Construction Of The Multi-function ?-hemolysin Protein Nanopore And Its Application For Single-molecule Sensor

Nanopore analysis is one of the attractive techniques which has been applied extensively in different fields including single-molecule detection,single-molecule chemical reaction and DNA sequencing because of its specific merits,such as lable-free,high tolerance to interference etc.However,the detection of small moleeules and analytes with diameter larger than the inner diameter of adopted channel are constrained by protein nanopore which did not engineered.The protein nanopore can be modified by genetically engineered recognition sites inside the channel and noncovalently bound internal host molecules such as cyclodextrin inside the channel lumen,greatly expands the research field of nanopore single-molecule technique.Here,we report a new nanoreactor and detector,utilized a newly synthesized per-6-quatemary ammonium-?-cyclodextrin(p-QABCD)as an adaptor in the mutant aHL protein(M113R)7 nanopore,to study reaction pathways,intermediates and products between GSH molecules and Cd2+ ions at single-molecule level.Furthermore,we report a strategy for introducing liposome signal magnification system into nanopore analysis to expand the research field of nanopore single-molecule technique.The main contributions of this dissertation are summarized as following:1.Protein nanopore sensor for real-time monitoring single-molecule chemistry pathways between divalent cadmium ion and glutathioneThe chelating reaction between glutathione peptides and divalent cadmium ion has been used as a typical model for investigating the coordination chemistry of SH-containing peptides and heavy metal ions,which is essential to understand the mechanism of intracellular cadmium detoxification.Here,we used the mutant(M113R)7 ?HL protein nanopore equipped with a first synthesized per-6-quaternary ammonium-?-cyclodextrin(p-QABCD)as nanoreactor and detector to study the single-molecule reaction of GSH molecules and Cd2+ ions.Different reaction pathways,intermediates,and products can be recognized.Cd2+-GSH reaction is highly dependent on the solution pH.The Cd(GSH)2 was formed at pH 7.4,and the Cd(GSH)2 and Cd2(GSH)2 were formed at pH 9.0 respectively.Cd2(GSH)2 is formed by two possible pathways:1.A Cd2+ ion primarily coordinates with the thiol group of two GSH molecules to form Cd(GSH)2,and then the second Cd2+ ion quickly incorporates with the deprotonated amino group of Cd(GSH)2 to form Cd2(GSH)2·2,Two Cd2+ions separately coordinate with the thiol and deprotonated amino group of one GSH molecule to form Cd2(GsH),,and the second GSH molecule quickly bound to Cd2+ions to form Cd2(GSH)2.Our work studied the chelating reaction between metal ions and bioactive glutathione at single-molecule level without labeling and chemical modification,and would be important to further understand intracellular mechanisms of detoxification of heavy metals and greatly expand the research field of nanopore single-molecule technique.2.A nanopore sensing strategy based on liposome signal magnificationWe have developed a nanopore sensing strategy based on liposome signal magnification for the detection of avidin which is too large to transfer through nanopore.Based on the fact that the mutant(M113R)7?HL protein nanopore can release substance encapsulated in lipsome through implantation in lipid bilayer membrane,when surface of the liposome was modified by a larger analytes,the steric hinderance of approach of aHL is formed.Hence the release of capsulated marker molecules is inversely proportional to the biomolecules.In the proposed strategy,we used IP6 as the signal magnification marker and synthesized IP6 encapsulated liposome.Biotin was modified on the surface of liposome as the biorecognition element to which avidin can bind with high affinity.The steric hindrance formed on liposomal surface through avidin-biotin conjugation plays crucial role in repelling hemolysin attack.The release of signal maker IP6 decreased as avidin concentration increased.The strategy provides a new way for protein nanopore detection technology and greatly expands the research field of nanopore single-molecule technique.