Optical Recognition of Converted DNA Nucleotides for Single-Molecule DNA Sequencing Using Nanopore Arrays

Abstract

We demonstrate the feasibility of a nanopore based single-molecule DNA sequencing method, which employs multi-color readout. Target DNA is converted according to a binary code, which is recognized by molecular beacons with two types of fluorophores. Solid-state nanopores are then used to sequentially strip off the beacons, leading to a series of detectable photon bursts, at high speed. We show that signals from multiple nanopores can be detected simultaneously, allowing straightforward parallelization to large nanopore arrays.Nanopore based DNA sequencing is widely considered to be a promising next generation sequencing platform [1,2]. Two main features of the nanopore method make it exceptionally useful for single molecule-based genome analyses: First, the method's ability to electrophoretically focus and thread extremely long DNA molecules from the bulk into the pore, making it possible to analyze minute DNA samples [3]. Second, sub-5 nm pores are now routinely used to linearize long DNA coils, thus in principle, nanopores can be used to effectively 'scan' information along a long genome. These features, as well as the fact that solid-state nanopores can be fabricated in a highly dense array [4,5], allow the development of massively parallel detection, and are crucial for the realization of an amplification-free, lowcost and high-throughput sequencing [2,[6][7][8][9].A nanopore is a nanometer-sized pore in an ultra-thin membrane that separates two chambers containing an ionic solution. An external electrical field applied across the membrane creates an ionic current and a local electrical potential gradient near the pore, which draws in and threads biopolymers through the pore in a single file manner [3,10]. As a biopolymer enters the pore, it displaces a fraction of the electrolytes, giving rise to a change in the pore conductivity, which can be measured directly using an electrometer. A number of nanopore based DNA sequencing methods have recently been proposed and highlight two major challenges [1,11]: 1) The ability to discriminate among individual nucleotides (nt). The system must be capable of differentiating among the four bases on a single-molecule level.2) The method must enable parallel readout. As a single nanopore can probe only a single molecule at a time, a strategy for manufacturing an array of nanopores and simultaneously monitoring them is needed. To date, parallel readout through any nanopore-based method has not yet been demonstrated. A large number of current, and future, generation sequencing methodologies rely on the use of an enzyme (polymerase, exonuclease, etc) in the readout process. The kinetics of enzymatic activity, however, is a major bottleneck for increasing readout speed, and these + Corresponding author. Email: ameller@bu.edu. * These authors contributed equally to this work. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2011 June 9. Published in final edited form as:Nano Lett. 2010 June 9; 10(6): 2237-2244. doi:10.1021/nl1012147. NI...

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