Sequential Multi-Step Nanoimprint Lithography Fabrication of Zero-Mode Waveguide Nanoaperture Arrays to Enhance Single Molecule Fluorescence Detection
Zero-mode waveguides (ZMWs) enable single-molecule fluorescence detection at micromolar concentrations by confining light to nanoscale volumes, overcoming the diffraction limit of confocal microscopy. However, their widespread adoption is hindered by high fabrication costs and limited throughput of traditional methods like focused ion beam or electron-beam lithography. Here, we introduce a scalable cost-effective approach using sequential nanoimprint lithography (NIL) combined with hydrofluoric acid etching to fabricate ZMW arrays with tunable diameters from a single initial master. In our sequential nanoimprint approach, each stamped NIL output serves as a master for the next nanoimprint generation. By leveraging the shrinkage of sol-gel nanopatterns during annealing, we achieve a cumulative diameter reduction from 230 nm to 115 nm over four successive imprints, all based on the same initial master. The resulting ZMWs exhibit detection volumes reduced by up to 1000-fold and fluorescence enhancement exceeding 16x, achieving performance comparable to state-of-the-art focused ion beam-fabricated devices. Eliminating the need for multiple master structures significantly expands the scalability of nanoimprint lithography approaches. By lowering the nanofabrication barriers and making ZMW arrays more accessible, the sequential NIL method paves the way towards broader adoption of nanophotonic devices in single-molecule biophysics, biosensing, and surface patterning applications.