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A fourth generation PAMAM dendrimer has been successfully employed for the development of a single step synthesis strategy for self-assembled Ag-Au nanohybrid structures. The surface plasmon resonance properties and the degree of self-assembly of the nanohybrid are strongly correlated with the stoichiometry of the metals which gives rise to enhanced plasmonic properties. The enhanced plasmonic response of the nanohybrids is modeled and is validated experimentally in a model HRP (horseradish peroxidise) bioassay carried out on an SPR-based biochip platform.

In this work, gold-silica plasmonic nanohybrids have been synthesized as model systems which enable tuning of dye fluorescence enhancement/quenching interactions. For each system, a dye-doped silica core is surrounded by a 15 nm spacer region, which in turn is surrounded by gold nanoparticles (GNPs). The GNPs are either covalently conjugated via mercapto silanization to the spacer or encapsulated in a separate external silica shell. The intermediate spacer region can be either dye doped or left undoped to enable quenching and plasmonic enhancement effects respectively. The study indicates that there is a larger enhancement effect when GNPs are encapsulated in the outer shell compared to the system of external conjugation. This is due to the environmental shielding provided by shell encapsulation compared to the exposure of the GNPs to the solvent environment for the externally conjugated system. The fluorescence signal enhancement of the nanohybrid systems was evaluated using a standard HRP-anti-HRP fluorescence based assay platform.

We present a novel approach to the enhancement of surface plasmon-coupled emission (SPCE) using surface plasmon excitation in a bimetal (Ag/Au) layer and we validate the enhancement by presenting the results of a model human IgG immunoassay. Theoretical calculations using Fresnel's equations have been carried out to determine the optimum bimetallic composition and the resulting electric field enhancement. Signal enhancement of SPCE was confirmed using a range of bimetallic layers which were deposited on the surface of a high collection efficiency polymer array biochip and subsequently immobilized with Alexa Fluor 647 labeled anti-human IgG. The bimetallic film of Ag/Au (36/10nm) was determined as an optimum substrate for maximum SPCE signal which was a compromise between the long-term stability of the metal layer and the optimized evanescent field enhancement. An enhanced dose-dependent response was also demonstrated which was ∼3 times greater than that detected with a pure gold layer. A human IgG immunoassay showed a dose-dependent response yielding a limit of detection of 1pg/ml by the 3σ rule. The improved performance of the bimetal layer compared to that of an assay carried out on a pure gold layer is attributed to the enhanced evanescent field intensity of surface plasmons in the bimetal combination which excites more fluorescence hence producing an enhanced SPCE signal. This result demonstrates the potential of the SPCE-based array biochips as a sensitive and high-throughput analysis platform for biomolecular interactions., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The technique of surface plasmon-coupled emission (SPCE) involves the coupling of light which is emitted from a fluorophore into the surface plasmon of an adjacent thin metal film, giving rise to highly directional emission. We have combined the advantages of SPCE with the high light collection efficiency of supercritical angle fluorescence by carrying out an immunoassay on a paraboloid array biochip in the absence of the conventional SPCE spacer layer normally used to minimize metal quenching of the fluorescence. In this work, we have successfully demonstrated an SPCE-based assay by utilizing the protein assay layer as the spacer layer. A novel 3 × 3 injection molded polymer biochip with paraboloid elements was used. The paraboloid elements served to enhance the light collection efficiency while the top surface was coated with a gold layer to use excitation of surface plasmons and detection of SPCE emission. Theoretical modeling of the gold-protein layer structure showed that the surface plasmon resonance angles were located in the detection range of the paraboloid biochip. The polarization dependence of SPCE emission was also demonstrated. Finally, a human IgG sandwich immunoassay was carried out which exhibited a limit of detection of ~10 ng/ml using 3σ. The results demonstrate the potential of the SPCE-based paraboloid array biochip as a novel platform for high-throughput analysis of biomolecular interactions.

We have carried out a human IgG immunoassay on a novel disposable optical array biochip using surface plasmon-coupled emission (SPCE) detection. The work successfully combines the advantages of the highly directional SPCE emission profile and enhanced surface plasmon excitation with the high light collection efficiency achieved using supercritical angle fluorescence (SAF). This is achieved using an array of transparent paraboloid polymer elements which have been coated with a thin gold layer to facilitate SPCE. Moreover, since only the emission of molecules which are close to the metal surface couple into the surface plasmon, the detection is highly surface-specific leading to background suppression and increased signal-to-noise ratio. Theoretical calculations have been carried out in order to match the surface plasmon resonance angles and SPCE emission angles to the paraboloid array features for light collection. A sandwich assay format was used and a dose response curve was obtained in the concentration range 2 ng/ml to 200 microg/ml yielding a limit of detection of 20 ng/ml. This is the first demonstration of an SPCE-based assay on a disposable biochip platform and indicates the potential of SPCE-based arrays for high-throughput analysis of biomolecular interactions., (Crown Copyright 2009. Published by Elsevier B.V. All rights reserved.)

Surface plasmon-coupled emission (SPCE) is a phenomenon whereby the light emitted from a fluorescent molecule can couple into the surface plasmon of an adjacent metal layer, resulting in highly directional emission in the region of the surface plasmon resonance (SPR) angle. In addition to high directionality of emission, SPCE has the added advantage of surface selectivity in that the coupling diminishes with increasing distance from the surface. This effect can be exploited in bioassays whereby a fluorescing background from the sample can be suppressed. We have investigated, both theoretically and experimentally, the SPCE effect for a Cy5-spacer-Ag layer system. Both the angular dependence of emission and the dependence of SPCE emission intensity on Cy5-metal separation were investigated. It is demonstrated that SPCE leads to lower total fluorescence signal than that obtained in the absence of a metal layer. This is the first experimental verification of the reduction in SPCE intensity compared to the metal-free case. Our results are in a good agreement with theoretical models. The validation of the theoretical model provides a basis for optimizing biosensor platform performance, particularly in the context of the advantages offered by SPCE of highly directional emission and surface selectivity.

Abstract: In this work we report on the so-called plasmonic enhancement effect, whereby the presence of metallic surfaces or particles in the vicinity of a fluorophore can dramatically alter the fluorescence emission and absorption properties of a fluorophore. The effect, which is associated with the surface plasmon resonance of the metallic surface, depends on parameters such as metal type, particle size, fluorophore type, and fluorophore-particle separation. This work focuses on the creation of metal nanoparticle arrays by a lithographic process and on optimisation strategies to maximise the fluorescence enhancement of dyes in the vicinity of the nanoparticles, for important applications such as fluorescence-based biochip platforms. Ordered arrays of metallic nano-islands were fabricated on glass substrates by a process of natural lithography using monodisperse polystyrene nanospheres. The metal particle dimensions were tailored in order to tune the plasmon resonance wavelength to match the spectral absorption of the fluorophore. The fluorophore, Cy5 dye, which is widely used in optical immunoassays and has a medium quantum efficiency (∼0.3), was used in this preliminary study of the plasmonic enhancement effect. The morphology of the metallic arrays was investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Absorption and emission spectroscopies were used to elucidate the enhancement effect and its dependence on metal island morphology. Results were correlated with existing theoretical models. The applicability of this important technique to sensor platforms, such as fluorescence-based biochips, is also discussed. [Copyright &y& Elsevier]