DNA self-assembly sample pretreatment method for scanning electron microscope imaging

Abstract

The invention relates to the technical field of biology, in particular to a DNA self-assembly sample pretreatment method for scanning electron microscope imaging. According to the method, the DNA self-assembly structure is treated by adopting the nickel ion solution, the dehydration shrinkage deformation of the DNA self-assembly structure on a solid substrate is effectively inhibited, nickel ions react with a DNA phosphoric acid skeleton to generate a nickel phosphate compound with semiconductor characteristics, the electrical conductivity and thermal conductivity of the DNA self-assembly structure are remarkably enhanced, and the electrochemical performance of the DNA self-assembly structure is improved. Therefore, the DNA self-assembly structure can be directly subjected to SEM imaging without metal sputtering pretreatment, and disturbance of a metal sputtering layer to the morphology and composition of a DNA sample is effectively avoided. Compared with a traditional metal sputtering SEM imaging measurement method, the method has the advantages that the statistical deviation of DNA line width measurement is reduced by 50%, measurement artifacts are avoided, the original morphology size and physicochemical properties of the sample are kept unchanged, and an important supplementary means is expected to be provided for an existing nanometer manufacturing technology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for pre-processing DNA self-assembly samples for scanning electron microscopy imaging. Background Technology

[0002] DNA self-assembly is one of the technologies capable of obtaining high-resolution patterned fabrication templates. However, its practical application faces two major bottlenecks: first, the severe deformation (shrinkage, collapse) caused by dehydration during the transfer of DNA nanostructures from solution to the fabrication substrate; second, SEM measurements of the cured insulating DNA structures typically require sputtering a conductive metal layer. For DNA self-assembled structures with critical dimensions reduced to the 10-nanometer scale, the metal sputtering layer may introduce artifacts such as critical size broadening and uneven deposition. Furthermore, the bombardment process by metal particles may damage the DNA structure and alter the film's chemical composition, failing to meet the requirements for non-destructive characterization and subsequent processing. Therefore, achieving low-deformation curing of DNA nanopatterns on the surface of the fabrication substrate and their precise morphological characterization is a crucial prerequisite for advancing DNA self-assembly-mediated nanofabrication technology. Summary of the Invention

[0003] To overcome the aforementioned technical difficulties, this invention provides a DNA self-assembly sample pretreatment method for scanning electron microscopy imaging. The purpose is to enable DNA self-assembly structures to be directly imaged by SEM without metal sputtering pretreatment, avoiding charge accumulation and thermally induced structural deformation during the imaging process. The measured samples can be directly connected to subsequent nanofabrication steps such as reactive ion etching, chemical vapor deposition, and atomic layer deposition.

[0004] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for preprocessing DNA self-assembly samples for scanning electron microscopy imaging, comprising: S1. Dilute the DNA self-assembled structure with MgCl2 solution, drop the diluted homogenate onto the substrate, and incubate at room temperature for 0.5-2 hours until the DNA nanopattern is deposited on the substrate surface; S2. Add NiCl2 solution to the substrate surface obtained in S1 and incubate at room temperature for 0.5-2 hours until ion exchange is completed; S3. The substrate obtained in S2 is subjected to gradient dehydration with ethanol solution and then dried.

[0005] Further, in S1, the concentration of the MgCl2 solution is 1-50 mM, or 1-40 mM, or 1-30 mM, or 1-20 mM, or 5-20 mM, or 5-15 mM.

[0006] Furthermore, in S1, the substrate is a highly conductive inorganic substrate.

[0007] Furthermore, in S1, the inorganic substrate is selected from one of a silicon substrate, a graphene-silicon substrate, and a graphene-thermally oxidized silicon dioxide substrate.

[0008] Furthermore, in S2, the soluble nickel salt is selected from one of NiCl2, Ni(NO3)2, NiSO4, and Ni(CH3COO)2.

[0009] Further, in S2, the concentration of the soluble nickel salt solution is 10-500 mM, or 10-400 mM, or 10-300 mM, or 10-250 mM, or 20-200 mM.

[0010] Further, in S3, the gradient dehydration conditions are as follows: the substrate is sequentially rinsed in 70%, 90%, and 99.5% ethanol solutions, with each step involving 10-60 minutes of soaking, to complete the gradient dehydration.

[0011] Furthermore, in S3, the soaking time is 10-50 min, or 10-40 min, or 15-35 min, or 20-35 min, or 30 min.

[0012] Compared to existing technologies, this invention effectively suppresses the dehydration and shrinkage deformation of DNA self-assembled structures on solid substrates by treating them with nickel ion solution. Furthermore, the reaction of nickel ions with the DNA phosphate backbone generates nickel phosphate compounds with semiconductor properties, significantly enhancing the electrical and thermal conductivity of the DNA self-assembled structures. This allows for direct SEM imaging of the DNA self-assembled structures without metal sputtering pretreatment, effectively avoiding charge accumulation and thermally induced structural deformation during imaging. Compared to traditional metal sputtering methods, this invention reduces the statistical bias of DNA linewidth measurement by 50% while essentially maintaining the original morphology, size, and physicochemical properties of the sample, potentially providing an important supplementary method to existing nanofabrication technologies. Attached Figure Description

[0013] Figure 1 The effect of different nickel ion concentrations on SEM imaging of DNA self-assembly structures; where 1a is 50 mM Ni 2+ SEM image of the self-assembled DNA structure after treatment; 1b is 100 mM Ni 2+ SEM image of the self-assembled DNA structure after treatment; 1c is 150mM Ni 2+ SEM image of the self-assembled DNA structure after treatment; 1 day is 200 mM Ni 2+ SEM images of the processed DNA self-assembled structures; 1e is a comparison of grayscale cross-sectional curves of DNA self-assembled structures prepared with different nickel ion concentrations; Figure 2 The effects of metal sputtering pretreatment on the measurement of critical dimensions of DNA self-assembled structures are shown in the figures. 2a is the original SEM image of the Pt-sputtered DNA self-assembled structure on a silicon substrate; 2b is the grayscale cross-sectional curve of 2a; 2c is the statistical distribution of DNA linewidth in 2a; 2d is the statistical distribution of DNA line spacing in 2a; 2e is the original SEM image of the non-sputtered DNA self-assembled structure on a graphene-silicon substrate; 2f is the grayscale cross-sectional curve of 2e; 2g is the statistical distribution of DNA linewidth in 2e; and 2h is the statistical distribution of DNA line spacing in 2e. Figure 3 The effects of different substrates on SEM imaging of DNA self-assembly structures are shown; where 3a is a silicon substrate; 3b is a thermally oxidized silica substrate; 3c is a graphene-silicon substrate; and 3d is a graphene-thermally oxidized silica substrate. Figure 4 The effect of different immobilized ions on SEM imaging of DNA self-assembly structures; Figure 5 Comparison of SEM images of DNA self-assembly structures on different substrates before and after 60-second continuous electron beam irradiation; among them, 5a is a silicon substrate with a 3 nm Pt metal layer sputtered on; 5b is a silicon substrate without conductive metal layer sputtering; 5c is a graphene-silicon substrate without conductive metal layer sputtering; 5d is a graphene-thermally oxidized silicon dioxide substrate without conductive metal layer sputtering. Figure 6 SEM image of a DNA self-assembled structure on a thermally oxidized silica substrate (without a conductive metal layer sprayed on) after 60 seconds of continuous electron beam irradiation. Detailed Implementation

[0014] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0015] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0016] This invention aims to develop a method for dehydrating and fixing DNA self-assembled structures for scanning electron microscopy imaging without the need for metal sputtering. The use of nickel ions can effectively suppress the dehydration and shrinkage of DNA self-assembled structures on the surface of a solid substrate. Furthermore, the nickel phosphate compounds generated by the reaction of nickel ions with the DNA phosphate backbone have semiconductor properties, thereby improving the conductivity and thermal conductivity of the DNA self-assembled structure. Therefore, the DNA self-assembled structure modified with nickel ions can effectively avoid charge accumulation and thermal deformation during SEM imaging without the need for a sputtered metal conductive layer.

[0017] Based on the present invention, a DNA self-assembly sample pretreatment method for scanning electron microscopy imaging includes: S2. Dissolve the DNA self-assembled structure in MgCl2 solution, dilute the homogenate with MgCl2 solution, drop the diluted homogenate onto the substrate, and incubate at room temperature for 0.5-2 hours until the DNA nanopattern is deposited on the substrate surface. In some embodiments of this implementation, the concentration of the MgCl2 solution is 1-50 mM, and the concentration of the MgCl2 solution can be selected as 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, or 50 mM; other specific values ​​within this range can also be selected, and will not be described in detail here. In some embodiments of this implementation, the substrate is a highly conductive inorganic substrate. In some preferred embodiments, the inorganic substrate is selected from one of a silicon substrate, a graphene-silicon substrate, and a graphene-thermally oxidized silicon dioxide substrate. S3. Ni is dropped onto the substrate surface obtained in S2. 2+ Incubate the solution at room temperature for 0.5-2 hours until ion exchange is complete; In some embodiments of this implementation, the Ni 2+ The solution concentration is 10-500 mM. The NiCl2 solution concentration can be selected from 10mM, 30mM, 50mM, 70mM, 100mM, 120mM, 150mM, 180mM, 200mM, 250mM, 300mM, 400mM, and 500mM. Other specific values ​​within this range can also be selected, and will not be elaborated here. S4. The substrate obtained in S3 is subjected to gradient dehydration with ethanol solution and then dried; In some embodiments of this implementation, the gradient dehydration conditions are as follows: the substrate is sequentially rinsed in 70%, 90%, and 99.5% ethanol solutions, with each step involving 10-60 minutes of soaking, to complete the gradient dehydration; in some preferred embodiments, the soaking time is 10-50 minutes, or 10-40 minutes, or 15-35 minutes, or 20-35 minutes, and in some more preferred embodiments, the soaking time is 30 minutes; specifically, gradient ethanol is selected as the dehydrating agent, and the final drying and curing is completed in an anhydrous ethanol vapor atmosphere, which can gently remove moisture and minimize the damage of surface tension to the capillary forces of the fine DNA nanostructure.

[0018] Surface imaging of DNA nanopatterns fixed on a substrate was performed using scanning electron microscopy.

[0019] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0020] Example 1 Self-assembly of DNA self-assembly structures: Synthetic single-stranded DNA (ssDNA, ~300 nM each) for the base module was mixed with 8 μL of TE / Mg 2+ A buffer solution (5 mM Tris, 1 mM EDTA, 40 mM MgCl2, pH 7.9) was mixed and incubated at 44°C for 12 hours, followed by a second incubation at 39°C for 24 hours to form a seed solution. Subsequently, 27 μL of TE / MgCl2 containing a 200 nM base module and a 280 nM line module was added. 2+ The buffer solution was added to the seed solution and mixed, and then incubated at 38°C for 48 hours, followed by incubation at 33°C for 8 hours to complete the epitaxial growth of the DNA self-assembled structure. The formed DNA self-assembled structure was stored in the reaction buffer at 4°C without further purification. Deposition of DNA nanopatterns on silicon substrates: The synthesized DNA brick self-assembled structures were diluted 100-fold in 15 mM MgCl2 solution and incubated overnight at 4°C; after homogenization, they were further diluted with an equal volume of 15 mM MgCl2 solution. 1 μL of the diluted solution was dropped onto a 5 mm × 5 mm silicon substrate and incubated for 1 hour to allow adsorption. Nickel ion stabilization: After the DNA nanopattern adsorption is complete, 2 μL of 50 mM NiCl2 solution is added and incubated for another hour to complete nickel ion exchange and structural stabilization. Gradient dehydration and curing: The nickel ion-treated sample was sequentially rinsed in 70%, 90%, and 99.5% ethanol solutions, with each step involving 30 minutes of soaking, to complete gradient dehydration and high-fidelity curing. Example 2

[0021] The same DNA self-assembly structure, nickel ion stabilization, deposition, and solidification procedures as in Example 1 were used, except that the NiCl2 solution concentration was 100 mM. Example 3

[0022] The same DNA self-assembly structure, nickel ion stabilization, deposition, and solidification procedures as in Example 1 were used, except that the NiCl2 solution concentration was 150 mM. Example 4

[0023] The same DNA self-assembly structure, nickel ion stabilization, deposition, and solidification procedures as in Example 1 were used, except that the NiCl2 solution concentration was 200 mM. Example 5

[0024] The same DNA self-assembly structure, nickel ion stabilization, deposition, and curing process as in Example 1 were used, except that the silicon substrate (Si) was replaced with a graphene-silicon substrate (Graphne-Si). The graphene-silicon substrate was prepared as follows: Silicon (Si) was cleaned in a piranha solution (H₂SO₄:H₂O₂ = 3:1 v / v). 2-5 wt% polymethyl methacrylate (PMMA) photoresist was spin-coated onto a single layer of graphene at 2000 rpm and baked at 120°C for 3 minutes. The layer was then treated with a copper etchant until the interlayer graphene film became optically transparent. Subsequently, the graphene film was sequentially cleaned with a hydrophilic glass slide in 15-20% HCl solution and deionized water (two cycles), capillarily covering the hydrophilic substrate. After annealing at 150°C for 30 minutes, PMMA residue was removed by three acetone immersions. Finally, the substrate was heated in a tube furnace at 300°C for 2 hours in a gas stream of 100 sccm hydrogen-argon mixture (10 vol% hydrogen) and 100 sccm argon to remove any residual PMMA, yielding the graphene-silicon substrate. Example 6

[0025] The same DNA self-assembly structure, nickel ion stabilization, deposition, and curing process as in Example 1 were used, except that the silicon substrate (Si) was replaced with a graphene-thermally oxidized silicon dioxide substrate (Graphne-Si-SiO2 / Si). The method for preparing the graphene-thermally oxidized silicon dioxide substrate is as follows: The thermally oxidized silicon dioxide (SiO2 / Si) substrate was cleaned in a piranha solution (H2SO4:H2O2 = 3:1 v / v). A 2-5 wt% polymethyl methacrylate (PMMA) photoresist was spin-coated onto the monolayer graphene at 2000 rpm and baked at 120°C for 3 minutes. The layered graphene film was treated with copper etchant until it became optically transparent. Subsequently, the graphene film was sequentially cleaned in 15-20% HCl solution and deionized water (two cycles) using a hydrophilic glass slide, capillarily coating the hydrophilic substrate. After annealing at 150°C for 30 minutes, PMMA residue was removed by three acetone immersions. Finally, the substrate was heated in a tube furnace at 300°C for 2 hours in a gas stream of 100 sccm hydrogen-argon mixture (10 vol% hydrogen) and 100 sccm argon to remove any residual PMMA, yielding a graphene-thermally oxidized silicon dioxide substrate.

[0026] Comparative Example 1 The same DNA self-assembly structure, nickel ion stabilization, deposition, and solidification procedure as in Example 1 were used, except that the NiCl2 solution was replaced with a MgCl2 solution.

[0027] Comparative Example 2 The same DNA self-assembly structure, nickel ion stabilization, deposition, and curing process as in Example 1 were used, except that the silicon substrate was replaced with a thermally oxidized silicon dioxide substrate (SiO2 / Si).

[0028] The above embodiments and comparative examples were subjected to SEM imaging tests and analyses.

[0029] Image processing employed quantitative analysis using image analysis software such as Gwyddion. The gray-level difference between the DNA line modules and their gaps was used as image contrast, and the maximum value of the first derivative of the periodic fluctuation curve of gray level along the x-direction was used as edge sharpness. Image contrast and image sharpness of the three-dimensional structure edges are indicators for evaluating image resolution. By extracting the half-width at half-maximum (FWHM) of the line cross-sections, the linewidth and spacing of the DNA parallel line nanopatterns were statistically analyzed to assess morphological fidelity and measurement accuracy.

[0030] Test Example 1 The cured samples from Examples 1-4 were directly placed in the Hitachi Regulus 8220 SEM sample chamber. Under high vacuum conditions, without platinum metal sputtering, imaging was performed using a secondary electron detector at an accelerating voltage of 10 kV, a beam current of 10 μA, and a working distance of 5 mm to obtain the original morphology of the DNA nanopatterns. The results are as follows: Figure 1 As shown.

[0031] Figure 1As can be seen, within the nickel ion concentration range of 50 mM to 200 mM, nickel ions effectively inhibit dehydration shrinkage and react with the DNA phosphate backbone to generate compounds with semiconductor properties. The sputter-free SEM image quality (contrast and sharpness) of DNA self-assembled structures incubated with different concentrations of nickel ions showed no significant difference, proving that 50 mM is sufficient to achieve saturation processing.

[0032] Test Example 2 The cured sample from Example 5 was directly placed in the Hitachi Regulus 8220 SEM sample chamber. Under high vacuum conditions, without platinum sputtering, imaging was performed using a secondary electron detector at an accelerating voltage of 10 kV, a beam current of 10 μA, and a working distance of 5 mm to obtain the original morphology of the DNA nanopatterns. The cured sample from Example 1 was sputtered with platinum using a combined sputtering and carbon coating system (Q150T ES plus) to form a conductive layer with a thickness of approximately 3 nm, serving as a control sample for characterization using conventional methods. The sample with the sputtered platinum conductive layer was placed in the Hitachi Regulus 8220 SEM sample chamber. Under high vacuum conditions, imaging was performed using a secondary electron detector at an accelerating voltage of 10 kV, a beam current of 10 μA, and a working distance of 5 mm to obtain the morphology of the platinum-coated DNA nanopatterns. The results are as follows: Figure 2 As shown.

[0033] Figure 2 As can be seen, the nickel-modified DNA self-assembled structure maintains a clear and regular parallel line morphology. Quantitative analysis of the grayscale values ​​of the SEM images shows that the linewidth of the Pt sputtered DNA self-assembled nanowires on the silicon substrate is 12.0 ± 2.4 nm, while the linewidth of the non-sputtered sample on the graphene-silicon substrate is 12.8 ± 1.1 nm. The statistical deviation of the linewidth of the non-sputtered sample (±1.1 nm) is only half that of the traditional Pt sputtering method (±2.4 nm). This is because the metal sputtering layer inevitably has micro-area deposition rate differences, forming metal particles of varying sizes, which is reflected in the SEM quantitative measurement of the parallel line pattern as an increase in the standard deviation of the linewidth. Therefore, this invention can significantly improve the measurement accuracy of ultra-small DNA structures, and the characterized original samples can still be used for various nanofabrication processes, demonstrating extremely high applicability.

[0034] Test Example 3 The cured samples from Examples 1, 5-6, and Comparative Example 2 were directly placed in the sample chamber of a Hitachi Regulus 8220 SEM. Under high vacuum conditions, without platinum sputtering, imaging was performed using a secondary electron detector at an accelerating voltage of 10 kV, a beam current of 10 μA, and a working distance of 5 mm to obtain the original morphology of the DNA nanopatterns. The results are as follows: Figure 3As shown.

[0035] Figure 3 As can be seen, Ni 2+ Stable DNA self-assembled structures can achieve sputter-free SEM imaging on various substrates, but their imaging quality is positively correlated with substrate conductivity. Compared to Comparative Example 2, the DNA self-assembled structure in Example 5 achieved the highest structural contrast and edge sharpness on the graphene-silicon substrate with the best conductivity, while the DNA self-assembled structure in Comparative Example 2 had the worst imaging quality on the insulating SiO2 / Si substrate. This demonstrates that improving the overall conductivity of the sample can effectively reduce surface electric field interference to the scanning electron beam, increase secondary electron yield, and thus improve SEM image quality.

[0036] Test Example 4 The cured samples from Example 1 and Comparative Example 1 were directly placed in the Hitachi Regulus 8220 SEM sample chamber. Under high vacuum conditions, without platinum sputtering, imaging was performed using a secondary electron detector at an accelerating voltage of 10 kV, a beam current of 10 μA, and a working distance of 5 mm to obtain the original morphology of the DNA nanopatterns. The cured samples from Example 1 and Comparative Example 1 were then subjected to platinum sputtering using a combined sputtering and carbon coating system (Q150T ES plus) to form a conductive layer approximately 3 nm thick, serving as a control sample for characterization using conventional methods. The samples with the sputtered platinum conductive layer were placed in the Hitachi Regulus 8220 SEM sample chamber. Under high vacuum conditions, imaging was performed using a secondary electron detector at an accelerating voltage of 10 kV, a beam current of 10 μA, and a working distance of 5 mm to obtain the morphology of the platinum-coated DNA nanopatterns. The results are as follows: Figure 4 As shown.

[0037] Figure 4 As can be seen, magnesium ions (Mg 2+ DNA self-assembly structures treated with nickel ions (Ni) collapse severely after dehydration, resulting in blurred lines; while those treated with nickel ions (Ni) 2+ The nickel-treated samples maintained clear and regular parallel line morphology, demonstrating the crucial role of nickel ions in suppressing the dehydration and deformation of DNA nanostructures. Furthermore, under sputter-free SEM images, the nickel-treated samples exhibited higher imaging resolution than magnesium-treated samples, proving that nickel ions react with the DNA phosphate backbone to generate nickel phosphate compounds with semiconductor properties. This significantly enhances the electrical and thermal conductivity of the DNA self-assembled structure, allowing for direct SEM imaging of the DNA self-assembled structure without metal sputtering pretreatment.

[0038] Test Example 5 Electron beam irradiation experiments were conducted on the cured samples from Examples 1, 5-6, and Comparative Example 2. Specifically, under conditions of 10 kV voltage and 10 μA current, the samples were first irradiated with 5.0 × 10⁻⁶ kV electron beam. 5 The SEM image before irradiation was obtained at a magnification of 2.0×10⁻⁶. 6 The area was irradiated with an electron beam for 60 seconds, and then the beam was reduced to the initial 5.0 × 10⁻⁶ s. 5 SEM images of the irradiated area were obtained at a magnification of [magnification level], and SEM images of the same location before and after irradiation were obtained accordingly. The results are as follows: Figure 5-6 As shown.

[0039] Figure 5 As can be seen, the stability of the nickel-ion-incubated sputter-free DNA samples depends on the substrate conductivity. The sputter-free samples from Examples 1 and 5-6 exhibit stable parallel line patterns with constant spacing on a highly conductive substrate. Although radiation-induced DNA dehydration and volatile redeposition occurred, the deformation was minimal, manifesting only as small fluctuations in the linewidth of the DNA self-assembly structure. Figure 6 As shown, the sample on the insulating substrate (such as SiO2 / Si) in Comparative Example 2 exhibited severe carbonization and redeposition problems, hindering subsequent re-imaging. On a highly conductive substrate, the nickel-ion-stabilized, non-sputtered sample showed electron beam tolerance comparable to that of a conventional Pt-sputtered sample, demonstrating that highly conductive substrates can provide protection comparable to metal capping layers.

[0040] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. A method for preprocessing DNA self-assembly samples for scanning electron microscopy imaging, characterized in that... include: S1. Dilute the DNA self-assembled structure with MgCl2 solution, drop the diluted homogenate onto the substrate, and incubate at room temperature for 0.5-2 hours until the DNA nanopattern is deposited on the substrate surface; S2. Add a soluble nickel salt solution to the surface of the substrate obtained in S1 and incubate at room temperature for 0.5-2 hours until ion exchange is completed; S3. The substrate obtained in S2 is subjected to gradient dehydration with ethanol solution and then dried.

2. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 1, characterized in that, In S2, the soluble nickel salt is selected from one of NiCl2, Ni(NO3)2, NiSO4, and Ni(CH3COO)2.

3. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 1, characterized in that, In S1, the concentration of the MgCl2 solution is 1-50 mM, or 1-40 mM, or 1-30 mM, or 1-20 mM. Or 5-20 mM, or 5-15 mM.

4. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 1, characterized in that, In S1, the substrate is a highly conductive inorganic substrate.

5. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 4, characterized in that, The inorganic substrate is selected from one of the following: silicon substrate, graphene-silicon substrate, and graphene-thermally oxidized silicon dioxide substrate.

6. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 1, characterized in that, In S2, the concentration of the soluble nickel salt solution is 10-500 mM, or 10-400 mM, or 10-300 mM, or 10-250 mM, or 20-200 mM.

7. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 1, characterized in that, In step S3, the gradient dehydration conditions are as follows: the substrate is sequentially rinsed in 70%, 90%, and 99.5% ethanol solutions, with each step involving 10-60 minutes of soaking, to complete the gradient dehydration.

8. The DNA self-assembly sample pretreatment method for scanning electron microscopy imaging according to claim 7, characterized in that, In S3, the soaking time is 10-50 min, or 10-40 min, or 15-35 min, or 20-35 min, or 30 min.

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