Anal. Chem. 2005, 77, 3897-3903
Accelerated Articles
Microfluidic Gradient-Generating Device for Pharmacological Profiling Johan Pihl,† Jon Sinclair,† Eskil Sahlin,‡ Mattias Karlsson,§ Fredrik Petterson,§ Jessica Olofsson,† and Owe Orwar*,†
Department of Chemistry and Bioscience, and Microtechnology Centre, Chalmers University of Technology, SE-412 96 Go¨teborg, Sweden, Department of Chemistry, Go¨teborg University, SE-412 96 Go¨teborg, Sweden, and Cellectricon AB, Fabriksgatan 7 SE-412 50 Go¨teborg, Sweden
We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cellbased sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K+ channels (hERG) and ligand-gated GABAA receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC50 and EC50 values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.
Microsystems engineering has during the past decade been instrumental for the realization of powerful research tools in chemistry and physics. Specifically, the area of single-cell analysis has benefited greatly, and examples of applications where microfabricated platforms are utilized for single-cell analysis include high-throughput screening,1 content analysis,2 viability assays,3 * Corresponding author. E-mail:
[email protected]. Phone: + 46 (0)31 772 30 60. Fax: + 46 (0)31 772 61 20. † Chalmers University of Technology. ‡ Go ¨teborg University. § Cellectricon AB. (1) Dunn, D. A.; Feygin, I. Drug Discovery Today 2000, 5, 84-91. (2) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Allbritton, N. L.; Sims, C. E.; Ramsey, J. M. Anal. Chem. 2003, 75, 5646-5655. 10.1021/ac050218+ CCC: $30.25 Published on Web 05/26/2005
© 2005 American Chemical Society
and disease diagnostics.4 One particular field that has witnessed a tremendous development from the implementation of microfabrication technology is ion channel research. To date, the standard method for deriving high-quality data on the function and behavior of ion channels is the patch-clamp technique.5 The technology is widely used in drug development for pharmacological profiling of ion channels, especially for target validation, lead optimization, and safety studies where data quality is of great importance. Unfortunately, patch-clamp is highly labor-intensive with a very low throughput, mainly limited by the number of cells that an operator can analyze per unit time. This fact underlies recent efforts in developing automized patch-clamp systems.6 We have previously reported on the use of a chip-based microfluidic device that addresses the throughput issue in patchclamp.7,8 This device produces well-defined solution environments in open volumes9 and enables solution exchange on the millisecond time scale around single-cell biosensors such as patchclamped cells. By utilizing a sequential screening concept, this device enables a massive increase in throughput in conventional patch-clamp by maximizing the number of data points extracted from each patch-clamped cell. In particular, this system is ideal (3) Wheeler, A. R.; Throndset, W. R.; Whelan, R. J.; Leach, A. M.; Zare, R. N.; Liao, Y. H.; Farrell, K.; Manger, I. D.; Daridon, A. Anal. Chem. 2003, 75, 3581-3586. (4) Shelby, J. P.; White, J.; Ganesan, K.; Rathod, P. K.; Chiu, D. T. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 14618-14622. (5) Hamill, O. P.; Marty, A.; Neher, E.; Sakmann, B.; Sigworth, F. J. Pflu ¨ gers Arch. 1981, 391, 85-100. (6) Willumsen, N. J.; Bech, M.; Olesen, S.-P.; Skaaning Jensen, B.; Korsgaard, M. P. G.; Christophersen, P. Recept. Channels 2003, 9, 3-12. (7) Sinclair, J.; Pihl, J.; Olofsson, J.; Karlsson, M.; Jardemark, K.; Chiu, D. T.; Orwar, O. Anal. Chem. 2002, 74, 6133-6138. (8) Sinclair, J.; Olofsson, J.; Pihl, J.; Orwar, O. Anal. Chem. 2003, 75, 67186722. (9) Olofsson, J.; Pihl, J.; Sinclair, J.; Sahlin, E.; Karlsson, M.; Orwar, O. Anal. Chem. 2004, 76, 4968-4976.
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for dose-response measurements where a large number of different concentrations of a single compound can be tested at the single-cell level, resulting in a high degree of reproducibility and a low cell-to-cell variance.7 Highly resolved dose-response curves are valuable since they tell you if there is single or multiple binding of the substance of interest and gives information on the efficiency and potency of a drug. Because most receptors and ion channels respond to drugs in concentrations spanning several orders of magnitude, doseresponse experiments require the preparation of a large number of solutions with different concentrations from one single stock solution. Needless to say, this is a time-consuming task and is, in addition, causing experimental error, since pipet stations or serial dilutions using micropipets will induce systematic dilution errors. Furthermore, the solutions must be delivered to the patch-clamped cell with full control of solution exchange and exposure times, since small variations in these parameters can change the response of ion channels dramatically (Sinclair, J.; et al., submitted). We here present a microfluidic device that solves these problems by combining the concept of open volume microfluidics for rapid solution exchange in patch-clamp with the automatic generation of gradients.10,11 By implementation of a novel microfabrication technology, we show that it is possible to manufacture microfluidic gradient generators with a dynamic range of nearly 5 orders of magnitude with excellent performance and reproducibility. EXPERIMENTAL SECTION Materials. The 150-mm, two sides polished, silicon on insulator wafers (SOI) with a device layer thickness of 61 ( 0.5 µm (TTV
