A capillary monolithic column modified with DNAzyme nanoflowers, its preparation method, and application in separating chiral molecules

Abstract

The present invention discloses a capillary monolithic column modified with a deoxyribozyme nanoflower, a preparation method thereof, and an application thereof in separating chiral molecules, belonging to the field of pharmaceutical analysis. The present invention synthesizes a deoxyribozyme-based nanoflower chiral selector, the nucleotide sequence of the single-stranded DNA template of the deoxyribozyme being shown in SEQ ID NO.1, and the synthesized deoxyribozyme nanoflower having good pH and temperature stability. The prepared deoxyribozyme nanoflower is bonded to a graphene oxide-functionalized capillary silica monolithic column, and chiral molecules are separated under electrophoretic conditions, achieving baseline separation of 2'-deoxyadenosine, atenolol, propranolol, tyrosine, and nafopam enantiomers.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to a capillary monolithic column modified with deoxyribozyme nanoflowers, a preparation method thereof, and an application thereof in separating chiral molecules. Background Art

[0002] During clinical medication, the enantiomers of chiral drugs have different absorption, distribution, and metabolic pathways in the human body, and the two enantiomers exhibit different pharmacological and toxicological effects. Common chiral selectors include cyclodextrins and their derivatives, chiral ionic liquids, chiral crown ethers, etc. The wide variety of enzyme molecules can circumvent the tedious reaction steps required for other chiral selectors to undergo derivatization, creating a microenvironment for the interaction between chiral drugs and enzyme molecules, which is more conducive to exploring the chiral recognition mechanism of drugs and enzymes at the molecular level. However, the immobilization efficiency and enzyme activity of traditional proteases are easily affected by various factors in the microenvironment, making the preparation of enzyme-modified chiral stationary phases with superior performance quite difficult.

[0003] Deoxyribozyme (DNAzyme) nanoflowers are highly stable chiral selectors. Immobilizing them within capillary columns can produce superior chiral chromatographic columns. Capillary electrophoresis technology demonstrates its unique advantages in applications in pharmacy, biology, medicine, and environmental science. Compared to traditional gel electrophoresis, high-performance capillary electrophoresis (HPCE), due to its use of thin inner diameter capillaries, can operate at high voltages without generating thermal effects. Compared to high-performance liquid chromatography (HPLC), HPCE offers the advantages of high efficiency and rapid analysis. However, there are few reports on the application of DNAzymes for chiral separations using capillary electrophoresis. Summary of the Invention

[0004] The present invention aims to expand the scope of currently available chiral selectors by providing a capillary monolithic column modified with DNAzyme nanoflowers, a preparation method thereof, and its application in separating chiral molecules. DNAzyme nanoflowers were selected as chiral selectors, and their stability was investigated. The resulting DNAzyme nanoflower-modified capillary column, bonded to a graphene oxide-modified capillary monolithic column, was applied to the separation of chiral molecules, achieving baseline separation of 2'-deoxyadenosine, atenolol, propranolol, tyrosine, and nafopam.

[0005] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows:

[0006] A capillary monolith modified with a DNAzyme nanoflower, comprising an amino-modified DNAzyme nanoflower bonded to a graphene oxide-modified silica monolith; wherein the DNAzyme nanoflower is a DNAzyme nanoflower structure formed by rolling circle amplification of a single-stranded DNA template and a primer closed into a circular shape under the action of DNA ligase;

[0007] The single-stranded DNA template is a single-stranded DNA with 5'-end phosphorylation modification, and its nucleotide sequence is: 5'-Phosphate-CAC AAC CAC CAC CAC CAA ATC GTC CAG GTC GTT GTA GCT AGC CTG GCC GCG GAA CCGGAG CGG TCG TTG TAG CTA GCC TGG CCGAGGAAC CAC CAC CAC-3';

[0008] The primer is a primer chain with a 5'-terminal amino modification, and its nucleotide sequence is: 5'-NH2-GTG GTG GTTGTG GTG GTG GTG GTT-3'.

[0009] The present invention further provides a method for preparing the aforementioned DNAzyme nanoflowers, comprising the following synthesis steps: mixing a template strand complementary to the DNA sequence and the DNAzyme and an amino-modified primer strand in 10× DNA ligation buffer, heating the strands (generally at 95°C for 5 minutes), and then slowly cooling the strands to room temperature to obtain an annealed product; ligating the annealed products using T4 DNA ligase, and reacting the strands at room temperature (generally for 4 hours) to obtain a circularized DNA template. The circularized DNA template is then incubated with Phi29 DNA polymerase, dNTPs, and BSA at 30°C for 3 hours, terminated by heating at 75°C for 10 minutes, and precipitated by centrifugation. The pellet is then rinsed with deionized water, and liquid crystallized to obtain the amino-modified DNAzyme nanoflowers.

[0010] In some exemplary embodiments of the present invention, the stability of the prepared DNAzyme nanoflowers was evaluated using agarose gel electrophoresis and dynamic light scattering. The stability of the DNAzyme nanoflowers was evaluated using agarose gel electrophoresis after exposure to solutions of different pH values ​​for 1 hour, 8 hours, 16 hours, 24 hours, 48 ​​hours, and 72 hours. The effects of temperature and urea on the stability of the hydrated particle size of the DNAzyme nanoflowers were also investigated. To evaluate the thermal stability of the DNAzyme nanoflowers, the hydrated particle size of the DNAzyme nanoflowers was measured after heat treatment at 50°C and 100°C.

[0011] The DNAzyme nanoflower chiral selectors prepared by the present invention exhibit high stability. Compared to traditional protease chiral selectors, DNAzyme is resistant to high temperatures and nuclease treatment. Furthermore, DNAzyme can be directly prepared into nanomaterials, resulting in a larger specific surface area, which increases its interaction with chiral molecules, thereby improving its chiral separation performance. Therefore, the application of DNAzyme nanoflowers in chiral separations offers unique advantages.

[0012] The present invention further provides a method for preparing the above-mentioned DNAzyme nanoflower-modified capillary monolithic column, which comprises the following steps: first, pretreating the capillary with alkali and acid, mixing polyethylene glycol, urea, acetic acid and tetramethoxysilane, and stirring in an ice bath to form a sol; ultrasonically degassing the sol formed in the previous step, injecting it into the pretreated capillary column, capping it, placing it in a 40°C water bath, reacting it for 40 hours, taking it out, heating it at 120°C for 3 hours, leaving it for one week, and then raising the temperature to 320°C and calcining it for 24 hours to obtain a capillary silica monolithic column. A 10% toluene solution of an amino-containing silane coupling agent (e.g., 3-(aminopropyl)triethoxysilane) was injected into an acidified silica monolith. After end-capping, the reaction was carried out at 110°C for 6 hours. The column was rinsed with toluene and anhydrous methanol to remove impurities, resulting in an amino-modified silica monolith. An aqueous solution of graphene oxide was ultrasonically dispersed, diluted with potassium hydroxide solution (the mass ratio of graphene oxide to potassium hydroxide was 1:2), injected into the amino-treated silica monolith. After end-capping, the reaction was carried out at 40°C for 2 hours. The column was rinsed with water until neutral, and then rinsed with anhydrous methanol to remove impurities, resulting in a graphene oxide-modified capillary silica monolith. The graphene oxide-modified silica monolith was functionalized with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (at a concentration of at least 20 mmol / L). A solution of amino-modified DNAzyme nanoflowers was then injected into the monolith (reaction time of at least 5 hours). Unreacted nanoflowers were then rinsed with buffer to obtain a DNAzyme-modified capillary silica monolith, which was then stored in electrophoresis buffer.

[0013] The present invention also provides an application of the DNAzyme nanoflower modified capillary monolithic column, namely, for capillary electrochromatographic chiral separation.

[0014] The method involves dissolving a chiral molecule in electrophoresis buffer to prepare a sample solution. After equilibration with electrophoresis buffer (typically for approximately 30 minutes), the DNAzyme nanoflower-modified capillary monolithic column is injected with the sample. Electrophoretic separation conditions are 10kV injection for 3 seconds, a separation voltage of 10kV, and a buffer concentration of 20mM. Under these conditions, the DNAzyme nanoflower-modified capillary monolithic column can achieve baseline separation for chiral molecules including 2'-deoxyadenosine, atenolol, propranolol, tyrosine, and nafopam.

[0015] The present invention provides a novel DNAzyme nanoflower, and uses the DNAzyme nanoflower as a chiral selector, combined with capillary electrophoresis technology, to construct a novel capillary electrochromatography chiral separation system, realizing the separation of multiple chiral drugs, and further expanding the application of DNAzyme as a chiral selector in the field of capillary electrophoresis chiral separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The hydrated particle size of the DNAzyme nanoflower synthesized in Example 1 of the present invention;

[0017] Figure 2 This is an agarose gel electrophoresis image of the DNAzyme nanoflower synthesized in Example 1 of the present invention;

[0018] Figure 3 Effects of different pH buffers on the stability of DNAzyme nanoflowers; (a) 1h; (b) 8h; (c) 16h; (d) 24h; (e) 48h; (f) 72h;

[0019] Figure 4 The effect of buffer pH on the hydrated particle size of DNAzyme nanoflowers; (a) pH 5; (b) pH 7; (c) pH 9;

[0020] Figure 5 The effect of temperature on the stability of hydrated particle size of DNAzyme nanoflowers; among them, (a) 25℃; (b) 50℃; (c) 100℃.

[0021] Figure 6 The effect of urea on the stability of DNAzyme nanoparticle size; (a) without urea treatment; (b) 5M urea treatment for 10 min;

[0022] Figure 7 This is the electrochromatogram of chiral separation of 2'-deoxyadenosine using DNAzyme nanoflower modified capillary monolithic column;

[0023] Figure 8 This is the electrochromatogram of chiral separation of atenolol using DNAzyme nanoflower modified capillary monolithic column;

[0024] Figure 9 This is the electrochromatogram of the chiral separation of propranolol using a DNAzyme nanoflower-modified capillary monolithic column;

[0025] Figure 10 This is the electrochromatogram of chiral separation of tyrosine using DNAzyme nanoflower modified capillary monolithic column;

[0026] Figure 11 This is the electrochromatogram of chiral separation of nafopamine on a DNAzyme nanoflower-modified capillary monolithic column;

[0027] Figure 12 Molecular docking prediction of the interaction mechanism between naphthopam enantiomers and DNA sequences, (A) Binding mode of R-naphthopam with DNA; (B) Binding mode of S-naphthopam with DNA, the dotted line represents π-π conjugation. DETAILED DESCRIPTION

[0028] The present invention will be further described with reference to the following examples. However, it should be understood that these examples are for illustration only and should not be construed as limiting the present invention.

[0029] Example 1

[0030] The DNAzyme template chain (10 μM, 6 μL) and the amino-modified primer chain (10 μM, 12 μL) were mixed in 10×T4 DNA ligase buffer, heated at 95°C for 5 minutes, and then slowly cooled to room temperature to obtain an annealed product; T4 DNA ligase was added and reacted at room temperature for 4 hours to obtain a circularized DNAzyme template. The circularized DNAzyme 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 DNAzyme nanoflowers were washed with deionized water and obtained by liquid crystallization to obtain amino-modified DNAzyme nanoflowers. The particle size of the prepared DNAzyme nanoflowers was analyzed using a laser particle size analyzer ( Figure 1 Since DNAzyme nanoflowers have a large molecular weight, their electrophoretic migration rate is close to zero. The template and primer have different chain lengths, and they also show different migration rates in agarose gel electrophoresis, which can achieve separation ( Figure 2 ). The above results demonstrate the successful synthesis of DNAzyme nanoflowers.

[0031] Example 2

[0032] The DNAzyme nanoflowers prepared according to the method of Example 1 were further investigated for their stability. The DNAzyme nanoflowers were treated with solutions of different pH values ​​(5-9) for a certain period of time to investigate the stability of the DNA in the DNAzyme nanoflowers. The stability of the DNAzyme nanoflowers was evaluated by agarose gel electrophoresis after the DNAzyme nanoflowers were treated with solutions of different pH values ​​for 1h, 8h, 16h, 24h, 48h and 72h. Figure 3 , (a) 1h; (b) 8h; (c) 16h; (d) 24h; (e) 48h; (f) 72h. After 72h of treatment with different pH solutions, there was no band breakage in the agarose gel electrophoresis diagram of DNAzyme nanoflowers, indicating that the DNA in the DNAzyme nanoflowers was still relatively stable. Dynamic light scattering was further used to evaluate the stability of DNAzyme nanoflowers. After treatment with solutions of different pH values, the hydrated particle size of DNAzyme nanoflowers did not change significantly, indicating that DNAzyme nanoflowers are relatively stable ( Figure 4At the same time, the effects of temperature and urea on the stability of the hydrated particle size of DNAzyme nanoflowers were also investigated. In order to evaluate the thermal stability of DNAzyme nanoflowers, the hydrated particle size of DNAzyme nanoflowers was measured after heat treatment at 50°C and 100°C. By comparison, it was found that DNAzyme nanoflowers have better thermal stability ( Figure 5 In addition, the DNAzyme nanoflowers were treated with 5M urea to measure their hydrated particle size, which showed good stability ( Figure 6 ).

[0033] Example 3

[0034] DNAzyme nanoflowers were prepared according to the method of Example 1 and further used to prepare a capillary silica monolith modified with DNAzyme nanoflowers. 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 rinsed with 0.1 mol / L hydrochloric acid for 45 minutes. The capillary was rinsed with pure water and anhydrous methanol for 30 minutes, and nitrogen was blown for 1 hour for standby 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 and cap it after 15 minutes. After 40 hours in a 40°C water bath, heat in an oven at 120°C for 3 hours, place at room temperature for one week, and then heat in a column oven at 320°C for 24 hours to obtain a silica monolith. The silica monolith was perfused with 1 mol / L hydrochloric acid 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 for 1 hour for end-capping. After reacting at 110°C for 6 hours, the column was 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 20 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride phosphate solution was injected into the graphene oxide-modified silica monolith for 1 hour, and then the DNAzyme nanoflower solution was injected and reacted for 5 hours. The unreacted DNAzyme nanoflowers were washed with buffer to obtain a DNAzyme nanoflower-modified capillary silica monolith, which was stored in electrophoresis buffer for later use.

[0035] Example 4

[0036] According to the method of Example 3, the prepared DNAzyme nanoflower-modified capillary silica monolithic column was used to separate the 2'-deoxyadenosine enantiomers. 20 mM phosphate buffer was prepared and the pH value was adjusted to 7.4. Purchased 2'-deoxyadenosine (McLean (Shanghai, China)) was dissolved in electrophoresis buffer and a 1 mg / mL sample solution was prepared at room temperature. Figure 7 As shown in the figure, the separation system has good chiral separation efficiency for 2'-deoxyadenosine.

[0037] Example 5

[0038] According to the method of Example 3, the prepared DNAzyme nanoflower-modified capillary silica gel monolithic column was used to separate the enantiomers of the rolol class of drugs. The rolol class of drugs belongs to β-adrenergic receptor blockers and can be used clinically to treat hypertension, angina pectoris, myocardial infarction and other diseases. Prepare 20mM phosphate buffer and adjust the pH value to 7.4. Dissolve the purchased atenolol and propranolol (McLean (Shanghai, China)) in the electrophoresis buffer and prepare a 1mg / mL sample solution at room temperature. Figure 8 As shown in Figure 9, the separation system has good chiral separation efficiency for both atenolol and propranolol.

[0039] Example 6

[0040] According to the method of Example 3, the prepared DNAzyme nanoflower-modified capillary silica gel monolithic column was used to separate tyrosine enantiomers. Tyrosine is the raw material of amino acid infusion and amino acid compound preparation. It is used as a nutritional supplement to treat poliomyelitis and hyperthyroidism. Prepare 20mM phosphate buffer and adjust the pH value to 7.4. Dissolve the purchased tyrosine (McLean (Shanghai, China)) in electrophoresis buffer and prepare a 1mg / mL sample solution at room temperature. Figure 10 As shown in the figure, the separation system has good chiral separation efficiency for tyrosine.

[0041] Example 7

[0042] According to the method of Example 3, the prepared DNAzyme nanoflower-modified capillary silica gel monolithic column was used to separate the enantiomers of nafopamine. As a non-addictive analgesic, nafopamine is commonly used for postoperative analgesia, cancer pain, and the treatment of acute traumatic pain. It is also suitable for visceral smooth muscle colic such as acute gastritis, biliary ascariasis, and ureteral stones. Prepare 20mM phosphate buffer and adjust the pH value to 7.4. Dissolve the purchased nafopamine (McLean (Shanghai, China)) in electrophoresis buffer and prepare a 1mg / mL sample solution at room temperature. Figure 11 As shown in the figure, the separation system has good chiral separation efficiency for nafopam.

[0043] Example 8

[0044] Using nafopam 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 both R-naphthopam and S-naphthopam interact with the pocket formed by DT-12, DT-11, DT-15, DG-18, and DC-19 on the DNA structure. The drug molecules form π-π conjugations with DT-15 and DG-18, indicating that DT-15 and DG-18 are key deoxyribonucleic acids. This indicates that the two small molecules bind to the same DNA site and exhibit similar interactions. The docking software assigns DNA binding affinity scores of -6.5 and -6.6 kcal / mol for R-naphthopam and S-naphthopam, respectively. Therefore, the strength of the interactions between R-naphthopam and S-naphthopam and DNA provides a foundation for the separation of naphthopam enantiomers by DNA.

[0045] It can be seen from the above examples that the capillary electrochromatography technology based on the DNAzyme nanoflower modified monolithic column provides a fast and efficient analytical platform for the separation of enantiomers.

[0046] 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.

Claims

1. Application of a capillary monolithic column modified with DNAzyme nanoflowers in capillary electrochromatographic separation of chiral molecules, characterized in that: The deoxyribozyme nanoflower-modified capillary monolithic column is an amino-containing deoxyribozyme nanoflower bonded to a graphene oxide-modified silica monolithic column. The deoxyribozyme nanoflower is a deoxyribozyme nanoflower structure formed by rolling circle amplification of a circular DNA template formed by closing a single-stranded DNA template and a primer under the action of DNA ligase. The nucleotide sequence of the single-stranded DNA template is: 5'-Phosphate-CAC AAC CAC CAC CAC CAC CAA ATC GTC CAG GTC GTT GTAGCT AGC CTG GCC GCG GAA CCG GAG CGG TCG TTG TAG CTA GCC TGG CCG AGG AAC CACCAC CAC-3'; the nucleotide sequence of the primer is: 5'-NH2-GTG GTG GTT GTG GTG GTG GTG GTT-3'; and the chiral molecule is one of 2'-deoxyadenosine, atenolol, propranolol, tyrosine and nafopam.

2. The use according to claim 1, characterized in that The deoxyribozyme nanoflower is prepared by the following method, which specifically includes the following steps: Step (1) mixing a template strand having a DNA sequence complementary to the deoxyribozyme and an amino-modified primer strand in a 10× DNA ligase buffer, heating the strands for reaction, and then slowly cooling the strands to room temperature to obtain an annealed product, ligating the annealed product using T4 DNA ligase, and reacting the annealed product at room temperature to obtain a circularized DNA template; Step (2) The circularized DNA template was mixed 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, and the pellet was precipitated by centrifugation and rinsed with deionized water to obtain DNAzyme nanoflowers.

3. The use according to claim 1, characterized in that The method for preparing the capillary monolithic column modified with DNAzyme nanoflowers specifically comprises the following steps: Step 1: 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 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, then removed and heated at 120°C for 3 hours. The column is then left for 7 days and then programmed to 320°C for calcination for 24 hours to obtain a capillary silica monolithic column. Step 2: injecting a toluene solution of an amino-containing silane coupling agent into an acidified silica monolith containing silanol functional groups, reacting at 110°C for 6 hours after end-capping, and rinsing to remove impurities to obtain an amino-modified silica monolith; ultrasonically dispersing an aqueous solution of graphene oxide and then diluting it with a potassium hydroxide solution, with a mass ratio of graphene oxide to potassium hydroxide of 1:2, injecting it into the amino-modified silica monolith, reacting at 40°C for 2 hours after end-capping, and then rinsing with water until neutral, and then rinsing with anhydrous methanol to remove impurities to obtain a graphene oxide-modified capillary silica monolith; Step 3: Use 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to functionalize the graphene oxide modified silica monolithic column, pour the solution containing the deoxyribozyme nanoflowers into the monolithic column for sufficient reaction, and rinse with buffer to remove the unreacted deoxyribozyme to obtain a deoxyribozyme nanoflower modified capillary monolithic column.

4. The use according to claim 3, characterized in that The molar concentration of the deoxyribozyme nanoflower solution is 0.001-1 μmol / L.

5. The use according to claim 1, characterized in that The electrophoretic separation conditions for capillary electrochromatographic chiral separation were as follows: 10 kV injection for 3 s; separation voltage of 10 kV; and buffer concentration of 20 mmol / L.

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