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Translational and clinical research requires sensitive sensors capable of high spectral resolution to observe the multitude of biomarkers present in biological systems. Biomarkers are molecules in biological systems that regulate or indicate the behavior of the biological system. Small oligonucleotides called microRNA (miRNA) are a class of biomarkers that regulate protein expression and cell behavior. Expression levels of miRNA’s in tissues are typically heterogeneous and have concentrations in the femtomolar to nanomolar range. Often the sensitivity of most commercially available in situ biosensors for tissue or cellular applications suffers from false signals due to nuclease degradation of the sensor. This puts a limit on the sensitivity of these sensing techniques. We have designed an innovative biosensor that aims to reduce false signals. The reporter-probe displacement biosensor for miRNA analysis uses a self-complementary reporter for signal change. In our design the reporter brings Cy5 and a quencher within proximity to allow for quenching of the fluorescence. Nuclease-related false positives can be reduced because the dyes must be forced together to create a change in analytical signal. As a model system we used a medically relevant miRNA, Lethal-7a (Let7a), for proof-of-principle studies. The speed, selectivity, sensitivity, and extent of false signals using a reporter-probe displacement biosensor will be discussed. The reporter-probe complex can selectively bind to miRNA targets within 15 minutes and reduce false positive signals from nuclease degradation by at least 15 % compared to molecular beacons. Preliminary data suggests picomolar limits of detection are obtainable. Studies on two-photon induced FRET enhancement mechanisms and use of spacers on linear oligonucleotides to improve sensor performance will be presented.
To improve spectral resolution of fluorescent species in two-photon applications we investigated the potential for synchronous scanning of the excitation and emission wavelengths. Thin-film tunable filters will be described for emission bandwidth control and scanning. The observed two-photon excitation spectrum of Rhodamine B without filters was about 90 nm broad at the full width half maximum (FWHM). Using linear variable filters at a fixed bandpass, the excitation wavelength and center wavelength of the emission bandpass filter were scanned at a constant wavelength off-set. The observed spectrum reveled the excitation bandwidth was reduced to about 52 nm FWHM with respect the no filters spectrum. Future studies will use multiple dyes to demonstrate potential of multiplex signaling with two-photon synchronous scanning excitation-luminescence.