Fully Integrated Miniature Device for Automated Gene Expression DNA Microarray Processing Robin Hui Liu,* Tai Nguyen, Kevin Schwarzkopf, H. Sho Fuji, Alla Petrova, Tony Siuda, Kia Peyvan, Michael Bizak, David Danley, and Andy McShea CombiMatrix Corporation, 6500 Harbor Heights Parkway, Mukilteo, Washington 98275 A DNA microarray with 12 000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Elec- trochemical micropumps were integrated into the car- tridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus elimi- nating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor- intensive and time-consuming fluidic handling steps in genomic analysis. Microarrays have become a widely used technology for studying mRNA levels and examining gene expression in biologi- cal samples. Investigators rely on data produced by microarray experiments to assess changes in gene expression levels among various experimental tissues and treatments. The applications of microarrays for gene expression profiling 1 include pathway dis- section, 2 drug evaluation, 3,4 discovery of gene function, 5 classifica- tion of clinical samples, 6-8 and investigation of splicing events, 9 among many others. 10 The highly parallel nature of microarrays has made them invaluable tools for monitoring gene expression patterns of numerous genes simultaneously. Biological experi- ments have a number of inherent variables making it imperative that the microarray platform be extremely reproducible, both to provide confidence in the data collected and to accurately identify small changes in gene expression patterns. Because the most interesting genes are often expressed at the lowest levels in the sample, it is equally important to use a highly sensitive microarray system. There are various microarray technologies and numerous commercially available sources of microarrays. Microarrays can be produced either by physical deposition of presynthesized DNA 1,11,12 or by in situ oligonucleotide synthesis. 13,14 The former requires labor-intensive preparation (and, hence, very significant * To whom correspondence should be addressed. E-mail: rliu@ combimatrix.com. (1) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467- 470. (2) Roberts, C. J.; Nelson, B.; Marton, M. J.; Stoughton, R.; Meyer, M. R.; Bennett, H. A.; He, Y. D.; Dai, H.; Walker, W. L.; Hughes, T. R.; Tyers, M.; Boone, C.; Friend, S. H. Science 2000, 287, 873-880. (3) Hughes, T. R.; Marton, M. J.; Jones, A. R.; Roberts, C. J.; Stoughton, R.; Armour, C. D.; Bennett, H. A.; Coffey, E.; Dai, H.; He, Y. D.; Kidd, M. J.; King, A. M.; Meyer, M. R.; Slade, D.; Lum, P. Y.; Stepaniants, S. B.; Shoemaker, D. D.; Gachotte, D.; Chakraburtty, K.; Simon, J.; Bard, M.; Friend, S. H. Cell 2000, 102, 109-126. (4) Gray, N. S.; Wodicka, L.; Thunnissen, A. M.; Norman, T. C.; Kwon, S.; Espinoza, F. H.; Morgan, D. O.; Barnes, G.; LeClerc, S.; Meijer, L.; Kim, S. H.; Lockhart, D. J.; Schultz, P. G. Science 1998, 218, 533-538. (5) Chu, S.; DeRisi, J.; Eisen, M.; Mulholland, J.; Botstein, D.; Brown, P. O.; Herskowitz, I. Science 1998, 282, 699-705. (6) Khan, J.; Simon, R.; Bittner, M.; Chen, Y.; Leighton, S. B.; Pohida, T.; Smith, P. D.; Jiang, Y.; Gooden, G. C.; Trent, J. M.; Meltzer, P. S. Cancer Res. 1998, 58, 5009-5013. (7) Perou, C. M.; Jeffrey, S. S.; van de Rijn, M.; Rees, C. A.; Eisen, M. B.; Ross, D. T.; Pergamenschikov, A.; Williams, C. F.; Zhu, S. X.; Lee, J. C.; Lashkari, D.; Shalon, D.; Brown, P. O.; Botstein, D. Proc. Natl Acad. Sci. U.S.A. 1999, 96, 9212-9217. (8) Golub, T. R.; Slonim, D. K.; Tamayo, P.; Huard, C.; Gaasenbeek, M.; Mesirov, J. P.; Coller, H.; Loh, M. L.; Downing, J. R.; Caligiuri, M. A.; Bloomfield, C. D.; Lander, E. S. Science 1999, 286, 531-537. (9) Hu, G. K.; Madore, S. J.; Moldover, B.; Jatkoe, T.; Balaban, D.; Thomas, J.; Wang, Y. Genome Res. 2001, 11, 1237-1245. (10) Schena, M. Microarray Biochip Technology; Eaton Publishing: Natick, MA, 2000. (11) Ramakrishnan, R.; Dorris, D.; Lublinsky, A.; Nguyen, A.; Domanus, M.; Prokhorova, A.; Gieser, L.; Touma, E.; Lockner, R.; Tata, M.; Zhu, X.; Patterson, M.; Shippy, R.; Sendera, T. J.; Mazumder, A. Nucleic Acids Res. 2002, 30, e30. (12) Yue, H.; Eastman, P. S.; Wang, B. B.; Minor, J.; Doctolero, M. H.; Nuttall, R. L.; Stack, R.; Becker, J. W.; Montgomery, J. R.; Vainer, M.; Johnston, R. Nucleic Acids Res. 2001, 29, e41. (13) Southern, E. M.; Maskos, U.; Elder, J. K. Genomics 1992, 13, 1008-1017. (14) Maskos, U.; Southern, E. M. Nucleic Acids Res. 1992, 20, 1679-1684. Anal. Chem. 2006, 78, 1980-1986 1980 Analytical Chemistry, Vol. 78, No. 6, March 15, 2006 10.1021/ac0518553 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/03/2006