A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.
A reusable electrochemical proximity assay for highly selective, real-time protein quantitation in biological matrices.
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DOI：
10.1021/ja503679q
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发表时间：
2014-06-11
影响因子：
15
通讯作者：
Easley, Christopher J.
中科院分区：
文献类型：
--
作者：
Hu, Jiaming;Yu, Yajiao;Brooks, Jessica C.;Godwin, Leah A.;Somasundaram, Subramaniam;Torabinejad, Ferdous;Kim, Joonyul;Shannon, Curtis;Easley, Christopher J.
在广泛的浓度范围内对多种蛋白质进行快速和特异性定量对于在护理点、临床实验室和研究环境中进行生物传感是非常理想的。我们最近开发的电化学邻近测定法（ECPA）是一种目标灵活，DNA导向，直接读出的蛋白质定量方法，检测限在低飞摩尔范围内，使其特别适合于即时检测。然而，在护理点需要在更复杂的基质中进行一致的定量，并且临床和研究环境需要提高测量速度。在这里，我们解决这些问题与可重复使用的ECPA，其中温和的再生的表面DNA单层（用于捕获的邻近复合物）是通过酶促实现的分子生物学和电化学的新组合。策略性地将尿嘧啶放置在DNA序列中触发主链的选择性切割，释放组装的邻近复合物。这允许通过方波伏安法（SWV）重复蛋白质定量-运行之间最快3分钟。该过程可以在单个电极上重复多达19次，而不会损失测定灵敏度，并且使用七个不同的电极进行类似的校准，电流显示出高度可重复性。通过在复杂基质中的两个重要应用证明了可重复使用的ECPA的实用性：（1）从少至5个鼠胰岛实时直接定量监测激素分泌和（2）在未加标血清中的标准添加实验，用于在临床相关水平直接定量胰岛素。两种应用程序的结果将ECPA区分为蛋白质定量的特殊工具。
Rapid and specific quantitation of a variety of proteins over a wide concentration range is highly desirable for biosensing at the point-of-care, in clinical laboratories, and in research settings. Our recently developed electrochemical proximity assay (ECPA) is a target-flexible, DNA-directed, direct-readout protein quantitation method with detection limits in the low femtomolar range, making it particularly amenable to point-of-care detection. However, consistent quantitation in more complex matrices is required at the point-of-care, and improvements in measurement speed are needed for clinical and research settings. Here, we address these concerns with a reusable ECPA, where a gentle regeneration of the surface DNA monolayer (used to capture the proximity complex) is achieved enzymatically through a novel combination of molecular biology and electrochemistry. Strategically placed uracils in the DNA sequence trigger selective cleavage of the backbone, releasing the assembled proximity complex. This allows repeated protein quantitation by square-wave voltammetry (SWV)—as quickly as 3 min between runs. The process can be repeated up to 19 times on a single electrode without loss of assay sensitivity, and currents are shown to be highly repeatable with similar calibrations using seven different electrodes. The utility of reusable ECPA is demonstrated through two important applications in complex matrices: (1) direct, quantitative monitoring of hormone secretion in real time from as few as five murine pancreatic islets and (2) standard addition experiments in unspiked serum for direct quantitation of insulin at clinically relevant levels. Results from both applications distinguish ECPA as an exceptional tool in protein quantitation.
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影响因子：
15.9
作者：
STEFAN, Y;MEDA, P;ORCI, L
通讯作者：
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