Zuck Paul, O'Donnell Gregory T, Cassaday Jason, Chase Peter, Hodder Peter, Strulovici Berta, Ferrer Marc
Department of Automated Biotechnology, Merck and Company, 502 Louise Lane, North Wales, PA 19454, USA.
Anal Biochem. 2005 Jul 15;342(2):254-9. doi: 10.1016/j.ab.2005.04.029.
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.
高通量筛选（HTS）分析的小型化具有几个明显的优点，包括通过减少试剂消耗提高通量和降低成本。尽管吸光度分析在研究实验室中广泛使用，但它们在低体积形式的HTS中的应用效果不一，因为它们难以小型化。吸光度分析小型化面临的挑战包括由于光程短导致的低信号以及小尺寸孔板中的弯月面变形。在这里，我们描述了一种使用荧光酶标仪进行检测，将吸光度分析小型化到标准的白色低体积384孔和1536孔微孔板的方法。这种吸光度分析的前提基于白色微孔板的荧光特性以及有色产物通过吸收激发光或发射光来淬灭板上荧光信号的能力。该方法应用于使用喹哪啶红和孔雀石绿染料检测无机磷酸盐以及监测对硝基苯磷酸酯的碱性磷酸酶水解。这些分析可以在低体积下进行，提供可靠的筛选统计数据，并且可以使用简单、廉价的荧光酶标仪完成。
