| Microgel-tethering is nontraditional method for oligonucleotide immobilization. Based on it, we have developed a method to control the oligonucleotide concentration on a microgel surface by patterning microgels from thin-film blends of end-functionalized and non-functional poly(ethylene glycol) [PEG]. We show that the activity of microgel-tethered molecular-beacon hybridization probes is significantly reduced as their concentration on a microgel surface is increased. This crowding phenomenon can largely prohibit hybridization, especially for structured probes like molecular beacons. Like untethered molecular beacons used in aqueous solutions, microgel-tethered beacons enable the real-time detection of both DNA and RNA. We demonstrate this property using a model for bloodstream infection that can differentiate between gram-positive and gram-negative bacteria. We use Nucleic Acid Sequence Based Amplification (NASBA) to isothermally amplify target RNA from cultures in a small volume over a low-density array of microgel-tethered molecular beacons on glass substrates. The real-time analysis enables us to follow one test at a time and determine results in a short time because of the high stability and low noise in the fluorescent signal. We show that this NASBA-on-microarray approach is able to detect target RNA from concentrations of bacteria in the range of 5-50 CFU/mL within only 20 minutes of assay time. We further exploit the microgel-tethering platform to integrate detection and oligonucleotide amplification in an array format. We realize this concept by tethering not only molecular-beacon detection probes but also the two primers required for the NASBA amplification process. Target RNA can bind to a tethered primer, which, in the presence of the three enzymes required for the NASBA process, leads to a double-stranded DNA (dsDNA) bridge tethered at each end and from which RNA- amplicons are produced. The RNA- amplicons can then hybridize to adjacent molecular-beacon probes within the same microgel leading to detectable fluorescence. The complex environment on surface introduces confinement for both oligonucleotides and enzymes, which limit their performance in reactions. To further develop the approach, future research includes exploring fundamental science in solid phase NASBA, highly multiplexed assay as well as combining this with microfluidic device. |
