標題: Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria
作者: Lee, Hui-Ju
Ho, Min-Rong
Tseng, Chih-Sian
Hsu, Ching-Yi
Huang, Meng-Shun
Peng, Hwei-Ling
Chang, Hwan-You
生物科技學系
Department of Biological Science and Technology
關鍵字: Adenosine 5 ' phosphosulfate;ADP-Glc pyrophosphorylase;Luciferase;ATP luminescence;ATP sulfurylase
公開日期: 1-十一月-2011
摘要: Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia cob is approximately 10(4) colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP(i) and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP(i)-recycling loop was completed using ATP sulfurylase and adenosine 5' phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water. (C) 2011 Elsevier Inc. All rights reserved.
URI: http://dx.doi.org/10.1016/j.ab.2011.07.013
http://hdl.handle.net/11536/18485
ISSN: 0003-2697
DOI: 10.1016/j.ab.2011.07.013
期刊: ANALYTICAL BIOCHEMISTRY
Volume: 418
Issue: 1
起始頁: 19
結束頁: 23
顯示於類別:期刊論文


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  1. 000294836300003.pdf