Counting and Dynamic Studies of the Small Unilamellar Phospholipid

Dec 9, 2014 - Chem. , 2015, 87 (1), pp 522–529 ... ranging from −0.6 to −1 V. The findings provide fundamental insights into the translocation a...
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Counting and Dynamic Studies of the Small Unilamellar Phospholipid Vesicle Translocation with Single Conical Glass Nanopores Lizhen Chen,†,‡ Haili He,†,‡ and Yongdong Jin*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Phospholipid vesicles are ubiquitous cellular organelles that perform vital functions including materials transport and information transmission and have found promising biomedical applications. Although the transmembrane translocation (via nanopores) of phospholipid vesicles, especially small unilamellar phospholipid vesicles (SUVs), is recognized to be very important for these processes and applications, the details and dynamics remain not very clear. Herein, we use single conical glass nanopores as a model platform to systematically investigate the translocation dynamics of SUVs (∼50−60 nm in diameter) through small nanopores with orifice diameters ranging from ∼14 to 72 nm. Dynamic translocation of individual SUVs one by one through the nanopores was clearly observed and was analyzed by the occurrence of periodic oscillation in ionic current blockage signal under a negatively applied voltage. Translocation behaviors of the SUVs, in terms of magnitude and duration of ionic current blockage signal, varied and can be modulated by changing nanopore size, solution pH, vesicle concentration, applied voltage, and inner surface charge properties of the nanopores. The translocation rate of the SUVs through an ∼72 nm nanopore is typically on a time scale of a few seconds (per SUV translocation event) and found nonlinearly proportional to the concentration of the SUVs. Moreover, the electrophoretic force has been verified as a main force to drive the SUVs through the nanopore since there is a nearly linear relationship between the current blockage frequency of SUVs translocation and the applied bias potentials ranging from −0.6 to −1 V. The findings provide fundamental insights into the translocation and interactions of SUVs with nanopores, and the reported nanopore platform may find potential useful bioapplications in single-cell and single-vesicle studies.

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molecule sensing and DNA sequencing.9−14 Among other artificial nanopores (such as the nanopores fabricated on the Si3N4, PI, PET membrane, etc.), glass nanopores have drawn enormous attention because of their properties of simplicity of preparation, (orifice) size tunability, repeatability, stability, and their unique conical shape. The glass nanopores have been used experimentally for the detections of single molecule, DNA segments as well as the ion currents and forces via different electrochemistry systems.15−18 Recently, Holden et al. reported investigations of the pressure-driven translocation behavior of multilamellar liposomes with radii between 190 and 450 nm (preprepared by extrusion through a polycarbonate membrane) through a single conical nanopore (100−575 nm in radius) embedded in the end of a glass capillary by the resistive-pulse method.19 In this work, we use artificial single conical glass

hospholipid vesicles are ubiquitous cellular organelles and have been widely used as a model biomimetic delivery and membrane system for a wide variety of biological and biomedical applications ranging from bioassay and drug delivery to cancer chemotherapy.1−5 Recently, as scientists have successfully revealed the fine structure and the controlled mechanisms of intracellular transport system by monitoring the accurate processes of the transportation of vesicles in the cells in real time,6−8 it will push tailor-made phospholipid vesicle systems for new breakthroughs in pharmacy, chemistry, and biology. Although phospholipid vesicle formation, delivery, and transmembrane translocation (via vesicle fusion or nanotunnels, such as nanopores) are recognized to be very important for these processes and bioapplications, the details and mechanisms (dynamics) remain not very clear due to the still lack of powerful in situ methodology and tools that are capable of mimicking and monitoring dynamic vesicle translocation process. In recent years, artificial nanopore-based analytics have emerged as powerful and ultrasensitive platforms for single© XXXX American Chemical Society

Received: August 5, 2014 Accepted: December 9, 2014

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to SUVs was approximately 1.5 and the applied bias potential was higher than −0.6 V.

capillary nanopores with remarkably smaller orifice diameters (∼14−72 nm) as a model platform to systematically investigate the in situ translocation dynamics of small unilamellar phospholipid vesicles (SUVs, ∼50−60 nm in diameter) through the solid-state nanopores. We chose SUVs system for nanopore investigation since their translocation behaviors may differ from that of big multilamellar liposomes. Moreover, SUVs are much more important in cell biology and they have been widely used as a model system to study the molecular interactions which occur in biological membranes.20 The SUVs are in situ translocated and detected by single conical glass nanopores under an applied potential across the nanopore, as illustrated in Scheme 1. The Ag/AgCl electrode inserted inside

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EXPERIMENTAL SECTION Chemicals. Cardiolipin and poly-L-histidine (PLH) were purchased from Sigma-Aldrich. Hydrogen peroxide (30% H2O2), sulfuric acid (H2SO4), ethanol (C2H5OH), Na2HPO4· 12H2O, KH2PO4, potassium chloride (KCl), and chloroform were purchased from Beijing Chemicals (Beijing China), and all of them were of analytical reagent grade. Glycidyloxypropyltrimethoxysilane (GPTMS) was purchased from Aladdin. The 10 mM phosphate buffer (PBS) solution containing 10 mM KCl was at pH 7.4. All solutions except 0.2% GPTMS solution were prepared by using deionized Milli-Q water (18.25 MΩ) with further purification with sterile membrane filter (0.22 μm). Fabrication and Surface Modification of Glass Nanopores. Conical glass nanopores were fabricated by using borosilicate capillaries with an outer diameter of 1.0 mm and inner diameter of 0.58 mm (BF100-58-10; Sutter Instrument Co.). Prior to the pulling process, all glass capillaries were thoroughly cleaned by immersing in a piranha solution (98% H2SO4/30% H2O2 = 3:1) for 2 h to remove organic impurities. (Caution: piranha solution is a powerf ul oxidizing agent and reacts violently with organic compounds. It should be handled with extreme care.) The cleaned capillaries were soaked in deionized Milli-Q water to remove residual acid and vacuum dried at 80 °C prior to use. This process could remove any contamination as well as obtain a high surface density of hydroxyl (−OH) groups in the inner side of the capillaries. The nanopores, with inner diameters ranging from ∼14 to 72 nm, were fabricated by using a P−2000 laser-based micropipette puller (Sutter Instrument Co.) preprogrammed with two-line program. In a typical preparation, parameters used were as follows: (line 1) heat = 350, fil = 3, vel = 30, del = 220, pul = 0; (line 2) heat = 330, fil = 2, vel = 27, del = 130, pul = 250.21 In order to obtain GPTMS-coated nanopore, 0.2% GPTMS in ethanol was injected into the glass nanopore and centrifuged at 3500 rpm for 5 min to help the solution get to the very tip of the nanopore, and then incubated at room temperature for 4 h. The nanopore was then filled with deionized water and centrifuged at least three times to remove unmodified GPTMS molecules and then dried at room temperature for subsequent experiment.22 For preparing PLH-decorated nanopore, glass nanopore was backfilled with 0.1 mg/mL PLH aqueous solution and centrifuged at 3500 rpm for 5 min and then incubated at room temperature for 2−3 h. The prepared PLHdecorated nanopore was then rinsed three more times with deionized water to remove excess PLH solution, and then baked at 50 °C for 1 h to stabilize the PLH coating.23 Preparation of Small Unilamellar Phospholipid Vesicles. The SUVs were prepared by using cardiolipin solutions from bovine heart (in ethanol). The cardiolipin solutions (100 μL) were added into a clean glass vial with 0.5 mL of chloroform. The chloroform mixture was then evaporated by using N2, leaving a thin film of lipid on the bottom of the vial. Then the sample was put into the vacuum oven to remove residual chloroform overnight at 26 °C. The dried lipids were then resuspended by vortexing in 1 mL of phosphate buffer (pH 7.4) for 3 h, followed by ultrasonication at less than 30 °C for 2 h. Final lipid concentration of the asprepared vesicles is ∼0.5 mg/mL. All vesicle suspensions were used immediately after the preparation.24

Scheme 1. Schematic Illustration of the Translocation of the SUVs through a Single Conical Glass Nanopore Detected by the Occurrence of Ionic Current Blockage

the glass capillary nanopore serves as working electrode, while the electrode that faces the outside of the nanopore in the bulk solution acts as reference/auxiliary electrode, and the bias negative potential is applied through the nanopore. As a vesicle translocates the sensing zone of the glass nanopore, it substitutes (partial of) the buffer solution in it and impedes the background ions flux, resulting in the decrease of ionic current detected (as illustratively shown in the bottom of Scheme 1). The SUVs were prepared by ultrasonication of a buffer solution with cardiolipin lipids containing four long hydrocarbon chains (chemical structure is shown in Figure S1a in the Supporting Information), which was an important component of the inner mitochondrial membrane. The in situ dynamic translocation of individual SUVs one by one through conical glass nanopores was clearly observed and counted by the occurrence of periodic oscillation in ionic current blockage signal under a constant applied voltage. Translocation behaviors of the SUVs, in terms of magnitude and duration of ionic current blockage signal, varied sensitively and can be modulated by varying different stimulation parameters including nanopore size, solution pH, vesicle concentration, applied voltage, and inner surface charge properties of glass nanopores. When the inner surfaces of the nanopores were neutral or negatively charged, the SUVs could translocate regularly. This was due to that the translocations of SUVs through the conical nanopore are driven mainly by electrophoretic force in the system, which had been verified briefly under different stimulus parameters. The translocation rate of the SUVs through an ∼72 nm nanopore was found nonlinearly proportional to the SUVs concentration. In addition, our results showed that the SUVs can translocate through the single conical glass nanopores in an amenable frequency when the diameter ratio of glass nanopore B

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Characterizations. The ionic transport properties of the translocation processes of vesicles were studied by measuring ionic current through a nanopore. The ionic currents were measured with Ag/AgCl electrodes (in 10 mM KCl) in a homemade electrolyte cell and recorded with the amplifier Axopatch 200B (Molecular Devices) in voltage-clamp mode using a low-pass Bessel filter of 5 kHz. The signals were digitized with DigiData 1440A digitizer (Molecular Devices) at 100 kHz and viewed with Clampfit 10.2 software (Molecular Devices). The Ag/AgCl electrode inserted in the glass nanopore backfilled with solution acts as working electrode, while another Ag/AgCl electrode setting outside in the bulk solution works as reference/auxiliary electrode. The main potential used for current−time recordings was set at −1 V in PBS buffer solution, while the current−voltage (I−V) curves are recorded by sweeping the voltage from −0.5 to +0.5 V with scanning rate of 50 mV/s in 0.1 M KCl electrolyte solution. The conductivity of the electrolyte solution was measured by using a DDS-11A electric conductivity meter (Inesa Instrument Ltd., Shanghai, China). All experiments were carried out at room temperature (∼22 °C). The dynamic light scattering (DLS) experiments to measure size distribution and ζ-potential of the phospholipid vesicles were performed with a Zetasizer Nano ZS Zen3600 Malvern (Malvern Instruments Ltd., Malvern, U.K.) by detecting for three times at an angle of 173°. The temperature of the clamber was controlled at 25 ± 0.1 °C. The transmission electron microscopy (TEM) images of the glass nanopores and SUVs were carried out by the use of a FEI TECNAI F20 EM at 200 kV accelerating voltage. TEM samples of nanopores were prepared by placing the very sharp tip of a glass capillary on a folding grid, while the TEM samples of the vesicles were prepared by placing a drop of the final suspension on a carbon-coated copper grid and drying under infrared light condition. The scanning electron microscopy (SEM) images of the bare nanopores with different diameters were taken with an XL30 ESEM FEG microscope (FEI Instrument Co.) at 20 kV accelerating voltage. To prevent charging effect, glass nanopores were sputter-coated with thin gold film prior to imaging.

detection (data not shown). Figure S1b (in the Supporting Information) shows the TEM image of the as-prepared SUVs with a closed shape and mean size of ∼53 nm, which was in agreement with the result of DLS.25 The single conical-shaped glass nanopores used in this study were prepared by a program-controlled microcapillary puller technology (see the Experimental Section).21 Figure S2a (in the Supporting Information) shows a typical TEM image of the asprepared conical nanopore. The diameter of the nanopore is ∼81 nm. Alternatively, the diameter of the glass conical nanopore could also be estimated electrochemically by using a typical two Ag/AgCl electrodes system in identical electrolyte solutions of 0.1 M KCl. Figure S2b in the Supporting Information shows the corresponding (I−V) curve. The nanopore diameter (∼77 nm) can be obtained electrochemically (details, see the Supporting Information), which is in close agreement with that visibly obtained from the TEM image (Supporting Information Figure S2a).26−28 Both TEM and electrochemical characterizations confirmed consistently the size of the nanopore used. The net surface charge of the glass nanopore can be monitored through the ionic current−voltage response of the nanopore as a function of the potential applied between two Ag/AgCl electrodes.29 Since the conical nanopore generates an interesting electrochemical behavior as it responds to a symmetric input potential with an asymmetric current output, the effect of which is referred to as ion current rectification (ICR), i.e., when the nanopore size is comparable with the thickness of the diffuse electrical double layer (ddl), the electrostatic interactions between charged nanopore surface and ionic species will alter ion transport properties, the characteristics of the I−V curves of nanopores will be strongly affected at the tip region of nanopore and hence useful for characterization of surface properties of nanopores. The degree of ICR of the I−V curves can be quantitated using the coefficient (r) through the equation r = lg|I+0.5/I−0.5|, where I−0.5 is the ionic current recorded at a given negative potential (−0.5 V) and I+0.5 is the ion current recorded at a given positive potential (+0.5 V).30,31 Both of the positive and negative rectifications are tackled equally according to the equation. As defined here, ICR coefficient (r) < 0 indicates transport ion current at −0.5 V applied potential is greater than ion current at +0.5 V. Similarly, ICR coefficient (r) > 0 indicates transport ion current at −0.5 V applied potential is smaller than ion current at +0.5 V. Ion current rectification coefficients of the nanopores are therefore used to confirm the surface properties of glass nanopores. Effect of Nanopore Size on SUVs Translocation. As depicted in Scheme 1, a suspension of the preformed phospholipid SUVs was first backfilled in a glass capillary nanopore for the translocation study. We first studied the effect of nanopore size on SUVs translocation, especially when the size of the nanopore is comparable with or even smaller than that of SUVs. Therefore, glass capillary nanopores with four different sizes (around 14, 31, 53, and 72 nm in diameter, respectively) were chosen for this study. The single glass nanopores with different diameters were controllably fabricated under different conditions (see Table S1 in the Supporting Information). Before SUVs translocation measurements, the sizes of the four single glass nanopores were characterized electrochemically in 0.1 M KCl solution at pH 7.0, and the corresponding I−V curves are shown in Figure S3a (see the Supporting Information). The electrochemically calculated

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RESULTS AND DISCUSSION With the use of single conical glass nanopores as self-sensing platforms for ion current measurements, we studied the in situ translocation dynamics of SUVs through the nanopores. Dynamic translocation of individual SUVs one by one through conical glass nanopores was clearly observed and counted by the occurrence of periodic oscillation in ionic current blockage signal under an applied voltage. The magnitude of the ionic current and the time duration for the translocation of SUVs through the glass nanopore depend on several factors, such as properties of the glass nanopore used (e.g., the nanopore diameter and its surface charges), experimental factors (e.g., pH condition as well as the applied potential), and SUVs themselves (e.g., concentration). Herein, we systematically investigate simple dynamics of SUVs translocation through small nanopores by investigating into these factors. Preparation and Characterization of SUVs and Single Conical Glass Nanopores. The SUVs used for this study were prepared by using cardiolipin in a PBS buffer solution (pH 7.4), which has been described in detail in the Experimental Section. The diameter of the as-prepared SUVs was typically ∼62 nm with negative surface charge according to the DLS C

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height of the translocation experiment relies on the relative size of the SUVs to be translocated to the nanopore used. An equation to estimate I/I 0 based on the ratio of the hydrodynamic cross section (diameter) of SUVs to the nanopore diameter can be given as32

diameters of the above four glass nanopores were in great agreement with the final SEM observation of the nanopores (Figure S3b−d in the Supporting Information). Figure 1 shows typical current−time recordings of the SUVs translocation through the four glass nanopores in 10 mM PBS

⎛ d ⎞2 I =1−⎜ ⎟ ⎝D⎠ I0

(1)

where d is the diameter of SUV in solution and D is the diameter of the glass nanopore. It is obvious that eq 1 yields a value of I/I0 = 0.26 for a nanopore with diameter of ∼72 nm, which shows good agreement with experimental data without involving any scaling factors or fitting parameters. From eq 1, we note that the value of I/I0 is proportional to the diameter of the SUV and in inverse proportion to the nanopore diameter. We therefore can expect that two or more SUVs assembly (due to, for example, vesicle fusion) should cause a smaller value of I/I0 due to the increase in value of (d/D)2 under the same experiment condition. However, the i−t trace dose not exhibit two peaks or smaller blockage current level (I), as shown in Figure 1d. This confirms that each ionic current blockage oscillation corresponds to an individual SUV translocation through the glass nanopore. The phenomenon is consistent with recent work done by Holden at al., in which the authors found that no translocation was observed when the pore radius is smaller than liposome’s radius, and the translocation behavior can be explained by pressure-driven flow theory when pore sizes larger than the particle size.33,34 The current oscillations in our system are likely caused by the SUVs translocation individually through the glass nanopore rather than SUVs formation at the tip of the nanopore and then budding off to give responses, since no regular current blockage oscillation can be seen when the diameter of the glass nanopore is smaller than that of the SUV (see Figure 1, parts a and b). However, it is noteworthy to mention that the nanopore will resize the translocated SUVs (due to orifice size restriction and flexibility of the SUVs) when the size of the nanopore is comparable to or slightly smaller than that of SUVs, resulting in more uniform responses in I/I0. The translocation times of SUVs in our experiments are much larger (Figure 1d) than that using significantly big-sized multilamellar liposomes and pore radius. This may probably be attributed to the following differences and reasons:19,34 First, since the diameters of our glass nanopores are significantly reduced and both the inner surface of a typical glass nanopore and SUVs are negatively charged, the resistant electrostatic repulsion force and electroosmosis force may contribute jointly to slow down the translocation process of SUVs (attributing to the effect of the ddl). Second, the taper of our conical glass nanopores is obviously larger, contributing an additional translocation resistance for SUVs. Our results from the nanopore size effect study showed that the SUVs could translocate through the single conical glass nanopores in an amenable frequency when the diameter ratio of glass nanopore to SUVs is approximately 1.5. And we found that the SUVs are quite stable during the translocation studies, since even at −1 V of applied potential, the size distribution of SUVs showed unobvious change before and after the translocation experiment (Supporting Information Figure S4). To further investigate whether other factors affect the translocation dynamics of the SUVs through the glass nanopore, glass nanopores fabricated in a typical preparation

Figure 1. (a−d) Representative current−time recordings of SUVs translocation for the bare glass nanopores with different diameters of (a) 14, (b) 31, (c) 53, and (d) 72 nm, respectively, examined in 10 mM PBS (pH 7.4) buffer solution. During all the experiments, phospholipid vesicles of 0.17 mg/mL in 10 mM PBS (pH 7.4) solution were injected into nanopores to carry out the measurements.

buffer solution under applied potential of −1 V at room temperature. Translocation events for the SUVs were typically characterized by their translocation dwell time (Δt), which was measured as the width of the translocation peak at half its maximum height, as well as the ratio of blockage current to the baseline current (I/I0). Δt and I/I0 here were used to determine the extent of the ionic current blockage oscillations and the degree of interaction between the SUVs and glass nanopore. As shown in Figure 1, parts a and b, the I/I0 ratios for the 14 nm diameter glass nanopore and 31 nm diameter nanopore are both approximately 1.00, indicating that there are almost no SUVs translocation events observed under the measurement conditions when the diameter of the glass nanopore is obviously smaller than that of SUVs. It is reasonable because the “big” SUVs might be sterically hindered and blocked in the inner wall of a smaller nanopore during the permeation, making the translocation resistance of SUVs larger than their translocation driving forces and therefore preventing effective translocation of SUVs through the nanopore. For glass nanopores with diameter close to or larger than the vesicle size, SUVs translocation occurred periodically as shown in Figure 1, parts c and d. The average values of the Δt and I/I0 for the translocation of SUVs through a bare glass nanopore with diameter of ∼53 nm were 6.7 ± 0.25 s and 0.09 ± 0.01, while the values of Δt and I/I0 with an ∼72 nm nanopore were 1.8 ± 0.4 s and 0.36 ± 0.02, respectively. This is indicative of the decrease of the translocation resistance with the increase of nanopore diameter, resulting in a faster rate of translocation and a smaller value of dwell time. The ionic blockage current D

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(see the Experimental Section) are used for subsequent experiments. Effect of Vesicle Concentration on the Permeation of SUVs through Glass Nanopores. In this system, the translocation experiments of SUVs were carried out at pH 7.4, for the pH values would affect the stability of the SUVs. According to the current−time curves shown in Figure S5a (in the Supporting Information), at pH 5.8, only small and irregular fluctuations in ionic current rather than pronounced and regular blockage oscillations were observed (top panel of Supporting Information Figure S5a). This might be strongly indicative of the formation of lipid aggregates or micelles, rather than uniform and stable SUVs. At alkaline solution (pH 8.0), the SUVs also turned out to be unstable and amenable to breakage and/or fusion, as reflected by the incomplete current−time blockage oscillations during the experiments within the same period of time (bottom panel of Supporting Information Figure S5a). Morphologies of SUVs at different pH solutions were further confirmed by TEM characterization (Supporting Information Figure S5c). Moreover, the control experiments performed in the absence of SUVs indicated that the nanopore was still stable at pH 5.8 and pH 8, as shown in Supporting Information Figure S5b. Since SUVs translocation behaviors were highly pH-dependent, we maintained the solution of pH 7.4 during the experiments. To inspect and verify vesicle concentration effect on the permeation of SUVs through glass nanopores, the vesicle suspensions with different concentrations had been examined with a glass nanopore in a buffer solution (pH 7.4) by using the two-electrode setup at room temperature. The vesicle concentrations studied here were set as 0.02, 0.17, and 0.5 mg/mL in terms of lipid content. All of the dilute vesicle suspensions were diluted with original 0.5 mg/mL suspension by using PBS buffer (pH 7.4). The average diameters of SUVs in these suspensions showed undetectable change by DLS characterizations (data not shown). Parts a−c of Figure 2 show the resulting current−time blockage oscillations of the three SUV samples recorded at an applied voltage of −1 V. As vesicle concentration changed, the profile of the ion current blockage oscillations changed accordingly. The average values of the translocation dwell time (Δt) and I/I0 for the permeation of SUVs (0.02 mg/mL) through a glass nanopore were 2.4 ± 0.5 s and 0.44 ± 0.03, respectively (Figure 2a). And the average values of Δt and I/I0 for SUVs (0.17 mg/mL) were 1.8 ± 0.4 s and 0.36 ± 0.02, respectively, while the Δt and I/I0 for SUVs (0.5 mg/mL) were 1.2 ± 0.3 s and 0.22 ± 0.01, respectively. As shown in Figure 2d, left panel, the SUVs translocation through the glass nanopore was in a slightly faster frequency in the case of 0.5 mg/mL concentration (blue square), while it took somewhat longer time for identical SUVs to translocate through the nanopore at low concentration of 0.02 mg/mL (green square). The translocation events increased with decreased translocation time (Δt, duration) as concentration of vesicles increased within a same period of time. Figure 2d, right panel, shows the concentration dependence in the translocation of SUVs through a glass nanopore. The number of translocated SUVs was counted for 3 min for each SUVs concentration. The translocation rate was found nonlinearly proportional to the concentration of the SUVs at the experiment conditions. The SUVs concentration-dependent translocation behavior was similar to that of a charged microgel due to lowering the deformation barrier as its concentration increased.34 A more

Figure 2. (a−c) Representative current−time blockage recordings of three different SUVs suspensions injected into a bare nanopore with diameter of ∼72 nm, with different vesicle concentrations in terms of lipid content of (a) 0.02, (b) 0.17, and (c) 0.5 mg/mL, respectively. (d) Left panel: scatter plot of the translocation events (normalized current blockage I/I0 vs duration time) caused by SUVs with concentration of 0.02 mg/mL (green squares), 0.17 mg/mL (red squares), and 0.5 mg/mL (blue squares), respectively. Right panel: histogram representation of SUVs translocation events in the presence of 0.02 mg/mL (green), 0.17 mg/mL (red), and 0.5 mg/mL (blue) SUVs recorded for continuous 3 min. All experiments were conducted in the buffer of 10 mM KCl and 10 mM phosphate solution at −1 V.

periodic pulse sequence (i.e., more regular interval between two adjacent events) was also closely observed when the concentration of the SUVs increased (cf. Figure 2a−c). One possible explanation for the concentration-dependent translocation phenomena in our case can be attributed to the increased intervesicle electrostatic repulsion force (and hence decreased translocation barrier) due to the crowding effect of SUVs at high concentration. Otherwise, the individual translocation events should be more randomly spaced in time although the vesicles are uniformly distributed in the solution. Effect of Applied Bias Potentials on the Permeation of SUVs through Glass Nanopores. Figure S6 (in the Supporting Information) shows the current−time blockage responses of a conical glass nanopore in 10 mM PBS buffer solution (pH 7.4) backfilled with PBS (SUVs-free) (Supporting Information Figure S6a) and with SUVs solution (Supporting Information Figure S6, parts b and c), respectively. No current−time blockage responses were observed when the nanopores were backfilled only with PBS solution and −1 V bias potential was applied (Supporting Information Figure S6a). When the SUVs solution was backfilled inside the nanopore, individual current−time blockage oscillations were detected at an applied potential of −1 V, as obviously shown in Supporting Information Figure S6b. Each current blockage E

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nanopore was linearly proportional to the electric field (applied bias potential).21 Influence of the Nanopore Inner Surface Properties on the SUVs Translocation through the Nanopores. Surface properties of single glass nanopores would significantly influence the translocation rate of SUVs as it was well-known that the interactions between charged particles and the nanopore internal surface affect the oscillating current blockages during the process.37−39 Thus, as shown in Figure 4b−d,

oscillation represents an individual SUVs translocation through the nanopore. However, when the bias potential switched from −1 to +1 V, no current blockage signals were observed (Supporting Information Figure S6c). The current blockage phenomenon could only be detected at negative voltage because of the lack of translocation driving force as the SUVs are negatively charged. The result suggests that the translocation of SUVs through the glass nanopore is driven mainly by electrophoretic force, just in the opposite direction of the electric field.35,36 Since the applied bias potential is a main driving force and dictates translocation behaviors of the SUVs, we therefore investigated the dependence of applied potential on translocation behaviors of SUVs over a wide range of applied potentials. Figure 3a shows the corresponding voltage-depend-

Figure 4. (a) I−V curves of the unmodified (black line), GPTMSmodified (blue line), and PLH-modified (red line) single glass nanopores in 0.1 M KCl. The voltage sweep was in the direction from −0.5 to +0.5 V with scanning rate of 50 mV/s. (b−d) Schematic representations of the interior surface properties of the as-prepared glass nanopores before (b) and after chemical modification with (c) GPTMS and (d) PLH. The inset showed the structures of PLH polymer (red) and GPTMS molecule (blue).

we used three glass nanopores with similar diameters but different inner surface properties, denoted as the unmodified, GPTMS-coated, and PLH-coated nanopores, respectively, to survey the translocation process of the SUVs. The electrochemical properties of single glass nanopores before and after the chemical modifications were examined by measuring the ionic current across the nanopore in 0.1 M KCl solution, and all corresponding current−voltage (I−V) curves are shown in Figure 4a. Before chemical modification (black line), the ICR ratio (r) of the bare glass nanopore was −0.11. This reflected the negatively charged internal surface of the as-prepared glass nanopore, as potassium ions present in solution were carried through the nanopore preferentially at negative applied bias potentials relative to corresponding positive potentials. The ICR ratio (r) turned out to be 0.49 after modification of the nanopore surface with PLH (red line), which indicated a reversal in the polarity of surface charge as well as the formation of positively charged PLH coating on the surface of the nanopore. After chemical modification with neutral GPTMS (blue line), a linear I−V curve was shown with the ICR ratio (r) of −0.01, indicating nearly uncharged inner surface of the nanopore coated with GPTMS.22 As expected and shown in Figure 5, the translocation behaviors of SUVs changed with changes in the surface property of nanopores. In the case of PLH-decorated nanopore, the direction of electroosmotic flow switched to the opposite direction relative to the case of the nonmodified nanopore, which will benefit for the translocation of SUVs through the glass nanopore. However, the translocation events are faint and

Figure 3. (a) Corresponding voltage-dependent current blockage frequency of SUVs translocation. (b) Histogram representation of SUVs translocation duration time through a glass nanopore with applied voltage of −600 mV (green), −800 mV (violet), and −1000 mV (red), respectively, recorded for continuous 2 min.

ent blockage frequency of SUVs (0.17 mg/mL) translocation through a glass nanopore (∼72 nm), and all measurements were performed in a 10 mM buffer solution (pH 7.4) with varied applied potential (their corresponding original current− time blockage oscillations are shown in Supporting Information Figure S7). No detectable translocation events were observed at the negative potential lower than approximately −0.6 V, while pronounced SUVs translocation signals occurred and could be analyzed as the applied negative potential higher than −0.6 V. With applied negative potential increased from −0.6 to −1 V, SUVs translocation frequency (events) increased (Figure 3a) and the translocation duration (Δt) decreased (Figure 3b). Figure 3a shows a nearly linear relation between the current blockage frequency of SUVs translocation and the applied potential ranging from −0.6 to −1 V. This is reasonable since the electrophoretic force for SUVs translocation through a F

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SUVs, in terms of magnitude and duration of ionic current blockage signal, vary and can be modulated by varying stimulus parameters (such as nanopore size, solution pH, vesicle concentration, applied voltage, and inner surface charge properties of glass nanopores). The glass nanopores with diameter larger than that of the SUVs will facilitate translocation of SUVs through them. No translocation was observed under −1 V applied potential when the glass nanopore diameter is smaller than the vesicle diameter. And the pH value of the precursor lipid suspension is crucial to SUVs formation, with optimal pH of 7.4, as revealed by in situ nanopore characterizations. The SUVs translocation turns out to be highly applied voltage dependent. The translocation of the SUVs through glass nanopores should be mainly driven by electrophoretic force in our case, which has been verified briefly under different stimulus parameters. In the present study, we show the first systematical investigation of the SUVs translocation through small single glass nanopores, providing a basis to study nanopore translocation of SUVs at a single-vesicle level. This sensing platform may open possibilities to distinguish different types of SUVs which may have different interactions with the nanopore under appropriate experimental conditions. It can be realized by modulating different stimulus parameters. The single glass capillary-based nanopore sensing platform may provide a powerful means to advance nanopore research into the territory of single-vesicle studies with interesting rheological properties and enable a previously unattainable range of applications for single-cell-based and vesicle-based biological and biomedical studies.

Figure 5. (a−c) Representative current−time blockage recordings of SUVs translocation through as-prepared single glass nanopores before (a) and after surface modification with (b) GPTMS and (c) PLH, respectively, examined in 10 mM PBS buffer solution (pH 7.4). During the experiments, the SUVs prepared with lipid content of 0.17 mg/mL were injected into nanopores to carry out the measurements.

irregular and the frequencies of the SUVs translocation through the PLH-decorated nanopore were not at a faster rate than that of the nonmodified nanopore, which means that the translocation of SUVs was mainly driven by the electrophoretic force instead of the electroosmotic flow. In fact, due to the existence of electrostatic interaction between the negatively charged vesicles and the positively charged nanopore surfaces, the SUVs cannot be dislodged easily from the PLH-decorated nanopore. The positively charged interior surface of the PLH-decorated nanopore might capture and destroy the complete structure of the SUVs because of strong electrostatic adsorption and therefore result in nanopore translocation of only eluted irregular smaller vesicles or broken patches (instead of intact SUVs), which was reflected as “inverted” some small sharpshaped blockage oscillations with short duration time (Figure 5c). When the nanopore interior surfaces change back to original negative charge, the current−time blockage oscillation curve turned out to be regular, indicative of one-by-one translocation of individual intact SUVs (Figure 5a).35 After modification with GPTMS, the interior surface of the glass nanopore was neutral charged and then formed a slightly hydrophobic surface, whereas the SUVs were hydrophilic in the aqueous solution due to the phosphate groups on the surface. As a result, it took a much longer time to drive an SUV out of the GPTMS-modified nanopore, as shown in Figure 5b. This suggests that different charged surfaces affect the behaviors of the SUVs translocation through the glass nanopore.

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ASSOCIATED CONTENT

S Supporting Information *

TEM and SEM images, I−V and current−time blockage oscillations curves of SUVs and unmodified conical glass nanopores. This material is available free of charge via the Internet at http://pubs.acs.org.

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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 This work was supported by the Hundred Talents Program of the Chinese Academy of Sciences, the National Science Foundation of China (no. 21175125), and the State Key Laboratory of Electroanalytical Chemistry (no. 110000R387).

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CONCLUSION In summary, we reported on using artificial single conical glass capillary-based nanopores as a model platform to systematically investigate the translocation behaviors and dynamics of SUVs (∼50−60 nm in diameter) through small artificial nanopores with orifice diameters ranging from ∼14 to 72 nm. Dynamic translocation of individual SUVs one by one through the nanopores was clearly observed and counted by the occurrence of periodic oscillation in ionic current blockage signal under an applied negative bias voltage. Translocation behaviors of the

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dx.doi.org/10.1021/ac5029243 | Anal. Chem. XXXX, XXX, XXX−XXX