ダイヤモンドナノ粒子の生体計測応用に関する研究

京都大学0048新制・課程博士博士(工学)甲第19002号工博第4044号新制||工||1622(附属図書館)31953京都大学大学院工学研究科分子工学専攻(主査)教授 白川 昌宏, 教授 田中 庸裕, 教授 濵地 格学位規則第4条第1項該当Doctor of Philosophy (Engineering)Kyoto UniversityDFA

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes

The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface

Comprehensive and quantitative analysis for controlling the physical/chemical states and particle properties of nanodiamonds for biological applications

The physical/chemical states and properties of nanodiamonds subjected to thermal annealing and air oxidation, which are indispensable processes for the preparation of fluorescent nanodiamonds, were investigated. Specifically, the weight loss, particle size, crystal quality, chemical bonding states of carbon and oxygen, zeta potential, dispersibility, and fluorescent and optically detected magnetic resonance (ODMR) properties were determined using X-ray diffraction (XRD) analysis, transmission electron microscopy (TEM), elemental analysis, dynamic light scattering, Raman analysis, X-ray photoelectron spectroscopy (XPS), IR spectroscopy, and a home-made fluorescence and ODMR microscope. The study focused on small-sized nanodiamonds ([similar]50 nm), which are applicable for biological research. The obtained results should be useful for controlling the mutually-related physical/chemical states and properties of diamond nanoparticles

Single-Step Metal-Free Grafting of Cationic Polymer Brushes on Fluorescent Nanodiamonds

Cationic polymers are often employed in conjugation with nanomaterials, and the resultant hybrids are useful for various bioapplications. Here, a single-step metal-free method for the synthesis of fluorescent nanodiamonds (FNDs) conjugated with cationic polymer brushes is reported. Distinct from the common methods such as atom transfer radical polymerization and reversible addition fragmentation chain transfer, our ring-opening-polymerization-based method is simple and less time consuming and hazardous. Infrared spectroscopy, thermogravimetric analysis, zeta potential, and dynamic light scattering confirmed the synthesis. The produced FND-polymer brushes showed markedly higher cell labeling and internalization efficiency without noticeable cytotoxicity. Our method is general and applicable to other nanoparticles as well for uses in diverse research areas

One-pot synthesis of highly dispersible fluorescent nanodiamonds for bioconjugation

Fluorescent nanodiamonds (FNDs) have been attracting much attention as promising therapeutic agents and probes for bioimaging and nanosensing. For their biological applications, several hydrophilizing methods to enhance FND colloidal stability have been developed to suppress their aggregation and the nonspecific adsorption to biomolecules in complex biomedical environments. However, these methods involve several complicated synthetic and purification steps, which prohibit the use of FNDs for bioapplications by biologists. In this study, we describe a simple one-pot FND hydrophilization method that comprises coating of the surface of the nanoparticles with COOH-terminated hyperbranched polyglycerol (HPG-COOH). HPG-COOH-coated FNDs (FND-HPG-COOHs) were found to exhibit excellent dispersibility under physiological conditions despite the thinness of 5-nm HPG-COOH layer. Biotinylated FND-HPG-COOHs specifically captured avidin molecules in the absence of nonspecific protein adsorption. Moreover, we demonstrated that FND-HPG-COOHs conjugated with antibodies can be used to selectively target integrins in fixed HeLa cells. In addition, intracellular temperature changes were measured via optically detected magnetic resonance using FND-HPG-COOHs conjugated with mitochondrial localization signal peptides. Our one-pot synthetic method will encourage the broad use of FNDs among molecular and cellular biologists, and pave the way for extensive biological and biomedical applications of FNDs

Effective production of fluorescent nanodiamonds containing negatively-charged nitrogen-vacancy centers by ion irradiation

Fluorescence from negatively-charged nitrogen-vacancy centers (NV[−]s) in diamonds has unique optical properties with none of the undesirable effects such as photo-bleaching and photo-blinking. In addition, the spin-dependent fluorescence intensity of NV[−]s allows us to perform optically detected magnetic resonance (ODMR) investigation for evaluating the presence of NV−s and for the electronic local environment. In this work, we irradiated H[+], He[+], Li[+] and N[+] ions to nanodiamonds with a median size of 26 nm at various irradiation energies and doses for improving the NV[−] concentration. ODMR observations of the nanodiamonds showed that ion irradiation increased the number of nanodiamonds containing NV[−]s up to 200 ppm, whereas without ion irradiation, only few NV[−]s were found. The number of nanodiamonds containing NV[−]s at various ion irradiation doses was not monotonous, but had maxima at certain irradiation doses. These results suggest a competition in two opponent roles of vacancies, effective for pairing with nitrogen atoms and as defects for developing damage in crystalline. We also found that sharp and strong ODMR signals were obtained from nanodiamonds irradiated at the optimal condition for the highest yield of NV−s. We concluded that He[+] ion irradiations with 60 or 80 keV at a dose of 1 × 10[13] ions cm[–2] are the conditions required for the most efficient production of a high quantity of nanodiamonds containing NV[−]s