Preparation of wearable microneedle sensor and its application in in-situ early visualization detection of melanoma

Abstract

This invention discloses the fabrication of a wearable microneedle sensor and its application in the early visual detection of in situ melanoma. The invention first prepares gold-etched cuprous oxide and silver-encapsulated nanoparticles, then loads them with the small molecule compound catechol, and finally combines this with microneedle technology to create a wearable microneedle sensor. This microneedle sensor integrates colorimetric visualization and surface-enhanced Raman scattering (SERS) dual-mode detection, enabling in-situ sampling of living organisms. It can reflect physiological states in real time and continuously, providing an effective approach for the early diagnosis of skin tumors such as melanoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to the preparation of a wearable microneedle sensor and its application in the early visualization detection of in situ melanoma. Background Technology

[0002] Cutaneous melanoma is a malignant tumor caused by the dysfunction and abnormal proliferation of melanocytes produced in the neural crest. The brown pigment produced by these cells protects the skin from harmful solar radiation. Prolonged exposure to ultraviolet radiation can cause abnormal proliferation of melanocytes, leading to cutaneous melanoma, which is a major contributing factor to its development. Cutaneous melanoma can metastasize to other organs through the lymphatic system and bloodstream, seriously threatening the patient's life and health.

[0003] Currently, traditional clinical diagnostic methods for melanoma include: (a) preliminary visual observation according to the ABCDE rule to assess the asymmetry, irregular borders, heterogeneity of color, diameter greater than 6 mm, and any tendency for progressive enlargement of the lesion; (b) dermoscopy and (c) imaging examinations to assist in the examination, observe whether the tumor has metastasized, and improve diagnostic accuracy; and (d) biopsy histopathology, performing biopsies at 1 to 3 mm lateral margins and full thickness to assess the depth of invasion (Breslow thickness). While the ABCED rule can provide a rapid initial screening for cutaneous melanoma, it relies heavily on the physician's expertise and experience, leading to a slight decrease in accuracy to some extent. Furthermore, the accuracy rate of visual observation is only around 65%, hampered by the observer's lack of experience and subjective bias. Although dermoscopy offers higher accuracy, it still results in a significant number of misdiagnoses or missed diagnoses. While biopsy histopathology is currently the gold standard for diagnosing melanoma, it can accurately diagnose cutaneous melanoma and classify the corresponding stages. However, it also has drawbacks such as long testing time, pain, trauma, and high testing costs, which can lead to reduced patient compliance.

[0004] In addition to commonly used clinical diagnostic methods, early, rapid, and accurate screening for cutaneous melanoma can be achieved by identifying disease-related biomarkers. Various common melanocyte markers, such as S100, HMB45, Melan A, tyrosinase (Tyr), MITF, and SOX10, are helpful in the detection and subtyping of melanoma, enabling accurate identification of cutaneous melanoma, precise treatment, and significantly improved patient survival rates. Tyrosinase, in particular, plays a crucial role in the early clinical management of cutaneous melanoma. As a specific molecular marker of melanocyte differentiation, its expression level in tissues aids pathologists in accurate diagnosis and subtyping. In disease monitoring, detecting tyrosinase mRNA in peripheral blood using high-sensitivity RT-PCR technology can detect circulating tumor cells and micrometastases earlier than imaging methods. This is critical for assessing postoperative recurrence risk, accurate staging, and prognosis. This "liquid biopsy" method provides an important molecular monitoring tool for early-stage melanoma patients, helping to guide clinical decision-making and achieve early intervention.

[0005] Currently, most tyrosinase-based detection methods rely on traditional human serum testing, which inevitably causes tissue damage, potential infection, and psychological stress for patients during the sampling process. Furthermore, blood samples contain various interfering components (such as immunoglobulin G, serum albumin, and myoglobin), severely affecting the sensitivity and reliability of the analysis. In contrast, interstitial fluid is located on the skin's surface, and its collection process is relatively non-invasive, avoiding the pain and infection risks associated with blood draws. Moreover, its concentration is highly correlated with blood, providing real-time and continuous reflection of physiological states. Its composition is also relatively simple (free of blood cells and some large molecular interfering substances), making it more suitable for developing miniaturized, highly sensitive point-of-care testing devices.

[0006] Microneedling is increasingly favored in the field of monitoring biomarkers on the skin surface and subcutaneous tissue due to its ability to physically penetrate the skin surface and reach deep tissues, as well as its advantages of simple, rapid, continuous, painless, and minimally invasive operation. However, microneedling technology still has limitations: (1) the content of interstitial fluid (ISF) in the dermis is low (accounting for about 15-30% of the skin volume), and the sample volume extracted by a single microneedling is usually only at the microliter (μL) or even nanoliter (nL) level, which limits the detection sensitivity for low-concentration biomarkers; (2) some enzymes or metabolites in ISF are easily degraded, requiring immediate analysis or special preservation conditions. Therefore, it is of great significance to develop new methods that combine high sensitivity and immediate analysis capabilities.

[0007] Currently, surface-enhanced Raman scattering (SERS) technology is increasingly being used in the fields of bacteria and biomarkers. It has advantages such as high sensitivity and molecular specificity. The colorimetric visualization method can detect color changes that are visible to the naked eye in a short time through the interaction between chemical substances and target analytes. It can also perform preliminary semi-quantification of the target analytes by the intensity of the color, thereby quickly indicating suspicious areas and judging the approximate disease progression, and can achieve a rapid and immediate initial screening.

[0008] Therefore, developing a wearable microneedle sensor that integrates porous microneedles (MN), colorimetric visualization, and surface-enhanced Raman scattering (SERS) technology to achieve early visual detection of in situ melanoma is of great practical significance. Summary of the Invention

[0009] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing gold-etched cuprous oxide and silver-encapsulated nanoparticles.

[0010] Another object of the present invention is to provide gold-etched cuprous oxide and silver-coated nanoparticles prepared by the method.

[0011] Another object of the present invention is to provide the application of the gold-etched cuprous oxide and silver-encapsulated nanoparticles.

[0012] The objective of this invention is achieved through the following technical solution:

[0013] A method for preparing gold-etched cuprous oxide and silver-encapsulated nanoparticles includes the following steps:

[0014] S1. Preparation of cuprous oxide nanoparticles:

[0015] Sodium hexadecyl sulfonate was dissolved in water, and then copper chloride aqueous solution, sodium hydroxide aqueous solution and hydroxylamine hydrochloride aqueous solution were added. The mixture was stirred at room temperature. After the reaction was completed, the mixture was centrifuged and washed, and then resuspended in anhydrous ethanol to obtain cuprous oxide nanoparticle ethanol solution.

[0016] S2. Preparation of gold-etched cuprous oxide nanoparticles:

[0017] Sodium hexadecyl sulfonate was dissolved in anhydrous ethanol. The cuprous oxide nanoparticle ethanol solution obtained in step S1, sodium hydroxide aqueous solution, and chloroauric acid aqueous solution were added sequentially. The mixture was stirred and reacted. After the reaction was completed, the mixture was centrifuged, washed, and resuspended in anhydrous ethanol to obtain the initial solution of gold-etched cuprous oxide nanoparticles. Tween-80 was then added and the mixture was stirred and reacted. After the reaction was completed, the mixture was centrifuged, washed, and resuspended in anhydrous ethanol to obtain the gold-etched cuprous oxide nanoparticle ethanol solution.

[0018] Preparation of S3, gold-etched cuprous oxide and silver-encapsulated nanoparticles (Cu2O@Au-Ag):

[0019] Add silver nitrate aqueous solution to the ethanol solution of gold-etched cuprous oxide nanoparticles obtained in step S2 to carry out the reaction. After the reaction is completed, centrifuge and wash to obtain gold-etched cuprous oxide and silver-coated nanoparticles (Cu2O@Au-Ag).

[0020] Further, the concentration of the copper chloride aqueous solution in step S1 is 50–500 mmol / L; preferably 250 mmol / L.

[0021] Further, the concentration of the sodium hydroxide aqueous solution in step S1 is 0.5–5 mol / L; preferably 2 mol / L.

[0022] Further, the concentration of the hydroxylamine hydrochloride aqueous solution in step S1 is 100–250 mmol / L; preferably 200 mmol / L.

[0023] Further, the molar ratio of sodium hexadecyl sulfonate, copper chloride, sodium hydroxide and hydroxylamine hydrochloride in step S1 is 1:(0.12-1.19):(0.50-4.95):(0.06-0.15); preferably 1:0.6:2.0:0.12.

[0024] Furthermore, the water mentioned in step S1 is deionized water.

[0025] Furthermore, the water mentioned in step S1 is used to dissolve sodium hexadecyl sulfonate, and the amount used can be adjusted according to the actual situation; preferably, it is calculated at 0.3 to 1 mL of water per milligram of sodium hexadecyl sulfonate; more preferably, it is calculated at 0.65 mL of water per milligram of sodium hexadecyl sulfonate.

[0026] Furthermore, the stirring reaction time in step S1 is 0.5 to 1.5 hours; preferably 1 hour.

[0027] Further, the concentration of the sodium hydroxide aqueous solution in step S2 is 1 to 7 mmol / L; preferably 3 mmol / L.

[0028] Further, the concentration of the chloroauric acid aqueous solution in step S2 is 0.3–0.9 mmol / L; preferably 0.7 mmol / L.

[0029] Further, the molar ratio of sodium hexadecyl sulfonate, cuprous oxide nanoparticles, sodium hydroxide and chloroauric acid in step S2 is 1 : 0.00379 : (0.0632~0.442) : (0.00758~0.0227); preferably 1 : 0.00379 : 0.190 : 0.0177.

[0030] Furthermore, the anhydrous ethanol mentioned in step S2 is used to dissolve sodium hexadecyl sulfonate, and its amount can be adjusted according to the actual situation. Preferably, it is calculated at 0.03 to 0.05 mL of anhydrous ethanol per milligram of sodium hexadecyl sulfonate; more preferably, it is calculated at 0.038 mL of anhydrous ethanol per milligram of sodium hexadecyl sulfonate.

[0031] Further, in step S2, the stirring reaction can be carried out by stirring for a period of time after each reactant is added. For example, when adding cuprous oxide nanoparticle ethanol solution, stir for 15-25 min (preferably 20 min), then add sodium hydroxide aqueous solution and stir for 25-35 min (preferably 30 min), and finally add chloroauric acid aqueous solution and stir for 4-6 min (preferably 5 min); the total stirring reaction time is 50-60 min (preferably 55 min).

[0032] Further, the volume ratio of Tween-80 to the initial solution of gold-etched cuprous oxide nanoparticles in step S2 is 0.05 mL to 0.25:2; preferably 0.2:2.

[0033] Furthermore, the time for continuing the stirring reaction in step S2 is 25 to 35 minutes; preferably 30 minutes.

[0034] Further, the molar ratio of gold-etched cuprous oxide nanoparticles to silver nitrate in step S3 is (3-7.5):1; preferably 3:1.

[0035] Furthermore, the concentration of the ethanol solution for etching cuprous oxide nanoparticles with gold in step S3 is 0.3–0.75 mmol / L.

[0036] Further, the concentration of the silver nitrate aqueous solution in step S3 is 0.4–1 mmol / L; preferably 1 mmol / L.

[0037] Furthermore, the reaction time in step S3 is 25 to 35 minutes; preferably 30 minutes.

[0038] Furthermore, the gold-etched cuprous oxide and silver-encapsulated nanoparticles (Cu2O@Au-Ag) described in step S3 can be resuspended in anhydrous ethanol for preservation.

[0039] A gold-etched cuprous oxide and silver-encapsulated nanoparticle is prepared by any of the preparation methods described above.

[0040] The application of gold-etched cuprous oxide and silver-encapsulated nanoparticles in loading small molecule compounds and / or preparing SERS probes.

[0041] The small molecule compound is preferably catechol (CL).

[0042] A gold-etched cuprous oxide nanoparticle and silver-encapsulated nanoparticle (SERS probe) loaded with a small molecule compound was prepared by the following method:

[0043] An ethanol solution of the above-mentioned gold-etched cuprous oxide and silver-coated nanoparticles (Cu2O@Au-Ag) was ultrasonically mixed with an aqueous solution of catechol (CL) and stirred at room temperature in the dark. After the reaction was completed, the solid was collected by centrifugation to obtain gold-etched cuprous oxide and silver-coated nanoparticles loaded with small molecule compounds (catechol-modified gold-etched cuprous oxide and silver-coated nanoparticles; Cu2O@Au-Ag-CL).

[0044] Furthermore, the concentration of the ethanol solution containing the gold-etched cuprous oxide and silver-encapsulated nanoparticles (Cu2O@Au-Ag) is 0.1–1 mmol / L; preferably 0.6 mmol / L.

[0045] Furthermore, the concentration of the catechol aqueous solution is 0.001–0.1 mol / L; preferably 0.01 mol / L.

[0046] Furthermore, the molar ratio of the gold-etched cuprous oxide and silver-encapsulated nanoparticles (Cu2O@Au-Ag) to catechol (CL) is (0.1~1):1; preferably 0.6:1.

[0047] Furthermore, the reaction time is 30 min to 2 h.

[0048] The application of the gold-etched cuprous oxide and silver-encapsulated nanoparticles and / or the gold-etched cuprous oxide and silver-encapsulated nanoparticles loaded with small molecule compounds in the fabrication of wearable microneedle sensors.

[0049] A wearable microneedle sensor is prepared by the following method:

[0050] (1) Preparation of porous microneedles:

[0051] Cellulose acetate was dissolved in dimethyl sulfoxide to obtain a polymer solution (cellulose acetate dimethyl sulfoxide solution); the polymer solution was then cast onto a PDMS master mold, and centrifugation was used to promote the filling of the mold with the polymer solution (viscous solution); the PDMS mold containing the polymer solution was then immersed in water to induce phase separation; finally, the cured polymer was peeled off from the PDMS mold and washed with water, and then freeze-dried to obtain porous microneedles.

[0052] (2) Preparation of hydrophilic porous microneedles:

[0053] The porous microneedles obtained in step (1) are immersed in an aqueous solution of hexadecyltrimethylammonium chloride, and then removed and dried to obtain hydrophilic porous microneedles (which are hydrophilic, insoluble and capable of extracting interstitial fluid from the skin).

[0054] (3) Fabrication of wearable microneedle sensors

[0055] A dimethyl sulfoxide solution of cellulose acetate was mixed with an ethanol solution of the above-mentioned gold-etched cuprous oxide and silver-coated nanoparticles (Cu2O@Au-Ag) to obtain a catechol-modified gold-etched cuprous oxide and silver-coated nanoparticle ethanol aqueous solution. Then, the nitrocellulose membrane was cut into microneedle-sized shapes and immersed in the catechol-modified gold-etched cuprous oxide and silver-coated nanoparticle ethanol aqueous solution. After immersion, it was taken out and dried, and then attached to the substrate of the hydrophilic porous microneedle obtained in step (2) to obtain a wearable microneedle sensor.

[0056] Further, the concentration of the polymer solution in step (1) is 10 wt% to 25 wt%; preferably 15 wt%.

[0057] Further, the centrifugation conditions described in step (1) are: 4000-5500 rpm, centrifugation for 5-10 min; preferably: 4000 rpm, centrifugation for 5 min.

[0058] Furthermore, in step (1), the PDMS mold containing the polymer solution is immersed in water for 8 to 12 minutes; preferably 10 minutes.

[0059] Further, the concentration of the hexadecyltrimethylammonium chloride aqueous solution in step (2) is 10 wt% to 25 wt%; preferably 25 wt%.

[0060] Furthermore, the soaking time in step (2) is 10 to 30 minutes; preferably 30 minutes.

[0061] Furthermore, the soaking time in step (3) is 10 to 30 minutes; preferably 30 minutes.

[0062] Further, in step (3), the concentration of the catechol-modified gold-etched cuprous oxide and silver-coated nanoparticle ethanol aqueous solution is 0.1–0.6 mmol / L; preferably 0.3 mmol / L.

[0063] Furthermore, the thickness of the nitrocellulose membrane described in step (3) is 50 mm.

[0064] The application of at least one of the gold-etched cuprous oxide and silver-coated nanoparticles, the gold-etched cuprous oxide and silver-coated nanoparticles loaded with small molecule compounds, and the wearable microneedle sensor in the preparation of products for detecting tyrosinase and / or skin melanoma.

[0065] The products mentioned include products for the early diagnosis of skin melanoma.

[0066] Technical principle of the invention:

[0067] This invention provides a porous microneedle with hydrophilicity and insolubility, capable of extracting interstitial fluid from the skin, as the sampling component, and a nitrocellulose membrane modified with a SERS probe as the detection component. The SERS probe comprises catechol, gold-etched cuprous oxide, and silver-coated nanoparticles. The porous microneedle and nitrocellulose membrane are assembled into a wearable microneedle sensor platform. This platform uses the characteristic peak of catechol as a qualitative standard and calculates the tyrosinase concentration based on the intensity of the characteristic peak as a quantitative standard. The microneedle sensor is attached to the suspected skin lesion area. The porous needle tip extracts interstitial fluid from the skin onto the nitrocellulose membrane via capillary force. When tyrosinase is present in the interstitial fluid, the catechol on the SERS probe is oxidized to catechol-diquinone. The characteristic peak of catechol weakens or disappears depending on the amount of tyrosinase in the interstitial fluid. Simultaneously, the nitrocellulose membrane with catechol changes from colorless to yellow. When tyrosinase is absent in the interstitial fluid, the characteristic peak of catechol on the SERS probe remains unchanged, and the color also remains unchanged. Therefore, colorimetry can be performed based on the color change of catechol after it is oxidized by tyrosinase, enabling real-time, visual detection.

[0068] The present invention has the following advantages and effects compared with the prior art:

[0069] (1) The cellulose acetate porous microneedle raw material provided by the present invention has low cost, simple preparation method, and can be mass-produced by compression molding. In addition, the colorimetric / SERS reaction is completed in nano-scale ISF, reducing the amount of reagents by 90%, which further reduces the cost.

[0070] (2) The porous microneedles provided by the present invention can penetrate only the stratum corneum to the epidermis as a sampler. They are almost painless and bleeding-free, significantly reducing patient pain and infection risk, improving compliance, and are suitable for frequent screening.

[0071] (3) The colorimetric visualization method provided by the present invention introduces colorimetry before SERS detection to produce visible color changes, which can quickly indicate suspicious areas and facilitate preliminary judgment by non-professionals. It is suitable for screening at the grassroots level.

[0072] (4) This invention provides a wearable microneedle sensor that integrates porous microneedles (MN), colorimetric visualization and surface-enhanced Raman scattering (SERS) technology. By using the “microneedle-colorimetry-SERS” combined method, it makes full use of the advantages of each and achieves breakthroughs in sensitivity, specificity, minimal invasiveness and convenience. It enables “sampling-detection” to be performed in situ. At the same time, the entire process (sampling + detection) can be completed within one hour without complicated sample pretreatment and laboratory analysis, realizing “on-site real-time detection” and accelerating diagnostic decision-making.

[0073] (5) The wearable microneedle sensor for in situ detection of melanoma provided by the present invention integrates colorimetric visualization and surface enhanced Raman scattering (SERS) dual-mode detection, enabling in situ sampling of living organisms. It has the advantages of simple, fast, continuous, painless, and minimally invasive operation, and can reflect physiological state in real time and continuously, providing a new paradigm for the early diagnosis of skin tumors (melanoma). Attached Figure Description

[0074] Figure 1 This is a transmission electron microscope image of Cu2O@Au-Ag obtained by the present invention;

[0075] Figure 2 This is the infrared spectrum of Cu2O@Au-Ag-CL prepared according to the present invention;

[0076] Figure 3 This is a scanning electron microscope image of the hydrophilic porous microneedles prepared according to the present invention;

[0077] Figure 4 This is the infrared spectrum of the hydrophilic porous microneedles prepared by the present invention;

[0078] Figure 5 This is an optical image of the wearable microneedle sensor obtained by the present invention;

[0079] Figure 6 This is a color change graph of pigskin after 30 minutes of microneedle sensor insertion treatment;

[0080] Figure 7 This is the SERS spectrum of pigskin after 30 minutes of microneedle sensor insertion treatment;

[0081] Figure 8 This is a surface-enhanced Raman spectrum of a microneedle sensor inserted into a patient's skin for 30 minutes.

[0082] Figure 9 This is a statistical result of the water extraction volume after 30 minutes of piercing the agarose gel with hydrophilic porous microneedles and the original porous microneedles. Detailed Implementation

[0083] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0084] Example 1

[0085] 1. A method for preparing gold-etched cuprous oxide and silver-encapsulated nanoparticles, comprising the following steps:

[0086] (1) Preparation of cuprous oxide nanoparticles:

[0087] Sodium hexadecyl sulfonate (87 mg) was dissolved in 56.4 mL of deionized water, followed by the addition of copper chloride aqueous solution (250 mM, 0.6 mL), then sodium hydroxide aqueous solution (2 M, 0.25 mL) and hydroxylamine hydrochloride aqueous solution (200 mM, 0.15 mL). The mixture was stirred at room temperature for 1 h, and then the reaction solution was centrifuged, washed, and resuspended in 5 mL of anhydrous ethanol to obtain cuprous oxide nanoparticles.

[0088] (2) Preparation of gold-etched cuprous oxide nanoparticles:

[0089] Sodium hexadecyl sulfonate (26 mg) was dissolved in 1 mL of anhydrous ethanol. A cuprous oxide nanoparticle ethanol solution (3 mmol / L, 0.1 mL) was added, and the mixture was stirred for 20 min. Then, a sodium hydroxide aqueous solution (3 mM, 5 mL) was added, and the mixture was stirred for 30 min. Finally, a chloroauric acid aqueous solution (0.7 mM, 2 mL) was added, and the mixture was stirred for 5 min. The reaction solution was then centrifuged, washed, and resuspended in 2 mL of anhydrous ethanol to obtain the initial solution for gold etching of cuprous oxide nanoparticles. Then, 0.1 mL of Tween-80 was added, and the mixture was stirred for 30 min. The reaction solution was then centrifuged, washed, and resuspended in 1 mL of anhydrous ethanol to obtain the gold etching of cuprous oxide nanoparticles.

[0090] (3) Preparation of gold-etched cuprous oxide and silver-encapsulated nanoparticles (Cu2O@Au-Ag):

[0091] Add silver nitrate aqueous solution (1 mM, 0.1 mL) to 1 mL of 0.3 mmol / L gold-etched cuprous oxide nanoparticle ethanol solution and stir for 30 min. Centrifuge and wash the reaction solution, and resuspend it in 0.5 mL of anhydrous ethanol solution to obtain gold-etched cuprous oxide and silver-coated nanoparticles, abbreviated as Cu2O@Au-Ag.

[0092] Figure 1The image shows a transmission electron microscope (TEM) image of the prepared Cu2O@Au-Ag. As can be seen from the image, the nanoparticles are hollow cubes with uniform morphology and an average particle size of about 700 nm.

[0093] 2. A method for preparing gold-etched cuprous oxide nanoparticles loaded with small molecule compounds and silver-encapsulated nanoparticles (Cu2O@Au-Ag-CL), comprising the following steps:

[0094] The above 1 mL 0.6 mmol / L Cu2O@Au-Ag ethanol solution was mixed with catechol (CL) aqueous solution (0.01 M, 0.1 mL) and sonicated until the solution was uniformly dissolved. The mixture was stirred for 30 min at room temperature and in the dark, and then the reaction solution was centrifuged to obtain catechol-modified gold-etched cuprous oxide and silver-coated nanoparticles, referred to as Cu2O@Au-Ag-CL.

[0095] Figure 2 The image shows the UV spectrum of the prepared Cu2O@Au-Ag-CL. As can be seen from the figure, a new absorption peak appears at 320 nm in the mixed solution of Cu2O@Au-Ag nanoparticles (Cu2O@Au-Ag NCs) and catechol, which proves that Cu2O@Au-Ag NCs and catechol form a surface complex or undergo plasmon resonance coupling.

[0096] 3. A method for preparing a porous microneedle that is hydrophilic, insoluble, and capable of extracting interstitial fluid from the skin, comprising the following steps:

[0097] (1) Preparation of porous microneedles:

[0098] First, 0.15 g of cellulose acetate (Shanghai Maclean Biochemical Technology Co., Ltd.) was dissolved in 1 mL of dimethyl sulfoxide. The resulting polymer solution (concentration 15% (w / v)) was then cast onto a PDMS master mold. The mold was then centrifuged at 4000 rpm for 5 min to promote the filling of the mold with the viscous solution. The PDMS mold containing the polymer solution was then immersed in water for 10 min to induce phase separation. The cured polymer was peeled off from the PDMS mold and washed with plenty of water, then dried using a freeze-drying process to obtain porous microneedles (CA MN).

[0099] (2) Preparation of hydrophilic porous microneedles:

[0100] First, dissolve 0.05 g of hexadecyltrimethylammonium chloride (CTAC) in 2 mL of deionized water, then soak the porous microneedles in the solution for 30 min, remove and dry them to obtain hydrophilic porous microneedles (CA@CTAC MN).

[0101] Figure 3The image shows a scanning electron microscope (SEM) image of the prepared hydrophilic porous microneedles. As can be seen from the image, the prepared hydrophilic porous microneedles have a regular rectangular conical geometry and a sharp tip. The height is 600 μm, the bottom diameter is 300 μm, the space between two adjacent centers is 600 μm, and pores of a few micrometers in size can be clearly observed.

[0102] Figure 4 The figure shows the infrared spectrum of the prepared hydrophilic porous microneedles. As can be seen, when the microneedles are modified with CTAC, the infrared spectrum is in the range of ~2900-2850 cm⁻¹. -1 It exhibits a strong absorption peak, which is typical of the CH stretching vibration of alkyl chains, indicating the presence of a long methylene chain in the molecule; furthermore, ~960-910 cm⁻¹ -1 Quaternary ammonium salt groups (N) appeared in the region. + The CN stretching vibration of (CH3)3 indicates that CTAC was successfully modified onto the microneedle.

[0103] 4. A method for fabricating a wearable microneedle sensor, comprising the following steps:

[0104] First, following the method in step 2 above, take 1 mL of 0.01 mol / L catechol aqueous solution and add it to 1 mL of 0.6 mmol / L ethanol solution of gold-etched cuprous oxide and silver-coated nanoparticles (Cu2O@Au-Ag) to prepare a catechol-modified gold-etched cuprous oxide and silver-coated nanoparticle ethanol aqueous solution (0.3 mmol / L). Then, cut a nitrocellulose membrane (Shanghai Xinya Purification Device Factory, 50 mm) into a shape of 1 cm × 1 cm, immerse the membrane in the above ethanol aqueous solution for 30 minutes, remove and dry it, and finally attach it to the hydrophilic porous microneedle substrate (bottom of the microneedle) prepared in step 3 to obtain the final product.

[0105] Figure 5 The image shows an optical image of the fabricated wearable microneedle sensor. As can be seen, the fabricated MN array is assembled in a square shape, making it easy to contact the skin.

[0106] 5. Colorimetric performance test of the prepared wearable microneedle sensor

[0107] The colorimetric performance of the wearable microneedle sensor prepared above was tested using the following steps: Tyrosinase at different concentrations (200, 150, 100, 50, 10, 5, 1, 0.5, 0.05, 0 U / ml) was added dropwise to 100 μL of different fresh pigskins and allowed to stand overnight. The prepared wearable microneedle sensor was then inserted into Tyr-treated pigskin for 30 min, and its color change was observed. The experiment was repeated three times.

[0108] Figure 6The figure shows the color change of the microneedle sensor after 30 minutes of Tyr-treated pigskin. As can be seen, the oxidation of catechol increases with the concentration of tyrosinase, and the color darkens accordingly. This indicates that the detection platform is feasible.

[0109] 6. Surface Enhanced Raman Scattering (SERS) Performance Testing of the Fabricated Wearable Microneedle Sensor

[0110] The SERS performance of the wearable microneedle sensor prepared above was tested using the following steps: Different concentrations (200, 150, 100, 50, 10, 5, 1, 0.5, 0.1, 0.05 U / ml) of tyrosinase were added to different pigskins and left to stand overnight. The microneedle sensor was then inserted into Tyr-treated pigskin for 30 min. The microneedle was then placed under a Raman spectrometer using a 532 nm laser at 50% power, with one integration, to obtain the SERS spectra of different concentrations of tyrosinase. The experiment was repeated three times.

[0111] Figure 7 The figure shows the surface-enhanced Raman spectrum of pigskin after 30 min of microneedle sensor insertion treatment. As can be seen, with increasing tyrosinase concentration, catechol at 775 cm⁻¹... -1 The characteristic peak at that location subsequently weakens, indicating that the detection platform is indeed feasible.

[0112] 7. The prepared wearable microneedle sensor was used to detect clinical samples.

[0113] The wearable microneedle sensor prepared above was used to detect clinical samples. The specific steps were as follows: The microneedle sensor was inserted into the skin of 5 different patients (diagnosed with cutaneous melanoma) collected from Shenzhen People's Hospital for 30 minutes. Interstitial fluid (ISF) was extracted. The microneedle was placed under a Raman spectrometer using a 532 nm laser at 50% power, and integrated once to obtain SERS spectra under different concentrations of tyrosinase. The same patient was measured three times.

[0114] Figure 8 The figure shows the surface-enhanced Raman spectrum of the microneedle sensor after 30 minutes of insertion into the patient's skin, indicating that the microneedle sensor has the ability to detect clinical samples.

[0115] Comparative Example 1

[0116] Hydrophilic porous microneedles were prepared according to the method in step 3 of Example 1, and compared with porous microneedles. The specific steps are as follows: First, agar was dissolved in water to prepare an agar gel with a concentration of 2% (w / v) as a skin model. Porous microneedles (CA MN) and hydrophilic porous microneedles (CA@CTAC MN) with cellulose acetate concentrations of 15%, 20%, and 25% (w / v) were inserted into the model agar gel at 37°C for 30 min, and the water extraction volume over time was recorded. The experiment was repeated three times.

[0117] Figure 9 The figures show the water extraction volumes of hydrophilic porous microneedles and original porous microneedles after 30 minutes of insertion into the model agarose gel. As shown in the figure, compared with the original porous microneedles, the hydrophilic porous microneedles exhibited a faster fluid extraction rate in agarose. The hydrophilic microneedles could extract 2.71 ± 0.10 mg of ISF within 10 minutes, which meets the volume required for ISF detection.

[0118] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing gold-etched cuprous oxide and silver-encapsulated nanoparticles, characterized in that, Includes the following steps: S1. Preparation of cuprous oxide nanoparticles: Sodium hexadecyl sulfonate was dissolved in water, and then copper chloride aqueous solution, sodium hydroxide aqueous solution and hydroxylamine hydrochloride aqueous solution were added. The mixture was stirred at room temperature. After the reaction was completed, the mixture was centrifuged and washed, and then resuspended in anhydrous ethanol to obtain cuprous oxide nanoparticle ethanol solution. S2. Preparation of gold-etched cuprous oxide nanoparticles: Sodium hexadecyl sulfonate was dissolved in anhydrous ethanol. The cuprous oxide nanoparticle ethanol solution obtained in step S1, sodium hydroxide aqueous solution, and chloroauric acid aqueous solution were added sequentially. The mixture was stirred and reacted. After the reaction was completed, the mixture was centrifuged, washed, and resuspended in anhydrous ethanol to obtain the initial solution of gold-etched cuprous oxide nanoparticles. Tween-80 was then added and the mixture was stirred and reacted. After the reaction was completed, the mixture was centrifuged, washed, and resuspended in anhydrous ethanol to obtain the gold-etched cuprous oxide nanoparticle ethanol solution. S3, Preparation of gold-etched cuprous oxide and silver-coated nanoparticles: Add silver nitrate aqueous solution to the ethanol solution of gold-etched cuprous oxide nanoparticles obtained in step S2 to carry out the reaction. After the reaction is completed, centrifuge and wash to obtain gold-etched cuprous oxide and silver-coated nanoparticles.

2. The method for preparing gold-etched cuprous oxide and silver-encapsulated nanoparticles according to claim 1, characterized in that: The molar ratio of sodium hexadecyl sulfonate, copper chloride, sodium hydroxide, and hydroxylamine hydrochloride in step S1 is 1:0.12-1.19:0.50-4.95:0.06-0.15; The molar ratio of sodium hexadecyl sulfonate, cuprous oxide nanoparticles, sodium hydroxide and chloroauric acid in step S2 is 1:0.00379:0.0632~0.442:0.00758~0.0227. The volume ratio of Tween-80 to the initial solution of gold-etched cuprous oxide nanoparticles in step S2 is 0.05 mL to 0.25:2; The molar ratio of gold-etched cuprous oxide nanoparticles to silver nitrate in step S3 is 3 to 7.5:

1.

3. The method for preparing gold-etched cuprous oxide and silver-encapsulated nanoparticles according to claim 1, characterized in that: The concentration of the copper chloride aqueous solution mentioned in step S1 is 50–500 mmol / L; The concentration of the sodium hydroxide aqueous solution mentioned in step S1 is 0.5–5 mol / L; The concentration of the hydroxylamine hydrochloride aqueous solution mentioned in step S1 is 100–250 mmol / L; The concentration of the sodium hydroxide aqueous solution mentioned in step S2 is 1–7 mmol / L; The concentration of the chloroauric acid aqueous solution mentioned in step S2 is 0.3–0.9 mmol / L; The concentration of the ethanol solution for etching cuprous oxide nanoparticles with gold in step S3 is 0.3–0.75 mmol / L; The concentration of the silver nitrate aqueous solution mentioned in step S3 is 0.4–1 mmol / L; The stirring reaction time in step S1 is 0.5 to 1.5 hours; The stirring reaction time in step S1 is 50–60 min; The stirring reaction time described in step S2 is 25–35 min; The reaction time described in step S3 is 25–35 min.

4. A method of etching cuprous oxide with gold and encapsulating silver nanoparticles, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 3.

5. The application of the gold-etched cuprous oxide and silver-encapsulated nanoparticles as described in claim 4 in loading small molecule compounds and / or preparing SERS probes.

6. A method for etching cuprous oxide nanoparticles with small molecule compounds on gold and encapsulating them with silver, characterized in that, It is prepared by the following method: The ethanol solution of gold-etched cuprous oxide and silver-coated nanoparticles as described in claim 4 was ultrasonically mixed with catechol aqueous solution and stirred at room temperature and in the dark. After the reaction was completed, the solid was collected by centrifugation to obtain gold-etched cuprous oxide and silver-coated nanoparticles loaded with small molecule compounds. The molar ratio of the gold-etched cuprous oxide and silver-encapsulated nanoparticles to catechol is 0.1 to 1:

1.

7. The application of the gold-etched cuprous oxide and silver-coated nanoparticles of claim 4 and / or the gold-etched cuprous oxide and silver-coated nanoparticles loaded with small molecule compounds of claim 6 in the preparation of wearable microneedle sensors.

8. A wearable microneedle sensor, characterized in that, It is prepared by the following method: (1) Preparation of porous microneedles: Cellulose acetate was dissolved in dimethyl sulfoxide to obtain a polymer solution; then the polymer solution was cast onto a PDMS master mold, and centrifugation was used to promote the filling of the mold with the polymer solution. The PDMS mold containing the polymer solution was then immersed in water to induce phase separation. Finally, the cured polymer was peeled off from the PDMS mold, washed with water, and freeze-dried to obtain porous microneedles. (2) Preparation of hydrophilic porous microneedles: The porous microneedles obtained in step (1) are immersed in an aqueous solution of hexadecyltrimethylammonium chloride, and then removed and dried to obtain hydrophilic porous microneedles. (3) Fabrication of wearable microneedle sensors A dimethyl sulfoxide solution of cellulose acetate is mixed with an ethanol solution of gold-etched cuprous oxide and silver-coated nanoparticles as described in claim 4 to obtain an ethanol aqueous solution of catechol-modified gold-etched cuprous oxide and silver-coated nanoparticles. Then, a nitrocellulose membrane is cut into the shape of microneedles, immersed in the ethanol aqueous solution of catechol-modified gold-etched cuprous oxide and silver-coated nanoparticles, removed and dried after immersion, and then attached to the substrate of the hydrophilic porous microneedles obtained in step (2) to obtain a wearable microneedle sensor.

9. The wearable microneedle sensor according to claim 8, characterized in that: The concentration of the polymer solution mentioned in step (1) is 10 wt% to 25 wt%; In step (1), the PDMS mold containing the polymer solution is immersed in water for 8 to 12 minutes; The concentration of the hexadecyltrimethylammonium chloride aqueous solution mentioned in step (2) is 10 wt% to 25 wt%; The soaking time described in step (2) is 10 to 30 minutes; The soaking time described in step (3) is 10 to 30 minutes; The concentration of the catechol-modified gold-etched cuprous oxide and silver-encapsulated nanoparticles in the ethanol aqueous solution in step (3) is 0.1–0.6 mmol / L.

10. The use of at least one of the gold-etched cuprous oxide and silver-coated nanoparticles of claim 4, the gold-etched cuprous oxide and silver-coated nanoparticles loaded with small molecule compounds of claim 6, and the wearable microneedle sensor of any one of claims 8 to 9 in the preparation of products for detecting tyrosinase and / or skin melanoma.

Images