A sers substrate with wettability gradient driven molecular enrichment and its preparation method and application

Abstract

This invention discloses a wettability gradient-driven molecular enrichment SERS substrate, its preparation method, and its applications, belonging to the field of surface-enhanced Raman scattering technology. Addressing the problem of molecules struggling to enter nanoscale interstitial hotspots, this invention modifies the surface wettability to construct a wettability gradient between hotspot and non-hotspot regions, forming micro / nano liquid transport channels. The SERS substrate prepared by this invention can utilize asymmetric capillary forces or evaporation-induced convection to drive trace molecules to spontaneously enrich into the nanoscale interstitial space, significantly increasing the probability of molecule detection. This method requires no complex equipment, can be used for batch preparation, is applicable to various plasmonic metal nanomaterials, and can be used for Raman spectroscopy detection of environmental pollutants, biomarkers, etc., exhibiting high sensitivity, strong quantification, and simple operation.

Description

Technical Field

[0001] This invention belongs to the field of surface-enhanced Raman scattering technology, and specifically relates to a wettability gradient-driven molecular enrichment type SERS substrate, its preparation method and application. Background Technology

[0002] In the fields of biomedicine, food safety, and environmental monitoring, there is an urgent need for highly sensitive and accurate quantitative detection and analysis of trace target analytes in complex matrices. Surface-enhanced Raman spectroscopy (SERS), based on the localized surface plasmon resonance effect of noble metals, has unique advantages such as label-free operation and the ability to provide molecular fingerprint spectra. However, SERS technology generally faces problems of insufficient detection sensitivity and poor quantification in practical applications, which seriously restricts its reliability and widespread adoption. Therefore, how to effectively improve the detection sensitivity and quantification of SERS technology has become a key technical problem that urgently needs to be solved in the field of SERS.

[0003] The fundamental reason for the insufficient sensitivity and poor quantification of SERS technology is that target molecules have difficulty entering the nano-interstitial hotspots. This is mainly because when the target molecule happens to be located at a nano-interstitial hotspot, its Raman signal can be enhanced by 10%. 6 -10 8 The signal enhancement is typically only 10 times for molecules that are away from the hotspot; while for molecules that are far from the hotspot, the signal enhancement is typically only 10 times. 2 The signal strength is often several times higher or even lower. Furthermore, the hotspot region typically occupies less than 1% of the total substrate area. This leads to several interconnected problems in actual detection: First, insufficient detection sensitivity: a large number of molecules are far from the hotspot and randomly adsorb in the non-enhanced region surrounding it, resulting in an actual detection limit far lower than theoretically expected. Second, poor quantification: the probability of molecules entering the hotspot is random; a small number of molecules that accidentally enter the hotspot can generate false high signals, while the contributions of a large number of molecules outside the hotspot are weak. Signal fluctuations at the same concentration can reach several orders of magnitude, making it difficult for the detection results to truly reflect the sample concentration.

[0004] Current SERS research largely focuses on designing SERS hotspot structures to improve detection sensitivity, neglecting the more critical issue of controlling the adsorption sites of target molecules on these hotspot structures. A significant technological gap remains in effectively driving the transport and enrichment of target molecules into the hotspot region. Therefore, developing a wettability gradient-driven method for preparing SERS substrates that facilitates molecule enrichment, and addressing the core challenge of target molecules entering nano-interstitial hotspots without requiring complex external equipment, is of significant research and application value. Summary of the Invention

[0005] The main objective of this invention is to address the problems of low detection sensitivity and poor quantification caused by the difficulty of target molecules entering the hot spots in the nano gaps in existing SERS technology. This invention provides a wettability gradient-driven molecular enrichment SERS substrate, its preparation method, and its application. This invention can obtain a wettability gradient-driven molecular enrichment SERS substrate that effectively drives the transport and enrichment of target molecules to the hot spot region without relying on complex external equipment.

[0006] To solve the above-mentioned technical problems, the present invention proposes the following technical solution: In a first aspect, the present invention provides a method for preparing a wettability gradient-driven molecular enrichment type SERS substrate, comprising the following steps: A low surface energy solid planar material with intrinsic hydrophobic properties is used as the first substrate, and the water contact angle of the surface of the first substrate is greater than 90°; or, a planar hydrophilic substrate with a surface water contact angle of less than 90° is subjected to surface hydrophobic modification treatment to obtain a second substrate, and the surface water contact angle of the second substrate is greater than 90°. Hydrophilic plasmonic metal nanoparticles are self-assembled at the solution interface to form a monolayer nanoparticle assembly layer, wherein the water contact angle of the hydrophilic plasmonic metal nanoparticles is less than 90°. The monolayer nanoparticle assembly layer is transferred to the surface of a first substrate or a second substrate, air-dried, and then heat-treated to obtain a hydrophobic-hydrophilic wettability gradient-driven molecular enrichment SERS substrate. In this process, the gaps between adjacent nanoparticles in the monolayer nanoparticle assembly layer form a nanohotspot structure with Raman signal enhancement effect. The SERS substrate can utilize the capillary force or evaporation-induced convection generated by the wettability gradient between the substrate surface and the surface of the metal nanoparticles to drive molecules in the solution to spontaneously accumulate into the interior of the nano gaps.

[0007] Furthermore, the low surface energy solid planar material with hydrophobic properties includes indium tin oxide, polydimethylsiloxane, polyacrylate, polyimide, polyethylene terephthalate, polypropylene, or polyethylene.

[0008] Furthermore, the planar hydrophilic substrate with a surface water contact angle of less than 90° includes silicon wafers, glass wafers, gold wafers, silver wafers, copper wafers, or aluminum wafers.

[0009] Furthermore, the surface hydrophobic modification is achieved through a combination of one or more of the following methods: chemical vapor deposition, surface chemical grafting, plasma treatment, and surface coating.

[0010] Furthermore, the hydrophilic plasmonic metal nanoparticles include silver nanoparticles, gold nanoparticles, porous silver nanoparticles, porous gold nanoparticles, gold nanocubes, silver nanocubes, gold nanorods, or silver nanorods.

[0011] Furthermore, the gap size between adjacent nanoparticles in the monolayer nanoparticle assembly layer is 1 nm to 100 nm.

[0012] Furthermore, the method also includes a step of preparing a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient driven molecular enrichment SERS substrate. Specifically, after obtaining the hydrophobic-hydrophilic wettability gradient driven molecular enrichment SERS substrate, a polymer brush is chemically grafted onto the surface of the monolayer nanoparticle assembly layer facing away from the substrate, thereby obtaining a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient driven molecular enrichment SERS substrate with further enhanced molecular enrichment effect.

[0013] Furthermore, the wettability gradient is used to enrich target molecules in the aqueous solution system within the hot spot gaps, thereby enhancing the test signal when detecting analytes in the aqueous solution system using Raman spectroscopy.

[0014] Secondly, the present invention provides a wettability gradient-driven molecular enrichment type SERS substrate prepared by any one of the methods.

[0015] Thirdly, the present invention also provides an application of a wettability gradient-driven molecular enrichment type SERS substrate prepared by any one of the methods in Raman spectroscopy detection.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention constructs a wettability gradient on the surface of a SERS substrate and uses capillary force or evaporation-induced convection to drive the spontaneous enrichment of target molecules into the interior of nano-interstic hot spots. This effectively overcomes the bottleneck of molecules relying on passive diffusion and having an extremely low probability of entering hot spots in traditional SERS substrates, and significantly improves the detection sensitivity and quantitative accuracy of SERS technology.

[0017] 2. This invention relies entirely on the wettability gradient design of the substrate surface to achieve molecular transport and enrichment, without the need for complex equipment such as external electric fields, flow fields, and acoustic fields. This avoids side effects such as Joule heating, bubble generation, and denaturation of biomolecules, reduces system costs and operational difficulty, and facilitates rapid on-site detection and batch application.

[0018] 3. The method of this invention does not depend on specific nanomaterials or hotspot structures, and is applicable to various plasmonic metal nanomaterials such as gold and silver nanoparticles, nanopillars, and porous structures, as well as various substrates such as silicon wafers, glass, and polymers. Simultaneously, it can enrich various types of target molecules, such as environmental pollutants and biomarkers, in aqueous or organic solutions, demonstrating broad applicability.

[0019] 4. This invention drives the controllable enrichment of molecules through wettability gradient, avoiding the "coffee ring effect" caused by traditional evaporation deposition, which leads to the aggregation of molecules on the periphery. This makes the distribution of target molecules more uniform and controllable in hot spot areas, significantly reducing signal fluctuations between different batches or different locations on the substrate at the same concentration, and improving the reproducibility and reliability of detection.

[0020] 5. The wettability modification methods used in this invention (such as chemical vapor deposition, self-assembled monolayers, plasma treatment, etc.) are all mature surface modification technologies that can be implemented under conventional laboratory conditions, are compatible with existing commercial nanomaterials, have the potential for mass production, and are cost-controllable.

[0021] 6. This invention solves the long-standing problem of molecular hotspots in SERS quantitative analysis from the perspective of molecular transport. It complements the existing hotspot structure plasmon electromagnetic coupling enhancement design, providing a new technical path for SERS quantitative detection with high sensitivity and high quantitative accuracy. Attached Figure Description

[0022] Figure 1 A schematic diagram of the spatial distribution of molecules on the substrate surface after drying of a droplet on a conventional hydrophilic SERS substrate without a wettability gradient; Figure 2 A schematic diagram of the spatial distribution of molecules on the substrate surface after drying of SERS substrate droplets driven by wettability gradient. Figure 3 The images show the morphological observation results of the isotropic gold nanoparticle assembly layer in Example 1; where a is the macroscopic morphology under an optical lens; b is the microscopic morphology under a field emission scanning electron microscope; and c is the microscopic morphology under a transmission microscope. Figure 4 This is a schematic diagram of the SERS substrate structure with a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient in Example 3. Figure 5 This is a schematic diagram of the spatial distribution of molecules on the substrate surface after drying of the SERS substrate droplets, which were enriched by a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient in Example 3. Figure 6 To verify the Raman detection performance of the glass slide-based hydrophilic SERS substrate without wettability gradient in Example 1; where a represents different concentrations (10-5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M) Raman spectrum of Rhodamine 6G solution (2 μL); b is 1510 cm⁻¹ -1 Linear fitting curve of the logarithm of characteristic peak intensity versus the logarithm of Rhodamine 6G concentration; Figure 7 To verify the Raman detection performance of the hydrophobic-hydrophilic wettability gradient SERS substrate based on indium tin oxide substrate in Example 1; where a represents different concentrations (10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M) Raman spectrum of Rhodamine 6G solution (2 μL); b is 1510 cm⁻¹ -1 Linear fitting curve of the logarithm of characteristic peak intensity versus the logarithm of Rhodamine 6G concentration; Figure 8 To verify the Raman detection performance of the hydrophobic-hydrophilic wettability gradient SERS substrate based on polydimethylsiloxane substrate in Example 2; where a represents different concentrations (10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M) Raman spectrum of Rhodamine 6G solution (2 μL); b is 1510 cm⁻¹ -1 Linear fitting curve of the logarithm of characteristic peak intensity versus the logarithm of Rhodamine 6G concentration; Figure 9 To verify the fluorescence distribution comparison of the enrichment effect of hydrophilic substrate glass and hydrophobic substrate polydimethylsiloxane on Rhodamine 6G in aqueous solution in Example 3, the following diagrams are provided: a) fluorescence distribution at the gap between microspheres on the glass surface; b) three-dimensional fluorescence distribution at the longitudinal position of the glass; c) fluorescence distribution at the gap between microspheres on the polydimethylsiloxane surface; and d) three-dimensional fluorescence distribution at the longitudinal position of the polydimethylsiloxane. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0025] This invention modifies the wettability of a substrate surface in a regionally selective manner, creating a wettability gradient between hot and non-hot spot regions. This forms micro / nano liquid transport channels, thereby utilizing capillary forces or evaporation-induced convection to drive the spontaneous and efficient accumulation of trace molecules within the nano-interstitial hot spots. The wettability modification is regionally selective, resulting in different surface wettability between hot and non-hot spot regions.

[0026] In this invention, the SERS substrate can enrich one or more molecules in an aqueous or organic solution containing environmental pollutants or biomarkers; the SERS substrate prepared using the method of this invention has a detection sensitivity higher than 10 after molecule enrichment. -7 M, and the coefficient of determination R for quantitative detection 2 A value greater than 0.95 significantly improves the sensitivity and quantification of the detection.

[0027] In a first aspect, the present invention provides a method for preparing a wettability gradient-driven molecular enrichment type SERS substrate, comprising the following steps: S1: A low surface energy solid planar material with intrinsic hydrophobic properties is used as the first substrate, and the water contact angle of the surface of the first substrate is greater than 90°; or, a planar hydrophilic substrate with a surface water contact angle of less than 90° is subjected to surface hydrophobic modification treatment to obtain a second substrate, and the surface water contact angle of the second substrate is greater than 90°. S2: Hydrophilic plasmonic metal nanoparticles are self-assembled at a liquid-liquid interface to form a single-layer nanoparticle assembly layer, wherein the water contact angle of the hydrophilic plasmonic metal nanoparticles is less than 90°. S3: Transfer the monolayer nanoparticle assembly layer to the surface of the first or second substrate, air dry it naturally, and then perform heat treatment to obtain a hydrophobic-hydrophilic wettability gradient-driven molecular enrichment SERS substrate.

[0028] After obtaining the hydrophobic-hydrophilic wettability gradient-driven molecular enrichment SERS substrate, the method further includes the step of preparing a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient-driven molecular enrichment SERS substrate. Specifically, a polymer brush is chemically grafted onto the surface of the monolayer nanoparticle assembly layer facing away from the substrate, thereby pointing to a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient-driven molecular enrichment SERS substrate with further enhanced molecular enrichment effect.

[0029] The order of steps S1 and S2 can be adjusted according to actual process requirements: either a nano-gap hotspot structure can be constructed on the substrate surface first, followed by wettability modification; or the substrate can be selectively wettable first, followed by the construction of a nano-gap hotspot structure in a designated area; both orders can achieve the present invention.

[0030] Secondly, the present invention provides a wettability gradient-driven molecular enrichment type SERS substrate obtained by the above method.

[0031] Thirdly, the present invention also provides an application of the wettability gradient-driven molecular enrichment type SERS substrate obtained by the above method in Raman spectroscopy detection.

[0032] Figure 1 This demonstrates the molecular distribution on a conventional hydrophilic SERS substrate lacking a wettability gradient. Due to the coffee ring effect, droplets spread completely on the substrate surface, and the detection molecules in the solution are randomly distributed on the nanoparticles and substrate surface. Molecules rely entirely on passive diffusion and are unlikely to actively enter the nano-interstitial hotspots. In contrast, the SERS substrate proposed and constructed in this invention possesses a hydrophobic-hydrophilic wettability gradient (…). Figure 2 This method effectively suppresses the non-specific adsorption of molecules in aqueous solutions on the substrate surface. The wettability gradient between the hydrophobic and hydrophilic hotspot regions of the substrate, combined with asymmetric capillary forces and thermal convection generated by the slow evaporation of the solution at 65% relative humidity, jointly drives the transport and enrichment of molecules into the nano-interstic spaces between gold nanoparticles, significantly improving the efficiency of molecule entry into hotspots.

[0033] Example 1 This embodiment is based on an indium tin oxide substrate to obtain a hydrophobic-hydrophilic wettability gradient SERS substrate.

[0034] (1) A commercially available hydrophobic indium tin oxide (ITO) substrate was cut into 0.8 cm × 0.8 cm squares and ultrasonically cleaned in anhydrous ethanol and deionized water for 10 min each. After air drying, the cleaned ITO substrate was obtained. The cleaned ITO substrate was then treated in a plasma cleaner under an oxygen atmosphere for 5 min. After treatment, the surface water contact angle decreased from the original 103.1° to 9.1°, exhibiting temporary hydrophilicity. The plasma-treated ITO substrate was then heated at 80 °C for 2 h, and its surface water contact angle recovered to 102.6°, essentially restoring the initial hydrophobic state. This step, through plasma treatment and thermal annealing, achieved a controllable transformation of the ITO substrate surface from hydrophobic to hydrophilic and back to hydrophobic.

[0035] (2) In this embodiment, gold nanoparticles synthesized in batches by wet chemical processes are selected as one of the examples of hydrophilic plasmonic metal nanoparticles.

[0036] Gold nanoparticles were uniformly dispersed in anhydrous ethanol. 250 μL of this dispersion was mixed thoroughly with 1 mL of dichloromethane in a 5 mL centrifuge tube. Then, 1.8 mL of ultrapure water was added, and the mixture was vigorously shaken for 30 s to ensure sufficient contact between the aqueous phase and the gold nanoparticles dispersed in the organic phase. After standing for approximately 1 min, a bright gold mirror layer was observed at the interface between the aqueous phase and dichloromethane, indicating that the gold nanoparticles self-assembled at the interface. Subsequently, 400 μL of n-hexane was added, and the 5 mL centrifuge tube was tilted at 45° and then slowly returned to the center. Utilizing the Marangoni effect between the mixed solvents, the gold nanoparticles were dragged to the interface between the aqueous phase and n-hexane, forming a dense monolayer of assembled gold nanoparticles, the morphology of which is shown in the figure. Figure 3 As shown.

[0037] (3) The above-mentioned monolayer gold nanoparticle assembly layer was transferred to the surface of the plasma-treated indium tin oxide substrate and air-dried overnight at 25 °C. The next day, after heat treatment at 80 °C for 2 h, a SERS substrate with a wettability gradient was obtained.

[0038] In other words, there is a SERS substrate with a hydrophobic (water contact angle of indium tin oxide surface is 103.1°) to a hydrophilic (water contact angle of gold surface is about 70.8°) wettability gradient between non-hotspot regions and hotspot regions.

[0039] Example 2 This embodiment is based on a polydimethylsiloxane substrate to obtain a hydrophobic-hydrophilic wettability gradient SERS substrate.

[0040] (1) The hydrophobic polydimethylsiloxane substrate was cut into squares of 0.8 cm × 0.8 cm and then placed in a plasma cleaner under an oxygen atmosphere for 5 min. After treatment, the surface water contact angle decreased from the original 124.2° to 11.7°, exhibiting temporary hydrophilicity. The plasma-treated polydimethylsiloxane substrate was then heated at 80 °C for 2 h, and the surface water contact angle recovered to 121.5°, basically restoring the initial hydrophobic state. That is, through plasma treatment and thermal annealing, the surface of the polydimethylsiloxane substrate can be controllably transformed from hydrophobic to hydrophilic and then back to hydrophobic.

[0041] (2) Repeat the steps in Example 1(2) to obtain a dense monolayer gold nanoparticle assembly layer.

[0042] (3) The monolayer gold nanoparticle assembly layer was transferred to the surface of the plasma-treated polydimethylsiloxane substrate and air-dried overnight at 25 °C. The next day, after heat treatment at 80 °C for 2 h, a SERS substrate with a wettability gradient was obtained.

[0043] The water contact angle in the non-hotspot region of the SERS substrate is 124.2° (hydrophobic), while the water contact angle on the gold surface in the hotspot region is 70.8° (hydrophilic), forming a hydrophobic-hydrophilic wettability gradient. Compared with the substrate prepared in Example 1, the difference in contact angle between the polydimethylsiloxane substrate and the gold nanoparticles in this example is larger (53.4° compared to 32.3° in Example 1), thus exhibiting a stronger molecular enrichment driving ability.

[0044] Example 3 This embodiment is based on a polydimethylsiloxane substrate to obtain a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient SERS substrate.

[0045] (1) Repeat the steps in (1) of Example 2 to obtain a polydimethylsiloxane substrate with a surface that can be controllably transformed from hydrophobic to hydrophilic and then back to hydrophobic.

[0046] (2) Repeat the steps in Example 1(2) to obtain a dense monolayer gold nanoparticle assembly layer.

[0047] (3) The monolayer gold nanoparticle assembly layer is transferred to the surface of the polydimethylsiloxane substrate after plasma treatment, and then naturally dried overnight at 25 °C to obtain the polydimethylsiloxane SERS substrate carrying the gold nanoparticle assembly layer.

[0048] (4) 15 μL of tetraethyl silicate, 100 μL of 0.1 M dilute hydrochloric acid aqueous solution, and 1 mL of anhydrous ethanol were thoroughly mixed to obtain a silica sol solution. Then, 10 μL of this silica sol solution was spin-coated onto the polydimethylsiloxane SERS substrate carrying the gold nanoparticle assembly layer at a speed of 3000 rpm. Under acidic conditions, tetraethyl silicate hydrolyzed and condensed to form a silica sol. After the ethanol evaporated, the sol further transformed into a gel. After complete drying at 25 °C, an extremely thin silica coating layer was formed on the surface of the gold nanoparticles, resulting in a polydimethylsiloxane SERS substrate with a silica shell on its surface.

[0049] (5) The polydimethylsiloxane SERS substrate with a silica shell was placed again in a plasma cleaner under an oxygen atmosphere for 5 min to activate the silica surface. Then, the substrate was immersed in a reaction solution composed of 1 g of dimethyldimethoxysilane monomer, 0.1 g of sulfuric acid, and 10 g of isopropanol, and removed after 10 s. It was then allowed to stand and dry at 25 °C for 30 min at a relative humidity of 65% to further graft polydimethylsiloxane polymer onto the silica shell of the SERS substrate. After the reaction, the SERS substrate was rinsed sequentially with toluene, isopropanol, and deionized water, air-dried at room temperature, and then heat-treated at 80 °C for 2 h the next day to obtain the desired product. Figure 4The SERS substrate shown has a hydrophobic-hydrophilic-superlubricating hydrophobic bilayer step wettability gradient.

[0050] The water contact angle in the non-hotspot region of the SERS substrate is 124.2° (hydrophobic), while the water contact angle on the gold surface in the hotspot region is 70.8° (hydrophilic). The water contact angle on the non-hotspot gold surface modified with a polydimethylsiloxane polymer brush is 112.1° (superlubricating hydrophobic). This creates a stepwise wettability gradient from bottom to top: hydrophobic-hydrophilic-superlubricating hydrophobic (structure as shown). Figure 4 (As shown).

[0051] Compared with the substrates prepared in Examples 1 and 2, this example further optimizes the gradient wettability in the micro / nano fluid channels and enhances the driving force for molecular enrichment, with the following effect: Figure 5 As shown.

[0052] Example 4 This embodiment is based on the preparation of a hydrophobic-hydrophilic wettability gradient SERS substrate by modifying a silicon wafer substrate with a polydimethylsiloxane polymer brush.

[0053] (1) Commercial silicon wafer substrates were cut into 0.8 cm × 0.8 cm squares and ultrasonically cleaned in anhydrous ethanol and deionized water for 10 min each, then air-dried for later use. The cleaned silicon wafer substrates were then immersed in a piranha solution of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 for 30 min to introduce abundant hydroxyl groups onto the surface. After treatment, the substrates were thoroughly cleaned with a large amount of deionized water to remove residual acid, and then purged with nitrogen to obtain silicon wafer substrates with a surface rich in hydroxyl groups.

[0054] (2) The silicon wafer substrate with hydroxyl-rich surface was immersed in a reaction solution composed of 1 g dimethyldimethoxysilane monomer, 0.1 g sulfuric acid, and 10 g isopropanol, and removed after standing for 10 s. It was then dried for 30 min under conditions of 65% relative humidity and 25 °C. The dimethyldimethoxysilane monomer underwent hydrolysis and condensation under acidic catalysis to graft polydimethylsiloxane polymer onto the silicon wafer surface, forming a polydimethylsiloxane polymer brush. The water contact angle of the modified silicon wafer surface was measured to be 113.4°, while the original silicon wafer substrate had a water contact angle of 43.5°, indicating that the silicon wafer substrate surface had successfully changed from hydrophilic to hydrophobic.

[0055] (3) Repeat the steps in (2) of Example 1 to obtain a dense monolayer gold nanoparticle assembly layer.

[0056] (4) Using the hydrophobic silicon wafer substrate modified with the above-mentioned polydimethylsiloxane polymer brush, a single layer of gold nanoparticle assembly was directly retrieved from the solution surface. The transferred substrate was air-dried overnight at 25 °C to obtain a hydrophobic-hydrophilic wettability gradient SERS substrate based on the polydimethylsiloxane polymer brush-modified silicon wafer.

[0057] In this substrate, the silicon wafer background region of the surface-modified polydimethylsiloxane polymer brush is hydrophobic (water contact angle 113.4°), while the gold nanoparticle assembly layer is hydrophilic (water contact angle approximately 70.8°). A hydrophobic-hydrophilic wettability gradient is formed between the two, which can be used to drive the spontaneous enrichment of target molecules in solution into the nano-interstitial hot spots.

[0058] Example 5 This embodiment is based on a polydimethylsiloxane coating to modify an aluminum substrate to obtain a hydrophobic-hydrophilic wettability gradient SERS substrate.

[0059] (1) Cut the commercial aluminum sheet substrate into squares of 0.8 cm × 0.8 cm, and place them in anhydrous ethanol and deionized water for ultrasonic cleaning for 10 min each, and let them air dry for later use.

[0060] (2) Polydimethylsiloxane prepolymer (Dow Corning 184) and crosslinking agent were mixed evenly at a mass ratio of 10:1 and spin-coated onto the cleaned aluminum substrate at a speed of 3000 rpm. The mixture was then cured at 60 °C for 3 h to form a thin polydimethylsiloxane coating on the aluminum substrate. The water contact angle of the aluminum substrate after the polydimethylsiloxane coating was measured to be 112.3°, while the water contact angle of the original aluminum substrate was 83.2°, indicating that the aluminum substrate surface had been successfully transformed from hydrophilic to hydrophobic.

[0061] (3) Then, wet chemically synthesized silver nanoparticles are used to replace gold nanoparticles as plasmonic metal nanoparticles in this embodiment. A dense monolayer silver nanoparticle assembly layer is prepared by self-assembly at the liquid-liquid interface according to the method described in step (2) of Example 1.

[0062] (4) The hydrophobic aluminum sheet substrate modified with the above polydimethylsiloxane coating was used to directly retrieve the single-layer silver nanoparticle assembly layer from the solution surface. The transferred substrate was air-dried overnight at 25 °C to obtain the hydrophobic-hydrophilic wettability gradient SERS substrate based on the polydimethylsiloxane coating-modified aluminum sheet.

[0063] Measurements showed that the water contact angle of the aluminum sheet background region with polydimethylsiloxane surface modification in the substrate was 112.3° (hydrophobic), while the water contact angle of the silver nanoparticle assembly layer was 85.6° (hydrophilic). A hydrophobic-hydrophilic wettability gradient was formed between the two, which can be used to drive the spontaneous enrichment of target molecules in the solution into the nano-interstitial hot spots.

[0064] Example 6 This embodiment is based on the hydrophobic-hydrophilic wettability gradient SERS substrate obtained by vapor deposition modification of glass substrate with perfluorodecyltrichlorosilane.

[0065] (1) Commercial glass substrates were cut into 0.8 cm × 0.8 cm squares and ultrasonically cleaned in anhydrous ethanol and deionized water for 10 min each, then air-dried for later use. The cleaned glass substrates were then immersed in a piranha solution of concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 3:1 for 30 min to introduce abundant hydroxyl groups onto the surface. After treatment, the substrates were thoroughly rinsed with plenty of deionized water to remove residual acid and then purged with nitrogen to dry.

[0066] (2) 1 g of perfluorodecyltrichlorosilane monomer was rapidly and thoroughly mixed with 10 g of anhydrous toluene. A glass slide rich in hydroxyl groups was placed in a sealed container containing the perfluorodecyltrichlorosilane / toluene solution, with the slide support fixed above the liquid surface to ensure complete non-contact between the slide and the reaction solution. The container was sealed and allowed to stand at a constant temperature of 25 °C for 60 min to allow the perfluorodecyltrichlorosilane molecules to chemically bond with the hydroxyl groups on the glass slide surface, forming a self-assembled monolayer hydrophobic layer. After the reaction was complete, the substrate was removed and ultrasonically cleaned for 5 min each with anhydrous toluene, anhydrous ethanol, and deionized water to remove unreacted monomers physically adsorbed on the surface. The substrate was then purged and dried with nitrogen. The water contact angle of the perfluorodecyltrichlorosilane-modified glass slide was measured to be 114.7°, indicating that the substrate surface had successfully transitioned from hydrophilic to hydrophobic.

[0067] (3) Repeat the steps in (2) of Example 1 to obtain a dense monolayer gold nanoparticle assembly layer.

[0068] (4) Using the hydrophobic glass substrate modified with the above-mentioned perfluorodecyltrichlorosilane, a monolayer of gold nanoparticle assembly was directly retrieved from the solution surface. The transferred substrate was air-dried overnight at 25 °C to obtain a hydrophobic-hydrophilic wettability gradient SERS substrate based on the perfluorodecyltrichlorosilane vapor deposition modified glass substrate.

[0069] Measurements showed that the water contact angle of the background region of the glass slide modified with perfluorodecyltrichlorosilane in the substrate was 114.7° (hydrophobic), while the water contact angle of the gold nanoparticle assembly layer was 70.8° (hydrophilic). A hydrophobic-hydrophilic wettability gradient was formed between the two, which can be used to drive the spontaneous enrichment of target molecules in the solution into the nano-interstitial hot spots.

[0070] Verification Example 1 Verification of molecular enrichment effect in aqueous system using SERS substrate with hydrophobic-hydrophilic wettability gradient based on indium tin oxide substrate.

[0071] (1) Cut the hydrophilic glass slide into squares of 0.8 cm × 0.8 cm, and ultrasonically clean them for 10 min each in anhydrous ethanol and deionized water, and then air dry them for later use. Assemble a monolayer of gold nanoparticles on the surface of the glass slide according to the methods described in steps (2) and (3) of Example 1, as a conventional hydrophilic SERS substrate (control substrate) without a wettability gradient.

[0072] (2) The hydrophobic-hydrophilic wettability gradient SERS substrate based on indium tin oxide substrate prepared in Example 1 and the above control substrate were used as test objects to conduct a molecular enrichment ability comparison experiment to verify the effect of wettability gradient on molecular enrichment ability.

[0073] The specific procedure is as follows: 2 μL of different concentrations (10 μL each) were dropped onto the surfaces of the hydrophobic-hydrophilic wettability gradient substrate based on indium tin oxide and the control substrate, respectively. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Rhodamine 6G aqueous solution (M) was prepared and allowed to stand at 65% relative humidity and 25 °C until the solution evaporated completely (approximately 20 min). Subsequently, Raman spectroscopy was performed on the dried substrate using a confocal laser Raman spectrometer. The excitation wavelength was 532 nm, the integration time was 10 s, and Raman spectra at 10 different locations were collected for each concentration. The average value was taken as the signal intensity for that concentration. The Raman spectroscopy results for the control substrate are shown below. Figure 6 As shown, the Raman detection results for the wettability gradient substrate are as follows: Figure 7 As shown. Comparing the data results of the two, it can be seen that in 10 -9 In the detection of low concentrations of rhodamine 6G, the characteristic Raman signal was almost undetectable on the control substrate, with particularly large signal fluctuations. However, the wettability gradient SERS substrate could still distinguish rhodamine 6G at 15-10 cm⁻¹ at the same concentration. -1 The characteristic peak at the location. In 10 -9 At M concentration, the signal intensity difference between the wettability gradient SERS substrate and the control substrate is much greater than 10. -5 The signal intensity difference at high concentrations of M indicates that the enrichment enhancement effect of the wettability gradient on low-concentration molecules is more pronounced. In other words, when the concentration of the target molecule in the solution is extremely low, the passive diffusion mechanism is insufficient to allow molecules to effectively enter the hot spot region, while the active enrichment mechanism driven by the wettability gradient can significantly increase the probability of molecules entering the hot spot, thereby greatly improving the detection sensitivity.

[0074] With 1510 cm -1Standard curves for the wettability gradient substrate and the control substrate were plotted with the logarithm of the characteristic peak intensity on the ordinate and the logarithm of the Rhodamine 6G concentration on the abscissa. The calculation results show that the linear correlation coefficient R of the wettability gradient substrate... 2 The linear correlation coefficient R0.978 was 0.978, while the linear correlation coefficient R0.978 of the control base was 0.978. 2 It's only 0.923. Normally, R... 2 A higher R value indicates higher accuracy and reliability of quantitative detection on SERS substrates. The wettability gradient substrate's R value... 2 The value (0.978) was significantly better than that of the control substrate (0.923), indicating that the wettability gradient-driven molecular enrichment not only improved the detection sensitivity but also improved the linear correlation between signal intensity and concentration, thereby enhancing the quantitative analysis capability of SERS technology. These results fully demonstrate that the hydrophobic-hydrophilic wettability gradient SERS substrate prepared in Example 1, based on an indium tin oxide substrate, has the advantages of higher detection sensitivity at low concentrations and better linear correlation for quantitative detection. This further confirms that the hydrophobic-hydrophilic wettability gradient can effectively drive target molecules to enrich towards nano-interstitial hotspots, significantly improving the molecular enrichment capability, detection sensitivity, and quantitative accuracy of the SERS substrate.

[0075] Verification Example 2 Verification of molecular enrichment effect in aqueous system using a hydrophobic-hydrophilic wettability gradient SERS substrate based on polydimethylsiloxane substrate.

[0076] The hydrophobic-hydrophilic wettability gradient SERS substrate based on polydimethylsiloxane substrate prepared in Example 2 was used as the test object. Following the Raman testing method described in step (2) of Verification Example 1, different concentrations (10... -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 Raman signals of Rhodamine 6G solution (M) were obtained by collecting spectra at 10 different locations for each concentration, and the average value was taken as the signal intensity for that concentration. Raman detection results are as follows: Figure 8 As shown. Figure 8 The results were compared with the Raman detection results of the control substrate and the hydrophobic-hydrophilic wettability gradient SERS substrate based on indium tin oxide substrate in Validation Example 1. The contact angle difference between the polydimethylsiloxane substrate and the gold nanoparticles was larger (53.4° compared to 32.3° in Example 1), indicating a larger step difference in the hydrophobic-hydrophilic wettability gradient, which further enhanced the molecular enrichment ability of the hydrophobic-hydrophilic wettability gradient SERS substrate based on the polydimethylsiloxane substrate. Specifically, at 10 -9In the detection of low concentrations of rhodamine 6G, the 1510 cm⁻¹ of a SERS substrate based on a hydrophobic-hydrophilic wettability gradient of a polydimethylsiloxane substrate was observed. -1 The characteristic peak signal intensity is the strongest. This directly confirms that a larger wettability gradient can generate stronger capillary driving force and evaporation-induced convection, thereby driving more target molecules into the nano-interstitial hotspot. Furthermore, the linear correlation coefficient R... 2 The value was further increased to 0.992. The molecular enrichment driving ability of the wettability gradient showed a significant positive correlation with the step wettability difference: a larger wettability gradient resulted in stronger detection capability for low-concentration signals and higher linear correlation for quantitative detection. Combined with the above results, it can be fully demonstrated that increasing the wettability difference between hot and non-hot regions can further improve the molecular enrichment efficiency and detection performance of SERS substrates.

[0077] Verification Example 3 Verification of molecular enrichment effect in aqueous system based on hydrophobic-hydrophilic wettability gradient substrate with fluorescence distribution.

[0078] In addition to Raman spectroscopy detection, this validation example visually assesses the distribution of molecules within the nanoconfined space formed by non-hotspot regions and hotspot regions from the perspective of fluorescence distribution, further verifying the enrichment and transport effect of the wettability gradient on molecules. Since the nano-gap size formed by plasmon gold nanoparticles is below the observation limit of super-resolution laser confocal fluorescence microscopy (approximately 130 nm), it is impossible to directly observe the distribution of molecules within the hotspot gaps using fluorescence microscopy. Therefore, this validation example uses 5 μm diameter polystyrene microspheres instead of gold nanoparticles to magnify the interparticle gap size, facilitating direct observation of the molecular distribution patterns within the microsphere gaps and on the surface, thereby indirectly verifying the enrichment effect of the wettability gradient on molecules. The specific experimental procedures are as follows: (1) Take 200 μL of a commercial polystyrene microsphere suspension with a diameter of 5 μm and a concentration of 10 wt%, mix it with 200 μL of anhydrous ethanol in an equal proportion, and ultrasonically disperse it for 10 min for later use. Clean a glass slide with a size of 2.5 cm × 2.5 cm and a crystallization dish with a diameter of 7.5 cm in sequence with anhydrous ethanol and deionized water for 10 min, and dry them completely at 60 ℃. Then, place both of them in a plasma cleaner under an oxygen atmosphere for 10 min to make their surfaces hydrophilic. Place the treated glass slide in the center of the crystallization dish, and add deionized water around the crystallization dish so that the water level is about 1 mm above the glass slide. Use a pipette to slowly add the diluted polystyrene microsphere dispersion to the center of the glass slide. Driven by the Marangoni effect at the gas-liquid interface, the microspheres spontaneously assemble into a monolayer. After all 400 μL of microsphere dispersion has been added, slowly inject deionized water along the inner wall of the crystallization dish to raise the water level and facilitate the transfer of the assembled layer. Before the formal transfer, 100 μL of sodium dodecyl sulfate aqueous solution with a density of 0.005 g / mL was added to make the polystyrene microspheres more densely packed.

[0079] (2) The hydrophilic glass slide and the hydrophobic polydimethylsiloxane substrate were cut into 1.5 cm × 2.0 cm pieces. The glass slide was ultrasonically cleaned with anhydrous ethanol and deionized water for 10 min, and then air-dried before being used to retrieve monolayer polystyrene microspheres from the liquid surface. The polydimethylsiloxane substrate was treated with oxygen plasma for 5 min before microsphere transfer. The transferred substrate was air-dried, then heat-treated at 80 °C for 2 h, cooled, and then treated at 100 °C for 1 min to slightly melt the bottom of the microspheres, thus stabilizing them on the substrate surface. As a control, the glass slide from which polystyrene microspheres were retrieved was air-dried and then treated at 100 °C for 1 min to fix the microspheres.

[0080] (3) Use a pipette to transfer 1 μL of a 10 concentration. -7 Aqueous solutions of Rhodamine 6G were dropped onto glass slides and polydimethylsiloxane substrates assembled with polystyrene microspheres, respectively. After slow drying at 65% relative humidity and 25°C, the samples were observed under a super-resolution laser confocal fluorescence microscope. Based on the excitation conditions of the Rhodamine 6G fluorescent dye, an excitation wavelength of 488 nm was selected for the fluorescence microscope, and the emitted light was collected through a 500-550 nm filter. The experimental results are as follows: Figure 9As shown, after monolayer polystyrene microspheres are assembled on a smooth substrate surface, the gaps between the microspheres spontaneously form nanoscale confined spaces. The distribution of Rhodamine 6G molecules in these confined spaces and on the microsphere surface is significantly affected by the wettability of the substrate. On hydrophilic substrate surfaces, due to the "coffee ring effect," solutes are transported towards the droplet edges during droplet drying, resulting in a dispersed and uneven distribution of Rhodamine 6G molecules in the gaps between microspheres and on the substrate surface. The molecules are difficult to effectively accumulate in the hotspot regions between the microspheres, resulting in weak fluorescence intensity. In contrast, the wettability gradient formed between the hydrophobic substrate and the microsphere surface generates asymmetric capillary forces, driving molecules to accumulate orderly within the gaps between microspheres, achieving controllable molecular positioning and efficient accumulation, resulting in stronger fluorescence intensity. This result further verifies the crucial role of the wettability gradient in molecular accumulation and transport described in this invention from an optical perspective, corroborating the Raman spectroscopy detection results in Verification Examples 1 and 2.

[0081] It should be noted that the low surface energy solid planar material with hydrophobic properties described in this invention is not limited to indium tin oxide, polydimethylsiloxane, silicon wafers, aluminum wafers, and glass wafers listed in the above embodiments. Those skilled in the art should understand that any solid planar material (polyacrylate, polyimide, polyethylene terephthalate, polypropylene, polyethylene, gold, silver, or copper wafers) with a flat surface capable of supporting a single-layer nanoparticle assembly and having a hydrophobic-hydrophilic wettability gradient constructed on its surface through plasma treatment or surface modification can be used to implement this invention. The substrate materials used in the above embodiments share common structural features in the technical solution of this invention, namely, they are all planar solid supports; they also have similar surface properties, all can achieve controllable adjustment of surface wettability through plasma treatment or surface modification, all can transfer a single-layer nanoparticle assembly layer through a liquid-liquid interface self-assembly method, and all can form a hydrophobic-hydrophilic wettability gradient between the substrate background and the nanoparticle surface. Therefore, those skilled in the art, inspired by the above embodiments, can reasonably extend the technical solution of the present invention to other similar planar substrate materials without creative effort, and these materials all fall within the protection scope claimed by the present invention.

[0082] The hydrophilic plasmonic metal nanoparticles described in this invention are not limited to the gold and silver nanoparticles listed in the above embodiments. Those skilled in the art should understand that other metal nanomaterials with different geometries (porous gold nanoparticles, porous silver nanoparticles, gold nanocubes, silver nanocubes, gold nanorods, or silver nanorods) exhibiting localized surface plasmon resonance effects also possess hydrophilic surfaces and can self-assemble into monolayers through liquid-liquid interfaces, forming nano-interstitial hotspots between adjacent particles. Therefore, the aforementioned metal nanoparticles can all replace the gold and silver nanoparticles in the embodiments for implementing this invention and are all within the scope of protection claimed by this invention.

[0083] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and include common knowledge or customary techniques in the art not disclosed herein. The specification and embodiments are to be considered exemplary only. It should be understood that this application is not limited to the precise structures described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope.

Claims

1. A method for preparing a SERS substrate of a wettability gradient-driven molecular enrichment type, characterized by, Includes the following steps: A low surface energy solid planar material with intrinsic hydrophobic properties is used as the first substrate, and the water contact angle of the surface of the first substrate is greater than 90°; or, a planar hydrophilic substrate with a surface water contact angle of less than 90° is subjected to surface hydrophobic modification treatment to obtain a second substrate, and the surface water contact angle of the second substrate is greater than 90°. Hydrophilic plasmonic metal nanoparticles are self-assembled at the solution interface to form a monolayer nanoparticle assembly layer, wherein the water contact angle of the hydrophilic plasmonic metal nanoparticles is less than 90°. The monolayer nanoparticle assembly layer is transferred to the surface of a first substrate or a second substrate, air-dried, and then heat-treated to obtain a hydrophobic-hydrophilic wettability gradient-driven molecular enrichment SERS substrate. In this process, the gaps between adjacent nanoparticles in the monolayer nanoparticle assembly layer form a nanohotspot structure with Raman signal enhancement effect. The SERS substrate can utilize the capillary force or evaporation-induced convection generated by the wettability gradient between the substrate surface and the surface of the metal nanoparticles to drive molecules in the solution to spontaneously accumulate into the interior of the nano gaps.

2. The SERS substrate preparation method of claim 1, wherein The low surface energy solid planar materials with hydrophobic properties include indium tin oxide, polydimethylsiloxane, polyacrylate, polyimide, polyethylene terephthalate, polypropylene, or polyethylene.

3. The SERS substrate preparation method of claim 1, wherein The planar hydrophilic substrate with a surface water contact angle of less than 90° includes silicon wafers, glass wafers, gold wafers, silver wafers, copper wafers, or aluminum wafers.

4. The SERS substrate preparation method of claim 1, wherein The surface hydrophobic modification is achieved through a combination of one or more of the following methods: chemical vapor deposition, surface chemical grafting, plasma treatment, and surface coating.

5. The SERS substrate preparation method according to claim 1, characterized in that, The hydrophilic plasmonic metal nanoparticles include silver nanoparticles, gold nanoparticles, porous silver nanoparticles, porous gold nanoparticles, gold nanocubes, silver nanocubes, gold nanorods, or silver nanorods.

6. The SERS substrate preparation method of claim 1, wherein The gap size between adjacent nanoparticles in the monolayer nanoparticle assembly layer is 1 nm to 100 nm.

7. The SERS substrate preparation method according to claim 1, characterized in that, The method further includes the step of preparing a hydrophobic-hydrophilic-superlubricated hydrophobic bilayer step wettability gradient driven molecular enrichment SERS substrate. Specifically, after obtaining the hydrophobic-hydrophilic wettability gradient driven molecular enrichment SERS substrate, a polymer brush is chemically grafted onto the surface of the monolayer nanoparticle assembly layer facing away from the substrate, thereby obtaining a hydrophobic-hydrophilic-superlubricated hydrophobic bilayer step wettability gradient driven molecular enrichment SERS substrate with further enhanced molecular enrichment effect.

8. The SERS substrate preparation method according to claim 1, characterized in that, The wettability gradient is used to enrich target molecules in the aqueous solution system within the hot spot gaps, thereby enhancing the test signal when detecting analytes in the aqueous solution system using Raman spectroscopy.

9. A wettability gradient-driven molecular enrichment SERS substrate prepared according to any one of claims 1-8.

10. An application of a wettability gradient-driven molecular enrichment SERS substrate prepared according to any one of claims 1-8 in Raman spectroscopy detection.

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