Survey of Redox-Active Moieties for Application in Multiplexed Electrochemical Biosensors
作者:Di Kang、Francesco Ricci、Ryan J. White、Kevin W. Plaxco
DOI:10.1021/acs.analchem.6b02376
日期:2016.11.1
Recent years have seen the development of a large number of electrochemical sandwich assays and reagentless biosensor architectures employing biomolecules modified via the attachment of a redox-active “reporter.” Here we survey a large set of potential redox reporters in order to determine which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter “sets” signaling at distinct, nonoverlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, we have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available commercially in forms that are readily conjugated to biomolecules or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters we conjugated it to one terminus of a single-stranded DNA “probe” that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an “E-DNA” sensor responsive to its complementary DNA target. We then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so we find that the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quantitative stability of such sensors against repeated scanning in even very complex sample matrices is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrrocene, is reasonable. Our work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale–we wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters we have investigated. Finally, we hope that our work also serves as an illustration of the pressing need for the further development of useful redox reporters.
近年来,大量的电化学夹心分析法和无试剂生物传感器架构得到了发展,这些架构采用了通过附着氧化还原活性"报告基团"修饰的生物分子。本文综述了一系列潜在的氧化还原报告基团,旨在确定哪些报告基团在巯基-金单层传感器中具有最佳的长期稳定性,并识别在不同、非重叠氧化还原电位上发出信号的报告基团"组合",以支持多重检测和校正比率或差分测量方法的错误。具体而言,我们对十几种潜在的报告基团进行了性能表征,这些报告基团首先在巯基-金单层的稳定电位窗口内具有氧化还原活性,其次以易于与生物分子偶联的形式商业化,或者可以通过一两个简单的合成步骤转化为这种形式。为了测试每个报告基团,我们将其与单链DNA"探针"的一端偶联,该探针的另一端通过六个碳的巯基连接到金电极上,形成了一个对互补DNA目标敏感的"E-DNA"传感器。然后,我们测量了每个传感器的信号特性以及其对抗重复循环伏安扫描和在血清中使用及重复使用的稳定性。通过这些测试,我们发现亚甲蓝基的巯基-金传感器性能无与伦比;即使在非常复杂的样品矩阵中,这种传感器对抗重复扫描的近似定量稳定性也是前所未有的。虽然较为有限,但使用少数其他报告基团(包括蒽醌、尼罗蓝和二茂铁)的传感器的稳定性是合理的。因此,我们的工作既突出了亚甲蓝在这些应用中作为氧化还原报告基团的卓越特性,也为其他研究人员提供了一个警示——我们希望帮助他们避免我们在调查看似有前景但最终不充分的报告基团时所经历的无用功。最后,我们希望我们的工作也展现了进一步开发有用的氧化还原报告基团的迫切需要。