Daniel Roitman

Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nanostructures

Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of ≈30% were obtained for a range of ZMW diameters (70–100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.

Improved fabrication of zero-mode waveguides for single-molecule detection

Metallic subwavelength apertures can be used in epi-illumination fluorescence to achieve focal volume confinement. Because of the near field components inherent to small metallic structures, observation volumes are formed that are much smaller than the conventional diffraction limited volume attainable by high numerical aperture far field optics (circa a femtoliter). Observation volumes in the range of 10−4fl have been reported previously. Such apertures can be used for single-molecule detection at relatively high concentrations (up to 20μM) of fluorophores. Here, we present a novel fabrication of metallic subwavelength apertures in the visible range. Using a new electron beam lithography process, uniform arrays of such apertures can be manufactured efficiently in large numbers with diameters in the range of 60–100nm. The apertures were characterized by scanning electron microscopy, optical microscopy, focused ion beam cross sections/transmission electron microscopy, and fluorescence correlation spectrosc...