Multiplexed nanoparticle-based immunoassays
Multiplexed immunoassays have been explored using the fluorescent and luminescent properties of fluorophores and nanoparticles. Epi-fluorescence microscopy, confocal laser scanning microscopy and programmable array microscopy were used to detect signals from mixtures of conventional organic fluorophores, quantum dots and silica nanoparticles doped with europium, samarium and terbium in single-welled multiplexed immunosorbent assays. Spectral unmixing was investigated using mixtures of fluorophores and cadmium selenide quantum dots. Mixtures of up to four dyes were separated quantitatively using least squares minimisation, with relative standard error ranging from 0.5 to 13 %. Silica nanoparticles doped with luminescent lanthanides were synthesised and used in a model immunoassay system for simultaneous, single-welled detection of human and mouse IgGs. The results indicated the lanthanides are well suited to multiplexed assays, mainly because of their atomic line emission bands. Analytes in a mixture could be quantified with < 5 % error. The multiplexed assay developed was applied to the detection of anti-dengue IgM and IgG in mouse sera, to differentiate primary and secondary dengue infection. The assay traced the kinetics of antibody production for both IgM and IgG with an IgM/IgG ratio of 1.