We investigate the voltage-driven translocation dynamics of individual DNA molecules through solid-state nanopores in the diameter range 2.7-5 nm. Our studies reveal an order of magnitude increase in the translocation times when the pore diameter is decreased from 5 to 2.7 nm, and steep temperature dependence, nearly threefold larger than would be expected if the dynamics were governed by viscous drag. As previously predicted for an interaction-dominated translocation process, we observe exponential voltage dependence on translocation times. Mean translocation times scale with DNA length by two power laws: for short DNA molecules, in the range 150-3500 bp, we find an exponent of 1.40, whereas for longer molecules, an exponent of 2.28 dominates. Surprisingly, we find a transition in the fraction of ion current blocked by DNA, from a length-independent regime for short DNA molecules to a regime where the longer the DNA, the more current is blocked. Temperature dependence studies reveal that for increasing DNA lengths, additional interactions are responsible for the slower DNA dynamics. Our results can be rationalized by considering DNA/pore interactions as the predominant factor determining DNA translocation dynamics in small pores. These interactions markedly slow down the translocation rate, enabling higher temporal resolution than observed with larger pores. These findings shed light on the transport properties of DNA in small pores, relevant for future nanopore applications, such as DNA sequencing and genotyping.

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...

Solid-state nanopores can be used to detect nucleic acid structures at the single molecule level. An e-beam has been used to fabricate nanopores in silicon nitride and silicon dioxide membranes, but the pore formation kinetics, and hence its final structure, remain poorly understood. With the aid of high-resolution TEM imaging as well as TEM tomography we examine the effect of Si 3 N 4 material properties on the nanopore structure. In particular, we study the dependence of membrane thickness on the nanopore contraction rate for different initial pore sizes. We explain nanopore formation kinetics as a balance of two opposite processes: (a) material sputtering and (b) surface-tension-induced shrinking.

We present a novel method for integrating two single-molecule measurement modalities, namely, total internal reflection microscopy and electrical detection of biomolecules using nanopores. Demonstrated here is the electrical measurement of nanopore based biosensing performed simultaneously and in-sync with optical detection of analytes. This method makes it possible, for the first time, to visualize DNA and DNA-protein complexes translocating through a nanopore with high temporal resolution ͑1000 frames/s͒ and good signal to background. This paper describes a detailed experimental design of custom optics and data acquisition hardware to achieve simultaneous high resolution electrical and optical measurements on labeled biomolecules as they traverse through a ϳ4 nm synthetic pore. In conclusion, we discuss new directions and measurements, which this technique opens up.

Nanopores have recently emerged as high-throughput tools for probing and manipulating nucleic acid secondary structure at the single-molecule level. While most studies to date have utilized protein pores embedded in lipid bilayers, solid-state nanopores offer many practical advantages which greatly expand the range of applications in life sciences and biotechnology. Using sub-2 nm solid-state nanopores, we show for the first time that the unzipping kinetics of individual DNA duplexes can be probed by analyzing the dwell-time distributions. We performed high-bandwidth electrical measurements of DNA duplex unzipping as a function of their length, sequence, and temperature. We find that our longer duplexes (>10 bp) follow Arrhenius dependence on temperature, suggesting that unzipping can be approximated as a single-barrier crossing, but the unzipping kinetics of shorter duplexes do not involve a barrier, due to the strong biasing electrical force. Finally, we show that mismatches in the duplex affect unzipping times in a positionsensitive manner. Our results are a crucial step towards sequence variability detection and our single-molecule nanopore sequencing technology, which rely on parallel detection from nanopore arrays. KeywordsDNA sequencing; nanopores; unzipping; single-molecule; mismatch; SNP Nanopores have recently emerged as a simple and unique tool for the manipulation and analysis of biopolymers at the single-molecule level. 1-3 A nanopore device consists of a molecularly-wide single pore through an impermeable membrane. Application of voltage across the membrane results in an ion-current signal, which transiently changes upon capture from solution of randomly diffusing biopolymers. Charged molecules, such as polynucleic acids, can be sequentially driven through the nanopore by the voltage-induced force. In addition to probing primary nucleic acid structure (length, sequence), 4-7 nanopores have been used to explore secondary structure by using the electrical force to unzip duplex regions. [8][9][10][11][12][13] The lipid-embedded α-hemolysin channel has been a model nanopore system for such experiments, although the fragility of the lipid bilayer and lateral diffusion of the channels in the membrane are major drawbacks that hinder prospective biotechnological nanopore applications. A more practical nanopore platform would ideally consist of a robust planar membrane design which contains nanopores at laterally-specified positions, allowing the incorporation of complementary probing approaches, such as optical imaging and transverse electron tunneling, thus enabling high-throughput parallel detection from many pores. 14,15 ameller@bu.edu. Supporting Information Available. Structures of the molecules used in all experiments, materials and methods, and the response time of our electrical measurements. 13 However, direct measurement of single-molecule unzipping kinetics through solid-state pores has yet to be reported. NIH Public AccessIn this letter, we employ high-bandwidth electrical measurements...