Study On DNA Translocation Within Solid-state Nanopores

DNA(Deoxyribonucleic acid)is the blueprint of life,contains the entire organism's genetic information.Accurate interpretation of the genetic information on the progress of life science and biological environment is of great significance,DNA sequencing technology arises at the historic moment.High efficiency and low cost of nanopore sequencing technology received gradually the favor of people.Inspired by Kurt's principle,the principle of nanopore sensing is as following:as charged DNA molecules are driven through the nanopore by an applied electric potential,the steric occupation of each nucleobase(A,T,C and G)or each short string of nucleobases,should correlate to a distinct measurable signal.By recording the ionic current when a DNA molecule passes through the nanopore,the sequence of nucleobases in this DNA molecule can be distinguished.nanopores are divided into biological nanopores and solid-state nanopores.Compared to the biological nanopores,the preparation of solid-state nanopores(the diameter,shape,thickness)is of high maneuverability.Solid-state nanopores are nanoscale apertures fabricated on two dimensional membranes,usually methods for preparing nanopores:Focused Ion Beam(FIB),Transmission Electron Microscope(TEM),chemical etching method and the dielectric breakdown method.Several kinds of traditional process for low yield,high cost and complex preparing nanopores,therefore,rapid and simple dielectric breakdown of the nanopore was born.Dielectric breakdown method is based on Lab VIEW software,which can make the communication between computer and Keithley2450,automatic control of the preparation of solid-state nanopores.The solid-state nanopore sequencing technology has been studied for several decades,but the translocation velocity of DNA through the nanopore is too fast and the thickness of the insulating film is too thick,which can't reach the purpose of distinguishing the single base in time resolution.Therefore,effective control of the DNA molecules' translocation behavior in nanopore and slow down the DNA translocation speed,which become the key to the further development of solid-state nanopore sequencing technology.In this paper,the research work mainly includes:Firstly,to prepare good stability,high sensitive solid-state nanopore device,we prepare solid-state nanopores based on the method of CSDB(Current-Stimulus Dielectric Breakdown).This method can precisely control the pore size and morphology(conical nanopores,cylindrical nanopore),so this method can meet the needs of individual molecules.Due to the diameter of the single DNA molecule is about 1.3 nm,to prevent folded DNA molecules jamming the pore and satisfy the ion current signal with high signal-to-noise ratio,so the diameter of nanopores are prepared in 2 to 3 nm.For 5'-biotinylation-DNA,considering the increase of the sample molecules in space structure,to prevent the DNA molecular jamming the pore,so the diameter of nanopores' size is about 3 nm.Then,we prepared 2 nm conical silicon nitride nanopores based on dielectric breakdown,four kinds of oligonucleotides chains of nucleotides(poly(dA)30,poly(dT)30,poly(dC)30 and poly(dG)30)through the conical nanopore in turn.The experimental results show that the conical nanopores can effectively identify the four oligonucleotides nucleotide chain molecules,but considering that these four types of nucleotides have the same number of nucleobase(30 nt),leading to a similar in the length(?6.8 nm)of the nucleobase chains.Therefore,the dwell time of the translocation behavior of the ssDNA through the same nano channel may not have a great difference,the translocation speeds all about 5.7?s/base.At the same time,we also prepared 2 nm MoS2 conical nanopores,containing the same experimental conditions,the same DNA molecules through the nanopore conical in turn.Experimental results show that the MoS2 conical nanopore also can effectively identify the four nucleotides molecular chains,and the translocation speed can delay to 11.3 us/base,which is twice as much as the translocation speed of ssDNA through the SiNx nanopore.Therefore,conical solid-state nanopore effectively identifies single molecule and provides an experimental foundation for the development of DNA sequencing.At last,in this work,we modified the DNA with biotin,hence to reduce the tranlocation velocity of DNA through the nanopore.Translocation behaviors of DNA through the nanopore before and after biotinylated-modification were then compared.We obtain an 8.50?s/base translocation speed for 100nt single-stranded DNA(ssDNA)with biotinylated-modification passing through a 3-nm diameter silicon nitride(SiNx)pore,which is twice as slow as that of the ssDNA without biotinylated-modification.Our results also indicated that the length of homopolymer chains may not affect the velocity of translocation,whether before or after biotinylated-modification.Moreover,we also studied the pH influence on the nucleotide translocation behavior after biotinylated modification.At acidic or alkaline conditions(pH 6 or pH 10),the mean current amplitude and translocation duration were all less than that at near-physiological condition(pH 8).