A DISPOSABLE MICROFLUIDIC ARRAY PLATFORM FOR AUTOMATIC ION CHANNEL RECORDING M. Rossi1*, F. Thei1, H. Morgan2 and M. Tartagni1 1
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ARCES - Advanced Research Center on Electronic Systems, University of Bologna, IT Nano Research Group, School of Electronics and Computer Science at the University of Southampton, UK
ABSTRACT High-throughput ion channel screening for drug discovery is the gold standard for investigating the function of channel proteins and it is at the base of the recent shift of resources in the pharmaceutical industry towards addressing drug safety issues earlier in the discovery process. This paper presents a versatile, low-cost and disposable microfluidic device realized using a micromilling process fabrication of polyoxymethilene homopolymer (Delrin™) substrates. The devices are suitable to host lipid bilayer membrane arrays for ion channel recording activities using a fully automated approach and are embedded in a parallel readout hybrid electronic system. KEYWORDS: Bilayer Lipid Membranes (BLM), Ion channels, High-Throughput Screening (HTS), Disposable biosensors. INTRODUCTION Membrane proteins are considered as a target for the majority of all pharmaceutical drugs. In this scenario, ionchannel HTS is promising and rapidly growing technology. Additionally, the integration of biological nanopores, such as ion channels with electronics is a promising approach for the development of novel biosensors to detect low concentrations of target molecules, or even identify differences between single DNA bases. In ion-channel HTS, proteins preserve their original structure and activity only within stable bilayer lipid membranes. Even if pioneering work on recording of single ion-channels started decades ago [1], the artificial formation of ion channel’s natural environment is a laborious process due to the complexity of the setup and very few examples of parallel ion-channel recording platforms are present in literature [2][3]. The main problems related to the mentioned platforms are: i) the above approaches are based on monolithic microfluidic structures where the yield of the array is linked to that of single spots; ii) the above operations frequently rely on microfluidic routing channels that do not scale efficiently with respect to the dimension of the array. PLATFORM STRUCTURE The section view of a single disposable monolithic block is shown in Fig.1 (left). The lateral dimensions of the block are 9 mm x 18 mm x 10 mm (WxLxH), designed to allow a limit-free scalable interface with the ANSI/SBS microplate standard multi-pipette or liquid handling robot in both planar directions. It is composed of two “operating chambers” (6 mm depth) separated by a thin (50 μm) septum containing a microhole (at 4 mm from the bottom) and two “inlet/outlet chambers” (at 9 mm of distance) for the infusion/withdrawal of buffer. The total volume of the chambers is 120 μl. The fabrication process is based on a computer numerical control (CNC) milling machine (Fig.1 right). Starting from a Delrin™ block milled on top and bottom surfaces for engraving the inlet/outlet and operating chambers on the top side, and the electrode slots on the bottom one (Fig.1a). Then side drilling, using a drill bit of 2mm (Fig.1b), allows to realize the interconnecting fluidic channels and the access hole for the creation of the microhole. Indeed, it is drilled using a 200 µm diameter drill bit (Fig.1c). The fabrication process ends by sealing the constructions holes with Delrin cylinders (Fig. 1d). Two Ag/AgCl electrodes are finally stuck into the bottom slots; the electrodes are fabricated by soldering a 0.5 mm diameter Ag wire into a socket connector and by immersion of the Ag wire into a fused AgCl solution. Microhole
Operating chambers
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Buffer infusion/ withdrawal chambers Ag/AgCl electrodes
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Figure 1: Section view of the Delrin block assembled on the PCB (left) and the four fabrication process steps (right)
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
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14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands
Fig.2 shows the final parallel readout hybrid electronic system [4] composed of the top “fluidic” side and the bottom “electronic” side connected only by the electrode sockets. Moreover each disposable Delrin block is directly interfaced with a dedicated low noise ADC converter [5] for ion channel recordings. Single Delrin blocks can be assembled on a PCB board to realize arrays of any size, with the final purpose to improve the acquisition capability of ion channels organized in parallel fashion.
TOP
BOTTOM
Figure 2: Top and bottom views of a 1x8 array platform (left and center). Black Delrin blocks are illustrated above, while the white one works as reservoirs for buffer and lipid solutions. Drawing showing the disposable characteristic of the approach (right). EXPERIMENTAL To test the platform, the approach is applied to an array of eight Delrin blocks and BLMs are formed using the automatic setup (Fig.3, right), which is composed of a MP-285 (three-axis micromanipulator) for pipette movements in the space and a NSC200 (single-axis micromanipulator) for pipette flux control. The phases for the BLM formation are described in Fig.3 (left), following the Montal-Mueller technique [6]. Briefly, the microhole is pre-treated with 10% hexadecane in pentane solution and after pentane evaporation the Delrin blocks are inserted in the platform. Then, buffer solution (1M KCl, 10mM HEPES, 1mM EGTA, PH 7) is infused in both intlet/outlet chambers beneath the microhole level. Afterwards, a lipid solution (10 mg/ml DPhPC in hexane) is deposited in both the operating chambers onto the surface of buffer solutions allowing the monolayer formation in about 15 minutes. Finally, the buffer levels are raised from the bottom infusing buffer in the inlet/outlet chambers at a velocity of 0,05 mm/s and the spreading of the two lipid monolayers on the microhole so as that the bilayer membrane is formed. To monitor the membrane formation, following a common technique [1], a triangular wave of 80 mVpp@6Hz is imposed by the electronic system and the signals coming from the parallel microfluidic Delrin blocks are visualized and recorded by means of a PC-based custom graphical user interface (GUI) [4].
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microhole pretreatment
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buffer injection
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lipids deposition & monolayer self-assembly
buffer rise and BLM formation
Figure 3: Four-steps BLM creation by automatic pipetting system (left) and picture of the overall automatic system with the PC-based custom graphical user interface (GUI) (right).
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RESULTS AND DISCUSSION Current signal squared waves response to the applied triangular wave are recorded and shown in Fig.4 (left) demonstrating the BLM formation during buffer rising. Due to the low frequencies regime of the input wave (6Hz) the current trace is not perfectly squared as expected. To demonstrate the effective bilayer formation, a constant voltage is then applied and α-hemolysin channel is inserted in the automatically formed lipid membrane. The current step, corresponding to the typical α-hemolysin channel ionic conductance of about 1 nS, is recorded after few minutes from the insertion of the toxin in solution (Fig.4, right). All the acquisitions are made using the presented disposable microfluidic devices embedded in the parallel readout hybrid electronic system.
Figure 4: Current traces measurement showing BLM automatic formation, with a zoom of square wave response(left) and a single α-hemolysin channel insertion demonstrated by the single current step of about 40pA versus an applied voltage of 40mV (right). CONCLUSION A new low-cost, disposable, microfluidic device able to host bilayer lipid membrane using Montal-Mueller technique has been presented and experimental results demonstrates the functionality of the array platform. The main advantages of the proposed approach over state-of-the-art are: 1) the microfluidic structure is selectively disposable at single spot level where faulty elements can be automatically revealed by electronic sensing; 2) the platform can be interfaced to any generic liquid-handling robot or multi-pipette and is fully scalable to any array size; 3) each microfluidic device is independent and directly interfaced with an electronic interface for a truly parallel readout signal acquisition. ACKNOWLEDGEMENTS This work was supported by the 6th Framework Programme of the European Commission under the contract NMP4CT-2005-017114 "RECEPTRONICS". We are alsRJUDWHIXOWRWKH³)RQGD]LRQH&DVVDGHL5LVSDUPLGL)RUOu´IRULWVVXpport. This paper is dedicated to the memory of the friend and colleague Prof. Silvio Cavalcanti. REFERENCES [1] “Bilayer Lipid Membranes (BLM) - Theory and practice”, H. Ti Tien, Marcel Dekker , INC., New York (1974). [2] “Lipid bilayer microarray for parallel recording of transmembrane ion currents”, B. Le Pioufle, H. Suzuki, K. V. Tabata, H. Noji, and S. Takeuchi, Anal. Chem., Vol. 80, no. 1, pp. 328–332 (2008). [3] “Microfluidic array platform for simultaneous lipid bilayer membrane formation”, M. Zagnoni, M.E. Sandison, and H. Morgan, Biosens. Bioelectron., Vol. 24, 1235-1240 (2009). [4] “Parallel Recording of Single Ion Channels: A Heterogeneous System Approach”, F. Thei, M. Rossi, M. Bennati, M. Crescentini, F. Lodesani, H. Morgan, and M. Tartagni, IEEE Transactions on Nanotechnology, Vol. 9, no. 3, pp. 295 - 302 (2010). [5] “A Sub-pA Delta-Sigma Current Amplifier for Single Molecule Nanosensors”, M. Bennati, F. Thei, M. Rossi, M. Crescentini, G. D’Avino, A. Baschirotto, and M. Tartagni, ISSCC Digest of Technical Papers, Vol. 52, 348-350 (2009). [6] “Formation of Bimolecular Membranes from Lipid Monolayers and a Study of Their Electrical Properties”, M. Montal and P. Mueller, Proc. Natl. Acad. Sci. U.S.A., Vol. 69, pp. 3561–3566 (1972). CONTACT *Michele Rossi, tel: +39 0547 339247; e-mail:
[email protected] 1915
