Surface Enhanced Fluorescence
Fluorescent nanotags are plasmonic silver or gold nanoparticles surrounded by a metal oxide shell containing a fluorophore. By tuning the optical properties of the metal core and the fluorophore location within the silica shell, the brightness of the nanotags can be increased through a process known as surface enhanced fluorescence (SEF). The SEF nanotags can be engineered to provide bright, stable fluorescence throughout the visible and near-IR spectrum. Further functionalization of the metal oxide shell provides compatibility with different solvents and composites, or for biological targeting applications.
Surface Enhanced Fluorescence
While fluorescent molecules are among the most popular biosensing reagents, they have significant drawbacks, including low optical cross sections which make individual fluorophores difficult to detect, and a poor photostability which can degrade emission complicating detection and quantification. Quantum dots which are brighter and more robust than individual organic dyes are a potential alternative, but also have drawbacks including cost, toxicity, irregular blinking, and an excitation wavelength (365-405 nm) that results in significant autofluorescence in many biological systems.
Metal nanoparticles support localized surface plasmon resonances, collective oscillations of conduction electrons that strongly couple to light at specific wavelengths and produce extremely high electromagnetic fields near the nanoparticle surface (see our Knowledge Base article on plasmonics). The surface plasmon resonance makes these particles extremely efficient absorbing and scattering materials in the visible and near-infrared regions of the electromagnetic spectrum. Surface enhanced fluorescence (SEF) is a phenomenon first observed in the 1970’s that occurs when a fluorophore is placed near the high electromagnetic fields at the surface of a plasmonic metal nanoparticle, enhancing the fluorophore emission intensity by orders of magnitude. The enhancement can be attributed to two effects: 1) the focusing of the incoming light due to the large absorption and scattering cross sections of the plasmonic particle and 2) a decrease in the fluorescence lifetime of the fluorophore that allows the excited state to return to the ground state at a higher frequency. While the first effect increases the emission intensity only, the second effect increases both the emission intensity (by allowing the fluorophore to cycle more quickly, outputting more light in a given amount of time), and also improves photostability (by reducing the time the fluorophore spends in the excited state).
The SEF effect is most pronounced when the plasmon resonance of the metal nanoparticle is spectrally coincident with the absorbance/emission of the fluorophore near the surface. NanoComposix has extensive experience fabricating and functionalizing monodisperse and unagglomerated silver and gold nanoparticles with precisely engineered sizes, shapes, and optical properties. Changing the size and shape of the nanoparticle has a dramatic effect on the optical properties, allowing the plasmon resonance to be shifted across the visible and near-IR regions of the spectrum for enhancement of a variety of different fluorophores.
The above plot demonstrates how the emission from fluorescein dye molecules varies as the size of the silver core – and hence the optical properties – are varied. The plasmon resonance of the 70 nm-diameter silver cores overlaps strongly with the absorbance and emission of the dye, resulting in the highest level of brightness enhancement.
In addition to negatively-charged hydroxyl groups and positively-charged amines, the surface of the particles can be functionalized with other chemical groups including carboxylic acids or thiols, and larger species such as PEG or other polymers can be grafted to the surface on request.
In addition to tailoring the surface functionalization for your needs, we can coat our plasmonic nanomaterials – including gold nanospheres and silver nanoplates – with fluorescent metal oxide shells, and custom fluorophores can be incorporated for your specific application. The plasmonic metal core can provide a strong scatter signal when observed using dark field microscopy; dual-purpose fluorescent/scattering labels can easily be fabricated by selecting the desired optical properties of the silver core and the fluorophore, with some decrease in the enhancement effect as the overlap between the dye and metal particle optical properties is reduced.
Performance and Applications
SEF nanotags were compared against dye-loaded latex and silica nanoparticles of similar size. Below, fluorescence microscope images of the corresponding SEF particles under the same illumination, particle concentration and detector conditions. The SEF nanotags are clearly much brighter than the corresponding commercial particles, and individual particles can be clearly distinguished. The fabricated SEF nanotags are approximately the same size as the commercial particles while generating between 6X and 30X the brightness. In fact, the fluorescence from the SEF nanotags can be easily imaged with a standard halogen microscope bulb, even when turned right down!
The combination of brighter emission and increased photostability makes SEF-nanotags an extremely promising alternative to standard organic fluorophore or quantum dot-based fluorescent tags for microarray- and microscopy-based detection techniques where a high degree of photostability is required.
The SEF-nanotags have immediate applicability in the improvement of existing fluorescent based detection assays including flow cytometry assays, microarray based assays, plate-based assays, bead-based assays. The nanotags are sufficiently bright to be individually visualized with standard fluorescent microscopy and are ideal as tracers and markers in in-vitro and in-vivo assay systems.