Over the past 15 years, fluorescence has become the dominant detection/sensing technology in medical diagnostics and biotechnology. Although fluorescence is a highly sensitive technique, where single molecules can readily be detected, there is still a drive for reduced detection limits. The detection of a fluorophore is usually limited by its quantum yield, autofluorescence of the samples and/or the photostability of the fluorophores; however, there has been a recent explosion in the use of metallic nanostructures to favorably modify the spectral properties of fluorophores and to alleviate some of these fluorophore photophysical constraints. The use of fluorophore-metal interactions has been termed radiative decay engineering, metal-enhanced fluorescence or surface-enhanced fluorescence.

The use of fluorescent nanoparticles as indicators in biological applications such as imaging and sensing has dramatically increased since the 1990s. 1 These applications require that the fluorescent nanoparticles are monodisperse, bright, photostable, and amenable to further surface modification for the conjugation of biomolecules and/or fluorophores. Among the many types of fluorescent nanoparticles available today, nanoparticles with core-shell architecture fulfill all these requirements, combining diverse functionalities into a single hybrid nanocomposite. 2 In this work, we have developed core-shell (silver core-silica shell) nanoparticles with various shell thicknesses featuring a variety of fluorophores, to show the versatility of the core-shell architecture, and have demonstrated their applicability for two platform technologies, metal-enhanced fluorescence (MEF) and single nanoparticle sensing. We demonstrate the broad potential applications of our technology by employing near-infrared emitting probes (Rh800) for potential applications in cellular imaging and the use of highly photostable long lifetime (μS) lanthanide probes, probes suitable for off-gating biological autofluorescence. The use of Alexa 647 serves to demonstrate that fluorophores can be readily covalently linked to the core-shell particles also, for metal-enhanced benefits.MEF is an established technology, 3a-d where the interactions of fluorophores with metallic nanoparticles results in fluorescence enhancement, increased photostability, decreased lifetime owing to increased rates of system radiative decay, reduced blinking in single molecule fluorescence spectroscopy, 3b and increased transfer distances for fluorescence resonance energy transfer. 3c Single-molecule fluorescence spectroscopy is the prime tool in single nanoparticle sensing, and it provides several advantages over ensemble measurements, such as, the elimination of averaging of the spectral properties over all members of the ensemble, which can reveal fundamental features otherwise masked in

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.KEY WORDS: Metal-enhanced fluorescence; radiative decay rate; increased excitation rate; radiative decay engineering; surface-enhanced fluorescence; silver island films; fluorescence spectroscopy.

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.

Surface modification of colloidal gold with 11-mercaptoundecanoic acid or 16-mercaptohexadecanoic acid was performed in the absence or in the presence of the nonionic surfactant polyoxyethylene (20) sorbitan monolaurate (Tween 20). The stability of the colloidal systems was assessed with optical absorption spectroscopy. The surface-modified nanoparticles were stable only within a narrow range of intermediate pH values when chemisorption of alkanethiols was performed in the absence of Tween 20. This was explained in terms of partial ionization of the surface carboxylic groups and charge neutralization at high pH values by counterions present in the buffer solutions. Formation of a physisorbed monolayer of Tween 20 onto the nanoparticles prior to chemisorption of alkanethiols resulted in surface-modified colloidal gold that was stable over a broader range of pH values. Parallel experiments demonstrated that self-assembled monolayers could form on flat substrates in the presence of Tween 20. Therefore, possible incorporation of alkanethiols within micelles or the presence of a physisorbed layer of Tween 20 on gold surfaces did not prevent their chemisorption. The chemisorption of alkanethiols on gold surfaces with a physisorbed layer of Tween 20 is slow and may be due to a decreased sticking coefficient of the alkanethiols on gold with a physisorbed layer of surfactant. Nanoparticles whose surface was modified in the presence of Tween 20 do not appear to undergo irreversible aggregation. They can be frozen or dried and resuspended again with mild sonication.

Surface plasmons are collective oscillations of free electrons at metallic surfaces. These oscillations can give rise to the intense colors of solutions of plasmon resonance nanoparticles and/or very intense scattering. While the use of plasmonic particle absorption based bioaffinity sensing is now widespread throughout biological research, the use of their scattering properties is relatively ill explored. We refer to the use, utility and control of surface plasmons as plasmonics. In this review and forward-looking article, we discuss the current opinions and uses of plasmonics, as well as speculate on areas of future research. These include the use of plasmon scatter for long-range immunosensing and macromolecular conformation studies, as well as the ability to Stokes shift plasmon scatter, a plasmonics phenomenon recently referred to as metal-enhanced fluorescence.

Biotinylated gold nanoparticles were prepared by using a two-step surface modification procedure. First, a carboxyl-terminated alkanethiol was chemisorbed onto the surface of gold nanoparticles in the presence of a stabilizing agent. Subsequently, the carboxyl groups were reacted with (+)-biotinyl-3,6,9,-trioxaundecanediamine and 2-(2-aminoethoxy)ethanol. This procedure resulted in stable, ligand-modified gold nanoparticles. Upon interaction with streptavidin, the biotinylated gold nanoparticles aggregated by means of specific biomolecular recognition. Their aggregation was studied by optical absorption spectroscopy. Controlled aggregation of biotinylated gold nanoparticles resulted in a shift in the surface plasmon resonance peak and broadening of the absorption spectrum of the nanoparticles. The spectral changes were used to assess the extent of aggregation. Aggregation was found to be dependent on the concentrations of streptavidin, biotinylated gold nanoparticles, and the surface mole fraction of biotin groups on the nanoparticles. Maximum aggregation was observed when 24 nM streptavidin and 0.80 nM biotinylated gold nanoparticles were used. Reversal of nanoparticle aggregation was accomplished by the addition of soluble biotin to the streptavidin−nanoparticle aggregates. Kinetic analysis of the absorbance data showed that streptavidin-induced aggregation of biotinylated gold nanoparticles could be interpreted in terms of a Reaction-Limited Colloidal Aggregation (RLCA) model. This indicates that optical absorption spectroscopy can provide a quantitative measurement of the aggregation process.

In this article, we report metal-enhanced singlet oxygen generation (ME 1 O2). We demonstrate a direct relationship between the singlet oxygen yield of a common photosensitizer (Rose Bengal) and the theoretical electric field enhancement or enhanced absorption of the photosensitizer in proximity to metallic nanoparticles. Using a series of photosensitizers, sandwiched between silver island films (SiFs), we report that the extent of singlet oxygen enhancement is inversely proportional to the free space singlet oxygen quantum yield. By modifying plasmon coupling parameters, such as nanoparticle size and shape, fluorophore/particle distance, and the excitation wavelength of the coupling photosensitizer, we can readily tune singlet oxygen yields for applications in singlet oxygen-based clinical therapy.metal-enhanced fluorescence ͉ metal-enhanced phosphorescence ͉ photodynamic therapy ͉ surface-enhanced fluorescence P hotodynamic therapy (PDT) has been widely used in both oncological (e.g., tumors and dysplasias) and nononcological (e.g., age-related macular degeneration, localized infection, and nonmalignant skin conditions) applications (1-4). Three primary components are involved in PDT: light, a photosensitizing drug, and oxygen. The photosensitizer adsorbs light energy, which it then transfers to molecular oxygen to create an activated form of oxygen called singlet oxygen (1). The singlet oxygen is a cytotoxic agent and reacts rapidly with cellular components to cause damage that ultimately leads to cell death and tumor destruction (4). PDT treatments are only effective within a specific range of singlet oxygen supply (5). For example, for solid tumors, too little singlet oxygen cannot effectively treat the tumor cells, but too much singlet oxygen can damage and kill surrounding healthy cells (6). Currently, the intensity of light is commonly adjusted to control the extent of singlet oxygen generation, but there are some limitations to this method. High fluency rates of the exposure light will lead to oxygen depletion and photosensitizer photobleaching (3). However, low fluency rates of exposure light lends to a long exposure time and can cause vascular shutdown, a precursory condition to hypoxia in the tissue (5, 7). One notable approach to controlling the fluency rate of exposure light is called interstitial PDT, where a precise amount of light is delivered locally to tumors through inserted optical fibers (8). The interstitial PDT also allows the real-time monitoring of the progression of the treatment via online collection of assessment parameters through the optical fibers (8). It is important to note that despite the better control over fluency rate, the photobleaching of the photosensitizers remains an issue. In this regard, our laboratory has introduced a metalenhanced phenomenon as a means to control the extent of singlet oxygen generation via metal-photosensitizer interactions, an alternative approach as compared with exposure settings and sensitizer dose, which we believe is a significant improvement...