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Suspended Planar Phospholipid Bilayers on Micromachined Supports Simon D. Ogier,† Richard J. Bushby,‡ Yaling Cheng,† Stephen D. Evans,*,† Stuart W. Evans,‡ A. Toby A. Jenkins,‡ Peter F. Knowles,‡ and Robert E. Miles§ Molecular Physics and Instrumentation Group, Department of Physics and Astronomy, Centre for Self-Organising Molecular Systems, and Institute of Microwaves and Photonics, Department of Electronic Engineering, University of Leeds, Leeds, LS2 9JT, U.K. Received October 15, 1999. In Final Form: March 3, 2000 Micromachined structures consisting of an aperture between a buried reservoir and ambient have been produced using the photoresist SU8. The external surface of the structure, surrounding the aperture, was coated in a gold layer to allow surface modification with self-assembled monolayers. These structures were assessed as substrates for the assembly of “suspended” phospholipid bilayers. We report that phospholipid bilayer membranes could be formed over such apertures and that they are highly resistive to the conduction of metal anions (K+). Resistance values are typically found to be ∼1010 Ω (∼106 Ω cm2). These values represent a significant improvement over our membranes supported directly on gold substrates (105 Ω cm2). Fluorescence microscopy images are presented to provide evidence that the lipid bilayer spans the 100 µm aperture. Given the ease of producing large numbers of devices using microlithographic techniques, the proposed structures could form a sensing element for studying membrane transport phenomena in general and for ion-channel assays in particular.
1. Introduction The cytoplasmic membrane (CM) consists of a phospholipid bilayer that provides an impermeable barrier to the transport of metal ions and large water-soluble molecules such as sugars and hormones. In naturally occurring membranes the CM hosts numerous proteins and carbohydrates which play an important role in regulating cellular activity. Of particular interest to us are the naturally occurring ion transporters in the form of ion channels and ion carriers. These play a role in regulating the ion concentration gradient between the cell interior and the exterior. For example the binding of an acetylcholine to the acetylcholine receptor opens a channel for a few milliseconds during which time ∼106 Na+ ions are transported across the membrane giving rise to an action potential at a neuromuscular junction. The currents associated with the ion flow through a single channel are typically of the order of a few picoamperes and can be readily detected with sensitive electronics. Our goal is to create artificial systems which mimic the “natural plasma membrane” in both its structure and function. To this end we have been investigating several routes for the attachment of phospholipid bilayers to planar substrates. A number of approaches have been followed for the creation of such bilayer architectures on solid supports including adsorption and subsequent unrolling of vesicles on self-assembled monolayer (SAM) functionalized surfaces or directly onto glass/SiO2 supports.1-9 While such * To whom correspondence should be addressed. E-mail:
[email protected]. † Molecular Physics and Instrumentation Group, Department of Physics and Astronomy. ‡ Centre for Self-Organising Molecular Systems. § Institute of Microwaves and Photonics, Department of Electronic Engineering. (1) Plant, A. L. Langmuir 1999, 15, 5128-5135. (2) Sackmann, E. Science 1996, 271, 43-48. (3) Lingler, S.; Rubinstein, I.; Knoll, W.; Offenha¨usser, A. Langmuir 1997, 13, 7085-7091.
approaches provide a simple method of bilayer formation, the resulting bilayers are in general too permeable to permit the detection of low levels of ion channel activity and the exact nature of the “leakiness” of these bilayers is not clear. Currently, there are numerous research groups trying to improve the ion-transport impermeability of such layers, and it is necessary for the specific resistance of the layers to be improved by between 2 and 3 orders of magnitude if single ion channel activity is to be observed. The alternative approaches of suspending lipid bilayers across apertures, e.g., using Langmuir-Blodgett transfer, or solvent spreading methods have received less attention but perhaps have greater promise for the generation of insulating bilayers. One such approach employs a micromachined hole in a silicon wafer which is bonded to a glass slide with a silver/silver chloride electrode patterned onto it.10 Nikolelis et al. have used a method of “solvent spreading” over a small hole (0.32 mm diameter) punctured in a plastic film and found specific resistances as high as 1 × 108 Ω cm2. Using a filter membrane version of this, they were able to detect acetylcholine, urea, and penicillin.11 In this paper we introduce a new design of micromachined support for bilayers (MSB). The device consists of (4) Jenkins, A. T. A.; Bushby, R. J.; Boden, N.; Evans, S. D.; Knowles, P. F.; Liu, Q. Y.; Miles, R. E.; Ogier, S. D. Langmuir 1998, 14, 46754678. (5) Jenkins, A. T. A.; Boden, N.; Bushby, R. J.; Evans, S. D.; Knowles, P. F.; Miles, R. E.; Ogier, S. D.; Schonherr, H.; Vancso, G. J. J. Am. Chem. Soc. 1999, 121, 5274-5280. (6) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751-757. (7) Raguse, B.; Braach-Maksvytis, V.; Cornell, B. A.; King, L. G.; Osman, P. D. J.; Pace, R. J.; Wieczorek, L. Langmuir 1998, 14, 648659. (8) Groves, J. T.; Ulman, N.; Boxer, S. G. Science 1997, 275, 651653. (9) Cremer, P. S.; Boxer, S. G. J. Phys. Chem. B 1999, 103, 25542559. (10) Osborn, T. S.; Yager, P. Langmuir 1995, 11, 8-12. (11) Nikolelis, D. P.; Siontorou, C. G. Anal. Chem. 1995, 67, 936944.
10.1021/la991367o CCC: $19.00 © 2000 American Chemical Society Published on Web 06/01/2000
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Figure 1. Cross section of the fabrication of one element of the MSB: (a) gold patterned on silicon (100 µm hole); (b) SU8 layer similarly patterned; (c) SU8 “O-ring” patterned around the hole; (d) evaporation and lift-off of silver on glass; (e) SU8 “O-ring” patterned around working area of electrode; (f) the two halves are brought together (first half inverted); (g) bonding of the two halves by capillary filling with epoxy; (h) separation of the final MSB from the silicon surface by pulling the silicon with a pair of tweezers; (i) MSB redrawn to scale to show relative dimensions.
a micromachined hole in a gold surface suspended over an aqueous reservoir (Figure 1h). A silver/silver chloride electrode at the bottom of this reservoir enables current flow through the bilayer to be measured. Self-assembled monolayers of alkanethiols can be formed around the hole, due to the presence of the gold surface, thus making it more compatible with the phospholipids in the bilayer. The procedure for fabricating the MSBs is relatively simple utilizing photolithography and thermal evaporation of metals and avoids the lengthy etches necessary when silicon is used as a substrate. A recently introduced photoresist, SU8, is used in the fabrication of the MSB; SU8 is a negative tone photoresist based upon an epoxy resin and can be used to make structures up to several hundred micrometers thick in a single coating.12 In this report we demonstrate that MSBs can be used to support lipid bilayers that are impermeable to ions and are thus suitable candidates for development into biosensors based upon ion-channel conduction. (12) Lee, K. Y.; LaBianca, N.; Rishton, S. A.; Zolgharnain, S.; Gelorme, J. D.; Shaw, J.; Chang, T. H. P. J. Vac. Sci. Technol., B 1995, 13, 30123016.
2. Experimental Section Materials and Apparatus. n-Type silicon wafers were purchased from Exsil wafer reclaim (U.K., 〈100〉 orientation, thickness 400-600 µm, resistivity, 0.5 Ω cm). Positive photoresist and developer (Shipley1813 resist, microposit developer) were purchased from Chestech (Rugby, U.K.). Two viscosities of the negative photoresist SU8 (Chestech) were used, these are termed 10 µm and 250 µm SU8 (due to their intended thickness at a particular spin speed). These resists were developed in propylene glycol monomethyl ether acetate (PGMEA) (Chestech). Microscope slides 0.8-1.0 mm thick were purchased from BDH (U.K.). Poly(dimethylsiloxane) (PDMS) (Sylgard 184, Dow Corning) and an epoxy EPO-TEK 377 (Polyscience, Switzerland) were used for bonding elements of the structure together. The solvents used in the microfabrication (acetone, methanol, and chlorobenzene) were either Aristar or AnalaR grade (BDH). Millipore water (resistivity 18.2 MΩ cm) was used in the cleaning procedures, in the rinsing after photoresist development, and for making KCl solutions. Photolithography was performed using soft contact in a mask aligner (MJB3, Karl Suss). The masks used were generated using photographic reduction from a rubylith and hence had an edge resolution of, at best, 1 µm. Metal evaporations of silver 99.99% and gold 99.99% (Advent, U.K.) were performed at 1 × 10-6 Torr (Edwards Auto 306). Electrical measurements
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were made using a high input impedance (1013 Ω) current amplifier designed to measure the conductance and capacitance of black lipid membranes and for use in patch clamps experiments (Industrial Developments type ID562; Bangor, Wales). The electrodes were positioned in a grounded Faraday cage mounted on an air-suspended table. Egg-phosphatidylcholine (egg-pc) was used without further purification (Lipid Products, U.K.). 2-(6(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl- 1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC), a fluorescently labeled lipid, was purchased from Molecular Probes Europe (Holland). HPLC grade dichloromethane and methanol (Aldrich, U.K.) were used in the SAM formation. The alkanethiols used in this study were octadecanethiol (ODT) and mercaptoethanol (EO1) (98% purity, Aldrich). Prior to SAM formation, structures were treated in an argon plasma cleaner 100 W, 20 s (Plasmatic Systems, NJ, USA). KCl (AnalaR) for electrode chloridation was purchased from BDH. Procedures. A detailed description of the fabrication of the MSB is given as Supporting Information but involves the following principal steps. The MSBs are fabricated in two parts that are subsequently bonded together. Figure 1 shows the process schematically. The upper half of the device (Figure 1a-c) consists of a gold layer deposited on silicon with well-defined holes, made using an evaporation “lift-off” technique. A polymeric (SU8) layer was deposited and patterned to be commensurate with the gold layer (Figure 1b). A second SU8 layer was deposited and patterned to produce an “O-ring” (internal diameter 560 µm, external diameter 760 µm) concentric with the hole in the gold/SU8 layer (Figure 1c). The second half of the MSB was fabricated on a glass microscope slide (Figure 1d,e). Silver was deposited using “liftoff” onto the glass slide to make an electrode in a pattern that consists of a working area of diameter 600 µm, which is connected via a track of width 200 µm to a contact pad at the edge of the slide (Figure 1d). The working area and some of the track was then chloridized in a 0.1 M KCl solution by passing a small (1 mA) current between it and a platinum electrode for 20 s. Subsequently a SU8 “O-ring” (internal diameter 820 µm, external diameter 1020 µm), slightly larger than the one on the silicon, was patterned around the working area of the electrode (Figure 1e). The two halves of the MSB were assembled by hand with the aid of a binocular microscope so that the “O-ring” on the glass fitted around the one on the silicon (Figure 1f). PDMS or epoxy was then placed at the interface between the two halves, and all areas outside the SU8 “O-ring” were filled by capillary action. Bonding is achieved either over a period of days at room temperature or at elevated temperatures (65 °C for PDMS, 150 °C for the epoxy) for 1 h (Figure 1g). The final MSB structure is revealed by pulling the silicon away from the gold as this adhesion is much weaker than the cohesion of the structure. SAM Formation on a Gold Surface. Prior to SAM formation, the MSBs were subjected to a short (20 s) argon plasma clean, on low power (100 W), to remove contamination that may have been left behind from the initial “lift-off” of gold. Hydrophobic and hydrophilic SAMs were formed on these gold surfaces by immersing the structures in a 1 mM solution of ODT or EO1, in methanol for 1 h (a small amount of DCM was used to enhance the solubility of ODT).13 Electrical Measurements. Figure 2 shows the arrangement for bilayer formation and electrical characterization. A typical device consisted of eight structures each of which could be addressed individually. The MSB formed one wall of a small PTFE reservoir which was filled with 0.1 M KCl. To fill the holes with electrolyte, the MSB was placed in a container containing the electrolyte and a vacuum was applied and released until all the air had escaped from the holes. Bilayers were formed using a procedure proposed by Nikolelis et al. adapted from Montal and Mueller, in which 10 µL of lipid in hexane (0.5% w/v) was added dropwise to the electrolyte/air interface (area ∼1.4 cm2) near to the gold surface while the fluid level was above one of the MSB elements (Figure 2b).14 The air/electrolyte interface (13) Pure DCM causes undercutting of the PDMS leading to the separation of the MSB into two halves. Using an epoxy resin to bond the device instead of PDMS was found to greatly increase its resistance to harsh solvents. (14) Nikolelis, D. P.; Krull, U. J. Talanta 1992, 39, 1045-1049.
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Figure 2. (a) The flowcell is clamped onto the MSB as shown. Parts b and c show the equivalent circuits and cross section of MSB in electrolyte before and after lipid spreading: Re, electrolyte and electron-transfer resistance; Rb, bilayer resistance. supporting the lipid multilayer was lowered below and then raised above a MSB element by withdrawing and reinjecting electrolyte, with a syringe connected to the flow cell. At this stage the aperture was coated with a multilayer film (Figure 2c). Over a period of a few minutes this film thinned to form a bilayer structure surrounded by a torus containing both solvent and lipid. Electrical measurements were made using a two-electrode configuration. The Ag/AgCl microfabricated electrode was used as the working electrode and a “chloridized” silver wire was used as the counter electrode. The ID562 unit was used to supply a voltage across the electrodes and to record the resulting current flow. Atomic Force Microscopy. Topographical measurements of a silicon template stripped gold film were performed in contact mode using a Nanoscope II (Digital Instrument Co.) under ambient conditions. A standard wide-legged silicon nitride cantilever with a notional spring constant of 0.12 N/m was used, and images were obtained at a scan rate of 2 Hz over a 1 µm × 1 µm region. Only zero-order flattening was applied to the image, and the resolution is 512 × 512 pixels. Fluorescence Microscopy. Fluorescence microscopy was used to show that the apertures in the MSB were covered with a lipid film. This required the MSB to be fabricated without a lower Ag/AgCl electrode to permit optical access to the membrane (in all other aspects the MSBs were the same as those described above). A lipid mixture containing egg-pc and the fluorescently tagged NBD C6-HPC (100:1 by mol) was dissolved in hexane (1 mg/mL) and smeared across the MSB surface using a pipet (with a right-angled bend near its tip). Fluorescence images were obtained using a Leitz epifluorescence microscope. Filters were selected to permit broad excitation between 450 and 500 nm and to collect emitted light at wavelengths greater than 510 nm. The aperture was imaged using a 50× ultralong working distance
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Figure 3. (a) SEM image of one element of an MSB that has been cut away to reveal the cavity below the hole in the gold/SU8 surface. (b) SEM of SU8 “O-ring” surrounding the hole in gold/SU8 layer. (N.B.: the silicon had been removed from the photopatterned structure and the material in the center of the hole is conducting epoxy that is used to fix the sample to the SEM stub.) (c) SEM image of the SU8 “O-ring” around the Ag/AgCl electrode on the glass substrate. (d) SEM of the hole in the gold/SU8 layer showing edge roughness (due to poor edge definition of the emulsion mask). objective (Olympus), and the images were photographed using a camera mounted on the microscope (Fuji 200 ISO film).
Results and Discussion The SEM images, Figure 3a-c, show different aspects of the MSB devices. Figure 3a shows a top view of a complete MSB following removal from the silicon substrate. The device was cut to allow the cavity below the gold substrate to be visualized and shows that the epoxy does not penetrate this region during the bonding process. Figure 3b shows a rear view of the upper half of the device, made on silicon, with the SU8 “O-ring” surrounding the hole in SU8/gold. The structure was removed from the silicon prior to imaging, and the conducting epoxy used to attach the sample to the SEM stub is clearly visible in the center of the 100 µm aperture. Figure 3c is an image of the “O-ring”, on the lower half of the device, surrounding the microfabricated Ag/AgCl electrode. A close-up view of one of the holes in the MSB can be seen in Figure 3d. The method by which the MSBs are made results in the formation of smooth gold surfaces, since the gold is evaporated directly onto a bare silicon surface and subsequently “stripped” from this surface.15 Figure 4 shows a topographic AFM image of such a silicon “template stripped” gold surface and shows a typical root mean square roughness of 4 Å over an area of 1 µm2. The formation of electrically insulating lipid layers across these structures was most successful when the gold surface had been modified with a SAM of ODT whereas lipid films could not be formed over holes in structures whose surfaces were derivatized with EO1. It is unclear as to the reason the surface hydrophobicity affects the process of forming a lipid/solvent film over the hole in the MSB; however, it is not unexpected since BLM experiments have traditionally been formed on apertures in PTFE or other hydrophobic materials. Klingler and Fromherz also report that they could only coat micromachined silicon grooves with free-standing BLM (using a similar solvent-spreading procedure) if the silicon was rendered hydrophobic with octadecylchlorosilane.16 Figure 5 shows the current/voltage characteristics of a device in the electrolyte before and after bilayer formation.
Figure 4. AFM image of a silicon template stripped gold surface and the corresponding cross section.
(15) Stamou, D.; Gourdon, D.; Liley, M.; Burnham, N. A.; Kulik, A.; Vogel, H.; Duschl, C. Langmuir 1997, 13, 2425-2428. (16) Klingler, J.; Fromherz, P. Ber. Bunsen-Ges. Phys. Chem. 1995, 99, 958-964.
In the best cases, the current flowing between the electrodes decreased by 5 orders of magnitude following bilayer formation (measured at 20 mV applied voltage). This corresponds to a resistance of the order of 1 × 1010
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Figure 5. Current/voltage characteristics for the device in 0.1 M KCl before (squares) and after (circles) membrane formation.
Ω (∼1 × 106 Ω cm2) and therefore shows that the bilayer effectively blocks the passage of ions through the hole. The capacitance of the lipid layer, on which the measurements for Figure 5 were made, was 0.56 ( 0.04 µFcm-2 and is close to that expected for a lipid bilayer.17 The capacitance of a 10 µm thick layer of SU8, assuming a dielectric constant of 3.65 for epoxy resin, is 3.23 × 10-10 F cm-2. Therefore despite having a surface area ∼30 times greater than that of the bilayer, the MSB structure is only expected to contribute ∼2% of the total measured capacitance. This has been subtracted from the total measured capacitance to give the value for the bilayer. Applying 500 mV to one electrode caused membrane rupture following which the current/voltage characteristics rapidly returned to that found for an uncovered hole in electrolyte. This shows that the changes observed during the bilayer formation process were not due to electrode failure. In addition, the formation procedure was repeated without adding the lipid in hexane and on no occasions were any changes in current observed (except for the period where the hole in the MSB was above the level of the electrolyte). From this we can conclude that the observed changes in current upon membrane formation are not due to trapped air bubbles. Indeed, if an air bubble were to be trapped in the hole, the measured capacitance would also drop to a very low level, vide supra. The average bilayer lifetime was approximately 20 min although some lasted considerably longer and some broke almost immediately. It is possible that the longevity and electrical resistance of the bilayers could be improved by making the holes smaller and the edges smoother. Figure 3d shows that, at present, the edges of the hole are quite rough and this roughness is due to the limited resolution of the photographically generated emulsion mask. Eray et al. demonstrated that lipid bilayers formed upon smooth 40 µm diameter micromachined polyimide apertures had lifetimes of up to 50 h, a significant improvement upon the traditional black lipid membrane support.18 Fluorescence microscopy. Figure 6 shows images of a hole in the MSB before (Figure 6a) and 15 min after (17) By use of a dielectric constant of 2.25 and a bilayer thickness of 48 ( 2 Å (from ref 6) the theoretical value is 0.40 ( 0.02 µF cm-2, but “solvent-free” membranes have been reported with capacitances of up to 1.0 µF cm-2 (see ref 15). It is likely that there is some residual solvent in the layers formed upon the MSB thus reducing the measured capacitance. (18) Eray, M.; Dogan, N. S.; Reiken, S. R.; Sutisna, H.; Van Wie, B. J.; Koch, A. R.; Moffett, D. F.; Silber, M.; Davis, W. C. BioSystems 1995, 35, 183-188.
Figure 6. (a) Fluorescence image of the hole in a MSB before adding lipid mixture (the autofluorescence of the SU8 layer enables it to be visualized). (b) Image 15 min after membrane formation. The less intense region toward the center of the hole corresponds to a thinner lipid film.
(Figure 6b) application of the lipid using the pipet. Prior to membrane formation the area in the hole appears dark, and the light regions are due to the autofluorescence of the SU8 layer. Immediately after membrane formation the image appears uniformly bright within the hole but, over a period of time (several minutes), a dark area grows from the center (as can be seen in Figure 6b). This is due to the solvent thinning in the center and draining toward the rim of the hole. At present our imaging medium is not sensitive enough to determine whether the dark area is a bilayer or a multilayer. Upon rupture of the membrane the image appears very similar to that shown in Figure 6a. Conclusions In this paper we have demonstrated a novel method for fabricating 3D structures which can be used as supports for creating “suspended” phospholipid bilayers. This approach, i.e., moving away from “supported” bilayers to “suspended” bilayers, is of potential technological interest since it reduces the problems of current leakage and irreproducibility found in bilayers formed on solid surfaces. The existence of a large ionic reservoir below the bilayer will permit the incorporation of membrane proteins with large extramembraneous segments. By incorporating ligand-gated ion-channels into this bilayer, we hope to develop a biosensor with very high sensitivity. The processes used in fabricating this structure are common in the microelectronic industry making it possible to fabricate large arrays of devices on a single slide, thus improving the chances of isolating a bilayer with good electrical properties. Work is currently underway in our laboratory to improve the method of fabricating these structures so that holes
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with submicrometer dimensions can be created. Once this has been achieved, it is hoped that the self-assembly of lipid vesicles into planar bilayers that span these holes will provide a convenient method of creating solvent-free fluid lipid bilayers with a high electrical resistance. Acknowledgment. This work was funded by grants from the Engineering and Physical Sciences Research Council (EPSRC). Y.C. acknowledges funding from EPSRC
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(Grant No. GR/M17037). S.D.O. acknowledges receipt of an EPSRC studentship and also a CASE award from the Defence Evaluation and Research Agency (DERA, Malvern). A.T.A.J. acknowledges funding from BBSRC. Supporting Information Available: A detailed description of the fabrication of the MSB. This material is available free of charge via the Internet at http://pubs.acs.org. LA991367O