Use of Lanthanide-Containing Polyoxometalates to Sensitise the Emission of Fluorescent Labelled Serum Albumin.

Monitoring the interaction of biomolecules is important, and the use of energy transfer is a principal technique in elucidating nanoscale interactions. Lanthanide compounds are promising luminescent probes for biological samples as their emission is longer-lived than any native autofluorescence. Polyoxometalates (POMs) are interesting structural motifs to incorporate lanthanides, offering low toxicity and a size pertinent for biological applications. Here, we employ iso-structured POMs containing either terbium or europium and assess their interaction with serum albumin by sensitisation of a fluorescent tag on the protein via LRET (luminescence resonance energy transfer) by exciting the lanthanide. Time-resolved measurements showed energy transfer with an efficiency of over 90% for the POM-protein systems. The Tb-POM results were relatively straightforward, while those with the iso-structured Eu-POM were complicated by the effect of protein shielding from the aqueous environment.

Viability of Saccharomyces cerevisiae incorporated within silica and polysaccharide hosts monitored via time-resolved fluorescence.

The viability of Saccharomyces cerevisiae in biocompatible polymers under different growth conditions and studied using time-resolved fluorescence techniques is presented. Two fluorophores, the viscosity sensitive probe 4-(4-(dimethylamino)styryl)-N-methyl-pyridiniumiodine (DASPMI) and the yeast viability stain 2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene)-1-phenylquinolinium iodide (FUN-1) are used to elucidate information on the incorporated yeast cell viability. Variations in cell viscosity, which are indicative of the cell state, were obtained using DASPMI. Prior to observing FUN-1 in yeast cells using fluorescence lifetime imaging, its photophysics in solution and heterogeneous media were investigated. Time-resolved emission spectra were measured and analysed to associate lifetimes to the spectral emission. Preliminary results show that monitoring the fluorescence lifetime of FUN-1 may give a useful insight into cellular metabolism. The results indicate that both fluorophores may be used to monitor the entrapped yeast cell viability, which is important for in vitro studies and applications, such as that in the biofuel industry, where Saccharomyces cerevisiae are required to remain active in high ethanol environments.

The influence of silver nanostructures formed in situ in silica sol-gel derived films on the rate of Förster resonance energy transfer.

The efficiency of Förster resonance energy transfer (FRET) can be enhanced in the presence of a metal. Herein, we demonstrate the increased efficiency for a novel model sensor system where FRET is shown to occur between Rhodamine 6G in the bulk sol-gel matrix and Texas Red, which is held a fixed distance away by covalent attachment onto a silane spacer. Silver colloids are formed using light to initiate the reduction of a silver salt, which can be achieved at controlled locations within the film. Both the fluorescence intensity and lifetime maps and analysis indicate that an enhanced FRET efficiency has been achieved in the presence of silver nanoparticles. An increase in efficiency of 1.2-1.5 times is demonstrated depending on the spacer used. The novelty of our approach lies in the method of silver-nanoparticle formation, which allows for the accurate positioning of the silver nanoparticles and hence selective fluorescence enhancement within a biocompatible host material. Our work gives a practical demonstration of metal-enhanced FRET and demonstrates the ability of such systems to be developed for molecular-recognition applications that could find use in lab-on-a-chip technologies.

In situ formation of silver nanostructures produced via laser irradiation within sol-gel derived films and their interaction with a fluorescence tagged protein.

The presence of a conducting metal surface is known to affect the emission of a fluorophore in its proximity. This can lead to an enhancement in its fluorescence intensity along with a decrease in the fluorescence lifetime. This phenomenon, sometimes known as metal enhanced fluorescence, has implications in the area of sensing and "lab on a chip" applications. Here controlled, localised use of metallic structures can be advantageous in enhancing the detection of a fluorescent signal. The sol-gel technique has been demonstrated as a useful method by which to produce a biocompatible material. The versatility of the reaction allows for the inclusion of metal ions, which can form metallic nanostructures permitting the potential enhancement of fluorescence to be exhibited. In this work we incorporate silver nitrate within silica sol-gel derived films produced using a simple procedure at relative low temperatures (close to ambient). A compact time-resolved fluorescence microscope equipped with a semiconductor laser was used to photoactivate the silver ions to form localised metallic structures within the films. Patterning was achieved by computer control of the microscope stage and using the laser in CW mode. The films were characterised using AFM and UV-vis spectroscopy to ascertain the presence of the photoactivated silver nanostructures. The effect of the presence of these structures was elucidated by studying the time-resolved fluorescence of FITC labelled bovine serum albumin adsorbed to the films, where a decrease in the lifetime of the FITC label was observed in the location of the nanostructures.

Effect of polymer strengtheners on the local environment of biocompatible glass as probed by fluorescence.

Mixed silica-calcite matrices were prepared by developing a "low" temperature (sol-gel) method in presence of several biocompatible polymers, thus providing samples with adequate porosity for the flow of biological fluids and also mechanically robust. In order to analyse and characterise the sample's microenvironments, the highly solvatochromic probe Nile red was used, which enabled the role of polymer addition upon local environmental effects in the host media to be elucidated. The polymers used were polyethylene glycol, polymethylmethacrylate and polyethylene. Each matrix was also characterized with respect to microstructure, morphology and pore size via the use of X-ray diffractometry and scanning electron microscopy. The results show that is was possible to obtain, in a controlled way, mixed silica-calcite matrices with a wide range of porosities (important if the material is to be used for scaffold or drug release applications, for example). The spectroscopic behaviour of Nile red when incorporated has confirmed the existence of distinct and specific local polarities within each type of matrix that may determine to a large extent the mechanism of interaction between these matrices and biological molecules.