Molecular Beacons are self-reporting DNA hairpin probes that have been used in a variety of real-time detection applications. When they hybridize to an oligonucleotide target they fluoresce, thus removing the additional requirement of fluorescently labeling the target itself. Consequently, molecular beacons have found substantial scientific success over the past two decades. Molecular beacons have rarely been used in microarray-based applications. When tethered to solid substrates a large background emerges due to beacon-substrate interactions. This large background is absent when beacons are used un-tethered in an aqueous solution. Researchers have discovered that the signal-to-background ratio of molecular beacons can remain relatively high if they are tethered to a solid substrate by means of a hydrogel. This high performance has been attributed to the fact that the crosslink density of surface-patterned microgels gradually approaches infinity at the microgel-water interface, and the beacons conjugated to the microgel surface find themselves in as water-like an environment as possible. This project is designed to demonstrate the effective use of surface-tethered molecular beacons in clinically relevant DNA assays.
Since their invention in the mid-1990's, molecular beacons and beacon-based assay platforms have served multiple scientific and commercial roles. However, one large applications area where beacons have rarely been used is in microarray technology where the oligonucleotide probes must be tethered to a solid surface. Site-specific surface tethering is important, because the nature of a specific probe and its target can be identified by position rather than by fluorophore color. Thousands of discrete surface positions can be interrogated simultaneously whereas the practical number of different fluorophores is less than ten. Applications involving the rapid identification of bacterial strains responsible for a broad range of different infections, the ability to simultaneously probe for tens or hundreds of genes can have substantial impact in health-care settings has the potential to be enhanced through the results of this project.
This project supported a team of three scientists from the Stevens Institute of Technology to explore the commercialization of a technology developed in part based on research funding previously provided by the National Science Foundation. Our basic scientific discovery has potential application to problems associated with detecting and identifying bloodstream infection in a clinical hospital environment. As part of this I-Corps project we learned the significance of talking to potential customers and end-users of our proposed technology. Because of dozens of such conversations, we realized that we need to still demonstrate another next important scientific concept before commercialization becomes a serious possibility. Consequently, we have developed new ideas, submitted a provisional patent application protecting our new approach, and secured a new (competitively reviewed) NSF grant to pursue experiments going forward. When a patient enters a hospital with symptoms suggesting some form of infection, determining whether an infection is present can take as long as 24 hours, and identifying the specific infectious species can take 72 hours or more. During that period, patients can suffer severely, because the lack of a clear and rapid diagnosis means that a patient may not receive the most appropriate treatment, such as the administration of the correct antibiotic, for hours or even days. While new technologies based on molecular diagnostics are beginning to mitigate this problem by rapidly identifying the DNA of infecting species, these new approaches are unable to keep up with the throughput required by major hospitals where dozens of such tests must be run every day. This research project is thus studying a new technology that has the potential to not only make a rapid diagnosis - the focus of our I-Coirps grant - but also make many such diagnoses for many different patients - the focus of our next NSF grant. This combined detection and amplificatiuon technology uses hydrogels - similar to the materials in soft contact lenses and in disposal diapers – that are microscopic in size, so only a very small amount of target DNA is required for each test. The engineering and science questions that must be addressed center, first, on how to make these microscopic hydrogels and, second, how to modify them, so the chemical reactions needed for DNA detection can proceed accurately and quickly.
