Abstract
A nanoimmunosensor based on wavelength-dependent dark-field illumination with enhanced sensitivity was used to detect a disease-related protein molecule at zeptomolar (zM) concentrations. The assay platform of 100-nm gold nanospots could be selectively acquired using the wavelength-dependence of enhanced scattering signals from antibody-conjugated plasmonic silver nanoparticles (NPs) with on–off switching using optical filters. Detection of human thyroid-stimulating hormone (hTSH) at a sensitivity of 100zM, which corresponds to 1–2 molecules per gold spot, was possible within a linear range of 100zM–100fM (R=0.9968). A significantly enhanced sensitivity (~4-fold) was achieved with enhanced dark-field illumination compared to using a total internal reflection fluorescence immunosensor. Immunoreactions were confirmed via optical axial-slicing based on the spectral characteristics of two plasmonic NPs. This method of using wavelength-dependent dark-field illumination had an enhanced sensitivity and a wide, linear dynamic range of 100zM–100fM, and was an effective tool for quantitatively detecting a single molecule on a nanobiochip for molecular diagnostics.
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