Intelligent deformation microneedle and manufacturing method thereof

Abstract

The invention belongs to the technical field of microneedles, and particularly relates to an intelligent deformation microneedle and a manufacturing method thereof. The device comprises a supporting seat; the counter electrode is arranged above the supporting seat; the elastomer has a compressed state and a natural state, a working electrode is arranged on the outer surface of the elastomer, and a specific enzyme capable of reacting with an analyte to be detected is arranged on the working electrode; and the soluble needle-shaped body is fixed on the supporting seat, the soluble needle-shaped body completely wraps the counter electrode and the elastic body from the outer side, and the soluble needle-shaped body is provided with an inner cavity structure which enables the elastic body to be in a compressed state. An elastomer is arranged in the microneedle, after the microneedle is punctured into the skin, the soluble needle-shaped body is dissolved, the elastomer in the soluble needle-shaped body is exposed, the length is increased, and a working electrode of an electrochemical sensor is attached to the soluble needle-shaped body. In this way, the length of the punctured microneedle is very short, but the obtained working electrode can penetrate into the skin, and corresponding substances can be accurately detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of microneedles, and in particular relates to an intelligent deformable microneedle and a manufacturing method thereof. Background Art

[0002] The use of microneedles combined with biomaterials to detect the concentration of corresponding components has been widely used in some treatments; for example, glucose oxidase is set on the microneedle, and then the microneedle penetrates the stratum corneum of the skin to reach the dermis to complete the detection of glucose concentration in the body of diabetic patients.

[0003] The structure of the existing microneedles is mostly in the shape of a needle, and the height of the microneedle is relatively small, so that the length of entering the dermis is relatively short. For example, when detecting the concentration of glucose, the detection structure may be inaccurate.

[0004] Therefore, how to provide a microneedle that can solve the above-mentioned drawbacks has become a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0005] The present invention aims at the technical problem that the microneedles in the prior art have poor accuracy in detecting the concentration of a substance to be detected, and further provides an intelligent deformable microneedle provided with an elastic body, so as to improve the detection accuracy.

[0006] The technical solution adopted to solve the technical problem of the present invention is:

[0007] An intelligent deformable microneedle, comprising:

[0008] Support seat;

[0009] A counter electrode arranged above the support seat and in a fixed state;

[0010] An elastic body having a compressed state and a natural state, the elastic body is arranged above the support seat and is set in a fixed state, one end of the elastic body adjacent to the support seat is the fixed end of the elastic body, and the end away from the support seat is the free end of the elastic body, and the free end is in a compressed state when it is close to the fixed end, and a working electrode is arranged on the outer surface of the elastic body, the working electrode is electrically insulated from the counter electrode, and a specific enzyme capable of reacting with the analyte to be measured is arranged on the working electrode;

[0011] A soluble needle-shaped body is fixed on the support base with the needle tip away from the support base. The soluble needle-shaped body completely wraps the counter electrode and the elastomer from the outside and has an inner cavity structure that makes the elastomer in a compressed state.

[0012] Preferably, the support seat has a bearing surface, the elastomer is extended and arranged in a direction perpendicular to the bearing surface, one end of the elastomer connected to the bearing surface is the fixed end so that the elastomer is set in the fixed state, and when the elastomer has the compressed state: the bottom end of the soluble needle-shaped body is fixed on the bearing surface and the direction of its needle tip is perpendicular to the direction of the bearing surface, the inner cavity structure of the soluble needle-shaped body is a chamber, the pair of electrodes and the elastomer are located in the chamber, and the area enclosed by the inner side wall of the soluble needle-shaped body adjacent to its needle tip is smaller than the cross-sectional area of ​​the free end.

[0013] Preferably, it also includes a reference electrode arranged corresponding to the working electrode and the counter electrode to form a three-electrode system.

[0014] Preferably, the elastic body is a spring.

[0015] Preferably, it further comprises a support body fixed on the carrying surface, and the counter electrode and the reference electrode are arranged on the support body so as to be arranged in the fixed state.

[0016] Preferably, the support body is a second solid needle-shaped body, the bottom end of the second needle-shaped body is fixed to the bearing surface and the needle tip is oriented perpendicular to the bearing surface, and the second needle-shaped body is located in the internal space of the spring.

[0017] Preferably, the inner cavity structure corresponds to the structure of the spring and the second needle-shaped body in a compressed state.

[0018] Preferably, the support seat is in the shape of a cuboid.

[0019] Preferably, the soluble needle-shaped bodies are water-soluble needle-shaped bodies.

[0020] Preferably, the specific enzyme is arranged on the working electrode near the free end of the elastomer.

[0021] The technical effects achieved by the present invention are specifically as follows:

[0022] The microneedle is equipped with an elastic body. After piercing the skin, the soluble needle-shaped body dissolves, exposing the elastic body inside, which becomes longer and has the working electrode of the electrochemical sensor attached to it. The length of the inserted microneedle is very short, but the obtained working electrode can penetrate deep into the skin to obtain accurate detection of the corresponding substance.

[0023] Preferably, a spring is selected as the elastic body, which is easy to deform.

[0024] The present invention also provides a method for manufacturing an intelligent deformable microneedle, comprising the following steps:

[0025] Providing a support base;

[0026] A counter electrode is arranged above the support seat in a fixed state;

[0027] An elastic body having a compressed state and a natural state is provided, and the elastic body is arranged above the support seat and is set in a fixed state, one end of the elastic body adjacent to the support seat is the fixed end of the elastic body, and the end away from the support seat is the free end of the elastic body, and the free end is in a compressed state when it is close to the fixed end, and a working electrode is arranged on the outer surface of the elastic body, and the working electrode is electrically insulated from the counter electrode, and a specific enzyme capable of reacting with an analyte to be detected is arranged on the working electrode;

[0028] A soluble needle-shaped body is provided and fixed on the support base with its needle tip away from the support base. The soluble needle-shaped body completely wraps the counter electrode and the elastomer from the outside. The soluble needle-shaped body has an inner cavity structure that makes the elastomer in a compressed state.

[0029] As a preference, the method comprises the following steps:

[0030] Providing the support base having a bearing surface;

[0031] The elastic body is fixedly mounted on the bearing surface, the elastic body is extended in a direction perpendicular to the bearing surface, one end of the elastic body connected to the bearing surface is the fixed end, and the other end is the free end;

[0032] A working electrode is disposed at the free end of the elastic body;

[0033] The specific enzyme is arranged on the working electrode near the free end of the elastomer;

[0034] A fixed counter electrode is arranged above the carrying surface;

[0035] A soluble needle-shaped body with a chamber inside is provided, the bottom end of the soluble needle-shaped body is fixed on the supporting surface and the direction of the needle tip is arranged to be perpendicular to the supporting surface, the soluble needle-shaped body is covered with the counter electrode and the working electrode, and the soluble needle-shaped body is squeezed against the free end of the elastomer so that the free end is compressed.

[0036] Preferably, the step of "providing the support base having a bearing surface; fixing the elastomer on the bearing surface, wherein the elastomer extends in a direction perpendicular to the bearing surface and one end of the elastomer connected to the bearing surface is the fixed end and the other end is the free end; arranging a working electrode at the position of the free end of the elastomer; arranging the specific enzyme on the working electrode near the free end of the elastomer; and arranging a counter electrode in a fixed state above the bearing surface" includes the following:

[0037] The following model is designed using 3D modeling software: a rectangular parallelepiped is designed as a support base, and a second needle-shaped body is designed to be solid on the surface of the rectangular parallelepiped, and the bottom end of the second needle-shaped body is fixed on the surface of the rectangular parallelepiped and the needle tip is oriented perpendicular to the surface;

[0038] Using a 3D printer to make the second needle-shaped body according to the model, and then forming the counter electrode on the surface of the second needle-shaped body;

[0039] A spring is provided as the elastic body, the spring is sleeved on the outside of the second needle-shaped body and fixedly mounted on the surface of the rectangular parallelepiped, the spring is extended in a direction perpendicular to the surface, one end of the spring connected to the surface is a fixed end, and the other end is a free end, and the working electrode on the spring is not electrically connected to the counter electrode, and the working electrode and the counter electrode are externally connected using non-electrically connected connection pastes respectively;

[0040] The specific enzyme is then immobilized on the working electrode.

[0041] Preferably, a reference electrode is formed on the surface of the second needle-shaped body, the reference electrode is formed by coating silver / silver chloride slurry, and the working electrode and the counter electrode are both formed by coating carbon slurry or gold slurry or platinum slurry or carbon-Prussian blue composite slurry or gold-Prussian blue composite slurry or platinum-Prussian blue composite slurry.

[0042] Preferably, the method further includes forming a reference electrode on the surface of the second needle-shaped body, which is formed by the following steps: evaporating or sputtering chromium or titanium as an adhesion layer, evaporating or sputtering a gold or platinum electrode thereon, evaporating or sputtering a silver film to obtain a silver electrode, and then immersing it in ferric chloride to obtain a silver / silver chloride electrode. The working electrode is formed by the following steps: evaporating or sputtering chromium or titanium as an adhesion layer, evaporating or sputtering a gold or platinum electrode thereon, and then electroplating Prussian blue thereon.

[0043] Preferably, glucose oxidase is provided as the specific enzyme, comprising: dissolving glucose oxidase in a phosphate buffer solution to prepare a solution with a concentration of 10u / μL, mixing it evenly with a 0.5% glutaraldehyde solution in a volume ratio of 1:1, dripping it onto the working electrode, drying it at 4°C for 24 hours, and then washing away the unfixed glucose oxidase with a phosphate buffer solution.

[0044] Preferably, the step of "providing a soluble needle-shaped body, fixing it on the support base with its needle tip away from the support base, and completely wrapping the counter electrode and the elastomer from the outside with the soluble needle-shaped body, and the soluble needle-shaped body having an inner cavity structure that makes the elastomer in a compressed state" includes the following:

[0045] This step uses a soft lithography method as follows:

[0046] First, a 3D printer is used to print and manufacture a first mold, the first mold comprising an open outer shell in the shape of a rectangular parallelepiped, a needle-shaped body model protrudes from the center of the inner bottom surface of the outer shell in a direction perpendicular to the inner bottom surface, the needle tip of the needle-shaped body model is oriented in a direction perpendicular to the inner bottom surface, the height of the needle-shaped body model is greater than the height of the second needle-shaped body, and the area enclosed by the outer side wall of the needle-shaped body model near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring;

[0047] Making a PDMS concave mold: mixing the material PDMS and the curing agent in a mass ratio of 10:1, stirring thoroughly in the middle to generate a large number of bubbles, then evacuating in a vacuum for 30 minutes to remove all bubbles, then pouring the resulting mixed liquid into the first mold, picking out the bubbles generated in the middle with a needle, and then drying in an oven at 65° C. for 4 hours under a blast condition to obtain a PDMS concave mold having a needle-shaped concave shape corresponding to the needle-shaped body model; the height of the needle-shaped concave shape is greater than the height of the second needle-shaped body, and the area enclosed by the outer side wall of the needle-shaped concave shape near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring;

[0048] A liquid soluble material is placed in the needle-shaped concave shape, and the previously manufactured support seat is covered on one side of the PDMS concave shape having the needle-shaped concave shape, and the free end of the spring is extended into the needle-shaped concave shape and compressed. After drying, the support seat is separated from the PDMS concave shape, and the soluble needle-shaped body formed by the soluble material is fixedly connected to the surface of the support seat.

[0049] Preferably, the soluble material is a PVP-PVA composite material, and the manufacturing method is as follows:

[0050] Weigh 0.6 g PVA and 3.0 g PVP, dissolve in 20 mL deionized water, heat at 95 °C for 3 h to completely dissolve, and cool to room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1 A schematic diagram of the structure of a specific embodiment of the intelligent deformable microneedle provided by the present invention in a natural state;

[0053] Figure 2 Schematic diagram of the specific deformation process of the microneedle;

[0054] Figure 3 A schematic diagram of measuring the specific dimensions of a microneedle in a top view;

[0055] Figure 4 A schematic diagram of measuring the specific dimensions of a microneedle in a side view;

[0056] Figure 5 is a side view of the needle tip of the dissolved microneedle;

[0057] Figure 6 A schematic diagram of measuring the specific dimensions of the dissolved microneedle in a side view;

[0058] Figure 7 CV curve of microneedle detection of hydrogen peroxide;

[0059] Figure 8 This is the relationship between the scan rate and oxidation peak current of the microneedle detection of hydrogen peroxide;

[0060] Fig. 9 It curve of microneedle detecting hydrogen peroxide at -0.1V;

[0061] Fig.10 Calibration curve for microneedle detection of hydrogen peroxide at -0.1 V;

[0062] Fig.11 CV curve of glucose detection by microneedle;

[0063] Fig.12 This is the relationship between the scanning rate and oxidation peak current of the microneedle detection of glucose;

[0064] Fig.13It curve of glucose detection by microneedle at -0.1V;

[0065] Fig.14 This is the calibration curve for microneedle detection of glucose at -0.1 V;

[0066] Fig.15 It curves of glucose detection by microneedle at -0.1V and different temperatures;

[0067] Fig.16 The current and temperature curve of glucose detection by microneedle at -0.1V from 22℃ to 52℃;

[0068] Fig.17 It curves of creatinine, uric acid and glucose detected by microneedle at -0.1V;

[0069] Fig.18 It curves of continuous dripping of creatinine, uric acid and glucose on the microneedle;

[0070] Fig.19 This is a graph showing the change in current value after repeated glucose measurements;

[0071] Fig. 20 The step of "providing the support base having a bearing surface; fixing the elastic body on the bearing surface, wherein the elastic body extends in a direction perpendicular to the bearing surface and one end connected to the bearing surface is the fixed end and the other end is the free end; arranging a working electrode at the position of the free end of the elastic body; arranging the specific enzyme on the working electrode near the free end of the elastic body; arranging a fixed counter electrode above the bearing surface" is a flow chart;

[0072] Fig.21 A schematic flow chart of the step of "providing a soluble needle-shaped body, fixing it on the support base with its needle tip away from the support base, and completely wrapping the counter electrode and the elastomer from the outside with the soluble needle-shaped body, wherein the soluble needle-shaped body has an inner cavity structure that places the elastomer in a compressed state".

[0073] Figure 1-21 The accompanying drawings are denoted as follows:

[0074] 1 support seat, 2 bearing surface, 3 spring, 4 second needle-shaped body, 5 soluble needle-shaped body, 6 fixed end, 7 free end. DETAILED DESCRIPTION

[0075] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0076] In a specific embodiment, the intelligent deformable microneedle includes a support seat 1 in the shape of a rectangular parallelepiped, which has a bearing surface 2. The spring 3 serves as an elastic body and is extended in a direction perpendicular to the bearing surface 2. One end of the spring 3 connected to the bearing surface 2 is a fixed end 6, and the other end is a free end 7. In a natural state, a second needle-shaped body 4 fixed to the bearing surface 2 is arranged in the internal space of the spring 3. A working electrode is arranged on the spring 3, and a reference electrode / counter electrode is arranged on the second needle-shaped body 4. It also includes a soluble needle-shaped body 5, which is fixed on the support seat 1 and the needle tip is away from the support seat 1. The soluble needle-shaped body 5 completely wraps the spring 3 from the outside, and the soluble needle-shaped body 5 has an inner cavity structure that makes the spring 3 in a compressed state.

[0077] For the inner cavity structure, in a specific embodiment, the inner cavity structure corresponds to the structure of the spring 3 and the second needle-shaped body 4 in a compressed state; in this specific embodiment, the soluble needle-shaped body 5 is formed by solidifying a liquid soluble material, and there is no gap inside it, that is, except for the compressed spring 3 and the second needle-shaped body 4, the inside is all solid soluble material. Specifically, refer to Fig. 20 , formed by the following method:

[0078] First, a 3D printer is used to print and manufacture a first mold, the first mold comprising an open outer shell in the shape of a rectangular parallelepiped, a needle-shaped body model protrudes from the center of the inner bottom surface of the outer shell in a direction perpendicular to the inner bottom surface, the needle tip of the needle-shaped body model is oriented in a direction perpendicular to the inner bottom surface, the height of the needle-shaped body model is greater than the height of the second needle-shaped body 4, and the area enclosed by the outer side wall of the needle-shaped body model near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring 3;

[0079] Making a PDMS concave mold: mixing the material PDMS and the curing agent in a mass ratio of 10:1, stirring thoroughly in the middle to generate a large number of bubbles, then evacuating in a vacuum for 30 minutes to remove all bubbles, and then pouring the resulting mixed liquid into the first mold, using a needle to pick out the bubbles generated in the middle, and then drying in an oven at 65°C for 4 hours under a blast condition to obtain a PDMS concave mold with a needle-shaped concave corresponding to the needle-shaped body model; the height of the needle-shaped concave is greater than the height of the second needle-shaped body 4, and the area enclosed by the outer side wall of the needle-shaped concave near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring 3;

[0080] A liquid soluble material is placed in the needle-shaped concave shape, and the previously manufactured support seat (a rectangular support seat 1 provided with a spring 3 and a second needle-shaped body 4) is covered on one side of the PDMS concave shape having the needle-shaped concave shape, and the free end of the spring 3 is extended into the needle-shaped concave shape and compressed. After drying, the support seat 1 is separated from the PDMS concave mold, and the soluble needle-shaped body 5 formed by the soluble material is fixedly connected to the surface of the support seat 1.

[0081] Regarding the inner cavity structure, in another specific embodiment, the inner cavity structure is a chamber, the counter electrode and the spring 3 are located in the chamber, and the area enclosed by the inner wall of the soluble needle-shaped body 5 adjacent to its needle tip is smaller than the cross-sectional area of ​​the free end.

[0082] For the electrode setting, one method is:

[0083] On all electrodes, chromium (Cr) or titanium (Ti) is evaporated or sputtered as an adhesion layer, and gold or platinum electrodes are obtained by evaporation or sputtering, and then Prussian blue is electroplated on the working electrode. On the reference electrode, a silver film is evaporated or sputtered to obtain a silver electrode, which is then immersed in ferric chloride to form a silver / silver chloride electrode.

[0084] Another way is:

[0085] The working electrode and the counter electrode are made of slurry, which is generally carbon slurry, gold slurry, platinum slurry, carbon-Prussian blue composite slurry, gold-Prussian blue composite slurry, or platinum-Prussian blue composite slurry. The reference electrode is made of silver / silver chloride slurry.

[0086] The spring 3 is fixed in the smart deformable microneedle in a compressed state. In actual operation, the soluble needle-shaped body of the smart deformable microneedle dissolves, and the spring 3 is no longer forced to stretch. The length of the spring 3 is greater than the original length of the smart deformable microneedle (see the state diagram). Figure 1 ), so as to achieve the purpose of extending the microneedle during the entire dissolution deformation process. The specific deformation process is as follows Figure 2 shown.

[0087] Regarding the characterization of the size of the smart deformable microneedle according to a specific embodiment of the present application, in order to more clearly and intuitively display the specific size information, an optical microscope is additionally used to characterize the size of the obtained smart deformable microneedle:

[0088] like Figure 3 As shown, it can be seen that the size of the microneedle actually produced (bottom diameter 703.6 μm, spring 3 diameter 483.1 μm, bottom diameter of the second needle 4 193.1 μm) is consistent with the designed size (bottom diameter 700 μm, spring diameter 450 μm, inner microneedle bottom diameter 200 μm).

[0089] like Figure 4 As shown in the figure, it can be seen that the actual microneedle size (bottom diameter 662.4μm, top diameter 367.7μm, height 1109.1μm) and the designed size (bottom diameter 700μm, top diameter 200μm, height 1100μm) are also consistent. The only big difference is the top diameter, which may be caused by excessive stress on the microneedle.

[0090] Characterization of the dissolution deformation performance of the smart deformable microneedle:

[0091] The obtained microneedles were completely immersed in deionized water and observed every 2 minutes. The microneedles were completely dissolved within about 10 minutes.

[0092] The microneedle was then taken out of the water, and after drying, the tip of the microneedle was observed under SEM and optical microscope, with a focus on comparing the length changes, such as Figure 5 and Figure 6 As shown in the figure, it can be found that the length of the microneedle changes from 1109.1μm before dissolution to 1368.2μm, which is close to the spring length of 1400μm, and the total elongation rate reaches more than 20%. This shows that the intelligent deformable microneedle has a certain deformation ability and can realize the elongation function of the working electrode after dissolution.

[0093] Test using the intelligent deformable microneedle to detect hydrogen peroxide:

[0094] 1. Cyclic voltammetry analysis of hydrogen peroxide

[0095] Hydrogen peroxide is the product of glucose oxidation reaction and is also the actual detection object of microneedles. Therefore, hydrogen peroxide is first used to test the performance of microneedles. The first step is to use cyclic voltammetry to find a suitable reaction potential.

[0096] Dilute hydrogen peroxide with phosphate buffer solution to prepare a concentration of 10 mmol / L hydrogen peroxide. Connect the two electrodes of the microneedle (i.e., working electrode and reference electrode / counter electrode) to a power source. Add 10 μL of the test solution to the tip of the microneedle (on the working electrode, which can also cover the reference electrode / counter electrode). Select a voltage range of -0.6V-0.6V and a scan rate of 0.01V / s. Measure the relationship between current and voltage. Then change the scan rate to 0.03V / s, 0.05V / s, 0.07V / s, and 0.09V / s. The measurement results are as follows: Figure 7 shown.

[0097] observe Figure 7It can be found that the CV curves for detecting hydrogen peroxide at different rates have roughly the same shape, but the peak value and voltage of the current peak are different. By comparison, it is found that the current peak value decreases as the scan rate decreases, and the corresponding voltage increases as the scan rate decreases, gradually approaching -0.1V, indicating that the optimal potential for constant voltage current time analysis of hydrogen peroxide is around -0.1V. Subsequent tests can be performed at -0.1V. Record the current peak value at each scan rate, as shown in the following table:

[0098] Detection of the current value of hydrogen peroxide

[0099]

[0100] It can be found that the current has a positive correlation with the scan rate, which can be further verified by the Randles-Sevcik equation:

[0101]

[0102] Among them, i p is the peak current, n is the number of half-reaction electron transfers, and D is the diffusion coefficient (cm 2 / s), v is the voltage scanning rate (v / s), A is the area of ​​the electrode (cm 2 ), c is the concentration of the substance being measured (mol / cm 3 ), k is a constant, which is 2.69×10 5 .

[0103] Substitute the data into the fitting graph and get Figure 8 It was found that the square of the current was roughly proportional to the scan rate, which was consistent with the results, indicating that the sensing process of hydrogen peroxide was mainly controlled by diffusion.

[0104] 2. Current-time curve analysis of hydrogen peroxide detection:

[0105] The measurement is carried out using the it-Amperometric method. The specific principle of the experiment is: a constant voltage is provided between the two electrodes of the microneedle as an excitation, which causes the hydrogen peroxide to undergo oxidation and reduction on the electrode to gain and lose electrons, generating a current signal, and the concentration of the generated hydrogen peroxide can be determined based on the strength of the current signal.

[0106] 30% hydrogen peroxide (corresponding to a molar concentration of 9.9 mol / L) was dissolved in a phosphate buffer solution to prepare a series of hydrogen peroxide solutions with concentrations of 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, and 25mmol / L. The two electrodes of the microneedle were connected to a power source, 10 μL of the test solution was dripped onto the tip of the microneedle, a voltage of -0.1 V was selected, and the relationship between the current and time was measured. The measurement results are shown in FIG. Fig. 9 shown.

[0107] It can be found that the current generally shows a downward trend and the rate of decline gradually decreases. After 30 seconds, the current tends to be stable. The current values ​​at each concentration at this time are recorded, and the measurement is repeated 3 times to take the average value, as shown in the following table:

[0108] Detection of the current value of hydrogen peroxide

[0109]

[0110] The obtained current and the corresponding concentration were then fitted using the least squares method. The fitting results are shown in Fig.10 As shown, it can be found that the current increases with the increase of hydrogen peroxide concentration, and there is a good linear relationship between the two, indicating that the fabricated microneedle can measure the concentration of hydrogen peroxide more accurately, which also provides a basis for its detection of glucose concentration.

[0111] Tests using the smart deformable microneedle to detect glucose:

[0112] 1. Cyclic voltammetry curve analysis of glucose detection

[0113] Dilute glucose with phosphate buffer solution to prepare a glucose solution with a concentration of 30 mmol / L. Connect the two electrodes of the microneedle to the power supply, add 30 μL of the test solution to the tip of the microneedle, select the voltage range of -0.6V-0.6V, the scan rate of 0.01V / s, measure the relationship between current and voltage, and then change the scan rate to 0.03V / s, 0.05V / s, 0.07V / s, 0.09V / s. The measurement results are as follows: Fig.11 shown.

[0114] observe Fig.11 It can be found that the CV curves of the microneedle for detecting glucose at different scanning rates are similar in shape. The current peak and the corresponding voltage have the same trend as the scanning rate decreases. The current peak gradually decreases and the corresponding voltage gradually increases. In order to find the appropriate reaction voltage, the current peak and the corresponding voltage at each scanning rate are recorded, as shown in the following table:

[0115] Peak current corresponding to voltage and scan rate for glucose detection

[0116]

[0117]

[0118] It can be found that as the scan rate decreases, the voltage corresponding to the peak current gradually approaches -0.1V, indicating that the optimal potential for constant voltage current time analysis of glucose is around -0.1V, which is consistent with the conclusion obtained by the cyclic voltammetry of hydrogen peroxide. Therefore, a voltage of -0.1V was used in the subsequent chronoamperometry test of glucose concentration. Further analysis of the current square and scan rate fitting yields Fig.12 It can be found that the square of the current and the scan rate have a good linear relationship, which conforms to the Randles-Savcik equation, indicating that similar to hydrogen peroxide, the sensing of glucose is also mainly determined by diffusion.

[0119] 2. Analysis of the current-time curve of glucose detection

[0120] Similar to the detection of hydrogen peroxide, the chronoamperometric method is used for measurement. The specific principle of glucose detection in this experiment is: hydrogen peroxide is generated during the oxidation of glucose by glucose oxidase on the microneedle. Under a given excitation voltage, hydrogen peroxide undergoes oxidation-reduction on the electrode to gain and lose electrons, generating a current signal. The strength of the current signal can be used to determine the concentration of the generated hydrogen peroxide. Combined with the 1:1 coefficient ratio of glucose to hydrogen peroxide in the glucose oxidation reaction, the concentration of glucose can be inferred.

[0121] Glucose was dissolved in phosphate buffer solution to prepare a series of glucose solutions with concentrations of 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L, and 30mmol / L. The two electrodes of the microneedle were connected to a power source, 10μL of the test solution was dripped onto the tip of the microneedle, a voltage of -0.1V was selected, and the relationship between the current and time was measured. The measurement results are shown in the figure. Fig.13 shown.

[0122] Since the redox of glucose takes a certain amount of time, the test time is longer than that of hydrogen peroxide. Fig.13 It can be found that, similar to hydrogen peroxide, the current for detecting glucose also decreases with time, and the rate of decrease gradually decreases, and finally tends to be stable. Then, the current at 130s when the current tends to be stable is selected, and the current value at each concentration is recorded. The measurement is repeated 3 times and the average value is taken, as shown in the following table:

[0123] Detecting the current value of glucose

[0124]

[0125] The current and the corresponding glucose concentration are fitted, and the fitting results are as follows: Fig.14As shown, it can be found that the magnitude of the current has a good linear relationship with the glucose concentration, indicating that the prepared soluble smart microneedle can measure the glucose concentration well.

[0126] Unlike the detection of hydrogen peroxide, one thing to note in this experiment is that since the enzyme-catalyzed reaction will be affected by the contact area, each time the glucose solution is added to the microneedle, it should be added to the same area and the area of ​​the droplet contacting the microneedle should be kept the same as much as possible, ensuring that the concentration of glucose each time and the concentration of hydrogen peroxide obtained by enzyme-catalyzed oxidation are as consistent as possible, thereby ensuring the accuracy of the experimental results.

[0127] Effects of different temperatures on the microneedle detection of glucose:

[0128] The human body temperature is generally 37°C, so the working temperature of the microneedle is also roughly around 37°C. Therefore, the range of 37±15°C was selected to use the microneedle to detect glucose concentration and explore the effect of temperature on microneedle detection of glucose.

[0129] A glucose solution with a concentration of 30 mmol / L was selected for measurement. The electrodes and the glucose solution were placed on a hot plate for heating. The two electrodes of the microneedle were connected to a power source. 10 μL of the solution to be tested was dripped onto the tip of the microneedle. The voltage was -0.1 V. The temperature was heated from 22°C. The temperature was stopped every 5°C and kept warm for a period of time to stabilize the temperature. The it curve was measured from 22°C to 55°C. The results are shown in Figure 1. Fig.15 shown.

[0130] It can be found that the IT curve of glucose detected by microneedle at different temperatures is also a curve with a gradually decreasing rate of decline. The current at 130s when the current tends to be stable is still selected, and the current values ​​at various concentrations are recorded, as shown in the following table:

[0131] Current values ​​of glucose detected by microneedles at different temperatures at 130s

[0132]

[0133] Plotting the temperature against the corresponding current, we get Fig.16 ,observe Fig.16 It can be found that within the experimental temperature range (22°C to 55°C), as the temperature rises, the detected current value gradually increases, indicating that the concentration of hydrogen peroxide produced by glucose oxidase oxidizing glucose increases, that is, the activity of glucose oxidase increases. On the other hand, the activity increase rate of glucose oxidase increases significantly starting from 40°C.

[0134] According to the data, the working temperature of glucose oxidase is 30-60℃, and the most suitable working temperature is 50-55℃, which is consistent with the experimental results.

[0135] Effects of different interfering substances on microneedle detection of glucose:

[0136] Common interfering substances in blood glucose testing include: creatinine, uric acid, lactic acid, ascorbic acid, dopamine, etc. (see the table below). The effects of these interfering substances on glucose testing can be detected by using two methods: separate addition and continuous addition.

[0137]

[0138] Creatinine and uric acid, which have a high concentration in the blood, were selected as interfering substances for the detection of glucose. Creatinine, uric acid and glucose were dissolved in phosphate buffer solution to prepare solutions with a concentration of 30 mmol / L, and then tested by dripping them separately. The two electrodes of the microneedle were connected to a power source, 10 μL of creatinine solution was dripped on the tip of the microneedle, the voltage was selected to be -0.1 V, and the relationship between the current and time was measured. The remaining solution was then sucked away with a pipette, and the uric acid solution was dripped again after it was dried for measurement. Finally, the current and time curve of the glucose solution was measured. The results are as follows Fig.17 shown.

[0139] observe Fig.17 It can be found that the current of the microneedle detecting glucose is much larger than the current of detecting creatinine and uric acid, indicating that the microneedle has a higher selectivity for glucose.

[0140] In order to further verify the selectivity of microneedle detection of glucose, a continuous dripping method can be used for testing. Using a voltage of -0.1V, 50μL of phosphate buffer solution is first dripped on the electrode. After the current stabilizes (about 100s), 5μL of creatinine is dripped. After 30s, 5μL of uric acid is dripped. After another 30s, 5μL of glucose is dripped. After the current stabilizes (about 270s), 5μL of uric acid is dripped again. After 30s, 5μL of creatinine is dripped again. The results are as follows: Fig.18 As shown in the figure, since it takes a certain amount of time to diffuse, glucose does not make the current reach its peak value at the moment of dripping. The greater change in current when uric acid and creatinine were dripped for the second time may be due to the fact that some glucose is still reacting.

[0141] Combining the two experiments, it can be found that the microneedle's response to glucose is significantly greater than that to the two interfering substances, creatinine and uric acid, indicating that the microneedle has a high specificity for glucose and can effectively avoid the influence of some other interfering substances.

[0142] Stability test for glucose detection:

[0143] In order to test the stability of the microneedle, repeated experiments were performed, and the glucose solution with a concentration of 30 mmol / L was measured multiple times, and the current value at 130 s was recorded. Before each repetition, the microneedle was fully dried after the previous test, and each drop was added to the same position of the microneedle to ensure the same contact area as much as possible. After repeating the experiment 20 times, the current values ​​obtained were plotted to obtain Fig.19 .

[0144] It can be found that within 15 tests, the deviation of the current value of the microneedle detecting glucose was within 10%, indicating that the prepared microneedle can measure the glucose concentration well and multiple measurements can still guarantee a certain degree of accuracy.

[0145] The microneedle produced in this application can be completely dissolved in water within 10 minutes, and can complete the stretching deformation of the spring 3, with an elongation of more than 20%. And the effect of the working electrode of the microneedle in actually detecting hydrogen peroxide and glucose was tested. The appropriate voltage of the microneedle for glucose sensing was tested using cyclic voltammetry (CV), and the detection of hydrogen peroxide and glucose concentrations by the working electrode of the microneedle was characterized by the chronoamperometric method. At the same time, the effects of temperature and other interfering substances on the detection of glucose by the microneedle were also studied. In addition, the current attenuation of repeated glucose detection by the microneedle was characterized. The results show that the microneedle can detect glucose concentration more accurately, and the detection sensitivity also changes with temperature. It can also exclude the influence of interfering substances such as creatinine and uric acid on glucose detection, and has a high detection accuracy within a certain number of detections.

[0146] In a specific implementation of the manufacturing method of the intelligent deformable microneedle used above, the support seat 1 and the second needle-shaped body 4 fixedly connected thereto are manufactured by 3D printing, and then silver / silver chloride slurry is coated thereon as a reference electrode / counter electrode, and then a spring 3 is sleeved on the outside of the second needle-shaped body 4, and a working electrode is set on the spring 3.

[0147] like Fig. 20 As shown, a 3D printer is used to print and manufacture a first mold, the first mold comprising an outer shell body which is open and in the shape of a rectangular parallelepiped, a needle-shaped body model protrudes from the center of the inner bottom surface of the outer shell body in a direction perpendicular to the inner bottom surface, the needle tip of the needle-shaped body model is oriented in a direction perpendicular to the inner bottom surface, the height of the needle-shaped body model is greater than the height of the second needle-shaped body, and the area enclosed by the outer side wall of the needle-shaped body model near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring;

[0148] Making a PDMS concave mold: mixing the material PDMS and the curing agent in a mass ratio of 10:1, stirring thoroughly in the middle to generate a large number of bubbles, then evacuating in a vacuum for 30 minutes to remove all bubbles, then pouring the resulting mixed liquid into the first mold, picking out the bubbles generated in the middle with a needle, and then drying in an oven at 65° C. for 4 hours under a blast condition to obtain a PDMS concave mold having a needle-shaped concave shape corresponding to the needle-shaped body model; the height of the needle-shaped concave shape is greater than the height of the second needle-shaped body, and the area enclosed by the outer side wall of the needle-shaped concave shape near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring;

[0149] A liquid soluble material is placed in the needle-shaped concave shape, and the previously manufactured support seat is covered on one side of the PDMS concave shape having the needle-shaped concave shape, and the free end of the spring is extended into the needle-shaped concave shape and compressed. After drying, the support seat is separated from the PDMS concave shape, and the soluble needle-shaped body formed by the soluble material is fixedly connected to the surface of the support seat.

[0150] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A smart deformable microneedle, characterized in that: include: Support seat; A counter electrode arranged above the support seat and in a fixed state; An elastic body having a compressed state and a natural state, the elastic body is arranged above the support seat and is set in a fixed state, one end of the elastic body adjacent to the support seat is the fixed end of the elastic body, and the end away from the support seat is the free end of the elastic body, and the free end is in a compressed state when it is close to the fixed end, and a working electrode is arranged on the outer surface of the elastic body, the working electrode is electrically insulated from the counter electrode, and a specific enzyme capable of reacting with the analyte to be measured is arranged on the working electrode; A soluble needle-shaped body is fixed on the support base with the needle tip away from the support base. The soluble needle-shaped body completely wraps the counter electrode and the elastomer from the outside and has an inner cavity structure that makes the elastomer in a compressed state.

2. The intelligent deformable microneedle according to claim 1, characterized in that: The support seat has a bearing surface, the elastomer is extended and arranged in a direction perpendicular to the bearing surface, one end of the elastomer connected to the bearing surface is the fixed end so that the elastomer is set in the fixed state, and when the elastomer has the compressed state: the bottom end of the soluble needle-shaped body is fixed on the bearing surface and the direction of its needle tip is perpendicular to the direction of the bearing surface, the inner cavity structure of the soluble needle-shaped body is a chamber, the pair of electrodes and the elastomer are located in the chamber, and the area enclosed by the inner side wall of the soluble needle-shaped body adjacent to its needle tip is smaller than the cross-sectional area of ​​the free end.

3. The intelligent deformable microneedle according to claim 1 or 2, characterized in that: It also includes a reference electrode arranged corresponding to the working electrode and the counter electrode to form a three-electrode system.

4. The intelligent deformable microneedle according to claim 3, characterized in that: The elastic body is a spring.

5. The intelligent deformable microneedle according to claim 4, characterized in that: It also includes a support body fixed on the carrying surface, and the counter electrode and the reference electrode are arranged on the support body so as to be arranged in the fixed state.

6. The intelligent deformable microneedle according to claim 5, characterized in that: The support body is a second solid needle-shaped body, the bottom end of which is fixed to the bearing surface and the needle tip is oriented perpendicular to the bearing surface. The second needle-shaped body is located in the internal space of the spring.

7. The intelligent deformable microneedle according to claim 1, characterized in that: The inner cavity structure corresponds to the structure of the spring and the second needle-shaped body in a compressed state.

8. The intelligent deformable microneedle according to claim 1, characterized in that: The support seat is in the shape of a cuboid.

9. The intelligent deformable microneedle according to claim 1, characterized in that: The soluble needle-shaped body is a water-soluble needle-shaped body.

10. The intelligent deformable microneedle according to claim 1, characterized in that: The specific enzyme is arranged on the working electrode near the free end of the elastomer.

11. A method for manufacturing an intelligent deformable microneedle, characterized in that: The steps include: Providing a support base; A counter electrode is arranged above the support seat in a fixed state; An elastic body having a compressed state and a natural state is provided, and the elastic body is arranged above the support seat and is set in a fixed state, one end of the elastic body adjacent to the support seat is the fixed end of the elastic body, and the end away from the support seat is the free end of the elastic body, and the free end is in a compressed state when it is close to the fixed end, and a working electrode is arranged on the outer surface of the elastic body, and the working electrode is electrically insulated from the counter electrode, and a specific enzyme capable of reacting with an analyte to be detected is arranged on the working electrode; A soluble needle-shaped body is provided and fixed on the support base with its needle tip away from the support base. The soluble needle-shaped body completely wraps the counter electrode and the elastomer from the outside. The soluble needle-shaped body has an inner cavity structure that makes the elastomer in a compressed state.

12. The method for manufacturing the intelligent deformable microneedle according to claim 11, characterized in that: The steps include: Providing the support base having a bearing surface; The elastic body is fixedly mounted on the bearing surface, the elastic body is extended in a direction perpendicular to the bearing surface, one end of the elastic body connected to the bearing surface is the fixed end, and the other end is the free end; A working electrode is disposed at the free end of the elastic body; The specific enzyme is arranged on the working electrode near the free end of the elastomer; A fixed counter electrode is arranged above the carrying surface; A soluble needle-shaped body with a chamber inside is provided, the bottom end of the soluble needle-shaped body is fixed on the supporting surface and the direction of the needle tip is arranged to be perpendicular to the supporting surface, the soluble needle-shaped body is covered with the counter electrode and the working electrode, and the soluble needle-shaped body is squeezed against the free end of the elastomer so that the free end is compressed.

13. The method for manufacturing the intelligent deformable microneedle according to claim 12, characterized in that: The step of "providing the support base having a bearing surface; fixing the elastomer on the bearing surface, wherein the elastomer extends in a direction perpendicular to the bearing surface and one end of the elastomer connected to the bearing surface is the fixed end and the other end is the free end; arranging a working electrode at the position of the free end of the elastomer; arranging the specific enzyme on the working electrode near the free end of the elastomer; arranging a fixed counter electrode above the bearing surface" includes the following: The following model is designed using 3D modeling software: a rectangular parallelepiped is designed as a support base, and a second needle-shaped body is designed to be solid on the surface of the rectangular parallelepiped, and the bottom end of the second needle-shaped body is fixed on the surface of the rectangular parallelepiped and the needle tip is oriented perpendicular to the surface; Using a 3D printer to make the second needle-shaped body according to the model, and then setting the counter electrode on the surface of the second needle-shaped body; A spring is provided as the elastic body, the spring is sleeved on the outside of the second needle-shaped body and fixedly mounted on the surface of the rectangular parallelepiped, the spring is extended in a direction perpendicular to the surface, one end of the spring connected to the surface is a fixed end, and the other end is a free end, and the working electrode on the spring is not electrically connected to the counter electrode, and the working electrode and the counter electrode are externally connected using non-electrically connected connection pastes respectively; The specific enzyme is then immobilized on the working electrode.

14. The method for manufacturing the intelligent deformable microneedle according to claim 13, characterized in that: It also includes forming a reference electrode on the surface of the second needle-shaped body, wherein the reference electrode is formed by applying silver / silver chloride slurry, and the working electrode and the counter electrode are both formed by applying carbon slurry, gold slurry, platinum slurry, carbon-Prussian blue composite slurry, gold-Prussian blue composite slurry, or platinum-Prussian blue composite slurry.

15. The method for manufacturing the intelligent deformable microneedle according to claim 13, characterized in that: It also includes forming a reference electrode on the surface of the second needle-shaped body, which is formed by the following steps: evaporating or sputtering chromium or titanium as an adhesion layer, evaporating or sputtering a gold or platinum electrode thereon, evaporating or sputtering a silver film to obtain a silver electrode, and then immersing it in ferric chloride to obtain a silver / silver chloride electrode. The working electrode is formed by the following steps: evaporating or sputtering chromium or titanium as an adhesion layer, evaporating or sputtering a gold or platinum electrode thereon, and then electroplating Prussian blue thereon.

16. The method for manufacturing the intelligent deformable microneedle according to claim 13, characterized in that: Glucose oxidase is provided as the specific enzyme, including: dissolving glucose oxidase in a phosphate buffer solution to prepare a solution with a concentration of 10u / μL, mixing it evenly with a 0.5% glutaraldehyde solution at a volume ratio of 1:1, dripping it onto the working electrode, drying it at 4°C for 24 hours, and then washing away the unfixed glucose oxidase with a phosphate buffer solution.

17. The method for manufacturing the intelligent deformable microneedle according to claim 11, characterized in that: The step of "providing a soluble needle-shaped body, fixing it on the support base with its needle tip away from the support base, and completely wrapping the counter electrode and the elastomer from the outside with the soluble needle-shaped body, wherein the soluble needle-shaped body has an inner cavity structure that makes the elastomer in a compressed state" includes the following: This step uses a soft lithography method as follows: First, a 3D printer is used to print and manufacture a first mold, the first mold comprising an open outer shell in the shape of a rectangular parallelepiped, a needle-shaped body model protrudes from the center of the inner bottom surface of the outer shell in a direction perpendicular to the inner bottom surface, the needle tip of the needle-shaped body model is oriented in a direction perpendicular to the inner bottom surface, the height of the needle-shaped body model is greater than the height of the second needle-shaped body, and the area enclosed by the outer side wall of the needle-shaped body model near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring; Making a PDMS concave mold: mixing the material PDMS and the curing agent in a mass ratio of 10:1, stirring thoroughly in the middle to generate a large number of bubbles, then evacuating in a vacuum for 30 minutes to remove all bubbles, then pouring the resulting mixed liquid into the first mold, picking out the bubbles generated in the middle with a needle, and then drying in an oven at 65° C. for 4 hours under a blast condition to obtain a PDMS concave mold having a needle-shaped concave shape corresponding to the needle-shaped body model; the height of the needle-shaped concave shape is greater than the height of the second needle-shaped body, and the area enclosed by the outer side wall of the needle-shaped concave shape near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring; A liquid soluble material is placed in the needle-shaped concave shape, and the previously manufactured support seat is covered on one side of the PDMS concave shape having the needle-shaped concave shape, and the free end of the spring is extended into the needle-shaped concave shape and compressed. After drying, the support seat is separated from the PDMS concave shape, and the soluble needle-shaped body formed by the soluble material is fixedly connected to the surface of the support seat.

18. The method for manufacturing the intelligent deformable microneedle according to claim 17, characterized in that: The soluble material is a PVP-PVA composite material, and the manufacturing method is as follows: Weigh 0.6 g PVA and 3.0 g PVP, dissolve in 20 mL deionized water, heat at 95 °C for 3 h to completely dissolve, and cool to room temperature.

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