Quantification Of Single-molecule Of Protein And Investigation On Binding, Motion Of Ligands With Receptor In Living Cells Surface

In chapter one,significance of single molecule detection(SMD)and techniques were reviewed briefly.These techniques were powerful tools for investigation of dynamics and kinetics of molecules.New information can be obtained from the single molecule research,which is otherwise hidden or averaged out.Single molecule detection is a technology of studying biomolecules with high spatial and temporal resolution.Fluorescence microscopy was rapidly expanding from single molecule detection techniques into all fields of cell and molecular biology.Total internal reflection fluorescence microscopy(TIRFM)was introduced.An intimate summary of their applications was also provided with single-molecule analysis in vitro and single-molecule analysis in vivo.In chapter two,we developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy(TIRFM) with adsorption equilibrium.Rhodamine 6G(RtG)and DNA molecules labeled by R6G had been detected by TIRFM in literature.The limit of detection(LOD)was only 2.5×10^-9mol/L.We choosed Alexa Fluor 488-labeled goat anti-rat IgG(H+L) (IgG(H+L)-488)as the model protein.First,the solution of IgG(H+L)-488 was incubated with a glass microscope coverslip for a certain time.In this case,the adsorption equilibrium of protein was achieved between the solution and the coverslip.Then,fluorescence images of protein molecules in an evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally,the number of fluorescent spots corresponding to the protein molecules in the images was counted.The spot number showed an excellent linear relationship with protein concentration.The concentration linear range was 5.4×10^-11to 8.1×10^-10 mol/L.The low end of the linear relationship was 46-fold lower than reported in literature. In chapter three,the total internal reflection fluorescence microscopy combined with Epi-FM was used to study the early events of Transferrin and Transferrin Receptor-mediated transcytosis in living cells at single molecule level.The early stages of Transferrin and Transferrin Receptor processes included moving, congregation and internalization of Transferrin-Transferrin Receptor complexes, which have already been studied by morphological methods and biochemical methods. But most researches made judgement by the direct measurement of radioactive marker or the stability of fluorescent labelling marker after a large number of cytolysis.Due to the limitation of equation measuring method,these processes could not be observed dynamically.We has carried out dynamic study on the early events of Transferrin and Transferrin Receptor-mediated transcytosis at single molecule level, and the moving and congregation of Transferrin and Transferrin Receptor complexes in the real time.And there were no reports about real time tracking until now.These early processes using single molecule fluorescence detection were as follow.1.The process that Transferrin-QD(Tf-QD)in the solution bound to its receptor (TfR)on the cell membrane to produce a complex(TfR-Tf)was visualized in real time.2.The lateral moving and the congregation of TfR-Tf complexes on the plasma membrane were traced at single molecule level.Then,the congregation of two TfR-Tf complexes happened.3.At the temperature of 4℃and 37℃,the analysis of TfR-Tf complexes and the lateral moving on the plasma membrane were observed through surveying the movement of fluorescence spots.On the previous condition,the suffused modes of TfR on living MGC832 cell were free diffusion mode and restricted diffusion mode. Consequently,the transient diffusion coefficients of TfR-Tf complexes were calculated respectively by pixel coordinates correspond to sequential movements of fluorescence spots in pictures.In chapter four,we report the incorporation of biotin onto small drug molecules, the antagonist prazosin,and the agonist phenylephrine,through a series of reactions. Prazosin and phenylephrine were respectively labeled with quantum dot (QD)-streptavidins through the strong affinity between biotin and streptavidin to observe the binding between small drug molecules andα_1A-AR.Because of the unique optical properties of QD,more and more studies have been reported now concerning such fluorescent QD-marked drug molecules and application in fluorescence imaging of cells.However,most of such processes included complicated modifications on drug molecules and therefore gave rise to the problems of yield and separation. Moreover,the attached amount of drug molecules to QD is difficult to control. Another method is to biotinylate small drug molecules and then to bind them to QD-streptavidin.Though there is now report that the antagonist of dopamine transporter can bind to QD in such way,application in fluorescence imaging on living cell has not been seen.Motivated by these considerations,we have performed investigations as follows:1.Using a novel synthetic clue,we have attached biotin to prazosin and phenylephrine molecules.Then,QD-streptavidins were introduced to bind prazosin and phenylephrine specifically and effectively.In such way,the problems of yield and separation usually met in covalent binding were avoided.Also,the amount of small molecules attached to QD became much controllable because the number of streptavidin coated on QD was invariable.Because QD-streptavidin is available commercially,it is convenient to apply to the life science studies.Obtained results indicated that the QD-labeled prazosin and phenylephrine molecules still had pharmacological activity.That is,QD-streptavidin could work effectively as fluorescent probe in imaging of living cell.2.Comparisons are made on the binding abilities respectively betweenα_1A-AR on the cell surface and four biotinylated phenylephrines with different chain lengths. It is found that the phenylephrine with longer chain between biotin and phenylephrine can bind toα_1A-AR on the cell surface more easily.Correspondingly,more fluorescent spots would appear under the same condition.This reflected the possible steric hindrance on the binding ability between acceptor on the cell surface and ligands.3.The phenylephrine-QD and prazosin-QD were used to observe their binding and anchoring status with the acceptor on the cell surface ofα_1A-AR HEK293A. Several methods have adopted to support the binding specificity of prazosin-QD and phenylephrine-QD toα_1A-AR respectively.Furthermore,we also observed the function difference when agonist-phenylephrine and antagonist-prazosin were respectively bound toα_1A-AR.