Abstract
We describe here a method for the continuous assessment of enzymatic activity using microarrays. By uniformly coating fluorogenic substrates on slides, we generated surfaces capable of detecting enzymatic activity. The enzymes were deposited on the arrays in segregated droplets using standard microarrayers. Surfaces were developed for assessing the activities of both proteases and phosphatases, hence capitalizing on microarray technology to perform miniaturized high-throughput screens for these, as well as potentially any other, classes of enzyme. This offers an unprecedented ability for performing solution-phase enzymatic assays in nanoliter volumes on microarrays, in contrast to microliter volumes typically required in microplate-based assays, thereby reducing the amounts of reagent(s) required by anywhere from a hundred to a thousand-fold. This new approach thus provides a potentially more cost-effective, label-free enzyme screening technique. A single slide is able to accommodate several thousand assays, facilitating the assessment of both dose and time-dependent inhibition parameters in a single run.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Uttamchandani, M., Lu, C. H., and Yao, S. Q. (2009) Next generation chemical proteomic tools for rapid enzyme profiling, Acc Chem Res 42, 1183–1192.
Evans, M. J., and Cravatt, B. F. (2006) Mechanism-based profiling of enzyme families, Chem Rev 106, 3279–3301.
Uttamchandani, M., Huang, X., Chen, G. Y., and Yao, S. Q. (2005) Nanodroplet profiling of enzymatic activities in a microarray, Bioorg Med Chem Lett 15, 2135–2139.
Wang, J., Uttamchandani, M., Sun, L. P., and Yao, S. Q. (2006) Activity-based high-throughput profiling of metalloprotease inhibitors using small molecule microarrays, Chem Commun (Camb), 717–719.
Babiak, P., and Reymond, J. L. (2005) A high-throughput, low-volume enzyme assay on solid support, Anal Chem 77, 373–377.
Salisbury, C. M., Maly, D. J., and Ellman, J. A. (2002) Peptide microarrays for the determination of protease substrate specificity, J Am Chem Soc 124, 14868–14870.
Gosalia, D. N., Salisbury, C. M., Ellman, J. A., and Diamond, S. L. (2005) High throughput substrate specificity profiling of serine and cysteine proteases using solution-phase fluorogenic peptide microarrays, Mol Cell Proteomics 4, 626–636.
Gosalia, D. N., and Diamond, S. L. (2003) Printing chemical libraries on microarrays for fluid phase nanoliter reactions, Proc Natl Acad Sci U S A 100, 8721–8726.
Uttamchandani, M., Wang, J., Li, J., Hu, M., Sun, H., Chen, K. Y., Liu, K., and Yao, S. Q. (2007) Inhibitor fingerprinting of matrix metalloproteases using a combinatorial peptide hydroxamate library, J Am Chem Soc 129, 7848–7858.
Chan, W. C., and White, P. D. (2000) Fmoc solid phase peptide synthesis: a practical approach, Oxford University Press, New York.
Acknowledgments
This work is supported by the National University of Singapore (NUS), the Agency of Science Technology and Research (A*Star) of Singapore and the DSO National Laboratories.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Aw, K.L.D., Yao, S.Q., Uttamchandani, M. (2010). Nanodroplet Microarrays for High-Throughput Enzyme Screening. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 669. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-845-4_7
Download citation
DOI: https://doi.org/10.1007/978-1-60761-845-4_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-844-7
Online ISBN: 978-1-60761-845-4
eBook Packages: Springer Protocols