The overall aim of this research is to develop enhanced surface plasmon resonance imaging (SPRI) methodologies that employ RNA aptamer microarrays for the multiplexed, ultrasensitive detection of both high and low molecular weight protein biomarkers in biological fluids down to femtomolar concentrations. The proposed research consists of two major areas: (i) the development of new RNA aptamer microarrays and (ii) the creation of new enhanced SPRI sensing methodologies. The creation of new RNA aptamer microarrays requires both the development of novel microarray fabrication strategies as well as the creation of new RNA aptamers specifically designed for the enhanced SPRI measurements. The array fabrication methods will employ surface enzyme reaction strategies using T4 RNA ligase and T7 RNA polymerase in conjunction with microfluidics to attach both synthetic oligonucleotides and in vitro transcribed RNA onto gold surfaces. The microarrays will use both previously discovered aptamers such as those for vascular endothelial growth factor (VEGF) and theophylline, as well as new RNA aptamers identified by the in vitro SELEX process. SPR and SPRI will be used in the screening of target-aptamer interactions during the SELEX selection. Some of these RNA sequences will be incorporated into combined aptamers which will include molecular switch sequences whose 3D structure changes upon target binding. The new enhanced SPRI sensing methodologies will employ various surface enzyme reactions of the nucleic acid microarrays such as T7 RNA polymerase, RNase H and poly(A) polymerase in conjunction with the adsorption of functionalized nanoparticles to either improve the specificity or the sensitivity of the surface bioaffinity measurements. The initial targets will be two growth factors that are potential cancer biomarkers, VEGF and platelet-derived growth factor. The initial molecular switch aptamers will employ known riboswitch sequences that bind Glucosamine-6-phosphate or theophyilline;these will be combined with other sequences that can bind to functionalized gold nanoparticles. Once the proposed enhanced SPRI biosensing methodologies have been realized, they will be applied to the microarray detection of the cancer biomarker VEGF, three cardiac biomarkers (troponin T, NT-proBNP, B-type natriuretic peptide) and two renal function biomarkers (cystatin C and beta 2-microglobulin) in serum and other biological fluids.
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