A Novel, Wafer-Level Method to Fabricate Zero-Mode Waveguides for Single Molecule Detection

Abstract

Zero-mode waveguides (ZMWs) are hollow, metal apertures that are substantially smaller than the wavelength of light typically used in fluorescence spectroscopy. Consequently, ZMWs restrict excitation illumination to sub-diffraction limited (zeptoliter-scale) volumes. Such small confinement volumes enable single molecule detection at micromolar concentrations of fluorescently-labeled target molecules, and hence offer a powerful tool for single molecule observations under near-physiological conditions. The primary limitation to the widespread use of ZMWs in modern biological laboratories is that well-defined nanoapertures currently require expensive fabrication methods to produce. Here, we present a novel method to create ZMWs that employs conventional, low-cost, wafer-level fabrication techniques. We first use conventional photolithography to pattern larger, microapertures (800-1000 nm diameter) into a gold film covering a glass microscope slide. The microapertures are then subjected to metal electrodeposition, where the metal-of-interest is plated onto all accessible metal surfaces. Consequently, the diameter of the nanoaperture array can be controlled by simply varying the electrodeposition time. As a proof-of-principle demonstration of the fabrication method, we fabricated waveguide arrays (300 μm x 300 μm) onto standard microscope cover glasses with diameters ranging from 70±20 nm to 1000±17 nm (mean±std, as determined by SEM). To determine the attenuation of the excitation light within the ZMWs we characterized the fluorescence emission as a function of diameter (D) and found that it is proportional to D3.4 in the zero-mode regime (where no modes can propagate), D2.35 in the transition regime (modes increasingly propagate as a function of diameter), and D∼2 in the super-wavelength regime (all modes exist). In addition, we have directly observed the diffusion of single molecules at a solution concentration of 1 μM. These results demonstrate that our fabrication method can produce usable waveguide geometries using low-cost processing techniques.