Tsuneo Urisu, Md. Mashiur Rahman, Hidetaka Uno, Ryugo Tero, PhD, Yoichi Nonogaki, “Formation of high resistance supported lipid bilayer on the surface of Si substrate with micro electrodes” Nanomedicine 1 (2005) 317-322.
Published on: Mar 3, 2016
Transcripts - Nanomedicine 2005
Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317 – 322 www.nanomedjournal.com Experimental Formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes Tsuneo Urisu, PhD,4 Md. Mashiur Rahman, Hidetaka Uno, Ryugo Tero, PhD, Yoichi Nonogaki, PhD Department of Vacuum UV Photoscience, Institute for Molecular Science, The Graduate University for Advanced Studies, Myodaiji, Okazaki, Japan Received 28 August 2005; accepted 10 October 2005Abstract We have developed two basic technologies for fabrication of supported planar lipid bilayer membrane ion channel biosensors: a defect-free lipid bilayer formation on the substrate surface with electrode pores and a patterning technique for the hydrophobic self-assembled-monolayer to form the guard ring that reduces the lipid bilayer edge-leak current. The importance of the supported- membrane structure to achieve low noise and high-speed performance is suggested on the basis of the observed relation between the single-ion-channel current noise and the pore size. D 2005 Published by Elsevier Inc.Key words: Supported membrane; Lipid bilayer; Membrane protein; Gramicidin; Self-assembled monolayer; Ion channel; Biosensor Signal transmission and processing in the living body many applications . The ion channel and/or receptor-takes place via life body molecules, specifically neuronal reconstructed lipid bilayer as a key component of thetransmitter molecules, as the signal carrier. It is a unique detector of neuronal transmitter molecules is useful notcommunication system comparable to electrical and optical only as a biosensor but also in an in vitro study of cellcommunication, which use electrons and photons, respec- membrane biological functions. In reports of single-ion-tively, as the signal carriers. Thus it can be called channel biosensors, membrane protein reconstructed lipidbmolecular communication.Q Neurotransmitter molecules bilayers are suspended in a small pore on the substratedischarged from the presynaptic membrane are received by made by Si, SiO2, glass, or other materials [2-13].ion channels on the surface of postsynaptic membranes, However, technological problems still exist with stabilityand the electrical signal (ie, the depolarization of the of the single-ion-channel biosensor. Development of themembrane) is generated by the channel current flowing supported-membrane sensor is considered to be one way tothough ion channel pores. The development of molecular solve these problems. However, single-ion-channel record-communication devices such as a detector and a transmitter ing has not yet been successful in supported-membraneof neurotransmitter molecules has the potential to facilitate devices. The challenges are believed to consist in theimportant medical applications such as diagnostics, treat- fabrication of a defect-free supported planar lipid bilayerment of diseases, and screening in drug development. (SPLB) on the substrate surface and the reduction in edge-Combination with the Si LSI technology allows a leak current through the thin water layer under the lipidsignificant scale-down to nanosized devices suitable for bilayer .insertion into the body, or efficient integrations useful in Here we have developed two basic process technolo- gies necessary to fabricate the ion channel supported- membrane biosensors: formation of a defect-free lipid bilayer on the Si surface with microelectrode pores, and No conflict of interest was reported by the authors of this paper. 4 Corresponding author. Institute for Molecular Science, Myodaiji, the deposition and patterning of the hydrophobic self-Okazaki, 444-8585 Japan. assembled monolayer (SAM) as a guard ring to reduce the E-mail address: email@example.com (T. Urisu). edge-leak current.1549-9634/$ – see front matter D 2005 Published by Elsevier Inc.doi:10.1016/j.nano.2005.10.002
318 T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322Materials Palmitoyl-2-oleoyl-sn-3-[phosphor-l-serin] (POPS), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DfPC), andfluorescence-labeled lipid diacyl phosphoethanolamine-N-lissamine rhodamine B sulfonyl (Rb) were purchased fromAvanti Polar Lipids Co. (Alabaster, AL) Dipalmitoylphos-phatidylcholine (DPPC) was provided by Nippon FineChemical Co. (Osaka, Japan) HF, H2O2, H2SO4, HCl, andHNO3 solutions, as well as CaCl2 and KCl, wereanalytical grade and purchased from Sigma-Aldrich(St. Louis, MO). Octadecyltrichlorosilane (OTS) andtoluene were also purchased from Sigma-Aldrich. All ofthe chemicals and solvents were used without furtherpurification. The purities of Co and SiO2 sputter targetsand Ag wires (0.5 mm diameter) were 99.99%. Spin-onglass (SOG) was purchased from Rasa Industries Co.(Tokyo, Japan) Si(100) wafers (p type, B doped, 0.018 Vcm, and 525 Am in thickness) were purchased fromMiyoshi Co. Water (Kanagawa, Japan) with a typicalresistivity of greater than 18 MV cm was produced using aMilli-Q purification system (Millipore Co., Billerica, MA)Formation of defect-free SPLB Formation of the SPLB with sufficiently high resistivity(greater than gigaohms) is the necessary condition forsingle-ion-channel recordings. To make a defect-free Fig 1. Schematics of the fabrication process of the supported-membraneSPLB, It is crucial that the surface roughness be substrate with AgCl/Ag microelectrodes and the SPLB.minimized. In this work we have successfully made thepore with about 1 Am diameter for the microelectrode,keeping the Si substrate surface extremely flat (Ra b 1 (0.05 torr) and O2 (0.002 torr) as the etching gas. The SRnm), by using femtosecond laser ablation patterning and etching provides a vertical side wall and completely stops atsynchrotron radiation (SR) etching. the CoSi 2 surface . The Co contact mask was successfully removed without damaging the substrate by immersion into 0.1 M HNO3 aqueous. Ag (50 nm thickness)Methods and results was deposited on CoSi2 electrode surfaces by electroplating. Fig. 1 shows the fabrication process of the well structures Then, AgCl/Ag was also formed by electroplating.with microelectrodes. Co (10 nm thick) and Ag (100 nm Unilamellar giant vesicles  of DPPC/POPS/Rb (ratiothick) thin films were sputter-deposited on the mirror- 89.5:10:0.5) were prepared as follows. A chloroformpolished and reverse-side rough Si(100) surfaces after solution of a lipid mixture (10 mg/mL) was dried underconventional wet cleaning. After that, a SiO2 thin film N2 flow using a rotary evaporator for about 30 minutes andconsisting of SOG (400 nm thickness) and sputtered SiO2 subsequently vacuum-dried for 10 hours to completely(200 nm thickness) were deposited and the sample was remove the solvent; a buffer solution (10 mM KCl) wasannealed at 5408C for 10 minutes. By this process the Co added to the lipid thin film obtained and gently agitated. Thelayer was changed to CoSi2 and the Co/Si interface became lipid concentration of the suspension obtained was 0.1 mg/ohmic. The sample was then annealed by SR irradiation to mL. All the processes were carried out at room temperatureremove gas from the SOG . A Co layer as an etching (RT). After incubation at 488C for 10 hours, dialysis wascontact mask was deposited on the SOG surface by then carried out for the suspension of giant vesicles using asputtering, and electrode hole mask patterns were made 5-Am filter for 1 hour in the buffer solution (10 mM KCl,using a femtosecond laser (E = 1560 nm, average power = pH 6.6) at RT. For the deposition of lipid bilayer250 mW, frequency = 258 kHz, pulse width = 900 femto- membranes, the substrate was incubated for 1 hour atseconds, and irradiation time = 4 milliseconds). The 508C under a buffer solution formed by mixing 200 AL ofdiameter of an electrode hole was about 1 Am. SR etching the vesicle suspension and 50 AL of a 50 mM solution ofof the SiO2 layer for making the well on the electrode was CaCl2. Then the sample was washed five times at RT withcarried out at beam line 4A2 of the SR facility (UVSOR) at the buffer solution. Atomic force microscopy (AFM)the Institute for Molecular Science, using a mixture of SF6 observations were carried out using a SPI3800 scanning
T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322 319 Fig 2. A, The surface morphology measured by AFM around the electrode holes of the substrate after AgCl electroplating. B, Cross-sectional profile.probe microscopy system (Seiko Instrument Inc.) in thedynamic force mode (tapping mode) using a Si cantilever.The spring constant of the cantilever for measuring thesurface roughness of the substrate in air was 43 N/m, and1.5 N/m for the in situ characterization of the lipid bilayer. Fig. 2, A shows the AFM topography of the substratesurface around the electrode wells made by the processshown in Fig. 1. The cross-sectional profile in line with X-Yis also shown in Fig. 2, B. These data show that the surfacearound the electrode well is very flat (Ra ~0.8 nm). Toobtain such a flat surface, it was important to control theirradiation power of the femtosecond laser such that only theCo film was removed while causing negligible damage tothe SiO2 layer beneath. If the SiO2 layer was also sputteredby the laser, particles (composition unknown) of 100 to200 nm diameter, which were difficult to remove using theusual etching solutions such as HF, HCl, H2SO4, and HNO3, Fig 3. I-V characteristics of the substrate measured in 10 mM KCl solutionwere deposited around the wells. The electric characteristics (A) before and (B) after SPLB formation, and (C) the equivalent circuit ofwere determined using a patch clamp amplifier (CEZ-2400, the system.Nihon-Koden, Tokyo, Japan) through the AgCl/Ag elec-trode in conjunction with the eCell (Version 2.12) software. lipid thin film. Because the bilayer was formed using theLine a in Fig. 3 shows the current-voltage (I-V) character- same protocol as that used in the formation of the singleistics of the substrate under the buffer solution before bilayer patches shown in Fig. 4, A, it is considered from thevesicle fusion. From these data, the series resistance Rs in fluorescence microscopy image that the single bilayer wasthe equivalent circuit shown in Fig. 3, C is given to be 10 F formed on the microelectrode area. From the I-V character-3 MV. Mixing of negatively charged lipid POPS with istics of the system measured after the lipid bilayerneutral lipid DPPC was essentially effective in forming formation (shown as line b in Fig. 3), the resistance of theunilamellar giant vesicles without aggregation. Addition of lipid bilayer (Rm in Fig. 3, C) was estimated to be 1.2 GV.Ca2+ was also necessary to induce the rupture of vesicles on The capacitance (Cm in Fig. 3, C) of the system measuredthe SiO2 surface . Fig. 4, A shows a fluorescence using a patch clamp amplifier was 10.7 pF. These valuesmicroscopy image of a single SPLB formed on the were observed with extremely good reproducibility duringSiO2/Si(100) surface by the rupture of the giant vesicles. our experiments for more than 5 hours. Because theThe diameter of the bilayer was typically about 200 to resistance almost completely returned to the original value300 Am, large enough to cover the electrode area (10 to 30 Am of 10 MV when the bilayer was broken by adding 5 AL ofdiameter). The thickness of the bilayer, 4.5 nm, observed gramicidin solution (1 mg/mL), the high resistance observedby AFM corresponds to the height of the single bilayer . subsequent to formation of the bilayer was considered not to The lipid bilayer covering the electrode well was formed be due to small vesicles clogging the electrode hole. Darkby giant vesicle fusion. A fluorescence microscopy image spots at the electrode holes in Fig. 4, B are not due to the(Fig. 4, B) after lipid bilayer formation on the microelec- nonexistence of the bilayer on the well. In the region oftrode area clearly shows the existence of the homogeneous 600-nm-thick SiO2, the fluorescence microscopy image is
320 T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322Fig 4. Fluorescence microscopy image of the lipid bilayer formed by the rupture of the giant vesicle on (A) the SiO2/CoSi2/Si surface and (B) the electrode area.very bright because of the fluorescence interference contrasteffect . On the other hand, the electrode hole area, inwhich there is no back surface reflection, is relatively dark. Because the observed capacitance of 10.7 pF is almostexplained by the calculated capacitance of 10 pF due to theSiO2 thin film (600 nm in thickness and 0.5 mm in diameter,determined from the upper electrode, were assumed), thecapacitance due to the lipid bilayer formed on the well isconsidered to be almost equal to the value estimated fromthe specific capacitance of the single bilayer, 0.5 to 0.8 AF[4,20]. From these considerations, it is concluded that thegigaohm seal by the single bilayer was formed on themicroelectrode area.Patterning of OTS-SAM and vesicle fusion on the surface Resistance of 1.2 GV is still insufficient to permit asingle-channel recording with a level of 1 pA channelcurrent. Much higher resistance is expected to be obtainedby reducing the edge-leak current. Working from the Fig 5. The AFM image after the patterning of OTS-SAM on SiO2/Si.concept of using the hydrophobic SAM guard ring toreduce the edge-leak current, we have developed a SiO2/Si surface is very flat (Ra = 0.22 nm). OTS-SAM waspatterning technique for SAM of OTS, of which the deposited by immersing the SiO2/Si(100) substrate in a 10hydrophobic carbon chain length is close to that of the lipid. mM water-saturated toluene solution of OTS for 5 seconds at The sample treatment and the OTS deposition were RT. The OTS/SiO2 sample was sonicated in toluene, acetone,carried out according to our earlier work . Briefly, a ethanol, and pure water to remove the excess OTS moleculesmirror-polished Si(100) wafer covered with the native oxide from the OTS/SiO2 surface.was first sonicated in acetone, ethanol, and Milli-Q water A negative-resist pattern was formed on the OTS/SiO2(N 18 MV cm; Millipore Co.) for 5 minutes each. Then surface by using conventional photolithographic technique.the substrate was boiled in a solution of concentrated H2SO4 A 50-Am line-and-space conventional photomask patternand H2O2 (30%) (7:3 in volume ratio) at 1208C for 5 minutes was used. The sample was then exposed to UV light in airto remove the organic contaminants and immersed in a HF for 30 minutes to remove the OTS-SAM from the opensolution (2.5%) for 2 minutes to remove the surface oxide area, where the distance between the sample and the lamplayer. After this cleaning, the SiO2 film of 120 nm in was also 3 cm. Finally, the photoresist was removed usingthickness was deposited on the Si(100) surface by sputtering. a negative-resist remover (NS, Tokyo Ohka Kogyo Co.,The sample was then exposed to UV light (UVL20US-60, Nakagawa, Japan) followed by rinsing with distilled waterSen Lights Co.) for 10 minutes. The distance between the and drying by blowing N2. The OTS line-and-space patternsample and the lamp was 3 cm. AFM images show that the obtained and measured by AFM is shown in Fig. 5.
T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322 321 Fig 6. A, Fluorescence microscopy image of SPLB formed on the patterned OTS-SAM. B, Intensity distribution on the A-B line in A. Lipid bilayers were deposited on the patterned OTS-SAM area by the rupture of giant unilamellar vesicles.When the substrate was immersed in an aqueous solution oflipid vesicles, the vesicles adhered to the surface, broke up,and spread to form a bilayer on hydrophilic surfaces and amonolayer on hydrophobic surfaces . Fig. 6, A shows afluorescence microscopy image of the OTS-SAM–patternedSiO2 surface after immersing in the giant vesicle suspen-sion. Fig. 6, B shows the intensity distribution on the A-Bline in Fig. 6, A. Earlier study had shown that after thevesicle fusion, a bilayer forms on hydrophilic SiO2 surfacesand a monolayer forms on OTS-SAM hydrophobic surfaces. In the present case, a monolayer was formed on thehydrophobic OTS-SAM area (i region in Fig. 6) and abilayer was formed on the hydrophilic SiO2 area (ii regionin Fig. 6), so the fluorescence intensity from the lipid layercontaining Rb is different between these areas. In the iiiregion it is very dark, because neither a bilayer nor amonolayer has formed on the SiO2 surface. The fluorescence microscopy image of Fig. 6 confirms Fig 7. Total root mean square current noise as a function of the porethat the bilayer and the monolayer are successfully 5 diameter. The bandwidths of the patch clamp amplifier were (O) 5 kHz, (5 ) 10 kHz, and (D) 20 kHz.deposited on the bare SiO2 and the OTS/SiO2 regions,respectively. A substantial body of evidence suggests that athin water layer of approximately 1 to 2 nm in thickness is considered that the OTS-SAM surface and the lipidtrapped between the substrate surface and the head-groups monolayer undersurface adhere completely to each otherlayer in the lower leaflet of the bilayer . This water layer in the i region because of the strong hydrophobic interactioncauses the edge-leak current of the SPLB system. The of both surfaces. This indicates that OTS-SAM patterns cancalculated length of the OTS molecule is 2.75 nm . On be used sufficiently as a guard ring to reduce the leakthe other hand, the lengths of the DPPC acyl group and head current from the SPLB edge.group are 2.0 nm and 1.5 nm, respectively. The lengthmismatch seems to exist between the OTS and DPPC Discussionmolecules. However, in our previous experiments withDPPC monolayer deposition by the Langmuir-Blodgett We have investigated the relation between single-ion-method on the SiO2 surface with OTS-SAM islands, the channel current noise and pore size using the conventionalheight of the DPPC monolayer area was observed to agree arrangement of the black membrane formed at the pore ofwith that of the OTS-SAM island area . Therefore, in the Teflon chip partitioning the two chambers. A lipidthe lipid layer–OTS-SAM structure shown in Fig. 6, it is bilayer was formed at the pore of the chip by contacting
322 T. Urisu et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 317–322droplets of a decane solution (0.01 M) of DfPC to the  Wilk SJ, Petrossian L, Goryll M, Thornton TJ, Goodnick SM, Tangpore. Gramicidin A was incorporated into the lipid bilayer JM, et al. Ion channels on silicon. Surf Sci Nanotech [serial on the Internet] 2005;3:184 - 9 [available from: http://www.sssj.org/ejssnt.].by mixing droplets of a KCl 1 M solution containing  Schats A, Linke-Hommes A, Neubert J. Gravity dependency of thegramicidin A (3–30 mg/L) with the chamber solution gramicidin A channel conductivity—a model for gravity perception(1 M KCl) close to the pore. Single-channel current mea- on the cellular level. Eur Biophys J 1996;25:37 - 41.surements were carried out using a patch clamp amplifier  Ide T, Yanagida T. An artificial lipid bilayer formed on an agarose-(CEZ-2400, Nihon-Koden, Japan). The observed noise was coated glass for simultaneous electrical and optical measurement of single ion channels. Biochem Biophys Res Commun 1999;265:reduced with decreasing the pore size as expected (Fig. 7) 595 - 9.. As shown in these data, small pore sizes (less than  Cheng Y, Bushby RJ, Evans SD, Knowles PF, Miles RE, Ogier SD.several microns) are necessary to create stable, low-noise, Single ion channel sensitivity in suspended bilayers on micro-and high-speed single-ion-channel current devices. With machined supports. Langmuir 2001;17:1240 - 2.respect to these small-pore-size chips, it is considered that  Schmidt C, Mayer M, Vogel H. Chip-based biosensor for the functional analysis of single ion channels. Angew Chem Int Edsupported membranes, in which the stability is not so 2000;39:3137 - 40.sensitive to the fine structure of the pore, can be more  Pantoja R, Ngarah JM, Starace DM, Melosh NA, Blunck R, Bezanillaeasily formed than can suspended membranes . F, et al. Silicon chip-based patch-clamp electrodes integrated withTherefore, we consider that stable, low-noise, and high- PDMS microfluidics. Biosens Bioelectron 2004;20:509 - 17.speed ion-channel biosensors can be created using the  Bayley H, Cremer PS. Stochastic sensors inspired by biology. Nature 2001;413:226 - 30.supported-membrane structure, for which defect-free bilay-  Urisu T, Kyuragi H. Synchrotron radiation-excited chemical-vaporer formation and edge-leak current reduction techniques deposition and etching. J Vac Sci Technol 1987;B5:1436 - 40.described here are essential.  Akashi K, Miyata H, Itoh H, Kinosita Jr K. Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. J Biophys 1996;71:3242 - 50.Acknowledgment  Wilschut J, Duezguenes N, Papahadjopoulos D. Calcium/magnesium specificity in membrane fusion: kinetics of aggregation and fusion of The authors thank Nippon Fine Chemical Co. for the gift phosphatidylserine vesicles and the role of bilayer curvature.of DPPC. This research was supported by Grants-in-Aid for Biochemistry 1981;20:3126 - 33.Scientific Research from the Ministry of Education, Culture,  Leonenko ZV, Carnini A, Cramb DT. Supported planar bilayerSports, Science and Technology (13gs0016) of Japan. formation by vesicle fusion: the interaction of phospholipid vesicles with surfaces and the effect of gramicidin on bilayer properties using atomic force microscopy. Biochim Biophys Acta 2000;1509:131 - 47.References  Lambacher A, Fromherz P. Fluorescence interference-contrast mi- croscopy on oxidized silicon using a monomolecular dye layer. Appl  Fromherz P. Neuroelectronic interfacing: semiconductor chips with Physics 1996;A63:207 - 16. ion channels, nerve cells and brain. In: Waser R, editor. Nano-  Terrettaz S, Mayer M, Vogel H. Highly electrically insulating tethered electronics and information technology. Berlin7 Wiley-VCH Verlag; lipid bilayers for probing the function of ion channel proteins. 2003. pp. 781 - 810. Langmuir 2003;9:5567 - 9.  Fertig N, Tike A, Blick RH, Kotthaus JP, Behrends JC, Bruggencate  Tero R, Takizawa M, Li YJ, Yamazaki M, Urisu T. Lipid membrane GT. Stable integration of isolated cell membrane patches in a formation by vesicle fusion on silicon dioxide surfaces modified nanomachined aperture. Appl Phys Lett 2000;77:1218 - 20. with alkyl self-assembled monolayer islands. Langmuir 2004;20:  Pantoja R, Sigg D, Blunck R, Bezanilla F, Heath JR. Bilayer 7526 - 7531. reconstitution of voltage-dependent ion channels using a micro-  Bayerl TM, Bloom M. Physical properties of single phospholipid fabricated silicon chip. Biophys J 2001;81:2389 - 94. bilayers adsorbed to micro glass beads. A new vesicular model  Fertig N, Meyer CH, Blick RH, Trautmann CH, Behrends JC. system studied by 2H-nuclear magnetic resonance. Biophys J 1990; Microstructured glass chip for ion-channel electrophysiology. Phys 58:357 - 62. Rev E 2001;64:040901-1-4.  Wasserman SR, Tao YT, Whitesides GM. Structure and reactivity of  Romer W, Steinem C. Impedance analysis and single-channel alkylsiloxane monolayers formed by reaction of alkyltrichlorosilanes recordings on nano-black lipid membranes based on porous alumina. on silicon substrates. Langmuir 1989;5:1074 - 87. Biophys J 2004;86:955 - 65.  Takizawa M, Kim YH, Urisu T. Deposition of DPPC monolayers by  Goryll M, Wilk S, Laws GM, Thornton TJ, Goodnick S, Saraniti M, the Langmuir-Blodgett method on SiO2 surfaces covered by et al. Silicon-based ion channel sensor. Superlattices Microstruct octadecyltrichlorosilane self-assembled monolayer islands. Chem 2003;34:451 - 7. Phys Lett 2004;385:220 - 4.  Wilk SJ, Goryll M, Laws GM, Goodnick SM, Thornton TJ, Saraniti  Mayer M, Kriebel JK, Tosteson MT, Whitesides GM. Microfabricated M, et al. Teflon (TM)-coated silicon apertures for supported lipid Teflon membranes for low-noise recordings of ion channels in planar bilayer membranes. Appl Phys Lett 2004;85:3307 - 9. bilayers. Biophys J 2003;85:2684 - 95.