Preparation method and application system of microelectrode sensor based on polymer osmium ions

Abstract

The invention discloses a preparation method and an application system of a microelectrode sensor based on polymer osmium ions. The method comprises the following steps: constructing opposite sensor electrodes on the surface of each microelectrode of a microelectrode array; the sensor electrode comprises a working electrode and a counter electrode; the microelectrode sensor is obtained by modifying the sensor electrode with a carbon nanotube, a hydrogel osmium electron mediator and an enzyme composite material. According to the microelectrode sensor based on the polymer osmium ion complex electron mediator and the minimally invasive transdermal sensing technology based on the microneedle array, various subcutaneous biological indexes can be detected highly sensitively and safely, and the sensitivity and stability of blood glucose detection can be improved. The method can be widely applied to the technical field of biomedical engineering micro devices.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering micro-devices, and particularly to a preparation method and application system of a microelectrode sensor based on polymer osmium ions. Background Art

[0002] In recent years, with the continuous increase in the number of global diabetes patients, the demand for blood glucose detection has also been growing continuously. Electrochemical sensors have the advantages of high sensitivity, good selectivity, fast response, and simple operation, so they show very broad application prospects in the fields of biomedicine, environmental detection, food industry, etc. Currently, the mainstream technical means of blood glucose concentration detection methods is electrochemical enzyme electrode technology. Among them, implantable blood glucose sensing technology has received extensive attention because it can achieve continuous blood glucose monitoring and has the potential to be used in combination with insulin pumps. The first-generation continuous blood glucose monitoring technology uses natural oxygen as an electron mediator, but such sensors have poor responsiveness and are easily affected by the oxygen concentration in the environment and interfered by other electroactive substances. The second-generation continuous blood glucose monitoring technology strengthens the direct electron transfer between the enzyme and the electrode by introducing an electron mediator to replace oxygen to transfer electrons between the enzyme and the electrode, overcoming the problem of oxygen deficiency in the first-generation continuous blood glucose monitoring technology. However, the commonly used electron mediator easily diffuses out of the enzyme layer into the body tissue solution, resulting in poor stability of the sensor, thus limiting the use of the sensor in the body. Therefore, high-sensitive electron transfer has not been achieved in related technologies, resulting in low sensitivity and poor stability of blood glucose detection. Summary of the Invention

[0003] An object of the present invention is to solve at least to some extent one of the technical problems existing in the prior art.

[0004] To this end, an object of the present invention is to provide a preparation method and application system of a high-sensitivity microelectrode sensor based on polymer osmium ions.

[0005] To achieve the above technical object, on the one hand, an embodiment of the present invention provides a preparation method of a microelectrode sensor based on polymer osmium ions, including the following steps: constructing opposing sensor electrodes on the surface of each microelectrode of a microelectrode array; the sensor electrodes include a working electrode and a counter electrode; modifying the sensor electrodes with a carbon nanotube, a hydrogel osmium electron mediator, and an enzyme composite material to obtain a microelectrode sensor. The present application proposes a microelectrode sensor based on a polymer osmium ion complex electron mediator. Based on the minimally invasive transdermal sensing technology of a micro-needle array, it can achieve highly sensitive and safe detection of various subcutaneous biological indicators, which is beneficial to improving the sensitivity and stability of blood glucose detection.

[0006] In some embodiments, the method for preparing a microelectrode sensor based on polymer osmium ions of an embodiment of the present invention, wherein the sensor electrode is modified by carbon nanotubes, hydrogel osmium electron mediators and enzyme composite materials to obtain a microelectrode sensor, comprises:

[0007] According to the covalent coupling effect between -NH2 connected to the hydrogel osmium electron mediator side chain and glutaraldehyde, the materials are connected and blended, and are uniformly modified on the surface of the sensor electrode.

[0008] In some embodiments, in one embodiment of the present invention, the hydrogel osmium electron mediator material is synthesized by the following steps:

[0009] The vinyl pyrrolidone is polymerized to generate polyvinyl pyrrolidone;

[0010] The structure and cross-linking degree of the polyvinyl pyrrolidone are designed to obtain the hydrogel osmium electron mediator.

[0011] In some embodiments, in one embodiment of the present invention, the method further comprises:

[0012] Mixing a first concentration of hydrogel osmium electron mediator, an aqueous solution of single-walled carbon nanotubes, an aqueous solution of glucose oxidase, and a second concentration of glutaraldehyde solution to form a blended solution;

[0013] Adding the blended solution into a container, immersing the sensor electrode into the blended solution, and determining that the immersion length is less than or equal to the first length;

[0014] Raising the pulling machine to pull and dry the sensor electrode;

[0015] The unreacted substances on the sensor electrode are washed away to obtain the microelectrode sensor.

[0016] In some embodiments, in one embodiment of the present invention, the method further comprises:

[0017] Producing polyvinyl pyrrolidone, and adding cis-bis(dichloroosmium(II)) and poly(4-vinyl-pyridine) into ethylene glycol, respectively, and reflux heating treatment under nitrogen; introducing amino groups into the obtained polymer;

[0018] N,N-dimethylformamide was added to the mixture, and 2-bromoethylamine hydrobromide was added. After stirring, the solution was poured into rapidly stirred acetone to precipitate a crude polymer;

[0019] The hygroscopic precipitate is collected, dissolved in deionized water, and filtered; ammonium hexafluorophosphate solution is added to precipitate to form PF6-salt;

[0020] Dissolve the PF6- salt in acetonitrile to prepare a solution of the third concentration, dilute it with deionized water to the fourth concentration, and stir it on an anion exchange column. Filter the solution and evaporate it under vacuum;

[0021] Add concentrated hydrochloric acid, adjust the pH, and drop the solution into rapidly stirred acetonitrile to obtain a white powder of the reaction product, which is determined to be a hydrogel osmium electron mediator.

[0022] In some embodiments, in one embodiment of the present invention, the working electrode is prepared by the following steps:

[0023] Perform laser etching on the microneedles and carry out cutting treatment;

[0024] Soak, clean, and dry the cut microneedles with absolute ethanol;

[0025] Install a stainless steel flux prepared by mixing ZnO, NH4Cl, HCl, CH3COOH, H2O, and a surfactant in a preset volume ratio, and immerse the microneedles in the stainless steel flux for ultrasonic treatment;

[0026] Take out the microneedles, connect an electrochemical workstation, use the microneedles as the first working electrode, a gold sheet electrode as the first counter electrode, and a silver / silver chloride electrode as the first reference electrode;

[0027] Use a multi-step constant current method to electrochemically deposit gold on the microneedles and dry them naturally to obtain the working electrode.

[0028] In some embodiments, in one embodiment of the present invention, the counter electrode is prepared by the following steps:

[0029] Take the microneedles that have completed electrochemical deposition of gold, connect the electrochemical workstation, use the microneedles as the second working electrode, a platinum sheet electrode as the second counter electrode, and a silver / silver chloride electrode as the second reference electrode;

[0030] Immerse the second working electrode, the second counter electrode, and the second reference electrode in an aqueous solution of sodium platinous sulfite, and use a multi-step constant current method to electrochemically deposit platinum on the microneedles; dry them naturally to obtain the counter electrode.

[0031] In some embodiments, in one embodiment of the present invention, the reference electrode is prepared by the following steps:

[0032] Take the microneedles that have completed electrochemical deposition of gold, coat them with Ag / AgCl ink, and dry them;

[0033] Perform immersion coating with a methanol solution and air dry to obtain the reference electrode.

[0034] On the other hand, an evaluation method based on a microelectrode sensor is proposed in an embodiment of the present invention, including:

[0035] The microelectrode sensor is used under the subcutaneous skin of ex vivo skin. The skin tissue is sliced and stained, and the transdermal depth of the microelectrode sensor is determined by evaluating the performance of the skin.

[0036] On the other hand, an application system of a microelectrode sensor based on a polymer osmium ion is provided in an embodiment of the present invention. The microelectrode sensor prepared by the above preparation method is used as a working electrode to detect glucose in vivo or ex vivo.

[0037] The embodiments of the present application at least include the following beneficial effects: Opposite sensor electrodes are constructed on the surface of each microelectrode of the microelectrode array; the sensor electrodes include a working electrode and a counter electrode; the sensor electrodes are modified by a carbon nanotube, a hydrogel osmium electron mediator and an enzyme composite material to obtain a microelectrode sensor. The present application proposes a microelectrode sensor based on a polymer osmium ion complex electron mediator. Based on the minimally invasive transdermal sensing technology of a micro-needle array, it can achieve highly sensitive and safe detection of various subcutaneous biological indicators, which is beneficial to improving the sensitivity and stability of blood glucose detection. Description of the Drawings

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following introduces the accompanying drawings of the relevant technical solutions in the embodiments of the present invention or the prior art. It should be understood that the accompanying drawings below only conveniently and clearly show some embodiments of the technical solutions in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0039] Figure 1 It is a schematic diagram of an embodiment of the coating structure of a continuous blood glucose sensing electrode in the related art;

[0040] Figure 2 It is a schematic diagram of another embodiment of the coating structure of a continuous blood glucose sensing electrode in the related art;

[0041] Figure 3 It is a schematic flow diagram of an embodiment of the preparation method of the microelectrode sensor based on a polymer osmium ion provided by the present invention;

[0042] Figure 4 It is a schematic diagram of the principle of the three-generation electrochemical sensor provided by the present invention;

[0043] Figure 5 It is a schematic structural diagram of the molecular formula of the osmium polymer mediator NH2-PVP-Os provided by the present invention. Detailed Embodiments

[0044] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, where like or similar reference numerals denote like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention. For the step numbers in the following embodiments, they are only set for the convenience of elaboration and explanation, and no limitation is imposed on the order between the steps. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0045] In recent years, with the continuous increase in the number of global diabetes patients, the demand for blood glucose detection has also been growing continuously. Electrochemical sensors have the advantages of high sensitivity, good selectivity, fast response, simple operation, etc., and thus show very broad application prospects in the fields of biomedicine, environmental detection, food industry, etc. Currently, the mainstream technical means for blood glucose concentration detection methods is electrochemical enzyme electrode technology. Among them, implantable blood glucose sensing technology has received extensive attention because it can achieve continuous blood glucose monitoring and has the potential to be used in combination with insulin pumps. The first-generation continuous blood glucose monitoring technology uses natural oxygen as an electron mediator, but such sensors have poor responsiveness and are easily affected by the oxygen concentration in the environment and interfered by other electroactive substances. The second-generation continuous blood glucose monitoring technology strengthens the direct electron transfer between the enzyme and the electrode by introducing an electron mediator to replace oxygen to transfer electrons between the enzyme and the electrode, overcoming the problem of oxygen deficiency in the first-generation continuous blood glucose monitoring technology. However, the commonly used electron mediators are easily diffused out of the enzyme layer into the body tissue solution, resulting in poor stability of the sensor, thus limiting the use of the sensor in the body.

[0046] Clinically, the detection of glucose mainly relies on blood sampling for testing. Frequent, invasive, and painful blood sampling tests bring inconvenience to the daily life of patients, increase the risk of wound infection, and reduce the compliance of treatment. Moreover, the blood sampling detection method can only obtain biological indicators at a few time points, cannot continuously monitor, and is difficult to be deeply applied to track the blood glucose fluctuations of diabetes.

[0047] In recent years, significant progress has been made in dynamic glucose monitoring (CGM) technology based on implantable electrodes. Since there is a close exchange of substances between subcutaneous capillaries and interstitial fluid, CGM electrodes implanted subcutaneously in patients can frequently and continuously (every 5 - 30 minutes) detect the glucose value in subcutaneous tissue fluid, which is then converted into a blood glucose value. However, the implantable electrodes used in CGM systems are usually up to 1 cm long, which easily induces subcutaneous inflammatory reactions and tissue fibrosis, resulting in deviations in the detected accuracy. Moreover, the implantation of electrodes is accompanied by risks of pain, bleeding, and infection, and the long-term wearing comfort and compliance are insufficient. Currently, for in-situ detection of internal physiological parameters in the human body, non-invasive or minimally invasive physiological signal monitoring methods relying on new materials and cutting-edge technologies have become the focus of research worldwide. Various innovative non-invasive detection technologies have emerged, commonly including sweat detection, near-infrared spectroscopy detection, tear detection, saliva detection, and detection methods based on iontophoresis, etc. For example, in one embodiment, a new concept of "laboratory on the skin" was proposed, and a multi-mode skin sensing patch was fabricated by all-laser technology, combined with microfluidic and flexible circuit board technologies, to achieve in-situ accurate detection of various trace biological information in sweat and real-time metabolism management. However, there are significant differences in the concentrations of physiological indicators in body fluids such as sweat, tears, and saliva compared to those in subcutaneous tissue fluid and blood. Therefore, large deviations may occur when measuring the fluctuations of actual physiological parameters. As for non-invasive detection methods such as near-infrared spectroscopy and iontophoresis, since they have not yet reached the accuracy required for clinical detection, they have not been able to be applied to the field of clinical monitoring.

[0048] Referring Figure 1 and Figure 2 As shown, in the first-generation continuous glucose sensing technology, oxygen is used as an electron acceptor. The oxidized glucose oxidase GOx(FAD) oxidizes glucose to gluconolactone acid, while the reduced enzyme GOx(FADH2) reduces oxygen in the solution to hydrogen peroxide. The glucose concentration is indirectly measured by determining the change in the concentration of oxygen or hydrogen peroxide during the reaction process. However, the sensors in this stage are extremely susceptible to oxygen in the environment and have poor anti-interference ability. Especially for in-vivo sensing, due to the lack of oxygen in the body's tissue fluid, the ratio of glucose concentration to oxygen concentration is too high during electrochemical detection, resulting in the linear range of glucose sensing not meeting the application requirements. Using an external diffusion membrane to limit the ratio of glucose to oxygen concentration can, to a certain extent, improve the linear range of the sensing electrode, but it is likely to affect the sensitivity of the electrode, especially posing a great challenge to the detection sensitivity of the fine electrode area of microneedles. At the same time, the outer diffusion membrane material is also likely to affect the in-vivo stability of the microneedle electrode.

[0049] Therefore, the second-generation continuous blood glucose sensing technology emerged as the times require. A mediator layer for electron transfer was introduced between glucose oxidase and the electrode, replacing oxygen as the electron acceptor and overcoming the problem of interference. A mediator material that can quickly undergo oxidation-reduction reactions is used as an intermediate for electron transfer between the enzyme active center and the electrode surface. The oxidized enzyme oxidizes the substrate to convert it into the reduced enzyme, and at the same time, the process of reducing and oxidizing the mediator substance transfers the reaction charge to the electrode surface, and the substrate concentration of the reaction is represented by the amount of charge. When measuring glucose, the interference of other electroactive substances can be avoided, improving the sensitivity and accuracy of the measurement. Currently, electrochemical sensing electron mediator materials mainly include hydrogel electron mediators based on osmium complexes, wired enzymes, etc. However, it is challenging to prepare a uniform thin layer of these electron mediator materials on the surface of fine microneedles. The adhesion of the electron mediator material to the microneedle surface is not firm, and it is very easy to fall off during the process of inserting the microneedle into the skin, ultimately resulting in the easy diffusion and loss of the hydrogel mediator material in the body tissue. Therefore, higher requirements are put forward for the selection and fixation of the mediator material.

[0050] In this regard, how to break through the outer limiting membrane and solve the problem of the decline in the sensitivity and stability of the implanted electrode when inserted subcutaneously.

[0051] Implantable electrochemical enzyme electrodes usually rely on synthetic materials of inorganic / organic electron mediators and have not yet achieved highly sensitive "enzyme-electrode" electron transfer. Moreover, the invasiveness of the implantable electrode to tissues easily leads to subcutaneous rejection reactions, generating various inflammatory factors and resulting in inaccurate signal detection. In particular, many synthetic electron mediator materials are prone to inducing inflammatory reactions and tissue fibrosis in the body, thus affecting the stability and accuracy of the electrode. Usually, there are potential safety hazards in the body, limiting the feasibility of clinical applications.

[0052] How to achieve real-time and continuous monitoring of subcutaneous blood glucose with high sensitivity, low detection limit, and high stability through process innovation.

[0053] Implantable enzyme electrodes need to be inserted subcutaneously for long-term detection, and the microfabrication on the surface of the microneedles is difficult. The process of constructing an enzyme electrochemical sensing electrode is complex, especially for the detection sensitivity of the fine electrode area of the microneedles, which poses a great challenge.

[0054] How to innovate through processes to solve the problem that the electron mediator on the surface of the microneedle easily diffuses from the enzyme layer.

[0055] Commonly used electron mediators easily diffuse out of the enzyme layer and into the body tissue solution, resulting in poor stability of the sensor, thus limiting the scope of use of the biosensor. Moreover, the microneedle scale is small, and it is very difficult to make the electron mediator layer uniform. The electron mediator layer adheres unevenly and is easy to fall off, etc., thus affecting the stability of the sensing electrode and the intensity of the detection signal.

[0056] In response to this, the present invention constructs a microelectrode sensor based on a polymer osmium ion complex electron mediator, and a minimally invasive transdermal sensing technology based on a microneedle array to achieve highly sensitive and safe detection of various subcutaneous biological indicators, thereby revealing the dynamic change rules of various metabolic indicators in different stages of diabetes. The present invention proposes a method for preparing a carbon nanotube / hydrogel osmium electron mediator / enzyme composite material system as an electron mediator layer on the surface of a conductive microneedle array, and fixing the composite material system to the surface of the microelectrode to form a highly sensitive and highly stable micro-needle enzyme sensing electrode. By means of laser micro-cutting or micro-fabrication, a conductive stainless steel microneedle array is prepared, and the enzyme is fixed on a porous electro-polymer modified electrode, so that the enzyme redox active center is close to the electrode, and direct electron transfer can be relatively easily carried out, thereby improving the responsiveness and sensitivity of the electrode, and providing a new strategy for the development of implantable continuous glucose monitoring technology.

[0057] The following will describe in detail the preparation method and system of the microelectrode sensor based on the polymer osmium ion according to the embodiments of the present invention with reference to the accompanying drawings. First, the preparation method of the microelectrode sensor based on the polymer osmium ion according to the embodiments of the present invention will be described with reference to the accompanying drawings. Refer to Figure 3 as shown, which includes the following steps:

[0058] S100: Construct opposing sensor electrodes on the surface of each microelectrode of the microelectrode array; the sensor electrodes include a working electrode and a counter electrode;

[0059] S200: Modify the sensor electrodes with a carbon nanotube / hydrogel osmium electron mediator / enzyme composite material to obtain a microelectrode sensor.

[0060] In some possible implementation manners, refer to Figure 4 as shown, since the first-generation continuous blood glucose sensing technology does not use an electron mediator, its in-vivo detection performance is poor, which greatly limits the linear detection range of the sensor. Because of the lack of oxygen, the linearity is not good, and using an outer membrane to improve the linearity will result in too weak a signal. This project uses a redox hydrogel / carbon nanotube / osmium electron mediator as an electron transfer mediator for directly transferring a specific enzyme and an electrode, realizes an enzyme electrode sensing mode with efficient redox electron migration, fundamentally solves the problem of "lack of oxygen" in interstitial fluid, and improves the sensitivity and accuracy of implantable glucose electrodes.

[0061] A hydrogel osmium electron mediator coating is adopted. By designing the structure and crosslinking degree of the electron mediator material, a high-performance electrochemical enzyme sensing system is effectively constructed. And the best scheme is optimized to immobilize glucose oxidase on the surface of the microneedles to form a uniform coating, and a microneedle blood glucose sensing electrode is prepared. The detection linear range of the microneedle blood glucose electrode is improved through the electron mediator layer of the composite material, and the electrode has excellent uniformity and mechanical stability. A uniform coating can be formed on the surface of the microneedles, solving the common problems of poor adhesion, easy detachment, and non-uniformity of the electron mediator in the surface treatment process of the microneedle electrode.

[0062] The minimally invasive characteristics of the microneedles can better avoid the pain caused by transdermal penetration and problems such as tissue inflammation and tissue fibrosis caused by tissue trauma. In this project, a microfabrication method for a new type of high-precision multi-channel microneedle array is explored, and the invasive nature of the transdermal electrode is reduced by using the minimally invasive physical structure of the microneedle electrode. By independently sensing each single microneedle, the signal interference between different sensing electrodes is effectively avoided, and the stability of continuous monitoring by the microneedles is improved. In addition, the microneedle array is based on the planar distribution of multiple microneedles. As a sensing electrode, it has the potential to detect signals of various biochemical indicators in a planar region, and has better signal diversity than the implanted single-point electrode.

[0063] Optionally, in an embodiment of the present invention, the sensor electrode is modified by a composite material of carbon nanotubes, hydrogel osmium electron mediator and enzyme to obtain a microelectrode sensor, including:

[0064] According to the covalent coupling effect of -NH2 connected to the branched chain of the hydrogel osmium electron mediator and glutaraldehyde, the materials are connected and blended, and uniformly modified on the surface of the sensor electrode.

[0065] In some possible implementation manners, referring to Figure 5 the schematic diagram of the molecular structure of the hydrogel osmium electron mediator shown, according to the covalent coupling effect of -NH2 connected to the branched chain and glutaraldehyde, the materials are connected and blended, and uniformly modified on the surface of the sensor electrode.

[0066] Optionally, in an embodiment of the present invention, the hydrogel osmium electron mediator material is synthesized by the following steps:

[0067] Vinylpyrrolidone is polymerized to generate polyvinylpyrrolidone;

[0068] The structure and crosslinking degree of the polyvinylpyrrolidone are designed to obtain the hydrogel osmium electron mediator.

[0069] Optionally, in an embodiment of the present invention, the method further includes:

[0070] Mix an aqueous solution of a hydrogel osmium electron mediator at a first concentration, an aqueous solution of single-walled carbon nanotubes, and an aqueous solution of glucose oxidase with an aqueous solution of glutaraldehyde at a second concentration to form a blend solution;

[0071] Add the blend solution into a container, immerse the sensor electrode into the blend solution, and ensure that the immersion length is less than or equal to a first length;

[0072] Lift the lifter to perform a lifting treatment and a drying treatment on the sensor electrode;

[0073] Wash away the unreacted substances on the sensor electrode to obtain the microelectrode sensor.

[0074] In some possible embodiments, the preparation parameters in the present application can be adjusted according to actual needs and are not specifically limited.

[0075] Optionally, in an embodiment of the present invention, the method further includes:

[0076] Generate polyvinylpyrrolidone, add cis-dichloroo osmium (II) and poly(4-vinylpyridine) to ethylene glycol respectively, and perform a reflux heating treatment under nitrogen; introduce amino groups onto the obtained polymer;

[0077] Mix and add N,N-dimethylformamide, add 2-bromoethylamine hydrobromide, stir and then pour the solution into rapidly stirred acetone to precipitate a crude polymer;

[0078] Collect the hygroscopic precipitate, dissolve it in deionized water, and filter; add an ammonium hexafluorophosphate solution for precipitation to form a PF6-salt;

[0079] Dissolve the PF6-salt in acetonitrile to prepare a solution at a third concentration, dilute it with deionized water to a fourth concentration, stir it on an anion exchange column, filter the solution and evaporate it under vacuum;

[0080] Add concentrated hydrochloric acid, adjust the pH, and drop the solution into rapidly stirred acetonitrile to obtain a white powder of the reaction product determined as the hydrogel osmium electron mediator.

[0081] Optionally, in an embodiment of the present invention, the working electrode is prepared by the following steps:

[0082] Perform laser etching on the microneedles and perform a cutting treatment;

[0083] Soak, clean, and dry the cut microneedles with absolute ethanol;

[0084] Prepare a stainless-steel soldering flux with a preset volume ratio containing ZnO, NH4Cl, HCl, CH3COOH, H2O, and a surfactant. Immerse the microneedles in the stainless-steel soldering flux and perform ultrasonic treatment.

[0085] Take out the microneedles, connect an electrochemical workstation, use the microneedles as the first working electrode, a gold sheet electrode as the first counter electrode, and a silver / silver chloride electrode as the first reference electrode.

[0086] Use the multi-step constant current method to electrochemically deposit gold on the microneedles, and naturally dry them to obtain the working electrode.

[0087] Optionally, in an embodiment of the present invention, the counter electrode is prepared by the following steps:

[0088] Take the microneedles that have completed the electrochemical deposition of gold, connect the electrochemical workstation, use the microneedles as the second working electrode, a platinum sheet electrode as the second counter electrode, and a silver / silver chloride electrode as the second reference electrode.

[0089] Immerse the second working electrode, the second counter electrode, and the second reference electrode in an aqueous solution of sodium platinous sulfite, and use the multi-step constant current method to electrochemically deposit platinum on the microneedles; naturally dry them to obtain the counter electrode.

[0090] In some possible implementation manners, the working electrode, the reference electrode, and the counter electrode can be prepared by other feasible methods, and the present application does not make specific limitations.

[0091] Optionally, in an embodiment of the present invention, the reference electrode is prepared by the following steps:

[0092] Take the microneedles that have completed the electrochemical deposition of gold, coat them with Ag / AgCl ink, and dry them.

[0093] Use methanol solution for immersion coating and air dry to obtain the reference electrode.

[0094] The following introduces the preparation method provided by the present application in detail with a specific embodiment:

[0095] The present invention constructs a microelectrode sensor based on a polymer osmium ion complex electron mediator, and a minimally invasive transdermal sensing technology based on a microneedle array to achieve highly sensitive and safe detection of multiple subcutaneous biological indicators, thereby revealing the dynamic change laws of multiple metabolic indicators in different courses of diabetes.

[0096] Develop a polymer electron mediator electrode coating material system based on hydrogel osmium complexes, optimize the composite material systems with different ratios and crosslinking degrees, as well as the electrode processing techniques and parameters, and select the best solution; explore the preparation method of enzyme electrochemical sensing electrodes based on microneedle arrays, and construct a hydrogel osmium electron mediator electrochemical enzyme sensing electrode with high sensitivity, low detection limit and high stability; construct a minimally invasive continuous blood glucose sensor based on hydrogel osmium electron mediators to achieve minimally invasive, real-time and continuous monitoring of subcutaneous glucose concentration fluctuations.

[0097] The minimally invasive characteristics of microneedles can better avoid the pain caused by transdermal penetration and problems such as tissue inflammation and tissue fibrosis caused by tissue trauma. Based on the second-generation blood glucose sensing principle of electron mediators, electrons are directly transferred from the enzyme active center to the electrode without passing through hydrogen peroxide as an intermediate electron carrier, so it is not dependent on the oxygen concentration and can overcome the problem of lack of oxygen in tissues during in vivo sensing. Modify the sensing electrode with carbon nanotubes, hydrogel osmium electron mediators and enzyme composites. By preparing a nanocomposite structure with good electrical properties on the surface of the conductive microneedles, carbon nanotubes form additional conductive paths in the hydrogel network, which has a positive effect on improving the sensitivity and performance of electron mediators.

[0098] Through innovation in microfabrication techniques, each microelectrode in the microelectrode array senses independently. This method of independent data transmission can effectively overcome the mutual interference of signals between different types of sensing electrodes. By constructing counter electrodes and reference electrodes on the surface of each microelectrode in the microelectrode array, the transmission distance of electrical signals between a group of working circuits is reduced, effectively reducing the influence caused by the low conductivity of biological tissues between electrodes, improving the signal strength and stability, and enhancing the sensor sensitivity. Moreover, the microneedle array is distributed in a planar shape based on multiple microneedles. As a sensing electrode, it has the potential to detect signals of various biochemical indicators in a planar area and has better signal diversity than implanted single-point electrodes.

[0099] Through a composite material system of hydrogel osmium polymer mediators and single-walled carbon nanotubes, a three-dimensional network structure is formed through cross-linking reactions to fix the hydrogel osmium polymer mediators and specific proteases on the sensor surface. By designing the structure of the polymer composite electrode material and different crosslinking degrees, continuously optimizing the process and adjusting the performance parameters, select the best ratio solution with excellent uniformity and mechanical stability.

[0100] Preparation of microneedle electrodes:

[0101] S21, Pretreatment of the working electrode microneedles: Take 12 syringe needles with a length of 20 mm and a GB of 30 g. Use a laser marking machine to perform laser etching on the microneedles and cut the hollow microneedles into uniform lengths of 6 mm. Immerse the cut microneedles in absolute ethanol, clean them in ultrasonic for 40 min, take them out and put them into an 80 °C oven to dry for later use. Prepare a stainless steel soldering flux with a volume ratio of 24% ZnO, 30% NH4Cl, 6% HCl, 30% CH3COOH, 12% H2O, and 3% surfactant. Immerse the microneedles in the stainless steel soldering flux and ultrasonicate. After 200 s, take them out and gently suck away the excess solution on the surface of the microneedles with lint-free paper. Take out the microneedles, connect an electrochemical workstation, use the microneedles as the working electrode, a gold sheet electrode as the counter electrode, and a silver chloride electrode as the reference electrode. Add 8 mL of an aqueous solution of sodium gold sulfite with a concentration of 2 mmol / L to a 10 mL beaker, immerse the three electrodes in the solution, and use the multi-step constant current method to electrochemically deposit gold on the microneedles in the sodium gold sulfite electroplating gold solution. After deposition, gently rinse them in deionized water, take them out and dry them naturally;

[0102] S22, Preparation of the counter electrode: Take the microneedles that have completed the electrochemical deposition of gold, connect an electrochemical workstation, use the microneedles as the working electrode, a platinum sheet electrode as the counter electrode, and a silver chloride electrode as the reference electrode. Add 8 mL of an aqueous solution of sodium platinum sulfite with a concentration of 2 mmol / L to a 10 mL beaker, immerse the three electrodes in the solution, and use the multi-step constant current method to electrochemically deposit platinum on the microneedles in the sodium platinum sulfite electroplating platinum solution. After deposition, gently rinse them in deionized water, take them out and dry them naturally;

[0103] S23, Preparation of the reference electrode: Take the microneedles that have completed the electrochemical deposition of gold, coat them with Ag / AgCl ink, and dry them in an 80 °C oven. After drying, take them out and perform immersion coating with a methanol solution of 1% polyvinyl butyral by mass fraction, and let it dry at room temperature for more than 12 hours.

[0104] Synthesis of hydrogel osmium electron mediator material

[0105] Vinylpyrrolidone and ionic water were mixed at a ratio of 1:1 (v / v), and then 2,2'-Azobisisobutyronitrile, a radical initiator, was added to make the final concentration of the radical initiator 2 mg / mL. The mixture was stirred at room temperature for about 20 minutes to completely dissolve the radical initiator AIBN in water. The reactants were microwave-heated for 3 minutes to complete the polymerization reaction and generate Polyvinylpyrrolidone (PVP). Cis-bis(2,2'-bipyridine-N,N')di-chloroosmium(II) (0.864 mmol) and poly(4-vinyl-pyridine) (4.09 mmol) were added to ethylene glycol such that their concentrations were 27 mg / mL and 48 mg / mL respectively, and the mixture was reflux-heated under nitrogen for 2 hours. An amino group (-NH2) was further introduced onto the polymer for subsequent crosslinking of the polymer to form a hydrogel network. After the solution was cooled to room temperature, N,N-Dimethylformamide (DMF) was mixed with the original solution at a ratio of 5:3 (v / v), and 2-bromoethylamine hydrobromide (7.3 mmol) was added. The solution was stirred overnight at 45 °C. After overnight stirring, the solution was poured into rapidly stirred acetone to precipitate the crude polymer. The hygroscopic precipitate was collected, dissolved in deionized water, and filtered. Ammonium hexafluorophosphate solution (NH4PF6) was added for precipitation to form PF6-salt. After drying, the PF6-salt was dissolved in acetonitrile to prepare a solution with an initial concentration of 24.5 mg / mL, and then diluted to 7 mg / mL with deionized water. The solution was stirred on a 5.2 g anion exchange column (chloride form) for 2 hours, the solution was filtered and evaporated to about 10 mL under vacuum. Then concentrated hydrochloric acid was added to adjust the pH to 2, and the solution was dropped into rapidly stirred acetonitrile to obtain a white powder of the reaction product. The obtained hydrogel osmium polymer mediator was named NH2-PVP-Os (as Figure 5 shown).

[0106] Construction of a hydrogel osmium electron mediator electrochemical enzyme sensing electrode:

[0107] According to the covalent coupling effect of -NH2 connected to the polymer side chain and glutaraldehyde, each material was connected and blended, and uniformly modified on the surface of the sensor. An aqueous solution of osmium polymer mediator PVP-Os-NH2, an aqueous solution of single-walled carbon nanotubes, an aqueous solution of glucose oxidase, and a 2.5% glutaraldehyde solution (final volume ratio of 0.04) were proportioned to form a blended solution. Take 8 mL of the blended solution and add it to a 10 mL beaker. Slowly immerse the prepared sensor electrode into the solution, controlling the immersion length not to exceed 2 mm. Use a puller to vertically pull the electrode evenly 4 times, with a pulling speed of 5 mm / s and a pulling distance of 10 mm. After pulling, dry it overnight at room temperature (>16 hours). After preparation, place the sensing electrode in PBS phosphate buffer solution (pH 7.4) and let it stand to wash away the unreacted substances. Take it out after 8 hours and air-dry it naturally at room temperature for 8 hours. Store the sensor electrode in an incubator at 25°C. Take the prepared metal micro-needle electrode and observe the size and surface morphology of the micro-needles using a scanning electron microscope (SEM).

[0108] In vitro glucose detection performance evaluation:

[0109] Take a 10 mL beaker, use the modified working electrode as the working electrode, a commercial platinum electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode. Place them in the beaker and add 10 mL of PBS buffer solution to the beaker. Immerse the electrodes in the solution. Use an electrochemical workstation to observe the measured peak value. Add glucose solutions with different concentrations to the solution every 60 seconds, so that the concentration change of the solution is 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16 mM. Measure the glucose response at different concentrations, and test the CV peak and the it response curve of the sensor electrode to glucose. At the most obvious bias voltage of the CV, test the sensitivity and stability of the sensor electrode for glucose detection. By adding substances such as lactic acid, uric acid, and cholesterol to the solution, test the anti-interference performance of the glucose sensing electrode.

[0110] Example 1:

[0111] Microelectrode sensor based on polymer osmium ion complex electron mediator, realizing in vivo glucose detection performance evaluation

[0112] Mechanism and application performance of a microelectrode sensor based on polymer osmium ion complex electron mediator for detecting blood glucose.

[0113] Construction of diabetic rat model: 10-week-old STZ-Type 1DR rats were used. After examination, no obvious abnormalities were found. After 7 days of adaptive feeding, 3 rats with blood glucose levels exceeding 250 mg / dL for two consecutive days and 3 rats with blood glucose levels not exceeding 160 mg / dL for two consecutive days were selected for the experiment. Evaluation of in vitro glucose detection performance

[0114] After the diabetic rat model was successfully established, the rat was fixed securely and general anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution (0.2mL / 100g). A constant temperature electric heating plate was used to keep the rat's body temperature constant. After the experimental device (microneedle sensor) was placed, it continued to be placed on the constant temperature electric heating plate until it woke up. The awakening solution can be injected intramuscularly to reduce the chance of accidental death from anesthesia. After the rat was fully anesthetized, the skin on its back was prepared, and the preparation area was about 3.5*3.5cm 2 . After skin preparation, the microneedle sensor was applied to the dorsal skin and fixed with a small mouse vest. The subcutaneous tissue fluid glucose signal was measured every 15 minutes for the sensing pathway. The microneedle sensor was worn continuously for 72 hours, during which time the rats ate and drank water normally, and glucose solution was injected into the rats at specific time intervals to induce blood sugar fluctuations. Blood was collected from the rats' tails every 30 minutes for the first 24 hours and every 1 hour for the next 48 hours. A commercial blood glucose meter was used to detect the actual blood sugar value for control, and the accuracy, sensitivity and other performance indicators of blood sugar monitoring of the microneedle sensor were evaluated.

[0115] Transdermal biosafety assessment: After the microneedle sensor array is inserted into the skin of an isolated animal (pig skin, mouse skin, rabbit skin, etc.) and then pulled out, the tissue at the microneedle insertion site is sliced ​​and stained with H&E. The tissue slices of the same area of ​​normal rats are used as the control group to study the possible allergic reactions, tissue inflammation, and tissue fibrosis of the skin tissue of acute diabetic rats, and then evaluate the transdermal depth and biosafety of the electrode array.

[0116] On the other hand, an embodiment of the present invention provides an evaluation method based on a microelectrode sensor, comprising:

[0117] The microelectrode sensor is applied to the subcutaneous part of the skin in vitro, the skin tissue is sliced ​​and stained, and the skin performance is evaluated to determine the skin penetration depth of the microelectrode sensor.

[0118] On the other hand, an embodiment of the present invention provides an application system of a microelectrode sensor based on polymer osmium ions, wherein the microelectrode sensor prepared by the above preparation method is used as a working electrode to detect glucose in vivo or in vitro.

[0119] It can be seen that the content in the above method embodiments is applicable to the system embodiments of the present invention. The functions specifically implemented by the system embodiments of the present invention are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those of the above method embodiments.

[0120] In some alternative embodiments, the functions / operations mentioned in the block diagrams may not occur in the order mentioned in the operation diagrams. For example, depending on the functions / operations involved, two consecutive blocks shown may actually be executed substantially simultaneously or the blocks can sometimes be executed in the reverse order. In addition, the embodiments presented and described in the flowcharts of the present invention are provided by way of example for the purpose of providing a more comprehensive understanding of the technology. The disclosed methods are not limited to the operations and logical flows presented herein. Alternative embodiments are contemplated, where the order of various operations is changed and where sub-operations described as part of a larger operation are executed independently.

[0121] In addition, although the present invention has been described in the context of functional modules, it should be understood that, unless otherwise stated to the contrary, one or more of the functions and / or features may be integrated in a single physical device and / or software module, or one or more functions and / or features may be implemented in separate physical devices or software modules. It can also be understood that a detailed discussion of the actual implementation of each module is not necessary for understanding the present invention. Rather, considering the attributes, functions, and internal relationships of the various functional modules in the devices disclosed herein, the actual implementation of the modules will be understood within the ordinary skills of an engineer. Therefore, those skilled in the art can implement the present invention as set forth in the claims without undue experimentation using ordinary skills. It can also be understood that the specific concepts disclosed are merely illustrative and are not intended to limit the scope of the present invention, which is determined by the full scope of the appended claims and their equivalents.

[0122] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a sequenced list of executable instructions for implementing logical functions, which can be specifically implemented in any computer-readable medium for use by or in connection with a program execution system, apparatus, or device, such as a computer-based system, a system including a processor, or other systems that can fetch and execute the program from the program execution system, apparatus, or device. For the purposes of this specification, a "computer-readable medium" can be any device that can contain, store, communicate, propagate, or transport the program for use by or in connection with the program execution system, apparatus, or device.

[0123] It should be understood that the various parts of the present invention can be implemented by hardware, software, firmware or a combination thereof. In the above embodiments, multiple steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable program execution system. For example, if implemented by hardware, as in another embodiment, any one or a combination of the following techniques well known in the art can be used: discrete logic circuits having logic gate circuits for implementing logical functions on data signals, application specific integrated circuits having appropriate combinational logic gate circuits, programmable gate arrays (PGAs), field programmable gate arrays (FPGAs), and the like.

[0124] In the foregoing description of this specification, the descriptions with reference to the terms "one embodiment / example", "another embodiment / example" or "certain embodiments / examples", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0125] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can 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, and the scope of the present invention is defined by the claims and their equivalents.

[0126] The above has specifically described the preferred embodiments of the present invention, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent deformations or substitutions without departing from the spirit of the present invention, and these equivalent deformations or substitutions are all included within the scope defined by the claims of the present invention.

Claims

1. A preparation method of a microelectrode sensor based on polymer osmium ions, characterized in that, It includes the following steps: Construct opposing sensor electrodes on the surface of each microelectrode of the microelectrode array; the sensor electrodes include a working electrode and a counter electrode; Modify the sensor electrodes with a composite material of carbon nanotubes, hydrogel osmium electron mediator and enzyme to obtain a microelectrode sensor.

2. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 1, characterized in that, The step of modifying the sensor electrodes with a composite material of carbon nanotubes, hydrogel osmium electron mediator and enzyme to obtain a microelectrode sensor includes: According to the covalent coupling effect of -NH2 connected to the side chain of the hydrogel osmium electron mediator and glutaraldehyde, the materials are connected and blended, and uniformly modified on the surface of the sensor electrodes.

3. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 2, characterized in that, The hydrogel osmium electron mediator is synthesized through the following steps: Polyvinylpyrrolidone is generated by the polymerization reaction of vinylpyrrolidone; The structure and crosslinking degree of polyvinylpyrrolidone are designed to obtain the hydrogel osmium electron mediator.

4. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 2, characterized in that, The method further includes: Mix a first concentration of hydrogel osmium electron mediator, an aqueous solution of single-walled carbon nanotubes, an aqueous solution of glucose oxidase and a second concentration of glutaraldehyde solution to form a blended solution; Add the blended solution into a container, immerse the sensor electrodes into the blended solution, and ensure that the immersion length is less than or equal to a first length; Raise the lifting machine, and perform lifting treatment and drying treatment on the sensor electrodes; Wash away the unreacted substances on the sensor electrodes to obtain the microelectrode sensor.

5. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 3, characterized in that, The method further includes: Generate polyvinylpyrrolidone, and respectively add cis-dichloroo osmium and poly(4-vinylpyridine) into ethylene glycol, and perform reflux heating treatment under nitrogen; introduce amino groups onto the obtained polymer; Mix and add N,N-dimethylformamide, and add 2-bromoethylamine hydrobromide. After stirring, pour the solution into rapidly stirred acetone to precipitate a crude polymer; Collect the hygroscopic precipitate, dissolve it in deionized water, and filter; add ammonium hexafluorophosphate solution for precipitation to form PF6-salt; Dissolve the PF6-salt in acetonitrile to prepare a solution with a third concentration, dilute it with deionized water to a fourth concentration, stir it on an anion exchange column, filter the solution and evaporate it under vacuum; Add concentrated hydrochloric acid, adjust the pH, and drop the solution into rapidly stirred acetonitrile to obtain a white powder of the reaction product, which is determined to be the hydrogel osmium electron mediator.

6. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 1, characterized in that The working electrode is prepared through the following steps: Perform laser etching on the microneedles and carry out cutting treatment; Soak, clean and dry the cut microneedles with absolute ethanol; Install a stainless steel soldering flux prepared according to a preset volume ratio containing ZnO, NH4Cl, HCl, CH3COOH, H2O and a surfactant, and immerse the microneedles into the stainless steel soldering flux for ultrasonic treatment; Take out the microneedles, connect an electrochemical workstation, use the microneedles as the first working electrode, a gold sheet electrode as the first counter electrode, and a silver chloride electrode as the first reference electrode; Perform electrochemical deposition of gold on the microneedles using a multi-step constant current method and dry it naturally to obtain the working electrode.

7. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 6, characterized in that, The counter electrode is prepared through the following steps: Take the microneedles that have completed the electrochemical deposition of gold, connect the electrochemical workstation, use the microneedles as the second working electrode, a platinum sheet electrode as the second counter electrode, and a silver / silver chloride electrode as the second reference electrode; Immerse the second working electrode, the second counter electrode, and the second reference electrode in an aqueous solution of sodium platinous sulfite, and perform electrochemical deposition of platinum on the microneedles using a multi-step constant current method; dry naturally to obtain the counter electrode.

8. The preparation method of the microelectrode sensor based on polymer osmium ions according to claim 1, characterized in that, The reference electrode is prepared by the following steps: Take the microneedles that have completed the electrochemical deposition of gold, coat them with Ag / AgCl ink, and dry; Perform immersion coating with a methanol solution and air dry to obtain the reference electrode.

9. An evaluation method based on a microelectrode sensor, characterized in that, Including: Use the microelectrode sensor subcutaneously on ex vivo skin, perform slicing and staining on the skin tissue, and determine the transdermal depth of the microelectrode sensor through the performance evaluation of the skin; the microelectrode sensor is a microelectrode sensor prepared by the preparation method according to any one of claims 1 to 8.

10. An application system of a microelectrode sensor based on polymer osmium ions, characterized in that, Use the microelectrode sensor prepared by the preparation method according to any one of claims 1 to 8 as the working electrode to detect glucose in vivo or ex vivo.

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