Tethered Catalytic Hairpin Assembly with Plasmon-Enhanced Fluorescence Readout for Single Molecule Detection.

Here a novel digital bioassay readout concept is reported that does not rely on enzymatic amplification nor compartmenting of an analyzed liquid sample. Rather, it is based on counting individual affinity-captured target biomolecules via the use of a tethered catalytic hairpin assembly (tCHA) deployed on a solid sensor surface with spatial confinement utilized by a flexible polymer linker (FPL). Wide-field plasmon-enhanced fluorescence (PEF) imaging is employed for optical real-time probing of the reaction kinetics, where affinity-captured target molecules are manifested as spatially distinct bright fluorescent spots. The effect of the length of the FPLs is investigated, and the analytical performance of the dual amplification tCHA-PEF concept is tested by using a model short single-stranded DNA analyte. When applied in a sandwich immunoassay, the detection of target proteins at sub-femtomolar concentrations is demonstrated. The reported experiments are supported by diffusion-limited mass transfer models and document the potential of tCHA-PEF as a new class of generic enzyme-free bioanalytical tools enabling the ultrasensitive analysis of trace amounts of protein and nucleic acid analytes, making it attractive for future molecular diagnostics and research applications.