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Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reductive amination to evaluate the multiplexing capacity of luminescence resonance energy transfer (LRET) between UCNPs and quantum dots (QDs). This is the first account of a multiplexed bioassay strategy that demonstrates the principle of use of a single form of UCNP as donor and three different color emitting QDs as acceptors to concurrently determine three analytes. Broad absorbance profiles of green, orange and red QDs that spanned from the first exciton absorption peak to the UV region were in overlap with a blue emission band from UCNPs composed of NaYF 4 that was doped with 30% Yb 3+ , 0.5% Tm 3+ , allowing for LRET that was stimulated using 980 nm near-infrared radiation. The characteristic narrow and well-defined emission peaks of UCNPs and QDs allowed for the collection of luminescence from each nanoparticle using a band-pass optical filter and an epi-fluorescence microscope. The LRET system was used for the concurrent detection of uidA, Stx1A and tetA gene fragments with selectivity even in serum samples, and reached limits of detection of 26 fmol, 56 fmol and 76 fmol, respectively. [ABSTRACT FROM AUTHOR]

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 min. [ABSTRACT FROM AUTHOR]