Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate
vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the
change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using this
method to observe fluorescence from a single ribosomal particle in a project aimed at acquiring sequence information
from the translating ribosome (NIH's $1000 Genome Initiative). Several quartz slides with silver nanostructures were
made using electron beam lithography techniques. The structures were approximately 50 nm high silver tiles measuring
400-700 nm on the side, and were spaced differently over a total area of 1 mm x 1 mm on any given quartz slide. In a
preliminary experiment, we coated this surface with the Alexa 647-labeled antibodies and collected single molecule
images using the MicroTime 200 (PicoQuant) confocal system. We showed that the fluorescence intensity measured
over the silver islands film was more than 100-fold higher than fluorescence from a comparable site on uncoated section
of the quartz slide. No noticeable photobleaching was seen. The fluorescence lifetime was very short, about 200 ps or
less (this is the resolution limit of the system). The method has great promise for investigations of biologically relevant
single molecules.
Surface plasmon-coupled emission (SPCE) has been used to reduce the detection volume in fluorescence measurements. The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With the excitation through the glass prism at SPR angle (Kretschmann configuration), the detection volume is a composition (product) of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that using Kretchmann configuration in a microscope with high numerical aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL, which is necessary to observe a single cross-bridge in the muscle. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be also used to study the small conformational changes of macromolecules.
We present a novel approach for performing fluorescence immunoassay in serum and whole blood using fluorescently labeled anti-rabbit IgG. This approach, which is based on Surface Plasmon-Coupled Emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the
fluorophores located near a bio-affinity surface. Effective coupling range for SPCE is only couple of hundred nanometers from the metallic surface. Excited fluorophores outside the coupling layer do not contribute to SPCE, and their free-space emission is not transmitted through the opaque metallic film into the glass substrate. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal is discussed. The kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately 2- and 3-fold, respectively (compared to buffer), resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Both glass and plastic slides can be used for SPCE-based assays. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood, without any need for washing steps.
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.
A multi-analyte multi-color immunoassay utilizing enhanced fluorophores fluorescence in the proximity of silver island film (SIF) coated surface is described. A mixture of four different model analyte proteins was immobilized on the surface, and fluorescence signal of four corresponding antibodies labeled with different color fluorophores (emission maxima varied from 570 to 700 nm) was monitored using appropriate cut-off filters. To localize the excitation volume within close proximity of the SIF surface we used the total internal reflection (TIR) mode of excitation. Use of SIF coating resulted in approximately 5-10 fold increase in the intensity of the fluorophores signal if compared to the non-coated glass substrate surface. We suggest application of this methodology for multiplex immunoassays for simultaneous detection of several different analytes in medicine and biotechnology.
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.
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.
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