Angular-Dependent Metal-Enhanced Fluorescence from Silver Island Films

In this letter we report the observation of angular-dependent Metal Enhanced Fluorescence (MEF) from fluorophores deposited onto silver island films (SiFs). When illuminated with laser light (473 nm) at angles of 45 and 90 degrees from the surface, SiFs scattered light at wide observation angles biased by the direction of the incident light. We observed angular-dependent MEF (10-fold) from FITC-HSA immobilized onto the SiFs, again slightly biased with respect to the direction of the incident light. We also measured the photostability of FITC from the back of the glass substrate at angles of 225 and 340 degrees.     

Metal-enhanced fluorescence using anisotropic silver nanostructures: critical progress to date

In this critical and timely review, the effects of anisotropic silver nanostructures on the emission intensity and photostability of a key fluorophore that is frequently used in many biological assays is examined. The silver nanostructures consist of triangular, rod-like, and fractal-like nanoparticles of silver deposited on conventional glass substrates. The close proximity to silver nanostructures results in greater intensity and photostability of the fluorophore than for fluorophores solely deposited on glass substrates. These new anisotropic silver nanostructure-coated surfaces show much more favorable effects than silver island films or silver colloid-coated substrates. Subsequently, the use of metal-enhanced fluorescence (MEF) for biosensing applications is discussed.     

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.     

Sulforhodamine Adsorbed Langmuir-Blodgett Layers on Silver Island Films: Effect of Probe Distance on the Metal-Enhanced Fluorescence

Metal-Enhanced Fluorescence (MEF) has become an important method in biomedical sensing. In this paper, we present the distance-dependent MEF of sulforhodamine B (SRB) monolayer on silver island films (SIFs). SRB is electrostatically incorporated into the Langmuir-Blodgett (LB) layers of octadecylamine (ODA) deposited on glass and SIFs substrates. The distances between SRB and SIFs or glass surfaces are controlled by depositing a varied number of inert stearic acid (SA) spacer layers. SRB is incorporated into positively charged LB layers of ODA by immersing the ODA deposited substrates into aqueous solution of SRB. Dye incorporated ODA layers with 10 nm separation distance from the SIFs surface show maximum metal-enhanced fluorescence intensity; ~7-fold increase in intensity as compared to that from the glass surface. The corresponding enhancement factor is reduced with increasing or decreasing the probe distance from the SIFs surface. Additionally, SRB on SIF surfaces show reduced lifetimes. We observed the shortest lifetime from the SRB with 5 nm distance from the SIF surfaces and the lifetime increased consistently with increasing the distances between the fluorophore and the SIFs surface. These observed spectral changes, increase in fluorescence intensity and decreased fluorescence lifetimes, are in accordance with the expected effects due to near-field interactions between the silver nanoparticles and fluorophores. We have also analyzed the complex fluorescence heterogeneous decays on metallic nanostructured surfaces using continuous distributions of decay times. The decay-time distributions appear to be sensitive to the distance between the metal and fluorophore and represent the underlying heterogeneity of the samples. The present systematic study provides significant information on the effect of fluorophore distance on the metal-enhanced fluorescence phenomenon.     

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.     

Angular-ratiometric plasmon-resonance based light scattering for bioaffinity sensing

We describe an exciting opportunity for affinity biosensing using a ratiometric approach to the angular-dependent light scattering from bioactivated and subsequently aggregated noble metal colloids. This new model sensing platform utilizes the changes in particle scattering from very small colloids, which scatter light according to traditional Rayleigh theory, as compared to the changes in scattering observed by much larger colloidal aggregates, formed due to a bioaffinity reaction. These larger aggregates no longer scatter incident light in a Cos(2) theta dependence, as is the case for Rayleigh scattering, but instead scatter light in an increased forward direction as compared to the incident geometry. By subsequently taking the ratio of the scattered intensity at two angles, namely 90 degrees and 140 degrees , relative to the incident light, we can follow the association of biotinylated bovine serum albumin-coated 20 nm gold colloids, cross-linked by additions of streptavidin. This new model system can be potentially applied to many other nanoparticle assays and has many advantages over traditional fluorescence sensing and indeed light-scattering approaches. For example, a single nanoparticle can have the equivalent scattered intensity as 10(5) fluorescing fluorescein molecules substantially increasing detection; the angular distribution of scattered light from noble metal colloids is substantially easier to predict as compared to fluorescence; the scattered light is not quenched by biospecies; the ratiometric measurements described here are not dependent on colloid concentration as are other scattering techniques; and finally, the noble metal colloids are not prone to photodestruction, as is the case with organic fluorophores.     

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.     

Silver-Gold Nanocomposite Substrates for Metal-Enhanced Fluorescence: Ensemble and Single-Molecule Spectroscopic Studies

In recent years, there has been a growing interest in the studies involving the interactions of fluorophores with plasmonic nanostructures or nanoparticles. These interactions lead to several favorable effects such as increase in the fluorescence intensities, increased photostabilities, and reduced excited-state lifetimes that can be exploited to improve the capabilities of present fluorescence methodologies. In this regard, we report the use of newly developed silver-gold nanocomposite (Ag-Au-NC) structures as substrates for metal-enhanced fluorescence (MEF). The Ag-Au-NC substrates have been prepared by a one-step galvanic replacement reaction from thin silver films coated on glass slides. This approach is simple and suitable for the fabrication of MEF substrates with large area. We have observed about 15-fold enhancement in the fluorescence intensity of ATTO655 from ensemble fluorescence measurements using these substrates. The fluorescence enhancement on the Ag-Au-NC substrates is also accompanied by a reduction in the fluorescence lifetime of ATTO655, which is consistent with the fluorophore-plasmon coupling mechanism. Single-molecule fluorescence measurements have been performed to gain more insight into the metal-fluorophore interactions and to unravel the heterogeneity in the interaction of individual fluorophores with the fabricated substrates. The single-molecule studies are in good agreement with the ensemble measurements and show maximum enhancements of ~50-fold for molecules located in proximity to the "hotspots" on the substrates. In essence, the Ag-Au-NC substrates have a very good potential for various MEF applications.     

Metal-enhanced fluoroimmunoassay on a silver film by vapor deposition

We studied a fluoroimmunoassay using metal-enhanced fluorescence (MEF) detection on silver film generated by vapor deposition method. The morphology of the silver film was controlled through the thickness of the film. A silica layer was coated on the silver film to protect the film and separate the fluorophore from the metal surface. Rabbit immunoglobulin G (IgG) was adsorbed on the silica by physiosorption and then dye-labeled anti-rabbit IgG was bound to the immobilized rabbit IgG. It was observed that the fluorophore was quenched on a thin silver film (2 nm), enhanced on a thick film (>5 nm), and reached saturation (ca. 10 times enhancement) at 20 nm. The MEF was also dependent on the thickness of the silica with a maximum at 10 nm. The lowest lifetime was observed on the 20 nm silver film, which was consistent with the saturation of MEF. These results showed the properties of a silver film needed for a maximum increase of fluorescence intensity in a fluoroimmunoassay. Dependence of the MEF on the emission wavelength was also studied using different dye-labeled anti-rabbit IgGs.     

Enhanced fluorescence images for labeled cells on silver island films

Silver island films (SIFs) were deposited on glass substrates to serve as supports. T-Lymphocytic (PM1) cell lines were labeled by Alexa Fluor 680-dextran conjugates on the membranes or by YOYO in the nuclei. The fluorescence images of the cell lines were recorded in the emission intensity and lifetime using scanning confocal microscopy. The fluorescence signals by the fluorophores bound on the cell membranes were enhanced significantly by SIF supports as compared with those on the glass. In addition to the increase in the intensity, there was a dramatic shortening of the emission lifetime. In contrast to the Alexa Fluor 680 fluorophores on the membranes, the YOYO fluorophores intercalated in the cell nuclei were not influenced significantly by the silver islands. This result can be interpreted by an effect of the distance on coupling between the fluorophores and metal particles: the fluorophores on the cell membranes are localized within, but the fluorophores in the cell nuclei are beyond the region of metal-enhanced fluorescence. Thus, the metal supports can be used to improve the detection sensitivity for target molecules on cell surfaces when they are fluorescently labeled.     

Surface-enhanced Raman spectroscopy based on conical holed enhancing substrates

In this contribution, surface-enhanced Raman spectroscopy (SERS) based on conical holed glass substrates deposited with silver colloids was reported for the first time. It combines the advantages of both dry SERS assays based on plane films deposited with silver colloids and wet SERS assays utilizing cuvettes or capillary tubes. Compared with plane glass substrates deposited with silver colloids, the conical holed glass substrates deposited with silver colloids exhibited five-to ten-folds of increase in the rate of signal enhancement, due to the internal multiple reflections of both the excitation laser beam and the Raman scattering photons within conical holes. The application of conical holed glass substrates could also yield significantly stronger and more reproducible SERS signals than SERS assays utilizing capillary tubes to sample the mixture of silver colloids and the solution of the analyte of interest. The conical holed glass substrates in combination with the multiplicative effects model for surface-enhanced Raman spectroscopy (MEM_SERS) achieved quite sensitive and precise quantification of 6-mercaptopurine in complex plasma samples with an average relative prediction error of about 4% and a limit of detection of about 0.02 μM using a portable i-Raman 785H spectrometer. It is reasonable to expect that SERS technique based on conical holed enhancing substrates in combination with MEM_SERS model can be developed and extended to other application areas such as drug detection, environmental monitoring, and clinic analysis, etc.     

Enhanced fluorescence of Cy5-labeled oligonucleotides near silver island films: a distance effect study using single molecule spectroscopy

We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.     

Metal-Enhanced Fluorescence of Phycobiliproteins from Heterogeneous Plasmonic Nanostructures

We report here the use of plasmonic metal nanostructures in the form of silver island films (SiFs) to enhance the fluorescence emission of five different phycobiliproteins. Our findings clearly show that the phycobiliproteins display up to a 9-fold increase in fluorescence emission intensity, with a maximum 7-fold decrease in lifetime when they are assembled as a monolayer above SiFs, as compared to a monolayer assembled on the surface of amine-terminated glass slides of the control sample. The study was also repeated with a thin liquid layer of the phycobiliproteins sandwiched between two glass substrates (and a SiFs and a glass substrate) clamped together. Similarly, the results show a maximum 10-fold increase in fluorescence emission intensity coupled with a 2-fold decrease in lifetime of the phycobiliproteins in the SiF-glass setup as compared to the glass control sample, implying that near-field enhancement of phycobiliprotein emission can be attained both with and without chemical linkage of the proteins to the SiFs. Hence, our results clearly show that metal-enhanced fluorescence (MEF) can potentially be employed to increase the sensitivity and detection limit of the plethora of bioassays that employ phycobiliproteins as fluorescence labels, such as in fluoro-immunoassays where the assay can be tethered on the surface of SiFs, and also in flow cytometry where analytes in the liquid phase could potentially flow through channels coated with SiFs without actually being attached to the silver.     

Preparation and study of polyacryamide-stabilized silver nanoparticles through a one-pot process

A one-pot route was illustrated to synthesize stable well-dispersed silver colloids stabilized by polyacrylamide on a large scale. Reduction of silver ions and polymerization of acrylamide occurred almost simultaneously in the absence of a commonly used reducing agent and initiator. A possible mechanism for the formation of silver nanoparticles with bimodal size distribution was proposed. The structure and composition of the obtained nanoparticles were characterized carefully. Furthermore, light scattering simulation and UV-vis absorption studies confirmed that the obtained colloids were the mixture of Ag and Ag2O nanoparticles. The presence of silver oxide layers on the nanoparticle surface should be responsible for the broadening of the surface plasmon band of silver nanoparticles. Ag2O layers could be added or removed from Ag nanoparticle surfaces by the addition of HNO3, HAc, or NaCl solution to the as-obtained silver colloids.     

Design and synthesis of fluorescent core–shell nanoparticles with tunable lower critical solution temperature behavior and metal‐enhanced fluorescence

In this article, the preparation of fluorescent nanohybrids with core–shell structure and metal‐enhanced fluorescence (MEF) effect was presented. The fluorescent core–shell nanohybrids were prepared using silver nanoparticles (AgNPs) as cores and fluorophore tethered thermoresponsive copolymers with tunable lower critical solution temperature (LCST) from 15 to 90 °C as shells. These thermoresponsive copolymers were synthesized by the random copolymerization of oligo(ethylene oxide) acrylate and di(ethylene oxide) ethyl ether acrylate using reversible addition–fragmentation chain transfer polymerization and grafted on to AgNPs surface via Ag–S coordination interaction. By thermal manipulation of polymer spacer between AgNPs and fluorophores, the tunable MEF was achieved. It was also revealed that the fluorescent nanohybrids would exhibit maximal MEF when the polymerization degree was tuned to 350. The manipulation of the solution temperatures below and above LCST resulted in switchable MEF behavior. In addition, the phase transition process of the thermoresponsive copolymer was also studied by MEF effect using this fluorescent core–shell nanohybrid design. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 87–95     

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.     

Feasibility of Using Bimetallic Plasmonic Nanostructures to Enhance the Intrinsic Emission of Biomolecules

Detection of the intrinsic fluorescence from proteins is important in bio-assays because it can potentially eliminate the labeling of external fluorophores to proteins. This is advantageous because using external fluorescent labels to tag biomolecules requires chemical modification and additional incubation and washing steps which can potentially perturb the native functionality of the biomolecules. Hence the external labeling steps add expense and complexity to bio-assays. In this paper, we investigate for the first time the feasibility of using bimetallic nanostructures made of silver (Ag) and aluminum (Al) to implement the metal enhanced fluorescence (MEF) phenomenon for enhancing the intrinsic emission of biomolecules in the ultra-violet (UV) spectral region. Fluorescence intensities and lifetimes of a tryptophan analogue N-acetyl-L-tryptophanamide (NATA) and a tyrosine analogue N-acetyl-L-tyrosinamide (NATA-tyr) were measured. Increase in fluorescence intensities of upto 10-fold and concurrent decrease in lifetimes for the amino acids were recorded in the presence of the bimetallic nanostructures when compared to quartz controls. We performed a model protein assay involving biotinylated bovine serum albumin (bt-BSA) and streptavidin on the bimetallic nanostructured substrate to investigate the distance dependent effects on the extent of MEF from the bimetallic nanostructures and found a maximum enhancement of over 15-fold for two layers of bt-BSA and streptavidin. We also used finite difference time domain (FDTD) calculations to explore how bimetallic nanostructures interact with plane waves and excited state fluorophores in the UV region and demonstrate that the bimetallic substrates are an effective platform for enhancing the intrinsic emission of proteins and other biomolecules.     

Thermoresponsive silver/polymer nanohybrids with switchable metal enhanced fluorescence

In this communication we describe a new approach to the fabrication of fluorescent silver/polymer nanohybrids with thermo-switchable metal enhanced fluorescence (MEF). By manipulating a soft polymer spacer between the silver nanoparticles and the fluorophores a tunable MEF was achieved.     

Effect of Glass Substrates on the Formation of Gold-Silver Colloids in Nanocomposite Thin Films

A sol-gel route to synthesize nanocomposite thin films containing phase separated metal colloids of gold (Au) and silver (Ag) was developed. Ag—Au colloids were prepared in silica films using dip coating technique. The annealing of the samples in air results in the formation of phase separated Ag and Au colloids in SiO₂ thin films, showing the surface plasmon peaks at 410 nm and 528 nm. For the synthesis of phase separated Ag and Au colloids on float glass substrates, formation of the silver colloids was found strongly dependent on the surface of the float glass. On the tin rich surface formation of both gold and silver colloids took place, whereas, on the tin poor surface the formation of only gold colloids was observed. The surface dependence of the formation of silver colloids was attributed to the presence of tin as Sn²⁺ state on the glass surface, which oxidizes into Sn⁴⁺ during heat treatment, reducing Ag⁺ into silver colloids.     

Installation/modulation of the emission response via click reaction

We have demonstrated the installation of a fluorescence property into a nonfluorescent precursor and modulation of an emission response of a pyrene fluorophore via click reaction. The synthesized fluorophores show different solvatochromicity and/or intramolecular charge transfer (ICT) feature as is revealed from the UV-visible, fluorescence photophysical properties of these fluorophores, and DFT/TDDFT calculation. We observed that some of the synthesized fluorophores showed purely ICT character while emission from some of them arose from the LE state. A structureless and solvent polarity-sensitive dual emission behavior was observed for one of the triazolylpyrene fluorophores that contains an electron-donating -NMe(2) substituent (fluorophore, 7a). Conversely, triazolylpyrene with an electron-withdrawing -CN group (fluorophore, 7b) showed a solvent polarity-independent vibronic emission. The effect of ICT on the photophysical properties of these fluorophores was studied by fluorescence emission spectra and DFT/TDDFT calculations. Fluorescence lifetimes were also measured in different solvents. All of our findings revealed the delicate interplay of structure and emission properties and thus having broader general utility. As the CT to LE intensity ratio can be employed as a sensing index, the dual emissive fluorophore can be utilized in designing the molecular recognition system too. We envisage that our investigation is of importance for the development of new fluorophores with predetermined photophysical properties that may find a wide range of applications in chemistry, biology, and material sciences.