High‐Throughput Fabrication of Zero‐Mode Waveguide Nanoaperture Arrays with Sol‐Gel Nanoimprint Lithography for Enhanced Single Molecule Fluorescence Detection

Abstract Zero‐mode waveguides (ZMWs) are subwavelength metallic nanoapertures enabling enhanced single‐molecule fluorescence detection at micromolar concentrations in conditions far beyond the diffraction‐limited capabilities of confocal microscopes. However, their widespread use remains limited by the complexity and cost of the nanofabrication techniques, such as focused ion beam and electron‐beam lithography. Here, a scalable, cost‐effective, and high‐throughput method for fabricating high‐performance ZMW arrays is presented, which combining sol‐gel nanoimprint lithography (NIL) with hydrofluoric acid (HF) vapor‐phase etching. This approach enables the parallel fabrication and massive replication of ZMW nanoapertures with attoliter volumes, without requiring expensive equipment. The optical performance of the resulting ZMWs is validated through a series of single‐molecule fluorescence experiments, including burst analysis, fluorescence correlation spectroscopy (FCS), and single‐molecule Förster resonance energy transfer (smFRET). The ZMW nanoapertures demonstrate up to 8× fluorescence brightness enhancement, sub‐millisecond temporal resolution, and broadband spectral operation across the visible range. This method represents a significant advance in making nanophotonic devices more accessible, paving the way for a broader adoption of ZMWs in single‐molecule biosensing and integrated nanophotonic systems.