This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of the American Chemical Society, copyright ©American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jacs.5b10162. / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19611号 / 医博第4118号 / 新制||医||1015(附属図書館) / 32647 / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 渡邊 直樹, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21128号 / 工博第4492号 / 新制||工||1698(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 吉田 潤一, 教授 杉野目 道紀, 教授 松田 建児 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Fluorescence spectroscopy has been considered to be one of the most important research techniques in modern analytical chemistry, biochemistry, and biophysics. At present, fluorescence is a dominant methodology widely used in a great number of research domains, including biotechnology, medical diagnostics, genetic analysis, DNA sequencing, flow cytometry, and forensic analysis, to name just a few. In the past decade, with the rapid development of fluorescence microscopy, there has been a considerable growth in applying fluorescence technique to cellular imaging. The distinguished merits of fluorescence techniques, such as high sensitivity, non-invasiveness, low cytotoxicity, low cost, and convenience, make it a promising tool to replace radioactive tracers for most biochemical measurements, avoiding the high expense and difficulties of handling radioactive tracers. Among the wide range of applications of fluorescence technique, fluorescent sensing of various cations and anions is one of the most important and active areas. This dissertation is all about developing fluorescent sensors for physiologically significant ions, including F-, K+, Fe3+, and Ca2+. All of these sensors demonstrate fluorescence "turn-on" response upon interacting with their respective ions, which makes them much more appealing than those based on fluorescence quenching mechanisms. In Chapter II, a novel highly selective fluorescence turn-on F- sensor (FS), comprised of a fluorene platform serving as the chromophore, and two 1,2,3-triazolium groups functioning as the signaling moieties, is described. The function of FS is established on the basis of deprotonation of the C-H bonds of 1,2,3-triazolium groups, which makes FS the first reported anion sensor based on the deprotonation of a C-H bond. Easy-to-prepare test strips were prepared for determining F- in aqueous media, providing an inexpensive and convenient approach to estimate whether the concentration of F- contained in drinking water is at a safe level. Chapter III contains an optimized synthesis of a reported K+-selective group (TAC), and the development of two TAC-based fluorescence turn-on K+ sensors (KS1 and KS2). The synthetic route of TAC is shortened and its overall yield is enhanced from 3.6% to 19.5%. Both KS1 and KS2 exhibited excellent selectivity toward K+ over other physiological metal cations, high sensitivity for K+ sensing, and pH insensitivity in the physiological pH range. Confocal fluorescence microscopy experiments demonstrate that they are capable of sensing K+ within living cells. 2PA determination reveals that KS2 has a desirable 2PA cross section of 500 GM at 940 nm, which makes it a two-photon red-emitting fluorescent sensor for K+. Chapter IV describes the development of a novel BODIPY-based fluorescence turn-on Fe3+ sensor (FeS). FeS is a conjugate of two moieties, a BODIPY platform serving as the fluorophore and a 1,10-diaza-18-crown-6 based cryptand acting as the Fe3+ recognition moiety. FeS displays good selectivity, high sensitivity, reversibility, and pH insensitivity toward Fe3+ sensing. Based on its excellent performance in determining Fe3+ and very low cytotoxicity, FeS was effectively applied to sensing Fe3+ in living cells. In Chapter V, a new BODIPY-based fluorescence turn-on sensor (CaS) was designed and synthesized for selectively and sensitively determining Ca2+. CaS is comprised of two moieties, a BODIPY fluorophore and a Ca2+ complexing unit. CaS demonstrated selective fluorescence turn-on response towards Ca2+ over other biological metal cations. Moreover, CaS exhibited desirable sensitivity for Ca2+ detection, which makes it more suitable for extracellular Ca2+ determination. In addition, CaS was insensitive to the pH of the physiological environment, especially in the pH range of blood and serum. Therefore, CaS has potential to be applied to sensing Ca2+ ions in extracellular environments. Chapter VI discusses potential future work of KS2 and CaS, following the results achieved in this dissertation. Based on the desirable performances of both sensors in sensing their respective ions, future work could largely be focused on their applications in cellular imaging.

Electrodialysis is a membrane separation process that uses an electrical potential to drive the separation. The performance of these systems is largely based on the performance of their ion exchange membranes (IEMs). This research focused on enhancing the performance of IEMs for electrodialysis through surface modification techniques involving chitosan bonded to the surface of commercially available cation exchange membranes (CEMs). The surface functionalization techniques resulted in membranes with improved electrodialysis performance. This research also explored the processing framework to produce functionalized sulfonated PEEK (sPEEK) nanofibers for future consideration as cation exchange membranes. Chitin was deacetylated to form the functionalized biopolymer chitosan, then applied to the surface of CEMs, rendering them more hydrophilic. These membranes were evaluated across several electrodialysis performance metrics. Results demonstrate that adjusting the degree of deacetylation of chitosan to enhance membrane hydrophilicity positively impacted electrodialysis performance. Furthermore, this research evaluated the effectiveness of similarly functionalized membranes to extract Lithium from brine solutions. The chitosan-coated membranes showed improved electrodialysis performance, including enhanced flux, limiting current density, system resistance, selectivity, and fouling resistance. Lastly, the sPEEK nanofibers were produced for the fabrication of ion exchange membranes by manipulating operational parameters to assess their impact. This research presents the successful functionalization of PEEK via sulfonation and electrospinning of the resulting sPEEK. These nanofibers were then pressed to form a solid sPEEK membrane. It was observed that changes in electrical potential and rotational speed influenced fiber diameter and spinnability. A correlation was established between membrane surface hydrophilicity and electrodialysis performance metrics in desalination and lithium extraction applications. This research advanced the understanding of structure-property relationships for CEMs. The research herein proposes techniques for industries such as desalination and lithium extraction that can meet growing demands for clean water and sustainable methods for producing high-value raw material streams.

Actin is a highly abundant protein in most eukaryotic cells. The assembly of actin monomers to double helical filaments is crucial for many cellular functions, including cell movement and cell division. Actin filament assembly in cells occurs in a crowded intracellular environment consisting of various molecules, including cations and organic osmolytes. Recent studies show that cation binding stiffens actin filaments, and a small organic osmolyte trimethylamine-N-oxide (TMAO) modulates filament assembly. However, how cations and TMAO combined affect actin filament mechanics is not understood. We hypothesize that depending on the concentrations of cations and osmolytes, there will be different effects on the stiffness and assembly of actin filaments. In this study, using TIRF we evaluate actin filament mechanics and assembly. Our findings indicate that when TMAO is present alone, it can increase the elongation rate and stiffness of actin filaments, however the inclusion of potassium levels alongside TMAO reduces the persistence length of actin filaments, suggesting a decrease in filament stiffness compared to the influence of TMAO alone. Furthermore, the elongation rate of actin filaments decreases when both TMAO and potassium ions are present. This study will help us better understand how cations and osmolytes together can affect actin filament mechanics in the living cells.

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