Highly Sensitive Quantitation of Human Serum Albumin in Clinical Samples for Hypoproteinemia using Metal-Enhanced Fluorescence

In this paper, we have explored metal-enhanced fluorescence (MEF) of the Human serum albumin indicators: Albumin Blue 580, Merocyanine 540 and Bromophenol Blue in close proximity to silver nano-particles, SiFs, from both buffered and clinical samples. The photostability of the Albumin Blue 580 is shown to be much more prolonged from the SiFs as compared to glass (a control sample), potentially allowing for longer detection times to further improve assay statistics. Our findings suggest the widespread use of nanoparticulate SiFs surfaces for the enhanced detection of HSA, particularly for Hypoproteinemia, where an enhanced assay performance at low protein abundance is required.     

Low Temperature Metal-Enhanced Fluorescence

In this short letter, we describe the effects of low temperature on the Metal-Enhanced Fluorescence (MEF) phenomenon. Fluorophores close to Silver Island Films (SiFs) show on average two- to ten-fold enhancements in their fluorescence signatures at room temperature. However, at 77 K, we have observed that MEF is even more pronounced as compared to an identical glass control sample. We also demonstrate that the further enhancements in MEF occur at low temperature over a range of visible wavelengths for different fluorophores, for both SiFs and 20 nm surface deposited gold colloids.     

Advances in Surface-Enhanced Fluorescence

We report recent achievements in metal-enhanced fluorescence from our laboratory. Several fluorophore systems have been studied on metal particle-coated surfaces 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 shaped silver particle deposition on surfaces.     

Fluorescence enhancement of the conjugated polymer films based on well-ordered Au nanoparticle arrays

In this paper, well-ordered Au nanoparticle arrays on silicon substrates were employed as efficient metal-enhanced fluorescence (MEF) substrates for investigating the fluorescence properties of the conjugated polymer poly(3-hexylthiophene) (P3HT). The ordered Au nanoparticle arrays were fabricated by block copolymer self-assembly technology, and the particle sizes were controlled by adjusting the molar ratios of HAuCl₄ precursor to vinyl pyridine units. The approach is economical and suitable to fabricate large-area MEF substrates. The results about fluorescence properties of P3HT showed that the fluorescence intensities of the P3HT films were improved on ordered Au nanoparticle arrays compared to those on bare silicon substrate and were significantly enhanced with the Au nanoparticle sizes increasing. The mechanism is based on localized surface plasmon resonances, coupling and propagating surface plasmons, and the emission enhancement mainly resulted from the increase of the excitation rate. This work provides a new way to prepare efficient MEF substrates for high-performance fluorescence-based devices.     

Metal-Enhanced Fluorescence Solution-Based Sensing Platform

In recent years our laboratories have reported the favorable effects for fluorophores placed in close proximity to surface immobilized silver nanostructures. These include; greater quantum yields, reduced lifetimes (increased photostability) and directional emission. However, while these findings are likely to find multifarious applications for surface assays based on enhanced fluorescence detection, a solution based enhanced sensing platform has yet to be realized. In this short, note, we show how SiO2-coated silver colloids, indeed provide for a solution based enhanced fluorescence sensing platform with a 3-5 fold enhancement typically observed.     

Fluorescence Enhancements on Silver Colloid Coated Surfaces

We observed a strong, more than 16-fold, enhancement of Texas Red-labeled BSA fluorescence emission when deposited on silver colloid coated surfaces (SCCS). The same labeled protein deposited on silver island films (SIFs) showed an approximate 8-fold fluorescence enhancement. The lifetimes of Texas Red-BSA fluorescence are significantly shorter on silvered surfaces than on uncoated quartz substrate indicating a strong change in radiative decay rate of the dyes. We also observed a 36-fold increased brightness of overlabeled fluorescein-HSA deposited on silver colloid coated surface. Stronger enhancement observed for overlabeled Fl-HSA protein indicates that presence of silver particles partially decreased self-quenching. Our results indicate that surfaces coated with silver colloids are valuable substrates for metal-enhanced fluorescence.     

Silver Fractal-Like Structures for Metal-Enhanced Fluorescence: Enhanced Fluorescence Intensities and Increased Probe Photostabilities

Substantial increases in fluorescence emission from fluorophore-protein–coated fractal-like silver structures have been observed. We review two methods for silver fractal structure preparation, which have been employed and studied. The first, a roughened silver electrode, typically yielded a 100-fold increase in fluorophore emission, and the second, silver fractal-like structures grown on glass between two silver electrodes, produced a 500-fold increase. In addition, significant increases in probe photostability were observed for probes coated on the silver fractal like structures. These results further serve to compliment our recent work on the effects of nobel metal particles with fluorophores, a relatively new phenomenon in fluorescence we have termed both metal-enhanced fluorescence [1] and radiative decay engineering [2,3]. These results are explained by the metallic surfaces modifying the radiative decay rate () of the fluorescent labels. We believe that this new silver-surface preparation, which results in ultrabright and photostable fluorophores, offers a new generic technology platform for increased fluorescence signal levels, with widespread potential applications to the analytical sciences, imaging, and medical diagnostics.     

Metal-Enhanced Chemiluminescence

In this short paper we report the interactions of silver island films with chemiluminescing species. Our findings show that silver island films can increase the detectability of chemiluminescent reactions/species, with an approximately 5-fold increase in signal intensity. This finding not only suggests the use of silver nanostructures to amplify chemiluminscent signatures in assay platforms, and therefore increase the detectability of analytes or biospecies, but more importantly, suggests that surface plasmons can be directly excited by chemically induced electronically excited molecules. This finding is of significance towards our understanding of fluorophore–metal interactions, a relatively new near-field fluorescence concept, recently named metal-enhanced fluorescence and also radiative decay engineering.     

Voltage-Gated Metal-Enhanced Fluorescence II: Effects of Fluorophore Concentration on the Magnitude of the Gated-Current

In this letter we report further findings on the ability of an applied direct current to modulate Metal-Enhanced Fluorescence (MEF). Fluorophores in close-proximity to just-continuous silver films (JCS) show significantly enhanced fluorescence intensities. However, when a current is applied to the films, the enhanced fluorescence can be gated in a manner that depends on both the fluorophore concentration, the magnitude of the applied current and the extent of the protein mono to multi-layer surface coverage. Our results are consistent and indeed further support our previous hypothesis and model that fluorophore-metal near-field interactions can be influenced by an applied direct current.     

Enhanced Fluorescence from Periodic Arrays of Silver Nanoparticles

Electron beam lithography was used to fabricate silver nanoparticle arrays and study the effects of geometrical properties of particles on metal-enhanced fluorescence. Nanoparticle size, shape, interparticle spacing, and nominal thickness were varied in a combinatorial pattern for investigation of the particle plasmon resonance effect on enhancement of fluorescence from three different fluorophores; Fluorescein, Cy3, and Cy5. A specific geometric property for optimal enhancement from each fluorophore was determined. For interparticle spacings greater or equal to 270 nm, the enhancement matched what is expected for a single-particle model. For those particles smaller than 210 nm, the enhancement was lower than for the larger spacing in the range studied. Triangular-shaped particles gave similar enhancement to those of square-shaped particles. This combinatorial pattern by e-beam lithography was found to be useful for studying how individual parameters enhance the fluorescence that are important for rational design of enhanced fluorescence sensors.     

Integral fluorescence enhancement by silver nanoparticles controlled via PMMA matrix

The metal-enhanced fluorescence is measured with different thickness of emission film. Silver nanoparticles are immobilized on glass slide by chemical self-assembly method. Rhodamine B molecules are dispersed in the polymer matrix of Poly(methyl methacrylate) (PMMA), then spin coated on prepared silver particles substrate with different thickness from 15 nm to 70 nm. The enhanced fluorescence is observed depending on the thickness of emission film since the average distance between rhodamine B molecules and silver nanoparticles is altered by the PMMA matrix. The 5-fold enhancement is attained. The experiment was explained qualitatively by an integral fluorescence enhancement.     

Annealed Silver-Island Films for Applications in Metal-Enhanced Fluorescence: Interpretation in Terms of Radiating Plasmons

The effects of thermally annealed silver island films have been studied with regard to their potential applicability in applications of metal-enhanced fluorescence, an emerging tool in nano-biotechnology. Silver island films were thermally annealed between 75 and 250^∘C for several hours. As a function of both time and annealing temperature, the surface plasmon band at ≈420 nm both diminished and was blue shifted. These changes in plasmon resonance have been characterized using both absorption measurements, as well as topographically using Atomic Force Microscopy. Subsequently, the net changes in plasmon absorption are interpreted as the silver island films becoming spherical and growing in height, as well as an increased spacing between the particles. Interestingly, when the annealed surfaces are coated with a fluorescein-labeled protein, significant enhancements in fluorescence are osbserved, scaling with annealing temperature and time. These observations strongly support our recent hypothesis that the extent of metal-enhanced fluorescence is due to the ability of surface plasmons to radiate coupled fluorophore fluorescence. Given that the extinction spectrum of the silvered films is comprised of both an absorption and scattering component, and that these components are proportional to the diameter cubed and to the sixth power, respectively, then larger structures are expected to have a greater scattering contribution to their extinction spectrum and, therefore, more efficiently radiate coupled fluorophore emission. Subsequently, we have been able to correlate our increases in fluorescence emission with an increased particle size, providing strong experiment evidence for our recently reported metal-enhanced fluorescence, facilitated by radiating plasmons hypothesis.     

Metal-Enhanced Fluorescence from Gold Surfaces: Angular Dependent Emission

The first observation of Metal-Enhanced Fluorescence (MEF) from large gold colloids is presented. Gold colloids, 40 and 200 nm diameter, were deposited onto glass substrates in a homogeneous fashion. The angular-dependent fluorescence emission of FITC-HSA, adsorbed onto gold colloids, was measured on a rotating stage which was used to evaluate MEF at all spatial angles. The emission intensity of FITC-HSA was found to be up to 2.5-fold brighter than the emission on bare glass substrates at an angle of 270 degrees. This is explained by the Radiating Plasmon Model, whereby the combined system, composed of the fluorophore and the metal colloids, emits with the photophysical characteristics of the fluorophore, after the excitation and the partial radiationless energy transfer between the excited states of the fluorophore and the surface plasmons of the gold colloids. The fluorescence enhancement was found to be higher with 200 nm gold colloids as compared to 40 nm colloids due to the increased contribution of the scattering portion of the 200 nm gold colloid extinction spectrum. These observations suggest that gold colloids could be used in MEF applications, offering more stable surfaces than the commonly used silvered surfaces, for applications requiring longer term storage and use.     

Fluorescence properties of dyes adsorbed to silver islands,investigated by picosecond techniques

The fluorescence properties of dye molecules (rhodamine 6G and erythrosin) adsorbed on pure glass surfaces and on silver islands films are investigated by cw and picosecond time-resolved methods. On pure glass surfaces we observe concentration quenching below a critical intermolecular distance (reduction of the fluorescence power per molecule as well as shortened and non-exponential fluorescence decay). On silver islands films the shortening in fluorescence lifetime is more drastic and is nearly independent of the intermolecular distance. This behavior suggests an electrodynamic interaction between dye monomers and plasmons in the metal particles, modified by a damping influence of dye dimers.     

Enhanced Ratiometric pH Sensing Using SNAFL-2 on Silver Island Films: Metal-enhanced Fluorescence Sensing

We have explored the opportunities for enhanced ratiometric pH sensing using the well-known carboxy seminaphthofluorescein (SNAFL-2) and silver island films (SiFs). Our results show that the metallic surfaces can provide up to a 40-fold increase in probe fluorescence intensity as compared to nonmetallic surfaces with the same probe coverage. However, while the S/N is significantly better for pH sensing, the emission wavelength ratiometric values are similar to that obtained in solution, due to the fact that the emission of both the acidic and basic forms of the probe are enhanced to similar extents. To the best of our knowledge this is the first report of enhanced ratiometric fluorescence sensing on metallic surfaces.     

Metal Enhanced Fluorescence Solution-based Sensing Platform 2: Fluorescent Core-Shell Ag@SiO<Subscript>2</Subscript> Nanoballs

In this Rapid Communication, we present the development of monodisperse core-shell (silver core-silica shell) nanoparticles with various shell thicknesses featuring a fluorophore, subsequently named Metal-Enhanced Fluorescence (MEF) nanoballs. MEF nanoballs consist of a ≈130 nm silver nanoparticle core, a silica shell with up to 35 nm thickness and fluorophores doped within the silica shell. Fluorescent nanobubbles where the silver core is removed by chemical etching are used as control samples to show the benefits of using silver nanoparticles, i.e, Metal-Enhanced Fluorescence. Finally, we demonstrate the broad potential biological applications of MEF nanoballs by employing near-infra red emitting probes (Rhodamine 800) within the silica shell, for potential applications in cellular imaging and solution-based sensing. Kadir Aslan, Meng Wu, Contributed equally     

Voltage-Gated Metal-Enhanced Fluorescence

We demonstrate the influence of electrical current on the ability of surface plasmons to amplify fluorescence signatures. An applied direct current across Silver Island Films (SIFs) of low electrical resistance perturbs the fluorescence enhancement. For a given applied current, surface plasmons in just-continuous films are sparsely available for fluorophore dipole-coupling and hence the enhanced fluorescence is gated as a function of the applied current. For thicker, low resistance films, sufficient charge carriers are now present in the metal that metal-enhanced fluorescence (MEF) is perturbed to a lesser extent, induced surface plasmons readily formed on the surface by the close-proximity dipole.     

Glass‐embedded silver nanoparticle patterns by masked ion‐exchange process for surface‐enhanced Raman scattering

Glass‐embedded silver nanoparticle patterns were fabricated by masked silver–sodium ion‐exchange process followed by etching to reveal the particles for surface‐enhanced Raman scattering (SERS). The intensity of the enhanced Raman signal is comparable to that of the fluorescence, and the detection limit of 1 nM for Rhodamine 6G has been achieved. Raman images at different etching depths and corresponding morphological images are compared to find optimal SERS signal. Our results demonstrate that silver nanoparticle patterns embedded in glass can be used as SERS‐active substrates. Nanoparticles can be formed in a glass of high optical quality and have potential to be integrated with optical waveguides for a sensor chip. Copyright © 2010 John Wiley & Sons, Ltd.     

Metal-enhanced Singlet Oxygen Generation: A Consequence of Plasmon Enhanced Triplet Yields

In this Rapid Communication, we report the first observation of Metal-Enhanced singlet oxygen generation (ME¹O₂). Rose Bengal in close proximity to Silver Island Films (SiFs) can generate more singlet oxygen, a three-fold increase observed, as compared to an identical glass control sample but containing no silver. The enhanced absorption of the photo-sensitizer, due to coupling to silver surface plasmons, facilitates enhanced singlet oxygen generation. The singlet oxygen yield can potentially be adjusted by modifying the choice of MEF (Metal-Enhanced Fluorescence) & MEP (Metal Enhance Phosphorescence) parameters, such as distance dependence for plasmon coupling and wavelength emission of the coupling fluorophore. This is a most helpful observation in understanding the interactions between plasmons and lumophores, and this approach may well be of significance for singlet oxygen based clinical therapy.     

Reduced Lifetimes are Directly Correlated with Excitation Irradiance in Metal-Enhanced Fluorescence (MEF)

We describe a fundamental observation in Metal-Enhanced Fluorescence (MEF), which has become a leading technology in the life sciences today, namely, how the lifetime of fluorophores near-to metallic plasmon-supporting silver islands/nanoparticles, modulates as a function of excitation power irradiance. This finding is in stark contrast to that observed in classical far-field fluorescence spectroscopy, where excitation power does not influence fluorophore radiative decay/lifetime.