Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis

Abstract

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

“…2g–i and 4 – 6 ), illustrate the generally improved capabilities of the assay. The improvements will benefit a growing spectrum of single-molecule fluorescence-based techniques for studies of actin and myosin, e.g., single-molecule FRET 71 , fluorescence polarization 72 , and use of nanofabricated surfaces (e.g., zero-mode waveguides) for enhanced fluorescence background suppression 73 , 74 . The methodological developments are also of value for the very active field of single-molecule fluorescence studies beyond myosin and molecular motors 75 .…”

Recent advances in plasmonic nanocavities for single-molecule spectroscopy

“…Furthermore, besides affecting the excitation rate of the molecules inside the zeptolitre detection volume, ZMWs can also modify the uorescence photokinetics decay rates, 32,33 improving the net detected photon count rate per molecule. 22,34 Although the most explored ZMW geometry is a circular hole prepared on a metallic lm, 35 recently several groups investigated alternative ZMW designs. In particular, rectangular ZMWs realized either on Al or Au-Si bilayers ( Fig.…”

Zero-mode waveguides can be made better: fluorescence enhancement with rectangular aluminum nanoapertures from the visible to the deep ultraviolet

“…[37][38][39][40][41] So far, most studies on zero-mode waveguide nanoapertures consider only circular shapes, as this is an intuitive form which appears also simple to fabricate. [42][43][44] Few reports have considered non-circular aperture shapes such as rectangles, [45][46][47][48][49][50] triangles, 51,52 bowties, 53,54 C-or H-apertures. 55,56 While these more advanced shapes offer more parameters to tune the nanoaperture response, in the absence of clear guidelines the parameter space to explore becomes also larger, and the performance gain as compared to a circular nanoaperture is oen unclear or marginal.…”