Using Atomic Force Microscopy To Study Celluar Structure And Property And To Discriminate The Single Base Mismatch

Cell envelope, including cell wall and cell membrane is the interface between cell and outer environment for material and energy exchange. It also provides protection of cell organelle and target sites from chemical or physical stimulations. The investigation of cell envelope at the nanometer level is very important in understanding the relationship between its structure and function, comprehensively evaluating the drug efficacy and providing evidence toward the development of medicines. On the other hand, the detection of single base mismatch is critical to illuminate the nosogenesis of some disease and realize the gene therapy, which appeals us to develop convenient, accurate and sensitive assays. Both of the above two issues are the promising directions in bio-analytical chemical research, and deserve devotion of analytical chemists.In this dissertation, utilizing the advantage of the atomic force microscopy (AFM), including the characterization of the surface topography at the atomic level and the measurement of intermolecular forces as low as 10-12 N (pN)grade, we investigated the effect of different antibiotics and the pulsed electric field (PEF) on cell envelope, established the optimized condition for the cell imaging and improved the detection ability toward the single base mismatch on the basis of the hairpin probe. The main researches of this dissertation are summarized as follows:1. AFM study of different effects of natural and semisyntheticβ-lactam on the cell envelope of E.coli. The distinct effect of amoxycillin and penicillin has been investigated using tapping mode imaging in air. The results indicated that although amoxycillin and penicillin could both induce nanoporous damage to the envelope of E. coli, the distribution of the pores was different: those induced by penicillin were randomly distributed on the cell surface, while those induced by amoxycillin were far more numerous and mainly on the two ends of the cell. These findings could explain why the effect of amoxycillin is stronger than that of native penicillin. Ofloxacin was used as a control due to its inactive membrane, and no cell wall damages were observed. It has been demonstrated that AFM is a useful tool in discerning and verifying antibiotic mechanisms at nanometer level and can be helpful to explain the relationship between chemical structure and the function of antibiotics.2. The effect of PEF on the cell envelope components of S. epidermidis was investigated by the combination of AFM imaging and force measurement under PBS. The topography of bacteria and interaction force between bacterial envelope and tip was probed in situ before and after different dosages of applied PEF. The results showed that the introduction of PEF would decrease the amount of the immobilized cell and induce the multi-unbinding force between AFM tip and the cell surface, both of which were more obvious with the increasing dosage of PEF. It was deduced that PEF could induce the collapse of the peptidoglycan layer and then the exposure of the plasma membrane, which was further confirmed by the effect of the lysozyme and heat on the bacterial envelope. The above conclusions offered strong evidence for the bactericidal mechanism of PEF at the molecular level, and demonstrated that the AFM is a powerful technique to explain the relationship between the chemical component change of the cellular envelope and the external stimulation.3. cell imaging was realized in air and under liquid, and the experimental conditions were optimized. First, three kinds of cell fixed with different fixatives were imaged in air and the qualities of image were evaluated individually. Among them, 0.5% glutaraldehyde kept the fine structure of all cells. Then, all these cells were successfully imaged under physiological media. The effect of the scan rate and different medium on cell imaging was investigated. Both MC and NRK cell could be imaged with clear cell cytoskeleton in the culture medium and the duration was about one hour. These results paved the way for the further cell investigation based on AFM technique.4. Using force spectroscopy analysis to improve the properties of the hairpin probe. The sensitivity of hairpin-probe-based FRET fluorescence analysis was sequence-dependent in detecting single base mismatches with different positions and identities. Through modified tip and substrate with hairpin probe and complementary sequence individually, the detection sensitivity of single base mismatch was systematically investigated by force spectroscopy analysis. The uneven fluorescence analysis sensitivity was obviously influenced by the GC contents as well as the location of the mismatched base. However, force spectroscopy analysis displayed a high and even sensitivity in detecting differently mismatched targets. This could be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe and to enhance the probe ability in mismatch detection.