Investigation And Application Of Novel Nanopore Single-molecule Analytical System

Nanopore single-molecule technology is a powerful analytical tool.Due to its some characteristics such as label-free,amplification-free,high throughput and high selectivity,it has been widely used in DNA sequencing,protein conformational analysis,biological macromolecular interaction and various single-molecule detections including DNA/RNA,peptides,proteins,organic molecules,metal ions and so on.The detecting technology for analytes is based on recording the change of ion conductance in nanopore triggered by analyte translocation through the pore.In nanopore analytical application,?-hemolysin(?-HL)protein nanopore is most often used because of its specific structure and easily genetic modification.KCl/NaCl is traditionally used as nanopore electrolyte,which possesses high ionic conductivity,and does not interact specifically with the analyte or protein nanopore.But the system noise and signal resolution are not controllable,and it is difficult to detect a variety of analytes with high selectivity.Here we used tetramethylammonium chloride(TMA-Cl)and guanidine hydrochloride(GdnHCl),instead of KCl,as novel analysis systems for nanopore analysis.Based on the interaction of electrolyte ions with analyte or phospholipid bilayer,we studied the properties of the novel nanopore detection system and detected the specific analytes.The main research contents are included as follows:(1)Research Review.A brief review is given to the development history of nanopore analytical technology,analytical principle and nanopore classification.Moreover,the research progress is summarized,especially in DNA sequencing,epigenetic detection and protein conformational analysis.(2)Preparation of a-HL Monomer Protein and Nanopore Assembly.a-HL monomer protein was prepared and assembled to heptamer nanopore.The prokaryotic expression system was used to replace the in vitro transcription and translation(?TT)system,and obtained higher protein yield.The used expression vector was E.coli BL-21(DE3)pLysS,and the a-HL monomer protein was purified by ion exchange gel purification method,size exclusion chromatography and ultrafiltration membrane purification method to obtain high purity a-HL.a-HL monomers were assembled on rabbit erythrocyte membrane to form heptamer(a-HL)7 nanopore.The activity of the heptamer protein,the stability and properties of the nanopore were examined by recording the current of a-HL single nanopore by patch-clamp amplifier.This method provides a basis for the understanding of protein nanopore and the application of single-molecule analysis.(3)Establishment of(?-HL)7/TMA-Cl Nanopore Analytical System.We used TMA-Cl,instead of KCl,as a novel analysis system for nanopores.Some unique characteristics of(?-HL)7/TMA-Cl nanopore analytical system were observed:(1)The stability of the planar lipid bilayer for embedding the protein pores was elevated at least 6 times.(2)The TMA-Cl system could effectively reduce the noise of single-channel recording.(3)It was easy to control the insertion of protein pores into the lipid bilayer,and the formed single nanopore could last for a sufficiently long time.(4)TMA-Cl could be used as a DNA speed bump in the nanopore to slow DNA translocation speed.(5)The ability of nanopores to capture DNA(capture rate)increased significantly and simultaneously increased the translocation time of DNA in the pore.(6)The TMA-filled nanopore could discriminate between various polynucleotides.(4)Application of(?-HL)7/TMA-Cl Nanopore Analytical System-Fast and Precise Detection of DNA Methylation.We used tetramethylammonium-based nanopore(termed TMA-NP)sensor that can quickly and accurately detect locus-specific DNA methylation.The advantages of methylation detection by TMA-NP sensor were observed:(1)Because of its methyl-philic nature,TMA can insert into the methylcytosine-guanine(mC-G)bond and then effectively unfasten and reduce the mC-G strength by 2.24 times.(2)Simultaneously,TMA can increase the stability of A-T to the same level as C-G.The abilities of TMA(removing the base pair composition dependence of DNA strands,yet highly sensing for methylated base sites)endow the TMA-NP sensor with high selectivity and high precision.Using nanopore to detect dsDNA stability,the methylated and unmethylated bases are easily distinguished.This method can directly detect DNA methylation at single-molecule level,which need either special recognizing elements,chemical modification or amplification of DNA,and should be applicable to the rapid analysis in epigenetic research.(5)Establishment and Application of(?-HL)7/GdnHCl Nanopore Analytical System.We used GdnHCl,instead of KCl,as a novel electrolyte for nanopores,based on the interruption of hydrophobic and hydrogen bonds by GdnHCl.A stable ?-HL nanopore was formed without any background signals,and the open current of ?-HL nanopore is linear and symmetrical with the voltage.We found the hydrophobic structure of the internal cavity changes by analysis of ?CD signals.Based on this system,the direct capture of ?CD and 1-Amantadine hydrochloride(1-AdNH2·HCl)complex by ?-HL nanopore was realized,which provided a new method for targeted drug delivery of drug-encapsulation system.Furthermore,the system was used to successfully distinguish the folded and unfolded states of Trp-cage miniprotein in GdnHCl denaturation.