Title

探討近紅外光螢光染劑-矽羅丹明衍生物及花菁-5在溫度應答型高分子內的螢光現象

Translated Titles

Exploration of Fluorescent Behaviors of Near-Infrared Dyes Si-Rhodamine Derivative and Cy5 Applied in Thermo-Responsive Polymers

DOI

10.6342/NTU.2015.00097

Authors

林富倉

Key Words

近紅外光螢光染劑 ; 矽羅丹明 ; 螢光共振能量轉移 ; 溫度應答型高分子 ; 低臨界相轉變溫度高分子 ; 高臨界相轉變溫度高分子 ; 溫度感測分子 ; near-infrared dyes ; Si-Rhodamine ; FRET ; thermo-responsive polymer ; LCST ; UCST ; thermometer

PublicationName

臺灣大學化學研究所學位論文

Volume or Term/Year and Month of Publication

2015年

Academic Degree Category

碩士

Advisor

陳昭岑

Content Language

繁體中文

Chinese Abstract

目前常見的螢光溫度感測分子放射波長大部分位於可見光的波段,但此類型的感測分子很容易受到生物組織的自體螢光干擾,若將螢光分子的放射光源波長增長至光學窗口 (optical window, 600-900 nm) 範圍便可降低生物組織的自體螢光干擾,且長波長光源具有較佳的細胞穿透深度及較低的細胞傷害強度。然而至今僅少數近紅外光螢光染劑修飾的溫度感測分子被報導,且大部分皆為單放射峰螢光輸出。單放射峰螢光強度易受到染劑濃度以及光漂白影響,因此發展出具有雙放射峰螢光輸出的螢光共振能量轉移式溫度感測分子,再佐以近紅外光螢光輸出,可提升活體內 (in vivo) 溫度感測的靈敏度。本文以矽羅丹明衍生物 (Si-rhodamine,SiR) 做為近紅外光螢光訊號輸出分子,並將其修飾在UCST與LCST高分子上,研究其作為近紅外光螢光溫度感測分子之潛力。 其中以本文合成出之PNIcoHI作為UCST高分子,將SiR及淬滅劑 (black hole quencher,BHQ) 分別作為FRET供體及受體,但是研究結果顯示無法如預期使SiR與BHQ達到螢光共振能量轉移現象。LCST高分子則以PNIPAM作為骨架,共合成出三種不同螢光表現之溫度感測高分子:(1) PNIPAM-N關開式溫度感測高分子、(2) PNIPAM-N-Q開關式溫度感測高分子、(3) PNIPAM-Cy-N雙螢光輸出溫度感測高分子,其溫度感測區間皆調控於攝氏35-41 度之間。在感測區間內,PNIPAM-N系統螢光有2.8倍的增幅,PNIPAM-N-Q系統螢光則有24倍降幅。而PNIPAM-Cy-N有比例螢光輸出,其分子的設計概念如圖所示。在室溫下,高分子膨脹使螢光團間距離拉大,FRET無法進行而螢光主要以Cy5 dye為主;當溫度高於LCST時,高分子收縮使螢光團距離靠近,FRET現象產生而螢光主要以SiR dye為主。此外,PNIPAM-Cy-N之研究結果可觀察到螢光雙峰的變化,利用兩峰的比例進行內部校正,具有活體內溫度感測或顯像的潛力。

English Abstract

Most of fluorescent thermosensors fluoresces in the visible spectral range, and one associated challenge is the strong interference from autofluorescence of the background tissue when used for tissue imaging/sensing. It is well-known that near-infrared (NIR) dyes with excitation/emission spectra within the “optical window” of biological tissues (i.e., 600-900 nm) show much reduced interference from tissue autofluorescence. In addition, NIR fluorescent dyes can also provide deep imaging/sensing penetration depth and low phototoxicity. There are few reports of NIR thermosensors, and most of them were based on the monitoring of changes in the relative fluorescence emission intensity of a single emission peak. However, the fluorescence intensity at a single wavelength can be affected by many parameters (e.g., dye concentration, photobleaching, background signals). The Förster resonance energy transfer (FRET) has been employed for the construction of ratiometric fluorescent thermosensors to effectively eliminate the interference from background signals and improve the detection limit in vivo. In this dissertation, NIR fluorescent dyes Si-rhodamine (SiR) derivatives were covalently incorporated into thermo-responsive polymers based on upper critical solution temperature (UCST) and lower critical solution temperature (LCST) polymers to exploit the feasibility of using them as NIR thermosensors. PNIcoHI was chosen as UCST polymer backbone, and SiR as FRET donor and black hole quencher (BHQ) as FRET acceptor were incorporated as side arms. My studies indicated that there is no effective FRET between SiR and BHQ. Three types of thermosensors derived from PNIPAM were synthesized and studied: (1) off-on thermosensor PNIPAM-N, (2) on-off thermosensor PNIPAM-N-Q, (3) ratiometric thermosensor PNIPAM-Cy-N. Within a sensing range of 35 to 41 oC, the fluorescent intensity was increased 2.8-fold in the case of PNIPAM-N, decreased 24-fold in the case of PNIPAM-N-Q. The sensing mechanism of PNIPAM-Cy-N was schematically depicted in figure A. The swollen state of PNIPAM-Cy-N emits the fluorescence of Cy5 at room temperature. Whereas the local temperature exceeds the LCST of PNIPAM-Cy-N, the polymer shrinks accompanied with enhancing FRET process between Cy5 and SiR resulting in an increase of emission intensity of SiR. Fluorescence ratiometric change was observed in the case of PNIPAM-Cy-N, which displays the potential for in vivo temperature sensing or imaging.

Topic Category 基礎與應用科學 > 化學
理學院 > 化學研究所
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