Integrated Microfluidic CustomArray™ Biochips for Gene Expression and Genotyping Analysis

  • Robin Hui Liu
  • Mike Lodes
  • H. Sho Fuji
  • David Danley
  • Andrew McShea
Part of the Biotechnology Intelligence Unit book series (BIOIU)


DNA microarray technology has become one of the most promising analytical tools in molecular biology. It has been widely used for studying mRNA levels and examining gene expression in biological samples. It is becoming a powerful tool in the arena of diagnostics and personalized medicine. In this chapter, we present a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps required to carry out microarray-based gene expression or genotyping analysis. 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 oligonucleotides were synthesized on an array of electrodes on a semiconductor chip using phosphoramidite chemistry under electrochemical control. The microfluidic 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.


Microarray Chip Hybridization Chamber Storage Chamber Fluidic Handling Microfluidic Array 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Schena M, Shalon D, Davis RW et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995; 270:467–470.PubMedCrossRefGoogle Scholar
  2. 2.
    Hughes TR, Marton MJ, Jones AR et al. Functional discovery via a compendium of expression profiles. Cell 2000; 102:109–126.PubMedCrossRefGoogle Scholar
  3. 3.
    Gray NS, Wodicka L, Thunnissen AM et al. Exploiting chemical libraries, structure and genomics in the search for kinase inhibitors. Science 1998; 218:533–538.CrossRefGoogle Scholar
  4. 4.
    Chu S, DeRisi J, Eisen M et al. The transcriptional program of sporulation in budding yeast. Science 1998; 282:699–705.PubMedCrossRefGoogle Scholar
  5. 5.
    Schena M. Microarray BiochipTechnology. Natick, MA: Eaton Publishing 2000.Google Scholar
  6. 6.
    Roberts CJ, Nelson B, Marton MJ et al. Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 2000; 287:873–880.PubMedCrossRefGoogle Scholar
  7. 7.
    Khan J, Simon R, Bittner M et al. Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays. Cancer Res 1998; 58:5009–5013.PubMedGoogle Scholar
  8. 8.
    Perou CM, Jeffrey SS, van de Rijn M et al. Distinctive gene expression patterns in human mammary epithelial cells and breast cancers. Proc Natl Acad Sci USA 1999; 96:9212–9217.PubMedCrossRefGoogle Scholar
  9. 9.
    Golub TR, Slonim DK, Tamayo P et al. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999; 286:531–537.PubMedCrossRefGoogle Scholar
  10. 10.
    Hu GK, Madore SJ, Moldover B et al. Predicting splice variants from DNA chip expression data. Genome Res 2001; 11:1237–1245.PubMedCrossRefGoogle Scholar
  11. 11.
    Dill K, McShea A. Recent advances in microarrays. Drug Discovery Today: Technologies 2005; 2(3):261–266.CrossRefGoogle Scholar
  12. 12.
    Gershon D. DNA Microarrays. Nature 2005; 437:1195–1200.PubMedCrossRefGoogle Scholar
  13. 13.
    Ellis JS, Zambon MC. Molecular diagnosis of influenza. Rev Med Virol 2002; 12(6):375–389.PubMedCrossRefGoogle Scholar
  14. 14.
    Ivshina AV, Vodeiko GM, Kuznetsov VA et al. Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method. J Clin Microbiol 2004; 42(12):5793–5801.PubMedCrossRefGoogle Scholar
  15. 15.
    Kessler N, Ferraris O, Palmer K et al. Use of the DNA flow-thru chip, a three-dimensional biochip, for typing and subtyping of influenza viruses. J Clin Microbiol 2004; 42(5):2173–2185.PubMedCrossRefGoogle Scholar
  16. 16.
    Li J, Chen S, Evans DH. Typing and subtyping influenza virus using DNA microarrays and multiplex reverse transcriptase PCR. J Clin Microbiol 2001; 39(2):696–704.PubMedCrossRefGoogle Scholar
  17. 17.
    Sengupta S, Onodera K, Lai A et al. Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization. J Clin Microbiol 2003; 41(10):4542–4550.PubMedCrossRefGoogle Scholar
  18. 18.
    Lodes MJ, Suciu D, Elliott M et al. Influenza A Subtype Identification and Sequencing with Semiconductor-based Oligonucleotide Microarrays. J Clin Microbiol 2006; 44:1209–1218.PubMedCrossRefGoogle Scholar
  19. 19.
    Hay A, Gregory V, Douglas AR et al. The evolution of human influenza viruses. Phil Trans R Soc Lond 2001; B356:1861–1870.Google Scholar
  20. 20.
    Fouchier RA, Munster V, Wallensten A et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 2005; 79(5):2814–2822.PubMedCrossRefGoogle Scholar
  21. 21.
    Hoffmann E, Stech J, Guan Y et al. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 2001; 146:1–15.CrossRefGoogle Scholar
  22. 22.
    Lipatov AS, Govorkova EA, Webby RJ et al. Influenza: Emergence and control. J Virol 2004; 78(17):8951–8959.PubMedCrossRefGoogle Scholar
  23. 23.
    Scholtissek C, Burger H, Kistner O et al. The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses. Virology 1985; 147(2):287–294.PubMedCrossRefGoogle Scholar
  24. 24.
    Mizuta K, Katsushima N, Ito S et al. A rare appearance of influenza A(H1N2) as a reassortant in a community such as Yamagata where A(H1N1) and A(H3N2) cocirculate. Microbiol Immunol 2003; 47(5):359–361.PubMedGoogle Scholar
  25. 25.
    Webby RJ, Webster RG. Emergence of influenza A viruses. Phil Trans R Soc Lond 2001; B356:1815–1826.Google Scholar
  26. 26.
    Allwinn R, Preiser W, Rabenau H et al. Laboratory diagnosis of influenza-virology or serology? Med Microbiol Immunol (Berl) 2002; 191(3–4):157–160.Google Scholar
  27. 27.
    Amano Y, Cheng Q. Detection of influenza virus: traditional approaches and development of biosensors. Anal Bioanal Chem 2005; 381(1):156–184.PubMedCrossRefGoogle Scholar
  28. 28.
    Ueda M, Macda A, Nakagawa N et al. Application of subtype-specific monoclonal antibodies for rapid detection and identification of influenza A and B viruses. J Clin Microbiol 1998; 36(2):340–344.PubMedGoogle Scholar
  29. 29.
    Ramakrishnan R, Dorris D, Lublinsky A et al. An assessment of Motorola CodeLink microarray performance for gene expression profiling applications. Nucleic Acids Res 2002; 30:e30.PubMedCrossRefGoogle Scholar
  30. 30.
    Yue H, Eastman PS, Wang BB et al. An evaluation of the performance of cDNA microarrays for detecting changes in global mRNA expression. Nucleic Acids Res 2001; 29:e41.PubMedCrossRefGoogle Scholar
  31. 31.
    Southern EM, Maskos U, Elder JK. Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics 1992; 13:1008–1017.PubMedCrossRefGoogle Scholar
  32. 32.
    Maskos U, Southern EM. Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesized in situ. Nucleic Acids Res. 1992;20:1679–1684.PubMedCrossRefGoogle Scholar
  33. 33.
    Fodor SPA, Read JL, Pirrung MC et al. Light-directed, spatially addressable parallel chemical synthesis. Science 1991; 251:767–773.PubMedCrossRefGoogle Scholar
  34. 34.
    Hughes TR, Mao M, Jones AR et al. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 2001; 19:342–347.PubMedCrossRefGoogle Scholar
  35. 35.
    Singh-Gasson S, Green RD, Yue Y et al. Maskless fabrication of lightdirected oligonucleotide microarrays using a digital micromirror array. Nat Biotechnol 1999; 17:974–978.PubMedCrossRefGoogle Scholar
  36. 36.
    Pellois JP, Zhou X, Srivannavit O et al. Individually addressable parallel peptide synthesis on microchips. Nat Biotechnol 2002; 20:922–926.PubMedCrossRefGoogle Scholar
  37. 37.
    Dill K, Montgomery DD, Ghindilis AL et al. Immunoassays and sequence-specific DNA detection on a microchip using enzyme amplified electrochemical detection. Biochem Biophys Methods. 2004; 59:181–187.CrossRefGoogle Scholar
  38. 38.
    Nittler MP, Hocking-Murray D, Foo CK et al. Identification of Histoplasma capsulatum Transcripts Induced in Response to Reactive Nitrogen Species. Mol Biol Cell 2005 (in press).Google Scholar
  39. 39.
    Maurer K, McShea A, Strathmann M et al. The Removal of the t-BOC Group by Electrochemically Generated Acid and Use of an Addressable Electrode Array for Peptide Synthesis. J Comb Chem 2005 (in press).Google Scholar
  40. 40.
    Oleinikov AV, Gray MD, Zhao J et al. Self-Assembling Protein Arrays Using Electronic Semiconductor Microchips and in Vitro Translation. J Proteome Res 2003; 2:313.PubMedCrossRefGoogle Scholar
  41. 41.
    Kelly RT, Woolley AT. Microfluidic Systems for Integrated, High-Throughput DNA Analysis. Anal Chem 2005; 77:97A–102A.Google Scholar
  42. 42.
    harrison DJ, Manz A, Fan Z et al. Capillary Electrophoresis and Sample Injection Systems Integrated on a Planar Glass Chip. Anal Chem 1992; 64:1926–1932.CrossRefGoogle Scholar
  43. 43.
    Wilding P, Pfahler J, Bau HH et al. Manipulation and Flow of Biological-Fluids in Straight Channels Micromachined in Silicon. Clin Chem 1994; 40(1):43–47.PubMedGoogle Scholar
  44. 44.
    Xia YN, Whitesides GM. Soft lithography. Annual Review of Materials Science 1998; 28:153–184.CrossRefGoogle Scholar
  45. 45.
    Piner RD, Zhu J, Xu F et al. “Dip-pen” nanolithography. Science 1999; 283(5402):661–663.PubMedCrossRefGoogle Scholar
  46. 46.
    Alonso-Amigo MG, Becker H. Microdevices fabricated by polymer hot embossing. Abstr Pap Am Chem Soc 2000; 219:468–COLL.Google Scholar
  47. 47.
    Becker H, Dietz W, Dannberg P, Microfluidic Manifolds by Polymer Hot Embossing for Micro Total Analysis System Applications. Paper presented at: uTas 98, 1998; Banff, Canada.Google Scholar
  48. 48.
    Boone T, Fan ZH, Hooper H et al. Plastic advances microfluidic devices. Anal Chem 2002; 74(3):78A–86A.PubMedCrossRefGoogle Scholar
  49. 49.
    Grodzinski P, Liu RH, Chen H et al. Development of Plastic Microfluidic Devices for Sample Preparation. Biomed Microdevices 2001; 3(4):275.CrossRefGoogle Scholar
  50. 50.
    Harrison DJ, Fluri K, Seiler K et al. Micromachining a Miniaturized Capillary Electrophoresis-Based Chemical-Analysis System on a Chip Science 1993; 261(5123):895–897.Google Scholar
  51. 51.
    Burns MA, Johnson BN, Brahmasandra SN et al. An integrated nanoliter DNA analysis device. Science 1998; 282(5388):484–487.PubMedCrossRefGoogle Scholar
  52. 52.
    Waters LC, Jacobson SC, Kroutchinina N et al. Microchip Device for Cell Lysis, Multiplex PCR Amplification and Electrophoretic Sizing. Anal Chem 1998; 70:158–162.PubMedCrossRefGoogle Scholar
  53. 53.
    Woollery AT, Hadley D, Landre P et al. Functional integration of PCR amplification and capillary electrophresis in a microfabricated DNA analysis device. Anal Chem 1996; 68(23):4081–4086.CrossRefGoogle Scholar
  54. 54.
    Kopp M, Mello AD, Manz A. Chemical amplification: Continuous-Flow PCR on a chip. Science 1998; 280:1046–1048.PubMedCrossRefGoogle Scholar
  55. 55.
    Ibrahim MS, Lofts RS, Jahrling PB et al. Real-time microchip PCR for detecting single-base differences in viral and human DNA. Anal Chem 1998; 70(9):2013–2017.PubMedCrossRefGoogle Scholar
  56. 56.
    Yuen PK, Kricka LJ, Fortina P et al. Microchip module for blood sample preparation and nucleic acid amplification reactions. Genome Res 2001; 11(3):405–412.PubMedCrossRefGoogle Scholar
  57. 57.
    Taylor MT, Belgrader P, Furman BJ et al. Lysing bacterial spores by sonication through a flexible interface in a microfluidic system. Anal Chem 2001; 73(3):492–496.PubMedCrossRefGoogle Scholar
  58. 58.
    Anderson RC, Su X, Bogdan GJ et al. A miniature integrated device for automated multistep genetic assays. Nucleic Acids Res 2000; 28(12):e60.CrossRefGoogle Scholar
  59. 59.
    Liu RH, Yang J, Lenigk R et al. Self-contained, Fully Integrated Biochip for Sample preparation, PCR amplification and DNA Microarray Detection. Anal Chem 2004; 76:1824–1832.PubMedCrossRefGoogle Scholar
  60. 60.
    Liu RH, Lodes MJ, Nugyen T et al. Validation of A Fully Integrated Microfluidic Array Device for Influenza A Subtype Identification and Sequencing. Anal Chem 2006; 78:4184–4193.PubMedCrossRefGoogle Scholar
  61. 61.
    Macken C, Lu H, Goodman J et al. The value of a database in surveillance and vaccine selection. In: A.D.M.E. Osterhaus NCAWH, ed. Options for the Control of Influenza IV. Amsterdam: Elsevier Science 2001; 103–106.Google Scholar
  62. 62.
    Wang D, Urisman A, Liu Y-T et al. Viral discovery and sequence recovery using DNA microarrays. PloS Biology 2003; 1(2):257–260.CrossRefGoogle Scholar
  63. 63.
    Allawi HT, Jr. JS. Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G and T.T mismatches. Biochemistry 1999; 38:3468–3477.PubMedCrossRefGoogle Scholar
  64. 64.
    Altschul SF, Madden TL, Schaffer AA et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25:3389–3402.PubMedCrossRefGoogle Scholar
  65. 65.
    Beebe DJ, Moore JS, Bauer JM et al. Functional Structures For Autonomous Flow Control Inside Microfluidic Channels. Nature 2000; 404:588–590.PubMedCrossRefGoogle Scholar
  66. 66.
    Liu RH, Yu Q, Beebe DJ. Fabrication and Characterization of Hydrogel-based Microvalves. J Microelectromechan Syst 2002; 11:45–53.CrossRefGoogle Scholar
  67. 67.
    Jerman H. Electrically-activated, normally-closed diaphragm valves. J Micromech Microeng 1994; 4:210–216.CrossRefGoogle Scholar
  68. 68.
    Ray CA, Sloan CL, Johnson AD et al. A Silicon-based Shape Memory Alloy Microvalve. Mater Res Soc Symp 1992; 276:161–166.Google Scholar
  69. 69.
    Liu RH, Stremler M, Sharp KV et al. A Passive Micromixer: 3-D C-shape Serpentine Microchannel. J Microelectromechan Syst 2000; 9(2):190–197.CrossRefGoogle Scholar
  70. 70.
    Su YC, Lin LW, Pisano AP. A water-powered osmotic microactuator. J Microelectromechan Syst 2002; 11(6):736–742.CrossRefGoogle Scholar
  71. 71.
    Zengerle R, Skluge S, Richter M et al. A Bidirectional Silicon Micropump. Sens Actuators A Phys 1995; 50:81–86.CrossRefGoogle Scholar
  72. 72.
    Unger MA, Chou H, Thorsen T et al. Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography. Science 2000; 288:113–116.PubMedCrossRefGoogle Scholar
  73. 73.
    Bohm S, Olthuis W, Bergveld P. An Integrated Micromachined Electrochemical Pump and Dosing System. Biomed Microdevices 1999; 1(2):121–130.PubMedCrossRefGoogle Scholar
  74. 74.
    Richter G. Device for Supplying Medicines. U.S. Patent, 3,894,538. 1975.Google Scholar
  75. 75.
    Munyan JW, Fuentes HV, Draper M et al. Electrically actuated, pressure-driven microfluidic pumps. Lab Chip 2003; 3:217–220.PubMedCrossRefGoogle Scholar
  76. 76.
    Liu RH, Lenigk R, Yang J et al. Hybridization Enhancement Using Cavitation Microstreaming. Anal Chem 2003; 75:1911–1917.PubMedCrossRefGoogle Scholar
  77. 77.
    Liu RH, Nguyen T, Schwarzkopf K et al. A Fully Integrated Miniature Device for Automated Gene Expression DNA Microarray Processing. Anal Chem 2006; 78:1980–1986.PubMedCrossRefGoogle Scholar
  78. 78.
    Iwatsuki-Horimoto K, Kanazawa R, Sugii S et al. 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 2004; 85:1001–1005.PubMedCrossRefGoogle Scholar
  79. 79.
    Dobbin KK, Beer DG, Meyerson M et al. Interlaboratory comparability study of cancer gene expression analysis using oligonucleotide microarrays. Clin Cancer Res 2005; 11:565–572.PubMedGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2007

Authors and Affiliations

  • Robin Hui Liu
    • 1
  • Mike Lodes
    • 2
  • H. Sho Fuji
    • 2
  • David Danley
    • 2
  • Andrew McShea
    • 2
  1. 1.Osmetech Molecular DiagnosticsPasadenaUSA
  2. 2.CombiMatrix Corp.MukilteoUSA

Personalised recommendations