Graphene-based microneedle sensing electrode preparation method and blood fat detection system

Abstract

The invention discloses a graphene-based microneedle sensing electrode preparation method and a blood fat detection system. The method comprises the following steps: preparing a microneedle array, preparing a graphene structure on the surface of the microneedle array by utilizing a chemical vapor deposition technology, and modifying cholesterol oxidase on the surface of the microneedle array; and coating the microneedle array with a water-soluble polymer to obtain the microneedle sensing electrode. According to the application, a graphene nanostructure is constructed on the surface of the microneedle, and cholesterol oxidase is fixed on the surface of the electrode, so that a great specific surface area and high electron mobility of the sensor are realized, the biocompatibility and detection precision of the sensor are improved, and accurate and convenient subcutaneous in-situ detection of cholesterol is realized. 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 in particular to a method for preparing a graphene-based micro-needle sensing electrode and a blood lipid detection system. Background Art

[0002] The prevalence and mortality of cardiovascular diseases show a severe trend of continuous increase and younger age. This trend not only poses a serious threat to personal health, but also brings a heavy burden to the social medical system. A large number of clinical research results have consistently shown that there is a close and complex correlation between skin cholesterol levels and the occurrence and development of atherosclerotic cardiovascular diseases. More importantly, compared with blood cholesterol levels, which are easily affected by various factors and produce short-term fluctuations, skin cholesterol levels show relatively stable characteristics in a shorter period of time. Therefore, the skin cholesterol level indicator can provide patients with cardiovascular diseases with more reliable and long-term blood lipid level monitoring, thereby providing a highly innovative detection method for medication evaluation and blood lipid management in patients with cardiovascular diseases.

[0003] As an emerging painless transdermal technology, microneedle technology has made optimistic progress in the field of transdermal drug delivery and has shown great application potential due to its unique advantages such as painlessness, minimal invasion, and convenience of use. In related technologies, the complex subcutaneous microenvironment and the inherent characteristics of metal electrodes will cause certain trauma to the skin and may even induce adverse reactions such as infection. These adverse reactions will not only affect the patient's comfort, but may also cause the sensitivity of the electrode to decrease, affecting the normal operation of the sensor device. In addition, metal electrodes themselves also have some inherent limitations, such as susceptibility to biomolecule contamination and poor biocompatibility. Summary of the invention

[0004] The purpose of the present invention is to solve one of the technical problems existing in the prior art to at least a certain extent.

[0005] To this end, the purpose of the present invention is to provide a high-performance graphene-based microneedle sensing electrode preparation method and a blood lipid detection system.

[0006] In order to achieve the above technical objectives, one aspect of the embodiments of the present invention provides a method for preparing a graphene-based microneedle sensing electrode, comprising the following steps: preparing a microneedle array, preparing a graphene structure on the surface of the microneedle array using chemical vapor deposition technology, and modifying cholesterol oxidase on the surface of the microneedle array; coating the microneedle array with a water-soluble polymer to obtain a microneedle sensing electrode. The present application constructs a graphene nanostructure on the surface of the microneedle and fixes cholesterol oxidase on the surface of the electrode to achieve a large specific surface area and high electron mobility of the sensor, which is beneficial to improving the biocompatibility and detection accuracy of the sensor, and realizing accurate and convenient subcutaneous in situ detection of cholesterol.

[0007] In some embodiments, the method for preparing a graphene-based microneedle sensing electrode according to an embodiment of the present invention, wherein the microneedle array is prepared, and a graphene structure is prepared on the surface of the microneedle array using chemical vapor deposition technology, comprising:

[0008] Preparation of stainless steel microneedle arrays;

[0009] A graphene structure is prepared on the surface of the stainless steel microneedle array by inductively coupled plasma enhanced chemical vapor deposition technology.

[0010] In some embodiments, in one embodiment of the present invention, the graphene structure is prepared on the surface of the stainless steel microneedle array by inductively coupled plasma enhanced chemical vapor deposition technology, comprising:

[0011] The stainless steel microneedle is placed on the cathode in the quartz reaction chamber for heating, and the quartz reaction chamber is evacuated;

[0012] Introducing a first preset flow rate of hydrogen and argon into the quartz reaction chamber, setting a first radio frequency power and a first bias voltage between the anode and the cathode, and performing pretreatment;

[0013] A second preset flow rate of hydrogen and methane is introduced into the quartz reaction chamber, a second radio frequency power and a second bias voltage between the anode and the cathode are set, and a growth process is performed to obtain a microneedle array with a graphene structure prepared on the surface.

[0014] In some embodiments, in one embodiment of the present invention, the preparation of the stainless steel microneedle array comprises:

[0015] Stainless steel was selected as the material for the microneedles;

[0016] Microneedle arrays were cut out on stainless steel plates by laser microcutting.

[0017] In some embodiments, in one embodiment of the present invention, the cholesterol oxidase is modified on the surface of the microneedle array, comprising:

[0018] Prepare cholesterol oxidase precursor solution;

[0019] The electrodes of the microneedle array are pulled in the cholesterol oxidase precursor solution for a preset number of times using the pulling method, and then dried to obtain a microneedle array with surface-modified cholesterol oxidase.

[0020] In some embodiments, in one embodiment of the present invention, the preparation of cholesterol oxidase precursor solution comprises:

[0021] Dissolving cholesterol oxidase in PBS to obtain a first solution;

[0022] dissolving bovine serum albumin in PBS to obtain a second solution;

[0023] dissolving glutaraldehyde in PBS to obtain a third solution;

[0024] The first solution, the second solution and the third solution are mixed according to a preset volume ratio to obtain a cholesterol oxidase precursor solution.

[0025] In some embodiments, in one embodiment of the present invention, the microneedle array is coated with a water-soluble polymer to obtain a microneedle sensing electrode, comprising:

[0026] A polyvinyl pyrrolidone aqueous solution with a preset mass percentage is prepared, and the polyvinyl pyrrolidone is coated on the electrode surface of the microneedle array using a spray gun at a preset spraying pressure, a preset distance, and a preset spraying time; the preset distance is the distance between the nozzle and the microneedle array;

[0027] The solution was allowed to stand for evaporation of the water to obtain a microneedle sensing electrode coated with a water-soluble polymer.

[0028] On the other hand, an embodiment of the present invention provides a method for preparing an electrochemical sensor, the method comprising:

[0029] Using the microneedle sensing electrode obtained by the above-mentioned graphene-based microneedle sensing electrode preparation method as a working electrode;

[0030] Prepare a microneedle array and deposit titanium and platinum to obtain a counter electrode;

[0031] Prepare a microneedle array, deposit titanium and silver, and form a silver chloride film on the electrode by electrochemical oxidation to obtain a reference electrode;

[0032] The working electrode, the counter electrode and the reference electrode are packaged to obtain an electrochemical sensor.

[0033] In some embodiments, in one embodiment of the present invention, the working electrode, the counter electrode, and the reference electrode are packaged to obtain an electrochemical sensor, comprising:

[0034] Put the working electrode, the counter electrode and the reference electrode into a mold and fix them with a PDMS thin layer;

[0035] Pour liquid photosensitive resin into the mold, and perform curing and demoulding treatment under ultraviolet light;

[0036] The PDMS thin layer is removed, and a wire is connected to each electrode to obtain an electrochemical sensor.

[0037] On the other hand, an embodiment of the present invention provides a blood lipid detection system, including an electrochemical sensor obtained by the above-mentioned preparation method of the electrochemical sensor.

[0038] The embodiments of the present application include at least the following beneficial effects: The method provided by the embodiments of the present invention includes: preparing a microneedle array, preparing a graphene structure on the surface of the microneedle array using chemical vapor deposition technology, and modifying cholesterol oxidase on the surface of the microneedle array; coating the microneedle array with a water-soluble polymer to obtain a microneedle sensing electrode. The present application constructs a graphene nanostructure on the surface of the microneedle and fixes cholesterol oxidase on the surface of the electrode to achieve a large specific surface area and high electron mobility of the sensor, which is beneficial to improving the biocompatibility and detection accuracy of the sensor, and realizing accurate and convenient subcutaneous in situ detection of cholesterol. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the embodiments of the present invention or the drawings of related technical solutions in the prior art are introduced below. It should be understood that the drawings introduced below are only for the convenience of clearly describing some embodiments of the technical solutions of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0040] Figure 1 A schematic flow chart of an embodiment of a method for preparing a graphene-based microneedle sensing electrode provided by the present invention;

[0041] Figure 2 A schematic diagram of an application of an embodiment of the electrochemical sensor provided by the present invention;

[0042] Figure 3 A schematic diagram of another embodiment of a method for preparing a graphene-based microneedle sensing electrode provided by the present invention;

[0043] Figure 4 A schematic diagram of a process flow of an embodiment of a method for preparing an electrochemical sensor provided by the present invention. DETAILED DESCRIPTION

[0044] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and are not to be construed as limitations of the present invention. For the step numbers in the following embodiments, they are only provided for the convenience of explanation, and the order between the steps is not limited in any way, and the execution order of each step in the embodiment can be adaptively adjusted according to the understanding of those skilled in the art.

[0045] With the rapid development of social economy and the continuous improvement of national living standards, people's material life is becoming increasingly rich. However, at the same time, unhealthy living habits, such as high-fat diet, long-term sitting, and excessive mental stress, have gradually become common, which has directly led to the severe trend of the morbidity and mortality of cardiovascular diseases continuing to rise and becoming younger. This trend not only poses a serious threat to personal health, but also brings a heavy burden to the social medical system. A large number of clinical research results have consistently shown that there is a close and complex correlation between skin cholesterol levels and the occurrence and development of atherosclerotic cardiovascular diseases. More importantly, compared with blood cholesterol levels, which are susceptible to short-term fluctuations due to various factors, skin cholesterol levels show relatively stable characteristics in a shorter period of time. Therefore, the skin cholesterol level indicator can provide more reliable and long-term blood lipid level monitoring for patients with cardiovascular diseases, thereby providing a highly innovative detection method for the evaluation of medication and blood lipid management of patients with cardiovascular diseases. This detection method can not only effectively evaluate the efficacy of drugs, but also help patients better understand their own blood lipid status, so as to make more targeted lifestyle adjustments and disease management. Therefore, in situ monitoring of skin cholesterol levels has great application value in the chronic management of patients with cardiovascular diseases. However, the current clinical detection of cholesterol mostly relies on in vitro methods, which requires blood or tissue fluid to be taken out of the body for analysis, or biological blood or tissue fluid droplets are extracted using a metal needle tube and then dripped onto the sensor for detection. These traditional methods are not only complicated and time-consuming, but also invasive and easy to cause discomfort to patients. More importantly, there is still a lack of a technology that can directly detect the cholesterol content in subcutaneous tissue in situ. This lack of technology makes it inconvenient to conduct early screening and daily blood lipid management for patients with cardiovascular diseases. The development of an innovative technology for in situ, minimally invasive, and real-time detection of subcutaneous cholesterol content is expected to completely change the existing detection mode of cardiovascular diseases, provide strong technical support for early screening, risk assessment, and daily blood lipid management of patients with cardiovascular diseases, and has broad application prospects and far-reaching social significance.

[0046] As an emerging painless transdermal technology, microneedle technology has made optimistic progress in the field of transdermal drug delivery due to its unique advantages such as painlessness, minimally invasiveness, and convenience of use, showing great application potential. Compared with traditional injection drug delivery, microneedle technology can significantly reduce the pain of patients and effectively avoid adverse reactions such as infection, greatly improving the patient's medication experience. However, although microneedle technology has made good progress in the field of transdermal drug delivery, it still faces some important challenges in the development and application of microneedle array-based sensing technology. Among them, a major problem is that the complex subcutaneous microenvironment and the inherent characteristics of metal electrodes will cause certain trauma to the skin, and may even induce adverse reactions such as infection. These adverse reactions will not only affect the patient's comfort, but may also cause the sensitivity of the electrode to decrease, affecting the normal operation of the sensor device. In addition, metal electrodes themselves also have some inherent limitations, such as susceptibility to biomolecule contamination and poor biocompatibility, which limit the development of microneedle array-based sensing technology in practical applications. By integrating nanostructures into the surface of microneedle electrodes, the specific surface area, conductivity and catalytic activity of the sensor can be effectively increased, thereby greatly improving the sensitivity and detection performance of the sensor. In addition, nanomaterials can also improve the biocompatibility of electrodes to a certain extent, reduce biological contamination, and improve the stability and life of the sensor. Therefore, combining nanostructures with microneedle technology to construct a high-performance, high-sensitivity microneedle sensor is of great significance and far-reaching value in promoting the further development of subcutaneous in situ cholesterol detection technology.

[0047] Specifically, the microneedle materials currently commonly used in microneedle array sensors, such as metals (such as stainless steel, titanium), silicon, and certain polymers, may cause a series of adverse reactions when implanted for a long time or in contact with biological tissues. Although metal materials have good conductivity and mechanical strength, they are prone to corrosion and release of metal ions, which in turn lead to tissue inflammation, allergic reactions or cytotoxicity, affecting the normal operation and biosafety of the sensor. Although silicon materials are widely used in the field of microelectronics, they have poor biodegradability, and long-term implantation may lead to tissue fibrosis and even cause foreign body reactions. Some traditional polymer materials may have problems such as insufficient biodegradability, low mechanical strength, and poor surface bioactivity, which are difficult to meet the needs of long-term implantation. The limitations of these materials make microneedle array sensors face many challenges in biocompatibility, limiting their long-term and safe application in the biomedical field. There is an urgent need to develop new and more biocompatible microneedle materials to improve the biocompatibility and clinical application potential of microneedle sensors.

[0048] Due to the limitation of microneedle size, its sensing area is usually small, resulting in a limited number of biomolecules that can be captured by the sensor, which leads to insufficient detection sensitivity, especially when detecting biomolecules with low concentrations in subcutaneous tissue, it is more likely to have problems of weak detection signal and low signal-to-noise ratio. Secondly, the sensing materials and structures used in traditional microneedle sensors are often not highly selective and are easily interfered by other biomolecules in the environment, resulting in deviation and inaccuracy in the detection results. For example, electrochemical sensors may be sensitive to other electroactive substances, and optical sensors may be affected by stray light. These non-specific interferences will reduce the selectivity of the sensor and make it difficult to accurately distinguish the target analyte. Therefore, how to improve the sensitivity of microneedle sensors so that they can effectively detect trace biomolecules in subcutaneous tissue, and how to improve the selectivity of sensors so that they only respond to target analytes, are key issues that need to be urgently addressed in the current microneedle array sensor technology.

[0049] Sensors based on microneedle arrays face multiple challenges in terms of stability, mainly in terms of biological contamination and mechanical strength. First, microneedle sensors are extremely susceptible to adsorption by biomolecules such as proteins, cells, and lipids in complex biological environments such as subcutaneous tissue, forming a biological contamination layer. This biological contamination will cover the sensor surface, hindering the effective contact between the target analyte and the sensor surface, resulting in decreased sensor sensitivity, prolonged response time, drift in detection performance, and even failure. Especially for sensors implanted for a long time, the cumulative effect of biological contamination is more significant, seriously affecting the stability and service life of the sensor. Secondly, microneedle sensors need to withstand mechanical forces from tissue structures during insertion into the skin or long-term use, including puncture force, friction, etc. Due to the small structure of microneedles, their mechanical strength is relatively low, and they are prone to breakage, bending, deformation, and other problems, resulting in sensor failure or inaccurate measurement results. In particular, some flexible microneedle sensors have weaker mechanical strength and are more susceptible to external mechanical forces. Therefore, how to improve the anti-biological contamination ability and mechanical strength of microneedle sensors and ensure the long-term stable operation of sensors in complex biological environments is an important challenge facing the development of microneedle array sensor technology.

[0050] The following describes in detail a method and system for preparing a graphene-based microneedle sensing electrode according to an embodiment of the present invention with reference to the accompanying drawings. First, the method for preparing a graphene-based microneedle sensing electrode according to an embodiment of the present invention will be described with reference to the accompanying drawings.

[0051] Reference Figure 1 In an embodiment of the present invention, a method for preparing a graphene-based microneedle sensing electrode is provided, comprising:

[0052] S100: preparing a microneedle array, preparing a graphene structure on the surface of the microneedle array by chemical vapor deposition technology, and modifying cholesterol oxidase on the surface of the microneedle array;

[0053] S200: coating the microneedle array with a water-soluble polymer to obtain a microneedle sensing electrode.

[0054] In some possible implementations, the microneedle array in the present application may be in any form, and the present application does not make any specific limitation.

[0055] Optionally, in one embodiment of the present invention, the step of preparing the microneedle array by using chemical vapor deposition technology to prepare a graphene structure on the surface of the microneedle array comprises:

[0056] Preparation of stainless steel microneedle arrays;

[0057] A graphene structure is prepared on the surface of the stainless steel microneedle array by inductively coupled plasma enhanced chemical vapor deposition technology.

[0058] Optionally, in one embodiment of the present invention, the graphene structure is prepared on the surface of the stainless steel microneedle array by inductively coupled plasma enhanced chemical vapor deposition technology, comprising:

[0059] The stainless steel microneedle is placed on the cathode in the quartz reaction chamber for heating, and the quartz reaction chamber is evacuated;

[0060] Introducing a first preset flow rate of hydrogen and argon into the quartz reaction chamber, setting a first radio frequency power and a first bias voltage between the anode and the cathode, and performing pretreatment;

[0061] A second preset flow rate of hydrogen and methane is introduced into the quartz reaction chamber, a second radio frequency power and a second bias voltage between the anode and the cathode are set, and a growth process is performed to obtain a microneedle array with a graphene structure prepared on the surface.

[0062] In some possible implementations, the first preset flow rate and the second preset flow rate, the first RF frequency and the second RF frequency, the first bias voltage and the second bias voltage can be set according to actual needs. This application does not limit the specific value of each parameter and the size relationship between their values.

[0063] Optionally, in one embodiment of the present invention, the preparation of the stainless steel microneedle array comprises:

[0064] Stainless steel was selected as the material for the microneedles;

[0065] Microneedle arrays were cut out on stainless steel plates by laser microcutting.

[0066] In some possible implementations, stainless steel may be selected as the base material of the microneedles.

[0067] Optionally, in one embodiment of the present invention, the step of modifying cholesterol oxidase on the surface of the microneedle array comprises:

[0068] Prepare cholesterol oxidase precursor solution;

[0069] The electrodes of the microneedle array are pulled in the cholesterol oxidase precursor solution for a preset number of times using the pulling method, and then dried to obtain a microneedle array with surface-modified cholesterol oxidase.

[0070] Optionally, in one embodiment of the present invention, the preparation of the cholesterol oxidase precursor solution comprises:

[0071] Dissolving cholesterol oxidase in PBS to obtain a first solution;

[0072] dissolving bovine serum albumin in PBS to obtain a second solution;

[0073] dissolving glutaraldehyde in PBS to obtain a third solution;

[0074] The first solution, the second solution and the third solution are mixed according to a preset volume ratio to obtain a cholesterol oxidase precursor solution.

[0075] Optionally, in one embodiment of the present invention, the microneedle array is coated with a water-soluble polymer to obtain a microneedle sensing electrode, comprising:

[0076] A polyvinyl pyrrolidone aqueous solution with a preset mass percentage is prepared, and the polyvinyl pyrrolidone is coated on the electrode surface of the microneedle array using a spray gun at a preset spraying pressure, a preset distance, and a preset spraying time; the preset distance is the distance between the nozzle and the microneedle array;

[0077] The solution was allowed to stand for evaporation of the water to obtain a microneedle sensing electrode coated with a water-soluble polymer.

[0078] In summary, the method provided in the embodiment of the present application includes: preparing a microneedle array, preparing a graphene structure on the surface of the microneedle array using chemical vapor deposition technology, and modifying cholesterol oxidase on the surface of the microneedle array; coating the microneedle array with a water-soluble polymer to obtain a microneedle sensing electrode. The present application constructs a graphene nanostructure on the surface of the microneedle and fixes cholesterol oxidase on the surface of the electrode to achieve a large specific surface area and high electron mobility of the sensor, which is conducive to improving the biocompatibility and detection accuracy of the sensor, and realizing accurate and convenient subcutaneous in situ detection of cholesterol.

[0079] On the other hand, an embodiment of the present invention provides a method for preparing an electrochemical sensor, the method comprising:

[0080] The microneedle sensing electrode obtained by the above-mentioned preparation method of the graphene-based microneedle sensing electrode is used as a working electrode;

[0081] Prepare a microneedle array and deposit titanium and platinum to obtain a counter electrode;

[0082] Prepare a microneedle array, deposit titanium and silver, and form a silver chloride film on the electrode by electrochemical oxidation to obtain a reference electrode;

[0083] The working electrode, the counter electrode and the reference electrode are packaged to obtain an electrochemical sensor.

[0084] In some embodiments, in one embodiment of the present invention, the working electrode, the counter electrode, and the reference electrode are packaged to obtain an electrochemical sensor, comprising:

[0085] Put the working electrode, the counter electrode and the reference electrode into a mold and fix them with a PDMS thin layer;

[0086] Pour liquid photosensitive resin into the mold, and perform curing and demoulding under ultraviolet light;

[0087] The PDMS thin layer is removed, and a wire is connected to each electrode to obtain an electrochemical sensor.

[0088] The implementation method provided by this application is described in detail below with a specific embodiment:

[0089] This application is based on minimally invasive subcutaneous cholesterol detection technology: the entire sensing system is based on a microneedle array, with minimally invasive, painless, real-time detection transdermal technology, which can easily carry out in vivo real-time biochemical signal sensing research. By exploring its in situ, painless transdermal, high-sensitivity detection of subcutaneous cholesterol signals, it provides a technical basis for early screening and prevention of cardiovascular diseases.

[0090] The present application relates to the precision processing of microneedle electrodes and surface modification with graphene nanomaterials: the precision preparation and processing technology of microneedle electrodes are complex. The present invention achieves a large specific surface area and high electron mobility of the sensor by constructing a graphene nanostructure on the surface of the microneedle and fixing cholesterol oxidase on the surface of the electrode, thereby providing a new idea and strategy for achieving accurate and convenient subcutaneous in situ detection of cholesterol.

[0091] The present application provides a soluble sensing protective coating: the existing research on microneedle patch technology mostly uses a single material or a simple composite structure. The microneedles with fine surface structures are easily damaged by mechanical friction during the transdermal process, which limits the application of microneedle technology to a certain extent. The present invention uses a water-soluble coating to protect the graphene structure on the surface of the microneedle without affecting its detection sensitivity.

[0092] Specifically, the enzyme immobilization technology of graphene structure: preparing graphene nanostructure on the surface of microneedles can expand the specific surface area of ​​microneedle electrodes, so that in the electrochemical process, the increase in reaction area leads to an enhancement of the detection signal. At the same time, due to the unique electronic structure and high electron mobility of graphene, it can also improve the signal-to-noise ratio of the detection signal. Allowing cholesterol oxidase to fully combine with the graphene structure and give full play to the advantages of this structure is the key to obtaining high-sensitivity sensing performance.

[0093] Protection of fine surface structures of microneedles during transdermal penetration: During transdermal penetration, the microneedle array will generate mechanical friction with the skin tissue, causing the surface nanostructure to peel off and break. Therefore, how to protect the surface nanostructure becomes a key factor in achieving high performance and stability of the sensor. The present invention intends to explore the use of water-soluble polymers to coat graphene-microneedle electrodes to protect the electrode surface nanostructure from damage during transdermal penetration without affecting its detection sensitivity.

[0094] Improved performance of subcutaneous real-time detection of cholesterol signals: Real-time detection in vivo faces many uncertainties and interference conditions, such as the temperature, humidity, pH of epidermal tissue, and subcutaneous inflammation and tissue fibrosis leading to electrode failure. Therefore, microneedle sensing electrodes need to have good biocompatibility and anti-interference performance.

[0095] Reference Figure 2 As shown, the present invention intends to construct an electrochemical sensor based on graphene-microneedle electrodes, prepare stainless steel microneedles by laser microcutting technology, prepare scale-arranged graphene structures on the surface of microneedles by chemical vapor deposition technology, and fix cholesterol oxidase on the surface of microneedles, explore water-soluble polymers to coat graphene-microneedle electrodes, protect the fine nanostructure on their surface during transdermal transmission, and test their sensitivity, detection limit and response time for detecting cholesterol in simulated fluid in vitro and subcutaneously in mice, and explore an in situ, subcutaneous cholesterol detection technology for early screening and prevention of cardiovascular diseases.

[0096] This application explores the preparation process of graphene-microneedle composite electrodes:

[0097] a) Explore the preparation of stainless steel microneedles by laser microcutting technology;

[0098] b) preparing a scale-like graphene structure on the surface of the microneedle by chemical vapor deposition technology, and modifying cholesterol oxidase on the surface;

[0099] c) Characterize the materials, morphology, and electrical properties of the graphene-microneedle composite electrode.

[0100] This application explores the use of water-soluble coatings to protect the fine structures on the surface of microneedles:

[0101] a) using water-soluble polymers to coat graphene-microneedles to protect the fine nanostructure on their surface;

[0102] b) explore the dissolution rate of the water-soluble coating after the microneedles are inserted subcutaneously;

[0103] c) Characterize the surface morphology changes of the microneedle before and after subcutaneous insertion.

[0104] Study on the performance of microneedle sensor for in-situ detection of cholesterol in this application:

[0105] a) Construction of electrochemical sensor based on graphene-microneedle electrode;

[0106] b) Evaluation and optimization of the cholesterol detection performance of graphene-microneedle sensor in in vitro simulated fluid;

[0107] c) Performance evaluation of in situ detection of cholesterol in mouse subcutaneous tissue.

[0108] Reference Figure 3 Schematic diagram of the preparation process of graphene-microneedle electrode and water-soluble coating. The process includes the following steps: (a) laser micro-cutting technology is used to carve out a microneedle array on a stainless steel plate. (b) graphene structure is grown on the surface of the microneedle array using ICPCVD technology. (c) cholesterol oxidase is fixed on the surface. (d) PVP coating is sprayed to protect the fine structure of the surface.

[0109] Specifically, preparation of graphene-microneedle composite electrode:

[0110] S21, first select corrosion-resistant 316L stainless steel as the material of the microneedle, and use laser micro-cutting technology to cut out the microneedle array on a stainless steel plate with a thickness of 0.1mm. The size, shape design and number of microneedles of the microneedle array can be flexibly adjusted according to experimental requirements. Among them, in order to maintain the transdermal effect and sensing performance of the microneedle, each microneedle is kept 700μm long and the distance between each microneedle is about 300μm.

[0111] S22, graphene structure on the surface of microneedles, was prepared by inductively coupled plasma enhanced chemical vapor deposition (ICPCVD) technology. First, the stainless steel microneedles were placed on the cathode in a quartz reaction chamber and heated to 900°C. At the same time, the chamber was evacuated to 5×10 -5 Torr or less. Then, H 2 The pretreatment was completed by igniting the anode and cathode at 900 W of RF power and 100 V bias voltage between the anode and cathode for 15 min. Next, the gas was changed to 60 sccm CH 4 and 10 sccm of H 2, the RF power was increased to 1100W, the bias voltage between the cathode and cathode was kept unchanged at 100V, and the growth process was completed after 20 minutes. After the system was cooled to room temperature in a vacuum, the sample was taken out to complete the preparation of the graphene structure. The micromorphology of the graphene-microneedle electrode was characterized by scanning electron microscopy (SEM). Graphene materials have good conductivity, large specific surface area and good biocompatibility, which can improve the performance of the sensor. If replaced with other carbon materials, on the one hand, it is difficult to guarantee the reliability of the material without experimental verification; on the other hand, the sensing performance of the sensor is also difficult to improve.

[0112] S23, prepare cholesterol oxidase (COx) precursor solution: dissolve cholesterol oxidase (50 mg mL -1 ), bovine serum albumin (80 mg mL -1 ), and glutaraldehyde (2.5wt% PBS), the above solutions are mixed in a volume ratio of 1:5:2 (sealed and stored at 4°C when not in use) to achieve the fixation and cross-linking of cholesterol oxidase on the surface of the microneedle. The graphene-microneedle electrode prepared in the present application is pulled three times in the above 200μL cholesterol oxidase precursor solution by the pulling method and then dried overnight. After drying at room temperature for 8h, the graphene-microneedle composite sensing electrode can be prepared.

[0113] Preparation and characterization of water-soluble protective coatings:

[0114] S31, prepare a 10% mass percent polyvinyl pyrrolidone (PVP) aqueous solution, and use a spray gun to coat the surface of the graphene-microneedle composite electrode obtained in step S23 with PVP. The diameter of the spray gun nozzle is 0.2mm, the spraying pressure is 0.2MPa, the distance between the nozzle and the microneedle is 10cm, and the spraying time is 30s. After the spraying is completed, let it stand for more than 1 hour to allow the water to evaporate, and the PVP to dry and harden. The preparation of the PVP coating is completed, and the morphology of the microneedle array is characterized by SEM. The role of PVP is to improve the mechanical strength of the microneedle array, and it can be replaced by polyimide (PI), polymethyl methacrylate (PMMA), etc.

[0115] S32, insert the coated microneedle into the subcutaneous tissue of a mouse (or replace it with pig skin) and then quickly pull it out. At this time, the PVP coating has not yet begun to dissolve. Then soak it in water for a certain period of time. After the PVP is fully dissolved, take it out and dry it. Observe the integrity of the nanostructure on the surface of the microneedle by SEM. If the PVP coating can better protect its surface nanostructure during the microneedle transdermal process, a surface morphology similar to that before the PVP coating should be observed.

[0116] S33, insert the coated microneedle into the subcutaneous tissue and keep it for a period of time (more than 5 minutes) before pulling it out, and use SEM to observe the residual PVP on the surface of the microneedle. If the PVP coating can be mostly dissolved in a short time, the surface morphology similar to that before the PVP coating should be observed.

[0117] Reference Figure 4 As shown, the preparation of graphene-microneedle composite sensor:

[0118] The sensor developed by the present invention adopts a three-electrode electrochemical sensing system.

[0119] Preparation of working electrode: The graphene-microneedle electrode prepared in step S23 is used as the working electrode.

[0120] Preparation of the electrode: Take a piece of stainless steel microneedle array of the same size, use magnetron sputtering to first deposit a layer of Ti of about 100nm as an adhesion layer, and then deposit a layer of Pt of about 300nm.

[0121] Preparation of reference electrode: Take a piece of stainless steel microneedle array of the same size, first use magnetron sputtering to deposit 100nm of Ti as an adhesion layer, then deposit 300nm of Ag, and then use electrochemical methods to apply 1μA current for 1min in a 1M concentration of KCl / HCl buffer solution to complete the preparation of the Ag / AgCl reference electrode.

[0122] Sensor packaging: (a) Place three prepared microneedle electrodes vertically into a PMMA mold, fix the microneedles with a 1 mm thick PDMS layer, and insert the electrode tails vertically downward with a spacing of 3 mm; (b) Pour liquid photosensitive resin into the mold; (c) 405 nm, 1.25 mW / cm 2 After curing under ultraviolet light for 2 minutes, demolding; (d) finally removing the PDMS thin layer and connecting wires to each microneedle electrode to complete the preparation of the sensor.

[0123] Embodiment 1:

[0124] In situ detection technology of blood lipids based on high-performance graphene microneedle composite sensing electrode.

[0125] In vitro cholesterol assay performance evaluation and optimization:

[0126] First, the sensor was assembled with a microfluidic channel made of PDMS, which was filled with 0.1M PBS solution (PH = 7.4). Cholesterol solutions with different concentration gradients (0–10mM) were prepared, and the sensing electrode was placed on the surface of the solution. Since cholesterol has a low solubility in PBS, 15% Triton was added as a dispersant, and the volume ratio of the two was 1:3. At the same time, glucose, galactose, urea, H2 O 2 et al., evaluated the selectivity and anti-interference performance of cholesterol sensors.

[0127] An electrochemical workstation was used and a constant potential of 0.1 V was applied on Ag / AgCl for steady-state current response. The detection limit of the sensor was analyzed by cyclic voltammetry or differential pulse voltammetry, and the sensitivity of the sensor was analyzed by step voltammetry.

[0128] Evaluation of the performance and biosafety of in situ detection of cholesterol in mouse subcutaneous tissue;

[0129] After the experimental mice were anesthetized and fixed, the microneedle tip of the graphene-microneedle composite sensor was pierced into the subcutaneous tissue of the mice to measure the electrical signal of subcutaneous cholesterol, and readings were taken every 15 minutes.

[0130] Blood was collected from the mouse tails at each measurement, and the mouse serum cholesterol concentration was calibrated with a commercial kit for comparison and evaluation of the accuracy of microelectrode detection. By comparing the concentrations of various indicators measured by the microelectrode sensor array and the standard detection method, the anti-interference, sensitivity, accuracy, and stability of the microelectrode array sensor for various detection indicators were evaluated.

[0131] Transdermal biosafety assessment: After the microneedle array is inserted into the skin of an ex vivo 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 mice are used as a control group to study the possible allergic reactions, tissue inflammation, and tissue fibrosis of the mouse skin tissue, and then evaluate the transdermal depth and biosafety of the electrode array.

[0132] On the other hand, an embodiment of the present invention provides a blood lipid detection system, including an electrochemical sensor obtained by the above-mentioned preparation method of the electrochemical sensor.

[0133] Similarly, the contents of the above method embodiments are all applicable to the system embodiments. The functions specifically implemented by the system embodiments are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those achieved by the above method embodiments.

[0134] In some selectable embodiments, the function / operation mentioned in the block diagram may not occur in the order mentioned in the operation diagram. For example, depending on the function / operation involved, the two boxes shown in succession can actually be executed substantially simultaneously or the boxes can sometimes be executed in reverse order. In addition, the embodiment presented and described in the flow chart of the present invention is provided by way of example, for the purpose of providing a more comprehensive understanding of technology. The disclosed method is not limited to the operation and logic flow presented herein. Selectable embodiments are expected, wherein the order of various operations is changed and the sub-operation of a part for which is described as a larger operation is performed independently.

[0135] In addition, although the present invention is described in the context of functional modules, it should be understood that, unless otherwise specified, one or more of the functions and / or features can be integrated into a single physical device and / or software module, or one or more functions and / or features can be implemented in a separate physical device or software module. It is also understood that a detailed discussion of the actual implementation of each module is unnecessary for understanding the present invention. More specifically, in view of the properties, functions and internal relationships of the various functional modules in the device disclosed herein, the actual implementation of the module will be understood within the conventional skills of the engineer. Therefore, those skilled in the art can implement the present invention set forth in the claims without excessive experimentation using ordinary techniques. It is also 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.

[0136] The logic and / or steps represented in the flowchart or otherwise described herein, for example, may be considered as an ordered list of executable programs for implementing the logical functions, and may be embodied in any computer-readable medium for use by a program execution system, device or apparatus (such as a computer-based system, a system including a processor, or other system that can fetch and execute a program from a program execution system, device or apparatus), or in conjunction with such program execution systems, devices or apparatuses. For purposes of this specification, a "computer-readable medium" may be any device that can contain, store, communicate, propagate or transmit a program for use by a program execution system, device or apparatus, or in conjunction with such program execution systems, devices or apparatuses.

[0137] In the above description of this specification, the description with reference to the terms "one embodiment / example", "another embodiment / example" or "certain embodiments / examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does 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.

[0138] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

[0139] The above is a specific description of the preferred implementation of the present invention, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of the present invention.

Claims

1. A method for preparing a graphene-based microneedle sensing electrode, characterized in that: The method comprises: preparing a microneedle array, preparing a graphene structure on the surface of the microneedle array using a chemical vapor deposition technique, and modifying cholesterol oxidase on the surface of the microneedle array; The microneedle array is coated with a water-soluble polymer to obtain a microneedle sensing electrode.

2. The method for preparing a graphene-based microneedle sensing electrode according to claim 1, characterized in that: The method of preparing the microneedle array, using chemical vapor deposition technology to prepare a graphene structure on the surface of the microneedle array, comprises: Preparation of stainless steel microneedle arrays; A graphene structure is prepared on the surface of the stainless steel microneedle array by inductively coupled plasma enhanced chemical vapor deposition technology.

3. The method for preparing a graphene-based microneedle sensing electrode according to claim 2, characterized in that: The method of preparing a graphene structure on the surface of the stainless steel microneedle array by inductively coupled plasma enhanced chemical vapor deposition technology comprises: The stainless steel microneedle is placed on the cathode in the quartz reaction chamber for heating, and the quartz reaction chamber is evacuated; Introducing a first preset flow rate of hydrogen and argon into the quartz reaction chamber, setting a first radio frequency power and a first bias voltage between the anode and the cathode, and performing pretreatment; A second preset flow rate of hydrogen and methane is introduced into the quartz reaction chamber, a second radio frequency power and a second bias voltage between the anode and the cathode are set, and a growth process is performed to obtain a microneedle array with a graphene structure prepared on the surface.

4. The method for preparing a graphene-based microneedle sensing electrode according to claim 2, characterized in that: The method for preparing the stainless steel microneedle array comprises: Stainless steel was selected as the material for the microneedles; Microneedle arrays were cut out on stainless steel plates by laser microcutting.

5. The method for preparing a graphene-based microneedle sensing electrode according to claim 1, characterized in that: The cholesterol oxidase is modified on the surface of the microneedle array, comprising: Prepare cholesterol oxidase precursor solution; The electrodes of the microneedle array are pulled in the cholesterol oxidase precursor solution for a preset number of times using the pulling method, and then dried to obtain a microneedle array with surface-modified cholesterol oxidase.

6. The method for preparing a graphene-based microneedle sensing electrode according to claim 5, characterized in that: The preparation of the cholesterol oxidase precursor solution comprises: Dissolving cholesterol oxidase in PBS to obtain a first solution; dissolving bovine serum albumin in PBS to obtain a second solution; dissolving glutaraldehyde in PBS to obtain a third solution; The first solution, the second solution and the third solution are mixed according to a preset volume ratio to obtain a cholesterol oxidase precursor solution.

7. The method for preparing a graphene-based microneedle sensing electrode according to claim 1, characterized in that: The method of coating the microneedle array with a water-soluble polymer to obtain a microneedle sensing electrode comprises: A polyvinyl pyrrolidone aqueous solution with a preset mass percentage is prepared, and the polyvinyl pyrrolidone is coated on the electrode surface of the microneedle array using a spray gun at a preset spraying pressure, a preset distance, and a preset spraying time; the preset distance is the distance between the nozzle and the microneedle array; The solution was allowed to stand for evaporation of the water to obtain a microneedle sensing electrode coated with a water-soluble polymer.

8. A method for preparing an electrochemical sensor, characterized in that: The method comprises: Using the microneedle sensing electrode obtained by the method for preparing a graphene-based microneedle sensing electrode according to any one of claims 1 to 7 as a working electrode; Prepare a microneedle array and deposit titanium and platinum to obtain a counter electrode; Prepare a microneedle array, deposit titanium and silver, and form a silver chloride film on the electrode by electrochemical oxidation to obtain a reference electrode; The working electrode, the counter electrode and the reference electrode are packaged to obtain an electrochemical sensor.

9. The method for preparing an electrochemical sensor according to claim 8, characterized in that: The step of encapsulating the working electrode, the counter electrode, and the reference electrode to obtain an electrochemical sensor comprises: Put the working electrode, the counter electrode and the reference electrode into a mold and fix them with a PDMS thin layer; Pour liquid photosensitive resin into the mold, and perform curing and demoulding treatment under ultraviolet light; The PDMS thin layer is removed, and a wire is connected to each electrode to obtain an electrochemical sensor.

10. A blood lipid detection system, characterized in that: include: The electrochemical sensor is obtained by the method for preparing the electrochemical sensor according to any one of claims 8 to 9.

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