Detection of nucleic acids plays an important role in diagnosis of various diseases. Some of the common methods used for DNA detection rely on fluorescence emission as means of transduction of target hybridization. While effective, these techniques require labeling of the target which complicates the sample preparation step. A new assay is proposed which explores the potential for quantum dots (QDs) to be used as donors in the fluorescence resonance energy transfer (FRET) to excite thiazole orange derivatives. Oligonucleotide probes were doubly labeled with TO derivatives that form an H-aggregate in the absence of the fully complementary (FC) target. The formation H-aggregate leads to excitonic interaction between the dyes which in turn causes to quenching of the fluorescence emission. The hybridization of the FC target causes restoration of fluorescence emission as it leads to the dissociation of the dimeric structure and disruption of the excitonic interaction. The proposed assay forgoes the need for labeling the target as well as allows for ratiometric detection and provides with improved sensitivity. The viability of the assay as well as various parameters affecting the ratiometric signal have been investigated in solution phase first. The viability of the assay was then assessed in solid phase format. The intrinsically labeled QD probe conjugates were immobilized on the surface of biotin functionalized paper and the quantitative response and selectivity of the assay were investigated. The limit of detection for a 34mer FC target was found to be 0.1 pmol. The selectivity of the assay was assessed by comparing the ratiometric signal associated with a FC target and a one base pair mismatch (1BPM) target. It was found that the ratiometric signal of the 1BPM was lower than the background signal which suggests that the presence of the mismatch affects the environment of the intercalating dyes.

Towards paper based assays with intinsically labeled oligonucleotide probes on QDs for detetction of selective hybridization

253rd National Meeting (2017)

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