On single-molecule DNA sequencing with atomic force microscopy using functionalized carbon nanotube probes

Abstract

A novel DNA sequencing method is proposed based on the specific binding nature of nucleotides and measured by an atomic force microscope (AFM). A single molecule of DNA is denatured and immobilized on an atomically fiat surface, and a force probe functionalized with a nucleotide is scanned along the molecule to detect locations of the probe nucleotide's complement. To increase the spatial resolution of the atomic force microscope so that individual bases can be distinguished, a single-walled carbon nanotube is grown from the AFM probe and functionalized with a single nucleotide. The carbon nanotube diameter is of the order as the nucleotide base spacing--providing the necessary spatial resolution for single molecule sequencing. The absolute force detection limit of the microscope is thermal noise-limited and derived herein from the equipartition theorem. The calculated minimum detectable force is less than experimentally obtained nucleotide binding forces, indicating that the AFM is capable of directly measuring single nucleotide interactions. A model of the oscillating AFM probe dynamics is developed, allowing a methodical approach to determining attractive forces with a chemically-specific sensor. This attractive force detection is performed by measuring the phase lag of the oscillating probe near the sample surface as compared to the resonating probe in free air. As grown, the carbon nanotubes are too long to be used as reliable force probes, therefore a method for shortening carbon nanotubes is presented utilizing high voltages to remove material. Measuring the length of the nanotube is performed with a novel technique that exploits the nanotube's unique elastic buckling property.

(cont.) This measurement technique characterizes the length of the nanotube while the probe is still mounted on the AFM and alleviates the need for a secondary microscope. The shortening procedure developed is performed in conjunction with the nucleotide functionalization, creating a precise and chemically-specific force probe. Experiments are performed on synthetic DNA of a known sequence to validate the proposed approach. A functionalized carbon nanotube force probe is scanned along single molecules of synthetic DNA to determine locations of target bases.