CN117379048A

CN117379048A - Microneedle electrode array, preparation method thereof and microneedle array patch type sensing device

Info

Publication number: CN117379048A
Application number: CN202311283034.1A
Authority: CN
Inventors: 李迎春; 刘家希; 刘江; 杨娇; 刘育坚; 彭锌
Original assignee: Shenzhen Graduate School Harbin Institute of Technology
Current assignee: Shenzhen Graduate School Harbin Institute of Technology
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-01-12

Abstract

The application relates to the technical field of electrochemical biosensors and provides a microneedle electrode array, a preparation method and a microneedle array patch type sensing device, wherein the microneedle electrode array comprises a plurality of working electrodes, counter electrodes and reference electrodes; each working electrode takes the counter electrode as a center and is uniformly distributed around the counter electrode, the reference electrode and any one of the working electrodes are arranged at intervals, and the reference electrode and the counter electrode are arranged at intervals; the surface of the tip end parts of the working electrode, the counter electrode and the reference electrode are respectively provided with a swelling resin layer used for isolating the external environment, and the swelling resin layers are in a three-dimensional porous three-dimensional structure after absorbing water. The micro-needle electrode is a core element of the electrochemical sensing device, and the swelling resin layers are coated on the needle tip sensing areas of the micro-needle electrode, so that the electrode interface can be effectively protected from pollution, and subcutaneous interference is reduced.

Description

Microneedle electrode array, preparation method thereof and microneedle array patch type sensing device

Technical Field

The application belongs to the technical field of electrochemical biosensors, and particularly relates to a microneedle electrode, a preparation method and a microneedle array patch type sensing device.

Background

Electrochemical biosensors have important applications in medical science, biological research and clinical diagnosis, and in recent years, wearable sensors with simple structures and convenient use become research hotspots, and have great potential in early disease diagnosis, daily medical care and the like. Wearable sensors can generally be divided into two categories, namely non-invasive devices and invasive devices. Recent studies have focused mainly on the former, where the original biological sample is mainly sweat, saliva or extracted interstitial fluid. Although non-invasive devices have the advantage of being non-invasive and portable, they are still currently in the laboratory stage, mainly because the correlation between biomarker concentration in epidermal secretions and biomarker concentration in endogenous body fluids (e.g. blood) has not been fully verified. Notably, since the content of the bio-small molecular markers in the subcutaneous intercellular fluid is highly correlated with the content of the small molecular markers in the blood, the minimally invasive wearable devices for tissue fluid analysis are attracting attention of researchers.

Among them, the electrochemical biosensor based on the micro needle is a typical representative of the minimally invasive device, and because the micro needle is an invasive action form, the micro needle can perform functions deep into tissues, benefit from a micro size and a fine structure, has a small dependence on the sample size, can realize high-efficiency detection of interstitial fluid, and has been widely used for in-situ monitoring of subcutaneous biomarkers at present. For example, J.Wang et al developed a fully integrated wearable microneedle array for monitoring glucose, lactate and alcohol in interstitial fluid with good correlation to standard methods in blood or breath. In addition, the great success of glucose monitoring systems in the international market also verifies the effectiveness of microneedle technology. However, most of the current micro-needle electrochemical sensors are complex to prepare, are not easy to produce in large scale, and are easily polluted by substances such as skin cutin, grease, skin care products and the like when penetrating into the skin, so that the performance of a sensing interface is reduced. In addition, the components in tissue fluids are complex, and electrochemical sensing of current subcutaneous biomarkers is susceptible to complex components. Therefore, the development of the micro-needle electrochemical sensor which is simple to prepare, low in cost, strong in anti-fouling performance and strong in anti-interference performance has important significance.

Disclosure of Invention

The application aims to provide a microneedle electrode array, a preparation method and a microneedle array patch type sensing device, and aims to solve the problem that an electrochemical sensor of a subcutaneous biomarker is poor in anti-interference performance.

In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows:

in a first aspect, the present application provides a microneedle electrode array comprising a plurality of working electrodes, a counter electrode, and a reference electrode; each working electrode takes the counter electrode as a center and is uniformly distributed around the counter electrode, the reference electrode and any one of the working electrodes are arranged at intervals, and the reference electrode and the counter electrode are arranged at intervals; any one of the working electrode and the counter electrode comprises a first metal microneedle and a noble metal layer modified on the surface of the first metal microneedle; the reference electrode comprises second metal micro-needles which are arranged at intervals with any of the first metal micro-needles, and a silver/silver chloride layer which is modified on the surface of the second metal micro-needles; the surface of the tip end parts of the working electrode, the counter electrode and the reference electrode are respectively provided with a swelling resin layer used for isolating the external environment, and the swelling resin layers are in a three-dimensional porous three-dimensional structure after absorbing water.

In a second aspect, the present application provides a method for preparing a microneedle electrode array, comprising the steps of:

surface modification of the metal microneedle: forming a noble metal layer on the surface of the first metal microneedle by an ion sputtering method; forming a silver/silver chloride layer on the surface of the second metal microneedle by an electrochemical deposition method to obtain a first metal microneedle and a second metal microneedle with surface modified;

swelling resin modified metal microneedle: coating a swelling resin precursor on the tip parts of the surface modified first metal micro-needle and the second metal micro-needle, and vacuum drying to obtain the micro-needle electrode.

In a third aspect, the present application provides a patch-type electrochemical sensing device with a microneedle array, which comprises a microneedle electrode, wherein the microneedle electrode is a microneedle electrode according to the first aspect or a microneedle electrode obtained by a preparation method according to the second aspect.

The microneedle electrode array is characterized in that a swelling resin layer is coated on a needle tip sensing area of the microneedle electrode, so that an electrode interface is protected from pollution, and subcutaneous interference is reduced. From the effect point of view, the unswollen resin is used as a protective layer of the microneedle electrode, and the surface of the electrode is isolated from skin secretion, exogenous skin care products and epidermis horny layer when the microneedle is inserted into skin, so that the microneedle is kept clean, the sensing device with the microneedle electrode has excellent anti-fouling performance, and the swelling resin has excellent water absorption, so that when the microneedle enters subcutaneous tissue, the resin naturally expands and rapidly forms a three-dimensional porous character structure, small molecules and ions including oxygen reach the interface of the microneedle, and the transportation of biological macromolecules is blocked due to the molecular sieve effect of the gel swelling resin, which is likely to be beneficial to ensure the specificity of the micro-needle for the oxygen and reduce potential matrix interference, and the sensing device with the microneedle electrode has excellent anti-interference performance.

The preparation method of the microneedle electrode array is simple and easy to operate. The surface of the metal microneedle is modified, the sensing area and the sensing performance of the microneedle are increased, then the swelling resin is coated on the tip sensing area, the electrode interface is protected from pollution, and subcutaneous interference is reduced.

The patch type sensing device for the microneedle array provided by the third aspect of the application is low in cost and simple to manufacture, can be successfully applied to monitoring subcutaneous oxygen content during exercise, and can timely distinguish the exercise state. The sensing device provides a general device strategy for detecting other biomarkers in interstitial fluid. It is envisioned that the microneedle array patch sensing devices provided herein will play a positive role in respiratory disease assessment, surgical monitoring, and public health care.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following description will briefly introduce the drawings that are needed in the embodiments or the description of the prior art, it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an SEM image of a bare microneedle (panel a) and EDS spectrum of a bare microneedle (panel b);

FIG. 2 is an SEM image (panel a) of a gold-modified microneedle provided in comparative example 1 and an EDS spectrum (panel b) of the gold-modified microneedle;

FIG. 3 is an SEM image of a swollen resin modified microneedle provided in example 1 of the present application (panel a) and an EDS spectrum of a swollen resin modified microneedle (panel b);

FIG. 4 is a microneedle electrode unit at 5mM K at various modification stages ₃ [Fe(CN) ₆ ]CV curve in solution (a graph); electrochemical impedance diagram (b-diagram) of different modified microneedle electrodes

FIG. 5 is a CV response of microneedle electrode units at different modification stages in 150mmHg oxygen solution;

FIG. 6 is a schematic illustration of the swelling phase of a microneedle array patch sensor device (MNAP) in the epidermis (i, swelling resin modified microneedles (SR/Au/MN) penetrating the skin; ii, swelling resin modified microneedles SR/Au/MN in the subcutaneous swelling phase; iii, swelling resin modified microneedles (SR/Au/MN) removing phase from the epidermis);

FIG. 7 is an SEM image of a swollen resin modified microneedle (SR/Au/MN) after lyophilization in the swelling phase ii (panel a); EDS spectra of swollen resin modified microneedles (SR/Au/MN) after lyophilization in the swelling ii phase (b plot);

FIG. 8 is an SEM image of a swollen resin modified microneedle (SR/Au/MN) at the swelling iii stage (panel a); EDS energy spectrum of swelling resin modified microneedle (SR/Au/MN) at swelling iii period (b plot);

FIG. 9 is a graph of swelling properties of a swelling resin, a swelling ratio curve (a graph) of a swelling resin material; resistance change curve of swelling resin modified microneedle (SR/Au/MN) during swelling (b plot);

FIG. 10 is an electrochemical impedance plot (a plot) of a swollen resin modified microneedle (SR/Au/MN) before and after full swelling; CV curves before and after SR/Au/MN fully swelled in the probe solution (b plot); CV response curves to oxygen before and after SR/Au/MN fully swelled (c plot);

FIG. 11 is a CV curve (a graph) of a microneedle array patch sensor device (MNAP) in a probe solution at a scanning rate of 10 to 200 mv/s; graph of redox peak current versus square root of scan rate in each curve (b graph);

FIG. 12 is a graph of response to oxygen in a hydrogel with or without petrolatum for swollen resin modified microneedles (SR/Au/MN) and gold modified microneedles (Au/MN);

FIG. 13 is a graph of response of swollen resin modified microneedles (SR/Au/MN) and gold modified microneedles (Au/MN) to oxygen in PBS with and without BSA interfering substrates;

FIG. 14 is a graph showing the current response of a microneedle array patch electrochemical sensor (MNAP) to a range of oxygen concentrations in the range of 0 to 150mmHg (a graph). The potential was set at-0.8V and the electrolyte solution was 0.1M PBS; a calibration fit curve between current and oxygen content (b plot);

FIG. 15 is a drawing of Na added to a saturated oxygen solution ₂ SO ₃ Oxygen ampere curves obtained by adopting a microneedle array patch type sensing device (MNAP) test before and after deoxidization;

FIG. 16 is a reaction of a microneedle array patch sensor device (MNAP) to oxygen in the presence of different interfering substrates. And (3) injection: i and I ₀ Representing the current of MNAP in the interfering solution containing the target of analysis and the current obtained from the initial solution, respectively. (potentially interfering substances include 50mmHg CO ₂ 、680mmHg N ₂ 3mg/mL KCl, 0.9% NaCl, 12mg/mL IgG, 140mg/dL glucose and 15mg/dL lactic acid);

FIG. 17 is a graph showing the detection of oxygen concentration using a microneedle array patch sensor (MNAP) and a commercial instrument, respectively (the inset shows the linear relationship between the detection results of the MNAP and the commercial instrument);

FIG. 18 is a graph of oxygen detection of 150mmHg using a microneedle array patch sensor (MNAP) for 7 days (notes: I and I) ₀ Representing the current values obtained for a given day and the first day, respectively);

FIG. 19 is an in vivo performance of a microneedle array patch sensor device (MNAP); a photograph of MNAP being worn on the arm of the participant (a-picture); a comparison plot (b plot) of MNAP oxygen partial pressure measurements versus sports smart watch oxygen saturation and heart rate reference measurements;

FIG. 20 is a schematic illustration of a modification process from a commercial microneedle to a workable microneedle electrode unit;

FIG. 21 is a photograph of the front and back sides of a microneedle array patch sensor device (MNAP) (panel a); exploded subassembly view (b-view) of fully integrated MNAP (i, one custom micro electrochemical workstation; ii, microneedle electrode array fixed on resin plate; iii, adhesive layer; iv, gas barrier layer);

FIG. 22 is a schematic view of a microneedle electrode array provided in example 1 of the present application (a-view); schematic structural diagram of microneedle electrode unit (b-drawing).

Wherein, each reference sign in the figure:

1-working electrode 2-counter electrode 3-reference electrode 4-first metal micropin or second metal micropin 5-noble metal layer or silver/silver chloride layer 6-insulating layer 7-swelling resin layer

Detailed Description

In order to make the technical problems, technical schemes and beneficial effects to be solved by the present application more clear, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application.

In this application, the term "and/or" describes an association relationship of an association object, which means that there may be three relationships, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.

In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.

It should be understood that, in various embodiments of the present application, the sequence number of each process does not mean that the sequence of execution is sequential, and some or all of the steps may be executed in parallel or sequentially, where the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weights of the relevant components mentioned in the embodiments of the present application may refer not only to specific contents of the components, but also to the proportional relationship between the weights of the components, and thus, any ratio of the contents of the relevant components according to the embodiments of the present application may be enlarged or reduced within the scope disclosed in the embodiments of the present application. Specifically, the mass described in the specification of the examples of the present application may be a mass unit known in the chemical industry such as μ g, mg, g, kg.

The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.

A first aspect of the present embodiment provides a microneedle electrode array, as shown in fig. 22, including a plurality of working electrodes 1, a counter electrode 2, and a reference electrode 3; each working electrode 1 takes a counter electrode 2 as a center, is uniformly arranged around the counter electrode 2, the reference electrode 3 is arranged at intervals with any working electrode 1, and the reference electrode 3 is arranged at intervals with the counter electrode 2; each of the working electrode 1 and the counter electrode 2 comprises a first metal microneedle 4 and a noble metal layer 5 modified on the surface of the first metal microneedle 4; the reference electrode 3 comprises second metal micro-needles which are arranged at intervals with any first metal micro-needle 4, and a silver/silver chloride layer which is modified on the surface of the second metal micro-needle; the surfaces of the tips of the working electrode 1, the counter electrode 2 and the reference electrode 3 are each provided with a swelling resin layer 7 for isolating the external environment, and the swelling resin layer 7 assumes a three-dimensional porous three-dimensional structure after absorbing water (as shown in fig. 3, 6 and 7).

According to the microneedle electrode array provided by the first aspect of the application, the metal microneedles (commercial acupuncture needles) modified by the swellable resin and the noble metal are used as the sensing units, and the sensing area of the microneedles is remarkably increased by the three-dimensional porous precious metal modified microneedles, so that the sensing performance is greatly improved. In addition, the swelling resin exhibits excellent anti-fouling and anti-interference properties in the sensing platform. The swelling resin layer is coated on the sensing area of the tip of the microneedle electrode, which can play the following roles: (1) The unswollen resin serves as a protective layer of the microneedle electrode, and separates the electrode surface from skin secretions (endogenous oil and sweat secretion), exogenous skin care and epidermis stratum corneum when the microneedles pierce the skin, thereby keeping the microneedle electrode clean. (2) The fully swelled resin is used as electrolyte gel to provide liquid environment for sensing, and the electrode surface is fully exposed to form a good oxygen electroreduction interface. When the microneedle electrode penetrates the skin and enters the subcutaneous tissue, a swelling process naturally occurs by absorbing the liquid matrix in the interstitial fluid of the skin. Since the resin has excellent water absorption properties, the resin rapidly forms a three-dimensional porous structure so that small molecules including oxygen and ions can be freely dispersed to the interface of the microneedle electrode, and the molecular sieve effect of the gel-like swelling resin hinders the transportation of biological macromolecules. This may be advantageous to ensure oxygen specificity of the microneedle electrodes, reducing potential matrix interference.

In some embodiments, as shown in fig. 22, the middle surfaces of the working electrode 1, the counter electrode 2, and the reference electrode 3 are all provided with an insulating layer 6; the middle part is a region with the width of 2-3 mm and is positioned 2-4 mm away from the tip part. After sputtering noble metal on the surface of the microneedle, an insulating layer is then provided in the middle area in order to form a constant sensing area at the tip. Further, the insulating layer material is selected from insulating materials with good biocompatibility and unidirectional adhesion, for example: medical epoxy resins.

In some embodiments, the noble metal layer includes a gold layer and/or a platinum layer. A gold layer or a platinum layer is arranged on the surface of the metal microneedle, so that the sensing area of the metal microneedle is increased, and the sensing performance is improved; further, the noble metal layer is a gold layer, so that the sensing performance is good, and the material is easy to obtain.

In some embodiments, the diameters of the first metal micro-needle and the second metal micro-needle are 0.12-0.3 mm, and the metal micro-needle of the invention adopts commercial superfine stainless steel needles, and the diameters of the metal micro-needles mainly comprise: 0.12mm, 0.14mm, 0.16mm, 0.18mm, 0.20mm, 0.25mm, 0.30mm.

In some embodiments, the total number of the first metal micro-needles and the second metal micro-needles is 4-10, the number of the micro-needle electrodes can be selected to be 4, 5, 6, 7, 8, 9 and 10, when the number of the micro-needles is too small, the subcutaneous sensing position is single, the detection accuracy is reduced, when the number of the micro-needles is too large, the pain of a wearer is aggravated, and further, 6-8 micro-needle electrodes are selected to be optimal, wherein 1 counter electrode and 1 reference electrode are respectively adopted, and 4-6 working electrodes are adopted.

In some embodiments, the distance between every two first metal microneedles is 2-6 mm, wherein the working electrodes are connected in series, the counter electrode is placed in the middle of the working electrode and is equidistant from the working electrodes, the distance between the two microneedles can be 2-6 mm, and excessive or insufficient distance between the microneedles can lead to signal reduction or crosstalk. Thus, further, the microneedle electrode spacing is selected to be 3 to 5mm.

In some embodiments, the first metal micro-needle and the second metal micro-needle are selected from one of steel needle, silver plated needle, gold needle and gold plated needle, wherein the first metal micro-needle is used as a working electrode and a counter electrode, and steel needle, silver plated needle and the like can be selected, and more preferably, in one embodiment, a commercially available stainless steel needle moxibustion needle is selected, so that the manufacturing cost is low, and the manufacturing is simple. The second metal microneedle is used as a reference electrode, a silver-plated needle, a silver needle, or the like may be selected for cost reasons, and more preferably, a silver-plated stainless steel needle is selected in one embodiment.

In some embodiments, the swelling resin layer comprises a polymer of a polymethylvinyl ether/maleic acid copolymer with polyethylene glycol, or a polymer of hyaluronic acid with methacrylic anhydride, or a polystyrene-polyacrylamide based resin.

A second aspect of the embodiments of the present application provides a method for preparing a microneedle electrode array, including the following steps:

In some embodiments, the method further comprises the step of cleaning, degreasing and rust removing the metal microneedles before the surface modification of the metal microneedles. The method comprises the following steps: respectively soaking the metal micro-needles in ethanol, a metal degreasing agent and a metal rust remover, and ultrasonically cleaning the metal micro-needles in the solution for 5-10 min. Further, each step needs to be rinsed with deionized water, and finally the dry and clean micro-needles are taken for standby.

In some embodiments, the method further comprises the step of coating the central surfaces of the first metal microneedles and the second metal microneedles with an insulating layer material after the metal microneedle surface modification step and before the swelling resin modification metal microneedles step. The method comprises the following steps: the remainder of the microneedle, except the sensing area of the tip and the contact area of the tip, is coated with an insulating material in order to form a constant sensing area at the tip of the microneedle electrode. Further, the insulating material is medical epoxy resin.

In some embodiments, the specific steps of forming the noble metal layer on the surface of the first metal microneedle by an ion sputtering method are as follows: and sputtering noble metal on the surface of the first metal microneedle by adopting an ion sputtering instrument with a noble metal target source, wherein the sputtering time is 1-6 min. Further, sputtering gold on the surface of the first metal microneedle by adopting an ion sputtering instrument with Jin Bayuan, wherein the sputtering time is 2-4 min. If the sputtering time is too short or too long, the sensing performance is easily affected, so the sputtering time is optimally 2-4 min.

In some embodiments, the specific steps of forming a silver/silver chloride layer on the surface of the second metal microneedle by electrochemical deposition are: the second metal micro-needle is a silver plating needle, and then the silver plating needle is immersed into a mixed solution of chloride and HCl for electrochemical deposition treatment, and is washed by deionized water and placed in the chloride solution in a dark place; the conditions of the electrochemical deposition treatment are as follows: the scanning speed is 0.01-0.2V/s, the voltage is-0.10V-1.00V or-0.15V-1.05V or 0.2V-1.1V, and the scanning is carried out for 1-6 periods. Further, the scanning speed is 0.05V/s, the scanning voltage range is-0.15V to 1.05V, and the number of scanning turns is 3-5, so that the chlorination effect is optimal.

In some embodiments, the step of swelling the resin-modified metal microneedle is specifically: coating the swelling resin precursor solution on the tip parts of the surface modified first metal micro-needle and the second metal micro-needle, and carrying out vacuum drying treatment for 8-10 h at 25-35 ℃.

In some embodiments, the swelling resin precursor solution comprises: a polymer of a polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol; or, a polymer of hyaluronic acid and methacrylic anhydride; or, a polystyrene-polyacrylamide-based resin.

In some embodiments, the method of preparing the swollen resin precursor solution is: the polymethyl vinyl ether/maleic acid copolymer (PMVE/MA), polyethylene glycol 8000 (polyethylene glycol with average molecular weight of 8000), sodium carbonate and sodium polyacrylate are stirred for 1 to 3 hours at the temperature of 40 to 80 ℃. The concentration of the polymethyl vinyl ether/maleic acid copolymer (PMVE/MA) is 2.5 to 15wt% based on 100% total mass of the swollen resin precursor solution, such as: may be selected to be 15wt%, 12.5wt%, 10wt%, 7.5wt%, 5wt%, 2.5wt%, etc.; the concentration of polyethylene glycol is 2.5-10wt%, such as: 10wt%, 7.5wt%, 5wt%, 2.5wt% and the like can be selected, and the concentration of sodium carbonate is 1 to 5wt%, such as: the concentration of sodium polyacrylate can be selected to be 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, and the concentration of sodium polyacrylate is 0.1-0.3 wt%, such as: may be selected to be 0.1wt%, 0.2wt%, 0.3wt%.

In one embodiment, the method of preparing the swelling resin precursor solution is: based on 100% of the total mass of the swelling resin precursor solution, 5wt% of polymethyl vinyl ether/maleic acid copolymer (PMVE/MA), 5wt% of polyethylene glycol 8000, 3wt% of sodium carbonate and 0.2wt% of sodium polyacrylate are stirred and mixed for 2-3 hours at 60-80 ℃.

In some embodiments, the swelling resin precursor solution comprises a polymer of hyaluronic acid and methacrylic anhydride (MeHA) prepared by: n, N-Dimethylformamide (DMF) and Methacrylic Anhydride (MA) were added sequentially to an aqueous solution of Hyaluronic Acid (HA), and stirred overnight to give a precursor gel (MeHA). Subsequently, naOH was added and the precursor gel (MeHA) was washed several times with ethanol while maintaining pH 8-9.

In some embodiments, the swelling resin precursor solution includes a method of preparing a polystyrene-polyacrylamide-based resin: the preparation method comprises the steps of taking poly (tert-butyl acrylate) -polystyrene block copolymer (PSb-ptBA) and methylene dichloride as raw materials, taking trifluoroacetic acid as a catalyst, hydrolyzing for 48-72 h to prepare polystyrene-polyacrylamide (PS-b-PAA), precipitating the PS-b-PAA by using normal hexane, filtering and washing for several times. The PS-b-PAA is dissolved in N, N-Dimethylformamide (DMF) by adopting a solvent casting method, and then is degassed.

In a third aspect, the present application provides a microneedle array patch-type sensing device, including a microneedle electrode array, where the microneedle electrode array is a microneedle electrode array according to the first aspect or a microneedle electrode array obtained by a preparation method according to the second aspect.

In some embodiments, a microneedle array patch sensing device comprises: a substrate; a microneedle electrode array fixedly disposed on the substrate; the electrochemical workstation is provided with a through hole; the substrate sequentially comprises a resin plate, an adhesion layer and an air-proof layer; one surface of the resin plate, which is away from the microneedle electrode array, is connected with the electrochemical workstation, and the microneedle electrode array is electrically connected with the electrochemical workstation through the through hole.

In some embodiments, as shown in fig. 21, the electrochemical workstation is a custom micro potentiostat having through holes corresponding to the number and diameter of the microneedle electrodes, which are electrically connected to the micro electrochemical workstation through the through holes thereon. The microneedle electrodes were fixed in a resin plate, which was integrally molded by 3D printing. The internal dimensions of the resin plates are consistent with those of the micro potentiostat, ensuring their close fit. The adhesive layer can be made of medical non-woven fabrics, and the medical non-woven fabrics can be cut into the required shape and size, such as: can be cut into capsules of 60X 40 mm.

In order to prevent oxygen in the external environment from interfering with the test, a layer of breathable film is integrated in the microneedle array patch-type electrochemical sensing device. In some embodiments, the gas barrier layer may be selected from Polyethylene (PE), polyvinylidene chloride (PVDC), chlorinated polyvinyl chloride (CPVC), polytetrafluoroethylene (PTFE), and the like. In a preferred embodiment, polyvinylidene chloride (PVDC) film is selected because polyvinylidene chloride (PVDC) is relatively impermeable to air. Cutting polyvinylidene chloride films into desired shapes and sizes, such as: a wafer with the diameter of 20mm is stuck on the medical non-woven fabric of the adhesive layer.

The microneedle array patch is formed by combining microneedles with wearable technology, and taking commercial acupuncture needles decorated by swellable resin and noble metal as sensing units, and can continuously monitor the subcutaneous oxygen content in the movement process on line. The introduction of commercial microneedles provides array patches with many advantages, such as ease of mass production, stable performance, and cost effectiveness. The three-dimensional porous noble metal modified microneedle significantly increases the sensing area of the microneedle, thereby greatly improving the sensing performance. In addition, the swelling resin exhibits excellent anti-fouling and anti-interference properties in the sensing platform. After the patch is integrated with the microneedle sensing unit, the sensor has the advantages of large sensing area, high signal intensity, large detection area and the like. The microneedle array patch is integrated with a small-sized working station with a Bluetooth transmission function, so that data can be transmitted to a mobile terminal in real time, and subcutaneous oxygen change in the field continuous monitoring movement process is realized. The micro-needle array patch type electrochemical sensing device has the advantages of simple manufacture, low price, easy storage and the like, plays a good role in real-time analysis of subcutaneous oxygen content, and can be used as a universal tool for analyzing other substances in interstitial fluid (ISF).

The following description is made with reference to specific embodiments.

Example 1

A microneedle electrode array is divided into 6 working electrodes, 1 reference electrode and 1 counter electrode. The working electrode and the counter electrode are both made of commercial stainless steel needle moxibustion needles with the diameter of 0.16mm, and the surfaces of the working electrode and the counter electrode are modified with gold layers; the reference electrode is made of silver-plated acupuncture needle with diameter of 0.16mm, and the surface is modified with silver/silver chloride layer. The surfaces of the tip end parts of the working electrode, the counter electrode and the reference electrode are respectively provided with a swelling resin layer used for isolating the external environment, the swelling resin layer is in a three-dimensional porous three-dimensional structure after absorbing water, and the swelling resin layer comprises a polymer of polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol. The working electrodes are uniformly distributed around the counter electrode by taking the counter electrode as the center, the reference electrode is arranged at intervals with any working electrode, and the reference electrode is arranged at intervals with the counter electrode.

A method of preparing a microneedle electrode array comprising the steps of:

s1: putting a commercially available stainless steel needle moxibustion needle into an ion sputtering device with Jin Bayuan to sputter for 2-4 min to obtain a gold-modified stainless steel needle moxibustion needle (Au/MN); immersing a commercially available silver-plated acupuncture needle into a mixed solution of 0.1M potassium chloride and 0.01M hydrochloric acid, scanning for 3-5 periods from-0.15V to 1.05V at a scanning rate of 0.05V/s, then flushing with deionized water, and placing in a 0.1M potassium chloride solution in a dark place for 24-36 hours to obtain a silver-plated acupuncture needle (Ag-AgCl/MN) after chlorination;

S2: coating the swelling precursor solution on the Au/MN and the tip sensing area of the Ag-AgCl/MN, and vacuum drying for 8-10 h at the temperature of 25-30 ℃ to obtain the microneedle electrode. The preparation method of the swelling resin precursor solution comprises the following steps: based on 100% of the total mass of the swelling resin precursor solution, a polymethyl vinyl ether/maleic acid copolymer (PMVE/MA) with a concentration of 5wt%, a polyethylene glycol (average molecular weight 8000) with a concentration of 5wt%, a sodium carbonate with a concentration of 3wt%, a sodium polyacrylate with a concentration of 0.2wt%, and stirring and mixing at 60-80 ℃ for 2-3 hours.

A micro-needle array patch type sensing device is assembled by a customized micro-potentiostat, the micro-needle electrode array fixed on a resin plate, medical non-woven fabrics and a polyvinylidene chloride film. The custom made micro potentiostat had 8 through holes of 0.16mm diameter through which the microneedles were electrically connected to the micro electrochemical workstation. The microneedle electrode array was fixed in a resin plate, the distance between every two microneedles was 4mm, and the resin plate was integrally formed by 3D printing. The internal dimensions of the resin plates are consistent with those of the micro potentiostat, ensuring their close fit. The medical non-woven fabric layer is cut into capsules with the diameter of 60 multiplied by 40mm by a laser cutting machine. In addition, the polyvinylidene chloride film was cut into a disc having a diameter of 20mm by laser and stuck to the nonwoven fabric layer. Finally, the four components are assembled in sequence, so that the assembling work of the micro-needle array patch type electrochemical sensing device is completed.

Example 2

A microneedle electrode array is divided into 6 working electrodes, 1 reference electrode and 1 counter electrode. The working electrode and the counter electrode are both made of commercial stainless steel needle moxibustion needles with the diameter of 0.14mm, and the surfaces of the working electrode and the counter electrode are modified with gold layers; the reference electrode is made of silver-plated acupuncture needle with diameter of 0.14mm, and the surface is modified with silver/silver chloride layer. The surfaces of the tip end parts of the working electrode, the counter electrode and the reference electrode are respectively provided with a swelling resin layer used for isolating the external environment, the swelling resin layer is in a three-dimensional porous three-dimensional structure after absorbing water, and the swelling resin layer comprises a polymer of polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol. The working electrodes are uniformly distributed around the counter electrode by taking the counter electrode as the center, the reference electrode is arranged at intervals with any working electrode, and the reference electrode is arranged at intervals with the counter electrode.

Insulating layers are arranged on the surfaces of the middle parts of the working electrode, the counter electrode and the reference electrode; the middle part is a region 3mm away from the tip part and 2mm wide.

A method of preparing a microneedle electrode array comprising the steps of:

s1: before the surface of the microneedle electrode is modified, the microneedle electrode is firstly pretreated, respectively soaked in 75% ethanol, a metal degreasing agent and a metal rust remover, and respectively ultrasonically cleaned in the solvent for 5-10 min. It should be noted that each step requires a deionized water rinse and finally a dry and clean microneedle is taken for use.

S2: putting a commercially available stainless steel needle moxibustion needle into an ion sputtering device with Jin Bayuan to sputter for 2-4 min to obtain a gold-modified stainless steel needle moxibustion needle (Au/MN); immersing a commercially available silver-plated acupuncture needle into a mixed solution of 0.1M potassium chloride and 0.01M hydrochloric acid, scanning for 3-5 periods from-0.15V to 1.05V at a scanning rate of 0.05V/s, then flushing with deionized water, and placing in a 0.1M potassium chloride solution in a dark place for 24-36 hours to obtain a silver-plated acupuncture needle (Ag-AgCl/MN) after chlorination;

s3: except for a sensing area at the tip of the micro needle and a contact area at the tail end, the rest parts of Au/MNs and Ag-AgCl/MN are coated with medical epoxy resin as an insulating layer;

s4: coating the swelling precursor solution on the Au/MN and the tip sensing area of the Ag-AgCl/MN, and vacuum drying for 8-10 h at the temperature of 25-30 ℃ to obtain the microneedle electrode. The preparation method of the swelling resin precursor solution comprises the following steps: based on 100% of the total mass of the swelling resin precursor solution, a polymethyl vinyl ether/maleic acid copolymer (PMVE/MA) with a concentration of 5wt%, a polyethylene glycol (average molecular weight 8000) with a concentration of 5wt%, a sodium carbonate with a concentration of 3wt%, a sodium polyacrylate with a concentration of 0.2wt%, and stirring and mixing at 60-80 ℃ for 2-3 hours.

A micro-needle array patch type sensing device is assembled by a customized micro-potentiostat, the micro-needle electrodes fixed on a resin plate, medical non-woven fabrics and a polyvinylidene chloride film. The custom made micro potentiostat had 8 through holes of 0.14mm diameter through which the microneedles were electrically connected to the micro electrochemical workstation. The electrode array is fixed in the resin board, and every two distance between the microneedle is 4mm, and the resin board is by 3D printing integrated into one piece. The internal dimensions of the resin plates are consistent with those of the micro potentiostat, ensuring their close fit. The medical non-woven fabric layer is cut into capsules with the diameter of 65 multiplied by 40mm by a laser cutting machine. In addition, the polyvinylidene chloride film was cut into a disc having a diameter of 20mm by laser and stuck to the nonwoven fabric layer. Finally, the four components are assembled in sequence, so that the assembling work of the micro-needle array patch type electrochemical sensing device is completed.

Example 3

A microneedle electrode array is divided into 4 working electrodes, 1 reference electrode and 1 counter electrode. The working electrode and the counter electrode are both made of commercial stainless steel needle moxibustion needles with the diameter of 0.18mm, and the surfaces of the working electrode and the counter electrode are modified with gold layers; the reference electrode is made of silver-plated acupuncture needle with diameter of 0.18mm, and the surface is modified with silver/silver chloride layer. The surfaces of the tip end parts of the working electrode, the counter electrode and the reference electrode are respectively provided with a swelling resin layer used for isolating the external environment, the swelling resin layer is in a three-dimensional porous three-dimensional structure after absorbing water, and the swelling resin layer comprises a polymer of polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol. The working electrodes are uniformly distributed around the counter electrode by taking the counter electrode as the center, the reference electrode is arranged at intervals with any working electrode, and the reference electrode is arranged at intervals with the counter electrode.

Insulating layers are arranged on the surfaces of the middle parts of the working electrode, the counter electrode and the reference electrode; the middle part is a region 3mm away from the tip part and 3mm wide.

A method of preparing a microneedle electrode array comprising the steps of:

S2: placing a stainless steel needle moxibustion needle sold in the market into an ion sputtering device with a platinum target source to sputter for 2-4 min to obtain a platinum modified stainless steel needle moxibustion needle (Pt/MN); immersing a commercially available silver-plated acupuncture needle into a mixed solution of 0.1M ammonium chloride and 0.01M hydrochloric acid, scanning for 3-5 periods from-0.15V to 1.05V at a scanning rate of 0.05V/s, then flushing with deionized water, and placing in 0.1M ammonium chloride solution in a dark place for 24-36 hours to obtain a silver-plated acupuncture needle (Ag-AgCl/MN) after chlorination;

s3: the rest parts of Pt/MNs and Ag-AgCl/MN are coated with medical epoxy resin as an insulating layer except a sensing area at the tip of the micro needle and a contact area at the tail end of the micro needle;

S4: coating the swelling precursor solution on the Pt/MN and Ag-AgCl/MN tip sensing area, and vacuum drying at 25-30 deg.c for 8-10 hr to obtain the micro needle electrode. The preparation method of the swelling resin precursor solution comprises the following steps: the polymethyl vinyl ether/maleic acid copolymer (PMVE/MA) at a concentration of 8wt%, polyethylene glycol (average molecular weight 8000) at a concentration of 7.5wt%, sodium carbonate at a concentration of 2wt%, sodium polyacrylate at a concentration of 0.2wt% were mixed with stirring at 60 to 80 ℃ for 2 to 3 hours, based on 100% of the total mass of the swollen resin precursor solution.

A micro-needle array patch type sensing device is assembled by a customized micro-potentiostat, the micro-needle electrodes fixed on a resin plate, medical non-woven fabrics and a polyvinylidene chloride film. The custom made micro potentiostat had 6 through holes of 0.18mm diameter through which the microneedles were electrically connected to the micro-electrochemical workstation. The electrode array is fixed in the resin board, and every two distance between the microneedle is 5mm, and the resin board is by 3D printing integrated into one piece. The internal dimensions of the resin plates are consistent with those of the micro potentiostat, ensuring their close fit. The medical non-woven fabric layer is cut into capsules with the diameter of 55 multiplied by 40mm by a laser cutting machine. In addition, the polyvinylidene chloride film was cut into a disc having a diameter of 20mm by laser and stuck to the nonwoven fabric layer. Finally, the four components are assembled in sequence, so that the assembling work of the micro-needle array patch type electrochemical sensing device is completed.

Example 4

This example differs from example 2 in that the swelling resin precursor solution of this example comprises a polymer of hyaluronic acid and methacrylic anhydride (MeHA) prepared by: to 50ml of an aqueous solution of Hyaluronic Acid (HA) having a mass-volume fraction of 2% were added sequentially 50ml of N, N-Dimethylformamide (DMF) and 9.3ml of Methacrylic Anhydride (MA), and the mixture was stirred overnight to give a precursor gel (MeHA). Subsequently, the precursor gel (MeHA) was washed 3 times with ethanol with the pH adjusted to 8-9 by dissolution with 1mol/L NaOH. The air-proof layer is made of chlorinated polyvinyl chloride film.

Example 5

The difference between this example and example 3 is that the swelling resin precursor solution of this example comprises a polystyrene-polyacrylamide-based resin, and the preparation method thereof is as follows: polystyrene-polyacrylamide (PS-b-PAA) was prepared by hydrolysis of 10g of t-butyl polyacrylate-polystyrene block copolymer (PSb-ptBA) and 50ml of methylene chloride as starting materials with 10g of trifluoroacetic acid as catalyst for 48 hours, followed by precipitation of PS-b-PAA with n-hexane, filtration and washing several times. PS-b-PAA was dissolved in N, N-Dimethylformamide (DMF) by solvent casting and then degassed. The gas-proof layer is made of polytetrafluoroethylene film.

Comparative example 1

A microneedle electrode is made of commercial stainless steel needle moxibustion needle with diameter of 0.16mm, and has a gold layer modified on its surface. The comparative example 1 is different from example 1 in that the tip portion of the microneedle electrode of comparative example 1 has no swollen resin layer.

Effect example 1

The prepared Microneedle (MN) electrode was characterized by Scanning Electron Microscopy (SEM) and energy spectroscopy (EDS).

SEM/EDS test method: and adopting a scanning electron microscope to perform morphological structure characterization and element measurement. The bare Microneedle (MN) and the gold-modified microneedle (Au/MN) tip areas are intercepted for testing, and the swelling resin-modified microneedle (SR/Au/MN) needs to be subjected to freeze drying in a vacuum freeze dryer for a period of time until the swelling resin-modified microneedle (SR/Au/MN) is completely dehydrated and then is characterized.

Test results: compared to the bare Microneedle (MN) with smooth surface (a in fig. 1) and the gold-modified microneedle (Au/MN) with a nanopore structure of comparative example 1 (a in fig. 2), the surface of the swollen resin-modified microneedle (SR/Au/MN) of example 1 (a in fig. 3) was more rough and porous, and the thickness of the swollen resin was about 25 μm. In addition, from the elemental composition data of EDS, the bare MN is mainly composed of metallic elements such as Fe, cr, and the like (b in fig. 1). After sputtering Au, a special peak of Au element (b in fig. 2) appears in the energy spectrum. Also, EDS data for SR/Au/MN showed a substantial increase in the percentage of C and a substantial increase in the percentage of Na, with O element present on Au/MN, because MN surface was modified with (MA/AA) Na copolymer swelling resin (b in FIG. 3).

Effect example 2

The electrochemical performance of the prepared bare Microneedle (MN) electrode was studied using classical Cyclic Voltammetry (CV) and Electrochemical Impedance (EIS) techniques.

Cyclic Voltammetry (CV) test method: and (3) testing by adopting an electrochemical workstation, immersing the microneedle electrode sensing area in a potassium ferricyanide probe solution with a certain concentration, and scanning at a certain scanning speed in a certain potential interval.

Electrochemical Impedance (EIS) test method: and (3) testing by adopting an electrochemical workstation, immersing the microneedle electrode sensing area in a potassium ferricyanide probe solution with a certain concentration, testing in a certain potential interval, and obtaining an electrode interface impedance value by adopting software fitting.

The oxidation-reduction peak current of the gold-modified microneedle (Au/MN) is significantly increased compared to the bare Microneedle (MN), which may be explained by that the gold layer having a three-dimensional porous interconnection structure formed on the surface of MN effectively increases the specific surface area and active area of the electrode interface, promoting the transfer of electrons (a in fig. 4 and a in fig. 2). The peak current of the swollen resin modified microneedle (SR/Au/MN) is lower than Au/MN because the swollen resin has poor conductivity. In addition, the EIS diagram also shows a similar trend in terms of the charge transfer resistance of the MN, au/MN and SR/Au/MN electrode interfaces (b in fig. 4). Furthermore, we also studied the response capability of MN electrodes to oxygen (fig. 5). Unfortunately, bare MN fails to electrocatalytic reduction of this species. It is notable that Au/MN has excellent oxygen reduction performance at a potential of around-1.0V. Although the response current of SR/Au/MN to oxygen is slightly lower than that of Au/MN, the friendly reduction potential of-0.8V on SR/Au/MN has better selectivity.

Effect example 3

Recovered swelling resin modified microneedle (SR/Au/MN) was used for SEM image and EDS spectrum.

Example 1 microneedle electrode surfaces were decorated with a swelling resin based on a polymethylvinyl ether/maleic acid copolymer (PMVE/MA swelling resin). The swelling process is shown in FIG. 6 (i, SR/Au/MN penetrating the skin; ii, SR/Au/MN swelling subcutaneously; iii, SR/Au/MN removing from epidermis). PMVE/MA resin can function in a microneedle array patch sensor (MNAP) system by (i) unswollen resin acting as a protective layer for the Microneedle (MN) array, isolating the electrode surface from skin secretions (endogenous oil and sweat), exogenous skin care and epidermal stratum corneum as the microneedle pierces the skin, thereby maintaining cleanliness of the MN array. (2) The fully swelled resin is used as electrolyte gel to provide liquid environment for sensing, and the electrode surface is fully exposed to form a good oxygen electroreduction interface. When MN penetrates the skin and enters the subcutaneous tissue, a swelling process naturally occurs by absorbing the liquid matrix in the interstitial fluid of the skin. Since the resin has excellent water absorption properties, the resin rapidly forms a three-dimensional porous structure (a in fig. 7, b in fig. 7) so that small molecules including oxygen and ions can be freely dispersed to MN interfaces, and the molecular sieve effect of the gel-like swelling resin hinders the transportation of biomacromolecules. This may be advantageous to ensure the specificity of the MN array for oxygen, reducing potential matrix interference. (iii) After monitoring is completed, the microneedles are removed and the non-biotoxic and biodegradable PMVE/MA resin is retained in the subcutaneous tissue. The SEM image and EDS spectra of the recovered SR/Au/MN after use are shown as a in fig. 8 and b in fig. 8, and the microstructure morphology and composition are similar (a in fig. 2, b in fig. 2) as compared to Au-MN. This means that only a small amount of resin remains on the gold surface, indicating that the interaction of the resin with the electrode surface is weak. Notably, weak interactions may also be beneficial for the response of the MN array to oxygen.

Effect example 4

Swelling Property test of the swollen resin in example 1

After inserting the swelling resin modified microneedle (SR/Au/MN) into the agar gel, the swelling capacity of SR/Au/MN s was calculated by using the weight of SR/Au/MN s before and after insertion as follows:

as shown in a of fig. 9, the resin had a water absorption swelling equilibrium time of about 50s and a maximum swelling ratio of about 500%. At the same time, the continuous resistance change during swelling of the SR/Au/MN array patch was recorded with a digital source table (b in fig. 9). The resistance of the SR/Au/MN array patch was continuously reduced during swelling until reaching a near constant value comparable to the Au/MN array at around 500s, indicating that the resin was almost completely separated from the interface of MN after complete swelling.

Effect example 5

Electrochemical characterization of the partially and fully swollen SR/Au/MN array was performed in terms of interfacial charge transfer resistance (a in fig. 10), electrochemically active region (b in fig. 10), response capability to oxygen (c in fig. 10), and the like.

The results indicate that the partially swollen microneedle electrodes respond poorly to oxygen, probably due to the higher electrical resistance of the partially swollen resin and poor penetration. However, fully swollen microneedles are able to restore the interface and significantly reduce the interfacial resistance (a in fig. 10). Thus, the conductivity is significantly improved and the response to oxygen is restored to a level comparable to Au-MNs. In addition, electrochemical studies of fully swollen microneedle array patch sensor devices (MNAP) were conducted The sensing interface was learned and electron transport at the electrode interface was found to be mainly affected by diffusion control (a in fig. 11 and b in fig. 11). As shown in a of fig. 11, MNAP has a peak redox current (I _pa And I _pc ) Increases with increasing sweep rate (10 mV/s to 200 mV/s) and the current value is proportional to the square root of the sweep rate (b in FIG. 11). This indicates that the electrochemical reaction of the probe ions at the MNAP sensing interface is affected by diffusion control.

Effect example 6

Anti-fouling properties of swollen resin modified microneedles (SR/Au/MN):

in the anti-fouling study of microneedle-penetrating skin, furan-based hydrogel is adopted to simulate human skin tissue, vaseline is coated on the hydrogel, and pollutants such as skin secretion, skin care products and the like on the skin are simulated. As shown in FIG. 12, the SR/Au/MN response to dissolved oxygen was not very different in the neat hydrogel and the petrolatum-coated hydrogel, whereas the Au/MN response to both hydrogels was very different. This phenomenon can be explained by the fact that Au/MN is easily contaminated with potential contaminants such as vaseline, resulting in a significant decrease in oxygen response. The resin adsorbed on the Au/MN surface effectively protects Au, which is a potential contaminant, as an electro-reduction region during the microneedle penetration of the skin.

Detection of contamination resistance and interference resistance of swollen resin modified microneedle (SR/Au/MN):

bovine Serum Albumin (BSA) was incorporated into the gel to mimic interfering substances in subcutaneous interstitial fluid. As shown in fig. 13, the swelling resin also exhibits excellent performance in protecting MN electrodes from biomacromolecules. This is due to the microporous structure formed after swelling of the resin, which effectively isolates the biological macromolecules in interstitial fluid (ISF) while allowing passage of small biological molecules such as oxygen.

Effect example 7

Current response of microneedle array patch sensor device to oxygen concentration.

at-0.8V potential, the microneedle array patch sensor (MNAP) had a significant electrochemical reduction of oxygen (c in fig. 10). Thus, we use amperometric techniques to study MNAP at the same electricityAt the site, different concentrations of oxygen versus reduction current (a in FIG. 14) for PBS (pH 7.4) containing 0.1M KCl. It is apparent that the current value gradually increases with an increase in the oxygen concentration, and the response current is proportional to the oxygen amount in the range of 6 to 150mmHg, the sensitivity is 0.3817. Mu.A/mmHg, and the detection Limit (LOD) is 5.06mmHg (b in FIG. 14). In addition, to demonstrate that the current is generated by oxygen and not other potential substrates, a special dissolved oxygen scavenger Na was used in the measurement ₂ SO ₃ Added to the test sample. Notably, after addition of the scavenger, the current drops sharply due to Na ₂ SO ₃ Reacts rapidly with dissolved oxygen, further resulting in an immediate decrease in oxygen content (fig. 15).

Effect example 8

Specificity evaluation of microneedle array patch-type sensing device.

The present invention also investigated several inorganic gases and several endogenous substances distributed in the environment, including inorganic electrolytes, nutrients and immune proteins, to evaluate the specificity of the microneedle array patch sensor device. The sensor device was used for measuring physiological levels (50 mmHg CO) in a 150mmHg oxygen standard solution and an oxygen solution in the coexistence of related interfering compounds ₂ 、680mmHg N ₂ The amperometric response at 3mg/mL KCl, 0.9% NaCl, 12mg/mL IgG, 140mg/dL glucose and 15mg/dL lactic acid) is shown in FIG. 16. The results clearly show that these potential disturbances have negligible effect on the oxygen response, indicating that the devices produced have good selectivity.

Effect example 9

And detecting the accuracy of the micro-needle array patch type electrochemical sensing device.

The accuracy of microneedle array patch sensing devices (MNAP) was explored by detecting a range of dissolved oxygen concentrations using a commercial dissolved oximeter as a control. The test results show that there is good agreement between the two methods of the proposed MNAP and commercial instrument (fig. 17). Subsequently, the good stability of the fabricated sensor array was studied by continuously detecting 150mmHg oxygen for 7 days, and the results showed that the consistency between each test was good, the Relative Standard Deviation (RSD) was 1.8%, and there was no significant drop after 7 days of use compared to the first test response (fig. 18).

Effect example 10

The application of the micro-needle array patch type electrochemical sensing device in human motion monitoring.

During real subcutaneous oxygen monitoring, microneedle array patch sensing device (NMAP) patches were worn at the upper arm of healthy subjects, using stationary bicycles for continuous low intensity exercise (a in fig. 19). Intermittently acquiring indexes such as oxygen content, heart rate, blood oxygen saturation and the like of the organism on line in the exercise process. As can be seen from b in fig. 19, the oxygen content in interstitial fluid (ISF) increased significantly by about 33.88% over rest for 40min of continuous exercise, the heart rate reached 126 times/min and the oxygen saturation increased relatively by about 2.10%, indicating that the oxygen supplementation and consumption of the subject may reach a relatively balanced state, and the optimal oxygenation condition was achieved. However, when the participants continued to exercise for 60min, the ISF oxygen partial pressure was significantly reduced by about 37.27% compared to that in the resting state, and the heart rate reached or even exceeded 140 times/min, at which time the blood oxygen saturation was reduced by about 1.03%. The symptoms of sweating, shortness of breath and the like appear in the subject, which indicates that oxygen supply can not meet the oxygen consumption of the organism any more and indicates that excessive oxygen consumption is generated in exercise. The volunteer's oxygen partial pressure, oxygen saturation and heart rate gradually recovered to the original basal values within 30min of rest after exercise. This demonstrates the significant ability of MNAP to accurately and continuously track dynamic changes in oxygen content in vivo.

The foregoing description of the preferred embodiments of the present application is not intended to be limiting, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the present application.

Claims

1. The microneedle electrode array is characterized by comprising a plurality of working electrodes, counter electrodes and reference electrodes; each working electrode takes the counter electrode as a center and is uniformly distributed around the counter electrode, the reference electrode and any one of the working electrodes are arranged at intervals, and the reference electrode and the counter electrode are arranged at intervals;

any one of the working electrode and the counter electrode comprises a first metal microneedle and a noble metal layer modified on the surface of the first metal microneedle;

the reference electrode comprises second metal micro-needles which are arranged at intervals with any of the first metal micro-needles, and a silver/silver chloride layer which is modified on the surface of the second metal micro-needles;

the surface of the tip end parts of the working electrode, the counter electrode and the reference electrode are respectively provided with a swelling resin layer used for isolating the external environment, and the swelling resin layers are in a three-dimensional porous three-dimensional structure after absorbing water.

2. The microneedle electrode array according to claim 1, wherein the middle surfaces of the working electrode, the counter electrode and the reference electrode are provided with insulating layers, and the middle is a region with a width of 2-3 mm located 2-4 mm away from the tip end; and/or the number of the groups of groups,

The noble metal layer comprises a gold layer and/or a platinum layer; and/or the number of the groups of groups,

the diameters of the first metal micro-needle and the second metal micro-needle are 0.12-0.3 mm; and/or the number of the groups of groups,

the total number of the first metal micro-needles and the second metal micro-needles is 4-10; and/or the number of the groups of groups,

the distance between every two first metal micro-needles is 2-6 mm; and/or the number of the groups of groups,

the first metal micro-needle and the second metal micro-needle are selected from one of steel needles, silver-plated needles, gold needles and gold needles; and/or the number of the groups of groups,

the swelling resin layer includes a polymer of a polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol, or a polymer of hyaluronic acid and methacrylic anhydride, or a polystyrene-polyacrylamide-based resin.

3. The microneedle electrode array of claim 2, wherein the insulating layer is an epoxy layer; and/or the number of the groups of groups,

the noble metal layer is a gold layer; and/or the number of the groups of groups,

the diameters of the first metal micro-needle and the second metal micro-needle are 0.16-0.25 mm; and/or the number of the groups of groups,

the total number of the first metal micro-needles and the second metal micro-needles is 6-8; and/or the number of the groups of groups,

the distance between every two first metal micro-needles is 3-5 mm; and/or the number of the groups of groups,

the first metal micro-needle is a steel needle; and/or the number of the groups of groups,

The second metal micro-needle is a silver-plated needle or a silver needle; and/or the number of the groups of groups,

the swelling resin layer includes a polymer of a polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol.

4. A method for preparing a microneedle electrode array, comprising the steps of:

swelling resin modified metal microneedle: and coating a swelling resin precursor on the tip parts of the surface modified first metal micro-needle and the second metal micro-needle, and vacuum drying to obtain the micro-needle electrode array.

5. The method for preparing the microneedle electrode array according to claim 4, wherein the specific step of forming the noble metal layer on the surface of the first metal microneedle by the ion sputtering method comprises the following steps: sputtering noble metal on the surface of the first metal microneedle by adopting an ion sputtering instrument with a noble metal target source, wherein the sputtering time is 1-6 min; and/or the number of the groups of groups,

the specific steps of forming the silver/silver chloride layer on the surface of the second metal microneedle by an electrochemical deposition method are as follows: the second metal micro-needle is selected from silver needle or silver-plated needle, then the second metal micro-needle is immersed into chloride solution for electrochemical deposition treatment, and after washing, the second metal micro-needle is placed in the chloride solution in a dark place; and/or the number of the groups of groups,

The step of swelling resin to modify the metal microneedle specifically comprises the following steps: coating the swelling resin precursor solution on the tip parts of the surface modified first metal micro-needle and the second metal micro-needle, and carrying out vacuum drying treatment.

6. The method for preparing a microneedle electrode array according to claim 4 or 5, further comprising the step of subjecting the metallic microneedles to a cleaning, degreasing, and rust removal treatment before the surface modification of the metallic microneedles; and/or the number of the groups of groups,

the method further comprises the step of coating the middle surfaces of the first metal micro-needle and the second metal micro-needle with an insulating layer material after the metal micro-needle surface modification step and before the swelling resin modification metal micro-needle step.

7. The method for preparing the microneedle electrode array according to claim 6, wherein the specific step of forming the noble metal layer on the surface of the first metal microneedle by the ion sputtering method comprises the following steps: sputtering gold on the surface of the first metal microneedle by adopting an ion sputtering instrument with Jin Bayuan, wherein the sputtering time is 2-4 min; and/or the number of the groups of groups,

the specific steps of forming the silver/silver chloride layer on the surface of the second metal microneedle by an electrochemical deposition method are as follows: the second metal micro-needle is a silver plating needle, and then the silver plating needle is immersed into a mixed solution of chloride and HCl for electrochemical deposition treatment, and is washed by deionized water and placed in the chloride solution in a dark place; the conditions of the electrochemical deposition treatment are as follows: scanning speed is 0.01-0.2V/s, voltage is-0.10-1.00V or-0.15-1.05V or 0.2-1.1V, scanning is carried out for 1-6 periods; and/or the number of the groups of groups,

The swelling resin precursor solution includes: a polymer of a polymethyl vinyl ether/maleic acid copolymer and polyethylene glycol; or, a polymer of hyaluronic acid and methacrylic anhydride; or, a polystyrene-polyacrylamide-based resin.

8. The method of preparing a microneedle electrode array according to claim 7, wherein the method of preparing the swelling resin precursor solution comprises: stirring the polymethyl vinyl ether/maleic acid copolymer, polyethylene glycol, sodium carbonate and sodium polyacrylate for 1-3 h at 40-80 ℃; the concentration of the polymethyl vinyl ether/maleic acid copolymer is 2.5-15 wt%, the concentration of the polyethylene glycol is 2.5-10 wt%, the concentration of the sodium carbonate is 1-5 wt%, and the concentration of the sodium polyacrylate is 0.1-0.3 wt%, based on 100% of the total mass of the swelling resin precursor solution; or alternatively, the first and second heat exchangers may be,

sequentially adding N, N-dimethylformamide and methacrylic anhydride into an aqueous solution of hyaluronic acid with the mass volume fraction of 2%, stirring overnight, washing with ethanol under an alkaline environment, and dialyzing; or alternatively, the first and second heat exchangers may be,

the preparation method comprises the steps of taking a poly (tert-butyl acrylate) -polystyrene block copolymer and methylene dichloride as raw materials, taking trifluoroacetic acid as a catalyst, preparing polystyrene-polyacrylamide by hydrolysis, then precipitating the polystyrene-polyacrylamide by using n-hexane, filtering and washing for a plurality of times.

9. A microneedle array patch sensor comprising a microneedle electrode array, wherein the microneedle electrode array is a microneedle electrode array according to any one of claims 1 to 3 or a microneedle electrode array obtained by the method of any one of claims 4 to 8.

10. The microneedle array patch sensor device of claim 9, comprising: a substrate;

the microneedle electrode array fixedly arranged on the substrate;

an electrochemical workstation provided with a through-hole;

the substrate comprises a resin plate, an adhesive layer and an air-proof layer which are sequentially connected; one surface of the resin plate, which is away from the microneedle electrode array, is connected with the electrochemical workstation, and the microneedle electrode array is electrically connected with the electrochemical workstation through the through hole.