This Small Business Innovation Research (SBIR) Phase I project proposes to examine high throughput methods to quantify intacellular microRNA (miRNA) concentrations in cells that have shown to be associated with normal physiological processes as well as diseases including cancer. Currently, there are no rapid, quantitative methods available to measure miRNA expression in living cells or tumor tissue. All current in vitro approaches require extensive preparation involving extraction, reverse transcription of miRNA into cDNA, and amplification. These methods are not only time consuming, but require that the low abundance miRNA be several fold greater than background to give a significant result. To meet the demand for a diagnostic/prognostic tool, we propose development of a biomolecular detection device based on a single electron transistor to bind and measure the concentration of miRNAs, giving a researcher or clinician an accurate profile to make proper clinical assessments. In addition, we propose development of fluorescent probes designed to bind to miRNAs intra-cellularly and fluoresce upon recognition. Developing these high-throughput methods to detect miRNA at the single cell level will give us direct information on intracellular miRNA levels, miRNAs that are essential for identifying tumor maintenance or metastasis, thus creating new diagnostic and therapeutic opportunities.
The broader/commercial impact of this project will be to enhance current diagnostic and prognostic tools for early detection of disease. Today, early cancer detection and treatment offers the best outcome for patients. This has driven the search for effective diagnostics. The identification of a universal tumor-specific epitope or marker has remained elusive. While many types of serological and serum markers have included enzymes, proteins, hormones, mucin, and blood group substances, at this time there are no effective diagnostic tests for cancer that are highly specific, sensitive, economical and rapid. This deficiency means that many cases of malignancy go undetected long past the time of effective treatment. The goal of this research is to develop clinical diagnostic tools where miRNA profiles can be examined from patient samples immediately in a hospital or clinical setting. The current size of the in vitro diagnostic market is estimated to be over $40 billion. Unique diagnostic kits developed from this technology will likely fulfill an unmet market opportunity with the potential to exceed $100 million in the first 3 - 5 years.
Changes in microRNA (miRNA) expression have been shown to be associated with a variety of normal physiological processes as well as diseases including cancer. Studies have already shown that miRNAs may provide useful markers for the development of disease diagnostic and prognostic assays. Currently there are no rapid, quantitative methods available to measure miRNA expression in living cells or tumor tissue. In phase I, the primary objective was to develop a simple platform for rapid, label-free measurement of microRNA (miRNA) using a hand-held electronic device. The device, a nanometric transistor is coupled to miRNA capture probes (antimers). Upon binding of miRNA to capture probe, changes in the molecules are transduced into an electrical signal that signifies analyte capture at the single molecule level. At the end of our Phase I research we successfully demonstrated: 1) development of a single electron transistor (SET) labeled with miRNA capture probes, 2) optimal hybridization conditions for miRNA detection, 3) accurate SET quantification of both synthetic and cellular microRNAs, 4) accurate multiplex detection. As a result of funding, we now have a working prototype and reader for measuring microRNA levels in tissues. We will continue to develop this prototype for use in the research market. Once mature, we will seek CLIA laboratory approval and further develop this technology for clinical markets.
