A microneedle patch technology for in vivo detection of collagen IgG in interstitial fluid and its preparation method

Abstract

This invention provides an in vivo microneedle patch technology for detecting collagen IgG in interstitial fluid and its preparation method, relating to the field of biomedical materials and devices. The invention provides a method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid, comprising the following steps: coating a solid microneedle patch in a collagen solution with a concentration of 2-100 μg / mL to obtain an in vitro microneedle patch; the solid microneedle patch is selected from polystyrene microneedle patches or epoxy resin microneedle patches. The preparation method of this invention enables the quantitative detection of trace amounts of specific collagen IgG in interstitial fluid using in vitro microneedle patches, exhibiting high sensitivity and high accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and devices technology, specifically relating to an in vivo microneedle patch technology for detecting collagen IgG in interstitial fluid of the skin and its preparation method. Background Technology

[0002] Collagen is a protein widely found in animal-derived foods. Ingestion may trigger the production of IgG antibodies in some individuals, leading to chronic symptoms such as gastrointestinal discomfort, headaches, and skin allergies. This reaction is usually delayed and difficult to diagnose through routine allergy tests. Collagen IgG testing is crucial in the diagnosis of food intolerances, especially in assessing collagen-related food allergies or intolerance reactions. Therefore, accurate detection of collagen IgG is essential for functional medicine, nutritional assessment, and the diagnosis of food intolerances. Currently, enzyme-linked immunosorbent assay (ELISA) is the standard method for collagen IgG detection due to its high sensitivity and specificity. However, this method requires drawing venous blood to prepare serum for testing, a relatively complex process that is susceptible to sample contamination. Furthermore, blood tests are invasive and painful for subjects, leading to strong aversion to traditional blood tests; some even experience fainting from blood or needles. In addition, ELISA testing is time-consuming, requires a high level of expertise from the testing personnel, and is costly.

[0003] Interstitial fluid (ISF) is a tissue fluid abundant in the dermis, obtained by filtering blood through capillaries. The types and quantities of small molecules, electrolytes, and proteins in ISF are remarkably similar to those in blood. Current research suggests that the vast majority of relevant biomarkers in blood can be detected in ISF. Furthermore, ISF contains some unique biomarkers that are not found in blood and can also be detected.

[0004] Microneedles have the unique advantages of being minimally invasive and painless, but existing microneedle-based detection technologies still have the following shortcomings: (1) The volume of interstitial fluid that can be collected is usually only microliters, and the sample volume is extremely limited. The subsequent marker separation and extraction steps required for detection consume a large amount of sample, which is difficult to meet the detection requirements of conventional ELISA and other methods; (2) Due to the small sample volume, the separation and extraction of target markers are significantly more difficult, which can easily lead to insufficient detection sensitivity; (3) During sample transfer and separation, external environment or operation steps can easily introduce contamination, affecting the specificity and accuracy of detection; (4) Existing methods require the separation of sampling and detection steps, making it difficult to achieve in-situ, rapid, and direct detection of micro-volume samples. Therefore, it is urgent to develop a device that can directly, sensitively, and specifically detect collagen IgG in micro-volume interstitial fluid samples to avoid loss and contamination during sample transfer and separation, and to meet the urgent need for rapid quantitative detection of collagen IgG in clinical and scientific research. Summary of the Invention

[0005] To address the aforementioned technical problems, the primary objective of this invention is to provide a method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid. This method enables the quantitative detection of trace amounts of specific collagen IgG in interstitial fluid using an in vitro microneedle patch, exhibiting high sensitivity and high accuracy.

[0006] A second objective of the present invention is to provide an in vitro microneedle patch prepared by the above-described preparation method.

[0007] A third objective of this invention is to provide the application of the above-described preparation method or the above-described in vitro microneedle patch in the preparation of products for detecting collagen IgG in interstitial fluid of the skin.

[0008] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: This invention provides a method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin, comprising the following steps: coating a solid microneedle patch in a collagen solution with a concentration of 2~100 μg / mL to obtain an in vitro microneedle patch; The solid microneedle patch is selected from polystyrene microneedle patches or epoxy resin microneedle patches.

[0009] As one implementation method, the polystyrene microneedle patch is immersed in an L-polylysine solution with a concentration of 0.01~0.1% for 30~120 min, and then coated.

[0010] As one implementation method, the solid microneedle patch is plasma treated and then coated; the plasma treatment power is 40~60 W, the gas pressure is 20~50 Pa, and the time is 1~5 min.

[0011] In one embodiment, after plasma treatment, the solid microneedle patch is first soaked in an L-polylysine solution with a concentration of 0.01~0.1% for 30~120 min, and then coated.

[0012] In one embodiment, after plasma treatment, the solid microneedle patch is surface-enhanced by carboxylation before coating. The method of surface carboxylation enhancement modification includes: immersing the plasma-treated solid microneedle patch in a mixed solution for 50-70 min, then shaking it with MES buffer containing 40-60 mM EDC and 20-30 mM NHS for 25-35 min, and then reacting it in a PBS solution containing 1-2% NH2-PEG-COOH for 4-6 h to form a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends. The mixed solution is a solution of 0.5-1.5% malonic acid and 25-35% H2O2 in a volume ratio of (0.5-1.5):1, or a solution of 0.5-1.5% citric acid and 25-35% H2O2 in a volume ratio of (0.5-1.5):1. In one embodiment, the MES buffer has a pH of 5-6 and a concentration of 30-70 mM; the PBS solution has a pH of 6-8 and a concentration of 0.01-0.02 M.

[0013] As one implementation method, the PEG-modified solid microneedle patch is immersed in an L-polylysine solution with a concentration of 0.01~0.1% for 30~120 min, and then coated.

[0014] As one implementation method, the solid microneedle patch can be prepared by molding or 3D printing.

[0015] The present invention also provides an in vitro microneedle patch prepared by the above-described preparation method.

[0016] The present invention also provides the application of the above-described preparation method or the above-described in vitro microneedle patch in the preparation of products for detecting collagen IgG in interstitial fluid of the skin.

[0017] The advantages of this invention compared to existing technologies are as follows: This invention provides a method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid. The method utilizes special surface modification to enable the needles of the in vitro microneedle patch to specifically capture collagen IgG from the interstitial fluid. The in vitro microneedle patch prepared by this method enables in vivo detection, significantly reducing the required sample size. It eliminates the need for venous blood collection and the separation and extraction of collagen IgG, allowing for collection and quantitative detection on the skin surface. Furthermore, this in vitro microneedle patch enables quantitative detection of minute samples, exhibiting high sensitivity and accuracy. This method can also be applied to the preparation of in vitro microneedle patches for other target protein biomarkers, enabling the quantitative detection of minute samples of other target proteins in interstitial fluid. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the overall process of preparing and testing in vitro microneedle patches.

[0019] Figure 2 This describes the molding process for preparing solid microneedle patches.

[0020] Figure 3 This is a schematic diagram of the preparation process of in vitro microneedle patches.

[0021] Figure 4 The figures show the load-displacement curves of microneedles made of different materials, where A is a polystyrene microneedle patch and B is an epoxy resin microneedle patch.

[0022] Figure 5 The effect of surface hydrophilic modification on in vitro microneedle patches is analyzed. 1 represents no surface hydrophilic modification, and 2 represents surface hydrophilic modification.

[0023] Figure 6 The grayscale standard curve for in vitro detection of collagen IgG using microneedle patches.

[0024] Figure 7 The image grayscale results are for in vitro detection of diluted mouse serum samples using microneedle patches.

[0025] Figure 8 The image shows the grayscale results of in vivo detection of collagen IgG in the interstitial fluid of mouse skin.

[0026] Figure 9 This is a diagram showing the usage state of the microneedle fixation device, where 5 is the fixing block, 7 is the microneedle, 8 is the skin, 22 is the fixing post, and 41 is the piston. Detailed Implementation

[0027] This invention provides a method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin, comprising the following steps: coating a solid microneedle patch in a collagen solution with a concentration of 2~100 μg / mL to obtain an in vitro microneedle patch; wherein the solid microneedle patch is selected from polystyrene microneedle patches or epoxy resin microneedle patches.

[0028] In this invention, the preparation method of the solid microneedle patch includes molding or 3D printing; the microneedles of the solid microneedle patch are conical or pyramidal.

[0029] The molding method for epoxy resin microneedle patches includes: thoroughly mixing epoxy resin and toluene dimethylamine in a ratio of 100g:15~20g, preferably 100g:17g; then pouring the mixture into a silicone microneedle mold. In this invention, the length of the silicone microneedle mold needle is 400~1500 µm, preferably 600 µm, 800 µm, 1000 µm, 1200 µm or 1400 µm; the number of needle arrays is 8×8~14×14, preferably 9×9, 10×10, 11×11, 12×12 or 13×13; the bottom of the needle is square or circular, the side length of the square is 200~600 µm, preferably 300 µm, 400 µm or 500 µm; the diameter of the circle is 200~400 µm, preferably 300 µm. In this invention, the silicone microneedle molds include models A104, A105, A106, and Y124. The silicone microneedle molds are then defoamed under vacuum for 5-10 minutes, preferably for 6, 7, 8, or 9 minutes. After removal, they are left to dry naturally for 24-30 hours, preferably for 26 or 28 hours. After drying, an epoxy resin microneedle patch is formed, which is rectangular or square, with a side length of 8-15 mm, preferably 10 mm, 12 mm, or 14 mm.

[0030] The molding method for polystyrene microneedle patches includes: dissolving polystyrene in dichloromethane to prepare a polystyrene-dichloromethane solution with a concentration of 15-25%, preferably 20%; then pouring the solution into a silicone microneedle mold. In this invention, the length of the silicone microneedle mold needle is 400-1500 µm, preferably 600 µm, 800 µm, 1000 µm, 1200 µm, or 1400 µm; the number of needle arrays is 8×8-14×14, preferably 9×9, 10×10, 11×11, 12×12, or 13×13; the bottom of the needle is square or circular, the side length of the square is 200-600 µm, preferably 300 µm, 400 µm, or 500 µm; the diameter of the circle is 200-400 µm, preferably 300 µm. In this invention, the silicone microneedle molds include models A104, A105, A106, and Y124. The silicone microneedle molds are then defoamed under vacuum for 5-10 minutes, preferably for 6, 7, 8, or 9 minutes. After removal, they are left to dry naturally for 6-12 hours, preferably for 8 or 10 hours. After drying, a polystyrene microneedle patch is formed, which is rectangular or square, with a side length of 8-15 mm, preferably 10 mm, 12 mm, or 14 mm.

[0031] In this invention, solid microneedle patches are plasma-treated and then immersed in a collagen solution for protein coating. The plasma treatment is performed using a plasma treatment machine. The distance between the solid microneedle patch and the plasma treatment machine nozzle is 0.5–2.5 cm, preferably 1.5 cm. The plasma treatment power is 40–60 W, preferably 50 W. The plasma treatment pressure is 20–50 Pa, preferably 30 Pa or 40 Pa. The plasma treatment time is 1–5 min, preferably 3 min. The plasma treatment gas is air. The plasma treatment temperature is room temperature, preferably 20–28°C, preferably 25°C. This invention introduces active functional groups onto the surface of solid microneedles through plasma treatment, thereby improving the ability to coat proteins.

[0032] In this invention, the concentration of the collagen solution is 2-100 μg / mL. The preferred concentration of the collagen solution is 10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, or 90 μg / mL. The collagen used in this invention is purchased from Solarbio, catalog number C8061, type I collagen (soluble). When the coating temperature is room temperature, the coating time is 1-3 hours, preferably 2 hours; when the coating temperature is 2-8°C, the coating time is 6-10 hours, preferably 7 hours, 8 hours, or 9 hours. After coating, washing is performed. The washing solution is a PBS solution containing Tween-20 (PBS+T solution), and the mass concentration of Tween-20 in the washing solution is 0.02%-0.1%, preferably 0.05% or 0.08%. In this invention, the washing is performed 2 to 5 times, preferably 3 or 4 times; each washing session lasts 3 to 8 minutes, preferably 4, 5, 6, or 7 minutes. After washing, the sample is dried at room temperature for 5 to 48 hours, preferably 12, 24, 36, or 40 hours; the drying method is natural air drying. After drying, the sample is blocked for 1 to 3 hours, preferably 2 hours; the blocking solution is a PBS solution containing bovine serum albumin, and the mass concentration of bovine serum albumin in the blocking solution is 1 to 3%, preferably 2%. After blocking, the sample is washed and air-dried; the washing is performed 2 to 5 times, preferably 3 or 4 times; each washing session lasts 3 to 8 minutes, preferably 4, 5, 6, or 7 minutes; the air drying is natural air drying. This invention obtains an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin through protein coating.

[0033] As an optional implementation, the solid microneedle patch of the present invention, after plasma treatment, is immersed in a 0.01-0.1% L-polylysine solution for 30-120 min before protein coating. The mass concentration of the L-polylysine solution is preferably 0.05%; the solvent for the L-polylysine solution is deionized water or PBS solution. The immersion time is 30-120 min, preferably 50 min, 70 min, 90 min, or 110 min. In the present invention, the molecular weight of the L-polylysine is 15-300 kDa, preferably 75 kDa, 135 kDa, 195 kDa, or 255 kDa. After soaking, the microneedle patch is washed with a PBS solution containing Tween-20 (PBS+T solution). The mass concentration of Tween-20 in the washing solution is 0.02%~0.1%, preferably 0.05% or 0.08%. The washing is performed 2~5 times, preferably 3 or 4 times. Each washing session lasts 3~8 minutes, preferably 4 minutes, 5 minutes, 6 minutes or 7 minutes. After washing, the patch is dried at room temperature for 5~48 hours, preferably 12 hours, 24 hours, 36 hours or 40 hours. The drying method is natural air drying. In this invention, by soaking the solid microneedle patch in L-polylysine solution, the hydrophilicity of the microneedle patch surface can be improved, the protein coating ability of the microneedle surface can be improved, thereby improving the microneedle protein coating efficiency and detection sensitivity.

[0034] As an optional implementation, the solid microneedle patch of the present invention is plasma-treated, then surface-enhanced by carboxylation before protein coating; the method of surface-enhanced carboxylation includes: immersing the plasma-treated solid microneedles in a mixed solution for 50-70 min, then shaking with MES buffer containing 40-60 mM EDC and 20-30 mM NHS for 25-35 min, and then reacting in PBS solution containing 1-2% NH2-PEG-COOH for 4-6 h to form a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends.

[0035] In this invention, the mixed solution is a mixture of 25-35% H2O2 solution and 0.5-1.5% malonic acid solution at a volume ratio of 1:(0.5-1.5), preferably 1:1; or the mixed solution is a mixture of 25-35% H2O2 solution and 0.5-1.5% citric acid solution at a volume ratio of 1:(0.5-1.5), preferably 1:1; the concentration of the H2O2 solution is preferably 30%; the concentration of the malonic acid solution is preferably 1%; and the concentration of the citric acid solution is preferably 1%. This invention increases the density of carboxyl groups on the surface of the solid microneedle patch through chemical oxidation. After soaking, ultrasonic cleaning is performed in water, preferably deionized water; the ultrasonic power is 80-120 W, preferably 100 W; the ultrasonic frequency is 30-50 Hz, preferably 40 Hz; the number of cleaning cycles is 2-4, preferably 3; and the time for each ultrasonic cleaning cycle is 3-5 min, preferably 4 min.

[0036] After cleaning, the sample is placed in a MES buffer containing 40-60 mM EDC and 20-30 mM NHS and shaken for 25-35 min. In this invention, the shaking time in the MES buffer is preferably 30 min. The pH of the MES buffer is preferably 5-6, more preferably 5.5; the concentration of the MES buffer is 30-70 mM, more preferably 50 mM; the concentration of the EDC is preferably 45-55 mM, more preferably 50 mM; and the concentration of the NHS is preferably 25 mM. Finally, the sample is transferred to a PBS solution containing 1-2% NH2-PEG-COOH and reacted for 4-6 h to form a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends. In this invention, the reaction time in the PBS solution is preferably 5 h; the pH of the PBS solution is 6-8, more preferably 7; the concentration of the PBS solution is 0.01-0.02 M; and the concentration of the NH2-PEG-COOH is preferably 1.5%. After the reaction is complete, the present invention performs ultrasonic cleaning to remove unreacted PEG. Ultrasonic cleaning is performed in water, preferably deionized water; the ultrasonic power is 80-120W, preferably 100W; the ultrasonic frequency is 30-50 Hz, preferably 40 Hz; the number of cleaning cycles is 2-4, preferably 3; and the duration of each ultrasonic cleaning cycle is 3-5 min, preferably 4 min. In this invention, by performing carboxylation enhancement modification on the microneedles to form a PEG layer with terminal carboxylic acid groups for subsequent protein coating, the coating efficiency and detection sensitivity can be further improved.

[0037] As an optional implementation, the present invention involves immersing the solid microneedle patch with the PEG layer in a 0.01-0.1% L-poly-L-lysine solution for 30-120 min before protein coating. The method of immersion in the L-poly-L-lysine solution is the same as described above and will not be repeated here.

[0038] As an optional implementation, the present invention involves immersing polystyrene microneedle patches in a 0.01-0.1% L-polylysine solution for 30-120 min before protein coating. The preferred mass concentration of the L-polylysine solution is 0.05%; the solvent for the L-polylysine solution is deionized water or PBS solution. The immersion time is 30-120 min, preferably 50 min, 70 min, 90 min, or 110 min. In this invention, the molecular weight of the L-polylysine is 15-300 kDa, preferably 75 kDa, 135 kDa, 195 kDa, or 255 kDa. After soaking, the microneedle patch is washed with a PBS solution containing Tween-20 (PBS+T solution). The mass concentration of Tween-20 in the washing solution is 0.02%~0.1%, preferably 0.05% or 0.08%. The washing is performed 2~5 times, preferably 3 or 4 times. Each washing session lasts 3~8 minutes, preferably 4 minutes, 5 minutes, 6 minutes or 7 minutes. After washing, the patch is dried at room temperature for 5~48 hours, preferably 12 hours, 24 hours, 36 hours or 40 hours. The drying method is natural air drying. In this invention, by soaking the polystyrene microneedle patch in L-polylysine solution, the hydrophilicity of the microneedle patch surface can be improved, the protein coating ability of the microneedle surface can be improved, thereby improving the protein coating efficiency and detection sensitivity of the microneedle.

[0039] The present invention also provides an in vitro microneedle patch prepared by the above-described preparation method.

[0040] The in vitro microneedle patch prepared according to this invention can detect the content of collagen IgG in interstitial fluid in minute quantities and with specificity. This invention also provides the above-described preparation method or the application of the above-described in vitro microneedle patch in the preparation of products for detecting collagen IgG in interstitial fluid. The products described in this invention include in vivo and in vitro detection products.

[0041] In this invention, the method of using the in vitro microneedle patch includes the following steps: (1) Serially diluted collagen IgG standards are added to 1% agarose solution, wherein the number of serially diluted collagen IgG standards is ≥5, preferably 6, 7, 8 or 9. As an optional embodiment, in this invention, the concentrations of the serially diluted collagen IgG standards are 46.8 pg / ml, 93.7 pg / ml, 187.5 pg / ml, 375 pg / ml, 750 pg / ml, 1500 pg / ml, and 3000 pg / ml. After solidification, an agarose gel containing collagen IgG standards is obtained, which serves as a standard curve for simulating skin to draw microneedle patches.

[0042] (2) In this invention, the needles of the in vitro microneedle patch are inserted into an agarose gel containing collagen IgG standards of different concentrations, and the collagen IgG in the standards is extracted after 5-15 min. After extraction, the in vitro microneedle patch is washed with a PBS solution containing Tween-20 (PBS+T solution), wherein the mass concentration of Tween-20 in the solution is 0.02%-0.1%, preferably 0.05% or 0.08%. In this invention, the number of washes is 2-5 times, preferably 3 or 4 times; the time for each wash is 3-8 min, preferably 4 min, 5 min, 6 min or 7 min. After washing, the patch is dried at room temperature for 5-48 h, preferably 12 h, 24 h, 36 h or 40 h; the drying method is natural air drying.

[0043] (3) After extraction, the needles of the in vitro microneedle patch are immersed in the solution and incubated for 15-90 min, wherein the incubation time is preferably 30 min, 45 min, 60 min or 75 min. In this invention, the solution is selected from horseradish peroxidase-linked rabbit anti-human IgG antibody solution or fluorescein-linked rabbit anti-human IgG antibody solution; in this invention, the concentration of the horseradish peroxidase-linked rabbit anti-human IgG antibody solution is 0.1-10 µg / mL, wherein the concentration is preferably 0.5 µg / mL, 2 µg / mL, 5 µg / mL or 8 µg / mL; the concentration of the fluorescein-linked rabbit anti-human IgG antibody solution is 0.1-10 µg / mL, wherein the concentration is preferably 0.5 µg / mL, 2 µg / mL, 5 µg / mL or 8 µg / mL. After incubation, the rabbit is washed with a PBS solution containing Tween-20 (PBS+T solution). The mass concentration of Tween-20 in the solution is 0.02%~0.1%, preferably 0.05% or 0.08%. In this invention, the number of washes is 2~5 times, preferably 3 or 4 times; the washing time for each wash is 3~8 minutes, preferably 4 minutes, 5 minutes, 6 minutes, or 7 minutes. After washing, the rabbit is dried at room temperature for 5~48 hours, preferably 12 hours, 24 hours, 36 hours, or 40 hours; the drying method is natural air drying. The stock solutions of horseradish peroxidase-linked rabbit anti-human IgG antibody and fluorescein-conjugated rabbit anti-human IgG antibody are both 0.5~2 mg / mL. The rabbit anti-human IgG antibody of this invention was purchased from Solarbio. The horseradish peroxidase-linked rabbit anti-human IgG antibody was obtained using a conventional ELISA method, and the fluorescein-conjugated rabbit anti-human IgG antibody was obtained using a conventional conjugation method.

[0044] After drying, the in vitro microneedle patch treated with horseradish peroxidase-linked rabbit anti-human IgG antibody solution was placed in TMB chromogenic solution for 5-15 min, preferably 10 min; after chromogenic development, the grayscale of the image was measured and a standard curve was plotted.

[0045] After drying, the in vitro microneedle patch treated with fluorescein-conjugated rabbit anti-human IgG antibody solution was dried at room temperature for 5–48 h, preferably 12 h, 24 h, 36 h, or 40 h; the drying method was natural air drying. After drying, the fluorescence intensity was measured, and a standard curve was plotted.

[0046] (4) Replace the agarose gel containing collagen IgG standard in step (2) with the sample to be tested, and repeat steps (2) to (3) to determine the gray value or fluorescence intensity of collagen IgG in the interstitial fluid of the skin, and obtain the concentration of collagen IgG in the interstitial fluid of the skin. Compare and analyze the detection results of the sample to be tested with the standard curve to obtain the in vitro and in vivo quantitative detection results of collagen IgG in the interstitial fluid of the skin.

[0047] In this invention, a microneedle fixation device is used to fix the microneedle patch onto the skin for quantitative detection. The patch is secured to a fixed post of the microneedle fixation device by attaching a magnetic patch to its backing. The needles of the microneedle patch are then inserted into the skin surface for initial fixation, achieved through magnetic attraction. Rotation of the microneedle gripping device creates negative pressure on the inner wall of the device, drawing the microneedles deeper into the skin for further fixation. This invention enables quantitative detection of collagen IgG in interstitial fluid both in vitro and in vivo, exhibiting high specificity and sensitivity without the need for separation and extraction of collagen IgG from the interstitial fluid.

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0049] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.

[0050] Example 1 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: 1. Preparation of solid microneedle patches (The overall flowchart of the preparation and detection of the in vitro microneedle patches of this invention is shown in the figure below). Figure 1 ) Preparation of epoxy resin microneedle patches (molding process as follows) Figure 2 As shown): Epoxy resin and toluene dimethylamine were thoroughly mixed at a ratio of 100 g: 15 g, and poured into a silicone microneedle mold with a needle length of 1200 µm, a base side length of 500 µm, and an array size of 10×10. The mold was placed in a vacuum chamber and defoamed at -0.1 MPa for 20 min. After removal, the mold was allowed to dry naturally for 30 h to obtain an epoxy resin microneedle patch with a patch side length of 14.5 mm.

[0051] 2. Plasma treatment of solid microneedle patches The epoxy resin microneedle patch was placed 1.5 cm below the spray gun of the plasma treatment machine and plasma treated for 3 min at a power of 50 W, a pressure of 40 Pa and a temperature of 25 °C to obtain a plasma-treated solid microneedle patch.

[0052] 3. Protein coating The solid microneedle patch treated with plasma in step 2 was immersed in a 50 µg / mL collagen solution and coated at 25°C for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time and air-dried at 25°C for 30 h. Then, the needle body was immersed in a PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin was obtained.

[0053] Example 2 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: 1. Preparation of solid microneedle patches Preparation of epoxy resin microneedle patches: Epoxy resin and toluene dimethylamine were thoroughly mixed at a ratio of 100 g: 15 g and poured into a silicone microneedle mold with a needle length of 1000 µm, a base side length of 390 µm, and an array number of 10×10. The mold was placed in a vacuum chamber and defoamed at -0.095 MPa for 30 min. After removal, the mold was allowed to dry under natural conditions for 30 h to obtain epoxy resin microneedle patches with a side length of 11.7 mm.

[0054] 2. Plasma treatment of solid microneedle patches The epoxy resin microneedle patch was placed 1.5 cm below the spray gun of the plasma treatment machine and plasma treated for 3 min at a power of 50 W, a pressure of 40 Pa and a temperature of 25 °C to obtain a plasma-treated solid microneedle patch.

[0055] 3. L-Poly-L-Lysine Treatment The microneedle patch tips treated with plasma in step 2 were immersed in a 0.1% L-polylysine solution (L-polylysine with a molecular weight of 200 kDa dissolved in deionized water) at 25°C for 60 min. After standing, the tips were washed and air-dried at 25°C for 25 h.

[0056] 4. Protein coating The solid microneedle patch treated with L-polylysine in step 3 was immersed in a 50 µg / mL collagen solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and air-dried at 25°C for 30 h. Then, the needle body was immersed in a PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin was obtained.

[0057] Example 3 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: 1. Preparation of solid microneedle patches Preparation of epoxy resin microneedle patches: Epoxy resin and toluene dimethylamine were thoroughly mixed at a ratio of 100 g: 15 g and poured into a silicone microneedle mold with a needle length of 720 µm, a base side length of 320 µm, and an array number of 12×12. The mold was placed in a vacuum chamber and defoamed at -0.095 MPa for 30 min. After removal, the mold was allowed to dry naturally for 30 h to obtain epoxy resin microneedle patches with a side length of 11.8 mm.

[0058] 2. Plasma treatment of solid microneedle patches The epoxy resin microneedle patch was placed 1.5 cm below the spray gun of the plasma treatment machine and plasma treated for 3 min at a power of 50 W, a pressure of 40 Pa and a temperature of 25 °C to obtain a plasma-treated solid microneedle patch.

[0059] 3. Carboxylation enhancement treatment of solid microneedle patches The plasma-treated solid microneedle patch from step 2 was immersed in a 1:1 (v / v) mixture of 1% malonic acid and 30% H₂O₂ for 60 min. It was then ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times. After cleaning, it was placed in a 50 mM MES buffer (pH 5.5) containing 50 mM EDC and 25 mM NHS and shaken for 30 min. Next, it was transferred to a 0.01 M PBS solution (pH 7) containing 1.5% NH₂-PEG-COOH (PEG molecular weight 5 kDa) and reacted for 5 h to complete PEG grafting. Finally, it was ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times, forming a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends, for subsequent protein coating.

[0060] 4. Protein coating The solid microneedle patch, which underwent carboxylation enhancement treatment in step 3, was immersed in a 50 µg / mL collagen solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and air-dried at 25°C for 30 h. Then, the needle body was immersed in a PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin was obtained.

[0061] Example 4 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: 1. Preparation of solid microneedle patches Preparation of epoxy resin microneedle patches: Epoxy resin and toluene dimethylamine were thoroughly mixed at a ratio of 100 g: 15 g and poured into a silicone microneedle mold with a needle length of 430 µm, a cone bottom diameter of 220 µm, and an array number of 11×11. The mold was placed in a vacuum machine and defoamed at -0.09 MPa for 30 min. After removal, the mold was allowed to dry naturally for 12 h to obtain epoxy resin microneedle patches with a side length of 9.8 mm.

[0062] 2. Plasma treatment of solid microneedle patches The epoxy resin microneedle patch was placed 1.5 cm below the spray gun of the plasma treatment machine and plasma treated for 3 min at a power of 50 W, a pressure of 40 Pa and a temperature of 25 °C to obtain a plasma-treated solid microneedle patch.

[0063] 3. Carboxylation enhancement treatment of solid microneedle patches The plasma-treated solid microneedle patch from step 2 was immersed in a 1:1 (v / v) mixture of 1% malonic acid and 30% H₂O₂ for 60 min. It was then ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times. After cleaning, it was placed in a 50 mM MES buffer (pH 5.5) containing 50 mM EDC and 25 mM NHS and shaken for 30 min. Next, it was transferred to a 0.01 M PBS solution (pH 7) containing 1.5% NH₂-PEG-COOH (PEG molecular weight 5 kDa) and reacted for 5 h to complete PEG grafting. Finally, it was ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times, forming a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends, for subsequent protein coating.

[0064] 4. L-Poly-L-Lysine Treatment The microneedle patch tips that have undergone carboxylation enhancement treatment in step 3 were immersed in a 0.1% L-polylysine solution (L-polylysine with a molecular weight of 200 kDa dissolved in deionized water) at 25°C for 60 min. After standing, the tips were washed and air-dried at 25°C for 25 h.

[0065] 5. Protein coating The solid microneedle patch treated with L-polylysine in step 4 was immersed in a 50 µg / mL collagen solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and air-dried at 25°C for 30 h. Then, the needle body was immersed in a PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin was obtained.

[0066] Example 5 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: 1. Preparation of solid microneedle patches Preparation of epoxy resin microneedle patches: Epoxy resin and toluene dimethylamine were thoroughly mixed at a ratio of 100 g: 15 g and poured into a silicone microneedle mold with a needle length of 720 µm, a base side length of 320 µm, and an array number of 12×12. The mold was placed in a vacuum chamber and defoamed at -0.095 MPa for 30 min. After removal, the mold was allowed to dry naturally for 30 h to obtain epoxy resin microneedle patches with a side length of 11.8 mm.

[0067] 2. Plasma treatment of solid microneedle patches The epoxy resin microneedle patch was placed 1.5 cm below the spray gun of the plasma treatment machine and plasma treated for 3 min at a power of 50 W, a pressure of 40 Pa and a temperature of 25 °C to obtain a plasma-treated solid microneedle patch.

[0068] 3. Carboxylation enhancement treatment of solid microneedle patches The plasma-treated solid microneedle patch from step 2 was immersed in a 1:1 (v / v) mixture of 1% citric acid and 30% H₂O₂ for 60 min. It was then ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times. After cleaning, it was placed in a 50 mM MES buffer (pH 5.5) containing 50 mM EDC and 25 mM NHS and shaken for 30 min. Then, it was transferred to a 0.01 M PBS solution (pH 7) containing 1.5% NH₂-PEG-COOH (PEG molecular weight 5 kDa) and reacted for 5 h to complete PEG grafting. Finally, it was ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times, forming a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends, for subsequent protein coating.

[0069] 4. Protein coating The solid microneedle patch, which underwent carboxylation enhancement treatment in step 3, was immersed in a 50 µg / mL collagen solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and air-dried at 25°C for 30 h. Then, the needle body was immersed in a PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin was obtained.

[0070] Example 6 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: 1. Preparation of solid microneedle patches Preparation of epoxy resin microneedle patches: Epoxy resin and toluene dimethylamine were thoroughly mixed at a ratio of 100 g: 15 g and poured into a silicone microneedle mold with a needle length of 430 µm, a cone bottom diameter of 220 µm, and an array number of 11×11. The mold was placed in a vacuum machine and defoamed at -0.09 MPa for 30 min. After removal, the mold was allowed to dry naturally for 12 h to obtain epoxy resin microneedle patches with a side length of 9.8 mm.

[0071] 2. Plasma treatment of solid microneedle patches The epoxy resin microneedle patch was placed 1.5 cm below the spray gun of the plasma treatment machine and plasma treated for 3 min at a power of 50 W, a pressure of 40 Pa and a temperature of 25 °C to obtain a plasma-treated solid microneedle patch.

[0072] 3. Carboxylation enhancement treatment of solid microneedle patches The plasma-treated solid microneedle patch from step 2 was immersed in a 1:1 (v / v) mixture of 1% citric acid and 30% H₂O₂ for 60 min. It was then ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times. After cleaning, it was placed in a 50 mM MES buffer (pH 5.5) containing 50 mM EDC and 25 mM NHS and shaken for 30 min. Then, it was transferred to a 0.01 M PBS solution (pH 7) containing 1.5% NH₂-PEG-COOH (PEG molecular weight 5 kDa) and reacted for 5 h to complete PEG grafting. Finally, it was ultrasonically cleaned in deionized water at 100 W and 40 Hz for 4 min, with this process repeated three times, forming a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends, for subsequent protein coating.

[0073] 4. L-Poly-L-Lysine Treatment The microneedle patch tips that have undergone carboxylation enhancement treatment in step 3 were immersed in a 0.1% L-polylysine solution (L-polylysine with a molecular weight of 200 kDa dissolved in deionized water) at 25°C for 60 min. After standing, the tips were washed and air-dried at 25°C for 25 h.

[0074] 5. Protein coating The solid microneedle patch treated with L-polylysine in step 4 was immersed in a 50 µg / mL collagen solution and coated at room temperature for 2 h. After removal, it was washed three times with 0.05% PBS+T solution for 5 min each time, and air-dried at 25°C for 30 h. Then, the needle body was immersed in a PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin was obtained.

[0075] Example 7 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 1 is that in step 1, the preparation of the solid microneedle patch is replaced by the preparation of a polystyrene microneedle patch instead of an epoxy resin microneedle patch: a 20% polystyrene-dichloromethane solution is prepared by dissolving polystyrene in dichloromethane, which is then poured into a silicone microneedle mold with a needle length of 1200 µm, a base side length of 500 µm, and an array size of 10×10. The mold is then placed in a vacuum chamber and defoamed at -0.09 MPa for 25 min. After removal, the mold is allowed to dry naturally for 12 h to obtain a polystyrene microneedle patch with a side length of 14.5 mm. All other steps are the same as in Example 1.

[0076] Example 8 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 2 is that in step 1, the preparation of the solid microneedle patch is replaced with the preparation of a polystyrene microneedle patch instead of an epoxy resin microneedle patch: a 20% polystyrene-dichloromethane solution is prepared by dissolving polystyrene in dichloromethane, which is then poured into a silicone microneedle mold with a needle length of 1000 µm, a bottom cone diameter of 390 µm, and an array size of 10×10. The mold is then placed in a vacuum chamber and defoamed at -0.09 MPa for 30 min. After removal, the mold is allowed to dry naturally for 12 h to obtain a polystyrene microneedle patch with a side length of 11.7 mm. All other steps are the same as in Example 2.

[0077] Example 9 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 3 is that in step 1, the preparation of the solid microneedle patch is replaced with the preparation of a polystyrene microneedle patch: polystyrene is dissolved in dichloromethane to prepare a 20% polystyrene-dichloromethane solution, which is poured into a silicone microneedle mold with a needle length of 720 µm, a base side length of 320 µm, and an array number of 12×12. The mold is then placed in a vacuum chamber and defoamed at -0.09 MPa for 30 min. After removal, it is left to dry naturally for 12 h to obtain a polystyrene microneedle patch with a side length of 11.8 mm. All other steps are the same as in Example 3.

[0078] Example 10 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 4 is that in step 1, the preparation of the solid microneedle patch is replaced with the preparation of a polystyrene microneedle patch: polystyrene is dissolved in dichloromethane to prepare a 20% polystyrene-dichloromethane solution, which is poured into a silicone microneedle mold with a needle length of 430 µm, a cone bottom diameter of 220 µm, and an array number of 11×11. The mold is then placed in a vacuum chamber and defoamed at -0.09 MPa for 30 min. After removal, it is left to dry naturally for 12 h to obtain a polystyrene microneedle patch with a side length of 9.8 mm. All other steps are the same as in Example 4.

[0079] Example 11 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 5 is that in step 1, the preparation of the solid microneedle patch is replaced with the preparation of a polystyrene microneedle patch: polystyrene is dissolved in dichloromethane to prepare a 20% polystyrene-dichloromethane solution, which is poured into a silicone microneedle mold with a needle length of 430 µm, a cone bottom diameter of 220 µm, and an array number of 11×11. The mold is then placed in a vacuum chamber and defoamed at -0.09 MPa for 30 min. After removal, it is left to dry naturally for 12 h to obtain a polystyrene microneedle patch with a side length of 9.8 mm. All other steps are the same as in Example 5.

[0080] Example 12 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 6 is that in step 1, the preparation of the solid microneedle patch is replaced by the preparation of a polystyrene microneedle patch instead of an epoxy resin microneedle patch: a 20% polystyrene-dichloromethane solution is prepared by dissolving polystyrene in dichloromethane, which is then poured into a silicone microneedle mold with a needle length of 430 µm, a cone bottom diameter of 220 µm, and an array size of 11×11. The mold is then placed in a vacuum chamber and defoamed at -0.09 MPa for 30 min. After removal, the mold is allowed to dry naturally for 12 h to obtain a polystyrene microneedle patch with a side length of 9.8 mm. All other steps are the same as in Example 6.

[0081] Example 13 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 7 is that step 2 was not performed; all other steps are the same as in Example 7.

[0082] Example 14 A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin: The difference from Example 8 is that step 2 was not performed; all other steps are the same as in Example 8.

[0083] Schematic diagrams of the preparation process of in vitro microneedle patches in Examples 1-14 are shown below. Figure 3 .

[0084] Comparative Example 1 The difference from Example 1 is that step 2 was not performed; all other steps are the same as in Example 1.

[0085] Experimental Example 1 The epoxy resin microneedle patches from Examples 1-4 and the polystyrene microneedle patches from Examples 7-10 were placed in a Shimadzu mechanical testing instrument to test the load-displacement curves of microneedles made of different materials. The results are as follows: Figure 4 As shown, A106 represents the silicone microneedle molds in Examples 1 and 7, A105 represents the silicone microneedle molds in Examples 2 and 8, A104 represents the silicone microneedle molds in Examples 3 and 9, and Y124 represents the silicone microneedle molds in Examples 4 and 10. Figure 4 As can be seen, the two materials and four mold models of microneedles all meet the mechanical requirements for skin penetration, and the mechanical properties of the materials and microneedle structures are good.

[0086] The epoxy resin microneedle patches from Examples 1-4 and the polystyrene microneedle patches from Examples 7-10 were used to insert four different types of microneedles of two different materials into fresh pigskin. The morphology of the needle tip and needle body during and after insertion was observed. The results showed that the morphology of the needle tip and needle body remained intact during and after insertion, without any breakage.

[0087] Experimental Example 2 The epoxy resin microneedle patches treated with plasma in Example 1 and the epoxy resin microneedle patches in Comparative Example 1 were respectively immersed in a 5 µg / mL fluorescein-conjugated rabbit anti-human IgG antibody solution and incubated overnight at 4°C in the dark. After coating, they were washed with 0.05% PBS+T solution for 5 min, and the washing was repeated 3 times. After air drying at 25°C, they were placed under an inverted fluorescence microscope for imaging. Fluorescence images of the microneedle area were acquired using a confocal microscope, and the average fluorescence intensity was measured. The results are as follows: Figure 5 As shown, the fluorescence intensity of the epoxy resin microneedle patch in Comparative Example 1 was 92.71 au, while the fluorescence intensity of the epoxy resin microneedle patch in Example 1 after plasma treatment was 673.68 au. The results indicate that the plasma treatment of the solid microneedle patch according to this invention enhances the binding capacity of the microneedle surface to proteins. In other words, this invention can improve the coating ability of collagen IgG, facilitating subsequent capture and sensitive detection of collagen IgG protein in the interstitial fluid of the skin.

[0088] Experimental Example 3 1. Collagen IgG standards were serially diluted with PBS to 46.8 pg / ml, 93.7 pg / ml, 187.5 pg / ml, 375 pg / ml, 750 pg / ml, 1500 pg / ml, and 3000 pg / ml. The serially diluted collagen IgG standards were then added to 1% agarose solution, and after solidification, agarose gels containing collagen IgG standards were obtained.

[0089] 2. The needles of the in vitro microneedle patch prepared in Example 1 were inserted into agarose gel containing different concentrations of collagen IgG standards, and the collagen IgG in the standards was extracted after 10 min. After extraction, the needles were washed with 0.05% PBS+T solution for 5 min, and the washing was repeated 3 times. After air drying at 25°C, the needles of the in vitro microneedle patch were immersed in 10 µg / mL horseradish peroxidase-linked rabbit anti-human IgG antibody solution and incubated for 30 min. After washing with 0.05% PBS+T solution for 5 min, the washing was repeated 3 times. After air drying at 25°C, the needles of the obtained in vitro microneedle patch were immersed in TMB single-component chromogenic solution for 5 min and then quickly placed under an imaging system to take pictures. The grayscale values ​​of the images were analyzed to obtain the grayscale standard curve of the microneedle in vitro detection (see...). Figure 6 ).

[0090] 3. A mouse model of enhanced collagen IgG secretion was constructed by intraperitoneal injection of collagen with aluminum hydroxide adjuvant in two doses. Tail vein blood from these mice was collected and diluted with Tris buffer at 100, 200, 400, 800, 1600, and 3200 times. The in vitro microneedle patches from Example 1 were then replaced with mouse serum diluent in step 2, and the grayscale values ​​of the mouse serum diluent detection results were measured. Figure 6 The standard curve in the figure yielded the quantitative results of collagen IgG in diluted mouse serum, such as... Figure 7 As shown, Figure 7 The horizontal axis represents the dilution factor of mouse serum, and the vertical axis represents the image grayscale value. The results show that the in vitro microneedle patch provided in this invention can accurately detect the content of collagen IgG in serum without the need for extraction and separation of collagen IgG, and can also accurately detect the target protein in trace blood samples diluted 1600 to 3200 times, demonstrating high sensitivity.

[0091] Test Example 4 In vivo testing was conducted using model mice with enhanced collagen IgG secretion (experimental group) and wild-type mice (control group). Hair was removed from the backs of both model and wild-type mice. The microneedle patch prepared in Example 1 was pressed into the hairless skin and held for 10 min. Collagen IgG was extracted from the interstitial fluid of the mouse skin. The patch was then washed with 0.05% PBS+T solution for 5 min, repeated three times. After washing and drying, the microneedle patch needles were immersed in a 1 mg / mL horseradish peroxidase-linked rabbit anti-mouse IgG antibody solution (rabbit anti-mouse collagen IgG was purchased from Solarbio and obtained according to conventional ELISA methods) for 30 min. After incubation, the patch was washed three times with 0.05% PBS+T solution for 5 min each time. After air-drying at 25℃, the needles of the in vitro microneedle patches were immersed in a horseradish peroxidase-linked rabbit anti-human IgG antibody solution diluted 800 times (1 mg / mL) and incubated for 30 min. They were then washed with 0.05% PBS+T solution for 5 min, repeated three times. After air-drying at 25℃, the needles of the resulting in vitro microneedle patches were immersed in TMB single-component chromogenic solution for 5 min and then quickly placed under an imaging system to take pictures. The grayscale values ​​of the interstitial fluid detection results for the experimental and control groups were measured. Based on... Figure 6 The standard curve in the figure yielded the quantitative results of collagen IgG in the interstitial fluid of the experimental and control groups, such as... Figure 8 As shown in the figure. The results indicate that the in vitro microneedle patch prepared in this invention can achieve quantitative detection of collagen IgG in the interstitial fluid of the skin in vivo.

[0092] Experimental Example 5 To apply the in vitro microneedle patch of this invention to the detection of collagen IgG in human interstitial fluid, this invention uses a microneedle fixation device to fix the patch to the human skin. In this invention, a magnetic patch is adhered to the backing of the in vitro microneedle patch prepared in Example 1 and fixed in the fixing groove of the microneedle fixation device. The skin contact device of the microneedle fixation device is tightly fitted to the subject's skin, with the microneedle tips slightly penetrating the skin. Initial fixation is achieved using magnetic attraction. Rotating the microneedle gripping device creates negative pressure on the inner wall of the skin contact device, causing the microneedles to penetrate deeper into the skin, achieving deep fixation. The usage state of the microneedle fixation device is described in [details omitted]. Figure 9 This indicates that the present invention can achieve in vivo detection of collagen IgG content in human skin interstitial fluid using a microneedle fixation device.

[0093] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing an in vitro microneedle patch for detecting collagen IgG in interstitial fluid of the skin, characterized in that, Includes the following steps: Solid microneedle patches were coated with collagen solutions with a concentration of 2-100 μg / mL to obtain in vitro microneedle patches. The solid microneedle patch is selected from polystyrene microneedle patches or epoxy resin microneedle patches.

2. The preparation method according to claim 1, characterized in that, The polystyrene microneedle patch was immersed in a 0.01-0.1% L-polylysine solution for 30-120 minutes before being coated.

3. The preparation method according to claim 1, characterized in that, The solid microneedle patch is plasma treated and then coated; the plasma treatment power is 40~60 W, the gas pressure is 20~50 Pa, and the time is 1~5 min.

4. The preparation method according to claim 3, characterized in that, After plasma treatment, the solid microneedle patch is first soaked in a 0.01-0.1% L-polylysine solution for 30-120 minutes, and then coated.

5. The preparation method according to claim 3, characterized in that, After plasma treatment, the solid microneedle patch is surface-enhanced by carboxylation before coating. The surface carboxylation enhancement method includes: immersing the plasma-treated solid microneedle patch in a mixed solution for 50-70 min, then shaking it with MES buffer containing 40-60 mM EDC and 20-30 mM NHS for 25-35 min, and then reacting it in PBS solution containing 1-2% NH2-PEG-COOH for 4-6 h to form a solid microneedle patch with a PEG layer retaining carboxylic acid groups at the ends. The mixed solution is a solution of 0.5~1.5% malonic acid and 25~35% H2O2 in a volume ratio of (0.5~1.5):1, or a solution of 0.5~1.5% citric acid and 25~35% H2O2 in a volume ratio of (0.5~1.5):

1.

6. The preparation method according to claim 5, characterized in that, The MES buffer has a pH of 5-6 and a concentration of 30-70 mM; the PBS solution has a pH of 6-8 and a concentration of 0.01-0.02 M.

7. The preparation method according to claim 5, characterized in that, The PEG-modified solid microneedle patch was immersed in a 0.01-0.1% L-polylysine solution for 30-120 min before being coated.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The solid microneedle patch can be prepared by molding or 3D printing.

9. An in vitro microneedle patch prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the preparation method according to any one of claims 1 to 8 or the in vitro microneedle patch according to claim 9 in the preparation of a product for detecting collagen IgG in interstitial fluid of the skin, characterized in that, The products include in vivo testing products and in vitro testing products.

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