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

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 Forster 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.