High-throughput arrayed biomimetic superwetting sample holder for electron microscopy characterization and preparation method thereof

Abstract

This disclosure provides a high-throughput arrayed biomimetic superwetting stage for electron microscopy characterization and its fabrication method. The stage includes: a conductive substrate; a superhydrophobic layer formed on the surface of the conductive substrate, wherein multiple superhydrophilic sites are formed at multiple locations of the superhydrophobic layer, and the boundary of the superhydrophobic layer and the boundary of the superhydrophilic sites constitute a heterogeneous interface, wherein the surface energy gradient generated by the heterogeneous interface is used to drive the target sample to accumulate in the superhydrophilic sites during the evaporation process; and a biofunctional anchoring layer formed inside each of the multiple superhydrophilic sites, used to specifically anchor the target sample accumulated in the superhydrophilic sites to complete the electron microscopy characterization of the target sample.

Description

Technical Field

[0001] This disclosure relates to the field of life science technology, specifically to the field of microstructure observation technology, and in particular to a high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization and its preparation method. Background Technology

[0002] Scanning electron microscopy (SEM), a core tool for observing subcellular structures and biomolecular morphology in life science research, relies heavily on sample preparation for its imaging quality. Traditional sample preparation methods for "low-load liquid biological samples" such as microorganisms, single cells, exosomes, and synthetic biomolecules suffer from problems like sample loss, morphological distortion, and charge accumulation. As structural biology enters the era of in-situ, high-throughput characterization, achieving precise anchoring and high-resolution characterization of trace and rare samples under "near-physiological conditions" has become a bottleneck restricting the development of bioelectron microscopy technology. Summary of the Invention

[0003] In view of this, the present disclosure provides a high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization and its preparation method, which at least partially solves the above-mentioned technical problems.

[0004] The first aspect of this disclosure provides a high-throughput arrayed biomimetic superwetting stage for electron microscopy characterization, comprising: a conductive substrate; a superhydrophobic layer formed on the surface of the conductive substrate, wherein multiple superhydrophilic sites are formed at multiple locations of the superhydrophobic layer, and the boundary of the superhydrophobic layer and the boundary of the superhydrophilic sites constitute a heterogeneous interface, wherein the surface energy gradient generated by the heterogeneous interface is used to drive the target sample to accumulate in the superhydrophilic sites during the evaporation process; and a biofunctional anchoring layer formed inside each of the multiple superhydrophilic sites, used to specifically anchor the target sample accumulated in the superhydrophilic sites to complete the electron microscopy characterization of the target sample.

[0005] A second aspect of this disclosure provides a method for fabricating a high-throughput arrayed biomimetic superwetting stage for electron microscopy characterization, comprising: constructing a superhydrophobic layer on a conductive substrate; selectively etching the superhydrophobic layer using a mask to form multiple superhydrophilic sites at multiple locations in the superhydrophobic layer, wherein the boundary of the superhydrophobic layer and the boundary of the superhydrophilic sites constitute a heterogeneous interface, and the surface energy gradient generated by the heterogeneous interface is used to drive the target sample to accumulate in the superhydrophilic sites during evaporation; modifying the interior of each superhydrophilic site to form a biofunctional anchoring layer, the biofunctional anchoring layer being used to specifically anchor the target sample accumulated in the superhydrophilic sites to complete the electron microscopy characterization of the target sample.

[0006] The high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization provided in this disclosure has the following beneficial effects:

[0007] This stage forms a superhydrophobic layer on a conductive substrate, with multiple superhydrophilic sites at various locations within the superhydrophobic layer. The boundaries of the superhydrophobic layer and the superhydrophilic sites constitute a heterogeneous interface. The surface energy gradient generated at this interface drives the target sample to accumulate at the superhydrophilic sites during evaporation. This geometric arrangement of a "superhydrophobic background + arrayed hydrophilic sites" enables self-driven accumulation of the target sample, achieving "single-layer, high-density" sample stacking. This avoids blurring of the underlying image due to excessive stacking thickness, thus improving image quality.

[0008] By modifying the superhydrophilic sites with nanoscale biological functional anchoring layers, the samples are stably anchored in a "near-physiological state" using charge attraction or covalent bonding. This eliminates the need for additional gold sputtering or conductive adhesives, achieving in-situ, non-destructive fixation. It maximizes the restoration of the natural ultrastructure of biological samples (such as cell membrane cilia and intact vesicle morphology), avoids artificial artifacts from obscuring the true microscopic morphology, and improves imaging quality.

[0009] The self-driven enrichment and non-spraying process of this stage differs from centrifugal enrichment and spraying processes, and will not generate huge physical stress. For sensitive samples such as exosomes, it avoids structural collapse or morphological changes.

[0010] The geometric arrangement of superhydrophobic background and arrayed hydrophilic sites can meet the needs of biomedical research for large sample size statistics and high-throughput screening, and realize automated scanning and big data statistical analysis.

[0011] By modifying chip sites, using conductive adapter bases, and employing spring-loaded grounding mechanisms, the problem of continuous conductivity in biological samples from the nanometer to the centimeter scale is solved, and the charging effect is significantly suppressed.

[0012] This stage can simultaneously achieve self-driven enrichment, in-situ non-destructive fixation, and high-throughput automatic sampling.

[0013] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0014] The above and other objects, features and advantages of this disclosure will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0015] Figure 1 One of the structural diagrams of a high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization according to an embodiment of the present disclosure is illustrated schematically.

[0016] Figure 2 A second schematic diagram illustrates the structure of a high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization according to an embodiment of the present disclosure.

[0017] Figure 3 The flowchart illustrates the fabrication process of a high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization according to an embodiment of the present disclosure. Detailed Implementation

[0018] Embodiments of the present disclosure will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the disclosure. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0020] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein are to be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0021] The accompanying drawings show some block diagrams and / or flowcharts. It should be understood that some blocks or combinations thereof in the block diagrams and / or flowcharts can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device, so that when executed by the processor, these instructions can create means for implementing the functions / operations described in these block diagrams and / or flowcharts.

[0022] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this disclosure. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0023] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.

[0024] In the technical solution disclosed herein, all data involved (including but not limited to data used for analysis, data stored, data displayed, etc.) are information and data authorized by the user or fully authorized by all parties. Furthermore, the collection, storage, use, processing, transmission, provision, disclosure, and application of the relevant data comply with relevant laws, regulations, and standards, necessary confidentiality measures have been taken, and they do not violate public order and good morals. Corresponding operation entry points are provided for users to choose to authorize or refuse.

[0025] In related technologies, the natural drop-coating / conductive adhesive patch preparation method is a common SEM sample preparation technique. This method involves directly dropping a biological sample dilution onto a polished silicon wafer, conductive adhesive, or carbon support film, and then fixing the sample to the substrate surface using natural evaporation or vacuum drying. Subsequently, to enhance conductivity, the sample surface is usually subjected to gold sputtering.

[0026] In the process of developing this disclosure, it was discovered that in the natural drop-coating / conductive adhesive patch sample preparation method, rare samples (such as extremely dilute biological solutions) are driven by the evaporation kinetics at the droplet edge during natural evaporation, causing target particles to tend to deposit randomly at the droplet edge. This "coffee ring effect," resulting in random distribution and sparse disorder, makes electron microscopy sample searching akin to "finding a needle in a haystack," significantly wasting the time of large instruments. Furthermore, there is a lack of effective chemical / physical anchoring between the sample and the substrate. Under vacuum conditions and high-energy electron beam bombardment, biological tissues are prone to shrinkage and displacement. Simultaneously, the insulating properties of the conductive adhesive and the biological material itself lead to severe charge accumulation (charging effect), producing artifacts that obscure the true microstructure. In addition, the centrifugation enrichment and gold sputtering processes used in this sample preparation method generate enormous physical stress, often causing structural collapse or morphological changes in sensitive samples such as exosomes.

[0027] In related technologies, to solve the problem of sample dehydration distortion, a microfluidic encapsulation system (liquid electron microscope) can be used to seal the sample in a silicon nitride (SiN) thin window, or liquid ethane can be used to rapidly freeze and seal the sample in a glassy ice layer (cryo-electron microscope), so that the near-physiological state of the sample can be observed under an electron microscope.

[0028] In the process of developing this disclosure, it was discovered that cryo-electron microscopy requires maintaining an ultra-low temperature chain (≤-140°C) throughout the entire process, while liquid electron microscopy requires a complex microfluidic sealing design. The cost of sample preparation per operation is high, and equipment investment is often in the tens of millions of dollars, making it difficult to promote in research laboratories. Furthermore, liquid electron microscopy is affected by the thickness of the liquid layer and the scattering of the SiN thin window, resulting in imaging contrast and resolution far lower than that of vacuum-dried samples. Cryo-electron microscopy still faces the problem of "low sample capture efficiency" when processing extremely rare samples, failing to achieve active directional enrichment and a balance between imaging contrast and resolution. In addition, this technology typically only characterizes a very small number or even a single sample per operation, failing to meet the needs of modern biomedical research for large-scale statistical analysis and high-throughput screening, and hindering automated scanning and big data statistical analysis.

[0029] In view of this, this disclosure proposes a high-throughput arrayed biomimetic super-immersion stage and its preparation method for electron microscopy characterization. This method addresses the problem of random distribution and sparse disorder of concentration samples on conventional substrates due to the "coffee ring effect," achieving high-magnification directional enrichment of target samples. It avoids the mechanical damage and structural distortion caused to the ultrastructure of biological samples (such as exosomes and single cells) by traditional centrifugation, dehydration, and gold sputtering. It also solves the problem of charge accumulation (charge effect) in non-conductive biological samples under high-energy electron beams, eliminating background artifacts and improving imaging clarity. Furthermore, it changes the current situation where traditional SEM can only process a very small number of samples at a time, enabling high-throughput parallel characterization of a large number of samples.

[0030] The following detailed description, with reference to specific embodiments, provides an overview of the high-throughput array-based biomimetic superimmersion stage and its fabrication method for electron microscopy characterization.

[0031] It should be noted that the sequence numbers of the operations in the following methods are for descriptive purposes only and should not be considered as indicating the execution order of the operations. Unless explicitly stated otherwise, the method does not need to be executed in the exact order shown.

[0032] Figure 1 One of the structural diagrams of a high-throughput arrayed biomimetic superimmersion stage for electron microscopy characterization according to an embodiment of the present disclosure is shown schematically.

[0033] like Figure 1 As shown, the high-throughput arrayed biomimetic superwetting stage 100 for electron microscopy characterization in this embodiment may include a conductive substrate 110, a superhydrophobic layer 120, and a biological functional anchoring layer 130.

[0034] A superhydrophobic layer 120 is formed on the surface of a conductive substrate 110. Multiple superhydrophilic sites 140 are formed at multiple locations of the superhydrophobic layer 120. The boundary of the superhydrophobic layer and the boundary of the superhydrophilic sites constitute a heterogeneous interface. The surface energy gradient generated by the heterogeneous interface is used to drive the target sample 160 to accumulate in the superhydrophilic sites 140 during the volatilization process.

[0035] The biological function anchoring layer 130 is formed inside each of the multiple superhydrophilic sites 140, and is used to specifically anchor the target sample 160 enriched in the superhydrophilic sites 140 to complete the electron microscopy characterization of the target sample 160.

[0036] According to embodiments of this disclosure, a heterogeneous wetting interface of "superhydrophobic background - superhydrophilic array sites" is constructed based on a flat conductive substrate 110 to achieve self-driven directional enrichment of droplets. The superhydrophilic array sites, selectively modified within the heterogeneous wetting pattern layer, are located at the top layer of the entire structure, directly contacting the target sample 160, thus achieving specific anchoring of target samples such as miRNAs and exosomes.

[0037] According to an embodiment of this disclosure, the heterogeneous interface is the direct contact boundary region between the superhydrophobic background and the arrayed superhydrophilic sites. It is not a geometric splicing line, but a molecular-level transition region with a thickness of only a few nanometers, which is completely located in the superhydrophobic layer 120 of the chip, and is connected to the biological functional anchoring layer 130 and the conductive substrate 110 above and below, respectively.

[0038] Because molecules in a superhydrophobic background contain a large number of low surface energy groups, their surface energy is extremely low. In contrast, the superhydrophilic sites exposed after selective etching are high-surface-energy bare substrates, and the subsequently modified biofunctional layer also belongs to a high-surface-energy interface. The surface energy difference between the two can reach tens of mJ / m², naturally forming a continuous surface energy gradient from the superhydrophobic region to the superhydrophilic core. This gradient can drive the automatic directional contraction of trace biological droplets towards the hydrophilic core during evaporation. Combined with the enhanced Marangoni effect under the superwetting system, it inhibits outward flow from the droplet edges, ultimately achieving monolayer high-density directional enrichment of samples such as miRNA and exosomes.

[0039] Figure 2 The diagram illustrates the second structural diagram of a high-throughput arrayed biomimetic superimmersion stage for electron microscopy characterization according to an embodiment of the present disclosure.

[0040] like Figure 2 As shown, the high-throughput arrayed biomimetic super-immersion stage 100 for electron microscopy characterization in this embodiment may also include an adapter base 150.

[0041] The conductive substrate 110 is mounted on the adapter base 150, which serves as the mechanical support and conductive pathway carrier for the high-throughput arrayed biomimetic super-immersion stage and docks with the sample holder of the scanning electron microscope.

[0042] According to embodiments of this disclosure, the adapter base 150 serves as the mechanical support and conductive path carrier for the entire stage. Located at the bottom of the stage, it directly connects to the SEM sample rod and acts as the mounting base for all upper structures. This structure ensures a continuous conductive path from the SEM sample rod to the top target sample, preventing charge interference during imaging. It also allows each functional layer to perform its respective function without interference, effectively controlling the droplets formed by the target sample and anchoring the target sample.

[0043] In some embodiments, the adapter base 150 has a grounding compensation mechanism. The grounding compensation mechanism is used to: create a conductive path by eliminating the contact resistance between the adapter base and the sample rod to drain the accumulated charge on the surface of the target sample; align the adapter base and the sample rod so that the superhydrophilic sites fall within the field of view of scanning electron microscopy imaging; and buffer vibrations during the movement of the sample rod.

[0044] According to embodiments of this disclosure, the grounding compensation mechanism can eliminate the contact resistance between the adapter base 150 and the SEM sample rod, constructing a continuous low-resistance conductive path to quickly remove accumulated charges from the target sample surface and avoid charge interference artifacts during SEM imaging. The grounding compensation mechanism can also automatically adapt to stage alignment deviations within ±0.2 mm, ensuring the coaxiality of each functional layer structure and the sample rod, allowing the arrayed superhydrophilic sites to accurately fall within the SEM imaging field of view. Furthermore, the grounding compensation mechanism can buffer minor vibrations during the insertion and removal of the SEM sample rod and its movement within the chamber, preventing structural damage to the upper heterogeneous wetting pattern layer and the biological functional anchoring layer, and ensuring the functional stability of droplet self-driven enrichment.

[0045] For example, the grounding compensation mechanism can consist of three parts: an array of elastic conductive springs, a micron-level alignment guide groove, and a vacuum adsorption locking module, which are integrated inside the bottom adapter base 150 and directly connected to the conductive contacts of the SEM sample rod.

[0046] In some embodiments, the conductive substrate 110 may include a conductive silicon wafer, ITO conductive glass, or a stainless steel substrate, that is, the conductive substrate 110 needs to be conductive.

[0047] In some embodiments, the superhydrophobic layer 120 is made of a loose network structure of hydrophobic carbon particles modified with long-chain alkyl-modified hydrophobic silica or modified with octadecyltrichlorosilane.

[0048] In some embodiments, the heterogeneous interface is configured such that the difference between the contact angle of the droplet formed by the target sample on the superhydrophobic layer surface and the contact angle on the superhydrophilic site surface is greater than 150°. The contact angle of the droplet formed by the target sample on the superhydrophobic layer surface can be greater than 150°, and the contact angle of the droplet formed by the target sample on the superhydrophilic site surface can be less than 10°.

[0049] In some embodiments, the material of the biofunctional anchoring layer 130 may include polylysine or aminosilane.

[0050] In some embodiments, the size of the superhydrophilic site 140 is 50 μm to 200 μm.

[0051] In some embodiments, 20 to 50 superhydrophilic sites 140 can be integrated on the superhydrophobic layer 120 to support simultaneous loading and continuous automatic characterization of multiple samples and gradients, thereby improving characterization efficiency.

[0052] The embodiments of this disclosure also provide a preparation method for preparing the above-mentioned high-throughput arrayed biomimetic super-immersion stage for electron microscopy characterization.

[0053] Figure 3 A flowchart illustrating a method for fabricating a high-throughput arrayed biomimetic superimmersion stage for electron microscopy characterization according to an embodiment of the present disclosure is shown.

[0054] like Figure 3 As shown, the preparation method of this embodiment may include operations S310 to S330.

[0055] In operation S310, a superhydrophobic layer is constructed on a conductive substrate.

[0056] In operation S320, a mask template is used to selectively etch the superhydrophobic layer, forming multiple superhydrophilic sites at multiple locations in the superhydrophobic layer.

[0057] In operation S330, the interior of each superhydrophilic site in multiple superhydrophilic sites is modified to form a biofunctional anchoring layer. The biofunctional anchoring layer is used to specifically anchor the target sample enriched in the superhydrophilic sites in order to complete the electron microscopy characterization of the target sample.

[0058] According to the embodiments of this disclosure, conductive substrates such as conductive silicon wafers and stainless steel can be used as carriers. Through processes such as gas phase molecular self-assembly, a fluorosilane superhydrophobic layer with both micro-nano rough structure and low surface energy can be constructed on the substrate surface. The water contact angle can reach more than 150°, which not only ensures the continuous conductivity of the substrate, but also provides a superhydrophobic background for subsequent droplet self-driven enrichment.

[0059] According to the embodiments disclosed herein, a superhydrophobic layer in a designated area can be precisely removed by using a patterned mask with a precision mask and processes such as plasma etching, exposing the original high surface energy hydrophilic surface of the substrate, forming an array of superhydrophilic sites of 50μm~00μm, realizing a heterogeneous wetting pattern of "superhydrophobic background-superhydrophilic site array", allowing droplets to be directionally enriched to the designated sites.

[0060] According to embodiments of this disclosure, the boundary of the superhydrophobic layer and the boundary of the superhydrophilic site constitute a heterogeneous interface, and the surface energy gradient generated by the heterogeneous interface is used to drive the target sample to accumulate towards the superhydrophilic site during the evaporation process.

[0061] According to embodiments of this disclosure, only the exposed superhydrophilic sites are selectively modified with biocompatible materials such as aminosilane and PLL to form a biofunctional anchoring layer that covers only the inside of the sites. This layer can specifically capture target biological samples such as miRNA and exosomes, avoiding the adsorption of non-target impurities in the superhydrophobic background and ensuring a continuous conductive pathway from the substrate to the sample, thus completely eliminating charge interference in SEM imaging and achieving high-quality electron microscopy characterization.

[0062] In some embodiments, the preparation method may further include: establishing a droplet dynamics model; and using the droplet dynamics model to determine the size of each superhydrophilic site among multiple superhydrophilic sites.

[0063] According to embodiments of this disclosure, a droplet dynamics model is used to describe the dynamic behavior of droplets formed from a target sample at a heterogeneous interface formed by the boundary of the superhydrophobic layer and the boundary of the superhydrophilic site.

[0064] Droplet dynamics models can focus on the interface between the superhydrophobic fluorosilane layer and the exposed conductive substrate, characterizing the spread, pinning, and directional enrichment of droplets at this heterogeneous interface, adapting to scenarios with superhydrophilic sites ranging from 50μm to 200μm. Driven by Laplace pressure, droplets spontaneously spread rapidly from the superhydrophobic background to the superhydrophilic site at this interface. The three-phase contact line pins to the heterogeneous boundary, preventing droplet overflow and achieving precise confined enrichment of the target sample. Droplet dynamics models can support parameter optimization for self-driven droplet manipulation, ensuring precise anchoring of biological samples such as miRNAs and exosomes at designated sites, avoiding cross-site contamination issues during SEM imaging.

[0065] In some embodiments, constructing a superhydrophobic layer on a conductive substrate includes: depositing candle ash at a constant rate onto the surface of the conductive substrate to form a loose network structure of carbon particles on the conductive substrate, wherein the diameter of the loose network structure of carbon particles is 30 nm to 40 nm; in a dryer, using ammonia water to catalyze tetraethyl orthosilicate (TEOS) to deposit silica on the loose network structure of carbon particles, forming a carbon-silica core-shell nanocomposite material; calcining the carbon-silica core-shell nanocomposite material to remove the carbon core, obtaining a conductive substrate modified with a porous nano-silica structure; immersing the conductive substrate modified with a porous nano-silica structure in an octadecyltrichlorosilane (OTS) solution for a preset time for surface modification, and then sequentially cleaning, drying, and thermally curing the nano-silica substrate to form a superhydrophobic layer on the conductive substrate.

[0066] In another embodiment, the superhydrophobic layer is constructed on the conductive substrate, which further includes: forming a silica nanoparticle layer on the conductive substrate, wherein the silica nanoparticles have a particle size of 30 nm to 40 nm; and hydrophobically modifying the silica particle layer with octadecyltrichlorosilane to form a superhydrophobic layer on the conductive substrate.

[0067] In some embodiments, selective etching of the superhydrophobic layer using a photomask is employed to form multiple superhydrophilic sites at multiple locations within the superhydrophobic layer. This includes: forming the superhydrophobic layer by tightly attaching a photomask; irradiating the superhydrophobic layer with ultraviolet light in the deep ultraviolet band through the photomask for etching; and destroying the silane coupling agent monolayer in the irradiated area through photo-oxidative degradation, thereby forming multiple superhydrophilic sites at multiple locations within the superhydrophobic layer, i.e., a hydrophilic silicon dioxide substrate. The etching method can be oxygen plasma etching.

[0068] For example, the candle ash is pushed at a constant speed (e.g., 5 cm / s). -1 The material is deposited on a clean conductive glass sheet (e.g., 5 cm thick) for 10 seconds.

[0069] In a desiccator, TEOS (2 mL) was catalyzed with ammonia (e.g., 2 mL, 98% by mass) and silica was deposited on a loose network of carbon particles by chemical vapor deposition (37°C, 24 h).

[0070] The obtained carbon-silica core-shell nanocomposite material was calcined at 550°C for 1.5 h to remove the carbon core, resulting in a conductive substrate modified with a porous nano-silica structure.

[0071] After cooling to room temperature, the conductive substrate modified with nano-silica porous structure was immediately immersed in OTS solution (1% OTS anhydrous toluene solution). After 10 min, it was cleaned with toluene and ethanol, dried with nitrogen, and then heat-cured in an oven at 120°C for 20 min.

[0072] The photomask was placed tightly against the OTS-modified nano-silica (OTS-SiO2) substrate, and ultraviolet light (approximately 150 mW / cm²) was used. -2 After irradiation for 30 minutes, a micropattern of "superhydrophobic background - superhydrophilic array site" was obtained.

[0073] It should be noted that the embodiments of the stage disclosed herein are compared with the embodiments of the preparation method, and the specific embodiments can be referred to each other, which will not be repeated here.

[0074] Those skilled in the art will understand that the features described in the various embodiments and / or claims of this disclosure can be combined or combined in various ways, even if such combinations or combinations are not explicitly described in this disclosure. In particular, the features described in the various embodiments and / or claims of this disclosure can be combined or combined in various ways without departing from the spirit and teachings of this disclosure. All such combinations and / or combinations fall within the scope of this disclosure.

[0075] The embodiments of this disclosure have been described above. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of this disclosure. Although various embodiments have been described above, this does not mean that the measures in the various embodiments cannot be used advantageously in combination. The scope of this disclosure is defined by the appended claims and their equivalents. Various substitutions and modifications can be made by those skilled in the art without departing from the scope of this disclosure, and all such substitutions and modifications should fall within the scope of this disclosure.

Claims

1. A high-throughput arrayed biomimetic superwetting stage for electron microscopy characterization, characterized in that, include: Conductive substrate; A superhydrophobic layer is formed on the surface of the conductive substrate. Multiple superhydrophilic sites are formed at multiple locations of the superhydrophobic layer. The boundary of the superhydrophobic layer and the boundary of the superhydrophilic sites constitute a heterogeneous interface. The surface energy gradient generated by the heterogeneous interface is used to drive the target sample to be directionally enriched towards the superhydrophilic sites during the volatilization process. A biofunctional anchoring layer is formed inside each of the multiple superhydrophilic sites, and is used to specifically anchor the target sample enriched at the superhydrophilic sites in order to complete the electron microscopy characterization of the target sample.

2. The high-throughput arrayed biomimetic super-immersion stage according to claim 1, characterized in that, Also includes: An adapter base is provided, on which the conductive substrate is mounted. The adapter base serves as the mechanical support and conductive pathway carrier for the high-throughput arrayed biomimetic super-immersion stage, and is connected to the sample holder of the scanning electron microscope.

3. The high-throughput arrayed biomimetic super-immersion stage according to claim 2, characterized in that, The adapter base has a grounding compensation mechanism; The grounding compensation mechanism is used for: A conductive path is constructed by eliminating the contact resistance between the adapter base and the sample rod to remove the accumulated charge on the surface of the target sample. Align the adapter base with the sample rod so that the superhydrophilic site falls within the field of view of the scanning electron microscope; This buffers the vibrations during the movement of the sample rod.

4. The high-throughput arrayed biomimetic superimmersion stage according to any one of claims 1 to 3, characterized in that, The conductive substrate includes a conductive silicon wafer, ITO conductive glass, or a stainless steel substrate; the superhydrophobic layer is made of a loose network structure of hydrophobic carbon particles modified with long-chain alkyl groups or modified with octadecyltrichlorosilane; the biofunctional anchoring layer is made of polylysine or aminosilane.

5. The high-throughput arrayed biomimetic superwetting stage according to any one of claims 1 to 3, characterized in that, The size of the superhydrophilic site is 50 μm to 200 μm.

6. The high-throughput arrayed biomimetic superimmersion stage according to any one of claims 1 to 3, characterized in that, The wettability gradient at the heterogeneous interface is configured such that the difference between the contact angle of the droplet formed by the target sample on the surface of the superhydrophobic layer and the contact angle on the surface of the superhydrophilic site is greater than 150°.

7. A preparation method, characterized in that, For fabricating high-throughput arrayed biomimetic superwetting stages for electron microscopy characterization, including: Constructing a superhydrophobic layer on a conductive substrate; The superhydrophobic layer is selectively etched using a mask template to form multiple superhydrophilic sites at multiple locations on the superhydrophobic layer. The boundary of the superhydrophobic layer and the boundary of the superhydrophilic sites constitute a heterogeneous interface. The surface energy gradient generated by the heterogeneous interface is used to drive the target sample to accumulate in the superhydrophilic sites during the evaporation process. The interior of each of the multiple superhydrophilic sites is modified to form a biofunctional anchoring layer. The biofunctional anchoring layer is used to specifically anchor the target sample enriched at the superhydrophilic site in order to complete the electron microscopy characterization of the target sample.

8. The preparation method according to claim 7, characterized in that, Also includes: A droplet dynamics model is established to describe the dynamic behavior of droplets formed by the target sample at the heterogeneous interface formed by the boundary of the superhydrophobic layer and the boundary of the superhydrophilic site. The size of each superhydrophilic site among the plurality of superhydrophilic sites is determined using the droplet dynamics model.

9. The preparation method according to claim 7 or 8, characterized in that, The construction of a superhydrophobic layer on a conductive substrate includes: Candle ash is deposited at a constant rate on the surface of the conductive substrate, forming a loose network structure of carbon particles on the conductive substrate, wherein the diameter of the loose network structure of carbon particles is 30 nm to 40 nm. In a dryer, ammonia water is used to catalyze tetraethyl orthosilicate to deposit silica on the loose network structure of carbon particles, forming a carbon-silica core-shell nanocomposite material. The carbon-silica core-shell nanocomposite material was calcined to remove the carbon core, resulting in a conductive substrate modified with a porous nano-silica structure. After the conductive substrate modified with a porous nano-silica structure is immersed in an octadecyltrichlorosilane solution for a preset time to perform surface modification, the nano-silica substrate is then sequentially cleaned, dried, and thermally cured to form the superhydrophobic layer on the conductive substrate. Alternatively, the construction of a superhydrophobic layer on a conductive substrate includes: A layer of silica nanoparticles is formed on the conductive substrate; wherein the particle size of the silica nanoparticles is 30 nm to 40 nm. The silica nanoparticle layer was hydrophobically modified using octadecyltrichlorosilane to form the superhydrophobic layer on the conductive substrate.

10. The preparation method according to claim 9, characterized in that, The selective etching of the superhydrophobic layer using a mask template forms multiple superhydrophilic sites at multiple locations on the superhydrophobic layer, including: The mask is tightly attached to form the superhydrophobic layer. The superhydrophobic layer is irradiated through the mask using the deep ultraviolet band of ultraviolet light. The silane coupling agent monolayer in the irradiated area is destroyed by photo-oxidative degradation, forming multiple superhydrophilic sites at multiple locations in the superhydrophobic layer.

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