MicroRNAs are small nucleic acid strands that are becoming important in understanding fundamental biological pathways and disease diagnostics. The proposed work will attempt measuring them using a technique that is expected to yield very high sensitivity in biological samples.
MicroRNAs (miRs) are single-stranded RNAs, capable of regulating genomic processes. Their measurement is useful in disease diagnosis, including cancer. Thus circulating miRs have the potential to serve as diagnostic markers. The current state-of-the-art miR assay is by qRT-PCR, requiring reverse transcription, labeling and amplification. Preliminary studies suggest that hybridizing miR to single-strand DNA (ssDNA) that is attached to gold nanoprisms (GNPs) may induce a novel transduction mechanism via delocalization of conduction electrons. This electronic effect can be investigated utilizing localized surface plasmon resonance (LSPR) properties of GNPs bound to solid substrates. It is found that GNPs undergo a large LSPR wavelength shift upon ssDNA/miR hybridization, and is thought to be due to electronic delocalization, and is an unexplored methodology for analyzing miRs in biological fluids. Further, there is a potential to develop this new methodology into a label-free, low cost multiplexing array. This proposal seeks to validate the electronic delocalization hypothesis by investigating spacing/electronic conjugation between ssDNA and the GNP surface, a single mismatch at different positions in ssDNA/miR helix, chemical functionality attached to ssDNA, and number of nucleotides in miR strands, for structural characterization of the LSPR-based sensors. The proposed research will result in LSPR-based miR sensors capable of assaying at ultralow concentrations in biofluids (Objective 1) via UV-visible spectrometry by monitoring changes in the LSPR peak. To achieve multiplexing capability, portability and long-term stability, GNPs will be immobilized at the bottom of plastic multiwall plates. In this way, each well can be treated as an independent miR sensor (Objective 2). It is expected that assay of 50 distinct miRs excluding positive and negative controls directly from biofluids using a single 96-well plate, and will be characterized in absorption mode by a standard plate reader. Such a design is expected to impact both biomedical research and medical diagnostics. Research activities will involve high school, undergraduate, and graduate students through mentored projects, and the research data will be integrated into existing courses. Nanoscience will be more broadly promoted to middle and high schools through training school teachers in nanostructure synthesis and women in science symposium
