Assembly And Test Of MspA Nanopore Formed On Silicon Substrate

Nanopore has attracted wide attentions in the past two decades as a method of single molecule detection and characterization.Traditional nanopore platforms include biological nanopore and solid-state nanopore.Biological nanopore mainly uses channel proteins which contain a narrow constriction for sequencing such as alpha hemolysin(α-HL),mycobacterium smegmatis porin A(Msp A),Aerolysin(Ae L),etc.Solid-state nanopore is normally fabricated in thin membranes by using ion beam sculpting and the anodic oxidation method,etc.But there remains a number of limitations with these systems mentioned above.For biological nanopore,it is difficult to change the protein pore size and the lipid bilayer lacks stability under different conditions.However,solid-state nanopore does not have such low noise compared with biological nanopore.This paper focuses on research the stability and mobility of the pore-spanning lipid bilayer membrane formed on silicon nitride nanopore array and developing a novel hybrid nanopore using the method of producing lipid bilayer across nanopore array on a silicon nitride substrate by painting and inserting mutMspA M3.The main research contents are as follows:1.Fabrication of solid-state nanopore Based on the self-supporting silicon nitride chip designed by our lab,the solid-state nanopore array was processed by circle mode of Dual Beam Focused Ion Beam(DB-FIB)and we fabricated 90 nm,120 nm and 150 nm nanopore array.In this process,we observed that present diameter and processing time are proportional to the nanopore diameter after determining the preset diameter.Beyond this time range,the nanopore diameter did not change significantly with the bombardment time.This finding indicated that the ion beam had a high beam spot accuracy when processing nanopores.Although the ion dose can increase with the bombardment time,the sputtering range is limited.2.Formation of pore-spanning lipid bilayer membrane by vesicles We formed the pore-spanning lipid bilayer membrane on the surface of silicon nitride nanopore array by vesicles and incorporated of a certain proportion of phospholipids with carboxyl groups(DOPE)in the DOPC vesicles.Modify the amino group on the surface of silicon nitride,thereby forming an amide bond through a dehydration condensation reaction to achieve semi-fixed effect.Subsequently,the mobility of the four phospholipid-matched membranes was compared using fluorescence bleaching recovery(FRAP),including DOPC,DOPE,DOPC:DOPE=100:1,and DOPE adding activator.Patch clamp was used to detect the real-time changes of membrane resistance.We set 1.0 GΩ as the standard of the effective resistance for ensuring the pore protein to work normally,comparing the effective survival time of the four phospholipidmatched membranes.Among them,we can observe that DOPC membrane wan the highest,and the membrane mobility was reduced adding DOPE phospholipids.The membrane mobility was the lowest adding the activator.By patch clamp detection,we find that DOPE phospholipid-formed transmembrane after the addition of activator has the longest effective survival time.This result shows that after introducing the amide bond to form a semi-fixed effect,the stability of the phospholipid membrane can be improved,but at the same time,the fluidity of the membrane is also reduced.3.Hybrid nanopore assembled by painting and testBy using the silicon nitride nanopore array chip and the Teflon fluid independently designed by the laboratory,we assembled the hybrid nanopore and ensure that the nanopore was the only channel for ionic solution exchange in both chambers.Finally,we tested the platform by translocation of DNA sample.First,we hydrophobized the chip and characterized it,and assembled it into Teflon fluid to ensure that the nanopore was the only channel.Then the transmembrane was formed on the surface of the chip by painting with hospholipid(DPh PC)and inserted the M3 mutant Msp A protein with detecting the formation of single channel characteristic current signal.Through the detection of translocation signals of DNA samples,the signal characteristics of the platform and biological nanopores were found to be similar.Besides,we compared the effective survival time of membranes on different pore size nanopore arrays.When the pore size was reduced,the effective survival time of lipid bilayer membranes was prolonged.The hybrid nanopore platform has near-Msp A biological nanopore sequencing accuracy and reduces the sample translocation rate and increases the effective duration of the lipid bilayer membrane.