for ethyldimethylaminopropylcarbodiimide and 0.1 M for N-hydroxysulfonylsuccinimide. The activation time was 35 min. S4. Immerse the conductive microneedles obtained in S3 in glucose oxidase solution and dry them; S5. Immerse the conductive microneedles obtained in S4 in a glutaraldehyde solution for cross-linking. S6. After cleaning the conductive microneedles obtained in S5, immerse them in Nafion solution to form a protective layer, and then dry them to obtain the electrode.

3. The preparation method according to claim 2, characterized in that, The electrodeposition solution for electrodepositing the Prussian blue layer in S1 includes ferric chloride, potassium chloride, potassium ferricyanide, and hydrogen chloride.

4. The preparation method according to claim 2, characterized in that, The glucose oxidase solution in S4 was prepared by mixing 20 mg / mL glucose oxidase solution and 1 wt% chitosan solution at a volume ratio of 1:2, and the immersion time was 5 min.

5. The preparation method according to claim 2, characterized in that, In step S6, the conductive microneedles obtained in step S5 are immersed in a 0.05% Nafion solution for 30 seconds and then dried at 4°C.

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