Charged Particles Modulate Local Ionic Concentrations and Cause Formation of Positive Peaks in Resistive-Pulse-Based Detection

Charged Particles Modulate Local Ionic Concentrations and CauseFormation of Positive Peaks in Resistive-Pulse-Based DetectionJustin Menestrina,†,‡ Crystal Yang,§,‡ Matthew Schiel,∥ Ivan Vlassiouk,⊥ and Zuzanna S. Siwy*,†,§,∥

†Department of Physics and Astronomy, §Department of Chemistry, and ∥Department of Biomedical Engineering, University ofCalifornia, 210G Rowland Hall, Irvine, California 92697, United States⊥Oak Ridge National Laboratory, Bethel Valley Road, Oak Ridge, Tennessee, 37831, United States

*S Supporting Information

ABSTRACT: We study the effect of electrolyte concentrationon the shape of ion current pulses in resistive-pulse sensing.We show that electrokinetic passage of several hundrednanometers in diameter charged polystyrene particles througha micropore leads to formation of current increase when theparticles exit the pore. The particle entrance, as reportedbefore, causes formation of the current decrease, which is ameasure of the particle size. Formation of the double peak, i.e.,current decrease followed by a current increase, is especiallypronounced if the resistive-pulse experiments are carried out in KCl concentrations below 200 mM. In order to explain the pulseshape, experiments were designed in which the particles passed through the pore only by either electroosmosis orelectrophoresis. The presented experiments and modeling indicate that while both electroosmosis and electrophoresis affect theion current pulse, formation of the positive peak is mainly determined by the latter effect and the charged state of the particle.The importance of the findings for resistive-pulse analysis is discussed.

I. INTRODUCTION

Passage of single molecules and particles through pores is thebasis of resistive-pulse sensing.1−12 When an electric potentialor a pressure difference is applied across a single-poremembrane, the translocation of single particles is observed asa change of the transmembrane current (typically a decrease)called a resistive pulse.6−8 The relative amplitude of theresistive pulse, expressed as a percentage change of the baselinecurrent, carries information on the particle size. In order toobtain a high signal-to-noise ratio, experiments are oftenperformed at high ionic strengths of the background electrolytesolution.In cases when particle agglomeration becomes a problem,

solutions with low salt content (e.g., 10 mM KCl) must be usedto enhance electrostatic repulsion. Passage of hydrogelsthrough glass pipettes and polymer pores was successfullyreported only when performed in solutions of concentrationbetween 10 and 100 mM KCl.13−15 Detection of DNA in lowionic strength solutions led to observation of concentration-dependent magnitudes and even relative sign of the resistivepulses. The presence of DNA in the pore causes either acurrent decrease or a current increase depending on whetherthe number of ions excluded by the molecule is higher or lowerthan the number of DNA counterions brought into the pore tofulfill electroneutrality.16,17 Experiments of DNA sensing in lowaspect ratio solid state pores also led to a surprising find that forcertain pore diameters higher signal-to-noise ratios for DNAtranslocation can be obtained in experiments performed in lowsalt concentrations.18

In this article, we studied the effects of ionic strength on thedetection of negatively charged polystyrene particles in singlepores. We show the shape of the resistive pulses is influencedby the background electrolyte concentration. In all studiedsolutions, the particle entrance caused a current decrease, whichwas used to calculate the effective size of the particles. In saltconcentrations below ∼200 mM, the current decrease wasfollowed by a current increase whose amplitude decreased withincreasing KCl concentration. The current increase occurred atthe end of the translocation event, and its magnitude in somecases exceeded the amplitude of the current blockage. Theexistence of the positive peak indicates there are additional ionspassing through the pore compared to ionic transport in anempty pore.The presence of the positive current spike at the end of a

resistive pulse was reported before in experiments of DNAmolecules passing through sub-2-nm in diameter SiNpores.19,20 We show the effect is also present in the detectionof large, several hundred nanometer in diameter spherespassing through micropores. Ion current increase in theresistive pulse is largest when both the particles and the porewalls are charged but is also present when the pore walls areneutral. We provide experimental and theoretical evidence thatthe positive peak results from transient modifications of theelectrolyte concentration caused by the charged particle.

Received: December 11, 2013Revised: January 9, 2014Published: January 9, 2014

Article

pubs.acs.org/JPCC

© 2014 American Chemical Society 2391 dx.doi.org/10.1021/jp412135v | J. Phys. Chem. C 2014, 118, 2391−2398

pubs.acs.org/JPCC

The pores used in the experiments were prepared in 12 μmthick films of polyethylene terephthalate by the track-etchingtechnique.21−23 Track-etched pores in this material arecharacterized by an undulating pore diameter reflected in theshape of resistive pulses.23 During its translocation, a sphericallyshaped particle blocks a pore to a larger extent when passingthrough a section with a narrower constriction zone comparedto when translocating a wider region. The amplitude ofsubpeaks in a resistive pulse is considered a measure of the localpore diameter. We find that for bulk electrolyte concentrationsbelow 200 mM the relative amplitude of ion current changeswithin a pulse is also a function of the electrolyte concentration.The presented results are very important for interpretation of

resistive pulses and relating the current change with the size ofthe translocating particle and pore diameter.

II. EXPERIMENTAL SECTION

Preparation of Pores. Single pores in 12 μm thick films ofpolyethylene terephthalate (PET) were prepared by the track-etching technique as described before.21−23 Briefly, films wereirradiated with single energetic heavy ions (e.g., 11.4 MeV/u Auand U ions) at the UNILAC linear accelerator of the GSIHelmholtzzentrum fur Schwerionenforschung in Darmstadt,Germany. Wet chemical etching of the films in 0.5 M NaOH,70 °C and 2 M NaOH, 60 °C leads to preparation of pores withundulating pore diameter along the pore axis.23,24 Bothconditions were used to fabricate pores presented in thismanuscript. The mean pore diameter was estimated from thecurrent−voltage measurements performed in 1 M KCl andrelating the pore diameter with its conductance.25,26 Experi-ments presented here were performed with pores with anopening diameter between 500 and 1500 nm. The walls oftrack-etched PET pores contain carboxyl groups and arenegatively charged at pH values above 3.8.25

Particles. All carboxylated polystyrene particles and un-charged poly(methyl methacrylate) (PMMA) particles were

purchased from Bangs Laboratories. Sulfonated polystyreneparticles were obtained from Life Technologies. Sizes of thepurchased particles were measured by the Zetasizer Nano ZS(Malvern Instruments, Westborough, MA). All purchasedparticles had ∼15% variation in their diameter. Surface chargeof the carboxylated particles was between −0.16 and −0.22 C/m2 (or −1.0 and −1.4 e/nm2). Sulfonated particles were lesscharged with −0.06 C/m2 (−0.31 e/nm2).

Ion Current Recordings. Measurements of ion current intime were performed using the Axopatch 200B and 1322ADigidata system (Molecular Devices, Inc.). Membranes wereplaced in a custom-made conductivity cell, and two homemadeAg/AgCl electrodes were placed on both sides of themembrane. Data analysis was performed with Clampfit 9.2and Origin 9.0. Detection of particles was performed from KClsolutions between 10 and 400 mM. Concentration of particleswas ∼108 per mL. Solutions of carboxylated particles contained0.01% of Tween 80; sulfonated particles were detected fromsolutions containing 0.015% of Tween 80.

Comsol Modeling. Coupled Poisson−Nernst−Planck andNavier−Stokes equations were solved using the ComsolMultiphysics 4.3 package.15,27 Cylindrical pores that were 2μm long were connected with 20 μm long cylindrical reservoirs.A very fine triangular mesh of 0.1 nm was used close to thecharged walls. Surface charge density of the channel wall waseither 0 or −0.25 e/nm2, while surface charge of particles waseither 0 or ±0.75 e/nm2. In the remaining parts of the modeledstructures, the mesh was reduced to the point when no changein the observed concentration profiles and currents wasobserved upon further mesh decrease. The dielectric constantof the solution ε = 80, particle ε = 4, and diffusion coefficients 2× 10−9 m2/s were used for both potassium and chloride ions. Inall calculations, a potential difference of 0.1 V was applied tothe right reservoir while the left reservoir was grounded.

Figure 1. Example shapes of resistive pulses obtained when (a) 470 nm carboxylated particles and (b) 410 nm carboxylated particles translocated a1400 nm pore. KCl concentration and voltage for each pulse are indicated in the figure. Events are vertically offset to facilitate comparison.

The Journal of Physical Chemistry C Article

dx.doi.org/10.1021/jp412135v | J. Phys. Chem. C 2014, 118, 2391−23982392

III. RESULTS AND DISCUSSION

Polystyrene particles with diameters of 180, 200, 280, 410, and470 nm were detected during their voltage-induced passagethrough single pores in a polyethylene terephthalate (PET)film. The 180 nm particles were modified with sulfonategroups, which assured they were negatively charged in a widespectrum of pH values down to pH ≈ 1.0.28 The remainingparticles were modified with carboxyl groups. Since pores inpolyethylene terephthalate films used in the experiments alsocontain carboxyl groups on the walls, all studied particles andthe pore walls were negatively charged at pH values above ∼4.As a result, particles transport occurred by a combination ofelectrophoresis and electroosmosis. Particles were movingtoward a positively biased electrode, indicating the effect ofelectrophoresis dominated the particle passage. All solutions ofparticles for the resistive-pulse experiments were prepared inKCl as the background electrolyte. KCl concentration wasvaried between 10 and 400 mM. Additional experiments wereperformed with 400 nm in diameter uncharged poly-(methylmethacrylate) (PMMA) particles in 100 mM KCl.Example recordings of polystyrene particles passing through

a pore with an average opening diameter of 1400 nm are shownin Figure 1. Experiments were performed at pH 10 to maximizethe surface charge on the pore walls and particles. As observedby us and other groups before, entrance of the particles into apore causes a current decrease whose modulation reflects theshape of the pore.7,15,23,29−33 The roughness of pores in PEToccurs due to the laminar structure of the foils and overetchingof the strata at elevated temperatures.34 Larger modulations ofthe current indicate larger modulations of the pore diameter.Reproducibility of the current shapes when different particlespass through the same pore was reported by us before,15,23 andmore examples of resistive pulses are shown in the SupportingInformation.Recordings presented in Figure 1 reveal resistive pulses of

polystyrene particles recorded in KCl concentrations below∼300 mM have a double-peak character, i.e., an initial currentdecrease is followed by a current increase above the baselinelevel. Experiments in different KCl concentrations had to beperformed in different voltages, which will be discussed below.In the case of the 1400 nm pore and 470 nm particles in 10mM KCl, the amplitude of the current increase accompanying aparticle exit exceeded the amplitude of the current decrease.The positive peak at the end of resistive pulses recorded with470 nm particles was not present at 400 mM and higher KClconcentrations; passage of 410 nm particles through the samepore did not cause a current increase at KCl concentrations

equal to and higher than 300 mM. The small amplitude andabsence of the positive peak coincide with the convergence ofthe shape of the resistive pulses so that the pulse shapebecomes KCl concentration independent. Experiments inFigure 1 suggest that in order to image the pore topographyusing the resistive-pulse technique experiments have to beperformed in at least 200 mM KCl. More concentratedsolutions also increase the signal-to-noise ratio, facilitating theability to distinguish finer topography changes.Resistive-pulse experiments with polystyrene particles at low

KCl concentrations revealed that particle transport wasobserved at significantly higher voltages compared to therecordings at higher ionic strengths. Thus, when comparing theresistive-pulse shapes, one has to take into account a possibledependence of the pulse shape on the applied voltage. Figure 2shows the ratio of a maximum current decrease and themaximum current increase observed when the particles wereexiting the pore recorded for different voltages and particlediameters for two pores with opening diameters of 1400 and980 nm, respectively. Each point represents an average of atleast 200 pulses. The graph suggests the ratio is fairly voltageindependent for the 1400 nm pore but increases with increasingbulk electrolyte concentration and the diameter of thetranslocating spheres (Figure 2a). A weak voltage dependenceof the current decrease and current increase ratio was found forpores with diameters below 1 μm; however, the voltage effectwas less significant compared to the influence of KClconcentration and particle diameter (Figure 2b).In order to explain the presence of the positive peak, we first

found its area as a function of voltage and bulk KClconcentration. The peak area is equal to the number of ionsthat pass through the pore in addition to the ionic transportthat constitutes the baseline current in an empty pore. Foralmost all examined pores and particles sizes, the integratedarea under the peak is at least a few times larger than thenumber of ions that were displaced by a single sphere (Table1). Thus, the positive peak cannot be explained by only theinflux of ions into the pore when the particle exits.A similar shape of resistive pulses with a current blockage

followed by a current increase was observed with DNAmolecules passing through a subnanometer SiN pore.19,20

Experiments were subsequently modeled approximating a DNAmolecule as a charged cylinder whose transport through thepore was described by the coupled Poisson−Nernst−Planck(PNP) and Navier−Stokes (NS) equations.35 The positivepeak was predicted to occur in cases when the electrical double-layer thickness was comparable to the pore dimensions. Theauthors concluded that the positive peak was observed in

Figure 2. Average ratios of maximum current decrease and maximum current increase as a function of voltage, recorded for two pores with averageopening diameters of (a) 1400 and (b) 980 nm. Ratio was calculated for different particles and KCl concentrations as indicated in the figure.



simulated experiments only when the PNP and NS equationswere solved simultaneously, in other words, when both effectsof electrophoresis and electroosmosis were taken into account.Although electrophoresis dominated, since, e.g., negativelycharged particles moved toward a positively biased electrode,this effect alone could not lead to formation of a currentincrease at the end of the resistive pulse.Dimensions of our system are significantly larger than the

size of DNA molecules and solid state nanopores considered inthe simulations;35 thus, the model cannot be directly applied toexplain our results. We decided however to test experimentallywhich phenomenon, electrophoresis or electroosmosis, domi-nates formation of the positive peak in our experimentalsystem.The effect of electrophoresis was eliminated in the transport

of 400 nm in diameter uncharged PMMA particles. Experi-ments were performed at pH 10 to maximize the charge on thepore walls and enable electroosmotic passage of the unchargedparticles. The concentration of KCl solution used in theseexperiments was 100 mM KCl. Recordings in 10 mM KCl weretoo noisy and did not allow for reliable detection of the pulses.As expected, the PMMA particles passed through the pore atvoltages of opposite polarity compared to previous experiments

with carboxylated particles (Figure 3). Although passage of 410nm in diameter carboxylated polystyrene particles created aclear current increase at the pore exit, resistive pulses of PMMAparticles of similar diameter had only a current decrease.Elimination of electroosmosis was more challenging since

carboxylated polystyrene particles have similar surface chem-istry to the chemical characteristics of the pore walls. Theisoelectric point of the track-etched PET surface is 3.8.25 At thispH, carboxylated polystyrene particles would also be un-charged. Therefore, experiments were performed with particleswhose surfaces were modified with sulfonate groups which onlylose their charge at pH ≈ 1.Figure 4 compares resistive pulses obtained with sulfonated

particles at pH 3.5 and 5.5 as well as carboxylated particles of asimilar diameter at pH 10 (Figure 4). Although the exit of theparticles at pH 5.5 and 10 caused a clear current increase, thepositive peak was absent in the pulses of single sulfonatedparticles recorded at pH 3.5 (Figure 4a). However, we noticedthe recordings at pH 3.5 often had events with doubleamplitude, suggesting that two particles translocated the poretogether. Particle agglomeration also caused frequent blockingof the pores and probably originates from the low surfacecharge density of the particles (maximum of −0.06 C/m2).Since the diameter of the pore was below 600 nm and theparticles were 180 nm, our system did not detect translocationof triple particles. However, the double events did show anonzero current increase with a smaller amplitude than eventsrecorded at higher pH (Figure 4b and 4c). The presence of thecurrent increase with double particles is in agreement with ourearlier results (Figure 1), indicating the current increase is morepronounced with larger particles. The small amplitude of thecurrent increase most probably results from partial protonationof the sulfonate groups at pH 3.5.It is also important to mention that due to the low surface

charge density of sulfonated particles, their electrophoretictransport was only observed at pH values below 6 when thecarboxyl groups on the pore walls are partially protonated. Wewere successful in recording passage of the particles at pH 8.0,but transport occurred by electroosmosis since the surfacecharge density on the walls was higher at these conditions thanthat on the particles.Experiments with PMMA and sulfonated particles suggest

the current increase only occurs in experiments when the

Table 1. Number of Ions Obtained after Integration of aPositive Peak in Resistive Pulses Recorded when 470 nmParticles Were Exiting a 1400 nm in Diameter Pore(Npositive_peak)

a

KClconcentration

no. of ionsdisplaced by avolume of asingle particle(Nvolume)

no. of ions obtained fromintegrating positive peaksof resistive pulses atdifferent voltages(Npositive_peak)

Npositive_peak/Nvolume

10 mM 3.26 × 105 2 V: (1.28 ± 0.20) × 107 39 ± 63 V: (1.30 ± 0.21) × 107 40 ± 74 V: (3.11 ± 0.65) × 107 95 ± 20

100 mM 3.26 × 106 0.6 V: (8.28 ± 1.77) × 106 2.5 ± 0.61 V: (1.14 ± 0.15) × 107 3.5 ± 0.51.3 V: (9.81 ± 1.78) × 106 3.0 ± 0.6

aThese ions pass through the pore in addition to the ions constitutingthe baseline current. Values from recordings in 10 and 100 mM KCl atdifferent voltages are given. Number of ions displaced by a volume of asingle particle is shown as well.

Figure 3. Ion current pulses obtained when 410 nm carboxylated particles (1.5 V) and 400 nm uncharged PMMA particles (−1.5 V) passed througha pore with an opening diameter of 980 nm. Recordings were performed in 100 mM KCl, pH 10.



translocating particles carry surface charge. Formation of thepositive spike accompanying the particle’s exit does not requirecharges on the pore walls.In order to unravel the physical basis for the shape of

resistive pulses in low KCl concentrations, we performednumerical modeling of ionic concentrations and ionic currentthrough a single pore with and without a particle placed atdifferent locations along the pore axis (Figures 5 and 6).Modeling involved solving coupled PNP and NS equations,which is computationally very expensive.15,27 Therefore,modeling was performed with a 2 μm long pore (vs ∼11 μmexperimentally studied structures) and at a lower voltage of 0.1V. The surface charge density of the pore walls and the particlewas set −0.25 and −0.75 e/nm2, respectively. For simplicity, acylindrical geometry was considered. The opening diameter ofthe modeled pore was 510 nm, thus equal to the diameter ofthe structure experimentally examined in Figure 4a, 4b, and 4d.Figure 5c shows the profile of KCl concentration along the

axis of an empty pore and electrolyte reservoirs placed on bothsides of the membrane. On the side of the membrane with a

positively biased electrode, the salt concentration was depletedby ∼1% compared to the bulk concentration (10 mM KCl),while on the opposite side the concentration was enhanced.Due to the large opening diameter of the pore, the difference inconcentration of potassium and chloride was negligible alongthe pore center. We concluded the slight modulations of KClconcentration at the pore openings originate from concen-tration polarization caused by an enhanced sourcing ofpotassium ions through a negatively charged pore. Concen-tration polarization is typically associated with nanopores andionic selectivity, and indeed, depletion/enhancement of ions atpore entrances is small in micrometer size pores.36

Figure 5a and 5b shows how the distribution of ionicconcentrations along the pore axis changes when a 220 nm indiameter negatively charged particle is placed at one of the twopore entrances. When approaching the pore, the particledepletes the region adjacent to the pore entrance with co-ions,chloride in the case of negatively charged particles consideredhere (Figure 5a). Since co-ions are moving in the samedirection as the particle, the current carried by chloride

Figure 4. (a) Example of resistive pulses obtained with single 180 nm in diameter sulfonated particles passing through a 510 nm in diameter pore atpH 3.5, 2.2 V. (b) Resistive pulse of two sulfonated particles translocating the 510 nm pore together at 2 V. (c) Sulfonated particle passing through adifferent pore with an opening of a 540 nm pore at pH 5.5, 4 V. (d) As a comparison, a pulse of a 220 nm carboxylated particle at pH 10, 2.2 V in the510 nm pore is shown as well. Dashed line facilitates observation of the positive peak at the end of the pulses. Bulk KCl concentration was 50 mM.

Figure 5. (Left) Results of numerical modeling of ionic concentrations along an axis of a 510 nm in diameter pore and at pore entrances with (a,b)and without (c) a 220 nm charged particle placed at two pore entrances (a,b). Length of the modeled pore was 2 μm. Concentrations along the poreaxis are shown; thus, the discontinuity in a and b is due to the presence of the particle. Bulk KCl concentration was 10 mM. (Right) Schematicrepresentation of ionic distributions and direction of potassium and chloride ions movement (red and blue arrows, respectively) for all cases shownin the left column.



diminishes. The potassium current decreases as well, sincethese ions are sourced from the entrance where theconcentration is lowered by the concentration polarization.When the particle exits the pore (Figure 5b), the pore end

from which potassium is sourced contains a higher concen-tration of K+ brought about by the particle. Chloride is sourcedfrom the opposite entrance that is in contact with enhancedKCl concentration due to the concentration polarization effect.As a result, the particle exit leads to an increase of the recordedion current.Modulation of ionic concentrations induced by the charged

particle is larger than the modulation caused by theconcentration polarization; thus, the current increase isobserved even with micrometer size pores (Figure 1).One can calculate the corresponding ion current through the

pore/particle system when the particle is placed at differentlocations along the pore axis. This current vs position relation isequivalent to the signal of current vs time measured inexperiments.It is convenient to measure the ionic current blockage/

enhancement degree as a percentage of the baseline current, I:χ = ΔI/I (Figure 6), where ΔI is the difference of currentsmeasured without and with a particle.35 The ion current pulsespredicted by the modeling are in qualitative agreement with ourexperiments. In Figure 6a, a negatively charged particle moveselectrophoretically toward a positively biased electrode; thecurrent increase in the modeled pulse indeed occurs when theparticle leaves the pore. We also modeled electroosmotictransport of a neutral particle through a charged pore, and asexpected, the predicted resistive pulse consists only of thecurrent decrease (Figure 6b).

In addition, we considered a positively charged particletranslocating a negatively charged pore, a situation which wouldbe difficult to test experimentally due to electrostatic attractionand possible adsorption of the particles on the walls. Apositively charged particle would move in the oppositedirection compared to the transport of a negative particle(Figure 6c). Changing directions corresponds to swapping ofthe position of the blockage and enhancement peak. As a result,an exit of a positively charged particle is still accompanied bycurrent increase. The modeling suggests the charge state of thetranslocating particles is critical for the shape of resistive pulses.In order to further corroborate the finding that the positive

peak in resistive pulses is present independently of the chargedstate of the pore (as long as the particles are charged), wemodeled the ion current pulse of a charged particle in a neutralchannel (Figure 6d). Similar to what was obtained in theexperiments (Figure 4b), the modeled pulse contained bothnegative and positive parts. Finally, we “turned off” any possiblesolvent movement including electroosmosis by solving onlyPNP (Figure 6e) and not the full set of PNP−NS. The currentincrease was still present in the predicted pulse, although theamplitude of the current change was smaller than in the casewhen PNP and NS were solved together. This result points to afinite influence of the concentration polarization on the shapeof resistive pulses.The modeling presented in the manuscript was performed

for a pore whose shape is cylindrical. However, the pores usedin the experiments are characterized by an undulating diameter.Thus, one can expect modulation of ionic concentrations at theboundaries between segments of different opening diameter.We wanted to check whether the possible modulations could

Figure 6. Relative current change, χ, when a 220 nm particle is passing through a 510 nm diameter pore. (a−d) Results of numerical modeling basedon coupled PNP−NS equations; 0.1 V is applied across the membrane, with the right-hand side being at the positive potential. (a) Both the particleand the pore walls carry negative surface charge, and the particle moves from left to right. (b) Particle is uncharged; thus, it passes electroosmoticallyfrom right to left. (c) Particle and channel have opposite charges positive and negative, respectively. Particle travels from right to left, and the peakpositions are swapped. (d) Negative particle passes electrophoretically through a neutral pore (left to right). (e) Same as in a but modeled by PNPonly (no solvent movement, particle travels left to right). All simulations were performed in 10 mM KCl as the background electrolyte.



affect the particle sizing using the formula describing theresistive-pulse depth7,10,37

ρπ

− = ⎜ ⎟⎛⎝

⎞⎠R R

dD

SdD

4particle empty

3

4 (1)

where Rparticle and Rempty are resistances of a pore with a particleand an empty pore, respectively, and S is a factor that dependson the ratio of particle diameter, d, to pore diameter, D, whichcan be approximated by7,10,37

= −−

⎜ ⎟ ⎜ ⎟⎛⎝

⎞⎠

⎡⎣⎢

⎛⎝

⎞⎠

⎤⎦⎥S

dD

dD

1 0.83 1

(2)

Using the maximum current decrease to determine Rparticle, eq 1relates the minimum pore diameter with the particle size.Equation 1 was used before to analyze particle passage throughpores characterized by undulating opening diameter.7

For each pore, the diameter of the narrowest constrictionzone was determined from the maximum current decreasemeasured with 280 nm particles at 400 mM KCl; at theseconditions the modulation of ionic concentrations in the poreand at the pore entrances is expected to be the weakest. Usingthis constriction size, we calculated the effective particlediameter based on recordings at various KCl concentrations.A summary of experiments performed with 4 different pores isshown in Figure 7. Collected data indicate the size of the

particles can be reliably determined when the resistive-pulseexperiments are performed in KCl concentrations of 100 mMand higher. The largest discrepancy in the effective particlediameter, as determined by the resistive-pulse approach anddynamic light scattering, was recorded for the lowest examinedKCl concentration of 10 mM for 410 and 470 nm particles.Particles 410 nm in diameter appear smaller in lowerconcentrations since the additional ions they bring to thepore partly offset the pore occlusion. Surprisingly, the 470 nmparticles have a larger effective size in 10 mM KCl compared tothe resistive-pulse recordings in higher ionic strengths. We donot have an explanation for that yet.

IV. CONCLUSIONSIn this article we report a series of experiments which wereaimed at understanding how the concentration of thebackground electrolyte influences the shape of resistive pulsesobtained with polystyrene particles. We identified conditionswhen the exit of an electrophoretic passage of particles througha pore is accompanied by a current increase above the baselinelevel. In low concentrations and large ratios of the particle andpore diameters, the amplitude of the current increase exceededthe amplitude of the current decrease.Resistive pulses observed at low concentrations of KCl affect

the ability to measure size of the particles correctly if theparticles are charged.The article presents rather surprising results thata surface

charge of several hundred nanometer hard sphere particles canmodulate ionic concentrations at the pore entrance. This effectwas observed before at the nanoscale in conditions at which thesize of the system was comparable to the thickness of theelectrical double layer.19,20,35 Here we show the importance ofthe electrostatics in micrometer size pores.The article also reports resistive-pulse experiments per-

formed in conditions when only electroosmosis or electro-phoresis influenced particle transport. Our results indicate thatin order to understand passage of particles through pores theability to separate different modes of transport might be veryimportant.

■ ASSOCIATED CONTENT*S Supporting InformationReproducibility of resistive pulses recorded in two differentconcentrations. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: 949-824-8290. E-mail: [email protected] Contributions‡These authors contributed equallyAuthor ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSIrradiation with swift heavy ions was performed at the GSIHelmholtzzentrum fur Schwerionenforschung GmbH, Darm-stadt, Germany. This research was supported by the NationalScience Foundation (CHE-1306058) and UC National Lab FeeProgram 12-LF- 236772.

■ REFERENCES(1) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W.Characterization of Individual Polynucleotide Molecules Using aMembrane Channel. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770−13773.(2) Venkatesan, B. M.; Bashir, R. Nanopore Sensors for Nucleic AcidAnalysis. Nat. Nanotechnol. 2011, 6, 616−624.(3) Cherf, G. M.; Lieberman, K. R.; Rashid, H.; Lam, C. E.; Karplus,K.; Akeson, M. Automated Forward and Reverse Ratcheting of DNAin a Nanopore at 5-Å Precision. Nat. Biotechnol. 2012, 30, 344−348.

Figure 7. Effective size of translocating particles as a function of bulkelectrolyte concentration used in resistive-pulse experiments. Datafrom different pores are marked with different colors and types ofsymbol. Pores had the following average opening diameter: pore 1,1330 nm; pore 2, 890 nm; pore 3, 1400 nm; pore 4, 980 nm. Dashedhorizontal lines indicate particle size as determined by light-scatteringapproach.



http://pubs.acs.org

mailto:[email protected]

(4) Manrao, E. A.; Derrington, I. M.; Laszlo, A. H.; Langford, K. W.;Hopper, M. K.; Gillgren, N.; Pavlenok, M.; Niederweis, M.; Gundlach,J. H. Reading DNA at Single-Nucleotide Resolution With a MutantMspA Nanopore and Phi29 DNA Polymerase. Nat. Biotechnol. 2012,30, 349−354.(5) Movileanu, L. Squeezing a Single Polypeptide Through aNanopore. Soft Matter 2008, 4, 925−931.(6) Coulter, W. H. Means for Counting Particles Suspended in aFluid. U.S. Patent 2,656,508, 1953.(7) DeBlois, R. W.; Bean, C. P.; Wesley, R. K. A. ElectrokineticMeasurements with Submicron Particles and Pores by the ResistivePulse Technique. J. Colloid Interface Sci. 1977, 61, 323−335.(8) Berge, L. I.; Feder, J.; Jøssang, T. A Novel Method to StudySingle-Particle Dynamics with Resistive Pulse Technique. Rev. Sci.Instrum. 1989, 60, 2756−2763.(9) DeBlois, R. W.; Bean, C. P. Counting and Sizing of SubmicronParticles by the Resistive Pulse Technique. Rev. Sci. Instrum. 1970, 41,909−916.(10) DeBlois, R. W.; Wesley, R. K. A. Sizes and Concentrations ofSeveral Type C Oncornaviruses and Bacteriophage T2 by theResistive-Pulse Technique. J. Virol. 1977, 23, 227−233.(11) Harms, Z. D.; Mogensen, K. B.; Nunes, P. S.; Zhou, K.;Hildenbrand, B. W.; Mitra, I.; Tan, Z.; Zlotnick, A.; Kutter, J. P.;Jacobson, S. C. Nanofluidic Devices with Two Pores in Series forResistive-Pulse Sensing of Single Virus Capsids. Anal. Chem. 2011, 83,9573−9578.(12) Zhou, K.; Li, L.; Tan, Z.; Zlotnick, A.; Jacobson, S. C.Characterization of Hepatitis B Virus Capsids by Resistive-PulseSensing. J. Am. Chem. Soc. 2011, 133, 1618−1621.(13) Holden, D. A.; Hendrickson, G.; Lyon, L. A.; White, H. S.Resistive Pulse Analysis of Microgel Deformation During NanoporeTranslocation. J. Phys. Chem. C. 2011, 115, 2999−3004.(14) Holden, D. A.; Hendrickson, G. R.; Lan, W.-J.; Lyon, L. A.;White, S. H. Electrical Signature of the Deformation and Dehydrationof Microgels During Translocation Through Nanopores. Soft Matter2011, 7, 8035−8040.(15) Pevarnik, M.; Schiel, M.; Yoshimatsu, K.; Vlassiouk, I.; Kwon, J.S.; Shea, K. J.; Siwy, Z. S. Particle Deformation and ConcentrationPolarization in Electroosmotic Transport of Hydrogels through Pores.ACS Nano 2013, 7, 3720−3728.(16) Chang, H.; Kosari, F.; Andreadakis, G.; Alam, M. A.; Vasmatzis,G.; Bashir, R. DNA-Mediated Fluctuations in Ionic Current throughSilicon Oxide Nanopore Channels. Nano Lett. 2004, 4, 1551−1556.(17) Smeets, R. M. M.; Keyser, U. F.; Krapf, D.; Wu, M.-Y.; Dekker,N. H.; Dekker, C. Salt Dependence of Ion Transport and DNATranslocation through Solid-State Nanopores. Nano Lett. 2005, 6, 89−95.(18) Smeets, R. M. M.; Keyser, U. F.; Dekker, N. H.; Dekker, C.Noise in Solid-State Nanopore. Prod. Natl. Acad. Sci. U.S.A. 2008, 105,417−421.(19) Aksimentiev, A.; Heng, J. B.; Timp, G.; Schulten, K. MicroscopicKinetics of DNA Translocation Through Synthetic Nanopores.Biophys. J. 2004, 87, 2086−2097.(20) Heng, J. B.; Ho, C.; Kim, T.; Timp, R.; Aksimentiev, A.;Grinkova, Y. V.; Sligar, S.; Schulten, K.; Timp, G. Sizing DNA Using aNanometer-Diameter Pore. Biophys. J. 2004, 87, 2905−2911.(21) Fleischer, R. L.; Price, P. B.; Walker, R. M. Nuclear Tracks inSolids: Principles and Applications; University of California Press:Berkeley, CA, 1975.(22) Spohr, R. Methods and Device to Generate a PredeterminedNumber of Ion Tracks. German Patent DE2951376 C2, Sept 15, 1983;U.S. Patent 4369370, 1983.(23) Pevarnik, M.; Healy, K.; Toimil-Molares, M. E.; Morrison, A.;Letant, S. E.; Siwy, Z. S. Polystyrene Particles Reveal PoreSubstructure as They Translocate. ACS Nano 2012, 6, 7295−7302.(24) Muller, S.; Schotz, C.; Picht, O.; Sigle, W.; Kopold, P.; Rauber,M.; Alber, I.; Neumann, R.; Toimil-Molares, M. E. ElectrochemicalSynthesis of Bi1−xSbx Nanowires with Simultaneous Control on Size,

Composition, and Surface Roughness. Cryst. Growth Des. 2012, 12,615−621.(25) Ermakova, L. E.; Sidorova, M. P.; Bezrukova, M. E. Filtrationand Electrokinetic Characteristics of Track Membranes. Colloid J.1998, 52, 705−712.(26) Apel, P.; Korchev, Y. E.; Siwy, Z.; Spohr, R.; Yoshida, M. Diode-Like Single-Ion Track Membrane Prepared by Electro-Stopping. Nucl.Instrum. Methods Phys. Res., Sect. B 2001, 184, 337−346.(27) Vlassiouk, I.; Smirnov, S.; Siwy, Z. S. Ion Selectivity of SingleNanochannels. Nano Lett. 2008, 8, 1978−1985.(28) Lewis, S. R.; Datta, S.; Gui, M.; Coker, E. L.; Huggins, F. E.;Daunert, S.; Bachas, L.; Bhattacharyya, D. Reactive NanostructuredMembranes for Water Purification. Proc. Natl. Acad. Sci. U.S.A. 2011,108, 8577−8582.(29) Lan, W. J.; Holden, D. A.; Zhang, B.; White, H. S. NanoparticleTransport in Conical-Shaped Nanopores. Anal. Chem. 2011, 83,3840−3847.(30) Lan, W. J.; Holden, D. A.; Liu, J.; White, H. S. Pressure-DrivenNanoparticle Transport Across Glass Membranes Containing aConical-Shaped Nanopore. J. Phys. Chem. C 2011, 115, 18445−18452.(31) Lan, W. J.; White, H. S. Diffusional Motion of a ParticleTranslocating Through a Nanopore. ACS Nano 2012, 6, 1757−1765.(32) Krasilnikov, O. V.; Sabirov, R. Z.; Ternovsky, V. I.; Merzliak, P.G.; Muratkhodjaev, J. N. A Simple Method for the Determination ofthe Pore Radius of Ion Channels in Planar Lipid Membranes. FEMSMicrobiol. Immunol. 1992, 105, 93−100.(33) Henrickson, S. E.; DiMarzio, E. A.; Wang, Q.; Stanford, V. M.;Kasianowicz, J. J. Probing Single Nanometer-Scale Pores withPolymeric Rulers. J. Chem. Phys. 2010, 132, 135101 (1−8).(34) Mukaibo, H.; Horne, L. P.; Park, D.; Martin, C. R. Controllingthe Length of Conical Pores Etched in Ion-Tracked Poly(ethyleneterephthalate) Membranes. Small 2009, 5, 2474−2479.(35) Liu, H.; Qian, S.; Bau, H. H. The Effect of TranslocatingCylindrical Particles on the Ionic Current Through a Nanopore.Biophys. J. 2007, 92, 1164−1177.(36) Zangle, T. A.; Mani, A.; Santiago, J. G. Theory and Experimentsof Concentration Polarization and Ion Focusing at Microchannel andNanochannel Interfaces. Chem. Soc. Rev. 2010, 39, 1014−1035.(37) Ito, T.; Sun, L.; Crooks, R. M. Simultaneous Determination ofthe Size and Surface Charge of Individual Nanoparticles Using aCarbon Nanotube-Based Coulter Counter. Anal. Chem. 2003, 75,2399−2406.



Documents

Charged Particles Modulate Local Ionic Concentrations and Cause Formation of Positive Peaks in Resistive-Pulse-Based Detection