Single Molecule Fluorescence Imaging On Solid/liquid Interface Of Single DNA And Protein As Probes

Single molecule detection (SMD) refers a set of ultra sensitive techniques. In the past two decades, single molecule and single cell detection techniques have proved to be important methods to unveil the physics, chemistry and mechanisms of many important biological processes. Among them, single-molecule fluorescence imaging techniques are used most often.In the past one decade or so, single molecule fluorescence imaging has reached the regime of detecting and monitoring the properties of individual molecules in different nanoenvironments, including solid/liquid, liquid/liquid interfaces and in live cells. Solid/liquid interface is one of the most important models for investigating biomolecule behaviors in vitro. Single-molecule fluorescence imaging has great potential for real-time observation of the motion, aggregation, dynamic heterogeneity and kinetic states of molecules, revealing differences between individual behaviors and the ensemble average behavior, and catching rare events that are often hidden in the macroscopic range. The observation of single biomolecules can provide insights into molecular genetics, biochip assembly, biosensor design, biophysics and basic separation theories of liquid chromatography and capillary electrophoresis. This thesis contains the following three sections:(1) Optimization of image analysis for single molecule fluorescence imagingSuccessful image processing and analysis is very important for information extraction from single molecule fluorescence detection experiments. But it is often a daunting task given the huge amounts of data obtained. Free and open source image analysis software Image J provides a powerful tool for such tasks. Compared to manual operation, image analysis using Image J is fast and reproducible, so we can save a lot of time on data analysis. Here, we introduce the application of Image J to identify the molecular adsorption/desorption process, count adsorbed molecules, determine the fluorescence intensity and single-step bleaching of individual molecules. The effects of frame average, radius of 2-D rolling-ball background subtraction algorithm, the threshold and single particle size selection on the reliability of single molecule counting were also discussed. We have taken the movie of single B-phycoerythrin (B-PE) molecule adsorption on a glass surface as an example. It indicated that optimization of image analysis parameters are very important for the reliability of single molecule fluorescence imaging analysis.(2) Single molecule fluorescence imaging of DNA molecules at agarose surfaces Agarose gel has been widely used in gel electrophoresis to separate and purify biological molecules such as nucleic acids and proteins. However, relatively little is known about dynamic interactions between stationary phase and individual biomolecules at single molecule level. These fundamental properties are important for optimizing bio-separation methods such as size exclusion chromatography and electrophoresis. In the present work, we have studied dynamic behaviors of YOYO-1 labeled singleλ-DNA molecules on agarose-modified surface using objective-type total internal reflection fluorescence microscopy (TIRFM). The adsorption and motion of individual DNA molecules at the interface have been monitored as a function of pH, ionic strength and agarose concentrations. We concluded that the quantity and behavior of DNA adsorption on agarose surface vary with the change of pH and ionic strength. The behavior of DNA absorption can be attributed to a combination of hydrophobic attraction, electrostatic repulsion and surface roughness. Both electrostatic repulsion and hydrophobic attraction play a major role while the surface topography also affects DNA adsorption dynamics at agarose-modified glass surfaces.(3) Single molecule fluorescence imaging analysis of 2-mercaptoethanol induced adsorption of B-phycoerythrin on silica surfaceThe photobleaching and blinking of individual fluorophores have limited the application of single-molecule fluorescence imaging. In many single molecule fluorescence imaging studies, 2-mercaptoethanol (BME) is used to reduce the photobleaching rate, decrease blinking and improve the fluorescence intensity stability of fluorophores. But the effect of BME on other single-molecule behaviors has not been investigated in detail so far. Here, we investigate events of BME-induced adsorption of B-PE on silica surface. The effect of BME on the adsorption of B-PE is studied quantitatively using single-molecule fluorescence imaging, and the mechanism of BME-induced adsorption of B-PE on silica surface was also analyzed. It indicated that as the concentration of BME increased, the adsorption of B-PE on glass surface increased, the number of adsorbed B-PE increased to 16.0 times of that without BME, and the fluorescence intensity also enhanced. However, higher concentrations of BME cause many proteins to denature. As a result, the number of adsorbed B-PE decreased, and the average fluorescence intensity reduced. The results were consistent with that of fluorescence spectroscopy measurement.According to these results, it can be summarized that we have achieved the reliable and rapid single molecule fluorescence imaging analysis, observed the dynamic process of biomolecules at the liquid/solid interface in real time and investigated the fundamental interactions that affect the single molecule adsorption at surfaces. These offered us valuable insights into chromatography separation mechanisms and DNA micro array and biosensor fabrication.