Skip to main content

A Homogenous Microarray for Enzymatic Functional Assays

Chemical Compounds Microarray

  • Chapter
Book cover Frontiers in Biochip Technology

Abstract

Microarrays as an emerging research tool promises to play a pivotal role in the post genomic era. However, in spite of the fast development of this technology special requirements, such as the immobilization and delivery of bio-reagents on the chip surface limit the utilization of microarrays, especially for small chemical compound libraries. We have developed a unique homogenous microarray system that overcomes these limitations and can be used to array most biofunctional molecules, such as small chemical compounds, peptides and proteins without pre-immobilization. A standard microscope slide containing up to 5000 microarray dots, with volumes less than 2 nanoliter each and acting as individual reaction centers, can be printed with standard DNA arrayer. An aerosol deposition technology was adapted to deliver extremely small volumes of biofluids uniformly into each reaction center. The fluorescence based reaction signals were then scanned and analyzed with standard DNA scanner and DNA array analyzing software. With this platform, we demonstrated that this chip format could be used for not only screening individual but also multiple enzymatic activities simultaneously with different fluorescent tagged small peptide libraries. We further demonstrated that this system could be a very powerful ultra high throughput screening tool for drug discovery, with which we identified potential “hits” after screening chips printed with small chemical compounds against caspases 1 and 3. This highly sensible chip is also able to monitor caspase protein expression profiles by activating the peptide chips with cell lysates undergoing apoptosis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S, Houfek, T, et al. (2000). Analysis of yeast protein kinases using protein chips. Nature Genetics 26, 283–289.

    Article  Google Scholar 

  2. Zhu, H., Klemic, J.F., Chang, S., Bertone, P., Casamayor, A., Klemic, K.G., Smith, D., Gerstein, M., Reed, M.A. and Snyder, M. (2001). Global analysis of protein activities using proteome chips. Science 293, 2101–2105.

    Article  Google Scholar 

  3. Houseman, B.T., Huh, J.H., Kron, S.J. and Mrksich, M. (2002). Peptide chips for the quantitative evaluation of protein kinase activity. Nat Biotechnol. 20, 270–274.

    Article  Google Scholar 

  4. MacBeath, G. and Schreiber, S.L. (2000). Printing proteins as microarrays for high-throughput function determination. Science 289, 1760–1763.

    Google Scholar 

  5. Kuruvilla, F.G., Shamji, A.F., Sternson, S.M., Hergenrother, P.J. and Schreiber, S.L. (2002). Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays. Nature 416, 653–657.

    Article  Google Scholar 

  6. De Wildt, R.M.T., Mundy, C. R., Gorick, B.D. and Tomlinson, I.M. (2000). Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat. Biotechnol. 18, 989–994.

    Article  Google Scholar 

  7. Nicholson, D.W. and Thornberry, N.A. (1997). Caspases: killer proteases. Trends Biochem. Sci. 22, 299–306.

    Article  Google Scholar 

  8. Lee, D., Long, S.A., Adams, J.L., Chan, G., Vaidya, K.S., Francis, T.A., Kikly, K., Winkler, J.D, Sung, C.M, Debouck, C. et al. (2000). Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality. J. Biol. Chem. 275, 16007–16014.

    Article  Google Scholar 

  9. Webber, S.E., Tikhe, J., Worland, S.T., Fuhrman, S.A., Hendrickson, T.F., Matthews, D.A., Love, R.A., Patick, A.K., Meador, J.W., Ferre, R.A. et al. (1996). Design, synthesis, and evaluation of nonpeptidic inhibitors of human rhinovirus 3C protease. J. Med. Chem. 39, 5072–5082.

    Article  Google Scholar 

  10. Knezevic, V., Leethanakul, C., Bichsel, V.E., Worth, J.M., Prabhu, V.V., Gutkind, J.S., Liotta, L.A., Munson, P.J., Petricoin, E.F. 3rd, Krizman, D.B. et al. (2001). Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 10, 1271–1278.

    Article  Google Scholar 

  11. Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P. et al. (1997). A combinatorial approach defines specificities of members of the caspase family and Granzyme B. J. Biol. Chem. 272, 17907–17911.

    Article  Google Scholar 

  12. Amstad, P.A., Yu, G., Johnson, G.L., Lee, B.W., Dhawan, S. and Phelps, D.J. (2001). Detection of caspase activation in situ by fluorochrome-labeled caspase inhibitors. Biotechniques 3, 608–610.

    Google Scholar 

  13. Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D., Wong, W.W. et al. (1997). Substrate specificities of caspase family proteases. J. Biol. Chem. 272, 9677–9682.

    Article  Google Scholar 

  14. Garcia-Calvo, M., Peterson, E.P., Leiting, B., Ruel, R., Nicholson, D.W. and Thornberry, N.A. (1998). Inhibition of human caspases by peptide-based and macromolecule inhibitors. J. Biol. Chem. 273, 32608–32613.

    Article  Google Scholar 

  15. Mitchell, P. (2002). A prospective on protein microarrays. Nature Biotech. 20, 225–229.

    Article  Google Scholar 

  16. Morita, T., Kato, H., Iwanaga, S., Takada, K. and Kimura, T. (1977). New fluorogenic substrates for α-thrombin, factor Xa, kallikreins, and urokinase. J. Biochem. 82, 1495–1498.

    Google Scholar 

  17. Backes, B.J., Harris, J.L., Leonetti, F., Craik, C.S. and Ellman, J.A. (2000). Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. Nat. Biotechnol. 18, 187–193.

    Article  Google Scholar 

  18. Butenas, S., Drungilaite, V. and Mann, K.G. (1995). Fluorogenic substrates for activated protein C: substrate structure-efficiency correlation. Anal. Biochem. 225, 231–241.

    Article  Google Scholar 

  19. Butenas, S., Ribarik, N. and Mann, K.G. (1993). Synthetic substrates for human factor VIIa and factor VIIa-tissue factor. Biochem. 32, 6531–6538.

    Article  Google Scholar 

  20. Harris, J.L., Backes, B.J., Leonetti, F., Mahrus, S., Ellman, J.A, Craik. C.S. (2000). Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. U S A. 97, 7754–7759.

    Article  Google Scholar 

  21. Toth, M.V. and Marshall, G.R. (1990). A simple, continuous fluorometric assay for HIV protease. Intl. J. Pept. Protein Res. 36, 544–50.

    Article  Google Scholar 

  22. Nishikata, M., Suzuki, K., Yoshimura, Y., Deyama, Y. and Matsumoto, A. (1999). A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases. Biochem. J. 343, 385–391.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Springer Science+Business Media, Inc.

About this chapter

Cite this chapter

Ma, H., Wang, Y., Pomaybo, A.S., Tsai, C. (2006). A Homogenous Microarray for Enzymatic Functional Assays. In: Xing, WL., Cheng, J. (eds) Frontiers in Biochip Technology. Springer, Boston, MA. https://doi.org/10.1007/0-387-25585-0_1

Download citation

  • DOI: https://doi.org/10.1007/0-387-25585-0_1

  • Publisher Name: Springer, Boston, MA

  • Print ISBN: 978-0-387-25568-2

  • Online ISBN: 978-0-387-25585-9

  • eBook Packages: EngineeringEngineering (R0)

Publish with us

Policies and ethics