Study On The Technology And Applications Of Single Molecule Force Spectroscopy

Single molecule force spectroscopy(SMFS)has emerged as a powerful tool to investigate the intrinsic of life in a single molecule level,and also played an important role in the area of disease diagnosis,drug discovery,biomaterials and biomimetic engineering.The most common force spectroscopy techniques are atomic force microscopy(AFM),optical tweezers and magnetic tweezers.However,with different techniques and methods,the results would be different,which makes it hard to compare these measurements.This has been the key issues in SMFS,which definitely hindered the development of single molecule force spectroscopy.This dissertation focus on the measurement of forece and positon,with techniques of AFM and optical tweezers,discussed the effect of the tips spring constant and the sample design on the feasibility and veracity of single molecule force spectroscopy,and then investigate the mechanical unfolding and folding of proteins for applications.On the other hand,a new system for multiple single molecule force spectrum measurement in parallel was developed,which paved a new way to investigate the single molecule force spectrum.The main achievements of this dissertation work are listed in the following: 1.A review of development in single molecule force spectroscopy was presented,including the sample design,techniques and instruments,as well as the methods for measurement.Then elaborate the theory for calculating the dynamic parameters in mechanical unfolding and folding of proteins,and analyzed the influence factor in the measurement of force with different techniques.2.Using a polyprotein(Nu G2)8 as model system,we studied the effect of device stiffness in single molecule force spectrum experiments with single molecule AFM technique.Meanwhile,we employed AFM-based single molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and folding of a metalloprotein Ferredoxin.The results demonstrate that the unfoldingrefolding of individual ferredoxin is reversible,enabling new avenues of studying both folding mechanism and reactivity of the metal center of metalloproteins in vitro.3.Using a small protein Nu G2 and its polyprotein as model systems,here we use optical tweezers to investigate the mechanical unfolding and folding.It's the first time to demonstrate that indeed stretching a polyprotein of Nu G2 is equivalent tostretching single Nu G2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.In the meantime,we employed optical tweezers-based single molecule force microscopy and protein engineering techniques to study the mechanical unfolding and folding of a pair of protein GA/GB and their mutant proteins.It's the first time to demonstrate that the sequence determine the protein folding,structure and function through the kinetics of protein.4.A high-throughput single molecule force spectroscopy based on the centrifugal force was developed.Also we propounded a high resolution and fast method based on the digital holographic microscopy and radial profile image matching technique for 3D tracking of beads in liquid.In the meantime,a software based on CUDA parallel computing with GPU was developed,which will facilitate the development and use of high-throughput single molecule approaches.Then,the applicability of this system was tested by single molecule experiments.This system overcomes the limit of the measurement for only one molecule once a time in AFM,optical tweezers,which would improve the efficient of single molecule force spectroscopy experiments and avoid the effect on the veracity of measurements because of the diversity of sample or the environmental changes.