Single-cell sensor of flexible micro-nano cross-scale integrated metasurface and preparation method of single-cell sensor

Abstract

The invention discloses a flexible micro-nano cross-scale integrated metasurface single-cell sensor and a preparation method thereof, and relates to the field of biosensing. The sensor comprises a PDMS flexible substrate layer, a micro-nano cross-scale structure layer and a gold metal film layer, the micro-nano cross-scale structure layer adopts a nesting design of a micron-scale single cell capture unit and a nano-scale plasmon structure, so that single cell precise limiting and local electromagnetic field multi-stage enhancement are realized, and the problems of poor adaptability and low sensitivity of a traditional rigid sensor are solved. According to the preparation method, a silicon-based template-replication forming-magnetron sputtering process is adopted, a silicon template is prepared through deep ultraviolet lithography and deep reactive ion etching, a gold film is deposited after a flexible micro-nano structure is copied by PDMS, and large-scale mass production of the sensor is realized. The sensor has excellent flexibility, biocompatibility and high detection sensitivity, can realize single cell level accurate and in-situ detection, is suitable for the scenes of early disease screening, cell biological analysis, personalized medical treatment and the like, and is simple in process and low in cost.

Description

Technical Field

[0001] This invention relates to the field of biosensing, and in particular to a single-cell sensor with a flexible micro / nano multi-scale integrated metasurface and its fabrication method. Background Technology

[0002] With the increasing demand for single-cell analysis in the biomedical field, nanotechnology-based biosensors have attracted widespread attention due to their advantages such as high sensitivity, portability, and label-free operation. Traditional biosensors are usually based on rigid materials, making it difficult to adapt to complex biological environments, especially at the cellular scale, where their performance is often limited. To address this issue, flexible sensors have gradually become a new research direction. Polydimethylsiloxane (PDMS) has become one of the ideal materials due to its excellent biocompatibility, flexibility, and ease of processing, making it particularly suitable for single-cell-scale biosensing.

[0003] Furthermore, in recent years, plasmonic metasurface technology has emerged as an advanced sensing technology. By designing metallic structures at the nanoscale, it can significantly enhance local electromagnetic fields, thereby achieving highly sensitive biomolecular detection. Utilizing the optical properties of metasurfaces, researchers can perform efficient biosensing at the cellular or single-molecule level. However, existing plasmonic metasurface sensors generally suffer from two major drawbacks: first, the substrate is highly rigid, making it impossible to adhere to single-cell interfaces, hindering precise single-cell anchoring and in-situ detection; second, the microstructure is designed only at the nanoscale, resulting in limited distribution of electromagnetic field hotspots, low coupling efficiency, and uneven cell adsorption and severe signal interference due to structural planarization, failing to meet the requirements for high-precision and high-sensitivity single-cell detection.

[0004] Therefore, developing a metasurface sensor that combines flexibility, micro-nano cross-scale electromagnetic field enhancement, and precise single-cell capture, along with a fabrication process that can be mass-produced, is a technical challenge that urgently needs to be solved in the field of biosensing. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies, such as poor adaptability to rigid substrates, insufficient sensitivity of single-scale metasurfaces, and low single-cell capture accuracy. This invention aims to provide a single-cell sensor based on a flexible micro / nano-scale integrated metasurface and its fabrication method. By integrating a micron-scale single-cell capture structure with a nano-scale plasmon metasurface structure, combined with a flexible PDMS substrate, it achieves precise single-cell anchoring and multi-level enhancement of the local electromagnetic field, significantly improving the sensitivity, repeatability, and biocompatibility of single-cell detection. Simultaneously, it incorporates standardized micro / nano fabrication processes to achieve low-cost, large-scale, and stable fabrication of the sensor.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a flexible micro / nano-scale integrated metasurface for a single-cell sensor and its fabrication method. The single-cell sensor comprises a flexible substrate layer, a micro / nano-scale integrated structure layer, and a metal film layer. The flexible substrate layer is made of polydimethylsiloxane (PDMS). The micro / nano-scale integrated structure layer is disposed on the surface of the flexible substrate layer and includes microstructural units arranged in an array at the micrometer scale, and nanoscale plasmon structures constructed on the microstructural units. The nanoscale plasmon structures further include arrayed nanopores and smaller nanopores or nano-rough structures distributed on the surface of the nanopores. The metal film layer covers the surface of the micro / nano-scale integrated structure layer and is used to excite and modulate local surface plasmon resonance. By integrating the micrometer-scale structure with the nanoscale plasmon structure in a cross-scale design, this invention achieves multi-scale coupling and enhancement of electromagnetic fields in three-dimensional space while maintaining flexibility and biocompatibility, thereby significantly improving the biosensing sensitivity and detection accuracy at the single-cell level.

[0008] The nanostructure layer of the sensor of this invention is at the single-cell scale, thereby enabling the detection of single-cell organisms.

[0009] Furthermore, the sensor substrate is made of PDMS flexible material, which has excellent optical properties, good biocompatibility, unique elasticity, and flexible surface modification capabilities.

[0010] Furthermore, the nanopore structure has a period of 100–200 nm, a pore size of 100–300 μm, and a depth of 40–50 nm, which can capture single-celled organisms.

[0011] Furthermore, the metal film layer of the sensor is a gold film with nanopores.

[0012] A second aspect of this invention provides a single-cell sensor with a flexible micro / nano multi-scale integrated metasurface and a method for fabricating the same, comprising:

[0013] 1) Provide a silicon-based substrate layer, coat the upper surface of the substrate layer with photoresist, and form an array of single-cell-scale spaced pore structures on the photoresist surface through a mask and deep ultraviolet lithography process;

[0014] 2) A deep reactive ion etching process is used to etch downwards on a silicon substrate until a nanostructure layer is formed. The nanostructure layer includes an array of single-cell-scale nanopores and smaller nanopores distributed on the surface of the nanopores. The residual photoresist on the surface is removed by a development technique.

[0015] 3) PDMS is cast onto a silicon substrate with a nanostructure layer, and the PDMS is peeled off, revealing that its surface has the same nanostructure layer.

[0016] 4) A metal film layer is formed on the nanostructure layer.

[0017] Furthermore, step 1) specifically includes the following steps for mask and deep ultraviolet lithography processes:

[0018] S1.1, through graphic design and data processing technology, the circuit layout is converted into a data format that can be recognized by the mask manufacturing equipment in order to define the target graphic;

[0019] S1.2, through deep ultraviolet exposure process, light is selectively irradiated onto photoresist through a mask at a specific wavelength and energy dose to trigger photochemical reaction in the exposed area;

[0020] S1.3, through post-exposure baking and chemical development processes, the pattern defined by the mask is transformed into the physical three-dimensional structure of the photoresist to form the mask for subsequent etching or injection;

[0021] S1.4, through optical and electron beam inspection processes, key dimensions and defects of the formed photoresist pattern are measured to ensure that the pattern quality meets the requirements of subsequent processes.

[0022] Furthermore, step 2) of the deep reactive ion etching process specifically includes the following steps:

[0023] S1, through the etching step of fluorine-based gas (such as SF6), isotropic chemical etching and physical bombardment are performed on the exposed silicon material to open the vertical etching of the patterned area;

[0024] S2, through a passivation step using a fluorocarbon gas (such as C4F8), a polymer protective film is deposited on all surfaces formed by etching to protect the sidewalls from subsequent lateral etching;

[0025] S3, the etching and passivation steps are performed alternately and cyclically, and the number of cycles and the time of each step are optimized to form a deep trench or hole structure with a high aspect ratio and vertical sidewalls.

[0026] S4, a clean final silicon microstructure is obtained by plasma debinding process through oxygen introduction or by removing the residual polymer passivation layer in a wet chemical bath.

[0027] Furthermore, the specific process of developing the photoresist surface involves targeting the photoresist layer that has undergone chemical modification (such as photoacid catalytic reaction) after exposure. Through a chemical development process, under the conditions of a 2.38% TMAH aqueous solution, a temperature of 23±0.5℃, and a time of 30 to 60 seconds, the photoresist is selectively dissolved.

[0028] Furthermore, the formation of a PDMS layer with a microchannel structure on a silicon substrate with a nanostructure layer specifically involves using the silicon substrate as a template and, through a PDMS casting and curing process, casting and demolding the PDMS microstructure layer under the conditions of mixing the prepolymer and curing agent at a mass ratio of 10:1 and curing at 65°C for 2 hours.

[0029] Furthermore, the deposition of a continuous and dense gold film on the PDMS specifically involves using a high-purity gold (Au) target as the source material and employing a magnetron sputtering process at a base vacuum better than 5.0 × 10⁻⁶. -4 The gold film was formed under the following conditions: Pa, working pressure 0.5 Pa, sputtering power 150 W, and argon atmosphere for 300 s.

[0030] This invention, through the synergistic design of flexible substrate selection and micro / nano multi-scale structures, has the following outstanding advantages compared to traditional single-cell sensing technology:

[0031] 1. This invention uses a biocompatible PDMS flexible substrate to replace rigid substrates such as silicon and glass, which can perfectly fit the microscopic interface of single cells, avoid mechanical damage to cells, ensure cell physiological activity, and adapt to complex dynamic biological microenvironments, thus solving the industry pain points of interface mismatch and signal distortion of rigid sensors.

[0032] 2. By employing flexible PDMS material and an optimized micro-nano cross-scale metasurface structure, this invention enables the sensor to generate a significant local electromagnetic field enhancement effect at the single-cell scale, construct a three-dimensional electromagnetic field coupling enhancement channel, realize the focusing and amplification of local electromagnetic field hotspots, effectively improve the detection signal-to-noise ratio, and achieve label-free, high-precision, and efficient detection of trace biomolecules. It is especially suitable for real-time monitoring at the cellular level, improving the sensitivity and accuracy of sensor detection.

[0033] 3. This invention adopts a standardized process of silicon-based template replication molding combined with magnetron sputtering, which can realize the mass and stable production of sensors. The template can be reused and the processing cost is low. It can simplify the operation process, improve the convenience of detection, and effectively reduce the overall cost of biosensing detection, overcoming the defects of traditional micro-nano sensor fabrication processes that are complex and difficult to scale up.

[0034] 4. The sensor of this invention can achieve efficient biochemical detection and real-time monitoring at the single-cell level, and can be widely used in the biomedical field, especially in early disease diagnosis, single-cell biological analysis, personalized medicine, drug screening and other scenarios, providing a more advanced and practical technical platform for high-precision, low-cost and portable biosensing detection. Attached Figure Description

[0035] Figure 1The flowchart illustrates a method for fabricating a single-cell sensor using a flexible micro / nano multi-scale integrated metasurface, as provided by this invention.

[0036] Figure 2 This is a top view schematic diagram of a single-cell sensor based on a flexible micro / nano multi-scale integrated metasurface provided by the present invention.

[0037] Figure 3 This is a microscopic cross-sectional schematic diagram of a single-cell sensor based on a flexible micro / nano multi-scale integrated metasurface provided by the present invention.

[0038] Figure 4 This is a schematic diagram of the structure of a single-cell sensor based on a flexible micro / nano multi-scale integrated metasurface provided by the present invention.

[0039] Figure 5 This is a schematic diagram of a single-cell sensor on the micrometer scale of a flexible micro / nano multi-scale integrated metasurface provided by the present invention.

[0040] Figure 6 This is a schematic diagram of a single-cell sensor at the nanoscale of a flexible micro / nano multi-scale integrated metasurface provided by the present invention. Detailed Implementation

[0041] To make the technical problems, technical solutions, and beneficial effects of this invention clearer and more understandable, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of protection of the invention.

[0042] Example 1

[0043] See Figures 2 to 6 In this embodiment, a single-cell sensor of a flexible micro / nano multi-scale integrated metasurface includes a PDMS substrate, a nanostructure layer disposed on the substrate, and a metal film layer with nanopores.

[0044] The nanostructure layer is at the single-cell scale and can capture single-cell organisms, thereby enabling single-cell detection.

[0045] The hydrophilic plasmonic metasurface biosensing region is provided with nanopores and a metal thin film. The metal thin film covers the surface of the nanopores and is used for surface plasmonic biosensing.

[0046] The single-cell sensor of the flexible micro / nano multi-scale integrated metasurface described in this embodiment has a size of 1.5mm × 1.5mm. The shape of the biosensing region of the hydrophilic plasmon metasurface is a circular hole with a size of 2.5 to 10μm, of which 5μm is the most effective.

[0047] The PDMS nanostructure layer is cylindrical in shape, and the height of the nanopillar is 40-50 nm.

[0048] The material of the metal film is Au.

[0049] The nanopores are circular in shape, with a distance r1 between adjacent nanopores of 100–200 nm, a diameter r2 of 100–300 μm, and a depth h of 40–50 nm.

[0050] See Figure 5 At the micrometer scale, the hydrophilic plasmon metasurface biosensing region in this invention has uniform size and good morphology.

[0051] See Figure 6 At the nanoscale, the single-cell polariton metasurface biosensing region and the nanopore biosensing region in this invention have uniform size and good morphology.

[0052] See Figure 1 This embodiment describes a method for manufacturing a PDMS flexible single-cell-scale metasurface biosensor, comprising the following steps:

[0053] S1. Provide a silicon-based substrate layer, and coat the upper surface of the substrate layer with photoresist. Specifically, select a clean single-crystal polished silicon wafer as the silicon-based substrate layer, remove the surface oxide layer, particles and oil stains through a standard RCA cleaning process, and after drying, use a spin coating process to uniformly coat the upper surface of the substrate with deep ultraviolet photoresist. Control the spin coating parameters to ensure that the photoresist thickness is uniform and free of bubbles and pinholes.

[0054] S2. The photoresist is patterned using a mask and deep ultraviolet lithography to form an array of single-cell-scale spaced holes on the photoresist surface. Specifically, after aligning the pre-designed mask with the substrate coated with photoresist, the photoresist is exposed under deep ultraviolet light to transfer the mask pattern to the photoresist layer. Subsequently, the photoresist in the exposed (or unexposed) areas is removed by development, thereby forming a periodically arranged array of holes on the photoresist surface with apertures and spacings within the single-cell scale range, providing a template for the subsequent fabrication of metasurface structures.

[0055] S3. Using the patterned photoresist as a mask, a deep reactive ion etching process is used to etch downwards on the silicon substrate until a nanostructure layer is formed. The nanostructure layer includes arrayed single-cell-scale nanopores and smaller nanopores distributed on the surface of the nanopores, realizing the integrated molding of cross-scale structures.

[0056] S4. Residual photoresist on the photoresist surface is removed by developing technology to obtain a photoresist structure with clear patterns and complete boundaries. Specifically, the exposed substrate is immersed in the developing solution for development to remove residual photoresist in the unexposed areas, so that the photoresist pattern is fully revealed and the influence of surface residue on subsequent processes is avoided, thereby providing a clean pattern template for subsequent metal deposition or metasurface structure fabrication.

[0057] S5. By casting PDMS onto a silicon substrate with a nanostructure layer and curing it, a PDMS substrate with a nanostructure replication layer is obtained, thereby completing the preparation of the flexible substrate. Specifically, a pre-prepared and fully mixed PDMS precursor is cast onto the surface of the silicon substrate with the nanostructure layer, so that the PDMS precursor fully fills the gaps in the nanostructure, and then cured under a set temperature condition. After the PDMS is completely cross-linked, it is peeled off from the silicon substrate, thereby forming a structural layer on the PDMS surface that is consistent with the nanostructure of the silicon substrate.

[0058] S6. After the PDMS is fully cross-linked, it is peeled off from the silicon substrate, thereby forming a structural layer on the PDMS surface that is consistent with the nanostructure of the silicon substrate.

[0059] S7. A metal film layer is formed on the nanostructure layer. The metal film layer uniformly coats the surface of the structure, forming a stable plasmon response layer, and finally a complete flexible single-cell sensor is obtained.

[0060] This invention, starting from the principles of bio-interface adaptation and the mechanism of local electromagnetic field enhancement, achieves three core innovations: First, the innovation of micro-nano cross-scale nested structures, integrating micron-scale single-cell confinement structures with nano-scale plasmon resonance structures. The micron structure enables precise anchoring of single cells and eliminates cell overlap and drift, while the nanostructure constructs multi-level electromagnetic field hotspot channels. This allows for the prediction of three-dimensional electromagnetic field coupling enhancement without relying on performance testing, breaking through the sensitivity bottleneck of traditional single-scale metasurfaces from a structural perspective. Second, the innovation of flexible sensing systems, using PDMS flexible substrates to replace rigid materials such as silicon and glass. Utilizing its biocompatibility and mechanical flexibility, it achieves seamless adhesion to the micro-interface of single cells, protecting cell activity and solving the signal distortion problem caused by interface mismatch in rigid sensors. Third, the innovation of mass production processes, employing silicon-based template replication molding technology, transforms the fabrication of complex micro-nano structures into a standardized, batch-production process. This ensures controllable structural precision, high product consistency, and balances sensing performance with industrial feasibility. The overall solution achieves a synergistic improvement in flexible adaptation, precise capture, and high-sensitivity sensing through structural design and principle reconstruction, and has outstanding substantive features and significant progress compared with existing technologies.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention 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 of the technical features. Such modifications or substitutions do not cause the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A single-cell sensor with a flexible micro / nano multi-scale integrated metasurface, characterized in that, It includes a flexible substrate layer, a micro / nano multi-scale structure layer, and a metal film layer; The flexible substrate layer is made of polydimethylsiloxane. The micro / nano multi-scale structure layer is formed on the upper surface of a flexible substrate layer, including arrayed micron-scale trapping units and nanoscale plasmon structures constructed on the surface of the micron-scale trapping units; the nanoscale plasmon structures include periodic nanopores and secondary nanoroughness structures or secondary nanopores on the inner walls of the nanopores. The metal film layer is uniformly covered on the entire surface of the micro-nano cross-scale structure layer and is a plasmon-responsive film layer made of gold.

2. The single-cell sensor of a flexible micro / nano multi-scale integrated metasurface according to claim 1, characterized in that, The micron-scale trapping unit has a circular hole structure with a pore size of 2.5–10 μm.

3. A single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 2, characterized in that, The diameter of the circular hole structure is 5 μm.

4. A single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 1, characterized in that, In the nanoscale plasmon structure, the period of the nanopores is 100-200 nm, the pore size is 100-300 nm, and the depth is 40-50 nm; the secondary nano-rough structure or the secondary nanopore size is less than 50 nm.

5. A single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 1, characterized in that, The metal film is prepared by magnetron sputtering, resulting in a dense, continuous film with strong adhesion.

6. A method for fabricating a single-cell sensor based on a flexible micro / nano multi-scale integrated metasurface as described in any one of claims 1-5, characterized in that, Includes the following steps: (1) Cleaning of silicon substrate and spin-coating of deep ultraviolet photoresist; (2) The photoresist is patterned using a mask and deep ultraviolet lithography process, and then developed to form a photoresist mask; (3) A deep reactive ion etching process is used to etch and prepare micro-nano cross-scale silicon templates on the surface of a silicon substrate using photoresist as a mask; (4) The PDMS prepolymer and curing agent are mixed and cast into a silicon template, and after curing, the substrate is peeled off to obtain a flexible PDMS substrate with micro-nano structure; (5) A finished sensor is prepared by depositing a gold film on the micro-nano structure surface of a PDMS flexible substrate by magnetron sputtering.

7. The method for fabricating a single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 6, characterized in that, In step (2), the developing solution is a 2.38% TMAH aqueous solution, the developing temperature is 23±0.5℃, and the developing time is 30~60s.

8. The method for fabricating a single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 6, characterized in that, In step (3), deep reactive ion etching employs an alternating cycle of SF6 etching and C4F8 passivation to control the structure depth and perpendicularity.

9. The method for fabricating a single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 6, characterized in that, In step (4), the mass ratio of PDMS prepolymer to curing agent is 10:1, and the curing condition is 65℃ for 2 hours.

10. The method for fabricating a single-cell sensor with a flexible micro / nano multi-scale integrated metasurface according to claim 6, characterized in that, The magnetron sputtering process parameters in step (5) are: background vacuum better than 5.0 × 10⁻⁶. -4 Pa, working pressure 0.5 Pa, sputtering power 150 W, argon atmosphere.

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