Roughened silver electrodes for use in metal-enhanced fluorescence

Roughened silver electrodes are widely used for surface-enhanced Raman scattering (SERS). We tested roughened silver electrodes for metal-enhanced fluorescence. Constant current between two silver electrodes in pure water resulted in the growth of fractal-like structures on the cathode. This electrode was coated with a monolayer of human serum albumin (HSA) protein that had been labeled with a fluorescent dye, indocyanine green (ICG). The fluorescence intensity of ICG-HSA on the roughened electrode increased by approximately 50-fold relative to the unroughened electrode, which was essentially non-fluorescent and increased typically two-fold as compared to the silver anode. No fractal-like structures were observed on the anode. Lifetime measurements showed that at least part of the increased intensity was due to an increased radiative decay rate of ICG. In our opinion, the use of in situ generated roughened silver electrodes will find multifarious applications in analytical chemistry, such as in fluorescence based assays, in an analogous manner to the now widespread use of SERS. To the best of our knowledge this is the first report of roughened silver electrodes for metal-enhanced fluorescence.     

Controllable metal-enhanced fluorescence in organized films and colloidal system

In recent years, considerable efforts have been devoted to better understand the unique emission properties of fluorophores enhanced by the localized surface plasmon resonance of metal nanoparticles (NPs), due to the widespread applications of fluorescence techniques. It is demonstrated by experiment and theoretical calculation that the enhancement efficiency strongly depends on the morphology of the metal NPs, the spectral overlap between metal and fluorophores, the separation distance between them, and other factors. Among these aspects to be considered are suitable spacer material and assembling methods to control the spatial arrangement of plasmonic NPs and fluorophore with proper optical properties and interactions. In this contribution, we provide a brief overview on recent progress of metal-enhanced fluorescence in organized films and colloidal systems.     

Enhancing the analytical performance of immunoassays that employ metal-enhanced fluorescence

In this work, we used a model assay system (polyclonal human IgG–goat antihuman IgG) to elucidate some of the key factors that influence the analytical performance of bioassays that employ metal-enhanced fluorescence (MEF) using silver nanoparticles (NPs). Cy5 dye was used as the fluorescent label, and results were compared with a standard assay performed in the absence of NPs. Two sizes of silver NPs were prepared with respective diameters of 60 ± 10 and 149 ± 16 nm. The absorption spectra of the NPs in solution were fitted accurately using Mie theory, and the dipole resonance of the 149-nm NPs in solution was found to match well with the absorption spectrum of Cy5. Such spectral matching is a key factor in optimizing MEF. NPs were deposited uniformly and reproducibly on polyelectrolyte-coated polystyrene substrates. Compared to the standard assay performed without the aid of NPs, significant improvements in sensitivity and in limit of detection (LOD) were obtained for the assay with the 149-nm NPs. An important observation was that the relative enhancement of fluorescence increased as the concentration of antigen increased. The metal-assisted assay data were analyzed using standard statistical methods and yielded a LOD of 0.086 ng/mL for the spectrally matched NPs compared to a value of 5.67 ng/mL obtained for the same assay in the absence of NPs. This improvement of ∼66× in LOD demonstrates the potential of metal-enhanced fluorescence for improving the analytical performance of bioassays when care is taken to optimize the key determining parameters.      

Control of metal-enhanced fluorescence with pH- and thermoresponsive hybrid microgels

In this paper, we report on the Ag nanoparticle-containing hybrid poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-co-PAA) microgels with pH- and thermoresponsive metal-enhanced fluorescence (MEF). The hybrid microgels were prepared by in situ reducing Ag salts to Ag nanoparticles in the PNIPAM-co-PAA microgels. According to the interaction distance-dependent nature of MEF effects, we can realize a controllable MEF effect by adjusting the average interaction distance between fluorophores and Ag nanoparticles due to the good stimuli-responsive swelling-shrinking behavior of the hybrid microgels. The results show that MEF effect can be well tuned in the pH region 2-12 as well as the temperature region of 20-50 °C. By this method, an enhanced fluorescence detection can possibly be manipulated by adjusting external stimuli such as pH and temperature.     

Core-shell Ag@SiO2@mSiO2 mesoporous nanocarriers for metal-enhanced fluorescence

A novel mesoporous nanocarrier consisting of a silver core, a silica spacer with controlled thickness and a fluorophores-loaded mesoporous silica shell was fabricated for the metal-enhanced fluorescence (MEF) and F?rster resonance energy transfer (FRET) effects.     

Excitation energy transfer in poly(p-phenylphenylenevinylene) determined by polarized fluorescence spectroscopy

Polarized fluorescence spectroscopy is used to investigate the photophysical behavior of poly(p-phenylphenylenevinylene) (PPPV) in polystyrene matrix in comparison with oligomeric model compounds. For this purpose PPPV is modeled by a chain consisting of a distribution of independent oligomeric segments. Excitation energy transfer (EET) between the segments depends on the wavelength of excitation, not only for transfer along an isolated polymer chain, but also for intermolecular transfer at high concentration of PPPV. The spatial range of EET, as indicated by fluorescence depolarization, is reduced for excitation at the long wavelength edge of the absorption spectrum (“red-edge-effect”). © 1995 John Wiley & Sons, Inc.     

Microwave-accelerated metal-enhanced fluorescence: an ultra-fast and sensitive DNA sensing platform

In this paper, we investigated the effects of low-power microwave heating on the components of the recently described new approach to surface DNA hybridization assays, based on the Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF) platform technology. Thiolated oligonucleotides have been linked to surface-bound silver nanostructures which partially coat a glass slide. The addition of a complementary fluorescein-labeled oligonucleotide results in metal-enhanced fluorescein emission as the probe is brought into close proximity to the silver upon hybridization. In addition, the combined use with low-power microwave heating, which is thought to locally heat around the silvered surface, affords for both the assay kinetics and optical amplification to also be localized to the surface. In our model DNA target assay reported here, we can detect 23-mer targets in less than 20 s, up to a 600-fold decrease in the assay run time as compared to control samples hybridized to completion at room temperature. Importantly, the use of MAMEF also reduces the extent of unwanted non-specific DNA absorption, further increasing specific DNA target detection limits. It was also found that low-power microwave heating did not denature DNA and the bulk temperature increase near to silver nanoparticles was only ca. 1 degrees C.     

Rapid deposition of triangular silver nanoplates on planar surfaces: application to metal-enhanced fluorescence

A simple and rapid wet-chemical technique for the deposition of silver triangles on conventional glass substrates, which alleviates the need for lithography, has been developed. The technique is based on the seed-mediated cetyltrimethylammonium-bromide-directed growth of silver triangles on glass surfaces, where smaller spherical silver seeds that were attached to the surface were subsequently converted and grown into silver triangles in the presence of a cationic surfactant and silver ions. The size of the silver triangles was controlled by sequential immersion of silver seed-coated glass substrates into a growth solution and by the duration time of immersion. Atomic force microscopy studies revealed that the size of the silver triangles ranged between 100 and 500 nm. Interestingly, these new surfaces are a significant improvement over traditional silver island films for applications in metal-enhanced fluorescence. A routine 16-fold enhancement in emission intensity was typically observed, for protein-immobilized indocyanine green, with a relatively very low loading density of silver triangles on the glass surface.     

Angular-dependent metal-enhanced fluorescence from silver colloid-deposited films: opportunity for angular-ratiometric surface assays

We describe an exciting opportunity for Metal-Enhanced Fluorescence (MEF)-based surface assays using an angular-ratiometric approach to the observed enhanced emission from fluorophores in close proximity to silver colloids deposited on glass substrates. This approach utilizes the radiationless energy transfer (coupling) between the excited states of the fluorophore and the induced surface plasmons of the silver colloids, and the subsequent angular-dependent fluorescence emission from the fluorophore-silver colloid system. Since MEF is related to surface plasmons' ability to scatter light, angular-dependent light scattering from three different silvered surfaces and glass substrates were investigated using two common excitation angles, 45 and 90 degrees . The scattered light from silvered surfaces with a high loading was observed at wider angles on both sides of the glass substrates, while forward scattering (from the back of the glass) was dominant for the silvered surfaces with low loading, as explained by both Mie and Rayleigh theories. When silver colloids were placed between the fluorophore and glass interface, the coupled fluorescence emission through the higher refractive index glass (and in air), increased in an angular-dependent fashion, following closely the angular-dependent light scattering pattern of the silver colloids themselves. Similar observations for fluorescence emission from fluorophores deposited onto glass surfaces alone were made, but at much narrower angles on both sides of the fluorophore-glass interface and were simply explained by Lambert's cosine law. As the loading of silver on glass was increased, the enhanced fluorescence emission was observed at wider angles (towards 0 and 180 degrees ) at both sides of the silvered surfaces. Glass surfaces without silver colloids were used as control samples to demonstrate the benefits of MEF for enhancing fluorescence signatures in an elegant, angular-dependent fashion. Finally, the utility of the angular-dependent MEF phenomenon for intensity-based angular-ratiometric surface assays is demonstrated.     

Distance-dependent metal-enhanced fluorescence from Langmuir-Blodgett monolayers of alkyl-NBD derivatives on silver island films

We described the effect of fluorophore distance from the silver island films (SIFs) on the metal-enhanced fluorescence (MEF) from two newly developed long-chain nitrobenzoxadiazole derivatives (NBD-C16 and NBD-C18). The well-established Langmuir-Blodgett technique is used to deposit the fluorophores at defined distances from the SIFs surface, and an inert amphiphilic stearic acid is used to control the distance. NBD probes deposited directly on the SIFs surface show the highest metal-enhanced fluorescence of approximately 32-fold, and both of the probes that were studied show a consistent decrease in metal-enhanced fluorescence when increasing the distance from the fluorophore to the SIFs surface. The lowest fluorescence enhancement of approximately 4-fold is observed for the probes located 90 nm from the SIFs surface. Additionally, we also have noticed the shortest fluorescence lifetimes for the NBD probes deposited directly onto the SIFs surface, and the lifetimes are consistently increased when increasing the distances between the fluorophore and SIFs surfaces. These contrasting spectral changes, enhanced fluorescence, and decreased fluorescence lifetimes are in accordance with an increase in the rate of radiative decay for fluorophores near the silver particles. The present study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.     

A close analysis of metal-enhanced fluorescence of tryptophan induced by silver nanoparticles: wavelength emission dependence

In the last few years, silver nanoparticles have been proposed as a promising alternative for the label-free detection of proteins via metal-enhanced fluorescence. Generally, the aromatic amino acid tryptophan is most frequently used in this type of studies, because the intrinsic fluorescence of proteins is usually dominated by tryptophan emissions. In the present study, we evaluated the fluorescence behavior of tryptophan in the presence of a silver colloid with nanoparticles of 100 nm in diameter. The results showed that a nanoparticles concentration of 32 mg L^?1 induced maximum fluorescence enhancement. However, the metal-enhanced fluorescence was dependent on the emission wavelength of tryptophan, and this phenomenon was closely related to the metal surface reabsorption process (inner filter effect), suggesting that the plasmon resonance reabsorption effect should be taken into account in analyses involving protein studies by metal-enhanced fluorescence.     

Fast and slow deposition of silver nanorods on planar surfaces: application to metal-enhanced fluorescence

Two methods have been considered for the deposition of silver nanorods onto conventional glass substrates. In the first method, silver nanorods were deposited onto 3-(aminopropyl)triethoxysilane-coated glass substrates simply by immersing the substrates into the silver nanorod solution. In the second method, spherical silver seeds that were chemically attached to the surface were subsequently converted and grown into silver nanorods in the presence of a cationic surfactant and silver ions. The size of the silver nanorods was controlled by sequential immersion of silver seed-coated glass substrates into a growth solution and by the duration of immersion, ranging from tens of nanometers to a few micrometers. Atomic force microscopy and optical density measurements were used to characterize the silver nanorods deposited onto the surface of the glass substrates. The application of these new surfaces is for metal-enhanced fluorescence (MEF), whereby the close proximity of silver nanostructures can alter the radiative decay rate of fluorophores, producing enhanced signal intensities and an increased fluorophore photostability. In this paper, it is indeed shown that irregularly shaped silver nanorod-coated surfaces are much better MEF surfaces as compared to traditional silver island or colloid films. Subsequently, these new silver nanorod preparation procedures are likely to find a common place in MEF, as they are a quicker and much cheaper alternative as compared to surfaces fabricated by traditional nanolithographic techniques.     

A model for DNA detection by metal-enhanced fluorescence from immobilized silver nanoparticles on solid substrate

N-(2-Mercaptopropionyl)glycine (tiopronin)-coated silver nanoparticles (average diameter of metal core=5 nm) were prepared by a modified Brust method. Tiopronin ligands were partially displaced by thiolate single-stranded oligonucleotides via ligand exchange. These particles were immobilized onto a solid substrate through hybridization with target oligonucleotides in a layer-by-layer approach. The dye-labeled complementary oligonucleotides were bound to the particle layers through hybridization. Fluorescence intensity was enhanced with a simultaneous increase of plasmon absorbance from accumulated particles. A steady state was shown at the 10th particle layer and then the fluorescence enhancement showed a plateau. This result reveals that increasing the particle layer contributes to fluorescence enhancement. This novel method was used to detect DNA hybridization through both absorbance and emission spectral changes.     

An enzyme-responsive metal-enhanced near-infrared fluorescence sensor based on functionalized gold nanoparticles

Near-infrared (NIR) fluorescence imaging is promising due to the high penetration depths and minimal levels of autofluorescence in living systems. However, it suffers from low fluorescent quantum yield, and metal-enhanced fluorescence (MEF) is considered to be a promising technique to overcome this. Stimuli-responsive NIR fluorescence enhancement shows remarkable potential for applications in medical imaging and diagnosis. Herein, we successfully fabricated an enzyme-responsive near-infrared sensor based on MEF by functionalizing gold nanoparticles with NIR fluorophores and enzyme-responsive self-aggregation moieties. The NIR fluorescence of fluorophores on the gold nanoparticles was significantly enhanced due to increases both in the light scattering intensity and in the radiative decay rate (k _r) of the NIR fluorophores, along with relatively small variation in the nonradiative decay rate. This novel strategy for NIR fluorescent sensors should be particularly promising for NIR fluorescence imaging of enzyme activities and early diagnosis based on rationally designed nanomaterials.     

Microwave-accelerated metal-enhanced fluorescence: application to detection of genomic and exosporium anthrax DNA in <30 seconds

We describe the ultra-fast and sensitive detection of the gene encoding the protective antigen of Bacillus anthracis the causative agent of anthrax. Our approach employs a highly novel platform technology, Microwave-Accelerated Metal-Enhanced Fluorescence (MAMEF), which combines the use of Metal-Enhanced Fluorescence to enhance assay sensitivity and focused microwave heating to spatially and kinetically accelerate DNA hybridization. Genomic and exosporium target DNA of Bacillus anthracis spores was detected within a minute in the nanograms per microliter concentration range using low-power focused microwave heating. The MAMEF technology was able to distinguish between B. anthracis and B. cereus, a non-virulent close relative. We believe that this study has set the stage and indeed provides an opportunity for the ultra-fast and specific detection of B. anthracis spores with minimal pre-processing steps using a relatively simple but cost-effective technology that could minimize casualties in the event of another anthrax attack.     

Excitation and decay processes in helium clusters studied by fluorescence spectroscopy

Excitation and decay processes of helium clusters are investigated with fluorescence methods. The results differ remarkably from that obtained for the heavier rare gas clusters. They are discussed in view of the unusual structural and electronic properties of helium.     

Langmuir-Blodgett monolayers of long-chain NBD derivatives on silver island films: Well-organized probe layer for the metal-enhanced fluorescence studies

The metal-enhanced fluorescence (MEF) from the well-organized monolayers of two newly prepared long-chain alkylamine derivatives of nitrobenzoxadiazole (NBD-C16 and NBD-C18) deposited on silver island films (SIFs) has been investigated. The NBD derivatives were conveniently prepared by using a single step procedure in quantitative yields. The monolayers of the probes on SIFs as well as on bare-glass slides were obtained by using the Langmuir-Blodgett (LB) technique. Orientation of the NBD probe molecule in LB monolayer film was measured with polarized absorption spectroscopy. The observed tilt angle of the probe transition dipole moment with respect to the surface normal of approximately 67-68 degrees was evaluated. We observed that the NBD monolayers deposited in close proximity to silver islands show about a 16-fold increase in fluorescence intensity and shortened fluorescence lifetime compared to those on bare-glass, which are due to metal enhanced fluorescence. On the other hand, the corresponding MEF from randomly oriented film obtained by using spin coating of the probes on SIFs was only 2.5-fold. Further, we deposited mixed monolayers of NBD-C16 or NBD-C18 with various molar ratios of stearic acid to understand the polarity effect on MEF. Interestingly, we found a consistent increase in MEF efficiency with increasing molar ratio of stearic acid. Along with MEF, we also found a continuous blue-shift in emission band maxima of the probes with an increasing molar ratio of stearic acid. The observed increase in MEF efficiency is justified based on cooperative effects of (1) the modulations in electronic density of the surface plasmon absorption band of SIFs and (2) defined probe orientation that might lead to preferential excitation.     

A volumetric investigation of solvophobic effects in halide-n-alkanol-n-alkane ternary systems

The density of solutions in the methanol-heptane-sodium chloride and ethanol-octane-lithium chloride systems was measured at 298.15 K using a vibratory densimeter. From the experimental data, apparent and partial molar volumes were calculated accurate to 0.01 cm³/mol. It was shown that volume properties versus composition dependences behave extremely. This behavior was attributed to restructuring of the solutions, namely, to a closer packing of molecules of the alkanol-alkane mixed solvent at an alcohol mole fraction of 0.95. It was shown that, for larger hydrocarbon residues in both components of the mixed solvent, the extremum in the volume properties does not disappear and holds its position.