Experimental Study On DNA Deceleration Based On Solid-state Nanopores

Gene sequencing technology has played an important role in many fields,such as decoding gene expression and bioscience.At the same time,gene sequencing is also of great significance to the diagnosis and treatment of diseases.For example,at the end of 2019,a new type of coronavirus pneumonia(COVID-19)appeared worldwide and spread rapidly.Scientists quickly measured the nucleotide sequence of the new coronavirus through genetic sequencing,and at the same time,determined whether human body was infected with novel coronavirus through nucleic acid detection method.Another example is the use of gene sequencing technology to screen for Down's syndrome before childbirth.The development and maturity of DNA sequencing technology has brought great innovations to the medical field.At present,nanopore sensor technology can complete the task of low economic cost and short sequencing time.Compared with biological pores,solid-state nanopores have the advantages like adjustable pore size,reliable,stable quality and high integration,which has become the focus among the next-generation gene sequencing technologies.A major challenge in the process of solid-state nanopore sequencing is that the speed of biomolecules to be tested through the nanopores under the action of electric field forces is too fast,which makes it impossible to collect a sufficient number of effective signals to analyze the characteristics of the molecules to be tested.This article aims to reduce the translocation speed of DNA molecules in solid-state nanopores,and explores an experimental method that can effectively extend the duration of DNA molecular translocation in nanopores.The core research work and results of this paper are:(1)Using mechanical separation method to peel off massive graphene,and then using wet transfer of graphene film to achieve stable coverage of graphene film on silicon nitride substrate,finally using focused ion beam technology to successfully prepare graphene nanopores and cage structure nanopores.After special hydrophilic treatment of nanopores,experiments were carried out to detect DNA molecules.Comparing the experimental results using silicon nitride nanopore detection,it can be found that when DNA molecules pass through the graphene nanopore,a longer via duration and a more obvious blocking current amplitude can be obtained.In the process of using cage-like nanopores to detect DNA molecules,there are significant differences in the signal characteristics of the DNA molecules through the forward and backward through pores.The focus is on the chamber space that can be used to achieve singlemolecule level and repeat sequencing Broad prospects.(2)In-depth exploration of changing various experimental conditions to obtain effective methods to slow down the translocation speed of DNA molecules in nanopores.According to the control experiments,the conclusions are as follows: applying a smaller bias voltage can slow down the translocation speed of DNA molecules,but the amplitude of current changes will also decrease;reducing the pore size of nanopores slows down the speed of DNA molecules.At the same time,the amplitude of the blocking current will increase;when the electrolyte solution is Li Cl,DNA molecules can obtain a slower translocation speed than in the KCl and Na Cl solution;reduce the temperature of the solution environment and increase the electrolyte solution at both ends of the nanopore.The concentration gradient scheme can achieve the purpose of slowing down the movement speed of DNA molecules in the silicon nitride nanopores;increasing the concentration of the electrolyte solution can also reduce the passing speed of DNA molecules.At the same time,the posture of the DNA in the hole corresponding to the different current signals is analyzed.The conformation of the DNA will also affect the duration of the DNA molecule in the nanopore.Through comparison,it is found that when the DNA concentration increases,DNA molecules tend to fold themselves and intertwine through nanopores.(3)The method of binding the ends of DNA molecules with biotin,streptavidin and magnetic beads was studied.Obtain the current signals generated by the translocation of DNA molecules with different binding substances in the nanopore.When the end of the DNA molecule is bound to the magnetic bead through the hole,the duration of generation is relatively long,reaching about 200 ms.The speed of DNA molecules bound to streptavidin is slower than when only biotin is bound.DNA molecules only bound to biotin can also achieve the effect of slowing down the translocation speed of DNA molecules.In addition,we will focus on exploring the effects of different intensities of voltage on the binding bonds of streptavidin and biotin.The experimental results show that after applying a larger voltage,the survival time of the binding bond becomes significantly shorter,and the easier it is for DNA molecules and magnetic beads to separate.(4)A liquid pool and fixture specially adapted to the upper and lower structure of the magnetic tweezers system are designed,which can meet the space requirements of the magnetic tweezers while also facilitating the observation of the thinned area of the chip by the microscope.The important components of the magnetic tweezers system and the specific methods of using magnetic tweezers to control DNA molecules are introduced in detail.Explore the use of magnetic tweezers system to control micron magnetic beads,and then actively control the movement of DNA molecules through the silicon nitride nanopores,and finally realize the active regulation of the speed of DNA molecules.