Asymmetric wetting flexible SERS (Surface Enhanced Raman Scattering) detection substrate as well as preparation method and application thereof

Abstract

The invention discloses an asymmetric wetting flexible SERS (Surface Enhanced Raman Scattering) detection substrate, a preparation method and an in-situ detection method for an acupuncture substance catecholamine. The preparation method comprises the following steps: generating a flexible hole carrier of a through hole with a large end and a small end on a flexible film by using laser; preparing noble metal nano sol with the surface modified with a hydrophilic stabilizer; the preparation method comprises the following steps: dispersing precious metal nano sol into water, then adding dispersion liquid, and after shaking, spontaneously assembling nano particles on a gas-liquid interface to form a single-layer nano film; and transferring the single-layer nano film to the front surface where the open end of the through hole is larger on the flexible hole carrier to obtain the SERS detection substrate. The hydrophilic back surface is larger than the hydrophilic front surface, after the acupuncture action, the tissue fluid is adsorbed on the back surface of the SERS detection substrate, moisture is evaporated through pore channels when the tissue fluid is transmitted to the front surface, catecholamine components in the tissue fluid are left on the front surface of the SERS detection substrate, and the influence of macromolecular proteins and other interfering substances on catecholamine detection is avoided.

Description

Technical Field

[0001] The present invention relates to the field of in-situ detection of acupuncture effect substances, and in particular to an asymmetric wettable and flexible SERS detection substrate and a preparation method thereof, and an in-situ detection method for acupuncture substance catecholamines. Background Art

[0002] Surface-enhanced Raman scattering (SERS) refers to the phenomenon in which the Raman signal of molecules adsorbed on rough nanoscale metal or non-metallic compound substrates is enhanced. Compared with other detection technologies, SERS not only enables the detection of biomolecules but also offers multiple significant advantages, including narrow characteristic spectral signals, strong resistance to photodegradation, and low stray signals in complex environments. As a non-contact, non-destructive, and highly sensitive spectral analysis technique, SERS has a wide range of applications in disciplines such as materials science, chemistry, physics, geology, and life sciences. Compared to traditional rigid substrates, flexible SERS substrates enable in situ and real-time detection of analytes on non-planar surfaces. PDMS and PET films have been widely used in flexible SERS sensors due to their chemical stability, non-toxicity, mechanical flexibility, bendability, and hydrophobicity.

[0003] Catecholamines (such as epinephrine, norepinephrine, and dopamine) are primarily secreted by the adrenal medulla and sympathetic nerve endings. Acupuncture stimulation of specific acupoints influences catecholamine secretion through neuroreflex and endocrine regulation. Catecholamine monitoring can reveal the body's stress response and regulatory capacity after acupuncture, as well as its effects on the neuroendocrine system.

[0004] However, because the levels of catecholamines secreted by the human body during acupuncture are extremely low and susceptible to interference from oxidation and degradation, their sensitivity and specificity are insufficient. Existing fluorescence analysis and enzyme-linked immunosorbent assays (ELISAs) for detecting these substances have low sensitivity and are susceptible to interference from other substances in the sample, leading to biased results. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) requires complex, time-consuming, and labor-intensive sample pretreatment steps, requiring specialized equipment and personnel, limiting its widespread clinical adoption. Summary of the Invention

[0005] Based on the technical and methodological problems existing in the technical background, the present invention proposes an asymmetric wettable and flexible SERS detection substrate and a preparation method thereof, as well as an in-situ detection method for catecholamines, a needle-piercing substance.

[0006] The technical solutions of the present invention are as follows: The first object of the present invention is to provide a method for preparing an asymmetric wettable and flexible SERS detection substrate, which comprises the following steps: S1. Using laser to generate a through hole on the flexible film, wherein one end of the through hole is large and the other end is small, thereby obtaining a flexible porous carrier; S2, preparing a noble metal nanosol whose surface is modified with a hydrophilic stabilizer; S3, dispersing the noble metal nanosol into water to form a metal nanoparticle mixture, then adding the dispersion, and shaking the nanoparticles to spontaneously assemble at the air-liquid interface to form a single-layer nanofilm; S4. Transfer the single-layer nanofilm to the front surface of the flexible porous carrier prepared in step S1 where the through-hole opening is located, and cover the surface of the flexible porous carrier and the through-hole with the nanofilm to obtain a SERS detection substrate.

[0007] Laser cutting and drilling is an existing technology. It involves irradiating a material surface with a high-energy laser beam, causing it to rapidly melt, vaporize, or even reach its ignition point, thereby achieving cutting. By precisely controlling laser parameters (such as power density, pulse width, scanning speed, and focal depth), a series of microporous structures are created that extend completely through the flexible membrane from top to bottom. For example, relatively large pores are formed on the front side of the flexible membrane (i.e., the side directly exposed to the laser beam). These pores may exhibit a regular geometric pattern or a random distribution, depending on the laser beam scanning pattern and energy distribution. In contrast, smaller micropores are formed on the back side of the flexible membrane (i.e., the side not directly exposed to the laser beam but affected by heat conduction). The through-holes form an asymmetric pore structure, with one end larger than the other. This asymmetric design optimizes the surface wettability of the flexible membrane, for example, the front side is more hydrophobic than the back side, resulting in the final SERS detection substrate possessing asymmetric wettability and flexibility. In a further embodiment, the hydrophilic stabilizer is at least one of sodium citrate, ascorbic acid, and sodium borohydride.

[0008] In a further embodiment, the noble metal nanosol is a colloid formed by dispersing nano-spherical particles of gold, silver or gold-silver alloy with isotropic structure in a solvent.

[0009] In a further embodiment, the dispersion is formed by adding a thiol compound to an organic solvent, wherein the thiol compound is dodecyl mercaptan or 1H,1H,2H,2H-perfluorodecyl mercaptan; and the organic solvent is composed of ethanol and n-hexane in a volume ratio of 2:1. The volume ratio of the nanoparticle mixture to the thiol compound is 1:1-2.

[0010] In a further embodiment, the flexible film is polydimethylsiloxane (PDMS) or polyethylene terephthalate (PET).

[0011] In a further embodiment, the opening end of the through hole is in an irregular shape, such as a five-pointed star, a circle, or a square structure, and the through hole has an asymmetric wettability structure.

[0012] The second object of the present invention is to provide a SERS detection substrate prepared by the above preparation method, which has asymmetric wettability.

[0013] The third object of the present invention is to provide an application of the above-mentioned SERS detection substrate for in-situ detection of catecholamines, a substance that responds to acupuncture, in body fluids subjected to acupuncture.

[0014] In a further embodiment, the detection step includes: attaching the back side of the SERS detection substrate to the area to be punctured, and performing SERS detection on the catecholamines, which are the puncture effect substances.

[0015] In a further embodiment, the excitation light wavelength of the SERS detection is 785 nm.

[0016] The SERS detection substrate used for SERS detection of catecholamines, a substance that responds to acupuncture, is based on asymmetric through-pores in a flexible membrane. These through-pores exhibit unique transmission properties, influencing the microscopic fluid dynamics at the interface, enabling directional liquid transport and analyte enrichment. The detection principle is as follows: Serum or tissue fluid containing large amounts of protein can affect the detection of catecholamines. A method for synchronizing rapid pretreatment and SERS detection of catecholamines is as follows: First, utilizing the principle of interfacial assembly, a nanomembrane is transferred to the surface and pore walls of a flexible porous support. The nanomembrane and the flexible porous support assemble into an asymmetric, wettable, and flexible SERS detection substrate. Due to the asymmetric wettability structure of the front and back sides of the through-holes, the liquid infiltration and transmission efficiency differs. When tissue fluid is discharged to the skin surface after acupuncture and contacts the small opening on the back side of the through-hole, it is adsorbed on the back side of the SERS detection substrate where the more hydrophilic small opening is located because its hydrophilicity is greater than that of the large opening on the front side. When the liquid flux is increased, the difference in wettability from the small opening to the large opening of the through-holes enables directional liquid transmission. Liquid is transferred from the small opening to the large opening, and water evaporates through the pores, transporting small-molecule catecholamines in the tissue fluid and concentrating them on the nanomembrane surface at the large opening, achieving the separation of large-molecule proteins and small-molecule substances. In other words, the SERS detection substrate can effectively and quickly collect analytes. When the laser is detected from the front side of the SERS detection substrate, the signal of the catecholamine substance can be collected, reducing the influence of proteins and large-molecule targets.

[0017] The beneficial effects of the present invention are as follows: 1. In the present invention, metal nanoparticles whose surfaces are modified with a hydrophilic stabilizer are assembled at the gas-liquid interface to form a single-layer nanofilm, thereby forming a SERS-enhanced substrate with a large number of rich hotspot structures.

[0018] 2. The present invention attaches the back side of the SERS detection substrate to the area to be punctured, and performs in situ SERS detection of catecholamines, a substance that is a puncture effect, while the single-layer nanomembrane is located on the front surface where the opening end of the through hole is located on the flexible porous carrier, thereby avoiding direct contact between the precious metal nanoparticles on the single-layer nanomembrane and the skin, reducing the contamination of the SERS detection substrate and the irritation of the precious metals to the skin.

[0019] 3. The hydrophilicity of the SERS detection substrate of the present invention is such that its back side (the surface with the smaller opening end of the through-hole) is larger than its front side (the surface with the larger opening end of the through-hole). When the back side is attached to the skin, after acupuncture, tissue fluid is adsorbed on the back side of the SERS detection substrate. When the water is transmitted to the front side, the water evaporates through the pores, and the catecholamine components in the tissue fluid are retained on the front side of the SERS detection substrate, thereby avoiding the influence of large molecular proteins and other interfering substances on catecholamine detection.

[0020] 4. The nanomembrane interface assembly process of the present invention can be completed in approximately 1-2 minutes. This process is convenient and rapid, and can form a nanomembrane structure on the pore walls of asymmetric pores. By manipulating the size, morphology, and distribution density of the metal nanoparticles, an optimized flexible asymmetric porous membrane substrate with an exciton nanostructure is obtained. 5. The SERS detection substrate has high sensitivity, repeatability, mechanical strength and stability, and the detection limit of catecholamine substances is 10 -6 Below M. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 1 are scanning electron micrographs of Example 1, wherein a is a scanning electron micrograph of the flexible porous support, b is a scanning electron micrograph of the SERS detection substrate, and c is a scanning electron micrograph of the SERS detection substrate after contacting the liquid; Figure 2 This is the SERS detection spectrum of Application Example 1; Figure 3 This is the SERS detection spectrum of Application Example 2; Figure 4 This is the SERS detection spectrum of Application Example 3; Figure 5 This is the SERS detection spectrum of Application Example 4; Figure 6 This is the SERS detection spectrum of Application Example 5; Figure 7 This is the SERS detection spectrum of Example 7; Figure 8 This is the SERS detection spectrum of Comparative Example 1; Figure 9 This is the SERS detection spectrum of Comparative Example 2. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is described in detail below through specific embodiments.

[0023] Example 1: Preparation of asymmetric wettable and flexible SERS detection substrate: S1. Using laser to generate through holes on the flexible membrane PDMS, with one end of the through hole being larger and the other end being smaller, to obtain a flexible porous carrier PDMS; S2, preparing silver nanosol surface-modified with sodium citrate; S3. Dispersing the washed silver nanosol into an aqueous solution to form a silver nanoparticle mixture, and then adding dodecyl mercaptan to an organic solvent (the organic solvent is composed of ethanol and n-hexane in a volume ratio of 2:1) to form a mixture; then pouring the dispersion into the above-mentioned nanoparticle aqueous solution, wherein the volume ratio of the silver nanoparticle mixture to dodecyl mercaptan is 1:1; shaking for several seconds, the nanoparticles will spontaneously assemble at the air-liquid interface to form a single-layer silver nanofilm; S4. Clamp the flexible porous carrier PDMS with tweezers and insert it under the single-layer silver nanofilm at a certain angle, and transfer it to the front surface where the through-hole opening end of the flexible porous carrier is located by a transfer method; cover the surface of the flexible porous carrier and the through-hole with nanofilm to obtain a SERS detection substrate.

[0024] The method for preparing the silver nanoparticle sol surface-modified with sodium citrate in step S1 is as follows: Under room temperature stirring, add 1 mL of AgNO3 aqueous solution (1 wt%) to 99 mL of water, heat and stir, and after boiling, add 4 mL of sodium citrate aqueous solution (1 wt%) to the boiling water. After heating and stirring for one hour, cool to room temperature to obtain sodium citrate-silver nanoparticle sol.

[0025] like Figure 1 As shown, a is a scanning electron microscope image of the flexible porous carrier prepared in Example 1, and the flexible porous carrier is provided with through holes, and one end of the opening of the through hole is large (located on the front) and the other end is small (located on the back); the opening end of the through hole is an irregular shape, such as at least one of a five-pointed star, a circle, and a square, and has an asymmetric wettability structure.

[0026] b is a scanning electron microscope image of the SERS detection substrate prepared in Example 1, and c is a scanning electron microscope image of the SERS detection substrate prepared in Example 1 in contact with the liquid asymmetric pore membrane. Figure 1 It can be seen from the figure that the front side of the SERS detection substrate has a macroporous structure, and the back side has a microporous structure; and the hydrophobicity of the front side is greater than that of the back side, forming an asymmetric wettability structure.

[0027] Application Example 1: Containing 10 -4mol / L dopamine standard and serum tissue control without dopamine were tested. The tissue sample was prepared by dissolving 15.3 mg dopamine solid in 1000 ml deionized water to make 10 -4 mol / L dopamine aqueous solution; equal volumes of dopamine aqueous solution and equal volumes of tissue samples were mixed as spiked samples, and tissue samples without dopamine were used as controls.

[0028] The specific detection steps are as follows: S1. Mix the tissue sample and ethanol in a volume ratio of 1:2, centrifuge, and collect the supernatant; S2. The supernatant is added dropwise onto the agar gel. After the agar gel completely absorbs the supernatant, the back side of the SERS detection substrate prepared in Example 1 is covered on the agar gel. The difference in wettability on both sides of the SERS detection substrate and the through-hole channels can quickly separate large and small substances in the tissue fluid or serum after acupuncture, and then catecholamines are enriched on the front side of the SERS detection substrate. S3. After drying, SERS detection is performed on the front side of the SERS detection substrate, and the laser wavelength is 785 nm.

[0029] As above, the serum tissue control was subjected to SERS detection, and the detection spectrum was as follows Figure 2 As shown, from Figure 2 As can be seen from the figure, dopamine molecular signals were detected on the front surface and pore walls of the SERS detection substrate prepared in Example 1. That is, the SERS detection substrate prepared in the present invention can achieve separation and sensitive detection of macromolecules and small molecules in complex fluids through the asymmetric through-pore structure.

[0030] Application Example 2: Same as Application Example 1, the SERS detection substrate prepared in Example 1 was used to detect 10 -5 mol / L dopamine standard and tissue control without dopamine were tested, and the detection spectrum was as follows Figure 3 shown.

[0031] Application Example 3: Same as Application Example 1, the SERS detection substrate prepared in Example 1 was used to detect the -6 The tissue samples of mol / L dopamine standard and tissue control without dopamine were tested, and the detection spectrum was as follows Figure 4 As shown, the specific detection steps are the same as those in Application Example 1. Example 2: Preparation of asymmetric wettable and flexible SERS detection substrate: S1. Using laser to generate through holes on the flexible PET film, wherein one end of the through hole is large and the other end is small, thereby obtaining a flexible porous carrier PET; S2, preparing gold nanosols surface-modified with ascorbic acid; S3, dispersing the washed gold nanosol into an aqueous solution to form a gold nanomixture, and then adding 1H,1H,2H,2H-perfluorodecylmercaptan into an organic solvent (composed of ethanol and n-hexane in a volume ratio of 2:1) to form a dispersion; The dispersion was added to the gold nanoparticle aqueous solution, wherein the volume ratio of the gold nanoparticle mixture to 1H,1H,2H,2H-perfluorodecylmercaptan was 1:2; after shaking for a few seconds, the nanoparticles spontaneously assembled at the air-liquid interface to form a monolayer gold nanofilm; S4. Transfer the gold nanoparticle film to the surface of the flexible porous carrier PET by a transfer method. Specifically, first clamp the flexible porous carrier PET with tweezers and insert it under the gold nanoparticle film at a certain angle to transfer it to the front surface of the flexible porous carrier PET; dry it to obtain a SERS detection substrate.

[0032] The method for preparing the gold nanoparticle sol surface-modified with sodium citrate in step S1 is as follows: Under room temperature stirring, add 1 mL of a 1 wt% aqueous solution of chloroauric acid to 99 mL of water. Heat with stirring until boiling. Then, add 1 mL of a 1 wt% aqueous solution of sodium citrate to the boiling water. After heating and stirring for 30 minutes, cool to room temperature to obtain a sodium citrate gold nanoparticle sol.

[0033] Application Example 4: Containing 10 -4 mol / L epinephrine standard and a tissue control without epinephrine were tested. The specific steps are as follows: S1. Mix the tissue sample and ethanol in a volume ratio of 1:2, centrifuge, and collect the supernatant; S2. The supernatant is added dropwise onto the agar gel. After the agar gel completely absorbs the supernatant, the back side of the SERS detection substrate prepared in Example 2 is covered on the agar gel. The difference in wettability on both sides and the through-holes can quickly separate large and small substances in the tissue fluid or serum after acupuncture, and catecholamines are enriched on the front side of the SERS substrate.

[0034] S3. After drying, SERS detection is performed on the front side of the SERS sensor with a laser wavelength of 785 nm.

[0035] As above, the serum tissue control was subjected to SERS detection, and the detection spectrum was as follows Figure 5 As shown, from Figure 5As can be seen in the figure, the adrenaline molecule signal is detected on the front surface and the pore wall of the SERS detection substrate. That is, the SERS detection substrate prepared in Example 2 of the present invention can achieve separation and sensitive detection of macromolecules and small molecules in complex fluids through the asymmetric through-pore structure.

[0036] Application Example 5: Same as Application Example 4, the SERS detection substrate prepared in Example 2 was used to detect the presence of 10 -5 The tissue samples of 10 mol / L adrenaline standard and tissue control without adrenaline were tested, and the detection spectrum was as follows Figure 6 As shown, the specific detection steps are the same as those in Application Example 4.

[0037] Application Example 6: Same as Application Example 4, the SERS detection substrate prepared in Example 2 was used to detect the presence of 10 -6 The tissue samples of 10 mol / L adrenaline standard and tissue control without adrenaline were tested, and the detection spectrum was as follows Figure 7 As shown, the specific detection steps are the same as those in Application Example 4. Comparative Example 1: Preparation of non-porous SERS detection substrate: S1. preparing a gold nanoparticle sol surface-modified with ascorbic acid (same as in Example 2); S2. dispersing the washed gold nanoparticle sol into an aqueous solution to form a gold nanoparticle mixed solution, and then adding 1H,1H,2H,2H-perfluorodecylmercaptan into an organic solvent (composed of ethanol and n-hexane in a volume ratio of 2:1) to form a dispersion solution; The dispersion was added to the gold nanoparticle aqueous solution, wherein the volume ratio of the gold nanoparticle mixture to 1H,1H,2H,2H-perfluorodecylmercaptan was 1:2; after shaking for a few seconds, the nanoparticles spontaneously assembled at the air-liquid interface to form a monolayer gold nanofilm; S3. Transfer the gold nanoparticle film to the surface of the flexible thin film PDMS by a transfer method. Specifically, first, clamp the complete PDMS without openings with tweezers and insert it under the gold nanoparticle film at a certain angle to transfer the single-layer gold nanofilm to the front surface of the flexible thin film PDMS; and dry it to obtain the SERS detection substrate.

[0038] The SERS detection substrate prepared in Comparative Example 1 was used to detect the -4 The tissue samples of mol / L dopamine standard and tissue control without dopamine were tested, and the detection spectrum was as follows Figure 8 As shown, the specific detection steps are the same as those in Application Example 4.

[0039] Among them, because the flexible film PDMS in the SERS detection substrate is a non-porous structure, that is, it cannot transmit liquid, catecholamines cannot be transmitted to the front of the SERS detection substrate and enriched, and SERS detection cannot always detect catecholamine signals instantly.

[0040] Comparative Example 2: Preparation of SERS detection substrate with symmetrical through-holes: S1. Using laser to generate symmetrical through holes on the flexible PET film, i.e., the two opening ends of the through holes are the same size, to obtain a flexible porous carrier PET; S2. preparing a gold nanosol surface-modified with ascorbic acid (same as in Example 2); S3, dispersing the washed gold nanosol into an aqueous solution to form a gold nanomixture, and then adding 1H,1H,2H,2H-perfluorodecylmercaptan into an organic solvent (composed of ethanol and n-hexane in a volume ratio of 2:1) to form a dispersion; The dispersion was added to the gold nanoparticle aqueous solution, wherein the volume ratio of the gold nanoparticle mixture to 1H,1H,2H,2H-perfluorodecylmercaptan was 1:2; after shaking for a few seconds, the nanoparticles spontaneously assembled at the air-liquid interface to form a monolayer gold nanofilm; S3. Transfer the gold nanofilm to the surface of the flexible porous carrier PET by a transfer method. Specifically, first clamp the flexible porous carrier PET with tweezers and insert it under the gold nanofilm at a certain angle to transfer it to the front surface of the flexible porous carrier PET; dry it to obtain a SERS detection substrate.

[0041] The SERS detection membrane with symmetrical through-hole structure prepared in Comparative Example 2 and the asymmetric pore SERS detection membrane prepared in Example 2 were used as SERS detection substrates. -4 mol / L adrenaline standard tissue sample was tested, and the detection spectrum was as follows Figure 9 As shown, the specific detection steps are the same as those in Application Example 2.

[0042] Among them, the difference in hydrophilicity (or hydrophobicity) between the front and back surfaces of the SERS detection substrate with an asymmetric through-hole structure is greater than that of the SERS detection substrate with a symmetric through-hole structure, wherein the water transmission efficiency is higher, and the inverted trapezoidal structure of the asymmetric through-hole allows more nanoparticles to be assembled on the asymmetric pore walls. Therefore, the SERS signal intensity of the SERS detection substrate with an asymmetric through-hole structure prepared in Comparative Example 2 is weaker than the SERS signal of Example 2, demonstrating that the SERS detection substrate with an asymmetric through-hole structure prepared in the present invention is more efficient in detecting catecholamine substances than the SERS detection substrate with a symmetric through-hole structure prepared in Comparative Example 2.

[0043] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing an asymmetric wettable and flexible SERS detection substrate, characterized by: The following steps are involved: S1. Using laser to generate a through hole on the flexible film, wherein one end of the through hole is large and the other end is small, thereby obtaining a flexible porous carrier; S2, preparing a noble metal nanosol whose surface is modified with a hydrophilic stabilizer; S3, dispersing the noble metal nanosol into water to form a metal nanoparticle mixture, then adding the dispersion, and shaking the nanoparticles to spontaneously assemble at the air-liquid interface to form a single-layer nanofilm; S4. Transfer the single-layer nanofilm to the front surface of the flexible porous carrier prepared in step S1 where the through-hole opening is located, and cover the surface of the flexible porous carrier and the through-hole with the nanofilm to obtain a SERS detection substrate.

2. The preparation method according to claim 1, wherein: The hydrophilic stabilizer is at least one of sodium citrate, ascorbic acid, and sodium borohydride.

3. The preparation method according to claim 1, wherein: The noble metal nano-sol is a colloid formed by dispersing nano-spherical particles of gold, silver or gold-silver alloy with isotropic structure in a solvent.

4. The preparation method according to claim 1, wherein: The dispersion is formed by adding a thiol compound to an organic solvent, wherein the thiol compound is dodecyl mercaptan or 1H,1H,2H,2H-perfluorodecyl mercaptan; and the organic solvent is composed of ethanol and n-hexane in a volume ratio of 2:1; The volume ratio of the nanoparticle mixture to the thiol compound is 1:1-2.

5. The preparation method according to claim 1, wherein: The flexible film is polydimethylsiloxane or polyethylene terephthalate.

6. The preparation method according to claim 1, wherein: The opening end of the through hole is irregular in shape, and the through hole has an asymmetric wettability structure.

7. A SERS detection substrate prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the SERS detection substrate according to claim 7, characterized in that: Used for in situ detection of catecholamines, acupuncture effect substances in body fluids.

9. The use according to claim 8, characterized in that: The in-situ detection step includes: attaching the back side of the SERS detection substrate to the area to be punctured, and performing SERS detection on the catecholamine, a substance that is a puncture effect.

10. The use according to claim 9, characterized in that: The excitation light wavelength of the SERS detection is 785 nm.

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