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Multiplexed photoluminescence (PL) detection plays an important role in chemical and biological sensing. Here, it is shown that time-gated (TG) detection of a single terbium-donor-based Förster resonance energy transfer (FRET) pair can be used to selectively quantify low nanomolar concentrations of multiple DNAs or microRNAs in a single sample. This study demonstrates the applicability of single-TG-FRET-pair multiplexing for molecular (Tb-to-dye) and nanoparticle (Tb-to-quantum-dot) biosensing. Both systems use acceptor-sensitization and donor-quenching for quantifying biomolecular recognition and modification of the donor-acceptor distance for tuning the PL decays. TG intensity detection provides extremely low background noise and a quick and simple one-step assay format. Single-TG-FRET-pair multiplexing can be combined with spectral and spatial resolution, paving the way for biosensing with unprecedented high-order multiplexing capabilities., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The importance of microRNA (miRNA) dysregulation for the development and progression of diseases and the discovery of stable miRNAs in peripheral blood have made these short-sequence nucleic acids next-generation biomarkers. Here we present a fully homogeneous multiplexed miRNA FRET assay that combines careful biophotonic design with various RNA hybridization and ligation steps. The single-step, single-temperature, and amplification-free assay provides a unique combination of performance parameters compared to state-of-the-art miRNA detection technologies. Precise multiplexed quantification of miRNA-20a, -20b, and -21 at concentrations between 0.05 and 0.5 nM in a single 150 μL sample and detection limits between 0.2 and 0.9 nM in 7.5 μL serum samples demonstrate the feasibility of both high-throughput and point-of-care clinical diagnostics., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nanoparticle clusters (NPCs) have attracted significant interest owing to their unique characteristics arising from their collective individual properties. Nonetheless, the construction of NPCs in a structurally well-defined and size-controllable manner remains a challenge. Here we demonstrate a strategy to construct size-controlled NPCs using the DNA-binding zinc finger (ZnF) protein. Biotinylated ZnF was conjugated to DNA templates with different lengths, followed by incubation with neutravidin-conjugated nanoparticles. The sequence specificity of ZnF and programmable DNA templates enabled a size-controlled construction of NPCs, resulting in a homogeneous size distribution. We demonstrated the utility of magnetic NPCs by showing a three-fold increase in the spin-spin relaxivity in MRI compared with Feridex. Furthermore, folate-conjugated magnetic NPCs exhibited a specific targeting ability for HeLa cells. The present approach can be applicable to other nanoparticles, finding wide applications in many areas such as disease diagnosis, imaging, and delivery of drugs and genes., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Semiconductor quantum dot nanocrystals (QDs) for optical biosensing applications often contain thick polyethylene glycol (PEG)-based coatings in order to retain the advantageous QD properties in biological media such as blood, serum or plasma. On the other hand, the application of QDs in Förster resonance energy transfer (FRET) immunoassays, one of the most sensitive and most common fluorescence-based techniques for non-competitive homogeneous biomarker diagnostics, is limited by such thick coatings due to the increased donor-acceptor distance. In particular, the combination with large IgG antibodies usually leads to distances well beyond the common FRET range of approximately 1 to 10 nm. Herein, time-gated detection of Tb-to-QD FRET for background suppression and an increased FRET range is combined with single domain antibodies (or nanobodies) for a reduced distance in order to realize highly sensitive QD-based FRET immunoassays. The "(nano)(2) " immunoassay (combination of nanocrystals and nanobodies) is performed on a commercial clinical fluorescence plate reader and provides sub-nanomolar (few ng/mL) detection limits of soluble epidermal growth factor receptor (EGFR) in 50 μL buffer or serum samples. Apart from the first demonstration of using nanobodies for FRET-based immunoassays, the extremely low and clinically relevant detection limits of EGFR demonstrate the direct applicability of the (nano)(2-) assay to fast and sensitive biomarker detection in clinical diagnostics., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)