2025

Pathak, Abhishek; Raj, Amar; Larsson, Sylva; Patil, Ajay B.; Singh, Atul K.; Laajala, Mira; Kumpulainen, Tatu; Marjomäki, Varpu S.; Toppari, J. Jussi

Here, we present two label-free ratiometric fluorosensors using CDs for selective and sensitive detection of enteroviral RNA. Using CDs (reference fluorophore), EB (specific fluorophore) and single-stranded DNA fragment (Probe), two fluorosensors were developed and compared in identifying target DNA sequence. Func sensor consists of CDs covalently functionalized with Probe and mixed with EB, while Non-Func sensor contains only a mixture of CDs, Probe and EB. In both sensors, the addition of the target DNA noticeably enhanced EB fluorescence, while CDs fluorescence changed slightly due to electron transfer between CDs and EB, thus enabling ratiometric detection. Non-Func sensor exhibited a sensitivity range of 100 nM-3 μM (Limit of Detection ∼88.7 nM) with the target DNA but no usable sensitivity range with real enteroviral RNA samples. In contrast, Func sensor showed a higher sensitivity range of 0.5 nM-3 μM (LOD ∼270 pM) with DNA and 0.3 nM-5 nM (LOD ∼67 pM) with viral RNA, as well as improved selectivity. The superiority of the Func sensor is likely owing to enhanced charge transfer between CDs and EB due to covalent functionalization. Furthermore, the Func sensor could detect in situ, and real-time the release of enteroviral RNA in vitro. Hence, we established a novel, sensitive, and selective method for detecting viral RNA, supported by a charge transfer mechanism between CDs and fluorophores.