This Small Business Innovation Research Phase II project examines 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 meaningful result. To meet the demand for a diagnostic/prognostic tool, development of a biomolecular detection device is proposed based on a single electron transistor to bind and measure the concentration of miRNAs. This will provide a researcher or clinician an accurate profile to make proper clinical assessments. Bringing this device to market will provide scientists with direct information on intracellular miRNA levels, enhancing predictions of miRNAs that are essential for tumor maintenance or metastasis, and creating new diagnostic and therapeutic opportunities.
The broader 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 bring a device to market for the research market and a device that can examine miRNA profiles from patient samples immediately in a hospital or clinical setting. The current size of the in vitro diagnostic market was over $40 billion in 2008. 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.
Biomolecular Detection of microRNA The discovery of small (around 22 bases), non-coding RNA (called microRNA, or miRNA) as post-transcription genetic expression regulators has been widely used to profile different cancers by recording the expression level of select target sequences. For diagnostics purposes, patient microRNA expression levels then map to these known cancer profiles. Bioo Scientific’s aim was to use existing sensor technology, mass manufactured at low cost for different industries, to determine miRNA profiles. We selected advanced capacitive touch sensor technology, which has been incorporated into many devices, such as cell phones, kiosks, navigation systems and tablets. The research aimed to validate and package the technology as an impedance-based sensor for biomolecular detection. Several other investigators have researched and validated this type of sensor in the laboratory. It works by detecting a change in impedance (inversely proportional to capacitance) at the self-assembling layer bound to the glass surface. When more molecules bind, the impedance increases. The signal difference between capture and no capture of the target molecule indicates a positive or negative reading, respectively. The success of the technology depended on both the selectivity of the target capture procedure and the sensitivity, accuracy and precision of the sensor. The system performed with high selectivity; after changing one molecule in the microRNA sequence, target capture no longer occurred. For example, when a pool of microRNA sequences compete in solution – with least one molecule that’s a perfect match for our probe and many mismatched molecules– only the perfect match will bind and produce a signal. Further work is required to commercialize the device as a clinical point-of-care genetic profiling technology. In collaboration with the hardware manufacturer, Ocular LCD, Inc., an adjustment plan has been made to improve the sensor design. We’ve submitted a provisional patent for this bimolecular device and plan to submit another with our new redesign. We would like to acknowledge three undergraduate researchers from the University of Texas at Austin that Bioo Scientific hired as interns during the final reporting year of the project. There were also several companies and laboratory facilities that generously assisted in the research: Arrow Electronics, Ocular LCD, Atmel, Nanomics, Asuragen, the Institute for Cellular and Molecular Biology, and the JJ Pickle Research Facility.
