Chiral capillary electrochromatography based on DNA nanoflower-modified silica monolithic column and its application

Abstract

The present invention discloses a chiral capillary electrochromatography system based on a DNA nanoflower-modified silica monolithic column and its application, belonging to the field of pharmaceutical analysis. The system uses DNA nanoflowers as chiral selectors, immobilized on a graphene oxide-functionalized capillary silica monolithic column, to separate chiral drugs under electrophoretic conditions. This system can achieve baseline separation of nafopam, propranolol, atenolol, and tyrosine enantiomers. The separation method is simple to operate, establishing a novel, highly selective chiral separation and detection technology.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis, and in particular relates to a chiral capillary electrochromatography method based on a DNA nanoflower-modified silica gel monolithic column and an application thereof. Background Art

[0002] Chiral separation is of great practical significance because a pair of enantiomers exhibit distinct physiological effects and pharmacological toxicities. Among the numerous chiral separation methods, capillary electrochromatography (CEC) offers unique advantages. It combines the high selectivity of high-performance liquid chromatography with the efficiency of capillary electrophoresis and can rapidly separate a wide range of samples using microliters of reagents, making CEC more environmentally friendly and economical. As a rapid, efficient, and low-sample-consumption technique, CEC holds broad application prospects in chiral separations. The development of novel chiral selectors plays a crucial role in the construction of chiral CEC systems. Endogenous biomolecules, including DNA, proteins, and polysaccharides, serve as superior chiral selectors. DNA is a naturally self-assembling biomolecule. Studies have used DNA as a chiral donor to construct chiral sensors based on various nanomaterials. However, there are fewer reports on the application of DNA combined with nanomaterials in CEC chiral separations. Summary of the Invention

[0003] The present invention aims to expand the scope of currently available chiral selectors by providing a chiral capillary electrochromatography system based on a DNA nanoflower-modified silica monolithic column and its applications. DNA nanoflowers were selected as chiral selectors and bonded to a graphene oxide-modified capillary silica monolithic column. The resulting DNA nanoflower-modified capillary column was used for chiral separation, achieving baseline separation of tyrosine, propranolol, atenolol, and nafopam.

[0004] In order to solve the technical problems, the technical solutions adopted by the present invention are as follows:

[0005] A DNA nanoflower-modified silica monolithic column for capillary electrochromatographic chiral separation is a capillary silica monolithic column modified with graphene oxide modified by amino-modified DNA nanoflowers. The DNA nanoflowers have a flower-shaped structure.

[0006] Furthermore, the synthesis of amino-modified DNA nanoflowers includes the following steps: the DNA template chain is converted into a circular template through an amino-modified primer chain, and then rolling circle amplification is performed to obtain a long-chain DNA with multiple repetitive sequences, and the amino-modified DNA nanoflowers are obtained through liquid crystallization.

[0007] Furthermore, the circularized template is obtained by the following steps: the DNA template chain and the amino-modified primer chain are mixed in 10× DNA ligation buffer, heated for reaction (generally at 95°C for 5 minutes), and then slowly cooled to room temperature to obtain an annealed product; the annealed products are then ligated using T4 DNA ligase and reacted at room temperature (generally for 4 hours) to obtain a circularized DNA template.

[0008] Furthermore, the rolling circle amplification method includes: incubating the circularized DNA template with Phi29 DNA polymerase, dNTPs, and BSA at 30°C for 3 hours, terminating the reaction by heating at 75°C for 10 minutes, centrifuging and precipitating, and washing with deionized water to obtain amino-modified DNA nanoflowers.

[0009] The amino-modified DNA nanoflowers prepared by the present invention are formed by self-assembly of repetitive DNA sequences through liquid crystallization. Compared with traditional DNA nanomaterials, the synthesis of amino-modified DNA nanoflowers does not rely on complementary base pairing, thus avoiding the complex sequence design process. Furthermore, DNA nanoflowers are highly stable and can withstand high temperatures and nuclease treatment. Therefore, the application of DNA nanoflowers in CEC chiral separation has unique advantages.

[0010] Furthermore, the capillary silica monolithic column is prepared as follows: first, the capillary is pretreated with alkali and acid. Polyethylene glycol, urea, acetic acid, and tetramethoxysilane are mixed and stirred in an ice bath to form a sol. The sol formed in the previous step is ultrasonically degassed and injected into the pretreated capillary column. After end-capping, the column is placed in a 40°C water bath for 40 hours, removed, heated at 120°C for 3 hours, allowed to stand for one week, and then programmed to 320°C for calcination for 24 hours to obtain the capillary silica monolithic column. The capillary silica monolithic column used in this patent provides abundant silanol functional groups, facilitating the introduction of further reactive groups for bonding to DNA nanoflowers. Other commonly used capillary silica monolithic columns, such as capillary silica hybrid monolithic columns, contain different reactive groups suitable for different reactions. Moreover, hybrid monolithic columns are generally prepared using a one-pot process, which limits their tunability. In addition to selecting an appropriate chiral selector, the support used to immobilize the biomolecule is also a crucial factor in the construction of a chiral separation system. The support here refers to a silica monolithic column. Compared with capillary open tubular columns and packed columns, silica monolithic columns have a higher specific surface area for the immobilization of biomolecules, their porous structure is suitable for the separation of small molecules, and their high stability is conducive to the multi-step bonding reaction.

[0011] Furthermore, a graphene oxide-modified capillary silica monolithic column is obtained by the following steps: injecting a toluene solution (mass concentration generally 10%) of an amino-containing silane coupling agent (such as 3-(aminopropyl)triethoxysilane) into the acidified silica monolithic column, reacting at 110°C for 6 hours after end-capping, and rinsing with toluene and anhydrous methanol to remove impurities to obtain an amino-modified silica monolithic column; ultrasonically dispersing the graphene oxide aqueous solution and then diluting it with a potassium hydroxide solution (the mass ratio of graphene oxide to potassium hydroxide is 1:2), injecting it into the amino-treated silica monolithic column, reacting at 40°C for 2 hours after end-capping, rinsing with water to neutrality, and rinsing with anhydrous methanol to remove impurities to obtain a graphene oxide-modified capillary silica monolithic column.

[0012] A method for modifying a graphene oxide-modified capillary silica monolith with aminated DNA nanoflowers comprises the following steps: functionalizing a graphene oxide-modified silica monolith using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, then injecting an aminated DNA nanoflower solution into the monolith to allow for sufficient reaction (further, injecting a phosphate solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a concentration of at least 20 mmol / L into the graphene oxide-modified silica monolith for at least 1 hour, then injecting the DNA nanoflower solution and allowing the reaction to proceed for at least 5 hours), then rinsing with a buffer solution to remove unreacted DNA, thereby obtaining an aminated DNA-modified capillary silica monolith, which is then stored in an electrophoresis buffer.

[0013] The sequences of the DNA single strands are:

[0014] Template strand: 5'-Phosphate-CAC AAC CAC CAC CAC CAA ATC GTC CAG GTC GTT GTAGCT AGC CTG GCC GCG GAA CAG TAC CAG TCG TTG TAG CTA GCC TCA CGG CTG AAC CACCAC CAC-3'

[0015] Primer strand: 5'-NH2-GTG GTG GTT GTG GTG GTG GTG GTT-3'

[0016] Template complementary strand: 5'-NH2-GTG GTG GTG GTT CAG CCG TGA GGC TAG CTA CAA CGA CTGGTA CTG TTC CGC GGC CAG GCT AGC TAC AAC GAC CTG GAC GAT TTG GTG GTG GTG GTTGTG-3'

[0017] The present invention also provides an application of the DNA nanoflower modified silica gel monolithic column, namely, for capillary electrochromatographic chiral separation.

[0018] Furthermore, the method includes the following steps: dissolving the chiral molecule in electrophoresis buffer to prepare a sample solution; injecting the sample after the DNA nanoflower-modified silica gel monolithic column is equilibrated with electrophoresis buffer (generally about 30 minutes), and the electrophoresis separation conditions are 10kV injection 1-6s, separation voltage 5-15kV, and buffer concentration 10-30mM.

[0019] Further optimized separation conditions were: injection volume of 10 kV for 3 s, capillary electrophoresis separation voltage of 10 kV, and buffer concentration of 20 mM. Under these conditions, the DNA nanoflower-modified capillary silica monolithic column strongly interacted with the nafopamine enantiomers, achieving good chiral recognition.

[0020] Furthermore, the chiral molecule is one of tyrosine, atenolol, nafopam, and propranolol.

[0021] DNA nanoflowers have high stability. The large number of DCT units in them cooperate with the nano-effect of DNA nanoflowers, increasing the interaction between chiral drugs and them, thereby increasing the chiral separation efficiency.

[0022] This invention combines the advantages of DNA nanoflowers and capillary silica monoliths to construct a novel CEC chiral separation system. By employing the aforementioned technical solution, the present invention achieves beneficial results: the chiral separation method using a DNA nanoflower-modified capillary silica monolith further expands the application of DNA nanomaterials as chiral selectors in capillary electrochromatography chiral separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Scanning electron microscopy image of DNA nanoflower.

[0024] Figure 2 : Hydration particle size of DNA nanoflowers.

[0025] Figure 3 : Agarose gel electrophoresis of DNA nanoflowers.

[0026] Figure 4 : Scanning electron microscopy image of the cross section of the capillary silica monolithic column.

[0027] Figure 5 : Scanning electron microscopy image of the cross section of the DNA nanoflower-modified capillary silica monolith.

[0028] Figure 6Effect of injection volume on the separation of nafopam enantiomers using a DNA nanoflower-modified capillary silica monolithic column. Injection volume: a, 10 kV, 1 s; b, 10 kV, 3 s; c, 10 kV, 6 s.

[0029] Figure 7 : Effect of separation voltage on the separation of nafopam enantiomers using a DNA nanoflower-modified capillary silica monolithic column. Separation voltages a, 15 kV; b, 10 kV; c, 5 kV.

[0030] Figure 8 Effect of buffer concentration on the separation of nafopam enantiomers using a DNA nanoflower-modified capillary silica monolithic column. Buffer concentrations a, 10 mM; b, 20 mM; c, 30 mM.

[0031] Figure 9 : Electrochromatograms of the separation of various chiral molecules using a DNA nanoflower-modified capillary silica monolithic column. (a) Tyrosine; (b) Atenolol; (c) Nafopam; (d) Propranolol.

[0032] Figure 10 : Electrochromatograms of the separation of various chiral molecules using a DCT-modified capillary silica monolithic column. (a) Tyrosine; (b) Atenolol; (c) Nafopam; (d) Propranolol.

[0033] Figure 11 : Electrochromatograms of the separation of various chiral molecules using a capillary silica monolithic column with unmodified DNA. (a) Tyrosine; (b) Atenolol; (c) Nafopam; (d) Propranolol.

[0034] Figure 12 Molecular docking predictions of the interaction mechanisms between naphthopam enantiomers and DNA sequences. (A) Binding mode of R-naphthopam with DNA; (B) Binding mode of S-naphthopam with DNA. (a) Dashed lines represent salt bridge interactions, (b) dotted lines represent hydrogen bonding interactions, and (c) dotted lines represent π-π conjugation interactions. DETAILED DESCRIPTION

[0035] The present invention is not limited to the following specific embodiments. Based on the disclosure of the present invention, a person skilled in the art may adopt a variety of other specific embodiments to implement the present invention. Any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other unless they conflict.

[0036] The present invention is further described in detail below in conjunction with the embodiments:

[0037] Example 1

[0038] 1. Preparation and Characterization of DNA Nanoflowers

[0039] The DNA template chain (10 μM, 6 μL) and the amino-modified primer chain (10 μM, 12 μL) were mixed in 10× DNA ligation buffer, heated at 95°C for 5 minutes, and then slowly cooled to room temperature. The annealed products were connected using T4 DNA ligase, and the reaction was carried out at room temperature for 4 hours to obtain a circularized DNA template. The circularized DNA template was incubated with Phi29 DNA polymerase, dNTPs and BSA at 30°C for 3 hours, and the reaction was terminated by heating at 75°C for 10 minutes. The obtained DNA nanoflowers were centrifuged and rinsed with deionized water. The prepared DNA nanoflowers were characterized by scanning electron microscopy, agarose gel electrophoresis and laser particle size analyzer. Figure 1 As shown in Figure 2, the prepared DNA nanoflower has a flower-shaped structure. At the same time, the particle size of the DNA nanoflower was analyzed and its particle size was about 500nm ( Figure 2 During the formation of DNA nanoflowers, the template chain first forms a circular template for rolling circle amplification to obtain a long chain DNA with multiple repeating sequences, and then the DNA nanoflowers are obtained by liquid crystallization. Since DNA nanoflowers have a large molecular weight, their electrophoretic migration rate is close to zero ( Figure 3 The different chain lengths of the template and primers also show different migration rates in agarose gel electrophoresis, enabling separation. The above electrophoresis results demonstrate the successful synthesis of DNA nanoflowers.

[0040] 2. Preparation and Characterization of Capillary Silica Monolithic Column

[0041] The quartz capillary was rinsed with pure water for 15 minutes, 1 mol / L sodium hydroxide for 45 minutes, pure water for 1 hour, and then 0.1 mol / L hydrochloric acid for 45 minutes. The capillary was rinsed with pure water and anhydrous methanol for 30 minutes respectively, and nitrogen was blown for 1 hour for use. Weigh 0.22g of polyethylene glycol and 0.225g of urea, add 2.5mL of 0.01mol / L acetic acid, add 0.9mL of tetramethoxysilane, stir for 45 minutes and then slowly stop, inject into the pretreated capillary for 15 minutes, which means that after perfusion for 15 minutes, the end was capped, and the capillary was placed in a 40℃ water bath for 40 hours, and then heated in an oven at 120℃ for 3 hours. After being placed at room temperature for one week, the column oven was heated to 320℃ and calcined for 24 hours to obtain a silica gel monolithic column. The cross-sectional structure of the capillary silica gel monolithic column was characterized by scanning electron microscopy ( Figure 4 ).

[0042] Example 2

[0043] DNA nanoflowers and a capillary silica monolith were prepared according to the method of Example 1, and a capillary silica monolith modified with DNA nanoflowers was further prepared. 1 mol / L hydrochloric acid was poured into the silica monolith for 3 hours, rinsed with purified water until neutral, and then rinsed with anhydrous methanol for 30 minutes. A 10% (v / v) 3-(aminopropyl)triethoxysilane solution in toluene was then injected and rinsed for 1 hour. End-capping was performed, and the mixture was reacted at 110°C for 6 hours. The mixture was then rinsed with toluene and anhydrous methanol for 30 minutes each, and dried with nitrogen to obtain an amino-modified silica monolith. A 1 mg / mL aqueous dispersion of graphene oxide was diluted with potassium hydroxide solution to a graphene oxide concentration of 0.15 mg / mL. This was injected into the amino-modified silica monolith, reacted at 40°C for 2 hours, and rinsed with anhydrous methanol for 30 minutes to obtain a graphene oxide-modified silica monolith. A 20mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride phosphate solution was injected into the graphene oxide modified silica monolith for 1 hour, and then a DNA nanoflower solution was injected (the concentration of the injected DNA nanoflower solution is generally in the nM level, 1nM-1000nM, and the concentration change has little effect on the modification effect). The reaction was allowed to proceed for 5 hours, and the unreacted DNA nanoflowers were washed away with a buffer solution to obtain a DNA nanoflower modified capillary silica monolith, which was then stored in an electrophoresis buffer solution. The cross-sectional structure of the DNA nanoflower modified capillary silica monolith was characterized using a scanning electron microscope ( Figure 5 ).

[0044] Example 3

[0045] According to the method of Example 2, the prepared DNA nanoflower-modified capillary silica monolithic column was used for chiral separation. 20 mM phosphate buffer was prepared and the pH value was adjusted to 7.4. Purchased nafopam (McLean (Shanghai, China)), propranolol (McLean (Shanghai, China)), atenolol (McLean (Shanghai, China)) and tyrosine (McLean (Shanghai, China)) were dissolved in electrophoresis buffer and prepared into a 1 mg / mL sample solution at room temperature. The electrophoretic separation conditions, including the injection volume (10 kV, 1-6 s), separation voltage (5-15 kV), and electrophoresis buffer concentration (10-30 mM), were optimized to obtain the optimal electrophoresis conditions for separation of the nafopam enantiomers.

[0046] Example 4

[0047] According to the method of Example 3, the preferred value of the injection volume was confirmed. This example investigated the effect of the injection volume on the separation of nafopamine enantiomers using a DNA nanoflower-modified capillary silica monolithic column ( Figure 6Increasing the injection volume results in peak tailing and decreased column efficiency. The theoretical plate number for the electrophoretic patterns of naphthopan enantiomers decreases with increasing injection volume. Because higher sample volumes cause peak tailing and decreased column efficiency, the resolution of naphthopan enantiomers decreases with higher sample volumes. Ultimately, the optimal injection volume was determined to be 10 kV, 3 s.

[0048] Example 5

[0049] According to the method of Example 3, the preferred value of the operating voltage was confirmed. This example investigated the effect of the operating voltage on the separation of nafopamine enantiomers using a capillary silica monolith column modified with DNA nanoflowers. The separation voltages a, 15kV; b, 10kV; and c, 5kV. Figure 7 The optimized test results show that the retention time of nafopamine decreases with increasing voltage. A voltage of 15 kV yields higher column efficiency, but the weak interaction between the DNA nanoflower-modified capillary silica monolith and nafopamine results in reduced resolution. Taking into account the excellent chiral separation capability and high column efficiency, a voltage of 10 kV achieves the highest resolution.

[0050] Example 6

[0051] According to the method of Example 3, the preferred value of the buffer concentration was confirmed. This example investigated the effect of the buffer concentration on the separation of nafopamine enantiomers using a DNA nanoflower-modified capillary silica monolithic column ( Figure 8 ). Among them, the buffer concentrations a, 10mM; b, 20mM; c, 30mM. As the buffer concentration increases, the electroosmotic flow decreases, resulting in an increase in the retention of nafopamine. When the buffer concentration is 30mmol L -1 When the buffer concentration is lower than 10 mmol L -1 Therefore, the buffer concentration was selected to be 20 mmol L -1 is the optimal value, at which point baseline separation can be achieved and the peak shape is better.

[0052] Example 7

[0053] According to the method of Example 3, the prepared DNA nanoflower-modified capillary silica monolithic column was used to separate nafopam, propranolol, atenolol and tyrosine enantiomers. The optimal separation conditions were selected: electrophoresis separation voltage of 10 kV, buffer concentration of 20 mM, injection volume of 10 kV, 3 s. The baseline separation of the above four chiral molecules was achieved ( Figure 9 ).

[0054] Example 8

[0055] According to the method of Example 2, a graphene oxide modified capillary silica monolith column activated with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was prepared, and then a DNA chain (DCT) having a sequence complementary to the template chain was injected to react for 5 hours. The unreacted DNA was washed with a buffer solution to obtain a DCT modified capillary silica monolith column for chiral separation. 20 mM phosphate buffer was prepared and the pH value was adjusted to 7.4. The purchased nafopam (McLean (Shanghai, China)), propranolol (McLean (Shanghai, China)), atenolol (McLean (Shanghai, China)) and tyrosine (McLean (Shanghai, China)) were dissolved in electrophoresis buffer and prepared into a 1 mg / mL sample solution at room temperature. As Figure 10 As shown, the DCT-modified capillary silica monolithic column only partially separated the four chiral molecules mentioned above. However, the DNA nanoflower-modified capillary silica monolithic column in Example 7 achieved baseline separation of these four chiral molecules. This is presumably because the large number of DCT tandem repeats in the DNA nanoflowers enhances their interaction with chiral molecules, further improving the chiral separation performance of the DNA nanoflower-modified capillary silica monolithic column.

[0056] Because the formed nanoflowers are highly stable, if multiple nanoflowers are connected in series, a stable product cannot be obtained. Another advantage of DNA nanoflowers is their nano-effect. They have a high specific surface area, which can increase the interaction between chiral drugs and them, thereby increasing the efficiency of chiral separation.

[0057] The improvement in chirality is due to the combined effects of the nano-effect of the DNA nanoflower and the increased amount of DCT units in the DNA nanoflower. However, using only high concentrations of DCT does not achieve baseline separation.

[0058] Example 9

[0059] According to the method of Example 2, the prepared graphene oxide modified capillary silica monolithic column was applied to chiral separation. 20 mM phosphate buffer was prepared and the pH value was adjusted to 7.4. The purchased nafopam (McLean (Shanghai, China)), propranolol (McLean (Shanghai, China)), atenolol (McLean (Shanghai, China)) and tyrosine (McLean (Shanghai, China)) were dissolved in electrophoresis buffer and prepared into a 1 mg / mL sample solution at room temperature. Figure 11 As shown, the graphene oxide modified capillary silica monolithic column (unmodified with DNA) has no chiral separation efficiency for the above four chiral molecules ( Figure 11 ). Therefore, the chiral separation performance of the DNA nanoflower-modified capillary silica monolithic column can be attributed to the DNA nanoflowers.

[0060] Example 10

[0061] Docking simulation technology is a convenient and effective means to explore the interaction between small molecules and target sites. Using naphthopan as a template molecule, Vina 1.1.2 software was used to study the docking of its enantiomers with DNA structures ( Figure 12 The figure shows that the R-nefopam small molecule undergoes π-π conjugation and hydrogen bonding with DG-90 on the DNA structure, and also forms a salt bridge with DC-31. These interactions maintain the foundation for R-nefopam's binding to DNA. In contrast, S-nefopam only undergoes π-π conjugation with DG-90 and DG-91 on the DNA, resulting in a stronger binding force for R-nefopam. Therefore, the differences in the interactions between R-nefopam and S-nefopam and DNA pave the way for the separation of nefopam enantiomers by DNA.

[0062] It can be seen from the above examples that the capillary electrochromatography technology based on DNA nanoflower-modified silica monolithic column provides a fast and efficient analytical platform for the separation of enantiomers.

[0063] With the above-described preferred embodiments of the present invention as a guide, and with reference to the above description, relevant personnel are fully capable of making various changes and modifications without departing from the technical scope of this invention. The technical scope of this invention is not limited to the contents of the specification and must be determined according to the scope of the claims.

[0064] SEQ ID NO.1

[0065] Template strand nucleotide sequence

[0066] CAC AAC CAC CAC CAC CAA ATC GTC CAG GTC GTT GTAGCT AGC CTG GCC GCGGAACAG TAC CAG TCG TTG TAG CTA GCC TCA CGG CTG AAC CAC CAC CAC

[0067] SEQ ID NO.2

[0068] Primer strand nucleotide sequence

[0069] GTG GTG GTT GTG GTG GTG GTG GTT

[0070] SEQ ID NO.3

[0071] Template complementary chain nucleotide sequence

[0072] GTG GTG GTG GTT CAG CCG TGA GGC TAG CTA CAA CGA CTG GTA CTGTTC CGCGGC CAG GCT AGC TAC AAC GAC CTG GAC GAT TTG GTG GTGGTG GTT GTG

Claims

1. A chiral capillary electrochromatography based on a DNA nanoflower-modified silica monolithic column, characterized by: A capillary silica monolithic column modified with graphene oxide modified with amino-modified DNA nanoflowers, wherein the DNA nanoflowers have a flower-shaped structure; The circularized template is obtained by the following steps: mixing the DNA template strand and the amino-modified primer strand in 10× DNA ligation buffer, heating the reaction, and then slowly cooling to room temperature to obtain an annealed product; Then, T4 DNA ligase was used to connect the annealed products and the reaction was carried out at room temperature to obtain a circularized DNA template; The rolling circle amplification method includes: reacting the circularized DNA template with Phi29 DNA polymerase, dNTPs, and BSA at 30 o C for 3 h, and then incubated at 75 o The reaction was terminated by heating at C for 10 min, the precipitate was centrifuged, and the precipitate was washed with deionized water to obtain amino-modified DNA nanoflowers.

2. The chiral capillary electrochromatography based on DNA nanoflower modified silica monolithic column according to claim 1, characterized in that: The synthesis of amino-modified DNA nanoflowers includes the following steps: the DNA template chain is converted into a circular template through an amino-modified primer chain, and then rolling circle amplification is performed to obtain a long-chain DNA with multiple repeated sequences, and the amino-modified DNA nanoflowers are obtained through liquid crystallization.

3. The chiral capillary electrochromatography based on DNA nanoflower modified silica monolithic column according to claim 1, characterized in that: The graphene oxide modified capillary silica monolithic column is obtained by the following steps: injecting a toluene solution of an amino-containing silane coupling agent into an acidified silica monolithic column containing silanol functional groups, capping the ends and allowing the mixture to react fully, and then rinsing and removing impurities to obtain an amino-modified silica monolithic column; ultrasonically dispersing a graphene oxide aqueous solution, diluting it with a potassium hydroxide solution, injecting it into the amino-modified silica monolithic column, capping the ends and allowing the mixture to react fully, then rinsing with water to neutrality, and then rinsing with anhydrous methanol to remove impurities, thereby obtaining a graphene oxide modified capillary silica monolithic column.

4. The chiral capillary electrochromatography based on DNA nanoflower-modified silica monolithic column according to claim 3, characterized in that: In the preparation of amino-modified silica monolithic columns, the reaction conditions after end-capping were 110 °C for 6 hours; In the graphene oxide modification reaction, the reaction conditions after capping were 40 °C for 2 h; The preparation of a capillary silica monolithic column comprises: firstly pretreating a capillary with alkali and acid, mixing polyethylene glycol, urea, acetic acid and tetramethoxysilane, and stirring in an ice bath to form a sol; degassing the sol formed in the previous step by ultrasonic means, injecting the sol into the pretreated capillary column, capping the end of the capillary column, reacting the capillary column in a 40°C water bath for 40 hours, removing the capillary column from the water bath, heating the capillary column at 120°C for 3 hours, allowing the capillary column to stand for one week, and then heating the capillary column to 320°C and calcining the capillary silica monolithic column for 24 hours.

5. The chiral capillary electrochromatography based on DNA nanoflower modified silica monolithic column according to claim 1, characterized in that: The preparation method of a DNA nanoflower modified silica monolithic column comprises the following steps: The graphene oxide-modified silica monolith was functionalized using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and then the amino-modified DNA nanoflower solution was poured into the monolith to react fully. The unreacted DNA was then washed with buffer to remove the resulting amino-modified DNA capillary silica monolith, which was then stored in electrophoresis buffer.

6. The chiral capillary electrochromatography based on DNA nanoflower-modified silica monolithic column according to claim 5, characterized in that: The sequences of the DNA single strands are: Template strand: 5'-Phosphate-CAC AAC CAC CAC CAC CAA ATC GTC CAG GTC GTT GTA GCTAGC CTG GCC GCG GAA CAG TAC CAG TCG TTG TAG CTA GCC TCA CGG CTG AAC CAC CACCAC-3' Primer strand: 5'-NH2-GTG GTG GTT GTG GTG GTG GTG GTT-3' Template complementary strand: 5'-NH2-GTG GTG GTG GTT CAG CCG TGA GGC TAG CTA CAA CGA CTG GTACTG TTC CGC GGC CAG GCT AGC TAC AAC GAC CTG GAC GAT TTG GTG GTG GTG GTT GTG-3'.

7. The chiral capillary electrochromatography based on DNA nanoflower-modified silica monolithic column according to claim 5, characterized in that: The concentration of the phosphate solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is at least 20 mmol / L, the functionalization time is at least 1 hour, and the reaction time is at least 5 hours.

8. An application of chiral capillary electrochromatography based on a DNA nanoflower-modified silica monolithic column according to any one of claims 1 to 7, characterized in that: It is used for capillary electrochromatographic chiral separation, comprising the following steps: The chiral molecules were dissolved in electrophoresis buffer to prepare the sample solution. The DNA nanoflower-modified silica monolithic column was equilibrated with electrophoresis buffer and then injected. The electrophoresis separation conditions were 10 kV injection for 1-6 s, capillary electrophoresis separation voltage of 5-15 kV, and buffer concentration of 10-30 mM.

9. The application of chiral capillary electrochromatography based on DNA nanoflower-modified silica monolithic column according to claim 8, characterized in that: The electrophoretic separation conditions are as follows: injection at 10 kV for 3 s; capillary electrophoresis separation voltage of 10 kV; buffer concentration of 20 mM; and / or the chiral molecule is one of tyrosine, atenolol, nafopam, and propranolol.

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