LASER-ASSISTED SYNTHESIS OF GOLD AND SILVER NANOPARTICLES AND SPECTROSCOPIC STUDY OF THEIR INTERACTION WITH PROTEIN DEEPTI JOSHI

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1 LASER-ASSISTED SYNTHESIS OF GOLD AND SILVER NANOPARTICLES AND SPECTROSCOPIC STUDY OF THEIR INTERACTION WITH PROTEIN DEEPTI JOSHI DEPARTMENT OF PHYSICS INDIAN INSTITUTE OF TECHNOLOGY, DELHI FEBRUARY 2016

2 Indian Institute of Technology Delhi (IITD), New Delhi, 2016

3 LASER-ASSISTED SYNTHESIS OF GOLD AND SILVER NANOPARTICLES AND SPECTROSCOPIC STUDY OF THEIR INTERACTION WITH PROTEIN by DEEPTI JOSHI Department of Physics Submitted in fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY to the INDIAN INSTITUTE OF TECHNOLOGY DELHI February 2016

4 CERTIFICATE This is to certify that the Thesis entitled Laser-assisted synthesis of gold and silver nanoparticles and spectroscopic study of their interaction with protein being submitted by Ms. Deepti Joshi to the Department of Physics, Indian Institute of Technology Delhi, for the award of degree of Doctor of Philosophy is a record of bonafide work carried out by her. She has worked under my guidance and supervision and has fulfilled the requirements for the submission of this thesis, which in my opinion has reached the requisite standard. The results contained in this dissertation have not been submitted, in part or full, to any other university or institute for award of any degree or diploma. Date Professor R. K. Soni Department of Physics Indian Institute of Technology Delhi New Delhi , India

5 ACKNOWLEDGEMENTS I was eagerly waiting for the day, which makes the outset for another journey with the personified glory of the current one. I convey my thanks to all my dear ones as without their blessings and support, it was not possible to reach this juncture of fulfillment. First and foremost, I would like to express my gratitude to my supervisor Prof. R. K. Soni, for giving me opportunity to work with him. I am deeply indebted for his supervision, honest advice, guidance and positive criticism throughout my research work. This thesis would have not been completed without his enthusiastic encouragements, effortful inspiration and constant help. I would also like to thank Prof. Harpal Singh and his student Mr. Manu Dalela, Centre for Biomedical Engineering, IIT Delhi for Raman and fluorescence measurements and valuable interpretations of the data. I also acknowledge the help of Prof. A.K. Ganguly and Mr. J.P. Sharma Chemistry Department, IIT Delhi for FTIR measurements. I would like to thank Mr. Vinod Khanna for TEM measurements and Mr. Rishab Sharma for HRTEM measurements. I express my sincere thanks to my lab-colleagues, past and present especially Dr. Geetika Bajaj, Dr. Rina Singh, Dr. Jyoti Katyal, Deepanshu, Rakesh, Naresh, Navas and Rupali for their help and providing an excellent working environment. I would like to thank members of Remote Sensing Technology Division, Laser Science & Technology Centre (LASTEC) and my colleagues from Centre for High Energy Systems & Sciences (CHESS) for their direct or indirect support to continue my Ph.D. work. i

6 It s difficult to express gratitude for my husband, Dr. Vikas Joshi whose constant support, understanding and love helped me to complete my research. He always stood by my side in tough times providing strength and courage to handle the situations. I am thankful to my sons Aniruddha and Kovid who had been a source of relief and fun. There is a shortage of words to express my heartfelt thanks to my siblings, my sister (Divya Mishra) and brothers (Rakesh Mishra, Sandeep Mishra), I am thankful to my friend Dr. Aditya verma for her constant motivation and having many fruitful discussions. On a more personal note, an inexpressible, heartfelt thanks to my parents and in laws for their moral support, inspiration and unconditional love without which it would have not been possible to complete this work. Finally, I thank Shiva for giving me the strength to complete this task and blessing me with the people around who helped me during this journey. Date: Deepti Joshi ii

7 ABSTRACT Pulsed Laser Ablation in Liquid (PLAL) has gained increasing technological importance with specific attention to the fabrication of metal nanoparticles for biomedical applications. It is a chemically clean synthesis technique and generates ligand free metal nanoparticles. Due to purity of PLAL generated ligand free metal nanoparticles, higher amount of molecules can be attached to their surface, which is useful particularly if expensive biomolecules are conjugated. By extending metal target ablation in biomolecule solution, in situ bio conjugation with size reduction and stabilization of nanoparticles can be achieved. Laser generated metal nanoparticle biomolecule conjugates and laser induced photothermal heating under metal nanoparticle plasmon condition have developed into an increasingly important research area due to their wide spread application at the frontier of both material and biomedical science. When nanoparticles are exposed to bio fluids, first they interact with proteins, thus making protein nanoparticle interactions of particular interest. The protein-metal nanoparticle bio conjugates play vital role in the studies of biological systems. This thesis deals with the study of growth of metal nanoparticles (gold and silver) by unconventional pathway of pulsed laser ablation in liquid, with the final aim of studying their interaction with protein. Two approaches were exploited for this objective, the first one comprises laser ablation of metal in water and subsequent conjugation with protein (ex situ conjugation) and the second one comprises laser ablation of metal in protein solution (in situ conjugation). Three proteins bovine serum albumin (BSA), S-ovalbumin and fresh egg white protein have been considered. Gold nanoparticles are perfect converter of light to heat due to their large absorption cross section along with very low radiative decay. This property makes gold nanoparticles best iii

8 candidate for photothermal therapy of cancer cells. Therefore, photothermal heating of gold nanoparticles under plasmonic condition has been investigated theoretically and experimentally. To explore the morphology of the synthesized nanoparticles and protein nanoparticle conjugates, structural investigations are carried out by transmission electron microscopy (TEM) and High Resolution Transmission Electron Microscopy (HRTEM). Optical properties of synthesized nanoparticles and protein nanoparticle conjugates are investigated by UV-visible absorption spectroscopy. The modifications in protein s conformation after interaction with nanoparticles are studied by spectroscopic techniques like Raman, Fourier transform infrared (FTIR) and fluorescence spectroscopy. In present work, gold and silver nanoparticles have been successfully synthesized by laser ablation of gold and silver target in ultrapure water at varying laser fluence (energy per unit area). Metal nanoparticles were formed due to aqueous oxidation of active metal species by water molecule at high temperature and high pressure conditions. Due to the presence of oxidation states in laser generated metal nanoparticles these can be easily functionalized with electron donor moieties like SH, SS, NH2, and COOH etc. present in protein. Conjugation of synthesized gold and silver nanoparticles with protein showed critical dependence on laser fluence and protein concentration. The laser fluence affects the plasma density and distribution of species in the plasma. A rise in the laser fluence value causes an increase in the density and intensity of the hot plasma, which results in synthesis of larger nanoparticle. Experimental results demonstrated that both Au and Ag nanoparticles interact with protein via cysteine residue in protein via dative covalent bond. Gold makes stronger bonding with protein than silver indicating that gold is better metal than silver for conjugation with protein. Due to conjugation with metal nanoparticles via cysteine residue, increase in hydrophobicity around tryptophan residue has been observed. iv

9 In situ conjugation process has been explored for metal nanoparticle-protein conjugates (AuNP-S-ovalbumin and AgNP-S-ovalbumin) synthesis. This process involves ablation of metal target in S-ovalbumin solution and evidenced narrowing of the nanoparticle size distribution. This narrowing is accompanied by the reduction in the size of particle, due to quenching of growth in presence of S-ovalbumin. The decrease in mean particle size and narrowing of size distribution demonstrated dependence on S-ovalbumin concentration. The effect of various factors such as aging and presence of salt on the stability of conjugates has been studied. The UV-visible absorption spectra reveal that in situ conjugation provides stabilization to the nanoparticles against aggregation. On post irradiation with 532 nm of Nd:YAG laser, absorption spectrum of AuNP-Sovalbumin conjugate shows red shift of 9 nm as well as broadening in SPR peak. The origin of red shift and broadening is aggregation of AuNPs after thermal degradation of S-ovalbumin on post laser irradiation. With increasing time of post irradiation blue shift in SPR peak is observed due to laser induced fragmentation of AuNPs. On post laser irradiation under same experimental condition no significant change in AgNP-S-ovalbumin absorption spectra observed. Thus thermal degradation of protein and size reduction in nanoparticle is observed when the post laser irradiation laser wavelength is in proximity of the plasmon resonance peak. On in situ conjugation no change in protein secondary structure and integrity of S-ovalbumin has been observed. Finite difference method is employed for theoretical calculation of laser heating of gold metal sheet to understand the heat generation by gold nanoparticles. Further, the laser heating of gold nanoparticles has been compared with aluminum nanoparticles under plasmonic condition. The rate of heat generation and temperature rise showed dependence on the dielectric function of the nanoparticle and dipole moment of the surrounding medium. Maximum temperature was also found to increase with laser fluence and radius below melting point for both Au and AlNPs. v

10 Experimental investigation on interaction of AuNPs with nanosecond pulse laser demonstrated decrease in size of AuNPs on post irradiation under plasmon condition. The heating effect of AuNPs under plasmon condition was further confirmed by degradation of egg white protein by gold nano heaters. Protein absorption spectrum shows rise in intensity of the peak around 220 nm along with broadening, which is caused by protein s thermal degradation. Moreover, the intensity of the peak at 280 nm rises owing to the decrease in the size of the AuNPs. FTIR results demonstrate increase in the β sheet contribution of protein on laser assisted thermal denaturation in presence of AuNPs. This thesis provides valuable data useful to researchers for understanding conjugation behavior of laser generated nanoparticles with protein (ex situ and in situ) and photo thermal heating of metal nanoparticles for applications in biological sensing, drug delivery and photo thermal killing of cancer cells. vi

11 Contents Certificate Acknowledgements... i Abstract... iii List of Figures... x List of Tables... xvii List of Symbols... xviii List of Abbreviations... xxi Chapter 1. Introduction Status of the current research on bio-conjugation with metal nanoparticles Protein structure Metal nanoparticle protein interaction Metal nanoparticle synthesis Pulsed laser ablation in liquid Motivation and importance of the present work Chapter 2. Theoretical Background Colloidal nanoparticle growth by pulsed laser ablation in liquid Physical mechanism of pulsed laser ablation of solid in liquid Thermodynamic aspects Kinetic aspects Metal nanoparticle growth Gold Silver Plasmonic properties of metal nanoparticles Mie Theory: Quasi-static approximation Effect of size, shape and aggregation Effect of surrounding medium Vibrational spectroscopy of Protein Raman spectroscopy FTIR spectroscopy Photo-thermal heating of metal nanoparticles One-temperature model (OTM) vii

12 2.6.2.Two-temperature model (TTM) Chapter 3. Experimental Details Experimental setup Pulse Laser Ablation in Liquid Laser wavelength Laser Fluence Focusing conditions Ablation time Characterization techniques Structural characterization Transmission Electron Microscopy (TEM) High resolution transmission electron microscopy (HRTEM) Spectroscopic characterization UV-visible absorption spectroscopy Fourier transforms infrared spectroscopy Raman spectroscopy Fluorescence spectroscopy Methodology Preparation of spherical gold and silver nanoparticles Specifications of prepared samples Sample preparation for TEM analysis Sample preparation for UV-vis absorption and fluorescence spectroscopy Sample preparation for FTIR and Raman analysis Protocol for Lyophilization Chapter 4. Laser induced metal nanoparticle protein conjugation Protein silver nanoparticle interaction Protein gold nanoparticle interaction Influence of nanoparticle on protein AgNPs S-Ovalbumin conjugates AuNPs-BSA conjugates Summary viii

13 Chapter 5. Metal nanoparticle protein in situ conjugation Synthesis of metal nanoparticle- protein conjugates by in situ conjugation Nanoparticle size reduction by in situ conjugation AuNP-S-ovalbumin conjugates AgNP-S-ovalbumin conjugates Stabilization of nanoparticle by in situ conjugation Effect of post laser irradiation on conjugate AuNP-S-ovalbumin conjugates AgNP-S-ovalbumin conjugates Effect of in situ conjugation on protein conformation Summary Chapter 6. Photo thermal heating Photothermal heating Nanosecond pulse laser heating of gold sheet using finite difference method Nanosecond pulse laser heating of gold and aluminium nanoparticles Experimental investigation of nanosecond pulse laser interaction with gold nanoparticles Aggregation and post-irradiation effects Effect of laser fluence during post irradiation UV Visible absorption TEM analysis Laser assisted thermal denaturation of egg white protein by gold nano heaters Optical absorption FTIR study Summary Chapter 7. Summary and future scope Summary Scope for future work List of publications Bio-data of the author ix

14 List of Figures Figure 1.1: Human serum albumin (PDB1BJ5) tertiary structure. The inset shows double-loop linking pattern formed by disulfide bonds between cysteine residues... 9 Figure 1.2: The structure of S-ovalbumin. The α helices and β strands are shown in rose and pink respectively... 9 Figure 2.1: The laser induced plasma progression in the liquid. (a) The laser induced plasma formation because of the illumination of the front portion of the laser pulse on the target. (b) The plasma plume expansion in liquid as a result of absorbing the subsequent portion of the laser pulse and formation of the plasma-induced pressure via shock wave. (c) Four types of chemical reactions going on within the liquid and plasma along with the liquid and plasma interface. (d) Plasma plume condensations in liquid: one is utilized to generate nanoparticles in liquid and another one is employed for surface coatings on the target surface Figure 2.2: Schematic of plasmon oscillation for a metal sphere, presenting the conduction electron charge displacement with respect to the nuclei Figure 2.3: Absorption spectrum of gold nanoparticles at wavelengths between 400 and 800 nm wavelengths: the curve is obtained for the excitation of the conduction band electrons Figure 2.4: Real and imaginary parts of dielectric constant of gold as a function of wavelength Figure 2.5: Real and imaginary parts of dielectric constant of silver as a function of wavelength Figure 2.6 Extinction efficiency for spherical gold nanoparticles at changing radii at 10 nm, 25 nm and 50 nm Figure 2.7: Calculated absorption spectra at changing dielectric constant of surrounding medium x

15 Figure 2.8: Recorded Raman spectrum of BSA aqueous solution Figure 2.9: The vibrations associated with the Amide I (C=O bond stretching vibrations) and Amide II (bending vibrations associated with the N-H) bands in the protein s infrared spectra. 43 Figure 2.10: Recorded FTIR spectra of S-ovalbumin in KD2PO4, heated at 30 0 C, 60 0 C, 65 0 C and 70 0 C for duration of 15 min Figure 2.11: Nanoparticle temperature evolutions on irradiation by (a) femtosecond laser (b) picosecond laser anticipated by two-temperature model and one temperature model Figure 3.1: Schematic diagram of the pulsed laser ablation in liquid. Inset shows generation of plasma plume on laser target interaction inside the liquid Figure 3.2: (a) Absorption spectra of gold nanoparticles generated by pulsed laser ablation in water at decreasing concentrations. (b) Experimental data of maximum absorption at the surface plasmon resonance band vs. corresponding concentration linear fitting curve Figure 4.1: (a) TEM micrograph of AgNPs prepared in ultrapure water and (b) size distribution Figure 4.2: TEM image of silver nanoparticles in presence of S-ovalbumin. S-ovalbumin molecules are adsorbed on the silver nanoparticle surface Figure 4.3: Normalized UV visible absorption spectra of colloidal AgNPs (black line) and AgNPs with S-ovalbumin (red line) Figure 4.4: UV visible absorption spectra of silver nanoparticles at varying laser fluence (a) 0.76 (b)1.3 (c) 2.5 and (e) 6.1 J/cm 2.77 Figure 4.5: Observed SPR peak wavelength shift with nanoparticle size. Inset shows nanoparticle size variation on increasing laser fluence Figure 4.6: Peak absorbance of S-ovalbumin-AgNPs versus S-ovalbumin concentration. The AgNPs size is 10 nm, 17 nm and 38 nm. Solid lines are polynomial fits to experimental data Figure 4.7: (a) TEM image of laser generated gold nanoparticles in water and (b) size distribution xi

16 Figure 4.8: TEM images of gold nanoparticles in presence of BSA (CAu=4.6 nm,cbsa=0.041 μm) (a) gold nanoparticles aggregate in chain-like structure on functionalization with BSA molecules. (b) aggregated AuNPs in presence of BSA molecules. (c) AuNP with BSA at higher magnification Figure 4.9: Normalized UV visible absorption spectra of gold nanoparticles (black line) and gold nanoparticles with BSA (red line). In presence of BSA the SPR peak wavelength red shifts by 10 nm and broaden towards long wavelength side. Inset shows change in AuNPs colloidal solution color in presence of BSA Figure 4.10 : UV visible absorption spectra of gold nanoparticles at varying laser fluence (a) 0.7 (b) 1.7(c) 3.2 (d) 6.9 and (e) 8 J/cm Figure 4.11 : Observed SPR peak wavelength shift in AuNP-BSA assembly with laser fluence Figure 4.12: Peak absorbance of BSA-AuNPs versus BSA molar concentration. AuNPs size is (a) 12 nm (b) 16 nm and (c) 30 nm Figure 4.13: Absorption spectra of (a) S-ovalbumin (b) unconjugated AgNPs and (c) AgNPs -Sovalbumin conjugates Figure 4.14: Absorption spectra of 1.6 μm S-ovalbumin with (a) 0 (b) 0.25 nm (c) 0.34 nm (d) 0.73 nm AgNPs loading.89 Figure 4.15: (a) Raman spectra of S-ovalbumin (lower spectrum) and S-ovalbumin conjugate with silver nanoparticle (upper spectrum). (b) Raman spectra between cm -1 in the S S stretch region for S-ovalbumin and AgNP- S-ovalbumin..91 Figure 4.16: The fluorescence emission spectra of 1.6 μm S-ovalbumin aqueous solution with increasing silver nanoparticle concentration (a) 0 (b) 0.19 nm (c) 0.22 nm (d) 0.25 nm (e) 0.29 nm (f) 0.34 nm (g) 0.42 nm (h) 0.50 nm (i) 0.73 nm in solution Figure 4.17: Absorption and fluorescence spectra of S-ovalbumin AgNPs donor acceptor pair. Brown colored region is the spectral overlay of S-ovalbumin fluorescence spectrum and AgNPs absorption spectrum..94 xii

17 Figure 4.18: Plot of log [(F0-F)/(F-Fsat))] versus log (AgNPs concentration). Solid line is linear fit to experimental data Figure 4.19: UV visible absorption spectra of (a) BSA (b) unconjugated AuNPs (c) BSA- AuNPs conjugates.. 98 Figure 4.20: UV visible absorption spectra of BSA at increasing gold nanoparticle loading. The spectra are BSA μm with (a) 0 (b) 2.5 nm (c) 3.5 nm (d) 4.6 nm AuNPs Figure 4.21: Raman spectra of BSA (lower spectrum) and BSA conjugate with AuNPs (upper spectrum). Existence of a low wavenumber mode in the neighborhood of 290 cm -1 is a characteristic of S Au bond formation Figure 4.22: The fluorescence emission spectra of (a) BSA aqueous solution (0.041 μm) with increasing AuNPs concentration (a) 0 (b) 0.45 nm (c) 0.64 nm (d) 0.9 nm (e) 1.3 nm (f) 1.8 nm (g) 2.5 nm (h) 3.5 nm (i) 4.6 nm in solution. (b) Stern-Volmer plots of BSA with the increasing concentration of AuNPs Figure 4.23: Stern-Volmer plot of BSA with increasing concentration of gold nanoparticles. Solid line is linear fit to experimental data 103 Figure 4.24: Plot of log [(F0-F)/(F-Fsat)] versus log (AuNPs concentration, M). Solid line is linear fit to experimental data Figure 5.1: (a) TEM image of gold nanoparticles synthesized by PLA in water for 20 min. (b) Size distribution. (c) TEM image of gold nanoparticles synthesized by PLA in S-ovalbumin (100 nm) solution for 20 min. (d) Size distribution of gold nanoparticles synthesized in S-ovalbumin solution (100 nm)..114 Figure 5.2: Variation in peak broadening of S-ovalbumin-AuNPs conjugate as a function of S- ovalbumin concentration Figure 5.3: Variation of peak absorbance of gold nanoparticles with concentrations of S- ovalbumin added prior to laser ablation Figure 5.4: (a) TEM image of gold nanoparticles synthesized by PLA in S-ovalbumin (300 nm) solution for 20 min. (b) The size distribution Figure 5.5: UV visible spectra of gold nanoparticles synthesized by PLA in (a) water (b) S- ovalbumin (300 nm) solution xiii

18 Figure 5.6: Calculated absorption spectra of spherical AuNP with size (a) 4 nm and (b) 10 nm 121 Figure 5.7: Calculated absorption spectra of spherical AuNP in (a) water and (b) S- ovalbumin 122 Figure 5.8: The optical absorption spectra of Ag nanoparticles ablated in (a) water (b) S- ovalbumin solution (300 nm) Figure 5.9: (a) TEM image of silver nanoparticles synthesized by PLA in water for 20 min. (b) Corresponding size distribution (c) TEM image of silver nanoparticles synthesized by PLA in S- ovalbumin (300 nm) solution for 20 min. (d) Size distribution of silver nanoparticles synthesized in S-ovalbumin solution Figure 5.10: UV visible spectra of gold nanoparticles in situ conjugated with S-ovalbumin after (a) Freshly prepared (b) 1 day (c) 4 days (d) 8 days (e) 15 days of synthesis Figure 5.11: UV visible spectra of (a) Au nanoparticles, showing red shift (8 nm) and broadening at longer wave length due to aggregation of AuNPs in presence of NaCl (0.15 M). (b) In situ conjugated AuNP-protein conjugate with NaCl (0.15 M) Figure 5.12: Peak position versus the NaCl salt solution concentration for (a) 100 nm and (b) 300 nm S-ovalbumin concentrations Figure 5.13: UV-visible absorption spectra of the AuNPs-S-ovalbumin conjugate (a) before and (b) after 20 min (c) 30 min (d) 40 min (e) 50 min laser irradiation Figure 5.14: TEM images of the AuNPs- S-ovalbumin conjugate (a) before (c) after 20 min and (e) 40 min laser irradiation. (b), (d) & (f) are size distributions, respectively Figure 5.15: UV-visible absorption spectra at ph 4 for the AuNPs-S-ovalbumin conjugate (a) before and (b) after 20 min (c) 30 min (d) 40 min post laser irradiation Figure 5.16: The UV-visible absorption spectra of (a) pure S-ovalbumin in water, (b) AuNPs generated by ablation in ultrapure water and (c) AuNPs fabricated by ablation in S-ovalbumin solution xiv

19 Figure 5.17: FTIR spectra of pure S-ovalbumin (black) and S-ovalbumin gold nanoparticle in situ conjugate (red) Figure 5.18: Raman spectra of S-ovalbumin and S-ovalbumin-gold nanoparticle conjugates displaying presence of Au-S bond peak at 290 cm Figure 5.19: Raman spectra of S-ovalbumin and S-ovalbumin-silver nanoparticle conjugates displaying presence of Ag-S bond peak at 212 cm Figure 6.1: Schematic showing interaction of nanosecond laser with 1mm thickness gold sheet.145 Figure 6.2: Temporal profile of incident laser beam Figure 6.3: Time variation of temperature increase of gold sheet for 1ns and 2 ns laser pulse duration Figure 6.4: Depth variation of temperature increase of gold sheet for 1 ns and 2 ns laser pulse duration Figure 6.5: Time variation of gold sheet temperature increase at (a) 50 mj/cm 2 and (b) 100 mj/cm 2 laser fluence values and pulse duration 1 ns Figure 6.6: Depth variation of gold sheet temperature increase at (a) 50 mj/cm 2 and (b) 100 mj/cm 2 laser fluence values and pulse duration 1 ns Figure 6.7: Calculated temperature increase for a 30 nm radius single (a) AuNP and (b) AlNP as a function of distance from the NP center Figure 6.8: Effect of size on Au and Al nanoparticle maximum temperature increase (surrounding medium is water) Figure 6.9: Calculated temperature increase as a function of illumination laser power density at plasmon resonance at the surface of single (a) AuNP in water (b) AlNP in water xv

20 Figure 6.10: Schematic diagram for experimental investigation of nanosecond laser interaction with AuNPs Figure 6.11: Normalized optical absorption spectra of AuNP colloidal solution (a) Freshly prepared colloidal solution (b) After aggregation for 48 hours and (c) On post irradiation Figure 6.12: Optical absorption spectra of AuNP colloidal solution (a) Before post irradiation (b) After post irradiation at 50 mj/cm 2 (c) 100 mj/cm 2 (d) 800 mj/cm 2 (e) 1 J/cm 2 laser fluence for 15 min Figure 6.13: Variation of plasmon resonance peak of Au nanoparticle with post irradiation laser fluence Figure 6.14: (a) TEM micrograph of gold nanoparticles generated at 5.5 J/cm 2 laser fluence (b) Their size distribution (c) TEM micrograph of gold nanoparticles post irradiated at 800 mj/cm 2 laser fluence (d) their size distribution Figure 6.15: UV-visible spectra of (a) fresh egg-white protein in deionized water (b) egg-white protein in AuNP colloidal solution and (c) post laser irradiated protein-aunps solution Figure 6.16: FTIR spectra of (a) fresh egg-white protein (b) egg-white protein in AuNP colloidal solution and (c) post laser irradiated egg-white protein- AuNPs solution. Inset shows FTIR spectra in the range cm -1 of amide І band xvi

21 List of Tables Table 2.1: Important physical constants of gold and silver Table 4.1: Peak assignments for the Raman spectra of AgNP-S-ovalbumin xvii

22 α α M α m ε ε 1 ε 2 ε m λ ψ l, ξ l ψ ' ' l, ξ l π 3.14 ρ σ σ ext σ sc σ abs τ e-e τ e-ph τ e-d τ 0 τ pulse List of Symbols Fraction of internal energy devoted to thermal energy Molar extinction coefficient of nanoparticle Material absorption coefficient Static dielectric constant Real part of frequency dependent dielectric constant of metal Imaginary part of frequency dependent dielectric constant of metal Dielectric constant of the surrounding medium Laser fluence Wavelength of radiation Riccati-Bessel cylindrical functions First derivative of Riccati-Bessel cylindrical functions Density Gaussian beam standard deviation Extinction cross section Scattering cross section Absorption cross section Electron-electron relaxation time Electron- photon relaxation time Electron-defects relaxation time Lifetime of the fluorophore in the absence of quencher Laser pulse duration χ χ D ω ω p ω d ω bulk Background susceptibility Drude response Frequency Plasmon frequency Damping frequency Bulk damping frequency Laplace operator xviii

23 μ s μ γ a l, b l A λ C C e C l E 0 e F 0 F F sat I 0 j D K K SV K abs K e k k diss k q k m L l m m e * n n e N Heat conductivity Heat conductivity of the surrounding medium at normal temperature Electron-lattice coupling constant Coefficients in Mie theory Absorbance Specific heat Electronic specific heat Lattice specific heat Amplitude of the incident laser radiation Charge of electron Maximum fluorescence intensity in the absence of nanoparticle Maximum fluorescence intensity in the presence of nanoparticle Fluorescence intensity of fluorophore at the highest concentration of nanoparticle Incident intensity Heat lost from the surface of the nanoparticle Binding constant Stern-Volmer fluorescence quenching constant Absorption efficiency of the nanoparticle Thermal conductivity of metal Wave vector of incident radiation Reciprocal of the binding constant (K) Bimolecular quenching constant Thermal conductivity of surrounding matrix Evaporation heat Summation index of partial waves Ratio of particle refractive index to medium refractive index Effective mass of electron Number of binding sites per particle Electron density in metal Particle refractive index xix

24 N med Q [Q] R r r 0 S 0 S s T e T s V v f X Z Z water Z target Medium refractive index Heat flux Quencher nanoparticle concentration Reflectivity of metal Distance from the center of nanoparticle Radius of nanoparticle Nanoparticle surface area Laser heating source term Power exponent Electron temperature Lattice temperature Nanoparticle volume Fermi velocity Magnification Reduced shock impedance Water impedance Target impedance xx

25 List of Abbreviations BSA CD CTAB DDA DSNP EM FCC FDTD FTIR FWHM HRTEM HSA HTHP NADH NP NR OTM PDT PL PLA PLAL PSA RPM SDS SPR TEM TTM XPS Bovine serum albumin Circular dichroism Cetyltrimethyl ammonium bromide Discrete dipole approximation Dendrimer-stabilized nanoparticle Electromagnetic Face centered cubic Finite difference time domain Fourier transform infrared spectroscopy Full width at half maxima High resolution transmission electron microscopy Human serum albumin High temperature high pressure Nicotinamide adenine dinucleotide hydride Nanoparticle Nanorod One-temperature model Photodynamic therapy Photoluminescence Pulse laser ablation Pulse laser ablation in liquid Prostate specific antigen Rotation per minute Sodium dodecyl sulfate Surface plasmon resonance Transmission electron microscope Two-temperature model X-ray photoelectron spectroscopy xxi