A micro-needle patch technology for in vivo detection of melatonin in skin interstitial fluid and a preparation method thereof

Abstract

The application provides a kind of in vivo detection microneedle patch technology of skin interstitial fluid melatonin and its preparation method, and relates to the technical field of biomedical materials and instruments.The application provides a kind of preparation method of in vitro microneedle patch for detecting skin interstitial fluid melatonin, comprising the following steps: placing a solid microneedle patch into a rabbit anti-human melatonin IgG solution with a concentration of 2-100 μg / mL for coating to obtain an in vitro microneedle patch;the solid microneedle patch is selected from a polystyrene microneedle patch or an epoxy resin microneedle patch.The preparation method of the application can enable the in vitro microneedle patch to quantitatively detect trace amounts of specific melatonin protein in skin interstitial fluid, with 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 melatonin in interstitial fluid of the skin and its preparation method. Background Technology

[0002] Melatonin is a hormone secreted by the pineal gland that primarily regulates the sleep-wake cycle and plays a crucial role in maintaining the biological clock. Abnormal fluctuations in melatonin levels are closely associated with a variety of diseases, such as sleep disorders, depression, seasonal affective disorder, irregular circadian rhythms, certain cancers, and Alzheimer's disease. Measuring melatonin levels can help assess and diagnose these conditions, especially in sleep disorders, jet lag, or circadian rhythm disruption caused by long-term shift work, and has significant clinical value. Currently, enzyme-linked immunosorbent assay (ELISA) has become the standard method for melatonin detection due to its high sensitivity and specificity.

[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 the sample transfer and separation process, 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-samples. Therefore, it is urgent to develop a device that can achieve direct, sensitive, and specific detection of melatonin in micro-interstitial fluid samples, so as to avoid loss and contamination during sample transfer and separation, and meet the urgent need for rapid quantitative detection of melatonin 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 melatonin in interstitial fluid. This method enables the quantitative detection of trace amounts of specific melatonin protein in interstitial fluid using an in vitro microneedle patch, exhibiting high sensitivity and 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 melatonin 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 melatonin in interstitial fluid of the skin, comprising the following steps: coating a solid microneedle patch in a rabbit anti-human melatonin IgG 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 melatonin 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 melatonin in interstitial fluid (ISF). The invention utilizes special surface modification to enable the needles of the in vitro microneedle patch to specifically capture melatonin protein from ISF. 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 melatonin protein, allowing for collection and quantitative detection on the skin surface. Furthermore, the in vitro microneedle patch of this invention enables quantitative detection of minute samples, exhibiting high sensitivity and accuracy. This preparation 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 ISF. 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 melatonin using microneedle patches.

[0024] Figure 7The 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 melatonin 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 melatonin in interstitial fluid of the skin, comprising the following steps: coating a solid microneedle patch in a rabbit anti-human melatonin IgG 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 rabbit anti-human melatonin IgG 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; and 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 rabbit anti-human melatonin IgG solution is 2-100 μg / mL. The preferred concentration of the rabbit anti-human melatonin IgG solution is 10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, or 90 μg / mL. The rabbit anti-human melatonin IgG used in this invention is purchased from FineTest, catalog number FNab09931. When the coating temperature is room temperature, the coating time is 1-3 h, preferably 2 h; when the coating temperature is 2-8°C, the coating time is 6-10 h, preferably 7 h, 8 h, or 9 h. 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 product 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 product 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 product 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 provides an in vitro microneedle patch for detecting melatonin 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-carboxylated for enhancement modification, and then coated with protein. The method of surface carboxylation enhancement modification includes: immersing the plasma-treated solid microneedles in a mixed solution for 50-70 min, then shaking them with MES buffer containing 40-60 mM EDC and 20-30 mM NHS for 25-35 min, and then reacting them 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.

[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 an L-polylysine solution with a concentration of 0.01-0.1% for 30-120 minutes before protein coating. The method of immersion in the L-polylysine 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 melatonin content in the interstitial fluid of the skin 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 melatonin in the interstitial fluid of the skin. The products described in this invention include in vivo detection products 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 melatonin standards are added to 1% agarose solution, wherein the number of serially diluted melatonin standards is ≥5, preferably 6, 7, 8, or 9. As an optional embodiment, in this invention, the concentrations of the serially diluted melatonin standards are 4.6 pg / ml, 9.3 pg / ml, 18.7 pg / ml, 37.5 pg / ml, 75 pg / ml, 150 pg / ml, and 300 pg / ml. After solidification, an agarose gel containing melatonin standards is obtained, which serves as a standard curve for simulating skin microneedle patch application.

[0042] (2) In this invention, the needles of the in vitro microneedle patch are inserted into an agarose gel containing melatonin standards of different concentrations, and the melatonin 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 melatonin IgG solution or fluorescein-linked rabbit anti-human melatonin IgG solution; in this invention, the concentration of the horseradish peroxidase-linked rabbit anti-human melatonin IgG 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 melatonin IgG 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 mixture 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; each wash lasts 3~8 minutes, preferably 4 minutes, 5 minutes, 6 minutes, or 7 minutes. After washing, the mixture 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 melatonin IgG and fluorescein-conjugated rabbit anti-human melatonin IgG were both 0.5-2 mg / mL. In this invention, the rabbit anti-human melatonin IgG was purchased from FineTest. The horseradish peroxidase-linked rabbit anti-human melatonin IgG was obtained by conventional enzyme-linking method, and the fluorescein-conjugated rabbit anti-human melatonin IgG was obtained by conventional conjugation method.

[0044] After drying, the in vitro microneedle patch treated with horseradish peroxidase-linked rabbit anti-human melatonin IgG solution was placed in TMB chromogenic solution for 5-15 min, with the preferred chromogenic time being 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 patches treated with fluorescein-conjugated rabbit anti-human melatonin IgG solution were 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 melatonin 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 melatonin in the interstitial fluid of the skin, and obtain the concentration of melatonin 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 melatonin 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 melatonin levels in interstitial fluid both in vitro and in vivo, exhibiting high specificity and sensitivity without the need for separation and extraction of melatonin from the interstitial fluid.

[0048] Example 1 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0049] 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.

[0050] 3. Protein coating The solid microneedle patch treated with plasma in step 2 was immersed in 50 µg / mL rabbit anti-human melatonin IgG 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 was immersed in 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 melatonin in interstitial fluid of the skin was obtained.

[0051] Example 2 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0052] 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.

[0053] 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.

[0054] 4. Protein coating The solid microneedle patch treated with L-polylysine in step 3 was immersed in 50 µg / mL rabbit anti-human melatonin IgG 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 PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, the in vitro microneedle patch for detecting melatonin in the interstitial fluid of the skin was obtained.

[0055] Example 3 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0056] 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.

[0057] 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.

[0058] 4. Protein coating The solid microneedle patch, which underwent carboxylation enhancement treatment in step 3, was immersed in 50 µg / mL rabbit anti-human melatonin IgG 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 was immersed in 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 melatonin in interstitial fluid of the skin was obtained.

[0059] Example 4 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 5. Protein coating The solid microneedle patch treated with L-polylysine in step 4 was immersed in 50 µg / mL rabbit anti-human melatonin IgG 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 PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, the in vitro microneedle patch for detecting melatonin in the interstitial fluid of the skin was obtained.

[0064] Example 5 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0065] 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.

[0066] 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.

[0067] 4. Protein coating The solid microneedle patch, which underwent carboxylation enhancement treatment in step 3, was immersed in 50 µg / mL rabbit anti-human melatonin IgG 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 was immersed in 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 melatonin in interstitial fluid of the skin was obtained.

[0068] Example 6 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 5. Protein coating The solid microneedle patch treated with L-polylysine in step 4 was immersed in 50 µg / mL rabbit anti-human melatonin IgG 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 PBS solution of 2% bovine serum albumin and blocked at 25°C for 2 h. After washing and air-drying, the in vitro microneedle patch for detecting melatonin in the interstitial fluid of the skin was obtained.

[0073] Example 7 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0074] Example 8 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0075] Example 9 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0076] Example 10 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0077] Example 11 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0078] Example 12 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0079] Example 13 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0080] Example 14 A method for preparing an in vitro microneedle patch for detecting melatonin 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.

[0081] Schematic diagrams of the preparation process of in vitro microneedle patches in Examples 1-14 are shown below. Figure 3 .

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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 melatonin IgG 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 imaged under an inverted fluorescence microscope, and their 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 capacity for melatonin, facilitating subsequent capture and sensitive detection of melatonin protein in the interstitial fluid of the skin.

[0086] Experimental Example 3 1. Melatonin standards were serially diluted with PBS to 4.6 pg / ml, 9.3 pg / ml, 18.7 pg / ml, 37.5 pg / ml, 75 pg / ml, 150 pg / ml, and 300 pg / ml. The serially diluted melatonin standards were then added to 1% agarose solution, and after solidification, agarose gels containing melatonin standards were obtained.

[0087] 2. The needles of the in vitro microneedle patch prepared in Example 1 were inserted into agarose gels containing different concentrations of melatonin standards to capture and extract melatonin from the standards for 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 melatonin IgG 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 ).

[0088] 3. A mouse model of enhanced melatonin secretion was established by intraperitoneal injection of melatonin. 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 patch from Example 1 was replaced with mouse serum dilution in step 2, following the procedure where the agarose gel of the melatonin standard was used. The grayscale values ​​of the mouse serum dilution detection results were measured. Figure 6 The standard curve in the figure yielded quantitative results for melatonin 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 by this invention can accurately detect the melatonin content in serum without the need for melatonin extraction and separation, and can also accurately detect the target protein in trace blood samples diluted 1600 to 3200 times, demonstrating high sensitivity.

[0089] Test Example 4 In vivo testing was conducted using mouse models with enhanced melatonin 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. Melatonin 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 horseradish peroxidase-linked rabbit anti-mouse melatonin IgG solution for 30 min. After incubation, the needles were washed three times with 0.05% PBS+T solution for 5 min each time. After air drying at 25°C, the microneedle patch needles were immersed in 1 mg / mL horseradish peroxidase-linked rabbit anti-human melatonin IgG solution (rabbit anti-mouse melatonin IgG was purchased from FineTest and obtained according to conventional ELISA methods) for 30 min. The needles were then washed with 0.05% PBS+T solution for 5 min, repeated three times. After air drying at 25℃, the needles of the obtained in vitro microneedle patches were immersed in TMB single-component chromogenic solution for 5 minutes and then quickly placed under an imaging system to take pictures, measuring the grayscale values ​​of the interstitial fluid detection results images of the experimental group and the control group. Figure 6 The standard curve in the figure yielded quantitative results of melatonin in the interstitial fluid of the skin in 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 melatonin in the interstitial fluid of the skin in vivo.

[0090] Experimental Example 5 To apply the in vitro microneedle patch of this invention to the detection of melatonin in the interstitial fluid of human skin, 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 melatonin content in human skin interstitial fluid using a microneedle fixation device.

[0091] 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 melatonin in interstitial fluid of the skin, characterized in that, Includes the following steps: Solid microneedle patches were coated in rabbit anti-human melatonin IgG solution 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 end. 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 melatonin 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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