Surface-patterned microgel-tethered molecular beacons

Abstract

We use focused electron beams to create surface-patterned biotinylated poly(ethylene glycol) microgels to which molecular beacon probes are immobilized viabiotin–streptavidin binding. We hypothesize that immobilizing molecular beacon probes to highly hydrated microgels will maintain the high performance typical of untethered molecular beacon probes free in solution. Monte Carlo simulations show that the microgel mesh size resulting from focused radiation crosslinking is highly non-uniform and approaches infinity at the microgel–water interface. This structure is important because the extremely liquid-like environment at the diffuse microgel surface reduces the compromising effects that hard surfaces can exert on molecular beacon secondary conformation, accessibility of nucleic acid targets to the molecular beacons, and the efficiency of energy transfer between the molecular beacon fluorophore and quencher. We assess the performance of microgel-tethered molecular beacons using molecular beacons designed to distinguish between methicillin-sensitive and methicillin-resistant Staphylococcus aureus. We show that each microgel presents approximately 11 800 such molecular beacons and that each molecular beacon on average occupies an area of microgel surface corresponding to an individual streptavidin molecule. The signal-to-background ratio we measure ranges from 40–50, substantially higher than many other surface-tethering approaches. Furthermore, this platform exhibits both low non-specific background and high specific fluorescence when the microgel-tethered molecular beacons are exposed to multiple targets, and it thus promises to lend itself well to high-sensitivity, self-reporting oligonucleotide based assays.