Glucose biosensor based on ruthenium-based complex as well as preparation method and application of glucose biosensor

Abstract

The invention discloses a glucose biosensor based on ruthenium-based complexes and a preparation method and application thereof in the technical field of biological medicine, four ruthenium-based complexes are systematically screened through a molecular docking method, and the feasibility of the ruthenium-based complexes is verified through EDS and infrared spectroscopy. According to the glucose biosensor based on the ruthenium-based complex, the one-step method immobilized enzyme is constructed by taking the ruthenium-based complex [Ru (bpm) 3] [Cl] 2 as a redox medium and carrying out co-electric polymerization with GOx and PPy, low-potential measurement can be realized, interference of endogenous substances is reduced by applying the glucose biosensor based on the ruthenium-based complex, and the glucose biosensor based on the ruthenium-based complex has the advantages of high sensitivity, high sensitivity and the like. And the application potential can be reduced from 0.6 V to 0.3 V. And moreover, the electropolymerization polypyrrole and the ruthenium-based complex synchronously fix the enzyme, so that the immobilization stability of the enzyme is improved, the application potential is reduced, the service life of the working electrode is prolonged, and the sensitivity of the sensor is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a glucose biosensor based on ruthenium complexes, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a metabolic disease characterized by hyperglycemia caused by insulin secretion defects or impaired insulin action. In severe cases, it can lead to complications such as diabetic ketoacidosis, cardiovascular and cerebrovascular diseases, kidney disease, and eye diseases, seriously threatening people's lives and health. Diabetes is a lifelong disease with an increasing incidence rate. Strengthening the prevention, diagnosis, and screening of diabetes is of paramount importance. Effective detection and monitoring of glucose levels is an indispensable key step in diabetes treatment and has attracted widespread attention worldwide.

[0003] Electrochemical enzyme sensors, with their high sensitivity, good selectivity, fast response, and ease of miniaturization, are commonly used in commercial blood glucose meters. However, in practical use, they face several challenges: 1. Enzyme immobilization using physical embedding methods is prone to detachment; 2. When implanted in the body, they are susceptible to interference from endogenous substances and current signals; 3. The electrode surface, based on a two-dimensional structure, has a small specific surface area, resulting in insufficient enzyme loading and a risk of immobilization, leading to a short lifespan; 4. The redox active site (FAD) of glucose oxidase (GOx) is surrounded by a protein shell, hindering direct electron transfer between GOx and the electrode surface, preventing glucose detection at lower application potentials and significantly reducing interference from easily oxidized compounds in the real matrix; 5. Needle-implanted electrodes can easily damage tissues, causing inflammation and leading to patient aversion; 6. Although previous studies have demonstrated the use of some redox mediators as electron transfer mediators in glucose biosensors, these studies typically rely on small amounts of mediator materials to experimentally evaluate electron transfer parameters, lacking in-depth structure-activity relationship studies. Redox mediator materials are a core technology of CGM (Continuous Glucose Monitoring System), directly impacting the safety and reliability of the product. However, the range of redox mediator materials currently available for commercial CGM is very limited, highlighting the urgent need for the development of novel model-driven redox mediator materials. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned technical problems by providing a glucose biosensor based on ruthenium complexes, its preparation method, and its application.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] As a first aspect of the present invention, a glucose biosensor based on a ruthenium complex is provided, comprising a flexible double-sided needle-shaped electrode strip having a working electrode, a counter electrode and a reference electrode, wherein the surface of the working electrode is coated with a surface modification layer PPy-Ru-GOx by electropolymerization, and a mass transfer restriction membrane is coated on the surface modification layer; the materials used in the surface modification layer PPy-Ru-GOx include glucose oxidase, polypyrrole and the ruthenium complex [Ru(bpm)3][Cl]2.

[0007] As a further optimization of the present invention, the preparation method of the flexible double-sided needle-shaped electrode strip is as follows: first, carbon conductive ink is printed on one side of the PET substrate as an interconnect, then a layer of lead-doped carbon is printed and cured to obtain the working electrode, silver / silver chloride ink is printed and cured to obtain the reference electrode, and finally, carbon conductive ink is printed on the other side of the PET substrate and cured to obtain the counter electrode.

[0008] As a further optimization of the present invention, the mass transfer limiting membrane is obtained by spraying a polyurethane solution with a mass concentration of 1-5% onto the surface of the surface modification layer and drying it to form a film.

[0009] As a second aspect of the present invention, a method for preparing a glucose biosensor based on a ruthenium complex as described in any one of the above claims is also provided, comprising the following steps:

[0010] (1) Add glucose oxidase, polypyrrole and [Ru(bpm)3][Cl]2 to a 0.5M pH=7 PBS buffer solution containing 1.0M KCl. A constant potential is formed on the working electrode of the flexible double-sided needle electrode strip for one-step electropolymerization. The potential of the working electrode is maintained at +0.6V. After the polymerization is completed, the surface modification layer PPy-Ru-GOx is covered on the working electrode.

[0011] (2) Prepare a mass transfer limiting membrane solution, spray the mass transfer limiting membrane solution onto the surface of the surface modification layer, and dry it to form a film to obtain a glucose biosensor.

[0012] As a further optimization of the present invention, in step (1), the working electrode is first immersed in a 0.01M pH=7.4 PBS buffer solution and activated for 3 cycles at 100mV / S before electropolymerization.

[0013] As a further optimization of the present invention, in step (1), the electropolymerization conditions are: electropolymerization time of 550s, PPy concentration of 0.25M, GOx concentration of 6mg / ml and [Ru(bpm)3][Cl]2 concentration of 0.5mM.

[0014] As a third aspect of the present invention, an application of the ruthenium-based glucose biosensor as described above in the preparation of continuous blood glucose monitoring products is also provided.

[0015] As a further optimization of the present invention, the application potential of the glucose biosensor based on ruthenium complex is 0.3V.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention systematically screened four ruthenium-based complexes using a molecular docking method, and verified their feasibility using EDS and infrared spectroscopy. Based on the functional groups and electronic conductivity of conjugated polymers (CP), a ruthenium-based glucose biosensor was developed, employing the ruthenium-based complex [Ru(bpm)3][Cl]2 as a redox medium and co-polymerizing it with GOx and PPy to construct a one-step immobilized enzyme. This biosensor enables low-potential measurements, reducing interference from endogenous substances and lowering the application potential from 0.6V to 0.3V. Furthermore, the simultaneous electropolymerization of polypyrrole and the ruthenium-based complex to immobilize the enzyme improves enzyme immobilization stability, lowers the application potential, extends the lifespan of the working electrode, and enhances sensor sensitivity. Attached Figure Description

[0018] Figure 1 The present invention provides several ruthenium complexes for binding to glucose oxidase (GOx) according to Example 1; wherein, (A) is a schematic diagram of the ligand compounds (highlighted with colored bars): (a) Ru(bpy)3 2+ (b) [Ru(dtbbpy)3](PF6)2; (c) [Ru(bpm)3][Cl]2 bound to the active site of GOx (left), magnified view of key amino acid residues (right); (B) Ligand compound (a) Ru(bpy)3 2+ (b) [Ru(dtbbpy)3](PF6)2; (c) Molecular interaction diagrams of [Ru(bpm)3][Cl]2 with GOx. Note that the different colors and line styles in these diagrams represent different interaction types (defined by the legend in each panel).

[0019] Figure 2 (a)Ru(bpy)3 provided in Embodiment 2 of the present invention 2+ (b) The response of the electrode to the change in current difference (ΔI) under 5mM glucose solution for glucose biosensors prepared under different concentration gradients of [Ru(bpm)3][Cl]2;

[0020] Figure 3This is a schematic diagram of the fabrication process of the glucose biosensor provided in Embodiment 2 of the present invention;

[0021] Figure 4 This refers to the optimized electropolymerization time provided in Embodiment 2 of the present invention;

[0022] Figure 5 This is the result of the optimized concentration of glucose oxidase GOx provided in Example 2 of the present invention;

[0023] Figure 6 The optimized concentration of [Ru(bpm)3][Cl]2 provided in Example 2 of this invention;

[0024] Figure 7 The surface morphology of the unmodified working electrode (bare electrode) provided in Embodiment 2 of the present invention;

[0025] Figure 8 The surface morphology of the working electrode after electropolymerization of PPy provided in Embodiment 2 of the present invention;

[0026] Figure 9 The surface morphology of the working electrode after electropolymerization of PPy-GOx provided in Embodiment 2 of the present invention;

[0027] Figure 10 The surface morphology of the working electrode after electropolymerization of PPy-Ru-GOx provided in Embodiment 2 of the present invention;

[0028] Figure 11 The Fourier transform infrared spectroscopy results of the working electrode of electropolymerized PPy-GOx and electropolymerized PPy-Ru-GOx provided in Embodiment 2 of the present invention;

[0029] Figure 12 EDS spectrum of the unmodified working electrode (bare electrode) provided in Embodiment 2 of the present invention;

[0030] Figure 13 The EDS spectrum of electropolymerized PPy-GOx provided in Example 2 of this invention;

[0031] Figure 14 The EDS spectrum of the working electrode of the electropolymerized PPy-Ru-GOx provided in Embodiment 2 of the present invention;

[0032] Figure 15 The CV measurements for the unmodified working electrode (a), the electropolymerized PPy-GOx working electrode (b), and the electropolymerized PPy-Ru-GOx working electrode (c) range from -1V to 1V, with a scan rate of 100mV / s.

[0033] Figure 16The EIS plots show that the working electrode without any modification (a), the working electrode of electropolymerized PPy-GOx (b), and the working electrode of electropolymerized PPy-Ru-GOx (c) have an EIS measurement frequency range of 0.02Hz-100kHz and an amplitude of 5mV.

[0034] Figure 17 The glucose biosensor provided in Example 3 of this invention continuously detects glucose concentrations of 2-22 mM / L.

[0035] Figure 18 The standard curve of the glucose biosensor provided in Example 3 of the present invention at glucose concentration detection of 2-22 mM / L;

[0036] Figure 19 The glucose biosensor provided in Embodiment 3 of the present invention provides the current response in the presence of 0.15 mM ascorbic acid (AA), 0.5 mM uric acid (UA), and 0.5 mM glycine (Gly). Detailed Implementation

[0037] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0038] Unless otherwise specified, all experimental methods used in the following examples can be performed using conventional methods. Unless otherwise specified, all materials and reagents used can be obtained commercially.

[0039] Example 1: Screening of Ruthenium-based complexes

[0040] 1.1 Screening of Ruthenium-based Complexes Using Molecular Docking Methods

[0041] Four commercially available ruthenium-based complexes, namely tris(2,2'-bipyridine)ruthenium(II)Ru(bpy)3, were analyzed by molecular docking. 2+ A systematic screening was conducted on tris[4,4'-di-tert-butyl-(2,2')-bipyridine]ruthenium(III) dihexafluorophosphate [Ru(dtbbpy)3](PF6)2, tris(2,2'-bipyridine)ruthenium di(hexafluoroborate) salt [Ru(bpz)3][PF6]2, and tris(2,2'-bipyrimidine)ruthenium dihydrochloride [Ru(bpm)3][Cl]2.

[0042] Starting with the characteristic of redox mediators as electron transfer mediators for glucose biosensors, this study investigates the interaction between enzyme biomacromolecules and ruthenium-based ligand small molecules.

[0043] First, the 2D structures of small molecule ligands were obtained from the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ). These 2D structures were then input into Chem Office 20.0 software to create their 3D structures, which were saved as mol2 files. Next, the RCSB PDB database (http: / / www.rcsb.org / ) was used to screen for high-resolution crystal structures of protein targets as molecular docking acceptors. PyMOL 2.6.0 software was used to perform operations such as dehydration and dephosphaterization on the proteins, which were then saved as PDB files. Molecular Operating Environment 2019 software was used to minimize the energy of the compounds, pre-treat the target proteins, and locate active pockets. Finally, MOE 2019 was run for molecular docking, with 50 operations performed.

[0044] The binding activity of the two components was assessed based on their binding energies, and the results were visualized using PyMOL 2.6.0 and Discovery Studio 2019 software. The docking studies provided information on the type and number of interactions between the ruthenium ligand and the amino acid residues of glucose oxidase (hereinafter referred to as GOx).

[0045] Figure 1 The binding modes of several ruthenium-based complexes on GOx are shown in Table 1. Table 1 presents the binding results of several ruthenium-based complexes with GOx.

[0046] Table 1, GO X Binding results with several ruthenium complexes

[0047]

[0048] Note: [Ru(bpz)3][PF6]2 has no results.

[0049] The results showed that three groups of ruthenium-based complexes docked with GOx, with molecular docking energies ranging from -4.9017 to -6.8808 kcal / mol. However, the structural model of [Ru(bpz)3][PF6]2 showed intermolecular breakage, failing to complete normal molecular docking (blank cells indicate no results). When comparing binding energies calculated based on molecular docking, the binding energies of groups 1 and 4 appeared to be close, while group 2 showed a lower docking binding energy. Lower binding energies indicate potentially stronger binding activity between the small molecule active ingredient and the target. Furthermore, the number and strength of hydrogen bonds between the ligand and protein ultimately determine the binding affinity; smaller distances result in stronger hydrogen bond interactions, leading to higher stability of the ligand-protein complex.

[0050] 1.2. Electropolymerization of Ruthenium-based complexes with polypyrrole (hereinafter referred to as PPy-GOx)

[0051] Based on the molecular docking results, three ruthenium-based complexes Ru(bpy)3 that showed docking results with GOx were selected. 2+ Electropolymerization of [Ru(dtbbpy)3](PF6)2 and [Ru(bpm)3][Cl]2 with PPy-GOx.

[0052] The specific steps are as follows: In a 0.5M PBS buffer solution containing 1M KCl, add 6mg / ml of GOx and 0.25M of PPy. Then, add 1-5mM of different concentrations of ruthenium complexes to the working electrode of the glucose biosensor for one-step electropolymerization. During the polymerization process, the working electrode potential is maintained at +0.6V.

[0053] The experiment revealed that [Ru(dtbbpy)3](PF6)2 has poor solubility and forms a large amount of precipitate. This may be due to the hydrophobic effect of the tert-butyl groups in the three dtbbpy ligands of [Ru(dtbbpy)3](PF6)2. Therefore, the [Ru(dtbbpy)3](PF6)2 molecule was no longer considered in subsequent experiments.

[0054] Using the chronoamperometry method to study Ru(bpm)3 2+ The glucose biosensors prepared from [Ru(bpm)3][Cl]2 at different concentration gradients were electrochemically characterized. The response of the electrodes to changes in current difference (ΔI) was tested in 5 mM glucose solution. The effects of Ru(bpm)3 on the current were compared and evaluated. 2+ The suitability of [Ru(bpm)3][Cl]2 as a redox medium is shown in the results. Figure 2 As shown, 1 mM [Ru(bpm)3][Cl]2, after electropolymerization, results in higher electroactivity of the glucose biosensor.

[0055] The feasibility of [Ru(bpm)3][Cl]2 as a redox mediator for glucose oxidase was verified by molecular docking and electrochemical detection of current response, and the ruthenium complex [Ru(bpm)3][Cl]2 was determined to be the optimal redox medium for GOx.

[0056] Example 2: Fabrication of a glucose biosensor

[0057] The glucose biosensor was fabricated by sequentially activating the electrode surface, electropolymerizing, and spraying a polyurethane (PU) film onto a flexible double-sided needle-shaped electrode to slow diffusion and ensure sensitive response at low concentrations. The flexible double-sided needle-shaped electrode strip employs a typical three-electrode system. The three electrodes are asymmetrically distributed near the tip, with the reference electrode and working electrode located on the front side, while the counter electrode is located on the opposite side to reduce interference.

[0058] The specific preparation process flowchart is as follows: Figure 3 As shown, the preparation steps are as follows:

[0059] (1) Preparation of flexible double-sided needle-shaped electrodes: First, carbon conductive ink is printed on one side of the PET substrate as an interconnect. Then, a layer of lead-doped carbon is printed as the working electrode, and silver / silver chloride (Ag / AgCl) ink is printed as the reference electrode. Finally, carbon conductive ink is printed on the other side of the PET substrate as the counter electrode. Each layer is cured in an oven at 80°C for 10 min.

[0060] (2) The working electrode of the flexible double-sided needle electrode prepared in step (1) was first immersed in 0.01M PBS buffer solution with pH=7.4 and activated for 3 cycles at 100mV / S (electrode surface activation). Then, glucose oxidase GOx, polypyrrole PPy, and [Ru(bpm)3][Cl]2 were added to 0.5M PBS (pH=7) buffer containing 1.0M KCl to form a constant potential on the working electrode for one-step electropolymerization. During the polymerization process, the working electrode potential was maintained at +0.6V. After the polymerization was completed, the working electrode was rinsed with 0.1M phosphate buffer (pH=7.4) to remove loose and unattached substances, thus constructing a surface modification layer PPy-Ru-GOx on the working electrode.

[0061] (3) In order to maintain the stability and continuous monitoring of the sensor, after the working electrode with the surface modification layer PPy-Ru-GOx is dried, a layer of polyurethane solution with a mass concentration of 1-5% is sprayed on its surface to form an outer membrane, and a glucose biosensor is constructed. In this embodiment, a polyurethane solution with a mass concentration of 3% (using a solution of 98% tetrahydrofuran and 2% dimethylformamide as solvent) is preferred.

[0062] (4) The constructed glucose biosensor was dried and stored in PBS buffer solution (pH=7.4) at 4°C before use.

[0063] The electropolymerization time, glucose oxidase concentration, and [Ru(bpm)3][Cl]2 concentration were optimized using a single-variable control method. Details are as follows:

[0064] With PPy concentration fixed at 0.25 M, GOx concentration at 6 mg / ml, and [Ru(bpm)3][Cl]2 concentration at 1 mM, the electropolymerization time was adjusted to 500 s, 550 s, 600 s, 650 s, 700 s, and 750 s during the preparation of the glucose biosensor. The prepared glucose biosensor was electrochemically characterized using cyclic voltammetry (CV), and the results are as follows: Figure 4 As shown, the glucose biosensor exhibits optimal performance when the electropolymerization time is 550 s, at which point the response current to a 5 mM glucose solution is maximized. Therefore, the optimal electropolymerization time is 550 s.

[0065] With a fixed PPy concentration of 0.25 M, an electropolymerization time of 550 s, and a [Ru(bpm)3][Cl]2 concentration of 1 mM, the GOx concentration was adjusted to 2 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 9 mg / ml, 10 mg / ml, and 15 mg / ml during the preparation of the glucose biosensor. The prepared glucose biosensors were electrochemically characterized using cyclic voltammetry (CV). The results are as follows: Figure 5 As shown, the glucose oxidase concentration in the solution is 6 mg / ml, at which point the response current value to the 5 mM glucose solution is the largest. Therefore, the optimal glucose oxidase concentration is selected as 6 mg / ml.

[0066] With a fixed PPy concentration of 0.25 M, an electropolymerization time of 550 s, and a GOx concentration of 6 mg / ml, the [Ru(bpm)3][Cl]2 concentrations were adjusted to 0.1 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, and 2.5 mM. The resulting glucose biosensors were then electrochemically characterized using cyclic voltammetry (CV). The results are as follows: Figure 6 As shown, when the concentration of [Ru(bpm)3][Cl]2 increases from 0.1 mM to 5 mM, the maximum response current value for a 5 mM glucose solution is reached at a concentration of 0.5 mM. Therefore, the optimal concentration of [Ru(bpm)3][Cl]2 is selected as 0.5 mM.

[0067] In summary, the optimal electropolymerization conditions for preparing glucose biosensors are: electropolymerization time of 550 s, PPy concentration of 0.25 M, GOx concentration of 6 mg / ml, and [Ru(bpm)3][Cl]2 concentration of 0.5 mM. Under these conditions, the electrocatalytic activity of glucose biosensors can be improved while saving costs.

[0068] 2.2 Surface morphology characterization

[0069] Under the optimal electropolymerization conditions obtained in the above investigation, PPy, PPy-GOx and PPy-Ru-GOx were electropolymerized on the working electrode according to the electropolymerization method disclosed in Section 2.1.

[0070] The surface morphology of the unmodified working electrode (bare electrode) and the working electrodes after electropolymerization of PPy, PPy-GOx and PPy-Ru-GOx were characterized by scanning electron microscopy.

[0071] SEM images are as follows: Figure 7-10 As shown, Figure 7 The surface morphology of the unmodified working electrode (bare electrode) shows that the microstructure of the PPy film is typically cauliflower-shaped. Figure 8 ), PPy-GOx is a heterogeneous microsphere ( Figure 9 ), PPy-Ru-GOx has a denser sphere density ( Figure 10 The protrusion of the sphere increases the effective surface area, which is beneficial for rapid electron transfer.

[0072] 2.3 Fourier Transform Infrared Spectroscopy and Energy Dispersive X-ray Spectroscopy

[0073] Fourier transform infrared spectroscopy (FTIR) was performed on the working electrodes after electropolymerization of PPy-GOx and PPy-Ru-GOx, respectively. Energy dispersive X-ray spectroscopy (EDS) was performed on the unmodified working electrode and the working electrode after electropolymerization of PPy-Ru-GOx. The results are as follows: Figure 11-13 As shown.

[0074] like Figure 11 Fourier transform infrared spectroscopy of PPy-GOx shows that at 3407 cm⁻¹ -1 The distinct characteristic peak centered at cm⁻¹ describes the presence of OH stretching. The peak at 2972 ​​cm⁻¹ corresponds to CH stretching vibration. The peak at 1796 cm⁻¹ belongs to C=O stretching. The absorption peak at 1648 cm⁻¹ is close to the characteristic peak of the amide I band of GOx. Simultaneously, the C=C stretching vibration in PPy may also contribute to this region, with both superimposed to form this absorption peak. The absorption peak at 1550 cm⁻¹ corresponds to the characteristic peak of the amide II band of GOx (coupling of NH bending vibration and CN stretching vibration), and the peak at 1290 cm⁻¹... -1 The absorption peaks at these locations are related to the stretching vibrations of single bonds such as CO and CN. The absorption peaks at 1165 cm⁻¹, 1082 cm⁻¹, and 1040 cm⁻¹ originate from the out-of-plane bending vibrations of CH₄. (The remaining text appears to be unrelated and possibly machine-translated.) Figure 11 Fourier transform infrared spectroscopy of PPy-Ru-GOx methylene blue showed that at 2140 cm⁻¹ -1A more prominent peak appeared at 1639 cm⁻¹, which can be attributed to the stretching vibrations of bonds associated with the Ru-containing redox component, indicating the successful introduction of the Ru complex moiety. The vibrational peak of the C=C skeleton at 1639 cm⁻¹ also showed a change in position. The peaks at 1539 cm⁻¹ and 1295 cm⁻¹ were also related to the vibrations of CN and CO. Compared with PPy-GOx, the positions and intensities of these peaks changed, indicating that the presence of Ru altered the vibrational properties of these chemical bonds in the material.

[0075] like Figures 12-14 Compared to the unmodified working electrode and the electropolymerized PPy-GOx working electrode, the EDS spectrum of the working electrode after electropolymerization of PPy-Ru-GOx showed a clear Ru peak.

[0076] The above results further confirm that PPy-Ru-GOx was successfully electropolymerized on the surface of the working electrode.

[0077] 2.4 Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analysis

[0078] The CV measurement scan range is -1V to 1V, and the scan rate is 100mV / s. The CV measurement scan range of the unmodified working electrode (a), the electropolymerized PPy-GOx working electrode (b), and the electropolymerized PPy-Ru-GOx working electrode (c) is -1V to 1V, and the scan rate is 100mV / s.

[0079] The results are as follows Figure 15 As shown in curve a, the reversible redox peaks of the unmodified working electrode are as follows. The peak current of the working electrode modified with PPy-GOx (curve b) is significantly increased, which is due to the increased conductivity resulting from the electropolymerization of the conductive polymer PPy on the working electrode surface. The response current of the glucose biosensor modified with PPy-Ru-GOx (curve c) is significantly increased in the presence of a ruthenium-based redox medium. This is because the presence of the ruthenium-based redox medium promotes the improvement of electrode surface conductivity and electron transport rate. The CV results indicate that the glucose biosensor based on electropolymerized PPy-Ru-GOx has been successfully constructed.

[0080] EIS measurements are performed in the frequency range of 0.02Hz-100kHz with an amplitude of 5mV. In the EIS plot, the diameter of the semicircle represents the electron transfer resistance (Ret). Figure 16As shown, the unmodified EIS exhibits a relatively small semicircle (curve a). After electropolymerization of PPy-GOx, the impedance value decreases significantly (curve b), which is attributed to the good conductivity of PPy, reducing the electron transport resistance at the electrode surface. For PPy-Ru-GOx, the Ret value decreases even further, indicating that PPy-Ru-GOx promotes electron transfer at the electrode surface and improves charge transfer efficiency. The increased slope of the straight line confirms that the ruthenium-based complex successfully adheres to the working electrode surface material during the electropolymerization of PPy-Ru-GOx, thereby increasing the electron transfer and diffusion processes in the matrix. The EIS results are consistent with the CV results, further demonstrating the successful fabrication of the glucose biosensor based on electropolymerized PPy-Ru-GOx.

[0081] Example 3: Electrochemical Sensing Measurement of Glucose Concentration

[0082] The glucose concentration of the glucose biosensor prepared according to the method of Example 2 under optimal electropolymerization conditions was determined by electrochemical sensing using the current-time curve (it) method. Specifically, the selected application potential was a constant potential of 0.3 volts (V), such as... Figure 17 As shown, a step-current response image was obtained by linearly and gradually increasing the glucose concentration. Figure 18 As shown, the linear equation of the constructed glucose biosensor, obtained through simple linear fitting, is y = 0.13898x + 2.97299, with a linear correlation coefficient R0. 2 =0.9916, where y is the current response intensity in microamperes (μA); x is the glucose concentration in the electrolyte in millimoles per liter (mM). The sensitivity of the constructed glucose biosensor is 17.81 μA / mmol / cm² (µA·mM). -1 cm -2 The linear range reaches 2 mmol / L glucose concentration to 22 mmol / L glucose concentration (2 mM-22 mM).

[0083] Furthermore, the current response of the glucose biosensor to 0.15 mM ascorbic acid (AA), 0.5 mM uric acid (UA), and 0.5 mM glycine (Gly) was investigated, and the results are as follows: Figure 19 As shown, the small change in current caused by interference indicates that the sensor has satisfactory selectivity.

[0084] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A glucose biosensor based on a ruthenium-based complex comprising a flexible double-sided needle-shaped electrode strip having a working electrode, a counter electrode and a reference electrode, characterized in that, The working electrode surface is covered with a surface modification layer PPy-Ru-GOx by electro-polymerization, and a mass transfer limiting membrane is covered on the surface modification layer; the material used in the surface modification layer PPy-Ru-GOx comprises glucose oxidase, polypyrrole and a ruthenium-based complex [Ru(bpm)3][Cl]2.

2. The ruthenium-based complex-based glucose biosensor according to claim 1, characterized in that, The preparation method of the flexible double-sided needle-shaped electrode strip is as follows: first, carbon conductive ink is printed on one side of a PET substrate as an interconnection, then a layer of lead-doped carbon is printed to obtain a working electrode after curing, silver / silver chloride ink is printed to obtain a reference electrode after curing, and finally, carbon conductive ink is printed on the other side of the PET substrate to obtain a counter electrode after curing.

3. The ruthenium-based complex-based glucose biosensor according to claim 1, wherein The mass transfer limiting membrane is obtained by spraying a polyurethane solution with a mass concentration of 1-5% on the surface of the surface modification layer and drying to form a film.

4. A method for the preparation of a glucose biosensor based on ruthenium-based complexes according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: (1) adding glucose oxidase, polypyrrole and [Ru(bpm)3][Cl]2 into a 0.5M PBS buffer solution containing 1.0M KCl, forming a constant potential on the working electrode of the flexible double-sided needle-shaped electrode strip to perform one-step electro-polymerization, and keeping the potential of the working electrode at +0.6V, so that the surface modification layer PPy-Ru-GOx is formed on the working electrode after polymerization; (2) preparing a mass transfer limiting membrane solution, spraying the mass transfer limiting membrane solution on the surface of the surface modification layer, and drying to form a film to obtain a glucose biosensor.

5. The method for preparing a glucose biosensor based on a ruthenium-based complex according to claim 4, characterized by, In step (1), the working electrode is immersed in a 0.01M PBS buffer solution with pH=7.4 and activated at 100mV / S for 3 circles before electro-polymerization.

6. The method for preparing a glucose biosensor based on a ruthenium-based complex according to claim 4, characterized by, In step (1), the electro-polymerization conditions are as follows: the electro-polymerization time is 550s, the PPy concentration is 0.25M, the GOx concentration is 6mg / ml, and the [Ru(bpm)3][Cl]2 concentration is 0.5mM.

7. Use of the glucose biosensor based on a ruthenium-based complex as claimed in any one of claims 1-3 in the preparation of a blood glucose continuous monitoring product.

8. Use according to claim 7, characterized in that, The application potential of the glucose biosensor based on a ruthenium-based complex is 0.3V.

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