This Article Full Text (PDF) All Versions of this Article: 1087057109336592v1 14/6/668    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Scopus Google Scholar Articles by Sukumaran, S. M. Articles by Dordick, J. S. PubMed PubMed Citation Articles by Sukumaran, S. M. Articles by Dordick, J. S. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB ALGINIC ACID FLUORESCEIN Social Bookmarking                         What's this?

Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays

Department of Chemical and Biological Engineering and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York

Benjamin Potsaid

Center for Automation Technologies and Systems, Rensselaer Polytechnic Institute, Troy, New York

Moo-Yeal Lee

Department of Chemical and Biological Engineering and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, Solidus Biosciences, Inc., Troy, New York

Douglas S. Clark

Department of Chemical Engineering, University of California, Berkeley, clark{at}berkeley.edu

Jonathan S. Dordick

Department of Chemical and Biological Engineering and Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, Department of Biology, Rensselaer Polytechnic Institute, Troy, New York

Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20 x 20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z' factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity. (Journal of Biomolecular Screening 2009:668-678)

Key Words: cytochromes P450 • microarrays • high-throughput screening • wide-field imaging

This version was published on July 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 6, 668-678 (2009)
DOI: 10.1177/1087057109336592


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?