Detection and quantification of biomolecules within biological fluids and tissues is of fundamental importance to biomedical research and clinical diagnostics. We have recently demonstrated that ultrabright fluorescent nano-labels, called plasmonic-fluors, improve the sensitivity of fluoro-immunoassays by more than 1000-fold compared to conventional molecular fluorophores. However, the relatively large dimensions of the plasmonic-fluors (50-80 nm) impose diffusion-limitation on assay kinetics, extending the sample-to-answer time. Therefore, for achieving ultrafast and ultrasensitive immunoassays, it is imperative to overcome diffusion and mass-transfer limitations on reaction kinetics. With a goal of reducing assay time without compromising the sensitivity and specificity of immunoassays, this work discusses two methods to overcome the “nano-label depletion zone” above the assay surface, based on external force fields. In the first method, we utilize an external rotational force field to achieve a cyclical draining and replenishing of nano-labels which results in enhanced labeling efficiencies on the assay surface. We observed 100% increase in the labeling efficiency at shorter incubation times (5 minutes) as the assay surface is subjected to an external rotational field (8 RPM). In the second method, we demonstrate the design and synthesis of a novel class of magnetic-plasmonic nano-labels that exhibit strong fluorescence signal and respond to an external magnetic field. The plasmonic-fluors labelled with the detection antibodies mix rapidly in the test sample to capture the protein analytes in solution phase, before they are drawn to the bottom of microtiter well coated with capture antibodies. Both methods overcome the diffusion and mass-transfer limitations on reaction kinetics associated with ultrabright but bulky nano-labels.

Overcoming the diffusion-limited kinetics of fluorescent nano-labels for rapid and ultrasensitive bio-detection

ACS Spring 2022

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