Sensitive fluorometric nanoparticle assays for cell counting and viability

We have developed easy-to-use homogeneous methods utilizing time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence quenching for quantification of eukaryotic cells. The methods rely on a competitive adsorption of cells and fluorescently labeled protein onto citrate-stabilized colloidal gold nanoparticles or carboxylate-modified polystyrene nanoparticles doped with an Eu(III) chelate. In the gold nanoparticle sensor, the adsorption of the labeled protein to the gold nanoparticles leads to quenching of the fluorochrome. Eukaryotic cells reduce the adsorption of labeled protein to the gold particles increasing the fluorescence signal. In the Eu(III) nanoparticle sensor, the time-resolved fluorescence resonance energy transfer between the nanoparticles and an acceptor-labeled protein is detected; a decrease in the magnitude of the time-resolved energy transfer signal (sensitized time-resolved fluorescence) is proportional to the cell-nanoparticle interaction and subsequent reduced adsorption of the labeled protein. Less than five cells were detected and quantified with the nanoparticle sensors in the homogeneous microtiter assay format with a coefficient of variation of 6% for the gold and 12% for the Eu(III) nanoparticle sensor. The Eu(III) nanoparticle sensor was also combined with a cell impermeable nucleic acid dye assay to measure cell viability in a single tube test with cell counts below 1000 cells/tube. This sensitive and easy-to-use nanoparticle sensor combined with a viability test for a low concentration of cells could potentially replace existing microscopic methods in biochemical laboratories.