Article pubs.acs.org/Langmuir
Electrochemical Impedance Spectroscopy and Atomic Force Microscopic Studies of Electrical and Mechanical Properties of NanoBlack Lipid Membranes and Size Dependence Zai-Wen Zhu, Yang Wang, Xuan Zhang, Chun-Feng Sun, Mian-Gang Li, Jia-Wei Yan, and Bing-Wei Mao* State Key Laboratory of Physical Chemistry of the Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China S Supporting Information *
ABSTRACT: We present electrochemical impedance spectroscopic (EIS) and two-chamber AFM investigations of the electrical and mechanical properties of solvent-containing nano-BLMs suspended on chip-based nanopores of diameter of 200, 400, and 700 nm. The chips containing nanoporous silicon nitride membranes are fabricated based on low-cost colloidal lithography with low aspect ratio of the nanopores. BLMs of DPhPC lipid molecules are constructed across the nanopores by the painting method. Two equivalent circuits are compared in view of their adequacy in description of the EIS performances of the nano-BLMs and more importantly the structures associated with the nanoBLMs systems. The BLM resistance and capacitance as well as their size and time dependence are studied by EIS. The breakthrough forces, elasticity in terms of apparent spring constant, and lateral tension of the solvent-containing nanoBLMs are investigated by AFM force measurements. The exact relationship of the breakthrough force of the nano-BLM as a function of pore size is revealed. Both EIS and AFM studies show increasing lifetime and mechanical stability of the nano-BLMs with decreasing pore size. Finally, the robust 200 nm diameter nanopores are used to accommodate functional BLMs containing DPhPC lipid molecules and gramicidins by using a painting method with drop of mixture solutions of DPhPC and gramicidins. EIS investigation of the functional nano-BLMs is also performed. bonate membranes,16 and glass nanopores.17,18 It has been proved that the lifetime of BLMs is much longer than that of BLMs created over the micrometer-sized apertures in Teflon or other plastic septa. However, the high aspect ratio of the alumina nanopores is disadvantageous to mass transfer of matters. To solve the problem, thin layers of silicon nitride (Si3N4) nanopores fabricated by means of direct electron-beam writing19 or focused ion beam lithography20 are used for the formation of BLMs, yet with high cost. To investigate the electrical properties of pore-spanning nano-BLMs, the electrochemical impedance spectroscopy (EIS) is one of the commonly employed techniques.14,19−22 An appropriate equivalent circuit that can describe all EIS behaviors of a system is important, relying on which the electrical properties of pore-spanning nano-BLMs including, for example, the membrane resistance and the specific membrane capacitance, can be extracted. By far, some equivalent circuits have been used for fitting EIS data, but fitting to all types of impedance representations of a system requires careful refinement of the equivalent circuits.
1. INTRODUCTION Biological membranes play a key role in signal transduction and metabolism owing to the functional proteins embedded in the membranes. To study the function of biological membranes, bionic membranes of lipid bilayers are usually used in addition to native membranes.1,2 Black lipid membrane (BLM), as an ideal biomimetic model for complex biological membranes, provides a platform for investigating the function of selected protein-embedding biomembrane such as ion channels, ion pumps, and receptors.3−6 Traditional suspended BLMs are formed across micrometersized apertures in Teflon or other plastic septa by using the painting or folding method.7−9 The two chambers thus formed are biologically more favorable environments for BLMs and allow convenient investigations of the properties of BLMs in different surroundings via change of experimental conditions such as concentration and constituent of solutions on one or both chambers. However, one problem encountered with such a configuration is the short-term lifetimes of the BLMs. Great efforts have been devoted to introduce BLMs on solid substrates to form so-called supported membranes,10−13 which however lose the advantages of two-chamber configurations in terms of flexibility of experimental control and comparability with real biological systems. Recently, BLMs are spanned over anodized porous alumina,14,15 porous polycar© 2012 American Chemical Society
Received: July 27, 2012 Revised: September 14, 2012 Published: September 18, 2012 14739
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layer of SiO2 was grown on the side walls of the trapezia-shaped cavities under the exposure of 100 W oxygen plasma for 10 min. The nanopores were formed with size of 200, 400, and 700 nm, in accordance with the initial template PS particles of 200, 400, and 800 nm, respectively. Figure 1A shows a schematic illustration of the structure of the suspended nanoporous Si3N4 membranes. Since the depth of the
Atomic force microscopy (AFM) is a recognized powerful tool in the investigation of the mechanical and topographic properties of pore-spanning BLM under physiological environments or other optional conditions.15,23−26 In these studies, AFM works in a two-chamber setup, which is composed of a pore-spanning BLM and the two aqueous compartments separated by the BLM. It has been demonstrated that the mechanical stability of BLMs increases with decrease of the pore size.15,25,26 However, the relationship between breakthrough force and aperture size of BLMs has not been obtained. On the other hand, as the stability of BLMs increases along with the reduction of aperture size, the reconstitution of proteins into BLMs becomes more difficult. Although using porous substrates for BLMs can in some measure resolve the problem, it is time-consuming to wait for the successful reconstitution of proteins to complete, and the time required is dependent on the density and size of the pores. Herein, we present EIS and AFM studies of electrical and mechanical properties of nano-BLMs which are suspended on the nanopores of several different diameters embedded in the Si3N4 membranes on a chip. The chips and nanopores are fabricated by colloidal lithography-based approach,27 and therefore the aspect ratio of the nanopores is low and the cost of the chip is low as well. Within the framework of the present paper, the nanopores of 200, 400, and 700 nm in diameters are fabricated, based on which BLMs are constructed. Two equivalent circuits are compared in view of their adequacy in description of the EIS performance of the nano-BLMs and relationship of the breakthrough force of the nano-BLM as a function of pore size is revealed by AFM force measurement with careful analysis. Both studies show increasing long-term lifetime and high mechanical stability of the nano-BLMs with decreasing pore size. Finally, the nanopores of 200 nm are used to accommodate functional BLMs containing DPhPC lipid molecules and gramicidins, which are investigated by EIS.
Figure 1. (A) Schematic illustration of the suspended nanoporous Si3N4 membranes. (B) Bottom-view SEM image of Si3N4 membranes viewed from the side. (C) Top-view SEM images of nanopores of Si3N4 membranes with different diameters. nanopores, which is the thickness of the Si3N4 layer, is equal to or smaller than the pore diameters, the nanopores have low aspect ratios ranging from 1 to 0.288. A bottom-view SEM image (Figure 1B) shows the four trapezia-shaped cavities with smooth exposed walls of Si (111) faces. Top-view SEM images of nanopores of different diameters are shown in Figure 1C. Each chip consists of four units of the trapezia-shaped cavities, the size of each cavity being 100 × 100 μm2. The total numbers of nanopores contained in the four cavities are ca. 1.51 × 104, 4.02 × 103, and 1.63 × 103 nanopores with porosities of 1.18%, 1.28%, and 1.57% for 200, 400, and 700 nm diameter nanopores, respectively. 2.3. Formation of Nano-BLMs. To ensure the hydrophobicity for subsequent BLM formation, the chips were hydrophobically silanized with F13-OTCS (1 μL) by chemical vapor deposition28,29 in a vacuum container. Then, the chips were dipped into toluene for 1 h, followed by rinsing with ethanol and water and finally air-drying. Pore-spanning nano-BLMs were constructed by the painting method9 following the procedure by White and co-workers18 by applying a drop of ∼2 μL of DPhPC lipid molecules dissolved in decane (10 mg mL−1) across the nanoporous Si3N4 membranes from the flat side of the chip with plastic pipet. The excess solvent was blown away by a stream of N2 gas so that a layer of lipid molecules was formed onto the hydrophobically silanized nanoporous Si3N4 membrane surface. It is noted that such a lipid-modified free nanoporous Si3N4 membranes was also used for EIS measurement for comparison. Two chambers were formed, which were separated by the chip. The chamber with the cavity side of the chip was filled 10 mM PBS buffer solution containing 0.1 M KCl (pH 7.4). Finally, another drop of ∼2 μL of the same lipid/decane solution was applied followed by a drop of the same PBS solution across the flat side of the chip in the other chamber. The lipid/decane solution was swept laterally across the chip to promote BLM formation at the nanopores. A schematic illustration of solvent-containing porespanning nano-BLMs is shown in Figure 2. For the formation functional nano-BLMs, gramicidin with lipid-to-peptide mass ratio of 34:1 was dissolved in decane. 2.4. Electrical Recording and EIS Measurements. All of electrical recordings were performed on Autolab PGSTAT128N (Eco Chemie B.V.) with a two-electrode (Ag/AgCl) cell. Single-channel recordings of gramicidin were monitored upon current stepping with corresponding i−t curves recorded at sampling frequency of 200 Hz
2. EXPERIMENTAL SECTION 2.1. Materials. 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (Avanti Polar, Lipids, Inc.), F13-OTCS (tridecafluoro1,1,2,2-tetrahydrooctyltrichlorosilane) (ABCR, Germany), and ndecane (Alfa Aesar) were used to construct BLMs. Cr (Alfa Aesar), poly(diallyldimethylammonium) chloride (PDDA) 20% w/w, MW = 100 000−200 000 (Alfa Aesar), and negatively charged polystyrene colloidal particles (PS) (200, 400, and 800 nm in diameter, Duke) were used to fabricate nanoporous silicon nitride membranes with diameter of 200, 400, and 700 nm. Gramicidin (a mixture of gramicidins A, B, C, and D) obtained from Sigma-Aldrich was used to form functional pore-spanning nano-BLMs. Milli-Q (>18 MΩ cm) water was used to prepare the solutions. 2.2. Fabrication of Nanoporous Silicon Nitride Membranes Chips. The nanoporous silicon nitride membranes were fabricated by colloidal lithography-based approach as has been reported previously.27 In brief, 4 in. n-type Si (100) wafers of 320 μm thick were used with both sides grown successively with 300 nm thick silicon oxide (SiO2) and 200 nm thick silicon nitride (Si3N4) layers. On one side of the Si wafer, negatively charged PS colloidal particles of 200, 400, and 800 nm in diameter, respectively, were introduced to the surface of Si3N4 layer via electrostatic self-assembly, which was followed by deposition of a thin Cr mask layer. After stripping off the PS particles, inductively coupled plasma (ICP) etching was applied to remove the Si3N4 layer of the exposed regions. On the other side of the wafer, bulk Si and SiO2 layer were etched to create four trapeziashaped cavities by standard Si fabrication procedures including photolithography, Cr etching, anisotropic Si wet etching, and BOE (buffered hydrofluoric acid) wet etching, resulting in randomly distributed nanopores on the silicon nitride layers. Finally, a thin 14740
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Figure 2. Schematic illustration of solvent-containing pore-spanning nano-BLM. and filtering constant of 0.1 s. EIS were recorded mainly at zero-bias in a frequency range of 10−2−106 Hz with equally scaled data points per decade with the signal amplitude of 10 mV. Measurements at nonzero bias of 0.05 and 0.10 V were also made for comparison purposes. 2.5. AFM Measurements. The mechanical properties of porespanning nano-BLMs were investigated under 0.1 M KCl PBS buffer (pH 7.4) in a two-chamber AFM setup using a MI 5500 AFM (Agilent Technologies, Inc.) equipped with top-view optics. The images and force curves were obtained under contact mode using gold-coated silicon tips (CSG11/Au, NT-MDT, Russia) which have nominal spring constants of 0.03−0.2 and 0.01−0.08 N m−1 and tip curvature radii of ca. 35 nm and half cone angles of ≤11°. The suspended nanoporous silicon nitride membranes were observable with the topview optic, as shown in Figure S1, which facilitates the orientation of AFM tips to the BLMs suspended on the silicon nitride nanopores. The exact spring constants of the cantilevers were calibrated by the thermal noise method.
Figure 3. (A) AFM image of 200 nm diameter pore-spanning nanoBLMs recorded with increasing force load from 100 pN for lower part and to 270 pN for upper part (x, y scale: 5 μm). (B) Indention profile along the black dashed line shown in the AFM image.
nanopores with diameter of 200 nm. The cyclic voltammograms (CVs, Figure 4S) indicate that the resistance increases by 5 orders of magnitude for nanopores with BLMs, in comparison with the much lower resistance of the free nanopores. The EIS for 200 nm diameter pores measured at zero bias are displayed in Bode (Figure 4A,B), Nyquist (Figure 4C), and admittance (Figure 4D) representation in a frequency range of 10−2−106 Hz. The EIS of nano-BLMs at bias of 0.05 and 0.10 V were also made (Figure S5A,B), but no obvious changes were observed. Two different equivalent circuits are used to fit the data. The equivalent circuit one as depicted in Figure 4E is commonly used in the literature, wherein the resistance of the electrolyte solution Rel is in series with the parallel RC element (Rtotal and Ctotal), where Rtotal and Ctotal are the total contribution of resistance and capacitance, respectively, of Si3 N4 membranes and free nanopores or nano-BLMs, respectively. A good fitting of data is obtained for the impedance plot of absolute values (Figure 4A) while small deviation presents at high frequencies of the phase angle plot (Figure 4B), which are similar to the results shown in other reports.19 However, obvious fitting deviation is seen for the admittance plot (red lines in Figure 4D), indicating that the equivalent circuit one is not adequate to describe the structure of the BLMs and their surroundings. In practice, DPhPC is a phosphocholine kind of phospholipid molecules which have been proved to be negatively or positively charged depending on environment.31 Therefore, an electric double layer is expected to be present at the liquid−BLM interface,32 at least during ac impedance measurements. Here, we propose the equivalent circuit two, also depicted in Figure 4E, wherein an additional parallel RC element (Rdl and Cdl) is introduced in series with the equivalent circuit one. The Rdl and Cdl are the resistance and capacitance
3. RESULTS AND DISCUSSION 3.1. AFM Images of Nano-BLMs. The silanized chips show water-contact angle of 102° in comparison with that of 46° of unsilanized ones (Figure S2). On the other hand, the mean roughness of the silanized surface is 0.205 nm, which is even smaller than that of the unsilanized surface of 0.485 nm (Figure S2C,D). These parameters indicate that the silanization process make the surface sufficiently hydrophobic without degrading the surface smoothness, which is desirable for BLM formation as well as subsequent AFM characterization. Figure 3A is the AFM image of the pore-spanning nano-BLMs on 200 nm diameter pores recorded upward with increasing force load from 100 pN for lower part and to 270 pN for upper part of the image. The indention profile (Figure 3B) across the three holes shows different tip indention into the pore-spanning nanoBLMs from 15 nm at 100 pN and to 28 nm at 270 pN. These values are much smaller than those from uncovered pores, which can reach as high as 92 nm under a force load of 100 pN (Figure S3A). This means that nanopores with tip indention less than ca. 90 nm are covered with BLMs. Furthermore, the BLMs on the 200 nm diameter pores can sustain tip rastering with force up to at least 270 pN. 3.2. Impedance Investigation of Nano-BLMs. The electrical properties of nano-BLMs, described by such as membrane resistance (Rm) and membrane capacitance (Cm), can be investigated by electrochemical methods. The successful formation of nano-BLMs is approved by detecting a value of the resistance above 1.0 GΩ.22,30 By means of cyclic voltammetry and EIS, the electrical properties are investigated in 0.1 M KCl solution containing 10.0 mM PBS buffer (pH 7.4) before and after the formation of suspended nano-BLMs on 14741
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and Ctotal is the sum of capacitance from the nano-BLMs and the lipid-modified Si3N4 membrane support, while Rdl and Qdl are the resistance and CPE associated with BLM−electrolyte interface. Fitting the data with equivalent circuit two for 200 nm diameter nano-BLM results in Rdl of 1.8 KΩ and Y0(Qdl) of 0.855 nF sn−1 (ndl = 0.609) as well as Rtotal and Ctotal of 53.6 GΩ and 1.912 nF, respectively. The capacitance of the nano-BLMs can in principle be obtained from the formula Ctotal = CBLM + Cm‑SiN, where Cm‑SiN is the capacitance of the lipid-modified porous Si 3 N 4 membranes. However, separate EIS measurements performed to the lipid-modified nanoporous Si3N4 membranes give a capacitance Cm‑SiN of 1.864 nF (equivalent of specific capacitance C′m‑SiN of 4.78 μF cm−2), which is very close to Ctotal (1.912 nF) of the suspended nano-BLM system. Considering the low porosity of the Si3N4 membranes employed in the present work (
