Nanopore Opening at Flat and Nanotip Conical Electrodes during

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On the Nanopore Opening at Flat and Nano-Tip Conical Electrodes during Vesicle Impact Electrochemical Cytometry Xianchan Li, Lin Ren, Johan Dunevall, Daixin Ye, Henry S. White, Martin A. Edwards, and Andrew G. Ewing ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b00781 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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On the Nanopore Opening at Flat and Nano-Tip Conical Electrodes during Vesicle Impact Electrochemical Cytometry Xianchan Li,† Lin Ren,‡ Johan Dunevall,‡ Daixin Ye,† Henry S. White,§ Martin A. Edwards,*, § and Andrew G. Ewing*,†,‡

†

Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10,

41296 Gothenburg, Sweden ‡

Department of Chemical and Chemical Engineering, Chalmers University of Technology,

Kemivägen 10, 41296 Gothenburg, Sweden §

Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah

84112, USA

*

Corresponding Author E-mails: [email protected]; [email protected]

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ABSTRACT: The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modelling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ~100% oxidation efficiency is achieved when employing a 33 µm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nano-tip conical electrode (~4 µm length, ~1.5 µm diameter at base) is employed, as in intracellular VIEC (IVIEC), the current-time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (~75%) of the catecholamine are predicted, as compared to higher currents and ~100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell-line using nano-tip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nano-tip electrodes can directly assess vesicular content without correction. KEYWORDS: simulation, disk electrode, nano-tip electrode, catecholamine, nanometer vesicles, electrochemistry, collection efficiency

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Signal transmission between cells in the nervous system mainly occurs through exocytosis, where vesicles loaded with neurotransmitters, neurohormones, and/or neuropeptides are the main sub-cellular agent of communication.1-9 Understanding the properties of these sub-micrometer sized vesicles and the mechanism of their release is of general importance in neuroscience. Owing to its high temporal resolution and sensitivity, single cell amperometry has been widely used for direct quantification of neurotransmitter release from single vesicles in individual endocrine cells and neurons.6,7,10,11 Although exocytosis has been classically thought to be an “all-or-none” process, recent studies with single cell amperometry have shown that the quantity of neurotransmitter released during exocytosis is strongly dependent on the mode of stimulation and physiological conditions, such as pH, temperature and osmotic pressure.12-19 Analysis of the current decay from such experiments indicates that the intravesicle matrix remains covered by the vesicular membrane during the entirety of a neurotransmitter release event20 and post-spike ‘feet’,21 a stable plateau current during the decay of amperometric spikes in current-time recordings, indicate a residual quantity of catecholamine in the vesicle when release ceases. When combined, these pieces of evidence imply that exocytosis is not a simple “all-or-none” process. Hence, a technique for quantifying the total neurotransmitter content in sub-micrometer sized vesicles would provide valuable information for understanding vesicular neurotransmitter storage and exocytosis.22 Recently, a pair of related electrochemical techniques, vesicle impact electrochemical cytometry (VIEC) and intracellular VIEC (IVIEC), have been employed to assess the catecholamine

content 23-31

phagolysosomes).

of

a

variety

of

soft

nanoparticles

(vesicles,

liposomes,

In these approaches, shown schematically in Figure 1, vesicles (or their

artificial analog, liposomes) are adsorbed at a micro- or ultramicro-electrode where they subsequently release their contents (catecholamines). The catecholamine is then oxidized at the electrode, resulting in a current spike, in which the charge passed can be converted to catecholamine content. VIEC and IVIEC have been used to measure the catecholamine content of single vesicles, both in vesicle preparations and in situ in living cells.23-30,

32

Comparison of the vesicular catecholamine content, as assessed by IVIEC, with the quantity released during exocytosis, measured by single cell amperometry, has provided direct experimental evidence that most exocytosis results in release of a fraction of the entire vesicle contents.24 However, quantification of vesicular content relies on the assumption that all catecholamine released in VIEC is captured at the underlying electrode, or knowledge of the fraction collected, yet this is a function of the release mechanism (full collapse33 vs release through pore25, 31) and also the electrode geometry and pore location (vide infra). 3


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Experimental studies have given some insight into the mechanism of release from vesicles. With experimental results showing complete oxidation of vitamin C loaded liposomes at a carbon electrode during electrochemical recording, Cheng et al. proposed that the liposome immediately collapses and releases its entire cargo, so-called ‘full collapse fusion’.33 However, the adsorption of liposomes without rupture was observed by the blocking of Fe(CN)!! ! oxidation at a Pt microelectrode electrode; and only when surfactant was added was spontaneous rupture observed.34,35 Moreover, when similar experiments were performed using vesicles isolated from pheochromocytoma (PC12) cells, scanning electron micrographs showed intact vesicles adsorbed to carbon microelectrodes prior to the application of a potential.24 These results indicate that the rupturing mechanism of single liposomes and mammalian vesicles at the electrode surface in VIEC is much more complicated than a simple collapse mechanism. This was the topic of considerable discussion at the 2016 Faraday Discussion on single entity electrochemistry, published in Faraday Discussions.36 Furthermore, the existence of a pre-spike foot in current transients in VIEC indicates the formation of an initial pore on vesicle membrane through which vesicular catecholamine is collected at the electrode.37 While these results have hinted at the mechanism of release, a controversy exists about the location of release, and this information is necessary to quantify the extent to which the released neurotransmitter is collected at the electrode in VIEC. This is also important as it reveals critical data on the opening of vesicles in general, for example when in contact with a membrane during exocytosis. To determine the location of the release pore in VIEC and to quantify catecholamine collection at the electrode surface, we have used time-dependent numerical simulations, which describe the catecholamine concentration during vesicular release. By comparing currents from simulated and experimental (VIEC) vesicle release at electrodes of different geometries (disk and nano-tip conical; Figure 1 b and c, respectively), we deduce that a pore forms at or close to the electrode surface, which leads to essentially complete capture of the released catecholamine by the electrode and the key implication that: charge collected during VIEC is a direct measurement of the entire vesicular content. Furthermore, we have carried out simulations at nano-tip conical electrodes placed inside single cells and the results verify that the mechanism of vesicle opening must proceed via pore formation at the electrode surface, thus allowing full quantification of catecholamine content in intracellular vesicles.

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Figure 1. (a) Schematic illustration of vesicle impact electrochemical cytometry (VIEC). Vesicles containing catecholamine adsorb to the electrode, rupture, and release catecholamine. Subsequent oxidation of the catecholamine on the polarized electrode results in amperometric spikes. Scanning electron micrographs of representative 33 µm diameter disk (b) and nano-tip conical (c) electrodes used for VIEC and IVIEC, respectively.

RESULTS AND DISCUSSION Transport through the nanometer pore that forms in the vesicle during VIEC determines the electrochemical response and is related to vesicle opening in other situations, including exocytosis. Here, the transport of catecholamine was modeled as a diffusion process governed by Fick’s second law, as described in detail in the Methods section. Briefly, the initial catecholamine concentration of the 180 nm diameter vesicle was set to 100 mM, in agreement with previously reported values for vesicles from PC12 cells.24, 27, 38 The pore was formed by setting a portion of the vesicle membrane to have unhindered permeability, while the remainder was impermeable. The diffusion-limited oxidation of catecholamines at the electrode surface, which is biased at a large over-potential (+700 mV vs Ag/AgCl), was represented by setting the catecholamine concentration at the electrode surface to zero. The diffusion coefficient of the catecholamine inside the vesicle was set to Dint = 5.0×10-8 cm2/s, an intermediate value of the range of 10-8-10-7 cm2/s reported for chromaffin cells, from which the PC12 cell-line is derived.39,40 Note, this value represents a lumped diffusion coefficient, which incorporates differences in transport in the dense core and halo (a lucent solution surrounding the dense core) inside of vesicles, and takes into account the effect of binding and unbinding of catecholamines to the dense core on the apparent diffusion coefficient. The simulated current response is obtained by an integral of the catecholamine flux over the 5


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electrode surface multiplied by nF. To compare vesicular release at electrodes with different geometries, numerical simulation of vesicular catecholamine release through a nanopore was performed at both flat disk microelectrodes and nano-tip conical electrodes, which were used in VIEC and IVIEC experiments, respectively. It is worth noting that similar simulations were performed for the model of “full collapse fusion”,33 where the entire vesicle membrane was set as permeable. These simulations showed significantly different responses compared with experimental data; including ~2 orders of magnitude higher currents and much shorter current transients. This clearly shows that the “full collapse fusion” mechanism is inconsistent with the release behavior for mammalian vesicles we observe in VIEC (see more details in section S3 in Supporting Information). Simulations of Vesicular Release at a Disk Microelectrode. Initially, simulations were performed describing catecholamine release dynamics from a vesicle adsorbed at the center of a 33-µm-diameter disk microelectrode (Figure 1b), of the type used for VIEC measurements of vesicle preparations. Figure 2 shows the resultant time-series of simulated catecholamine concentration distributions following the pore opening (t = 0 s), taken on the x-z plane intersecting the vesicle center, showing only the ~0.85 µm region close to the vesicle (see Figure S4 for plots showing the entire electrode). Results are shown for the pore in two limiting scenarios: the left panel is a simulation with the pore diametrically opposite the electrode surface (on the top of the vesicle), and the right panel portrays simulations with the pore located where the vesicle contacts the electrode (on the bottom of vesicle). The initial concentration profiles (top-left parts in both panels) are identical in each case. The dark red color represents 100 mM catecholamine within the vesicle, whereas zero concentration outside the vesicle is shown as black. The diffusion coefficient of catecholamine in the buffer outside the vesicle was set as Dext = 6.4×10-6 cm2/s,41,42 which is significantly higher than that inside the vesicle, Dint = 5.0×10-8 cm2/s.39,40 This results in the concentration of catecholamine outside the vesicle being markedly lower than that inside, thus different concentration color scales are used inside (cint) and outside (cext) the vesicle (see legend). As the catecholamine leaves the vesicle through the pore, the concentration of catecholamine inside of the vesicle near the pore decreases. Initially the decrease occurs only close to the pore opening in a planar fashion (t = 0.001 ms; see top-right panes), but very quickly (t ≥ 0.05 ms) it extends in a hemispherical form, which maintains throughout the remainder of the release process. The concentration within the vesicle far from the pore opening remains approximately uniform at all times and decreases with time in an exponential 6


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fashion (Figure S1a). Visually, for a given time, one cannot easily distinguish whether more catecholamine has been discharged or has reached the electrode in one pore configuration or the other; this requires analysis of the current/charge (vide infra). However, it is apparent that the concentration distribution outside of the vesicle depends on the pore location. When the pore is on top of the vesicle (left panel in Figure 2), the concentration outside the vesicle increases, spreading outward in a radial fashion, with a magnitude that diminishes over time. In contrast, when the pore is located at the bottom of the vesicle (right panel in Figure 2) the catecholamine immediately encounters the electrode and is oxidized; therefore, there is no appreciable concentration of catecholamine clearly visible outside of the vesicle, although a small amount does exist in the tiny gap between the vesicle and the electrode (see inset). The white lines in Figure 2 represent the flux of catecholamine outside of the vesicle. When the nanopore opens on top of the vesicle (left panel), the lines initially spread out radially, but very quickly loop around to the electrode surface as the oxidation of catecholamine creates a concentration gradient that drives its diffusion to the electrode. It is interesting to note that these lines are essentially stable in direction after 0.5 ms. Thus, the flux follows the same path to the electrode; however, the magnitude of the flux decays exponentially with time (vide infra). If the nanopore opens towards the electrode (right panel), the lines of flux are only visible between the vesicle and the electrode (see inset), confirming the immediate oxidation of catecholamine upon leaving the vesicle. Note, Figure 2 only shows a small fraction (~0.85 µm) of the 33 µm diameter disk microelectrode; the flux lines that do not reach the electrode within these images loop around before the edge of the electrode (see Figure S4 and accompanying discussion). Notably, flux lines intersecting the electrode far from the vesicle carry a much smaller fraction of the flux than those reaching the electrode close to it. For instance, 90% of the current is collected within a region 2 µm of the vesicle when the pore is on top of the vesicle, while the equivalent collection region is smaller when the pore is positioned closer to the electrode surface. As the catecholamine is collected only in a region close to the vesicle, the above description of vesicle release with the vesicle in the center of the electrode applies to release from vesicle positioned away from the center. Indeed, essentially complete collection (>93%) is achieved when the vesicle is positioned just 0.5 µm from the edge of the 33 µm diameter electrode; thus the current response described above is representative of essentially all release events on the disk microelectrode (see section S5 Supporting Information for further details).

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Figure 2. Concentration profiles at selected times following formation of a pore on the top (left panel) or bottom (right panel) of a 180 nm diameter vesicle sitting at a planar disk microelectrode. White streamlines show the direction of diffusional flux, which rapidly develops to a pseudo-steady state; however, the magnitude of the flux (not shown) diminishes with time. Note, owing to their largely different ranges, the concentrations inside the vesicle, cint, and outside the vesicle, cext, are plotted using different color scales. The inset in the top-right frame of the right panel shows an enlarged view of the pore near the electrode at 0.001 ms. The diffusion coefficients in the interior of the vesicle (Dint) and the solution exterior to the vesicle (Dext) were 5.0×10-8 cm2/s and 6.4×10-6 cm2/s, respectively. The radius of pore (a) was 50 nm, and the initial catecholamine concentration within the vesicle (c0) was 100 mM. Intervening frames are shown in Movies S1 and S2 in the Supporting Information.

The current-time (i-t) response for vesicular release at a disk microelectrode is shown in Figure 3 for a range of pore positions, where an angle of 0° is defined as the pore on the opposite side of the vesicle from the electrode surface (left panel, Figure 2), while 180° represents the pore opening at the electrode surface (right panel, Figure 2). The i-t responses appear insensitive to the pore angle, except at the shortest times (98.5%) for all pore positions. This indicates that the entire content is captured and oxidized at the 33-µm diameter disk microelectrode, as would be predicted by the flux lines looping to the electrode surface (Figures 2 and S4). The lowest collection efficiency (98.5%) is observed when the pore is opposite the electrode surface (θ = 0°) and the collection efficiency approaches 100% as θ approaches 180° (Figure S15). A collection efficiency of ~100% indicates that VIEC, when performed with a disk microelectrode, directly measures of the entire vesicular catecholamine content, irrespective of the location of the vesicular pore. As expected from the rapid decay in the current with time (Figure 3), the majority of the catecholamine is rapidly collected with only a small fraction ( 0.045) very similar collection efficiencies are observed as a function of pore position. A 14


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collection efficiency of ~70-85% is observed when the pore is on the opposite side of the vesicle from the electrode surface (θ = 0°). However, when the pore is positioned closer to the electrode surface, the collection efficiency increases and when the pore is close to/at the electrode surface (θ ≥ 130°), the entire catecholamine content of the vesicle is oxidized (collection efficiency > 95%). This result implies that the experimentally measured collection efficiency using a nano-tip conical electrode can be used to discriminate the position of the vesicular pore.

Figure 6. Simulated collection efficiencies for the release of catecholamine via a vesicular pore as a function of the pore position at nano-tip conical electrodes (compared to a disk electrode, dashed line). 15


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Solid lines with points represent the results for vesicles at nano-tip conical microelectrodes at different fractional distances along the electrode (0 represents the vesicle at the narrow tip of the electrode and 1 abutting the cell membrane, as shown in the schematic - vesicle and pore are not to scale). Dashed lines represent the calculated collection efficiencies from a vesicle in the center of a 33 µm-diameter disk microelectrode. Parts a and b represent the results of simulations performed with different diffusion coefficients inside the vesicle, Dint, and the pore radii, a, as labeled on the plots. The diffusion coefficient outside of the vesicle was set at Dext = 3.3×10-6 cm2/s for release on a nano-tip conical microelectrode, representing the intracellular diffusion coefficient and Dext = 6.4×10-6 cm2/s for release on a disk microelectrodes, representing the diffusion coefficient in buffer. All collection efficiencies are taken from 0 to 100 ms.

Vesicle Content with VIEC Compared to IVIEC. The release of catecholamine from single vesicles was monitored by VIEC with electrode geometries comparable to those used in the simulations described above. Ex situ measurements using vesicles extracted from PC12 cells employed a 33 µm diameter disk electrode, whereas IVIEC was performed using a nano-tip conical microelectrode inserted into individual PC12 cells. Note, the parameters used in the simulations were chosen to be representative of vesicles from these cells (see above and Supporting Information, section S5). In the IVIEC experiment, the length of the nano-tip conical electrode exposed to vesicles in cytoplasm was controlled to be 2-6 µm by observation with a microscope, in agreement with the geometry used for simulations of IVIEC (vide supra). The average i-t response for a single release event at each kind of microelectrode is shown in Figure 7a (typical i-t curves and individual amperometric spikes for each case are shown in Figure S16). In each case the average i-t response is very similar, the current rapidly rises to a maximum (25.2 ± 6.9 pA vs 20.5 ± 5.8 pA, disk vs nano-tip conical microelectrode; mean ± standard deviation) before decaying exponentially over a period of ~10 ms. The catecholamine content in individual vesicles was calculated using Faraday’s Law, N=Q/nF, where Q is the charge calculated by integrating current for each amperometric spike recorded in i-t curves, n is the number of electrons transferred during the redox reaction (2 for catecholamines) and F is Faraday’s constant (96 485 C mol−1).The distribution of vesicular catecholamine content, plotted in Figure 7b, shows closely matched asymmetric distributions that are well fit to log normal distributions (solid lines). The mode of the catecholamine content distribution at nano-tip conical microelectrodes is higher than that at disk microelectrodes (89700 vs 64600 molecules); however, this is a small difference on the scale of the full distribution and the medians are not statistically different (vide infra). As 16


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numerical simulations indicate that nearly 100% collection efficiency is obtained at the disk microelectrodes, we conclude that the experimentally measured catecholamine content distribution faithfully reproduces the actual content. We attribute the distribution of catecholamine content to variability in the vesicle size and/or vesicular catecholamine concentration. To minimize the impact of sample-to-sample or cell-to-cell variation, i-t transients of vesicular release were grouped by electrochemical traces (10 min recording for each sample for VIEC or 2 min for one cell for IVIEC) instead of pooling all data together, and were analyzed by comparing the median vesicular catecholamine content. The total vesicular catecholamine content measured at the nano-tip conical microelectrodes [1.29×105 (1.04×105, 1.72×105), median (1st quartile−3rd quartile)] is greater than the amount detected at disk microelectrodes [1.16×105 (1.04×105, 1.23×105), median (1st quartile−3rd quartile)], but not statistically different (p = 0.112, Mann-Whitney rank-sum test). This non-statistically significant difference might result from a variation of the cell populations used in the VIEC and IVIEC experiments. Another more likely explanation is that larger vesicles with higher catecholamine content preferentially rupture during the homogenization stage of vesicle isolation and/or vesicular leakage occurs during preparation, both of which could lead to a slightly lower content measured in VIEC (again, this is not significantly different at the 95% level).

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Figure 7. Average i-t curves (a) and catecholamine content (b) for vesicular release measured at disk microelectrodes (red) and a nano-tip conical microelectrodes (black). (a) Average i-t responses were calculated by averaging the average response of each recording / trace (33 traces for disk microelectrodes, 37 traces for nano-tip conical microelectrodes) obtained with Igor software. The shaded regions represent ± 1 standard deviation. (b) N=5717 events (33 traces, 7 vesicle isolations) for disk microelectrodes. N=3101 events from (37 traces from 37 cells) for nano-tip conical microelectrodes. Bin size: 2×104 molecules. Fits to histograms are log normal distributions.

Numerical simulations of vesicular catecholamine release at nano-tip conical electrodes predicted collection efficiencies ranging from ~75% (pore opening opposite the electrode surface; θ=0°) to 100% (pore opening at/close to the electrode surface; θ ≥ 130°). A prediction from this result is: if a significant proportion of vesicular pores are formed far from 18


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the electrode surface the distribution of number of molecules collected in IVIEC will be shifted to lower values compared to the distribution from the disk electrode. However, statistical analysis of the catecholamine collected in each situation showed no significant difference. We therefore argue that during VIEC the pore is formed on, or close to the electrode surface. This conclusion has profound implications for the interpretation of IVIEC; it indicates that nano-tip conical electrodes provide an accurate, direct measurement of the total vesicular content and that no correction is necessary to take into account incomplete collection. Moreover, when taken in context with previously reported results: that the probability of vesicle release is potential dependent,25 a pore forms at the beginning of vesicle release,37 and the persistence of the vesicular membrane,25 we propose that electroporation is the main driving force for vesicle opening at a polarized electrode. This location of the pore coincides with the largest electric field across vesicle membrane. Additionally, the electroporation mechanism explains why only a small percentage of the vesicles that adsorb to the electrode appear to open allowing the contents to be exposed to the electrode.24,37

CONCLUSIONS We address the controversy of how nanometer-sized neurotransmitter-containing vesicles open during VIEC and IVIEC. We present numerical simulations of diffusion through a nanopore during both measurements. In VIEC, these show that release from vesicles on a 33 µm-diameter disk microelectrode results in ~100% oxidation of content and a nearly identical current-time response, irrespective of the pore position. In contrast, simulations employing a 4 µm-long, 1.5 µm diameter (at base) nano-tip conical microelectrode, as used for IVIEC, deliver a collection efficiency that ranges from ~75% (pore opposite the electrode surface) to ~100% (pore positioned at the electrode surface). Experiments measuring vesicular catecholamine content in intracellular and extracted vesicles, employing nano-tip and microdisk electrodes, respectively, give closely agreeing catecholamine distributions. This suggests that the pore predominantly opens at the electrode surface during VIEC measurements, providing evidence that electroporation is the primary driving force for vesicle rupture. These results demonstrate that IVIEC employing nano-tip conical electrodes provides a reliable measure of the entire vesicular catecholamine content. Furthermore, these results suggest that vesicle opening is not always spontaneous on surfaces.

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METHODS Simulation The flux of catecholamine out of a vesicle (or liposome) was calculated using finite element simulations. The dimensions of the vesicle and the size and location of the pore were kept constant within a single simulation. Example geometries are shown in Figures 2 and 4. Transport of catecholamine was modeled as a diffusion process as described by Fick’s second law: !" !"

= 𝐷∇! 𝑐

(2)

where c represents the concentration of catecholamine, and D its diffusion coefficient. The value of the diffusion coefficient outside the vesicle, Dext, was set equal to 6.4×10-6 cm2/s for VIEC at a disk microelectrode and 3.3×10-6 cm2/s for IVIEC at a nano-tip conical microelectrode. The value of the diffusion coefficient inside the vesicle, Dint, was set as a lower value to account for the dense core, taking 5.0×10-8 cm2/s and 2.9×10-7 cm2/s as two examples in the range reported before.39,40 The initial concentration of catecholamine within the vesicle was set as 100 mM, while that outside was set to 0 mM. Diffusion-limited oxidation of catecholamine was considered, thus a boundary condition of c = 0 mM was used for the electrode surface. A boundary condition of c = 0 mM was also applied to the boundaries representing bulk solution to account for the absence of catecholamine. Free diffusion (flux continuity) was permitted across the pore, as described by 𝐷!"# ∇𝑐!"# ∙ 𝑛!"# = −𝐷!"# ∇𝑐!"# ∙ 𝑛!"#

(3)

where 𝑛 represents the inward pointing unit normal and the subscripts, ‘int’ and ‘ext’, represent the values of the quantities in the internal and external domains, respectively. The remainder of the vesicle, the electrode sheath (ultramicroelectrode only), and the cell membrane were set as impermeable, as described by 𝐷∇𝑐 ∙ 𝑛 = 0

(4)

The current at the electrode, i, and the rate of catecholamine leaving the vesicle through the pore, J, were calculated through the following flux integrals: 𝑖 = 𝑛𝐹 𝐽=

!"#$

!"!#

𝐷∇𝑐 ⋅ 𝑛

𝐷!"# ∇𝑐!"# ∙ 𝑛!"#

(5) (6)

where F is the Faraday constant (96485 C/mol), n is the number of electrons transferred upon oxidation (2 for catecholamine).

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Equations were solved using the commercial finite element package Comsol Multiphysics (version 5.2a). The mesh used to discretize the problem was set to be finest around the edge of the pore. A boundary layer mesh was used to capture the transient planar flux during the initial period of simulation. The mesh was determined to be sufficiently fine when further refinement gave no meaningful change in the current. The infinite element feature of Comsol Multiphysics, which mapped the region beyond the electrode to infinity using a coordinate transform, was used to ensure the domain of simulation was large enough and also had the effect of representing the cell membrane as an infinite plane. Collection efficiency was evaluated by taking total charge collected (the integral of the current with respect to time) and dividing it by the equivalent charge for complete oxidation of the vesicle contents (0.1 M×nF 4/3π r3). Vesicle Impact Electrochemical Cytometry Vesicles were isolated from PC12 cells as previously reported.23,37,38 Briefly, PC12 cells cultured on mouse collagen (type IV, BD Biosciences, Bedford, MA) coated 75 cm2 flasks were detached using TrypLE Express (Gibco, USA) and then washed carefully with RPMI 1640. The cell suspension was then centrifuged at 300 g for 5 min to pellet the cells. The cells were then placed in a glass homogenizer (Wheaton, USA) to break the cell membrane until entire cells were not observed under microscopy. The homogenate was then centrifuged at 1000 g for 20 min at 4°C to remove the cell debris. The supernatant was subsequently centrifuged at 70000g for 45 min at 4 °C to pellet PC12 vesicles. The pellet was re-suspended in 0.1 mL of homogenizing buffer and used as a vesicle stock solution. For VIEC recording, a 33 µm disk microelectrode was first dipped in a stock PC12 vesicle suspension for 10 min at 4 °C. The electrode was then transferred to vesicle-free homogenizing buffer and release events recorded for 10 min at 37 °C. During this time a potential of +700 mV vs Ag/AgCl was applied using a patch-clamp amplifier (Axopatch 200B, Molecular Devices, USA). The current was filtered at 2 kHz using a 4-pole Bessel filter and digitized at 10 kHz using a Digidata model 1440A with Axoscope 10.3 software (Axon Instruments Inc., USA). IVIEC Prior to IVIEC, the cell media was removed and the PC12 cells were rinsed three times with HEPES physiological saline (150 mM NaCl, 5mM KCl, 1.2 mM MgCl2, 2 mM CaCl2, 5mM glucose, 10 mM HEPES, pH 7.4). The cells were maintained at 37 oC in a solution of HEPES physiological saline during the entire experimental process. Electrochemical recordings from 21


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single PC12 cells were performed on an inverted microscope with a 40× objective (IX81, Olympus), inside a Faraday cage. The nano-tip electrode, prepared as reported before,24 was first placed on top of a single PC12 cell and slowly moved to press through the cell membrane. Throughout the experiment the working electrode was held at a potential of +700 mV vs Ag/AgCl using a patch-clamp amplifier with same recording parameters as in VIEC. Data Analysis for VIEC The amperometric traces obtained from both VIEC and IVIEC were processed using an IgorPro 6.22 software script originating from David Sulzer’s group.44 The threshold for peak detection was five times the standard deviation of the noise. The standard deviations of the noise of amperometric traces for VIEC and IVIEC were 1.34 ± 0.05 pA and 1.10 ± 0.11 pA (mean ± SEM), respectively, with no significant difference (Mann-Whitney rank-sum test). The traces were carefully inspected after peak detection and false positives were manually rejected. To minimize the effect of extremes, the median of vesicular catecholamine content recorded in each amperometric trace is used for statistical analysis. The distributions of the samples were evaluated using the 1st quartile−3rd quartile (or 25%−75%) interval and pairs of data sets were compared using the Mann-Whitney test.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at … The authors declare no competing financial interest. Corresponding Author *E-mail: [email protected]; [email protected] ORCID Xianchan Li: 0000-0002-3256-6610 Lin Ren: 0000-0003-2686-8142 Johan Dunevall: 0000-0001-9188-9893 Daixin Ye: 0000-0001-6020-429X Henry S. White: 0000-0002-5053-0996 Martin A. Edwards: 0000-0001-8072-361X 22


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Andrew G. Ewing: 0000-0002-2084-0133 ACKNOWLEDGEMENTS The authors acknowledge funding from The European Research Council (ERC), Knut and Alice Wallenberg Foundation, the Swedish Research Council (VR), and the National Institutes of Health. M. Edwards was supported by AFOSR MURI on Electrochemical Imaging and Mechanistic Studies on the Nanometer Scale (award number FA9550-14-10003).

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TOC graphic 87x39mm (300 x 300 DPI)


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Nanopore Opening at Flat and Nanotip Conical Electrodes during

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