A Conductive Hydrogel Microneedle Patch and Its Application in Glucose Monitoring

Abstract

The present invention discloses a conductive hydrogel microneedle patch and its application in glucose monitoring. It is locked in the skin through microneedle phase change, and the swelling of the hydrogel microneedles is utilized to achieve non-invasive or minimally invasive extraction of interstitial fluid, and stably, sensitively and real-time detect the glucose content. In the present invention, glucose oxidase, an electron mediator and a conductive polymer are in-situ loaded into the hydrogel, so as to achieve glucose responsiveness, and the glucose signal can be real-time converted by connecting an external electrode. The hydrogel can provide a quasi-physiological hydration environment for glucose oxidase, which is beneficial to maintaining the enzyme activity, improving the stability of the enzyme, and avoiding the phenomenon that glucose oxidase in commercial test strips is easily detached.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel microneedles, and in particular relates to a conductive hydrogel microneedle patch and application thereof in glucose monitoring. Background Art

[0002] Developing devices that can continuously and in real time monitor glucose is an important part of accurate diabetes control. Currently, the most commonly used method for testing glucose is to analyze blood samples. Traditional glucose sensors using disposable test strips collect blood by piercing the skin, but blood collection causes local pain and increases the risk of infection. Interstitial fluid (ISF), which has similar biomarker content to blood, is currently being studied as a source of biomarkers. ISF is more suitable for real-time monitoring because it does not have cells and coagulation factors.

[0003] Continuous glucose meters (CGMs) monitor the glucose concentration of the interstitial fluid of the subcutaneous tissue through sensors, which can realize real-time monitoring of the blood glucose concentration of diabetic patients throughout the day. The scanning glucose monitoring system (FreeStyle Libre) can realize continuous blood glucose monitoring for 14 days, but the sensor probe has a 5mm long needle, which still causes pain. In addition, most current blood glucose meters rely on enzyme detection mechanisms. The activity and stability of the enzyme are affected by factors such as pH, temperature and environmental humidity levels, which directly affect the accuracy of CGMs.

[0004] Microneedle (MN) arrays are an emerging platform for minimally invasive extraction of ISF and transdermal biosensing. A variety of MN designs have been used for enzymatic or non-enzymatic continuous glucose monitoring, including solid, hollow, and porous MNs. Hollow and porous MNs can enhance fluid collection, but the manufacturing process is complex. Solid MN arrays are easier to manufacture and can act as sensing transducers, coated with a conductive layer for continuous glucose monitoring, but cannot extract fluids limiting their sensing capabilities. Summary of the invention

[0005] In response to the problems that existing blood glucose meters are prone to pain and have poor stability, the present invention provides a conductive hydrogel microneedle patch and its application in glucose monitoring. The microneedles are locked in the skin through phase change, and the swelling of the hydrogel microneedles is used to achieve non-invasive or minimally invasive extraction of interstitial fluid, thereby stably, sensitively and real-time detection of glucose content.

[0006] In a first aspect, the present invention provides a conductive hydrogel microneedle patch, which is prepared by chemically crosslinking and freeze-drying a hydrogel precursor solution. The hydrogel precursor solution comprises the following components: 100-300 mg / g of monomer, 5-10 mg / g of biopolymer crosslinking agent, 0.2-5 mg / g of photoinitiator, 1-2 mg / g of conductive polymer, 0.5-5 mg / mL of glucose oxidase, and 1-10 mg / mL of electron mediator.

[0007] In the present invention, immobilizing glucose oxidase in the hydrogel can achieve real-time conversion of glucose signals, while maintaining enzyme activity and improving stability. By incorporating an electron mediator and a conductive polymer, electron transfer can be achieved, which can be used for continuous glucose sensor devices, facilitating the capture and detection analysis of biomarkers, and having good stability and practicality.

[0008] Further, the monomer is acrylic acid or acrylamide, the biopolymer crosslinking agent is methacrylated gelatin or methacrylated hyaluronic acid, the photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, the conductive polymer is poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), the electron mediator is potassium ferricyanide, and the solvent is deionized water.

[0009] Gels prepared with pure biopolymers as monomers have relatively low mechanical properties. In the present invention, synthetic small molecules are used as monomers and methacrylated biopolymers are used as crosslinking agents, making the gel have better flexibility and achieving a balance between mechanical properties and biocompatibility.

[0010] Further, methacrylated gelatin is prepared as follows: Dissolve gelatin in PBS buffer at 50 °C, drop 10 mL of methacrylic anhydride into the PBS buffer, stir and then add PBS buffer to terminate the reaction, dialyze using a dialysis bag with a molecular weight of 8000-14000, and freeze-dry to obtain it.

[0011] Preferably, the preparation method of methacrylated gelatin is: Dissolve 10 g of gelatin in 100 mL of 0.01 M PBS buffer at 50 °C, drop 10 mL of methacrylic anhydride at a rate of 0.5 mL / min, stir vigorously at 50 °C for 3 h, add 5 times the amount of PBS buffer to terminate the reaction, dialyze using a dialysis bag with a molecular weight of 8000-14000 for 7 days, obtain sponge-like methacrylated gelatin by freeze-drying, and store it at -20 °C.

[0012] Furthermore, the methacrylated hyaluronic acid is prepared as follows: hyaluronic acid is dissolved in water, the pH value is adjusted to 8, methacrylic anhydride is dropped into the aqueous hyaluronic acid solution, after stirring, absolute ethanol is added, the white flocculates are collected, the white flocculates are dissolved in water, dialyzed using a dialysis bag with a molecular weight of 3500, and freeze-dried to obtain the product.

[0013] Preferably, the preparation method of the methacrylated hyaluronic acid comprises the following steps: 2 g of hyaluronic acid is magnetically stirred and dissolved in 100 mL of deionized water, the pH value is adjusted to 8 using 5 M sodium hydroxide, 2 mL of methacrylic anhydride is dropped in at a rate of 0.5 mL / min, vigorously stirred at 50 °C for 4 h, absolute ethanol is added to precipitate it, white flocculates precipitate out, the white flocculates are dissolved in 100 mL of deionized water, dialyzed using a dialysis bag with a molecular weight of 3500 for 7 days, and white methacrylated hyaluronic acid is obtained through freeze-drying and stored at -20 °C.

[0014] Furthermore, the preparation method of the conductive hydrogel microneedle patch comprises the following steps:

[0015] (1) A monomer, a biopolymer crosslinker, a photoinitiator, a conductive polymer, glucose oxidase, and an electron mediator are added to a solvent to obtain a hydrogel precursor solution as a composite system;

[0016] (2) The hydrogel precursor solution is dropped into a microneedle mold, left standing, centrifuged to uniformly disperse the hydrogel precursor solution in the mold;

[0017] (3) A hydrogel microneedle is prepared by photoinitiated chemical crosslinking, and the swelling but not dissolution of the microneedle can be achieved;

[0018] (4) The hydrogel microneedle is freeze-dried and demolded to obtain a dry conductive hydrogel microneedle patch.

[0019] Furthermore, in step (2), the centrifugation speed is 3000 - 5000 rpm, the centrifugation time is 5 - 10 min, and the number of centrifugation times is three times to make the hydrogel precursor solution completely enter the mold.

[0020] Furthermore, in step (3), ultraviolet light, violet light or blue light is used to initiate chemical crosslinking, and the chemical crosslinking time is 0.5 - 30 min.

[0021] Furthermore, in step (4), first the hydrogel microneedle is frozen at -80 °C or in liquid nitrogen, and then placed in a vacuum freeze-dryer and freeze-dried for 6 - 12 hours, and a conductive hydrogel microneedle patch is obtained after demolding.

[0022] Furthermore, the conductive hydrogel microneedle patch contains at least two hydrogel microneedles, and the arrangement is a single row or an array structure; the height of each microneedle is 100 - 1000 μm, and the spacing between the microneedles is 100 - 1000 μm.

[0023] In a second aspect, the present invention also provides an application of the conductive hydrogel microneedle patch in glucose monitoring. Specifically, it is used to prepare a glucose monitoring working electrode with the conductive hydrogel microneedle patch. When applying, the conductive hydrogel microneedle patch is inserted into the skin through a microneedle injector.

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) The conductive hydrogel microneedle patch of the present invention utilizes the design of the microneedle array to achieve non-invasive or minimally invasive extraction of interstitial fluid, with low infectivity, which is beneficial to improving the clinical compliance of patients.

[0026] (2) By in-situ loading glucose oxidase, an electron mediator, and a conductive polymer into the hydrogel, the present invention can achieve glucose responsiveness, and the glucose signal can be converted in real time by connecting an external electrode; the hydrogel can provide a quasi-physiological hydration environment for glucose oxidase, which is beneficial to maintaining enzyme activity, improving enzyme stability, and avoiding the phenomenon that glucose oxidase in commercial test strips is prone to fall off.

[0027] (3) The present invention uses the swelling property of the hydrogel microneedle to prepare phase change microneedles, which can quickly extract biological molecules such as glucose from interstitial fluid, lock itself in the skin by volume expansion to prevent the patch from falling off, and can also be painlessly removed without leaving residues; by means of an external electrode system, the hydrogel microneedle patch of the present invention can stably, sensitively, and real-time detect the glucose content, showing great potential in diagnosis.

[0028] (4) The preparation method of the hydrogel microneedle patch provided by the present invention has good operability, low cost, can be mass-produced, and has strong practicability. The prepared hydrogel microneedle patch has multifunctionality, can be used as a working electrode for glucose monitoring, and has broad application prospects in the development field of wearable real-time biosignal detection devices. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0030] Figure 1 It is a flow chart of the preparation method of the conductive hydrogel microneedle patch.

[0031] Figure 2 Schematic diagram for extracting body fluid using a conductive hydrogel microneedle patch.

[0032] Figure 3 Schematic diagram of the mechanism for detecting glucose using potassium ferricyanide as an electron mediator. Detailed implementation manners

[0033] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0034] The poly(3,4-ethylenedioxythiophene):poly(styrenesulfonic acid) used in the detailed implementation manners is in particulate form and is purchased from Macklin;

[0035] The gelatin is of type A with a gel strength of 300 g Bloom and is purchased from Sigma.

[0036] The molecular weight of hyaluronic acid is between 10k and 20k.

[0037] Example 1

[0038] As Figure 1 shown, a conductive hydrogel microneedle patch contains 18 hydrogel microneedles. The hydrogel microneedles are arranged in a 3×6 matrix, with the height of each microneedle being 1000 μm and the spacing between the microneedles being 1000 μm.

[0039] The preparation method includes the following steps:

[0040] (1) Add 300 mg of acrylamide and 10 mg of methacrylated gelatin to 0.7 mL of deionized water, place it in an oven at 50 °C to completely dissolve it, add 1 mg of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) dissolved in 10 μL of ethanol, add 2 mg of glucose oxidase, 5 mg of potassium ferricyanide, and 0.2 mg of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and shake to completely dissolve it to obtain a hydrogel precursor solution.

[0041] Among them, the methacrylated gelatin is prepared as follows: 10 g of gelatin is dissolved in 100 mL of 0.01 M PBS buffer at 50 °C, 10 mL of methacrylic anhydride is dropped in at a rate of 0.5 mL / min, and it is vigorously stirred at 50 °C for 3 h. 5 times the amount of PBS buffer is added to terminate the reaction. It is dialyzed for 7 days using a dialysis bag with a molecular weight of 8000 - 14000, and the sponge-like methacrylated gelatin is obtained through freeze-drying and stored at -20 °C.

[0042] (2) Drop the hydrogel precursor solution into the microneedle mold, let it stand for 5 min, centrifuge at 3000 rpm for 5 min, and centrifuge three times in total to make it completely enter the mold, and scrape off the excess liquid with a knife.

[0043] (3) At 25 °C, use a 365 nm ultraviolet lamp with an illumination intensity of about 4.5 mW / cm 2 , and react for 5 min to obtain the hydrogel microneedles.

[0044] (4) Place the hydrogel microneedles in a -80 °C refrigerator for freezing, then transfer them to a vacuum freeze-dryer for drying for 6 h, demold, and obtain the conductive hydrogel microneedle patch.

[0045] Example 2

[0046] A conductive hydrogel microneedle patch contains 18 hydrogel microneedles. The hydrogel microneedles are arranged in a 3×6 matrix. The height of each microneedle is 1000 μm, and the spacing between the microneedles is 1000 μm.

[0047] The preparation method includes the following steps:

[0048] (1) Add 300 mg of acrylic acid, 10 mg of methacrylated gelatin, and 0.7 mL of deionized water, place it in an oven at 50 °C to completely dissolve it, adjust the pH value to 7 with a 10 M sodium hydroxide concentration, add 1 mg of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) assisted by 10 μL of ethanol, add 2 mg of glucose oxidase, 5 mg of potassium ferricyanide, and 0.5 mg of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and completely dissolve them to obtain the hydrogel precursor solution.

[0049] Among them, the methacrylated gelatin is prepared as follows: 10 g of gelatin is dissolved in 100 mL of 0.01 M PBS buffer at 50 °C, 10 mL of methacrylic anhydride is dropped in at a rate of 0.5 mL / min, and it is vigorously stirred at 50 °C for 3 h. 5 times the amount of PBS buffer is added to terminate the reaction. It is dialyzed for 7 days using a dialysis bag with a molecular weight of 8000 - 14000, and the sponge-like methacrylated gelatin is obtained through freeze-drying and stored at -20 °C.

[0050] (2) Drop the hydrogel precursor solution into the microneedle mold, let it stand for 5 min, centrifuge at 3000 rpm for 5 min, and centrifuge three times in total to make it completely enter the mold. Scrape off the excess liquid with a knife.

[0051] (3) At 25 °C, use a 365 nm ultraviolet lamp with an illumination intensity of about 4.5 mW / cm 2 , and react for 5 min to obtain hydrogel microneedles.

[0052] (4) Put the hydrogel microneedles into a -80 °C refrigerator for freezing, then transfer them to a vacuum freeze dryer for drying for 6 h, and demold to obtain a conductive hydrogel microneedle patch.

[0053] Example 3

[0054] A conductive hydrogel microneedle patch contains 25 hydrogel microneedles. The hydrogel microneedles are arranged in a 5×5 matrix. The height of each microneedle is 100 μm, and the spacing between the microneedles is 100 μm.

[0055] The preparation method includes the following steps:

[0056] (1) Add 100 mg of acrylamide and 5 mg of methacrylated hyaluronic acid to 0.4 mL of deionized water, stir with a magnetic stirrer until completely dissolved, add 10 μL of 0.8 mg of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) dissolved in ethanol, add 1 mg of glucose oxidase, 2 mg of potassium ferricyanide, and 0.2 mg of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and shake until completely dissolved to obtain a hydrogel precursor solution.

[0057] Among them, methacrylated hyaluronic acid is prepared as follows: 2 g of hyaluronic acid is magnetically stirred and dissolved in 100 mL of deionized water, the pH value is adjusted to 8 with 5 M sodium hydroxide, 2 mL of methacrylic anhydride is dropped at a rate of 0.5 mL / min, and stirred vigorously at 50 °C for 4 h. Add absolute ethanol to precipitate it, and white flocculates will precipitate. Dissolve the white flocculates in 100 mL of deionized water, dialyze with a dialysis bag with a molecular weight of 3500 for 7 days, and obtain white methacrylated hyaluronic acid through freeze-drying, and store it at -20 °C.

[0058] (2) Drop the hydrogel precursor solution into the microneedle mold, let it stand for 5 min, centrifuge at 5000 rpm for 8 min, and centrifuge three times in total to make it completely enter the mold. Scrape off the excess liquid with a knife.

[0059] (3) At 25 °C, use a 365 nm ultraviolet lamp with an illumination intensity of about 4.5 mW / cm 2 , and react for 3 min to obtain hydrogel microneedles.

[0060] (4) Freeze the hydrogel microneedles in an -80 °C refrigerator, then transfer them to a vacuum freeze dryer and dry for 6 h, and demold to obtain a conductive hydrogel microneedle patch.

[0061] Example 4

[0062] A conductive hydrogel microneedle patch contains 36 hydrogel microneedles. The hydrogel microneedles are arranged in a 6×6 matrix. The height of each microneedle is 100 μm, and the spacing between the microneedles is 100 μm.

[0063] The preparation method includes the following steps:

[0064] (1) Add 100 mg of acrylic acid and 5 mg of methacrylated hyaluronic acid to 0.4 mL of deionized water, stir with a magnetic stirrer until completely dissolved, adjust the pH value to 7 with a 10 M sodium hydroxide concentration, add 10 μL of ethanol-assisted 0.8 mg of poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), add 1 mg of glucose oxidase, 3 mg of potassium ferricyanide, and 0.2 mg of lithium phenyl(2,4,6-trimethylbenzoyl)phosphate, and stir until completely dissolved to obtain a hydrogel precursor solution.

[0065] Among them, methacrylated hyaluronic acid is prepared as follows: 2 g of hyaluronic acid is magnetically stirred and dissolved in 100 mL of deionized water, the pH value is adjusted to 8 with 5 M sodium hydroxide, 2 mL of methacrylic anhydride is dropped in at a rate of 0.5 mL / min, and stirred vigorously at 50 °C for 4 h. Add absolute ethanol to precipitate it, and white flocculates will precipitate. Dissolve the white flocculates in 100 mL of deionized water, dialyze with a dialysis bag with a molecular weight of 3500 for 7 days, and obtain white methacrylated hyaluronic acid through freeze drying, and store it at -20 °C.

[0066] (2) Drop the hydrogel precursor solution into the microneedle mold, let it stand for 5 min, centrifuge at 5000 rpm for 8 min, and centrifuge three times in total to make it completely enter the mold, and scrape off the excess liquid with a knife.

[0067] (3) At 25 °C, use a 365 nm ultraviolet lamp with an illumination intensity of about 4.5 mW / cm 2 , and react for 3 min to prepare hydrogel microneedles.

[0068] (4) Freeze the hydrogel microneedles in an -80 °C refrigerator, then transfer them to a vacuum freeze dryer and dry for 6 h, and demold to obtain a conductive hydrogel microneedle patch.

[0069] Example 5

[0070] Use the conductive hydrogel microneedle patch of Example 1 as the working electrode for glucose monitoring, and pierce the conductive hydrogel microneedle patch into the skin through a microneedle injector, as Figure 2As shown, the dry microneedles can rapidly absorb body fluids and lock themselves in the skin through swelling.

[0071] Figure 3 It is a schematic diagram of the mechanism for monitoring glucose with potassium ferricyanide as an electron mediator. Glucose oxidase oxidizes glucose to gluconic acid, accompanied by electron transfer. Potassium ferricyanide, as an electron mediator, can transfer electrons to the electrode. The conductive hydrogel microneedle patch of the present invention can be used as a working electrode for glucose monitoring. With the help of an external electrode system, the glucose content can be sensitively and real-time detected.

[0072] Although the present invention has been described in detail by referring to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those of ordinary skill in the art can make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions should all be within the scope of the present invention. / Any person familiar with the technical field of the present invention can easily think of changes or substitutions within the technical scope disclosed by the present invention, and they should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope described in the claims.

Claims

1. A conductive hydrogel microneedle patch, characterized in that, the conductive hydrogel microneedle patch is prepared from a hydrogel precursor solution through chemical cross-linking and freeze-drying. The hydrogel precursor solution comprises the following components: 100 - 300 mg / g of monomer, 5 - 10 mg / g of biopolymer cross-linking agent, 0.2 - 5 mg / g of photoinitiator, 1 - 2 mg / g of conductive polymer, 0.5 - 5 mg / mL of glucose oxidase, and 1 - 10 mg / mL of electron mediator; the monomer is acrylic acid or acrylamide, the biopolymer cross-linking agent is methacrylated gelatin or methacrylated hyaluronic acid, the photoinitiator is lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate, the conductive polymer is poly(3,4 - ethylenedioxythiophene):poly(styrenesulfonate), the electron mediator is potassium ferricyanide, and the solvent is deionized water; methacrylated gelatin is prepared as follows: dissolve gelatin in PBS buffer at 50 °C, drop 10 mL of methacrylic anhydride into the PBS buffer, stir and then add PBS buffer to terminate the reaction, dialyze using a dialysis bag with a molecular weight of 8000 - 14000, and freeze-dry to obtain; methacrylated hyaluronic acid is prepared as follows: dissolve hyaluronic acid in water, adjust the pH value to 8, drop methacrylic anhydride into the hyaluronic acid aqueous solution, stir and then add absolute ethanol, collect the white floccules, dissolve the white floccules in water, dialyze using a dialysis bag with a molecular weight of 3500, and freeze-dry to obtain.

2. A conductive hydrogel microneedle patch according to claim 1, characterized in that, the preparation method of the conductive hydrogel microneedle patch comprises the following steps: (1) Add the monomer, biopolymer cross-linking agent, photoinitiator, conductive polymer, glucose oxidase, and electron mediator into the solvent to obtain a hydrogel precursor solution; (2) Drop the hydrogel precursor solution into a microneedle mold, let it stand, and centrifuge to make the hydrogel precursor solution evenly dispersed in the mold; (3) Prepare hydrogel microneedles through photoinitiated chemical cross-linking; (4) After freeze-drying and demolding the hydrogel microneedles, obtain a dry conductive hydrogel microneedle patch.

3. A conductive hydrogel microneedle patch according to claim 2, characterized in that, in step (2), the centrifugation speed is 3000 - 5000 rpm, the centrifugation time is 5 - 10 min, and the number of centrifugation times is three.

4. A conductive hydrogel microneedle patch according to claim 2, characterized in that, in step (3), ultraviolet light, violet light or blue light is used to initiate chemical cross-linking, and the chemical cross-linking time is 0.5 - 30 min.

5. A conductive hydrogel microneedle patch according to claim 2, characterized in that, in step (4), first freeze the hydrogel microneedles at -80 °C or in liquid nitrogen, then place them in a vacuum freeze-dryer and freeze-dry for 6 - 12 hours, and obtain a conductive hydrogel microneedle patch after demolding.

6. A conductive hydrogel microneedle patch according to claim 1, characterized in that, The conductive hydrogel microneedle patch contains at least two hydrogel microneedles, and the arrangement is a single row or an array structure; the height of each hydrogel microneedle is 100-1000 μm, and the distance between adjacent hydrogel microneedles is 100-1000 μm.

7. Application of the conductive hydrogel microneedle patch according to claim 1 in preparing a glucose monitoring working electrode, using the conductive hydrogel microneedle patch to prepare a glucose monitoring working electrode.

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