Construction Of Single-molecule Nanopore Sensors Based On The ?-hemolysin Nanopore

In recent years,with the development of single-molecule science,scientists have gradually shifted from the study of macroscopic substances and their reaction characteristics to that of the microscopic systems.A series of single-molecule detection techniques have been developed to study the characteristics of molecules and the differences between molecules.For example,single-molecule spectroscopy,single-molecule force spectroscopy and so on were used to monitor single-molecule behaviors,chemical kinetics processes and so on.Although these technologies have solved many problems that cannot be solved by traditional ensemble methods,they also have defects.For example,the detection based on the solid surface,high requirements for sample surface properties,the need for fluorescent labeling,cumbersome sample preparation processes and so on.In order to remedy the above defects,nanopore-based technique,which is a biomimetic nanotechnology that can simulate the transport of ions or small molecules by transport membrane proteins on the cell membrane has been developed.Nanopore-based technique is realized by monitoring and analyzing the blocking current generated by the analytes when they interacted with the nanopore channel.Compared with traditional single-molecule detection technologies,nanopore-based technique has such advantages as simple sample preparation,label-free,high sensitivity,high resolution,high-throughput analysis,and real-time monitoring and so on.Among various nanopores applied in the field of single-molecule detection based on biological nanopores,?-hemolysin(?-HL)nanopore has attracted much attention due to its structural stability and easy modification.So far,researchers have constructed various nanopore detectors by introducing recognition sites,aptamers,or probes into the lumen of?-hemolysin nanopore by means of genetic engineering and chemical modification.It not only realizes high-sensitivity detection of ions and molecules,and then is used in the fields of environmental detection and medicine,but also is able to monitor many kinetic processes that cannot be monitored by traditional methods,may providing important information for researches to study chemical kinetics in depth.This study attempts to continue to carry out more in-depth functional modification of?-HL nanopore,aiming to build more nanopore detectors and broaden the range of the single-molecule detection.In this thesis,by means of genetic engineering and chemical modification,three kinds of nanopore detectors were constructed and then used to detect the catalytic intermediates of selenoenzymes,the coordination interaction between selenium and platinum and the pesticide paraquat,respectively.1.Single-molecule observation of selenoenzyme intermediates in a semisynthetic seleno-?-hemolysin nanoreactorThe capture of unstable intermediates is the key to verify the kinetic mechanism of the enzymatic reaction.Based on the nanopore-based technique,unstable intermediates can be observed,providing vital information for researches to investigate the complicated mechanism of kinetics.Therefore,in this chapter,we constructed a semisynthetic selenoenzyme nanoreactor by introducing the catalytically essential active site-selenocysteine(Sec)of natural Glutathione peroxidase(GPx)into the position 147 of?-HL nanopore using the genetic mutation and the Escherichia coli cysteine auxotrophic system.Taking advantage of the confined space of the protein nanopore,the designed semisynthetic seleno-?-HL nanoreactor can be able to mimic the catalytic behavior of Selenocysteine(Sec)in an actual protein microenvironment that resembles the substrate-binding pocket of natural enzymes for the reduction of H₂O₂ by GSH with a high efficiency.Meanwhile,it can also act as a nanopore sensor to observe the catalytic process and capture the key intermediates of GPx,including selenol(ESe H),selenenyl sulfide(ESe SG)and even the very unstable selenenic(ESe OH)(E represented enzyme)can be directly characterized.Our results may inspire future studies to explore a more sophisticated enzymatic kinetic process at the single-molecule level,which could provide deep insights into the structure-activity relationship of natural enzymes.2.Single-molecule observation of coordination interaction between selenium and platinum based on?-hemolysin nanoporePlatinum(Pt)is a metal element widely used in cancer treatment.However,platinum-based anticancer drugs are toxic to normal cells as well as killing cancer cells.Therefore,the design of platinum-based anticancer drugs with a high anticancer activity and small side effects is a research hotspot for scientists.Meanwhile,as an essential trace element in the human body,selenium is mostly existence in the form of Sec in vivo,which regulates the concentration of reactive oxygen species(ROS)and plays a vital role in preventing cancer.Hence,the new kind of anticancer drugs with high anticancer activity and smaller side effects could be designed based on the selenium-platinum coordination interaction.However,up to date,the investigation on the coordination interaction between platinum and selenium at the single molecular level is rare.Based on these,in this chapter,using the genetic mutation and the Escherichia coli cysteine auxotrophic system,a new mutant selenium-containing?-HL(M-Seleno-?-HL)nanopore sensor was constructed to investigate the coordination interaction between selenium and platinum at the single-molecule level.Based on this nanopore sensor,we can observe the single-molecule reaction signal of platinum and selenocysteine in real time.In addition,this nanopore sensor has high sensitivity and the limit of detection(LOD)can reach at the level of nanomolar.In this study,nanopore-based technique was used to study the coordination interaction between platinum and selenium at the single-molecule level for the first time,which may be helpful in the design of anticancer drugs.3.The detection of paraquat with pillar[5]arene as an aptamer in an a-hemolysin nanoporeThe pesticide paraquat(PQ)is highly toxic to animals and humans.Therefore,it is of great significance and urgency to develop efficient,sensitive,and rapid detection methods for PQ.The macrocyclic host molecule-pillar[5]arene(P[5]A)can recognize the PQ through the host-guest interaction,and can well match with the lumen of?-HL nanopore.Based on these,in this chapter,by means of the genetic mutation and chemical modification,carboxyl-pillar[5]arene(CP[5]A)acting as the aptamer was introduced into the lumen of the?-HL nanopore for constructing a nanopore sensor to detect PQ.In addition,by introducing positively charged arginine(Arg)at 111 and 147of the?-HL nanopore,the nanopore sensor exhibits better stability.More importantly,the nanopore sensor has high sensitivity and the LOD for PQ can reach the level of nanomolar.This is the first time that macrocyclic host molecular-P[5]A was introduced into biological nanopore for the construction of new nanopore sensor.We expect that this nanopore sensor can be applied in the field of public health.