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.

Faculty of Mathematics and Physics Charles University Prague 121 16 Czech Republic

FZU Institute of Physics Czech Academy of Sciences Na Slovance 2 Prague 182 21 Czech Republic

LiST Laboratory for Life Sciences and Technology Danube Private University Viktor Kaplan Straße 2 Wiener Neustadt 2700 Austria

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