Gold nanoparticle-PMMA (polymethyl methacrylate) microneedle sensor based on graphene quantum dot modification as well as preparation method and application of gold nanoparticle-PMMA microneedle sensor

Abstract

The invention discloses a gold nanoparticle-PMMA (polymethyl methacrylate) microneedle sensor based on graphene quantum dot modification as well as a preparation method and application of the gold nanoparticle-PMMA microneedle sensor. According to the sensor, a PMMA microneedle array serves as a substrate, AuNPs and y-GQDs are sequentially immobilized on the surface through a liquid level self-assembly method and a soaking method, and a GQDs-AuNPs composite active interface is formed. According to the interface, analytes are specifically captured and enriched through functional groups such as amino groups and carboxyl groups on the surfaces of the y-GQDs, and SERS signals are remarkably improved through the charge transfer effect of the y-GQDs and the electromagnetic field enhancement synergistic effect of AuNPs. During detection, uric acid is directly detected through SERS spectrum; the indirect detection of the pH is realized by modifying the pH sensitive molecule 4-MBA. The sensor shows high linear correlation in detection of uric acid (40-640 [mu] mol / L) and pH (4.5-6.5), and realizes high-sensitivity, rapid, synchronous or separate detection of two key biomarkers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical sensing and detection technology, specifically relating to a surface-enhanced Raman scattering (SERS) microneedle sensor, its preparation method, and its application. Background Technology

[0002] Chronic metabolic diseases, such as gout, have become a major global public health problem. Effective management of these diseases relies on real-time, continuous, and accurate monitoring of key biomarkers such as uric acid and pH. Blood tests are the gold standard, but they are invasive and difficult to monitor continuously. In recent years, interstitial fluid (ISF) has emerged as a promising alternative sample due to its high correlation with analyte concentrations in blood and its minimally invasive sampling process. Microneedle (MN) technology can efficiently penetrate the stratum corneum of the skin, causing low pain and exhibiting good biocompatibility, making it an ideal carrier for ISF extraction. Surface-enhanced Raman scattering (SERS) technology, with its ultra-high sensitivity, unique "fingerprint" recognition capability, excellent photostability, and selectivity, demonstrates significant advantages in the field of biosensing.

[0003] Currently, research has attempted to combine SERS with microneedles. However, most existing SERS microneedle sensors are single-function (usually detecting only one analyte), rely on complex modification processes, or have shortcomings in sensitivity, stability, hydrophilicity, and ease of practical application. For example, the preparation process of the SERS active interface of some microneedles is cumbersome, involves toxic reagents, and has poor biocompatibility; others have failed to effectively solve the problem of efficient extraction and enrichment of ISF on the microneedle surface, resulting in limited detection sensitivity. Therefore, developing a SERS microneedle sensor that can simultaneously detect multiple biomarkers, has high sensitivity, excellent stability, good biocompatibility, and is easy to prepare, has important clinical needs and practical significance. In existing technologies, for example, CN117368177A proposes a flexible SERS sensor chip for detecting uric acid in sweat. Although it has the advantages of flexible bonding and structural design, it is still limited to the detection of a single analyte (uric acid) and cannot achieve simultaneous monitoring of multiple parameters; in addition, it relies on a complex patterned metal structure preparation process, which is costly, and does not involve efficient extraction of interstitial fluid and pH detection functions. Another technology, CN117907308A, provides a SERS-based quantitative uric acid detection method. It improves the accuracy of detection by introducing calibrated molecules. However, this method still requires liquid sampling and in vitro detection, and fails to achieve truly non-invasive, real-time, in vivo monitoring. Furthermore, it does not integrate pH detection capabilities, making its functionality relatively limited. Summary of the Invention

[0004] To address the shortcomings of existing SERS microneedle sensors in terms of functional integration, fabrication process, and detection performance, this invention provides a GQDs-AuNPs@PMMA microneedle SERS sensor and its fabrication method that is simple to prepare, low in cost, and easy to use, capable of simultaneously or separately detecting uric acid and pH values ​​with high sensitivity. The SERS sensor of this invention is a multifunctional integrated sensor that can non-invasively or minimally invasively detect pH values ​​and uric acid concentrations in interstitial fluid (ISF) simultaneously or separately. This sensor combines excellent mechanical strength, biocompatibility, and surface hydrophilicity, and can efficiently extract and enrich analytes in skin interstitial fluid (ISF).

[0005] The GQDs-AuNPs@PMMA microneedle SERS sensor provided by this invention utilizes the GQDs-AuNPs interface to specifically capture and enrich analytes using the amino and carboxyl functional groups on the surface of γ-GQDs. Furthermore, through the synergistic effect of charge transfer from γ-GQDs and electromagnetic field enhancement from AuNPs, the SERS signal is significantly improved. This sensor not only achieves simultaneous detection of both uric acid and pH parameters but also possesses excellent in-situ extraction capability of interstitial fluid, high mechanical strength, and good biocompatibility, demonstrating significant advantages in preparation process, functional integration, and ease of practical application.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution.

[0007] A graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor is disclosed. The sensor uses a polymethyl methacrylate (PMMA) microneedle array as a substrate. Gold nanoparticles (AuNPs) are uniformly immobilized on the surface of the PMMA microneedle array via a liquid surface self-assembly method to form an active interface with high-density SERS "hot spots". Then, γ-GQDs are loaded using an immersion method. Finally, 4-mercaptobenzoic acid (4-MBA) is modified at the GQDs-AuNPs active interface as a pH-sensitive probe.

[0008] The fabrication method of the GQDs-AuNPs@PMMA microneedle SERS sensor of the present invention includes the following steps: (1) Fabrication of PMMA microneedle array; (2) Synthesis of aqueous gold nanoparticle solution; (3) AuNPs@PMMA microneedles were obtained by immobilizing gold nanoparticles on the surface of PMMA microneedles using the liquid surface self-assembly method; (4) A y-GQDs solution was prepared by hydrothermal method. AuNPs@PMMA microneedles were immersed in the y-GQDs solution to immobilize GQDs and obtain a GQDs-AuNPs@PMMA microneedles forming a GQDs-AuNPs composite active interface. This interface utilizes the functional groups such as amino and carboxyl groups on the surface of y-GQDs to specifically capture and enrich analytes. Through the charge transfer effect of y-GQDs and the electromagnetic field enhancement of AuNPs, the SERS signal is significantly improved. (5) 4-Mercaptobenzoic acid (4-MBA) was modified on the GQDs-AuNPs@PMMA microneedles to obtain GQDs-AuNPs@PMMA microneedles SERS sensor.

[0009] Further, the method for preparing the PMMA microneedle array in step (1) is as follows: PMMA is dissolved in DMF solution to obtain PMMA solution, the PMMA solution is uniformly coated on silicon mold, and after vacuuming, it is centrifuged and dried to obtain PMMA microneedle array.

[0010] Furthermore, the concentration of the PMMA solution is 15-35 wt%, and the silicon mold is an array of 11×11 needles with a length of 800 μm and a needle tip spacing of 850 μm.

[0011] Furthermore, the method for synthesizing aqueous gold nanoparticles in step (2) is as follows: Prepare a HAucl4·3H2O solution of appropriate concentration and heat it to a gentle boil. Then add sodium citrate, using sodium citrate as a reducing agent and stabilizer to reduce Au³⁺ in HAucl4·3H2O to metallic Au, thus obtaining an aqueous gold nanoparticle solution. The gold nanoparticles are obtained through an aqueous synthesis method and are uniformly distributed on the surface of the microneedles.

[0012] Further, step (3) involves immobilizing gold nanoparticles onto the surface of microneedles using a liquid surface self-assembly method as follows: add n-hexane and ethanol sequentially to the aqueous gold nanoparticle solution and shake gently. After gold precipitates from the solution, immerse the PMMA microneedle array under the gold, then slowly lift it out and place it in an oven to dry. Repeat the above operation until the gold is evenly covered on the surface of the PMMA microneedle array.

[0013] Further, the steps of preparing y-GQDs by hydrothermal method in step (4) are as follows: orange peel powder and DMF solution are mixed and placed in a high-temperature reactor lined with polytetrafluoroethylene for hydrothermal reaction. After cooling, the mixture is filtered to obtain y-GQDs solution. The hydrothermal reaction conditions are 160-200℃ for 8-12 hours; the soaking time is 5-20 min to achieve the purpose of immobilizing GQDs. Preferably, the amount of orange peel is controlled at 0.03-0.05 g, and the amount of DMF solution is controlled at 6-10 g. Furthermore, the method for modifying 4-mercaptobenzoic acid (4-MBA) at the GQDs-AuNPs composite active interface in step (5) is as follows: GQDs-AuNPs@PMMA microneedles are immersed in a solution with a concentration of 1×10⁻⁶. -5 M-5×10 -5 Immerse the sample in a 4-MBA solution of M for 5-20 minutes, then allow it to stand at room temperature until dry. This allows 4-MBA to modify the surface of AuNPs via Au-S bonds, with its carboxyl group (-COOH) serving as a pH-sensitive probe.

[0014] This invention also provides the application of the aforementioned GQDs-AuNPs@PMMA microneedle SERS sensor for non-invasive detection of uric acid and pH values ​​in non-disease diagnosis. The microneedle SERS sensor is attached to the skin, interstitial fluid is extracted, uric acid is directly detected by SERS spectroscopy, and pH values ​​are indirectly detected by 4-MBA modified microneedles. Uric acid concentration and pH value are calculated based on a preset linear model. This sensor exhibits a high linear correlation (R² better than 0.995 and 0.991, respectively) in the detection of uric acid (40–640 μmol / L) and pH (4.5–6.5) solutions. In particular, the pH detection range accurately covers the uric acid crystallization risk range (pH 4.5–5.5 is high risk), achieving high sensitivity, rapid, simultaneous or separate detection of these two key biomarkers, and has significant application prospects in non-invasive monitoring of chronic metabolic diseases.

[0015] The advantages of this invention compared to existing technologies are as follows: This invention achieves highly sensitive detection of uric acid and pH in ISF. It utilizes gold nanoparticles rich in SERS "hot spots" to form a GQDs-AuNPs composite active interface with γ-GQDs. This interface specifically captures and enriches analytes using the amino and carboxyl functional groups on the γ-GQDs surface, and significantly enhances the SERS signal through the synergistic effect of charge transfer from γ-GQDs and electromagnetic field enhancement from AuNPs. The sensor exhibits high linear correlation (R² better than 0.995 and 0.991, respectively) for uric acid in the range of 40–640 μmol / L and for pH in the range of 4.5–6.5, meeting the clinical need for precise monitoring of key biomarkers. The platform uses PMMA as the microneedle matrix, ensuring excellent mechanical strength and easy penetration into simulated skin, while also possessing inherent biocompatibility and chemical stability, providing a safe basis for in vivo application. The introduction of AuNPs and γ-GQDs not only enhances the signal but also significantly improves surface hydrophilicity, enabling efficient extraction of interstitial fluid and targeted enrichment of analytes, thereby synergistically improving detection sensitivity. This invention has successfully completed penetration and signal acquisition tests in a simulated skin environment, verifying its feasibility and effectiveness in near-real-world applications. This approach provides a novel, comprehensive, and highly promising technical solution for non-invasive, real-time monitoring of chronic metabolic diseases. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Schematic diagram of the microneedle SERS sensor process.

[0018] Figure 2 Schematic diagram of microneedles loaded with and unloaded aqueous gold and γ-GQDs under an optical microscope.

[0019] Figure 3 Microneedle TEM images of aqueous gold and γ-GQDs loaded and unloaded.

[0020] Figure 4 Electron micrograph of aqueous gold.

[0021] Figure 5 AFM and TEM images of y-GQDs.

[0022] Figure 6 Fluorescence UV and IR images of y-GQDs.

[0023] Figure 7 Schematic diagram of the synergistic effect of optical enhancement and fluid manipulation at different locations of the microneedle. Figure 8 Concentration gradient Raman spectra of Rhodamine 6G, concentration gradient Raman spectra of 4-ATP, and three-dimensional Raman spectra and statistical histograms of 4-ATP reproducibility test.

[0024] Figure 9 A schematic diagram illustrating the SERS strategy for uric acid detection and the linear relationship between Raman peak intensity and concentration, and a schematic diagram illustrating the SERS strategy for pH detection and the linear relationship between Raman peak intensity and concentration.

[0025] Figure 10 Agar was used to simulate skin for testing.

[0026] Figure 11 A schematic diagram of a microneedle SERS sensor applied to the human body. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above-described invention.

[0028] The preparation method of the graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor provided by the present invention is as follows: (1) Prepare a PMMA solution with a mass concentration of 20% using PMMA solid reagent at 85℃ and 500 r / min; (2) Apply an appropriate amount of 20% PMMA solution evenly to the silicon mold of the required array type, vacuum for 3 min, then centrifuge at 3000 r / min for 3 min, repeat the above operation twice and dry in an oven at 40℃ for 8 h. (3) After the time is up, remove the PMMA microneedles and rinse them with ethanol for use in subsequent experiments; (4) Prepare a HAucl4·3H2O solution of appropriate concentration and heat it to a gentle boil; (5) Then sodium citrate is added to reduce Au³⁺ in chloroauric acid to metallic gold using sodium citrate as a reducing agent and stabilizer; (6) A liquid surface self-assembly method was used to load aqueous gold onto PMMA microneedles to obtain gold-loaded microneedles AuNPs@PMMA microneedles.

[0029] (7) Weigh 0.05 g and 10 g DMF into a 10 ml high-temperature reactor and react at 180 °C for 12 h. After standing and cooling, filter to obtain y-GQDs. Then take 10 ml of y-GQDs solution and soak the gold-loaded microneedles in it for 15 min to obtain GQDs-AuNPs@PMMA microneedles.

[0030] (8) Immerse GQDs-AuNPs@PMMA microneedles in 1×10 -5 The 4-MBA solution of M was used for 10 min to modify the active interface of GQDs-AuNPs through Au-S bond. The carboxyl group (-COOH) of the carboxyl group was used as a pH-sensitive probe to obtain the GQDs-AuNPs@PMMA microneedle SERS sensor.

[0031] This microneedle structure possesses excellent mechanical strength and biocompatibility, making it an ideal carrier for tissue fluid extraction. The introduction of AuNPs and γ-GQDs not only significantly enhances the SERS signal but also greatly improves surface hydrophilicity, thereby promoting the efficient extraction and enrichment of analytes.

[0032] Example 1 The preparation method of the graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor in this embodiment is as follows: 1) Place 2 g PMMA into 8 ml (8 g) DMF solution and react for 1 h at 85 ℃ with a rotor speed of 500 r / min to obtain a PMMA solution with a mass concentration of 20%.

[0033] 2) 100 μL of 20% PMMA solution was uniformly coated onto a silicon mold with an array of 11×11 needles, a needle length of 800 μm, and a needle tip spacing of 850 μm. The mold was then evacuated for 3 min, centrifuged at 3000 r / min for 3 min, and the above operation was repeated twice. The mold was then dried in an oven at 40 ℃ for 8 h to obtain a PMMA microneedle array.

[0034] 3) Prepare solution A: Add 57 mg of sodium citrate to 5 ml of water. Prepare solution B: Add 197 mg of HAucl4·3H2O to 50 ml of water. Heat solution B until it just boils, then add solution A. React for 15 minutes. The solution will first turn colorless and then slowly turn wine-red, indicating successful preparation of the aqueous gold solution. Add 6 ml of n-hexane and 6 ml of ethanol sequentially to 9 ml of the aqueous gold solution and shake gently. After gold precipitates, immerse the PMMA microneedle array under the gold and then slowly lift it into a 50°C oven to dry. Repeat the above operation until the gold is evenly covered.

[0035] 4) Weigh 0.05 g of orange peel and 10 g of DMF into a 10 ml high-temperature reactor and react at 180 ℃ for 12 h. After standing and cooling, filter to obtain y-GQDs solution. Then take 10 ml of y-GQDs solution and immerse the gold-loaded microneedles in it for 15 min to obtain GQDs-AuNPs@PMMA microneedles.

[0036] 5) Immerse GQDs-AuNPs@PMMA microneedles in a solution with a concentration of 1×10⁻⁶. -5 The GQDs-AuNPs@PMMA microneedle SERS sensor is obtained by soaking in 4-MBA solution for 10 min and allowing it to stand at room temperature until dry.

[0037] The complete flowchart in this embodiment is as follows: Figure 1 As shown. Figure 2 (b)-(d) are microneedle array images without AuNPs and y-GQDs under an optical microscope, and (e)-(g) are microneedle array images with AuNPs and y-GQDs. It can be seen that the microneedles are arranged in a neat array. They are of appropriate size and will not affect normal daily activities when worn on the body.

[0038] Example 2 Analysis of the detection capability of GQDs-AuNPs@PMMA microneedle sensor TEM testing was performed on Example 1. Figure 3 (a)-(d) are microneedles without AuNPs and y-GQDs loaded. (e)-(h) are TEM images of microneedles loaded with AuNPs and y-GQDs. It can be seen that the microneedles are intact and have sharp tips. By comparing the mapping of loaded and unloaded microneedles, it can be seen that the aqueous gold is uniformly loaded on the microneedles. Figure 4 This is a TEM image of aqueous gold, which is spherical in shape. Spherical AuNPs have a symmetrical structure, and their LSPR peak positions (typically in the 500-600 nm range) can be well matched with the excitation wavelengths of many commercial Raman lasers (such as 532 nm, 633 nm, 785 nm).

[0039] Figure 5 (a) and (b) are AFM and TEM images of y-GQDs. The AFM image shows that the average thickness of y-GQDs is 3.51 nm, indicating that they are composed of multiple layers of graphene sheets. Due to the coupling of electrons between the layers, this structure typically has high conductivity, which is beneficial for synergistic enhancement of electromagnetic effects with AuNPs. The multilayer structure provides abundant edge sites, and its good conductivity ensures efficient charge transfer. This makes them more suitable for application in SERS. The TEM image shows that the average particle size of y-GQDs is 0.41 nm, indicating very uniform size. Figure 5 (c) is a high-resolution electron microscope image of y-GQDs, which clearly reveals the crystallization properties of y-GQDs, showing a well-defined single-crystal structure with a quantum dot lattice spacing of 0.22 nm. The excellent single-crystal structure is the reason for its enhanced optical properties.

[0040] Figure 6 Images (a), (b), and (c) show the fluorescence, UV, and IR spectra of y-GQDs, respectively. It can be seen that y-GQDs are brown under natural light and emit a pale yellow light after UV irradiation. The optimal excitation wavelength is 450 nm, and the fluorescence intensity peak changes with the excitation wavelength, indicating that the fluorescence spectrum of y-GQDs is independent of the excitation wavelength due to their highly ordered graphite structure. The IR spectrum reveals numerous hydrophilic functional groups, which can be used for analyte enrichment in SERS detection.

[0041] Figure 7 (a), (b), and (c) are schematic diagrams illustrating the synergistic effect of optical enhancement and fluid manipulation at different locations on the microneedle, highlighting the dual functions of gold nanoparticles (AuNPs) within the microneedle structure. The left-hand diagrams (a, b, c) present different distribution / structural models of AuNPs on the microneedle surface, where the gray area represents the microneedle substrate and the yellow particles represent AuNPs, which are used to construct the "hot spot" regions for SERS enhancement. The optical simulation diagram on the right uses a color gradient (see the right-hand color scale; red → blue indicates decreasing optical enhancement or electric field intensity) to visually represent the localized surface plasmon resonance (LSPR) effect in different microneedle regions. Model (a) focuses on the AuNP assembly structure at the microneedle edge (i) and tilted surface (ii), demonstrating the SERS enhancement potential of these regions by simulating their electric field distribution; Model (b) presents the optical response of a large-scale AuNP array (iii, iv) on the microneedle surface, illustrating the effect of the array structure on extending the enhancement range; Model (c) analyzes the AuNP configuration at the microneedle tip (vi) and longitudinal arrangement (v), highlighting the strong enhancement characteristics at key locations such as the tip. In summary, this figure, through a combination of structural schematics and optical simulations, intuitively illustrates how AuNPs on the microneedle platform achieve SERS signal enhancement, providing a structural basis for fluid manipulation of biological samples (such as the precise capture and transport of biomolecules), ultimately serving high-sensitivity bioanalytical applications such as uric acid and pH detection.

[0042] Figure 8 Figures a and 8b show the SERS spectra of rhodamine 6G and 4-aminobenzylthiophenol (4-ATP), respectively. The solution concentrations decrease sequentially from top to bottom, with a concentration gradient of 1 × 10⁻⁶. -2 M to 1×10 -6M. The spectrum clearly shows that the intensity of the characteristic Raman peak gradually decreases with decreasing concentration. Both analytes exhibit similar spectral intensity decay patterns, confirming the considerable detection capability of this microneedle SERS platform. To further verify durability and reusability, 4-ATP Raman data acquired one week apart were used to construct a three-dimensional Raman spectrum. Figure 8 c). Located at 985 cm -1 1068 cm -1 and 1580 cm -1 The characteristic peaks exhibit a highly clustered distribution, providing evidence for its durability. Furthermore, a bar chart based on the intensity of the characteristic peaks from ten consecutive 4-ATP tests ( Figure 8 d) The intensity fluctuation is small. The calculated value is 985 cm. -1 1068 cm -1 With 1580 cm -1 The relative standard deviations (RSDs) of the three characteristic peak intensities were 4.4%, 6.7%, and 6.2%, respectively, further demonstrating its excellent reusability.

[0043] Example 3 Uric acid and pH values ​​were detected using a GQDs-AuNPs@PMMA microneedle SERS sensor. After verifying the detection performance of this SERS platform, actual measurements of uric acid and pH were performed. 0.5 g agarose was added to 50 ml of water and solidified at room temperature to simulate human skin. ISF was simulated by preparing pH and uric acid solutions of appropriate concentrations under agarose. Surface-enhanced Raman scattering (SERS) spectroscopy was acquired using a LabRAM HR Evolution spectrometer with an excitation wavelength of 785 nm. Uric acid was used for direct measurement. The uric acid gradient experiment ranged from 40 μmol / L to 640 μmol / L (40, 80, 160, 320, 640). The application range for uric acid showed a low uric acid limit of 202.4 μmol / L, a normal uric acid limit of 297.6 μmol / L, and a high uric acid limit of 357 μmol / L. pH was used for indirect measurement. AuNP-loaded microneedles were placed in a 1×10⁻⁶ spherical atmosphere. -5 Immersing M in a 4-MBA solution for 10 minutes allows 4-MBA to act as a trapping molecule. Gradient measurements were performed using solutions at different pH levels (4.5, 5, 5.5, 6, 6.5). pH was detected by measuring the different pH sensitivities of the carboxyl group (-COOH) in 4-MBA. A pH of 4.5-5.5 indicates severe uric acid crystallization, 5.5-6 indicates mild uric acid crystallization, and pH above 6 is considered normal.

[0044] Figure 9A schematic diagram of direct uric acid detection is shown. Tests were conducted using uric acid solutions with concentration gradients ranging from 40 to 640 μmol / L (40, 80, 160, 320, 640 μmol / L). Figure 9 The spectrum shown in b corresponds to the test results of decreasing concentration from top to bottom. The intensity of the characteristic peaks of uric acid (especially the strong peaks at 1400 cm⁻¹ and 1600 cm⁻¹) gradually weakens as the concentration decreases. Figure 9 c established the relationship between peak intensity and uric acid concentration, and obtained a linear correlation coefficient of 0.995, confirming that there is a strong linear correlation between the two: peak intensity increases with increasing uric acid concentration. Figure 9 d demonstrates a pH detection strategy that achieves indirect pH detection using microneedle-modified 4-mercaptobenzoic acid.

[0045] The carboxyl group of 4-MBA acts as a pH-sensitive probe, undergoing reversible protonation and deprotonation reactions depending on the ambient pH. Changes in the protonation state of the carboxyl group significantly affect the electron cloud distribution of the linked benzene ring, thus causing changes in the intensity of characteristic Raman peaks. By monitoring two representative characteristic peaks at 1050 cm⁻¹ and 1520 cm⁻¹, such as… Figure 9 The spectral changes under different pH conditions are shown in e. Figure 7 The linear relationship between peak intensity and pH value in graph f shows that the peak intensity gradually increases with increasing pH value, with a linear correlation coefficient of 0.991. These results fully demonstrate that this SERS platform has excellent detection capabilities for both uric acid and pH.

[0046] Application Example 1 This application example relates to a graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor, suitable for home or clinical real-time monitoring of chronic metabolic diseases (such as gout). It achieves dual-parameter, non-invasive, and highly sensitive detection of uric acid concentration and pH value by penetrating the stratum corneum of the skin to extract interstitial fluid (ISF).

[0047] A microneedle array with high mechanical strength and good biocompatibility was formed by centrifugal casting using a 20% polymethyl methacrylate (PMMA) solution through a silicon mold (11×11 array, needle height 800 μm, needle spacing 850 μm). Spherical gold nanoparticles (AuNPs) were synthesized in an aqueous phase and uniformly immobilized on the microneedle surface using a liquid surface self-assembly method, forming a high-density SERS "hot spot". Then, a γ-GQDs solution was synthesized using orange peel as a precursor. The AuNP-loaded microneedles were immersed in the γ-GQDs solution for 10 min, dried, and then immersed in a 1×10⁻⁶ solution. -5 The 4-MBA solution of M was used for 10 min to modify the surface of AuNPs with 4-MBA via Au-S bonds, and its carboxyl group (-COOH) was used as a pH-sensitive probe.

[0048] like Figure 10 As shown, when the microneedles in this application example extract ISF from the skin, they can pierce the skin and be arranged neatly, which also meets the requirements of minimally invasive surgery.

[0049] like Figure 11 As shown, SERS microneedle sensors are attached to the surface of human skin. The microneedle array penetrates the stratum corneum to the superficial dermis, extracting interstitial fluid. SERS signals are directly acquired from the microneedle surface. The intensity of uric acid characteristic peaks at 1400 cm⁻¹ and 1600 cm⁻¹ is used as an indicator, and uric acid concentration is calculated based on a pre-defined linear model (R² = 0.995). By monitoring the intensity changes of the characteristic peak of 4-MBA at 1050 cm⁻¹ and 1520 cm⁻¹, and combining this with a pH-intensity linear model (R² = 0.991), the environmental pH value is inferred.

[0050] The data is displayed in real time by an integrated spectrometer, showing uric acid levels (e.g., below 202.4 μmol / L is low uric acid, above 357 μmol / L is high uric acid) and pH status (e.g., pH < 5.5 indicates a high risk of uric acid crystallization).

[0051] The foregoing has shown and described the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor, characterized in that, The sensor uses a polymethyl methacrylate (PMMA) microneedle array as a substrate. Gold nanoparticles (AuNPs) and yellow graphene quantum dots (y-GQDs) are sequentially immobilized on the surface of the PMMA microneedles using a liquid self-assembly method and an immersion method to form a GQDs-AuNPs composite active interface. 4-Mercaptobenzoic acid (4-MBA) is modified on the GQDs-AuNPs composite active interface as a pH-sensitive probe, thus obtaining a gold nanoparticle-PMMA microneedle sensor based on graphene quantum dot modification, namely the GQDs-AuNPs@PMMA microneedle SERS sensor.

2. The method for preparing a graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 1, characterized in that... Includes the following steps: (1) Fabrication of PMMA microneedle array; (2) Synthesis of aqueous gold nanoparticle solution; (3) AuNPs@PMMA microneedles were obtained by immobilizing gold nanoparticles on the surface of PMMA microneedles using the liquid surface self-assembly method; (4) Prepare y-GQDs solution by hydrothermal method, and immerse AuNPs@PMMA microneedles in y-GQDs solution to immobilize GQDs to obtain GQDs-AuNPs@PMMA microneedles; (5) 4-Mercaptobenzoic acid (4-MBA) was modified on the GQDs-AuNPs@PMMA microneedles to obtain GQDs-AuNPs@PMMA microneedles SERS sensor.

3. The method for preparing a graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 2, characterized in that, The method for preparing the PMMA microneedle array in step (1) is as follows: PMMA is dissolved in DMF solution to obtain PMMA solution, the PMMA solution is uniformly coated on silicon mold, and after vacuuming, it is centrifuged and dried to obtain PMMA microneedle array.

4. The method for preparing a graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 3, characterized in that, The PMMA solution concentration is 15-35 wt%, and the silicon mold is an array of 11×11 needles with a length of 800 μm and a needle tip spacing of 850 μm.

5. The method for preparing a graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 2, wherein the method for synthesizing aqueous gold nanoparticles in step (2) is as follows: prepare a HAucl4·3H2O solution and heat it to a slight boil, then add sodium citrate, and use sodium citrate as a reducing agent and stabilizer to reduce Au³⁺ in HAucl4·3H2O to metallic Au to obtain an aqueous gold nanoparticle solution.

6. The method for preparing a graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 2, wherein step (3) involves immobilizing gold nanoparticles on the surface of microneedles using a liquid surface self-assembly method as follows: add n-hexane and ethanol sequentially to an aqueous gold nanoparticle solution and shake gently. After gold precipitates from the solution, immerse the PMMA microneedle array under the gold and then slowly lift it out and place it in an oven to dry. Repeat the above operation until the gold is evenly covered on the surface of the PMMA microneedle array.

7. The preparation method of gold nanoparticle-PMMA microneedle sensor based on graphene quantum dot modification according to claim 2, the steps of preparing y-GQDs by hydrothermal method in step (4) are as follows: orange peel powder and DMF solution are mixed and placed in a high-temperature reaction vessel with polytetrafluoroethylene lining for hydrothermal reaction. After cooling, the solution is filtered to obtain y-GQDs solution; the hydrothermal reaction conditions are 160-200℃ for 8-12 hours; the soaking time is 5-20 min.

8. The method for preparing the graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 2, wherein step (5) involves modifying the GQDs-AuNPs composite active interface with 4-mercaptobenzoic acid (4-MBA) as follows: GQDs-AuNPs@PMMA microneedles are immersed in an immersion solution with a concentration of 1×10⁻⁶. -5 M-5×10 -5 The 4-MBA solution of M is placed in the solution for 5-20 min, and then allowed to stand at room temperature until dry, so that 4-MBA is modified on the surface of AuNPs through Au-S bonds, and its carboxyl group (-COOH) serves as a pH-sensitive probe.

9. The application of the graphene quantum dot-modified gold nanoparticle-PMMA microneedle sensor according to claim 1 for non-invasive detection of uric acid and pH value for non-disease diagnostic purposes, characterized in that: The GQDs-AuNPs@PMMA microneedle SERS sensor was attached to the skin, and interstitial fluid was extracted. Uric acid was directly detected by SERS spectroscopy, and pH was indirectly detected by 4-MBA modified microneedles. Uric acid concentration and pH were calculated based on a preset linear model.

10. The application according to claim 9, characterized in that, The GQDs-AuNPs@PMMA microneedle SERS sensor showed a high linear correlation for the detection of uric acid at concentrations of 40–640 μmol / L and solutions with pH values ​​of 4.5–6.5, with R² values ​​better than 0.995 and 0.991, respectively.

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