through the nanopore. We find well defined ionic current blockades and translocation timesfor ss-DNA molecules that differ significantly from those of the same length ds-DNA
molecules. The room temperature denaturation of ds-DNA responsible for this difference isconfirmed by optical absorption measurements.
A single molecule translocating through a nanopore is schematically illustrated in Figure 1a.The experimental setup is shown in Figure 1b. It consists of an ionic solution divided into twoisolated reservoirs by an insulating silicon nitride membrane containing a single nanopore. Anionic current through the open nanopore is established by a voltage applied between silver/silver chloride electrodes placed in each of the two reservoirs. DNA molecules of interest areadded to the reservoir with the negative electrode. They diffuse towards the nanopore and byvirtue of their negative charge are captured by the local electric field near it18. The electricfield within the nanopore then induces the molecule to pass through it to the positive reservoir.A time recording of the nanopore current during this event reveals the history of the molecule’sinteraction with the nanopore.
Nanopores used in this study were fabricated in free standing 280 nm thick silicon nitridemembranes supported by a 380 2,19μm thick silicon chip using FIB milling followed by feedbackcontrolled ion beam sculpting. Figure 1c shows a TEM image of a 4 nm pore used in ourstudies. A voltage bias is applied and current measurements made with an Axopatch 200Bpatch clamp amplifier (Axon Instruments) operated with a 10 kHz low-pass Bessel filter.Additional features of our apparatus include an integrated flow system that allows for
continuous and rapid interchange of solutions in the reservoirs and an open reservoir designthat allows access to the solution for introduction of DNA and measurement of pH. For pH ~7 the nanopores can be operated stably in solution with ds-DNA for days while at pH ~ 13 theyare useable only for hours before increasing 1/f electronic noise, drifting baseline currents andpermanent ss-DNA molecule blockages terminate the experiments.
The starting molecular material was ~ 3 kilobase-pair (kbp) ds-DNA, obtained from pSP65plasmid (Promega Corp., Madison, WI). The plasmid was propagated in E. coli DH5α purifiedby Qiagen Plasmid Maxi kit (QIAGEN Inc., Valencia CA) and cleaved at a single site with theSmaI restriction enzyme to produce blunt-ended ds-DNA. The linearized plasmids werepurified by two sequential phenol:chloroform (1:1 ratio) extractions, followed by onechloroform extraction, and finally precipitation with 2 volumes of ethanol. The purity andquantity of the recovered DNA was confirmed by agarose gel electrophoresis and UV
absorbance. The prepared DNA was then re-suspended at a concentration of ~100 nM in TEbuffer (10 mM Tris, 1 mM EDTA pH 7.5) (RT) and stored at −20°C. The DNA was
subsequently diluted to ~10 nM concentration in the negative chamber. The solution in bothchambers was a 10 mM TE buffer (pH adjusted with KOH) with 1.6 M KCl, and 20% byvolume glycerol. The glycerol increased the viscosity of the solution, slowing the DNA
translocation through the nanopore20. This increased the length of single molecule events wellbeyond the rise time of the 10 kHz noise suppressing electronic filter.
Figure 2 shows nanopore DNA results for a bias voltage of 120 mV at pH 7 and 13 using thesame 8 nm diameter solid state nanopore. Panel a) shows an event density plot of 3782 singlemolecule translocation signatures for the pH 7 ionic environment. The horizontal and verticalaxes indicate the translocation time and mean ionic current blockage respectively, for eachmolecular event. The translocation time is the total time it takes an individual molecule totransit the pore. The ionic current blockage is the average decrease in nanopore ionic currentduring an event. The color represents the density of events at a given time and blockage. Thistwo-dimensional histogram characterizes all events in the aggregate, but each event has acurrent vs. time history that reveals information about the configuration of a single moleculeas it passes through the nanopore. These are all recorded, and representative single molecule
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current-time traces for several events selected from the indicated region of the density plot aredisplayed as insets of figure 2. No events are observed when no DNA is loaded in the solution.
The distribution of figure 2a has a well defined peak in translocation time at ~ 170
microseconds. The current blockage for these events is ~ 200 picoamperes (pA). When the pHis raised to 13 the results change dramatically, as indicated in Figure 2b (2905 events). Anoutline of the pH 7 data is included for comparison. The peak translocation time has droppedto ~ 120 microseconds and the peak current blockage to ~100 pA. Figure 2a corresponds tods-DNA and Figure 2b to ss-DNA in solution, a conclusion supported by optical experimentsdiscussed below. Changing the pH for our salt solution produces only slight changes in ionicconductivity (< 2%),
Inspection of Figure 2 shows that events whose translocation time falls below the most probablepeak value generally have increased average current blockage values. This is consistent withthe notion that some of the molecules pass through the pore folded (doubled over) on
themselves3. The net electric force and the viscous drag are both doubled for a folded part ofa molecule. This makes the velocity of a folded part of a molecule transiting the pore the sameas that of an unfolded one. Thus in a time dt twice the contour length passes for a folded regioncompared to an unfolded region. In addition the instantaneous current blockage is proportionalto the discrete number of strands of the folded molecule in the pore at any given time duringan event. The distinct levels of current blockage during an event characterize the instantaneousstate of the molecule’s folding in the nanopore. (See insets of figure 2 for examples of tracesfrom unfolded, partly doubled over, and completely doubled over molecules for each pH.) Thecompensating effects of decreased translocation time and increased current blockage for foldedmolecules means that all molecules of the same length, regardless of folding, will producecurrent traces whose areas (time integral of the deviation from the baseline current) are
equal3. We call this area the event charge deficit, ecd. Calling n the instantaneous number ofstrands in the pore, v the constant translocation velocity, ΔI1 and τ1 the current blockage andtranslocation time of an unfolded molecule respectively, and L the length of the molecule, theinvariance of ecd to folding follows from
Hyperbolae of constant ecd are shown as dashed lines in Figure 2. Events with translocationtimes at and below the most probable are seen to fall near this curve. This feature is a signatureof the translocation of molecules folded in various configurations through the pore and cannotbe explained by collisions of non-translocating molecules with the pore, random noise, or filtereffects. The detailed current-time traces provide information about where the folding occursduring the event. The peak in the distribution occurring on the long time low blockage part ofthe ecd curve corresponds to unfolded molecules.
The above argument applies only to molecules with viscosity limited motions through the porethat do not stick to its surface. We call these freely translocating. Those that do stick must havelonger transit times and fail to follow curves of constant ecd in the event plots. Such events areseen in Figure 2; they have the same current blockage as the most probable ones, but longerdurations. Thus we identify these events as molecules that temporarily stick to the walls of thenanopore during the translocation process. Earlier studies on ds-DNA show that this part ofthe distribution is greatly enhanced with very small nanopores3. The translocation times ofthese events is dominated by stochastic processes involving strong binding and unbindingbetween the DNA molecules and the walls of the pore. At this stage of nanopore science the
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Fologea et al.Page 4
study of such events will yield more information about pore-molecule chemistry than aboutthe properties of the biomolecules themselves.21
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAnalysis of event plots like those in figure 2 provides a great deal of evidence of, and
information about, individual molecular translocations. The actual physical passage of DNAmolecules between reservoirs through the nanopore is also established by observations ofevents for molecules returning to the starting reservoir upon bias voltage bias reversal. Thesesignals are observed only after hours of operation in the forward bias condition.
Figure 3 presents a plot of the most probable current blockage for unfolded translocationsobtained in a series of experiments where the pH was changed in steps between 7 and 13. Thesemeasurements were made with the expectation that a drop in this current blockage would occurabruptly at the room temperature pH where the DNA denatures, because a single strandedmolecule should be less effective in blocking the area of the nanopore. Although it is wellknown that the stability of the ds DNA helix is reduced at high pH, and at high enough pH thesingle stranded state is favored22, we were not able to find the value for the room temperaturedenaturing pH in the literature for the high salt concentrations of our experiments (see reference(22) for melting curves in 250 mM NaCl at pHs up to 10.6). Thus, we also measured the roomtemperature DNA optical absorbance at 260 nm as a function of pH in 1.6 M KCl with 20%glycerol. As seen in Figure 3 the absorbance increases abruptly by ~ 30% at pH 11.6. This iscaused by the well documented hyperchromicity of DNA22,23 upon denaturation. Thus therelatively constant blockade current up to pH 11.2 and its subsequent drop at pH 12.2 and 13,where no properties of the nanopore change dramatically, is associated with denaturation. Thisconfirms that ss-DNA is responsible for the results shown in figure 2b.
Discussion
The events we observe have a well defined duration and average current blockage and aresimilar to those that previous studies have identified with translocating molecules3,7. Thecurrent trace for each event shows quantized blockages with shorter events composed ofregions with deeper current blockage. Event distributions like those in figure 2 have a welldefined peak and a hyperbolic tail of events with constant ecd extending to lower translocationtimes and larger average blockages. We are unable to conceive of any explanation for thesefeatures other than the peak of the distribution corresponding to extended DNA moleculespassing single file through the nanopore and the tail to molecules having various foldedconfigurations where multiple strands of the same molecule can simultaneously occupy thepore at some time during the translocation. These features are observed at both low and highpH, confirming the translocation of double stranded and pH melted single stranded DNAthrough the same nanopore. The ubiquity of unfolded molecular events (> 50%) even fornanopores of ~10 nm in diameter suggests that long molecules are often unfolded by thenanopore capture process itself and that extremely small “molecule hugging” pores are notnecessary to linearize DNA molecules as they translocate through the nanopore3,5.We have referred above to events that fall on hyperbolas of constant ecd as “free
translocations.” For freely translocating molecules the translocation time and ecd is determinedprimarily by the identity, length and charge state of the molecule, the applied electric field andviscous drag in the fluid within and around the pore. The results presented here regardingtranslocation times and current blockages for freely translocating ds-DNA are consistent withprevious experiments3,7 when the effect of the glycerol is taken into account. The translocationtime ratio for 0% glycerol ds-DNA2 to 20% glycerol ds-DNA is 105 microseconds/170
microseconds which is remarkably close to the ratio of viscosities, 1.0 cp/1.68cp. For ss-DNAin alkaline conditions, both current drop and translocation time are reduced. Both parametersare related to the ratio of the ss to ds-DNA molecule areas which is nominally ~ 2/1. In this
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Fologea et al.Page 5
way we can account for reduced current drop for ss-DNA under the same ionic salt conditions.For translocation time other factors, including effective charge density, viscous drag forces,and persistence length differences should be considered in a further analysis of ss-DNAtranslocation times.
In conclusion our experiment demonstrates conclusively that long denatured single strandedDNA molecules can be observed to freely translocate in stretched out configurations througha solid state nanopore. This bodes well for the merging of nanopore based molecule
manipulation with molecular electronics methods for single base resolution sequencing via theincorporation of local nanoscale electrodes in the nanopore. Our results also demonstrate thenew and unique ability of a nanopore to detect single molecule hybridization without the useof fluorescent labels.
NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptAcknowledgements
We acknowledge Dr. Qun Cai, Dr. Toshiyuki Mitsui’s assistance with nanopore preparation and valuable discussionswith Prof. Daniel Branton. Support for this research has been provided by NIH, DOE, ABI, and the University ofArkansas start-up fund
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Figure 1.
a) Schematic illustration of DNA molecule translocating through a solid-state nanopore. b)Experimental setup for single molecule measurements with a nanopore detector. c) TEM of asilicon nitride nanopore, in this case 4 nm in diameter in a ~ 5–10 nm thick local membrane.
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Figure 2.
a) Event density plot vs. translocation time and average blockage current for 3 kb ss-DNA inpH 7 electrolyte solution. b) pH 13 electrolyte solution. The dashed lines are hyperbolae ofconstant event charge deficit (see text). In figure 2(b) the green outline of the data from 2(a)is presented to aid comparison. The color scale represents the probability density for a singleevent to occur at a given translocation time and blockage. Insets show representative time tracesfor individual events (i – unfolded molecule, ii – partially folded molecule, iii – completelydoubled over molecule). The bounding box on each inset event is 400 us wide and 500 pA tall.
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Figure 3.
Plot of mean current blockage (for simple, unfolded, single level) events and DNA opticalabsorbance as a function of pH showing transition from ds to ss-DNA at pH ~11.5. The squaremarkers represent the data from figure 2, while the triangle and diamond markers representexperiments in two other pores including that in Figure 1(b). The dashed line is a guide to theeye and not a fit.
Nano Lett. Author manuscript; available in PMC 2008 September 19.
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