Zero-mode waveguides: Sub-wavelength nanostructures for single molecule studies at high concentrations

Abstract

The study of single fluorescent molecules allows individual measurements which can reveal characteristics typically obscured by ensemble averages. Yet, single molecule spectroscopy through traditional optical techniques is hindered by the diffraction limit of light. This restricts the accessible concentrations for single molecule experiments to the nano- to picomolar range. Zero-mode waveguides (ZMWs), optical nanostructures fabricated in a thin aluminum film, confine the observation volume to the range of atto- to zeptoliters. Thus, they extend the accessible concentrations for single molecule spectroscopy to the micro- to millimolar regime. Through the combination of ZMWs and fluorescence correlation spectroscopy, a number of biologically relevant systems have been studied at physiological concentrations. In this review, the concept and implementation of ZMWs is outlined, along with their application to the study of freely diffusing, and membrane-bound fluorescent biomolecules.

Introduction

Since the advent of molecular biology and biotechnology, the need for high throughput, high sensitivity analysis systems has steadily increased. At the same time, single molecule and single cell measurements of relevant biomolecules have been sought because they can reveal behavior typically obscured by ensemble measurements (e.g. distributions of molecular properties and their dynamic behavior in short timescales) [1], [2], [3], [4]. Optical techniques, such as confocal microscopy, two-photon microscopy, total internal reflection fluorescence microscopy (TIRF), and fluorescence correlation spectroscopy (FCS), provide the capability of selectively detecting individual fluorescently tagged molecules with high sensitivity and in some cases high temporal resolution. Although such techniques have yielded a wealth of information in the biological and chemical sciences, they are inherently hindered by the diffraction limit of light, which limits the range of concentrations that can be studied at the single molecule level.

The use of diffraction-limited optics allows the excitation of fluorophores in observation volumes on the order of 1 fl. Thus, to observe single occupancy of the observation volume and distinguish individual fluorescently tagged molecules, solutions must be in the in the pico- to nanomolar concentrations. However, most biologically relevant systems involving binding or catalysis require the active molecules to be in the micro- to millimolar concentration regimes (Fig. 1) [5], [6]. This means that in order to perform single molecule measurements at physiological conditions the observation volume must be reduced by 3–6 orders of magnitude to insure single occupancy. This has spurred interest in nanofabricated structures for the reduction of the observation volume and the study of single molecule events at high molecular concentrations.

Multiple nanofabricated structures have been used in the past to conduct single molecule experiments. Among them are nano slits, nanofluidic channels, near-field apertures, and zero-mode waveguides (ZMWs). Nanofluidic channels and slits present the advantage of controlled flow through the observation volume, and have moderate observation volume confinement. In such structures, the observation volume is constrained in two dimensions by the cross-section of the channel, and in the third dimension by the illumination profile. This yields observation volumes on the order of tens of atto-liters, which require concentrations in the nano to micromolar regimes for single molecule measurements. Such structures have been successfully used in the study of the mechanical properties of single DNA molecules [7], [8], in DNA separation [9], [10], [11], [12], [13], [14], in fragment sizing [15], [16], and in DNA mapping [17], [18], [19]. Near-field scanning optical microscopy (NSOM), on the other hand, reduces the observation volume to approximately one atto-liter by shining light through a nanoaperture at the end of a tapered optical fiber. This technique has been used to image single molecules on surfaces [20], [21], but has the drawback of unreliable probe fabrication and complex implementation. Zero-mode waveguides (ZMWs), the subject of this review, are nanostructures that have been shown to effectively confine the observation volume to the range of atto- to zeptoliters, opening the possibility to perform single molecule studies at micro to millimolar concentrations. In the following pages, the fabrication, principle of operation, FCS theory, and applications of ZMWs are described.