Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
Anal Chem. 2011 May 15;83(10):3770-7. doi: 10.1021/ac200217d. Epub 2011 Apr 21.
We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic bead (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to the limited sites of anti-BChE antibody labeled with quantum dots (QD-anti-BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1-20 nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based sensor coupled with a microflow injection system after immunocapture by the MB-anti-BChE conjugate. Therefore, the formed phosphorylated BChE adduct (OP-BChE) can be estimated by the difference values of the total amount of BChE (including active and OP-inhibited) and active BChE from established calibration curves. This approach not only eliminates the difficulty in screening of low-dose OP exposure (less than 20% inhibition of BChE) because of individual variation of BChE values but also avoids the drawback of the scarce availability of OP-BChE antibody. It is sensitive enough to detect 0.5 nM OP-BChE, which is less than 2% BChE inhibition. This method offers a new method for rapid, accurate, selective, and inexpensive quantification of OP-BChE and enzyme inhibition for biomonitoring of OP and nerve agent exposures.
我们报告了一种新的电化学方法，用于定量检测有机磷（OP）农药和神经毒剂暴露的酶抑制和磷酸化，该方法基于磁珠（MB）免疫传感平台。该方法的原理基于 MB 免疫捕获酶活性测定和总酶量竞争免疫测定的结合，用于同时检测生物体液中的酶抑制和磷酸化。我们选择丁酰胆碱酯酶（BChE）作为模型酶。在竞争免疫测定中，样品中的靶 BChE 与固定在 MB 上的 BChE 竞争结合到标记有量子点（QD-抗 BChE）的抗 BChE 抗体的有限结合位点，随后对 MB 上结合的 QD 缀合物进行剥离伏安分析。该测定法在 0.1-20 nM 的总 BChE 浓度范围内呈线性响应。在免疫捕获后，通过 MB-抗 BChE 缀合物在基于电化学碳纳米管的传感器上实时测量同时的实时 BChE 活性，并与微流动注射系统耦合。因此，通过从建立的校准曲线中减去总 BChE（包括活性和 OP 抑制）和活性 BChE 的差值，可以估计形成的磷酸化 BChE 加合物（OP-BChE）。该方法不仅消除了由于 BChE 值个体差异导致的低剂量 OP 暴露筛选困难（BChE 抑制率小于 20%），而且避免了 OP-BChE 抗体稀缺的缺点。它足够灵敏，可以检测到 0.5 nM 的 OP-BChE，这相当于 BChE 抑制率的 2%。该方法为快速、准确、选择性和廉价地定量检测 OP-BChE 和酶抑制提供了一种新方法，可用于 OP 和神经毒剂暴露的生物监测。
