Microlens Enhancement of Surface-Tethered Molecular Beacons

The tethering of molecular beacon oligonucleotide detection probes to surface-patterned poly(ethylene glycol) (PEG) microgels has enabled the integration of molecular beacons into a microarray format. The microgels not only localize the probes to specific surface positions but also maintain them in a waterlike environment. Here we extend the concept of microgel tethering to include dielectric microlenses. We show that streptavidin-functionalized polystyrene microspheres (3 μm diameter) can be colocalized with molecular beacons using biotinylated PEG gels in patterns ranging from pseudocontinuous microgel pads with lateral dimensions on the order of tens of micrometers to individual microgels with lateral dimensions on the order of 400-500 nm. We use a simplex assay based on Influenza A detection to study the lensing behavior. The microspheres increase the effective numerical aperture of the collection optics, and we find that a tethered microsphere increases the peak intensity collected from hybridized beacons between 1.5 and 10 times depending on the specific pattern size and areal density of microgels. The highest signal increase occurs when a single microsphere is tethered to a single isolated microgel. The tethering is highly self-directed and occurs in the individual-microgel case only when the microgel is close to the optic axis of the microsphere. This alignment minimizes spherical aberration and maximizes coupling of emitted fluorescent intensity into the collection optics.