Flexible biosensor with microneedle array and application thereof

Abstract

The invention discloses a flexible biosensor with a microneedle array, and belongs to the technical fields of flexible wearable sensing technology, biosensing and medical detection. The biosensor comprises a hydrophilic microneedle array and a hydrophobic flexible substrate, the flexible substrate comprises a hydrophilic detection area based on the microneedle array and a hydrophobic background area based on the flexible substrate, the flexible substrate and the hydrophilic microneedle array are made of flexible materials, and the microneedle array is hydrophilic and can realize directional conveying of sweat; on the basis of the hydrophilic detection area constructed by the microneedle and the hydrophobic background area of the flexible substrate, the enrichment of sweat in the detection area of the sensor can be realized; the detection area is functionally modified, so that the specific fixed-point detection of sweat can be realized.

Description

Technical Field

[0001] The present invention belongs to the fields of flexible wearable sensing technology, biosensing and medical detection technology, and in particular relates to a flexible biosensor with a microneedle array and an application thereof. Background Art

[0002] Sweat, as a liquid secreted by sweat glands, is mainly composed of water and contains rich human physiological information, such as various electrolyte ions (such as potassium, calcium, sodium and chloride) and metabolites produced by the human body (such as glucose, lactic acid, uric acid, urea, etc.). Many clinical medical studies have shown that physiological dysfunction of the human body can cause changes in the composition of sweat (for example, high blood sugar concentration can lead to increased glucose concentration in sweat; cystic fibrosis can lead to increased salt content in sweat; drug use can also produce corresponding metabolites in sweat), and sweat does not need to be invasively extracted from the human body. Therefore, sweat is an important non-invasive detection object.

[0003] At present, sweat detection still faces some problems. From the perspective of the substrate material of the sweat sensing interface, sweat sensors made from purely rigid substrates are not easy to stretch, and are not comfortable or wearable. On the other hand, sweat detection mostly collects sweat from the surface of human skin and sends it to the laboratory for testing, which cannot be quickly detected in real time and provide timely feedback. In addition, sweat itself has the characteristics of small amount and dispersion, fast evaporation rate, easy contamination by impurities on the skin surface, and low content of ingredients, which are not conducive to detection. These problems make the collection of sweat face challenges that cannot be ignored.

[0004] Therefore, it is necessary to design a flexible wearable sweat sensor that integrates sweat transport, enrichment and real-time detection. Summary of the invention

[0005] One of the purposes of the present invention is to provide a flexible biosensor with a microneedle array, wherein the biosensor is made of flexible material, including a hydrophilic microneedle array and a hydrophobic flexible substrate; the flexible substrate includes a hydrophilic detection area based on the microneedle array and a hydrophobic background area based on the flexible substrate, the flexible substrate and the microneedle array are made of flexible material, the microneedle array is hydrophilic, and can achieve directional transport of sweat; the hydrophilic detection area constructed based on the microneedles and the hydrophobic background area of ​​the flexible substrate can achieve enrichment of sweat in the sensor detection area; the detection area is functionally modified to achieve specific point-to-point detection of sweat.

[0006] Preferably, the flexible material used is selected from one or more of polydimethylsiloxane, silicone, hydrogel, silicone rubber, and epoxy resin, and may also be doped with magnetic materials and responsive materials to obtain special functionalization.

[0007] Preferably, the preparation method of the biosensor comprises one or more of micromolding, 3D printing, etching, replication, hot pressing, and self-assembly. More preferably, the surface of the hydrophilic microneedle array and the detection area is smooth or has microstructure textures / grooves or structures with micron or nanometer features.

[0008] More preferably, the hydrophilic microneedle array and the structure constructed on the hydrophilic detection surface can be prepared by deposition, etching, evaporation, spraying, frosting, spin coating, scraping or mineralization.

[0009] More preferably, the structural material of the hydrophilic microneedle array and the surface of the hydrophilic detection area is one or more of metal organic frameworks, metal oxides, non-metal oxides, and mineral-based micro-nano materials.

[0010] Preferably, the hydrophilic microneedle array is vertical or has a certain tilt angle.

[0011] A second object of the present invention is to provide a method for preparing a flexible biosensor having a microneedle array, the preparation method comprising the following steps: 1) Prepare flexible substrates and microneedle arrays by one or more methods including micromolding, 3D printing, etching, replication, hot pressing, and self-assembly; 2) or further constructing microstructure textures / grooves or structures with micron or nano features on the surface of the microneedle array and the detection area; 3) Functional modification of the surface of the microneedle array detection area; 4) Add skin adhesion layer.

[0012] Preferably, the flexible substrate material is hydrophobic; the hydrophilic microneedle array can be designed according to demand, such as a 4*4 array, a 2*3 array; the microneedle length, taper, microneedle bottom diameter, and microneedle spacing can all be designed according to demand, such as a microneedle length height of 0.5 mm / 1.5 mm / 2.5 mm, a microneedle taper of 20° / 30° / 45°, a microneedle bottom diameter of 1.3 mm / 1.0 mm, and a microneedle spacing of 3.0 mm / 5.0 mm; the microneedle inclination angle can also be designed according to demand, such as a microneedle inclination angle of 60° / 45° / 30°, and the detection area of ​​the micron or nanometer characteristic structure can be designed and prepared according to demand, such as adding 0.15 g zinc nitrate hexahydrate, 0.07 g triethylamine hydrochloride, and 0.12 g dimethylimidazole to 50 mL deionized water in sequence for deposition to form a micro-nano structure.

[0013] The third object of the present invention is to provide the use of the above-mentioned biosensor in the preparation of sweat or glucose detection products.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses flexible materials to construct a microneedle array biosensor, and performs hydrophilic and hydrophobic modification and functional modification on the microneedles. The hydrophilic microneedles are used to transport sweat, and the detection area constructed based on the hydrophilic microneedles and the hydrophobic background substrate area of ​​the flexible substrate can achieve the enrichment of sweat in the sensor detection area. The functionally modified detection area can achieve specific detection of various physiological indicators in sweat. The transport and enrichment of microneedles can achieve sensitive detection of trace samples.

[0015] Furthermore, the surface of the microneedle array of the present invention may have a micro-nano structure, which can promote the transport, enrichment and sensitive detection of sweat. The invention integrates sweat transport, enrichment and detection, has low manufacturing cost, is easy to wear, and the detection process is non-invasive and painless. It can detect one or more physiological indicators at the same time and is suitable for real-time detection of human sweat. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the structure of the flexible biosensor prepared in Example 1.

[0017] Figure 2 This is a characterization diagram of the flexible biosensor microneedle in Example 1.

[0018] Figure 3 This is a picture of the hydrophobic contact angle of the flexible background substrate of Example 3.

[0019] Figure 4 This is a picture of the hydrophilic contact angle of the microneedle array with micro-nano structure and the detection area of ​​Example 3.

[0020] Figure 5 This is a diagram of the sweat transport process of Example 3.

[0021] Figure 6 This is a pH detection curve diagram of the microneedle array flexible biosensor in Example 4.

[0022] Figure 7 This is a glucose detection curve diagram of the microneedle array flexible biosensor in Example 6.

[0023] Figure 8 This is a linear fitting curve diagram of the G value in glucose detection in Example 7. DETAILED DESCRIPTION

[0024] Example 1 Preparation of a flexible substrate with a vertical microneedle array 1. The flexible substrate and microneedle array are made of polydimethylsiloxane (PDMS). The prepolymer (Syglard 184) and the crosslinker (Syglard 184) are fully mixed at a mass ratio of 10:1 and poured into a vertical microneedle array mold (height: 2.0 mm, bottom diameter: 0.9 mm, 2*3 array, microneedle spacing 5.0 mm, microneedle taper 25°), the mold is placed in a vacuum tank and evacuated to -0.08 MPa, and the vacuum is repeatedly evacuated 3-5 times. After standing for 1 hour, the mold is placed in an electric hot air drying oven at 65°C for 2 hours. Finally, the cured PDMS is demolded and taken out to obtain a flexible substrate and a vertical microneedle array; 2. Add 0.15 g zinc nitrate hexahydrate, 0.07 g triethylamine hydrochloride and 0.12 g dimethylimidazole to 50 mL deionized water in sequence and stir with a magnetic stirrer for 10 min to fully mix them. 3. The prepared flexible substrate with vertical microneedle array was placed in deionized water, 75% alcohol, and ultrapure water for ultrasonic cleaning for 10 min, and then a mask plate (thickness 0.1 mm) with a specific pattern was covered on the prepared flexible substrate and microneedle array; 4. Add the solution prepared in step 2 to a clean weighing bottle, put in the sensor substrate covered with the mask plate, and then put the weighing bottle into an electric hot air drying oven at 80°C for 24 hours; 5. Take out the sensor substrate covered with the mask plate, separate the mask plate from the flexible substrate and the microneedle array, and let it dry naturally to obtain a flexible substrate with a vertical microneedle array of micro-nano structures, as shown in the schematic diagram. Figure 1 , vertical microneedle Figure 2 shown.

[0025] Example 2 Preparation of a flexible substrate with an inclined microneedle array 1. The flexible substrate and microneedle array are made of PDMS. The polymer and crosslinker are fully mixed at a mass ratio of 10:1 and poured into an inclined microneedle array mold (height: 3.0 mm, bottom diameter: 1.0 mm, tilt angle: 60°, 6*6 array, microneedle spacing 8.0 mm, taper 30°), the mold is placed in a vacuum drying oven and evacuated to -0.09 MPa, and the vacuum is repeatedly evacuated 3-5 times. After standing for 30 minutes, the mold is placed in an electric hot air drying oven and cured at 100°C for 1 hour. Finally, the mold is demolded after curing to obtain a flexible substrate and an inclined microneedle array; 2. Add 0.15 g zinc nitrate hexahydrate, 0.07 g triethylamine hydrochloride and 0.12 g dimethylimidazole to 50 mL deionized water, and stir with a magnetic stirrer for 30 min to mix them thoroughly. 3. The prepared flexible substrate with tilted microneedle array was placed in deionized water, 75% alcohol, and ultrapure water for ultrasonic cleaning for 10 min. Then, a mask with a specific pattern was placed on the prepared flexible substrate and the tilted microneedle array, and placed in a plasma cleaner for cleaning for 5 min; 4. Add the solution prepared in step 2 to a clean weighing bottle, then put in the sensor substrate covered with the mask plate in step 3, and then put the weighing bottle into an electric hot air drying oven for deposition for 8 hours; 5. Take out the sensor substrate covered with the mask plate, let it stand and dry naturally, then separate the mask plate from the flexible substrate and the inclined microneedle array to obtain a flexible substrate with an inclined microneedle array.

[0026] Example 3 Sweat transport process of flexible substrate with microneedle array The PDMS-based flexible background substrate prepared in Example 1 has hydrophobicity, such as Figure 3 As shown, the contact angle of water on the flexible background area is about 106.7 ± 2.8°; the obtained microneedle array with micro-nano structure and the detection area are hydrophilic, as shown in FIG. Figure 4 As shown in FIG. 1 , the contact angle of water is about 15.6 ± 4.0°. Using the sensor substrate prepared in Example 1, the microneedle is contacted with 1 μL of sweat, and the process of sweat being adsorbed by the microneedle and transported along the microneedle can be observed, as shown in FIG. Figure 5 shown.

[0027] Example 4 pH detection Functional modification of the microneedle array detection area: Using the sensor substrate prepared in Example 1, 1 μL of purple litmus reagent was dropped on the microneedle array detection area. After the solvent evaporated, the component for detecting pH was loaded into the microneedle array detection area to complete the functional modification.

[0028] pH detection: Use the above functionalized microneedle array to detect 1 μL of pH standard solution (pH values ​​are 6.0, 6.5, 7.0, 7.5, and 8.0, respectively), put it in a water bath, react at 37°C for 3 min, then take it out, use a smartphone to shoot, extract, and record the RGB value of the colorimetric reaction under fixed light conditions, and draw the change curve under different pH conditions, such as Figure 6 .

[0029] Example 5 Detection of pH of human sweat Functional modification of the microneedle array detection area: Using the flexible sensor substrate prepared in Example 1, 1 μL of purple litmus reagent was dropped on the microneedle array detection area. After the solvent evaporated, the component for detecting pH was loaded into the microneedle array detection area to complete the functional modification.

[0030] Skin adhesive layer assembly: The prepared flexible sensor substrate with microneedle array is fixed to the medical bandage to achieve wearable detection: The flexible sensor is attached to the arm of the person being tested, and the person being tested is asked to do physical exercise. After the person being tested sweats, the RGB value of the microneedle array detection area is photographed, extracted, and recorded with a smartphone, and compared with the concentration fitting curve (such as Figure 6 ) to obtain the corresponding pH value of human sweat.

[0031] Example 6 Glucose Concentration Detection Functional modification of the microneedle array detection area: Using the sensor substrate prepared in Example 1, 1 μL of the working solution of the glucose detection kit (KB016-200T, Isejiu Bio, China) (phenol reagent and enzyme reagent mixed in a volume ratio of 1:1) was dropped on the microneedle array detection area. After the solvent evaporated, the active ingredient for detecting the glucose concentration was loaded into the microneedle array detection area to complete the functional modification.

[0032] Glucose detection: Take a clean test tube, add weighed anhydrous glucose and deionized water, and prepare glucose standard solutions with concentrations of 0 mM, 1.25 mM, 2.50 mM, 3.75 mM, and 5.00 mM, respectively, for later use. Use the above functionalized microneedle array to detect 1 μL of glucose standard solution, put it into a water bath, react at 37°C for 4 min, then take it out, use a smartphone to shoot, extract, and record the RGB value of the colorimetric reaction under fixed light conditions, and draw the change curve under different glucose concentration conditions, such as Figure 7 .

[0033] Example 7 Verification of Glucose Concentration Detection Accuracy Functional modification of the microneedle array detection area: Using the sensor substrate prepared in Example 1, 1 μL of the working solution of the glucose detection kit was dropped on the microneedle array detection area. After the solvent evaporated, the active ingredient for detecting the glucose concentration was loaded into the microneedle array detection area to complete the functional modification.

[0034] Verification: Take the green color with the most obvious color change and draw a linear fitting curve of the relationship between color change (G value) and concentration (such as Figure 8 ), the linear fitting equation is y = -10.88x + 135.55, R 2= 0.987. Take a clean test tube, add weighed anhydrous glucose and deionized water, prepare a glucose standard solution with a concentration of 1.00 mM, and set aside. Use the above functionalized microneedle array to detect the 1 μL glucose standard solution drop, put it into a water bath, react at 37°C for 4 min, then take it out, use a smartphone to shoot, extract, and record the colorimetric reaction under fixed light conditions. The G value is 124. The concentration of glucose is 1.062 mM when the fitting equation is brought back, with an error of about 6.2%.

[0035] Example 8 Detection of Glucose Concentration in Human Sweat Functional modification of the microneedle array detection area: Using the flexible sensor substrate prepared in Example 1, 1 μL of the working solution of the glucose detection kit was dropped on the microneedle array detection area. After the solvent evaporated, the active ingredient for detecting the glucose concentration was loaded into the microneedle array detection area to complete the functional modification.

[0036] Skin adhesive layer assembly: Fix the prepared flexible sensor substrate with microneedle array on the medical bandage to achieve wearable detection: Attach the flexible sensor to the arm of the person being tested, let the person be tested eat, and after the meal, when the person is sweating, use a smartphone to shoot, extract, and record the RGB value of the microneedle array detection area, and compare the G value with the concentration fitting curve (such as Figure 8 ) for comparison to obtain the corresponding glucose concentration.

[0037] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A flexible biosensor with a microneedle array, characterized in that: The biosensor is made of flexible materials, including a hydrophilic microneedle array and a hydrophobic flexible substrate; the flexible substrate includes a hydrophilic detection area based on the microneedle array and a hydrophobic background area based on the flexible substrate.

2. The flexible biosensor according to claim 1, characterized in that: The flexible material used is selected from one or more of polydimethylsiloxane, silicone, hydrogel, silicone rubber and epoxy resin.

3. The flexible biosensor according to claim 1, characterized in that: The preparation method of the biosensor includes one or more of micro-molding, 3D printing, etching, replicating, hot pressing, and self-assembly.

4. The flexible biosensor according to claim 1, characterized in that: The hydrophilic microneedle array and the hydrophilic detection area have smooth surfaces or are constructed with microstructure textures / grooves or structures with micro-nano features.

5. The flexible biosensor according to claim 4, characterized in that: The hydrophilic microneedle array and the structure constructed on the surface of the hydrophilic detection area are prepared by deposition, etching, evaporation, spraying, grinding, spin coating, scraping or mineralization.

6. The flexible biosensor according to claim 5, characterized in that: The structures constructed on the surface of the hydrophilic microneedle array and the hydrophilic detection area are one or more of metal organic frameworks, metal oxides, non-metal oxides, and mineral-based micro-nano materials.

7. The flexible biosensor according to claim 1, characterized in that: The hydrophilic microneedle array is vertical or has a certain tilt angle.

8. A method for preparing a flexible biosensor having a microneedle array, characterized in that: The preparation method comprises the following steps: 1) Prepare microneedle arrays and flexible substrates using one or more methods including micromolding, 3D printing, etching, replication, hot pressing, and self-assembly; 2) or further constructing microstructure textures / grooves or structures with micron or nano features on the surface of the microneedle array and the detection area; 3) Functional modification of the surface of the microneedle array detection area; 4) Add skin adhesion layer.

9. The preparation method according to claim 8, characterized in that: The flexible substrate is hydrophobic; the microneedles are arranged in an X*Y array.

10. Use of the flexible biosensor according to any one of claims 1 to 7 in preparing a wearable sweat or glucose detection product.

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