Silver and gold nanoparticle coated membranes applied to protein dot blots

Detection and identification of low abundance biomarker proteins is frequently based on various types of membrane-based devices. Lowering of the protein detection limits is vital in commercial applications such as lateral flow assays and in Western blots widely used in proteomics. These currently suffer from insufficient detection sensitivity and low retention for small 2–5 kDa proteins. In this study, we report the deposition of two types of metal nanoparticles: gold colloids (50–95 nm diameter) and silver fractals onto a range of commonly used types of membranes including polyvinylidene fluoride (PVDF). Due to strong affinity of proteins to noble metals, such modified membranes have the potential to effectively capture trace proteins preventing their loss. The membranes modified by metal particles were characterized optically and by SEM. The membrane performance in protein dot blots was evaluated using the protein—fluorophore conjugates Deep Purple-bovine serum albumin and fluorescein—human serum albumin. We found that the metal nanoparticles increase light extinction by metals, which is balanced by increased fluorescence, so that the effective fluorescence signal is unchanged. This feature combined with the capture of proteins by the nanoparticles embedded in the membrane increases the detection limit of membrane assays.12 page(s

Plasmonic platforms of self-assembled silver nanostructures in application to fluorescence

Fluorescence intensity changes were investigated theoretically and experimentally using self-assembled colloidal structures on silver semitransparent mirrors. Using a simplified quasi-static model and finite element method, we demonstrate that near-field interactions of metallic nanostructures with a continuous metallic surface create conditions that produce enormously enhanced surface plasmon resonances. The results were used to explain the observed enhancements and determine the optimal conditions for the experiment. The theoretical parts of the studies are supported with reports on detailed emission intensity changes which provided multiple fluorescence hot spots with 2–3 orders of enhancements. We study two kinds of the fluorophores: dye molecules and fluorescent nanospheres characterized with similar spectral emission regions. Using a lifetime-resolved fluorescence/reflection confocal microscopy technique, we find that the largest rate for enhancement (~1000-fold) comes from localized areas of silver nanostructures

Reduction of photobleaching and photodamage in single molecule detection: observing single actin monomer in skeletal myofibrils

Recent advances in detector technology make it possible to achieve single molecule detection (SMD) in a cell. SMD avoids complications associated with averaging signals from large assemblies and with diluting and disorganizing proteins. However, it requires that cells be illuminated with an intense laser beam, which causes photobleaching and cell damage. To reduce these effects, we study cells on coverslips coated with silver nanoparticle monolayers (NML). Muscle is used as an example. Actin is labeled with a low concentration of fluorescent phalloidin to assure that less than a single molecule in a sarcomere is fluorescent. On a glass substrate, the fluorescence of actin decays in a step-wise fashion, establishing a single molecule detection regime. Single molecules of actin in living muscle are visualized for the first time. NML coating decreases the fluorescence lifetime 17 times and enhances intensity ten times. As a result, fluorescence of muscle bleaches four to five times slower than on glass. Monolayers decrease photobleaching because they shorten the fluorescence lifetime, thus decreasing the time that a fluorophore spends in the excited state when it is vulnerable to oxygen attack. They decrease damage to cells because they enhance the electric field near the fluorophore, making it possible to illuminate samples with weaker light

Plasmon-enhanced fluorescence near metallic nanostructures : biochemical applications

Amplification of fluorescence is a nanoscale phenomenon which is particularly pronounced in close proximity to metal nanostructures. Due to its sharp distance dependence, it is ideally suited to monitor biorecognition reactions. Using this effect we have been able to demonstrate ultrasensitive bioassays. Two types of metal nanostructures have been employed, nanometric silver islands deposited over an ultrathin metal mirror and silver fractal structures. For the first type, metal mirrors (aluminum, gold, or silver protected with a thin silica layer) were coated with SIFs and an immunoassay (model assay for rabbit IgG or myoglobin immunoassay) was performed on this surface using fluorescently labeled antibodies. Our results show that SIFs alone (on a glass surface not coated with metal) enhance the immunoassay signal approximately 3 to 10-fold. Using a metal mirror instead of glass as support for SIFs leads to up to 50-fold signal enhancement. The second type of metal nanostructures, silver fractals, were produced by electrochemical reduction of silver nitrate deposited on sapphire covered with a thin conductive film of indium tin oxide. These structures were used as a substrate for a model rabbit IgG bioassay. The fluorescence resulting from the binding of antibody labeled with Rhodamine was highly nonuniform with distinctive hot spots. These highly fluorescent regions were correlated with areas of higher Ag thickness and coverage. Such high values of fluorescence amplification in both types of nanostructures have been interpreted by using time-resolved fluorescence data and by considering the radiative properties of plasmons in the environments which promote plasmon coupling.7 page(s

Long wavelength depolarized light scattering from silver nanoparticles

We report the depolarized light scattering from heterodisperse silver nanoparticles. The profile of the wavelength dependent anisotropy of the colloidal solution of silver nanoparticles extends to the red and near-infrared (NIR) spectral region. For long wavelengths, above 600 nm, the anisotropy drops below 0.5. The presence of such a strong orthogonal component in the scattering opens new opportunities for imaging in dispersive media when polarizers can be used to suppress the background. The anisotropy profile of the scattering of heterodisperse silver nanoparticles can be satisfactorily explained by a theory based on interference between two surface plasmon resonances. (c) 2007 Elsevier B.V. All rights reserved