Helium Scanning Transmission Ion Microscopy and Electrical

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Helium Scanning Transmission Ion Microscopy and Electrical Characterization of Glass Nanocapillaries with Reproducible Tip Geometries Ludovit P Zweifel, Ivan Shorubalko, and Roderick Y.H. Lim ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b05754 • Publication Date (Web): 19 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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Helium Scanning Transmission Ion Microscopy and Electrical Characterization of Glass Nanocapillaries with Reproducible Tip Geometries

Ludovit P. Zweifel1, Ivan Shorubalko2 and Roderick Y.H. Lim1 1

2

Biozentrum and the Swiss Nanoscience Institute, University of Basel, Switzerland

Laboratory for Reliability Science and Technology, EMPA, Swiss Federal Laboratories for Materials Science and Technology, Dübendorf, Switzerland

Corresponding Author Roderick Y. H. Lim Biozentrum and the Swiss Nanoscience Institute University of Basel Klingelbergstrasse 70 CH-4056 Basel, Switzerland Phone: +41 61 267 2083 Fax: +41 61 267 2109 E-mail: [email protected] ACS Paragon Plus Environment

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Abstract Nanopores fabricated from glass microcapillaries are used in applications ranging from scanning ion conductance microscopy to single molecule detection. Still, evaluating the nanocapillary tip by a non-invasive means remains challenging. For instance, electron microscopy characterization techniques can charge, heat and contaminate the glass surface, and typically require conductive coatings that influence the final tip geometry. Per contra, electrical characterization by the means of ion current through the capillary lumen provides only indirect geometrical details of the tips. Here, we show that Helium scanning transmission ion microscopy provides a non-destructive and precise determination of glass nanocapillary tip geometries. This enables the reproducible fabrication of axially asymmetric blunt, bullet and hourglass-shaped tips with opening diameters from 20 nm to 400 nm by laser-assisted pulling. Accordingly, this allows for an evaluation of how tip shape, pore diameter and opening angle impacts on ionic current rectification behavior and the translocation of single molecules. Our analysis shows that current drops and translocation dwell times are dominated by the pore diameter and opening angles regardless of nanocapillary tip shape.

Keywords: Helium Ion Microscope, Glass Micropipette, Nanopore Geometry, Ion Current Rectification, Plasma Cleaning, Protein Translocation, Single Molecule Detection

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Solid-state nanopores are useful biophysical tools for the label-free detection of single molecules in aqueous solutions.1, 2 Similar to the macroscopic Coulter counter technique, this works by connecting two salt solution-containing reservoirs via a single nanopore, across from which an electrical potential is applied. This establishes an ionic current that is primarily restricted by the small dimensions of the connecting pore. The ionic current then drops upon partial blockage of the pore as biomolecules are electrokinetically-driven from one reservoir to the other. In this manner, nucleic acids3 and proteins4 are identified by monitoring the “drop” length, amplitude and frequency. Nanopores of differing geometries can be produced within membranes of different materials by single ion-track etching, ion beam sculpting as well as focused ion or electron beam milling. Single ion-track etched PET films cover the widest range of geometries such as conical,5 cylindrical,6 hourglass,7 cigar8 and bullet-like9 shaped nanopores. On the other hand, ion beam sculpting of silicon nitride membranes leads to bowl shapes,10 while nanopores directly milled by a focused ion11 or electron12 beam take on hourglass shapes. Arguably, these fabrication methods can be technically demanding and time consuming. Thus, a current goal is to simplify and scale-up device production.13 Glass nanocapillaries (GNCs) fabricated by laser assisted thermal pulling14 of glass microcapillaries have emerged as a simple and low cost alternative for single molecule detection. Depending on the glass type and pulling parameters, GNC tips with opening diameters from micrometers down to nanometers15 and opening angles from 5° to 66° have been achieved.16 GNCs have been used to detect λ-phage DNA,14 single protein molecules17 and proteins via DNA carriers.18 GNCs were further able to discriminate between sizes of different proteins.19 Still, it remains challenging to characterize GNCs in a non-invasive manner due to the insulating nature of glass. For instance, the use of scanning electron microscopy (SEM) leads to charging effects and hydrocarbon contamination of the glass surface20 while heating the GNC changes its tip geometry.21 Notably, all these effects can greatly influence the current-to-voltage (I-V) characteristics and the detection of molecular translo-


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cation. Although metal coats can be used for SEM, their presence influences the tip geometry and limits surface functionalization protocols. Here, we show that scanning transmission ion microscopy with Helium ions22 (HeSTIM) provides a non-invasive means to resolve GNCs with unparalleled accuracy. This is achieved using very low He beam currents, which minimizes unfavorable heating effects and omits the need of metal coatings. Although some contamination may occur due to secondary electron release following He ion bombardment, plasma cleaning effectively removes this contamination such that the GNCs are not altered after HeSTIM imaging. HeSTIM further facilitates the determination of mild plasma conditions that preserve the integrity of the geometrical structure. By quantifying the inner opening angle, outer opening angle, opening diameter and overall shape of each GNC by HeSTIM, our work reveals the reproducible fabrication of hourglass, bullet-like and blunt-ended GNC tip geometries by laser assisted pulling. Subsequent I-V measurements validate the structural integrity and consistency between GNCs fabricated in a similar fashion based on their reproducible conductance and ion current rectification (ICR) behavior. Comparable I-V characteristics between imaged and non-imaged GNCs from the same pulling procedures further prove that the fabrication is reproducible and that HeSTIM imaging is non-destructive. Finally, molecular transport experiments show how differences in pore diameter and opening angle manifest in changes to current drops and dwell times. This opens a route towards the controlled functionalization of well-defined GNCs bearing different geometries.


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Results and Discussion HeSTIM characterization. GNC geometries were determined by the use of a He focused ion beam microscope. Briefly, a focused He ion beam scans over a sample similar to a traditional SEM23 (Fig. 1A and 1B) and generates an image by detecting secondary electrons (SE) that are excited by impinging He ions. However, in contrast to imaging techniques based on electrons (SEM, TEM and STEM) or Gallium ions (Gallium FIB), He ions enable the imaging of non-coated, insulating GNCs mainly due to their larger mass than electrons and lower mass than Gallium ions. First, beam currents as low as 0.1 pA are suitable for image generation since SE are mostly excited by the incoming He ions via kinetic emission, leading to an approximately 70 fold higher SE yield δ than electrons (δe≈ 0.1 and δHe≈7).24 The low beam current at the typical acceleration voltages of 25 and 30 kV used throughout this study ensured non-destructive image generation. Higher He beam currents would destroy the GNCs through significant sputtering and heating effects during image acquisition. Second, given the small He ion scattering cross section, He ion scattering with the sample nuclei is minimal, i.e. He ions are less likely to collide with sample atoms. This results in a considerable penetration depth before the beam diverges.25 Consequently, SEs from the GNCs are excited within a narrow conical interaction volume over hundreds of nanometers (unlike the bell-shaped interaction volume for electrons or Gallium ions).24 Third, the de Broglie wavelength is orders of magnitudes smaller than electrons, which allows for probing sizes as small as 0.25 nm.26 Since the escape depth of SE excited by electronic collisions with He ions is restricted to a few nanometers, the small probing size and low beam divergence predominantly defines the superior spatial resolution over electron and Gallium based imaging techniques. At different positions on the tip, He ions encounter a tapered glass surface of varying thickness (Fig. 1B and 1C). The number of He ions transmitting the GNCs therefore depends on the actual position the beam impinges on the tip as the number of nuclear collisions and hence the energy loss is given by the encountered wall thickness. Both the escaping SE and the incoming He ions contribute positive charge to the sample. However, given the insulating nature of the GNCs these contribu5 ACS Paragon Plus Environment

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tions are highly localized. Regarding their large penetration depth, non-transmitted He ions are mainly buried and neutralized deep within the GNC wall, while the accumulation of remaining positive holes due to SE emission is restricted to the escape depth near the surface. Because the interaction volume within the escape depth increases with the angle between the local surface normal and the incident beam, charge accumulation per incoming ion increases against the edges of the GNC. The transmitted component of the beam travels further and excites additional SE from the Aluminum background below the GNC tip. Those SE are attracted either towards the secondary electron detector or towards the positively charged GNC tip, thereby neutralizing the accumulated surface charge and suppressing major charging effects (Fig. 1C). Considering the low primary He ion beam current of 0.1-0.5 pA, charges buried in the wall can readily dissipate and have a negligible effect on the transmission of impinging He ions. Assuming full neutralization of the surface charge on the GNC, the total amount of detected SE is equal to the SE emitted from the Aluminum background, whereas δGlass 0), since RR + RP ≈ RP. Current mean values at a given voltage are therefore obtained from averaging over stable currenttime traces (t > 0). The stabilized current between the two reservoirs at a given V and ionic strength is then mainly defined by RP and hence dominated by the GNC pore diameter dP. For pores exhibiting Ohmic behavior, an approximation of RP solely based on the geometry of the GNCs can be described by the relationship29

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(* $, 12

(3)

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where σ is the solution conductivity, l is the length of the GNC base, Θ is the full inner opening angle and dB is the diameter of the base (200 µm). Nevertheless, it is the tip shape itself, which determines the electrical field line distribution and thus the ion transport properties at least as much as the pore size.30, 31 Accordingly, the I-V response of a GNC may provide a further means to interrogate the different tips such as blunt, bullet and hourglass shaped GNCs. In the present case however, exclusive geometric considerations to deduce the ionic current from equation (3) are not straightforward due to the departure of the I-V curve from Ohmic behavior for some of the GNCs (Fig. 4B). The origin of this ion current rectification (ICR) is uncertain and is being extensively debated.5, 15, 32 General agreement exists that ICR is inherent to charged axially asymmetric nanopores (either geometrically or in surface charge distribution) when the pore radius is of the order of the Debye screening length λD.33 Indeed, the GNCs shown in Figure 4B fall into this category as their tip geometries are asymmetric along their axes and their glass surfaces bear a fixed surface charge of -0.02 C/m2 at pH 7.2.34 To understand the origin of ICR, finite-element simulations based on Poisson-Nernst-Planck and Navier-Stokes equations have identified a voltage-dependent conductivity (σ → σ (V)) in the vicinity of the GNC pore mediating ICR.33, 35 As a consequence, the conductivity along the GNC axis shows maxima and minima as opposed to the bulk solution (σ → σ (x,V)). A maximum of σ (x,V) located next to the GNC pore opening will thus result in a high current state and a minimum in a low current state. The dependence of minima and maxima positions on the polarity of the applied voltage then leads to the fact that |I(+V)| ≠ |I(-V)|.


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Figure 4. (A) Schematic of a PDMS reservoir with an integrated GNC. The cis reservoir is kept at ground while the potential of the trans Ag/AgCl electrode is swept. The HeSTIM image shows a typical blunt ended tip. Dashed lines are guides to the eye that outline the inner cavity. For a transverse applied potential this system is best described by an equivalent model circuit with four main components: Rtrans, CP, RP and Rcis (see main text). (B) Comparison of I-V responses for bullet (N = 7, program 2), blunt (N = 5, program 3) and hourglass (N = 8, program 5) shaped GNCs. See Table 1 for details. In all cases, the high voltage regime at both polarities (-500 to -250 and 250 to 500 mV respectively) can be fitted linearly to obtain a lower and upper conductance limit (see Table 2). (C) ICR ratios ξ for the GNCs from (B). Deviations at a given voltage below the dashed line correspond to negative rectification (see main text). (D) Comparison of I-V responses between ensembles of hourglass shaped (N = 8, program 5 and N = 7, program 6) GNCs. Inset: N = 8, program 7. (E) Increasing pore size reduces ICR for hourglass GNCs.

Comparison of HeSTIM vs. Electrical Characterization. Although the I-V curves in Figure 4B show a moderate negative rectification |I(+V)| < |I(-V)| in all three tip shapes, notice that high voltage regimes (-500 to -250 and 250 to 500 mV respectively) exhibit close to Ohmic behavior. Using 13 ACS Paragon Plus Environment

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the properties outlined in Table 1, linear fits to these ranges allow estimates of the local voltagedependent conductivity in the vicinity of the GNC pores from pure geometrical considerations by equation (3), as summarized in Table 2. Table 2. Conductance values and calculated conductivity differences for each GNC shape shown in Figure 4B. Note: SE of the linear fits are all below ± 40 pS and are not considered. G500mV [nS]

G-500mV [nS]

∆ σ [mSm-1]

Blunt (program 3)

1.18

2.17

721

Bullet (program 2)

2.24

3.75

503

Hourglass (program 5)

3.81

4.42

235

GNC type

The rectification for GNCs shown in Figure 4B is moderate due to the large pore radii (from ~10 nm to ~20 nm) compared to 3 at 150 mM NaCl (~ 1 nm). To quantify differences in rectification, we define a weighted rectification ratio given as

4 =

|678 9|

|8|

|6:8 9| ; =

!?"

(5)

$#%

with l being the length of the taper and G the conductance, assuming a conical approximation from tip to base. This enables a comparison between the pore diameters deduced by HeSTIM imaging in Table 1 and the calculated diameters from the related conductance values, which are summarized in Table 3. Table 3. Calculated pore diameters dp for the different hourglass GNCs shown in Figure 4D. SE of the linear fits are all below ± 40 pS and are not considered. GNC type G [nS] l [mm] dp [nm] Program 5*

4.09

2.6

42

Program 6

7.02

2

56

Program 7

57.62

1.5

344

Note: Conductance value G of Program 5* is the average of G500mV and G-500mV from Table 2

Interestingly, the calculated values correspond approximately to the measured pore diameters (compare with Table 1). Moreover, it should be emphasized that the narrow distribution of the I-V curves supports the reproducibility for the different GNC types as found by HeSTIM imaging.

Single Molecule Translocation. Next, we measured the translocation of bovine serum albumin (BSA) through different GNCs. The baseline in Figure 5A shows a representative current-time trace 15 ACS Paragon Plus Environment

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IB(t) for an hourglass GNC (program 5) at 500 mV for 10 s, mainly fluctuating around its mean value due to intrinsic low-frequency ionic current noise. Upon application of 1 µM of BSA to the cis reservoir, current drops emerge from transient ion blockages ∆I(t) due to BSA translocation. Meanwhile, we did not observe any long-lived baseline current reduction that might be suggestive of BSA adsorption to the GNC walls for the duration of data collection.38 The translocation of the negatively charged BSA molecules (hydrodynamic diameter dhyd = 7 nm measured from dynamic light scattering, data not shown; and theoretical isoelectric point pI = 5.6) towards the positive potential (Φtrans > 0) indicates that electrophoretic transport dominates over electro-osmosis. As usual,17 single translocation events are characterized by their duration and mean current drop amplitude (Fig. 5B) and recognized as such when |∆I(t)| > 5 IB,rms , with IB,rms being the root-mean-square of the baseline current. The entire translocation process thereby consists of two asymmetric phases, the capture of the proteins on the cis side and the actual translocation through the GNC pore towards the trans side. The event duration is therefore a composition of the capture time (cis) and the residential time of the BSA molecules in the sensing volume (trans) while the maximal current drop depends on the ratio between dP and dhyd. Particle passage analysis of well-defined molecules (BSA) at a common set of parameters (V = 500 mV, 150 mM NaCl pH 7.2, BW = 10 kHz, sample rate = 100 kHz, 1 µM BSA) may thus open an additional way to investigate the GNC geometries. Similar studies to characterize nanopore geometries by their particle translocation properties have been performed recently for symmetric membrane embedded systems.39, 40 The current drops in Figures 5A and 5B can be directly converted into changes of conductance ∆G considering near-Ohmic behavior around 500 mV. To compare between GNCs with different IB(t), mean conductance changes of translocation events are normalized by the respective baseline conductance, / = /. The event times are defined as the duration of the first deviation from the baseline (|∆I(t)| > 0) until its recovery (|∆I(t)| → 0) and are directly comparable between pores. Individual BSA translocation events from within a 1 min trace are then represented as single points in a scatter plot as presented in Figure 5C for bullet, blunt and hourglass GNCs. The 16 ACS Paragon Plus Environment

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mean normalized current drops are 0.3 %, 3.9 %, 1.3 % and 1.2 % and the mean event durations 0.32 ms, 2.05 ms, 1.64 ms and 1.93 ms respectively. Log-normal fits of the event duration distributions (Fig. S10) and normal fits of the current drop distributions (Fig. S11 and Table S3) are in good agreement with previous reports of BSA translocation through GNCs17, 19 (Table S4). Yet, considerable differences in event frequency might stem from different shape effects (Fig. 5D) since all other parameters between the measurements remained unchanged. Regardless, we do observe large device-to-device variations in the event rates between GNCs fabricated from the same pulling parameters. The reason for this variation is not understood and awaits further study.

Figure 5. (A) Ion transport through an hourglass geometry leads to a current-time trace that fluctuates due to ionic current noise (baseline) while BSA translocation manifests in temporal current drops. (B) Zoom of a single translocation event as characterized by the event duration and the associated current drop. The event asymmetry around the maximal current drop may be due to the geo17 ACS Paragon Plus Environment

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metrical asymmetry of the tip. (C) Blunt-ended (red: program 3, n = 781; and green: program 4, n = 68), bullet-like (blue: program 2, n = 43), and hourglass (black: program 5, n = 2687) GNCs. Each point in the scatter plots corresponds to a single translocation event. All plots are obtained at 500 mV with 1 µM BSA applied to the cis chamber. (D) Pore geometry versus event distribution corresponding to the colors in (C). Smaller pore diameters (25 nm) lead to bigger current drops (4 %), smaller angles (4 °) to longer event durations (2 ms).

Conclusion Blunt-ended, bullet-like and hourglass-shaped GNC tips have been reproducibly fabricated by laser-assisted pulling. Non-destructive characterization using HeSTIM is evident by the analysis of the electrical conductance and their corresponding ICR properties. Variations in electrical transport characteristics largely originate from the physical shape of the tip, since all GNCs were fabricated from the same material and by the same technique. Nevertheless, we observed the least influence of ICR and highest capture rates for hourglass tips, suggesting their utility in biomimetic and selective nanochannels.41


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Methods GNC Fabrication. Asymmetrical GNCs were produced by laser-assisted capillary pulling (Sutter instruments P-2000 B). Similar quartz capillaries (Hilgenberg; O.D. 500 µm, I.D. 200 µm with inner filament) were heated with the same laser spot size and pulled with a constant load. The puller exposes the capillaries to a tunable CO2 (up to 10 W) IR laser (approx. λ = 10 µm) beam with a minimal spot size of 0.1 mm. Quartz glass absorbs firmly in the infrared and is heated up to the melting point (approx. 1700 °C) upon irradiation with the laser. Pulling and heating at the same time then leads to a shrinking of the exposed region of the capillary. A final pull separates the thinned middle region resulting in two GNCs. GNCs specified in Table S1 are on average reproducible up to 79% (see Table S2).

HeSTIM Imaging. Imaging was performed with a He ion microscope (OrionPlus, Carl Zeiss Microscopy GmbH). The GNCs were mounted more than 1 cm away from an Aluminum background on a custom-made sample holder allowing for scan angles from 0° (central capillary axis perpendicular to the beam) to 90° (central capillary axis parallel to the beam). Prior to the transfer of the GNCs to the main chamber a plasma cleaning procedure was performed (10W air plasma for 3 min at 0.8 mbar). The focused He ion beam was then rastered across the tip endings of the GNCs (Fig. 1B), where the wall thickness is either 100nm (field of view = 100 µm). Comparability between images was guaranteed with a constant image resolution of 0.94 nm2 per pixel (Fig. S12-S14). Standard acquisition parameters were ~8 mm working distance, 0.1 - 0.5 pA beam current, 25 and 30 kV acceleration voltage, a dwell time of 0.5 or 1 µs and 16 or 32 line averaging. Image processing was performed with ImageJ (ImageJ 1.48v) and by a custom written MATLAB code (MATLAB r2012b, The MathWorks).

Electrical Characterization. Individual GNCs were mounted as a sole connection between two reservoirs filled with filtered (0.22 um, TRP) PBS (pH 7.2, 150 mM NaCl, Gibko) in homemade 19 ACS Paragon Plus Environment

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PDMS cells (Fig. 4A). Wettability was increased by hydrophilizing the glass surface in an O2plasma (13.56 MHz, 12.5 W, 5 sccm, 5 min, Femto, DienerElectronics). The O2-plasma conditions have a strong influence on the I-V characteristics therefore the geometrical integrity of each GNC structure was preserved by a choice of mild plasma conditions (Fig. S15). A current amplifier (Axopatch 200B, Axon Instruments) was used to apply potentials (-500 mV to 500 mV) between the reservoirs and to measure the ionic current through the GNC. Custom-made Ag/AgCl electrodes were placed with the ground electrode in the reservoir with the tip of the GNC (cis) and with the reference electrode setting the potential in the reservoir with the base of the GNC (trans). Currents were low pass filtered (internal 4-pole Bessel) at 10 kHz or 100 kHz and sampled at 100 kHz or 1 MHz with a NI-PCIe-6251 card (National Instruments), respectively. I-V responses of individual GNCs from program 1 to 7 are shown in Figures S16 to S18. A custom written LabView (LabView v13.0, National Instruments) program was used to record and analyze I-V curves as well as translocation events of BSA (>98 %, lyophilized, Sigma-Aldrich) (Figs. 4 and 5). All graphs were produced in OriginPro (OriginPro v9.1.0, OriginLab Corp.) and all schemes designed in Adobe Illustrator (Adobe Illustrator CS5 v15.0.2, Adobe Systems Inc.)

Conflict of Interest. The authors declare no competing financial interest.

Acknowledgment. L.P.Z. and R.Y.H.L acknowledge support from the Swiss National Science Foundation grant 31003A_1466124, the Biozentrum and the Swiss Nanoscience Institute. I.S. thanks the Swiss National Science Foundation for support in equipment procurement (REquip 206021_133823). L.P.Z. and R.Y.H.L. are grateful to U. Keyser and his group for helpful discussions.


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Supporting Information. Summary of the fabrication parameters for laser-assisted pulling, secondary electron intensity versus altitude angle dependence, various transmission images of different GNCs fabricated with the same parameters, influence of oxygen plasma on I-V characteristics and single I-V curves of GNCs. This material is available free of charge via the internet at http://pubs.acs.org.


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Helium Scanning Transmission Ion Microscopy and Electrical

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