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IEEE TRANSACTIONS ON NANOTECHNOLOGY, VOL. 15, NO. 1, JANUARY 2016
Novel Nano-Device to Measure Voltage-Driven Membrane Transporter Activity Rikiya Watanabe, Naoki Soga, and Hiroyuki Noji
Abstract—The use of an arrayed lipid bilayer chamber system (ALBiC) enables highly sensitive quantitative analysis of membrane transporter activity, a major target of pharmaceutical research. Although membrane voltage is one of the main driving forces of transporters, the versatility of ALBiC is limited to transporter assays in the absence of membrane voltage, owing to technical limitations with voltage modulation. Here, we report a novel nano-device based on ALBiC (el-ALBiC) containing sub-million lipid bilayer chambers, each equipped with nano-sized electrodes. Since the nano-sized electrodes enable quantitative modulation of membrane voltage, the el-ALBiC is capable of performing highly sensitive detection of the voltage-driven membrane transporter activity. Thus, the novel nano-device el-ALBiC extends the versatility of ALBiC and has potential for further analytical and pharmacological applications, such as drug screening. Index Terms—Femtoliter chamber, membrane transporter, membrane voltage, phospholipid-bilayer membrane.
Fig. 1. ALBiC with nano-sized electrode (el-ALBiC). (a) A bright-field image of the through-hole structures on a fabricated nano-device. (b) Schematic illustration of el-ALBiC that displays through-hole structures (φ = 3 μm) on a double layer of fluororesin (h = 500 nm) and Au (h = 500 nm). Individual orifices on through-hole structures are sealed with lipid-bilayer membranes.
I. INTRODUCTION HE membrane proteins responsible for the transport of substrate molecules across cell membranes, i.e., membrane transporters, play pivotal physiological roles such as nutrient uptake, secretion of signaling molecules, and energy transduction [1]. Owing to their physiological importance, transporters have frequently been the targets of pharmaceutical research [2], [3]. A variety of massively parallel and highly quantitative systems for the analysis of transporters has been developed to contribute to high-throughput drug discovery [4], [5]. One of the most sensitive systems for transporter analysis is the arrayed lipid bilayer chamber system (ALBiC) [6]–[8], which measures the accumulation of transported substrate in femtoliter chambers with high sensitivity. ALBiC enables highthroughput analysis of transporter activity at the single-molecule level [6], [8]; however, it has not been utilized for a majority of membrane transporters, including voltage-driven transporter
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Manuscript received August 24, 2015; accepted October 31, 2015. Date of publication November 5, 2015; date of current version January 6, 2015. The work of R. Watanabe was supported by Grant-in-Aid for Scientific Research 15H05591 and 15H01312 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and Precursory Research for Embryonic Science from the Japan Science and Technology Agency. The review of this paper was arranged by Associate Editor L. G. Villanueva R. Watanabe is with the Department of Applied Chemistry, School of Engineering, University of Tokyo, Bunkyo-ku 113-8656, Japan, and also with the PRESTO, Japan Science and Technology Agency, Bunkyo-ku 113-8656, Japan (e-mail:
[email protected]). N. Soga and H. Noji are with the Department of Applied Chemistry, School of Engineering, University of Tokyo, Bunyo-ku 113-8656, Japan (e-mail:
[email protected];
[email protected]). Color versions of one or more of the figures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identifier 10.1109/TNANO.2015.2498167
proteins, owing to technical difficulties in modulating the voltage applied across lipid-bilayers on an ALBiC. In this study, we address this issue by developing a novel ALBiC system with nano-sized electrodes (el-ALBiC) to achieve highly sensitive detection of voltage-driven membrane transporter activity in a high throughput manner. II. MATERIALS AND METHODS A. Nano-Device Fabrication A nano-device containing 100000 femtoliter chambers equipped with nano-sized electrodes was fabricated using conventional vacuum metal deposition and photolithography. A clean cover glass (32 mm × 24 mm) was coated with 500nm-thick Au with 20-nm-thick chromium adhesion layers in a vacuum evaporator (VTR-350M, ULVAC, Japan). A carbonfluorine hydrophobic polymer (CYTOP CTX-809M, Asahiglass, Japan) was spin-coated onto the Au layer at 4000 r/min for 30 s and then incubated for 1 h at 180 °C. The thickness of the CYTOP layer was 500 nm. Photolithography was conducted using a positive photoresist (AZP4903, AZ Electronic Materials, Japan) to pattern mask structures onto the CYTOP layer. The resist-patterned substrate was dry-etched with O2 plasma using a reactive-ion etching system (RIE-10NR, Samco, Japan) to expose the Au surface. The substrate was then cleaned and rinsed with acetone and ethanol to remove the photoresist layer remaining on the substrate. Regions bare of Au and Cr were etched using Au and Cr etchants, respectively. A schematic illustration of the nanodevice is shown in Fig. 1. The Au and fluororesin layers are
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WATANABE et al.: NOVEL NANO-DEVICE TO MEASURE VOLTAGE-DRIVEN MEMBRANE TRANSPORTER ACTIVITY
Fig. 2. Formation of an asymmetric lipid-bilayer membrane. Schematic illustration of asymmetric lipid bilayer formation. First, an aqueous solution is injected into the device through an access port. Second, the lipid solution containing fluorescent-labeled lipids (fluorescein-DHPE) is injected. Third, another lipid solution without fluorescein-DHPE is injected. Finally, a second aqueous solution is injected to flush the second lipid solution. During this process, asymmetric lipid bilayers are formed on the orifices of the micro-chambers. The inset is the fluorescence image of fluorescein-DHPE on the asymmetric lipid bilayers.
used as an electrode to modulate membrane voltage and to support the lipid bilayer membrane, respectively. The through-hole structures on these layers are used as femtoliter chambers to detect biological reactions with high sensitivity as previously reported [9]. For the exchange of the sample solution, a flow cell was constructed from the fabricated nano-device, spacer sheet (FrameSeal SLF0601, Bio-Rad, USA), and CYTOP-coated glass block (CYTOP thickness: ∼1 μm), which had an access port for sample injection and an Au electrode for voltage modulation. B. Asymmetric Lipid Bilayer Formation Asymmetric lipid-bilayers were formed on each individual orifice of the femtoliter chambers via sequential injection of liquids by a micro pipette (see Fig. 2) [7]. First, an aqueous solution was infused into the flow cell, filled femtoliter chambers. Second, a lipid solution containing 20 μg·mL−1 lipid (a 1:10:10-by-weight mixture of fluorescein-DHPE, DOPE, and DOPG) in chloroform was infused to flush away the first aqueous solution. After the flushing, water-in-chloroform droplets were formed in the individual chambers. Third, a second lipid solution containing 2 mg·mL−1 lipid (a 1:1 mixture by weight of DOPE and DOPG) in chloroform was infused. Finally, a second aqueous solution was infused to flush the second lipid solution. In this process, the hydrocarbon tails of the residual second lipids zipped with those of the first lipids to form a membrane. Uniform-sized planar bilayers with an asymmetric lipid composition, wherein fluorescein-DHPE was located only in the inner leaflet of the bilayer, were thus formed on the opened orifice of the femtoliter chambers (see Fig. 2). C. Fluorescence imaging Fluorescence time-lapse recordings were obtained using a confocal microscope system with a 60× objective lens and photomultiplier tubes (A1R, Nikon, Japan). A laser was used to
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Fig. 3. Evaluation of membrane voltage using DiBAC4 . (a) Schematic illustration of membrane voltage monitoring. A membrane voltage indicator, DiBAC4 , was encapsulated in the chamber. Membrane voltage was modulated using electrodes on a femtoliter chamber and a top glass block. (b) Fluorescence intensity of DiBAC4 (green) against applied voltage using nano-sized electrodes (black). Error bars represent the standard deviations of 36 wells.
excite fluorescein-DHPE and DiBaC4 (λex = 488 nm). Analysis was performed using NIS Elements software (Nikon, Japan). D. Transporter Assay Using Fo F1 ATP Synthase A mutant Fo F1 ATP synthase (Fo F1 ) from E. coli that lacks two α-helices in the C-terminus of the ε subunit and has a His3 tag and a biotin-binding domain in the c and β subunits, respectively, was expressed and purified as previously reported [10]. To examine proton transport mediated by Fo F1 , a voltage-driven membrane transporter, the flow cell and femtoliter chambers were filled with buffer A (100 μM MES [pH 6.0] and 20 mM NaCl) and buffer A containing a solubilized Fo F1 . After the formation of the lipid bilayers, the solubilized Fo F1 was spontaneously reconstituted into the lipid bilayers of the ALBiC. Fluorescence images of fluorescein-DHPE on the asymmetric bilayers were recorded at intervals of 50 s. III. RESULTS A. Evaluation of Membrane Voltage The sealing of asymmetric lipid bilayers is extremely tight on ALBiC, i.e., the leakage of ions across the lipid bilayers is almost negligible for 2 h [6], which is a prominent advantage of ALBiC among the artificial lipid bilayer systems developed thus far. Therefore, ALBiC has been regarded as a suitable platform for quantitatively modulating the voltage applied across a lipid bilayer membrane. We attempted to evaluate the membrane voltage modulated by nano-sized electrodes using el-ALBiC. For this evaluation, the femtoliter chambers were filled with a solution containing DiBAC4 [11], a fluorescent indicator which increases fluorescence intensity in proportion to the amplitude of membrane voltage (see Fig. 3(a)). To monitor the membrane voltage, the fluorescence signal of DiBaC4 located proximate to the lipid bilayers was selectively recorded using a confocal microscope with a focal depth of ∼100 nm. As shown in Fig. 3(b), the fluorescence intensity of DiBAC4 changed following voltage modulation using nano-sized electrodes. In addition, the intensity change of DiBaC4 was highly reproducible for more than
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as a change in the fluorescence intensity of fluorescein-DHPE, which increases in alkaline conditions (see Fig. 4(b)). When a membrane voltage (200 mV) was applied to induce Fo F1 to transport protons from the inside to the outside of the femtoliter chambers, the fluorescence intensity of fluorescein-DHPE increased and reached a plateau at approximately 3000 s (see Fig. 4(c) and (d)), which corresponds to a pH of approximately 7.7 (see Fig. 4(d)). The generated transmembrane proton gradient (ΔpH) was approximately 2.0, which was similar to the membrane voltage applied by the nano-sized electrodes. As a control experiment, we conducted the same assay in the absence of Fo F1 . As expected, no fluorescence intensity changes were observed (see Fig. 4(d), gray), supporting the validity of the voltage-driven transport assay. Thus, the el-ALBiC achieved highly sensitive detection of voltage-driven membrane transport, facilitated by Fo F1 . IV. CONCLUSION Fig. 4. Voltage-driven transport of protons by Fo F1 . (a) Schematic illustration of voltage driven transport of Fo F1 reconstituted in the asymmetric lipid bilayer membrane in which fluorescein-DHPE is located on the inner leaflet for pH monitoring. Fo F1 transports protons from the inside to the outside of the femtoliter chambers by membrane voltage. (b) Fluorescence intensity of fluorescein-DHPE against pH. (c) Fluorescence images of the proton transport of Fo F1 in the chambers. The images were recorded just after applying a membrane voltage of 200 mV (left panel) and 4000 s later (middle panel). The right panel (Diff.) represents the intensity difference between the left and middle panels as a color gradient. (d) Time course of fluorescence intensity of fluorescein-DHPE in the presence (green) or absence (gray) of Fo F1 . Error bars represent the standard deviations of 405 (green) and 636 wells (gray).
10 min. Thus, the long-term quantitative modulation of membrane voltage was demonstrated using nano-electrodes on the el-ALBiC. B. Measurement of Voltage-Driven Membrane Transport Next, we explored the potential of the transporter assay using the voltage-driven transporter protein Fo F1 [12]. This enzyme mediates the in vivo energy conversion between the phosphoryl group transfer potential of ATP and the proton motive force (pmf) across membranes, which comprises membrane voltage and transmembrane proton gradient, via mechanical rotation of the inner rotor subcomplex [13]–[18]. When the pmf is small or diminishes, Fo F1 hydrolyzes ATP, actively pumping protons to form pmf. Under physiological conditions where pmf is sufficient, proton flux decreases pmf, pushing Fo F1 to work in the reverse direction and inducing ATP synthesis. The performance of Fo F1 in energy conversion surpasses that of artificial engines [19], [20]; therefore, it has been a longstanding goal of many researchers to understand the operating principles of Fo F1 . To measure voltage-driven membrane transport, Fo F1 was reconstituted into asymmetric lipid-bilayer membranes on elALBiC, where fluorescein-DHPE was located on the inner leaflet of bilayers, and the femtoliter chambers were filled with a buffer (pH 6.0) (see Fig. 4(a)). In this setup, the proton transport from the femtoliter chamber mediated by Fo F1 is detectable
In this study, we constructed a novel nano-device (el-ALBiC) for quantitative modulation of membrane voltage on an ALBiC. Moreover, we demonstrated highly sensitive measurements of voltage-driven membrane transport facilitated by Fo F1 . Voltagedriven membrane transport is a major target of pharmaceutical research, and el-ALBiC thus largely extends the versatility of the ALBiC system and has potential for further analytical and pharmacological applications. REFERENCES [1] M. H. Saier, Jr., “A functional-phylogenetic classification system for transmembrane solute transporters,” Microbiol. Mol. Biol. Rev., vol. 64, pp. 354–411, Jun. 2000. [2] C. International Transporter, K. M. Giacomini, S. M. Huang, D. J. Tweedie, L. Z. Benet, K. L. Brouwer et al., “Membrane transporters in drug development,” Nature Rev. Drug Discovery, vol. 9, pp. 215–236, Mar. 2010. [3] G. J. Kaczorowski, O. B. McManus, B. T. Priest, and M. L. Garcia, “Ion channels as drug targets: The next GPCRs,” J Gen. Physiol., vol. 131, pp. 399–405, May. 2008. [4] J. Dunlop, M. Bowlby, R. Peri, D. Vasilyev, and R. Arias, “Highthroughput electrophysiology: An emerging paradigm for ion-channel screening and physiology,” Nature Rev. Drug Discovery, vol. 7, pp. 358– 368, Apr. 2008. [5] M. Zagnoni, “Miniaturised technologies for the development of artificial lipid bilayer systems,” Lab Chip., vol. 12, pp. 1026–1039, Mar. 2012. [6] R. Watanabe, N. Soga, D. Fujita, K. V. Tabata, L. Yamauchi, S. Hyeon Kim et al., “Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity,” Nature Commun., vol. 5, p. 4519, 2014. [7] R. Watanabe, N. Soga, T. Yamanaka, and H. Noji, “High-throughput formation of lipid bilayer membrane arrays with an asymmetric lipid composition,” Sci. Rep., vol. 4, p. 7076, 2014. [8] N. Soga, R. Watanabe, and H. Noji, “Attolitre-sized lipid bilayer chamber array for rapid detection of single transporters,” Sci. Rep., vol. 5, p. 11025, 2015. [9] S. Sakakihara, S. Araki, R. Iino, and H. Noji, “A single-molecule enzymatic assay in a directly accessible femtoliter droplet array,” Lab Chip., vol. 10, pp. 3355–3362, Dec. 2010. [10] R. Iino, R. Hasegawa, K. V. Tabata, and H. Noji, “Mechanism of inhibition by C-terminal alpha-helices of the epsilon subunit of Escherichia coli Fo F1 -ATP synthase,” J. Biol. Chem., vol. 284, pp. 17457–17464, Jun. 2009. [11] D. E. Epps, M. L. Wolfe, and V. Groppi, “Characterization of the steadystate and dynamic fluorescence properties of the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBaC4 (3)) in model systems and cells,” Chem. Phys. Lipids., vol. 69, pp. 137–150, Feb. 1994. [12] M. Allegretti, N. Klusch, D. J. Mills, J. Vonck, W. Kuhlbrandt, and K. M. Davies, “Horizontal membrane-intrinsic alpha-helices in the
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Rikiya Watanabe received the B.S. degree from Waseda University, Tokyo, Japan, in 2004, the M.S. degree from the University of Tokyo, Tokyo, in 2006, and the Ph.D. degree in single-molecule biophysics from Osaka University, Osaka, Japan, in 2009. He was a Postdoctoral Fellow at International Society for Inventory Research, Osaka University, before joining the Department of Applied Chemistry, University of Tokyo, as an Assistant Professor. His research interests include mechanisms conserved among biomolecular machines, mainly through the development of novel single-molecule techniques, including microsystems fabricated at the micron- or nano-scale. His current focus is on using membrane transporters to extend the versatility of single-molecule techniques for further analytical and pharmacological applications, such as drug screening. He received the Young Scientists’ Prize from the Minister of Education, Culture, Sports, and Science and Technology of Japan in 2015.
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Naoki Soga received the B.S., M.S., and Ph.D. degrees in biophysics from Waseda University, Tokyo, Japan, in 2008, 2010, and 2013, respectively. He was a Postdoctoral Fellow of the Japan Society for the Promotion of Science in the Department of Applied Chemistry,University of Tokyo, Tokyo. His research interests include elucidating the structure-function relationship of membrane transporters, mainly through biophysical and biochemical approaches at the single-molecule level. He has received an Early Research Award from the Foundation for the Promotion of Engineering Research of Japan in 2015.
Hiroyuki Noji received the Ph.D. degree from the Tokyo Institute of Technology, Tokyo, Japan, in 1997. After the postdoctoral fellowship at Keio University, he was appointed as an Associate Professor at the Institute of Industrial Science, University of Tokyo, in 2001. In 2005, he moved to the Institute of Scientific and Industrial Research, Osaka University as a Full Professor. Since 2010, he has been a Professor at the Department of Applied Chemistry, University of Tokyo, Tokyo. He has been studying the chemomechanical coupling mechanisms of Fo F1 ATP synthase using single-molecule techniques. He is also an Inventor of the femtoliter chamber array system for single-molecule enzymatic assays that are currently used in single-molecule digital enzyme-linked immunosorbent assay. He received the Yomiuri Gold Medal in 2015, the Nakatani Incentive Prize in 2015, the Inoue Science Research Award in 2013, the Yamazaki Teiichi Prize in 2013, the JSPS Prize in 2006, the Tejima Prize in 1999, and the Grand Prize, Amersham Pharmacia Biotech and Science Prize for Young Scientists, in 1998.
