2015 Fiscal Year Research-status Report

単細胞・多細胞生物進化の熱力学的解明

Research Project

Project/Area Number	15K13617
Research Institution	Saitama University
Principal Investigator	中林誠一郎埼玉大学, 理工学研究科, 教授 (70180346)
Project Period (FY)	2015-04-01 – 2017-03-31
Keywords	C-dot / fluorescent probes / bioimaging
Outline of Annual Research Achievements	Highly luminescent ultrasmall carbon nanodots (CDs) have been prepared by one step microwave-assisted pyrolysis and functionalized with fluorescein photosensitizer by diazo-bond. The absorption edge of such prepared Fluorescein;CDs was red shifted in comparison with the bare one. Nevertheless, the emission signal induced by the nanoparticle quantum-sized graphite structure was quenched due to photo-isomerization of the diazo group at photoexcited state. In order to restrict the photoisomerization, i.e. rotation around nitrogen; nitrogen bond, the diazo group was fixed by metal cation to form complex compound or chelate. The obtained metal;complex of Fluorescein;N=N;CDs show absorbance maximum same as bare CDs, but recovered emission signal from nanoparticle moiety, which was bathochromic ; shifted. They exhibit lower quantum yield in comparison with the bare CDs but better photostability toward emission quenching in nutrition cell culture. The formed photosensitizer-conjugates nanoprobes were proposed as multifunctional fluorophores for intracellular in vivo imaging due to their attractive photophysical attributes, tunable and excitation-dependent emission. The bio-application of photosensitizer;conjugated CDs was demonstrated as fluorescent tracers for endocytosis pathways in cultured Tobacco cells. Their successful staining and lower toxicity to the plant cells were compared with conventional quantum dots (CdSe/ZnS core ; shell type, which caused acute toxicological in vivo effect).
Current Status of Research Progress	Current Status of Research Progress 2: Research has progressed on the whole more than it was originally planned. Reason We have reported new findings as follows; (1).Carbon nano-dots (CDs) were conjugated with dye molecule (Fluorescein).(2).The coupling between CDs and dye was through diazo bond.(3).When the N=N- rotation was prohibited by Ruthenium chelation, new electronic state was emerged between CDs and Fluorescein. [Fluorescein;CDs]Ru2 (4). [Fluorescein;CDs]Ru2 is used as bio-imaging marker in BY2 Tobacco plant cells.(5).The carbonbased nontoxic fluorescent nanoprobes was successful in non-breaching staining with tunable visible emission color.
Strategy for Future Research Activity	BY-2 cells stained with Fluorescein;CDs possessed significantly stronger emission signal than those stained with [Fluorescein;CDs]Ru2+ . This effect was explained with the different quantum yield of the nanoprobes , which effect their emission signal in the fluorescence microscope. In addition, in the staining with [Fluorescein;CDs]Ru2+ we observed some labeled details surrounding cell walls, which do not appear in the staining with Fluorescein;;CDs. We also noticed that after longer incubation, a strong fluorescence was observed at the nucleus. In addition, non homogeneous staining of dividing cells was discovered in the case of staining with Fluorescein;CDs. The investigation of these phenomena is still in progress All these features demonstrate that the photosensitizer-conjugated CDs have good biocompatibility and make them appropriate candidates for cell imaging.
Causes of Carryover	１９３円の余りは、消耗品の端数である。
Expenditure Plan for Carryover Budget	消耗品に使用する。