Electrically facilitated translocations of proteins through silicon nitride nanopores:

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1 SUPPORTING INFORMATION Electrically facilitated translocations of proteins through silicon nitride nanopores: conjoint and competitive action of diffusion, electrophoresis, and electroosmosis Matthias Firnkes, Daniel Pedone, Jelena Knezevic, Markus Döblinger, and Ulrich Rant,* Walter Schottky Institute, Technische Universitaet Munich, Am Coulombwall 3, Garching and Chemistry Department, Ludwig Maximilians Universitaet, Butenandtstrasse 11, Munich Materials and Methods Protein hydrodynamic diameter and zeta potential. Avidin and streptavidin were purchased from Thermo Fisher and Rockland Immunochemicals, respectively, dissolved in DI-water and stored in aliquots at -20 C if not used immediately. For the measurements without KCl, 10 mm Tris buffer was used (ph 8). Other ph values were adjusted by titrating NaOH or HCl to the desired value. For the measurements with 50 mm KCl the solution ph was adjusted by adding HCl (ph 2), sodium citrate (ph 4), sodium acetate (ph 6), Tris (ph 8) or sodium carbonate/bicarbonate (ph 10) as buffers in concentrations of 10 mm. The hydrodynamic diameter and zeta potential were measured by dynamic light scattering with a Zetasizer Nano instrument (Malvern Ltd, UK), employing laser Doppler velocimetry combined with phase analysis light scattering 1. Zeta potential measurement cells from Malvern were used; the protein concentration was 1.25 mg/ml and all solutions were filtered (100 nm pore size) prior to injection into the measurement cells. All samples were equilibrated for 15 min at 25 C before starting the measurement. The obtained intensity auto-correlation function was fitted with a number-

2 weighted distribution of exponential decays. The zeta potential was determined using the monomodal measurement mode with a maximum of 100 runs. Depending on the solution conductivity, the applied potential was adjusted to keep the current < 1 ma in order to avoid Joule heating of the sample (150 V without KCl, V for [KCl] > 30 mm). On the other hand, the necessity to apply low voltages impaired the signal-to-noise ratio which in turn limited the possibility to conduct measurements in high conductivity (high salinity) solutions. Moreover, the electrode surfaces corroded quickly in high salinity solutions, which required a frequent exchange of the measurement cells. To test the stability of avidin in high salinity solutions, we performed DLS experiments in 400 mm KCl solutions (ph 6). SI Figure 1 shows the auto-correlation function as well as the intensity and number distributions. A distinct scattering signal from monomeric protein (Dh = 6.2 ±1.2 nm) was found, indicating that avidin molecules do not coalesce in the presence of 400 mm KCl. SI Figure 1: Measurement of the hydrodynamic diameter of avidin in 400 mm KCl by dynamic light scattering. The correlation function is fitted with a distribution of exponential decays (red line). Inset: Number (red line) and intensity (black line) weighted size distribution.

3 Nanopore fabrication. DuraSiN chips (3x3 mm²) featuring a 30 nm thick Si 3 N 4 membrane (50x50 µm²) were purchased from Protochips Inc. (USA). Nanopores with a diameter of 20 nm were milled into the Si 3 N 4 membrane with a 300 kev energy electron beam using a FEI Titan transmission electron microscope 2 in nanoprobe mode with an extraction voltage of 4500 V and a convergence angle of 9.2 mrad. The holes were milled by moving the chip manually under the highly focused electron beam in circular paths until the desired diameter was reached. SI Figure 2: Polycarbonate measurement chamber. The chip was cleaned in piranha solution for 15 minutes prior to coating the membrane side with Polydimethylsiloxane (PDMS). The PDMS was painted manually around the Si 3 N 4 membrane (leaving a window of roughly 60 µm open) in order to electrically passivate the chip and reduce electrical noise 3. After curing the PDMS at 120 C for 1 hour, the chip was exposed to oxygen

4 plasma (Tepla 100-E Plasma System) for 30 seconds on each side, immediately afterwards put in ethanol, and thereafter stored in 400 mm KCl solution before mounting it in the measurement chamber. The polycarbonate measurement chamber (SI Figure 2) consists of two symmetrical parts in between which the chip is placed vertically between two silicon gaskets (0.7 mm inner diameter). The 400 µl reservoirs are sealed; static pressures up to 3 bars could be applied by a luer-lock connector at the end of the reservoir. Nanopore zeta potential measurement. An EPC 8 patch clamp amplifier and a LIH1600 DAQ interface (both from HEKA, Germany) were used for the nanopore zeta potential and protein translocation measurements. The EPC 8 four-pole Bessel filter was set to 3 khz and the sampling rate was 20 khz. The pressure was set by connecting pressurized air via a pressurereduction valve to the measurement chamber and varied between 1 and 2.5 bar in steps of 0.5 bar. For each pressure step, 4 cycles were performed, where the applied pressure was switched between 0 and designated value. Each of this 8 runs lasted for 2 seconds, and the measured streaming potential was recorded with the patch clamp amplifier in current clamp mode. Each run was averaged and the difference of the streaming potential for the two runs in each cycle was calculated. These four potential values were again averaged for each pressure and the du/dp dependence was obtained for each of the four pressure values. SI Figure 3 (a) shows raw data of a streaming potential measurement in 400 mm KCl (ph 9) for increasing and decreasing pressure. SI Figure 3 (b) shows a linear fit to the mean values of the streaming potential, from which the zeta potential of the nanopore was analyzed using Eq. 3.

5 SI Figure 3. (a) Representative streaming potential difference measurements for varying pressure in 400 mm KCl solution (ph 9). The red line depicts average values. b) Dependence of the streaming potential difference on the applied pressure, values taken from (a). Black squares indicate increasing pressure, red circles indicate decreasing pressure. The red solid line is a linear fit to the data. The zeta potential of the SiN nanopore depends on the solution salt concentration. SI Figure 4 shows a dilution series for which the solution salinity was decreased by subsequently adding DI water to a solution which initially contained 400 mm KCl and 20 mm Tris (ph 8.2). The magnitude of the zeta potential increases with decreasing KCl concentration, which is in accordance with other reports 4-6 and theoretical expectations 7.

6 SI Figure 4: Dependence of the nanopore zeta potential on the solution salt concentration. Protein translocation measurements. Trans-pore currents were recorded using the EPC 8 amplifier with the Bessel-filter set to 10 khz and a sampling rate of 200 khz. Protein was added in 40 nm concentrations to the cis chamber and voltages of +/- 150 mv were applied across the pore by immersed chlorinated Ag wires. Solution ph was adjusted with the same buffers that were used for the protein zeta potential experiments in 50 mm KCl, i.e. HCl (ph 2), sodium citrate (ph 4), sodium acetate (ph 6), Tris (ph 8), sodium carbonate/bicarbonate (ph 10). The buffer concentration was 50 mm for translocation experiments. The stability of the ph value was checked before and after translocation experiments inside the measurement chamber with a Biotrode (Metrohm GmbH, Germany). Traces which showed signs of transient pore clogging (events lasting for several hundred microseconds) were rejected and not considered for analysis. Pulses were analyzed with respect to their height and width by a self-written Matlab program which has been previously described 8.

7 Translocation of streptavidin. We observed EO driven translocations also for streptavidin (SA) at ph 4 and ph 6. At ph 4, we measured a positive zeta potential of SA, ζ SA (ph 4, 0 mm KCl) +20 mv. For a positive bias of +150 mv applied to the trans chamber, we detected an event rate of 36 s -1, whereas a low event rate of 0.7 s -1 for a bias of -150 mv was observed. At ph 6, SA is very close to its isoelectric point (ζ SA 0), yet still a considerable event rate of ~12 s -1 for -150 mv (0.5 s -1 for +150 mv) was observed. Thus, for ph 4 and 6, EO is the dominating transport mechanism. For ph 8, SA features a negative zeta potential of ζ SA (ph 4, 0 mm KCl) - 13 mv. Despite EO also dominates here, the observed event rate (0.4 s -1 at -150 mv) is lower than what would be expected from a zeta potential based estimation of the EO and EP velocities.

8 SI Figure 5: (a) Current-time traces of a streptavidin translocation experiment. The upper row depicts measurements with a positively biased trans side, for the lower row, the trans side was biased negatively. (b) Schematic illustration of the nanopore and protein charge and expected EO and EP velocities (indicated as arrows) for the case of a negatively biased trans chamber. 1. Berne, B.; Pecora, R., Dynamic Light Scattering, Dover Publications, New York, Storm, A. J.; Chen, J. H.; Ling, X. S.; Zandbergen, H. W.; Dekker, C. Nature Materials 2003, 2, (8), Cossa, T.; Trivedi, D.; Wiggin, M.; Jetha, N. N.; Marziali, A. NANOTECHNOLOGY 2007, 18, (30), Gustafsson, J.; Mikkola, P.; Jokinen, M.; Rosenholm, J. B. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2000, 175, (3),

9 5. Obi, I.; Ichikawa, Y.; Kakutani, T.; Senda, M. Plant Cell Physiol. 1989, 30, (1), Tangsuphoom, N.; Coupland, J. N. Journal of Food Science 2008, 73, (6), E274-E Smolyanitsky, A.; Saraniti, M. Journal of Computational Electronics 2009, 8, (2), Pedone, D.; Firnkes, M.; Rant, U. Analytical Chemistry 2009, 81, (23),