Nanoholes Improve Single-Molecule Analysis

Daniel S. Burgess

Researchers at Cornell University in Ithaca, N.Y., have developed a nanostructured sample holder that enables the analysis of single molecules at realistic concentrations. The device, which features arrays of tiny holes in a metal film, has potential for applications in single-molecule enzymology and in drug discovery.

Subwavelength holes in an aluminum film enable researchers to observe the dynamics of single molecules at more realistic concentrations. The holes act as zero-mode waveguides, permitting the excitation light for fluorescence correlation spectroscopy to enter into the sample chamber as an evanescent field so that only one molecule near the opening may be stimulated. The arrays of holes are chemically isolated on the chip, allowing the user to perform multiple experiments simultaneously.

Conventional single-molecule techniques employ femtoliter-scale observation volumes, requiring the use of pico- to nanomolar sample concentrations to ensure that only one molecule, on average will be present in the sampling volume. These concentrations are far lower than they are in nature, which can affect the dynamics of the molecules under test. In contrast, the new approach reduces the effective observation volume by several orders of magnitude so that it can hold only one molecule at micromolar concentrations.

The device incorporates approximately 50-nm-diameter holes produced by reactive ion etching in a <100-nm-thick aluminum film on a fused silica coverslip. In practice, the illumination passes through the back side of the coverslip to the opening of a hole. Because the diameter of the hole is much smaller than the excitation wavelength, the hole acts as a zero-mode waveguide, and the light generates an evanescent field that extends approximately 10 nm into the cavity, producing a zeptoliter-scale effective observation volume.

This enables researchers to observe single molecules in an environment that more closely approximates natural conditions, said Michael J. Levene, a postdoctoral researcher at the university. In a demonstration of the technique, the team used fluorescence correlation spectroscopy with tagged coumarin-dCTP to observe the enzymatic synthesis of double-stranded DNA. "This means we can see all the nuances of what the enzyme does," he said. "Does it pause? Does it have multiple ways of doing the same thing? Does it have a 'memory' of what it is doing that makes it behave differently, depending on its recent past? That would not be apparent in a bulk study."

The Cornell scientists hope to develop a DNA sequencing strategy based on the technique and plan to investigate other molecules. They also are considering alternative approaches to attaching single molecules to the bottom of the holes. "Currently, we rely on simple adsorption of the enzyme to the surface and use appropriate concentrations so that many holes contain just one enzyme," Levene said, "but we have some other ideas that we hope will guarantee that we have exactly one enzyme per hole."