DNA compaction enhances the sensitivity of fluorescence-based nucleic acid assays: a game changer in point of care sensors?

Abstract

Fluorescence-based nucleic acid assays frequently exhibit a feeble signal at low analyte concentrations, necessitating complex, expensive methods such as the development of sequence-specific oligo tags, molecular beacons, and chemical modifications to maintain high detection sensitivity. Hence, there is growing interest in accomplishing fluorescence enhancement in nucleic acid assays using robust and cost-effective strategies. The study exploits the use of two compaction agents, PEG 8000 and CTAB, to compact the ITS-2 amplicon of the fungus Candida albicans and evaluates the effect of both of these agents on the fluorescence intensity of SYTO-9 labelled nucleic acids. Conventional fluorometric measurements showed that both CTAB and PEG 8000 enhanced the emission intensity by ∼1.2- and 2-fold, respectively. Furthermore, we leveraged paper-based spot tests and distance-based assays to validate the effect of DNA compaction for enhancing sensitivity in the point-of-care context. The spot assay performed on paper with compacted samples showed an increase in the emission intensity of SYTO-9 and this was manifested by an elevated G channel intensity in the order of PEG 8000 compacted > CTAB compacted > amplified. Moreover, in the distance-based assay, the PEG 8000 compacted sample was found to migrate farther compared to CTAB compacted and amplified DNA samples at amplicon concentrations, 15 μg ml⁻¹ and 39.65 μg ml⁻¹. The limit of detection (LOD) for PEG 8000 and CTAB compacted samples on both paper-spot and distance-based assays were found to be 0.4 μg ml⁻¹ and 0.5 μg ml⁻¹, respectively. Hence our work provides an overview of employing DNA compaction as an approach for enhancing the sensitivity of fluorescence-based point-of-care nucleic acid assays without the need for cumbersome sensitivity enhancement methods.