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Miniaturization of absorbance assays using the fluorescent properties of white microplates

Academic Article
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Overview

authors

  • Zuck, P.
  • O'Donnell, G. T.
  • Cassaday, J.
  • Chase, P.
  • Hodder, Peter
  • Strulovici, B.
  • Ferrer, M.

publication date

  • July 2005

journal

  • Analytical Biochemistry  Journal

abstract

  • Miniaturization of high-throughput screening (HTS) assays has several obvious advantages, including increased throughput and lower cost by reduction in reagent consumption. Although absorbance assays are widely used in research laboratories, their application for HTS in a low-volume format has been met with mixed success because they are difficult to miniaturize. Challenges for the miniaturization of absorbance assays include low signal due to short path lengths and meniscus distortions in small well sizes. Here we describe a method to miniaturize absorbance assays to standard, white, low-volume 384-well and 1536-well microplates using a fluorometric plate reader for detection. The premise of this absorbance assay is based on the fluorescent properties of white microplates and the ability of a colored product to quench the fluorescence signal from the plate by absorbing either the excitation light or the emission light. This method was applied to the detection of inorganic phosphate using Quinaldine red and Malachite green dyes and to the monitoring of alkaline phosphatase hydrolysis of p-nitrophenyl phosphate. These assays can be carried out in low volumes, give robust screening statistics, and can be accomplished with a simple, inexpensive fluorometric plate reader.

subject areas

  • Combinatorial Chemistry Techniques
  • Fluorescence
  • Fluorometry
  • Miniaturization
  • Molybdenum
  • Phosphates
  • Phosphoric Acids
  • Quinaldines
  • Rosaniline Dyes
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Research

keywords

  • HTS
  • absorbance assays
  • colorimetric assays
  • high-throughput screening
  • malachite green
  • phosphate
  • quinaldine red
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Identity

International Standard Serial Number (ISSN)

  • 0003-2697

Digital Object Identifier (DOI)

  • 10.1016/j.ab.2005.04.029

PubMed ID

  • 15949786
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Additional Document Info

start page

  • 254

end page

  • 259

volume

  • 342

issue

  • 2

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