Fabrication of nanoscale zero-mode waveguides using microlithography for single molecule sensing

Abstract

We present a novel approach to the fabrication of zero-mode waveguides (ZMWs) using inexpensive processing techniques. Our method is capable of rapid fabrication of circular nanoapertures with diameters ranging from 70 nm to 2 μm, allowing us to perform a detailed characterization of the dependence of the fluorescence emission on the waveguide diameter. We also validated the use of the fabricated ZMWs by detecting single molecule binding events with a signal-to-noise ratio of ten.

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.…”

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence

“…However, this sophisticated technology has not been broadly available to the scientific community. Despite multiple efforts to develop ZMW instrumentation, the combined difficulties in fabricating the ZMW chips (17), the need for appropriate surface chemistry, and the required instrumentation development hindered their widespread use (18)(19)(20)(21). To circumvent these problems, we repurposed commercially available ZMW chips [single-molecule real-time (SMRT) Cells] and adapted a ZMW-based DNA sequencer for use as a versatile single-molecule real-time fluorescent microscope to allow multiplexed collection of single-molecule fluorescence events.…”

Dark-Field Illumination on Zero-Mode Waveguide/Microfluidic Hybrid Chip Reveals T4 Replisomal Protein Interactions

“…i v i d u a l n o t d i r e c t l y a s s o c i a t e d w i t h s c i t e i s p r o h i b i t e d .targets.51−57 The accessibility of a ZMW-based single-molecule fluorescence platform, however, remains limited to a handful of specialized laboratories due to the stringent technical requirements in nanofabrication, surface passivation, and microscopy instrumentation 58,59. Although these constraints have recently been relieved somewhat as more efforts have been directed to the optimization of ZMW nanofabrication protocols and the development of surface passivation methods, the simplification of a single-molecule fluorescence microscopy setup for ZMWs and the optimization of its performance have remained challenging.…”

“…Since its inception, this nanostructure has found ample utility in single-molecule investigations of a growing list of biological targets. − The accessibility of a ZMW-based single-molecule fluorescence platform, however, remains limited to a handful of specialized laboratories due to the stringent technical requirements in nanofabrication, surface passivation, and microscopy instrumentation. , Although these constraints have recently been relieved somewhat as more efforts have been directed to the optimization of ZMW nanofabrication protocols and the development of surface passivation methods, the simplification of a single-molecule fluorescence microscopy setup for ZMWs and the optimization of its performance have remained challenging. Holographic confocal fluorescence microscopy in which sophisticated optics are employed in complex light paths to generate thousands of subillumination spots and corresponding prism-dispersed emission spots that match the pattern of ZMW arrays was originally developed for ZMW-based single-molecule imaging .…”