CN114164442B

CN114164442B - Electrochemical preparation method and application of framework nucleic acid

Info

Publication number: CN114164442B
Application number: CN202111255391.8A
Authority: CN
Inventors: 沈建磊; 李茜; 翟婷婷; 孙晨蕴; 樊春海
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2023-07-14
Anticipated expiration: 2041-10-27
Also published as: CN114164442A

Abstract

The invention provides an electrochemical preparation method and application of framework nucleic acid, comprising the following steps: construction of an electrochemical device: the electrochemical device is formed by bonding a PDMS layer and a glass sheet layer, and comprises: the reaction area and the control area take silver/silver chloride as a working electrode, iridium oxide as a reference electrode and iridium as a counter electrode; self-assembly of framework nucleic acids: constructing frame nucleic acid with designable morphology and biochemistry property through an electrochemical strategy, providing a required DNA single chain, adding the frame nucleic acid into a reaction zone, applying a certain potential to a working electrode to enable oxidation reaction of water to occur in the reaction zone, reducing the pH value of a solution, and carrying out DNA denaturation; by applying another potential to the working electrode, a reduction reaction of water occurs in the reaction zone, the pH of the solution is raised, DNA renaturation occurs, and electrochemical self-assembly of the framework nucleic acid is realized. The invention utilizes the characteristic that the potential of the working electrode can be controlled to quickly and accurately regulate and control the pH of the solution, thereby obtaining a series of frame nucleic acids with the size and the modification of specific functional groups.

Description

Electrochemical preparation method and application of framework nucleic acid

Technical Field

The invention relates to the technical field of nano materials, in particular to an electrochemical preparation method and application of framework nucleic acid.

Background

The DNA nano technology realizes accurate two-dimensional and three-dimensional static or dynamic DNA nano structure by utilizing the DNA double helix with accurate structure and the strict Watson-Click base complementary pairing principle, and has wide application prospect in the fields of medical diagnosis, molecular machines and the like. The construction of the DNA nano structure mainly utilizes the base complementation principle among DNA single chains, and utilizes the binding energy difference among the DNA single chains with different lengths and sequences to realize the acquisition of the most stable configuration of the DNA nano structure. However, in DNA single strands, some secondary or tertiary structures (in kinetic traps) are often generated due to some nonspecific interactions, so DNA origami assembly often requires opening secondary or tertiary structures that may exist in DNA by means of heating or other means, and then controlled folding, hybridization and assembly of DNA strands are achieved by using the base complementarity principle between DNA.

Currently, the controllable assembly means for realizing the DNA nanostructure mainly comprise thermal denaturation technology, non-ionizing radiation (microwave and terahertz) technology, and the like. Among them, thermal denaturation is the most common and mature means for opening up non-specific interactions between the DNA itself or between different DNAs. Typically, at 90℃the DNA undergoes a thermal denaturation process, the secondary structure (in kinetic traps) is opened, the single strand of DNA is restored to its original loose state, and subsequent DNA renaturation occurs during the temperature reduction process, and the complementary paired DNA forms an ordered double helix structure. Among them, the commercial polymerase chain reaction (polymerase chain reaction, PCR) nucleic acid amplification instrument can realize precise control of the temperature of the reaction system, and thus is widely used for the assembly of DNA nanostructures. Commercial PCR is expensive and poorly portable, thus limiting its use in situations where resources are limited. Furthermore, in recent years, it has been reported that non-ionizing radiation in microwave and terahertz (Thz) systems can achieve assembly/disassembly of DNA nanostructures without any sequence modification. However, this system is not suitable for micro-DNA nanostructure synthesis systems and is costly. In general, the techniques currently available for opening nonspecific interactions in DNA and thus achieving controlled assembly of DNA nanostructures are limited.

Recent studies indicate that hydrogen bonds in the DNA duplex are very sensitive to the pH of the solution, where the hydrogen bonds are broken, the DNA duplex is opened, and the DNA duplex is hybridized again under alkaline conditions. Based on this, a series of DNA molecular machines based on pH regulation and the work of polymerase chain reaction were reported successively (Y. Zhang, Q. Li, L. Guo, Q.Huang, J.Shi, Y.Yang, D.Liu and C. Fan, ion-Mediated Polymerase Chain Reactions Performed with an Electronically Driven Microfluidic Device.Angew.Chem., int.Ed.Engl., 2016,55,12450-12454.). Therefore, a new idea is provided for assembling the DNA nanostructure by regulating the pH value of the solution system. While achieving pH regulation of solution systems is a routine laboratory operation, miniature DNA nanostructure synthesis systems present new requirements and challenges to pH regulation. The common "volume titration" method is not suitable for micro-DNA nanostructure synthesis because exogenous substances are introduced during pH adjustment and the total volume of the system is changed. The electrochemical method has the characteristics of high sensitivity, rapid response and easy control, and the electrochemical method electrolyzes water to generate protons, so that the pH value of the system is regulated and controlled, the volume of the system is slightly influenced, and no irrelevant accumulated waste is generated, so that the method is very suitable for regulating and controlling the pH value of a micro system. In 2008, the Hiroaki Suzuki subject group of japan reported a non-classical three-electrode electrochemical system. By utilizing the pH sensitivity characteristic of the iridium oxide electrode and the non-polarizable property of the silver/silver chloride electrode, the accurate and rapid adjustment and stabilization of the pH of the micro system are realized. (K.Morimoto, M.Toya, J.Fukuda and H.Suzuki, automatic Electrochemical Micro-pH-Stat for biochem. Anal. Chem.,2008,80,905-914) this work provides a new opportunity for accurate and rapid read-out of the pH-controlled solution by electrochemical means, and hence the controlled synthesis of DNA nanostructures.

Disclosure of Invention

The invention aims to provide an electrochemical preparation method of frame nucleic acid and application thereof, thereby solving the problems of expensive equipment, large equipment volume, difficult carrying and the like of an instrument and equipment for accurately controlling the temperature of a system used in the process of preparing the frame nucleic acid by the existing thermal denaturation technology.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to a first aspect of the present invention, there is provided a method of electrochemical preparation of a framed nucleic acid, the method comprising: construction of an electrochemical device: the electrochemical device is formed by bonding a PDMS layer and a glass sheet layer, and comprises: the reaction zone is connected with the control zone through a salt bridge, silver/silver chloride is used as a working electrode in the reaction zone, and iridium oxide is used as a reference electrode and iridium is used as a counter electrode in the control zone; self-assembly of framework nucleic acids: constructing framework nucleic acid with designable morphology and biochemistry property through an electrochemical strategy, providing a DNA single strand required by constructing the framework nucleic acid, adding a mixed solution of the DNA single strands into the reaction zone, applying a certain potential to the working electrode to enable water oxidation reaction to occur in the reaction zone, reducing the pH value of the solution, and carrying out DNA denaturation; and (3) applying another potential to the working electrode to enable the reduction reaction of water to occur in the reaction zone, raising the pH value of the solution, and carrying out DNA renaturation to finally realize electrochemical self-assembly of the framework nucleic acid.

The framework nucleic acid is a three-dimensional DNA nanostructure formed by denaturation and renaturation of a plurality of DNA short chains. It will be appreciated that the number of short strands will depend on the size of the framework nucleic acid designed.

More preferably, the shape of the framework nucleic acid is tetrahedral, but it is understood that other shapes of framework nucleic acids such as cubes, octahedra, triangular prisms, triangular bipyramids, etc., may also be used in the present invention.

The self-assembly of the framework nucleic acid further comprises: modification of functional groups is achieved on the structure of the framework nucleic acid by design of the DNA single strand.

More preferably, the functional group modified by the functional framework nucleic acid is a sulfhydryl group, but it is understood that other chemical groups and functional molecule modified framework nucleic acids such as ferrocene, amine groups, carboxyl groups, DBCO, benzene, cy3, cy5, cy7, cholesterol, commercial fluorescent molecules, and the like, may also be used in the present invention.

More preferably, the ratio of thiol groups in the thiol-modified framework nucleic acid to the framework nucleic acid is 3:1.

The modification sites of the functional groups on the framework nucleic acids include: the apex of the framing nucleic acid, the internal cavity of the framing nucleic acid, or the border of the framing nucleic acid.

During electrochemical self-assembly of the framework nucleic acids, the means for capturing the probes include: base pairing, high affinity between biotin and avidin, interaction between antigen and antibody, specific recognition between aptamer and specific target.

The reaction system for preparing the framework nucleic acid comprises: naCl reaction system or MgCl ₂ The reaction system, wherein the frame nucleic acid has the highest yield under the condition of 0.6M NaCl in the NaCl reaction system, and is MgCl ₂ 60mM MgCl in the reaction System ₂ The yield under the condition is highest.

The self-assembly of the framework nucleic acid further comprises: a biosensor based on the framework nucleic acid is constructed by modifying the framework nucleic acid modified by the functional group at the gold electrode interface.

According to a preferred embodiment of the present invention, a DNA biosensor is prepared in which thiol groups are modified at the top of a framed nucleic acid in order to densely modify the framed nucleic acid on the gold electrode surface by Au-S bond, but it should be understood that other framed nucleic acids of modified sites such as the inner cavity of the framed nucleic acid, the rim of the framed nucleic acid, etc. may be used in the present invention as well.

According to the method provided by the invention, the assembly principle of the framework nucleic acid in an electrochemical system is as follows: the potential of the reference electrode has a good linear relationship with the pH of the solution. The oxidation reaction of water is generated in the reaction area by applying a certain potential to the working electrode, the pH value of the solution is reduced, the hydrogen bonding action of DNA in an acid system is destroyed, and the DNA denaturation is generated; and the reaction zone generates a reduction reaction of water by applying another potential to the working electrode, the pH value of the solution is raised, DNA renaturation occurs in an alkaline system, and finally, the self-assembly of the framework nucleic acid is realized.

It will be appreciated that the probes of the present invention capture the target molecule by double-stranded hybridization, and in particular, extend a certain number of bases from the vertices of the framework nucleic acid of the DNA tetrahedron, and the portion extending is a single strand of DNA that can capture the target molecule by base-pairing. It will be appreciated that in addition to base complementary pairing, the capture probe means include high affinity between biotin and avidin, interaction between antigen and antibody, specific recognition between aptamer and specific target, etc. The emphasis is on the specific recognition that requires high affinity between the probe and the target. The target molecules detected by the invention are DNA, and other target molecules or ions, such as miRNA, mRNA, protein, glucose, sarcosine, metal ions and the like, can also be used in the system.

According to the method provided by the invention, the constant-temperature and rapid assembly of the framework nucleic acid is realized.

According to the method provided by the invention, the assembly of the framework nucleic acid is realized by regulating the pH value of the solution in the electrolytic cell by using the electric potential.

It will be appreciated that the different frame nucleic acids require different driving energies, i.e.different concentrations of H ⁺ Or OH (OH) ^- Denaturation and renaturation are achieved, and thus the potentials required for the different framework nucleic acids are different. According to different framework nucleic acids to be constructed, how to set the potential can be obtained according to experiments of specific practical situations.

According to a preferred embodiment of the present invention, example 3 of the present invention can further improve the yield of tetrahedral preparation by employing a plurality of gradient potential changes. However, it should be understood that tetrahedra can also be successfully prepared by merely changing from one potential to another, differing only in the level of yield. The present invention is not limited to the potential change of the embodiment 3.

According to a second aspect of the present invention there is provided the use of a method for electrochemical preparation of a framework nucleic acid in the fields of medical diagnostics and molecular machinery.

As described in the background section of the invention, the problems of expensive equipment, large equipment volume, difficult carrying and the like of the instrument and equipment for precisely controlling the system temperature used in the process of preparing the frame nucleic acid by the existing thermal denaturation technology are solved, and in order to overcome the problems, the invention realizes the regulation and control on the denaturation and renaturation process of DNA by rapidly and precisely regulating and controlling the pH of the solution in an electrochemical system, and meanwhile, the molecular information and the structural information of the DNA are reserved.

Experimental results show that the method provided by the invention is simple, quick and effective, and can be used for obtaining a series of frame nucleic acids with sizes and specific functional groups modified by utilizing the characteristic that the pH value of the solution can be quickly and accurately regulated by controlling the potential of the working electrode. The frame nucleic acid structure prepared by the invention has the advantages of good dispersibility, complete frame structure and effective regulation and control of a biosensing interface. The reagent medicine and instrument used in the invention are cheap and portable, and are easy to standardize and commercialize. Therefore, the invention provides a new idea and technical support for realizing the construction of the framework nucleic acid by an electrochemical means and breaking through the defects of the traditional thermal denaturation technology.

Drawings

FIG. 1 shows the principle of operation of the present invention, wherein a shows a schematic diagram of electrochemically regulating the pH of a solution, and b shows a schematic diagram of pH controlling framework nucleic acid formation;

FIG. 2 is a graph showing the results of electrochemical performance characterization of an iridium oxide electrode, wherein a is a cyclic voltammogram and b is the results of electrode stability performance characterization;

FIG. 3 is a graph showing the electrochemical performance of the prepared iridium oxide electrode and the experimental results of the relation analysis between the Tm value and pH of the DNA double strand, wherein a is a schematic diagram of a device for electrochemically preparing a framework nucleic acid, b is a linear relation experimental result between the potential of iridium oxide and pH, c is a potential regulation speed characterization of the iridium oxide electrode, and d is an experimental result of the Tm value of double strands with different lengths under different pH conditions;

FIG. 4 is a diagram showing experimental results of pH-controlled denaturation and renaturation states of DNA double strand, wherein a is a schematic diagram of fluorescent assay for verifying denaturation and renaturation states of DNA double strand at

pH

11 and 7, b is fluorescence spectrum of system at

pH

7 and 11, and c is analysis result of fluorescence intensity of system at

pH

7 and 11;

FIG. 5 shows the experimental parameters of the preparation of the framework nucleic acid in example 3, a is the experimental parameters of the electrochemical method, and b is the experimental parameters of the thermal denaturation method;

FIG. 6 shows gel electrophoresis characterization and yield analysis of framework nucleic acids in salt ions of different species and concentrations, where a is different Na ⁺ Gel electrophoresis characterization of framework nucleic acids in concentration System, b is different Na ⁺ Yield analysis of the framed nucleic acids in the concentration System, c is the different Mg ²⁺ Gel electrophoresis characterization of framework nucleic acids in concentration System, d is different Mg ²⁺ Analyzing the experimental result of the yield of the framework nucleic acid in the concentration system;

FIG. 7 shows the composition of Mg ²⁺ Gel electrophoresis characterization and yield analysis results of three size framework nucleic acids prepared by an electrochemical method and a thermal denaturation method in a system, wherein a is the framework nucleic acidGel electrophoresis characterization, b is the yield analysis result of the framework nucleic acid;

FIG. 8 shows that the composition contains Na ⁺ Gel electrophoresis characterization and yield analysis results of three-size framework nucleic acids prepared by an electrochemical method and a thermal denaturation method in a system respectively, wherein a is the gel electrophoresis characterization of the framework nucleic acids, and b is the yield analysis result of the framework nucleic acids;

FIG. 9 is an AFM characterization of three size-framed nucleic acids prepared using electrochemical and thermal denaturation methods, respectively;

FIG. 10 is an AFM characterization and size analysis result of three size-framed nucleic acids electrochemically prepared, wherein c is the AFM characterization of the three size-framed nucleic acids and d is the size analysis result of the three size-framed nucleic acids;

FIG. 11 is a gel electrophoresis characterization and AFM characterization of an electrochemically prepared sulfhydryl modified framework nucleic acid, wherein a is the gel electrophoresis characterization of the sulfhydryl modified framework nucleic acid and b is the AFM characterization of the sulfhydryl modified framework nucleic acid;

FIG. 12 is a schematic diagram of a frame nucleic acid biosensor, wherein a is a schematic diagram of the frame nucleic acid biosensor, b is a time-current experimental result of a sensor with different DNA structure modifications, and c is a current intensity analysis result of a sensor with different DNA structure modifications;

FIG. 13 is a cyclic voltammogram of a sensor modified by different DNA structures.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

According to the present invention, there is provided a method for electrochemical preparation of a framework nucleic acid. The invention selects DNA tetrahedrons with different sizes as an example, and then adjusts the pH value of the solution in the electrolytic cell by regulating and controlling the potential on the working electrode under the condition of room temperature. Specifically, when a certain potential is applied, the reaction zone undergoes a reduction reaction of water, producing a large amount of OH ^- The pH of the reaction area is increased, and single-stranded DNA is denatured; when applying a certain amount ofAt another potential, the reaction zone undergoes oxidation of water to produce a large amount of H ⁺ The reaction region is lowered in pH, and the complementary pair of DNA single strands are hybridized to double strands, whereby the frame nucleic acid is assembled (as shown in FIG. 1). Then, constructing sulfhydryl modified DNA tetrahedron framework nucleic acid, assembling on the surface of gold electrode by Au-S action, regulating hybridization thermodynamics of nucleic acid probe and target molecule, and further improving overall performance of biosensor.

Wherein the DNA is purchased from a biological organism (Shanghai); reagents such as potassium chloride, disodium hydrogen phosphate, iridium are purchased from national drug group reagent limited. Platinum electrode, silver/silver chloride electrode were purchased from wuhan gaoshirui technologies limited. Britton-Robinson buffer (0.01. 0.01M H) ₃ PO ₄ -H ₃ BO ₃ -CH ₃ COOH,0.1M KCl, and adjusting specific pH with hydrochloric acid and sodium hydroxide, hereinafter referred to as BR buffer) was purchased from Qingdao Jieshikang biotechnology Co.

The DNA sequences are shown in Table 1 below.

TABLE 1

Example 1 construction of electrochemical device

In this embodiment, an electrochemical device is constructed, comprising the steps of:

preparation of iridium oxide electrode: the iridium wire is used as a working electrode, a silver/silver chloride electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and 0.7M Na ₂ HPO ₄ The solution was an electrolyte and cyclic voltammetric oxidation was performed using Chenhua CHI1040c according to the parameters shown in Table 2. Iridium oxide subjected to cyclic voltammetric oxidation treatmentThe electrode was immersed in water and aged for 24 hours with hydration.

TABLE 2

Initial potential (V)	-0.5
		Highest potential (V)	0.8
Lowest potential (V)	-0.5
		Final potential (V)	0
Sweeping speed (V/s)	0.1
		Number of scan turns	300
Rest time (V)	0
		Sensitivity (A/V)	10 ^-4

Electrode performance testing process: britton-Robinson (BR) buffer solutions with the pH values of 1.7, 2.7, 3.7, 4.5, 5.5, 6.7, 7.7, 8.7, 9.5, 10.4 and 11.6 are sequentially added into a reaction zone, silver/silver chloride is used as a working electrode, an iridium oxide electrode is used as a reference electrode, the potential difference between the iridium oxide electrode and the silver/silver chloride electrode is measured, and a correlation curve of the potential difference and the pH value of the solution is obtained. After the corresponding relation between the potential difference and the pH value of the system is calibrated, setting up an iridium oxide reference electrode, a silver/silver chloride working electrode and an iridium electrode as a counter electrode, circularly applying-190 mV and 178mV according to the data of a correlation curve between the iridium oxide electrode and the pH value of the solution, and examining the rapid and reversible regulating and controlling capability of an electrochemical method on the pH value of the solution.

Preparation of PDMS: PDMS and curing agent with the mass ratio of 10:1 are weighed into a clean beaker, uniformly stirred by a glass rod, placed into a vacuum dryer, and vacuumized for 15min to extract bubbles in the mixture. Then, the mixture of PDMS and curing agent was poured into a horizontal glass tank, and the mixture was placed on a horizontal heating table to cure for 1 hour at 80℃to obtain a PDMS film.

Construction of an electrochemical device: silver/silver chloride is used as a working electrode, an iridium oxide electrode is used as a reference electrode, and iridium is used as a counter electrode. Two holes of about 1.5cm were punched in the PDMS film in advance using a punch, and then the PDMS was attached to the surface of a slide glass to assemble an electrolytic cell. 200. Mu.L of 0.1M KCl was added to the control zone, and 20. Mu.L of single-stranded mixture to be prepared into a nanostructure was added to the reaction zone, with 0.5% agarose as a salt bridge (as shown in a of FIG. 3).

Results: the cyclic voltammogram shows a pair of characteristic peaks of iridium oxide at about 0.1V and 0.5V (vs. ag/AgCl), respectively, demonstrating that we have successfully prepared an iridium oxide electrode (as shown by a in fig. 2). And the measurement result of the open circuit potential method shows that the potential of the iridium oxide electrode has good linear relation with the pH value of the solution, and the correlation coefficient R ² 0.999, slope 77mV pH ^-1 (as shown by b in figure 3). When the operating potential was changed from-190 mV (corresponding ph=12) to 178mV (corresponding ph=12), a significant oxidation current was observed and a steady state was reached within 20s (as shown by c in fig. 3), indicating that the electrochemical method can achieve rapid regulation of the pH of the solution. In addition, the electrochemical regulation system can realize several times of reversible regulation on the pH of the solution (shown as b in fig. 2).

EXAMPLE 2 control of the pH of the solution on the DNA denaturation and renaturation Process

Measurement procedure of DNA double strand Tm value under different pH conditions: the single-stranded DNA required was quantified using an ultraviolet-visible absorption spectrometer, and all DNA single-stranded concentrations were quantified to a concentration of 100. Mu.M. Two complementary paired single strands of 13 bp, 20, 26, 37 and 47bp in chain length were mixed in equal proportions and added to BR buffer solutions of different pH containing 1 XSYBR-Green to prepare samples with single strand final concentrations of 1. Mu.M. And (3) placing the prepared samples into an RT-PCR instrument, heating for 10min at 95 ℃, then suddenly reducing to 25 ℃, slowly heating to 95 ℃, and collecting fluorescent signals of each group of samples in the whole temperature change process in real time.

Verification of denaturation and renaturation process of DNA double strand in different pH systems: two single-stranded DNAs (F1-Alexa 488 and F2-BHQ 1) modified with Alexa488 and BHQ1, respectively, were quantified to 50. Mu.M using an ultraviolet-visible absorption spectrometer. Then, the two single-stranded DNAs were mixed in equal amounts and added to BR buffer

solutions having pH

7 and 11, respectively, and the final concentration of the single-stranded DNA was 100nM. The fluorescence test conditions were: the excitation wavelength was 488nm and the emission wavelength was 498-650 nm, with excitation and emission slits of 5nm.

Results: the DNA melting temperature results showed that the Tm value gradually increased as the length of the double-stranded DNA increased. And the Tm decreases with increasing pH of the solution for the same length of DNA strand (as shown by d in fig. 3). Thus, in an alkaline system, the energy of the double-helix structure becomes weak, and the occurrence of the unwinding behavior is facilitated.

In BR buffer solution with pH=7, F1-Alexa488 and F2-BHQ1 are hybridized into a double-chain structure, the fluorescent groups and the quenching groups at two ends are close, the fluorescence of Alexa488 is quenched, the double-chain structure formed by F1-Alexa488 and F2-BHQ1 is opened in alkaline environment with pH=11, the fluorescent groups and the quenching groups at two ends are far away, the fluorescence of Alexa488 is recovered, and in conclusion, the pH of the solution can realize the regulation of hybridization and de-rotation states of DNA double chains (as shown in FIG. 4).

Example 3 preparation of DNA tetrahedral framework nucleic acids of different sizes

Experimental procedure for screening electrochemical methods for preparing salt concentration of framework nucleic acids: optimal Mg for DNA tetrahedron preparation ²⁺ The concentration of the four single-stranded DNAs forming the DNA tetrahedron nanostructure was mixed in a final concentration of 1. Mu.M in a constant proportion in a TM buffer, 10mM MgCl, respectively ₂ 、20mM MgCl ₂ 、40mM MgCl ₂ And 60mM MgCl ₂ Heating at 95deg.C for 10min in a PCR instrument, and rapidly cooling to 4deg.C for more than 10 min. Optimal Na for DNA tetrahedron preparation ⁺ Concentration, four single-stranded DNAs forming the DNA tetrahedron nanostructure were mixed in a final concentration of 1. Mu.M in a constant proportion in TM buffer, 0.2M NaCl, 0.3M NaCl, 0.4M NaCl, 0.5M NaCl and 0.6M NaCl, respectively, and placed in a PCR instrument, heated at 95℃for 10min, and then rapidly cooled to 4℃and maintained for 10min or more.

Electrochemical preparation process of framework nucleic acid: picking up the required single-stranded DNAQuantification was performed using an ultraviolet-visible absorption spectrometer to quantify all DNA single strand concentrations to 100 μm. Equal amounts of four or eight single stranded DNA strands for preparing 20bp,26bp, and 37bp DNA tetrahedrons were mixed and added to 0.5M NaCl or 60mM MgCl ₂ In (2), the final concentration of each single-stranded DNA was 1. Mu.M. mu.L was added to the reaction zone, and the working potentials were set to 178mV, 104 mV, 31mV, -43mV, -117mV and-190 mV, respectively, each of which was maintained for 1min, to prepare an electrochemical-framed nucleic acid (E-TDNs) (as shown in a of FIG. 5).

The preparation process of the thermal denaturation of the framework nucleic acid comprises the following steps: the single-stranded DNA required was quantified using an ultraviolet-visible absorption spectrometer, and all DNA single-stranded concentrations were quantified to 100. Mu.M. Mixing four or eight single-stranded DNAs of the prepared DNA tetrahedron in equal amounts, adding into a reaction zone of 1×TM buffer (20mM Tris,50mM MgCl) ₂ pH 8.0), each single-stranded DNA was 1. Mu.M, and the resulting mixture was heated to 95℃for 10min, followed by rapid cooling to 4℃for more than 10min in 5min, to obtain heat-denatured framework nucleic acids (T-TDNs) (shown as b in FIG. 5).

Characterization by gel electrophoresis: characterization was performed using 8% polyacrylamide electrophoresis gel electrophoresis, run 2 h at 120V.

Atomic force microscope characterization: the freshly torn mica was treated with 0.5% (v/v) APTES solution for 1min, the unadsorbed APTES on the mica surface was removed by rinsing with a large amount of ultrapure water, and the mica surface was blown dry with ear-washing balls for use. 10 μL of 1nM sample was dropped onto the mica surface and adsorbed for 5min. Scanning and imaging under an atomic force microscope, wherein an imaging probe is SCANASYST-FLUID+, and an imaging mode is PeakForce QNM in Fluid.

Results: the gel electrophoresis result shows that the yield difference is not large under the condition of 0.4-0.6M in the NaCl reaction system, the yield of the framework nucleic acid is highest under the condition of 0.6M NaCl, and the framework nucleic acid is in MgCl ₂ 60mM MgCl in the reaction System ₂ Under the condition that the yield of the framework nucleic acid is highest, the reaction system selected in the subsequent framework nucleic acid assembly experiment is 0.5M NaCl or 60mM MgCl for the convenience of liquid preparation ₂ Solution (FIG. 6).

Furthermore, gel electrophoresis characterization was shown in the NaCl bodyLinking with MgCl ₂ The electrochemical method in the system can successfully prepare DNA tetrahedrons of 20bp,26bp and 37bp (FIG. 7 and FIG. 8). And atomic force microscopy characterization showed that the electrochemically prepared DNA tetrahedra also had good dispersibility and structural integrity (fig. 9) compared to the traditional thermal denaturation method, and atomic force image size analysis showed that the lateral dimensions of the 20bp,26bp, and 37bp DNA tetrahedra were 8.6nm,11.7nm, and 17.5nm (fig. 10).

EXAMPLE 4 preparation of thiol-modified framework nucleic acids

Preparation of thiol-modified framework nucleic acids: the single-stranded DNA required was quantified using an ultraviolet-visible absorption spectrometer, and all DNA single-stranded concentrations were quantified to 100. Mu.M. Four single stranded DNA strands from which DNA tetrahedra were prepared were mixed in equal amounts in 1 XTM buffer (20mM Tris,50mM MgCl) containing 3mM TCEP ₂ pH 8.0) to a final concentration of 1. Mu.M, a denaturing synthesis of the framework nucleic acid was performed. Denaturation procedure reference example 3.

Results: polyacrylamide electrophoresis shows that the electrochemically prepared framework nucleic acid lanes have clear bands on the target band, and that the preparation of DNA tetrahedral framework nucleic acids modified with sulfhydryl groups was successful (as shown by a in FIG. 11).

Atomic force microscope characterization showed that the electrochemically prepared thiol-modified framework nucleic acid had good monodispersity and structural integrity, indicating that the electrochemical method provided by the invention can be used to prepare a thiol-modified framework nucleic acid (as shown in b in fig. 11).

Example 5 preparation of DNA biosensors based on framed nucleic acid probes

Modification of tetrahedral DNA nanoprobe on gold electrode surface: three thiol-modified framework nucleic acids were prepared by electrochemical and thermal denaturation methods, respectively, as described in reference to example 4. And respectively taking 6 mu L of prepared frame nucleic acid to be dripped on the surface of the gold electrode, and putting the gold electrode into a wet box for assembly at normal temperature overnight. mu.L of thiol-modified single-stranded DNA was also dropped onto the gold electrode surface and assembled overnight at room temperature. Then, the reaction was blocked with 2mM MCH for 1 hour in order to reduce nonspecific adsorption of DNA on the surface and to help the DNA probe to maintain an upright state.

Detection of target DNA: the final concentration of 10nM T-probe and the final concentration of 100nM signal probe R-biotin were mixed in 10mM PB hybridization solution, heated at 80℃for 5 minutes, and cooled at room temperature for 20 minutes. Then soaking the assembled electrode into the mixed solution, and reacting for 2 hours at 37 ℃; the electrode was then rinsed with 0.01M PBS, N ₂ And (5) blow-drying. Subsequently, 3. Mu.L of avidin-HRP (0.5U/mL) was added and the mixture was labeled at room temperature for 15min. Then washed with 0.01M PBS for electrochemical measurement.

Electrochemical measurement: all electrochemical tests were signal read out by the Chenhua CHI660E electrochemical workstation. The three-electrode system comprises a gold electrode as a working electrode, a platinum wire electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode. The sweep rate of cyclic voltammetry was 100mV/s, ranging from-0.25V to 0.4V. In addition, the potential of the chronoamperometry was set to 100mV, and an electrochemical reduction current of 100s was taken as an output electric signal (equilibrium state of HRP catalytic reaction).

Results: the reduction current of both E-TDNs and T-TDNs sensors was significantly increased between-0.3V and 0V compared to ssDNA sensors (FIG. 13), and more TMB oxide was reduced from the E-TDNs and T-TDNs modified electrode surfaces. The time-current (i-t) curve shows that at a constant reduction voltage (-0.1V), the TDNs modified gold electrode experimental group significantly increased in reduction current (as shown by b in fig. 12). These results indicate that the E-TDNs and T-TDNs modified gold electrode interfaces have higher DNA hybridization efficiency. The signal of the single-stranded DNA group was only about 2-fold higher than that of the blank group, while the signals of both the E-TDNs and T-TDNs experimental groups were 186-fold higher than that of the blank group. These results show that the rigid three-dimensional framework structure allows precise adjustment of the distance between probes, thereby improving the efficiency of electrochemical interfacial hybridization. And there was no significant difference (about 2%) in the current signal values between the two groups of E-TDNs and T-TDNs, demonstrating that the electrochemical method is consistent with the framework nucleic acid structure prepared by the conventional thermal denaturation method (as shown in c in FIG. 12).

The foregoing description of the preferred embodiments of the present invention is not intended to limit the scope of the invention, and various modifications can be made to the above-described embodiments of the present invention. I.e. all simple and equivalent changes made in the content of the description of the claims according to the application of the invention fall within the scope of the claims of the patent of the invention. The present invention is not exhaustive of the conventional technology.

SEQUENCE LISTING

<110> Shanghai university of transportation

<120> an electrochemical preparation method of a framed nucleic acid and its application

<160> 34

<170> PatentIn version 3.5

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<212> DNA

<213> artificial sequence

<400> 3

ccgcagcgga gcaggagtcc tcggcc 26

<210> 4

<211> 37

<212> DNA

<213> artificial sequence

<400> 4

gcgtatgtgt tctgtgcggc ctgccgtccc gtgtggg 37

<210> 5

<211> 47

<212> DNA

<213> artificial sequence

<400> 5

gcgtatgtgt tctgtgcggc ctgccgtccc gtgtgggcag gccttgg 47

<210> 6

<211> 13

<212> DNA

<213> artificial sequence

<400> 6

tctgacgtag tgt 13

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<400> 7

cgaacattcc taagtctgaa 20

<210> 8

<211> 26

<212> DNA

<213> artificial sequence

<400> 8

ggccgaggac tcctgctccg ctgcgg 26

<210> 9

<211> 37

<212> DNA

<213> artificial sequence

<400> 9

cccacacggg acggcaggcc gcacagaaca catacgc 37

<210> 10

<211> 47

<212> DNA

<213> artificial sequence

<400> 10

ccaaggcctg cccacacggg acggcaggcc gcacagaaca catacgc 47

<210> 11

<211> 20

<212> DNA

<213> artificial sequence

<400> 11

gctgcaagct tactaatagg 20

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<400> 12

cctattagta agcttgcagc 20

<210> 13

<211> 62

<212> DNA

<213> artificial sequence

<400> 13

cgattacagc ttgctacacg attcagactt aggaatgttc gacatgcgag ggtccaatac 60

cg 62

<210> 14

<211> 62

<212> DNA

<213> artificial sequence

<400> 14

cgtatcacca ggcagttgag acgaacattc ctaagtctga aatttatcac ccgccatagt 60

ag 62

<210> 15

<211> 62

<212> DNA

<213> artificial sequence

<400> 15

cgtgtagcaa gctgtaatcg acgggaagag catgcccatc cactactatg gcgggtgata 60

aa 62

<210> 16

<211> 62

<212> DNA

<213> artificial sequence

<400> 16

ctcaactgcc tggtgatacg aggatgggca tgctcttccc gacggtattg gaccctcgca 60

tg 62

<210> 17

<211> 84

<212> DNA

<213> artificial sequence

<400> 17

gcctggagat acatgcacat tacggctttc cctattagaa ggtctcaggt gcgcgtttcg 60

gtaagtagac gggaccagtt cgcc 84

<210> 18

<211> 84

<212> DNA

<213> artificial sequence

<400> 18

cgcgcacctg agaccttcta atagggtttg cgacagtcgt tcaactagaa tgccctttgg 60

gctgttccgg gtgtggctcg tcgg 84

<210> 19

<211> 84

<212> DNA

<213> artificial sequence

<400> 19

ggccgaggac tcctgctccg ctgcggtttg gcgaactggt cccgtctact taccgtttcc 60

gacgagccac acccggaaca gccc 84

<210> 20

<211> 84

<212> DNA

<213> artificial sequence

<400> 20

gccgtaatgt gcatgtatct ccaggctttc cgcagcggag caggagtcct cggcctttgg 60

gcattctagt tgaacgactg tcgc 84

<210> 21

<211> 58

<212> DNA

<213> artificial sequence

<400> 21

ccctgtactg gctaggaatt cacgttttaa tctgggcttt gggttaagaa actccccg 58

<210> 22

<211> 59

<212> DNA

<213> artificial sequence

<400> 22

cgctggaggc gcatcaccgt ttgcgtatgt gttctgtgcg gcctgccgtc ccgtgtggg 59

<210> 23

<211> 58

<212> DNA

<213> artificial sequence

<400> 23

cggtgatgcg cctccagcgc ggggagtttc ttaacccttt ccgacttaca agagccgg 58

<210> 24

<211> 59

<212> DNA

<213> artificial sequence

<400> 24

gcgagactca ggtggtgcct ttggcattcg accaggagat atcgcgttca gctatgccc 59

<210> 25

<211> 59

<212> DNA

<213> artificial sequence

<400> 25

cccatgagaa taataccgcc gatttacgtc agtccggttt cccacacggg acggcaggc 59

<210> 26

<211> 58

<212> DNA

<213> artificial sequence

<400> 26

cgcacagaac acatacgctt tgggcatagc tgaacgcgat atctcctggt cgaatgcc 58

<210> 27

<211> 59

<212> DNA

<213> artificial sequence

<400> 27

gcccagatta aaacgtgaat tcctagccag tacagggttt ccggactgac gtaaatcgg 59

<210> 28

<211> 58

<212> DNA

<213> artificial sequence

<400> 28

cggtattatt ctcatgggtt tggcaccacc tgagtctcgc ccggctcttg taagtcgg 58

<210> 29

<211> 104

<212> DNA

<213> artificial sequence

<400> 29

catcttgcct aaaaaaaaaa gcctggagat acatgcacat tacggctttc cctattagaa 60

ggtctcaggt gcgcgtttcg gtaagtagac gggaccagtt cgcc 104

<210> 30

<211> 84

<212> DNA

<213> artificial sequence

<400> 30

cgcgcacctg agaccttcta atagggtttg cgacagtcgt tcaactagaa tgccctttgg 60

gctgttccgg gtgtggctcg tcgg 84

<210> 31

<211> 84

<212> DNA

<213> artificial sequence

<400> 31

ggccgaggac tcctgctccg ctgcggtttg gcgaactggt cccgtctact taccgtttcc 60

gacgagccac acccggaaca gccc 84

<210> 32

<211> 84

<212> DNA

<213> artificial sequence

<400> 32

gccgtaatgt gcatgtatct ccaggctttc cgcagcggag caggagtcct cggcctttgg 60

gcattctagt tgaacgactg tcgc 84

<210> 33

<211> 30

<212> DNA

<213> artificial sequence

<400> 33

tttttttttt aggcaagatg cacaacagca 30

<210> 34

<211> 10

<212> DNA

<213> artificial sequence

<400> 34

tgctgttgtg 10

Claims

1. A method for electrochemical preparation of a framed nucleic acid, the method comprising:

construction of an electrochemical device: the electrochemical device is formed by bonding a PDMS layer and a glass sheet layer, and comprises: the reaction zone is connected with the control zone through a salt bridge, silver/silver chloride is used as a working electrode in the reaction zone, and iridium oxide is used as a reference electrode and iridium is used as a counter electrode in the control zone; and

self-assembly of framework nucleic acids: constructing framework nucleic acid with designable morphology and biochemistry property through an electrochemical strategy, providing a DNA single strand required by constructing the framework nucleic acid, adding a mixed solution of the DNA single strands into the reaction zone, applying a certain potential to the working electrode to enable water oxidation reaction to occur in the reaction zone, reducing the pH value of the solution, and carrying out DNA denaturation; by applying another potential to the working electrode, a reduction reaction of water occurs in the reaction zone, the pH of the solution is raised, and DNA renaturation occurs; by adopting the potential changes of a plurality of gradients, the pH of the solution is rapidly and accurately regulated and controlled in an electrochemical system, the denaturation and renaturation processes of DNA are regulated and controlled, and finally the electrochemical self-assembly of the framework nucleic acid is realized;

wherein the framework nucleic acid is a three-dimensional DNA nanostructure formed from a plurality of DNA strands by denaturation and renaturation, the three-dimensional DNA nanostructure comprising: tetrahedron, cube, octahedron, triangular prism, or triangular bipyramid;

the reaction system for preparing the framework nucleic acid adopts: naCl reaction system or MgCl ₂ A reaction system, wherein the concentration of NaCl in the NaCl reaction system is 0.6M, theMgCl ₂ MgCl in the reaction system ₂ Is 60 mM.

2. The electrochemical preparation method of claim 1, wherein the self-assembly of the framework nucleic acid further comprises: modification of functional groups is achieved on the structure of the framework nucleic acid by design of the DNA single strand.

3. The electrochemical preparation method according to claim 2, wherein the functional group comprises: sulfhydryl, ferrocene, amine, carboxyl, DBCO, benzene, cy3, cy5, cy7, cholesterol, or commercial fluorescent molecules.

4. The method of electrochemical preparation of claim 3, wherein the modification site of the functional group on the framework nucleic acid comprises: the apex of the framing nucleic acid, the internal cavity of the framing nucleic acid, or the border of the framing nucleic acid.

5. The method of electrochemical preparation according to claim 1, wherein during electrochemical self-assembly of the framework nucleic acid, the capture probes are in the form of: base pairing, high affinity between biotin and avidin, interaction between antigen and antibody, specific recognition between aptamer and specific target.

6. The electrochemical preparation method of claim 2, wherein the self-assembly of the framework nucleic acid further comprises: a biosensor based on the framework nucleic acid is constructed by modifying the framework nucleic acid modified by the functional group at the gold electrode interface.