A biosensor and a method of manufacturing the same

Abstract

This application provides a biosensor and its fabrication method. The biosensor includes a flexible transparent substrate and a sensing and detection component formed on the flexible transparent substrate. The sensing and detection component includes a conical nanostructure layer, a first metal thin film layer, and a second metal thin film layer. The conical nanostructure layer is formed on the flexible transparent substrate, the first metal thin film layer is formed on the conical nanostructure layer, and the second metal thin film layer is formed on the first metal thin film layer. The first metal thin film layer and the second metal thin film layer are made of different metal materials. The sensing and detection component is used to excite local surface plasmon resonance of the metal layer on the conical nanostructure layer under laser irradiation, generating an electromagnetic field enhancement region. The electromagnetic field enhancement region is enhanced through the coupling between the first metal thin film layer and the second metal thin film layer, achieving a balance between high SERS activity and high transmittance, thereby improving the stability of the SERS signal and the detection efficiency.

Description

Technical Field

[0001] This application relates to the field of biosensor technology, and more specifically, to a biosensor and its preparation method. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) is a Raman spectroscopy technique that provides characteristic molecular fingerprint information with molecular-level sensitivity. This enhancement effect primarily originates from electromagnetic field enhancement regions (i.e., "hot spots") near metal nanostructures caused by plasmon interactions. Due to its non-destructive nature, high sensitivity, and low sample requirements, this technique has been applied in fields such as chemical sensing, food safety, environmental monitoring, and drug analysis.

[0003] Traditional SERS substrates include rigid substrates, flexible substrates, and flexible transparent substrates. Flexible transparent substrates enable them to operate in transmission mode, meaning that both excitation light and signal can be emitted from the back side of the substrate. This allows for in-situ detection on any surface to which the substrate can be attached, improving the applicability of non-destructive testing.

[0004] However, achieving a balance between high SERS activity and high optical transmittance remains a common technical challenge. While continuous and thick metal films are beneficial for enhancing the SERS signal, they significantly reduce substrate transmittance. Conversely, if the metal nanoparticles are too sparse, although transparency can be maintained, insufficient hotspot density leads to weak signal strength and poor stability. Therefore, it is necessary to construct a structural design that can simultaneously achieve high-density electromagnetic hotspots and high transmittance using a simple and scalable fabrication process, but this combination remains difficult to realize. Summary of the Invention

[0005] The purpose of this application is to provide a biosensor and its preparation method, which can achieve rapid, highly stable and highly repeatable in-situ detection of biological surface residues.

[0006] In a first aspect, this application provides a biosensor, including a flexible transparent substrate and a sensing and detection component formed on the flexible transparent substrate; the sensing and detection component includes a conical nanostructure layer, a first metal thin film layer, and a second metal thin film layer, wherein the conical nanostructure layer is formed on the flexible transparent substrate, the first metal thin film layer is formed on the conical nanostructure layer, and the second metal thin film layer is formed on the first metal thin film layer; wherein the first metal thin film layer and the second metal thin film layer are made of different metal materials; the sensing and detection component is used to excite local surface plasmon resonance of the metal layer on the conical nanostructure layer under laser irradiation to generate an electromagnetic field enhancement region, and to enhance the electromagnetic field enhancement region through the coupling between the first metal thin film layer and the second metal thin film layer.

[0007] The biosensor designed above firstly employs a flexible transparent substrate. Its bendable and foldable properties allow the sensor to be attached to any irregular surface (such as biological tissue, food surfaces, environmental sample surfaces, etc.). Simultaneously, the high light transmittance of the flexible transparent substrate enables transmission mode detection; excitation light and Raman signals can penetrate from the back of the substrate, allowing in-situ detection by attaching the sensor to the sample surface. Secondly, the conical morphology of the conical nanostructure layer in this application provides the substrate with more hot spots, a larger adhesion area, and a more robust solid support. Finally, a first metal thin film layer and a second metal thin film layer are designed and formed on the conical nanostructure layer. The thin film layer, on the one hand, generates coupling through the local surface electromagnetic fields of adjacent metal structures, which can further enhance the surface electromagnetic field, and strong plasma waves are also generated between adjacent metal layers, which are further conducted to the edge of the conical nanostructure layer to promote the enhancement of the electric field. On the other hand, the first thin film metal layer and the second metal thin film layer adopt a thin film layer design, which reduces the obstruction of light and ensures the high light transmittance of the flexible transparent substrate. The two work together to achieve a balance between high SERS activity and high light transmittance, thereby improving the stability of SERS signal and detection efficiency, and thus providing a biosensor that can achieve rapid, highly stable and highly repeatable in-situ detection of biological surface residues.

[0008] In an optional embodiment of the first aspect, the conical nanostructure layer includes a plurality of conical nanostructures, which are periodically spaced on a flexible transparent substrate to form a conical nanoarray, and both the first metal thin film layer and the second metal thin film layer cover the surface of the conical nanoarray.

[0009] In the above-described embodiments, the present application designs a conical nanostructure layer by periodically arranging multiple conical nanostructures 2 on a flexible transparent substrate to form a conical nanoarray. This allows the hot spots generated by each conical nanostructure to couple with each other, forming an array synergistic enhancement effect, resulting in an order-of-magnitude increase in the hot spot density of the entire sensing and detection component. Simultaneously, the array structure enables incident light to undergo multiple scattering and focusing on the surfaces of the multiple conical nanostructures, further exciting stronger local surface plasmon resonance, thereby significantly improving the SERS signal intensity and meeting the detection requirements of lower concentrations of biomolecules. Furthermore, the periodic spacing ensures that the size and spacing of each conical nanostructure are consistent. After the first and second metal thin film layers are applied, the metal layer thickness and coverage uniformity on the surface of each conical nanostructure are also consistent, resulting in essentially the same hot spot intensity generated by each conical nanostructure. The hot spots are uniformly distributed across the entire sensing and detection component surface, significantly improving the stability and repeatability of the SERS signal and reducing detection errors.

[0010] In an optional embodiment of the first aspect, each conical nanostructure is a regular square pyramidal nanostructure; wherein the base length of each regular square pyramidal nanostructure ranges from 900 nm to 1100 nm, the height of each regular square pyramidal nanostructure ranges from 650 nm to 750 nm, and the lateral period of the conical nanoarray ranges from 1100 nm to 1300 nm.

[0011] In the above embodiments, this application optimizes the array density, the electric field enhancement effect of individual structures, and the light transmission efficiency by limiting the specific dimensions and lateral period of the regular square pyramid nanostructure. Specifically, limiting the base length and height ensures that a single regular square pyramid can excite strong plasma resonance, generating high-intensity hotspots; limiting the lateral period ensures reasonable array spacing, avoiding light obstruction and achieving synergistic enhancement of hotspots. Ultimately, this allows the sensor to maintain high optical transmittance while possessing ultra-high SERS activity, and significantly improves performance stability. In addition, the regular square pyramid nanostructure has a symmetrical morphology and well-defined dimensions, making it easier to fabricate using mature processes such as nanoimprinting compared to irregular cone structures, with smaller dimensional errors. At the same time, the well-defined lateral period ensures a clear array arrangement pattern, which can be precisely controlled by molds to ensure the structural consistency of each sensor produced in batches, improving product repeatability and providing a reliable dimensional basis for large-scale production.

[0012] In an alternative embodiment of the first aspect, the angle between the sidewall of each conical nanostructure and the flexible transparent substrate ranges from 45° to 55°.

[0013] In the above embodiments, the angle α between the sidewall of each conical nanostructure and the flexible transparent substrate is designed to be in the range of 45° to 55°. Within this range, the tilt angle of the conical sidewall is moderate, and when incident light irradiates the sidewall, moderate reflection and scattering occur, which can effectively excite local surface plasmon resonance of the metal thin film on the sidewall surface. At the same time, the electromagnetic field of the sidewall can couple with the electromagnetic field of the conical tip, further enhancing the hot spot intensity. Furthermore, within this 45° to 55° angle range, the tilt angle of the sidewall facilitates the uniform coverage of the first metal thin film layer 220 and the second metal thin film layer 230. The metal thin film can tightly adhere to the sidewall surface, forming a continuous and uniform metal layer, avoiding problems such as metal layer detachment and uneven coverage caused by excessively steep sidewalls. This uniform coverage of the metal thin film ensures a consistent surface electromagnetic field distribution and uniform hot spot intensity for each conical nanostructure, thereby significantly improving the stability and repeatability of the SERS signal and reducing detection errors.

[0014] In an optional embodiment of the first aspect, the metal material of the first metal thin film layer can be any one of aluminum, copper, and aluminum oxide; the metal material of the second metal thin film layer can be any one of silver and gold.

[0015] In the above embodiments, the second metal thin film layer of this application is designed with highly active SERS metals such as silver and gold, which have high free electron concentration and strong local surface plasmon resonance effect, and can generate extremely strong hot spots, significantly improving the SERS signal intensity. The aluminum, copper and aluminum oxide of the first metal thin film layer can provide a stable adhesion substrate for the second metal thin film layer, enhance the bonding force between the metal layer and the conical nanostructure layer, and prevent the second metal thin film layer from falling off. At the same time, the local surface electromagnetic fields of adjacent metal structures generate a coupling effect, which can further enhance the surface electromagnetic field. In addition, strong plasma waves are also generated between adjacent metal layers, which are further conducted to the edge of the conical nanostructure layer to promote the enhancement of the electric field.

[0016] In an alternative embodiment of the first aspect, the thickness of the first metal thin film layer ranges from 10 nm to 20 nm, and the thickness of the second metal thin film layer ranges from 5 nm to 15 nm.

[0017] In the above-described embodiments, this application achieves a precise match between the coverage and transmittance of the metal thin films by limiting the specific thickness range of the two metal thin films. Specifically, the thickness of the first metal thin film layer, ranging from 10nm to 20nm, ensures continuous coverage and initial enhancement, while the thickness of the second metal thin film layer, ranging from 5nm to 15nm, ensures efficient enhancement. The total thickness of the two layers is controlled within a reasonable range, avoiding both the decrease in transmittance due to excessive thickness and the insufficient SERS intensity due to excessive thinness, thus achieving a precise balance between the two and optimizing the overall performance of the sensor.

[0018] In an alternative embodiment of the first aspect, the flexible transparent substrate is made of one of PET, PDMS, nanofibers and biofilms, and the thickness of the flexible transparent substrate ranges from 12 μm to 250 μm.

[0019] In the above embodiments, the selected PET, PDMS, nanofibers, and biofilms all have high light transmittance and good flexibility, which can ensure the smooth realization of transmission mode detection. At the same time, the thickness range of 12μm to 250μm allows the substrate to be flexibly attached to any irregular surface (such as biological tissue, food surface, environmental sample surface, etc.), without being damaged due to being too thin or unable to be attached due to being too thick, which greatly broadens the application scenarios of the sensor.

[0020] Secondly, this application provides a method for fabricating a biosensor, which includes forming a conical nanostructure layer on a flexible transparent substrate by nanoimprinting; sequentially forming a first metal thin film layer and a second metal thin film layer on the flexible transparent substrate on which the conical nanostructure layer is formed, such that the first metal thin film layer and the second metal thin film layer cover the surface of the conical nanostructure layer, thereby obtaining a biosensor.

[0021] The biosensor fabrication method described above utilizes nanoimprinting and metal thin film deposition to fabricate the biosensor. Compared to existing complex SERS substrate fabrication processes, this method eliminates the need for complex equipment, cumbersome operations, and expensive chemical reagents. The nanoimprinting process enables batch replication of tapered nanostructures, significantly improving fabrication efficiency, and the reusable molds greatly reduce fabrication costs. The metal thin film deposition method is mature, with strong process compatibility, and can be continuously integrated with the nanoimprinting process to achieve large-scale continuous production, solving the technical challenge of large-scale fabrication of existing flexible transparent SERS sensors. Furthermore, the fabricated biosensor can achieve rapid, highly stable, and highly repeatable in-situ detection of biological surface residues.

[0022] In an optional embodiment of the second aspect, a conical nanostructure layer is formed by nanoimprinting on a flexible transparent substrate, comprising: drop-coating a UV-curable adhesive onto the flexible transparent substrate; imprinting the UV-curable adhesive using a conical nanostructure mold, controlling the thickness of the imprinted residual layer to be in the range of 30 nm to 100 nm; and irradiating the imprinted UV-curable adhesive with UV light to solidify the imprinted conical nanostructure layer on the flexible transparent substrate.

[0023] In the above-described embodiments, this application achieves rapid and automated fabrication of cone-shaped nanostructure layers through three simple steps: dispensing, imprinting, and UV curing. The entire process eliminates the need for complex post-processing (such as etching and cleaning), resulting in a seamless workflow that significantly improves fabrication efficiency and meets the demands of large-scale mass production. Simultaneously, the limited residual layer thickness of 30nm to 100nm ensures a tight bond between the cone-shaped nanostructure and the flexible transparent substrate, preventing detachment during subsequent fabrication and use. It also avoids the decrease in transmittance and weakening of the electric field concentration effect caused by an excessively thick residual layer. Furthermore, precise UV irradiation ensures complete curing of the UV-curable adhesive, resulting in a stable morphology of the cured cone-shaped nanostructure that does not deform. This avoids fluctuations in the plasma resonance effect caused by structural size deviations or morphological deformation, thereby improving the stability and repeatability of the sensor's SERS signal.

[0024] In an alternative embodiment of the second aspect, a first metal thin film layer and a second metal thin film layer are sequentially formed on a flexible transparent substrate on which a tapered nanostructure layer is formed, including: using any one of electron beam evaporation, thermal evaporation, or magnetron sputtering to sequentially form the first metal thin film layer and the second metal thin film layer on the flexible transparent substrate on which the tapered nanostructure layer is formed.

[0025] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the overall structure of the biosensor provided in the embodiments of this application; Figure 2 This is a schematic diagram of the structure of the cone-shaped nanostructure array provided in the embodiments of this application; Figure 3 A schematic diagram illustrating parameter annotations for the biosensor provided in an embodiment of this application; Figure 4 This is a first flowchart of a biosensor fabrication method provided in an embodiment of this application; Figure 5 This is a second flowchart of the biosensor fabrication method provided in the embodiments of this application; Figure 6 Nine sets of surface-enhanced Raman spectra for in-situ detection of methylene blue on the surface of crucian carp using a biosensor.

[0028] Figure 7 (a) Detection concentration range of 10 in the back-side collection mode of the biosensor -7 ~10 -3 Surface-enhanced Raman spectrum of mol / L methylene blue solution; Figure 7 (b) Detection of concentration 10 in the back-side collection mode of the biosensor -5 Graph of repeatability test results at mol / L; Figure 8 (a) In the back-end excitation / collection mode of the biosensor at 10 -15 ~10 -10 Surface-enhanced Raman spectra of R6G in the mol / L concentration range; Figure 8 (b) Raman mapping diagram of the biosensor in frontal excitation / collection mode; Figure 8 (c) is a Raman mapping diagram of the back of the biosensor in excitation / collection mode.

[0029] Reference numerals: 10-Flexible transparent substrate; 20-Sensing and detection component; 210-Conical nanostructure layer; 220-First metal thin film layer; 230-Second metal thin film layer; 2110-Conical nanostructure; L1-Base side length; L2-Lateral period; h-Height; a-Included angle; d1-Thickness of flexible transparent substrate; d2-Thickness of imprinted residual layer; d3-Thickness of first metal thin film layer; d4-Thickness of second metal thin film layer. Detailed Implementation

[0030] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0032] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.

[0033] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0034] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0035] In the description of the embodiments of this application, the technical terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0036] In the description of the embodiments of this application, unless otherwise expressly specified and limited, technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. For those skilled in the art, the specific meaning of the above terms in the embodiments of this application can be understood according to the specific circumstances.

[0037] Surface-enhanced Raman scattering (SERS) is a Raman spectroscopy technique that provides characteristic molecular fingerprint information with molecular-level sensitivity. This enhancement effect primarily originates from electromagnetic field enhancement regions (i.e., "hot spots") near metal nanostructures caused by plasmon interactions. Due to its non-destructive nature, high sensitivity, and low sample requirements, this technique has been applied in fields such as chemical sensing, food safety, environmental monitoring, and drug analysis.

[0038] Traditional SERS substrates include rigid substrates, flexible substrates, and flexible transparent substrates. Flexible transparent substrates enable them to operate in transmission mode, meaning that both excitation light and signal can be emitted from the back side of the substrate. This allows for in-situ detection on any surface to which the substrate can be attached, improving the applicability of non-destructive testing.

[0039] However, achieving a balance between high SERS activity and high optical transmittance remains a common technical challenge. While continuous and thick metal films are beneficial for enhancing the SERS signal, they significantly reduce substrate transmittance. Conversely, if the metal nanoparticles are too sparse, although transparency can be maintained, insufficient hotspot density leads to weak signal strength and poor stability. Therefore, it is necessary to construct a structural design that can simultaneously achieve high-density electromagnetic hotspots and high transmittance using a simple and scalable fabrication process, but this combination remains difficult to realize.

[0040] To address the aforementioned issues, this application first provides a biosensor and its fabrication method. Firstly, this application employs a flexible transparent substrate, whose bendable and foldable properties allow the sensor to be attached to any irregular surface (such as biological tissue, food surfaces, environmental sample surfaces, etc.). Simultaneously, the high light transmittance of the flexible transparent substrate enables transmission mode detection; excitation light and Raman signals can penetrate from the back of the substrate, allowing in-situ detection by attaching the sensor to the sample surface. Secondly, the conical morphology of the conical nanostructure layer in this application enables the substrate to have more hot spots, a larger adhesion area, and a more robust solid support. Finally, a first metal thin film layer is designed and formed on the conical nanostructure layer, and a first metal... The second metal thin film layer on the thin film layer, on the one hand, can further enhance the surface electromagnetic field by coupling with the local surface electromagnetic field of the adjacent metal structure, and strong plasma waves are also generated between the adjacent metal layers, which are further conducted to the edge of the conical nanostructure layer to promote the enhancement of the electric field. On the other hand, the first thin film metal layer and the second metal thin film layer adopt a thin film layer design, which reduces the obstruction of light and ensures the high light transmittance of the flexible transparent substrate. The two work together to achieve a balance between high SERS activity and high light transmittance, thereby improving the stability of SERS signal and detection efficiency, and thus providing a biosensor that can achieve rapid, high stability and high repeatability in-situ detection of biological surface residues.

[0041] Based on the above ideas, this application first provides a biosensor, such as... Figure 1 As shown, the biosensor includes a flexible transparent substrate 10 and a sensing and detection component 20 formed on the flexible transparent substrate. The sensing and detection component 20 includes a conical nanostructure layer 210, a first metal thin film layer 220, and a second metal thin film layer 230. The conical nanostructure layer 210 is formed on the flexible transparent substrate 10, the first metal thin film layer 220 is formed on the conical nanostructure layer 210, and the second metal thin film layer 230 is formed on the first metal thin film layer 220.

[0042] The flexible transparent substrate 10 designed in this application refers to a substrate material with bendable and foldable properties and high transmittance to visible light, used to support sensing and detection components, while ensuring the smooth transmission of excitation light and Raman signals to achieve transmission mode detection.

[0043] The flexible transparent substrate 10 serves as the carrier substrate for the biosensor, and its material and thickness directly determine the sensor's flexibility, light transmittance, biocompatibility, and mechanical stability. Specifically, the flexible transparent substrate 10 designed in this application can be made of a material with high light transmittance and good flexibility, such as PET, PDMS, nanofibers, and biomembranes. To achieve a balance between flexibility and mechanical strength, such as... Figure 3As shown, the thickness d1 of the flexible transparent substrate designed in this application can be any thickness from 12μm to 250μm. Within the thickness range of 12μm to 250μm, the substrate can ensure good flexibility (bendable, foldable, and attachable) while possessing sufficient mechanical strength to avoid breakage, and simultaneously ensuring high light transmittance to ensure the smooth transmission of excitation light and Raman signals, thereby achieving efficient transmission mode detection. Specifically, the thickness d1 of the flexible transparent substrate designed in this application can be any thickness from 12μm, 20μm, 25μm, 30μm, 40μm, 50μm, 70μm, 80μm, 90μm, 100μm, 150μm, 200μm, 220μm, 240μm, and 250μm.

[0044] The conical nanostructure layer 210 refers to a nanoscale thin film layer with a conical protrusion structure formed on the surface of a flexible transparent substrate, which makes the substrate have more hot spots, a larger adhesion area and a more robust solid support.

[0045] The first metal thin film layer 220 refers to the metal thin film covering the surface of the conical nanostructure layer 210, which is used to initially enhance the electromagnetic field generated by the conical nanostructure layer, and at the same time provide a stable adhesion substrate for the second metal thin film layer 230.

[0046] The second metal thin film layer 230 refers to a metal thin film covering the surface of the first metal thin film layer 220. It is made of a different material than the first metal thin film layer 220 and is used to further enhance the electromagnetic field.

[0047] Localized surface plasmon resonance (LSPR) refers to the phenomenon where, when a laser (usually visible or near-infrared light) irradiates the surface of a metal nanostructure, the free electrons on the metal surface undergo collective oscillations under the influence of the light field, forming localized plasma waves. The direct result is the formation of a region (hot spot) with extremely strong electromagnetic field enhancement near the metal nanostructure.

[0048] The biosensor designed above, when attached to the sample standard to be tested, ensures that the excitation light can pass through the substrate smoothly and irradiate the sensing and detection component 20 on the surface due to the high light transmittance of the flexible transparent substrate 10. At the same time, it also ensures that the Raman signal generated by the sensing and detection component 20 can pass through the substrate smoothly and be captured by the detector, so as to realize in-situ detection in transmission mode. Specifically, when excitation light irradiates the sensing and detection component 20, the cone-shaped nanostructure layer 210, due to its special cone shape, causes the incident light to be focused and scattered on its surface, thereby exciting local surface plasmon resonance between the cone-shaped nanostructure layer 210 and the first metal thin film layer 220 and the second metal thin film layer 230, generating an initial electromagnetic field enhancement region (hot spot). At the same time, due to the presence of the first metal thin film layer 220 and the second metal thin film layer 230, the local surface electromagnetic fields of adjacent metal structures are coupled, which can further enhance the surface electromagnetic field. Strong plasma waves are also generated between adjacent first metal thin film layers 220 and second metal thin film layers 230, which are further conducted to the edge of the cone-shaped nanostructure layer 210 to promote the enhancement of the electric field. The above combined effects ultimately form a high-density, high-intensity electromagnetic field enhancement region, thereby significantly enhancing the Raman scattering signal captured by the detector.

[0049] The biosensor designed above firstly employs a flexible transparent substrate. Its bendable and foldable properties allow the sensor to be attached to any irregular surface (such as biological tissue, food surfaces, environmental sample surfaces, etc.). Simultaneously, the high light transmittance of the flexible transparent substrate enables transmission mode detection; excitation light and Raman signals can penetrate from the back of the substrate, allowing in-situ detection by attaching the sensor to the sample surface. Secondly, the conical morphology of the conical nanostructure layer in this application provides the substrate with more hot spots, a larger adhesion area, and a more robust solid support. Finally, a first metal thin film layer and a second metal thin film layer are designed and formed on the conical nanostructure layer. The thin film layer, on the one hand, couples with the local surface electromagnetic fields of adjacent metal structures, further enhancing the surface electromagnetic field. Strong plasma waves are also generated between adjacent metal layers, which are further conducted to the edge of the conical nanostructure layer, promoting electric field enhancement. On the other hand, the first and second thin film metal layers employ a layered design, reducing light obstruction and ensuring high light transmittance of the flexible transparent substrate. These two elements work synergistically to achieve a balance between high SERS activity and high transmittance, thereby improving the stability and detection efficiency of the SERS signal. This provides a biosensor capable of rapid, highly stable, and highly repeatable in-situ detection of biological surface residues. The sensing and detection component consists only of a conical nanostructure layer and two different metal thin film layers, with a clear structural hierarchy, eliminating the need for complex nano-assembly processes. Each layer can be fabricated using mature thin film preparation technologies (such as nanoimprinting, evaporation, and sputtering), requiring no special equipment or complex operations. Compared to existing complex SERS substrate structures, the structural design of this application is easier to scale up for production, reducing manufacturing costs.

[0050] In an optional implementation of this embodiment, such as Figure 2 As shown, the conical nanostructure layer 210 designed in this application includes multiple conical nanostructures 2110, which are periodically spaced on the flexible transparent substrate 10 to form a conical nanoarray. A first metal thin film layer 220 and a second metal thin film layer 230 both cover the surface of the conical nanoarray. The periodic spacing means that the distance (lateral period) between the centers of the multiple conical nanostructures 2110 remains consistent, and a uniform gap is left between adjacent conical nanostructures 2110. This arrangement allows incident light to form uniform scattering and focusing on the array surface, avoiding mutual interference of local electromagnetic fields, while ensuring a uniform distribution of hot spots.

[0051] In the above embodiments, the conical nanostructure layer 210 is designed as a conical nanoarray formed by the periodic arrangement of multiple conical nanostructures 2110, rather than a disordered conical structure. On the one hand, the periodically arranged conical nanoarray enables the incident light to be periodically scattered and focused on the array surface. Each conical nanostructure acts as an independent hot spot generating unit, and its generated local electromagnetic field can couple with the electromagnetic field generated by adjacent conical nanostructures to form an array synergistic enhancement effect, further improving the overall electromagnetic field strength and hot spot density. On the other hand, the periodic interval setting can ensure that there are uniform gaps between adjacent conical nanostructures 2110. These gaps can reduce the overall coverage of the metal material, avoid blocking light, ensure the high light transmittance of the substrate, and allow target biomolecules to smoothly enter the hot spot area in the gap, thereby improving the effectiveness of detection.

[0052] In addition, the first metal thin film layer 220 and the second metal thin film layer 230 are both covered on the surface of the conical nanoarray to form a uniform metal covering layer, so that a stable plasma resonance region can be formed on the surface of each conical nanostructure 2110, avoiding hot spot intensity fluctuations caused by uneven surface metal coverage of a single conical nanostructure, and further improving the stability and uniformity of the SERS signal.

[0053] In the above embodiments, the present application designs a tapered nanostructure layer by periodically spacing multiple tapered nanostructures 2 on a flexible transparent substrate to form a tapered nanoarray. This allows the hot spots generated on the surface of each tapered nanostructure to couple with each other, forming an array synergistic enhancement effect, resulting in an order-of-magnitude increase in the hot spot density of the entire sensing and detection component. Simultaneously, the array structure enables incident light to undergo multiple scattering and focusing on the surfaces of the multiple tapered nanostructures, further exciting stronger local surface plasmon resonance, thereby significantly improving the SERS signal intensity and meeting the detection requirements of lower concentrations of biomolecules. Furthermore, the periodic spacing ensures that the size and spacing of each tapered nanostructure are consistent. After the first and second metal thin film layers are applied, the metal layer thickness and coverage uniformity on the surface of each tapered nanostructure are also consistent, resulting in essentially the same hot spot intensity generated by each tapered nanostructure. The hot spots are uniformly distributed across the entire sensing and detection component surface, significantly improving the stability and repeatability of the SERS signal and reducing detection errors.

[0054] In an optional implementation of this embodiment, as a specific implementation method, such as Figure 2 and Figure 3As shown, each conical nanostructure 2110 designed in this application is a regular square pyramid nanostructure. The regular square pyramid nanostructure refers to a conical nanostructure with a regular quadrilateral base, four congruent isosceles triangles on all four sides, and the projection of the vertex onto the base coincides with the center of the regular quadrilateral base, which is similar to a pyramid-shaped structure. In this way, by using the regular square pyramid nanostructure as an array unit, its structure is symmetrical, which can uniformly scatter and focus incident light in all directions, avoiding the problem of uneven electromagnetic field distribution caused by asymmetrical conical structures, ensuring that the hot spot intensity is consistent in each direction, and improving the uniformity of detection. At the same time, the four sides of the regular square pyramid structure are all inclined surfaces, which can increase the contact area between the metal thin film and the incident light, and further excite strong local surface plasmon resonance.

[0055] The size of the conical nanostructure array is closely related to its hotspot generation effect. The size of the conical nanostructure array can include the base side length L1 and height h of each conical nanostructure, as well as the transverse period L2 of the conical nanostructure array.

[0056] Specifically, if the base side length L1 of the regular square pyramid nanostructure is too small, the surface area of ​​the structure will be too small, making it difficult to excite sufficiently rich plasmon resonance and resulting in insufficient hotspot density. If the base side length is too large, a single regular square pyramid structure will occupy too much space, leading to an overly dense array arrangement with too small gaps, blocking light and reducing substrate transmittance. This also significantly increases the fabrication difficulty of the nanoimprint mask. Therefore, to balance plasmon resonance excitation and transmittance, this application designs the base side length L1 of each regular square pyramid nanostructure to be in the range of 900 nm to 1100 nm. This ensures that the base side length L1 of the regular square pyramid satisfies the requirement of sufficiently strong plasmon resonance excitation without occupying excessive space and blocking light. As a specific embodiment, the base side length L1 of the regular square pyramid nanostructure in this application can be any length selected from 900 nm, 925 nm, 950 nm, 975 nm, 1000 nm, 1025 nm, 1050 nm, 1075 nm, or 1100 nm.

[0057] The ratio of height h to the base side length L1 determines the steepness of the regular square pyramid. In this application, the height h of each regular square pyramid nanostructure ranges from 650 nm to 750 nm. This moderate steepness ensures both the electric field concentration effect at the tip and a reasonable tilt angle on the sides, facilitating uniform coverage of the metal thin film. As a specific embodiment, the height h of the regular square pyramid nanostructure in this application can be any of 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, or 750 nm.

[0058] The transverse period L2 of the cone-shaped nanoarray designed in this application ranges from 1100 nm to 1300 nm. This transverse period L2 range matches the length of the base side, resulting in a gap of 200 nm to 300 nm between adjacent regular square pyramids. This gap size ensures sufficient light penetration while also guaranteeing effective coupling of the electromagnetic fields of adjacent structures, generating a synergistic enhancement effect. Simultaneously, it facilitates the entry of target biomolecules into the hotspot regions within the gaps. As a specific embodiment, the transverse period L2 of the regular square pyramid nanoarray of this application can be any transverse period value selected from 1100 nm, 1125 nm, 1150 nm, 1175 nm, 1200 nm, 1225 nm, 1250 nm, 1275 nm, or 1300 nm.

[0059] In the above embodiments, this application optimizes the array density, electric field concentration effect of individual structures, and light transmission efficiency by limiting the specific dimensions and lateral period of the regular square pyramid nanostructure. Specifically, limiting the base length and height ensures that a single regular square pyramid can excite strong plasma resonance, generating high-intensity hotspots; limiting the lateral period ensures reasonable array spacing, avoiding light obstruction and achieving synergistic enhancement of hotspots. Ultimately, this allows the sensor to maintain high optical transmittance while possessing ultra-high SERS activity, and significantly improves performance stability. In addition, the regular square pyramid nanostructure has a symmetrical morphology and well-defined dimensions, making it easier to fabricate using mature processes such as nanoimprinting compared to irregular cone structures, with smaller dimensional errors. At the same time, the well-defined lateral period ensures a clear array arrangement pattern, which can be precisely controlled by molds to ensure structural consistency of each sensor produced in batches, improving product repeatability and providing reliable dimensional basis for large-scale production.

[0060] In an optional embodiment of this invention, the angle α between the sidewall of each conical nanostructure and the flexible transparent substrate is designed to range from 45° to 55°. Here, the angle α between the sidewall of the conical nanostructure and the flexible transparent substrate represents the angle between the side face of the conical nanostructure (for a regular square pyramid, the plane containing the legs of the four isosceles triangles) and the surface (horizontal plane) of the flexible transparent substrate.

[0061] As a specific embodiment, the angle α between the sidewall of each conical nanostructure designed in this application and the flexible transparent substrate can be any angle among 45°, 46°, 47°, 48°, 49°, 50°, 51°, 52°, 53°, 54° and 55°.

[0062] In the above embodiments, the angle α between the sidewall of each conical nanostructure and the flexible transparent substrate is designed to be in the range of 45° to 55°. Within this range, the tilt angle of the conical sidewall is moderate, and when incident light irradiates the sidewall, moderate reflection and scattering occur, which can effectively excite local surface plasmon resonance of the metal thin film on the sidewall surface. At the same time, the electromagnetic field of the sidewall can couple with the electromagnetic field of the conical tip, further enhancing the hot spot intensity. Furthermore, within this 45° to 55° angle range, the tilt angle of the sidewall facilitates the uniform coverage of the first metal thin film layer 220 and the second metal thin film layer 230. The metal thin film can tightly adhere to the sidewall surface, forming a continuous and uniform metal layer, avoiding problems such as metal layer detachment and uneven coverage caused by excessively steep sidewalls. This uniform coverage of the metal thin film ensures a consistent surface electromagnetic field distribution and uniform hot spot intensity for each conical nanostructure, thereby significantly improving the stability and repeatability of the SERS signal and reducing detection errors.

[0063] In an optional embodiment of this example, the conical nanostructure layer 210 generates an imprinted residual layer during the formation process. The thickness d2 of the imprinted residual layer can range from 30 nm to 100 nm. The residual layer thickness of 30 nm to 100 nm can ensure the bonding force between the conical nanostructure and the substrate, avoid the influence on light transmittance and electric field concentration effect, and facilitate the uniform coverage of the subsequent metal thin film.

[0064] Specifically, as a specific embodiment, the imprint residual layer thickness d2 designed in this application can be any thickness value among 30nm, 35nm, 40nm, 45nm, 50nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm or 100nm.

[0065] In an optional embodiment of this invention, the metal material of the first metal thin film layer 220 designed in this application can be any one of aluminum, copper, and aluminum oxide; the metal material of the second metal thin film layer 230 can be any one of silver and gold.

[0066] In the above embodiments, the first metal thin film layer 220 and the second metal thin film layer 230 are designed to be made of different materials. The core is to utilize the performance differences between the two metal materials to achieve the functions of substrate support and efficient reinforcement, while improving the electromagnetic field enhancement effect through material coupling.

[0067] Specifically, the first metal thin film layer 220 (any one of aluminum, copper, or aluminum oxide) serves as the bottom metal layer, its core function being to provide a stable substrate for the second metal thin film layer 230, while simultaneously achieving initial enhancement of the electromagnetic field. Aluminum, copper, and aluminum oxide all possess good conductivity and compatibility with thin film fabrication processes, enabling them to be uniformly coated onto the surface of the conical nanostructure layer through processes such as evaporation and sputtering, forming a dense and uniform bottom layer. Among these, aluminum oxide, as a metal oxide, also exhibits good insulation and chemical stability, reducing charge transfer interference between the second metal thin film layer 230 and the conical nanostructure layer 210, while protecting the second metal thin film layer 230 from oxidation. Furthermore, the plasmonic resonance frequencies of these materials complement the materials of the second metal thin film layer 220 (silver or gold), broadening the response range of the excitation light and enhancing the applicability of the sensor.

[0068] The second metal thin film layer 230, as the top metal layer, has the core function of efficiently enhancing the electromagnetic field and improving the SERS signal strength and stability. Silver and gold are the two metals with the best performance in the SERS effect. They have a high concentration of free electrons and can generate strong localized surface plasmon resonance when irradiated by incident light, forming extremely strong hot spots. At the same time, silver and gold have good chemical stability, which can ensure the long-term stable use of the sensor in complex detection environments (such as body fluids, food samples, environmental water, etc.).

[0069] In the above embodiments, the second metal thin film layer designed in this application uses highly active SERS metals such as silver and gold, which have high free electron concentration and strong local surface plasmon resonance effect, and can generate extremely strong hot spots, significantly improving the SERS signal intensity. The aluminum, copper, and aluminum oxide of the first metal thin film layer can provide a stable adhesion substrate for the second metal thin film layer, enhance the bonding force between the metal layer and the conical nanostructure layer, and prevent the second metal thin film layer from falling off. At the same time, the local surface electromagnetic fields of adjacent metal structures generate a coupling effect, which can further enhance the surface electromagnetic field. In addition, strong plasma waves are also generated between adjacent metal layers, which are further conducted to the edge of the conical nanostructure layer to promote the enhancement of the electric field.

[0070] In an optional embodiment of this invention, the thickness of the metal thin film is one of the key factors affecting SERS activity and optical transmittance. This application achieves optimal matching between the two by limiting the specific thickness range of the first metal thin film layer 220 and the second metal thin film layer 230, while taking into account the enhancement effect, transmittance and film stability.

[0071] Specifically, the thickness d3 of the first metal thin film layer designed in this application ranges from 10 nm to 20 nm, and the thickness d4 of the second metal thin film layer ranges from 5 nm to 15 nm. Here, the thickness d3 of the first metal thin film layer represents the vertical thickness of the first metal thin film covering the surface of the conical nanostructure layer; and the thickness d4 of the second metal thin film layer represents the vertical thickness of the second metal thin film covering the surface of the first metal thin film layer.

[0072] If the thickness of the first metal thin film layer 220 is too small (<10nm), the metal thin film cannot form a continuous capping layer, resulting in gaps and breaks, which weakens the plasma resonance effect and fails to achieve effective initial enhancement of the electromagnetic field. It also fails to provide a stable substrate for the second metal thin film layer. If the thickness is too large (>20nm), the metal thin film coverage is too high, significantly blocking light and reducing substrate transmittance, while increasing fabrication costs. Furthermore, excessively thick films are prone to stress concentration, leading to detachment and cracking. This application designs a thickness range of 10nm to 20nm, enabling the metal thin film to form a continuous and dense capping layer, achieving both initial enhancement of the electromagnetic field and ensuring sufficient transmittance, while simultaneously providing a stable substrate for the second metal thin film layer.

[0073] As a specific example, the thickness d3 of the first metal thin film layer designed in this application can be any thickness value among 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm or 20nm.

[0074] If the thickness of the second metal thin film layer 230 is too small (<5nm), the metal distribution will be uneven, and the uniformity of the SERS signal will decrease significantly. If the thickness is too large (>15nm), the transmittance will decrease significantly, and the stress of the metal thin film will increase, making it prone to detachment and oxidation. In this application, the metal thin film is designed to generate strong local surface plasmon resonance within a thickness range of 5nm to 15nm, forming a very strong hot spot and achieving efficient enhancement of the SERS signal. At the same time, the thin-layer design can reduce the obstruction of light and ensure the high transmittance of the substrate.

[0075] As a specific example, the thickness d4 of the second metal thin film layer designed in this application can be any thickness value among 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm or 20nm.

[0076] In the above embodiments, this application achieves a precise match between the coverage and transmittance of the metal thin films by limiting the specific thickness range of the two metal thin films. Specifically, the thickness of the first metal thin film layer, ranging from 10nm to 20nm, ensures continuous coverage and initial enhancement, while the thickness of the second metal thin film layer, ranging from 5nm to 15nm, ensures efficient enhancement. The total thickness of the two layers is controlled within a reasonable range, avoiding both the decrease in transmittance caused by excessive thickness and the insufficient SERS activity caused by excessive thinness, thus achieving a precise balance between the two and optimizing the overall performance of the sensor.

[0077] This application also provides a method for preparing a biosensor, such as... Figure 4 As shown, the method includes: Step S400: A cone-shaped nanostructure layer is formed on a flexible transparent substrate by nanoimprinting.

[0078] Step S410: A first metal thin film layer and a second metal thin film layer are sequentially formed on a flexible transparent substrate on which a conical nanostructure layer is formed, and the first metal thin film layer and the second metal thin film layer cover the surface of the conical nanostructure layer to obtain a biosensor.

[0079] In the above-mentioned implementation method, nanoimprinting refers to the technology of precisely preparing nanostructures by imprinting a mold with a nanostructure onto the surface of a curable material (such as a UV-curable adhesive) and then fixing the nanostructure through a curing process.

[0080] This application employs nanoimprint lithography to solidify a conical nanostructure layer on a flexible transparent substrate. Then, a first metal thin film layer and a second metal thin film layer are sequentially formed on the flexible transparent substrate with the conical nanostructure layer. Specifically, a physical deposition method is used to uniformly deposit two different metal materials sequentially on the surface of the conical nanostructure layer, forming a continuous and dense bilayer metal thin film. Specifically, this application can utilize any of the methods selected from electron beam evaporation, thermal evaporation, or magnetron sputtering to sequentially form the first and second metal thin film layers on the flexible transparent substrate with the conical nanostructure layer.

[0081] In the above embodiments, this application achieves the fabrication of biosensors through nanoimprinting curing and metal thin film deposition. Compared with the existing complex SERS substrate fabrication process, it does not require complex equipment, cumbersome operations, and expensive chemical reagents. The nanoimprinting process can realize the batch replication of conical nanostructures, which significantly improves the fabrication efficiency, and the mold can be reused, greatly reducing the fabrication cost. The metal thin film deposition method is mature, has strong process compatibility, and can be continuously connected with the nanoimprinting process to achieve large-scale continuous production, solving the technical problem that existing flexible transparent SERS sensors are difficult to fabricate on a large scale.

[0082] In an optional embodiment of this example, the above step S400 process can be performed as follows: Figure 5 The steps shown are implemented in detail as follows: Step S500: Apply UV-curable adhesive onto a flexible transparent substrate.

[0083] Step S510: Imprint the UV-curable adhesive using a conical nanostructure mold, controlling the thickness of the imprinted residual layer to be between 30 nm and 100 nm.

[0084] Step S520: Irradiate the UV-curable adhesive after imprinting with UV light to cure the cone-shaped nanostructure layer obtained by imprinting onto the flexible transparent substrate.

[0085] In the above-described embodiments, this application first drips UV-curable adhesive onto a flexible transparent substrate. During the dripping process, it is necessary to ensure that the UV-curable adhesive evenly covers the substrate surface to avoid problems such as excessively thick or thin adhesive layers in certain areas, thus ensuring a tight fit between the mold and the adhesive layer during subsequent imprinting.

[0086] Then, using the mechanical pressure of the conical nanostructure mold, the conical nano-pit structure on the surface of the mold is imprinted into the liquid UV-curable adhesive, achieving precise transfer of the pattern. At the same time, by controlling the imprinting pressure, imprinting time, and adhesive layer thickness, the thickness of the residual layer is precisely controlled.

[0087] If the residual layer is too thin (<30nm), the adhesion between the bottom of the conical nanostructure and the substrate will be insufficient, making it prone to detachment during subsequent metal film fabrication or use. Furthermore, uneven residual layer thickness can lead to dimensional deviations in the conical nanostructure. If the residual layer is too thick (>100nm), it will block the propagation of subsequent incident light, reducing the substrate's transmittance and increasing the amount of metal film deposition required, thus increasing costs. Therefore, this application designs a residual layer thickness of 30nm to 100nm, which ensures the adhesion between the conical nanostructure and the substrate while avoiding any impact on transmittance, and also facilitates uniform coverage of the subsequent metal film.

[0088] Based on the above, this application uses ultraviolet light to irradiate the UV-curable adhesive after imprinting. The ultraviolet light irradiation excites the photoinitiator in the UV-curable adhesive, causing the UV-curable adhesive molecules to undergo a rapid polymerization reaction, changing from a liquid state to a solid state, thereby fixing the imprinted cone-shaped nanostructure on a flexible transparent substrate to form a stable cone-shaped nanostructure layer.

[0089] In the above-described embodiments, this application achieves rapid and automated fabrication of cone-shaped nanostructure layers through three simple steps: dispensing, imprinting, and UV curing. The entire process eliminates the need for complex post-processing (such as etching and cleaning), resulting in a seamless workflow that significantly improves fabrication efficiency and meets the demands of large-scale mass production. Simultaneously, the limited residual layer thickness of 30nm to 100nm ensures a tight bond between the cone-shaped nanostructure and the flexible transparent substrate, preventing detachment during subsequent fabrication and use, while also avoiding a decrease in transmittance due to an excessively thick residual layer. Furthermore, precise UV irradiation ensures complete curing of the UV-curable adhesive, resulting in a stable morphology of the cured cone-shaped nanostructure that does not deform. This avoids fluctuations in the plasma resonance effect caused by structural dimensional deviations or morphological deformation, thereby improving the stability and repeatability of the sensor's SERS signal.

[0090] This application also provides the application of the above-mentioned biosensor in the detection of biological residues, specifically including: dropping methylene blue solution onto the surface of crucian carp to simulate residues on the surface of crucian carp, covering the sample surface with the sensing and detection components of the biosensor facing down, and obtaining the Raman spectrum result of the residue to be tested after Raman detection.

[0091] Methylene blue is widely used in aquaculture for the prevention and treatment of fish diseases and for water disinfection. If methylene blue residues in aquatic products are ingested by humans, they can have adverse effects on human health, including damage to the blood system, abnormal liver and kidney function, and mutagenic toxicity. This application will utilize a biosensor to detect methylene blue residues on the surface of crucian carp in situ.

[0092] The present application will now be described in detail with reference to the embodiments.

[0093] The formula for calculating the SERS enhancement factor (EF) is as follows:

[0094] in, To detect the peak intensity of Raman spectra using SERS biosensors, Peak intensity of Raman spectra was detected on a common substrate. This represents the concentration of the analyte adsorbed on the surface of the SERS biosensor. This represents the concentration of the analyte adsorbed on a common substrate surface.

[0095] Example 1: In-situ detection of methylene blue residue on crucian carp surface This embodiment uses a SERS biosensor prepared using the above method for detection and identification. A pyramid structure with a lateral period L1 of 1.2 µm, a base side length L2 of 1 µm, and a height h of 700 nm is fabricated on a 50 µm thick PET film substrate. The residual layer thickness is 50 nm. The first metal layer is an Al layer, and the second metal layer is an Ag layer with thicknesses d3 and d4 of 10 nm and 5 nm, respectively. This biosensor is used for in-situ detection of a concentration of 10 -6 The residual methylene blue solution was measured at mol / L. The excitation wavelength was 532 nm, and the spectral acquisition time was 1 s.

[0096] Figure 6 Nine sets of surface-enhanced Raman spectra of in-situ detection of methylene blue on the surface of crucian carp using a biosensor according to an exemplary embodiment are shown. Figure 6 As shown, the surface-enhanced Raman spectra of the biosensors clearly identified the Raman characteristic structure of methylene blue, and the Raman enhancement effect was significant.

[0097] Simultaneously, the concentration gradient of methylene blue was detected in the back-side excitation / collection mode. 10 μL of methylene blue was dropped onto a glass slide under a 532 nm laser. -7 ~10 -3 A mol / L methylene blue solution was used to cover the droplet with the pyramid-shaped structure of the biosensor facing down for detection.

[0098] Figure 7 The following diagram illustrates the concentration range of 10 detected in the back-side excitation / collection mode of the biosensor according to an exemplary embodiment of this application. -7 ~10 -3 Surface-enhanced Raman spectrum (a) of mol / L methylene blue solution and concentration 10 -5 Repeatability test results at mol / L (b), as shown Figure 7 As shown in (a), this SERS biosensor can clearly identify 10 -7 ~10 -3 Methylene blue in the mol / L concentration range.

[0099] In addition, to study the reproducibility of this SERS biosensor, 10 -5 The experiment was repeated using mol / L methylene blue solution. Fifteen points were randomly selected for the repeat experiment. Figure 7 (b) shows a repeatability (relative signal deviation) of 8.11% at a Raman shift of 1623 cm⁻¹.

[0100] Example 2: This example also uses the biosensor from Example 1 above for detection and identification. The excitation wavelength is 532nm and the spectral acquisition time is 1s. Figure 8The following is an example of a biosensor in a rear-side excitation / collection mode according to an exemplary embodiment of this application, at 10 -15 ~10 -10 R6G surface-enhanced Raman spectra in the mol / L concentration range (a), Raman mapping in front-excitation / collection mode (b), and Raman mapping in back-excitation / collection mode (c).

[0101] like Figure 8 As shown in (a), the biosensor detects a concentration of 10 -15 ~10 -10 Surface-enhanced Raman spectroscopy of biosensors clearly identified the Raman characteristic structure of R6G at mol / L concentrations. Calculations showed that R6G exhibits Raman characteristics at 610 cm⁻¹. -1 The EF in the back-side excitation / collection mode is ~4.63×10⁻⁶. - ¹ 6 In addition, the uniformity of the biosensor under different modes was investigated, and Raman mapping detection was performed in a 10μm × 10μm range, such as... Figure 8 (b) and Figure 8 As shown in (c), the detection results show that both the front excitation / collection mode and the back excitation / collection mode have good uniformity.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any way. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A biosensor, characterized in that, Includes a flexible transparent substrate and a sensing and detection component formed on the flexible transparent substrate; The sensing and detection component includes a conical nanostructure layer, a first metal thin film layer, and a second metal thin film layer. The conical nanostructure layer is formed on the flexible transparent substrate, the first metal thin film layer is formed on the conical nanostructure layer, and the second metal thin film layer is formed on the first metal thin film layer. The first metal thin film layer and the second metal thin film layer are made of different metal materials. The sensing and detection component is used to excite local surface plasmon resonance of the metal layer on the conical nanostructure layer under laser irradiation, generate an electromagnetic field enhancement region, and enhance the electromagnetic field enhancement region through the coupling between the first metal thin film layer and the second metal thin film layer.

2. The biosensor according to claim 1, characterized in that, The conical nanostructure layer includes multiple conical nanostructures, which are periodically spaced on the flexible transparent substrate to form a conical nanoarray. The first metal thin film layer and the second metal thin film layer both cover the surface of the conical nanoarray.

3. The biosensor according to claim 2, characterized in that, Each of the aforementioned cone-shaped nanostructures is a regular square pyramidal nanostructure; The base length of each of the regular square pyramidal nanostructures ranges from 900 nm to 1100 nm, the height of each of the regular square pyramidal nanostructures ranges from 650 nm to 750 nm, and the lateral period of the pyramidal nanoarray ranges from 1100 nm to 1300 nm.

4. The biosensor according to claim 2, characterized in that, The angle between the sidewall of each of the conical nanostructures and the flexible transparent substrate ranges from 45° to 55°.

5. The biosensor according to claim 1, characterized in that, The metal material of the first metal thin film layer can be any one of aluminum, copper, and aluminum oxide; the metal material of the second metal thin film layer can be any one of silver and gold.

6. The biosensor according to claim 1, characterized in that, The thickness of the first metal thin film layer ranges from 10 nm to 20 nm, and the thickness of the second metal thin film layer ranges from 5 nm to 15 nm.

7. The biosensor according to claim 1, characterized in that, The flexible transparent substrate is made of one of PET, PDMS, nanofibers, and biofilms, and the thickness of the flexible transparent substrate ranges from 12 μm to 250 μm.

8. A method for preparing a biosensor, characterized in that, The method includes: A cone-shaped nanostructure layer was formed on a flexible transparent substrate by nanoimprinting. A first metal thin film layer and a second metal thin film layer are sequentially formed on a flexible transparent substrate on which the conical nanostructure layer is formed, such that the first metal thin film layer and the second metal thin film layer cover the surface of the conical nanostructure layer, thereby obtaining a biosensor.

9. The method according to claim 8, characterized in that, The process of solidifying a cone-shaped nanostructure layer on a flexible transparent substrate using nanoimprinting includes: UV-curable adhesive is drop-coated onto the flexible transparent substrate; The UV-curable adhesive is imprinted using a conical nanostructure mold, and the thickness of the imprinted residual layer is controlled to be in the range of 30 nm to 100 nm. The UV-curable adhesive that has been imprinted is irradiated with UV light, so that the cone-shaped nanostructure layer obtained by imprinting is cured and formed on the flexible transparent substrate.

10. The method according to claim 8, characterized in that, A first metal thin film layer and a second metal thin film layer are sequentially formed on a flexible transparent substrate on which the conical nanostructure layer is formed, including: The first metal thin film layer and the second metal thin film layer are sequentially formed on the flexible transparent substrate on which the conical nanostructure layer is formed using any one of electron beam evaporation, thermal evaporation, or magnetron sputtering.

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