Microneedle sensor based on cross-linked recombinant bovine serum albumin and preparation method thereof

Abstract

The invention discloses a microneedle sensor based on cross-linked recombinant bovine serum albumin and a preparation method of the microneedle sensor. The preparation method comprises the following steps: S1, providing a microneedle array electrode; s2, preparation of a working electrode: providing a bovine serum albumin solution, and adding beta-mercaptoethanol into the bovine serum albumin solution to reduce bovine serum albumin so as to obtain a reduced bovine serum albumin solution; then adding ethidene diamine and ethanol to crosslink the reduced bovine serum albumin to obtain a crosslinked recombinant bovine serum albumin polymer solution; coating the cross-linked recombinant bovine serum albumin polymer solution on the surface of the microneedle array electrode to form a buffer layer so as to obtain a working electrode; s3, preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin: providing a reference electrode and a field effect transistor; a working electrode and a reference electrode are fixed on the same substrate and connected with a field effect transistor through an external circuit, and the microneedle sensor based on the crosslinked recombinant bovine serum albumin is obtained.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a microneedle sensor based on cross-linked recombinant bovine serum albumin and a preparation method thereof. Background Art

[0002] The ion concentration in the human body is one of the most important human indicators, and the monitoring of ion concentration is an important part of human health management. At present, there are some ion concentration monitoring sensors on the market, which monitor the ion concentration in human tissue fluid by inserting implantable microneedle electrodes into the subcutaneous tissue instead of traditional blood testing methods. However, most of the existing ion concentration monitoring microneedle sensors are metal hard microneedle sensors. Although they have good skin puncture ability, the rigidity of the microneedle does not match the flexibility of the skin. When the wearer moves, the rigid microneedle sensor can easily fall off from the soft skin surface. At the same time, the hard microneedle can easily pierce the subcutaneous tissue when the skin is squeezed or during exercise, causing discomfort and the risk of infection, affecting the safety and comfort of the device. More importantly, long-term wearing of hard microneedle sensors may cause skin inflammation or even ulceration, which not only reduces the accuracy of the sensor itself but also affects human health. Summary of the invention

[0003] The purpose of the present invention is to solve the problem that the stiffness of the microneedle in the existing microneedle sensor does not match the flexibility of the skin, which causes the existing microneedle sensor to easily fall off and puncture the subcutaneous tissue when worn, affecting the safety and comfort of wearing.

[0004] In order to achieve the above object, the present invention provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, comprising the following steps:

[0005] S1, provide microneedle array electrodes.

[0006] S2, preparing a working electrode: providing a bovine serum albumin solution, adding β-mercaptoethanol thereto to reduce the bovine serum albumin, thereby obtaining a reduced bovine serum albumin solution; then adding ethylenediamine and ethanol to cross-link the reduced bovine serum albumins, thereby obtaining a cross-linked recombinant bovine serum albumin polymer solution; coating the cross-linked recombinant bovine serum albumin polymer solution on the surface of the microneedle array electrode to form a buffer layer, thereby obtaining a working electrode.

[0007] S3, preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin: providing a reference electrode and a field effect transistor; fixing the working electrode and the reference electrode on the same substrate, connecting them to the field effect transistor through an external circuit, to obtain the microneedle sensor based on cross-linked recombinant bovine serum albumin.

[0008] Optionally, in step S2, the bovine serum albumin solution is prepared by taking bovine serum albumin and dissolving the bovine serum albumin in a mixed solution of trifluoroethanol and water to obtain a bovine serum albumin solution.

[0009] Optionally, in the bovine serum albumin solution, the concentration of bovine serum albumin is 5wt% to 12wt%.

[0010] Optionally, the volume ratio of trifluoroethanol to water is 1:(2-9).

[0011] Optionally, in step S2, the volume ratio of ethylenediamine to ethanol is 1:(0.5-2).

[0012] Optionally, in step S1, the microneedle array electrode comprises: microneedle bodies arranged in an array, and a conductive layer and an ion selective layer sequentially disposed on the microneedle bodies; the ion selective layer is prepared by the following method:

[0013] Dissolving an ion carrier, an ion electrolyte, a stabilizer, and a plasticizer in an organic solvent to obtain an ion selective material mixture; coating the ion selective material mixture on the surface of the conductive layer to form an ion selective layer;

[0014] The ion carriers include: Na + Carrier, Ca 2+ Carrier, K + Carrier, Mg 2+ The invention relates to a carrier comprising at least one of: sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate; the ionic electrolyte comprises sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate; the stabilizer comprises at least one of polyvinyl chloride, polyurethane and polyvinyl acetate; the plasticizer comprises at least one of di(2-ethylhexyl)sebacate, nitrophenyl octyl ether and phthalate; the organic solvent comprises tetrahydrofuran and / or cyclohexanone.

[0015] Optionally, the mass ratio of the ion carrier, ion electrolyte, stabilizer and plasticizer is (10-30): (5-10): (20-50): (500-800).

[0016] Optionally, the conductive layer is formed on the surface of the microneedle body by at least one of magnetron sputtering, chemical deposition, and vacuum evaporation.

[0017] Optionally, the material of the conductive layer includes at least one of gold, platinum, copper and silver.

[0018] The present invention also provides a microneedle sensor based on cross-linked recombinant bovine serum albumin prepared by the above method.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, wherein a cross-linked recombinant bovine serum albumin polymer solution is coated on the surface of the microneedle, and the cross-linked recombinant bovine serum albumin polymer is used as a buffer layer to form a buffer between the microneedle array electrode and the skin tissue, thereby improving the safety and comfort of wearing the sensor.

[0021] The cross-linked recombinant bovine serum albumin polymer buffer layer provided by the present invention is in a dry state before use (when the microneedle has not pierced the skin), and has the properties of high mechanical strength and high rigidity. The cross-linked recombinant bovine serum albumin polymer buffer layer serves as the surface of the microneedle array electrode, making it easier for the microneedle array electrode to pierce the skin. After the microneedle array electrode pierces the skin, the cross-linked recombinant bovine serum albumin polymer absorbs moisture in the tissue fluid and becomes wet, with reduced rigidity, matching the flexibility of the skin, so that the microneedle sensor is not easy to fall off the skin surface during wearing; and when the microneedle sensor is squeezed, the cross-linked recombinant bovine serum albumin polymer with reduced rigidity can form a flexible buffer between the hard microneedle and the flexible skin tissue, so that the microneedle is not easy to pierce the subcutaneous tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the preparation process of a microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1.

[0023] Figure 2 This is a process flow chart for preparing the cross-linked recombinant bovine serum albumin polymer solution of Example 1.

[0024] Figure 3 The infrared spectra of bovine serum albumin and cross-linked recombinant bovine serum albumin polymer in Example 1 are shown.

[0025] Figure 4 This is a diagram of the mechanical properties of the cross-linked recombinant bovine serum albumin polymer in Example 1.

[0026] Figure 5 This is a diagram of the mechanical properties of the microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1.

[0027] Figure 6 This is the X-ray photoelectron spectrum of the cross-linked recombinant bovine serum albumin polymer of Example 1;

[0028] A is a full scan X-ray photoelectron spectrum of the cross-linked recombinant bovine serum albumin polymer of Example 1;

[0029] B is the C1s fine spectrum of the cross-linked recombinant bovine serum albumin polymer of Example 1;

[0030] C is the N1s fine spectrum of the cross-linked recombinant bovine serum albumin polymer of Example 1;

[0031] D is the S2p fine spectrum of the cross-linked recombinant bovine serum albumin polymer of Example 1;

[0032] E is the O1s fine spectrum of the cross-linked recombinant bovine serum albumin polymer of Example 1.

[0033] Figure 7 This is an atomic force microscope image of the cross-linked recombinant bovine serum albumin polymer in Example 1.

[0034] Figure 8 The scanning electron microscope images are of the microneedle of Example 1 (containing only polystyrene) and the microneedle of the finally manufactured microneedle sensor based on cross-linked recombinant bovine serum albumin.

[0035] Fig. 9 The Na of the microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1 + Detection performance graph;

[0036] A is the Na of the microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1 + Concentration-response current-time curves;

[0037] B is the current of the microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1 -Na + Concentration fitting curve;

[0038] C is the Na of the microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1 + Sensitivity test chart;

[0039] D is the stability test curve of the microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1.

[0040] Fig.10 This is a diagram of long-term wearing safety assessment of a microneedle sensor based on cross-linked recombinant bovine serum albumin in Example 1;

[0041] A is a picture of the skin condition of mice after they were momentarily wearing the microneedles of Example 1 (containing only polystyrene) and the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin;

[0042] B is a picture of the skin condition of mice after wearing the microneedles of Example 1 (containing only polystyrene) and the microneedles of the final microneedle sensor based on cross-linked recombinant bovine serum albumin for 7 days and 14 days. DETAILED DESCRIPTION

[0043] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0044] The present invention provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, comprising the following steps:

[0045] S1, provide microneedle array electrodes.

[0046] S2, preparing a working electrode: providing a bovine serum albumin solution, adding β-mercaptoethanol thereto to reduce the bovine serum albumin, thereby obtaining a reduced bovine serum albumin solution; then adding ethylenediamine and ethanol to cross-link the reduced bovine serum albumins, thereby obtaining a cross-linked recombinant bovine serum albumin polymer solution;

[0047] The cross-linked recombinant bovine serum albumin polymer solution is coated on the surface of the microneedle array electrode to form a buffer layer to obtain a working electrode.

[0048] S3, preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin: providing a reference electrode and a field effect transistor; fixing the working electrode and the reference electrode on the same substrate, connecting them to the field effect transistor through an external circuit, to obtain the microneedle sensor based on cross-linked recombinant bovine serum albumin.

[0049] The present invention provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, wherein a cross-linked recombinant bovine serum albumin polymer solution is coated on the surface of the microneedle, and the cross-linked recombinant bovine serum albumin polymer is used as a buffer layer to form a buffer between the microneedle array electrode and the skin tissue, thereby improving the safety and comfort of wearing the sensor.

[0050] The cross-linked recombinant bovine serum albumin polymer buffer layer provided by the present invention is in a dry state before use (when the microneedle has not pierced the skin), and has the properties of high mechanical strength and high rigidity. The cross-linked recombinant bovine serum albumin polymer buffer layer serves as the surface of the microneedle array electrode, making it easier for the microneedle array electrode to pierce the skin. After the microneedle array electrode pierces the skin, the cross-linked recombinant bovine serum albumin polymer absorbs moisture in the tissue fluid and becomes wet, with reduced rigidity, matching the flexibility of the skin, so that the microneedle sensor is not easy to fall off the skin surface during wearing; and when the microneedle sensor is squeezed, the cross-linked recombinant bovine serum albumin polymer with reduced rigidity can form a flexible buffer between the hard microneedle and the flexible skin tissue, so that the microneedle is not easy to pierce the subcutaneous tissue.

[0051] In some embodiments, in step S1, the microneedle array electrode comprises: microneedle bodies arranged in an array, and a conductive layer and an ion selective layer sequentially disposed on the microneedle bodies; the ion selective layer is prepared by the following method:

[0052] Dissolving an ion carrier, an ion electrolyte, a stabilizer, and a plasticizer in an organic solvent to obtain an ion selective material mixture; coating the ion selective material mixture on the surface of the conductive layer to form an ion selective layer;

[0053] The ion carriers include: Na + Carrier, Ca 2+ Carrier, K + Carrier, Mg 2+ The invention relates to a carrier comprising at least one of: sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate; the ionic electrolyte comprises sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate; the stabilizer comprises at least one of polyvinyl chloride, polyurethane and polyvinyl acetate; the plasticizer comprises at least one of di(2-ethylhexyl)sebacate, nitrophenyl octyl ether and phthalate; the organic solvent comprises tetrahydrofuran and / or cyclohexanone.

[0054] In some embodiments, in step S2, the bovine serum albumin solution is prepared by the following method:

[0055] Bovine serum albumin is taken and dissolved in a mixed solution of trifluoroethanol and water to obtain a bovine serum albumin solution.

[0056] The volume ratio of trifluoroethanol to water is 1:(2-9), and the concentration of bovine serum albumin in the solution is 5wt%-12wt%.

[0057] The bovine serum albumin used in the present invention has the advantages of low cost and good biocompatibility, and the microneedle sensor prepared therefrom has the advantage of high biosafety.

[0058] In some embodiments, the field effect transistor in step S3 is purchased from Song Teng, which is a SI2302 A2SHB SOT232.5A / 20V N-type channel field effect transistor, and the reference electrode is prepared by the following method:

[0059] (1) Providing a microneedle mold, pouring a polystyrene solution into the microneedle mold, and centrifugally curing and demoulding to obtain microneedles. The microneedles include: a substrate, and microneedle bodies arranged in an array.

[0060] The microneedle array electrode mold can be made of polydimethylsiloxane (PDMS) and is obtained by laser engraving technology. In other preferred embodiments, the microneedle array electrode mold can also be obtained by 3D printing technology.

[0061] (2) A conductive layer is prepared on the surface of the microneedle, and then an insulating material (such as epoxy resin glue) is coated on the substrate area of ​​the microneedle except the needle body to obtain a microneedle electrode.

[0062] The microneedle electrode was immersed in the Ag / AgCl slurry for 15 minutes, taken out and placed in an oven, and baked at 40° C. for 8 hours to fix the Ag / AgCl slurry on the microneedle electrode to obtain a reference electrode.

[0063] Example 1

[0064] like Figure 1 As shown, this embodiment provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, comprising the following steps:

[0065] (1) Preparation of microneedle electrodes:

[0066] A microneedle mold was provided, 3 g of polystyrene (Aladdin, P107085) was weighed and dissolved in 15 mL of ethyl acetate (Aladdin, E116132) to obtain a polystyrene solution. The polystyrene solution was dripped into the microneedle mold, and the polystyrene solution was filled into the microneedle mold under centrifugal conditions at 3500 rpm, and the microneedle was obtained by demolding after drying at room temperature.

[0067] The microneedle comprises: a substrate and arrayed microneedle bodies, wherein the microneedle bodies are conical (in other preferred embodiments, the shape of the microneedle bodies may also be pyramidal, cylindrical with a sharp tip, etc.), the length of the microneedle bodies is 550 μm, and the spacing between adjacent microneedle bodies is 600 μm.

[0068] The microneedle is placed in a chamber of a magnetron sputtering instrument, and magnetron sputtering is performed using a metal target. The sputtering environment is a 1.5Pa vacuum environment. The sputtering process is performed for 300 to 100 seconds under 10 to 30 mA conditions to form a conductive layer on the surface of the microneedle. Then, an insulating material (such as epoxy resin glue) is coated on the substrate area of ​​the microneedle except the needle body to obtain a microneedle electrode.

[0069] In addition to the magnetron sputtering process, the conductive layer can also be prepared by chemical deposition or vacuum evaporation. The conductive layer can be made of at least one of gold, platinum, copper and silver.

[0070] (2) Preparation of ion selective layer: The ion selective layer prepared in this example is Na + Selective layer.

[0071] 20 mg Na + The carrier (Merck, 71747-50MG) was added to 8 mL of tetrahydrofuran (Sino-pharm Shanghai Trial, 40058161), and then 6.5 mg of sodium tetrakis[3,5-di(trifluoromethyl)phenyl]borate (McLean, S832379), 35 mg of polyvinyl chloride (Aladdin, P107109), and 650 mg of di(2-ethylhexyl) sebacate (Aladdin, B105169) were added, and after they were completely dissolved, an ion-selective material mixture was obtained.

[0072] 30 μL of the above ion selective material mixture was drop coated on the surface of the microneedle electrode prepared in step (1) to form an ion selective layer to obtain a microneedle array electrode.

[0073] In other preferred embodiments, the ion selective material mixture can also be spin coated or dip coated on the microneedle electrode.

[0074] (3) Preparation of working electrode:

[0075] like Figure 2 As shown, 0.85 g of bovine serum albumin (McLean, B917420, molecular weight: 50000-100000) was weighed and dissolved in 10 mL of a mixed solution of trifluoroethanol (McLean, T819391) and water to obtain a bovine serum albumin solution. β-mercaptoethanol (McLean, M6230) was added thereto to open the protein structure of the bovine serum albumin and reduce the bovine serum albumin. Ethylenediamine (Shanghai Pharmaceutical Testing, 10009518) and ethanol (Shanghai Pharmaceutical Testing, 10009161) were then added to crosslink the reduced bovine serum albumins to obtain a crosslinked recombinant bovine serum albumin polymer solution.

[0076] The volume ratio of trifluoroethanol to water is 7:3. In other preferred embodiments, the volume ratio of trifluoroethanol to water may also be 8:2 or 9:1.

[0077] The amount of β-mercaptoethanol added is 500-900 μL. After the β-mercaptoethanol is added, the bovine serum albumin solution changes from transparent to cloudy, and the bovine serum albumin in the solution is reduced.

[0078] The total amount of ethylenediamine and ethanol is 800-1200 μL, and the volume ratio is 2: 1. In other preferred embodiments, the volume ratio of ethylenediamine and ethanol can also be 1: 1 or 1: 2.

[0079] After adding ethylenediamine and ethanol, the solution changes from cloudy to transparent again. At this time, the reduced bovine serum albumins in the solution are cross-linked with each other to become cross-linked recombinant bovine serum albumin polymers.

[0080] 30 μL of the cross-linked recombinant bovine serum albumin polymer solution was drop-coated on the surface of the microneedle array electrode to form a buffer layer, and then vacuum dried for 6 to 12 hours to obtain a working electrode.

[0081] (4) Preparation of a microneedle sensor based on cross-linked recombinant bovine serum albumin: Provide a reference electrode and a field effect transistor. Use a two-electrode system, fix the reference electrode and the working electrode prepared in step (3) on the same substrate with the needle tip facing upward, use the working electrode as an extended gate and connect it to the gate of the field effect transistor, connect the source of the field effect transistor to the source voltage output terminal of a test instrument (Keithle, 2636B), connect the gate voltage output terminal of the test instrument to the reference electrode, and connect the current input terminal of the test instrument to the drain of the field effect transistor, to obtain the microneedle sensor based on cross-linked recombinant bovine serum albumin of this embodiment.

[0082] The present invention does not limit the reference electrode and the field effect transistor. In other cases, the working electrode of this embodiment can also replace the working electrode in the existing ion concentration monitoring sensor to assemble a microneedle sensor based on cross-linked recombinant bovine serum albumin.

[0083] The principle of the microneedle sensor in this embodiment to detect ion concentration is as follows:

[0084] The sodium ion selective layer in the working electrode is used to selectively react with sodium ions and convert them into electrical signals. When the sodium ion concentration in the body changes, the potential difference between the working electrode and the reference electrode changes, thereby detecting the sodium ion concentration.

[0085] Example 2

[0086] This embodiment provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin. The difference from Embodiment 1 is that the ion selective layer of this embodiment is Ca 2+ Selective layer, ion carrier is Ca 2+ Vector (Merck, 21193-50MG-F).

[0087] The cross-linked recombinant bovine serum albumin polymer prepared in Example 1 of the present invention was tested below to verify its working performance.

[0088] like Figure 3 As shown in the figure, the bovine serum albumin and the cross-linked recombinant bovine serum albumin polymer of Example 1 were coated and dried to form films, which were then scanned using a Fourier transform infrared spectrometer in a range of 400-4000 cm -1 , and the spectrum results are as follows Figure 3 As shown, the results show that bovine serum albumin has a peak at 1643 cm -1 and 1543cm -1 The vibration peaks at the wavenumbers are changed into two vibration peaks after cross-linking and reorganization, which are located at 1647cm -1 、1621cm -1 and 1543cm-1 、1481cm -1 This result indicated that the secondary structure of the cross-linked recombinant BSA polymer was different from that of BSA, indicating that BSA was successfully reconstituted.

[0089] like Figure 4 As shown, the cross-linked recombinant bovine serum albumin polymer prepared in Example 1 was coated and dried to form a film, and the mechanical properties of the film in dry / wet state were measured using a universal testing platform for mechanical properties of materials. During the measurement process, the film was stretched longitudinally, and the stress change during the tensile fracture process was recorded to obtain a stress-strain curve as shown in FIG. Figure 4 As shown, the results show that in the dry state, the film hardly deforms with the increase of stress, indicating that the cross-linked recombinant bovine serum albumin polymer has a large stiffness in the dry state; in the wet state, the film undergoes a large deformation with the increase of stress, indicating that the stiffness of the cross-linked recombinant bovine serum albumin polymer decreases in the wet state, reflecting the "variable stiffness" feature of the cross-linked recombinant bovine serum albumin polymer of the present invention.

[0090] like Figure 5 As shown, microneedles need to have a certain mechanical strength to penetrate the skin. Studies have shown that a force of >0.058N / needle is required for microneedles to penetrate the skin, so microneedles should be able to withstand a force of >0.058N / needle.

[0091] The microneedle sensor is fixed at the center of the base of the universal test platform for mechanical properties of materials, with the needle tip facing the upper platform. During the test, the platform is slowly pressed down, and the instrument automatically records the displacement-force curve. The results are as follows: Figure 5 As shown, it can be seen from the displacement-force curve that the microneedle does not break slightly until it is subjected to a force of about 2N / needle, thus meeting the requirement of puncturing the skin.

[0092] like Figure 6 As shown, the cross-linked recombinant bovine serum albumin polymer prepared in Example 1 was coated and dried to form a film, and then scanned using an X-ray photoelectron spectrometer. The scanning content included full spectrum scanning, C 1s, N 1s, O 1s, and S2p fine spectrum scanning, and the spectrum results were as shown in FIG. Figure 6 The results show that the elements in the cross-linked recombinant bovine serum albumin polymer are the same as those in bovine serum albumin, and the chemical bonds formed between the elements exist in bovine serum albumin, indicating that the cross-linked recombinant bovine serum albumin is not doped with additional elements and does not produce new chemical bonds.

[0093] like Figure 7 As shown, the cross-linked recombinant bovine serum albumin polymer prepared in Example 1 was coated and dried to form a film, and the surface roughness of the film was tested using an atomic force microscope. The results are as follows Figure 7As shown, the results indicate that the surface of the cross-linked recombinant bovine serum albumin polymer coating is smooth and uniform, and no damage is caused during skin puncture.

[0094] like Figure 8 As shown in FIG. 1 , the microneedles (containing only polystyrene) prepared in Example 1 and the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin were fixed on a metal base with conductive tape, and the morphology of the microneedles was observed with a scanning electron microscope (JSM-6330F, Japan). Figure 8 The results show that the microneedle sensor prepared in Example 1 has a good structure, a sharp needle tip and a smooth surface.

[0095] like Fig. 9 As shown, the microneedle sensor prepared in Example 1 was placed in different concentrations of Na + Continuous current change in ionic solution, anti-interference (interference factor is K + Mg 2+ , Ca 2+ , Fe 3+ ), stability test. Fig. 9 A is the Na of the microneedle sensor + Concentration-responsive current-time curves. Fig. 9 B is the current of the microneedle sensor -Na + Concentration fitting curve. Fig. 9 A and Fig. 9 It can be seen from B that as Na + As the concentration increases, the output current of the sensor will also increase accordingly, showing a linear relationship. Therefore, the detected Na + Concentration information. Fig. 9 C is the Na of the microneedle sensor + Sensitivity test diagram. It can be seen from the figure that the microneedle sensor prepared in Example 1 has a good sensitivity to Na + It has high selectivity and high sensitivity. Fig. 9 D is the stability test curve of the microneedle sensor. It can be seen from the figure that within 7 days, the Na + The concentration response test curve did not change significantly, indicating that the microneedle sensor prepared in Example 1 had excellent stability.

[0096] like Fig.10 As shown in FIG. 1 , the microneedles prepared in Example 1 (containing only polystyrene) and the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin were respectively inserted into the mouse skin and then removed to study the recovery of the mouse skin. The results are shown in FIG. Fig.10As shown in A, the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin finally prepared in Example 1 were inserted into the mouse body and taken out within 20 minutes, and the mouse skin was completely restored, indicating that the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin finally prepared in Example 1 can smoothly penetrate the skin and cause very little damage to the skin. Further, the above two types of microneedles were inserted into the mouse skin and worn for 14 days, and the skin condition of the mice was studied on the 7th and 14th days of wearing. The results are as follows: Fig.10 As shown in Figure B, the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin finally prepared in Example 1 did not cause obvious damage to the skin, while the traditional microneedles (containing only polystyrene) caused skin ulcers and scar formation in mice. Therefore, it can be seen that compared with the traditional microneedles, the microneedles of the microneedle sensor based on cross-linked recombinant bovine serum albumin finally prepared in Example 1 have better safety and comfort.

[0097] In summary, the present invention provides a method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, by coating a cross-linked recombinant bovine serum albumin polymer solution on the surface of the microneedle, using the cross-linked recombinant bovine serum albumin polymer as a buffer layer, forming a buffer between the microneedle array electrode and the skin tissue, thereby improving the safety and comfort of wearing the sensor.

[0098] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin, characterized in that: The following steps are involved: S1, provides microneedle array electrodes; S2, preparing a working electrode: providing a bovine serum albumin solution, adding β-mercaptoethanol thereto to reduce the bovine serum albumin, thereby obtaining a reduced bovine serum albumin solution; then adding ethylenediamine and ethanol to cross-link the reduced bovine serum albumins, thereby obtaining a cross-linked recombinant bovine serum albumin polymer solution; Coating the cross-linked recombinant bovine serum albumin polymer solution on the surface of the microneedle array electrode to form a buffer layer to obtain a working electrode; S3, preparation of microneedle sensor based on cross-linked recombinant bovine serum albumin: providing reference electrode and field effect transistor; The working electrode and the reference electrode are fixed on the same substrate and connected to the field effect transistor via an external circuit to obtain the microneedle sensor based on cross-linked recombinant bovine serum albumin.

2. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 1, characterized in that: In step S2, the bovine serum albumin solution is prepared by: Bovine serum albumin is taken and dissolved in a mixed solution of trifluoroethanol and water to obtain a bovine serum albumin solution.

3. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 2, characterized in that: In the bovine serum albumin solution, the concentration of bovine serum albumin is 5wt% to 12wt%.

4. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 2, characterized in that: The volume ratio of trifluoroethanol to water is 1:(2-9).

5. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 1, characterized in that: In step S2, the volume ratio of ethylenediamine to ethanol is 1:(0.5-2).

6. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 1, characterized in that: In step S1, the microneedle array electrode comprises: microneedle bodies arranged in an array, and a conductive layer and an ion selective layer sequentially disposed on the microneedle bodies; The ion selective layer is prepared by the following method: Dissolving an ion carrier, an ion electrolyte, a stabilizer, and a plasticizer in an organic solvent to obtain an ion selective material mixture; coating the ion selective material mixture on the surface of the conductive layer to form an ion selective layer; The ion carriers include: Na + Carrier, Ca 2+ Carrier, K + Carrier, Mg 2+ The invention relates to a carrier comprising at least one of: sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate; the ionic electrolyte comprises sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate; the stabilizer comprises at least one of polyvinyl chloride, polyurethane and polyvinyl acetate; the plasticizer comprises at least one of di(2-ethylhexyl)sebacate, nitrophenyl octyl ether and phthalate; the organic solvent comprises tetrahydrofuran and / or cyclohexanone.

7. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 6, characterized in that: The mass ratio of the ion carrier, ion electrolyte, stabilizer and plasticizer is (10-30): (5-10): (20-50): (500-800).

8. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 6, characterized in that: The conductive layer is formed on the surface of the microneedle body by at least one of magnetron sputtering, chemical deposition and vacuum evaporation.

9. The method for preparing a microneedle sensor based on cross-linked recombinant bovine serum albumin according to claim 6, characterized in that: The material of the conductive layer includes at least one of gold, platinum, copper and silver.

10. A microneedle sensor based on cross-linked recombinant bovine serum albumin, characterized in that: Obtained by the preparation method according to any one of claims 1 to 9.

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