Photosensitive DNA-based pattern template and pattern preparation method

Abstract

The application provides a photosensitive DNA-based pattern template and a pattern preparation method. The pattern preparation method comprises the following steps: performing passivation treatment on a substrate surface; modifying an anchor DNA chain, an azobenzene-DNA chain and a guide assembly DNA chain on the substrate surface, wherein the first end of the azobenzene-DNA chain has a first hybridization fragment with the anchor DNA chain, and the second end of the azobenzene-DNA chain has a second hybridization fragment with the guide assembly DNA chain; performing light irradiation on the second hybridization fragments of the azobenzene-DNA chain and the guide assembly DNA chain according to a set pattern to make the second hybridization fragments denature; and assembling a modified material in a region where the guide assembly DNA chain is reserved to obtain the set pattern. The application can form a required pattern through a two-photon scanning mode, prevents thermal damage of biological molecules, and provides a feasible scheme for a multifunctional, mask-free, clean room-free and environmentally friendly photolithography method.

Description

Technical Field

[0001] This invention relates to the field of nanopattern fabrication, and in particular to a pattern template based on photosensitive DNA and a method for fabricating the pattern. Background Technology

[0002] Manipulating functional materials on surfaces with submicron precision is a key challenge in surface engineering, impacting applications from biology to optics and materials science. Surface patterning of biomaterials and micro / nanostructures plays a crucial role in constructing anti-wetting surfaces, cell migration studies, generating surface plasmon resonance sensors, and microfluidics research. The invention of various photolithography techniques has greatly accelerated the development of surface patterning methods for biomaterials and micro / nanostructures. However, complex surface structures possess multiple functions and contain a variety of materials ranging from molecules to nanoparticles, making the development of diverse etching patterning strategies for constructing such complex surface structures extremely challenging. Recent patterning strategies have become increasingly complex and require numerous large and expensive instruments, making them inaccessible to general researchers. On the one hand, methods for material patterning include scanning probe lithography, electron beam lithography, and nanoimprint lithography. These techniques typically involve invasive transfer of thermal, mechanical, electronic, and electrical energy to the substrate, relying heavily on disrupting the substrate surface to achieve material patterning. This limits the types and number of materials and substrates that can be used with them, which in turn limits the application scope of these patterning methods. On the other hand, these top-down material patterning methods require cleanroom environments, are time-consuming, require expensive consumables, and inevitably involve operation by specialized personnel or specialized training, increasing the barrier to practical application. Furthermore, for mask-based lithography, any slight modification to the pattern design requires the entire process to be restarted from scratch, resulting in significant delays between device iterations based on material patterning.

[0003] Furthermore, the deactivation of biomolecules caused by high vacuum and high-energy radiation has been used to pattern materials, for example, by selectively ablating biomolecules under electron beam irradiation. In contrast, patterning functional biomolecules (such as antibodies or DNA aptamers) on surfaces holds great promise for applications in high-throughput biosensors and biodiagnostics. These microarrays are increasingly used to construct protein and small molecule sensors for biomedical and environmental research, and their further development will enable system-wide proteomics and interactomics studies, as well as wider adoption in dynamic molecular analysis of living cells and tissues. This requires that the patterned biomolecules remain active and can be further used as templates for the secondary immobilization of various analytes, such as nucleic acids or proteins, biomolecules, and nanoparticles. In addition, in the colloid field, a series of plasma sensors with different physical properties have been developed by precisely controlling the structure of plasma nanoassemblies, applied to various fields from measuring mechanical pressure during manufacturing to detecting protein concentrations in biomedical samples. However, compared to molecular systems, nanoassemblies, due to their large size and mass, often cannot be well transferred to a designated surface. Despite advancements in cutting-edge nanolithography, the controlled patterning of nanoassemblies remains a significant challenge due to the difficulties in nanoparticle transfer systems.

[0004] To avoid these drawbacks, specific interface modifications, including chemical linkers and oligonucleotides, have attracted considerable attention in the engineering of biosensors based on surface nanoassemblies. The versatility of surface self-assembly can be used to form diverse nanoparticle systems, further driving the development of nanoparticle systems. Specifically, DNA has been used as a universal engineering template for a variety of submicron structures due to its predictable and programmable hybridization interactions. Recently, researchers have devoted considerable effort to designing DNA-modified surfaces with complex surface structures, including non-nucleic acid compounds such as proteins, as well as functional DNA devices and structural DNA nanotechnology. Importantly, due to the ease of modification of oligonucleotides, DNA has been widely used to guide the self-assembly of various nanoparticles, including carbon nanotubes, plasmonic nanoparticles, and various biomolecules, to form desired geometries. Furthermore, advances in DNA biochips have created interfaces for functional connections between artificial materials and living systems.

[0005] Despite these significant advances in the field of DNA-based surface modification, the need to develop maskless strategies for generating geometrically complex multi-component patterns with submicron resolution using only non-invasive and minimal additional inputs without generating environmental pollutants remains unmet.

[0006] It should be noted that the above introduction to the technical background is only for the purpose of providing a clear and complete explanation of the technical solutions of this application and facilitating understanding by those skilled in the art. It should not be assumed that these technical solutions are known to those skilled in the art simply because they have been described in the background section of this application. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a graphic template based on photosensitive DNA and a graphic preparation method, so as to achieve a graphic preparation method that does not damage the activity of biomolecules.

[0008] To achieve the above and other related objectives, the present invention provides a method for preparing patterns based on photosensitive DNA. The method includes the following steps: providing a substrate and passivating the surface of the substrate; modifying the substrate surface with an anchor-DNA strand, an azobenzene-DNA strand, and a guide assembly DNA strand, wherein a first end of the azobenzene-DNA strand has a first hybridization fragment with the anchor-DNA strand, and a second end of the azobenzene-DNA strand has a second hybridization fragment with the guide assembly DNA strand; irradiating the azobenzene-DNA strand and the second hybridization fragment of the guide assembly DNA strand according to a predetermined pattern, thereby causing the azobenzene-DNA strand and the guide assembly DNA strand to unwind based on the irradiation, so as to remove the guide assembly DNA strand from the azobenzene-DNA strand in the irradiated area; and assembling a modification material in the area where the guide assembly DNA strand is retained to obtain the predetermined pattern.

[0009] Optionally, the illumination is single-photon ultraviolet light with a wavelength between 300nm and 400nm or two-photon light with a wavelength between 650nm and 800nm. Under single-photon ultraviolet light or two-photon light illumination, azobenzene changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA strand to unwind from the guide assembly DNA strand.

[0010] Optionally, the illumination method includes one of two-photon scanning illumination mode, wide-field illumination mode with mask, and digital mirror device illumination mode.

[0011] Optionally, the substrate is a glass substrate, and the materials used to passivate the surface of the substrate include polyethylene glycol-silane and biotin-polyethylene glycol-silane, so that the anchored DNA strand can be specifically attached to the modified glass substrate.

[0012] Optionally, modifying the glass substrate with polyethylene glycol-silane and biotin-polyethylene glycol-silane includes the steps of: activating the surface of the glass substrate with HNO3 at a concentration of 40% or higher; and modifying the surface of the glass substrate with a mixed solution of biotin-polyethylene glycol-silane and polyethylene glycol-silane.

[0013] Optionally, the azobenzene-DNA chain comprises a first fragment and a second fragment. The first fragment is a natural DNA fragment whose sequence is complementary to the anchored DNA chain on the substrate, for anchoring the azobenzene-DNA chain onto the substrate. The second fragment comprises a photosensitive fragment composed of multiple oligonucleotides, wherein several azobenzene-modified bases serve as structure transition domains. Under light irradiation, the azobenzene in these structure transition domains changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain to unwind from the guide assembly DNA chain.

[0014] Optionally, in the photosensitive fragment sequence, an azobenzene molecule is modified every two bases.

[0015] Optionally, the guided assembly DNA strand includes a first fragment and a second fragment, the first fragment being complementary to the photosensitive fragment, and used to control the hybridization or unwinding of the guided assembly DNA strand with the azobenzene-DNA strand anchored on the substrate through the structural transition domain of the photosensitive fragment, thereby achieving the patterning of the guided assembly DNA strand.

[0016] Optionally, the second fragment can be used as a binding site to guide the assembly of the modified material by hybridization with the complementary DNA strand of the modified material, thereby achieving the re-patterning of the modified material.

[0017] Optionally, the modified material includes one of DNA-modified nanoparticles, biomolecules, and DNA-modified fluorescent groups.

[0018] Optionally, the modifying material comprises multiple layers of DNA modification connected in sequence, with adjacent DNA modification layers linked by complementary DNA. The material of the multiple DNA modification layers includes one or more of DNA-modified nanoparticles, DNA-modified fluorescent groups, and biomolecules.

[0019] Optionally, the multiple DNA modification layers are incubated sequentially or simultaneously.

[0020] Optionally, the preparation method further includes the step of: irradiating the azobenzene-DNA chain with visible light to transform it from a non-planar cis structure to a planar trans structure, and restoring its hybridization with the guide assembly DNA chain, thereby enabling the azobenzene-DNA chain to have rewriting capability.

[0021] The present invention also provides a graphic template based on photosensitive DNA, comprising: a substrate having a passivation layer on its surface; an anchoring DNA strand, an azobenzene-DNA strand, and a guide assembly DNA strand, sequentially modified on the surface of the passivation layer; wherein a first end of the azobenzene-DNA strand has a first hybridization fragment with the anchoring DNA strand, and a second end of the azobenzene-DNA strand has a second hybridization fragment with the guide assembly DNA strand; wherein the second hybridization fragment between the azobenzene-DNA strand and the guide assembly DNA strand can be defused under light to remove the guide assembly DNA strand from the azobenzene-DNA strand in the light-exposed area.

[0022] Optionally, the substrate is a glass substrate, and the passivation layer is made of polyethylene glycol-silane and biotin-polyethylene glycol-silane to enable the anchored DNA to be specifically attached to the modified glass substrate.

[0023] Optionally, the azobenzene-DNA chain comprises a first fragment and a second fragment. The first fragment is a natural DNA fragment whose sequence is complementary to the anchored DNA chain on the substrate, for anchoring the azobenzene-DNA chain onto the substrate. The second fragment comprises a photosensitive fragment composed of multiple oligonucleotides, wherein several azobenzene-modified bases serve as structure transition domains. Under light irradiation, the azobenzene in these structure transition domains changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain to unwind from the guide assembly DNA chain.

[0024] Optionally, in the photosensitive fragment sequence, an azobenzene molecule is modified every two bases.

[0025] Optionally, the guided assembly DNA strand includes a first fragment and a second fragment. The first fragment is complementary to the photosensitive fragment and is used to control the hybridization or unwinding of the guided assembly DNA strand with the azobenzene-DNA strand anchored on the substrate through the structural transition domain of the photosensitive fragment, thereby realizing the patterning of the guided assembly DNA strand. The second fragment serves as a binding site for hybridization with the complementary DNA strand of the modification material to guide the assembly of the modification material, thereby achieving secondary patterning.

[0026] Optionally, the modified material includes one of DNA-modified nanoparticles, biomolecules, and DNA-modified fluorescent groups.

[0027] Optionally, the modifying material comprises multiple layers of DNA modification connected in sequence, with adjacent DNA modification layers linked by complementary DNA. The material of the multiple DNA modification layers includes one or more of DNA-modified nanoparticles, DNA-modified fluorescent groups, and biomolecules.

[0028] Optionally, after the second hybridization fragment of the azobenzene-DNA chain and the guide assembly DNA chain is unwound under light, the azobenzene-DNA chain can be transformed from a non-planar cis structure to a planar trans structure by visible light irradiation, and hybridization with the guide assembly DNA chain can be restored, thereby enabling the azobenzene-DNA chain to have rewriting capability.

[0029] As described above, the graphic template and graphic preparation method based on photosensitive DNA of the present invention have the following beneficial effects:

[0030] This invention achieves the dissociation of azobenzene-DNA strands and guide assembly DNA strands using two-photon illumination with a longer wavelength. Two-photon illumination can satisfy the dissociation of azobenzene-DNA strands and guide assembly DNA strands with a longer wavelength, that is, the optimal DNA strand optical switching control efficiency can be achieved using a very low-power two-photon laser. This effectively avoids the inactivation of DNA strands or biomolecules caused by using shorter wavelength ultraviolet light, prevents thermal damage to biomolecules, and greatly improves the application scope of this invention.

[0031] This invention uses light to transform the azobenzene-DNA chain from a planar trans structure to a non-planar cis structure, thereby achieving dissociation from the guide assembly DNA chain. Subsequently, visible light irradiation can transform the azobenzene-DNA chain from a non-planar cis structure to a planar trans structure, restoring hybridization with the guide assembly DNA chain and enabling the azobenzene-DNA chain to have rewriting capabilities.

[0032] This invention can form the desired pattern through a two-photon scanning mode, providing a feasible solution for a multifunctional, maskless, cleanroom-free, and environmentally friendly photolithography method. Attached Figure Description

[0033] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the embodiments of this application and to illustrate the implementation of this application, together with the textual description, to explain the principles of this application. Obviously, the drawings described below are merely some embodiments of this application.

[0034] Figure 1 A shows a schematic diagram of the structure presented in each step of the photosensitive DNA-POINT method according to an embodiment of the present invention. Figure 1 B shows the JHU pattern as a template for the self-assembly of gold nanoparticles (AuNPs), and the dots in the magnified image represent individual AuNPs.

[0035] Figure 2 The diagram shown is a schematic representation of the light-modulated convertible DNA interface of the present invention, wherein, Figure 2A shows a schematic diagram of single-photon driven DNA double-helix hybridization switching at the interface. Dark-field images show AuNPs attached to the surface before and after 10 minutes of UV irradiation. Analysis of AuNP scattering intensity reveals the switching efficiency. Figure 2 B shows a schematic diagram of the switching of two-photon driven DNA double helix hybridization on the surface. Dark-field images show the AuNPs connected on the interface at laser powers of 0%, 0.2%, 0.5%, 1%, 2%, and 3%, respectively. Analysis of the AuNP scattering intensity gives the switching efficiency as a function of different laser powers.

[0036] Figure 3 This invention displays a maskless submicron pattern achieved in the scanning mode of its invention, wherein... Figure 3 A is shown as a dashed AuNP pattern formed using DNA-POINT, with widths of 1 μm, 2 μm, and 10 μm, respectively. The intensity distribution of the line cross-section was analyzed using Gaussian fitting. Figure 3 B is displayed as a symmetrical pattern of a linear grid and a block array connected to AuNPs, as well as an asymmetrical pattern of spiral lines.

[0037] Figure 4 The diagram shows a self-assembly schematic of different materials and multiple layers according to the present invention, wherein, Figure 4 A, acting as a complementary second DNA layer, further patterned the original light-driven DNA modification pattern using this complementary second DNA layer. Fluorescence (FL) images showed negative JHU patterns formed in DNA regions without Cy3 fluorescent labeling. Figure 4 B is shown as a three-layer pattern consisting of a patterned light-driven DNA primitive layer, a self-assembled AuNPs layer (second layer) on which it is assembled, and a DNA layer assembled using the AuNPs layer as a template (third layer). Corresponding dark-field (DF), fluorescence (FL), and overlay images show the green scattering of AuNPs, the pseudo-red fluorescence of Cy3-labeled DNA, and colocalization, respectively.

[0038] Figure 5 This invention demonstrates how DNA-POINT-controlled RGD clusters regulate cell adhesion and diffusion, wherein... Figure 5 A shows a schematic diagram of a high-density self-assembled AuNPs layer used for cell seeding, with an accompanying bright-field image showing small round cells after 6 hours of incubation; Figure 5 Image B shows RGD clusters formed after adding RGD-modified DNA linked to gold nanoparticles. The corresponding bright-field image clearly shows adherent (elongated) cells after 6 hours of incubation. Figure 5 An illustration showing the C-limiting effect of using light-controlled RGD cluster density to dynamically regulate cell adhesion and diffusion. Figure 5 D and Figure 5E represents bright-field images, showing cell size and shape before and after illumination. Figure 5 Statistical analysis, with F representing cell size, showed that the average cell size before and after light exposure was 3680±175 μm. 2 and 1150±48μm 2 .

[0039] Figure 6 A shows the structure of DNA modified with azobenzene. Figure 6 B shows the structure of Cy3-modified DNA.

[0040] Figure 7 The diagram illustrates the specific binding of DNA molecules to the DNA-POINT surface of this invention, wherein... Figure 7 A shows a schematic diagram of Cy5-DNA being incubated together with a surface modified with complementary anchoring DNA. Figure 7 B shows a schematic diagram of Cy5-DNA incubated together with a surface that has not been modified with complementary anchoring DNA.

[0041] Figure 8 The diagram shows the specific binding of gold nanoparticles to the DNA-POINT surface of this invention, wherein... Figure 8 A schematic diagram showing the incubation of DNA-modified 40nm gold nanoparticles with a surface modified with biotin-labeled complementary DNA. Figure 8 B shows a schematic diagram of incubating DNA-modified 40 nm gold nanoparticles together with a surface modified with complementary DNA without biotin labeling.

[0042] Figure 9 The diagram shows the binding dynamics of gold nanoparticles on a surface at different binding sites and incubation times. Figure 9 A is a dark-field image showing the binding density of gold nanoparticles on surfaces modified with different proportions of PEG and biotin-PEG after 3 hours of incubation. Figure 9 B shows a schematic diagram of Track Mate software integrated into Image J as a point detector for particle counting. Figure 9 C and Figure 9 D shows schematic diagrams of the density of gold nanoparticles on the surface at different binding sites at 10 minutes and 3 hours, respectively.

[0043] Figure 10 This is a schematic diagram showing the formation of the JHU pattern in a selectively unlit area within a black box.

[0044] Figure 11Electrophoretic analysis of the light conversion of DNA structures is shown. Each DNA structure was run on a 15% PAGE gel at 100V for 60 minutes. Inverted photographs of SybrGold stained gels are also shown. The left lane contains azobenzene-modified DNA and assembly guide DNA after visible (VIS) light irradiation, and the right lane contains azobenzene-modified DNA and assembly guide DNA after ultraviolet light irradiation. The switching efficiency was determined to be 85 ± 2.8%.

[0045] Figure 12 The diagram illustrates the rewriting capability of the DNA-POINT of this invention. The substrate is treated with visible light-ultraviolet-visible light irradiation cycles, and gold nanoparticles are incubated after each irradiation treatment. The surface is imaged by dark-field microscopy, and the AuNP density is used to calculate the binding efficiency.

[0046] Figure 13 Dark-field microscopy revealed a high density of AuNPs on the surface, confirming the functional preservation of the PEG and DNA layers after two-photon scanning. Figure 13 Image A shows the scanning of a biotin-PEG-silane modified surface using a two-photon excitation system, followed by sequential incubation with AuNPs modified with neutral avidin, anchored DNA, azobenzene-DNA, guide assembly DNA, and complementary DNA. Dark-field images show AuNP modification onto the glass substrate surface, indicating that the PEG layer was not damaged by the two-photon scan. Figure 13 B shows a schematic diagram of hybridization between the control DNA designed in this invention, replacing azobenzene-DNA and guide assembly DNA, and anchor DNA and DNA-modified AuNPs. Biotin-PEG-silane, neutral avidin, anchor DNA, and control DNA were used to modify the glass surface, followed by two-photon laser scanning. The DNA-modified AuNPs were then incubated on this glass substrate. Dark-field images show that the AuNPs were successfully modified onto the glass substrate surface, indicating that the DNA layer was not damaged by the two-photon scanning.

[0047] Figure 14 The diagram shows a cell size analysis performed as a bright-field image, a process consistent with binary and contour measurements.

[0048] Component designation explanation

[0049] 101 base

[0050] 102 Passivation layer

[0051] 103 Anchoring DNA strands

[0052] 104 Azobenzene-DNA strands

[0053] 105 Guided assembly of DNA strands

[0054] 106 Decorative Materials Detailed Implementation

[0055] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0056] It should be emphasized that the term "including / comprises" as used herein refers to the presence of a feature, whole, step, or component, but does not exclude the presence or addition of one or more other features, wholes, steps, or components.

[0057] Features described and / or illustrated for one embodiment may be used in the same or similar manner in one or more other embodiments, combined with features in other embodiments, or substituted for features in other embodiments.

[0058] In the detailed description of embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged and not to scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In actual fabrication, the three-dimensional spatial dimensions of length, width, and depth should be included.

[0059] For ease of description, spatial relation terms such as “below,” “under,” “lower than,” “below,” “above,” and “upper” may be used herein to describe the relationship between one element or feature shown in the accompanying drawings and other elements or features. It will be understood that these spatial relation terms are intended to include directions other than those depicted in the drawings for devices in use or operation. Furthermore, when a layer is referred to as being “between” two layers, it may be the only layer between the two layers, or there may be one or more layers in between.

[0060] In the context of this application, the structure described above the first feature may include embodiments in which the first and second features are formed in direct contact, or embodiments in which additional features are formed between the first and second features, such that the first and second features may not be in direct contact.

[0061] It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0062] Despite significant progress in DNA-based surface modification, the need to develop maskless strategies for generating geometrically complex multi-component patterns with submicron resolution using only non-invasive and minimal additional inputs without generating environmental pollutants remains unmet. Therefore, to address these pressing needs, this invention proposes a photosensitive DNA surface that bridges bottom-up self-assembly of nanomaterials and top-down engineering systems, providing an ideal platform for large-scale integration between the two fabrications. Azobenzene can be incorporated into DNA strands to achieve reversible regulation of DNA hybridization, and photoresponsive oligonucleotides containing azobenzene moieties can serve as efficient single-molecule switches. Notably, the process does not degrade or induce undesirable side reactions, and light is a clean, waste-free excitation source among various energy sources, inherently biocompatible, and readily available. This invention provides precise positional control of molecules and nanoparticles in the axial plane and templates for bottom-up self-assembly of multilayers along the vertical plane through a top-down light-driven DNA patterning. This method of DNA surface patterning opens up the possibility of integrating bottom-up DNA nanotechnology with top-down photolithography processes.

[0063] Based on the above, this invention provides a DNA patterning (DNA-POINT) technique for optical imprinting of nanomaterial morphology. This technique utilizes photoinduced biorecognition DNA patterns to construct nanomaterial surface structures in a short time. In this study, this invention developed a synthetic strategy based on the photo-driven molecular motion of azobenzene and integrated it into the DNA double helix, enabling photo-switching control of DNA hybridization on glass surfaces under a commercial scanning microscope. Therefore, by using templated DNA-modified surfaces assembled from various molecules or nanoparticles, this invention demonstrates multilayered hybrid structures constructed on DNA templates through the self-assembly of molecules and nanoparticles. Finally, this invention also demonstrates multiphoton conversion of DNA hybridization with azobenzene modification and develops a scanning mode for DNA-POINT, paving the way for the development of multifunctional, maskless, cleanroom-free, and environmentally friendly photolithography methods.

[0064] like Figure 1As shown, the present invention provides a pattern preparation method based on photosensitive DNA. The pattern preparation method includes the following steps: providing a substrate 101, passivating the surface of the substrate 101 to form a passivation layer 102; modifying the surface of the substrate 101 with an anchoring DNA strand 103, an azobenzene-DNA strand 104, and a guide assembly DNA strand 105, wherein the first end of the azobenzene-DNA strand 104 has a first hybridization fragment with the anchoring DNA strand 103, and the second end of the azobenzene-DNA strand 104 has a second hybridization fragment with the guide assembly DNA strand 105; irradiating the azobenzene-DNA strand 104 and the guide assembly DNA strand 105 according to a predetermined pattern, and causing the azobenzene-DNA strand 104 and the guide assembly DNA strand 105 to unwind based on the irradiation, so as to remove the guide assembly DNA strand 105 from the azobenzene-DNA strand 104 in the irradiated area; and assembling a modification material 106 in the area where the guide assembly DNA strand 105 is retained to obtain the predetermined pattern.

[0065] In one embodiment, the illumination is two-photon light with a wavelength between 650 nm and 800 nm. Under two-photon illumination, azobenzene changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain 104 to unwind from the guide assembly DNA chain 105.

[0066] In one embodiment, the illumination is single-photon ultraviolet light with a wavelength of 365 nm. Under 365 nm ultraviolet light, the azobenzene changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain 104 to unwind from the guide assembly DNA chain 105.

[0067] In one embodiment, the illumination method includes one of a two-photon scanning illumination mode, a masked wide-field illumination mode, and a digital mirror device illumination mode.

[0068] In one embodiment, the substrate 101 is a glass substrate, and the materials used to passivate the surface of the substrate include polyethylene glycol-silane and biotin-polyethylene glycol-silane, so that anchored DNA can be specifically linked to the modified glass substrate.

[0069] In one embodiment, modifying the glass substrate with polyethylene glycol-silane and biotin-polyethylene glycol-silane includes the steps of: activating the surface of the glass substrate with HNO3 at a concentration of 40% or higher; and modifying the surface of the glass substrate with the mixed solution of biotin-polyethylene glycol-silane and polyethylene glycol-silane.

[0070] In one embodiment, the anchored DNA strand 103 can be:

[0071] 5`-GAGCTGCACGCTGCCGTCAAAAAAAAAA-biotin-3`, where biotin represents a biotin molecule modified at the DNA strand end.

[0072] In one embodiment, the azobenzene-DNA chain 104 comprises a first fragment and a second fragment. The first fragment is a natural DNA fragment whose sequence is complementary to the anchored DNA chain 103 on the substrate, used to anchor the azobenzene-DNA chain 104 onto the substrate. The second fragment comprises a photosensitive fragment composed of multiple oligonucleotides, wherein several azobenzene-modified bases serve as a structure transition domain. Under light irradiation, the azobenzene in this structure transition domain changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain 104 to unwind from the guide assembly DNA chain 105. In a specific example, the sequence of the azobenzene-DNA chain 104 is 5`-GACGGCAGCGTGCAGCTCTGXAAXCTXAAXCG-3`, where X is an azobenzene molecule modified on the DNA chain, the sequence GACGGCAGCGTGCAGCTC is a natural DNA fragment that can hybridize and connect with the anchored DNA chain 103, and the sequence TGXAAXCTXAAXCG is a photosensitive fragment. A specific azobenzene-DNA strand 104 structure is as follows Figure 6 As shown in Figure A.

[0073] In one embodiment, in the photosensitive fragment sequence, an azobenzene molecule is modified every two bases.

[0074] In one embodiment, the guided assembly DNA strand 105 includes a first fragment and a second fragment, the first fragment being complementary to the photosensitive fragment, for controlling the hybridization or unwinding of the guided assembly DNA strand 105 with the azobenzene-DNA strand 104 anchored on the substrate through the structural transition domain of the photosensitive fragment, thereby achieving the patterning of the guided assembly DNA strand 105.

[0075] In one embodiment, the second fragment serves as a binding site, guiding the assembly of the modified material 106 by hybridization with the complementary DNA strand of the modified material 106, thereby achieving re-patterning of the modified material. In a specific example, the first sequence for guiding the assembly of the DNA strand 105 may be:

[0076] 5`-ATGATATAGACGTTGTGGCCGTTAGTTCA-3`;

[0077] Among them, the sequence CGTTAGTTCA is the light-sensitive complementary fragment, which can hybridize with the light-sensitive fragment of azobenzene-DNA strand 104, and the sequence ATGATATAGACGTTGTGGC is the assembly fragment. The second sequence to guide the assembly of DNA strand 105 can be:

[0078] 5`-CCACAACGTCTATATCATCGTTAGTTCA-3`;

[0079] Among them, the sequence CGTTAGTTCA is a light-sensitive complementary fragment, which can hybridize with the light-sensitive fragment of azobenzene-DNA chain 104, and the sequence CCACAACGTCTATATCAT is an assembly fragment.

[0080] In one embodiment, the modifying material 106 comprises one of DNA-modified nanoparticles, biomolecules, and DNA-modified fluorescent groups. The nanoparticles and biomolecules may be, for example, proteins, carbohydrates, silver-gold nanoparticles, and other nanoparticles, and are not limited to the examples listed herein. In a specific example, the first type of DNA-modified gold nanoparticle may be:

[0081] 5`-GCCACAACGTCTATATCATAAAAAAAA-thiol-3`;

[0082] Among them, the sequence GCCACACGTCTATATCAT is a complementary fragment, which can hybridize with the assembly fragment of the first type of guided assembly DNA strand 105 mentioned above.

[0083] Alternatively, the second type of DNA-modified gold nanoparticles could be:

[0084] 5`-ATGATATAGACGTTGTGGCAAAAAAAA-thiol-3`;

[0085] Among them, the sequence ATGATATAGACGTTGTGG is a complementary fragment, which can hybridize with the assembly fragment of the second type of guided assembly DNA strand 105 mentioned above;

[0086] Here, thiol represents the thiol molecule modified at the 3' end of the DNA strand. The DNA molecule is modified onto the surface of the gold nanoparticles by forming Au-S chemical bonds between the thiol and the surface of the gold nanoparticles, thereby achieving the patterning of the gold nanoparticles.

[0087] In a specific example, the preparation of DNA-modified gold nanoparticles includes the following steps:

[0088] (1) Before modifying DNA, concentrate the 40nm AuNPs (BBI) colloidal solution by centrifugation 10 times (40nm AuNPs: centrifuge at 8000rpm for 10 minutes).

[0089] (2) The disulfide bonds in the thiol-modified DNA strand were reduced to monothiol groups using TCEP (20 mM, 1 h) to form Au-S chemical bonds with the gold nanoparticle surface, thereby modifying the DNA molecules onto the gold nanoparticle surface. The reduced DNA was then purified using a size exclusion column (G-25, GE Healthcare) to remove excess TCEP and other small molecules generated during the reaction.

[0090] (3) Mix 800 μL of concentrated AuNPs colloidal solution with the freshly reduced thiol-modified DNA (100 μM) from step (2) in water at a ratio of 1:10,000, and incubate the mixture at room temperature (300 rpm) for 2 hours.

[0091] (4) Then add 100 μL of PB buffer (100 mM, pH = 7.4) to the mixture from step (3).

[0092] (5) After 30 minutes, we added 10 μL of NaCl solution (2M concentration) every 20 minutes for a total of 4 times, and then added 20 μL of NaCl solution (2M) every 30 minutes for a total of 3 times. The NaCl concentration was gradually increased to ensure that the thiol-modified DNA completely covered the AuNP. The final NaCl concentration was 200 mM, and the mixture was incubated overnight at room temperature (300 rpm).

[0093] (6) After overnight incubation, AuNP-DNA was purified by centrifugation.

[0094] (7) Freshly prepared 40 nm AuNPs completely covered with DNA did not precipitate in PBS buffer (100 mM, pH 8.0). This high salt tolerance of DNA-covered AuNPs allows them to be assembled at the interface via DNA hybridization.

[0095] In one embodiment, the modified material 106 comprises multiple layers of DNA-modified material linked together, with adjacent DNA-modified layers connected by complementary DNA molecules. The material of the multiple DNA-modified layers includes one or more of DNA-modified nanoparticles, DNA-modified fluorescent groups, and biomolecules. The DNA-modified fluorescent group may, for example, be a Cy3-DNA chain or a Cy5-DNA chain. The sequence of the Cy3-DNA chain may be:

[0096] 5`-GCCACAACGTCTATATCAT-Cy3-3`;

[0097] Here, Cy3 represents the Cy3 fluorescent molecule modified at the DNA strand end. The Cy3-DNA strand hybridizes with the assembly fragment of the first guide assembly DNA strand 105, immobilizing the Cy3 fluorescent molecule on the substrate, thus achieving the patterning of the Cy3 fluorescent molecule. A specific Cy3 fluorescent molecule structure is shown below. Figure 6 As shown in B

[0098] The sequence of the Cy5-DNA strand can be:

[0099] 5`-GACGGCAGCGTGCAGCTC-Cy5-3`

[0100] Cy5 represents a Cy5 fluorescent molecule modified at the DNA strand end. Cy5-DNA strands can anchor DNA strands for hybridization to immobilize them on a substrate.

[0101] In one embodiment, the multiple DNA modification layers are incubated sequentially or simultaneously.

[0102] In one embodiment, the preparation method further includes the step of: irradiating the azobenzene-DNA chain 104 with visible light to transform it from a non-planar cis structure to a planar trans structure, and restoring hybridization with the guide assembly DNA chain 105, thereby enabling the azobenzene-DNA chain 104 to have rewriting capability.

[0103] like Figure 1 As shown, this embodiment also provides a graphic template based on photosensitive DNA, including: a substrate, the surface of which has a passivation layer 102; an anchoring DNA strand 103, an azobenzene-DNA strand 104, and a guide assembly DNA strand 105, which are sequentially modified on the surface of the passivation layer 102. The first end of the azobenzene-DNA strand 104 has a first hybridization fragment with the anchoring DNA strand 103, and the second end of the azobenzene-DNA strand 104 has a second hybridization fragment with the guide assembly DNA strand 105. The second hybridization fragment between the azobenzene-DNA strand 104 and the guide assembly DNA strand 105 can be de-hysterized under light to remove the guide assembly DNA strand 105 from the azobenzene-DNA strand 104 in the light-illuminated area.

[0104] In one embodiment, the substrate is a glass substrate, and the material of the passivation layer 102 includes polyethylene glycol-silane and biotin-polyethylene glycol-silane, so that anchored DNA can be specifically linked to the modified glass substrate.

[0105] In one embodiment, the azobenzene-DNA chain 104 comprises a first fragment and a second fragment. The first fragment is a natural DNA fragment whose sequence is complementary to the anchored DNA chain 103 on the substrate, for anchoring the azobenzene-DNA chain 104 onto the substrate. The second fragment comprises a photosensitive fragment composed of multiple oligonucleotides, wherein several azobenzene-modified bases serve as a structure transition domain. Under light irradiation, the azobenzene in the structure transition domain changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain 104 to unwind from the guide assembly DNA chain 105.

[0106] In one embodiment, in the photosensitive fragment sequence, an azobenzene molecule is modified every two bases.

[0107] In one embodiment, the guide assembly DNA strand 105 includes a first fragment and a second fragment. The first fragment is complementary to the photosensitive fragment and is used to control the hybridization or unwinding of the guide assembly DNA strand 105 with the azobenzene-DNA strand 104 anchored on the substrate through the structure transition domain of the photosensitive fragment, thereby achieving patterning of the guide assembly DNA strand 105. The second fragment serves as a binding site for hybridization with the complementary DNA strand of the modification material 106, guiding the assembly of the modification material 106 to achieve secondary patterning.

[0108] In one embodiment, the modified material 106 includes one of DNA-modified nanoparticles, biomolecules, and DNA-modified fluorescent groups.

[0109] In one embodiment, the modified material 106 comprises multiple layers of DNA modified layers connected in sequence, with adjacent DNA modified layers linked by complementary DNA. The material of the multiple DNA modified layers includes one or more of DNA-modified nanoparticles, DNA-modified fluorescent groups, and biomolecules.

[0110] In one embodiment, after the second hybridization fragment of the azobenzene-DNA chain 104 and the guide assembly DNA chain 105 is unwound under light, the azobenzene-DNA chain 104 can be transformed from a non-planar cis structure to a planar trans structure by visible light irradiation, and hybridization with the guide assembly DNA chain 105 can be restored, thereby enabling the azobenzene-DNA chain 104 to have rewriting capability.

[0111] like Figure 1 As shown, in a specific implementation process, the pattern preparation method based on photosensitive DNA includes the following steps:

[0112] Step 1), passivating the glass substrate, including:

[0113] (1) The glass substrate surface is first cleaned with acetone and plasma for 5 minutes, then activated in concentrated nitric acid for 10 minutes, and then thoroughly rinsed twice with water and methanol.

[0114] (2) Preparation of a mixture of biotin-mPEG-silane and mPEG-silane with a concentration of 10 mg / mL: The mixture contains 80% mPEG-silane (concentration 8 mg / mL) and 20% biotin-mPEG-silane (concentration 2 mg / mL), and the solvent is methanol.

[0115] (3) The glass substrate surface was then modified with biotin-mPEG-silane and incubated for 2 hours in a freshly prepared mixture of biotin-mPEG-silane and mPEG-silane from (2). After 2 hours of incubation, the coverslip surface was rinsed twice with methanol and then washed three times with water in preparation for subsequent DNA coating modification.

[0116] Step 2), preparing the DNA coating, including:

[0117] (1) Incubate the passivated glass substrate surface in PBS buffer with 500 nM avidin for 20 minutes, and then wash the glass substrate surface with PBS 3 times for 5 minutes each time to remove excess avidin on the unmodified surface.

[0118] (2) Next, the glass substrate surface was incubated with the prepared 100 nM anchored DNA strand 103 in PBS buffer for 20 minutes, and then the glass substrate surface was washed with PBS 3 times for 5 minutes each time to remove excess anchored DNA strand 103 that was not modified.

[0119] (3) Finally, the premixed 50 nM azobenzene-modified DNA and its complementary DNA strand were incubated with the surface for 20 minutes, and then washed three times in PBS for 5 minutes each time.

[0120] Step 3), using two-photon mapping to pattern the DNA layer, including:

[0121] Two-photon lithography was performed on a commercial multiphoton laser scanning microscope, the Zeiss LSM510. This is an inverted microscope for non-invasive lithography and is equipped with a temperature-controlled chamber. This invention utilizes a 40xNA 1.0 water immersion objective and a 750nm chameleon femtosecond laser (Coherent Inc.) to achieve DNA layer patterning. The wavelength range of the two-photon laser used to achieve DNA layer patterning via azobenzene structural transition is 650nm-800nm.

[0122] Of course, without considering the activity of biomolecules or whether a mask is needed, DNA layer patterning can also be achieved using single photons, including:

[0123] The surface of the DNA-modified glass substrate covered with PBS buffer obtained in step 2) was exposed to a 5W UV LED diode (365nm) for 10 minutes, while a heating plate was placed under the glass substrate for temperature control at 37°C. The distance between the UV lamp and the surface was kept less than 10mm, thereby achieving DNA layer patterning.

[0124] Step 4), the self-assembly of the second and third layers of nanomaterials, includes:

[0125] (1) Wash the DNA pattern obtained by photon drive in step 3) or 4) twice with PBS buffer, each time for 5 minutes.

[0126] (2) Then, the 0.1 nM DNA-modified AuNPs or 100 nM Cy3-labeled DNA, which are the components of the second layer, are incubated together with the glass substrate surface with the patterned DNA layer in (1) at room temperature for 20 minutes. After that, the substrate is washed twice with PBS buffer to remove excess AuNPs or Cy3-DNA that have not bound to the surface, each wash lasting 5 minutes.

[0127] (3) Repeat the incubation and washing steps in (2) above to assemble the third layer.

[0128] The photo-driven DNA-POINT technology is based on the ability of azobenzene modified on the DNA strand to regulate DNA hybridization and dehybridization. Specifically, under irradiation with 300nm–400nm single-photon or ~750nm two-photon (2P) light, azobenzene transforms from a planar trans structure to a non-planar cis structure, thereby causing the DNA double helix to unwind. In the planar trans structure, intercalated planar azobenzene can stabilize the DNA helix through stacking interactions. Conversely, the non-planar cis structure of azobenzene makes the DNA helix unstable due to steric hindrance and facilitates DNA double helix unwinding. Based on this principle, this invention utilizes azobenzene-modified DNA to design a DNA photoswitch at the interface, such as… Figure 1 As shown in Figure A, it mainly consists of three DNA strands: the azobenzene-modified DNA strand 104, the biotin-DNA strand 103, and the assembly guide DNA strand. The following is a detailed description of this DNA photoswitch. First, the azobenzene-DNA strand 104 contains two DNA fragments, such as... Figure 6As shown in Figure A, the first is a photosensitive fragment consisting of ten oligonucleotides, with four azobenzene-modified bases serving as structural transition domains. This design follows previous findings that the sequences of two unmodified oligonucleotides can be separated by two azobenzene-modified residues. The second is a native DNA fragment whose sequence is complementary to the modified anchor DNA strand 103 on the substrate, used to anchor the azobenzene-DNA strand 104 to the substrate. Figure 1 As shown in Figure A, the guide assembly DNA strand also comprises two DNA fragments. One is complementary to the photosensitive fragment, thus allowing the hybridization or unwinding of the guide assembly DNA strand with the azobenzene-DNA strand 104 anchored on the substrate to be controlled via the structural transition domain of the photosensitive fragment, thereby achieving patterning of the guide assembly DNA strand. The other DNA fragment serves as a binding site, guiding the assembly of the nanomaterial by hybridizing with the complementary DNA strand modified on the nanomaterial surface, thereby achieving its secondary patterning.

[0129] This invention uses a transparent glass cover glass, widely used in biomedical sensing and imaging applications, as an example of a photolithographic surface. Here, a DNA-modified glass surface is prepared using a biotin-neutravidin-biotin sandwich assembly strategy. Biotin-mPEG-silane, neutral avidin, and anchored DNA strand 103 are sequentially linked to a glass substrate. This passivates the glass substrate with PEG molecules, resulting in ultra-low non-specific adsorption of other molecules and nanoscale particles, while the anchored DNA specifically binds to and modifies the glass substrate, forming a DNA layer on it. Figure 7 and Figure 8 As shown. The entire modification process is simplified as follows: the glass surface is activated in concentrated HNO3 for 5 minutes, then immersed in a freshly prepared mixed solution of biotin-PEG-silane and PEG-silane for 2 hours. Subsequently, neutral avidin, anchored DNA strand 103, azobenzene-DNA strand 104, and guide assembly DNA strand are sequentially incubated on the glass substrate in physiological phosphate-buffered saline (PBS, pH 7.4) solution for a total of 2 hours. The time used for DNA modification of the glass substrate surface is equivalent to the initial substrate cleaning time in a conventional cleanroom photolithography process. Furthermore, this invention measures and optimizes the binding efficiency of nanoparticles to the glass substrate surface to accommodate different incubation times and the ratio of biotin-PEG-silane to PEG-silane, such as... Figure 9 As shown.

[0130] Due to the optical switching properties of azobenzene-DNA, the design strategy of this invention can be widely combined with multiphoton scanning mode operation, wide-field masking, and digital mirror devices (DMDs) to construct complex geometric patterns on DNA-modified surfaces. Taking the scanning mode of DNA-POINT technology as a specific example, such as... Figure 1 B and Figure 10 As shown, this invention first employs a scanning laser planar handwritten JHU pattern on a commercial two-photon microscope (Zeiss LSM510Meta) for 10 minutes. Subsequently, DNA-modified gold nanoparticles (AuNPs) self-assemble onto the DNA pattern as a secondary pattern during a 30-minute incubation period. Then, the JHU pattern projected by the AuNPs is imaged using a dark-field microscope, utilizing the green colorimetric scattering of 40 nm AuNPs. The single green dot representing a single AuNP shown in the magnified image indicates high pattern resolution. It is worth emphasizing that the DNA-POINT scanning mode technique demonstrated in this invention is a maskless photolithography method, encompassing substrate preparation, optical scanning, and secondary patterning, all performed without the use of cleanrooms or the generation of hazardous chemicals.

[0131] like Figures 1 to 14 As shown below, the solution and effects of the present invention will be further described.

[0132] 1) Regarding the light-induced modulation of DNA switching on and off on the surface:

[0133] The photo-driven conversion efficiency of azobenzene-based DNA hybridization in solution is influenced by several factors, including the DNA strand density, light irradiation time, and temperature. However, the photomodulation efficiency of azobenzene-based DNA hybridization at interfaces has not been systematically studied. Furthermore, whether DNA-POINT technology can serve as a feasible surface micropatterning method, quantifying the photomodulation efficiency of azobenzene-based DNA hybridization, is a key factor in evaluating its potential. Therefore, after successfully achieving photo-conversion of DNA switching in solution, this invention first investigated the conversion capability of DNA-POINT on glass surfaces, determining its switching efficiency to be 85 ± 2.8%. Figure 11 As shown. During illumination, the DNA photoswitches modified on the glass substrate surface were incubated in PBS buffer at 37°C to ensure high light conversion efficiency while avoiding excessive temperature that could cause DNA double helix unwinding. Figure 2As shown in Figure A, this invention uses DNA-modified AuNPs as probes to display the hybridization state of azobenzene-DNA strand 104 with the guide assembly DNA strand. The DNA-modified AuNPs can hybridize with the guide assembly DNA strands at the interface, thus becoming anchored there. The anchored area is then observed using a dark-field microscope. Experimental results show that in the control experiment without UV irradiation, a high density of AuNPs was observed at the interface under a dark-field microscope. This indicates that a large number of guide assembly DNA strands were assembled at the interface. In contrast, after 10 minutes of irradiation with 5W UV light (365nm), the density of AuNPs at the interface significantly decreased, indicating a reduced density of guide assembly DNA strands assembled at the interface. By quantitatively analyzing the DNA switching efficiency using the scattering intensity of AuNPs at the interface, this invention calculated that only 20.4 ± 8% of the AuNP scattering intensity remained on the surface after light irradiation (n = 5 replicates). The results confirm the high efficiency of the azobenzene-based DNA hybridization photoconversion at the surface. Furthermore, azobenzene can convert from a cis to a trans structure and restore DNA hybridization after visible light irradiation. Therefore, the rewriting capability of azobenzene-modified DNA is a unique feature compared to photolysis-based DNA patterning (which leads to permanent DNA fragmentation). To demonstrate this unique feature, the DNA photoswitching modified interface was subjected to a visible light-ultraviolet-visible light irradiation cycle, and AuNPs were incubated with the surface after each treatment. Quantitative analysis showed no difference in AuNP binding between the two visible light irradiation treatments, such as... Figure 12 As shown, this demonstrates the rewriting capability of DNA-POINT.

[0134] In addition to the light-driven lithography described above, to fully realize the potential of azobenzene-based photoelectric switches, this invention also addresses a key issue: whether near-infrared lasers can be used for multiphoton scanning, besides the appropriate ultraviolet light, to achieve maskless patterning at sub-micron resolution. Previous studies have demonstrated the use of two-photon excitation with near-infrared wavelengths to achieve light switching of azobenzene. However, exploring multiphoton-excitation-driven DNA hybridization switching of azobenzene-modified DNA on surfaces is crucial for developing scanning modes. To address this issue, this invention uses a commercial confocal microscope (LSM510, Zeiss) equipped with a Ti:sapphire laser (150 fs, 80 MHz, coherent chameleon) for DNA-POINT operations, such as... Figure 2As shown in B. To determine the optimal laser power for efficient DNA double-helix switching under a 40X water immersion objective (NA1.0), the laser power was initially kept at a minimum of 0.2% (~4 mW) for a scanning area of ​​22.5 μm x 22.5 μm. Compared to high-density AuNPs under 0% laser power control, the density of AuNPs in these square patterns was observed to be significantly reduced at laser powers of 0.2%, 0.5%, 1%, 2%, and 3%, respectively. The invention also observed that the switching efficiency value did not change significantly as the laser power increased beyond 4 mW (up to 60 mW), suggesting an energy-independent switching process. Although femtosecond nonlinear lithography based on multiphoton lasers has been used to fabricate structures on glass surfaces, the required laser power is typically several orders of magnitude higher (1–100 W, 1 kHz repetition rate) than that used in the DNA-POINT method of this invention (4 mW, 80 MHz repetition rate).

[0135] To determine the integrity of PEG and native DNA under a 60 mW femtosecond laser (150 fs, 80 MHz) power, a control experiment was also conducted. Biotin-PEG or native DNA-modified surfaces were scanned in an aqueous medium and then sequentially incubated with biorecognition DNA and AuNPs. Images showed no significant difference in AuNP binding efficiency between the scanned and non-scanned regions. Figure 13 As shown, this confirms that biotin-PEG and natural DNA retain their biorecognition activity on the surface after laser scanning. In summary, these results demonstrate that optimal switching efficiency can be achieved even using very low-power multiphoton lasers, which is crucial for preventing thermal damage to biomolecules.

[0136] 2) Regarding maskless submicron patterning technology for DNA-POINT in scanning mode:

[0137] Because the DNA-POINT scanning method of this invention does not require a mask, and the laser spot size is limited by the diffraction limit, this means that the effective resolution of DNA-POINT is on the order of hundreds of nanometers. Therefore, the DNA-POINT scanning mode performed on a confocal system can achieve the construction of feature patterns with sub-micron resolution. This invention uses an objective lens with NA=1.0 to focus a 750nm two-photon laser (150fs, 80MHz, Coherent Chameleon), which theoretically should achieve an optical resolution of ~400nm. By utilizing the patterning of AuNPs on a glass surface, this invention further determines the printing capabilities of DNA-POINT, including feature size control and the ability to fabricate arbitrary patterns. Figure 3As shown in Figure A, this invention designed line patterns with widths of 1 μm, 2 μm, and 10 μm, and subsequently observed the micropatterns with attached AuNPs using dark-field microscopy. The results showed that the AuNPs were distributed within the designed linewidth region. Furthermore, analysis of the cross-sectional intensity distribution using optimal Gaussian curve fitting revealed a close correspondence between the full width at half maximum (FWHM) of the Gaussian fit and the designed linewidth. In summary, these results confirm the submicron patterning capability of DNA-POINT.

[0138] Furthermore, the DNA-POINT scanning mode can realize patterns of any design by the user. To demonstrate the ability of the DNA-POINT scanning mode to realize arbitrary patterns, using the same method described above, this invention successfully generated symmetrical and asymmetrical patterns using AuNP, such as... Figure 3 B showcases a line-based network with a linewidth of 4 μm and a spacing of 14 μm, a square array with an area of ​​12.5 μm x 12.5 μm and a spacing of 5 μm, and a hand-drawn coil pattern with an overall size of 200 μm x 200 μm. These patterns demonstrate the complexity of patterning using the DNA-POINT technology, and also demonstrate the ability of DNA-POINT technology to pattern nanoscale particles at microscale resolution in confocal scanning mode.

[0139] 3) Regarding the multilayer self-assembly of DNA-POINT:

[0140] Patterning multilayer functional materials with micron-level precision is crucial for a wide range of applications, from molecular system synthesis to high-throughput / high-content screening. For example, miniaturized biomolecule-embedded plasmonic nanoparticle arrays are invaluable for developing rapid and highly sensitive diagnostic analytical methods and biosensors. To investigate the ability of DNA-POINT technology to construct second-layer patterns of different materials, Cy3-labeled DNA strands (i.e., small molecules rather than DNA-modified AuNPs as shown above) were used as the second material layer on a light-driven DNA proto-pattern. Similar to the AuNP pattern, a handwritten JHU pattern with line widths of approximately 10 μm and an overall size of 60 μm x 120 μm was designed and scanned using a focused laser. Only the non-illuminated regions could then hybridize with the Cy3-labeled DNA, producing a negative pattern. Figure 4 A shows the negative JHU pattern as imaged by a fluorescence microscope.

[0141] Furthermore, AuNPs with densely packed DNA surface-modified via covalent bonds can also serve as templates for self-assembly. Therefore, this invention develops a three-layer photolithography technique in the DNA-POINT system, consisting of a primary layer of light-driven DNA patterned as a 12.5 μm x 12.5 μm square array with a spacing of 5 μm, a layer of self-assembled AuNPs on top, and a DNA layer assembled using the AuNPs layer as a template. Following the previously used assembly method, the AuNPs are incubated together with the surface of the patterned DNA primary layer, followed by incubation with Cy3-modified DNA to assemble the third layer. This invention uses in-situ imaging with dark-field and fluorescence microscopy to study the co-localization between AuNPs and Cy3-labeled DNA. Figure 4 As shown in Figure B, the high degree of colocalization between the green scattering of AuNPs and the pseudo-red fluorescence of Cy3-labeled DNA demonstrates that this invention achieves the self-assembly of three different materials. This model can also be readily used to generate bioactive surfaces to study fundamental questions concerning biomolecular interactions.

[0142] 4) Regarding the use of DNA-POINT to regulate cell adhesion and diffusion:

[0143] Focal adhesions (FAs) are large protein complexes that form on the cell basal surface, physically connecting the extracellular matrix (ECM) to the cytoskeleton, and have long been thought to mediate cell growth and migration. Previous studies have shown that upon binding to specific adhesive ECM epitopes, such as RGD sequences, several intracellular ankylosing proteins are recruited to adhesion sites, ultimately inducing the assembly of the FA complex and the associated cytoskeleton. Here, by characterizing the nanoscale variations in the spacing between adhesive RGD clusters on cell diffusion and focal adhesion dynamics, this invention investigates integrin lateral clustering as a mechanism controlling integrin function.

[0144] To evaluate cell adhesion and diffusion on PEG-modified DNA surfaces, the DNA surfaces were coated with a high density of 40 nm AuNPs, such as... Figure 5 As shown in Figure A, RGD-modified DNA (RGD-DNA for short) is used as the second layer of AuNPs. Figure 5 As shown in B. Initially, this invention attempted to seed HeLa cells onto a DNA surface modified only with a high density of AuNPs (without RGD-DNA on the AuNP surface). After 6 hours of incubation, as expected, this invention found that the HeLa cells remained round and did not adhere to the substrate surface, indicating that the PEG-modified DNA surface does not support cell adhesion and diffusion, as... Figure 5As shown in Figure A. Subsequently, after adding RGD-DNA with a sequence complementary to the surface DNA sequence of 40 nm AuNPs, 40 nm RGD clusters were formed through hybridization. (See Figure A for details.) Figure 5 As shown in Figure B, HeLa cells exhibited excellent adhesion and diffusion after being incubated on a surface modified with RGD clusters for 6 hours, demonstrating that high-density RGD clusters can support cell adhesion and diffusion.

[0145] This invention has demonstrated that the density of AuNPs on the DNA surface significantly decreases after 10 minutes of ultraviolet light irradiation. Therefore, in addition to using light to regulate the d-density of RGD clusters, the DNA-point erasure process can reduce RGD cluster density, thereby controlling cellular diffusion behavior. Figure 5 As shown in Figure C. The results showed that cells seeded on high-density RGD clusters could completely diffuse after 6 hours of incubation. Figure 5 As shown in D, after 10 minutes of UV light irradiation, the cell size and shape drastically changed to small and round. Cell size analysis, as shown... Figure 5 F and Figure 14 As shown, this further demonstrates that cell size significantly decreased after light exposure. These results indicate that the DNA-POINT method is an effective approach for dynamically regulating cell adhesion and diffusion.

[0146] As described above, the graphic template and graphic preparation method based on photosensitive DNA of the present invention have the following beneficial effects:

[0147] This invention achieves the unwinding of azobenzene-DNA strand 104 and guide assembly DNA strand 105 by using two-photon illumination with a longer wavelength. Two-photon illumination can satisfy the unwinding of azobenzene-DNA strand 104 and guide assembly DNA strand 105 by irradiating with a longer wavelength. That is, the optimal DNA strand optical switching control efficiency can be achieved using a very low-power two-photon laser, thereby effectively avoiding the inactivation effect of using shorter wavelength ultraviolet light on DNA strands or biomolecules, preventing thermal damage to biomolecules, and greatly improving the application scope of this invention.

[0148] This invention uses light to transform the azobenzene-DNA chain 104 from a planar trans structure to a non-planar cis structure, thereby achieving unwinding from the guide assembly DNA chain 105. Subsequently, visible light irradiation can transform the azobenzene-DNA chain 104 from a non-planar cis structure to a planar trans structure, restoring hybridization with the guide assembly DNA chain 105, thus enabling the azobenzene-DNA chain 104 to have rewriting capabilities.

[0149] This invention can form the desired pattern through a two-photon scanning mode, providing a feasible solution for a multifunctional, maskless, cleanroom-free, and environmentally friendly photolithography method.

[0150] Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0151] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing patterns based on photosensitive DNA, characterized in that, The graphic preparation method includes the following steps: A substrate is provided, and the surface of the substrate is passivated. An anchoring DNA strand, an azobenzene-DNA strand, and a guide assembly DNA strand are modified on the substrate surface. The azobenzene-DNA strand comprises a first fragment and a second fragment. The first fragment is a natural DNA fragment, and its sequence is complementary to the anchoring DNA strand on the substrate, used to anchor the azobenzene-DNA strand onto the substrate. The second fragment of the azobenzene-DNA strand comprises a photosensitive fragment composed of multiple oligonucleotides, wherein several azobenzene-modified bases serve as a structure transition domain. Under light irradiation, the azobenzene in this structure transition domain changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA strand to unwind from the guide assembly DNA strand. The guide assembly DNA strand... Chain A comprises a first segment and a second segment. The first segment of the guide assembly DNA chain is complementary to the photosensitive segment and is used to control the hybridization or unwinding of the guide assembly DNA chain with the azobenzene-DNA chain anchored on the substrate through the structural transition domain of the photosensitive segment, thereby achieving patterning of the guide assembly DNA chain. The second segment of the guide assembly DNA chain serves as a binding site, guiding the assembly of the modified material through hybridization with the complementary DNA chain of the modified material, thereby achieving re-patterning of the modified material. The first end of the azobenzene-DNA chain has a first hybridization segment with the anchored DNA chain, and the second end of the azobenzene-DNA chain has a second hybridization segment with the guide assembly DNA chain. The second hybridization fragment of the azobenzene-DNA chain and the guide assembly DNA chain is illuminated according to a set pattern. Based on the illumination, the azobenzene-DNA chain and the guide assembly DNA chain are unwound to remove the guide assembly DNA chain from the azobenzene-DNA chain in the illuminated area. Modification materials are assembled in the regions where the guide assembly DNA strands are retained to obtain the defined pattern.

2. The method for preparing patterns based on photosensitive DNA according to claim 1, characterized in that: The illumination is single-photon ultraviolet light with a wavelength between 300nm and 400nm or two-photon light with a wavelength between 650nm and 800nm. Under single-photon ultraviolet light or two-photon light illumination, azobenzene changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA chain to unwind from the guide assembly DNA chain.

3. The method for preparing patterns based on photosensitive DNA according to claim 2, characterized in that: The illumination method includes one of the following: two-photon scanning illumination mode, wide-field illumination mode with mask, and digital mirror device illumination mode.

4. The method for preparing patterns based on photosensitive DNA according to claim 1, characterized in that: The substrate is a glass substrate, and the materials used to passivate the surface of the substrate include polyethylene glycol-silane and biotin-polyethylene glycol-silane, so that the anchored DNA strand can be specifically attached to the modified glass substrate.

5. The method for preparing patterns based on photosensitive DNA according to claim 4, characterized in that: Modifying the glass substrate with polyethylene glycol-silane and biotin-polyethylene glycol-silane includes the following steps: The surface of the glass substrate was activated using HNO3 with a concentration of 40% or higher. The surface of the glass substrate is modified using a mixed solution of biotin-polyethylene glycol-silane and polyethylene glycol-silane.

6. The method for preparing patterns based on photosensitive DNA according to claim 1, characterized in that: In the sequence of the photosensitive fragment, an azobenzene molecule is modified every two bases.

7. The method for preparing patterns based on photosensitive DNA according to claim 1, characterized in that: The modified material includes one of DNA-modified nanoparticles, biomolecules, and DNA-modified fluorescent groups.

8. The method for preparing patterns based on photosensitive DNA according to claim 1, characterized in that: The modified material comprises multiple layers of DNA modified material connected in sequence, with adjacent DNA modified layers linked by complementary DNA. The material of the multiple DNA modified layers includes one or more of DNA-modified nanoparticles, DNA-modified fluorescent groups, and biomolecules.

9. The method for preparing patterns based on photosensitive DNA according to claim 8, characterized in that: The DNA modification layers are incubated sequentially or simultaneously.

10. The method for preparing patterns based on photosensitive DNA according to claim 1, characterized in that, The method also includes the step of: irradiating the azobenzene-DNA strand with visible light to transform it from a non-planar cis structure to a planar trans structure, and restoring its hybridization with the guide assembly DNA strand, thereby enabling the azobenzene-DNA strand to have rewriting capability.

11. A graphic template based on photosensitive DNA, characterized in that, include: A substrate, the surface of which has a passivation layer; An anchoring DNA strand, an azobenzene-DNA strand, and a guide assembly DNA strand are sequentially modified onto the surface of the passivation layer. The azobenzene-DNA strand comprises a first fragment and a second fragment. The first fragment is a natural DNA fragment. The sequence of the first fragment of the azobenzene-DNA strand is complementary to the anchoring DNA strand on the substrate, used to anchor the azobenzene-DNA strand onto the substrate. The second fragment of the azobenzene-DNA strand comprises a photosensitive fragment composed of multiple oligonucleotides, wherein several azobenzene-modified bases serve as structure transition domains. Under light irradiation, the azobenzene in these structure transition domains changes from a planar trans structure to a non-planar cis structure, thereby causing the azobenzene-DNA strand to unwind from the guide assembly DNA strand. The guide assembly DNA strand comprises a first fragment and a second fragment. The first fragment of the guide assembly DNA strand... A complementary fragment to the photosensitive fragment is used to control the hybridization or unhybridization of the guide assembly DNA strand with the azobenzene-DNA strand anchored on the substrate through the structure transition domain of the photosensitive fragment, thereby achieving patterning of the guide assembly DNA strand. A second fragment of the guide assembly DNA strand serves as a binding site for hybridization with the complementary DNA strand of the modification material, guiding the assembly of the modification material to achieve secondary patterning. The first end of the azobenzene-DNA strand has a first hybridization fragment with the anchored DNA strand, and the second end of the azobenzene-DNA strand has a second hybridization fragment with the guide assembly DNA strand. The azobenzene-DNA strand and the second hybridization fragment of the guide assembly DNA strand can be unhybridized under light to remove the guide assembly DNA strand from the azobenzene-DNA strand in the illuminated area. Modification materials are assembled in the regions where the guide assembly DNA strands are retained to obtain the defined pattern.

12. The graphic template based on photosensitive DNA according to claim 11, characterized in that: The substrate is a glass substrate, and the passivation layer is made of polyethylene glycol-silane and biotin-polyethylene glycol-silane, so that the anchored DNA can be specifically linked to the modified glass substrate.

13. The graphic template based on photosensitive DNA according to claim 11, characterized in that: In the sequence of the photosensitive fragment, an azobenzene molecule is modified every two bases.

14. The graphic template based on photosensitive DNA according to claim 11, characterized in that: The modified material includes one of DNA-modified nanoparticles, biomolecules, and DNA-modified fluorescent groups.

15. The graphic template based on photosensitive DNA according to claim 11, characterized in that: The modified material comprises multiple layers of DNA modified material connected in sequence, with adjacent DNA modified layers linked by complementary DNA. The material of the multiple DNA modified layers includes one or more of DNA-modified nanoparticles, DNA-modified fluorescent groups, and biomolecules.

16. The graphic template based on photosensitive DNA according to claim 11, characterized in that: After the second hybridization fragment of the azobenzene-DNA chain and the guide assembly DNA chain is unwound under light, the azobenzene-DNA chain can be transformed from a non-planar cis structure to a planar trans structure by visible light irradiation, and hybridization with the guide assembly DNA chain is restored, thus enabling the azobenzene-DNA chain to have rewriting capability.

Images