Intelligent deformable microneedle and manufacturing method thereof

Abstract

The present invention belongs to the field of microneedle technology, and specifically relates to an intelligent deformable microneedle and a manufacturing method thereof; the microneedle comprises: a support seat; a counter electrode arranged above the support seat; an elastomer having a compressed state and a natural state, a working electrode provided on the outer surface of the elastomer, and a specific enzyme capable of reacting with an analyte to be detected provided on the working electrode; a soluble needle-shaped body fixed on the support seat, the soluble needle-shaped body completely wraps the counter electrode and the elastomer from the outside, and the soluble needle-shaped body has an inner cavity structure that puts the elastomer in a compressed state. The microneedle is provided with an elastomer inside, and after piercing the skin, the soluble needle-shaped body dissolves, exposing the elastomer inside, and lengthening the elastomer, onto which the working electrode of the electrochemical sensor is attached. The length of the microneedle inserted in this way is very short, but the working electrode obtained can penetrate deep into the skin to obtain accurate detection of the corresponding substance.

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 microneedles is relatively small, so that the length of the microneedles 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 disadvantages has become a technical problem that technicians in this field urgently need to solve. Summary of the Invention

[0005] The present invention addresses the technical problem that 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 disposed above the support seat and fixedly arranged;

[0010] An elastomer having a compressed state and a natural state, the elastomer being arranged above the support seat and being in a fixed state, the end of the elastomer adjacent to the support seat being the fixed end of the elastomer, the end away from the support seat being the free end of the elastomer, the free end being in a compressed state when it is close to the fixed end, a working electrode being provided on the outer surface of the elastomer, the working electrode being electrically insulated from the counter electrode, and a specific enzyme being provided on the working electrode that can react with the analyte to be measured;

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

[0012] Preferably, the support seat has a bearing surface, the elastomer is extended 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 bearing surface, the inner cavity structure of the soluble needle-shaped body is a chamber, the counter electrode 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, a reference electrode is provided 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 solid second needle-shaped body, the bottom end of the second needle-shaped body is fixed to the bearing surface and the direction of the needle tip is 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 embedded with an elastomer. Upon penetration, the soluble needle dissolves, exposing the elastomer, which becomes longer and carries the working electrode of the electrochemical sensor. This allows the microneedle to penetrate deeply into the skin, allowing for accurate detection of the 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 elastomer having a compressed state and a natural state is provided, and the elastomer is arranged above the support seat and in a fixed state. The end of the elastomer adjacent to the support seat is the fixed end of the elastomer, and the end away from the support seat is the free end of the elastomer. When the free end is close to the fixed end, the elastomer is in a compressed state. A working electrode is provided on the outer surface of the elastomer, and the working electrode is electrically insulated from the counter electrode. A specific enzyme capable of reacting with an analyte to be measured is provided 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 an advantage, 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 extending in a direction perpendicular to the bearing surface, one end of the elastic body connected to the bearing surface being the fixed end and the other end being the free end;

[0032] A working electrode is provided 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 provided above the supporting 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 its 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 connected to the bearing surface is the fixed end, and the other end is the free end; disposing a working electrode at the position of the free end of the elastomer; disposing the specific enzyme on the working electrode near the free end of the elastomer; and disposing the fixed counter electrode above the bearing surface" includes the following:

[0037] Using 3D modeling software, design the following model: a rectangular parallelepiped as a support base, and a second solid needle-shaped object on the surface of the rectangular parallelepiped, with the bottom end of the second needle-shaped object fixed to the surface of the rectangular parallelepiped and the needle tip oriented perpendicular to the surface;

[0038] Using a 3D printer to manufacture 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 being sleeved on the outside of the second needle-shaped body and fixed to the surface of the rectangular parallelepiped, the spring extending in a direction perpendicular to the surface, with one end connected to the surface being a fixed end and the other end being a free end, and the working electrode on the spring being electrically non-connected to the counter electrode, and the working electrode and the counter electrode being externally connected using a non-electrically connected connection paste;

[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, wherein the reference electrode is formed by applying silver / silver chloride paste, and the working electrode and the counter electrode are both formed by applying carbon paste or gold paste or platinum paste or carbon-Prussian blue composite paste or gold-Prussian blue composite paste or platinum-Prussian blue composite paste.

[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 the solution evenly with a 0.5% glutaraldehyde solution in a volume ratio of 1:1, adding the solution dropwise to the working electrode, drying the solution 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, wherein the soluble needle-shaped body has an inner cavity structure that puts the elastomer in a compressed state" includes the following:

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

[0046] A first mold is first produced using a 3D printer. The first mold comprises an open, rectangular outer shell. A needle-shaped 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-shaped model has a tip perpendicular to the inner bottom surface. The needle-shaped model has a height greater than that of the second needle-shaped model. The area enclosed by the outer wall of the needle-shaped model near its tip is smaller than the cross-sectional area of ​​the free end of the spring.

[0047] Preparation of a PDMS concave mold: PDMS and a curing agent are mixed in a mass ratio of 10:1, stirred thoroughly to generate a large number of bubbles, and then vacuumed for 30 minutes to remove all bubbles. The resulting mixture is then poured into the first mold, and bubbles generated during the process are removed with a needle. The mixture is then dried in an oven at 65°C for 4 hours using a forced air conditioner 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 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 the side of the PDMS concave mold 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 mold, 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 them in 20 mL deionized water, heat at 95 °C for 3 h to completely dissolve them, 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0052] Figure 1 This is a schematic structural diagram of a specific embodiment of the intelligent deformable microneedle provided by the present invention in its natural state;

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

[0054] Figure 3 Schematic diagram of the measurement of the specific dimensions of the microneedle in a top view;

[0055] Figure 4 Schematic diagram of the measurement of the specific dimensions of the microneedle in side view;

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

[0057] Figure 6 Schematic diagram of the measurement of the specific dimensions of the dissolved microneedle in 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] Figure 9 It curve of hydrogen peroxide detected by microneedle at -0.1V;

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

[0062] Figure 11 CV curve of glucose detection by microneedles;

[0063] Figure 12 This is the relationship between the scan rate and oxidation peak current of the microneedle detection of glucose;

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

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

[0066] Figure 15 It curves of glucose detection by microneedles at -0.1V and different temperatures;

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

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

[0069] Figure 18 The IT curves of continuous dripping of creatinine, uric acid and glucose on the microneedle;

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

[0071] Figure 20 The steps 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; disposing a working electrode at the position of the free end of the elastic body; disposing the specific enzyme on the working electrode near the free end of the elastic body; and disposing the fixed counter electrode above the bearing surface" are schematic flow diagrams;

[0072] Figure 21 A 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, 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 puts the elastomer in a compressed state."

[0073] Figure 1-21 The accompanying drawings are numerals 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 with reference to 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 provided in the internal space of the spring 3. A working electrode is provided on the spring 3, and a reference electrode / counter electrode is provided 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] Regarding 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 Figure 20 , formed by the following method:

[0078] First, a first mold is printed using a 3D printer. The first mold includes an open rectangular outer shell. A needle-shaped 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 model is oriented perpendicular to the inner bottom surface. The height of the needle-shaped model is greater than the height of the second needle 4. The area enclosed by the outer wall of the needle-shaped model near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring 3.

[0079] Preparation of a PDMS concave mold: PDMS material and curing agent are mixed in a mass ratio of 10:1, stirred thoroughly during the process to generate a large number of bubbles, then vacuumed for 30 minutes to remove all bubbles, and then the resulting mixture is poured into the first mold. The bubbles generated during the process are picked out with a needle, and then dried in an oven at 65°C under blast conditions for 4 hours 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 4, and the area enclosed by the outer 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 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 the 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 side 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 electrode setup, one method is:

[0083] Chromium (Cr) or titanium (Ti) is evaporated or sputtered on all electrodes as an adhesion layer. Gold or platinum electrodes are evaporated or sputtered, and Prussian blue is electroplated on the working electrode. Silver film is evaporated or sputtered on the reference electrode 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 counter electrode are coated with slurries, typically 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 coated with silver / silver chloride slurry.

[0086] The spring 3 is fixed in the smart deformable microneedle in a compressed state. When the smart deformable microneedle is actually working, the soluble needle-like 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 ), thereby achieving the purpose of extending the microneedle during the entire dissolution and 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 was 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, the actual microneedle dimensions (base diameter 662.4μm, top diameter 367.7μm, height 1109.1μm) are consistent with the designed dimensions (base diameter 700μm, top diameter 200μm, height 1100μm). The only significant difference is in the top diameter, which may be caused by excessive stress on the microneedle.

[0090] Characterization of the dissolution and deformation properties 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 dried. The tip of the microneedle was observed under SEM and optical microscope, with a focus on comparing the length changes. Figure 5 and Figure 6 As shown in the figure, the length of the microneedle changed from 1109.1μm before dissolution to 1368.2μm, which is close to the spring length of 1400μm, and the total elongation reached over 20%. This shows that the intelligent deformable microneedle has a certain degree of deformation ability and can realize the extension function of the working electrode after dissolution.

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

[0094] 1. Cyclic voltammetry curve analysis for hydrogen peroxide detection

[0095] Hydrogen peroxide, a product of glucose oxidation, is also the actual detection target of the microneedles. Therefore, hydrogen peroxide was used to test the performance of the microneedles. The first step was to use cyclic voltammetry to find the appropriate 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 supply. 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 hydrogen peroxide detection at different rates have roughly the same shape, but the peak current and voltage are different. By comparison, it is found that the peak current decreases as the scan rate decreases, and the corresponding voltage increases as the scan rate decreases, gradually approaching -0.1V. This indicates 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. The peak current at each scan rate is recorded, as shown in the following table:

[0098] Detecting 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 to obtain Figure 8 It was found that the square of the current was roughly proportional to the scan rate, which is consistent with the results, indicating that the sensing process of hydrogen peroxide is mainly controlled by diffusion.

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

[0105] The measurement is performed using the it-Amperometric method. The specific principle of the experiment is that a constant voltage is applied between the two electrodes of the microneedle as an excitation, which causes the oxidation and reduction of hydrogen peroxide on the electrodes, gaining and losing electrons, generating a current signal. The strength of this current signal can be used to determine the concentration of the generated hydrogen peroxide.

[0106] Dissolve 30% hydrogen peroxide (corresponding to a molar concentration of 9.9 mol / L) in phosphate buffer solution to prepare a series of hydrogen peroxide solutions with concentrations of 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, and 25 mmol / L. Connect the two electrodes of the microneedle to a power source, add 10 μL of the test solution to the needle tip of the microneedle, select a voltage of -0.1 V, and measure the relationship between the current and time. The measurement results are shown in the figure. Figure 9 shown.

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

[0108] Detecting 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 Figure 2. Figure 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 microneedles can more accurately measure the concentration of hydrogen peroxide, 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] Glucose was diluted with phosphate buffer solution to prepare a glucose solution with a concentration of 30 mmol / L. The two electrodes of the microneedle were connected to a power supply. 30 μL of the test solution was dripped onto the tip of the microneedle. The voltage range was -0.6 V to 0.6 V, the scan rate was 0.01 V / s, and the relationship between current and voltage was measured. The scan rate was then changed to 0.03 V / s, 0.05 V / s, 0.07 V / s, and 0.09 V / s. The measurement results are as follows: Figure 11 shown.

[0114] observe Figure 11 It can be found that the CV curves of glucose detection by microneedles at different scan rates have similar shapes. The current peak and the corresponding voltage have the same trend as the scan 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 scan 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 cyclic voltammetry of hydrogen peroxide. Therefore, a voltage of -0.1V was used in the subsequent chronoamperometry test of glucose concentration. Further analysis was performed by fitting the square of the current and the scan rate to obtain Figure 12 It can be found that the square of the current and the scan rate have a good linear relationship, which is consistent with 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 for Glucose Detection

[0120] Similar to hydrogen peroxide detection, the chronoamperometry method is used for measurement. The specific principle of glucose detection in this experiment is that the glucose oxidase on the microneedles produces hydrogen peroxide during the oxidation of glucose. Under a given excitation voltage, hydrogen peroxide undergoes redox reactions at the electrode, gaining and losing electrons, generating a current signal. The strength of this current signal can be used to determine the concentration of the generated hydrogen peroxide. Combined with the 1:1 ratio of glucose to hydrogen peroxide in the glucose oxidation reaction, the glucose concentration can be estimated.

[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 supply. 10μL of the test solution was added to the needle tip of the microneedle. The voltage was selected to be -0.1V, and the relationship between the current and time was measured. The measurement results are shown in the figure. Figure 13 shown.

[0122] Since the redox of glucose takes a certain amount of time, the test time is longer than that of hydrogen peroxide. Figure 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, where 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: Figure 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 as consistent as possible. This ensures 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 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 glucose solution were placed on a hot plate for heating. The two electrodes of the microneedle were connected to a power supply. 10 μL of the test solution was dripped onto the tip of the microneedle. A voltage of -0.1 V was selected. Heating was started at 22°C, and the temperature was kept constant for a period of time after stopping the heating at 5°C intervals. The IT curve was measured from 22°C to 55°C. The results are shown in the figure below. Figure 15 shown.

[0130] It can be found that the IT curve of glucose detection 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 Figure 16 ,observe Figure 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 glucose detection:

[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 high concentrations 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 supply, 10 μL of creatinine solution was dripped onto the needle tip of the microneedle, a voltage of -0.1 V was selected, and the relationship between the current and time was measured. The remaining solution was then aspirated with a pipette, and uric acid solution was added after drying for measurement. Finally, the current and time curve of the glucose solution were measured. The results are as follows. Figure 17 shown.

[0139] observe Figure 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 high 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 onto 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: Figure 18 As shown in the figure, since glucose requires a certain amount of time to diffuse, the current does not reach its peak immediately upon addition. The greater current change during the second addition of uric acid and creatinine 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] Glucose stability test:

[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 placed at 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 Figure 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 can still ensure a certain accuracy after multiple measurements.

[0145] The microneedles made 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%. The actual detection effect of the working electrode of the microneedle on hydrogen peroxide and glucose was tested. The suitable 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 microneedle detection of glucose were also studied. In addition, the current attenuation of the microneedle repeated detection of glucose 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 eliminate 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 detection times.

[0146] In a specific implementation of the manufacturing method of the intelligent deformable microneedle adopted above, the support base 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 Figure 20 As shown, a first mold is first printed using a 3D printer. The first mold includes an open outer shell in the shape of a rectangular parallelepiped. A needle-shaped 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 model is oriented perpendicular to the inner bottom surface. The height of the needle-shaped model is greater than the height of the second needle. The area enclosed by the outer wall of the needle-shaped model near its needle tip is smaller than the cross-sectional area of ​​the free end of the spring.

[0148] Preparation of a PDMS concave mold: PDMS and a curing agent are mixed in a mass ratio of 10:1, stirred thoroughly to generate a large number of bubbles, and then vacuumed for 30 minutes to remove all bubbles. The resulting mixture is then poured into the first mold, and bubbles generated during the process are removed with a needle. The mixture is then dried in an oven at 65°C for 4 hours using a forced air conditioner 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 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 made support seat is covered on the side of the PDMS concave mold 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 mold, and the soluble needle-shaped body formed by the soluble material is fixedly connected to the surface of the support seat.

[0150] It will be understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present invention, and the present invention is not limited thereto. Those skilled in the art will appreciate that various modifications and improvements can be made without departing from the spirit and substance of the present invention, and such 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 disposed above the support seat and fixedly arranged; An elastomer having a compressed state and a natural state, the elastomer being arranged above the support seat and being in a fixed state, the end of the elastomer adjacent to the support seat being the fixed end of the elastomer, the end away from the support seat being the free end of the elastomer, the free end being in a compressed state when it is close to the fixed end, a working electrode being provided on the outer surface of the elastomer, the working electrode being electrically insulated from the counter electrode, and a specific enzyme being provided on the working electrode that can react with the analyte to be measured; A soluble needle-shaped body is fixed on the support seat with its needle tip away from the support seat. The soluble needle-shaped body completely wraps the counter electrode and the elastomer from the outside and has an inner cavity structure that puts 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, and the elastomer is extended 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. 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 bearing surface. The inner cavity structure of the soluble needle-shaped body is a chamber. The counter electrode and the elastomer are located in the chamber. 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 2, characterized in that: It also includes a reference electrode 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 solid second 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. The second needle-shaped body is located in the internal space of the spring.

7. The intelligent deformable microneedle according to claim 6, 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 elastomer having a compressed state and a natural state is provided, and the elastomer is arranged above the support seat and in a fixed state. The end of the elastomer adjacent to the support seat is the fixed end of the elastomer, and the end away from the support seat is the free end of the elastomer. When the free end is close to the fixed end, the elastomer is in a compressed state. A working electrode is provided on the outer surface of the elastomer, and the working electrode is electrically insulated from the counter electrode. A specific enzyme capable of reacting with an analyte to be measured is provided 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 extending in a direction perpendicular to the bearing surface, one end of the elastic body connected to the bearing surface being the fixed end and the other end being the free end; A working electrode is provided 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 provided above the supporting 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 its 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 steps 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 connected to the bearing surface is the fixed end, and the other end is the free end; disposing a working electrode at the free end of the elastomer; disposing the specific enzyme on the working electrode near the free end of the elastomer; and disposing the fixed counter electrode above the bearing surface" include the following: Using 3D modeling software, design the following model: a rectangular parallelepiped as a support base, and a second solid needle-shaped object on the surface of the rectangular parallelepiped, with the bottom end of the second needle-shaped object fixed to the surface of the rectangular parallelepiped and the needle tip oriented perpendicular to the surface; Using a 3D printer to make the second needle-shaped body according to the model, and then arranging the counter electrode on the surface of the second needle-shaped body; A spring is provided as the elastic body, the spring being sleeved on the outside of the second needle-shaped body and fixed to the surface of the rectangular parallelepiped, the spring extending in a direction perpendicular to the surface, with one end connected to the surface being a fixed end and the other end being a free end, and the working electrode on the spring being electrically non-connected to the counter electrode, and the working electrode and the counter electrode being externally connected using a non-electrically connected connection paste; 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, the reference electrode is formed by applying silver / silver chloride paste, and the working electrode and the counter electrode are both formed by applying carbon paste or gold paste or platinum paste or carbon-Prussian blue composite paste or gold-Prussian blue composite paste or platinum-Prussian blue composite paste.

15. The method for manufacturing the intelligent deformable microneedle according to claim 13, wherein: 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, wherein: 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 in a volume ratio of 1:1, adding it dropwise to 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 13, wherein: 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 with the soluble needle-shaped body from the outside, wherein the soluble needle-shaped body has an inner cavity structure that places the elastomer in a compressed state" includes the following: This step uses a soft lithography method as follows: A first mold is first produced using a 3D printer. The first mold comprises an open, rectangular outer shell. A needle-shaped 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-shaped model has a tip perpendicular to the inner bottom surface. The needle-shaped model has a height greater than that of the second needle-shaped model. The area enclosed by the outer wall of the needle-shaped model near its tip is smaller than the cross-sectional area of ​​the free end of the spring. Preparation of a PDMS concave mold: PDMS and a curing agent are mixed in a mass ratio of 10:1, stirred thoroughly to generate a large number of bubbles, and then vacuumed for 30 minutes to remove all bubbles. The resulting mixture is then poured into the first mold, and bubbles generated during the process are removed with a needle. The mixture is then dried in an oven at 65°C for 4 hours using a forced air conditioner 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 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 the side of the PDMS concave mold 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 mold, 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 them in 20 mL deionized water, heat at 95 °C for 3 h to completely dissolve them, and cool to room temperature.

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