Fabrication and Characterization of a K+-Selective Nanoelectrode and

9 Aug 2014 - A nanopipette containing a solution of bis(benzo-15-crown-5) dissolved in 1,6-dichlorohexane was used as an ion-selective electrode (ISE)...
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Fabrication and Characterization of a K+‑Selective Nanoelectrode and Simultaneous Imaging of Topography and Local K+ Flux Using Scanning Electrochemical Microscopy Hiroshi Yamada,* Daiki Haraguchi, and Kenji Yasunaga Department of Applied Chemistry, National Defense Academy, 1-10-20 Hashirimizu, Yokosuka, Kanagawa 239-8686, Japan S Supporting Information *

ABSTRACT: A nanopipette containing a solution of bis(benzo15-crown-5) dissolved in 1,6-dichlorohexane was used as an ionselective electrode (ISE) to probe K+ for shear force-based constant-distance scanning electrochemical microscopy (SECM). In a previous study, the ISE responded only at low K + concentrations ([K+] < 1 mM), due to the depletion of the bis(benzo-15-crown-5) at the oil/water interface at high K+ concentrations and the unstable response of the tip at the oil/ water interface for shear force and current detection. In the present study, a nanopipette reshaped by heating and with the hydrophobic layer removed was used as the ISE. This modified ISE enabled a rapid response to changes in K + flux at a physiological concentration of K+ and allowed SECM imaging on a nanometer scale. The fabricated nano-ISE was used as a probe for shear force-based SECM. Topography and K+ flux images were obtained simultaneously at a polycarbonate membrane filter with 5 μm pores and human embryonic kidney 293 cells (HEK293). Several areas containing a K+ flux larger than the surrounding areas were found in the SECM images of the HEK293 cells, which indicated the existence of K+ channels.

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Alternatively, shear force techniques have been utilized in scanning probe microscopy (SPM) systems, such as scanning near-field optical microscopy (SNOM),14,15 SICM,16,17 and SECM.18−31 A tuning fork,21,25−29 Fresnel diffraction,23,24,31 and simple piezoelectric plate18−20,30 have been used to detect dampening of the probe vibration caused by shear force between the probe and the sample surface. To regulate the tip/sample distance for conventional SPM, the vertical position of the tip is adjusted using a closed feedback loop to maintain a prefixed value of feedback signals while the tip moves at a constant speed across the surface. Because the tip of the probe is located very close to the sample surface, the tip could destroy the morphology of the sample surface or could be broken by contact with the sample if a fragile surface or a surface with a steep vertical slope was imaged. To overcome these obstacles, incorporating a method such as a tapping-like scanning mode,32 standing approach (SA) mode,25,26,29,33 and hopping probe ion conductance microscopy (HPICM)10,34 or backstep mode,35,36 featuring a repeated approach and retraction of the tip at each data point, has been proposed. For these methods, the tip of the probe approaches the sample until a predetermined threshold is reached. A soft sample such as a living cell is deformed easily by the tip of the probe during the scan.37 In a previous study on

on channels play an important role in maintaining the ionic balance and control of ionic fluxes across biological membranes. Although the functions of ion channels have been investigated extensively since the patch clamp method was proposed by Neher and Sakmann,1 challenges in the imaging of the distribution of ion channels have remained. Loose patch clamp2 and scanning ion conductance microscopy (SICM)3 have been used to investigate ion channel distributions. However, these methods do not allow for ion selectivity. Therefore, additional experiments were necessary to obtain the distribution of the specific ion channels. Micro/nanoscale ion-selective electrodes (ISEs) have been used to measure intracellular and extracellular K+ and Na+ concentrations.4−7 Messerli et al. used potentiometric micro-K+ ISE to achieve functional mapping of Ca2+-activated K+ channels.6,7 The ISE was fixed near the cell membrane, and an extracellular flux change in K+ caused by the single channel opening was observed. Scanning electrochemical microscopy (SECM) has been widely used for biological applications8,9 because electroactive species can be detected selectively using an ultramicroelectrode. The current at the SECM probe depends on the distance between the tip and sample surface. Therefore, control of the noncontact distance is critical. One promising system for maintaining constant distance SECM control involved ion conductance. 10−12 The diffusion-limited current of the mediator was also used as a feedback signal for constantdistance control using voltage-switching mode SECM.13 © 2014 American Chemical Society

Received: July 3, 2014 Accepted: August 9, 2014 Published: August 9, 2014 8547

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and goggles, should be worn when handling it.] This process is referred to as hydrophilization. The hydrophilized capillary was then filled with 140 mM NaCl solution, and a mixture of TOATCPB (25 mM) and bis (benzo15-crown-5) (75 mM) in DCH was drawn into the capillary to fill ∼300 μm from the tip with the organic solution. Then, a 0.1 mm silver chloride-covered silver wire was inserted into the capillary for electrical contact to form a K+-sensitive nano/micro-ISE. When the tip of the ISE was immersed into the aqueous solution, the oil/water interface at the tip was advanced into the capillary. A quartz crystal tuning fork (CFS-308, Citizen, Tokyo, Japan) was soldered onto the diaphragm of a commercially available piezoelectric transducer to excite mechanical resonance of the tuning fork, and the capillary was placed in contact with one prong of the tuning fork. Since the capillary was not permanently attached to the tuning fork, replacement of the capillary was easy. The voltage applied to the piezoelectric buzzer was adjusted to maintain the amplitude of the tuning fork such that the voltage produced was 0.95 mV for the SECM measurements. (The fabrication process of the ISE is illustrated in Figure S1 of the Supporting Information.) Microscopic Observation. The tip of the pulled capillary was observed using a digital microscope (VHX-1000, Keyence, Tokyo, Japan) and scanning electron microscope (SEM) (JCM-5000, Jeol, Tokyo, Japan). During SECM imaging, the cultured cells were observed using a CCD camera (L-830, Hozan, Tokyo, Japan) equipped with a macro lens (KCM-ZS, Kenko-Tokina, Tokyo, Japan). HEK293 Cell Culture. Human embryonic kidney 293 (HEK293) cells were grown on 3.5 cm polystyrene dishes (353001, BD Falcon, Franklin Lakes, NJ, USA) in DMEM medium supplemented with 10% fetal bovine sera for 3 days, as described previously.46 SECM Apparatus. An SECM system (HV404, Hokuto Denko, Tokyo, Japan) was modified to allow shear force-based tip positioning at nanometer resolution. A piezoelectric actuator (PSt 150/7/20 VS12, Piezomechanik GmbH, Munich, Germany) or a piezo-driven stage (NC301C, Nanocontrol, Tokyo, Japan) was mounted on a step motor-driven XYZ stage (KS701-20LHD, Suruga Seiki, Tokyo, Japan) to allow micromovements of the probe in the Z direction. The stroke of the piezoelectric actuator and the piezo-stage were 20 and 15 μm, respectively. Samples were moved with a piezo-driven XY stage (NS4214, Nanocontrol, Tokyo, Japan). The signal of the tuning fork was amplified with a lock-in amplifier (7264, Signal Recovery, Oak Ridge, TN, USA); the signal amplitude then was digitized and transferred to a computer (MT9000, Epson Direct, Tokyo, Japan) via a 16-bit AD/DA board [ADA16-32/ 2(PCI)F, Contec, Tokyo, Japan]. Measurements were obtained using a two-electrode configuration; the reference- and counterelectrode connectors were attached to the Ag/AgCl wire immersed directly into the solution, and the working electrode connector was attached to the Ag/AgCl wire inserted into the capillary. Electrochemical Characterization of ISE. When a negative potential was applied to the electrode in the capillary of the ISE, K+ transfer across the oil/water interface and the resulting current was proportional to the concentration of K+ in the aqueous phase in the presence of an excess concentration of crown ether in the organic phase. [Figure S2 in the Supporting Information shows the cyclic voltammogram of ISE with (red line) and without K+ (black line) in a 140 mM NaCl solution.] Current increase started at −0.2 V in the absence of K+. In the

shear force-based SICM imaging of a single living cell, the observed height of the cell depended on the threshold in SA mode. Therefore, a differential standing approach (d-SA) mode that used the differential of the amplitude of the tuning fork for constant-distance SICM imaging was employed.17 Micropipette-based micro/nanoelectrodes have been applied as SECM probes due to their ease of fabrication.38−45 A micropipette filled with an ionophore solution, such as valinomycin or another crown ether derivative dissolved in an organic solvent, has been used to probe nonelectroactive ions as an ion-selective electrode (ISE) for SECM.38,43,44 In a previous study, an amperometric ISE responded only at low K+ concentrations ([K+] < 1 mM) because a depletion of the ionophore occurred at the oil/water interface at high concentrations of K+.38 In addition, the oil/water interface existing at the tip of the ISE caused an unstable response to the shear force/current detection in shear force-based SECM imaging. In the present study, fabrication of the ISEs was modified. First, the tip of the capillary was reshaped by heating to reduce the inner radius of the capillary, which improved the resolution for SECM imaging. Second, the hydrophobic layer for 10−30 μm of the tip was removed to allow an oil/water interface inside the capillary. This recessed ISE enabled stable constantdistance imaging of a polycarbonate membrane filter, which was soluble in the organic solution used in the ISE. This ISE was also used for simultaneous topography and K+ flux imaging in living cells.

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EXPERIMENTAL SECTION Materials. All aqueous solutions were prepared with water purified using a Milli-Q Jr. system (Millipore, Billerica, MA, USA). Potassium chloride, sodium chloride, 1 M sodium hydroxide solution, ethanol, tetraoctylammonium (TOA) bromide, and octadecyltriethoxysilane were purchased from Wako Pure Chemical (Tokyo, Japan) and used as received. Tetrakis(4-chloroohenyl)borate (TCPB) potassium salt (Dojindo, Tokyo, Japan), bis(benzo-15-crown-5) (Dojindo), and 1,6-dichlorohexane (DCH) (Alfa Aesar, Ward Hill, MA, USA) were used as received. Tetraoctylammonium tetrakis(4chlorophenyl)borate (TCPB-TOA) was prepared by mixing saturated solutions of TOA-Br/ethanol and K-TCPB/ethanol, and the precipitate formed was recrystallized three times from ethanol. RPMI and DMEM media were purchased from Life Technologies (Carlsbad, CA, USA) and were used as received. Fabrication of ISE Probe. A glass tube (1B150-4, World Precision Instruments, Sarasota, FL, USA) was pulled using a capillary puller (PC-10, Narishige, Tokyo, Japan) to form a 2 to 3 μm radius capillary tip. The tip was fused using a microforge (MF-900, Narishige, Tokyo, Japan) with reduced pressure in the capillary. The inner (rin) and outer (rout) radius of the tip of the capillary were 0.2−0.3 and 1−2 μm, respectively. The hydrophobic capillary was prepared by silanizing the inner wall of the tip with octadecyltriethoxysilane to prevent penetration of the aqueous solution into the capillary. An aqueous mixture of sodium hydroxide then was introduced into the tip of the hydrophobic capillary, and the capillary was heated at 80 °C in saturated steam overnight. After washing with purified water three times, chromic acid was introduced into the tip of the capillary, and the capillary was stored at room temperature for 1 h to remove the hydrophobic layer from approximately 10 to 30 μm of the tip. [Caution: chromic acid solution can be dangerous to handle, and protective equipment, including gloves 8548

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Figure 1. (a) Optical and (b) SEM images of the reshaped tip of the glass capillary.

presence of K+, a steady-state current was observed at −0.3 to −0.5 V, indicating spherical diffusion of a microelectrode. To prevent the influence of Na+ on the diffusion current of K+, the SECM measurement was performed at −0.15 V. Scanning Mode for SECM Imaging. The standing approach (SA) and differential standing approach (d-SA) modes were used to image the local K+ flux and topography of the surface. Details of these modes have been described previously.17 In SA mode, the damped amplitude was used as a threshold. In d-SA mode, the threshold was the slope of the amplitude of the fork obtained by numerical differentiation. The slope of the approach curve was obtained from an approximation of the previous 50−100 data points using linear least-squares in the numerical differentiation. Current−Distance Profile. The substrate was immersed in RMPI or DMEM medium. The tip of the probe was placed above the substrate and adjusted to −0.15 V vs Ag/AgCl. ac voltage at the resonance frequency was applied to the diaphragm of the piezoelectric buzzer; adjustment of the voltage resulted in an amplitude of 0.95 mV generated by the tuning fork. The tip was lowered toward the surface at 1 μm/s with the piezoelectric actuator or the piezo-stage while also measuring the output signal of the tuning fork until its amplitude decreased to the threshold value (90−99%) of its original amplitude. Imaging of Polycarbonate Membrane Filters. A polycarbonate membrane filter with 5 μm pores (Isopore 5.0 μm TMTP, Millipore, Billerica, MA, USA) was placed at the bottom of a custom-made electrochemical cell that was then filled with RPMI medium. The electrochemical cell was covered, and humidified air was continuously introduced to prevent evaporation of the solution. Immersion depth of the tip of the capillary was 50−100 μm. The SA mode was used for imaging membranes with a threshold value of 97%. Imaging HEK293 Cells. Measurements were conducted in covered cell culture dishes, in which humidified air was introduced continuously to prevent evaporation of the solution. Immersion depth of the tip of the capillary was 50−100 μm. The d-SA mode was used for imaging membranes with a threshold value of 0.08 mV/μm.

while increasing the potassium ion concentration. Figure 2 shows the calibration curves for ISEs, which were fabricated

Figure 2. Dependence of current on K+ concentration obtained with the ISE using an unmodified (squares), reshaped (circles), and reshaped and hydrophilized (triangles) glass capillary in 50 mM MgCl2 (squares) and 140 mM NaCl (circles and triangles). The potential of the ISEs was set at −0.25 V (squares) and −0.15 V (circles and triangles).

with unmodified (squares), reshaped (circles), and reshaped/ hydrophilized (triangles) capillaries. When unmodified ISE was used, the current became nearly constant, with no significant increase at K+ concentrations greater than 1 mM. This phenomenon was considered to be a result of depletion of the crown ether dissolved in organic solvent at the oil/water interface, because diffusion of the crown ether was linear in the oil phase inside the capillary, whereas diffusion of the potassium ion phase outside the capillary in the water occurred in all directions. To measure the variation in K+ flux under physiological conditions, the current needed to be increased proportionally with K+ concentration to a maximum of ∼6 mM. Mass transfer of the crown ether in the organic phase of the reshaped ISE was increased compared to that of the unmodified ISE. Linearity of the calibration curve also was improved with the reshaped ISE. The K+ flux to the oil/water interface was decreased in the reshaped/hydrophilized ISE because the transfer of K+ was restricted by the tip filled with the aqueous solution. Therefore, linearity with the reshaped/hydrophilized ISE improved over that obtained with the reshaped ISE. Furthermore, the hydrophilization process prevented contact between the organic solution in the capillary and sample surfaces during scanning. To confirm the availability and resolution of the reshaped/hydrophilized ISE, the approach curve and SECM images for the polycarbonate membrane were obtained (Figures S3 and S4 in the Supporting Information).

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RESULTS AND DISCUSSION Characterization of ISE Probe. The tip of the capillary was reshaped to improve the resolution of the SECM image. Figure 1 shows optical and SEM images of the reshaped tip of the capillary. The rin and rout values were 230 nm and 1.8 μm, respectively. To evaluate the dependence of current on the concentration of K+ in aqueous solution, current was measured 8549

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Approach Curve for a HEK239 Cell. Figure 3 shows the amplitude and current approach curves and the differential of

|Δfmax | =

2 ⎛ Δzx ⎞2 ⎛ Δzy ⎞ ⎜ ⎟ +⎜ ⎟ ⎝ Δx ⎠ ⎝ Δy ⎠

where Δx and Δy are the distance between the data sampling points for the X and Y axes, respectively, and Δzx and Δzy are the difference in height between the data sampling points in the X and Y directions, respectively. The current at the edge of the cell was larger than that at the top of the cell or at the dish surface. Thus, the current depends on the |Δf max| value at the sampling point, indicated by comparing Figure 4, panels b and c. This was observed when a PC12 cell was imaged via the shear force-based SICM system.17 When the ISE approached a tilted surface, the sensing portion (inner part of the tip) does not approach the surface because one side of the outer wall of the tip interacts with the surface. Therefore, K+ diffused through the gap, and a larger current was observed at the edge of the cell. In the large response at the edge of the cell, the contribution of the K+ channels might be included. However, further investigation is needed to estimate the extent of this contribution. The current for most of the flat cell surface was the same as that at the dish surface, indicating that the cell membrane was practically impermeable to K+ over most of the cell surface. However, even at the flat cell surface (with small |Δf max| area), the current was larger than that in the surrounding areas in several places. A typical example of these areas has values of (X, Y) = (1 μm, 7 μm) and (7 μm, 15 μm) (enclosed areas with solid and broken lines in Figure 4b,c). The line profiles for topography and current at X = 1 and 7 μm and Y = 7 and 15 μm are shown in Figure 4d−g. At the flat cell surface, the current depends on the permeability of K+ through the membrane if K+ channels exist in the area. Therefore, the distribution density of the K+ channels must be high in these areas. The current in these areas was 5−10 pA greater than that in the surrounding areas. The current at the recessed electrode depends not only on the rin value but also on the recessed distance from the tip.47 Therefore, determining how many channels contributed to the increment of the current is difficult. Whole-cell voltage clamp experiments for HEK239 cells revealed endogenous voltage-gated K+ (KV) channels.48−50 Some K+ channels, which were unblocked by typical channel blockers such as tetraethylammonium or 4-aminopyridine, were also observed. One type of unblocked K+ channel was considered to be a two-pore domain (K2p) channel, known as a potassium leak channel. Although the K2p channels were always open to maintain the resting potential of the cell, no actual outward K+ transfer occurred because of the resting potential, despite the transmembrane concentration gradient of K+. Because the ISE induces a concentration gradient of K+ in the vicinity of the cell surface during SECM imaging, K+ ions penetrate outward through the K2p channels. As a result, the current increased in several areas where the distribution density of the K2p channels was high. Furthermore, once the K+ moves outward, the local decrease in K+ concentration in the cell may induce the opening of the KV channels, which facilitates K+ transfer to the outside.

Figure 3. (a) Approach curves showing amplitude of the tuning fork (dashed line) and current (solid line) as the tip approached a HEK293 cell in DMEM medium. (b) Differential of the amplitude approach curve. The differentiation of a data point was calculated to be the slope of the prior 100 data points, as determined via linear least-squares. One hundred data points correspond to a distance of 0.3 μm.

the amplitude approach curve as the ISE approached a HEK239 cell in DMEM medium. The Z = 0 μm distance in Figure 3 does not represent the cell surface or the dish surface, but rather the limit of the position for the approach curve. Although the approach curve to the polycarbonate membrane depicted in the Supporting Information (Figure S3) showed a sudden decrease near the surface, the amplitude and current decreased gradually upon approaching the cell surface. As described previously, soft samples, such as living cells, deform easily when the tip moves toward the surface without touching it. Therefore, the amplitude was differentiated with Z (Figure 3b) to recognize the exact position at which the tip began to push the cell surface away. The derivative of the amplitude increased upon approach to Z = 5 μm, and then the derivative value decreased as the tip approached Z = 3 μm. Therefore, the tip probably starts to deform the cell membrane at Z = 3 μm. To avoid pushing the cell surface away and to maintain a constant distance between the tip and cell surface, the differential of the amplitude (0.05−0.1 mV/μm) was used as the threshold for constant-distance SECM imaging for HEK239 cells in d-SA mode. Simultaneous Ion Current and Topographic Imaging of HEK293 Cells. Figure 4 shows the topographic, maximum gradient of the sample surface, and the SECM images of HEK293 cells in d-SA mode (set point: 0.08 mV/μm). The range of these images was 30 μm2, with a resolution of 1 μm. The maximum gradient (|Δf max|) at each point was obtained using the following equation

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CONCLUSIONS A nano-ISE was fabricated successfully and applied to a shear force SECM system for simultaneous imaging of the topography and K+ flux at the membrane filter and a HEK293 cell surface. The size of the pores in the membrane filter, observed in topographic and SECM images, was in reasonable agreement 8550

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Figure 4. (a) Topography, (b) maximum gradient of the sample surface, and (c) SECM images of HEK293 cells in d-SA mode. Calculation of the maximum slope of the sample is described in the text. Line profiles of topography (black) and current (red) at (d) X = 1 μm and (f) 7 μm and at (e) Y = 7 μm and (g) 15 μm.

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with the expected size. The SECM image of HEK239 cells revealed that most of the cell surface was impermeable to K+, but several areas with K+ flux greater than that of the surrounding areas were present. This finding suggested the existence of K+ channels, such as K2p or KV channels, in these areas. Unlike the potentiometric ISE, the amperometric ISE induced a concentration gradient of the analyte. Therefore, the amperometric ISE is considered to be suitable for the investigation of K+ leak channels, which cannot be detected using potentiometric methods. In this study, the current at each point in the SECM images was the average of a 0.4 s interval. The observation of a single-channel event in high K+ flux areas was needed to clarify whether the high flux of K+ was caused by efflux through the K+ channels. Analysis of the time course of the current at each sampling point in SECM imaging is underway.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Professor Ichimura, National Defense Academy, for providing HEK293 cells and for helpful advice about culturing the cells.

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ASSOCIATED CONTENT

S Supporting Information *

Schematic illustration of the fabrication process for ISE; optical images of ISE in air and in aqueous solution; cyclic voltammograms of an ISE with and without K+; approach curves of the amplitude of the tuning fork and current as the tip approached the isopore membrane filter in RPMI medium; and topography and SECM images with a reshaped/hydrophilized ISE. This material is available free of charge via the Internet at http://pubs.acs.org. 8551

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