A sers biosensing platform with nanocavity structure and preparation method and application thereof

Abstract

The application discloses a SERS biosensing platform with a nano-cavity structure and a preparation method and application thereof, and belongs to the technical field of biosensing. The preparation method of the SERS biosensing platform comprises the following steps: preparing a SERS active substrate with a gold nano-popcorn structure, performing surface functionalization treatment on the SERS active substrate by using 4-mercapto phenylboronic acid, adding a cell extracellular vesicle-dopamine mixed solution drop prepared by using a Tris-HCl buffer solution or adding a surface positively charged polystyrene nanoparticle suspension and a dopamine single drop prepared by using a Tris-HCl buffer solution drop by drop, forming a polydopamine imprint layer covering the cell extracellular vesicles or the polystyrene nanoparticles through limited self-oxidation polymerization, and removing the cell extracellular vesicles or the polystyrene nanoparticles. The SERS biosensing platform prepared by the application can inhibit non-specific interference in a complex matrix, and realizes high-specificity, label-free, rapid and trace detection of the cell extracellular vesicles.

Description

Technical Field

[0001] This invention relates to a SERS biosensing platform, its preparation method, and its application. Specifically, it relates to a SERS biosensing platform with a nanocavity structure, its preparation method, and its application in highly specific label-free detection of low-abundance biomarkers (such as extracellular vesicles in untreated urine), belonging to the field of biosensing technology. Background Technology

[0002] Extracellular vesicles (EVs) are widely distributed in blood, urine, and other body fluids. They can carry information about proteins, lipids, and nucleic acids from cells of origin, making them important candidate biomarkers for early cancer screening, treatment monitoring, and disease subtyping. However, EVs are typically low in abundance, small in size, and come in complex sample matrices. Conventional antibody methods often rely on cumbersome enrichment and pretreatment steps, and struggle to distinguish between intact vesicles and free proteins or non-vesicle particles, resulting in high detection costs, long processing times, and poor on-site applicability.

[0003] Molecularly imprinted polymers, as biomimetic alternatives to natural antibodies, offer advantages such as high stability, low cost, and customizable recognition sites. However, traditional immersion molecular imprinting for scarce biological particles typically requires large volumes of template solution and long polymerization times, resulting in low template utilization, insufficient process reproducibility, and a tendency to produce non-specific recognition in complex biological samples.

[0004] SERS (Sequencing-Effect Arrays) are characterized by high sensitivity, strong fingerprint recognition capabilities, and suitability for trace detection. Combining a highly reproducible SERS substrate with a high-quality nanocavity imprinted layer holds promise for achieving rapid, highly specific, label-free detection of low-abundance biological particles such as EVs.

[0005] Therefore, it is of great significance to develop a molecularly imprinted SERS platform that requires less template, is prepared rapidly, and can suppress non-specific interference in complex body fluids. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the first objective of this invention is to provide a SERS biosensing platform with a nanocavity structure, capable of suppressing non-specific interference in complex matrices, based on single microdroplet confined molecular imprinting. The second objective is to provide a method for preparing the aforementioned SERS biosensing platform that requires less template, has a simpler process, and a shorter polymerization time. The third objective is to provide a method for the highly specific, label-free, and rapid detection of low-abundance biomarkers (such as extracellular vesicles in untreated urine) using the aforementioned SERS biosensing platform.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a SERS biosensing platform with a nanocavity structure includes the following steps: (1) A nano-pit structure was sequentially etched on the surface of a flexible substrate, a gold layer was deposited, perfluorodecyl mercaptan was modified, and a gold layer was deposited to obtain a SERS active substrate with a gold nano-popcorn structure. (2) The surface of the SERS active substrate was functionalized with 4-mercaptophenylboronic acid to obtain a functionalized substrate; (3) Drop a mixture of extracellular vesicles and dopamine prepared with Tris-HCl buffer onto the surface of the functionalized substrate and perform confined auto-oxidative polymerization at room temperature and in windless conditions to form a polydopamine imprint layer covering the template. Alternatively, a virtual template suspension is first dropped onto the surface of a functionalized substrate, and after standing, rinsing, and drying, a single drop of dopamine prepared with Tris-HCl buffer is dropped on. Confined auto-oxidative polymerization is carried out at room temperature and under windless conditions to form a polydopamine imprinted layer covering the virtual template, wherein the virtual template is a positively charged polystyrene nanoparticle. (4) Remove extracellular vesicles or virtual templates, rinse thoroughly, and dry to obtain a SERS biosensing platform with a nanocavity structure.

[0008] Preferably, in step (1), the flexible substrate is a polyethylene naphthalate film with a thickness of 125±10μm; the etching of the nano-pit structure is carried out in an ion beam treatment system under an oxygen atmosphere; the gold layer is deposited by thermal evaporation, with the first gold layer having a thickness of 100nm and the second gold layer having a thickness of 80nm.

[0009] Preferably, in step (2), the surface functionalization process is performed by immersing the SERS active substrate in a 4-mercaptophenylboronic acid ethanol solution for 12 hours, rinsing with ethanol, and drying at 37°C to obtain the functionalized substrate.

[0010] Preferably, in step (3), the method of dropping a mixture of extracellular vesicles and dopamine prepared with Tris-HCl buffer onto the surface of the functionalized substrate is as follows: the extracellular vesicles are dispersed in Tris-HCl buffer to obtain an EVs suspension; dopamine hydrochloride is added to the EVs suspension and the mixture is shaken to obtain a mixture; 10-50 μL of the mixture is dropped onto the surface of the functionalized substrate.

[0011] Preferably, in step (3), the method of first adding a virtual template suspension to the surface of the functionalized substrate, allowing it to stand, rinse, and dry, and then adding a single drop of dopamine prepared with Tris-HCl buffer is as follows: the virtual template is dispersed in Tris-HCl buffer to obtain a virtual template suspension; 10-50 μL of the virtual template suspension is added to the surface of the functionalized substrate, allowed to stand at room temperature for 5 min, rinsed with deionized water and dried at 37°C, and then 10-50 μL of dopamine hydrochloride solution prepared with Tris-HCl buffer is added to the surface of the substrate.

[0012] Preferably, in step (4), the method for removing extracellular vesicles is as follows: the substrate is washed three times in the elution solution, and the last time is subjected to ultrasound to remove extracellular vesicles, and then rinsed with deionized water. The elution solution is a mixed solution of acetic acid and sodium dodecyl sulfate, with the concentration of acetic acid being 2.5 wt% and the concentration of sodium dodecyl sulfate being 5 wt%.

[0013] Preferably, in step (4), the method for removing the virtual template is as follows: first, clean the substrate with deionized water, then place the substrate in toluene and use ultrasound to remove the virtual template, and rinse it clean with isopropanol.

[0014] A SERS biosensing platform with a nanocavity structure was prepared by the aforementioned method.

[0015] The aforementioned SERS biosensing platform with a nanocavity structure is used to detect extracellular vesicles in samples, wherein the sample is any one of artificial urine, real urine, plasma, or buffer solution.

[0016] The advantages of this invention are: (1) The present invention uses a single microdroplet confined polymerization strategy to construct a polydopamine molecular imprinted layer. No external oxidant or initiator is required. It relies on oxygen in the air and can achieve rapid polymerization and high-fidelity imprinting under mild conditions. It effectively maintains the natural structure of the biological template, while significantly shortening the preparation time and reducing reagent consumption. (2) The polydopamine imprinted layer constructed in this invention has the characteristics of uniform structure, adjustable thickness and high stability. It can form a specific recognition cavity that is highly matched with the target molecule in terms of size and surface chemical properties, thereby significantly improving the selective recognition ability of the target molecule and reducing non-specific adsorption interference. (3) This invention proposes a label-free SERS detection mechanism based on Raman reporter molecule diffusion blocking. The signal change is achieved by regulating the transmission of reporter molecules through the imprinted cavity, avoiding complex labeling steps. It has the advantages of simple operation, high sensitivity and suitability for trace analysis. (4) The platform has good reproducibility, stability and scale-up manufacturing potential, and is suitable for conversion into preclinical screening and on-site testing devices. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the process for preparing the SERS biosensing platform with a nanocavity structure according to the present invention; Figure 2 These are schematic diagrams of the surface structure of the substrate after polystyrene nanoparticles are fixed on the substrate surface, after polydopamine imprinting, and after template removal, as well as corresponding microstructure characterization results. The top row shows schematic diagrams of the surface structure of the substrate, and the bottom row shows the corresponding microstructure characterization results. Figure 3 This is a schematic diagram of the surface structure of the substrate of SERS biosensing platform B in different states (no template, template present, template removed, target molecule bound), as well as the corresponding microstructure characterization results and SERS signal response diagrams. The top row shows the schematic diagram of the surface structure of the substrate in different states, the middle row shows the microstructure characterization results corresponding to the top row, and the bottom row shows the SERS signal response diagrams corresponding to the middle row. Figure 4 This is a schematic diagram of the SERS detection mechanism of the SERS biosensing platform with nanocavity structure prepared in this invention. Figure 5 This is a graph showing the SERS response and quantitative analysis of EV concentration by SERS biosensor platform A. Figure 6 This is a graph showing the SERS response and quantitative analysis of EV concentration by the SERS biosensor platform B. Figure 7 This is a graph showing the test results of the SERS biosensor platform B in a complex matrix. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] I. Fabrication of a SERS biosensing platform with a nanocavity structure The process for preparing the SERS biosensing platform with a nanocavity structure according to this invention is as follows: Figure 1 As shown.

[0020] 1. Preparation of SERS active substrate with gold nanoparticle popcorn structure A polyethylene naphthalate (PEN) film with a thickness of 125±10μm was selected as the flexible substrate.

[0021] First, the PEN film is placed in an ion beam processing system and subjected to nanostructure etching in an oxygen atmosphere to form a nano-pit structure on the surface of the flexible substrate.

[0022] Subsequently, a 100 nm thick gold layer was deposited on the surface of the flexible substrate using thermal evaporation to obtain a nanostructured substrate with high-density plasma hotspots.

[0023] Then, the flexible substrate was modified with the hydrophobic molecule perfluorodecyl mercaptan.

[0024] Finally, an 80nm thick gold layer is deposited on the surface of the flexible substrate using thermal evaporation to form a three-dimensional gold nanopopcorn structure, thus obtaining a SERS active substrate with a gold nanopopcorn structure.

[0025] The resulting substrate has abundant plasmon hotspots and good batch-to-batch consistency, and can be used as the active substrate for subsequent SERS biosensing platforms.

[0026] 2. Surface functionalization of SERS active substrates The previously obtained substrate was cut into 5mm×5mm sheets and immersed in a 1mmol / L 4-mercaptophenylboronic acid (4-MPBA) ethanol solution for 12h for functionalization. This allowed 4-MPBA to be modified onto the gold surface through Au-S bonds, giving the substrate a specific binding ability to the glycosyl groups on the surface of the target biological particles. After rinsing with ethanol, the substrate was dried at 37℃ for 10min to obtain the functionalized substrate.

[0027] 3. Fix the target bioparticle template or virtual template onto the surface of the functionalized substrate. (1) Fixing the target bioparticle template onto the surface of the functionalized substrate Extracellular vesicles (EVs) were used as templates for target biological particles with an average particle size of 60 nm-160 nm.

[0028] A template-dopamine mixture prepared with Tris-HCl buffer was dropped onto the surface of a functionalized substrate, and confined auto-oxidative polymerization was carried out at room temperature and in a windless environment to form a polydopamine (PDA) imprinted layer covering the template. Specifically: EVs were dispersed in Tris-HCl buffer (10 mmol / L, pH 8.5) to achieve a concentration of 1 × 10⁻⁶ EVs. 6 Particles / μL, to obtain an EVs suspension. Add 2.5 mg of dopamine hydrochloride to 0.5 mL of the EVs suspension, shake to mix, and obtain a mixture. Take 35 μL (within the range of 10 μL-50 μL) of the mixture and drop it onto the surface of the functionalized substrate. Incubate at room temperature without significant airflow for 2 h to allow dopamine to undergo auto-oxidative polymerization within the microdroplets.

[0029] (2) Fix the virtual template on the surface of the functionalized substrate. Positively charged polystyrene nanoparticles (PS NPs) were used as virtual templates with an average particle size of 80 nm-150 nm, preferably 122 nm.

[0030] A virtual template suspension was first added to the surface of a functionalized substrate. After standing, rinsing, and drying, a single drop of dopamine prepared with Tris-HCl buffer was added. Confined auto-oxidative polymerization was then carried out under room temperature and windless conditions to form a polydopamine imprinted layer covering the virtual template. Specifically: PS NPs were dispersed in Tris-HCl buffer (10 mmol / L, pH 8.5) to a concentration of 0.025 wt%, yielding a PS NP suspension. 35 μL (or within the range of 10 μL-50 μL) of the PS NP suspension was added dropwise to the surface of a functionalized substrate. The suspension was allowed to stand at room temperature for 5 min (allowing the PS NPs to be immobilized on the substrate surface through electrostatic interactions, hydrogen bonding, and boric acid-diol interactions), then rinsed with deionized water and dried at 37 °C for 20 min. Subsequently, an equal volume (35 μL) of a 5 mg / mL dopamine hydrochloride solution prepared in Tris-HCl buffer (10 mmol / L, pH 8.5) was added dropwise to the substrate surface, and the solution was incubated at room temperature without significant airflow for 2 h.

[0031] 4. Remove the target biological particle template or virtual template. (1) The template is the target biological particle After polymerization, the substrate was washed three times in an eluent (a mixed solution of acetic acid and sodium dodecyl sulfate, wherein the concentration of acetic acid was 2.5 wt% and the concentration of sodium dodecyl sulfate was 5 wt%). The last wash was performed with 1 min of sonication to remove EVs and form EV-imprinted nanocavities. Finally, the substrate was washed with deionized water and dried to obtain a SERS biosensing platform with a nanocavity structure, denoted as SERS biosensing platform A.

[0032] (2) The template is a virtual template After polymerization, the substrate was washed with deionized water, then placed in toluene and sonicated for 1 minute to remove PS NPs, forming a virtual template-imprinted nanocavity. Finally, it was rinsed with isopropanol and dried to obtain a SERS biosensing platform with a nanocavity structure, denoted as SERS biosensing platform B.

[0033] Compared to SERS biosensor platform A, SERS biosensor platform B has advantages in terms of cost and process scale-up.

[0034] During the fabrication of the SERS biosensing platform B, the morphology of the substrate was characterized after polystyrene nanoparticles were imprinted on the substrate surface, after polydopamine imprinting, and after template removal. The characterization results are shown in [Figure number missing]. Figure 2 .Depend on Figure 2 It can be seen that polystyrene nanoparticles (PS NPs) have successfully imprinted nanocavity structures on the surface of the substrate.

[0035] II. Label-free SERS detection method First, 35 μL of the sample to be tested (e.g., urine, plasma, buffer) is dropped onto the surface of the previously prepared SERS biosensor platform A or SERS biosensor platform B and incubated at room temperature for 1 h to allow the EVs in the sample to specifically bind to the nanocavities of SERS biosensor platform A or SERS biosensor platform B.

[0036] Subsequently, the identified SERS biosensor platform A or SERS biosensor platform B was immersed in a 0.1 mmol / L IR-792 perchlorate (Raman reporter molecule) solution for 10 s, allowing the Raman reporter molecule to diffuse to the surface of the imprint layer. The permeability of the nanocavity was then investigated by observing the diffusion behavior of the Raman reporter molecule in the imprint cavity.

[0037] Subsequently, SERS signals were acquired using a Raman microscope system equipped with a 780nm laser, with an exposure time of 0.1s, a laser power of 0.125mW, and an analysis depth of 1212cm. -1 Peak intensity is determined by the concentration of EVs. The higher the concentration of EVs, the more fully the nanocavities of SERS biosensor platforms A and B are sealed, making it more difficult for IR-792 perchlorate to reach the gold nanopopcorn hotspot, resulting in a lower SERS signal. This establishes a correlation between the target concentration and peak intensity, thereby enabling the qualitative or quantitative detection of EVs.

[0038] For SERS biosensing platform B, the surface microstructure characterization results of the imprinted substrate under different states (template-free, template-present, template-removed, target molecule-bound) and the corresponding SERS signal response diagrams are shown below. Figure 3 .Depend on Figure 3 It can be seen that there is a corresponding relationship between the concentration of EVs and the peak intensity of the SERS signal, and the SERS signal can be used for qualitative or quantitative detection of EVs.

[0039] III. Mechanism Explanation Taking SERS biosensor platform B as an example, such as Figure 4As shown, the polydopamine imprinted layer consists of a non-imprinted dense region and an imprinted cavity region. The non-imprinted region is thick and dense, essentially preventing Raman reporter molecules from penetrating; the imprinted cavity region is thinner and matches the template particle size, serving as a diffusion path for the Raman reporter molecule IR-792 perchlorate to enter the substrate hotspot region when not occupied by target biological particles (EVs). When only non-specific components such as small molecules or proteins are adsorbed, IR-792 perchlorate can still penetrate the cavity and diffuse to the substrate surface, generating a strong SERS signal, because these non-specific components cannot completely fill the cavity. However, when EVs specifically match and bind to the cavity and completely occupy it, the cavity channel is fully blocked, preventing IR-792 perchlorate from reaching the plasma hotspot, resulting in a significant quenching of the SERS signal. Therefore, it can effectively distinguish between specific and non-specific recognition.

[0040] For non-imprinted platforms, there are only non-imprinted dense regions and no imprinted cavity regions, which basically prevents Raman reporter molecules from penetrating. Therefore, regardless of whether the adsorbed components are non-specific components such as small molecules and proteins or target biological particles (EVs), the SERS signal will be significantly quenched, and thus it is impossible to effectively distinguish between specific and non-specific recognition.

[0041] IV. Applicable Samples and Detection Performance The SERS response and quantitative analysis curves of SERS biosensor platform A to EV concentrations are shown in the figure. Figure 5 The SERS response and quantitative analysis curves of SERS biosensor platform B to EV concentrations are shown in the figure. Figure 6 .Depend on Figure 5 and Figure 6 It is known that the detection limit of SERS biosensor platform A is 241 particles / μL, and the detection limit of SERS biosensor platform B is 70 particles / L. Both SERS biosensor platforms A and B can achieve quantitative and trace detection of EVs. Therefore, the SERS biosensor platforms (SERS biosensor platform A and SERS biosensor platform B) with nanocavity structures prepared in this invention can be used for the detection of EVs in samples such as urine (artificial urine and real urine), plasma, and buffer solutions (e.g., PBS), and have a wide range of applications.

[0042] Due to the synergistic effects of morphological recognition of the imprinted cavities, the interaction between 4-mercaptophenylboronic acid and glycosyl groups, and the multi-site interaction of the polydopamine layer, the SERS biosensing platforms (SERS biosensing platform A and SERS biosensing platform B) with nanocavity structures prepared in this invention still exhibit high specificity in complex matrices. Taking SERS biosensing platform B as an example, its detection performance in complex matrices is shown in […]. Figure 7 .Depend on Figure 7 It can be seen that this SERS biosensing platform has high specificity.

[0043] The preferred method for detecting untreated real urine samples is to use the SERS biosensor platform B, which reduces the pre-enrichment steps and improves operational convenience and field applicability.

[0044] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention and are not intended to limit the implementation of the present invention. It should be understood that, without departing from the spirit and essence of the present invention, those skilled in the art can make other variations or modifications to the template type, droplet volume, dopamine concentration, type of functional molecule, Raman reporter molecule, and substrate structure based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for preparing a SERS biosensing platform with a nanocavity structure, characterized in that, Includes the following steps: (1) A nano-pit structure was sequentially etched on the surface of a flexible substrate, a gold layer was deposited, perfluorodecyl mercaptan was modified, and a gold layer was deposited to obtain a SERS active substrate with a gold nano-popcorn structure. (2) The surface of the SERS active substrate was functionalized with 4-mercaptophenylboronic acid to obtain a functionalized substrate; (3) Drop a mixture of extracellular vesicles and dopamine prepared with Tris-HCl buffer onto the surface of the functionalized substrate and perform confined auto-oxidative polymerization at room temperature and in windless conditions to form a polydopamine imprint layer covering the template. Alternatively, a virtual template suspension is first dropped onto the surface of a functionalized substrate, and after standing, rinsing, and drying, a single drop of dopamine prepared with Tris-HCl buffer is dropped on. Confined auto-oxidative polymerization is carried out at room temperature and under windless conditions to form a polydopamine imprinted layer covering the virtual template, wherein the virtual template is a positively charged polystyrene nanoparticle. (4) Remove extracellular vesicles or virtual templates, rinse thoroughly, and dry to obtain a SERS biosensing platform with a nanocavity structure.

2. The method for preparing the SERS biosensing platform with a nanocavity structure according to claim 1, characterized in that, In step (1), the flexible substrate is a polyethylene naphthalate film with a thickness of 125±10μm; The etching of the nano-pit structure was carried out in an oxygen atmosphere using an ion beam treatment system; the gold layer was deposited by thermal evaporation, with the first gold layer having a thickness of 100 nm and the second gold layer having a thickness of 80 nm.

3. The method for preparing the SERS biosensing platform with a nanocavity structure according to claim 1, characterized in that, In step (2), the surface functionalization process is as follows: the SERS active substrate is immersed in 4-mercaptophenylboronic acid ethanol solution for 12 hours, rinsed with ethanol and dried at 37°C to obtain the functionalized substrate.

4. The method for preparing the SERS biosensing platform with a nanocavity structure according to claim 1, characterized in that, In step (3), the method of dropping extracellular vesicle-dopamine mixture prepared with Tris-HCl buffer onto the surface of the functionalized substrate is as follows: the extracellular vesicles are dispersed in Tris-HCl buffer to obtain an EVs suspension; dopamine hydrochloride is added to the EVs suspension and shaken to mix to obtain a mixture; 10-50 μL of the mixture is dropped onto the surface of the functionalized substrate.

5. The method for preparing the SERS biosensing platform with a nanocavity structure according to claim 1, characterized in that, In step (3), the method of first adding a virtual template suspension to the surface of the functionalized substrate, allowing it to stand, rinse, and dry, and then adding a single drop of dopamine prepared with Tris-HCl buffer is as follows: the virtual template is dispersed in Tris-HCl buffer to obtain a virtual template suspension; 10-50 μL of the virtual template suspension is added to the surface of the functionalized substrate, allowed to stand at room temperature for 5 min, rinsed with deionized water and dried at 37°C, and then 10-50 μL of dopamine hydrochloride solution prepared with Tris-HCl buffer is added to the surface of the substrate.

6. The method for preparing the SERS biosensing platform with a nanocavity structure according to claim 1, characterized in that, In step (4), the method for removing extracellular vesicles is as follows: the substrate is washed three times in the elution solution, and the last time is assisted by sonication to remove extracellular vesicles. The substrate is then rinsed with deionized water. The elution solution is a mixed solution of acetic acid and sodium dodecyl sulfate, with the concentration of acetic acid being 2.5 wt% and the concentration of sodium dodecyl sulfate being 5 wt%.

7. The method for preparing the SERS biosensing platform with a nanocavity structure according to claim 1, characterized in that, In step (4), the method for removing the virtual template is as follows: first, clean the substrate with deionized water, then place the substrate in toluene and use ultrasound to remove the virtual template, and rinse it clean with isopropanol.

8. A SERS biosensing platform with a nanocavity structure, characterized in that, It is prepared by the method described in any one of claims 1 to 7.

9. The application of the SERS biosensing platform with nanocavity structure as described in claim 8 in detecting extracellular vesicles in samples.

10. The application according to claim 9, characterized in that, The sample can be any one of artificial urine, real urine, plasma, or buffer solution.

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