Fluorometric Quantification of Natural Inorganic Polyphosphate
Fluorometric Quantification of Natural Inorganic Polyphosphate
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DOI：
10.1021/es100191h
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发表时间：
2010-06-15
影响因子：
11.4
通讯作者：
Ingall, Ellery D.
中科院分区：
文献类型：
--
作者：
Diaz, Julia M.;Ingall, Ellery D.
聚磷酸盐是一种线性的正磷酸盐聚合物，在环境中含量丰富，是废水处理和许多生物修复过程中的关键成分。尽管聚磷酸盐具有广泛的相关性，但目前量化它的方法存在重大缺陷。在这里，我们描述了一种直接定量复杂天然样品中无机聚磷酸盐的新方法。该方案依赖于4‘，6-二氨基-2-苯基吲哚(DAPI)与溶解的聚磷酸盐之间的相互作用。使用我们描述的基于DAPI的方法，可以在中性缓冲的淡水基质中以+/-0.03µM P的精度定量聚磷酸盐的浓度范围为0.5-3µM P。标准产生的聚磷酸盐浓度与荧光的模式不显示出大小为15-130个磷单位的聚磷酸盐的链长依赖性。在这种方法中，较短长度的聚磷酸盐分子(例如，长度为三个和五个磷单位的聚磷酸盐)对信号的贡献很小，因为这些分子在测试的浓度范围内只与DAPI发生轻微反应或根本不反应。对于较长的链长(例如，45-130个磷单位)，在电导率高达10ms/cm时，盐的存在抑制了来自中间聚磷酸链长(例如，15个磷单位)的荧光。我们的结果表明，标准聚磷酸盐应冷冻储存不超过10-15天，以避免与标准降解相关的不一致结果。我们已经将荧光分析方案应用于五个具有良好特性的自然样品的分析，以演示该方法的使用。
Polyphosphate, a linear polymer of orthophosphate, is abundant in the environment and a key component in wastewater treatment and many bioremediation processes. Despite the broad relevance of polyphosphate, current methods to quantify it possess significant disadvantages. Here, we describe a new approach for the direct quantification of inorganic polyphosphate in complex natural samples. The protocol relies on the interaction between the fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and dissolved polyphosphate. With the DAPI-based approach we describe, polyphosphate can be quantified at concentrations ranging from 0.5-3 mu M P in a neutral-buffered freshwater matrix with an accuracy of +/-0.03 mu M P. The patterns of polyphosphate concentration versus fluorescence yielded by standards exhibit no chain length dependence across polyphosphates ranging from 15-130 phosphorus units in size. Shorter length polyphosphate molecules (e.g., polyphosphate of three and five phosphorus units in length) contribute little to no signal in this approach, as these molecules react only slightly or not at all with DAPI in the concentration range tested. The presence of salt suppresses fluorescence from intermediate polyphosphate chain lengths (e.g., 15 phosphorus units) at polyphosphate concentrations ranging from 0.5-3 mu M P. For longer chain lengths (e.g., 45-130 phosphorus units), this salt interference is not evident at conductivities up to similar to 10mS/cm. Our results indicate that standard polyphosphates should be stored frozen for no longer than 10-15 days to avoid inconsistent results associated with standard degradation. We have applied the fluorometric protocol to the analysis of five well-characterized natural samples to demonstrate the use of the method.