Enriching And Detecting Biomolecules By Dielectrophoresis And Electrophoresis Based On Protein Nanopore

Protein nanopore-based analytical technique,which combines biotechnology,nanotechnology and single-molecule technology has become one of the core technology in interdisciplinary field.This technology is widely used in biology,chemistry and medicine based on α-HL.It has been used for real-time detection of different analytes including anion,cation,organic molecules,DNA,RNA,protein,peptides,polymers and et al,and investigating the interaction of host-guest molecules,enzyme kinetics and so on at single molecule level.Although the critical information obtained from nanopores comes from the signal collected during analytes translocation,the throughput of the method is determined by the rate at which molecules arrive and thread into the pores.Only when the signal acculation reaches a certain number can it be statistically significant.The dielectrophoresis(DEP)is defined that when a dielectric particle is suspended in a liquid media of different polarizabilities and subjected to a non-uniform electric field,the particle experiences a net force(DEP)and is either attracted to(positive DEP)or pushed away from(negative DEP)a high electric field region.Some workers have used DEP effect to separate the leukemia cell,tumour cell,living cells and dead cells,bacterium and virus.And the DEP can separate,trap or focus the whole analyte molecules at specific frequency and alternative voltage.Here,we took the advantages of good recognition ability by protein nanopore at single molecule level and focusing small molecule with DEP effect,and developing a new detection method:focusing analytes(single-stranded DNA,β-cyclodextrin,1-Amantadine hydrochloride)with DEP effect at alternative voltage field in order to satisfy the lowest concentration in statistical sense,then detecting the analytes based on protein nanopore.This method featured easy operation,high sensitivity and rapid speed,and it provided a brief idea to the research of single molecule detection and dielectrophoresis.The important conclusions of this work are summarized as follows:1.Focusing and deteting single-stranded DNA with α-HL.We designed specific single-stranded DNA sequence(5’-ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA GGG TCT GAG GGA-(C)30-3’)as analyte.The research showed that the optical conditions was at high alternative voltage and low frequency(15 Hz,16 V)or high frequency and low alternative voltage(100 kHz,10 V or 7 V)in(WT)7 nanopore system.The results demonstrated that the dwell time of ssDNA translocation of the pore increased from～0.2 ms to～1.0 ms and events frequency increased by 13 times at ＋120 mV direct voltage after focusing with high voltage and low frequency.The dwell time increased to 4.36±0.15 ms and events frequency increased by 60 times after focusing with high frequency and low voltage.2.Selecting the nanopore with a long binding time for β-cyclodextrin from(K8A/M113R)7 and(M113F)7,then focusing and detecting β-cyclodextrin.We detected)β-cyclodextrin by(K8A/M113R)7 and(M113F)7,the results showed that the responsible time is 0.55±0.033 ms by(K8A/M1113R)7 and 1.21 士 1.19 s by(M113F)7.We selected(M113F)7 with longer dwell time for β-cyclodextrin as the nanopore sensor to focusβ-cyclodextrin with DEP effect because of detecting host-guest compounds with the adapter of β-cyclodextrin in nanopore system.The bind numbers increased and maximum to 2 times with DEP effect at 2～5 MHz and 0.8 V or 1.6 V except 3 MHz and 0.8 V.3.Focusing detecting 1-Amantadine hydrochloride by(M113F)7 andβ-cyclodextrin.We detected 1-Amantadine hydrochloride by the nanopore of(M113F)7 and adapter ofβ-cyclodextrin.The results showed that the current blockage by β-cyclodextrin was partial(>60%)and further reduced open channel current by l-Amantadine hydrochloride.The responsible time of β-cyclodextrin/l-Amantadine hydrochloride decreased from 0.63±0.025 ms to 0.3 8±0.013 ms at detecting voltage from ＋40 mV to ＋100 mV and bind numbers decreased from 42 to 20.The responsible time of β-cyclodextrin/1-Amantadine hydrochloride increased to 2～3 times and bind numbers increased～2 times at detecting voltage of ＋40 mV with DEP effect at 1～5 MHz and 1.6 V.