A gold micropipette-based SERS biosensing probe, a preparation method and application thereof

Abstract

The application belongs to the technical field of biomedicine, and relates to a gold micrometer pipette-based SERS biosensing probe and a preparation method and application thereof. The preparation method comprises the following steps: preparing a chloroauric acid photochemical reduction solution; injecting the chloroauric acid photochemical reduction solution into a micrometer pipette channel, and in-situ reducing and depositing gold nanoparticles under the conditions of ultraviolet light irradiation and rotation, and then performing vacuum annealing treatment after washing; the parameters of the micrometer pipette include that the inner diameter of the tip is 30-70 mu m, the thickness of the tip wall is 10-40 mu m, the taper angle is 1-7 DEG, and the shape of the tip is circular. The prepared probe has excellent SERS detection sensitivity and signal reproducibility, and meanwhile, the provided probe array can realize three-dimensional in-situ detection in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a gold micropipette-based SERS biosensing probe, its preparation method, and its application. Background Technology

[0002] Surface-enhanced Raman scattering (SERS), a highly sensitive molecular fingerprinting technique, has demonstrated enormous application potential in the fields of chemistry, biology, and medicine since its discovery in 1974. The SERS effect can enhance the Raman scattering signal of molecules by 10-1. 6 -10 14 This advancement enables single-molecule detection, providing a revolutionary tool for the label-free, rapid, and in-situ detection of trace biomolecules. In the biomedical field, SERS technology has been successfully applied to disease biomarker detection, drug metabolism analysis, tumor tissue boundary identification, and rapid pathogen identification. Its high specificity, resistance to photobleaching, and aqueous compatibility give it unique advantages in the analysis of complex biological samples. Metal nanostructures (especially gold and silver) have become the most commonly used active substrate materials for SERS due to their localized surface plasmon resonance (LSPR) effect in the visible-near infrared region.

[0003] Although SERS technology shows great promise due to its single-molecule-level detection sensitivity and fingerprint-characteristic spectral information resolution capabilities, several key bottlenecks still exist in traditional noble metal nanostructure substrates, limiting their practical application. From the perspective of substrate performance, the self-assembly process of nanoparticles is difficult to precisely control the heterogeneous distribution of local surface plasmon resonance effects, leading to significant dispersion in the spatial location and density of active hotspots. This structural heterogeneity directly causes significant fluctuations in the detection signal (relative standard deviation often exceeding 30%), severely weakening the reproducibility of SERS technology. Regarding analyte capture, the enrichment efficiency of existing planar substrates for trace target molecules is limited by the solid-liquid interface interaction mechanism; when the analyte concentration is below 10... -9 When the signal intensity (M) is low, effective molecular adsorption and electromagnetic field coupling are often difficult to achieve, leading to a sharp decline in signal enhancement. For biomedical detection needs, traditional two-dimensional substrate structures struggle to overcome limitations in optical penetration depth and tissue compatibility, making in-situ dynamic monitoring of the three-dimensional microenvironment within living tissue impossible. These multi-dimensional technical shortcomings collectively constitute a substantial obstacle to the translation of SERS technology into clinical diagnostics and life science research. Summary of the Invention

[0004] To address the aforementioned problems in the existing technology, the present invention aims to provide a gold micropipette-based SERS biosensing probe, its preparation method, and its application, achieving highly sensitive and reproducible integrated SERS detection. It also provides a probe array that enables three-dimensional in-situ detection.

[0005] The present invention employs the following technical solutions to achieve its objective: One aspect of this invention provides a method for preparing a gold micropipette-based SERS biosensing probe, comprising the following steps: (1) Prepare chloroauric acid photochemical reducing solution; (2) Chloroauric acid photochemical reduction solution was injected into a micron-sized pipette channel. Under ultraviolet light irradiation and rotation, gold nanoparticles were obtained by in-situ reduction deposition. After rinsing, the nanoparticles were vacuum annealed. (3) Repeat step (2) 0 to 3 times; The parameters of the micron-sized pipette include: tip inner diameter of 30~70μm, tip wall thickness of 10~40μm, cone angle of 1~7°, and tip shape of round.

[0006] The tip refers to the very end of the pipette (i.e., the opening). The tip's inner diameter is the internal diameter of the hollow channel at the very end of the pipette. The tip's wall thickness is the thickness of the quartz glass wall at the very end of the pipette. The cone angle is the angle formed between the two side walls of the channel at the very end of the pipette. The tip shape refers to the geometric profile of the very end of the pipette.

[0007] Preferably, the parameters of the micron-sized pipette are: tip inner diameter of 40~60 μm, tip wall thickness of 10~30 μm, cone angle of 2~3°, and tip shape of circular.

[0008] Further preferred, the parameters of the micron-sized pipette are: tip inner diameter of 50 μm, tip wall thickness of 20 μm, cone angle of 2.29°, and tip shape of circular.

[0009] The geometric parameters of micrometer-sized pipettes have a significant impact on SERS signal intensity. Probes constructed from micrometer-sized pipettes with a tip inner diameter of 50 μm, a tip wall thickness of 20 μm, a cone angle of 2.29°, and a circular tip shape exhibit the best signal intensity and reproducibility at the characteristic peak. Probes prepared from pipettes with a tip inner diameter <1 μm show very weak or even negligible SERS enhancement.

[0010] Preferably, the micron-sized pipette is made of quartz glass. Quartz (silica) can minimize incident laser energy loss and reduce Raman signal attenuation. In addition, the quartz surface is rich in silanol groups (Si-OH), which provides favorable sites for the adsorption of gold precursors during photochemical reduction, and can form stable covalent bonds with gold nanoparticles, greatly improving the stability and crystallinity of the SERS active surface.

[0011] Preferably, the chloroauric acid photochemical reducing solution comprises a chloroauric acid solution and an alcohol solvent. The chloroauric acid solution is a solution formed by dissolving chloroauric acid in water, and the alcohol solvent is one or more of methanol, ethanol, and isopropanol. The concentration of the chloroauric acid solution is 5-20 mmol / L, and the volume ratio of the chloroauric acid solution to the alcohol solvent is 3-5:1.

[0012] Preferably, the micron-sized pipette is treated with aqua regia and then repeatedly cleaned with deionized water and anhydrous ethanol 2 to 5 times.

[0013] Utilizing the UV-induced reduction mechanism, gold ion reduction and deposition are preferentially promoted in the high-curvature region of the microchannel (the needle tip). Simultaneously, the micron-sized pipette rotates uniformly along its long axis, ensuring uniform irradiation across the inner wall, thereby forming a high-density, highly uniform distribution of crystalline gold nanoparticles on the inner wall of the needle tip. Preferably, the UV wavelength is 230–270 nm, and the irradiance is 8–15 μW / cm². 2 The irradiation time is 5-8 hours. Preferably, the rotation speed is 10-100 r / min.

[0014] Preferably, the vacuum annealing treatment is performed at a temperature of 60~90 ℃ for a time of 0.5~3 h.

[0015] Preferably, the particle size of the gold nanoparticles is in the range of 30~70 nm.

[0016] Preferably, the probe treated in step (2) is used as a new micron-sized pipette, and step (2) is repeated once for a second cycle of reduction deposition. That is, the preparation method of the gold micron-sized pipette-based SERS biosensing probe includes the following steps: (1) Prepare chloroauric acid photochemical reducing solution; (2) Chloroauric acid photochemical reduction solution was injected into the micron-sized pipette channel, and gold nanoparticles were obtained by in-situ reduction deposition under ultraviolet light irradiation and rotation. After rinsing, vacuum annealing was performed to obtain gold micron-sized pipette-based SERS biosensing probes obtained by one cycle process. (3) Inject chloroauric acid photochemical reduction solution into the channel of the gold micropipette-based SERS biosensing probe obtained by the first cycle process, and obtain gold nanoparticles by in-situ reduction deposition under ultraviolet light irradiation and rotation. After rinsing, vacuum annealing is performed to obtain the gold micropipette-based SERS biosensing probe obtained by the second cycle process.

[0017] The performance of SERS probes depends on the balance between nanostructure coverage and optical transmittance. A single reduction operation results in insufficient coverage; a triple reduction operation leads to a significant decrease in light transmission efficiency due to excessive gold nanolayer thickness. Two cycles of processing can precisely achieve an ideal electromagnetic "hot spot" density while maintaining high transmittance, thus maximizing the detection signal.

[0018] A second aspect of the present invention provides a gold micropipette-based SERS biosensing probe, which is prepared by the above-described preparation method.

[0019] Preferably, the gold micropipette-based SERS biosensing probe comprises a micrometer-sized pipette and gold nanoparticles bonded to the inner wall of the micrometer-sized pipette; The parameters of the micron-sized pipette are: tip inner diameter of 30~70μm, tip wall thickness of 10~40μm, cone angle of 1~7°, and tip shape of circular. The gold nanoparticles have a particle size range of 30~70 nm.

[0020] Preferably, the gold micropipette-based SERS biosensing probe detects surface-enhanced Raman scattering (SERS) characteristic peak signals, with detection limits ≤1×10⁻⁶ for direct blue and crystal violet, respectively. -8 mol / L.

[0021] A third aspect of the present invention provides a three-dimensional array-type SERS biosensing probe, comprising 3 to 1000 of the gold micropipette-based SERS biosensing probes.

[0022] Preferably, each gold micropipette-based SERS biosensing probe is arranged via a support base.

[0023] Preferably, the spacing between each gold micropipette-based SERS biosensing probe is 0.3~2 mm.

[0024] Preferably, each gold micropipette-based SERS biosensor probe is prepared using the same method, but the tips of each probe are arranged at different depths in three-dimensional space. Because the tips of each probe are positioned differently, when inserted into living tissue, surface-enhanced Raman scattering signals from tissue sites at different depths can be simultaneously acquired through the different depth arrangements of the tips.

[0025] The arrangement of tips at different depths in three-dimensional space can be achieved in the following ways: The support base has mounting sites with a gradient height distribution. By fixing each gold micropipette-based SERS biosensing probe to mounting sites at different heights, different depths of the tips can be achieved. Alternatively, after preparing each gold micropipette-based SERS biosensing probe, the length can be cut to obtain probes of different lengths, thus achieving different depths of the tips.

[0026] A fourth aspect of this invention provides a SERS detection method not intended for disease diagnosis and treatment, comprising the following steps: inserting the gold micropipette-based SERS biosensing probe or the three-dimensional array SERS biosensing probe into the sample to be tested, ex vivo tissue, or living tissue; after drying, focusing a laser at the tip to acquire the surface-enhanced Raman scattering spectral signal at the tip; or, A single cell is drawn into the gold micropipette-based SERS biosensing probe, and the tip position is scanned axially to obtain the surface-enhanced Raman scattering spectral signal along the cell axis.

[0027] The sample to be tested can be a solution of Raman signal molecules, which can be one or more of the following: Direct Blue 86 (DB), 4-nitrobenzenethiophenol (4-NTP), 4-mercaptobenzoic acid (4-MBA), Rhodamine 6G (R6G), Alizarin Red (AR), 4-mercaptopyridine (4-MPY), 4-aminobenzenethiophenol (4-ATP), and Crystal Violet (CV). Preferably, the Raman signal molecules are Direct Blue 86 (DB) and Crystal Violet (CV). The gold micron pipette-based SERS biosensing probe of the present invention has extremely low detection limits for Direct Blue 86 and Crystal Violet, as low as 1×10⁻⁶. -9 mol / L.

[0028] The fifth aspect of this invention provides the application of the gold micropipette-based SERS biosensing probe or the three-dimensional array SERS biosensing probe in the preparation of products for disease biomarker detection, cell analysis or in vivo tissue analysis.

[0029] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention uses a micron-sized pipette as a carrier, internally loaded with gold nanoparticles, to successfully construct a SERS sensing platform integrating sampling, enrichment, and detection. The conical channel of the micron-sized pipette guides the analyte to migrate directionally towards the tip through hydrodynamic effects. During the nanoparticle modification process, the surface tension gradient and local charge distribution promote the preferential deposition of gold nanostructures in high curvature regions, forming a high-density, high-uniformity SERS "hot spot" array. When the laser is focused on the tip, a "triple enrichment" effect of analyte molecule enrichment, nanostructure aggregation, and light field energy focusing is simultaneously achieved, significantly improving detection sensitivity and signal stability.

[0030] 2. This invention uses a micron-sized pipette as a carrier and employs an ultraviolet photochemical reduction method to deposit gold nanoparticles in situ, combined with vacuum annealing to enhance interfacial bonding. This eliminates the need for complex processes such as seed modification and multi-step silanization bonding, significantly simplifying the preparation process. Furthermore, this invention precisely controls the parameters of the micron-sized pipette (preferably: 50 μm inner diameter, 20 μm wall thickness, 2.29° cone angle, circular tip, and quartz material) to achieve multi-dimensional coupling with the gold nanoparticles prepared by the photochemical reduction method, resulting in a substantial improvement in the probe's SERS performance.

[0031] 3. The SERS probe constructed in this invention has a micrometer-scale size, allowing for minimally invasive insertion into living tissues (such as the tumor microenvironment or brain regions). By precisely controlling the insertion depth and spatial coordinates, SERS spectra are collected point-by-point, reconstructing the three-dimensional distribution map of biochemical components (metabolites, proteins, etc.), providing a visual basis for tumor boundary identification and lesion gradient analysis. At the single-cell level, intact cells can be drawn into microchannels, and axial scanning can be performed in the nano-modified region to non-destructively obtain the three-dimensional distribution information of intracellular biochemical components, achieving dynamic analysis at the subcellular organelle level and opening new pathways for research on cell heterogeneity, drug response mechanisms, and other areas.

[0032] 4. This invention perfectly integrates microfluidic precision sampling with SERS high-sensitivity optical detection into a single carrier, significantly simplifying the cumbersome procedures and complex sample pretreatment steps in traditional detection. This integrated design drastically reduces detection time from hours to minutes, greatly improving the efficiency and reliability of trace disease biomarker identification, and providing a highly valuable technical solution for point-of-care testing and long-term dynamic real-time monitoring of live samples.

[0033] 5. This invention further provides a three-dimensional array-type SERS biosensing probe, comprising 3 to 1000 gold micropipette-based SERS biosensing probes, achieving a systematic leap in detection mode from point to surface to three-dimensional space. The tips of each gold micropipette-based SERS biosensing probe are arranged at different depths in three-dimensional space. When inserted into living tissue, the probe tips simultaneously cover multiple preset depth layers from the superficial to the deep part of the sample, realizing parallel in-situ sampling at multiple points in three-dimensional space and synchronous acquisition of SERS signals. Attached Figure Description

[0034] Figure 1 The graph shows the comparison results of SERS performance of different preparation methods in Example 1, where (A) is the photochemical reduction method, (B) is the in-situ seed growth method, (C) is the external synthesis perfusion method, and (D) is a comparison graph of the three methods after two cycles.

[0035] Figure 2 The images show the microscopic characterization of the gold micropipette-based SERS biosensing probe in Example 2. (A) is an optical image of the micropipette before UV irradiation, (B) is an optical image of the micropipette after UV irradiation, (C) is an X-ray diffraction image of the probe, and (D), (E), and (F) are transmission electron microscopy and energy dispersive spectroscopy images of the probe washing solution.

[0036] Figure 3 The image shows the SERS performance of the micron-pipette-based SERS biosensing probe for various Raman signal molecules in Example 3. (A) is the Raman curve of the probe's response to various Raman signal molecules, (B) is the detection limit of the probe for DB, and (C) is the detection limit of the probe for CV.

[0037] Figure 4 The figure shows the SERS detection reliability results of the micron-pipette-based SERS biosensing probe in Example 4, where (A) is the Raman spectrum thermogram of the probe binding to DB; (B) is ten sets of spectral data of the probe binding to DB; (C) and (D) are 747cm -1 and 1533 cm -1 The spectral stability and relative standard deviation (RSD) of a specific peak.

[0038] Figure 5 The images show the Raman spectra of probes obtained by different preparation methods in Example 5, where (A) is the detection limit of DB on a conventional colloidal gold nanoparticle substrate (n=10); and (B) is the detection limit of DB on a micropipette-based SERS biosensing probe (n=10).

[0039] Figure 6 This is a diagram of the micrometer pipette array constructed in Example 6.

[0040] Figure 7 The SERS performance of gold micropipette-based SERS biosensing probes with different tip inner diameters is shown in the figure. (A) is the SERS curve of the probe (n=5); (B) is the Raman intensity of a specific peak at 1533 cm-1 (****p<0.0001).

[0041] Figure 8 The diagram shows the influence of different micron-sized pipette structural parameters on the performance of SERS. (A) Raman spectra of the response DB of SERS biosensing probes based on gold micron-sized pipettes with different wall thicknesses (20, 40, 60, 80, 100 μm) (n=15); (B) Raman spectra of the response DB of SERS biosensing probes based on gold micron-sized pipettes with different tip cone angles (2.29°, 6.87°, 11.42°, 15.94°, 22.62°) (n=15); (C) Raman spectra of the response DB of SERS biosensing probes based on gold micron-sized pipettes with different tip morphologies (circular, triangular, square, pentagonal, hexagonal) (n=15); (D) Raman spectra of the response DB of SERS biosensing probes based on gold micron-sized pipettes with different substrate materials (n=15). Detailed Implementation

[0042] In the description of this invention, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, and includes both a and b. "Multiple" includes two or more types, and can be two, three, four, five, or more.

[0043] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used in the art, and the methods used in the embodiments are all conventional methods in the art.

[0044] Example 1: Screening of gold nanostructure synthesis process and cycle number To evaluate the impact of different gold nanostructure synthesis processes and cycle numbers on the SERS performance of micropipettes, this invention systematically compared three preparation processes: photochemical reduction, in-situ seed growth, and external synthesis-infusion, along with the corresponding synthesis cycle number parameters for each process. In this embodiment, the parameters of the micropipette include: material of quartz glass, tip inner diameter of 50 μm, tip wall thickness of 20 μm, cone angle of 2.29°, and tip shape of circularity. The specific preparation methods for the three processes are as follows: Option 1: Preparation of gold micropipette-based SERS biosensing probes using the in-situ seed growth method. (1) Pretreatment of micron pipettes Prepare a 30% (v / v) ethanol solution of 3-aminopropyltrimethoxysilane (APTMS). Inject the 30% (v / v) APTMS ethanol solution into the cleaned micropipette channels and circulate for 1 hour at room temperature, then place in a vacuum desiccator and let stand at room temperature for 4 hours. After the reaction is complete, rinse the pipette channels with ethanol and dry under vacuum for 2 hours to obtain APTMS-functionalized micropipettes.

[0045] (2) Seed solution preparation Add 99 mL of ultrapure water to a 250 mL Erlenmeyer flask, followed by 1 mL of 1% (w / v) chloroauric acid (HAuCl4) solution. Stir gently to mix thoroughly to obtain the initial precursor solution. Heat the solution to boiling (approximately 100°C). Under rapid stirring, quickly add 2.7 mL of 1% trisodium citrate solution (prepared by dissolving 0.1 g of sodium citrate in 10 mL of ultrapure water) and reflux for 15 minutes while maintaining boiling. Stop heating and continue stirring until the solution cools naturally to room temperature. Then, make up the reaction volume to 100 mL with ultrapure water (as a small amount of water evaporates during boiling) to maintain a consistent concentration. After cooling, filter the solution using a 0.22 μm microporous membrane. Store the resulting colloidal gold seed solution in a clean brown glass bottle at 4°C in the dark for later use.

[0046] (3) Seed adsorption process Colloidal gold seed solution was infused into APTMS-modified pipette channels using a microinjection pump at a flow rate of 2 μL / min. The channels were circulated at 25°C for 2 hours to allow specific adsorption of gold seed particles onto the inner wall surface. After adsorption was complete, the channels were flushed with 0.1 mM PBS buffer (pH 7.4) at a flow rate of 5 μL / min for 15 minutes.

[0047] (4) Preparation of growth solution Preparation of growth solution: Chloroauric acid (HAuCl4), hexadecyltrimethylammonium bromide (CTAB), and ascorbic acid (AA) are mixed, with HAuCl4 concentration of 0.5 mM, CTAB concentration of 80 mM, and ascorbic acid concentration of 1.5 mM. Let stand for 10 minutes.

[0048] (5) In-situ growth process The newly prepared growth solution was continuously infused into the micropipette channel with adsorbed seeds at a flow rate of 3 μL / min using a micropipette pump, and the reaction was carried out at room temperature and in the dark for 6 hours. After the reaction, the channel was rinsed with ultrapure water at a flow rate of 5 μL / min for 1 hour, and finally gold micropipette-based SERS biosensing probe was obtained.

[0049] Repeat steps (3)-(5) 0 times, 1 time, and 2 times respectively to obtain gold micropipette-based SERS biosensing probes prepared by single-cycle, double-cycle, and triple-cycle processes respectively.

[0050] Option 2: Preparation of gold micropipette-based SERS biosensing probes using the photochemical reduction method. (1) Preparation of chloroauric acid solution Take 1.28 mL of 50 mM HAuCl4 stock solution (prepared with ultrapure water), add 5.12 mL of ultrapure water, mix well to obtain a total volume of 6.4 mL and a concentration of 10 mM HAuCl4 solution, and set aside.

[0051] (2) Preparation of photochemical reducing solution Measure 1.6 mL of anhydrous ethanol and mix it with 6.4 mL of the above 10 mM HAuCl4 solution in a clean glass container. Shake well to obtain a HAuCl4 ethanol solution with a volume ratio of 4:1 (v / v), which will be used for subsequent filling.

[0052] (3) Filling of micron-sized pipettes and photoreduction The pre-prepared photochemical reducing solution was carefully injected into the channels of a micropipette treated with aqua regia using a micropipette pump. Air bubbles were gently removed, ensuring the channels were completely filled without any gaps. After filling, the micropipette was fixed to a rotating stand and placed in a light-protected reaction chamber. An Analytikjena UVG-11 ultraviolet lamp (254 nm, 4 W) was used as the light source, and the sample was continuously irradiated at room temperature (25±1°C) (ensuring the ultraviolet irradiance on the sample surface was controlled at 10 μW / cm²). 2 Simultaneously, a rotator was activated to rotate the micropipette along its long axis at a constant speed of 50 r / min, ensuring uniform UV irradiation across all areas of the inner wall and promoting the uniform reduction and nucleation of gold ions on the glass surface. The entire photoreduction process lasted 6 hours, during which the reaction system was kept in a light-proof, sealed state to prevent interference from external light and solution evaporation. After the reaction, the sample was removed from the reaction apparatus and the inner channel was slowly rinsed with ultrapure water at a flow rate of 5 μL / min for 30 minutes to remove unbound gold ions and reaction byproducts, yielding a gold micropipette substrate with SERS activity.

[0053] (4) Processing and stability enhancement The pipette was then placed in a vacuum oven and annealed at 80°C for 1 hour to improve the crystallinity and interfacial bonding strength of the gold nanoparticles, thereby enhancing their long-term stability. A gold micropipette-based SERS biosensing probe was thus prepared using a single-cycle process.

[0054] The pre-prepared photochemical reducing solution was carefully injected into the gold micropipette-based SERS biosensing probe prepared by a single-cycle process using a micropipette pump. Air bubbles were gently removed to ensure that the channel was completely filled and without gaps. The subsequent steps were the same as steps (3)-(4) to obtain the gold micropipette-based SERS biosensing probe prepared by two-cycle process.

[0055] The pre-prepared photochemical reducing solution was carefully injected into the gold micropipette-based SERS biosensing probe prepared by two cycles using a micropipette pump. Air bubbles were gently removed to ensure that the channel was completely filled and without gaps. The subsequent steps were the same as steps (3)-(4) to obtain the gold micropipette-based SERS biosensing probe prepared by three cycles.

[0056] Option 3: Preparation of gold micropipette-based SERS biosensing probes using the external synthesis perfusion method. (1) Synthesis of gold nanoparticles 150 mL of ultrapure water was added to a 250 mL round-bottom flask, which was then fitted with a condenser and placed on a magnetically stirred heater. The solution was heated to a vigorous boil under continuous stirring. Then, 183 μL of a 10 mg / mL HAuCl4 solution was rapidly added, resulting in a pale yellow solution. Immediately afterward, 6.5 mL of a 68 mM trisodium citrate solution was rapidly added while stirring. Upon addition of the reducing agent, the solution color changed sequentially: from pale yellow to grayish-purple within 1-3 minutes, to purplish-red after approximately 5 minutes, and finally stabilized as a wine-red color within 6-10 minutes, indicating successful formation of gold nanoparticles. After color stabilization, the reaction mixture was refluxed for 10 minutes to ensure complete reduction and sufficient surface functionalization. Heating was then stopped, and the system was allowed to cool slowly while stirring for 15 minutes, followed by standing at room temperature for 1 hour. The resulting colloidal gold solution was transferred to a brown glass bottle and stored at 4°C protected from light for later use.

[0057] (2) Irrigation of micropipettes The synthesized gold nanoparticle solution (approximately 0.05 nM) was injected into the plasma-cleaned micropipette channels at a constant flow rate of 10 μL / min using a microinjection pump. The injection process was carried out at room temperature (25 ± 1°C) and in the dark until the pipette channels were completely filled and free of air bubbles. Subsequently, the injected micropipettes were carefully transferred to a vacuum drying oven and dried under vacuum at 60°C for 8 hours to promote effective adsorption and immobilization of the gold nanoparticles on the glass inner wall. After drying, the sample was equilibrated in a desiccator at room temperature for 12 hours, and then the inner wall was slowly rinsed with ultrapure water at a flow rate of 2 μL / min for 1 hour to remove any loosely bound gold nanoparticles, ultimately obtaining a gold micropipette-based SERS biosensing probe.

[0058] Repeat step (2) 0 times, 1 time, and 2 times respectively to obtain gold micropipette-based SERS biosensing probes prepared by single-cycle, double-cycle, and triple-cycle processes respectively.

[0059] All samples were taken at 1*10 -5 mol / L Direct Blue 86 (DB) was used as the signal molecule for SERS performance characterization (a gold micron-based SERS biosensor probe was immersed in DB solution, and the solution was aspirated into the pipette via capillary action; it was then removed, placed directly onto the Raman stage without drying, and Raman detection was performed with the laser focused on the tip region). All SERS performance tests in this paper were conducted under strictly uniform experimental conditions (633 nm laser, 1 mW power, 2 s integration time). Figure 1 As shown in Figure D, the gold micropipette substrates prepared by the three methods all exhibit characteristic Raman peaks, but the signal intensities differ significantly. This performance difference can be attributed to the different mechanisms by which the three methods construct gold nanostructures within the complex geometry of the microchannel. The photochemical reduction method utilizes a UV-induced surface-selective reduction mechanism to form a uniform, high-density distribution of gold nanoparticles on the inner wall of the micropipette, particularly in the high-curvature tip region. This process benefits from the enhanced local electric field effect in the tip region, preferentially reducing and depositing gold ions in this area, forming dense SERS "hot spots," thus producing the best signal intensity. In contrast, the in-situ seeding method involves multiple steps, among which the APTES silanization modification step is difficult to achieve uniform and stable covalent bonding within the microchannel, leading to uncontrollable subsequent seed adsorption quantity and distribution. The external synthesis and infusion method faces the challenge of uneven distribution in microfluidic dynamics; the synthesized gold nanoparticles exhibit a uniform distribution within the microchannel, failing to fully utilize the geometric conical structure of the micropipette to achieve a tip enrichment effect.

[0060] This invention also investigated the effect of the number of reduction cycles on SERS performance. For example... Figure 1As shown in AC, the sample treated in a single cycle had a lower signal intensity, indicating insufficient coverage of gold nanoparticles. While the sample treated in three cycles had high coverage, the Raman signal actually decreased due to the significantly reduced light transmission efficiency caused by the excessively thick gold nanolayer. The sample treated in two cycles, however, achieved optimal SERS performance while maintaining high transmittance. These results confirm that a balance must be struck between nanostructure coverage and optical transmittance when constructing SERS active surfaces within micrometer-scale channels. The two-step cycle process of photochemical reduction precisely achieves this optimization, providing an ideal strategy for the fabrication of highly sensitive and reproducible gold micropipette-based SERS probes.

[0061] Example 2: Verification of the effectiveness of photochemical reduction method in constructing gold nanostructures on the inner wall of a micrometer pipette The gold micropipette-based SERS biosensing probe prepared by two cycles of photochemical reduction in Example 1 was characterized at the microscale. Figure 2 As shown in AB, during ultraviolet light irradiation, the color of the solution in the micron-sized pipette channel gradually changed from initially colorless to a distinct orange-red. This macroscopic change initially indicates that gold ions were successfully reduced and deposited on the inner wall to form a nanostructure.

[0062] To characterize the properties of the gold nanostructure crystals attached to the inner wall of the pipette, the modified micropipette was ground into powder for X-ray diffraction (XRD) analysis. Figure 2 As shown in Figure C, the XRD pattern shows a distinct diffraction peak at 38.2°, corresponding to the (111) crystal plane of face-centered cubic gold. Although only the strongest (111) crystal plane diffraction peak was detected due to the limited sample size, its position perfectly matches that of the standard gold crystal, confirming the crystalline gold characteristics of the inner wall deposits. Notably, no other impurity phase peaks were found in the XRD analysis of the ground sample, indicating that the photochemical reduction process has good selectivity and did not introduce obvious impurity phases.

[0063] To further analyze the properties of the generated nanoparticles, the eluent after rinsing the gold micropipette-based channels with deionized water was collected and characterized by transmission electron microscopy (TEM). Figure 2 As shown in Figure D, the TEM image clearly shows the presence of spherical gold nanoparticles with a particle size distribution ranging from 45 to 55 nm. Energy-dispersive X-ray spectroscopy (EDS) elemental analysis confirmed the presence of gold in the particles. Figure 4 (EF), confirming that the eluent was mainly gold nanoparticles.

[0064] These characterization results collectively confirm that photochemical reduction can successfully construct well-crystallized gold nanostructures on the inner wall of a micrometer-sized pipette. TEM analysis showed the presence of gold nanoparticles of approximately 50 nm in the rinsing solution, and XRD and EDS results clearly confirmed the successful loading of high-purity crystalline gold nanostructures onto the inner wall, providing a reliable material basis for subsequent SERS performance studies.

[0065] Example 3: Study on the SERS response characteristics of a gold micropipette-based SERS biosensing probe prepared by photochemical reduction to various Raman signal molecules. The gold micropipette-based SERS biosensing probes prepared by two cycles of photochemical reduction in Example 1 were subjected to 1*10... -5 SERS performance was characterized using mol / L concentrations of DB, 4-nitrophenylthiophenol (4-NTP), 4-mercaptobenzoic acid (4-MBA), Rhodamine 6G (R6G), Alizarin Red (AR), 4-mercaptopyridine (4-MPY), 4-aminophenylthiophenol (4-ATP), and crystal violet (CV) as signal molecules. (The gold micron-based SERS biosensor probe was immersed in the signal molecule solution, and the solution was aspirated into the pipette via capillary action. After removal, it was placed directly onto the Raman stage for Raman detection without drying, with the laser focused on the tip region.) The detection method remained consistent. Figure 3 As shown in Figure A, the sensor exhibits a significant enhancement effect on a variety of typical Raman probe molecules (DB, 4-NTP, 4-MBA, R6G, AR, 4-MPY, 4-ATP, CV), among which DB and CV molecules show the best SERS response characteristics, significantly higher than other tested molecules.

[0066] To quantitatively evaluate the sensor's detection capability, the limit of detection (LOD) for DB and CV molecules was determined using a serial dilution method. Figure 3 As shown in BC, the intensity of the characteristic Raman peak decreases linearly with decreasing analyte concentration. This gold micropipette substrate achieves a detection limit of 1*10⁻⁶ for both DB and CV. -9 M. It is noteworthy that this high sensitivity is mainly attributed to two factors: (1) the high-density, uniformly distributed gold nanoparticle structure constructed by photochemical reduction in the tip region of the micrometer pipette forms abundant electromagnetic "hot spots"; (2) the fluid focusing effect generated by the conical geometry of the microchannel effectively enriches analyte molecules in the detection area, achieving a synergistic effect of optical enhancement and spatial enrichment. This result confirms that by precisely controlling the number of cycles and the gold nanostructure preparation process, ideal plasma nanostructures can be constructed in micrometer-scale channels, providing a reliable technical platform for ultra-high sensitivity in-situ detection of biomolecules.

[0067] Example 4: Reliability Study of SERS Detection of Gold Micropipette-Based SERS Biosensing Probe Prepared by Photochemical Reduction Method To systematically evaluate the SERS detection reliability of the gold micropipette-based SERS biosensing probe prepared by two cycles of photochemical reduction in Example 1, DB was used as the signal molecule (concentration of 1*10). -5 M), repeatability tests are conducted under strictly controlled experimental conditions. For example... Figure 4 As shown in Figures AB, ten different detection points were randomly selected at the tip of a gold micropipette, and Raman spectra were acquired using a 633 nm laser. The results show that all spectra exhibit the characteristic vibrational modes of the DB molecule, particularly at 747 cm⁻¹. -1 (CN stretching vibration) and 1533 cm -1 A strong and stable characteristic peak is observed at the (benzene ring skeleton vibration) location.

[0068] Quantitative analysis showed that ( Figure 4 CD), 747 cm -1 and 1533 cm -1 The relative standard deviations (RSDs) of the two characteristic peaks were 9.679% and 10.56% (n=10), respectively, both below the threshold of 10.6%. This result is significantly better than the RSD of conventional SERS substrates reported in the literature (RSD is usually >15%), indicating that the gold micropipette substrate prepared in this invention has excellent signal reproducibility.

[0069] Example 5: Comparison of SERS detection sensitivity between gold micropipette-based SERS biosensing probes prepared by photochemical reduction and conventional gold nanoparticles. To comprehensively evaluate the detection advantages of the optimized gold micropipette substrate, a control experiment was designed. The performance of the gold micropipette-based SERS biosensing probe prepared in Example 1 using a two-cycle photochemical reduction method was compared with that of a conventional colloidal gold nanoparticle substrate. The control group used 50 nm gold nanoparticles synthesized under the same conditions (prepared using the citrate reduction method, specifically: 5 mL of 5 mmol / L HAuCl4•4H2O solution and 5 mL of 1% sodium citrate solution were added to 40 mL of pure water, stirred and heated to boiling. After 10 minutes of reaction, the solution turned purple, and the resulting gold nanoparticles were cooled to room temperature). These were mixed with DB solutions of varying concentrations and then drop-coated onto a silicon wafer to form a conventional SERS substrate.

[0070] like Figure 5 As shown in Figures AB, under the same testing conditions, the detection limits for DB molecules differ significantly between the two substrates. The gold micropipette substrate clearly detects 10... -9 Characteristic Raman signal at M concentration (747 cm⁻¹)-1 1533 cm -1 (where the signal is masked by noise at the same concentration), while the signal of a traditional SERS substrate is almost completely masked by noise, and its reliable detection limit is only 10. -7 M.

[0071] This performance difference can be attributed to the following mechanisms: First, the tapered microchannel structure of the gold micropipette base generates a significant hydrodynamic focusing effect, enriching the analyte in the tip detection region and increasing the local concentration by about two orders of magnitude; second, the gold nanostructure constructed on the inner wall through photochemical reduction forms a stable covalent bond with the glass substrate, effectively avoiding the "coffee ring effect" and particle aggregation caused by solvent evaporation during testing on traditional colloidal gold substrates; third, the confined space of the microchannel prolongs the residence time of target molecules in the SERS active region, enhancing the probability of molecule-hotspot interactions.

[0072] These results clearly demonstrate that the gold micropipette substrate not only improves detection sensitivity by two orders of magnitude compared to traditional colloidal gold substrates, but also offers significant advantages in signal reproducibility and spatial uniformity. This performance enhancement provides a reliable technological platform for the in-situ, real-time detection of trace molecules in complex biological samples, overcoming key limitations of traditional SERS substrates in practical applications.

[0073] Example 6: Optimization of Micrometer-scale Pipette Structural Parameters (1) A 4×9 cylindrical micropipette array (36 units in total) was constructed on a single substrate by 3D printing, such as Figure 6 As shown, this array design orthogonally combines four key geometric parameters: tip inner diameter (20, 30, 50, 70, 100 μm), tip shape (circular, triangular, square, pentagonal, hexagonal), tube wall thickness (20, 40, 60, 80, 100 μm), and cone angle (2.29°, 6.87°, 11.42°, 15.94°, 22.62°). Gold micropipette-based SERS biosensing probes were prepared using the photochemical reduction method described in Example 1 through two cycles. Then, under strictly uniform experimental conditions, at 10... -5 mol / L DB was used as the standard Raman signal molecule to evaluate the SERS performance of the entire probe array. Figure 7 , 8 As shown, probes with different geometries exhibit significantly different signal response characteristics.

[0074] like Figure 7As shown, the SERS signal intensity exhibits a significant nonlinear dependence on the tip inner diameter. A micropipette with a 50 μm tip inner diameter demonstrates optimal performance. Notably, when the tip inner diameter is less than 50 μm (20-30 μm), the signal intensity decreases with decreasing size, primarily due to increased fluid resistance and reduced molecular transport efficiency caused by the narrower microchannel. Conversely, when the tip inner diameter is greater than 50 μm (70-100 μm), the signal intensity also significantly decreases, as the increased radius of curvature of the conical structure weakens the fluid focusing effect and local electromagnetic field enhancement. These results indicate that the tip inner diameter of the micropipette is a key parameter for controlling SERS performance. A 50 μm inner diameter achieves synergistic optimization in terms of molecular enrichment efficiency, electromagnetic field enhancement intensity, and spatial uniformity, making it an ideal geometric configuration for achieving high-sensitivity in-situ detection.

[0075] Figure 8 A shows the Raman spectral comparison results for different tube wall thicknesses (20, 40, 60, 80, 100 μm) (this group of probes has the same parameters except for the wall thickness of the micrometer pipettes, which include: a tip inner diameter of 50 μm, a tip cone angle of 2.29°, a circular tip morphology, and a substrate material of photocurable resin). Experimental data show that the sample with a 20 μm wall thickness has a Raman spectral density of 1536 cm⁻¹. -1 The signal intensity at the characteristic peak is significantly higher than that of other thickness groups, indicating that a thinner tube wall structure is beneficial for obtaining a stronger SERS response. Figure 8 B presented performance differences for different cone angles (2.29°, 6.87°, 11.42°, 15.94°, 22.62°) (this group of probes had the same parameters except for the wall thickness of the micropipette, which included a tip inner diameter of 50 μm, a wall thickness of 20 μm, a circular tip shape, and a substrate material of photocurable resin). The sample with a small cone angle of 2.29° had the highest characteristic peak intensity, the sharpest peak shape, and the smallest half-peak width, indicating that it had the best signal resolution and signal-to-noise ratio. Figure 8 C compared the Raman spectral characteristics of different tip shapes (circular, equilateral triangle, square, regular pentagon, and regular hexagon) (this group of probes had the same parameters except for the wall thickness of the micropipette, which included: a tip inner diameter of 50 μm (for equilateral triangles, the height; for squares, the diagonal; for regular pentagons, the height from a vertex to the corresponding side; and for regular hexagons, the diagonal); a wall thickness of 20 μm; a tip cone angle of 2.29°; and a substrate material of photocurable resin). The results showed that the circular tip not only performed best in terms of signal intensity but also had the most stable spectral baseline and the lowest background noise. Comprehensive analysis indicated that the optimal performance combination was a wall thickness of 20 μm, a circular tip shape, and a cone angle of 2.29°. This configuration was found at 1536 cm⁻¹.-1 The signal strength reaches its peak at the characteristic peak, and the signal reproducibility is optimal.

[0076] (2) Further investigation into the influence of the material of the micropipette on SERS performance: Micropipettes were constructed using 3D printing with the following parameters: tip inner diameter of 50 μm, wall thickness of 20 μm, tip cone angle of 2.29°, tip morphology of circular shape, and substrate material of photocurable resin; quartz capillaries were drawn to form micropipettes with the following parameters: tip inner diameter of 50 μm, wall thickness of 20 μm, tip cone angle of 2.29°, tip morphology of circular shape, and substrate material of quartz. Gold micropipette-based SERS biosensing probes were prepared by two cycles of photochemical reduction as described in Example 1, and then, under strictly uniform experimental conditions, at 10 -5 mol / L DB was used as the standard Raman signal molecule to evaluate the SERS performance of probes made of two different materials.

[0077] like Figure 8 As shown in Figure D, under the same synthesis conditions and geometric parameters, quartz microneedles exhibit superior SERS performance compared to microneedles made of 3D-printed photocurable resin. This difference mainly stems from the fundamental differences between the two materials in terms of optical properties and surface chemical properties. From an optical perspective, quartz contains abundant silica, which, as an inorganic amorphous material, possesses extremely high optical transparency and extremely low light absorption coefficient in the visible to near-infrared band. In contrast, photocurable resin, as an organic polymer material, typically contains residual photoinitiators, incompletely reacted double bonds, and other organic additives in its molecular structure. These components produce a certain degree of intrinsic absorption in the visible light region, resulting in a pale yellow hue. This difference in optical absorption directly affects the transmission efficiency of laser light within the microneedle: silica can minimize the energy loss of the incident laser, ensuring that more photons reach the metallized region at the tip, thereby more effectively exciting surface plasmon resonance; simultaneously, the Raman scattering signal experiences less attenuation during its return to the detector, ultimately achieving a higher signal-to-noise ratio and detection sensitivity.

[0078] From the perspective of surface chemistry, silica surfaces are rich in silanol groups (Si-OH). These active hydroxyl groups not only form stable chemical bonds with subsequently modified gold nanoparticles but also provide favorable reaction sites for the adsorption and reduction of gold precursors during photochemical reduction. In the photochemical reduction preparation of gold nanostructures, silica exhibits excellent transmittance to ultraviolet light, preventing interference with the activation process of photoinitiators and ensuring uniform nucleation and growth of gold nanoparticles on the tip surface. Conversely, photocurable resins have relatively inert surface chemistry, lacking active groups that strongly interact with metal nanoparticles. Furthermore, their organic components may competitively absorb ultraviolet light, interfering with the kinetics of the photochemical reduction reaction, resulting in lower deposition density, size distribution, and crystallinity of gold nanoparticles compared to silica substrates. In addition, photocurable resins may undergo photodegradation or thermal degradation under long-term laser irradiation, further affecting the stability of the SERS active surface.

[0079] All aspects, embodiments, and features of this invention should be considered illustrative in all respects and not limiting of the invention; the scope of the invention is defined only by the claims. Other embodiments, modifications, and uses will become apparent to those skilled in the art without departing from the spirit and scope of the invention as claimed.

[0080] In the preparation method of this invention, the order of the steps is not limited to the listed order. For those skilled in the art, variations in the order of the steps without creative effort are also within the scope of protection of this invention. Furthermore, two or more steps or actions can be performed simultaneously.

[0081] Finally, it should be noted that the specific embodiments described herein are merely illustrative examples of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A method for preparing a gold micropipette-based SERS biosensing probe, characterized in that, Includes the following steps: (1) Prepare chloroauric acid photochemical reducing solution; (2) Chloroauric acid photochemical reduction solution was injected into a micron-sized pipette channel. Under ultraviolet light irradiation and rotation, gold nanoparticles were obtained by in-situ reduction deposition. After rinsing, the nanoparticles were vacuum annealed. (3) Repeat step (2) 0 to 3 times; The parameters of the micron-sized pipette include: tip inner diameter of 30~70μm, tip wall thickness of 10~40μm, cone angle of 1~7°, and tip shape of round.

2. The preparation method according to claim 1, characterized in that, The parameters of the micron-sized pipette are: tip inner diameter of 50 μm, tip wall thickness of 20 μm, cone angle of 2.29°, and tip shape of circular. And / or, the micropipette is made of quartz glass.

3. The preparation method according to claim 1, characterized in that, The particle size range of gold nanoparticles is 30~70nm; And / or, repeat step (2) once.

4. The preparation method according to claim 1, characterized in that, The chloroauric acid photochemical reducing solution consists of a chloroauric acid solution and an alcohol solvent. The concentration of the chloroauric acid solution is 5~20 mmol / L, and the volume ratio of the chloroauric acid solution to the alcohol solvent is 3~5:

1. And / or, ultraviolet light with a wavelength of 230–270 nm and an irradiance of 8–15 μW / cm². 2 The irradiation time is 5-8 hours; And / or, the rotational speed is 10~100 r / min; And / or, the vacuum annealing treatment is performed at a temperature of 60~90 ℃ for a time of 0.5~3 h.

5. A gold micropipette-based SERS biosensing probe, characterized in that, It is prepared by the preparation method as described in any one of claims 1-4.

6. A gold micropipette-based SERS biosensing probe, characterized in that, The gold micropipette-based SERS biosensing probe includes a micropipette and gold nanoparticles bonded to the inner wall of the micropipette. The parameters of the micron-sized pipette are: tip inner diameter of 30~70μm, tip wall thickness of 10~40μm, cone angle of 1~7°, and tip shape of circular. The gold nanoparticles have a particle size range of 30~70 nm.

7. A three-dimensional array-type SERS biosensing probe, characterized in that, It includes 3 to 1000 gold micropipette-based SERS biosensing probes as described in claim 5 or 6.

8. The three-dimensional array-type SERS biosensing probe according to claim 7, characterized in that, Each gold micropipette-based SERS biosensing probe is arranged via a support base; And / or, the spacing between each gold micropipette-based SERS biosensing probe is 0.3~2 mm; And / or, each gold micropipette-based SERS biosensing probe is prepared using the same method, but the tips of each gold micropipette-based SERS biosensing probe are arranged at different depths in three-dimensional space.

9. A SERS detection method not intended for disease diagnosis and treatment, characterized in that, The procedure includes the following steps: inserting the gold micropipette-based SERS biosensing probe of claim 5 or 6 or the three-dimensional array SERS biosensing probe of claim 7 into the sample to be tested or ex vivo tissue, drying it, focusing a laser on the tip of the probe, and collecting the surface-enhanced Raman scattering spectral signal at the tip. or, A single cell is drawn into the gold micropipette-based SERS biosensing probe, and the tip position is scanned axially to obtain the surface-enhanced Raman scattering spectral signal along the cell axis.

10. The application of the gold micropipette-based SERS biosensing probe as described in claim 5 or 6, or the three-dimensional array SERS biosensing probe as described in claim 7, in the preparation of products for disease biomarker detection, cell analysis, or in vivo tissue analysis.

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