PROBING SINGLE DNA MOLECULE TRANSPORT USING FABRICATED NANOPORES

Abstract

Nanopores can serve as high throughput, single molecule sensing devices that provide insight into the distribution of static and dynamic molecular activities, properties, or interactions. We have studied double stranded DNA electrophoretic transport dynamics through fabricated nanopores in silicon nitride. A fabricated pore enables us to interrogate a broader range of molecules with a wider range of conditions than can be investigated in a self-assembled protein pore in a lipid membrane.

Figure 1

Fabricated nanopores and nanochannels. (a) TEM picture of an ion-beam sculpted nanopore with 3 nm alumina ALD coating (top) and schematic of its cross section (bottom). The Al₂O₃ layer is represented in gray. (b) TEM picture of a nanochannel produced from a ~100 nm FIB pore plus 40 nm Al₂O₃ ALD coating (top) and schematic of its cross section (bottom).

Figure 2

Translocation events. (a) Examples of blockade events produced by λ DNA molecules translocating through an ion-beam sculpted nanopore (as in Figure 1a) in either strictly single-file order (left, as diagramed in box) or with the leading portion of the molecule folded on itself such that two parallel lengths of the same double stranded DNA translocated through the pore simultaneously (right). (b) Examples of similar blockade events produced by λ DNA molecules translocating through a nanochannel fabricated by extensive ALD treatment of a ~100 nm FIB pore (as in Figure 1b). All recordings were taken from a nanopore or nanochannel biased at 200 mV, trans side positive. (c) A panel of 7 translocation events (λ DNA through a nanopore) to further illustrate our observations and interpretations. The line diagram above each event indicates our interpretation. Arrows indicate the levels corresponding to blockades due to translocation of one, two, or three parallel lengths of DNA. The event at the far right can be interpreted by assuming two DNA molecules translocating simultaneously, but such events were considered ambiguous.

Figure 3

The percentage of unfolded events through ion-beam sculpted nanopores increases with applied voltage level and decreases with DNA length. Squares represent the average percentage of single-level events of λ DNA through 6 nanopores. Circles represent the average of 10 kbp DNA through 6 nanopores. The diamond is the average of 3 kbp DNA through 3 nanopores. The standard deviation of each point is < 5%. Dashed lines are exponential fitting.

Figure 4

Electrophoretic velocity versus voltage bias. Each data point is the average of 100 –1000 translocations. Squares (48.5 kbp λ DNA), circles (10 kbp), and diamonds (3 kbp) represent DNA translocation through ion-beam sculpted nanopores. Stars represent λ DNA translocation through a nanochannel.

Figure 5

Translocation kinetics in ion-beam sculpted nanopores. Each symbol represents statistics of >2000 translocation events (except the square, which represents only 212 events of λ DNA). (a) The time histogram of 10 kbp DNA translocation events (n = 4247) at 120 mV with a Gaussian fit. It corresponds to the same data point as indicated by an arrow in panel b. (b) The standard deviation σ_T of translocation time linearly scales with its mean <T>. Open circles represent 10 kbp DNA data at 120, 200, 300, and 400 mV. Dark circles are 10 kbp DNA data from another pore at 60 mV and 120 mV. The diamond and square represent 3 kbp DNA and λ DNA (48.5 kbp) at 200 mV, respectively. A dashed zero-crossing line is plotted to guide your eyes.

Figure 6

A typical example of the DNA capture and translocation rate as a function of the voltage applied across the nanopore. The measured number of λ DNA (5 μg/ml) translocations per second at different voltages are fitted by a line.

Figure 7

Time resolved fluorescence study (50 ms resolution) shows distinct phases of DNA diffusion and capture by a biased pore (500 mV). (a) A view of fluorescently labeled DNA molecules on the cis side of a nanopore at time = 0 ms. The capture area surrounding the location of the nanopore (dotted circle) is typically depleted of DNA molecules. One fluorescently labeled molecule whose trajectory can be followed in images b and c is indicated by an arrow. (b) The same nanopore 4,400 ms later. The molecule identified by the arrow has slowly diffused into or very near the capture area surrounding the nanopore. (c) At t= 4,450, within 50 ms of the image shown in b, the molecule appears somewhat elongated as it was rapidly captured by, and translocated through, the nanopore. By t = 4,500 ms (not shown) the molecule has disappeared from view and the region around the nanopore again appears depleted of DNA molecules.