Chapter 7 - Single molecule analysis in nanofluidic devices

Abstract

Observation of single molecules requires probing a sample volume that contains no more than one molecule at a time. For diffraction-limited probe volumes this requires molecular concentrations that may be incompatible with the biological system being interrogated. In this chapter we describe the use of nanofluidic devices as an alternative approach to restrict probe volumes below diffraction-limited values. In particular, we will focus on the use of nanoscale devices for the detection and optical mapping of single DNA molecules. The chapter describes unique electrokinetic phenomena that emerge on the nanoscale as well. The driving forces for fluid flow and interactions with the fluid via walls are qualitatively different on the nanoscale as compared to the microscale. Overlap of the electric double layers generated by opposite walls on the nanoscale alters the velocity profile (plug-like on the microscale to parabolic on the nanoscale for electrokinetic flows). The conformation of DNA is sensitive to channel dimensions. In nanochannels, DNA molecules stretch to nearly their full contour length when the channel dimensions are comparable to the persistence length of the DNA molecule. Methods of fabrication of nanochannel devices are surveyed. Electron beam lithography, focused ion beam milling, and nanoimprint lithography have been used to generate nanoscale devices. Finally, we describe recent applications of fluorescence imaging of single molecules in nanochannels for optical mapping of DNA and the detection of sequence variations.