Nuclease Hydrolysis Does Not Drive the Rapid Signaling Decay of DNA Aptamer-Based Electrochemical Sensors in Biological Fluids.
Nuclease Hydrolysis Does Not Drive the Rapid Signaling Decay of DNA Aptamer-Based Electrochemical Sensors in Biological Fluids.
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DOI：
10.1021/acs.langmuir.1c00166
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发表时间：
2021-05-04
期刊：
影响因子：
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通讯作者：
Arroyo-Currás N
中科院分区：
文献类型：
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作者：
Shaver A;Kundu N;Young BE;Vieira PA;Sczepanski JT;Arroyo-Currás N
基于电化学适配体（E-AB）传感器是一种能够直接实时监测体内药物浓度的技术。这些传感器通过表面附着的适体实现其选择性，这些适体在目标结合时改变其构象，从而导致适体结合的氧化还原报告体和电极表面之间的电子转移动力学发生变化。因为，理论上，适体可以选择几乎任何感兴趣的目标，E-AB传感器在诊断和生物医学应用方面具有深远的潜力。然而，该平台的一个关键弱点在于生物电子界面的时间依赖性和自发降解。这种渐进式降解——部分表现为附着在适体上的氧化还原报告体的法拉第电流的持续下降——限制了E-AB传感器的体内工作寿命，使其无法长期应用于人体的连续分子监测。在这项工作中，我们研究了核酸酶作用对E-AB传感器信号寿命的影响，以确定信号的逐渐丢失是否由DNA适体的水解引起，从而导致传感器表面信号部分的丢失。我们在37°C下连续询问部署在几种未稀释生物液体中的传感器，并注入核酸酶以达到生理相关浓度。通过使用天然存在的d-DNA和抗核酸酶对映体l-DNA，我们确定在目前最先进的E-AB传感器的使用寿命内，在我们测试的条件下，核酸酶水解不是传感器信号丢失的主要原因。相反，信号损失主要是由电极表面的单层元件（包括阻挡烷硫醇和适体单层）的损失引起的。虽然l-DNA适配体的使用可以延长E-AB的长期使用寿命，但在这些影响可见之前，必须解决被动单层损失的关键问题。
Electrochemical aptamer-based (E-AB) sensors are a technology capable of real-time monitoring of drug concentrations directly in the body. These sensors achieve their selectivity from surface-attached aptamers, which alter their conformation upon target binding, thereby causing a change in electron transfer kinetics between aptamer-bound redox reporters and the electrode surface. Because, in theory, aptamers can be selected for nearly any target of interest, E-AB sensors have far-reaching potential for diagnostic and biomedical applications. However, a remaining critical weakness in the platform lies in the time-dependent, spontaneous degradation of the bioelectronic interface. This progressive degradation—seen in part as a continuous drop in faradaic current from aptamer-attached redox reporters—limits the in vivo operational life of E-AB sensors to less than 12 h, prohibiting their long-term application for continuous molecular monitoring in humans. In this work, we study the effects of nuclease action on the signaling lifetime of E-AB sensors, to determine whether the progressive signal loss is caused by hydrolysis of DNA aptamers and thus the loss of signaling moieties from the sensor surface. We continuously interrogate sensors deployed in several undiluted biological fluids at 37 °C and inject nuclease to reach physiologically relevant concentrations. By employing both naturally occurring d-DNA and the nuclease-resistant enantiomer l-DNA, we determine that within the current lifespan of state-of-the-art E-AB sensors, nuclease hydrolysis is not the dominant cause of sensor signal loss under the conditions we tested. Instead, signal loss is driven primarily by the loss of monolayer elements—both blocking alkanethiol and aptamer monolayers—from the electrode surface. While use of l-DNA aptamers may extend the E-AB operational life in the long term, the critical issue of passive monolayer loss must be addressed before those effects can be seen.
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DOI：
10.1038/mtna.2012.34
发表时间：
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期刊：
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