Microarrays pp 67-93 | Cite as

Integrated Microfluidic Devices for Automated Microarray-Based Gene Expression and Genotyping Analysis

  • Robin H. Liu
  • Mike Lodes
  • H. Sho Fuji
  • David Danley
  • Andrew McShea
Part of the Integrated Analytical Systems book series (ANASYS)


Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray® chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The micro-fluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. 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. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.


Check Valve Microarray Chip Hybridization Chamber Storage Chamber Fluidic Handling 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Acknowledgment The authors thank Tai Nguyen, Kevin Schwarzkopf, Tony Siuda, Alla Petrova, Kia Peyvan, Michael Bizak, Jeff Kemper, Al Pierce, and Mike Slota for technical support and useful discussions. This work has been sponsored by DoD contract #1999011104A.


  1. 1.
    Kelly, R.T. and A.T. Woolley (2005). Microfluidic systems for integrated, high-throughput DNA analysis. Anal. Chem. 77: 97A–102A.Google Scholar
  2. 2.
    Harrison, D.J., A. Manz, et al. (1992). Capillary electrophoresis and sample injection systems integrated on a planar glass chip. Anal. Chem. 64: 1926–1932.CrossRefGoogle Scholar
  3. 3.
    Wilding, P., J. Pfahler, et al. (1994). Manipulation and flow of biological-fluids in straight channels micromachined in silicon. Clin. Chem. 40(1): 43–47.Google Scholar
  4. 4.
    Xia, Y.N. and G.M. Whitesides (1998). Soft lithography. Ann. Rev. Mater. Sci. 28: 153–184.CrossRefGoogle Scholar
  5. 5.
    Piner, R.D., J. Zhu, et al. (1999). “Dip-pen”p nanolithography. Science 283(5402): 661–663.CrossRefGoogle Scholar
  6. 6.
    Becker, H., W. Dietz, et al. (1998). Microfluidic manifolds by polymer hot embossing for micro total analysis system applications. uTas 98, Banff, Canada; Dordrecht, Kluwer Academic.Google Scholar
  7. 7.
    Alonso-Amigo, M.G. and H. Becker (2000). Microdevices fabricated by polymer hot embossing. Abstracts of Papers of the American Chemical Society 219: 468–COLL.Google Scholar
  8. 8.
    Grodzinski, P., R.H. Liu, et al. (2001). Development of plastic microfluidic devices for sample preparation. Biomed. Microdevices 3(4): 275.CrossRefGoogle Scholar
  9. 9.
    Boone, T., Z.H. Fan, et al. (2002). Plastic advances microfluidic devices. Anal. Chem. 74(3): 78A–86A.CrossRefGoogle Scholar
  10. 10.
    Harrison, D.J., K. Fluri, et al. (1993). Micromachining a miniaturized capillary electrophoresis-based chemical-analysis system on a chip. Science 261(5123): 895–897.CrossRefGoogle Scholar
  11. 11.
    Woolley, A.T., D. Hadley, et al. (1996). Functional integration of PCR amplification and capillary electrophresis in a microfabricated DNA analysis device. Anal. Chem. 68(23, Dec. 1996): 4081–4086.CrossRefGoogle Scholar
  12. 12.
    Burns, M.A., B.N. Johnson, et al. (1998). An integrated nanoliter DNA analysis device. Science 282(5388): 484–487.CrossRefGoogle Scholar
  13. 13.
    Waters, L.C., S.C. Jacobson, et al. (1998). Microchip device for cell lysis, multiplex pcr amplification and electrophoretic sizing. Anal. Chem. 70: 158–162.CrossRefGoogle Scholar
  14. 14.
    Emrich, C.A., H.J. Tian, et al. (2002). Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis. Anal. Chem. 74(19): 5076–5083.CrossRefGoogle Scholar
  15. 15.
    Ibrahim, M.S., R.S. Lofts, et al. (1998). Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal. Chem. 70(9): 2013–2017.CrossRefGoogle Scholar
  16. 16.
    Kopp, M., A.D. Mello, et al. (1998). Chemical amplification: Continuous-Flow PCR on a chip. Science 280: 1046–1048.CrossRefGoogle Scholar
  17. 17.
    Lagally, E.T., I. Medintz, et al. (2001). Single-molecule DNA amplification and analysis in an integrated microfluidic device. Anal. Chem. 73: 565–570.CrossRefGoogle Scholar
  18. 18.
    Taylor, M.T., P. Belgrader, et al. (2001). Lysing bacterial spores by sonication through a flexible interface in a microfluidic system. Anal. Chem. 73(3): 492–496.CrossRefGoogle Scholar
  19. 19.
    Yuen, P.K., L.J. Kricka, et al. (2001). Microchip module for blood sample preparation and nucleic acid amplification reactions. Genome Res. 11(3): 405–412.CrossRefGoogle Scholar
  20. 20.
    Anderson, R.C., X. Su, et al. (2000). A miniature integrated device for automated multistep genetic assays. Nucleic Acids Res. 28(12): e60.CrossRefGoogle Scholar
  21. 21.
    Liu, R.H., J. Yang, et al. (2004). Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection. Anal. Chem. 76: 1824–1832.CrossRefGoogle Scholar
  22. 22.
    Hay, A., V. Gregory, et al. (2001). The evolution of human influenza viruses. Phil. Trans. R. Soc. Lond. B356: 1861–1870.Google Scholar
  23. 23.
    Fouchier, R.A., V. Munster, et al. (2005). Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79(5): 2814–2822.CrossRefGoogle Scholar
  24. 24.
    Scholtissek, C., H. Burger, et al. (1985). The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses. Virology 147(2): 287–294.CrossRefGoogle Scholar
  25. 25.
    Hoffmann, E., J. Stech, et al. (2001). Universal primer set for the full-length amplification of all influenza A viruses. Arch. Virol. 146: 1–15.CrossRefGoogle Scholar
  26. 26.
    Lipatov, A.S., E.A. Govorkova, et al. (2004). Influenza: Emergence and control. J. Vi ro l. 78(17): 8951–8959.CrossRefGoogle Scholar
  27. 27.
    Webby, R.J. and R.G. Webster (2001). Emergence of influenza A viruses. Phil. Trans. R. Soc. Lond. B356: 1815–1826.Google Scholar
  28. 28.
    Mizuta, K., N. Katsushima, et al. (2003). A rare appearance of influenza A(H1N2) as a reassortant in a community such as Yamagata where A(H1N1) and A(H3N2) co-circulate. Microbiol. Immunol. 47(5): 359–361.Google Scholar
  29. 29.
    Ueda, M., A. Maeda, et al. (1998). Application of subtype-specific monoclonal antibodies for rapid detection and identification of influenza A and B viruses. J. Clin. Microbiol. 36(2): 340–344.Google Scholar
  30. 30.
    Allwinn, R., W. Preiser, et al. (2002). Laboratory diagnosis of influenza-virology or serology? Med. Microbiol. Immunol. (Berl) 191(3–4): 157–160.Google Scholar
  31. 31.
    Amano, Y. and Q. Cheng (2005). Detection of influenza virus: Traditional approaches and development of biosensors. Anal. Bioanal. Chem. 381(1): 156–184.CrossRefGoogle Scholar
  32. 32.
    Li, J., S. Chen, et al. (2001). Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR. J. Clin. Microbiol. 39(2): 696–704.CrossRefGoogle Scholar
  33. 33.
    Ellis, J.S. and M.C. Zambon (2002). Molecular diagnosis of influenza. Rev. Med. Virol. 12(6): 375–389.CrossRefGoogle Scholar
  34. 34.
    Ivshina, A.V., G.M. Vodeiko, et al. (2004). Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method. J. Clin. Microbiol. 42(12): 5793–5801.CrossRefGoogle Scholar
  35. 35.
    Kessler, N., O. Ferraris, et al. (2004). Use of the DNA flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses. J. Clin. Microbiol. 42(5): 2173–2185.CrossRefGoogle Scholar
  36. 36.
    Sengupta, S., K. Onodera, et al. (2003). Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization. J. Clin. Microbiol. 41(10): 4542–4550.CrossRefGoogle Scholar
  37. 37.
    Lodes, M.J., D. Suciu, et al. (2006). Influenza A subtype identification and sequencing with semiconductor-based oligonucleotide microarrays. J. Clin. Microbiol. 44: 1209–1218.CrossRefGoogle Scholar
  38. 38.
    Liu, R.H., M.J. Lodes, et al. (2006). Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing. Anal. Chem. 78: 4184–4193.CrossRefGoogle Scholar
  39. 39.
    Liu, R.H., K. Dill, et al. (2006). Integrated microfluidic biochips for DNA microarray analysis. Expert Rev. Molec. Diagnostics 6: 253–261.CrossRefGoogle Scholar
  40. 40.
    Oleinikov, A.V., M.D. Gray, et al. (2003). Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation. J. Proteome Res. 2: 313.CrossRefGoogle Scholar
  41. 41.
    Dill, K., D.D. Montgomery, et al. (2004). Immunoassays and sequence-specific DNA detection on a microchip using enzyme amplified electrochemical detection. J. Biochem. Biophys. Meth. 59: 181–187.CrossRefGoogle Scholar
  42. 42.
    Macken, C., H. Lu, et al. (2001). The value of a database in surveillance and vaccine selection. Options for the Control of Influenza IV. N. C. A. W. H. A.D.M.E. Osterhaus. Amsterdam, Elsevier Science: 103–106.Google Scholar
  43. 43.
    Wang, D., A. Urisman, et al. (2003). Viral discovery and sequence recovery using DNA microarrays. PLoS Biol. 1(2): 257–260.CrossRefGoogle Scholar
  44. 44.
    Allawi, H.T. and J. Santa Lucia Jr. (1999). Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches. Biochemistry 38: 3468–3477.CrossRefGoogle Scholar
  45. 45.
    Altschul, S.F., T.L. Madden, et al. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402.CrossRefGoogle Scholar
  46. 46.
    Ray, C.A., C.L. Sloan, et al. (1992). A silicon-based shape memory alloy microvalve. Proc. Mater. Res. Soc. Symposium 276: 161–166.Google Scholar
  47. 47.
    Jerman, H. (1994). Electrically-activated, normally-closed diaphragm valves. J. Micromech Microeng. 4: 210–216.CrossRefGoogle Scholar
  48. 48.
    Beebe, D.J., J.S. Moore, et al. (2000). Functional structures for autonomous flow control inside microfluidic channels. Nature 404: 588–590.CrossRefGoogle Scholar
  49. 49.
    Liu, R.H., Q. Yu, et al. (2002). Fabrication and characterization of hydrogel-based microvalves. J.Microelectromech.Syst. 11: 45–53.CrossRefGoogle Scholar
  50. 50.
    Liu, R.H., M. Stremler, et al. (2000). A passive micromixer: 3-D C-shape serpentine microchannel. J. Microelectromech. Syst. 9(2): 190–197.CrossRefGoogle Scholar
  51. 51.
    Zengerle, R., S. Skluge, et al. (1995). A bidirectional silicon micropump. Sensors Actuators A-Physical 50: 81–86.CrossRefGoogle Scholar
  52. 52.
    Unger, M.A., H. Chou, et al. (2000). Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 288: 113–116.CrossRefGoogle Scholar
  53. 53.
    Su, Y.C., L.W. Lin, et al. (2002). A water-powered osmotic microactuator. J. Microelectromech. Syst. 11(6): 736–742.CrossRefGoogle Scholar
  54. 54.
    Richter, G. (1975). Device for Supplying Medicines. U.S. Patent, 3,894,538.Google Scholar
  55. 55.
    Bohm, S., W. Olthuis, et al. (1999). An integrated micromachined electrochemical pump and dosing system. J. Biomed. Microdevices 1(2): 121–130.CrossRefGoogle Scholar
  56. 56.
    Munyan, J.W., H.V. Fuentes, et al. (2003). Electrically actuated, pressure-driven microfluidic pumps. Lab Chip 3: 217–220.CrossRefGoogle Scholar
  57. 57.
    Liu, R.H., R. Lenigk, et al. (2003). Hybridization enhancement using cavitation microstreaming. Anal. Chem. 75: 1911–1917.CrossRefGoogle Scholar
  58. 58.
    Liu, R.H., T. Nguyen, et al. (2006). A fully integrated miniature device for automated gene expression DNA microarray processing. Anal. Chem. 78: 1980–1986.CrossRefGoogle Scholar
  59. 59.
    Iwatsuki-Horimoto, K., R. Kanazawa, et al. (2004). The index influenza A virus subtype H5N1 isolated from a human in 1997 differs in its receptor-binding properties from a virulent avian influenza virus. J. Gen. Virol. 85: 1001–1005.CrossRefGoogle Scholar
  60. 60.
    Schena, M., D. Shalon, et al. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270.: 467–470.CrossRefGoogle Scholar
  61. 61.
    Chu, S., J. DeRisi, et al. (1998). The transcriptional program of sporulation in budding yeast. Science 282: 699–705.CrossRefGoogle Scholar
  62. 62.
    Gray, N.S., L. Wodicka, et al. (1998). Exploiting chemical libraries, structure and genomics in the search for kinase inhibitors. Science 218: 533–538.CrossRefGoogle Scholar
  63. 63.
    Hughes, T.R., M.J. Marton, et al. (2000). Functional discovery via a compendium of expression profiles. Cell 102: 109–126.CrossRefGoogle Scholar
  64. 64.
    Schena, M. (2000). Microarray BiochipTechnology. Natick, MA, Eaton.Google Scholar
  65. 65.
    Roberts, C.J., B. Nelson, et al. (2000). Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287: 873–880.CrossRefGoogle Scholar
  66. 66.
    Khan, J., R. Simon, et al. (1998). Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays. Cancer Res. 58: 5009–5013.Google Scholar
  67. 67.
    Golub, T.R., D.K. Slonim, et al. (1999). Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 286: 531–537.CrossRefGoogle Scholar
  68. 68.
    Perou, C.M., S.S. Jeffrey, et al. (1999). Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc. Natl Acad. Sci. USA 96: 9212–9217.CrossRefGoogle Scholar
  69. 69.
    Hu, G.K., S.J. Madore, et al. (2001). Predicting splice variants from DNA chip expression data. Genome Res. 11: 1237–1245.CrossRefGoogle Scholar
  70. 70.
    Dill, K. and A. McShea (2005). Recent advances in microarrays. Drug Discovery Today: Technol. 2(3): 261–266.CrossRefGoogle Scholar
  71. 71.
    Gershon, D. (2005). DNA microarrays. Nature 437: 1195–1200.CrossRefGoogle Scholar
  72. 72.
    Dobbin, K.K., D.G. Beer, et al. (2005). Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays. Clin. Cancer Res. 11: 565–572.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Robin H. Liu
    • 1
  • Mike Lodes
    • 2
  • H. Sho Fuji
    • 2
  • David Danley
    • 2
  • Andrew McShea
    • 2
  1. 1.CombiMatrix Corporation, IncOsmetech Molecular, DiagnosticsPasadena, CAUSA
  2. 2.CombiMatrix Corp.Mukilteo

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