A Basic Investigation of Cytochrome c and Upconverting Nanoparticles through Correlated Microscopic and Spectroscopic Analysis

Abstract

This thesis investigates the fundamental properties of Cytochrome c (cyt c) and Lanthanide-doped Upconverting Nanoparticles (UCNPs), exploring their potential applications spanning bioelectrochemistry to biomedical imaging. Cyt c is a versatile redox protein, serves multiple functions ranging from Cellular respiration to Programmed cell death. Its study has been extensively pursued through various methodologies due to its redox properties, making cyt c a focal point in Bioelectrochemistry Research. Researchers have explored immobilizing cyt c onto metal electrodes, either electrostatically or covalently, to manipulate its redox properties. Understanding the molecular dynamics that directly influence cyt c's functions holds promise for enhancing the efficiency of bioelectronic devices such as Biosensors and Biofuel cells. Various Spectro-Electrochemical techniques, such as Electrochemical-High-Speed Atomic Force Microscopy (EC-HSAFM), Surface-Enhanced Resonance Raman Spectroscopy (SERRS), and Surface-Enhanced Infrared Absorption (SEIRA), have been employed to probe the correlation between cyt c's dynamic nature and its redox properties when absorbed onto electrodes. This study employs a range of Fundamental Optical and Microscopic Techniques to explore the basic properties of cyt c, shedding light on its structural and functional attributes. Key methods such as Resonance Raman Micro-Spectroscopy, UV-visible spectroscopy, and FT-IR spectroscopy characterize the heme environment of cyt c, revealing the redox state of the heme group and detecting conformational changes upon ligand binding. Dynamic Light Scattering (DLS) determines the Hydrodynamic radius and distribution of cyt c in DI water, offering insights into the protein's physical state and stability in solution. Additionally, Microscopic techniques such as Atomic Force Microscopy (AFM) and correlated Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy (SEM-EDS) allows for direct visualization of the dynamic molecular behavior of cyt c uniformly deposited on Si wafers and analyzes their elemental composition of cyt c, revealing uniformly distributed cyt c with significant variations in elemental composition across the sample. Parallelly, Nanoparticles, particularly Lanthanide-doped Upconverting Nanoparticles (UCNPs), have garnered significant attention in biomedical sciences due to their unique properties. UCNPs are composed of inorganic crystalline host matrices doped with rare-earth lanthanide ions like Yb3+, Nd3+, and Tm3+, can upconvert low-energy near-infrared (NIR) radiation into higher-energy visible luminescence. This anti-Stokes shift luminescence avoids competition from autofluorescent background signals in biological systems, making UCNPs ideal for optical imaging. This study reports the use of NdGdF4 core UCNPs doped with 20% Yb3+ and 2% Tm3+ ions as multimodal optical imaging nanoprobes for single nanoparticle and HeLa cell imaging. The synthesized UCNPs exhibit resistance to photobleaching and no photoblinking, with cellular uptake visualized by background-free optical imaging under a NIR continuous-wave laser excitation source at 980