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Today, 40 % of the world’s population live in areas with a significant risk of dengue infection. Early and reliable diagnosis of dengue virus (DENV) is essential to provide the patients with the required medical care and prevent spreading of the disease. Conventional methods for DENV diagnosis like PCR and virus isolation can be used in laboratory settings, yet are difficult to implement in point-of-care diagnostics, requiring simple, selective, fast, and sensitive detection schemes. We present here a novel approach for the detection of DENV, via its RNA, with optical read-out that relies on RNA-catalyzed fluorophore transfer onto a semiconductor quantum dot (QD) and Förster resonance energy transfer (FRET).
For this RNA assay, peptide nucleic acid (PNA) oligomers were used as highly specific capture and reporter probes. PNA exhibits remarkable affinity towards RNA as well as extremely high chemical and enzymatic stability. The capture probe, which is immobilized on a QD acting as FRET donor, bears a nucleophile at the N-terminus and the reporter probe is modified with an organic dye acting as FRET acceptor. The presence of DENV genomic RNA in the sample triggers a transfer of the dye onto the QD, signaled by FRET between the QD and the dye. A unique advantage of this system is the ability of one RNA molecule to trigger multiple transfer reactions, thereby amplifying the fluorescence signal. This assay together with the exceptional brightness of QDs and outstanding hybridization properties of PNA allows for highly specific and sensitive detection of DENV RNA in the sub-nM range.
The optical properties of semiconductor nanocrystals (SCNC) are controlled by constituent material, particle size, and surface chemistry, specifically the number of dangling bonds favoring nonradiative deactivation, and hence also by particle synthesis. In this respect, the fluorescence properties of coreshell CdSe SCNCs with different shells and surface chemistries were studied on ensemble and single particle level, using steady state and timeresolved fluorometry and confocal microscopy with time correlated single photon counting detection. Special emphasis was dedicated to correlate ensemble photoluminescence (PL) quantum yields and decay kinetics with particle brightness, PL time traces, and the Ontime fraction of the single SCNCs. Additionally, the confocal PL images were correlated with AFM measurements in order to derive the amount of absorbing, yet nonemisssive ”dark” SCNCs, the presence of which leading to an underestimation of ensemble PL quantum yields. The results of this study can help to identify synthetic routes and surface modifications minimizing the fraction of dark SCNC, thereby closing the gap to the ultimate goal of colloidally and photochemically stable SCNCs with a PL quantum yield of close to unity.