Gold Nanorods Synthesis, Engineering And Their Applications In Single Molecule Imaging As Orientation Probes

Single molecule techniques are significant for investigating the basic science. They can obtain not only the common properties of the objects of research but also the individual events, which can reveal the unprecedented information that the traditional method can not achieve. As single molecule optical probes, gold nanorods (AuNRs) have several obvious advantages in contrast to traditional fluorescent dyes. They have strong optical signals, high photostability and can be easily synthesized and engineered. More importantly, the absorption and scattering of light by AuNRs are polarized, which make them be suitable to probe orientations. However, there are few studies to investigate the biological and physical events by using AuNRs as orientation probes. There are three crucial reasons that contribute to the result, that is, the lack of well preparing and engineering methods for obtaining AuNRs of specific sizes, the deficiency of suitable techniques to decipher their orientations and the lack of appropriate research systems.In this paper, we did several studies to attempt to address the above problems. The main investigations are as follows.(1) In chapter2, we synthesized high-quality AuNRs by using H2O2as the reducing agent through seed-mediated methods under alkaline conditions for the first time. The impacts of solution pH value, H2O2/Au3+ratio and the concentration of silver ion on the size and morphology of AuNRs were all investigated by UV-Vis spectroscopy and transmission electron microscope (TEM). It was found the alternations of three factors would lead to the change of aspect ratio of AuNRs and the corresponding longitude localized surface plasmon resonance (LLSPR) peak. pH higher than～6.0is essential for preparing AuNRs. Compared with the AuNRs through traditional method using ascorbic acid as the reducing agent, the AuNRs by H2O2have good morphology and improved monodispersity. In addition, our approach aided to understand the mechanism that how pH variation affect the morphology of nanomaterials.(2) Based on AuNRs synthesis, in chapter3, we further engineered AuNRs by post-processing procedures, which have high light scattering signals and are suitable for single molecular optical probes. At the beginning, the theories on light absorption and scattering of AuNRs were presented and the simulated results of aspect ratio and environmental medium on LLSPR were also demonstrated. AuNRs with aspect ratio around2.2have been proved to have strongest scattering efficiency and be suitable for single molecule imaging by using CCD. In the process, AuNRs with small diameter were synthesized first by traditional seed-mediated method and then large AuNRs were obtained through overgrowth process by using them as seeds. By controlling the synthetic conditions, the desired AuNRs were obtained. The alternations of UV-Vis spectra, the length, and the diameter were investigated by spectrophotometer and TEM. At last, single AuNRs scattering images under dark-field microscopy were obtained. By rotating a polarizer in the optical path, the question whether the scattering intensity of AuNRs through overgrowth is polarized was addressed, and the AuNRs are proved to be appropriate to probe orientations in single molecule imaging.(3) In chapter4, we developed new techniques to study the transmembrane dynamics of single AuNRs during their endocytosis. Through theoretical calculations, we provided the approach to determine the orientation of AuNRs in three-dimensional space under different illumination schemes, especially dark-filed oblique illumination. The impacts of focus plane placed at cell top and at cell sidewall on studying the transmembrane dynamics were investigated. Cell sidewall imaging mode would improve the obtained angle accuracy, be more proper to get the angle of AuNRs against membrane and be convenient to capture the whole transmembrane process. Finally, the real-time transmembrane processes verified that cell sidewall imaging under dual-channel darkfield microscopy was versatile to study the transmembrane dynamics of single AuNRs.(4) With excellent materials and techniques, we further investigated the translational and rotational behaviors of single AuNRs on membrane in chapter5. We presented the theories on translation and rotation of single AuNRs. We mostly focused on the variation of translation and rotation of AuNRs during their translocation on membrane, analyzed the corresponding biological events, and preliminarily revealed the decoupling of translation and rotation. At last, we given the statistical results of translational diffusion coefficients and rotational diffusion coefficients of41AuNRs on membrane and found the translation and rotation of AuNRs on membrane are independent based on the statistical parameter-correlation coefficient.(5) The azimuthal and polar angles of AuNRs obtained through optical techniques are defined with respected to the polarization direction of crystal and the imaging plane, respectively. Therefore, in chapter6, we studied the spatial distributions of AuNRs in polar coordinate system. Three rotational modes-partially confined rotation, restricted rotation and freely Brownian rotations, were found, which were closely related to endocytosis process. The rotational modes, combined with azimuthal and polar angle, out of plane angle, trajectories, the translational and rotational diffusion coefficients perfectly disclosed the detailed endocytosis dynamics. It is found that the mode variations can determine the exact time of the end of the endocytosis.(6) The orientation dynamics of single protein-coated AuNRs at solid-liquid interface were investigated in chapter7. There are different rotational dynamics at interface, which reflect the interaction affinity between single protein-coated AuNRs and surface. The protein-coated AuNRs were absorbed on C18surface at high concentration salt solution first and then eluted with solution containing low concentration of salt to investigate orientation dynamics during desorption process. The distributions of these dynamics states changed when decreasing the salt concentration. The real-time desorption process of single AuNRs further illustrated that the desorption of molecule from surface must be preceded by rotation.