Ratiometric SPCE Detection of miRNA

DESCRIPTION (provided by applicant): Fluorescence technologies are key research tools in the life sciences, biotechnology, medical diagnostics, forensics, and other fields. Fluorescence-based measurements include ELISA, PCR, microarray gene expression chips, other medical diagnostics, forensics tests and biohazard detection technologies. More sensitivity is often desired or needed in many of these techniques to permit reliable detection of a smaller number of sample molecules within in a smaller sample volume. The ideal technology for this should not require expensive complicated equipment. Detection of SPCE Fluorescence, the proposed project will use an emerging surface plasmon- coupled emission (SPCE) technology in a novel ratiometric, confocal format to detect micro-RNA (mi-RNA) analogs. The miRNA to be detected are small, 21-23-mers that are involved in regulating mRNA function and are of considerable interest to many researchers. The very limited, surface confined detection volumes of SPCE (~10-18 liters) create an opportunity to develop a very low background, highly sensitive, yet inexpensive detection technology. The limit of detection (LOD) will be reduced by at least ~10-fold over current methodologies through the use of highly directional fluorescence, low background, and surface only excitation of SPCE. The detector will be inexpensive and useful for other systems. The general objectives are to demonstrate the feasibility a new generic SPCE technology using an innovative ratiometric confocal signal collection device for the detection of miRNA. This technique/device will be useful for low-cost, highly sensitive detection of mi-RNA in complex biological matrixes such as plasma and cell extracts. The ratiometric signal will be between a fluorophore from the sample of interest and an internal standard fluorophore. This ratiometric detection will cancel out many variables in the detector and samples. This will allow for the use of inexpensive detectors with simple calculations. The technical objectives are: (1) To demonstrate that wavelength-resolved SPCE is a sensitive and reliable technology for ratiometric sensing with a novel confocal format (e.g. pinhole). We will determine the detection limits of surface bound oligo-DNA strands (as analogs of mi-RNA) in clean buffers and in a 'dirty matrix' such as reconstituted plasma and cell extracts. We expect improvements of 100-fold and 10-fold in the LOD over solution and current state of the art surface techniques, respectively. (2) To develop two prototypes of a small, simple, inexpensive ratiometric SPCE sensing device (Fig. 1b and 12). Prototypes will utilize laser pointer excitation with fluorescence focused to a pinhole chamber connected to an Ocean Optics USB4000 detector. In Phase II we will further develop the prototype device for sale along with an array capable of sensing miRNAs expressed by the fruit fly and other organisms of interest. PUBLIC HEALTH RELEVANCE: Fluorescence technologies are key tools for research in many areas. A new surface fluorescence technology called Surface Plasmon Coupled Emission (SPCE) will be developed to sense oligonucleotides in a ratiometric method that will be sensitive, reliable and relatively inexpensive. The device and method will be useful in applications in the life sciences, medical diagnostics, forensics and other fields.