A method for preparing molecular patterned surface based on mechanochemical reaction

Abstract

The present invention discloses a method for preparing a molecular patterned surface based on a mechanochemical reaction, comprising the following steps: (1) modification of a template and a substrate: using a binary structure molecule having a force-responsive functional group at one end and a bio-affinity at the other end as a printing ink molecule; using a template modified with a bio-affinity molecule as a pattern template for mechanochemical printing, which can be used to enrich ink molecules; using a substrate modified with a force-responsive functional group as a pattern transfer object, which can undergo a mechanochemical reaction with the force-responsive ink to obtain a patterned surface; (2) mechanochemical imprinting: contacting the template with the substrate in an ink environment, applying pressure to force the ink molecules to covalently bind to the functionalized substrate, thereby fixing the ink molecules to the substrate according to the patterning of the template. The present invention has the characteristics of low cost and high throughput, high molecular pattern resolution, high stability, and can maintain the activity of biological molecules.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for preparing molecularly patterned surfaces based on mechanochemical reactions. Background Technology

[0002] With the development of micro- and nano-patterning fabrication technology, integrated circuits have been miniaturized on semiconductors, greatly satisfying people's needs for electronic information devices. This has also inspired scientists to apply micro- and nano-patterning technology to the biological field. Micro- and nano-precision fabrication technology makes it possible to regulate the extracellular microenvironment and even manipulate individual cells. Early explorations focused on using patterned surfaces fabricated using micro- and nano-fabrication technology to study basic cell biology research such as cell adhesion and growth. In addition, research has shown that biomolecules are patterned and incorporated onto surfaces using micro- and nano-fabrication technology. Miniaturized biomolecule arrays can serve as miniature biochemical reaction laboratories (i.e., biochips) for the detection and analysis of biological samples. The miniaturization of biomolecule patterns can increase the detection signal density, thereby significantly improving the sensitivity of biochips.

[0003] Early biomolecular arrays were fabricated using methods such as micro-dotting, with pattern precision controlled only at tens of micrometers. However, the most advanced extreme ultraviolet (EUV) lithography machines have achieved a precision of 13nm. Lithography technology boasts extremely high resolution and high throughput, making it one of the most important processes in the semiconductor industry. Therefore, scientists naturally applied lithography to the surface patterning of biomolecules. Lithography directly patterns the photoresist spin-coated onto the wafer surface, but cannot directly attach biomolecules to the surface. Instead, a silane coupling agent is first bound to the patterned area of ​​the exposed substrate after development, and then biomolecules are grafted through subsequent reactions. Similarly, electron beam lithography and nanoimprint lithography directly pattern the surface resist, exposing the patterned area before modifying it with biomolecules. Both of these technologies have their advantages over lithography, but due to their different underlying principles, they inevitably have some limitations. From de Broglie's matter-wave theory, we know that electrons are waves with wavelengths shorter than ultraviolet light. According to the diffraction-limiting formula, using electron beams to process resists can improve precision to the nanometer scale. However, electron beam lithography suffers from drawbacks such as long processing time and low yield. Directly transferring a template with a pre-defined nanopattern onto the substrate can improve yield and reduce cost while maintaining pattern resolution. Nanoimprint lithography uses thermoplastic materials as resists, spin-coated onto the substrate surface. At high temperatures, the pattern is transferred to the substrate surface medium through contact between the template and the substrate under high pressure. However, even small particles or wrinkles between the rigid template and the substrate can severely affect processing quality. Therefore, pattern transfer technology using elastic templates has emerged. Microcontact printing technology uses softer polydimethylsiloxane as the template material, directly transferring ink molecules onto the substrate by dipping the template in ink. Compared to the three patterning techniques using resists mentioned above, the direct transfer of ink molecules avoids harsh conditions such as high temperature, high pressure, and vacuum, making it more suitable for the patterning and fixation of biomolecules. Biomolecules (especially proteins) require buffer solutions that simulate physiological environments to maintain their biological activity. During the transfer of ink solution from the template to the substrate, severe diffusion occurs, ultimately leading to a significant reduction in the precision of the biomolecular pattern. Ink diffusion is affected by factors such as solution viscosity and contact time. Using an atomic force microscope probe to dip into ink for writing allows for precise control of contact time and writing speed. This dip pen etching technology can achieve an accuracy equivalent to the probe tip size (about 30nm), but it has the inherent defect of low yield, making it almost impossible to commercialize.

[0004] There are many technologies that can perform patterning processing at present. These technologies all have unique advantages and have unique advantages in specific fields. However, no technology can maintain the activity of biomolecules while having the advantages of high pattern accuracy, high quality, high yield, and low cost. Judging from the number of relevant patents of global biochips from 2010 to 2019, the United States has 250,000 published patents, occupying an absolute leading position. Although China has 12% of the number of published academic papers related to biochips, the number of related patents is less than 50,000, and the conversion rate of scientific research results is relatively low. Since the COVID-19 pandemic, the demand for biochips in China has continued to expand, and the market scale has grown rapidly. Therefore, it is urgent to develop the upstream manufacturing technology of biochips, and the research and development of new biomolecule patterning technology can promote the development of related technologies of biochips in China. Summary of the Invention

[0005] In view of the above defects, the present invention provides a method for preparing a molecularly patterned surface based on mechanochemical reactions. This technology covalently binds force-responsive inks in a solution environment to the surface by contacting a template with a substrate and applying pressure.

[0006] In order to achieve the above invention purpose, the technical solution adopted by the present invention is as follows: A method for preparing a molecularly patterned surface based on mechanochemical reactions according to the present invention includes the following steps:

[0007] (1) Modification of the template and the substrate: Use a binary structure molecule with a force-responsive functional group at one end and biocompatibility at the other end as a printing ink molecule; Use a template modified with a biocompatible molecule as a mechanochemical printing pattern template, which can be used to enrich ink molecules; Use a substrate modified with a force-responsive functional group as a pattern transfer object, which can undergo a mechanochemical reaction with the force-responsive ink to obtain a patterned surface.

[0008] (2) Mechanochemical imprinting: In an ink environment, contact the template with the substrate and apply pressure to force the covalent binding of ink molecules to the functionalized substrate, thereby fixing the ink molecules to the substrate according to the pattern of the template.

[0009] Further, in step (1), for silicon and silicon nitride templates, the surface is first cleaned with acetone, then immersed in chromic acid and piranha solution at 70-100°C, rinsed with pure water and dried, and then modified with an ethanol solution of aminosilanizing agent and an aqueous solution of glutaraldehyde, and then modified with streptavidin or nickel-containing affinity resin; for polydimethylsiloxane templates, the surface is first cleaned with ethanol and then treated in an ultraviolet ozone environment, then coated with dopamine hydrochloride, and finally modified with streptavidin or nickel-containing affinity resin; for silicon substrates, the surface is first ultrasonically cleaned in acetone, then immersed in chromic acid and piranha solution at 70-100°C, rinsed with pure water and dried, and then modified with aminosilanizing agent.

[0010] Further, in step (2), mechanochemical imprinting: ink molecules with force-responsive ends and bio-affinity ends are dissolved in a buffer solution simulating a physiological environment, immersed between the functionalized template and the substrate described in step (1), pressure is applied to bind the ink molecules to the substrate, release allows the template to re-enrich ink molecules, and then imprinting is repeated to increase the amount of surface ink molecules bound.

[0011] Furthermore, the patterned surface prepared in step (2) is characterized: the patterned surface bound to ink molecules can be characterized by atomic force microscopy or fluorescence microscopy. For atomic force microscopy, the patterned surface can be directly washed and dried before scanning. For fluorescence microscopy, the non-specific binding sites on the patterned surface need to be blocked with bovine serum albumin or Tween 20, then fluorescently stained with streptavidin or quantum dots, and finally characterized by fluorescence microscopy.

[0012] Furthermore, in step (1), the template enriches the ink through bioaffinity.

[0013] Further, in step (1), the bioaffinity effect includes:

[0014] Affinity interaction between S1 biotin and streptavidin;

[0015] The affinity interaction between the S2 histidine tag and the nickel-containing affinity resin.

[0016] The two bioaffinity mechanisms mentioned above have been reported and widely used before, but the use of bioaffinity mechanisms to enrich ink molecules is a first proposed invention.

[0017] Furthermore, in step (1), the template material is silicon, silicon nitride, or polydimethylsiloxane.

[0018] Furthermore, in step (2), the pattern transfer process involves a mechanochemical reaction.

[0019] Furthermore, in step (2), the mechanochemical reaction involves functional groups including:

[0020] The mechanochemical reaction of S1 maleimide and amino group

[0021]

[0022] The mechanochemical reaction between the S2 carbon-carbon double bond and the thiol group

[0023]

[0024] The mechanochemical reaction between the S3 carbon-carbon triple bond and the thiol group;

[0025]

[0026] The mechanochemical reaction of S4 phenylboronic acid and hydroxyl groups

[0027]

[0028] Furthermore, in step (2), the substrate material is silicon or polydimethylsiloxane.

[0029] In this invention, the polydimethylsiloxane (PDMS) used is Dow Corning's SYLGARD 184, which contains basic components and a curing agent. The preparation method is as follows:

[0030] S1 weighs the basic components and curing agent at a weight ratio of 10:1 and mixes them in a container. Then, it is placed in a vacuum dryer and vacuumed to remove air bubbles.

[0031] S2 Pour the mixture into a glass petri dish and place it in a vacuum desiccator to remove excess air bubbles;

[0032] S3 is placed in a 100℃ heat to cure for 35 minutes, then removed and cooled before demolding.

[0033] Beneficial effects: This invention features low cost and high throughput. The molecular patterns prepared by this technology have high resolution, high stability, and can maintain the activity of biomolecules. This invention can use templates with different degrees of hardness to meet different needs.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] (1) The template used in this invention can be prepared by photolithography, electron beam exposure and etching, and the template can be reused; the pressure device used in this invention is very simple, and only a trace amount of ink molecule solution is needed in the mechanochemical imprinting described in technical step (2). These features make this invention have the advantage of low cost.

[0036] (2) The mechanochemical imprinting process described in step (2) of this invention can obtain a patterned surface in just 30 seconds. It is fast and has a high throughput. This invention can improve the amount of ink bonded to the surface by repeating the imprinting process in step (2).

[0037] (3) The molecular patterns prepared by this invention can reach a precision of 100 nm or even higher; the patterned molecules prepared by this invention are connected to the substrate surface by covalent bonds, and have high stability; this invention is carried out in a biomolecular solution, which can well maintain the activity of biomolecules. Attached Figure Description

[0038] Figure 1 This is a mechanism diagram of the method for preparing molecularly patterned surfaces based on mechanochemical reactions according to the present invention.

[0039] Figure 2 The present invention describes the use of silicon template imprinting to fabricate patterned biotin surfaces and fluorescence characterization.

[0040] Figure 3 This invention aims to investigate the molecular binding stability of ink.

[0041] Figure 4 To verify the present invention, repeated embossing enriches the ink and increases the amount of ink molecules bound together.

[0042] Figure 5 This invention utilizes a mechanochemical reaction-based patterning technique to directly process various proteins.

[0043] Figure 6 The present invention uses a polydimethylsiloxane template for pattern transfer.

[0044] Figure 7 The present invention uses a silicon nitride material template for pattern transfer.

[0045] Figure 8 The present invention uses a rigid template to explore the accuracy of patterning technology. Detailed Implementation

[0046] To facilitate understanding of the present invention, a more comprehensive description of the present invention will be given below with reference to the accompanying drawings, which illustrate several embodiments of the present invention. However, the present invention can be implemented in different forms and is not limited to the embodiments described in the text. Rather, these embodiments are provided to make the disclosure of the present invention more thorough and complete.

[0047] The invention will now be described in further detail with reference to the accompanying drawings.

[0048] Example 1

[0049] like Figure 1 As shown, this novel method for preparing molecularly patterned surfaces based on mechanochemical reactions has the following characteristics in terms of design principle:

[0050] Feature 1. This novel biomolecular patterning technology is based on surface mechanochemical reactions: functional groups capable of undergoing mechanochemical reactions are modified onto both the substrate and ink molecules. Under stress, the force-responsive functional groups on the ink molecules and the substrate will undergo mechanochemical reactions, while no reaction occurs in the unstressed areas of the surface. Using a template with a pre-designed pattern, pressure is applied. The raised patterned areas contact the substrate and generate pressure, while the recessed, unpatterned areas do not generate pressure. Therefore, ink molecules can be patterned and bonded to the surface (e.g., ...). Figure 2 shown).

[0051] Feature 2. In this method for preparing molecularly patterned surfaces based on mechanochemical reactions, a stable covalent bond is formed between the ink molecules and the substrate: various mechanochemically responsive functional groups can be used on the substrate and ink molecules, including maleimide and amino groups, phenylboronic acid and silanol groups, and double bonds and thiol groups. These mechanochemically responsive functional groups undergo mechanochemical reactions under stress, forming covalent interactions stronger than bioaffinity, thereby stably connecting biomolecules to the surface (e.g., ...). Figure 3 shown).

[0052] Feature 3. This novel method for preparing molecularly patterned surfaces based on mechanochemical reactions utilizes bioaffinity to enrich ink, thereby increasing the amount of ink molecules bound to the surface. Bioaffinity receptors and ligands are modified onto the template and ink molecules, respectively. In the ink environment, the receptors on the template enrich ink molecules by binding to the ligands of the ink molecules. During the imprinting process, the ink molecules form covalent bonds with the surface, and weaker bioaffinity interactions break down when the template is removed, leaving a large number of ink molecules in the patterned area. These bioaffinity interactions include the affinity of streptavidin and biotin, the role of nickel-containing affinity resins, and histidine tags, etc.

[0053] Feature 4. This novel method for preparing molecularly patterned surfaces based on mechanochemical reactions can increase the amount of ink molecules bound to the surface through multiple imprinting steps: As described in Feature 3, the template can enrich ink through bioaffinity, thus allowing it to be released after one imprinting step, enabling it to re-bind ink molecules in the ink environment, and then imprinted again at the same location to increase the amount of ink molecules bound to the patterned area. This repeated imprinting step can be performed multiple times (e.g., ...). Figure 4 shown).

[0054] Feature 5. This novel method for preparing molecularly patterned surfaces based on mechanochemical reactions allows for the direct patterning of biomolecules as inks: As mentioned above, ink molecules are binary structures modified with mechanoresponsive functional groups and bioaffinity ligands, while the molecular chains connecting the mechanoresponsive and bioaffinity ends can be polyethylene glycol, or biomolecules such as peptides, proteins, and DNA. Therefore, this method allows for direct patterning onto surfaces (such as...) using this mechanochemical reaction-based molecularly patterned surface preparation method. Figure 5 shown).

[0055] Feature 6. The precision of the biomolecular patterns processed by this novel mechanochemical reaction-based molecular patterning surface preparation method depends on the template: Compared to microcontact printing technology, the principle of this novel biomolecular patterning technology is not ink transfer, but rather that ink molecules in the patterned area undergo a mechanochemical reaction under force, thus covalently bonding to the surface, thereby eliminating the problem of ink diffusion. The pattern size processed by this novel biomolecular patterning technology depends on the contact area between the pattern protrusions in the template and the substrate. For elastic templates, the greater the pressure, the greater the template deformation, thus increasing the contact area between each micropattern on the template and the substrate, ultimately causing the ink molecular pattern to enlarge accordingly (e.g., ...). Figure 6 (As shown); For rigid templates, pressure changes do not significantly alter the contact area between the micropatterns on the template and the substrate, therefore the final ink molecular pattern can completely replicate the pattern on the template (e.g. Figure 7 As shown). The higher the precision of the pattern on the template, the higher the precision of the final ink molecule pattern (e.g.). Figure 8 shown).

[0056] Example 2

[0057] The process of this novel patterning technology includes modification of the template and substrate, mechanochemical imprinting, and characterization of the patterned surface, as follows:

[0058] In step (1), the template and substrate are modified as follows: For silicon and silicon nitride templates, the surface is first cleaned with acetone, then immersed in chromic acid and piranha solution at 80°C, rinsed with pure water and dried, and then modified with an ethanol solution of aminosilanizing agent and an aqueous solution of glutaraldehyde, and then modified with streptavidin or nickel-containing affinity resin; For polydimethylsiloxane templates, the surface is first cleaned with ethanol and then treated in an ultraviolet ozone environment, then coated with dopamine hydrochloride, and finally modified with streptavidin or nickel-containing affinity resin; For silicon substrates, they are first ultrasonically cleaned in acetone, then immersed in chromic acid and piranha solution at 80°C, rinsed with pure water and dried, and then modified with aminosilanizing agent.

[0059] The template enriches ink through bioaffinity.

[0060] The aforementioned bioaffinity includes:

[0061] Affinity interaction between S1 biotin and streptavidin;

[0062] The affinity interaction between the S2 histidine tag and the nickel-containing affinity resin.

[0063] The template is made of silicon, silicon nitride, or polydimethylsiloxane.

[0064] In step (2), mechanochemical imprinting: ink molecules with force-responsive ends and bio-affinity ends are dissolved in a buffer solution that simulates a physiological environment, and then immersed between the functionalized template and the substrate described in (1). Pressure is applied to bind the ink molecules to the substrate, and the template is released to re-enrich the ink molecules. The imprinting is repeated to increase the amount of ink molecules bound to the surface.

[0065] Characterization of patterned surfaces: Patterned surfaces bound to ink molecules can be characterized by atomic force microscopy or fluorescence microscopy. For atomic force microscopy, the patterned surface obtained in step (2) can be directly rinsed and dried for scanning. For fluorescence microscopy, the non-specific binding sites on the patterned surface need to be blocked with bovine serum albumin or Tween 20, then fluorescently stained with streptavidin or quantum dots, and finally characterized by fluorescence microscopy.

[0066] The pattern transfer process involves a mechanochemical reaction. The mechanochemical reaction involves functional groups including:

[0067] The mechanochemical reaction of S1 maleimide and amino group

[0068]

[0069] The mechanochemical reaction between the S2 carbon-carbon double bond and the thiol group

[0070]

[0071] The mechanochemical reaction between the S3 carbon-carbon triple bond and the thiol group

[0072]

[0073] The mechanochemical reaction of S4 phenylboronic acid and hydroxyl groups

[0074]

[0075] The substrate material is silicon or polydimethylsiloxane.

[0076] This invention uses Dow Corning's SYLGARD 184 to prepare PDMS, which comprises basic components and a curing agent. The preparation method is as follows:

[0077] Weigh the basic components and curing agent at a weight ratio of 10:1 and mix them in a container. Place the container in a vacuum dryer and evacuate to remove air bubbles.

[0078] Pour the mixture into a glass petri dish and place it in a vacuum desiccator to remove excess air bubbles;

[0079] Heat at 100℃ for 35 minutes to cure, then remove and cool before demolding.

[0080] The polydimethylsiloxane described in this invention is used in molecularly patterned surfaces based on mechanochemical reactions.

[0081] Example 3

[0082] The difference between Example 3 and Example 2 is as follows: In step (1), the modification of the template and the substrate is as follows: For silicon and silicon nitride templates, the surface is first cleaned with acetone, then immersed in chromic acid and piranha solution at 100°C, rinsed with pure water and dried, and then modified with an ethanol solution of aminosilanizing agent and an aqueous solution of glutaraldehyde, and then modified with streptavidin or nickel-containing affinity resin; For polydimethylsiloxane templates, the surface is first cleaned with ethanol and then treated in an ultraviolet ozone environment, then coated with dopamine hydrochloride, and finally modified with streptavidin or nickel-containing affinity resin; For silicon substrates, they are first ultrasonically cleaned in acetone, then immersed in chromic acid and piranha solution at 100°C, rinsed with pure water and dried, and then modified with aminosilanizing agent.

[0083] The following are examples of experiments demonstrating the various features of the present invention:

[0084] Experimental Example 1

[0085] This invention enables molecular patterning based on mechanochemical reactions.

[0086] According to step (1), streptavidin and amino groups are modified on the square array template and the silicon substrate, respectively. A molecule with biotin and maleimide connected by polyethylene glycol at the bioaffinity and mechanoresponsive ends, respectively, is used as the ink molecule for the mechanochemical imprinting described in step (2). The substrate with the transferred biotin pattern is then subjected to fluorescence imaging after the aforementioned fluorescent staining, as shown in the figure. Figure 2 As shown in c, the pattern on the template was successfully transferred to the substrate using this molecular patterning technique, while no obvious fluorescence signal was observed in the area outside the pattern.

[0087] Experimental Example 2

[0088] The biomolecular patterns produced by this invention exhibit good stability.

[0089] The patterned fluorescent protein surface obtained in Example 1 was treated with acidic, alkaline, and mercaptoethanol solutions, such as... Figure 3 As shown in b, the fluorescence signal did not show significant attenuation, verifying the good stability of the biomolecular pattern. Figure 3 c. The amino-functionalized surface obtained in step (1) is then imprinted with ink molecules. For example... Figure 3 As shown in d, compared to inks that are simply physically adsorbed, the N1s spectrum of nitrogen on the surface of the embossed ink shows a binding energy peak of secondary amines, indicating that an addition reaction occurred between the amino group and maleimide, which directly verifies the covalent connection between ink molecules and the surface.

[0090] Experimental Example 3

[0091] This invention can increase the amount of ink molecules bound by repeated printing steps.

[0092] like Figure 4 As shown in Figure a, by repeating the mechanochemical imprinting process described in step (2), ink molecules enriched multiple times in the template can be bound to the same pattern area, thus greatly increasing the number of ink molecules bound to the pattern area. Using the same experimental conditions as in Example 1, the imprinting and release process was repeated 1, 3, and 6 times. After fluorescent staining, the fluorescence intensity of the fluorescent patterns under different conditions was statistically analyzed. Figure 4 As shown in b, the fluorescence intensity of the pattern increases with the number of repetitions, indicating that repeated imprinting increases the amount of ink molecules bound to the surface.

[0093] Test Example 4

[0094] This invention can directly pattern and process various proteins.

[0095] As described in feature 5, this biomolecular patterning technology can directly pattern biomolecules as ink. Figure 5 As shown in a, in step (1), maleimide and nickel-containing affinity resin are used to modify the substrate and two templates respectively. In step (2), red fluorescent protein and green fluorescent protein with histidine tags are used as ink molecules to be sequentially imprinted onto the substrate. The patterned multi-protein array is stained with quantum dots using the fluorescence staining step described in step (3). Figure 5 As shown in b, this novel method for preparing molecularly patterned surfaces based on mechanochemical reactions successfully immobilized two proteins in different patterns on the same surface.

[0096] Experimental Example 5

[0097] This invention can achieve a precision of hundreds of nanometers.

[0098] As described in feature 6, the precision of the biomolecular patterns produced by this biomolecular patterning technique depends on the precision of the template and the material. Polydimethylsiloxane is used as the elastic template material, and streptavidin is modified on its surface according to the method described in step (1). Mechanochemical imprinting as described in step (3) is performed under a pressure of 600 kPa. The ink and subsequent staining characterization methods are the same as in Example 1. Figure 6 As shown, the size of the fluorescent pattern is larger than that of the elastic template, indicating that the elastic template deformed under pressure. However, when silicon nitride was used as the rigid template, the fluorescent pattern obtained according to the method steps of Example 1 matched the pattern size of the template, indicating that the rigid template did not undergo significant deformation (e.g., Figure 7 (As shown). Templates with a precision of hundreds of nanometers were prepared using silicon as a rigid template material, such as... Figure 8 As shown, fluorescent patterns with a precision of hundreds of nanometers were obtained by following the method steps in Example 1.

[0099] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a molecular patterned surface based on mechanochemical reaction, characterized in that The steps include: (1) Modification of template and substrate: using a binary structure molecule with a force-responsive functional group at one end and a bio-affinity at the other end as the printing ink molecule; The template modified with bioaffinity molecules is used as a pattern template for mechanochemical printing to enrich ink molecules; The substrate modified with force-responsive functional groups is used as a pattern transfer object, which can undergo a mechanochemical reaction with the force-responsive ink to produce a patterned surface; (2) Mechanochemical imprinting: The template is brought into contact with the substrate in an ink environment, and pressure is applied to promote covalent bonding between the ink molecules and the functionalized substrate, thereby fixing the ink molecules to the substrate according to the pattern of the template.

2. The method for preparing a molecular patterned surface based on a mechanochemical reaction according to claim 1, characterized in that: In step (1), for silicon and silicon nitride templates, firstly, their surfaces are cleaned with acetone, then they are immersed in chromic acid and piranha washing solution at 70-100°C in turn, rinsed with pure water and then blown dry, and then modified with ethanol solution of aminosilanization reagent and aqueous solution of glutaraldehyde in turn, and then modified with streptavidin or nickel-containing affinity resin; for polydimethylsiloxane templates, firstly, their surfaces are cleaned with ethanol and then placed in an ultraviolet ozone environment for treatment, then dopamine hydrochloride is coated on their surfaces, and finally modified with streptavidin or nickel-containing affinity resin; for silicon substrates, firstly, they are placed in acetone for ultrasonic cleaning, then they are immersed in chromic acid and piranha washing solution at 70-100°C in turn, rinsed with pure water and then blown dry, and then modified with aminosilanization reagent in turn.

3. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 2, characterized in that: In step (2), mechanochemical imprinting: ink molecules having a force-responsive end and a bio-affinity end are dissolved in a buffer solution simulating a physiological environment, immersed between the template functionalized in step (1) and the substrate, pressure is applied to allow the ink molecules to bind to the substrate, and released to allow the template to re-enrich the ink molecules, and then repeatedly imprinted to increase the amount of ink molecules bound to the surface.

4. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 3, characterized in that: In step (2), the surface patterned with ink molecules is characterized by atomic force microscopy or fluorescence microscopy. For atomic force microscopy characterization, the prepared patterned surface is directly rinsed and blown dry for scanning characterization; for fluorescence microscopy characterization, the non-specific binding sites on the patterned surface must first be blocked with bovine serum albumin or Tween 20 reagent, and then fluorescently stained with fluorescently labeled streptavidin or quantum dots, and finally characterized by fluorescence microscopy.

5. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 1, characterized in that: In step (1), the template is enriched with ink by bioaffinity.

6. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 2, characterized in that: In step (1), the bioaffinity comprises: S1 affinity interaction between biotin and streptavidin; or affinity interaction between the S2 histidine tag and nickel-containing affinity resin.

7. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 1, characterized in that: In step (1), the material of the template is silicon, silicon nitride or polydimethylsiloxane; in step (2), the pattern transfer process involves a mechanochemical reaction.

8. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 1, characterized in that: In step (2), the functional groups involved in the mechanochemical reaction include: Mechanochemical reaction between S1 maleimide and amino group or the mechanochemical reaction between the S2 carbon-carbon double bond and the thiol group or the mechanochemical reaction between the S3 carbon-carbon triple bond and the thiol group or the mechanochemical reaction of S4 phenylboronic acid and hydroxyl groups 9. The method for preparing a molecular patterned surface based on mechanochemical reaction according to claim 1, characterized in that: In step (2), the substrate material is silicon or polydimethylsiloxane.

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