In Situ Nanopore Fabrication and SingleMolecule Sensing with Microscale Liquid Contacts Christopher E. Arcadia, Carlos C. Reyes, and Jacob K. Rosenstein* School of Engineering, Brown University, Providence, Rhode Island 02912, United States S Supporting Information *
ABSTRACT: In this article, we introduce a flexible technique for high-throughput solid-state nanopore analysis of single biomolecules. By confining the electrolyte to a micron-scale liquid meniscus at the tip of a glass micropipette, we enable automation and reuse of a single solid-state membrane chip for measurements with hundreds of distinct nanopores per day. In addition to overcoming important experimental bottlenecks, the microscale liquid contact dramatically reduces device capacitance, which is a key limiting factor to the speed and fidelity of solid-state nanopore sensor recordings. KEYWORDS: solid-state nanopore, in situ, meniscus contact, pipette, dielectric breakdown, capacitance, array
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automation, and in situ use of buffer solutions commonly used for nanopore studies. Here, we introduce an experimental approach which enables the formation and measurement of hundreds of in situ fabricated solid-state nanopores per day, at high speed, while at the same time dramatically reducing the device capacitance which often limits measurement quality.
he past several years have seen nanopores emerge as a powerful platform for DNA sequencing, and hardly a week goes by without news of an exciting new sequencing achievement. Long read lengths, real-time output, portability, and low capital costs are driving nanopore sequencing into new applications and real-world environments. Even so, these achievements may prove to be just the leading edge of nanopore technology. A nanopore sensor is a single hole in an insulating membrane that bridges two electrolyte chambers. When an analyte molecule occludes the pore, its presence can be detected by transient changes in ionic conductance. Thus, far, commercial nanopore platforms (such as Oxford Nanopore’s MinION 1) rely on engineered membrane ion channel proteins,2,3 but comparable sensors can also be created in solid-state materials, such as silicon nitride,4−7 graphene,8−10 molybdenum disulfide,11 and hafnium oxide.12 While they are still catching up with their biological counterparts, solid-state nanopore sensors have been demonstrated to detect DNA4,5,8,9,11−16 and RNA,17 distinguish proteins,18 and elicit single-molecule binding kinetics.19 Still, despite high promise, solid-state nanopores have yet to graduate from the laboratory. This is in large part due to practical challenges with solid-state nanopore experiments, which often result in limited signal-to-noise ratios, lowthroughput fabrication, and significant time investments. Most successful solid-state nanopore experiments have used pores drilled with focused electron sources5,20,21 or ion beams,22,23 which are capital intensive methods. More recently, a nanopore fabrication technique was introduced based on the dielectric failure of a thin membrane under high electric fields.7,24 The benefits of this strategy include its low cost, readiness for © 2017 American Chemical Society
RESULTS AND DISCUSSION System Overview. Our experimental system is comprised of a glass micropipette which is filled with electrolyte, held by a motorized positioning stage, and positioned directly above a thin insulating solid-state membrane. This arrangement is illustrated in Figure 1a. The bottom side of the membrane is in contact with a solution reservoir. Both the pipette and reservoir electrolytes are connected to custom sensing electronics via silver/silver-chloride electrodes. To proceed with an experiment, the pipette is slowly lowered until its tip forms a liquid meniscus that contacts a small region of the membrane. Micrographs of the pipette during this approach can be seen in Figure 1b. After establishing contact, a nanopore is fabricated under the small patch of liquid contact by applying a high electric field until the SiN experiences dielectric breakdown. The nanopore is then conditioned and grown using pulsed electric fields, until it reaches a desired size and exhibits a linear I−V curve. Finally, the solid-state Received: March 3, 2017 Accepted: May 9, 2017 Published: May 9, 2017 4907
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Figure 1. System overview. (a) An illustration of the experimental setup depicting a micropipette with its tip forming a liquid contact to a silicon nitride membrane. A nanopore is formed within the meniscus area and used for single-molecule experiments. (See SI for a more detailed diagram.) (b) A series of microscope images capturing the position of the pipette tip as it approaches, contacts, and retracts from the surface. (c) A flow-chart of the sequence of events for conducting automated meniscus contact nanopore experiments.
Figure 2. Pipette approach. (a) Equivalent circuit models of the experimental setup during approach, while in contact, and after a pore has been formed. Typical values are Rpipette ≈ 500 kΩ, Rpore ≈ 100 MΩ, Rreservoir ≈ 100 Ω, Capproach ≈ 35 fF, and Ccontact ≈ 65 fF. (b) Real-time measurements of pipette position and capacitance during the initial approach, showing the lowering of the pipette and the abrupt capacitance jump ΔC that occurs on contact. (c) A zoomed-in rendering of the electric field (gray) and equipotential lines (colored) between the pipette and membrane, as obtained from finite element simulations when the pipette is 9.2 μm from the surface. (d) A comparison of the simulated capacitance approach curve overlaid with the measured capacitance. Inset: Measured data including the capacitance after contact. See SI for model details.
measure dozens or hundreds of nanopores per day on a single solid-state membrane chip. Meniscus Contact. In the first step of each experiment, the pipette is slowly lowered until it establishes a liquid contact with the membrane surface. Other systems that utilize scanned glass pipettes rely on faradaic currents to detect when contact is made.25,26 However, the surfaces used here are electrically insulting and will not support this method. Instead, the meniscus contact is detected by monitoring the mutual
nanopore can be used for single-molecule detection of analyte biomolecules introduced into either the top or bottom electrolyte. When data collection for a pore is complete, the micropipette is retracted and moved laterally to a new position on the membrane, where the process is repeated. An overview of the entire experimental procedure can be seen in Figure 1c. Since each trial wets only a small fraction of the top membrane surface, we can use this automated system to fabricate and 4908
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Figure 3. Array of contacts. (a) A plot showing the recorded locations and size of sequentially obtained contacts on a 13 × 13 grid with a 5 μm pitch. The dots have been scaled to match the measured diameters of contact. (b) An image of the silicon nitride membrane and remaining contact residues after completing 169 automated sequential experiments. (c) A histogram of the capacitance jumps ΔC and inferred contact diameters d from the array.
a diameter of d ≈ 2 μm, for instance, will typically exhibit contact capacitances of ΔC ≈ 30 fF (0.03 pF). During the tip approach phase, capacitances are monitored in real time with a lock-in style technique by applying a periodic voltage waveform to the pipette electrode while recording the induced periodic current in the bottom electrode. The parasitic capacitance contributed by instrument wiring in our current setup is approximately 150 fF, which appears in parallel with the true device capacitance. A baseline measurement is taken before each approach, and when an appropriate capacitance jump (ΔC) is detected, the approach is halted and the system proceeds to nanopore fabrication. Contact Arrays. A valuable feature of the proposed technique is that experiments can be repeated many times per device. To demonstrate this concept, we programmed the system to sequentially perform experiments at locations on a 13 × 13 grid (5 μm spacing) on a single SiN membrane. The average measured capacitance jump was ΔC = 29.04 ± 1.56 fF, which corresponds to a liquid contact diameter of d = 2.442 ± 0.065 μm. Figure 3 shows the recorded positions and sizes of the contacts, along with a micrograph of the salt residues remaining on the membrane at the conclusion of the experiment. Nanopore Fabrication. After establishing a liquid contact, the system proceeds to form a nanopore in the membrane by dielectric breakdown. Dielectric breakdown is a recently introduced method7 for nanopore fabrication which relies on the failure of an insulating material under high electric fields. A key advantage of this technique is that it does not require harsh chemicals, vacuum systems, or complex optics. Most examples24,29,30 of dielectric breakdown nanopore formation have applied a constant voltage prior to breakdown, but our system instead utilizes a custom electronic circuit which sources a constant current. The benefit of this configuration is that it is inherently self-limiting;31 when the pore forms, the conductance increases and the electric field automatically reduces. This allows us to be more aggressive with the applied electric field strength, reducing the formation time while limiting the risk of runaway pore growth. Initially, the applied current flows through tunneling leakage paths in the thin membrane. These tunneling pathways have high transport barriers, hence sustaining a nanoampere-scale current requires considerable voltage. In our experiments, a 10
capacitance between the top and bottom electrolytes. A typical measurement during the approach phase is shown in Figure 2b. The monitored capacitance is quite small, on the order of femto-farads, yet we can observe a clear and abrupt increase in capacitance when the meniscus forms. Before the liquid contact, there is weak capacitive coupling across the air gap between the pipette and the surface. Since we are interested in the eventual contact of the round pipette tip with the membrane, it is tempting to consider the air gap capacitance as a parallel plate capacitance between the tip area and the membrane, or as the capacitance between a conducting disk and plane.27 However, both of these models neglect the pipette sidewalls and thus significantly underestimate the capacitance. Instead, we numerically model the air gap using finite element simulations (COMSOL). The electric field solution, shown in Figure 2c, captures the fringe fields emanating from the side of the pipette. In both the simulated and measured data, we find that as the pipette comes within 10 μm of the surface, the capacitance steadily increases by about 0.04 fF/μm until the separation distance becomes less than the tip diameter. At a separation distance less than the tip diameter, we observe a rapid jump-to-contact, similar to what is seen in scanning electrochemical microscopy systems.28 This jump is apparent in Figure 2d; where, as the 2 μm tip nears the membrane’s surface, the measured capacitance abruptly increases instead of following the smooth rise of the simulated capacitance. After contact, the capacitance can be summarized by the sidewall fringing and meniscus contact components. Since prior to contact the capacitance is dominated by the fringing field term, the sudden change in capacitance after contact (ΔC) is approximately equal to the capacitance due to the meniscus, which is modeled as a simple parallel plate capacitor formed by the circular liquid contact area, the insulating membrane, and the fluid reservoir below: ΔC = ϵrϵo Ah−1, where ϵr,SiN = 7 and h = 10 nm are the relative permittivity and thickness of the membrane, ϵo is the permittivity of vacuum, and A is the contact area of the liquid π meniscus. The observed contact area ( A = 4 d 2 ) correlates well to the diameter of the glass pipette (d), with a correction factor attributable to the wetting angle of the electrolyte. Pipettes with 4909
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Figure 4. Nanopore formation. (a) A typical membrane breakdown curve showing the current stimulus and resulting voltage. An abrupt drop in voltage signifies the creation of a nanopore, after which the stimulus is halted. (b) Overlaid curves from an array of sequential breakdown experiments (N = 169) highlighting the stochastic nature of the time-to-breakdown. (c) A histogram of the time-to-breakdown and its Weibull distribution fit. See SI for distribution details. (d) A Weibull plot of the time-to-breakdown. (e) A gallery of I−V curves associated with each nanopore in the array, as recorded immediately after breakdown. Each I−V uses the same voltage and current scale, ± 500 mV and ±0.1 nA, respectively. (f) A histogram of the initial conductances of the formed nanopores. The conductance of a 0.5 nm cylindrical nanopore is noted for comparison.
field, and pH.24,29 Figure 4b shows a collection of breakdown events recorded sequentially with one pipette on one membrane using the same 10 nA formation recipe. Among these tests, the median time-to-breakdown was 7.7 s. Since breakdown is a weakest-link process, the pore formation times can be modeled with a Weibull distribution,29,33,34 as shown in Figure 4c and d. When the sudden drop in voltage is observed, the current stimulus is ceased and the system proceeds to nanopore conditioning. Immediately after breakdown, the nanopores are quite small. Figure 4e shows the post breakdown I−V curves of the sequentially fabricated pores in the 13 × 13 array. A histogram of the linear conductance fits to these curves is shown in Figure 4f. The peak of the distribution occurs at 76 pS, and more than
nA command can cause the potential to rise from 0 V to 23 V within a few seconds. After the initial charging period, we observe a gradual increase in voltage (less than 300 mV/s), which we attribute to the slower accumulation of charge into traps or defects in the dielectric material. Eventually, some of these traps align to form a dominant localized path of lower resistance for leakage current. Presumably, the resulting joule heating causes the membrane to fail,32,33 forming a nanoscale pore. This abruptly increases the conductance of the membrane, which causes a rapid drop in voltage (>100 V/s). Figure 4a shows a representative breakdown event. The exact mechanism is not well understood and timing of these failures are stochastic, but the time-to-breakdown kinetics have been shown to depend strongly on the dielectric material, electric 4910
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Figure 5. Controlled nanopore growth. (a) A typical curve of the voltage induced by exposing a nanopore to a pulsed current stimulus (53 nA, 200 ms period, 50% duty cycle), showing the voltage reduce as the pore enlarges. Inset: A magnified portion of the overall trace showing one cycle of pulsed current growth. (b) An overlay of growth measurements from the array of sequential experiments (N = 169), during which the stimulus amplitude was varied. For clarity, only the envelopes of the induced voltage pulses are plotted. Inset: A color legend indicating trial number and peak applied current. (c) I−V curves measured from several of the pores after one growth interval. Larger pulsed currents tend to produce pores with higher conductance. (d) A scatter plot of the estimated nanopore diameters after 20 s of growth pulses, plotted against the amplitude of the current stimulus.
recipe was used for each pore and the pulsed growth current was varied between trials from −10 nA to −150 nA. Larger conditioning currents tended to produce larger final conductances. This trend can be seen in Figure 5c where the I−V curves of several of the pores are overlaid. The final diameters for the array of nanopores were estimated by applying the conductance model to the measured I−V curves and are plotted in Figure 5d as a function of stimulus current. The resulting diameters increase by approximately 0.01 nm/nA, but with a nearly 50% variance. For applications requiring subnanometer diameter control, it would be important to choose a relatively cautious growth protocol which proceeds more slowly over multiple growth intervals and automatically stops when a target diameter is reached. An example of this type of iterative procedure is included in the SI. Aside from their size, additional important features of nanopores that can be inferred from their I−V responses are linearity and stability. It is common for breakdown-induced nanopores to initially exhibit a nonlinear response, as can be seen in a few of the postbreakdown I−V curves in Figure 4e. Nonlinear behavior is typically a result of an asymmetric shape or charge distribution. Exposing pores to high-voltage pulses can often improve their linearity37 and can also help to reduce 1/f noise.38 Single-Molecule Detection. Figure 6 shows an example data set in which single 1 kbp dsDNA molecules were detected
70% of the pores exhibit an initial conductance between 30 pS and 180 pS. For comparison, an 0.5 nm-diameter cylindrical pore would be expected to have a conductance of 300 pS. These pores are smaller than is typically desired for nanopore sensing applications, such as in DNA transport studies where the molecule’s hydrodynamic diameter is larger than 1 nm.35 Nanopore Conditioning and Growth. After its initial formation, a solid-state nanopore can be grown to a desired size with further application of high electric fields. This is a distinct physical process from breakdown, but pore growth is empirically voltage-dependent and can be performed iteratively while monitoring its progress. For this procedure we again utilize the custom constant−current circuit used for breakdown. A pulsed current stimulus is applied for a short interval (20 s), while the induced voltage is measured. After the stimulus we perform an I−V sweep and estimate the diameter of the nanopore according to its conductivity G via G = σ (4lπ−1d−2 + d−1)−1, where d is the pore diameter, l = 10 nm is the membrane thickness, σ = 7.1 S/m is the conductivity of the 1 M LiCl solution, and G is the pore conductance.36 Figure 5a shows an example of the induced voltage during one growth interval. The initial current pulses produce a large voltage, but the pore quickly grows and soon arrives at a roughly constant voltage response. A collection of 169 sequentially performed nanopore growth experiments are shown in Figure 5b. For these trials, a single interval growth 4911
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Figure 6. Single molecule detection. (a) Detection of single DNA molecules with a silicon nitride nanopore at the tip of the scanned glass pipette. Transient reductions in current correspond to the passage of single 1 kbp dsDNA molecules from the reservoir (cis) to the pipette (trans) under the influence of an applied electrical potential (Vreference − Vclamp = 150 mV). The signal was digitally filtered to a bandwidth of 100 kHz. (b) The I−V curve of the nanopore. Its equivalent resistance is 52.9 MΩ, which yields a diameter estimate of d = 7.30 nm. (c) Three selected events from this data set, showing two discrete blockage levels which are attributed to unfolded (shallow) and folded (deep) conformations of the DNA molecule. (d) An all-points histogram of the blockage current (ΔI) from a concatenated series of the events. The two highlighted peaks correspond to the discrete levels visible in the current traces.
can improve more advanced techniques like active recapture47 and force spectroscopy.48,49 Early solid-state nanopore experiments were conducted in systems with total capacitances in the hundreds of picofarads. With thoughtful passivation, this value is often reduced to 30−50 pF50−52 and with more aggressive techniques devices as low as 0.69 pF have been demonstrated.53 In contrast, nanopores on the meniscus contact platform routinely have a capacitance on the order of 0.2 pF. We have taken advantage of this system to make meaningful electrochemical impedance spectroscopy (EIS) measurements beyond 10 kHz, by far the widest reported range for single nanopores (see SI). Additionally, these extraordinarily low capacitances may also allow researchers to get the most out of lowcapacitance amplifier designs.54−56 Since meniscus contact nanopores are produced in micropipette-confined liquid contacts, their location is known to within ±1.2 μm (the contact radius). This may be useful for performing nanopore experiments colocated within prefabricated microdevices, electrodes, optical structures, or small-area two-dimensional materials.10,11 If circumstances allow, breakdown nanopore localization can also be achieved by targeted membrane thinning57 or plasmonic heating.58,59 However, the meniscus contact method has an added benefit of enabling interrogation of many locations on a sample. The fact that the top chip surface remains dry may also simplify passivation requirements for metallic contacts. The use of a motorized positioning stage offers the opportunity to perform spatially resolved single molecule studies. Additionally, since the meniscus contact nanopore system shares many properties with scanning ion-conductance60−62 and scanning electrochemical microscopes,25,26,28 it would be feasible to create a multimode system which supports both SICM and SECM recordings as well as solid-state nanopore sensing. Since meniscus-contact nanopores can be formed at specified locations, the technique may prove useful as a means of manufacturing nanopore arrays for later use. We have not yet
with a nanopore grown to a diameter of d = 7.30 nm. The DNA was loaded into the bottom reservoir and the potential between the two reservoirs was held at 150 mV while its current was measured. The electric field draws the negatively charged DNA molecules from the bottom chamber into the nanopore,39 and after detection they diffuse into the micropipette. With an open pore current of 3 nA, transient current blockades were observed at discrete levels of 24% and 48%, corresponding to unfolded and folded conformations of the molecules, respectively, as they pass through the pore. Discussion. Although they represent the ultimate in sensitivity, experimental single-molecule techniques can be frustratingly slow and challenging, and thus strategies which increase experimental throughput are highly valuable. With the meniscus contact nanopore approach, a single researcher can fabricate and measure hundreds of solid-state nanopores per day, which can dramatically accelerate data collection. The small working area, defined by the size of the meniscus at the tip of the glass micropipette, also allows for efficient use of valuable devices, enabling more measurements per chip and more data points per sample. While commercial diagnostics may trend toward parallelized nanopore sensors, such as those based on microwell arrays,40 microfluidic networks41,42 or simultaneous optical detection,43−46 the meniscus-contact configuration offers a valuable compromise between throughput and investment in system complexity. Despite providing a significantly greater throughput than systems commonly used for single nanopore studies, a meniscus contact setup does not require complicated lithography, fluidics, optics, or vacuum systems, and instead only calls for a single patch clamp amplifier and positioning stage. The low capacitance inherent to micron-sized meniscus contacts is a key advantage of this method, as it translates both to lower noise measurements and to faster transient response. Reductions in noise allow for better resolved molecular signals, while a faster transient response enables rapid I−V sweeps and 4912
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ACS Nano investigated this extensively, and typically the membrane chips are disposed after use. However, in a few experiments we recontacted pores after several minutes of exposure to air. These nanopores remained conductive, but tended to exhibit a lower conductance than was initially recorded. This suggests salts or other residues may have collected around the pore, but after appropriate cleaning steps it may be possible to reuse a nanopore array at a later time. Lastly, an interesting consequence of using a micropipette as a trans electrolyte chamber is that analyte molecules will naturally collect in the tip after translocating through the nanopore. It may be possible to selectively deliver these accumulated molecules to other instruments or regions on a sample for further analysis.
Additional details about approach capacitance models, comments on meniscus stability and membrane durability, impedance spectroscopy, a brief overview of Weibull distributions, an iterative pore growth demonstration, and a more detailed diagram of the experimental setup (PDF)
AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. ORCID
Jacob K. Rosenstein: 0000-0001-9791-704X Notes
The authors declare no competing financial interest.
CONCLUSIONS By leveraging the confined liquid contact and precise positioning of a glass micropipette, combined with the rapid in situ nanopore formation enabled by dielectric breakdown, a meniscus contact system provides an exceptionally large degree of freedom in conducting nanopore studies. We have demonstrated the potential of this setup by measuring an array of 169 nanopores on a single suspended solid-state membrane in less than 10 h. The presented system uses many components already found in modern research laboratories, and its increased throughput, automation, and simplicity make it an attractive paradigm for continued advancement of solid-state nanopore sensing technology.
ACKNOWLEDGMENTS We would like to thank Zi Yang for his contribution to the data acquisition system and Rukshan T. Perera for his support in conducting experiments. REFERENCES (1) Jain, M.; Olsen, H. E.; Paten, B.; Akeson, M. The Oxford Nanopore MinION: Delivery of Nanopore Sequencing to the Genomics Community. Genome Biol. 2016, 17, 239−250. (2) Astier, Y.; Braha, O.; Bayley, H. Toward Single Molecule DNA Sequencing: Direct Identification of Ribonucleoside and Deoxyribonucleoside 5′-Monophosphates by Using an Engineered Protein Nanopore Equipped with a Molecular Adapter. J. Am. Chem. Soc. 2006, 128, 1705−1710. (3) Butler, T. Z.; Pavlenok, M.; Derrington, I. M.; Niederweis, M.; Gundlach, J. H. Single-Molecule DNA Detection with an Engineered MspA Protein Nanopore. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 20647−20652. (4) Li, J.; Gershow, M.; Stein, D.; Brandin, E.; Golovchenko, J. A. DNA Molecules and Configurations in a Solid-State Nanopore Microscope. Nat. Mater. 2003, 2, 611−615. (5) Storm, A. J.; Chen, J. H.; Ling, X. S.; Zandbergen, H. W.; Dekker, C. Fabrication of Solid-State Nanopores with Single-Nanometre Precision. Nat. Mater. 2003, 2, 537−540. (6) Kim, M.; Wanunu, M.; Bell, D.; Meller, A. Rapid Fabrication of Uniformly Sized Nanopores and Nanopore Arrays for Parallel DNA Analysis. Adv. Mater. 2006, 18, 3149−3153. (7) Briggs, K.; Kwok, H.; Tabard-Cossa, V. Automated Fabrication of 2-nm Solid-State Nanopores for Nucleic Acid Analysis. Small 2014, 10, 2077−2086. (8) Merchant, C. A.; Healy, K.; Wanunu, M.; Ray, V.; Peterman, N.; Bartel, J.; Fischbein, M. D.; Venta, K.; Luo, Z.; Johnson, A. T. C.; Drndic, M. DNA Translocation through Graphene Nanopores. Nano Lett. 2010, 10, 2915−2921. (9) Schneider, G. F.; Kowalczyk, S. W.; Calado, V. E.; Pandraud, G.; Zandbergen, H. W.; Vandersypen, L. M. K.; Dekker, C. DNA Translocation through Graphene Nanopores. Nano Lett. 2010, 10, 3163−3167. (10) Kuan, A. T.; Lu, B.; Xie, P.; Szalay, T.; Golovchenko, J. A. Electrical Pulse Fabrication of Graphene Nanopores in Electrolyte Solution. Appl. Phys. Lett. 2015, 106, 203109. (11) Feng, J.; Liu, K.; Bulushev, R. D.; Khlybov, S.; Dumcenco, D.; Kis, A.; Radenovic, A. Identification of Single Nucleotides in MoS2 Nanopores. Nat. Nanotechnol. 2015, 10, 1070−1076. (12) Larkin, J.; Henley, R.; Bell, D. C.; Cohen-Karni, T.; Rosenstein, J. K.; Wanunu, M. Slow DNA Transport through Nanopores in Hafnium Oxide Membranes. ACS Nano 2013, 7, 10121−10128. (13) Chen, P.; Gu, J.; Brandin, E.; Kim, Y.-R.; Wang, Q.; Branton, D. Probing Single DNA Molecule Transport Using Fabricated Nanopores. Nano Lett. 2004, 4, 2293−2298.
METHODS Materials. All buffer solutions were prepared using deionized water (Millipore Milli-Q) having a resistivity of 18.2 MΩ·cm at 25 °C. All data presented was measured using 1 M LiCl (99%, Sigma-Aldrich) with pH 8 or pH 10. AgCl/Ag electrodes were made from silver wire (99.9%, Sigma-Aldrich) roughened with 120-grit sandpaper and chlorinated in sodium hypochlorite (Sigma-Aldrich). Glass pipettes were pulled in a Sutter P-2000 laser micropipette puller from borosilicate capillary tubes (BF100−78−10, Sutter) using the following recipe: Heat: 450, Velocity = 30, Filament = 4, Delay = 200, Pull = 0. Typically, tips made this way have inner diameters of about 2 μm and a resistance of around 500 kΩ in 1 M LiCl. The experiments used 100 μm × 100 μm 10-nm SiN membranes that were supported by a 200 μm thick silicon frame (TA301Z, Norcada). Translocation experiments used 1 kbp DNA fragments (SM1671, Fisher) diluted in buffer to a final concentration of 8 nM. The membrane chip was mounted on a custom Teflon fluid cell using a silicone elastomer (Ecoflex 5, Smooth On). Measurements. The data acquisition system was built-in house and includes voltage-clamp and current-clamp circuits with a 6 MSa/s raw sample rate. The waveform for capacitance measurements is produced by a programmable arbitrary waveform generator (33500B, Keysight). Alignment and optical monitoring of experiment progression was performed using a small custom lens-tube microscope with a 10× objective (Nikon) and CMOS camera (DCC1645C, Thorlabs). The micropipette is positioned using a three-axis stepper motor stage (MMP3, MadCityLabs). The entire experimental setup was contained in a large custom Faraday cage on a vibration isolation table (Newport). Data acquisition and processing were performed in MATLAB. The OpenNanopore software package was used to detect translocation events.63 Capacitance simulations were performed in COMSOL.
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S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b01519. 4913
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DOI: 10.1021/acsnano.7b01519 ACS Nano 2017, 11, 4907−4915
