A built-in micro-pipe microneedle for Raman spectrum detection and a preparation method and application thereof

Abstract

The application provides a built-in micro-pipe micro-needle for Raman spectrum detection and a preparation method and application thereof, and belongs to the technical field of Raman spectrum detection. First, the capillary force of the micro-pipe is used to extract the sample to be detected in a sub-surface biochemical system, the liquid to be detected is transmitted to the bottom of a sensing micro-pipe, and then the metal nanoparticles assembled at the bottom of the sensing micro-pipe are used to enhance the Raman scattering signal of the detected molecule, so that the signal is collected by a Raman spectrometer. The SERS enhancement substrate is modified on the inner surface of the micro-pipe micro-needle instead of the outer surface, so as to avoid the flow of the enhancement substrate into the biochemical system to be detected, cause biological safety and pollution, and avoid the flow of nanoparticles into the biochemical system to be detected from the root, and meanwhile, in-situ extraction and Raman measurement are realized. The aperture of the through hole above the sensing micro-pipe is much larger than the laser spot, so that the Raman laser can be almost losslessly transmitted to the detected object, and the influence of the micro-needle material on the laser attenuation is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of Raman spectroscopy detection, and particularly relates to a micro-needle with a built-in micro-pipe for Raman spectroscopy detection, a preparation method and application thereof. BACKGROUND

[0002] In the current frontier of medical research, the detection and monitoring of biochemical markers in interstitial fluid (ISF) has become a highly regarded and crucial research field. This exploration not only provides us with a deep insight into the dynamic physiological processes within the human body, but also has a profound impact on the scientific community. ISF, due to its close association with the cellular microenvironment, has become a repository of biochemical markers, capable of reflecting real-time changes in key physiological parameters such as metabolic activity and neurotransmitter levels.

[0003] However, traditional sampling techniques have limitations in capturing the dynamic changes of ISF biochemical markers, such as chromatography, fluorescence, electrophoresis, and electrochemical methods. These methods often involve complex extraction steps, which not only cause pain to patients but also are very cumbersome for doctors. Moreover, such methods are subject to time delays, insufficient sensitivity, and other issues. In this challenging context, the introduction of surface-enhanced Raman scattering (SERS) combined with micro-needle technology is particularly important. SERS not only provides high sensitivity and resolution of molecular spectral information, but also, in combination with micro-needle technology, creates the possibility of real-time and precise biochemical molecule detection.

[0004] In recent years, in the detection of various indicators in interstitial fluid (ISF), the use of micro-needle patches to extract ISF from the subcutaneous tissue for biochemical marker detection has been widely applied. Currently, many research teams are trying to use micro-needles and SERS technology to solve the pain points of traditional detection methods. However, the research of these teams cannot offset the influence of micro-needle materials on Raman signals, and cannot eliminate the health risks or pollution caused by the SERS enhancement substrate on the outer surface of the micro-needle to the measured biochemical system.

[0005] Wang et al. from Guangdong University of Technology designed a 3D printed ladder-shaped microneedle for sensing and detecting pesticide residues on the surface and mesophyll cells of tea leaves (Yi X, Yuan Z, Yu X, et al. Novel microneedle patch-based surface-enhanced Raman spectroscopy sensor for the detection of pesticide residues [J]. ACS Applied Materials & Interfaces, 2023, 15(4): 4873-4882); Chen et al. from the Chinese Academy of Sciences used a core-satellite structure of gold nanoparticles to modify the microneedle, successfully detected the cyanin in the mouse dermal ISF, and in subsequent work designed two different microneedles for detecting the process of drug diffusion in mice (Shi S, Wang Y, Mei R, et al. Revealing drug release and diffusion behavior in skin interstitial fluid by surface-enhanced Raman scattering microneedles [J]. Journal of Materials Chemistry B, 2023, 11(14): 3097-3105). Liu et al. from Nanyang Technological University used polymethyl methacrylate (PMMA) to design a microneedle for traction light path, which can detect glucose in mice after the microneedle is inserted into the skin for 3 min (Ju J, Hsieh CM, Tian Y, et al. Surface enhanced Raman spectroscopy based biosensor with a microneedle array for minimally invasive in vivo glucose measurements [J]. ACS Sensors, 2020, 5(6): 1777-1785).

[0006] However, Wang et al. can detect subcutaneously, but cannot detect in situ, and need to detect after removing the microneedle. Liu et al. from Nanyang Technological University can detect in situ, but cannot offset the influence of microneedle material on Raman signal, and these methods will directly modify nanoparticles on the outer surface of the microneedle, although they will be removed after being inserted into the measured biochemical system, but cannot avoid the residual nanoparticles in the measured biochemical system, thereby endangering biological health or causing pollution. SUMMARY

[0007] Therefore, the present application aims to provide a micro-needle with built-in micro-pipe for Raman spectrum detection, and a preparation method and application thereof.

[0008] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0009] The present application provides a micro-needle with built-in micro-pipe for Raman spectrum detection, comprising a micro-needle substrate and a micro-needle body arranged on the lower surface of the micro-needle substrate; a sensing micro-pipe and an extraction micro-pipe connecting the micro-needle body and the sensing micro-pipe are arranged in the micro-needle substrate.

[0010] The sensing micro-pipe is in the shape of a funnel, the top of which penetrates the upper surface of the micro-needle substrate, and the top diameter is 750-1500 μm; the bottom of the sensing micro-pipe is closed, and a surface-enhanced Raman substrate nanoparticle is assembled on the bottom surface.

[0011] The extraction micro-pipe is in the shape of a ladder, the inlet end of the extraction micro-pipe penetrates the micro-needle body, and the outlet end is located on the side wall of the sensing micro-pipe.

[0012] Preferably, the material of the surface-enhanced Raman substrate nanoparticle is gold, silver or copper, and the particle size is 50-200 nm.

[0013] The number of layers of the surface-enhanced Raman substrate nanoparticle assembled on the bottom of the sensing micro-pipe is 1-3 layers.

[0014] Preferably, the material of the micro-needle substrate and the micro-needle body is photosensitive resin.

[0015] The thickness of the micro-needle substrate is 800-2000 μm.

[0016] Preferably, the inner diameter of the extraction micro-pipe is 60-160 μm.

[0017] The micro-needle body is in the shape of an inverted cone, the height is 600-2000 μm, and the maximum diameter is 120-400 μm.

[0018] The present application provides a preparation method of the above-mentioned micro-needle with built-in micro-pipe for Raman spectrum detection, comprising the following steps:

[0019] A 3D model of the micro-needle with built-in micro-pipe is established in a 3D modeling software, and the 3D model is 3D printed to obtain a micro-needle with built-in micro-pipe precursor;

[0020] The surface-enhanced Raman substrate nanoparticle sol is added to the bottom of the sensing micro-pipe, and after solidification, the built-in micro-pipe microneedle for Raman spectrum detection is obtained.

[0021] Preferably, the material used for 3D printing is photosensitive resin, the laser wavelength of 3D printing is 405nm, the thickness of each layer is 5-20um, and the solidification time is 3-120s.

[0022] The application provides application of the built-in micro-pipe microneedle in surface-enhanced Raman spectrum detection of sub-surface biochemical indexes.

[0023] Preferably, the biochemical indexes include one or more of levodopa, uric acid and glucose.

[0024] The application provides a surface-enhanced Raman spectrum detection method of sub-surface biochemical indexes, which comprises the following steps:

[0025] The built-in micro-pipe microneedle is pricked into the sub-surface of a biochemical system to be detected, so that the sample to be detected enters the bottom of the sensing micro-pipe;

[0026] The single-wavelength laser of a Raman spectrum detection instrument is focused on the bottom of the sensing micro-pipe, and Raman spectrum detection is performed; and the sub-surface biochemical indexes are obtained according to the obtained Raman spectrum.

[0027] Preferably, the Raman spectrum detection laser wavelength is 500-800nm, the laser power is 30-100mW, the integration time is 1-120s, and the integration frequency interval is 1-20 times.

[0028] The application provides a micro-needle with built-in micro-pipe for Raman spectrum detection, comprising a micro-needle base and a micro-needle body arranged on the lower surface of the micro-needle base; a sensing micro-pipe and an extraction micro-pipe connecting the micro-needle body and the sensing micro-pipe are arranged in the micro-needle base; the sensing micro-pipe is in a "funnel" shape, the top of the sensing micro-pipe penetrates the upper surface of the micro-needle base, and the top diameter is 750-1500 microns; the bottom of the sensing micro-pipe is closed, and a surface-enhanced Raman substrate nanoparticle is assembled on the bottom surface; the extraction micro-pipe is in a "ladder" shape, the inlet end of the extraction micro-pipe penetrates the micro-needle body, and the outlet end is located on the side wall of the sensing micro-pipe. The micro-needle with built-in micro-pipe and the assembled gold nanoparticles are combined with the micro-needle and surface-enhanced Raman spectroscopy (SERS) to realize the extraction and SERS detection of the sub-surface (subcutaneous) sample in the same structure, and realize the in-situ optical sensing of the subcutaneous biochemical index. Thanks to the design of the micro-pipe micro-needle, the SERS enhancement substrate such as gold nanoparticles can be prevented from entering the human body to cause potential health risks, and the safety of the method is greatly improved. Specifically, the micro-pipe micro-needle is used to extract the sub-surface biochemical system, and the extracted sample is transmitted to the bottom of the sensing micro-pipe through the connecting pipe between the extraction micro-pipe and the sensing micro-pipe, and then the Raman scattering signal of the sample is enhanced by the metal nanoparticles assembled on the bottom of the sensing micro-pipe, so as to be collected by the Raman spectrometer.

[0029] The SERS enhancement substrate is modified on the inner surface of the micro-pipe micro-needle instead of the outer surface, so as to avoid the flow of the enhancement substrate into the biochemical sample system and cause biological safety and pollution. The extraction micro-pipe and the sensing micro-pipe are used to separate the extraction of the sample and the Raman spectrum detection, so as to avoid the flow of the nanoparticles into the detected biochemical system from the root, and realize the in-situ extraction and Raman measurement. A through hole with an aperture much larger than the laser spot is designed above the built-in sensing micro-pipe, so that the Raman laser can be transmitted to the sample almost without loss, and the influence of the micro-needle material on the laser attenuation is solved. Therefore, the micro-needle with built-in micro-pipe provided by the application can realize in-situ, non-interfering, high-sensitivity and safe detection of sub-surface biochemical indexes.

[0030] The application provides a preparation method of the micro-needle with built-in micro-pipe for Raman spectrum detection. The 3D printing technology is used to realize the preparation of the micro-needle with built-in micro-pipe, which is convenient to manufacture, simple to operate, high in practicability, and easy to realize industrialized batch production.

[0031] The application provides a surface-enhanced Raman spectrum detection method of a sub-surface biochemical index, and the application extracts a biochemical index or a to-be-measured solution in a biochemical to-be-measured system to the bottom of a sensing micro-pipe in a micro-needle by using an extraction micro-pipe in the micro-needle; a single-wavelength laser is focused on the bottom of the sensing micro-pipe, surface plasmons of a SERS enhanced substrate are excited, and the Raman signal of a to-be-measured biochemical molecule is enhanced. The method provided by the application has realized the ultra-sensitive detection of a plurality of biochemical indexes such as levodopa, uric acid and glucose, and the detection limit reaches 1 muM. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is an array profile schematic diagram of the micro-needle with the built-in micro-pipe.

[0033] Figure 2 It is a structure schematic diagram of a single micro-needle with the built-in micro-pipe.

[0034] Figure 3 It is an electron scanning microscope graph of the micro-needle with the built-in micro-pipe in a top view.

[0035] Figure 4 It is an electron scanning microscope graph of the micro-needle with the built-in micro-pipe in a profile view.

[0036] Figure 5 It is an electron scanning microscope graph of the 55nm gold nanoparticles obtained in Example 1.

[0037] Figure 6 It is an electron scanning microscope graph of the 120nm gold nanoparticles obtained in Example 1.

[0038] Figure 7 It is a Raman spectrum graph of the 1 muM, 5 muM, 10 muM, 50 muM and 100 muM levodopa solution extracted by the micro-pipe micro-needle.

[0039] Figure 8 It is a linear calibration curve drawn by selecting the 780cm-1 peak value in the Raman spectrum graph of the 5 muM, 10 muM, 50 muM and 100 muM levodopa solution. -1

[0040] Figure 9 It is a Raman spectrum graph of the 200 muM levodopa solution extracted by the micro-pipe micro-needle assembled with 120nm, 55nm and non-assembled gold nanoparticles.

[0041] Figure 10 It is a measurement result of the 10mM uric acid solution extracted by the micro-pipe micro-needle.

[0042] Figure 11 It is a measurement result of the 100mM glucose solution extracted by the micro-pipe micro-needle. DETAILED DESCRIPTION ​

[0043] The application provides a micro-needle with built-in micro-pipe for Raman spectrum detection, comprising a micro-needle base and a micro-needle body arranged on the lower surface of the micro-needle base; a sensing micro-pipe and an extraction micro-pipe connecting the micro-needle body and the sensing micro-pipe are arranged in the micro-needle base.

[0044] The sensing micro-pipe is in a "funnel" shape, the top of which penetrates the upper surface of the micro-needle base, and the top diameter is 750-1500 μm; the bottom of the sensing micro-pipe is closed, and a surface-enhanced Raman substrate nanoparticle is assembled on the bottom surface.

[0045] The extraction micro-pipe is in a "ladder" shape, the inlet end of the extraction micro-pipe penetrates the micro-needle body, and the outlet end is located on the side wall of the sensing micro-pipe.

[0046] The micro-needle with built-in micro-pipe for Raman spectrum detection provided by the application comprises a micro-needle base. In the application, the material of the micro-needle base is preferably photosensitive resin, and the specific material preferably comprises one or more of polymethyl methacrylate, polypropylene oxide, epoxy acrylate and polyurethane. In the application, the thickness of the micro-needle base is preferably 800-2000 μm, and more preferably 1000-1500 μm.

[0047] The micro-needle with built-in micro-pipe for Raman spectrum detection provided by the application comprises a micro-needle body arranged on the lower surface of the micro-needle base. In the application, the material of the micro-needle body is preferably photosensitive resin, and the specific material is preferably the same as that of the micro-needle base. In the application, the micro-needle body is in an "inverted cone" shape, the height thereof is preferably 600-2000 μm, and more preferably 1000-1500 μm, and the maximum diameter thereof is preferably 120-400 μm, and more preferably 200-300 μm.

[0048] In the application, a plurality of micro-needle bodies are preferably arranged on the lower surface of the same micro-needle base in an array, and the specific number is designed according to the actual use. In the application, the distance between adjacent micro-needle bodies is preferably 1000-1500 μm, and more preferably 1300 μm.

[0049] The micro-needle with built-in micro-pipe for Raman spectrum detection provided by the application comprises a sensing micro-pipe arranged in the micro-needle base. In the application, the sensing micro-pipe is in a "funnel" shape, the top of which is in a hollow "inverted circular table" shape, and the lower part is in a cylindrical shape, and the diameter thereof is preferably 450-600 μm, and more preferably 500 μm. In the application, the top of the sensing micro-pipe penetrates the upper surface of the micro-needle base, and the top diameter is 750-1500 μm, and preferably 900-1200 μm.

[0050] In the present application, the bottom of the sensing micro-pipe is closed, and the bottom surface is assembled with surface-enhanced Raman substrate nanoparticles. In the present application, the surface-enhanced Raman substrate nanoparticles are metal nanomaterials with surface plasmon resonance effect, preferably gold, silver or copper, and preferably one or more of nanospheres, nanocubes and nanopolyhedrons. In the present application, the particle size of the surface-enhanced Raman substrate nanoparticles is preferably 50-200 nm, and more preferably 100-150 nm. The present application does not have special requirements for the source of the surface-enhanced Raman substrate nanoparticles, and commercially available metal nanomaterials with surface plasmon resonance effect or self-prepared ones can be used. When self-prepared, taking gold nanoparticles as an example, the preparation method is preferably sodium citrate reduction method, which specifically includes the following steps:

[0051] Mix chloroauric acid with boiling water, add sodium citrate, and perform reduction reaction to obtain gold nanoparticles.

[0052] In the present application, the concentration of the chloroauric acid in boiling water is preferably 0.01wt%, and the concentration of the sodium citrate is preferably 1wt%. In the present application, the particle size of the gold nanoparticles is preferably 30-60 nm.

[0053] When the particle size of the gold nanoparticles needs to be provided, the present application preferably uses the above gold nanoparticles as seeds, mixes the gold nanoparticles with a chloroauric acid solution in a stepping motor, and performs reduction reaction.

[0054] In the present application, the concentration of the chloroauric acid solution is preferably 1wt%, and the addition time of the chloroauric acid solution is preferably 3h. The present application controls the particle size of the nanoparticles by controlling the amount of chloroauric acid.

[0055] In the present application, the number of layers of the surface-enhanced Raman substrate nanoparticles assembled on the bottom of the sensing micro-pipe is preferably 1-3 layers, and more preferably 2 layers.

[0056] The in-built micro-pipe microneedle for Raman spectrum detection provided by the present application comprises an extraction micro-pipe connecting the microneedle body and the sensing micro-pipe. In the present application, the extraction micro-pipe is in a "ladder" shape, the inlet end of the extraction micro-pipe penetrates the microneedle body, and the outlet end is located on the side wall of the sensing micro-pipe. In the present application, the outlet height of the extraction micro-pipe is higher than the highest point of the surface-enhanced Raman substrate nanoparticles. In the present application, the inner diameter of the extraction micro-pipe is preferably 60-160μm, and more preferably 80-120μm.

[0057] In the present application, the array profile schematic diagram of the in-built micro-pipe microneedle for Raman spectrum detection is as shown in Figure 1 , and the structure schematic diagram of a single in-built micro-pipe microneedle is as shown in Figure 2 .

[0058] The application provides a preparation method of the micro-needle with the built-in micro-pipe for Raman spectrum detection.

[0059] A 3D model of the micro-needle with the built-in micro-pipe is established in a 3D modeling software, and the 3D model is subjected to 3D printing to obtain a micro-needle with the built-in micro-pipe precursor.

[0060] The surface-enhanced Raman substrate nanoparticle sol is added to the bottom of the sensing micro-pipe, and after solidification, the micro-needle with the built-in micro-pipe for Raman spectrum detection is obtained.

[0061] The application establishes a 3D model of the micro-needle with the built-in micro-pipe in a 3D modeling software, and the 3D model is subjected to 3D printing to obtain a micro-needle with the built-in micro-pipe precursor. The 3D modeling software used in the application is a 3D modeling software well known in the art, and the method for establishing the 3D model is a method well known in the art, which will not be described here.

[0062] In the application, the material for 3D printing is a photosensitive resin raw material, specifically including one or more of polymethyl methacrylate, polypropylene oxide, epoxy acrylate and polyurethane. In the application, the laser wavelength of 3D printing is preferably 405 nm, the thickness of each layer is preferably 5-20 μm, more preferably 10-15 μm; the solidification time is preferably 3-120 s, more preferably 10-90 s, and further preferably 30-60 s.

[0063] In the application, the surface-enhanced Raman substrate nanoparticle sol is added to the bottom of the sensing micro-pipe, and after solidification, the micro-needle with the built-in micro-pipe for Raman spectrum detection is obtained. In the application, the surface-enhanced Raman substrate nanoparticle sol is added to the top of the sensing micro-pipe. In the application, the solid content of nano-gold in the surface-enhanced Raman substrate nanoparticle sol stock solution is preferably 6.94 mg / mL. In the application, the solidification is preferably carried out under a baking lamp, the temperature of the solidification is preferably 50-75℃, more preferably 60℃, and the time is preferably 5-20 min, more preferably 10 min.

[0064] The application provides an application of the micro-needle with the built-in micro-pipe in surface-enhanced Raman spectrum detection of sub-surface biochemical indicators.

[0065] In the application, the sub-surface is preferably one or more of a human or animal skin surface, a polymer film surface and an oxide film surface. In the application, the biochemical indicators preferably include one or more of levodopa, uric acid and glucose.

[0066] The application provides a surface-enhanced Raman spectrum detection method of sub-surface biochemical indicators, which comprises the following steps:

[0067] The built-in micro-pipeline microneedle is used to prick the sub-surface of the biochemical system to be tested, so that the sample to be tested enters the bottom of the sensing micro-pipeline;

[0068] A single-wavelength laser of a Raman spectrum detection instrument is focused on the bottom of the sensing micro-pipeline, and Raman spectrum detection is performed, and the sub-surface biochemical index condition is obtained according to the obtained Raman spectrum.

[0069] In the present application, the Raman spectrum detection laser wavelength is preferably 500-800 nm, more preferably 600-700 nm; the laser power is preferably 30-100 mW, more preferably 50-80 mW; the integration time is preferably 1-120 s, more preferably 5-100 s, and further preferably 20-80 s; and the integration time interval is preferably 1-20 times, more preferably 5-10 times. The objective lens for Raman spectrum detection is selected from one of 50x, 20x and 10x.

[0070] In the present application, the biochemical index preferably includes one or more of levodopa, uric acid and glucose, and the detection limit is 1 μM.

[0071] The built-in micro-pipeline microneedle for Raman spectrum detection, the preparation method and the application thereof provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0072] Example 1

[0073] The thickness of the microneedle substrate of the built-in micro-pipeline microneedle for Raman spectrum detection is 1000 μm, and the material is polymethyl methacrylate. The size is as follows:

[0074] The length of the needle tip of the microneedle body is 800 μm, the bottom radius is 120 μm, the diameter of the extraction micro-pipeline is 120 μm, the diameter of the top of the sensing micro-pipeline is 1000 μm, and the lateral spacing between adjacent needle tips is 1300 μm.

[0075] The preparation method of the built-in micro-pipeline microneedle for Raman spectrum detection adopts the following steps:

[0076] (1) A designed microneedle and built-in micro-pipeline model is established in a 3D modeling software; the 3D model is imported into a 3D printer, a photosensitive resin material polymethyl methacrylate is used to print the designed microneedle structure, the 3D printing solidification laser wavelength is 405 nm, each layer is 5 μm thick, and the solidification time is set to 3 s, to obtain a built-in micro-pipeline microneedle precursor.

[0077] The electron scanning microscope picture of the obtained built-in micro-pipeline microneedle is shown in Figure 3 、 4 , wherein Figure 3 is a top view of the built-in micro-pipeline microneedle, Figure 31 is the needle body part, and 2 is the extraction microchannel lower opening. By Figure 3 The micro-needle tip opening can be clearly seen.

[0078] Figure 4 is a micro-needle profile diagram with an embedded microchannel, Figure 4 1 and 2 are extraction microchannels, 3 is a sensing microchannel, 5 is a sensing microchannel upper opening, which facilitates the flow of nanoparticle solution into the sensing microchannel and assembly in 4, and 6 is Figure 3 The arrow 2 in points to the direction. By Figure 4 The micro-needle internal microchannel structure can be seen.

[0079] (2) First, 55 nm gold nanoparticle seeds were synthesized using a sodium citrate reduction method. A chloroauric acid solution (HAuCl4, 200 mL, 0.01 wt%) was reduced in boiling (100°C) ultrapure water by sodium citrate (C6H5Na3O7, 1.4 mL, 1 wt%). Then, in the 55 nm gold nanoparticle solution, 55 nm gold nanoparticles were used as seeds to obtain 120 nm gold nanoparticles through secondary reduction. Under ice bath conditions, 3 mL of the above-mentioned 55 nm gold nanoparticles were taken as seeds, 20 mL of ultrapure water was added for dilution, and after the addition of a chloroauric acid solution (HAuCl4, 654 μL, 1 wt%), it was reduced by ascorbic acid (C6H8O6, 400 μL, 1 wt%) under the protection of sodium citrate (C6H5Na3O7, 100 μL, 1 wt%). After waiting for 30 min, it was heated in a 70°C water bath for 2 h, and finally 120 nm gold nanoparticles were obtained. Subsequently, a centrifuge was used at a speed of 5000 rpm for 5 min, and 100 mL of nanoparticle sol was concentrated to 3 mL to obtain a concentrated colloid of 120 nm gold nanoparticles.

[0080] The electron scanning microscope image of the obtained 55 nm gold nanoparticles is shown in Figure 5 The electron scanning microscope image of the obtained 120 nm gold nanoparticles is shown in Figure 6

[0081] After the embedded microchannel micro-needle precursor was dried, 1.5 μL of 120 nm gold nanoparticles were added to its surface. In order to ensure sufficient assembly, the micro-needle was placed under a 60°C baking lamp for 10 min of drying. By repeating this step 10 times, an embedded microchannel micro-needle modified with 120 nm gold nanoparticles was obtained.

[0082] ​(3) Dissolve 140 mg of agar powder in 10 mL of deionized water, then heat and stir at a constant temperature of 120°C for 40 min. Put the obtained transparent agar solution into a mold and solidify at room temperature. Then, add a 200 mM levodopa solution into the mold and let it diffuse in the agar gel at room temperature for 2 h, and finally cover a sealing film to replicate the skin properties of the organism to obtain a simulated skin gel.

[0083] After the micro-needle with the built-in micro-pipe prepared in step (2) is pricked into the simulated skin gel, it is allowed to stand for 3 min. Subsequently, the micro-needle is placed under a Raman spectrometer for measurement, with a laser power of 60 mW, an integration time of 5 s, and an integration number of 2. After removing the dark current, high-quality Raman spectrum data are obtained.

[0084] (4) Then, repeat the process of step (3) to reduce the concentration of the levodopa solution to 100 μM, 50 μM, 10 μM, 5 μM, and 1 μM, respectively.

[0085] Among them, since 1 μM is difficult to measure, the measurement conditions of 1 μM are optimized to an integration time of 12 s and an integration number of 10 times in the present application, and the figure is enlarged by 5 times for easy analysis.

[0086] The Raman spectrum graphs of the 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM levodopa solutions extracted by the micro-needle with the built-in micro-pipe are shown in FIG. 2. Figure 7 Figure 7 The middle curve i is the spectrum of 1 μM levodopa after being enlarged by 5 times, and ii-vi are 5 μM, 10 μM, 50 μM, and 100 μM, respectively. Figure 7 As can be seen from FIG. 2, as the concentration of the levodopa solution increases, the peak intensity of the Raman spectrum also increases, and the peak intensity and the concentration are in a linear relationship, so the content of levodopa can be quantitatively measured. The linear calibration curve of the peak intensity and the concentration is shown in FIG. 3. Figure 8 As can be seen from FIG. 3, R 2 = 0.996, and from Figure 8 it can be seen that the method of the present application can realize quantitative analysis of biochemical indicators.

[0087] Example 2

[0088] (1) Prepare the micro-needle precursor with the built-in micro-pipe according to the method of step (1) of Example 1, and the size and material are the same as those of Example 1.

[0089] ​(2) Synthesis of 55 nm gold nanoparticle seeds using sodium citrate reduction method. 200 mL of 55 nm gold nanoparticle solution was prepared by reducing HAuCl4 (200 mL, 0.01 wt%) with sodium citrate (C6H5Na307, 1.4 mL, 1 wt%) in boiling (100 °C) ultrapure water, followed by concentration of 200 mL of gold nanoparticle solution to 3 mL in a centrifuge.

[0090] After the microneedle was dried, 1.5 μL of the above-mentioned concentrated 55 nm gold nanoparticle solution was added on the upper surface (i.e. above the sensing microchannel). To ensure sufficient assembly, the microneedle was placed under a baking lamp at 60 °C for 10 min of drying. By repeating this step 10 times, a microchannel microneedle modified with 55 nm gold nanoparticles was obtained.

[0091] (3) 140 mg of agar powder was dissolved in 10 mL of deionized water, then heated and stirred at a constant temperature of 120 °C for 40 min. The transparent agar solution obtained was placed in a mold and solidified at room temperature. Then, 200 mM of levodopa solution was added to the mold and allowed to diffuse in the agar gel at room temperature for 2 h, and finally a sealing film was covered to replicate the skin properties of the organism, obtaining a simulated skin gel.

[0092] After the built-in microchannel microneedle prepared in step (2) was inserted into the simulated skin gel, it was allowed to stand for 3 min. Subsequently, the microneedle was placed under the Raman spectrometer for measurement, with a laser power of 60 mW, an integration time of 5 s, and an integration number of 10 times.

[0093] As a comparison, a blank microchannel microneedle without gold nanoparticles was used. After the blank microchannel microneedle was inserted into the simulated skin gel, it was allowed to stand for 3 min. Subsequently, the microneedle was placed under the Raman spectrometer for measurement, with a laser power of 60 mW, an integration time of 5 s, and an integration number of 10 times.

[0094] The Raman spectra of 200 μM levodopa solution extracted by microchannel microneedles assembled with 120 nm nanoparticles (dotted line), 55 nm nanoparticles (dashed line), and without nanoparticles (solid line) are shown in Figure 9 As can be seen from Figure 9 For all the characteristic peaks in the figure, the Raman signal measured with 120 nm nanoparticles is stronger than that with 55 nm nanoparticles, which proves that the corresponding plasmonic enhancement effect is also stronger, with an intensity of about 100 times.

[0095] Example 3

[0096] Built-in microchannel microneedles modified with 120 nm gold nanoparticles were prepared according to the method in Example 1.

[0097] 140 mg of agar powder was dissolved in 10 mL of deionized water, and then heated and stirred at a constant temperature of 120 °C for 40 min. The resulting transparent agar solution was placed in a mold and allowed to solidify at room temperature. Then, 10 mM uric acid solution was added to the mold and allowed to diffuse in the agar gel at room temperature for 2 h. Finally, a sealing film was applied to replicate the skin properties of a living organism, resulting in a simulated skin gel.

[0098] After inserting the prepared microneedles with built-in microchannels into the corresponding skin gel, the mixture was allowed to stand for 3 minutes. Subsequently, the microneedles were placed under a Raman spectrometer for measurement. The laser power was selected as 60mW, the integration time was 5s, and the integration was performed twice. The measurement results of extracting 10mM uric acid solution using the microneedles are as follows: Figure 10 As shown. By Figure 10 It can be seen that the present invention successfully detected the characteristic peak of uric acid in a 10 mM uric acid solution, of which the peak at 810 cm⁻¹ is significant. -1 and 998cm -1 The Raman displacement is attributed to ring vibration, 1154 cm. -1 The Raman shift is attributed to the vibration between carbon and nitrogen.

[0099] Example 4

[0100] Built-in microchannel microneedles modified with 120 nm gold nanoparticles were prepared according to the method in Example 1.

[0101] 140 mg of agar powder was dissolved in 10 mL of deionized water, and then heated and stirred at a constant temperature of 120 °C for 40 min. The resulting transparent agar solution was placed in a mold and allowed to solidify at room temperature. Then, 100 mM glucose solution was added to the mold and allowed to diffuse in the agar gel at room temperature for 2 h. Finally, a sealing film was applied to replicate the skin characteristics of a living organism, resulting in a simulated skin gel. The prepared microneedles with built-in microchannels were inserted into the corresponding skin gels and allowed to stand for 3 min. Subsequently, the microneedles were placed under a Raman spectrometer for measurement. The laser power was selected as 60 mW, the integration time was 5 s, and the integration was performed twice. The measurement results of extracting 100 mM glucose solution using the microneedles are shown below. Figure 11 As shown. By Figure 11 It can be seen that the present invention successfully detected the characteristic peak of glucose in a 100mM glucose solution, of which the peak at 926 cm⁻¹ is significant. -1 The Raman shift is attributed to carbon-oxygen bond vibration, 1126 cm. -1 The Raman shift is attributed to vibrations between carbon-carbon bonds.

[0102] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A built-in microchannel microneedle for Raman spectroscopy detection, comprising a microneedle substrate and microneedle bodies disposed on the lower surface of the microneedle substrate; multiple microneedle bodies are disposed on the lower surface of the same microneedle substrate and arranged in an array; The microneedle substrate is provided with a sensing microchannel and an extraction microchannel connecting the microneedle body and the sensing microchannel. The sensing microchannel is funnel-shaped, with its top extending through the upper surface of the microneedle substrate, and its top diameter is 750~1500μm; the bottom of the sensing microchannel is closed, and surface-enhanced Raman substrate nanoparticles are assembled on the bottom surface; the number of assembly layers of the surface-enhanced Raman substrate nanoparticles at the bottom of the sensing microchannel is 1~3. The extraction microchannel is stepped, with the inlet end of the extraction microchannel penetrating the microneedle body and the outlet end located on the side wall of the sensing microchannel; the inner diameter of the extraction microchannel is 60~160 μm; the microneedle body is inverted conical, with a height of 600~2000 μm and a maximum diameter of 120~400 μm.

2. The built-in microchannel microneedle for Raman spectroscopy detection according to claim 1, characterized in that, The surface-enhanced Raman substrate nanoparticles are made of gold, silver, or copper, and have a particle size of 50~200 nm.

3. The built-in microchannel microneedle for Raman spectroscopy detection according to claim 1, characterized in that, The microneedle substrate and the microneedle body are made of photosensitive resin; The thickness of the microneedle substrate is 800~2000 μm.

4. The method for preparing the built-in microchannel microneedle for Raman spectroscopy detection according to any one of claims 1 to 3, comprising the following steps: A 3D model of the built-in microchannel microneedle is created in 3D modeling software, and the 3D model is 3D printed to obtain the built-in microchannel microneedle precursor. A surface-enhanced Raman substrate nanoparticle sol was added to the bottom of a sensing microchannel, and after curing, a built-in microchannel microneedle for Raman spectroscopy detection was obtained.

5. The preparation method according to claim 4, characterized in that, The material used in the 3D printing is photosensitive resin, the laser wavelength of the 3D printing is 405 nm, the thickness of each layer is 5~20 μm, and the curing time is 3~120 s.

6. The application of the microneedle with built-in microchannels as described in any one of claims 1 to 3 or the microneedle with built-in microchannels prepared by the preparation method described in any one of claims 4 to 5 in the detection of subsurface biochemical indicators by surface-enhanced Raman spectroscopy.

7. The application according to claim 6, characterized in that, The biochemical indicators include one or more of levodopa, uric acid, and glucose.

8. A surface-enhanced Raman spectroscopy method for detecting subsurface biochemical indicators, comprising the following steps: The microneedle with built-in microchannel as described in any one of claims 1 to 3 or the microneedle with built-in microchannel prepared by the preparation method described in any one of claims 4 to 5 is inserted into the subsurface of the biochemical system to be tested, so that the sample to be tested enters the bottom of the sensing microchannel. The single-wavelength laser of the Raman spectroscopy instrument is focused on the bottom of the sensing microchannel to perform Raman spectroscopy detection, and the subsurface biochemical indicators are obtained based on the obtained Raman spectra.

9. The detection method according to claim 8, characterized in that, The Raman spectroscopy detection laser wavelength is 500~800 nm, the laser power is 30~100 mW, the integration time is 1~120 s, and the number of integrations is 1~20.

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