Design, Development And Application Of Fluorescent Probes Of The Composite System Of Gold Nanoparticles

Fluorescence resonance energy transfer (FRET) is one of the most powerful and widely used fluorescence techniques available for probing structure and dynamics in media. Applications are multifarious and span numerous disciplines, all linked by the need to interpret FRET data in terms of improved fundamental understanding of the task at hand. For example, FRET has been used as an ? ruler in proteins, to probe dynamics in synthetic polymers, to monitor membrane fluidity, to describe the morphology of silica and in sensors for metal ions as well as glucose detection. FRET is a powerful spectroscopic technique that allows biologically relevant distances between 20 and 80 ? to be quantified under physiological conditions with near-angstrom resolution due to the strong distance dependence of the transfer process. The efficiency of FRET is dependent upon donor-acceptor proximity and spectral overlap, whether the acceptor partner is fluorescent or not. The most used donor is fluorescein due to its high quantum yield. The use of quenching acceptors is becoming increasingly popular in FRET systems. The principal advantage that these molecules offer over their fluorescent counterparts is the elimination of background fluorescence originating from direct acceptor excitation or re-emission. As a photoluminescent quencher, gold nanoparticles (Au NPs) can ultra-efficiently quench the molecular-excitation energy in chromophore-Au NP composites. Au NPs have been of great interest because of their high extinction coefficient and a broad absorption spectrum in a visible light that is overlapped with the emission wavelength of usual energy donors. In comparison with the organic quencher, Au NPs have unique structural and optical properties for new applications in biosensing and molecular engineering.Based on the FRET principle in designing new-typed fluorescent probes for trapping biological active species, we have carried out three aspects of investigation:First is the design of Au NPs as a quencher module of fluorescent probes for DNA damages caused by intracellular hydroxyl radicals (?OH). Au NPs possessing high extinction coefficient function as excellent fluorescent quenchers in fluorescein-Au NP composites. FRET switches off by a factor of ?OH-induced strand breaks in single-stranded DNAs, which restores the fluorescence of the quenched fluorescein. In vitro assays with an OH radical generating Fenton reagent demonstrate an increase in fluorescence intensity with a linear range from 8.0 nM to 1.0μM and the detection limit as low as 2.4 nM. Confocal microscopic imaging of macrophages and HepG2 reveals that the probe is cell-permeable and intracellular ?OH-responsive.Second, Au NPs-β-CDs-fluorescein has been designed as a selective fluorescent chemosensor for detecting cholesterol content utilizing FRET. Inclusion of fluorescein intoβ-CDs makes FRET occur through donor and quencher nearby. FRET switches off by the addition of the competitive CD binder cholesterol, which restores the fluorescence of the quenched fluorescein. This phenomenon is explained by the guest-induced location change of the fluorescein from inside to outside of the cavity, suggesting that the Au NPs-β-CDs-fluorescein is effective as a fluorescent chemosensor for cholesterol recognition. The fluorescence increase is proportional to the concentration of cholesterol in the range of 30 nM~15μM. A concentration of cholesterol as low as 9 nM would be readily detected. The recovery and precision of the method applied to determine cholesterol in serum extracts were satisfactory.Third is the design of Au NPs as a quencher module of fluorescent probe for hydrolysis of amide bonds in peptides caused by Type IV collagenase. In the present study, high sensitivity was obtained using the fluorescent europium chelate as label, internally quenched by suitable quencher Au NPs and released upon enzymatic reaction. In vitro assays with Type IV collagenase demonstrate an increase in fluorescence intensity with a linear range from 0.1 to 5.0μg/mL and the detection limit as low as 5.76 ng/mL. This approach would allow sensitive monitoring of low Type IV collagenase activities in normal and cancerous cell cultures using FRET pair-labeled peptide substrate.