Single Particle Tracing Technology Based On Dark Field Imaging To Study The Interaction Between Gold Nanorods And Cells

In recent years,in order to meet the needs of development of disease diagnosis and precision medicine,the interaction between nanoparticles and cells has attracted wide attention.The interaction between nanoparticles and cells is mysterious and complex due to the complexity of cells and the diversity of nanoparticles.However,it is no longer enough of researchers to study the complex interaction between nanoparticles and cells more comprehensively with the traditional macro-average method.Therefore,single-molecule/single-particle tracking technology,tracking dynamics information of single-molecule/single-particle in real time,has been favored by researchers.In this thesis,a single particle tracer technology using gold nanorods(AuNRs)as a probe was constructed based on the traditional dark-field imaging method,with which interaction between gold nanorods and cells was further analyzed.Through analyzing the translation and rotational dynamics of tracing particles with various methods and means,the constructed single particle tracking method can be used to pry into the movement of cellular material,thereby uncovering the mystery of complex cellular mechanism.This thesis is mainly discussed from the following three aspects:(1)Construction of the translational and rotational single particle tracking technology based on gold nanorod probeBased on the scattering polarization of AuNRs,we constructed a simple but robust single particle tracking method with traditional dark-field microscopy,which can track the translational and rotational information of single AuNRs.The principle of this method is that under dark-field imaging conditions,AuNR appears as a scattering spot in the color camera,and the intensity of the spot is related to the angle between long axis of AuNR and the optical axis(polarization angle),that is,the polar angle of AuNR can be calculated with the its scattering intensity values in the three imaging channels of the color cameras(R,G and B channels).Here,we use G as internal reference and calculate polar angles with(R-G).Compared to other methods with AuNRs as probe,the advantages of the two-channel differential method are simple,lower systematic errors and higher measurement accuracy of polar angles.Furthermore,the feasible and reliable of the two-channel differential method to calculate the polar angle was verify through experiments and data simulations.Finally,we used this method to analyze the rotational behavior of CTAB-AuNRs when they interact with cells with different receptor protein(PD-L1)expression,demonstrating that PD-L1 receptor proteins affect the interaction of AuNRs with cells.(2)Study on the dynamics of gold nanorod diffused on cell membrane with single particle tracking technologyWith the two-channel differential method,we investigated the diffusion behavior of functionalized AuNRs on MCF-7 cell membrane.In order to meet the experimental requirements,we rationally designed the AuNRs probe in advance.The results of the research indicate that functionalized AuNRs undergo “random-waiting-time confinement” and “instantaneous long-range hop-motion” on the cell surface,which is caused by the dynamic picket-fence structure of the membrane.The complex interaction between AuNRs and cell membranes makes the translational and rotational behaviors of AuNRs on the cell membrane to be spatially and temporally heterogeneous.Similarly,the single particle tracking method provides a new direction for the study of complex cell membrane structures.(3)Study on interaction between various modified gold nanorod and cell with single particle tracking technologyWe used the two-channel differential method to monitor the interaction of different modified AuNRs with MCF-7 cells.We found that various modified AuNRs have distinct dynamic properties when they interact with cell,including the adsorption phase,membrane-diffusion phase,transmembrane phase and intracellular transport phase,due to the differences in ligand-receptor interaction,cell-membrane distribution of receptor,cell working mechanism and so on.Therefore,a detailed analysis of the interaction between nanoparticles and cells helps us understand and control the mechanism of cell function,which in turn advance the development of disease diagnosis and precision medicine.