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Title Page 2

Declaration 5

Contents 8

Abstract 10

Abbreviations 12

Chapter 1. Cytochrome c: Multifunctional redox protein for Life and Death 16

1. Introduction 16

2. Cytochrome c: composition and structure 18

3. Cell Localization and Functions of Cytochrome c 20

3.1. Mitochondrial Cytochrome c 20

3.2. Cytoplasm Cytochrome c 24

3.3. Nucleus Cytochrome c 28

3.4. Extracellular space Cytochrome c 30

4. Conclusion 31

5. Bibliography 32

Chapter 2. Exploring Cytochrome c: Microscopic and Spectroscopic Insights 35

1. Introduction 35

2. Materials and Methods 36

2.1. Materials 36

2.2. Sample Preparation 37

2.3. Characterization Techniques 39

3. Results and Discussions 42

3.1. ATR-FTIR and UV-visible absorption spectrum of Cytochrome c 42

3.2. Resonant Raman Spectrum of Cytochrome c 44

3.3. Topography studies of Cytochrome c on Si wafer via Atomic Force Microscopy 49

3.4. Measurement of Zeta Potential and Particle Size Distribution of Cytochrome c in DI water 52

3.5. Correlated SEM-EDS analysis of Cytochrome c 54

4. Conclusion 57

5. Bibliography 57

Chapter 3. Synthesis of Tm³⁺-doped Upconverting Nanoparticles as a Multimodal Optical Imaging Probe for HeLa cell Imaging via Photoluminescence Spectroscopy 59

1. Introduction 59

2. Photoluminescence Spectroscopy (Basic Principle and Instrumentation) 62

3. Materials and Methods 64

3.1. Materials 64

3.2. Sample Preparation 65

3.3. Experimental Techniques 69

4. Results and discussions 70

4.1. Photoluminescence Spectrum of tim³⁺ -doped UCNPs (stock solution) 70

4.2. Power-dependent Photoluminescence Spectra of Tim³⁺ -doped UCNPs 71

4.3. Photoluminescence Imaging of Individual Tim³⁺ -doped UNCPs 72

4.4. Cellular uptake and NIR-Excited Fluorescent Imaging of HeLa-cell by using Tim³⁺ -doped UCNPs 75

5. Conclusion 77

6. Bibliography 78

List of Tables 15

Table 1. Experimental conditions and measurement techniques for the analysis of cytochrome c... 38

Table 2. Assignments of the main Raman bands in oxidized Cytochrome c and their corresponding... 45

List of Figures 14

Figure 1. Different types of cytochromes based on their Heme group and binding patterns 17

Figure 2. Three-dimensional (3D) illustration of Equine heart Cytochrome c showcasing the heme... 19

Figure 3. The Role of Cytochrome c in the Electron Transport Chain. ETC comprises four main... 22

Figure 4. The Role of Cytochrome c in Apoptosis, Pyroptosis, and Persister Phenotype (a) In... 27

Figure 5. The Role of Cytochrome c in the Nucleus. Cytochrome c enters the nucleus and directly... 29

Figure 6. The Role of Cytochrome c in the Extracellular space. Cyt c moves from damaged cells... 31

Figure 7. (a) ATR-FTIR spectra of Cytochrome c display transmittance bands at 3350 cm⁻¹,1650... 44

Figure 8. Time-resolved Resonance Raman Spectrum of cytochrome c (0.1. nM in 1 mL distilled... 47

Figure 9. In situ Raman mapping of cytochrome c distributed onto the Si wafer. Illustrated the... 48

Figure 10. 2 and 3D AFM images of Si wafer (a) polished side (b) unpolished side,... 50

Figure 11. 2D and 3D AFM images of free Cytochrome c at various positions (a) Schematic... 52

Figure 12. Hydrodynamic size measurement of Cytochrome c (1 mM) dispersed in deionized... 54

Figure 13. Correlated SEM-EDS measurement of powder form of Cytochrome c (left) EDS... 55

Figure 14. SEM images of cytochrome c deposited at various positions on the Si wafer,... 56

Figure 15. Different types of nanoparticles 61

Figure 16. Energy diagram illustrates the absorption of light and the subsequent emission... 63

Figure 17. Block diagram of the PL Spectroscopy 64

Figure 18. Synthesis of Oleic acid coated NdGdF₄: 20%Yb³⁺, 2%Tm³⁺ core UCNPs by co-... 67

Figure 19. Modification of oleic acid-coated core UCNPs with methoxy-PEG polymers resulting... 68

Figure 20. Bright-field imaging of HeLa cells at different positions 69

Figure 21. Schematic illustration of the Energy level diagram of Tm³⁺ion (a) and their PL spectrum... 71

Figure 22. Laser-power resolved upconversion luminescence spectrum of NdGdF₄: 20%Yb³⁺,... 72

Figure 23. Single nanoparticles imaging of Tm³⁺-doped NdGdF₄ core UCNPs via... 74

Figure 24. Schematic illustration of cellular uptake of Tm³⁺-doped NdGdF₄ UCNPs in the HeLa... 76

Figure 25. Schematic illustration of cellular uptake of Tm³⁺-doped NdGdF₄UCNPs in the HeLa... 77

초록보기

 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, that 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) allow for direct visualization of the dynamic molecular behavior of cyt c uniformly deposited on Si wafers and analyze 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⁺, which 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㎚.

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