WO2019090886A1 - Method for preparing pattern visible under polarized light - Google Patents

Abstract

A method for preparing a pattern visible under polarized light and a method for preparing a DNA patterned substrate. The method for preparing a DNA patterned substrate comprises: respectively providing a substrate, a PDMS stamp having a patterned surface, a 12-mercaptododecanoic acid solution, and a DNA that can be combined with the surface of the substrate; obtaining a processed PDMS stamp; making the processed PDMS stamp contact the surface of the substrate to obtain a hydrophilic substrate having a patterned surface; and obtaining a DNA patterned substrate having a DNA surface layer.

Description

Method for preparing a visible pattern under polarized light

Priority information

The present application claims priority to and the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit of the benefit.

Technical field

The present invention relates to the field of biotechnology, and in particular to the field of liquid crystal droplet patterning imprinting technology, and more particularly to a method of preparing a visible pattern under polarized light.

Background technique

Under external stimuli, changes in liquid crystal orientation can be transmitted and amplified to the micrometer scale, and the accompanying optical signal changes can be used for chemical and biological detection. In the presence of the liquid crystal droplets, the liquid crystal orientation change can be observed by an optical microscope to achieve the detection purpose. How to make liquid crystal realize the fields of important application significance such as stimulus response, detection, anti-counterfeiting and even information display function is a scientific problem that requires long-term exploration and research.

At present, the use of liquid crystals to construct a potentially responsive smart surface, and then to prepare a visible pattern under polarized light remains to be studied.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a means for constructing a potentially responsive smart surface using liquid crystals to prepare a visible pattern under polarized light.

It should be noted that the present invention has been completed based on the following findings and work of the inventors:

With the steady development of science and technology, people's requirements for intelligent life are getting higher and higher, and the system with intelligent response and detection functions has received more and more attention. In addition to being used as a display material, liquid crystals have been gradually used in the field of response and detection in recent years. Liquid crystal is a phase of a special substance between a solid crystal and an amorphous liquid. The substance in this phase combines the fluidity of the liquid with the anisotropy of the crystal. Under the external stimulus, the change of orientation of liquid crystal can be transmitted and amplified to the micrometer scale, and the accompanying optical signal changes can be used for chemical and biological detection. Therefore, the application of liquid crystal as a special detection element to construct biosensors has caused the scientific community. s concern.

Since Abbott Laboratories first proposed the use of liquid crystals in the field of biosensors in 1998, important research results have been achieved in the use of liquid crystal-water phase interfaces for detection. Liquid crystal biosensing is based on the change of the orientation of the liquid crystal molecules on the surface of the functional film, changing the refractive power of the liquid crystal molecules to the light, converting the reaction change information generated at the interface into optical signal changes, and realizing the detection of the target, such as achievable Detection of protein macromolecules, bacteria and viruses, enzymatic reactions, DNA hybridization, stem cell culture, and the like. Recently, Professor JJde Pablo of the Institute of Molecular Engineering (IME) at the University of Chicago reported that liquid crystals have been used as detectors to successfully detect amyloid, a protein associated with neurodegenerative diseases such as Alzheimer's disease (also known as Alzheimer's disease). The disease of Alzheimer's disease is closely related. This research shows that liquid crystal is expected to be used in the early diagnosis of Alzheimer's disease in the near future.

As a form of another liquid crystal-water phase interface with high curvature, liquid crystal droplets have received more and more attention from researchers. When the liquid crystal droplets are assembled with the amphiphilic molecules, the liquid crystal molecules will change in orientation, and the liquid crystal droplets will be converted into a central defect by the bipolar defect induced by the amphiphilic molecules. This orientation transition can be clearly observed under a polarizing microscope. The inventors have found that when a droplet of 4 μ-n-pentyl-4-cyano-biphenyl (5CB) having a diameter of 8 μm is in water, liquid crystal molecules are aligned along the axial direction of the droplet due to the orientation of the liquid crystal itself, due to The high curvature of the droplets, the liquid crystal molecules at the two poles of the droplets can not be arranged in an orderly manner, showing a disordered state, that is, a bipolar defect appears on the surface of the droplet, forming a bipolar configuration, which can be observed under an optical microscope. Both ends are dark. When the amphiphilic endotoxin molecule is assembled with the liquid crystal droplets, six hydrophobic alkyl chains are embedded in the liquid crystal droplets, and the liquid crystal molecules are induced to be arranged in a radial direction to form a radial configuration, and the droplets can be observed under white light. The center is darker, and the liquid crystal droplets can be found to have a "ten" pattern under polarized light. When the liquid crystal droplets interact with a polymer or a biomolecule, the orientation changes. The detection characteristics brought by this orientation change make liquid crystal droplets have more important research value and application prospects.

As a carrier of genetic information, DNA is mainly focused on biology. Until the 1980s, Professor Seeman of New York University proposed a new concept for DNA from the perspective of polymer. He believed that DNA is a kind of linear polymer with precise structure, certain composition and shape control. A new field of "Structural DNA Nanotechnology". Since then, the unique structure and properties of DNA molecules have attracted the attention of chemists and materials scientists. As a new type of assembly material, DNA molecules have gradually realized new functions outside the biological system. The unique properties of DNA mainly include the following aspects: 1 DNA is a type of structurally defined nanomaterial, and the diameter and period of DNA double helix are accurate nanometer size. For example, the DNA double helix molecule has an average diameter of 2 nm, 10.4 nucleotides per revolution around the central axis, and a pitch of 3.4 nm; 2DNA has sequence programmability, highly accurate recognition ability and structural controllability, Among the DNA molecules, common bases are adenine (A), thymine (T), guanine (G), and cytosine (C). Among them, A and T, G and C can be hydrogen-bonded. Complementary pairing. The base sequence of DNA determines the structure formed by its complementation with other DNA; 3 The functionality of the DNA sequence, the aptamer sequence can selectively bind to the corresponding ligand, in the external environment such as temperature, pH Under the stimulation of ionic strength and biomolecules, the conformation changes and produces specific responsiveness. 4 The synthesis and modification techniques of DNA molecules are making great progress, and DNA synthesis of less than 100 bases can be completed in the laboratory. And DNA has now been commercialized to facilitate the acquisition of the required DNA sequences for related research. Based on the above special properties of DNA, after nearly three decades of efforts, DNA has become a new building unit of intelligent response materials.

Surface patterning technology is a research hotspot in science and technology in recent years. It has a wide range of applications in data information storage, optoelectronic devices and biochips. There are many ways to construct a patterned surface, in which Microcontact Printing (μCP) is a micro-pattern on a stamp by a simple imprint process using molecules or particles capable of forming a self-assembled film on a substrate. A technique for transferring to a substrate. The poly-dimethylsiloxane (PDMS) stamp is the primary seal material for μCP, which can be used to imprint submicron to micron surface patterns. The specific process of μCP is as follows: Firstly, a patterned silicon template is prepared by photolithography, then PDMS prepolymer and cross-linking agent are cast on the template, and PDMS is peeled off from the template after cross-linking curing, in elastic PDMS. The surface of the stamp is coated with a solution of molecules or particles. After the solution is volatilized, the PDMS is closely attached to the surface of the substrate. In this way, the molecules or particles can be transferred to the substrate in the contact portion, and the pattern corresponding to the template is left on the surface of the substrate after the stamp is removed.

μCP technology is an effective method for substrate pattern printing. It not only has the advantages of fast and low cost, but also does not need to have the harsh conditions such as ultra-clean room, and does not even need an absolutely flat surface. It requires low experimental conditions and does not require expensive. The experimental instrument has simple process flow, fast and flexible operation, can form a patterned surface with fine structure, and can be applied on different chemical surfaces to realize the modification of the surface of the substrate. Therefore, μCP is a more flexible and effective method for preparing patterned surfaces, and has important applications in materials science, biotechnology, optical systems, and microelectronic systems.

After a series of studies, the inventors have introduced DNA into liquid crystal droplet systems for the first time, and have carried out related work on liquid crystal droplets modified by DNA-parent molecules, including DNA-parent molecule synthesis and DNA-parent molecule modified liquid crystal liquid. Preparation of drops, etc. By designing the DNA sequence, the inventors have realized the dispersion of the liquid crystal droplets under the external stimulus of the target, and tried the stimulation response study of temperature, mercury ion, pH, restriction endonuclease and ATP.

Furthermore, through the accumulation of previous research, the inventors first proposed the idea of combining liquid crystal droplets, DNA and μCP technology, and patterned and assembled liquid crystal droplets on the substrate by utilizing the design and specific binding characteristics of the DNA sequence. It has the potential to change the pattern of the surface of the substrate when there is an external stimulus, thereby obtaining a responsive surface. The method can be applied not only to a variety of liquid crystals, but also to the response of the analyte by using DNA designability, and introducing a responsive element makes the system more versatile, such as azobenzene. This method has the advantages of simpler, less used, and reusable than the detection in solution, thus providing new methods and new ideas for stimuli response, detection and product anti-counterfeiting.

Thus, in a first aspect of the invention, the invention provides a method of preparing a DNA pattern substrate. According to an embodiment of the present invention, the method comprises the steps of separately providing a substrate, a PDMS stamp having a pattern on the surface, a 12-decyldodecanoic acid solution, and a DNA capable of binding to the surface of the substrate; and having the surface patterned with a PDMS stamp Extracting the 12-mercaptododecanoic acid solution to obtain a treated PDMS stamp; contacting the treated PDMS stamp with the surface of the substrate, and then removing the stamp to obtain a patterned hydrophilic surface a substrate; a hydrophilic substrate having a pattern on the surface is contacted with the DNA capable of binding to a surface of the substrate to obtain the DNA pattern substrate having a DNA surface layer.

The inventors have surprisingly found that a DNA pattern substrate having a DNA surface layer can be efficiently prepared by this method. Further, a DNA liquid crystal droplet prepared by using a DNA amphiphilic molecule having at least a 12 bp sequence complementary to DNA of the DNA surface layer of the DNA pattern substrate is brought into contact with the DNA pattern substrate, and the DNA pattern substrate for adsorbing liquid crystal droplets is placed At room temperature, the surface of the DNA pattern substrate can be rendered in a desired pattern under polarized light. Thereby, it is effective to obtain a pattern which is visible under polarized light.

According to an embodiment of the invention, the substrate is a gold substrate, a glass substrate or a silicon substrate. Wherein, the gold substrate can be operated using thiol-modified DNA; the glass substrate can be subjected to a series of treatments in advance, including azo modification, aldehyde modification, hydrophobic modification, etc.; for silicon substrate, thiol chemical modification can be performed in advance.

According to some specific examples of the invention, the substrate is a gold substrate, and the DNA capable of binding to the surface of the substrate is a sulfhydryl DNA. The term "gold substrate" as used herein refers to a substrate having a gold surface. In addition, it needs to be explained Yes, when thiol-based DNA is used as the DNA capable of binding to the surface of the substrate, the SH-DNA does not replace 12-mercaptododecanoic acid because the longer the thiol carbon chain, the smaller the adsorption free energy ΔG (negative The value, the larger the absolute value, the easier it is to adsorb on the Au substrate. Moreover, from the literature on co-modification using DNA and thiol, it can be found that co-modification with thiol helps to promote the thiol DNA to be vertically aligned on the substrate, which is more conducive to the complementary pairing of DNA segments. There is no mention of the problem that thiol DNA will replace the modified thiol. In one embodiment of the invention, 2 mM 12-mercaptododecanoic acid is imprinted first, followed by SH-DNA (ie, sulfhydryl DNA) over the entire area at a concentration of 1 μM so that The region imprinted with 12-mercaptododecanoic acid can be modified with DNA. The inventors believe that sulfhydryl DNA has the potential to replace 12-mercaptododecanoic acid (because the molecular size of sulfhydryl DNA is much larger than 12-mercaptododecanoic acid), but in experimental design, the concentration of thiol DNA is 12-mercapto 1/2000 of diacid, and 2 mM 12-mercaptododecanoic acid imprinting operation can be coated on the Au substrate with 12-mercaptododecanoic acid molecule, which can further modify the thiol DNA substrate and the substituted 12-mercapto 10 There are few diacid molecules, so the inventors believe that even if the sulfhydryl DNA undergoes a very small part of the substitution of 12-mercaptododecanoic acid, there is very little incorporation in the region where the original imprinted 12-mercaptododecanoic acid is imprinted. The effect of sulfhydryl DNA is negligible, and it can still form a significant contrast with the thiol-modified DNA on the Au in the bare area (how much liquid crystal droplets are adsorbed).

According to an embodiment of the present invention, the thiol DNA may be an ethynyl modification, whereby it may undergo a Click reaction with an azo-modified glass substrate; the thiol DNA may be an amino modification, whereby it may be modified with an aldehyde group An aldiamine condensation occurs in the glass substrate; the sulfhydryl DNA may be an alkyl chain modification whereby it may interact hydrophobically with a hydrophobically modified glass substrate; the thiol DNA may be biotin modified, thereby, in avidin Specific interactions with biotin-modified silicon substrates can occur under the conditions present. Thus, according to some specific examples of the invention, the thiol DNA is modified with at least one selected from the group consisting of a thiol group, an ethynyl group, an amino group, an alkyl chain, or biotin.

According to some specific examples of the invention, the nucleic acid sequence of the thiol DNA is: TACACATCTACTTCACCA.

According to an embodiment of the invention, the thiol DNA is pretreated. According to some specific examples of the invention, the pretreatment is a disulfide bond reduction reaction of the sulfhydryl DNA using dithiothreitol.

According to an embodiment of the invention, the 12-mercaptododecanoic acid solution is a solution of 12-mercaptododecanoic acid in ethanol.

According to an embodiment of the invention, the treated PDMS stamp is brought into contact with the surface of the substrate for 20-40 seconds, preferably 30 seconds. Thereby, the contact effect is good, and the obtained DNA pattern substrate is more favorable for the preparation of the subsequent polarized visible pattern.

According to an embodiment of the present invention, the hydrophilic substrate having the pattern on the surface is contacted with the sulfhydryl DNA by immersing the hydrophilic substrate having the pattern on the surface in the PBS solution of the thiol DNA for 8 to 24 hours. , preferably achieved in 16 hours. Thereby, the contact effect is good, and the obtained DNA pattern substrate is more advantageous for preparation with a polarized visible pattern.

In a second aspect of the invention, the invention provides a DNA pattern substrate. According to an embodiment of the present invention, it is obtained by the method for preparing a DNA pattern substrate as described above. Thereby, the obtained DNA pattern substrate can be effectively contacted with the DNA liquid crystal droplets prepared by the DNA amphiphilic molecule in which the DNA of the DNA surface layer of the DNA pattern substrate is at least 12 bp complementary, thereby preparing a pattern visible under polarized light.

In a third aspect of the invention, the invention provides a DNA liquid crystal droplet. According to an embodiment of the present invention, the DNA liquid crystal droplets are prepared by mixing a DNA amphiphilic molecule with a liquid crystal material and emulsifying, To obtain the DNA liquid crystal droplets, wherein the DNA amphiphilic molecule is complementary to at least a 12 bp sequence of the thiol DNA of the DNA surface layer of the DNA pattern substrate described above. Thereby, the obtained DNA liquid crystal droplets can be effectively brought into contact with the DNA pattern substrate having the DNA surface layer complementary to the sequence in which the DNA amphiphilic molecule is at least 12 bp, thereby preparing a pattern visible under polarized light.

According to an embodiment of the present invention, the DNA amphiphilic molecule is DNA-C18, the hydrophilic end of which is a DNA strand end, and the hydrophobic end of which is a C18 chain end. Thereby, the contact of the DNA liquid crystal droplets with the DNA pattern substrate is facilitated.

According to some specific examples of the invention, the nucleic acid sequence of the DNA amphiphilic molecule is TGGTGAAGTAGATGTGTA. Thus, the genomic DNA sequence of the DNA amphiphilic molecule and the DNA surface layer of the DNA pattern substrate described above (TACACATCTACTTCACCA) satisfies the requirement of at least 12 bp of sequence complementation, facilitating contact of the DNA liquid crystal droplets with the DNA pattern substrate and polarizing. Preparation of a visible pattern underneath.

According to an embodiment of the invention, the liquid crystal material is 5CB liquid crystal, TL205 liquid crystal or E7 liquid crystal, preferably 5CB liquid crystal. It should be noted that 5CB, 4'-n-pentyl-4-cyanobiphenyl, is a commonly used nematic liquid crystal, and E7 and TL205 are two commercially available mixed liquid crystals. Wherein E7 is from 4'-n-heptyl-4-cyanobiphenyl (7CB), E7 is from 4'-n-heptyl-4-cyanobiphenyl (7CB), 4'-n-pentyl-4 -Cyanobiphenyl (5CB), 4-n-octylcyano-4'-cyanobiphenyl (80CB) and 4'-n-pentyl-4-cyanoterpene (5CT) in a certain proportion Phase liquid crystal. TL205 is a mixed thermotropic liquid crystal containing F.

In a fourth aspect of the invention, the invention provides a method of preparing a pattern that is visible under polarized light. According to an embodiment of the present invention, the method comprises the steps of: obtaining a PDMS stamp having a target pattern on a surface; preparing a DNA pattern substrate using the PDMS stamp having the desired pattern on the surface according to the method for preparing a DNA pattern substrate as described above; The DNA liquid crystal droplet described above, wherein the DNA amphiphilic molecule is complementary to at least a 12 bp sequence of the thiol DNA of the DNA surface layer of the DNA pattern substrate; and the DNA liquid crystal droplet and the DNA surface of the DNA pattern substrate are The layer is contacted to obtain a DNA pattern substrate that adsorbs liquid crystal droplets; and the DNA pattern substrate of the adsorbed liquid crystal droplets is placed at room temperature, and the surface of the DNA pattern substrate can exhibit the pattern under polarized light.

According to an embodiment of the present invention, the DNA liquid crystal droplets are previously diluted with PBS buffer before contacting the DNA liquid crystal droplets with the DNA surface layer of the DNA pattern substrate. Thereby, the contact effect is good.

According to an embodiment of the present invention, the DNA liquid crystal droplets were diluted 10-fold with PBS buffer.

According to an embodiment of the invention, the DNA liquid crystal droplets are contacted with the DNA surface layer of the DNA pattern substrate for 3-8 minutes, preferably 5 minutes. Thereby, the contact effect is good.

It is emphasized that the present invention has been achieved based on the findings of the inventors and a series of scientific experimental designs and studies. The inventors believe that under external stimuli, changes in liquid crystal orientation can be transmitted and amplified to the micrometer scale, and the accompanying optical signal changes can be used for chemical and biological detection. In the presence of the liquid crystal droplets, the liquid crystal orientation change can be observed by an optical microscope to achieve the detection purpose. In addition to being a carrier of genetic information in organisms, DNA has become a new type of assembly material due to its sequence programmability, functionality, highly accurate recognition capabilities, and the development of synthetic modification techniques. The field of responsive materials is beyond doubt. How to combine the excellent properties of liquid crystals with DNA functionality to achieve areas of important application significance such as stimulus response, detection, anti-counterfeiting and even information display functions is a scientific problem that requires long-term exploration and research. The inventor first made DNA, liquid crystal droplets and micro contacts Printing technology combines the design of DNA sequences and DNA patterning of substrates to introduce DNA-modified liquid crystal droplets onto the surface of the substrate to create potentially responsive (such as temperature, ions, pH, biomolecules, etc.) intelligence. The surface lays the foundation for the visualization of surface information, readable and writable functions, product anti-counterfeiting and even the construction of test kits.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 is a flow chart showing a method of preparing a visible pattern under polarized light (ie, an imprint experiment) according to an embodiment of the present invention;

Figure 2 shows the DNA-C18 mass spectrum in Example 1;

3 is a schematic view showing the assembly of a DNA-parent molecule on the surface of a liquid crystal droplet in Example 1, in which the complementary pairing causes liquid crystal droplets to aggregate in the presence of R1 and R2;

4 is a photograph showing the contact angle of the Au surface, the gold surface after imprinting 12-decyldodecanoic acid, and the gold surface after modifying the SH-DNA in Example 1;

Fig. 5 is a photograph showing a polarized photograph of the pattern of the Au surface exhibited by the adsorption of 5CB droplets in Example 1.

Detailed ways

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

Example 1

According to the method of the present invention for preparing a pattern visible under polarized light, a pattern visible under polarized light is prepared. Including: using emulsification method to prepare 4'-n-pentyl-4-cyanobiphenyl (5CB) liquid crystal droplets modified by DNA-parent molecule, and then using 12-mercaptododecanoic acid and DNA by microcontact printing technology Binding to the gold substrate is achieved by a sulfur-gold bond, and finally liquid crystal droplets having a DNA segment complementary to the DNA segment on the substrate are assembled on the surface of the gold substrate by the principle of complementary base pairing of DNA, as shown in FIG. The method can realize the visualization of the substrate pattern and the selective display of the substrate information, and combines the patterned substrate with the liquid crystal material to provide a new idea and application prospect for preparing the portable nanometer detecting device.

Among them, FIG. 1 shows a schematic diagram of an imprint experiment. As shown in Figure 1, (A) DNA-C18 emulsified 5CB droplets are applied to the patterned gold substrate, and (B) the DNA segment on the 5CB droplet is complementary to the DNA segment on the substrate, (C) The pattern regions modified with complementary SH-DNA segments exhibited a bright color when observed under polarized light.

Specifically, the steps of preparing a visible pattern under polarized light are as follows:

1. Preparation of DNA amphiphilic DNA-C18 emulsified 5CB droplets

1. Synthesis and characterization of DNA-parent molecule DNA-C18

The DNA design and synthesis part has mature software and synthesis technology. The connection between the hydrophobic molecule and the DNA is carried out by Solid Phase Synthesis. The hydrophobic molecule having a core of a hydroxyl group is reacted with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite to obtain a phosphoramidite activating reagent. Subsequently, the hydrophobic molecule phosphoramidite activating reagent is subjected to solid phase synthesis with DNA supported on a controllable pore glass (CPG) pellet. The product is finally obtained by activation of tetrazole, oxidation of iodine water, deprotection of concentrated ammonia water, and cleaving of CPG beads. It was subsequently characterized by HPLC purification, denaturing polyacrylamide gel electrophoresis and MALDI-TOF MS. The inventors have completed the synthesis of the DNA-parent molecule DNA-C18.

2, 5CB droplet preparation

10 μL of 5CB was added to 50 μL of 20 μM DNA amphiphilic DNA-C18, and emulsified by a cell disrupter at 20% power for 30 s to prepare liquid crystal droplets; 1 μL of 100 μM R1 and 1 μL of 100 μM R2 were mixed, and 8 mL of PBS solution (pH=7.40) was added. After mixing, the solution was prepared into a 20 μM linker (R1+R2) solution, and allowed to stand for about 5 minutes to form a linker. Take 5 μL of 20 μM linker (R1+R2) solution, add 5 μL of 5CB droplets, wait for about 30 s, and observe with naked eyes.

The DNA sequences used in this experiment are shown in Table 1 below.

Table 1 DNA sequence

Among them, the numbers 1-5 in the above table are the DNA sequences used in the experiment, in which 1 and 2 are used as experimental groups, 2 and 3 are used as control groups, and 4 and 5 are used to verify that the prepared 5CB droplets can realize DNA bases. The performance of complementary pairing. In this experiment, the linker formed by R1 and R2, which is complementary to DNA-C18, is added to the 5CB droplets and left to stand for about 30 s. The liquid crystal droplets will show obvious macroscopic aggregation.

Second, the preparation of DNA pattern substrate

1. Pretreatment of thiol DNA

(1) Dithiothreitol (DTT) treatment

The SH-C18, SH-NC18 sulfhydryl DNA was subjected to a reduction reaction of disulfide-s-s-. In order to ensure the effective progress of the reaction, DTT was used in a large excess, about 5 mg of DTT powder was added to each thiol DNA, and the mixture was thoroughly mixed and dissolved at 4 ° C overnight.

(2) Filtering

The sample was transferred to an ultrafiltration tube with a molecular weight cutoff of 3000, and ultrapure water was added to 400 μL, and the two were trimmed, centrifuged at 14,000 rcf for 25 min; and washed with 400 μL of water for 3 times, each time for centrifugation for 25 min. Take the liquid on the ultrafiltration membrane, about 50 μL.

(3) Set the thick

The thiol DNA was diluted with water at an appropriate multiple, such as 100-fold or 10-fold, and the Abs value of the UV-vis spectrum at 260 nm was determined, and the Abs value was in the range of 0.2-0.8.

2, Au substrate preparation

The slides were washed using "piranha", N _{2 was} blown dry and sealed for storage. An Au substrate was prepared on a glass slide by vapor deposition: a 10 nm thick Cr adhesive layer and a 20 nm thick Au layer. Sealed and stored. The Au substrate was cut into a size of 1 cm × 1 cm using a glass knife.

3. Preparation of PDMS seal

The PDMS polymer prepolymer and the crosslinking agent were mixed at a volume ratio of 10:1, and after stirring for 10 minutes, they were cast on the surface of the lithographic silicon template having a pattern on the surface. After degassing for 30 min using a vacuum pump, it was cured in an oven at 120 ° C for 2 h. The cured PDMS is stripped from the surface of the lithographic silicon template to obtain a PDMS stamp having a pattern on the surface. The PDMS was cut into a 1 cm x 1 cm stamp for use with a blade.

4, micro-contact printing 12-decyl dodecanoic acid molecule

The patterned PDMS stamp was subjected to mid-range intensity processing for 1 min. Microcontact pattern printing was performed on a gold plated glass substrate. 12-mercaptododecanoic acid was imprinted with patterned PDMS, 2 mM solution of 12-mercaptododecanoic acid in ethanol, 15 μL was applied onto PDMS, and the coating was completed for about 5 s to ensure that the PDMS surface was fully adsorbed. - Mercapto dodecanoic acid molecule, N ₂ blown dry. Gently place the stamp on the Au surface, so that the PDMS is in full contact with the gold, stay for 30s, and remove the seal.

5. Patterned assembly DNA sequence modification SH-DNA

The hydrophobic pattern gold substrate assembled with 12-mercaptododecanoic acid was immersed in a thiolated "SH-DNA" (1 μM) PBS solution (1 mM, pH 7.4) overnight (16 h), and the surface was washed three times with PBS.

Third, the liquid crystal droplets combined with the base DNA

The prepared liquid crystal droplets are dropped on the DNA patterned substrate, and based on the principle of DNA base complementary pairing, the liquid crystal droplets anchored with the DNA-parent molecule will be complementary to the DNA strand immobilized on the gold substrate. The segment is combined to realize the visualization of the substrate pattern, and the process is as shown in FIG. Take 5 μL of 5CB droplets, add 45 μL of PBS solution (pH=7.40), and make a 10-fold dilution. The diluted 5CB droplets were spread in the modified area, allowed to stand for 5 min, and washed carefully with PBS.

Fourth, experimental results and discussion

1. Characterization of DNA-C18 and 5CB droplets of DNA amphiphiles

The inventors emulsified 5CB using the DNA amphiphilic DNA-C18. The amphiphilic DNA-C18 consists of an 18-carbon alkyl chain as a hydrophobic end and an 18-base DNA strand as a hydrophilic end. The mass spectrometry results are shown in Fig. 2. As can be seen from the mass spectrum, there are two peaks with molecular weights of 5632 and 5964, the peak at 5632 corresponds to the molecular weight of 18 bases, and 5964 corresponds to the total molecular weight of C18-DNA. Explain that the parental DNA-C18 was successfully synthesized, but in DNA-C18 It is inevitable that a trace amount of 18 base DNA fragments will be present.

In order to verify the performance of the complementary pairing of DNA by the prepared 5CB droplets, the inventors added a linker formed of R1 and R2 complementary to DNA-C18 into the 5CB droplet, and left it for about 30 seconds. Significant macroscopic aggregation occurs (see Figure 3).

Specifically, FIG. 3 shows a schematic diagram in which liquid crystal droplets are assembled with DNA-parent molecules on the surface thereof, and in the presence of R1 and R2, their complementary pairing causes aggregation of liquid crystal droplets. As shown in Fig. 3, (A) the liquid crystal droplets are uniformly dispersed in the macroscopic emulsion, (B) the liquid crystal droplets are aggregated after the addition of R1+R2linker, (C) the liquid crystal droplet emulsion microscope white light photograph, (D) A liquid crystal droplet emulsion microscope polarized photograph, (E) a microscope white light photograph after the liquid crystal droplets are aggregated, and (F) a microscope polarized photograph after the liquid crystal droplets are aggregated.

It can be seen from Fig. 3 that the DNA-C18 on the surface of the 5CB droplet has a base pairing with the linker, so that a large number of liquid crystal droplets are aggregated and settled, and it is further proved that the liquid crystal droplet prepared by the method has base complementary pairing. Performance.

2, patterned assembly of liquid crystal droplets

After the 12-mercaptododecanoic acid molecule was imprinted on the surface of Au using PDMS, the contact angle of the Au surface changed significantly. The results are shown in Fig. 4. 4A and 4B, 12-mercaptododecanoic acid was embossed on the surface of Au. At the same time, the inventors also directly modified the Au surface (16h) using 1μM SH-DNA, and the contact angle was also significantly changed compared with the Au surface, indicating that SH-DNA was also modified on the Au surface. Laid the foundation for subsequent experiments.

Here, in Fig. 4, the graphs A, B, and C are photographs of the Au surface, the gold surface after imprinting 12-decyldodecaic acid, and the contact angle of the gold surface after modifying the SH-DNA, respectively.

The inventors chose SH-NC18, which was not complementary to DNA-C18, to also modify Au as a control group for the experiment. The embossing of 12-mercaptododecanoic acid was chosen to avoid the non-specific adsorption of 5CB droplets on the Au surface, after imprinting 12-mercaptododecanoic acid, modifying SH-DNA and adsorbing 5CB droplets, and washing, Au surface was presented. The pattern on the PDMS stamp is shown in Figure 5. It can be seen from Fig. 5 that the 5CB droplets which are imprinted with 12-mercaptododecanoic acid have almost no adsorption, and a large amount of droplets can be adsorbed in the region where SH-C18 which is complementary to DNA-C18 is modified, and In contrast, in the region where SH-NC18 which is not complementary to DNA-C18 is modified, as in the region of 12-mercaptododecanoic acid, adsorption of droplets hardly occurs, indicating that 5CB droplets rely on DNA complementary pairing. The principle-specific adsorption is in the pattern area. After heating the pattern, 5CB loses the liquid crystal phase and becomes dark under polarized light. Such pattern visualization using optical forms of liquid crystals has potential applications in display and surface patterning.

As shown in Fig. 5, the Au surface is polarized by a pattern showing the adsorption of 5CB droplets, the SH-C18 is modified by the figures (A) and (C), and the SH-NC18 is modified by the figures (B) and (D). .

V. Conclusion

The inventors succeeded in using the microcontact printing technology to realize the patterning modification of 12-mercaptododecanoic acid on a gold substrate, and then modifying the thiol DNA on a bare gold substrate, and the complementary DNA modified on the surface of the liquid crystal droplet can be complementary to the substrate. The thiol DNA, thereby showing the pattern on the substrate. This method is not only suitable for a variety of liquid crystals, but also can be used to complete the response to the detector using DNA designability. The introduction of responsive elements will make the system more versatile, such as azobenzene. At the same time, the realization of the visualization of the substrate pattern and the selective display of the substrate information, the combination of the patterned substrate and the liquid crystal material provides a new idea and application prospect for the preparation of the portable nanometer detection device.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims (10)

1. A method of preparing a DNA pattern substrate, comprising the steps of:

   Providing a substrate, a patterned PDMS stamp, a 12-mercaptododecanoic acid solution, and a DNA capable of binding to the surface of the substrate;

   Drawing the surface of the PDMS stamp having the pattern on the surface to extract the 12-mercaptododecanoic acid solution to obtain a processed PDMS stamp;

   Passing the treated PDMS stamp to the surface of the substrate, and then removing the stamp to obtain a hydrophilic substrate having a pattern on the surface;

   A hydrophilic substrate having a pattern on the surface is brought into contact with the DNA capable of binding to the surface of the substrate to obtain the DNA pattern substrate having a DNA surface layer.
2. The method of claim 1 wherein the substrate is a gold substrate, a glass substrate or a silicon substrate.

   Optionally, the substrate is a gold substrate, and the DNA capable of binding to the surface of the substrate is sulfhydryl DNA,

   Optionally, the thiol DNA is modified with at least one selected from the group consisting of a thiol group, an ethynyl group, an amino group, an alkyl chain, or biotin.
3. The method according to claim 1, wherein the nucleic acid sequence of the thiol DNA is: TACACATCTACTTCACCA.
4. The method of claim 1 wherein said thiol DNA is pretreated.

   Optionally, the pretreatment is a disulfide bond reduction reaction of the sulfhydryl DNA using dithiothreitol,

   Optionally, the 12-mercaptododecanoic acid solution is an ethanol solution of 12-mercaptododecanoic acid,

   Optionally, contacting the treated PDMS stamp with the surface of the substrate for 20-40 seconds, preferably 30 seconds,

   Optionally, contacting the hydrophilic substrate having a pattern on the surface with the sulfhydryl DNA by immersing the hydrophilic substrate having the pattern on the surface in the PBS solution of the thiol DNA for 8-24 hours, preferably 16 Realized in hours.
5. A DNA pattern substrate obtained by the method of any one of claims 1-4.
6. A DNA liquid crystal droplet obtained by the following method:

   Mixing the DNA amphiphile with the liquid crystal material and emulsifying to obtain the DNA liquid crystal droplets,

   Wherein the DNA amphiphilic molecule is complementary to the thiol DNA of the DNA surface layer of the DNA pattern substrate of claim 5 at least in a sequence of 12 bp.
7. The DNA liquid crystal droplet according to claim 6, wherein the DNA amphiphilic molecule is DNA-C18, the hydrophilic end of which is a DNA strand end, and the hydrophobic end of which is a C18 chain end.

   Optionally, the nucleic acid sequence of the DNA amphiphilic molecule is TGGTGAAGTAGATGTGTA.
8. The DNA liquid crystal droplet according to claim 6, wherein the liquid crystal material is 5CB liquid crystal, TL205 liquid crystal or E7 liquid crystal, preferably 5CB liquid crystal.
9. A method for preparing a visible pattern under polarized light, comprising the steps of:

   Obtaining a PDMS stamp having a target pattern on the surface;

   A method according to any one of claims 1 to 4, wherein a DNA pattern substrate is prepared using a PDMS stamp having a target pattern on the surface;

   The DNA liquid crystal droplet according to any one of claims 6 to 8, wherein the DNA amphiphilic molecule is complementary to a thiol DNA of a DNA surface layer of the DNA pattern substrate by at least 12 bp;

   Contacting the DNA liquid crystal droplets with a DNA surface layer of the DNA pattern substrate to obtain a DNA pattern substrate that adsorbs liquid crystal droplets;

   When the DNA pattern substrate that adsorbs the liquid crystal droplets is placed at room temperature, the surface of the DNA pattern substrate can exhibit the pattern under polarized light.
10. The method according to claim 9, wherein the DNA liquid crystal droplets are previously diluted with PBS buffer before contacting the DNA liquid crystal droplets with the DNA surface layer of the DNA pattern substrate.

    Optionally, the DNA liquid crystal droplets are diluted 10-fold with PBS buffer,

    Optionally, the DNA liquid crystal droplets are contacted with the DNA surface layer of the DNA pattern substrate for 3-8 minutes, preferably 5 minutes.

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