A blood glucose sensing electrode based on multi-channel microneedle array and preparation method thereof

Abstract

The present invention discloses a blood glucose sensing electrode based on a multi-channel microneedle array and a preparation method thereof, a microneedle array substrate and a flexible gasket attached to the surface of the microneedle array substrate, the microneedle array substrate and the flexible gasket are both made by a printing process, and the flexible gasket is provided with a hollow pattern array corresponding to the microneedle array; the microneedle array substrate comprises a substrate containing a microneedle array, a functionalized modification layer attached to the surface of the microneedle array, and a patterned conductive layer connecting each microneedle, the functionalized modification layer modifies the microneedle array into a blood glucose enzyme sensor including a counter electrode, a working electrode and a reference electrode, the patterned conductive layer is arranged on the surface of the substrate, and the patterned conductive layer transmits the signal of each microneedle separately. The blood glucose sensing electrode prepared by the present invention responds separately to realize the measurement of the spatial distribution of local glucose concentration, and has practical value for blood glucose measurement of wounds, inflammatory sites, etc.

Description

Technical Field

[0001] The present invention belongs to the field of biosensor technology, and in particular relates to a blood sugar sensing electrode based on a multi-channel microneedle array and a preparation method thereof. Background Art

[0002] Accurate detection and analysis of human blood sugar concentration, development of advanced diabetes precision monitoring systems, and timely acquisition of patient conditions will help reduce the impact of blood sugar fluctuations on diabetic patients.

[0003] Commonly used blood sugar testing methods in clinical practice include: collecting venous blood and using a fully automatic biochemical analyzer for testing and analysis; pricking the fingertips to draw blood and using a rapid blood sugar meter for testing. The above methods are invasive and discontinuous, and the blood sugar test method of blood sampling reflects the overall blood sugar level of the whole body. In some application scenarios where attention needs to be paid to local blood sugar concentration, such as biomarker monitoring of wounds and inflammatory sites, a real-time biosensor technology is needed that can accurately measure local blood sugar levels and reflect their temporal and spatial variation trends.

[0004] Therefore, a wearable continuous blood glucose monitor has been developed. It monitors blood glucose by continuously inserting a needle electrode under the skin. It can efficiently pierce the human skin epidermis in a minimally invasive manner without reaching the pain nerves. The wound is small and the patient's pain is reduced. It can be stably applied to the surface of human skin, facilitating continuous blood glucose monitoring sensing, which is of great positive significance in improving the current inconvenience of blood glucose testing.

[0005] For example, patent CN110558993A provides a Prussian blue microneedle electrode for blood glucose monitoring, its preparation method, blood glucose monitoring patch and its preparation method. The provided Prussian blue microneedle electrode includes: a gold microneedle electrode, the gold microneedle electrode includes a gold microneedle electrode substrate and a plurality of gold microneedles fixed at the bottom on the gold microneedle electrode substrate; a Prussian blue layer compounded on the surface of the gold microneedle; and a chitosan-immobilized glucose oxidase layer compounded on the surface of the Prussian blue layer. This solution solves the problem of cumbersome work caused by the need to draw blood for blood glucose monitoring in diabetes. It only needs to penetrate the stratum corneum of the skin to test the glucose concentration in the tissue fluid quickly and accurately for a long time. The microneedle electrode is small in size and easy to carry, and has the potential to be a good, safe, portable and painless blood glucose electrochemical sensor.

[0006] However, the above scheme uses the microneedle array as a whole as a blood glucose sensing electrode, and mainly performs "point" measurement, which reflects the comprehensive blood glucose level of a local tissue of the human body, and does not give full play to the characteristics of the microneedle array structure that can realize multi-point synchronous sensing. Moreover, as a micron-level sensing structure, the preparation and processing technology of the microneedle array is also important and difficult. At present, the traditional silicon-based semiconductor process has many and complex processes, and the production environment requirements are also relatively strict. Through the silicon-based semiconductor process, it is necessary to ensure the high cleanliness of the working environment to avoid impurities from contaminating the product, and it is also necessary to continuously cycle through a series of processes such as film, photoresist, development, etching, photoresist removal, etc. dozens of times to obtain the final product. The laboratory process strategy with high manual degree and low automation degree has problems such as low efficiency, poor repeatability, and low yield, which is not conducive to the transformation of the research results of microneedle blood glucose electrodes, and it is also not in line with the development trend of engineering research. This is difficult to bear for the microneedle array blood glucose electrode, which is currently in rapid iterative development, in terms of cost, energy consumption and environmental protection pressure.

[0007] Therefore, how to overcome the measurement reliability problem of microneedle array electrodes and the inability to meet the application scenario requirements of reflecting the spatiotemporal distribution of blood glucose in order to achieve efficient, low-consumption, stable and controllable blood glucose sensing electrode preparation is an urgent problem to be solved by technical personnel in this field. Summary of the invention

[0008] In view of the defects in the above-mentioned prior art, the present invention provides a blood glucose sensing electrode based on a multi-channel microneedle array and a preparation method thereof, using a 3D printed microneedle array of high molecular polymer materials, and surface-modifying the microneedle array by inkjet printing technology to obtain a working electrode, a counter electrode and a reference electrode, in-situ drawing of a patterned conductive layer that can connect a single microneedle tip, using the automatic dispensing technology in inkjet printing technology to obtain a packaged microneedle array, attaching an inkjet-printed flexible gasket, and constructing an antibacterial and anti-interference sensor-skin interface. The blood glucose sensing electrode based on a multi-channel microneedle array prepared by the present invention realizes blood glucose monitoring that is minimally invasive, continuous, accessible in time and space distribution, anti-multiple interference and comfortable to wear.

[0009] In a first aspect, the present invention provides a blood glucose sensing electrode based on a multi-channel microneedle array, comprising: a microneedle array substrate and a flexible gasket attached to the surface of the microneedle array substrate, wherein the microneedle array substrate and the flexible gasket are both made by an in-situ printing process, and the flexible gasket is provided with a hollow pattern array corresponding to the microneedle array;

[0010] The microneedle array matrix includes a substrate containing a microneedle array, a functional modification layer attached to the surface of the microneedle array, and a patterned conductive layer connecting each microneedle. The functional modification layer modifies the microneedle array into a blood glucose enzyme sensor including a counter electrode, a working electrode, and a reference electrode. The patterned conductive layer is arranged on the surface of the substrate, and the patterned conductive layer transmits the signal of each microneedle individually.

[0011] Furthermore, a substrate containing a counter electrode, a substrate containing a working electrode and a substrate containing a reference electrode are spliced ​​in the left and right directions to form a microneedle array matrix, the size of any substrate is (0.5×1.5)-(1.5×4.5) mm, the number of microneedles in the microneedle array of any substrate is 2-15, the needle tip length of the microneedle is 600-800 μm, the width of each microneedle is 100-300 μm, and the distance between two adjacent microneedles of any substrate is 100-300 μm.

[0012] Furthermore, the functional modification layer of the counter electrode is a conductive carbon material layer, the functional modification layer of the reference electrode is a conductive carbon material layer and a silver / silver chloride ink layer attached in sequence, and the functional modification layer of the working electrode is a conductive carbon material layer, a serum protein layer containing glucose oxidase, and a polyurethane layer attached in sequence, and the thickness of the conductive carbon material layer is ≤400 μm.

[0013] Furthermore, an insulating isolation layer is included between the microneedle array substrate and the flexible gasket. The insulating isolation layer is made of a photocurable resin and has the same shape as the flexible gasket. The thickness of the insulating isolation layer is 1-5 μm.

[0014] Furthermore, the thickness of the patterned conductive layer is 0.5-5 μm, and the line width is 0.5-5 μm.

[0015] In a second aspect, the present invention further provides a method for preparing the above-mentioned blood sugar sensor electrode, comprising the following steps:

[0016] The arrangement of the microneedle array is preset, and the substrate is prepared by using resin material and digital light processing 3D printing of light-hardening resin;

[0017] The microneedle array is subjected to corona treatment, and a functional modification layer is prepared by spraying on the surface of the microneedle array by inkjet printing, and the microneedle array is modified into a blood glucose measuring enzyme sensor including a counter electrode, a working electrode and a reference electrode;

[0018] The shape of the conductive pattern corresponding to the arrangement of the microneedle array is preset, and a conductive carbon material is used to inkjet print a patterned conductive layer on the surface of the substrate;

[0019] The ink material of nanosilver viscous hydrogel is used to prepare a flexible gasket by inkjet printing to form a blood glucose sensing electrode based on a multi-channel microneedle array.

[0020] Furthermore, a functionalized modification layer is prepared by spraying on the surface of the microneedle array by inkjet printing, and the microneedle array is modified into a glucose measuring enzyme sensor including a counter electrode, a working electrode and a reference electrode, specifically comprising:

[0021] Conductive ink is sprayed on the surface of the microneedle array, and a conductive carbon material layer is formed after low-temperature sintering, wherein the solute of the conductive ink is a carbon material, the solvent is water or an organic solvent, the ratio of the solute to the solvent is (1:1)-(1:10), the viscosity of the conductive ink is 1-30 cP, the carbon material is at least one of multi-walled carbon nanotubes, single-walled carbon nanotubes and graphene, and the spraying speed is 1-500 m·min -1 , low temperature sintering temperature ≤120℃;

[0022] The microneedle array substrate is divided into a left substrate, a middle substrate and a right substrate, and a silver / silver chloride ink layer is sprayed on the surface of the conductive carbon material layer of the left substrate;

[0023] A serum protein layer containing glucose oxidase and a polyurethane layer are sequentially sprayed on the surface of the conductive carbon material layer of the middle substrate;

[0024] The right substrate obtains a counter electrode with a conductive carbon material layer attached to the surface of the microneedle array, the left substrate obtains a reference electrode with a conductive carbon material layer and a silver / silver chloride ink layer attached to the surface of the microneedle array in sequence, and the middle substrate obtains a working electrode with a conductive carbon material layer, a serum protein layer and a polyurethane layer attached to the surface of the microneedle array in sequence.

[0025] Furthermore, a serum protein layer containing glucose oxidase and a polyurethane layer are sequentially sprayed on the surface of the conductive carbon material layer of the middle substrate, specifically comprising:

[0026] The serum protein layer containing glucose oxidase is sprayed on the surface of the conductive carbon material layer of the middle substrate by inkjet printing process, and then dried at room temperature for at least 8 hours;

[0027] Spray a polyurethane layer on the surface of the serum protein layer and dry it at room temperature for at least 16 hours;

[0028] The inkjet ink of the serum protein layer is composed of 30-100 mg / mL glucose oxidase solution, 50-200 mg / mL bovine serum protein solution and 1-5wt% glutaraldehyde solution in a volume ratio of 1:5:2, and the inkjet ink of the polyurethane layer is 3-10wt% polyurethane solution.

[0029] Furthermore, the conductive carbon material is used to inkjet print a patterned conductive layer on the substrate surface, specifically comprising:

[0030] Conductive carbon material is used to inkjet on the substrate surface, wherein the inkjet of the pattern conductive layer is a mixture of conductive carbon material and photosensitive resin, the mixing ratio is (1:1)-(1:10), the conductive carbon material is at least one of multi-walled carbon nanotubes, single-walled carbon nanotubes and graphene, and the inkjet printing speed of the pattern conductive layer is 1-500m·min -1 , ink viscosity is 1-30cP;

[0031] Curing is performed by irradiating with ultraviolet light having a wavelength of 200-400nm and an intensity of 1-5mW for 1-30min;

[0032] A patterned conductive layer with a thickness of 0.5-5 μm and a line width of 0.5-5 μm is obtained.

[0033] Furthermore, the flexible gasket is prepared by inkjet printing using the ink material of nanosilver viscous hydrogel, which specifically includes:

[0034] The nano silver viscous hydrogel is composed of 1-30wt% polymer material, 0.05-0.3wt% nano silver, a cross-linking agent and water, wherein the polymer material is at least one of cellulose, alginic acid, hyaluronic acid, chitosan, polyacrylic acid, polymethacrylic acid, polyacrylamide and poly-N-polyacrylamide.

[0035] The present invention provides a blood glucose sensing electrode based on a multi-channel microneedle array and a preparation method thereof, which at least includes the following beneficial effects:

[0036] (1) The fully in-situ printing process of 3D printing and inkjet printing meets the requirements of model complexity design and high precision standards. The blood glucose sensing electrode based on the multi-channel microneedle array is prepared. The advantage of microneedles as an array structure can realize multi-point synchronous sensing. A multi-channel blood glucose sensing electrode in which each microneedle tip can work independently is developed. The difficulty of preparing microneedle blood glucose electrodes with independent sensing channels is overcome, and blood glucose monitoring with minimally invasive, continuous, accessible spatiotemporal distribution, resistance to multiple interferences and comfortable wearing experience is realized.

[0037] (2) The conductive pattern of the patterned conductive layer of the microneedle array is planned and designed to achieve the spatial distribution and overall analysis test of blood glucose concentration; the comprehensive response of multiple independent electrodes can make the measurement results more accurate and avoid the influence of aging or partial failure of a single microneedle tip on the overall sensing performance; the individual response of multiple independent electrodes can realize the measurement of the spatial distribution of local glucose concentration, which has practical value for blood glucose measurement in wounds, inflammatory sites, etc.

[0038] (3) The flexible gasket is provided to insulate the substrate of the microneedle array matrix, thereby ensuring the reliability of the blood glucose test results; at the same time, the flexible gasket also serves to isolate the bottom of the blood glucose sensor electrode from the skin, avoiding direct contact and reducing the effects of skin sweat, temperature, surface charge, etc. on electrode sensing; a certain proportion of nanosilver is added to the flexible gasket to play an antibacterial role and achieve resistance to multiple interferences, and can be applied to special skin environments such as wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of the structure of a blood glucose sensing electrode based on a multi-channel microneedle array provided by the present invention;

[0040] Figure 2 A schematic diagram of wearing a blood sugar sensor electrode provided by the present invention for transdermal glucose detection;

[0041] Figure 3 A process diagram of preparing a blood glucose sensing electrode based on a multi-channel microneedle array provided by the present invention;

[0042] Figure 4 A process flow chart of preparing a substrate containing a microneedle array by 3D printing provided by the present invention;

[0043] Figure 5 A process flow chart of preparing a functionalized modification layer and a patterned conductive layer by inkjet printing technology provided by the present invention;

[0044] Figure 6 A process flow chart of preparing a flexible gasket by inkjet printing technology provided by the present invention;

[0045] Figure 7 This is a graph showing in vitro test results of the blood sugar sensor electrode provided by the present invention;

[0046] Figure 8 A diagram showing the simulated in vivo test results of the blood glucose sensor electrode provided by the present invention;

[0047] Fig. 9 This is a graph showing the antibacterial performance test results of the blood glucose sensor electrode provided by the present invention. DETAILED DESCRIPTION

[0048] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0049] like Figure 1 and Figure 2As shown, a blood glucose sensing electrode based on a multi-channel microneedle array comprises: a microneedle array substrate 1 and a flexible gasket 2 attached to the surface of the microneedle array substrate, the microneedle array substrate 1 and the flexible gasket 2 are both made by an in-situ printing process, and the flexible gasket 2 is provided with a hollow pattern array corresponding to the microneedle array;

[0050] The microneedle array substrate 1 includes a substrate containing a microneedle array, a functional modification layer attached to the surface of the microneedle array, and a pattern conductive layer connecting each microneedle. The functional modification layer modifies the microneedle array into a blood glucose enzyme sensor including a counter electrode, a working electrode, and a reference electrode. The pattern conductive layer is arranged on the surface of the substrate, and the pattern conductive layer transmits the signal of each microneedle individually.

[0051] The blood glucose sensing electrode based on multi-channel microneedle array provides a more general solution for the core technology of in-vivo and continuous monitoring of blood glucose - blood glucose electrode. It takes the multi-channel sensing of microneedle array with single microneedle working independently as an innovation to realize richer and more accurate sensing functions. It takes in-situ printing as a process innovation to cooperate with blood glucose sensing electrode, breaks through the bottleneck of preparation of microneedle array electrode with single microneedle working independently, and realizes efficient, low-consumption, stable and controllable electrode preparation.

[0052] Specifically, a substrate containing a counter electrode, a substrate containing a working electrode and a substrate containing a reference electrode are spliced ​​in the left-right direction to form a microneedle array matrix 1, the size of any substrate is (0.5×1.5)-(1.5×4.5) mm, the number of microneedles in the microneedle array of any substrate is 2-15, the needle tip length of the microneedle is 600-800 μm, the width of each microneedle is 100-300 μm, and the distance between two adjacent microneedles of any substrate is 100-300 μm.

[0053] The overall size of the microneedle array substrate can be adjusted according to the area of ​​the required test site and the surface flatness. Of course, the size of each substrate containing the counter electrode, the substrate containing the working electrode, and the substrate containing the reference electrode can also be adjusted. The length of the microneedle, the width of a single microneedle, and the distance between two adjacent microneedles on each sheet must meet the set numerical range and form a certain numerical ratio. If the microneedle array is arranged too densely, the independence of the single measurement will be affected. If it is too sparse, mutual verification cannot be achieved to improve the accuracy of the measurement results. However, the specific size of the microneedle can be appropriately adjusted within a given numerical range. For example, on a flat skin surface, the size of the microneedle can be large or small. In the joint area, a smaller size of the microneedle is more appropriate.

[0054] Specifically, the functional modification layer of the counter electrode is a conductive carbon material layer, the functional modification layer of the reference electrode is a conductive carbon material layer and a silver / silver chloride ink layer attached in sequence, and the functional modification layer of the working electrode is a conductive carbon material layer, a serum protein layer containing glucose oxidase, and a polyurethane layer attached in sequence, and the thickness of the conductive carbon material layer is ≤400μm.

[0055] The thickness of the conductive carbon material layer is ≤400nm, the thickness of the pattern conductive layer is 0.5-5μm, and the line width is 0.5-5μm. After a large number of tests, the more preferred thickness of the pattern conductive layer is 3-5μm, and the line width is 3-5μm. Too narrow a line width will affect the reliability of conductivity, while too wide a line width will affect the overall design and arrangement of the conductive pattern.

[0056] An insulating layer is also included between the microneedle array substrate and the flexible gasket. After the functional modification is completed, a 1-5 μm thick photocurable resin is printed on the substrate surface (excluding the microneedle array area) by inkjet printing process, and then cured by low-intensity light to form an insulating layer. The shape of the insulating layer is consistent with the flexible gasket and is used to protect the functional modification layer.

[0057] like Figure 3 As shown, a method for preparing the above-mentioned blood sugar sensor electrode comprises the following steps:

[0058] The arrangement of the microneedle array is preset, and the substrate is prepared by using resin material and digital light processing 3D printing of light-hardening resin;

[0059] The microneedle array is subjected to corona treatment, and a functional modification layer is prepared by spraying on the surface of the microneedle array by inkjet printing, and the microneedle array is modified into a blood glucose measuring enzyme sensor including a counter electrode, a working electrode and a reference electrode;

[0060] The shape of the conductive pattern corresponding to the arrangement of the microneedle array is preset, and a conductive carbon material is used to inkjet print a patterned conductive layer on the surface of the substrate;

[0061] The ink material of nanosilver viscous hydrogel is used to prepare a flexible gasket by inkjet printing to form a blood glucose sensing electrode based on a multi-channel microneedle array.

[0062] The full printing technology has obvious advantages such as high efficiency, low energy consumption, environmental friendliness, convenient process adjustment, and is suitable for rapid iteration and small-batch trial production. Specifically, the full in-situ printing process of 3D printing and inkjet printing meets the requirements of model complexity design and high precision standards. The prepared blood glucose sensing electrode based on multi-channel microneedle array takes advantage of the microneedle as an array structure to achieve multi-point synchronous sensing. A multi-channel blood glucose sensing electrode in which each microneedle tip can work independently is developed, breaking through the difficulty of preparing microneedle blood glucose electrodes with independent sensing channels, and realizing minimally invasive, continuous, accessible spatiotemporal distribution, anti-multiple interference, and comfortable wearing experience blood glucose monitoring.

[0063] like Figure 4 As shown, the photocurable resin digital light processing technology cures the resin material by irradiating it with ultraviolet light, and projects the cross-section of the 3D model onto the photosensitive liquid polymer to cure it, which can meet the requirements of model complexity design and high precision standards. Specifically, the digital light processor processes the light source into ultraviolet light, focuses it through a lens and irradiates it into a container containing the photosensitive liquid polymer. Based on the preset arrangement of the microneedle array, the photosensitive liquid polymer is cured to form a substrate containing the microneedle array.

[0064] The resin material can not only meet the demand for medical-grade materials for microneedle transdermal detection of glucose, but also has appropriate mechanical strength (on the order of 10-50MPa), good biocompatibility, and can support the use of common disinfection and sterilization methods.

[0065] like Figure 5 As shown, the microneedle array is subjected to corona treatment to make its surface partially charged, which is convenient for the adsorption and fixation of ink when preparing the functional modification layer. Inkjet printing sprays ink droplets to form a dot matrix image. In continuous inkjet, the nozzle continuously sprays ink, and the ink that does not participate in the imaging is recovered by deflection.

[0066] A functional modification layer is prepared by spraying on the surface of a microneedle array by inkjet printing, and the microneedle array is modified into a glucose measuring enzyme sensor including a counter electrode, a working electrode and a reference electrode, specifically comprising:

[0067] Conductive ink is sprayed on the surface of the microneedle array, and a conductive carbon material layer is formed after low-temperature sintering, wherein the solute of the conductive ink is a carbon material, the solvent is water or an organic solvent, the ratio of the solute to the solvent is (1:1)-(1:10), the viscosity of the conductive ink is 1-30 cP, the carbon material is at least one of multi-walled carbon nanotubes, single-walled carbon nanotubes and graphene, and the spraying speed is 1-500 m·min -1 , the temperature of low temperature sintering is ≤120℃; after a large number of tests, the more preferred viscosity of the conductive ink is 10-30cP, the ratio of solute to solvent is (1:5)-(1:10), and the spraying speed is 50-500m·min -1, low temperature sintering temperature is 80-120℃.

[0068] If the ink viscosity is too low, it will affect the sintering efficiency. If the ink viscosity is too high, it will require additional spraying pressure, resulting in larger ink droplets and affecting the spraying accuracy. The spraying speed should consider both the spraying efficiency and the spraying speed not being too high to affect the accuracy.

[0069] The microneedle array substrate is divided into a left substrate, a middle substrate and a right substrate, and a silver / silver chloride ink layer is sprayed on the surface of the conductive carbon material layer of the left substrate;

[0070] A serum protein layer containing glucose oxidase and a polyurethane layer are sequentially sprayed on the surface of the conductive carbon material layer of the middle substrate;

[0071] The right substrate obtains a counter electrode with a conductive carbon material layer attached to the surface of the microneedle array, and the right substrate is a substrate containing a counter electrode. The left substrate obtains a reference electrode with a conductive carbon material layer and a silver / silver chloride ink layer attached to the surface of the microneedle array in sequence, and the left substrate is a substrate containing a reference electrode. The middle substrate obtains a working electrode with a conductive carbon material layer, a serum protein layer and a polyurethane layer attached to the surface of the microneedle array in sequence, and the middle substrate is a substrate containing a working electrode.

[0072] After the right substrate is sprayed to form a conductive carbon material layer, it can meet the requirements for the electrode without additional treatment.

[0073] For the middle substrate, a serum protein layer containing glucose oxidase and a polyurethane layer are sprayed sequentially on the surface of the conductive carbon material layer of the middle substrate, specifically comprising:

[0074] The serum protein layer containing glucose oxidase is sprayed on the surface of the conductive carbon material layer of the middle substrate by inkjet printing process, and then dried at room temperature for at least 8 hours;

[0075] Spray a polyurethane layer on the surface of the serum protein layer and dry it at room temperature for at least 16 hours;

[0076] The inkjet ink of the serum protein layer is composed of 30-100 mg / mL glucose oxidase solution, 50-200 mg / mL bovine serum protein solution and 1-5wt% glutaraldehyde solution in a volume ratio of 1:5:2, and the inkjet ink of the polyurethane layer is 3-10wt% polyurethane solution.

[0077] The inkjet ink of the serum protein layer is preset with a fixed ratio to ensure that the bovine serum protein solution and the glutaraldehyde solution fix the glucose oxidase enzyme in the form of a serum protein layer and attach it to the surface of the conductive carbon material layer. The polyurethane layer is a porous mesh structure that can play a certain isolation role. The polyurethane layer is attached to the surface of the working electrode, and then, when the blood sugar concentration of the test solution is too high, the penetration speed of the solution is delayed and reduced to prevent the working electrode from being impacted by the high concentration of blood sugar and failing.

[0078] In addition, in order to integrate the prepared three electrodes into a microneedle array blood glucose sensing electrode, the automatic dispensing process in inkjet printing technology is used to spray the precursor of the insulating elastomer material into the gap between the two adjacent substrates of the left substrate, the middle substrate and the right substrate, thereby realizing the encapsulation between the substrates of the three microneedle electrodes.

[0079] The insulating elastic material is at least one of polydimethylsiloxane, platinum catalyzed silica gel, polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymer and copolymers thereof.

[0080] Based on the mechanical properties of insulating elastomer materials, the obtained microneedle array sensing electrode is flexible and can be better attached to the skin surface. At the same time, the automatic dispensing process can flexibly layout the number and arrangement of the encapsulated microneedle electrode substrates according to actual needs.

[0081] The characteristic of this microneedle array structure is that it can realize the overall and local sensing detection of blood sugar. The comprehensive response of multiple independent microneedle electrodes can make the measurement results more accurate, avoiding the influence of the aging or partial failure of a single microneedle tip on the overall sensing performance; multiple independent microneedle electrodes can realize the measurement of the spatial distribution of local glucose concentration, which has practical value for the measurement of wounds, inflammatory sites, etc.

[0082] like Figure 6 As shown, a conductive carbon material is used to inkjet print a patterned conductive layer on the surface of a substrate, specifically comprising:

[0083] Conductive carbon material is used to inkjet on the substrate surface, wherein the inkjet of the pattern conductive layer is a mixture of conductive carbon material and photosensitive resin, the mixing ratio is (1:1)-(1:10), the conductive carbon material is at least one of multi-walled carbon nanotubes, single-walled carbon nanotubes and graphene, and the inkjet printing speed of the pattern conductive layer is 1-500m·min -1 , ink viscosity is 1-30cP;

[0084] Similarly, the more preferred inkjet viscosity of the pattern conductive layer is 10-30 cP, the ratio of the conductive carbon material to the photosensitive resin mixture is (1:5)-(1:10), and the spraying speed is 50-500 m·min -1 .

[0085] Curing is performed by irradiating with ultraviolet light having a wavelength of 200-400nm and an intensity of 1-5mW for 1-30min;

[0086] A patterned conductive layer with a thickness of 0.5-5 μm and a line width of 0.5-5 μm is obtained.

[0087] The thickness of the obtained more preferred pattern conductive layer is 3-5 μm, and the line width is 3-5 μm.

[0088] Through the patterned design of the patterned conductive layer, the spatial distribution and overall analysis test of blood glucose concentration can be realized. The individual responses of multiple independent electrodes can realize the measurement of the spatial distribution of local glucose concentration, which has practical value for blood glucose measurement in wounds, inflammatory sites, etc.

[0089] like Figure 7 As shown, the ink material selected for printing the flexible gasket is nanosilver viscous hydrogel, which is obtained through the automatic dispensing process in inkjet printing technology.

[0090] The flexible gasket is prepared by inkjet printing using nanosilver viscous hydrogel ink material, which specifically includes:

[0091] The nano silver viscous hydrogel is composed of 1-30wt% polymer material, 0.05-0.3wt% nano silver, a cross-linking agent and water, wherein the polymer material is at least one of cellulose, alginic acid, hyaluronic acid, chitosan, polyacrylic acid, polymethacrylic acid, polyacrylamide and poly-N-polyacrylamide. The polymer material is more preferably contained in an amount of 5-30wt%, and the water content is controlled accordingly. Too high a water content in the hydrogel affects the formability and mechanical stability of the flexible gasket; too low a water content leads to excessive viscosity, which makes the spraying parameters more demanding and affects the process stability.

[0092] The flexible gasket based on multifunctional hydrogel material was prepared in situ by inkjet printing, which is highly consistent with the overall process of the microneedle array matrix of the blood glucose sensing electrode, ensuring the efficiency and repeatability / consistency of the process.

[0093] The direct contact of the microneedle array substrate with the test skin will introduce a variety of interferences, including test artifacts caused by temperature, humidity changes, surface charge and movement, which will affect the results of the accurate blood sugar test experiment. The flexible gasket is attached to the surface of the microneedle array blood sugar sensor electrode in situ, acting as an insulating pattern conductive layer, maintaining the stability of the sensing signal, avoiding environmental interference, and is beneficial to the accurate sensing of blood sugar.

[0094] The flexible gasket of the hydrogel has good adhesion to the human skin and can isolate the influence of the human skin surface (such as sweat, temperature fluctuations, motion artifacts, surface charge, etc.) to a certain extent; at the same time, the hydrogel is also an ideal dressing. After the microneedle array blood glucose sensor electrode penetrates the skin, it has a moist healing effect on the micropore wound formed, inhibits allergic and inflammatory reactions, and promotes skin recovery after the electrode is removed.

[0095] Silver nanoparticles were added to the flexible gasket as an effective antibacterial agent with good biocompatibility to inhibit bacterial metabolism at the sensing electrode-skin interface.

[0096] Performance Testing

[0097] Test Example 1:

[0098] The in vitro working performance of the blood glucose sensor electrode provided by the present invention was tested, and the specific process and results are as follows:

[0099] Glucose was added to deionized water to prepare the solution. For the entire glucose solution addition process, the blood glucose sensor electrode provided by the present invention was used for in vitro experimental testing. First, the glucose concentration was increased from 0mM to 10mM with a gradient of 1mM, and then the glucose concentration was increased from 10mM to 20mM with a gradient of 5mM. After each glucose was added to the solution, it was stirred for 1 minute using a magnetic device, and then the glucose level of the solution was measured using a portable electrochemical workstation. Each acquisition lasted for 30 seconds. The collected current signal was composed of all microneedles in the microneedle array (left figure) and a specific single microneedle in the microneedle array (right figure), realizing the overall and local glucose sensing test.

[0100] like Figure 7 As shown, the experimental results show that the glucose concentration data measured by all microneedles and a specific single microneedle blood glucose sensor electrode are accurate and highly stable during the duration of the collection.

[0101] Test Example 2:

[0102] The simulated in vivo working performance of the blood glucose sensor electrode provided by the present invention was tested, and the specific process and results are as follows:

[0103] The blood glucose detection of human skin was simulated in a 2mM gradient from 0mM to 10mM glucose agar medium. The blood glucose sensing electrode provided by the present invention was placed on the surface of the glucose agar medium, and the sensing signal was connected to a portable electrochemical workstation with a thin wire to measure the glucose level in the agar medium, and each acquisition lasted for 30 seconds. The collected current signal is composed of all microneedles in the microneedle array (left figure) and a specific single microneedle in the microneedle array (right figure), realizing the test of overall and local glucose sensing.

[0104] like Figure 8 As shown, the experimental results show that the glucose concentration data measured by all microneedles and a specific single microneedle blood glucose sensor electrode are accurate and highly stable during the duration of the collection.

[0105] Test Example 3:

[0106] The antibacterial performance of the blood glucose sensor electrode provided by the present invention was tested, and the specific process and results are as follows:

[0107] Add 10 μL of mixed bacterial suspension (1×10 8CFU / mL of Escherichia coli 5μL and 1×10 8 CFU / mL Staphylococcus aureus 5μL). The blood glucose sensing electrode provided by the present invention (middle picture) and the blood glucose sensing electrode without doped nanosilver pad (right picture) were respectively attached to the mixed bacteria agar culture medium, and the blank control group (left picture) of the mixed bacteria was added for 6 hours, and then the electrode was rinsed with PBS solution, and then immersed in PBS solution for 6 hours. The electrode soaking liquid was added dropwise to the blank agar culture medium, incubated at 37°C for 24 hours, and the growth of the colonies on the surface of the culture medium was observed. No colonies appeared in the left picture, there were spotted colonies in the middle picture, and the colonies in the right picture had grown all over the surface.

[0108] like Fig. 9 As shown, the experimental results show that the antibacterial performance of the blood glucose sensor electrode provided by the present invention is better than that of the blood glucose sensor without the nano-silver doped flexible gasket.

[0109] The performance test results show that the comprehensive response of multiple independent microneedle electrodes based on the multi-channel microneedle array provided by the present invention can make the measurement results more accurate and effectively avoid the influence of the aging or partial failure of a single microneedle tip on the overall sensing performance. At the same time, multiple independent microneedle electrodes can realize the measurement of the spatial distribution of local glucose concentration.

[0110] Silver nanoparticles were added to the flexible gasket as an effective antibacterial agent with good biocompatibility to inhibit bacterial metabolism at the sensing electrode-skin interface.

[0111] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a blood glucose sensing electrode based on a multi-channel microneedle array, characterized in that: The blood glucose sensing electrode comprises: a microneedle array substrate and a flexible gasket attached to the surface of the microneedle array substrate, the microneedle array substrate and the flexible gasket are both made by an in-situ printing process, and the flexible gasket is provided with a hollow pattern array corresponding to the microneedle array; The microneedle array substrate includes a substrate containing a microneedle array, a functional modification layer attached to the surface of the microneedle array, and a pattern conductive layer connecting each microneedle, wherein the functional modification layer modifies the microneedle array into a blood glucose enzyme sensor including a counter electrode, a working electrode, and a reference electrode, and the pattern conductive layer is arranged on the surface of the substrate, and the pattern conductive layer transmits the signal of each microneedle separately; The method comprises the following steps: The arrangement of the microneedle array is preset, and the substrate is prepared by using resin material and digital light processing 3D printing of light-hardening resin; The microneedle array is subjected to corona treatment, and a functional modification layer is prepared by spraying on the surface of the microneedle array by inkjet printing, and the microneedle array is modified into a blood glucose measuring enzyme sensor including a counter electrode, a working electrode and a reference electrode; The shape of the conductive pattern corresponding to the arrangement of the microneedle array is preset, and a conductive carbon material is used to inkjet print a patterned conductive layer on the surface of the substrate; The ink material of nanosilver viscous hydrogel is used to prepare a flexible gasket by inkjet printing to form a blood glucose sensing electrode based on a multi-channel microneedle array.

2. The method according to claim 1, characterized in that A substrate containing a counter electrode, a substrate containing a working electrode and a substrate containing a reference electrode are spliced ​​in the left-right direction to form a microneedle array matrix. The size of any substrate is (0.5×1.5)-(1.5×4.5) mm, the number of microneedles in the microneedle array of any substrate is 2-15, the needle tip length of the microneedle is 600-800μm, the width of each microneedle is 100-300μm, and the distance between two adjacent microneedles of any substrate is 100-300μm.

3. The method according to claim 2, characterized in that The functional modification layer of the counter electrode is a conductive carbon material layer, the functional modification layer of the reference electrode is a conductive carbon material layer and a silver / silver chloride ink layer attached in sequence, and the functional modification layer of the working electrode is a conductive carbon material layer, a serum protein layer containing glucose oxidase, and a polyurethane layer attached in sequence, and the thickness of the conductive carbon material layer is ≤400μm.

4. The method according to claim 1, characterized in that An insulating isolation layer is also included between the microneedle array substrate and the flexible gasket. The insulating isolation layer is made of photocurable resin. The shape of the insulating isolation layer is consistent with that of the flexible gasket. The thickness of the insulating isolation layer is 1-5 μm.

5. The method according to claim 1, characterized in that The pattern conductive layer has a thickness of 0.5-5 μm and a line width of 0.5-5 μm.

6. The method according to claim 1, characterized in that A functional modification layer is prepared by spraying on the surface of a microneedle array by inkjet printing, and the microneedle array is modified into a glucose measuring enzyme sensor including a counter electrode, a working electrode and a reference electrode, specifically comprising: Conductive ink is sprayed on the surface of the microneedle array, and a conductive carbon material layer is formed after low-temperature sintering, wherein the solute of the conductive ink is a carbon material, the solvent is water or an organic solvent, the ratio of the solute to the solvent is (1:1)-(1:10), the viscosity of the conductive ink is 1-30cP, the carbon material is at least one of multi-walled carbon nanotubes, single-walled carbon nanotubes and graphene, and the spraying speed is 1-500m·min -1 , low temperature sintering temperature ≤120℃; The microneedle array substrate is divided into a left substrate, a middle substrate and a right substrate, and a silver / silver chloride ink layer is sprayed on the surface of the conductive carbon material layer of the left substrate; A serum protein layer containing glucose oxidase and a polyurethane layer are sequentially sprayed on the surface of the conductive carbon material layer of the middle substrate; The right substrate obtains a counter electrode with a conductive carbon material layer attached to the surface of the microneedle array, the left substrate obtains a reference electrode with a conductive carbon material layer and a silver / silver chloride ink layer attached to the surface of the microneedle array in sequence, and the middle substrate obtains a working electrode with a conductive carbon material layer, a serum protein layer and a polyurethane layer attached to the surface of the microneedle array in sequence.

7. The method according to claim 6, characterized in that A serum protein layer containing glucose oxidase and a polyurethane layer are sequentially sprayed on the surface of the conductive carbon material layer of the middle substrate, specifically comprising: The serum protein layer containing glucose oxidase is sprayed on the surface of the conductive carbon material layer of the middle substrate by inkjet printing process, and then dried at room temperature for at least 8 hours; Spray a polyurethane layer on the surface of the serum protein layer and dry it at room temperature for at least 16 hours; The inkjet ink of the serum protein layer is composed of 30-100 mg / mL glucose oxidase solution, 50-200 mg / mL bovine serum protein solution and 1-5wt% glutaraldehyde solution in a volume ratio of 1:5:2, and the inkjet ink of the polyurethane layer is 3-10wt% polyurethane solution.

8. The method according to claim 1, characterized in that The conductive carbon material is used to inkjet print a patterned conductive layer on the substrate surface, specifically comprising: Conductive carbon material is used to inkjet on the substrate surface, wherein the inkjet of the pattern conductive layer is a mixture of conductive carbon material and photosensitive resin, the mixing ratio is (1:1)-(1:10), the conductive carbon material is at least one of multi-walled carbon nanotubes, single-walled carbon nanotubes and graphene, and the inkjet printing speed of the pattern conductive layer is 1-500m·min -1 , ink viscosity is 1-30cP; Curing is performed by irradiating with ultraviolet light having a wavelength of 200-400nm and an intensity of 1-5mW for 1-30min; A patterned conductive layer with a thickness of 0.5-5 μm and a line width of 0.5-5 μm is obtained.

9. The method according to claim 1, characterized in that The flexible gasket is prepared by inkjet printing using nanosilver viscous hydrogel ink material, which specifically includes: The nano silver viscous hydrogel is composed of 1-30wt% polymer material, 0.05-0.3wt% nano silver, a cross-linking agent and water, wherein the polymer material is at least one of cellulose, alginic acid, hyaluronic acid, chitosan, polyacrylic acid, polymethacrylic acid, polyacrylamide and poly-N-polyacrylamide.

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