A tyrosinase in-situ sensor based on carbon quantum dot microneedle composite structure, a preparation method and application thereof

Abstract

This invention discloses an in-situ tyrosinase sensor based on a carbon quantum dot microneedle composite structure, its preparation method, and its application. Carbon quantum dots (Cate-CQDs) with surface-modified catechol groups were synthesized using urea, anhydrous citric acid, and catechol as raw materials. The Cate-CQDs were uniformly dispersed in a hydrogel and then fixed inside the microneedles after drying. When the microneedles pierce the target skin containing TYR, due to the polyphenol oxidase properties of TYR, catechol is specifically oxidized to benzoquinone by TYR, quenching the original fluorescence of the Cate-CQDs. This invention uses Cate-CQDs as a probe, possessing sensitive tyrosinase detection capabilities and resisting interference from common amino acids and proteases. Compared to small molecule fluorescent probes, carbon quantum dots exhibit stronger resistance to photobleaching. With a fluorescence detection limit as low as 0.052 U / mL and a colorimetric detection limit of 0.10 U / mL, and a linear range covering 0.5–100 U / mL, it can complete the detection within 30 minutes and is suitable for rapid analysis of tyrosinase concentrations in the skin or other matrices.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to an in-situ tyrosinase sensor based on a carbon quantum dot microneedle composite structure, its preparation method, and its application. Background Technology

[0002] Cutaneous melanoma is a malignant tumor originating from mutations in melanocytes, accounting for approximately two-thirds of all skin cancer-related deaths. Treatment options for advanced melanoma remain limited, and the disease carries a high risk of metastasis; therefore, early diagnosis and surgical resection are crucial for improving survival rates. While gross examination is the most convenient diagnostic method, the morphological similarity between melanoma and benign nevi can lead to misdiagnosis. Tyrosinase (TYR), an enzyme involved in melanin production, is frequently overexpressed in melanoma patients and is recognized as an important biomarker for diagnosis and prognosis. Therefore, monitoring TYR levels is of significant value for the early diagnosis and preventative management of melanoma.

[0003] Skin tissue fluid (ISF)—which surrounds the cells and tissues within the skin—is rich in biomarkers, providing a promising medium for the accurate diagnosis and monitoring of skin diseases. Given that melanoma primarily occurs on the skin surface, the concentration of TYR in the skin interstitial fluid is comparable to, or even higher than, that in the blood. Therefore, developing novel methods for direct TYR detection on the skin holds great potential for improving the early diagnosis and prognosis of melanoma.

[0004] Microneedle patches are a popular ISF extraction technology that can penetrate the stratum corneum without touching nerve endings and capillaries, offering advantages such as painlessness, ease of operation, and low risk. Carbon quantum dots exhibit strong resistance to photobleaching, good biocompatibility, and unique fluorescence / colorimetric dual-mode response characteristics, requiring no additional substrate and thus providing better safety and reliability. By combining carbon quantum dots with in-situ microneedle detection, detection can be performed at the treatment point using a smartphone. The colorimetric / fluorescence modes provide more detection options and result verification, reducing false positive / negative results and accurately and promptly reflecting changes in TYR levels in ISF. Therefore, developing a non-invasive, in-situ, and rapid dual-mode sensor for detecting TYR in skin tissue fluid has significant clinical application value.

[0005] The existing technology also has the following limitations:

[0006] 1) Relying on serum as a test sample, current technologies typically require blood samples to be collected from veins or fingertips. These methods require specially trained personnel to collect and analyze samples, and often cause discomfort to patients. Furthermore, interfering substances present in blood samples may reduce the sensitivity and accuracy of the test.

[0007] 2) High testing costs (including time, equipment, and learning costs). Existing technologies, including enzyme-linked immunosorbent assay (ELISA) and laboratory tests, involve expensive reagents, complex procedures, specialized testing equipment, and long waiting times.

[0008] 3) Limited testing modes. Existing technologies typically only offer one detection mode, lacking means to verify the results. Summary of the Invention

[0009] To overcome the shortcomings of the prior art, the primary objective of this invention is to provide an in-situ tyrosinase sensor based on a carbon quantum dot microneedle composite structure, its preparation method, and its application.

[0010] This invention combines carbon quantum dot probes with hydrogel microneedles to develop a colorimetric / fluorescence dual-mode sensor for early monitoring and screening of melanoma through in-situ detection of TYR. Carbon quantum dots (Cate-CQDs) with surface-modified catechol groups were synthesized using urea, anhydrous citric acid, and catechol as raw materials. The Cate-CQDs were uniformly dispersed in a hydrogel and then fixed inside the microneedles after drying. When the microneedles pierce the target skin containing TYR, due to the polyphenol oxidase properties of TYR, catechol is specifically oxidized to benzoquinone by TYR, quenching the original fluorescence of the Cate-CQDs. The color of the microneedle patch gradually changes from yellow to reddish-brown, resulting in the fluorescence signal being "off" and the colorimetric signal being "on." At this point, the logarithm of the TYR concentration is linearly correlated with the fluorescence / colorimetric signal.

[0011] The above-mentioned objective is achieved through the following technical solution:

[0012] This invention discloses an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure, comprising a carbon quantum dot probe and a microneedle patch, including:

[0013] The carbon quantum dot probe is modified with catechol groups on its surface, giving it fluorescence and color response capabilities to tyrosinase.

[0014] Carbon quantum dots are embedded in the backing layer of microneedle patches.

[0015] As a further improvement, the carbon quantum dots of the present invention have a maximum excitation wavelength between 460-470 nm, produce yellow fluorescence with a maximum emission wavelength of 540-550 nm, and have a fluorescence quantum yield between 10-20%.

[0016] As a further improvement, the microneedle patch of the present invention is a hydrogel crosslinked with methacrylic acid modified gelatin and polyvinyl alcohol under ultraviolet light.

[0017] This invention also discloses a method for preparing an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure, comprising the following steps:

[0018] 1) Carbon quantum dots and their synthesis:

[0019] Anhydrous citric acid, urea, and catechol were dissolved in anhydrous ethanol and dispersed by ultrasonication to obtain a solution.

[0020] The ultrasonicated solution was placed in a hydrothermal reactor and heated at 180-190℃ for 8-10 hours to obtain the crude product.

[0021] The crude product was removed by rotary evaporation and ultrasonically dispersed in deionized water to obtain the dispersed crude product.

[0022] The dispersed crude product was filtered and dialyzed to obtain a purified product, which was then freeze-dried to obtain carbon quantum dot powder.

[0023] 2) Preparation of microneedle array patches crosslinked with methacrylic acid-modified gelatin and polyvinyl alcohol:

[0024] Carbon quantum dot powder was dissolved in methacrylic acid modified gelatin and polyvinyl alcohol prepolymer, with the methacrylic acid modified gelatin having a mass fraction of 75%-100%, the polyvinyl alcohol having a mass fraction of 0%-25%, and the carbon quantum dots having a mass volume fraction of 0.1-1%. A photoinitiator, lithium phenyl-2,4,6-trimethylbenzoyl phosphate, was added. The prepolymer solution with the initiator was coated into a designed PDMS mold, and the mold was degassed under vacuum at -0.08 to -0.09 MPa for 5-10 minutes to remove bubbles, concentrated, and dried. The prepolymer was then added to the mold, cured under ultraviolet light for 10-20 minutes, dried, and demolded to obtain a microneedle array patch.

[0025] As a further improvement, the hydrothermal reaction temperature in step 1) of the present invention is preferably 185°C, and the time is preferably 10 hours.

[0026] As a further improvement, in step 2) of the present invention, the mass fraction of methacrylic acid modified gelatin is preferably 75%, the mass fraction of polyvinyl alcohol is preferably 25%, and the mass volume fraction of carbon quantum dots is preferably 0.5%.

[0027] As a further improvement, the vacuum degassing pressure in step 2) of the present invention is preferably -0.09 MPa, the vacuum degassing time is preferably 5 minutes, and the ultraviolet irradiation curing time is preferably 15 minutes.

[0028] This invention also discloses an application of an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure, which is used for the detection of tyrosinase activity for non-disease diagnostic purposes, and is used to detect tyrosinase activity in isolated skin tissue, skin models or food simulants.

[0029] As a further improvement, when the application of this invention is for the detection of tyrosinase activity for non-disease diagnostic purposes, the detection concentration range of tyrosinase is 0.1 - 200 U / mL.

[0030] The present invention has the following advantages and beneficial effects compared with the prior art:

[0031] 1) This invention uses Cate-CQDs as probes, possessing sensitive tyrosinase detection capabilities and resisting interference from common amino acids and proteases. Carbon quantum dots exhibit stronger photobleaching resistance compared to small-molecule fluorescent probes. The fluorescence detection limit is as low as 0.052 U / mL, the colorimetric detection limit is 0.10 U / mL, and the linear range covers 0.5–100 U / mL. Detection can be completed within 30 minutes, making it suitable for rapid analysis of tyrosinase concentrations in skin or other matrices. Compared to commercially available ELISA kits, the detection time is shorter, the linear range is wider, and the cost is lower, as detailed in Table 1.

[0032] Table 1

[0033] Detection time 30 minutes 2-3 hours Linear range 0.5–100 U / mL 2.5-80 U / mL Analytical equipment smartphones ELISA reader cost Less than 10 yuan 1000-2000 yuan Dual signal readout yes no

[0034] 2) This invention fixes carbon quantum dot probes inside a microneedle patch, achieving integrated sampling and detection. The material ratio of the microneedles has been optimized, allowing for the extraction of sample solution more than five times its own weight in as little as 30 minutes. Skin tissue fluid is collected as a test sample via capillary force, eliminating the need for epidermal damage and blood collection, offering the advantages of being painless and non-invasive, and leaving no scars on the sample surface.

[0035] 3) This invention is simple to operate. Microneedles can be inserted into the skin by applying even pressure with the fingertips. After data collection, the needles are removed and read. This can be done by a single person. During the reading stage, a photo of the microneedle backing is taken with a smartphone and a light source, and the signal is read using color analysis software. The operation is simple, requires no professional analysis instruments, and can be used in a home environment.

[0036] 5) Cate-CQDs probes and microneedles have simple manufacturing processes, readily available raw materials, and low manufacturing costs, making them more economical than general instrumental analysis techniques.

[0037] 4) The Cate-CQDs prepared in this invention combine fluorescence and colorimetric properties, achieving dual signal readout. They inherit the complementary advantages of both, providing more options for detection and result verification, enhancing the reliability and sensitivity of the analysis, providing a new approach for early prediction of skin melanoma, and making a new attempt for transdermal, painless, and rapid detection of TYR. Attached Figure Description

[0038] Figure 1This is a transmission electron microscope image of the Cate-CQDs obtained by this invention;

[0039] Figure 2 This is a graph showing the fluorescence quenching of Cate-CQDs under different amino acid interferences;

[0040] Figure 3 This is a graph showing the fluorescence quenching of Cate-CQDs under different protein and enzyme interferences;

[0041] Figure 4 This is a graph showing the swelling properties of microneedles composed of different materials;

[0042] Figure 5 This is a scanning electron microscope image of the MN microneedle patch prepared according to the present invention;

[0043] Figure 6 This is a scanning electron microscope image showing the morphological changes of MN microneedles after multiple punctures;

[0044] Figure 7 This is a diagram showing the restoration process of pigskin after the needle is removed;

[0045] Figure 8 This is a fluorescence signal diagram of Cate-CQDs after catalysis with TYR at different concentration gradients;

[0046] Figure 9 This is a colorimetric signal diagram of Cate-CQDs after catalysis with TYR at different concentration gradients. Detailed Implementation

[0047] This invention discloses an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure, comprising a carbon quantum dot probe and a microneedle patch, including:

[0048] The carbon quantum dot probe is modified with catechol groups on its surface, giving it fluorescence and color response capabilities to tyrosinase; the carbon quantum dots are embedded in the backing layer of the microneedle patch.

[0049] The maximum excitation wavelength of carbon quantum dots is between 460-470 nm, producing yellow fluorescence with a maximum emission wavelength of 540-550 nm, and the fluorescence quantum yield is between 10-20%. The microneedle patch is a hydrogel crosslinked with methacrylic acid-modified gelatin and polyvinyl alcohol under ultraviolet light.

[0050] This invention also discloses a method for preparing an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure, comprising the following steps:

[0051] 1) Carbon quantum dots and their synthesis:

[0052] Anhydrous citric acid, urea, and catechol were dissolved in anhydrous ethanol and dispersed by ultrasonication to obtain a solution.

[0053] The ultrasonicated solution was placed in a hydrothermal reactor and heated at 180-190℃ for 8-10 hours to obtain the crude product.

[0054] The crude product was removed by rotary evaporation and ultrasonically dispersed in deionized water to obtain the dispersed crude product.

[0055] The dispersed crude product was filtered and dialyzed to obtain a purified product, which was then freeze-dried to obtain carbon quantum dot powder.

[0056] 2) Preparation of microneedle array patches crosslinked with methacrylic acid-modified gelatin and polyvinyl alcohol:

[0057] Carbon quantum dot powder was dissolved in methacrylic acid modified gelatin and polyvinyl alcohol prepolymer, with the methacrylic acid modified gelatin having a mass fraction of 75%-100%, the polyvinyl alcohol having a mass fraction of 0%-25%, and the carbon quantum dots having a mass volume fraction of 0.1-1%. A photoinitiator, lithium phenyl-2,4,6-trimethylbenzoyl phosphate, was added. The prepolymer solution with the initiator was coated into a designed PDMS mold, and the mold was degassed under vacuum at -0.08 to -0.09 MPa for 5-10 minutes to remove bubbles, concentrated, and dried. The prepolymer was then added to the mold, cured under ultraviolet light for 10-20 minutes, dried, and demolded to obtain a microneedle array patch.

[0058] Step 1) The preferred hydrothermal reaction temperature is 185℃, and the preferred reaction time is 10 hours. Step 2) The preferred mass fraction of methacrylic acid-modified gelatin is 75%, the preferred mass fraction of polyvinyl alcohol is 25%, and the preferred mass-volume fraction of carbon quantum dots is 0.5%.

[0059] Step 2) The vacuum degassing pressure is preferably -0.09 MPa, the vacuum degassing time is preferably 5 minutes, and the UV curing time is preferably 15 minutes.

[0060] This invention also discloses an application of an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure. The application is for non-disease diagnostic purposes, specifically for detecting tyrosinase activity in isolated skin tissue, skin models, or food simulants. When used for non-disease diagnostic purposes, the detection concentration range for tyrosinase is 0.1–200 U / mL.

[0061] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0062] Example 1:

[0063] A tyrosinase in-situ sensor based on a carbon quantum dot-microneedle composite structure comprises a carbon quantum dot probe and a microneedle patch. The carbon quantum dot probe is modified with catechol groups on its surface, enabling it to exhibit fluorescence and color responses to tyrosinase. The carbon quantum dots are embedded in the backing layer of the microneedle patch. The maximum excitation wavelength of the carbon quantum dots is between 460-470 nm, producing yellow fluorescence with a maximum emission wavelength of 540-550 nm, and a fluorescence quantum yield between 10-20%.

[0064] (1) Preparation of Cate-CQDs:

[0065] 0.24 g anhydrous citric acid, 0.15 g urea, and 0.6 g catechol were dissolved in 10 mL anhydrous ethanol and sonicated for 5 minutes to form a homogeneous solution. The solution was placed in a 25 mL hydrothermal reactor and heated at 185 °C for 10 hours. After the solution cooled naturally to room temperature, the ethanol solvent was separated using a rotary evaporator, and the product was then sonicated and dispersed in 20 mL deionized water. To further purify Cate-CQDs, large-size impurities were removed by filtration through a 0.22 μm microporous membrane, followed by dialyzing with a dialysis bag with a molecular cutoff of 500 Da for 24 hours. Finally, the solution was freeze-dried under vacuum for 48 hours to obtain purified Cate-CQDs powder. The prepared Cate-CQDs probe solution was directly dropped onto an ultrathin carbon film support, dried, and observed under a transmission electron microscope. The results are as follows: Figure 1 As shown in the figure, the nanoparticles are uniformly dispersed with an average particle size of 2.63 nm.

[0066] (2) Specificity assessment of Cate-CQDs for tyrosinase

[0067] To examine the specificity of the prepared Cate-CQDs in response to tyrosinase, different interfering substances were added: three proteins—peanut protein isolate (PPI), soy protein isolate (SPI), and ovalbumin (OVA)—at a concentration of 3 mg / mL; three enzymes—glucose oxidase (GOX), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH)—at a concentration of 100 U / mL; and seven amino acids—histidine (His), serine (Ser), alanine (Ala), cysteine ​​(Cys), cysteine ​​(Cys-Cys), threonine (Thr), and tyrosine (Tyr)—at a concentration of 1 mM. Their effects on the fluorescence emission of Cate-CQDs were observed. The results are as follows: Figure 2 and Figure 3 As shown, the quenching rate of interfering substances on Cate-CQDs was <10%, while the quenching rate of tyrosinase on Cate-CQDs was >75%, indicating that Cate-CQDs have excellent specificity for tyrosinase.

[0068] (3) Preparation of MN microneedle patches

[0069] Polyvinyl alcohol and methacrylic acid-modified gelatin were mixed at a mass ratio of 25% to 75% to form a 10% (w / w) solution (20 mL). 0.05 g of photoinitiator phenyl-2,4,6-trimethylbenzoyl phosphate lithium and 0.1 mL of a 50 μg / mL Cate-CQDs aqueous solution were added to obtain a prepolymer solution. 600 μL of the prepolymer solution was poured into a PDMS mold and degassed under a vacuum of -0.09 MPa. After complete elimination of foam, the hydrogel patch was concentrated and dried overnight at room temperature. Before complete drying, 400 μL of the prepolymer solution was added, and the mold was placed under ultraviolet light at a wavelength of 365 nm for crosslinking for 15 min, followed by drying at room temperature for 24 hours. After two castings, Cate-CQDs were mainly enriched in the backing layer of the microneedles.

[0070] (4) Microneedle patch swelling capacity test

[0071] To evaluate the swelling capacity of the microneedle patch, the microneedle patch (mass M0) from step (3) was immersed in physiological saline at certain time intervals (1 min, 3 min, 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, and 100 min). The patch was then removed, and excess water was gently blotted off with filter paper. The mass M of the microneedle after swelling was recorded, and the swelling rate was (M – M0) × 100%.

[0072] (5) Microneedle patch penetration test

[0073] To evaluate the skin penetration ability of the microneedle patch, the microneedle patch prepared in step (3) was inserted into the pigskin and gently pressed for 2 minutes before being removed with the needle facing upwards for further scanning electron microscopy observation to examine its mechanical stability. Figure 5 and Figure 6 These are scanning electron microscope images of the microneedles after they have passed through the skin multiple times. The microneedle structure remained intact and did not break, indicating that the microneedles can withstand necessary mechanical deformation. Figure 4 The process of pigskin recovery after the needle was removed was demonstrated. Within 20 minutes, all the pores created by the microneedles on the pigskin closed, leaving no obvious trauma or scars.

[0074] (6) Microneedles for the construction of TYR in-situ detection system

[0075] Using pig skin as a model for in vivo studies, the pig skin was cut into 4 cm pieces. 2The pigskin was squared and the surface grease was removed and disinfected with 70% alcohol. Then the pigskin was soaked in TYR solutions of different concentrations (0, 0.1 U / mL, 0.5 U / mL, 2.5 U / mL, 10 U / mL, 50 U / mL, 200 U / mL) and placed in a refrigerator at 4°C overnight to allow it to diffuse completely in the pigskin.

[0076] With the microneedle tip facing down, the microneedle patch was manually pressed onto the TYR-treated pigskin surface. After 5 minutes of fixation, it was left to stand for 25 minutes to allow for sufficient reaction. Then, with the tip facing up, an inverted fluorescence microscope with a filter wavelength of approximately 465 nm was used to capture fluorescence images of the back of the microneedle patch. The microneedle patch was moved to collect data from at least three areas. The fluorescence intensity before use was recorded as F0, and the remaining fluorescence intensity after use was recorded as F. The fluorescence quenching rate Qr was defined as (F0-F) / F0. Simultaneously, a smartphone was used to record an image of the microneedle patch under natural light, and the average value of the red channel R in at least three RGB color samples was collected using Gradient software.

[0077] As the concentration of TYR increased from 0.05 U / mL to 200 U / mL, the fluorescence signal of the microneedle patch gradually decreased, and the color gradually darkened, leading to a decrease in the value of the red channel in the RGB colorimetric signal. The regression equations are Qr = 0.1129 log[TYR] + 0.5425 and Ir = -33.22 log[TYR] + 174.8, where Qr and Ir represent the fluorescence signal quenching rate and the value of the red channel in the colorimetric signal, respectively. Figure 8 and Figure 9 Based on the ratio of the standard deviation of the blank to the slope of the linear equation of the calibration plot (LOD = 3SD / slope, n = 3), the limit of detection (LOD) for fluorescence is 0.052 U / mL, and the limit of detection (LOD) for colorimetry is 0.10 U / mL. These limits of detection are comparable to or lower than those reported for TYR assays.

[0078] Example 2:

[0079] Unlike Example 1, in step (3), the ratio of polyvinyl alcohol to methacrylic acid modified gelatin is 0% to 100%.

[0080] Comparative Example 1:

[0081] Unlike Example 1, in step (3), the ratio of polyvinyl alcohol to methacrylic acid modified gelatin is 100% to 0%.

[0082] Comparative Example 2:

[0083] Unlike Example 1, in step (3), the ratio of polyvinyl alcohol to methacrylic acid modified gelatin is 75% to 25%.

[0084] Comparative Example 3:

[0085] Unlike Example 1, in step (3), the ratio of polyvinyl alcohol to methacrylic acid modified gelatin is 50% to 50%.

[0086] The results are as follows Figure 4 As shown in Table 2, the swelling capacity of the microneedle patch increases with the addition of methacrylic acid modified gelatin. When the proportion of methacrylic acid modified gelatin is >75%, the microneedle exhibits the best swelling performance, reaching a swelling rate of 573% within 60 minutes. In contrast, when the proportion of methacrylic acid modified gelatin is <75%, the swelling rate is less than 400% within 60 minutes. Therefore, a mass fraction of 75%-100% methacrylic acid modified gelatin and a mass fraction of 0%-25% polyvinyl alcohol are suitable for this invention.

[0087] Table 2

[0088] 0 100 502.7 25 75 573.0 50 50 409.7 75 25 352.9 100 0 137.5

[0089] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent substitutions, improvements, or variations made within the spirit and principles of the present invention should fall within the scope of protection of the present invention.

Claims

1. A tyrosinase in-situ sensor based on a carbon quantum dot-microneedle composite structure, characterized in that, Includes carbon quantum dot probes and microneedle patches, including: The carbon quantum dot probe is modified with catechol groups on its surface, giving it fluorescence and color response capabilities to tyrosinase. The carbon quantum dots are embedded in the backing layer of the microneedle patch.

2. The in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure according to claim 1, characterized in that, The carbon quantum dots have a maximum excitation wavelength between 460-470 nm and produce yellow fluorescence with a maximum emission wavelength of 540-550 nm, with a fluorescence quantum yield between 10-20%.

3. The in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure according to claim 1, characterized in that, The microneedle patch is a hydrogel crosslinked with methacrylic acid modified gelatin and polyvinyl alcohol under ultraviolet light.

4. A method for preparing an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure as described in any one of claims 1 to 3, characterized in that, Includes the following steps: 1) Carbon quantum dots and their synthesis: Anhydrous citric acid, urea, and catechol were dissolved in anhydrous ethanol and dispersed by ultrasonication to obtain a solution. The ultrasonicated solution was placed in a hydrothermal reactor and heated at 180-190℃ for 8-10 hours to obtain the crude product. The crude product was removed by rotary evaporation and ultrasonically dispersed in deionized water to obtain the dispersed crude product. The dispersed crude product was filtered and dialyzed to obtain a purified product, which was then freeze-dried to obtain carbon quantum dot powder. 2) Preparation of microneedle array patches crosslinked with methacrylic acid-modified gelatin and polyvinyl alcohol: Carbon quantum dot powder was dissolved in methacrylic acid modified gelatin and polyvinyl alcohol prepolymer, with the methacrylic acid modified gelatin having a mass fraction of 75%-100%, the polyvinyl alcohol having a mass fraction of 0%-25%, and the carbon quantum dots having a mass volume fraction of 0.1-1%. A photoinitiator, lithium phenyl-2,4,6-trimethylbenzoyl phosphate, was added. The prepolymer solution with the initiator was coated into a designed PDMS mold, and the mold was degassed under vacuum at -0.08 to -0.09 MPa for 5-10 minutes to remove bubbles, concentrated, and dried. The prepolymer was then added to the mold, cured under ultraviolet light for 10-20 minutes, dried, and demolded to obtain a microneedle array patch.

5. The preparation method according to claim 4, characterized in that, The preferred hydrothermal reaction temperature in step 1) is 185°C, and the preferred reaction time is 10 hours.

6. The preparation method according to claim 4, characterized in that, In step 2), the methacrylic acid modified gelatin preferably has a mass fraction of 75%, the polyvinyl alcohol preferably has a mass fraction of 25%, and the carbon quantum dots preferably have a mass volume fraction of 0.5%.

7. The preparation method according to claim 4, characterized in that, The vacuum degassing pressure in step 2) is preferably -0.09 MPa, the vacuum degassing time is preferably 5 minutes, and the UV curing time is preferably 15 minutes.

8. An application of an in-situ tyrosinase sensor based on a carbon quantum dot-microneedle composite structure as described in any one of claims 1 to 3, characterized in that, The application described is for the detection of tyrosinase activity for non-disease diagnostic purposes, and is used to detect tyrosinase activity in isolated skin tissue, skin models, or food simulants.

9. The application according to claim 8, characterized in that, When the application is for the detection of tyrosinase activity for non-disease diagnostic purposes, the detection concentration range of tyrosinase is 0.1 - 200 U / mL.

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