Osmotically-Driven Transport in Carbon Nanotube Porins - Nano

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Osmotically-Driven Transport in Carbon Nanotube Porins Kyunghoon Kim,†,§,∥ Jia Geng,†,‡,∥ Ramya Tunuguntla,†,∥ Luis R. Comolli,⊥ Costas P. Grigoropoulos,§ Caroline M. Ajo-Franklin,∥,# and Aleksandr Noy*,†,‡,∥ †

Biology and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550, United States ‡ School of Natural Sciences, University of California, Merced, California 95340, United States § Mechanical Engineering Department, University of California, Berkeley, California 94720, United States ∥ The Molecular Foundry, ⊥Life Sciences Division, and #Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States S Supporting Information *

ABSTRACT: We report the measurements of transport of ions and uncharged species through carbon nanotube (CNT) porins short segments of CNTs inserted into a lipid bilayer membrane. Rejection characteristics of the CNT porins are governed by size exclusion for the uncharged species. In contrast, rejection of ionic species is governed by the electrostatic repulsion and Donnan membrane equilibrium. Permeability of monovalent cations follows the general trend in the hydrated ion size, except in the case of Cs+ ions.

KEYWORDS: Molecular transport, nanopores, carbon nanotubes, CNT porins, ion selectivity

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Liposomes serve as a convenient system for studying transport through pore channels. A phospholipid membrane, which is virtually impermeable to ions and large molecules, has limited permeability for water, protons, and small, uncharged molecules, forming a highly efficient nanoscale barrier system.13 Liposomes are also inherently flexible and will deform when they are subjected to stress caused, for example, by a difference between the osmotic pressure inside and outside the vesicle.14 In pure liposomes, these deformations primarily reflect slow nonspecific leakage of water and osmolytes through the lipid bilayer.15 However, when efficient transport conduits, such as protein pores, are embedded in the liposome walls, monitoring the size change of these vesicles can give us information about the transport through these pores. Although these measurements lack the sensitivity of the electrophoretic transport measurements in individual pores, they are much simpler and take advantage of large-scale averaging inherent in bulk measurements. Here we used dynamic light scattering (DLS) to monitor the size charge of vesicles with embedded CNT porins after they were subjected to weak osmotic gradients. From the measurements of the osmotically induced transport of water, ions, and sugars through the CNT porins, a picture

mong the large number of biological and artificial nanopores,1−4 inner channels of carbon nanotubes stand out due to their extraordinary transport efficiency. Both simulation and experimental studies have shown that water transport through CNT channel is almost frictionless and can be comparable to the transport efficiency of aquaporins.1,5,6 Water filling and ion transport processes in nanotubes have been studied using transmission electron microscopy,7 electrophoretic transport measurements,8,9 field-effect transistor device based measurements,10 and X-ray diffraction (XRD) measurements.11 However, for the vast majority of these studies, the nanotube pore length was much longer than that of its biological counterparts, and a number of questions remained on how these transport properties translate to CNT geometries with a much shorter pore length. Recently, we have synthesized and characterized such a structureCNT porin12which consists of a carbon nanotube pore with a diameter of ∼1.5 nm and length ranging between 5 and 20 nm. Single pore transport measurements12 show that CNT porins can self-incorporate into a lipid membrane forming a close analog of an ion channel (Figure 1a) that exhibits many of the same transport characteristics. In this work, we use bulk measurements to explore selectivity of ionic and uncharged species transport through CNT porins embedded in the walls of phospholipid vesicles. © XXXX American Chemical Society

Received: September 8, 2014 Revised: October 20, 2014

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we prepared showed a relatively narrow size distribution (see Supporting Information, Figure S3) centered around 200 nm. The average size of the liposomes prepared with and without the CNT porins differed by less than 25% (188.9 ± 4.4 nm/236 ± 2.5 nm, respectively) with the nanotube-loaded vesicles consistently showing smaller sizes. We hypothesize that traces of longer uncut CNT fragments that remain in the solution after centrifugation contribute to partial clogging of the extrusion membrane pores, effectively reducing the size of these pores and producing smaller liposomes. Osmotically Induced Transport Measurements. To induce the transport across the lipid bilayer, we subjected the liposomes to mild osmotic shock conditions. In a typical measurement, we added an aliquot of liposomes containing pure water in the lumen into an excess of solution containing a defined concentration of an ionic or nonionic osmolyte, such as KCl or sucrose (Figure 2a). The difference in the osmolyte concentrations between the solution inside and outside the vesicle created an osmotic differential across the lipid bilayer. When the lipid bilayer contained water-permeable CNT porins, the difference in the osmotic pressure would quickly drive some water outside the vesicle lumen and shrink the vesicle sizes (Figure 2a). Critically, the outcome of this process also depends on the permeability of the CNT porins to the osmolyte molecules. When the membrane pores are permeable to the osmolyte molecules, then the inward flux of the osmolyte from the outside into the vesicle lumen space would quickly neutralize the osmotic pressure gradient and effectively prevent the vesicles from shrinking. If the pores reject the osmolyte, then the osmotic pressure can drive water out of the vesicle, which should result in much greater shrinkage. Significantly, these measurements are predicated on the ability of the lipid vesicles to resist the osmotic gradient in absence of membrane pores. As no lipid bilayer is strictly impermeable to water and small molecules,15 we can only consider the vesicles to be leak-free over a limited period of time and a limited concentration range. To establish these boundaries we have performed control experiments where we exposed pure DOPC lipid vesicles to a range of osmolytes and monitored the DLS signal over time and determined size change as a function of osmolyte concentration (Figure 2b). We have found that, when pure DOPC vesicles were exposed to a high concentration of KCl or sucrose osmolytes, they exhibited gradual shrinkage over a period of almost an hour. As a control experiment, we added vesicles to pure water and did not observe any measurable shrinkage, proving that the shrinkage observed in osmolyte-containing solutions was indeed due to the effect of osmotic pressure. Curiously, the shrinkage kinetics that we observe in our experiments using pure DOPC lipids occurred at a slower time scale than what was predicted by some of the literature bilayer permeability values;18 however, this kinetics was consistent in control experiments taken over the period of many months and using several batches of lipids. Subjecting the vesicles to 10 consecutive freeze−thaw cycles to ensure formation of unilamellar structures19 also did not change the time scale of the shrinkage kinetics (see Supporting Information, Figure S4); thus we cannot attribute the slow time scale to the presence of a significant number of multilamellar liposomes in the system. Significantly, control measurements on pure DOPC vesicles also showed that solutions in the concentration range of 0.2− 40 mOsm lost water at a rate that was slow enough that these liposomes did not show a significant size change over a period

Figure 1. CNT porins in lipid membranes. (a) A photograph of a 3D model showing a CNT porin in a patch of lipid membrane (model and photo by M. McDaniel and K. Hadley, LLNL). (b) Histograms of CNT porin lengths determined from TEM images. (c−f) TEM images of individual CNT porins.

emerged where the rejection of the charged species was controlled by the electrostatic repulsion at the pore mouth and the rejection of the uncharged species was controlled by the pore size. CNT Porins: Preparation and Characterization. We have prepared CNT porins using a lipid-assisted cutting procedure similar to what we described previously11 (see also Supporting Information). Briefly, prolonged sonication of purified single wall carbon nanotubes in the presence of excess 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC) lipid produced short carbon nanotubes with lengths on the order of 5− 20 nm (Figure 1b−f). After separating longer uncut CNT fragments by centrifugation, we collected the supernatant containing CNT porins stabilized by the lipid coating. Raman spectra of the CNT porins (see Supporting Information, Figure S2) showed typical carbon nanotube bands16 at ∼1600 cm−1 (G band) and ∼1300 cm−1 (D band)17 with the typical ratio of G/D band intensities of around 8, indicating that CNT porin walls still possess graphitic structure. In the final step, we extruded hydrated lipid−CNT porin mixture through a 200 nm polycarbonate membrane, producing liposomes that had carbon nanotube pores in the liposome walls.12 We characterized the hydrodynamic radius of extruded liposomes using a dynamic light scattering setup (Zetasizer NanoZS90, Malvern Instruments). The vesicle solutions that B

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size. This behavior is consistent with the expected quick equilibration of osmotic gradient by rapid reverse flux of ions into the vesicle lumen through the CNT porins embedded in the lipid membrane. The shrinkage pattern was strikingly different when the vesicles were exposed to the low concentration of KCl osmolyte (Figure 2c). While for the pure DOPC vesicles, lower KCl concentrations predictably caused less shrinkage, vesicles with CNT porins actually shrank more in low salt than they did in high salt. This behavior is a clear indication that the ion permeability of CNT porins changes at different levels of ionic strength (we will discuss a detailed picture of this behavior in the following sections). Kinetics of water leakage observed in these measurements was faster (ca. 5 min) than background water leakage through the DOPC bilayer (ca. 20 min), which is consistent with the much faster rates of water transport though the CNT pores. Note that each vesicle incorporates only 10− 15 porins,20 and thus the combined nanotube pore area is still much smaller than the total vesicle surface area. Quantitative Description of Osmotic Transport. The basic features of the osmotically induced transport process can be captured in a simple kinetic model (Figure 2a). In this model, a vesicle of initial volume, V0, filled with water is placed into the osmolyte solution of concentration C0. The vesicle also contains Np pores of radius r and length L (although the CNT porins have a relatively wide distribution of lengths, for this simplified model we consider all the porins to have identical dimensions). Since the volume of the outer solution is much larger than the volume of the vesicles, we assume that the concentration C0 always remains constant. Diffusion of the osmolyte through the CNT pores follows Fick’s Law: Js = −Ds ×

Ci − C0 L

(1)

where Ds is the effective osmolyte diffusion coefficient through the CNT pore. For relatively small vesicle size changes (V ≈ V0) the equation describing the time evolution of the osmolyte concentration inside the vesicle will be

Figure 2. Vesicle size change kinetics under osmotic stress. (a) Schematic illustrating the mechanism of vesicle size change under osmotic stress, as well as the main parameters of the model that describes pressure-driven water transport in this system. (b,c) Time courses of the normalized light scattering values measured after water containing vesicles were exposed to different osmolyte solutions (concentrations as indicated in the legend). Dashed lines correspond to the fit of the kinetic data to eq 6. (b) Control experiments showing the size change of pure DOPC vesicles after exposure to water (black circles), KCl (red squares), and sucrose (blue diamonds) solutions. (c) Comparison of the vesicle size response to two different concentrations of KCl. Filled symbols on the left graph correspond to kinetics obtained for the DOPC vesicles that also contained CNT porins in the bilayer, and empty symbols on the right graph correspond to pure DOPC vesicles.

Js Npπr 2 NpDsπr 2 dCi = =− × (Ci − C0) dt V0 V0L = −ks(Ci − C0)

(2)

where ks is an effective rate constant for osmolyte transport, and Npπr2 is the total area of all CNT pores in a vesicle. Thus, the osmolyte concentration inside the vesicle evolves as Ci(t ) = C0(1 − e−kst )

of 10 min (see Supporting Information, Figure S5). Thus, to ensure that we probe the transport through the CNT porins as opposed to probing background water leakage, we conducted all measurements of CNT porin transport within this concentration window and limited the observation time to 10 min, except when indicated otherwise. Remarkably, the DOPC vesicles that contained CNT porins and the pure DOPC vesicles exhibited qualitatively different responses when we placed them in the KCl osmolyte solutions of varying concentrations (Figure 2c). While pure DOPC vesicles shrank gradually under the 30 mOsm gradient, the vesicles that contained CNT porins maintained their original

(3)

Darcy’s law describes the vesicle shrinkage due to osmotically driven transport of water as k wNpπr 2 dV =− Δπosm dt μL

(4)

where kw is the water permeability of the CNT pores, μ is water viscosity, and Δπosm is the difference in the osmotic pressure between the outer and inner vesicle space, that is equal to Δπosm = iRT(C0 − Ci), where i is the van’t Hoff factor. We then can rewrite this equation as: C

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−k wNpπr 2iRT dV = × (C0 − Ci) = −K w(C0 − Ci) dt μL (5)

where Kw is an effective rate constant for water transport. Equations 3 and 5 produce the final expression describing the vesicle volume evolution with time: V (t ) = V0 −

KW C0(1 − e−kst ) ks

(6)

Equation 6 provides an excellent fit for our data on the kinetics of the vesicle shrinkage (see dotted lines on Figure 2b,c). Finally, the final vesicle size change, D∞/D0, which we measure in our experiments, is given by 1/3 1/3 ⎛ C0 ⎞ KW D∞ ⎛ V∞ ⎞ = ⎜ ⎟ = ⎜1 − × ⎟ D0 ks V0 ⎠ ⎝ V0 ⎠ ⎝

⎛ ⎞1/3 k iRT = ⎜1 − × w × C 0⎟ μ Ds ⎝ ⎠

Figure 3. Transport of uncharged species through CNT porins. Plot of the size change of vesicles containing CNT porins after exposure to solution containing different concentrations of sucrose and dextran. Schematic of CNT porin in lipid bilayer and hydrodynamic radii of dextran and sucrose. Dashed lines are guides to the eye.

(7)

Equation 7 indicates that, while the liposome shrinkage is proportional to the rate of water transport through CNT pores, it is also inversely proportional to the rate of osmolyte leakage. Thus, this analysis indicates that higher shrinkage corresponds to higher ion rejection by the CNT porins. Transport of Uncharged Species. We first investigated how the molecular size affects the transport of uncharged molecules through the CNT porins. For these measurements, we chose two uncharged sugar molecules of different sizes, sucrose (molecular weight 342 Da) and dextran (molecular weight ca. 12 000 Da). A comparison of the approximate hydrodynamic radii of dextran, 2.4 nm,17 and sucrose, 0.46 nm,21 with the 1.5 nm diameter of the CNT pores suggests that only sucrose molecules should pass through them. Thus, if we subjected the liposomes containing CNT porins to the osmotic pressure created by the sucrose and dextran, we would expect to see significant shrinkage in case of dextran and very little shrinkage in case of sucrose. This is indeed what we observed in the measurement (Figure 3): water-filled DOPC vesicles with CNT porins placed into a mild sucrose solution showed almost no shrinkage, indicating that small sucrose molecule readily permeated through the CNT porins and quickly equalized the osmotic pressure gradient. In contrast, vesicles placed in the dextran solution showed appreciable shrinkage up to 12%, indicating that dextran molecules cannot cross to the inside of the vesicle through the CNT pore. These experiments confirm the prediction that the molecule physical size (e.g., hydrodynamic size) remains the primary determinant of the rejection characteristics in the CNT pores. Transport of Charged Species. We also probed the CNT porin rejection for a series of mono-, di-, and trivalent salts that shared the same monovalent cation, K+. For these measurements we replaced uncharged osmolyte solution (sugar) with the electrolyte (salt) solution. The hydrodynamic radii of most ions are typically smaller than the inner diameter of the CNT porin; thus, we would not expect the size exclusion to play a significant role. Instead, the rejection should be dominated by the electrostatic interactions of the ions with the charged groups at the CNT porin rim.22 In our experiments (Figure 4), the vesicles incorporating CNT porins in the lipid bilayer exhibited a significant shrinkage of up to 20% at lower ionic

Figure 4. Transport of charged species in CNT porins. Plot of the vesicle size change as a function of the solution Debye length after exposure to the different 1:1, 1:2, and 1:3 electrolyte solutions of different concentrations. Electrolytes used for each experiment are indicated on the graph legend. Dotted lines indicate a fit to a sigmoidal function. Schematic on the graph illustrates ion rejection at the charged tips of the CNT porins. (Inset) Comparison of the CNT porin ion rejection data (markers and solid lines, same as indicated on the main figure legend) with the predictions of the Donnan model (eq 8) for 1:1, 1:2, and 1:3 electrolyte solution (dashed and dotted lines). See text for model parameter description.

strengths, which corresponded to high Debye length values (i.e., low osmotic pressure differentials). The most striking feature of the data is that as the ionic strength increased the vesicle shrinkage reduced drastically to the levels well below 5%, approaching the shrinkage level seen in control experiments using pure DOPC vesicles. As we discussed in the previous sections, it is clear that the observed effect reflects the different levels of reverse diffusion of KCl osmolyte into the vesicle at those conditions. Similar to the D

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Figure 5. Transport of charged species with different cations through CNT porins. (a) Plot of the vesicle size change as a function of the solution Debye length after exposure to a series of 1:1 chloride salt solution with different cations, as indicated on the graph. (b) Plot of the highest observed vesicle size shrinkage as a function of the hydrated cation radius for the electrolyte used in the experiment. The dashed line added as a guide to the eye.

nanotube is markedly shorter and is comparable to the range of Debye length values used in the experiment. It is then reasonable to assume that charges at both CNT ends contribute to the effective charge density, which gives us an effective membrane charge density of 6 mM. Indeed, the rejection curves predicted by eq 2 for 1:1, 1:2, and 1:3 electrolytes match the transitions observed in the data very closely (Figure 4, inset), which confirms that the electrostatic rejection mechanism is indeed responsible for the observed behavior. Cation Transport through the CNT Porins. We have also investigated the different counterion permeabilities of varying 1:1 salts through the CNT porins. The vesicle size change curves obtained for solutions of alkali metal chlorides (Figure 5a) showed a qualitatively similar trend where the experiments showed significant vesicle shrinkage (ion rejection) at high Debye lengths and transitioned to the low shrinkage (low ion rejection) regime at lower Debye length values. The observed rejection decreased as Li+ > Na+ > K+ > Cs+, which is not surprising given that the hydrated ion radii decrease with the same trend (Figure 5b). Indeed, the measured vesicle size shrinkage was proportional to the hydrated ion size; however, a surprising observation was the very low rejection observed in the solutions of CsCl. Cs+ is different from the other cations because it has a very weakly bound hydration shell.25 We hypothesize that Cs+ ions can form a weakly bound pair with the hydrated Cl− ions and this neutral complex can squeeze through the electrostatic barrier at the mouth of the CNT porins even at low ionic strengths, contributing to the low observed rejection. In summary, we report the observations of selective transport through the CNT porins embedded in the lipid bilayer membranes. Osmotically driven transport experiments show that CNT porins transport ions and small molecules and reject large uncharged species. Unlike the rejection of the uncharged species that is determined by the pore size, ion rejection in CNT channels is determined by charge repulsion at the CNT rim, and changes in the electrostatic screening can significantly modulate ion rejection. Because CNT porins readily incorporate into the lipid membranes, they provide a convenient model system for studying transport in nanofluidic pores. Such biomimetic channels also open up ways to develop novel applications for biosensors, nanofluidic devices, molecular filtration, and artificial cells and cell-like structures.

effect observed for ion transport through macroscopically long CNT pores,23 the ion permeability of the CNT porins is determined by the electrostatic interactions that an ion experiences at the pore mouth. That rim is lined with −COOH groups (created during the cutting process), and at neutral pH values these groups create a ring of negative charge that impedes the passage of anions though the pore at low ionic strength. At high ionic strength (and correspondingly shorter Debye lengths), the charge at the pore is screened, and the ions can pass through unimpeded. This explanation is also supported by a comparison between 1:1 (KCl), 1:2 (K2SO4), and 1:3 (K3Fe(CN)6) electrolytes (Figure 4). Since divalent SO42− ions experience stronger rejection than monovalent Cl− ions, the transition between high and low rejection regime for 1:2 electrolyte is shifted toward higher salt concentrations (smaller Debye lengths). The same logic explains why the transition for 1:3 electrolyte is shifted even more to the smaller Debye length (higher salt concentration) region. We can also model the observed ion rejection trends using the Donnan model, which predicts the ion rejection in charged pores as22 ⎛ |zi|ci ⎞|zi / zj| R=1−⎜ m m⎟ ⎝ |zi|ci + cx ⎠

(8)

Cim

where Ci and are the concentrations of the mobile co-ions (ions of the same charge as the membrane charge) in the solution and in the membrane, Cxm is the membrane stationary charge density, and Zi and Zj are the charges of the co-ion and counterions, respectively. We note that CNT porins embedded in a lipid membrane differ in a few aspects from the classic uniformly charged porous membrane of the Donnan model; thus its use requires making several assumptions. First, the pore is very short (ca. 5−20 nm) and the transport through it highly efficient and nearly frictionless.5 If we also take into account that the concentration range we investigated corresponds to relatively low salt concentrations, it will be reasonable to assume that Cim ≪ Cxm at all times, and thus we could neglect the contribution from the mobile co-ions in the membrane. Previous work studying macroscopically long channels in a CNT membrane geometry estimated the effective stationary membrane charge density as 3 mM.24 Since the charge in the CNT pores is localized at the ends of the pore, and those pores were 3 μm long, that charge density effectively corresponded to the contribution of one CNT end. In our case, the length of the E

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(16) Saito, R.; Dresselhaus, G.; Dresselhaus, M. S. Physical Properties of Carbon Nanotubes; Imperial College Press: London, 1998. (17) Granath, K. A. J. Colloid Sci. 1958, 13, 308−328. (18) Mathai, J. C.; Tristram-Nagle, S.; Nagle, J. F.; Zeidel, M. L. J. Gen. Physiol. 2008, 131 (1), 69−76. (19) Traikia, M.; Warschawski, D. E.; Recouvreur, M.; Cartaud, J.; Devaux, P. F. Eur. Biophys. J. 2000, 29 (3), 184−195. (20) Tunuguntla, R.; Noy, A. 2014, unpublished data. (21) Walstra, P. Food Science and Technology; CRC Press: Boca Raton, FL, 2002; Vol. 121. (22) Schaep, J.; Bruggen, B. V. d.; Vandecasteele, C.; Wilms, D. I. Sep. Purif. Technol. 1998, 14, 155−162. (23) Perkin, S.; Goldberg, R.; Chai, L.; Kampf, N.; Klein, J. Faraday Discuss. 2008, 141, 399. (24) Fornasiero, F.; Park, H.-G.; Holt, J. K.; Stadermann, M.; Grigoropoulos, C.; Noy, A.; Bakajin, O. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (45), 17250−17255. (25) Goldberg, R.; Chai, L.; Perkin, S.; Kampf, N.; Klein, J. Phys. Chem. Chem. Phys. 2008, 10 (32), 4939−4945.

ASSOCIATED CONTENT

S Supporting Information *

Experimental details, TEM image of CNT, Raman spectrum of CNT, histograms of liposome size distribution, and size change of liposomes as a function of osmolyte concentration. 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 U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering. The LDRD program at LLNL supported CNT porin synthesis. Work at the Lawrence Livermore National Laboratory was performed under the auspices of the U.S. Department of Energy under Contract DE-AC5207NA27344. Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC0205CH11231. R.T. acknowledges support from the Lawrence Scholar program at LLNL. We thank Dr. Frances I. Allen (LBNL) for the help with electron microscopy images of CNT porins.

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