Talanta 144 (2015) 268–274

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Talanta

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Nanopore detection of double stranded DNA using a track-etchedpolycarbonate membrane

Kaan Kececi a,n, Nevim San b, Dila Kaya b

a Istanbul Medeniyet University, Department of Chemistry, Uskudar 34700, Istanbulb Yildiz Technical University, Department of Chemistry, Esenler 34220, Istanbul

a r t i c l e i n f o

Article history:Received 11 February 2015Received in revised form31 May 2015Accepted 3 June 2015Available online 10 June 2015

Keywords:Resistive-pulseTrack-etched polymer membraneDNAConically shaped nanoporeBiosensing

x.doi.org/10.1016/j.talanta.2015.06.00540/& 2015 Elsevier B.V. All rights reserved.

esponding author.ail address: kaan.kececi@medeniyet.edu.tr (K.

a b s t r a c t

We investigate the resistive-pulse sensing of 50-bp DNA using track-etched polycarbonate (PC) nano-pores and show the translocation dynamics originating from the electrophoretic transport of DNAs.Conically shaped PC nanopore membranes have been prepared with asymmetric chemical etchingtechnique. We show the potential and concentration dependence of DNA translocation through a PCnanopore. We find that the translocation of DNA scales linearly with both potential and concentration.Additionally, the threshold potential is determined to complete the translocation. Finally, by in-vestigating the current-pulse amplitudes of nanopores with different tip sizes, we show that the na-nopore size can be successfully used to distinguish the DNA molecules. These results suggest greatpromise for the sensing of short DNAs and understanding the dynamics of the translocation processusing chemically-etched PC nanopores.

& 2015 Elsevier B.V. All rights reserved.

1. Introduction

The detection of individual molecules without labeling is anemerging field and resistive-pulse sensing is a powerful techniqueboth for detection and analysis of molecules [1–6]. The sensingparadigm of resistive-pulse was built on the Coulter-counterprinciple, which was used to count particles and measure theirsizes [7]. Basically, a micron (or sub-micron) sized pore is placedbetween two electrolyte solutions and particles are electro-phoretically driven to the oppositely charged electrode. During thetranslocation of particles, the ionic current momentarily drops andthis signal is used for counting particles and identifying theirproperties. In the mid-1990s, the technique was miniaturized byusing biological nanopores and enabled detection of individualmolecules without labelling and discriminate different types ofmolecules or slight differences inside the molecules [8].

Several resistive-pulse studies have been conducted usingbiological nanopores [9–12]. One of such nanopores, α-Hemolysin,has been widely used in determining the translocation propertiesand detection of proteins [13], RNA [14] and DNA [15] or evencontrolling reactions by analyzing the current [16]. However, thefixed size, instability and fragility of lipid bilayer at high potentialsand pH limit the usage of biological nanopores. Especially forbiomolecule sensing the demands are that the sensor is low cost,

Kececi).

user friendly and enable rapid analysis while the detection ishighly sensitive and specific [17]. So the researches for syntheticnanopores that could meet these qualifications were in demand.Alternative techniques such as e-beam lithography [18,19], nano-pipettes [20,21], ion-beam sculpting [22–24], micromolding[25,26], laser melting [27,28] and track-etch method [29–32] havebeen developed to fabricate and engineer synthetic nanopores toovercome the limitations of biological nanopores. The ability tocontrol the pore size very precisely, to change the surface char-acteristics and to integrate with electronics or optical systemsmade the synthetic nanopores advantageous over the biologicalones [33]. Also, surface functionalization is more diverse in syn-thetic systems, which enhances the sensibility and selectivity forcertain molecules [34,35]. However, the necessities of high costinstrumentation and trained personnel to operate the fabricationprocess limit the lithography and laser based techniques. On theother hand, micromolding is a simple and easy method for na-nopore fabrication but the size of the nanopore is not as preciseand reproducible as others. Therefore compared to all these na-nopore fabrication techniques, track-etch method emerges as analternative technique to obtain the desired nanopore in size andshape with uniform pore density without the limitations of theothers [36].

The track-etch method is basically irradiation of membranes (orfilms) with accelerated heavy ions and creating latent tracks insidethese membranes. As the irradiated membranes are exposed toappropriate etching solution, the latent tracks turn to nanoporesand the etching process is controlled by neutralization of etching

Fig. 1. SEM image of multipore PC membrane surface.

K. Kececi et al. / Talanta 144 (2015) 268–274 269

solution. The chemical etching of a single-ion irradiated film (i.e.,1 ion/membrane) leads to the formation of single-pore mem-branes, which are optimal for studying the detection and identi-fication of individual molecules [37]. The control of nanopore sizeswith a good reproducibility in track-etched membranes was suc-cessfully shown by Wharton et al. [38]. The poly(ethylene ter-ephthalate) (PET), polycarbonate (PC) and polyimide (PI) mem-branes have been widely used to fabricate nanopores and detectdifferent types of biomolecules such as proteins [39,40] and DNAs[31,41]. In addition, several surface modification techniques havebeen developed to promote the translocation of analytes[20,31,39,42,43]. However, these techniques are mostly labor-in-tense and may also cause undesired interactions between surfaceand analytes; especially for the small size nanopores.

Having an insight into the factors effecting the translocation ofshort DNAs is still critical for understanding the transport me-chanism through a single nanopore. Several studies have beenconducted to characterize the dynamics of the DNA translocationprocess by using synthetic nanopores. However, the studies usingtrack-etched nanopores and PC are still limited to DNA sensing andits transport characteristics. In this study, we aimed to show theelectrophoretic transport of short DNA strand as a model moleculeand examine the effect of voltage, concentration and tip size onthe current-pulse characteristics. To the best of our knowledge,this study is the first to detect such a short DNA strand and showtranslocation dynamics using track-etched PC nanoporemembranes.

2. Materials and methods

Polycarbonate (PC) membranes (3 cm diameter, 10 mm thick-ness) were obtained from Gesellshaft für Schwerionenforschung(GSI, Darmstadt-Germany). The membranes were irradiated withheavy ions (i.e., Au ion, 11.4 MeV) at various ion densities evendown to 1 ion/membrane. This was succeeded by defocusing theion beam and using a metal mask with a 0.1 mm diameter aper-ture with a shutter system which shuts down the ion beam as thesingle ion passage was detected. All the membranes were exposedto 4 h of UV irradiation (254 nm) to saturate the damages in tracks.The ds-DNA was obtained from Integrated DNA Technologies (IDTDNA, Germany) and suspended in Tris-EDTA buffer (10 mM Tris,0.1 M EDTA, pH¼7.5). The sequence of the DNA chain is 5ʹ-AATTCG AGC TCG GTA CCC GGG GAT CCT CTA GAG TCG ACC TGC AGGCAT GC with its complimentary strand. All solutions have beenprepared from deionized water (Millipore Direct-Q 5, MilliporeCo.). Formic acid (HCOOH), sodium hydroxide (NaOH) and po-tassium chloride (KCl) were purchased from Sigma Aldrich. Allchemicals were used as received without further purification.

Fig. 2. Current–voltage curve of PC nanopore in 1 M KCl.

3. Chemical etching and pore size calculations

Asymmetric chemical etching was used to fabricate conicallyshaped nanopores. Briefly, the single-track membranes wereplaced between two halves of the conductivity cell. One half of thecell was filled with etching solution (9 M NaOH) and the other halfwith stopping solution (1 M HCOOH and 1 M KCl). Two platinum(Pt) electrodes were immersed into each cell and potential wasapplied (1 V) to monitor the breakthrough moment. The etchingprocess was stopped as breakthrough current was observed andthe etching solution was replaced with stopping solution forneutralization. Then both cells were rinsed with di-water to re-move residues from the membrane surface. After this etchingprocess, a conically shaped nanopore was obtained with two dif-ferent sized openings called base (large opening) and tip (small

opening) [31].The large opening of the nanopore (dbase) was determined by

electron microscopy of multipore membranes(108 nanopores/cm2). A base diameter of 410735 nm was ob-tained after chemical etching, indicating good reproducibility forthe nanopore fabrication process [31,39]. A SEM image of multi-pore membrane was given in Fig. 1. The base diameter was cal-culated by the averaging the diameters of 20 nanopores. All thesingle-pore membranes were chemically etched under the sameetching conditions of the multipore membranes. The small open-ing (dtip) was calculated using electrochemical measurements.Briefly, each side of the conductivity cell was filled with electrolytesolution (i.e., 1 M KCl, 10 mM PBS buffer at pH¼7). Ag/AgCl elec-trodes were immersed into both sides of the cell and potential wasstepped (50 mV) between �1 V and þ1 V (Keithley 6487 pi-coammeter/voltage source, Cleveland, OH, USA). The resistance ofthe nanopore (R) is proportional to the conductivity of solution (ρ),the length of nanopore (l, thickness of the membrane), dbase anddtip. In order to calculate the tip diameter (dtip) Eq. (1) was used; allthe values were determined and the R value was the reciprocal ofthe slope of I–V curve (see Fig. 2). Less rectification was observeddue to the high electrolyte concentration, which compensated thesurface charge of nanopore and diminished the influence of thesurface charge on rectification.

Rl

d d4

.....1tip base

ρπ

=( )

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The average of three sequential measurements has been usedfor the calculations. The conductivity of the solution was measuredusing a conductivity meter (Mettler-Toledo FE 30, Colombus, OH,USA). The agreement between the electrochemical measurementsand SEM images was for the tip size calculation was also given inprevious studies [41].

4. Resistive-pulse experiments

The chemically etched nanopore membrane was placed be-tween two halves of a conductivity cell and each half-cell wasfilled with 1 M KCl in Tris, 0.1 mM EDTA. The ds-DNA sampleswere added to the tip side of the conductivity cell. Ag/AgCl (BAS,West Lafayette, IN) electrodes were used to measure the current-pulses (events) [31,44]. An Axopatch 200B (Molecular DevicesCorporation, CA, USA) was used in voltage-clamp mode to apply atransmembrane potential between Ag/AgCl electrodes with a low-pass Bessel filter at 2 kHz bandwidth. The signal was digitizedusing a Digidata 1440 analog-to-digital converter (Molecular De-vices Corporation, CA, USA) at a sampling frequency of 10 kHz.Data were analyzed using pClamp 10.2 (Molecular Devices

Fig. 3. Current–time traces of 50-bp DNA (10 nM) at 1000 mV (Buffer only, no DNdiameter¼27 nm. (base diameter¼410 nm).

Corporation). Minimum 100 data point were evaluated forcalculations.

In order to characterize the current-pulses, the current-pulseamplitude (Δi) and duration (τ) were used. The duration of thecurrent-pulse (τ) is defined as the time between the drop of cur-rent and recovery to the initial value. The current-pulse amplitude(Δi) is defined as the difference between the lowest point of thepulse and initial value (see Fig. 3B inset). The average Δi and τvalues were obtained by fitting the histograms to a Gaussiandistribution.

5. Results and discussion

5.1. Resistive-pulse sensing of 50-bp DNAs

In order to prove the electrophoretic transport of DNA mole-cules, the effect of potential and concentration were examined.After chemical etching, the ionic current was recorded prior to theaddition of 50-bp DNAs, which was stable and showed no current-pulses (Fig. 3A). Then the 50-bp DNA was added to the tip side ofthe membrane and as a result of electrophoretic transport of DNAs

A)(A), 1000 mV (B), 800 mV (C), 600 mV (D), 400 mV (E), and 200 mV (F). Tip

Fig. 4. DNA current-pulse frequency versus potential. DNA concentration¼10 nM. Fig. 5. DNA current-pulse frequency versus concentration. Appliedpotential¼1000 mV.

Fig. 6. Scatter plot of current-pulse magnitude (Δi) versus current-pulse duration(τ) for 50-bp DNA (10 nM).

K. Kececi et al. / Talanta 144 (2015) 268–274 271

to the counter electrode; current-pulses were observed accord-ingly under same potential (i.e., 1000 mV) (Fig. 3B). The potentialwas stepped down to 200 mV and no current-pulses were ob-served under 200 mV (Fig. 3C–F). Fig. 4 illustrates the potentialdependence of DNA translocation. The current-pulse frequencywas calculated by averaging the number of current-pulses of threecurrent-time recordings (5 min each). A linear correlation wasobserved between 400 mV and 1000 mV. The data obtained at200 mV is excluded and will be discussed later in detail. The linearpotential dependency of double strand (ds) DNA has been alsodiscussed in both experimental [41,45] and theoretical [46] stu-dies. The dynamics of translocation in track-etched nanoporeshave been explained in detail by Cabello-Aguilar and co-workers[47]. Since the translocation phenomenon is based on the elec-trophoretic transport of particles (or molecules) inside the solutionunder electric field, they simply derived an equation giving therelationship between the effective velocity (νeff) and electric field(E) where, ξ is the zeta potential of particle/DNA, ε is the dielectricpermittivity and η is the viscosity of fluid. (see Eq. (2)).

E.....

2effν ε

ηξ=

( )

The previous studies showed that the electric field strength (E)was ranged between 1.3 and 2 MV/m for the conically shapedtrack-etched nanopore [39], which forms a trapping zone to cap-ture the molecule easily at the tip [48]. So the increase in theelectric field at the nanopore entrance causes a linear increase inthe current-pulse frequency. The linear dependence and thresholdvoltage suggest that the translocation frequency was dominatedby the transport of DNA through the nanopore. The concentrationdependence was also analyzed within a range of 1 nM to 20 nM.The data points were obtained by averaging the number of countsin five (5 min) current–time traces. No current-pulses were ob-served under 1 nM and the current-pulse frequency increasedsignificantly from 15 min�1 to 156 min�1 at 1000 mV. A linearrelation between current-pulse frequency and concentration wasfound (Fig. 5). The linear concentration dependence of DNA wasalso shown by several studies using both synthetic [41,49] andbiological [50] nanopores.

To gain insight into the translocation process, the current-pulses were characterized via the scatter plot of current-pulseamplitude (Δi) and duration (τ) (Fig. 6). The duration values wereall in millisecond range. The average duration and current-pulseamplitudes were found as 1.3570.13 ms, 7777 pA at 1000 mV;1.8170.28 ms, 6472 pA at 800 mV; 2.7770.28 ms, 5574 pA at

600 mV; 3.9270.51 ms, 3671 pA at 400 mV and 0.7770.19 ms,27.670.8 pA at 200 mV. For current-pulse durations and ampli-tude, it is hard to make a quantitative estimation, however, bothvalues at 1000 mV are correlated and in close proximity to thepreviously reported values [31] (i.e., 1.371 ms, 50712 pA). Theresults confirmed the inverse relationship between the current-pulse duration (τ) and the potential. The duration got longer as thepotential decreased. This inverse correlation is formulated in Eq.(3), where η is the viscosity of solution, r is the radius of ion, e isthe electronic charge of ion, ld is the length of the detection zoneand E is the electric field [41].

l z eE6 r / ..... 3dτ πη= ( )

In addition to the longer durations, the spread is also broader inlower potentials due to the energy barrier that the DNA moleculeneeds to overcome. Dragging the molecule inside the nanopore isharder at lower potentials, which also reinforce the interactionsbetween surface and molecule.

On the other hand, as reported above, the average durationvalue for current-pulses at 200 mV is shorter than 400 mV with asmaller spread which is against the relationship given in Eq. (3).This can be attributed to the approach of DNA molecules to thepore opening but not completing the translocation. In other words,

K. Kececi et al. / Talanta 144 (2015) 268–274272

it can be accepted as incomplete translocation (or ‘bumping’) ofmolecules at the tip opening. So, rather than a complete thread ofDNAs, the tip opening is partially blocked or screened by DNAmolecules causing brief current-pulses. An entropic barrier appliesto any kind of molecules but especially large or complex moleculesfaces a large entropic barrier due to the loss of number of availableconfigurations. Physical or geometrical restrictions, complexstructure of molecule or surface–molecule interactions can in-crease the energy requirement for a complete thread and limitsthe passage of such molecules. In order to pass the entropic bar-rier, an additional external field, a chemical potential or a selectiveadsorption is required to drive a molecule from one side to theother. Since each nucleotide in the DNA carries roughly one unitcharge, depending on the pH of solution, it is possible to drive theDNA molecules through the nanopore under electric field. Kasia-nowicz and co-workers showed the effect of electrical field on thetranslocation of DNA and RNA molecules and discussed the posi-tive its contribution to overcome the entropic barrier [8,50]. Chenand Sen confirmed the same observation using artificial nanoporesensors [26,45]. In addition to the The same phenomena alsoshowed in addition to experimental studies, the entropic barrieralso discussed theoretically based on different parameters such aseffect of solvent, nanopore size and adsorption [51–53]. For ourstudy, an entropic barrier applies to 50-bp DNA molecules tocomplete a translocation that a threshold voltage above 200 mVwould be necessary to drive the DNA through the PC nanopore[41,54].

Of particular interest, a final experiment was performed undersame conditions to show the effect of tip size on the translocation.The duration and magnitude of current-pulses were investigatedwith a smaller nanopore having a tip size of 18 nm (basediameter¼360 nm) and the current-pulse characteristics werecompared to the 27 nm nanopore. The current–time traces forboth nanopores are given in Fig. 7. The current-pulses were ob-served upon addition of 50-bp DNAs with a lower backgroundcurrent. The duration and current-pulse amplitudes were

Fig. 7. Current–time traces of 50 bp DNA (10 nM) for 18 nm tip (base diamete

summarized in a scatter plot (see Fig. 8A). The duration valueswere relatively scattered and broadly distributed for the 18 nmnanopore respect to the values obtained by 27 nm nanopore (SeeFig. 8A). No significant difference has been observed in averageduration values, which was 1.2270.25 ms for 18 nm nanopore(see Fig. 8B). The scatter in duration may originate from the ag-gregation of DNA molecules or limitations due to the smaller sizeof the nanopore. In this case, the peak shape would help us tounderstand the translocation dynamics. In both sizes of nanopores,similar spike shape current-pulses were observed, which wouldindicate single level translocation of molecules without folding.Basically, the size of 50-bp DNA is �2.2 nm in diameter, �17 nmin length and its persistence length is 50 nm. Since the length ofthe molecule is less than its persistence length, the shape of themolecule is essentially rod-like and it is hard to consider the fol-ded conformations of such a short DNA.

As the nanopore size decrease and become in comparable sizes,the surface charge, electrical double-layer and electro-osmoticflow also become dominant and play role during the translocationprocess [55]. However, in our case, the diameter of the nanopore isnot critically small. Nevertheless, the orientation of the moleculealso gains more importance and the translocation of the moleculeis more restricted compared to the larger nanopores. The spread induration for the 18 nm nanopore was relatively larger, which canbe due to the less rotational degree of freedom and possible in-teractions between DNA and nanopore walls [49,56,57]. Theaverage current-pulse amplitude increased in respect to the 27 nmnanopore and was found as 11777 pA for the 18 nm tip (Fig. 8C),which has a close correlation among the nanopore sizes. The in-verse correlation between the Δi and dtip was discussed by Itoet al. [58] The current-pulse amplitude is correlated to the block-age ratio of the nanopore by the molecule. In other words, theoccupied volume inside the nanopore determines the amplitude ofthe current-pulses [58,59]. The results show that by fine tuningthe tip size, different types of analytes can be identified based ontheir sizes and Δi can be used to differentiate molecules. The shape

r¼360 nm) (A), and 27 nm tip (base diameter¼410 nm) (B) at 1000 mV.

Fig. 8. Scatter plot of current-pulse magnitude (Δi) versus current-pulse duration (τ) for 50-bp (10 nM) for 18 nm tip (red) and 27 nm tip (black) (A). Histogram of current-pulse duration (τ) of 18 nm tip (red) and 27 nm tip (green) (B). Histogram of current-pulse amplitude (Δi) of 18 nm tip (red) and 27 nm tip (green) (C). Potential¼1000 mV.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

K. Kececi et al. / Talanta 144 (2015) 268–274 273

of the current-pulses also helps us to understand the dynamics oftranslocation, which were in spike shape indicating single-leveltranslocations without folding. As indicated above, the length ofDNA and its persistence length limit such a possibility. In case ofany aggregation, DNA molecules should occupy more volume withlarger current-pulse amplitude, more spread and cause second orthird group on the scatter plot. Such results have been shown forthe longer DNA strands or proteins having multi-level events withvarious peak shapes [49,56,60,61]. In conclusion, DNA-surface in-teraction [49] and limited orientation of DNA with less degree offreedom are more responsible for the spread in 18 nm nanopore.

6. Conclusion

In this paper, we showed the detection of 50-bp DNA and thepotential use of PC nanopores as a tool for resistive-pulse sensing.The track-etched PC membranes were used to fabricate the na-nopores. Unlike PET, PC was directly used without any surfacemodification. This resulted shorter nanopore preparation time andeliminated undesired surface interaction with analytes. The cur-rent-pulse frequency showed a linear correlation with DNA con-centration and applied potential, which is in agreement withelectrophoretic transport theory. We also showed quantitativeresults of duration and current-pulse magnitude as a function ofpotential and threshold potential to overcome the energy barrierfor a complete translocation. The effect of pore size on current-pulses was also given to support the translocation paradigm. Weshowed the change in current-pulse amplitude as a function of tipdiameter, which can be used as a critical parameter for detectionand discrimination of molecules [31]. The track-etched nanoporemembranes stand as a powerful tool to be integrated to otherplatforms for the analysis of viruses, nanoparticles and biomole-cules. The detection of such short DNA strands can be potentiallyused for fragment sizing, PCR studies and tracking enzymatic di-gestion without any labeling.

Acknowledgement

This work is financially supported by the The Scientific andTechnological Research Council of Turkey (TUBITAK) (P.no:113Z335).

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