We present a new method for multi-color fluoroimmunoassays based on directional surface plasmon-coupled emission (SPCE). SPCE is coupling of excited fluorophores with a nearby thin metal film (silver) resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagates through the prism depends on emission wavelength and makes possible measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using two antibodies labeled with different fluorophores, binding to an antigen protein immobilized on the silver surface. We observed independent emission at a different angle on the glass prism, resulting of the surface binding of each antibody. This methodology can be readily extended to 3 or more fluorophores. This technology presents opportunity to develop highly sensitive multiplex assay format for biological agents' detection.
We have recently shown that metallic particles or colloids when deposited on the transparent surface can enhance fluorescence properties of nearby fluorophores. We obtained the fluorophore-metal colloid complexes that display significant fluorescence signal enhancement in solution.
Silver nanoparticles (about 20-50 nm size) were synthesized as a stable yellow colloidal solution, and coated with proteins labeled with fluorophores. A several-fold amplification of the fluorescence signal in presence of colloid Ag nanoparticles in solution was observed. Such fluorophore-metal complex presents a unique opportunity for developing of new class of contrast agents for optical imaging and fluorescence based sensing. Solution of silver nanoparticles with enhanced fluorescence can be used in various assays such as DNA hybridization or immunoassays for high sensitivity detection.
We describe the development of a novel generic approach to fluorescence sensing based on metal-enhanced fluorescence (MEF). This work follows our initial reports of radiative decay engineering (RDE), where we experimentally demonstrated dramatic signal enhancements of fluorophores positioned close to surface-bound silver nanostructures. The attractive changes in spectral properties of fluorophores includes increased rates of excitation, increased quantum yields, decreased fluorescence lifetimes with an increased photostability, and drastically increased rates of multi-photon excitation. In this report we present a new class of fluorescent biomarkers which are strongly enhanced by metallic particles. This has afforded the development of a novel generic approach for ultra-sensitive fluorescence assay technology. The assay platform utilizes metal particles deposited on glass/quartz surfaces, covered with sub-nanometer layers of a fluorescent biomarker. As such the fluorescence signal of the composite is strongly enhanced. This readily allows easy, quantitative and inexpensive fluorescence detection of minimal traces of specific antigens. We also explore different sensing geometries, such as using evanescent wave excitation.
We described a new approach to measuring DNA hybridization using surface plasmon-coupled emission (SPCE). This phenomenon occurs for fluorophores within few hundreds of nanometers of a thin metal film on a glass substrate, in our case a 50 nm thick silver film. Excited fluorophores coupled with the surface plasmons in the metal resulting in directional emission through the glass substrate. We studied the emission of Cy3-labeled DNA oligomers bound to complementary unlabeled biotinylated-oligomers, which were bound to the metal surface via a streptavidin-BSA monolayer. Hybridization resulted in directional emission of Cy3-DNA into the prism. Additionally, the use of SPCE resulted in suppression of interfering emission from non-complementary Cy5-DNA oligomers due to weaker coupling of the more distant fluorophores with the surface plasmons. A large fraction of the total potential emission can couple to the surface plasmon resulting in improved sensitivity. We expect SPCE to have numerous applications to nucleic acid analyses.
Directional fluorescence emission of a sulforhodamine 101 in polyvinyl alcohol film has been observed from samples deposited on semi-transparent silver mirror. The fully p-polarized fluorescence emerges through the glass prism in form of hollow cone. The angle of this cone of emission depends on the thickness of the sample, and does not depend on the mode of excitation. The angular dependence of surface plasmon-coupled emission (SPCE) on the sample thickness has been discussed as well as its relevance to the surface plasmon resonance (SPR) analysis.
We report recent achievements in metal-enhanced fluorescence. Several fluorophore systems have been studied on metal particle-coated surface and in colloid suspensions. In particular, we describe a distance dependent enhancement on silver island films (SIFs), release of self-quenching of fluorescence near silver particles, and the applications of fluorescence enhancement near metalized surfaces to bioassays. We discuss a number of methods for various shpae silver particle deposition on surfaces.
In this presentation we describe a novel methodology for ultra-sensitive fluorescence immunoassays based on a new class of fluorescent biomarkers, which are strongly enhanced by nano-size metallic particles. Specifically, we discuss development of the immunoassay on the surfaces coated with metallic particles for high sensitivity detection of cardiac markers. This technology will allow detection of the biomarkers in serum and blood without separation and amplification steps. We present an experimental platform that uses front-face excitation in total internal reflection mode for efficient rejection of background fluorescence.
Fluorescence spectroscopy is a widely used research tool in biochemistry and has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing and genomics. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by a nearby metallic surface, the most important effect being an increase in the radiative decay rate. We describe the usual effects expected form increase in the radiative rates with reference to the biomedical applications of immunoassay and DNA hybridization. We also present experiments which show that metallic particles can increase the quantum yield of low quantum yield fluorophores, increase fluorophore photostability and increase the distance for resonance energy transfer. And finally we show that proximity to silver particles can increase the intensity of the intrinsic fluorescence from DNA.