Professor Sean Burrows
Oregon State University
2:00PM in the OCO Conference Room – 240 Willamette
“Creative Spectrochemical Biosensing and Techniques to Improve Resolution for
Two-Photon Luminescence Applications”
Abstract:
Translational and clinical research requires sensitive sensors capable of high spectral resolution to observe the multitude of biomarkers describing biological systems. Biomarkers are molecules in biological systems that regulate or indicate the behavior of the biological system. Small ligonucleotides called messenger RNA (mRNA) and microRNA (miRNA) are a class of biomarkers that regulate protein expression and ultimately cell behavior. Typically tissues express these RNA heterogeneously at 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 and poor spectral resolution. False signals are due to nuclease and other forms of biosensor degradation. This puts a limit on the sensitivity of these techniques. To address this problem we have designed an innovative biosensor that aims to reduce false signals and improve sensitivity by forcing dyes together. Our reporter-probe displacement biosensor for miRNA analysis uses a self-complementary reporter for signal change. 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 our reporter-probe displacement biosensor will be discussed. False positive signals from nuclease degradation were reduced by at least 16 % points compared to molecular beacons. Preliminary data suggests picomolar limits of detection are obtainable. We will also present studies to improve sensor performance in terms of two-photon induced Förster Resonance Energy Transfer (FRET) enhancement and the effect of spacer length and orientation on the FRET enhancement.
The ability to simultaneously analyze multiple fluorescent markers that cannot be spatially resolved remains a challenging area in spectroscopy. While multi-color analysis is possible when colors are spatially separated, the broad spectral profile inherent to fluorescence limits the ability to isolate colors that cannot be spatially separated. As a result a mixing of colors limits the number of dyes that can be simultaneously detected from the same focal volume. Synchronous scanning luminescence (SSL) improves multi- color detection by simultaneously scanning the excitation and emission wavelengths at a defined wavelength offset. The wavelength offset is responsible for SSL peak position and peak width. The benefit of this method is reduced peak-width, allowing for multiple dyes to be interrogated simultaneously by significantly reducing mixing of colors. The proof of concept for multicomponent SSL was demonstrated by Vo-Dinh in the late 1970’s. To date, SSL has found many uses in multicomponent chemical analysis, but to the best of our knowledge two-photon SSL has yet to be explored. We will present the use of a tunable excitation source in conjunction with tunable thin-film filters to achieve two-photon induced SSL. The thin-film tunable filters control emission bandwidth, peak wavelength of emission, and contribute to the SSL peak width. The two-photon excitation spectrum of Rhodamine B without SSL has a bandwidth of about 90 nm broad. We will show that SSL reduces the bandwidth to about 52 nm. Future studies will use multiple dyes to demonstrate potential for multiplex detection with two-photon synchronous scanning excitation-luminescence.