NANO LETTERS
Microfluidic System for Planar Patch Clamp Electrode Arrays
2006 Vol. 6, No. 4 815-819
Xiaohui Li,† Kathryn G. Klemic,‡ Mark A. Reed,†,§ and Fred J. Sigworth*,‡ Departments of Electrical Engineering, Cellular and Molecular Physiology, and Applied Physics, Yale UniVersity, New HaVen, Connecticut 06520 Received January 24, 2006; Revised Manuscript Received March 3, 2006
ABSTRACT We present a microfluidic system integrated with disposable cell interface partitions for simultaneous patch clamp recordings. Glass-supported poly(dimethylsiloxane) (PDMS) partitions, having a 2 µm air-blown aperture, were reversibly sealed to a microfluidic system including PDMS channels with isolation valves and microfabricated Ag/AgCl electrodes. Gigaseal recordings from RBL-1 cells were obtained with a 24% success rate. Simultaneous whole cell recordings from valve-isolated electrodes were obtained.
Patch clamp technology has proven to be the accepted standard1 for fundamental studies of ion channel proteins and the discovery of drugs that affect them. Initially developed by Neher and Sakmann,2 the traditional patch clamp system consists of a fire-polished glass pipet with a 1-2 µm diameter tip, which is carefully pressed onto a cell membrane with a micromanipulator. The membrane patch is sealed to the pipet with suction and hence electrically isolated.3,4 Additional suction or a voltage pulse breaks the patch membrane, yielding the whole cell recording configuration. The patch clamp setup thus measures the ionic current through the membrane patch or the entire cell membrane area. This technique, however, is very laborintensive and requires expensive equipment. To meet high-throughput screening requirements, many efforts have been taken to improve the patch clamp system. Planar patch clamp electrodes, scalable and easy to use, have been fabricated using silicon oxide coated nitride membranes,5 deep RIE etched silicon holes coated with PECVD oxide,6 polyimide films,7 track-etched quartz,8 silicon oxide nozzles,9 glass substrates,10,11 and oxygen plasma treated PDMS poly(dimethylsiloxane).12 Most of them have a proven reliability for obtaining patch clamp recordings but have a high cost due to microfabrication. PDMS has the potential of much lower cost, and the air-molding technique proves to be a simple way to fabricate PDMS cell-patch interfaces (partitions) for planar patch clamp measurements.13 Microfluidics have been integrated with patch clamp systems using PDMS6 or glass.14 Those devices have made possible fast fluid exchange for single electrodes. However, * Corresponding author. E-mail:
[email protected]. † Department of Electrical Engineering. ‡ Department of Cellular and Molecular Physiology. § Department of Applied Physics. 10.1021/nl060165r CCC: $33.50 Published on Web 03/22/2006
© 2006 American Chemical Society
dense arrays of electrodes also need microfluidics to allow for common fluid lines. These lines need, in turn, isolation valves to electrically isolate neighboring electrodes during measurement. The requirements of a patch clamp measurement demands that the dc electrical isolation between neighboring electrodes be greater than about 10 GΩ. The development of multilayer PDMS technology15 makes it possible to fabricate microfluidic system with valves. Here we present a microfluidic system for array applications, integrated with planar Ag/AgCl electrodes and with disposable air-blown PDMS patch partitions. This system allows simultaneous planar patch clamp measurements. A disposable planar PDMS patch partition is micromolded using a micrometer-sized stream of nitrogen to define an aperture in the silicone elastomer, by the method of Klemic et al.13 However, instead of using a PDMS secondary support for the partition, here we use a glass washer as secondary support, which provides a robust support to the elastomeric partition and a surface that seals well to PDMS microfluidics. Figure 1 shows a schematic of the fabrication process. A metal plate (fabricated by Dynamic Research, Wilmington, MA) was used as the air-blown hole master form, with 2 µm holes defined by a high ratio electroplating process. This metal plate was mounted onto a chamber with resistive heaters and a compressed nitrogen supply. In a thin PDMS layer a 2 µm diameter hole was formed by a continuous gas flow through the plate. The resultant PDMS partition was mounted onto a disposable glass washer support. Using our current manual approach, one can fabricate about 60 PDMS partitions per day. After being heated in a 180 °C oven, the planar PDMS patch partitions were placed on their sides in an oxygen plasma system (Anatech SP100 plasma system, Anatech, Springfield, VA) so that both sides of the partitions were oxidized.
Figure 1. The process of making disposable planar PDMS patch partitions. A metal plate with an array of 2 µm holes is mounted in a chamber with a resistive heater and compressed air supply (A). The primary PDMS support washer is prepared by punching 13-gauge and 20-gauge needles into a 450 µm PDMS sheet. After being painted with PDMS prepolymer, the primary support is placed onto the metal plate and aligned carefully to the hole. The flow of compressed air defines a tiny hole in the prepolymer as it is cured by heating (B). The glass washer is prepared by drilling a 1 mm hole in 0.15 mm thick glass disk. After curing, the PDMS partition is peeled off, flipped over and bonded onto the glass washer with fresh PDMS prepolymer. An SEM picture (C) shows that the air-blown hole has a smooth surface. Picture (D) shows two variants of patch partitions.
The process flow of making the PDMS microfluidic system is shown in Figure 2A. Four layers of PDMS were molded individually from photoresist masters and bonded together after oxygen plasma treatment of the surfaces. A 16 µm layer of positive photoresist (Shipley SPR 220-7i; Microchem, Newton, MA) was patterned using a 3600 dpi resolution transparency film (Silverline Studio, Madison, WI) as a mask. The photoresist was reflowed at 110 °C for 5 min. To promote PDMS release, the photoresist master was exposed for 1 min to trimethochlorolosilane (TMCS) vapor before PDMS molding. Casting and peeling off the PDMS gave a microchannel structure with a high-fidelity negative replication of the photoresist pattern. Electrode solution connection holes were punched with a sharpened 19-gauge needle. A channel in the second layer and a channel in the third layer form a valve at their crossover point. A positive pressure (10 psi) in the top channel presses the thin elastomer film and pinches off the ionic solution in the bottom channel (Figure 3A-C). The electrical resistance of the blocked channel was measured to be greater than 10 GΩ, as measured 816
by the patch clamp amplifier. The success rate of our valves (resistance >10 GΩ) is higher than 90%. Although the valve isolation is stable over several hours, a decrease of dc resistance with time is observed, presumably due to contamination. A protocol of cleaning by flowing deionized water followed by methanol after each daily use of 6 h yielded a valve lifetime of 1 week. Planar Ag/AgCl electrodes were fabricated with traditional microfabrication methods (Figure 2A). A shadow mask (Fotofab Corp, Chicago, IL) was used to selectively coat nickel (50 Å) and silver (0.5 µm) on the cleaned glass slide. A 0.5 µm layer of Spin-on-Glass polymer (SOG 500F, Filmtronics, Butler, PA) was deposited and patterned with a 0.5 × 0.5 mm square window over the metallization. A Clorox bleach droplet was used to react with the exposed silver and generate an AgCl coating. The PDMS microfluidic and planar Ag/AgCl electrode subsystems were then carefully aligned and bonded together after treating both surfaces with UV ozone. Tin-coated copper wire was glued onto the silver leads with silver conductive Nano Lett., Vol. 6, No. 4, 2006
Figure 3. Microfluidic system for simultaneous planar PDMS patch clamp measurement. (A) Two micromolded channels form a valve at the crossover point. Channel widths are 200 µm and channel depths are 15 µm. (B) The 35 mm long flow channel is filled with ionic solution containing blue dye and has a resistance of 3 MΩ when the pressure in the control channel is zero. (C) The flow channel is closed when there is a positive pressure (10 psi) in the control channel (filled with air in this example). The measured electrical resistance in this case is higher than 10 GΩ. (D, E). A microfluidic device for simultaneous planar patch clamp measurement. Eight patch partitions are mounted onto the PDMS chamber through reversible bonding. Figure 2. (A) Microfluidic device fabrication procedure. Four layers of PDMS with thicknesses of 4 mm, 0.5 mm, 30 µm, and 60 µm are molded from patterns in reflowed photosensitive resist. They are cut, treated briefly with oxygen plasma, and bonded together to generate a monolithic fluidic device. Metal electrodes are formed by shadow evaporation of 50 Å thick nickel and 0.5 µm thick silver onto a cleaned glass slide. A coating of Spin-onGlass is applied and cured. After the SOG is patterned with standard lithography, small droplets of bleach are applied onto the open windows to chemically react with exposed silver and generate a thin layer of silver chloride coating. After opening holes are punched, the PDMS monolithic piece is bonded onto the microfabricated Ag/AgCl electrodes with the help of UV ozone treatment. (B) Schematic cross-section view of the device. A dense suspension of cells is dropped onto the PDMS partition, which readily seals to the reusable microfluidic system.
paste. Tubing was plugged into the connection holes in the PDMS and 1:1 epoxy glue was applied at the connection. Planar PDMS patch partitions, treated by oxygen plasma, were placed onto the PDMS microfluidic device. The glass support seals to PDMS microfluidic layer in a tight but reversible way. This seal also forms an electrical barrier between the bath solution and electrode solution. Positive pressure (20 mmHg) on the flow channel was used to force electrode solution to fill the cavity below the partition and the partition aperture. A bath solution droplet was subsequently dropped onto the planar patch partition. An AgClcoated silver wire connected the bath solution to ground. The planar Ag/AgCl electrode was connected to the signal input of the amplifier (Multiclamp 700A patch clamp Nano Lett., Vol. 6, No. 4, 2006
amplifier, Molecular Devices, Sunnyvale, CA). The signal was recorded with pClamp8.1 acquisition software using the Digidata 1322A interface (both from Molecular Devices). All fluidic channels were filled before measurement: control channels were filled with deionized water; flow channels and vacuum suction channels were filled with electrode solution. RBL-1 cells were used to investigate the system’s suitability for patch clamp measurements. The cell line was maintained at 37 °C, 5% CO2 in 75 mL culture bottles containing Minimum Essential Medium (MEM), 1% MEM sodium pyruvate solution, 1% MEM nonessential amino acids solution, 1% penicilin streptomycin, 15% fetal bovine serum. The bath (extracellular) solution contained 130 mM KCl, 4.4 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 5 mM dextrose, adjusted to pH 7.4 with NaOH; the electrode (intracellular) solution contained 130 mM KCl, 10 mM NaCl, 4 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM EGTA, adjusted to pH 7.4 with NaOH. Once RBL-1 cells reached 3.0 × 105 per mL in the culture bottle, they were washed twice with recording solution and harvested into a dense cell suspension. For each recording, 5 µL of cell suspension was dropped onto the patch partition (Figure 2B). A gentle (
