A plasmonic ‘antenna-in-box’ platform for enhanced single-molecule analysis at micromolar concentrations

Single-molecule fluorescence techniques 1 , 2 , 3 are key for a number of applications, including DNA sequencing 4 , 5 , molecular and cell biology 6 , 7 and early diagnosis 8 . Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur 2 . This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes 9 , 10 , 11 , 12 , 13 . Although these nanoantennas enhance fluorescence brightness 14 , 15 , 16 , 17 , 18 , 19 , 20 , large background signals 20 , 21 , 22 and/or unspecific binding to the metallic surface 23 , 24 , 25 have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel ‘antenna-in-box’ platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules. A plasmonic nanoantenna enables a thousand fold-enhanced fluorescence brightness allowing single-molecule analysis to be carried out in a zeptolitre volume at physiological concentrations.