Fluorescence enhancement in an over-etched gold zero-mode waveguide.

The fluorescence enhancement in an over-etched gold zero-mode waveguide (ZMW) was investigated through both numerical simulation and experiments. Using Cy3 and Cy5 as the fluorescent probes, the simulation showed that the undercut not only enhances the fluorescence signals of both fluorophores, but also greatly improves the radial uniformity of the excitation fields in the ZMW. Furthermore, using a focused-ion-beam tool, we fabricated Au-ZMW arrays with different radius and undercut. The fluorescence enhancement per molecule and the effective excitation volume of the Au-ZMW were then measured as functions of its radial size and over-etching depth by using fluorescence correlation spectroscopy. It was found that the undercut can significantly enhance the fluorescence signal per molecule in the ZMW, but it also slightly increased the excitation volume. Decreasing the radial size of the ZMW can efficiently reduce the excitation volume and also further enhance the fluorescence per molecule. These results together indicate that combining the undercut and reduction of radius of the ZMW can serve as a simple and effective way to essentially improve the performance of an Au-ZMW for single molecule fluorescence detection.