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WIREs Nanomed Nanobiotechnol
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Molecular diagnostics for personal medicine using a nanopore

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Abstract Semiconductor nanotechnology has created the ultimate analytical tool: a nanopore with single molecule sensitivity. This tool offers the intriguing possibility of high‐throughput, low cost sequencing of DNA with the absolute minimum of material and preprocessing. The exquisite single molecule sensitivity obviates the need for costly and error‐prone procedures like polymerase chain reaction amplification. Instead, nanopore sequencing relies on the electric signal that develops when a DNA molecule translocates through a pore in a membrane. If each base pair has a characteristic electrical signature, then ostensibly a pore could be used to analyze the sequence by reporting all of the signatures in a single read without resorting to multiple DNA copies. The potential for a long read length combined with high translocation velocity should make resequencing inexpensive and allow for haplotyping and methylation profiling in a chromosome. WIREs Nanomed Nanobiotechnol 2010 2 367–381 This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices

A nanopore larger than double‐stranded DNA in thin membrane. (a) A transmission electron micrograph of a 3.6 × 3.2 nm cross‐section nanopore in a 31.5‐nm thick Si3N4 membrane. (b) Blockades in the electrolytic current are measured in a 100 mM KCl as a function of membrane bias through the pore shown in (a) as a function of time. (c) Distributions illustrating the frequency as a function of the duration of a current blockade, tD, at 400 mV (red); 200 mV (black) and 100 mV (green). The distribution depends sensitively on the voltage. Inset: The reciprocal of the duration, 1/tD, as a function of the applied voltage. 1/tD seems to depend linearly on the voltage.

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Nanopores in thin membranes used for single molecule detection. (a) A transmission electron image of a pore with a diameter of 2.2 nm in a 15‐nm thick Si3N4 membrane. (b) A cross‐section through a 2‐nm nanopore in a silicon nitride membrane showing a dsDNA in the pore. (c) Distribution of the electrostatic potential in a nanopore at 2.6‐V bias across the membrane in 1 M KCl. (d) A measurement of the electrolytic current through the 2.2‐nm pore interacting with a λ‐DNA with 1.0 V applied across the silicon nitride membrane. The current blockades are observed as DNA translocates through the pore. The figure on the right is an expanded view of a blockade. Figures (b) and (c) courtesy of A. Aksimentiev.

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Streptavidin bound biotin DNA duplex trapped by the electric field in a nanopore. (a) Model of biotinylated dsDNA bound to streptavidin in a 2.6 nm × 2.1 nm cross‐section pore in a 23‐nm thick membrane. (b) A current blockade measured at 1.0 V transmembrane bias in 1 M KCl in a 2.6 × 2.1 nm pore associated with C–G and A–T duplexes biotinylated to streptavidin. The difference in blockade current can be used to discriminate C–G from A–T.

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Trapping a single γ‐DNA molecule in a nanopore. (a) Triggered by the onset of a current blockade indicating that dsDNA is translocating through the 2.5‐nm pore, the voltage across the pore is switched from 800 mV (above the threshold for the stretching transition) to 200 mV (below threshold). As a result, the mean width of the current transient (blue) increases from about 200 µs to about 0.5 s. The red trace, which is offset by 0.5 nA shows the current after the switch is manually triggered—in the absence of a blockade—without DNA translocating through the nanopore. Inset: an example of a current blockade observed in the same pore as a function of time at a constant V = 800 mV bias. (b) Triggered by the onset of a current blockade indicating that dsDNA is translocating through a 2.6‐nm diameter pore, the voltage is switched from 600 mV (above the stretching threshold) to 100 mV (below threshold). As a result, the molecule is trapped in the pore till t = 23.4 s. (c) Histogram showing the distribution of the current during the blockade in an interval t = 0.5 s when λ–DNA is trapped for 53.2 s and (d) the open pore after the molecule exits the trap. The distribution for the trapped molecule must be fit to at least two Gaussians: one (solid blue) offset from the median (ΔI = 0) by + 1.8 pA with a width of 14.4 pA and another (solid red line) offset by − 2.1 pA with a width of 12.9 pA. The black line represents the sum. In contrast, the data in (d) representing the open pore can be fit by a single Gaussian with a width 8.6 pA.

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Voltage threshold for permeation through a solid‐state nanopore depends on the sequence. (a) Molecular dynamics simulations of EcoRI DNA complex encountering synthetic nanopore. Snapshots illustrating simulated dissociation of an EcoRI–DNA complex due to the electric field in a nanopore. The Si3N4 membrane is shown in gray, two strands of DNA in blue and green, a protein dimer in pink and purple, water and ions are not shown. Time elapsed from the moment a 4‐V transmembrane bias was applied as indicated. (b) Quantitative PCR (qPCR) results indicating the number of 105 base pair (bp) copies from EcoRI–DNA that permeates a 3.4 × 4.7 nm pore with a threshold voltage that depends on the DNA sequence. Superimposed on the data are fits to the curve used to determine the threshold. The cognate sequence GAATTC (blue) has a threshold voltage of about 2.1 V, but in contrast, with a substitution for the first base, TAATTC (green) has a threshold of only 1.1 V. (c) qPCR results indicating the number of 900 bp DNA copies from either EcoRI–DNA or BamHI–DNA that permeates a pore depends on the enzyme. The inset shows a transmission electron micrograph of the 3.4 × 4.5 nm pore. The threshold for the EcoRI and BamHI rupture scales with the bulk dissociation energies.

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(a) A transmission electron micrograph of a 2.3 × 2.0 nm nanopore in a 15‐nm thick silicon nitride membrane. (b) Electrolytic current measured in 100 mM KCl at 800 mV (blue) and 200 mV through the pore shown in (a) as a function of time. The frequency of blockades decreases dramatically with voltage; at 200 mV (red) no transients are observed. (c) The frequency of blockades observed with the same pore as a function of membrane voltage illustrating the frequency drop as voltage decreases below 0.5 V. The dotted line represents a fit to the data. (d) Distributions illustrating the frequency as a function of the duration of a current blockade, tD, above threshold at 800 mV (red), 600 mV (black), and 400 mV (green). The distribution depends on the voltage. Inset: 1/tD as a function of the applied voltage indicating a threshold.

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Stretching DNA in a nanopore. (a) Quantitative PCR (qPCR) results obtained for 2‐nm pores showing the copy number versus voltage. 622 base pair dsDNA permeates the 2‐nm pore in 10‐nm thick Si3N4 membrane (top inset) for V > 2.5 V and the 1.8 × 2.2 nm in 20‐nm thick Si3N4 membrane (bottom inset) pore for V > 5 V. (b) qPCR results indicating the number of MS3 DNA copies that permeates through the 1.8 ± 0.2 nm pore shown in the inset as a function of the membrane voltage. Insets on the left and right are snapshots of methylated and unmethylated MS3 translocating through the 1.8‐nm pore. Both DNA exhibit an ordered B‐DNA form, but there is a significant degree of disorder for unmethylated DNA. The highlighted region of the strand shows the portion of the DNA where methylated cytosines are located. The same region is also highlighted in the unmethylated strand for comparison. (c) qPCR results indicating the number of MS3 and BRCA1 DNA copies that permeates through the 1.7 ± 0.2 nm pore shown in the inset as a function of the membrane voltage. Unmethylated MS3 and BRCA1 permeate the U > 3.8 V and U > 3.6 V, respectively while the threshold for fully methylated MS3 and BRCA1 is U > 2.5 V and U > 2.7 V, respectively.

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