Preparation And Application Of The MspA Protein Nanosensor In Single Molecule Detection

Nanopore-based single molecule detection technology has great development potential in many fields such as chemistry, biology, medicine and environment. As the basic sensors, researchs of protein nanopores are significantly important for promoting the development of single molecule detection technology. Recently, many proteins such as ?-hemolysin (?-HL), MspA, ClyA, OmpF, SPI, phi29 and others have been explored and are playing their roles in detections of different analytes including ssDNA, dsDNA, biological macromolecules, small organic molecules and inorganic metal ions. Among these biosensors, MspA octamer protein nanopore has an excellent structure thus becomes another powerful tool besides the ?-HL nanopore in promoting the development of nanopore detection technology.In this paper, we developed a new method to prepare the protein nanopore MspA and studied the host-guest chemistry between the cyclodextrin molecules and small organic molecules using the MspA biosensor in order to detect the organic molecules. The main contents are as follows.1. Preparation and purification of the MspA nanoporeWe used the pT7(?-HL) plasmid as the template to give pT7 empty plasmid by double digestions enzyme reaction with Hind? and Nde? enzymes and ligated the MspA synthetic gene into the pT7 empty plasmid by ligase reaction to obtain the pT7(MspA) recombinant plasmid. This recombinant plasmid was transformed into E.coil BL-21(DE3)pLysS strain.Culturing the host bacteria under suitable conditions and inducing the high expression of MspA protein by isopropyl thiogalactoside (IPTG). Based on the super stability of MspA protein, we used the surfactant Triton-100 to extract MspA protein nanopore at a high temperature and found that the concentration of 0.5% extraction agent,90? and 30min are the most optimistic extraction conditions. Finally, we obtained a high yield, high activity MspA protein further by acrylamide gel and dialysis purification. The perforated patch clamp experiment showed the channel activity is good, which lay a foundation for subsequent single molecule detection researchs.2. Study of the cyclodextrins responses in MspA nanopore and detection of 1-Amantadine hydrochlorideWe detected the responses of cyclodextrins in the MspA protein nanopore including ?-CD,?-CD,?-CD,s7-?-CD,cm7-?-CD,am7-?-CD,per-6-N+(CH3)3-?-CD,per-6-NH3Cl-?-CD and so on. The results showed that only the cyclodextrins such as am7-?-CD, per-6-N+(CH3)3-?-CD and per-6-NH3Cl-?-CD have response signals in MspA nanopore. We presume this result due to the dense negative charge region in the wild-type MspA protein nanopore. We further studied the response of am7-?-CD in MspA under variations in voltage and buffer salt concentration. The results showed that the dwell time of am7-?-CD stuck in MspA reduces with the increase of the voltage whereas the blocking current remains constant. The experiments also showed that both the dwell time and the blocking current decrease when the buffer concentration increases.We further detected 1-Amantadine hydrochloride by the MspA nanopore based on the host-gust chemistry between am7-?-CD and 1-Amantadine hydrochloride, which is important for promoting the development of nanopore detection technology.