京都大学 / 新制・課程博士 / 博士(工学) / 甲第25296号 / 工博第5255号 / 新制||工||2000(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 田中 勝久, 教授 三浦 清貴, 教授 藤田 晃司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

When exposed to light, metal nanoparticles exhibit a phenomenon known as LSPR, Localized Surface Plasmon Resonance. The wavelengths at which LSPR occurs is very dependent on the refractive index of the surrounding medium. Binding of biomolecules to the surface of gold nanoparticles result in a change in the refractive index that can be detected spectrophotometrically by monitoring the LSPR peak shift. When functionalized with the corresponding ligand(s), gold nanoparticles can be utilized in biosensors to detect the presence and concentration of a predetermined analyte. However, the system must exhibit high specificity and give rise to a detectable shift for analytes in the desired concentration range to be of commercial interest. The aim of the diploma project was to investigate and optimize the biofunctionalization and performance of a fiber optics based LSPR biosensor.  Three ligand systems were investigated for detection of antibodies (IgG), insulin and avidin. Binding of the analyte to the ligand caused a shift of a few nanometers when using spherical gold nanoparticles. The shifts were significantly larger when using gold nanorods. When using the IgG and insulin ligands, only minor unspecific binding was observed. The setup thus shows great potential for use in a wide range of sensing applications.

This dissertation comprises experimental studies on the synthesis and applications of metallic nanoshells. These are a class of nanoparticles composed of a dielectric core and a thin metallic shell. Metallic nanoshells play an important role in nanotechnology, particularly in nanomedicine, due to their peculiar optical properties. The overall objectives of the dissertation were to improve the fabrication of these nanoparticles, and to demonstrate new applications of these materials in cancer research and spectroscopy. The fabrication of nanoshells is a multi-step process. Previously to our work, the procedures for the synthesis of nanoshells reported in the literature lacked systematic characterization of the various steps. The procedure was extremely time-consuming and the results demonstrated a high degree of size variation. In Chapter 3, we have developed characterization tools that provide checkpoints for each step of the synthesis. We demonstrated that it is possible to control the degree of coverage on the shell for a fixed amount of reagents, and also showed important differences on the shell growth phase for gold and silver. The synthetic optimization presented in Chapter 3 led to an overall faster protocol than those previously reported. Although the improvements presented in Chapter 3 led to a higher degree of control on the synthesis of nanoshells, the variations in the resulting particle population were still too large for applications in single particle spectroscopy and imaging. In Chapter 4, the synthesis was completely reformulated, aiming to narrow the size distribution of the nanoshell colloids. Through the use of a reverse microemulsion, we were able to fabricate ultramonodisperse silica (SiO2) cores, which translate into nanoshell colloids with narrow extinction bands that are comparable to those of a single nanoshell. We then fabricate a library of colloids with different core sizes, shell thicknesses and composition (gold or silver). The localized surface plasmon resonance (LSPR) of these colloids span across the visible range. From this library, two nanoshells (18nm silver on a 50nm SiO2 core, and 18nm gold on a 72nm SiO2 core) were selected for a proof of principle cell imaging experiment. The silver nanoshells were coated with a nuclear localization signal, allowing it to target the nuclear membrane. The gold nanoshells were coated with an antibody that binds to a receptor on the plasma membrane of MCF-7 human breast cancer cells. The nanoshells were easily distinguishable by eye in a dark field microscope and successful targeting was demonstrated by hyperspectral dark field microscopy. A comparison was made between fluorescent phalloidin and nanoshells, showing the superior photostability of the nanoparticles for long-term cell imaging. The results from Chapter 4 suggest that the nanoshells obtained by our new synthetic route present acceptable particle-to-particle variations in their optical properties that enables single particle extinction spectroscopy for cell imaging. In Chapter 5 we explored the use of these nanoshells for single-particle Surface-enhanced Raman spectroscopy (SERS). Notice that particle-to-particle variations in SERS are expected to be more significant than in extinction spectroscopy. This is because particle-to-particle SERS variabilities are driven by subtle changes in geometric parameters (particle size, shape, roughness). Two types of gold nanoshells were prepared and different excitation wavelengths (λex) were evaluated, respective to the LSPR of the nanoshells. Individual scattering spectra were acquired for each particle, for a total of 163 nanoshells, at two laser excitation wavelengths (632.8 nm and 785 nm). The particle-to-particle variations in SERS intensity were evaluated and correlated to the efficiency of the scattering at the LSPR peak. Chapter 6 finally shows the application of gold nanoshells as a platform for the direct visualization of circulating tumor cells (CTCs). 4T1 breast cancer cells were transduced with a non-native target protein (Thy1.1) and an anti-Thy1.1 antibody was conjugated to gold nanoshells. The use of a transduced target creates the ideal scenario for the assessment of nonspecific binding. On the in vitro phase of the study, non-transduced cells were used as a negative control. In this phase, parameters such as incubation times and nanoshell concentration were established. A murine model was then developed with the transduced 4T1 cells for the ex vivo portion of the work. Non-transduced cells were implanted in a control group. Blood was drawn from mice in both groups over the course of 29 days. Antibody-conjugated nanoshells were incubated with the blood samples and detection of single CTCs was achieved in a dark field microscope. Low levels of nonspecific binding were observed in the control group for non-transduced cells and across different cell types normally found in peripheral blood (e.g. lymphocytes). All positive and negative subjects were successfully identified. Chapter 7 provides an outlook of the work presented here and elaborates on possible directions to further develop the use of nanoshells in bioapplications and spectroscopy. / Graduate / 2019-05-03

No description available.

This research investigates an extension of the glancing angle deposition (GLAD) technique and a biosensing application of films produced by GLAD. The extension to GLAD, called phi-sweep (PS), improves column isolation compared to films grown by traditional GLAD (TG) as well as modifies the column tilt angle, , of the slanted columns according to tan(_{PS}) = tan(_{TG}) cos(), where is the sweep angle. The biosensing application makes use of localized surface plasmon resonance in noble metal GLAD films functionalized with rabbit immunoglobulin G (rIgG) to detect binding of anti-rabbit immunoglobulin G (anti-rIgG) to the films' surface. The extinction peak red-shifts a distance dependent on the concentration of anti-rIgG solution in a manner described by the Langmuir isotherm with a saturation value, _{max}, of 29.4 0.7 nm and a surface confined thermodynamic binding constant, K, of (2.7 0.3)10 M. / Microsystems and Nanodevices

No description available.

Localized surface plasmon resonance (LSPR) is induced in metal nanoparticles by resonance between incident photons and conduction electrons in nanoparticles. For noble metal nanoparticles, LSPR can lead to strong absorbance of ultraviolet-violet light. Although it is well known that LSPR depends on the size and shape of nanoparticles, the inter-particle spacing, the dielectric properties of metal and the surrounding medium, the temperature dependence of LSPR is not well understood. By thermally annealing gold nanoparticle arrays formed by nanosphere lithography, a shift of LSPR peak upon heating has been shown. The thermal characteristics of the plasmonic nanoparticles have been further used to detect chemicals such as explosive and mercury vapors, which allow direct visual observation of the presence of mercury vapor, as well as thermal desorption measurements

No description available.

This dissertation is about fabrication and functionalization of metal nanoparticles for use in plasmonic and nonlinear optical (NLO) applications. In the first two chapters, I describe a series of experiments, where I combined silver nanoparticles fabricated by nanosphere lithography with ionic self-assembled multilayer (ISAM) films, tuning the geometry of the particles to make their plasmonic resonances overlap with the frequency of optical excitation. The designed hybrid metallic/organic nanostructures exhibited large enhancements of the efficiency of second harmonic generation (SHG) compared to conventional ISAM films, causing a modified film with just 3 bilayers to be optically equivalent to a conventional 700-1000 bilayer film. SHG responses from Ag nanoparticle-decorated hybrid-covalent ISAM (HCISAM) films were investigated as the next logical step towards high-Ï ²⁺ ISAM films. I found that the plasmonic enhancement primarily stems from interface SHG. Interface effects were characterized by direct comparison of SHG signals from PAH/PCBS ISAM films and PAH/PB HCISAM films. Though interface &chi²⁺ is substantially smaller in PAH/PCBS than in PAH/PB, plasmonically enhanced PAH/PCBS films exhibit stronger NLO response. I propose that the structure of PAH/PB film makes its interface more susceptible to disruptions in the nanoparticle deposition process, which explains our observations. During the fabrication of monolayer crystals for nanosphere lithography, I developed a variation of the technique of convective self-assembly, where the drying meniscus is restricted by a straight-edge located approximately 100 μM above the substrate adjacent to the drying zone. This technique can yield colloidal crystals at roughly twice the growth rate compared to the standard technique. I attribute this to different evaporation rates in the thin wet films in the two cases. I also found that the crystal growth rate depends strongly on the ambient relative humidity. Finally, dithiocarbamate (DTC)-grafted polymers were synthesized and employed to functionalize surfaces of Au nanopartciles. PAH-DTC shows greater stability in different environments than PEI-DTC. I also investigated the stability of PAH-DTC coated particles in suspensions with UV-Vis spectroscopy and autotitration. The covalently bonded PAH-DTC enhances the colloidal stability of the Au nanoparticles and enables subsequent ISAM film deposition onto the particles. / Ph. D.

Anisotropic plasmonic noble metallic nanoparticles (APMNs) have received enormous attention due to their distinct geometric features and fascinating physicochemical properties. Owing in large part to their tailored localized surface plasmon resonance (LSPR) and the intensive electromagnetic field at the sharp corners and edges, APMNs are exceptionally well suited for biomedical applications such as biosensing, bioimaging, diagnostics and therapeutics. Although a rich variety of surfactant-assisted colloidal routes have been developed to prepare well-defined APMNs, biomedical applications necessitate tedious and rigorous purification processes for the complete removal of toxic surfactants. In this dissertation, we aim to develop generic bioenabled green synthetic methodologies towards APMNs. By applying a series of thermodynamic, kinetic and seed quality control, a series of APMNs with varied morphologies such as branched nanostars and triangular nanoprisms have been successfully prepared. We first presented the preparation of gold nanostars (Au NSTs) through a two-step approach utilizing a common Good's buffer, HEPES, as a weak reducing agent. Single crystalline Au NSTs with tunable branches up to 30 nm in length were produced and the halide ions rather than the ionic strength played a significant roles on the length of the branches of Au NSTs. Then consensus sequence tetratricopetide repeat (CTPR) proteins with increasing number of repeats were used as model proteins to probe the effects of concentration as well as the protein shape on the morphology and resulting physicochemical properties of plasmonic gold nanoparticles. Since the underlying growth mechanism for the biomimetic synthesis of APMNs remains elusive and controversial, the other objective is to elucidate the molecular interactions between inorganic species and biopolymers during the course of NP evolution. Fluorescent quenching and 2D NMR experiments have confirmed the moderate binding affinity of CTPR to the Au(0) and Au(III). We observed that the initial complexation step between gold ions and CTPR3 is ionic strength dependent. Furthermore, we also found that NPs preferentially interact with the negatively charged face of CTPR3 as observed in 2D NMR. Knowledge of binding behavior between biospecies and metal ions/NPs will facilitate rational deign of proteins for biomimetic synthesis of metallic NPs. A modified seed-mediated synthetic strategy was also developed for the growth of silver nanoprisms with low shape polydispersity, narrow size distribution and tailored plasmonic absorbance. During the seed nucleation step, CTPR proteins are utilized as potent stabilizers to facilitate the formation of planar-twinned Ag seeds. Ag nanoprisms were produced in high yield in a growth solution containing ascorbic acid and CTPR-stabilized Ag seeds. From the time-course UV-Vis and transmission electron microscopy (TEM) studies, we postulate that the growth mechanism is the combination of facet selective lateral growth and thermodynamically driven Ostwald ripening. By incorporation of seeded growth and biomimetic synthesis, gold nanotriangles (Au NTs) with tunable edge length were synthesized via a green chemical route in the presence of the designed CTPR protein, halide anions (Br⁻) and CTPR-stabilized Ag seeds. The well-defined morphologies, tailored plasmonic absorbance from visible-light to the near infrared (NIR) region, colloidal stability and biocompatibility are attributed to the synergistic action of CTPR, halide ions, and CTPR-stabilized Ag seeds. We also ascertained that a vast array of biosustainable materials including negatively charged lignin and cellulose derivatives can serve as both a potent stabilizers and an efficient nanocrystal modifiers to regulate the growth of well-defined Ag nanoprisms using a one-pot or seeded growth strategy. The influential effects of reactants and additives including the concentration of sodium lignosulfonate, H2O2 and NaBH4 were studied in great detail. It implies that appropriate physicochemical properties rather than the specific binding sequence of biomaterials are critical for the shaped-controlled growth of Ag NTs and new synthetic paradigms could be proposed based on these findings. Last but not the least, we have demonstrated the resulting APMNs, particularly, Au NSTs and Ag NTs exhibit remarkable colloidal stability, enhanced SERS performance, making them promising materials for biosensing and photothermal therapy. Since the Ag nanoprisms are susceptible to morphological deformation in the presence of strong oxidant, they also hold great potential for the colorimetric sensing of oxidative metal cation species such as Fe3+, Cr3+, etc. / Ph. D. / When a beam of light impinges on the surface of noble metallic nanoparticle (NP), particularly gold (Au) and silver (Ag), the conduction electrons are excited which induces a collective oscillatory motion, resulting in an intense localized surface plasmon resonance (LSPR) absorbance as well as the amplified localized electromagnetic filed. Owing in large part to the tailored LSPR and the intensive electromagnetic field at the sharp corners and edges, anisotropic plasmonic noble metallic nanoparticles (APMNs) can be utilized to span an array of applications such as biosensing, bioimaging, diagnostics and therapeutics. Although great advancement has been made to prepare well-defined APMNs through versatile surfactant-assisted colloidal methodologies, biomedical applications necessitate tedious and rigorous purification processes for the complete removal of toxic surfactants. To address this ubiquitous challenge, biomimetic and bioinspired green synthesis have been extensively explored to fabricate APMNs under mild and ambient conditions. In this dissertation, we aim to develop generic bioenabled synthetic strategies towards APMNs, particularly, Au nanostars and Au/Ag nanoprisms. Herein, protein mediated shape-selective synthesis of APMNs were presented, in which consensus sequence tetratricopetide repeat (CTPR) proteins and biological Good’s buffers were employed as nanocrystal growth modifiers and mild reducing agents, respectively. The dramatic implications of repeat proteins on the morphological and optical properties of the Au NPs were explicitly discussed. The other objective of this dissertation is to elucidate the molecular interactions between inorganic species and biopolymers to further unravel the underlying growth mechanism during the course of APMNs evolution. By incorporation of seeded growth and biomimetic synthesis, Ag/Au nanotriangles (Au NTs) with tunable edge length were synthesized in the presence of the designed CTPR protein, halide anions (Br⁻) and CTPR-stabilized Ag seeds. The well-defined morphologies, tailored plasmonic absorbance from visible-light to the near infrared (NIR) region, colloidal stability and biocompatibility are attributed to the synergistic action of each components in the synthetic system. Last but not the least, we have demonstrated the resulting NPs exhibit remarkable colloidal stability, mitigated cytotoxicity and surface enhanced Raman spectroscopy (SERS) performance, making them good candidates for biosensing and photothermal therapy. This work might shed light on the roles biomolecules play in green synthesis of APMNs, along with rationalizing the design of biomimetic systems to bridge the gap between the bioenabled technique and traditional colloidal synthesis.