Department of Chemistry and Biomolecular Science, Clarkson University, 8 Clarkson Avenue, Potsdam, NY, 13699-5810, USA.
U.S. Army Institute of Surgical Research, Blood and Shock Resuscitation, Fort Sam Houston, TX, 78234, USA.
Biosens Bioelectron. 2023 Nov 15;240:115664. doi: 10.1016/j.bios.2023.115664. Epub 2023 Sep 2.
Oxidative stress and excessive accumulation of the superoxide (O) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O) release is important to assess the extent of oxidative stress in these conditions. Herein, we present the design, fabrication and characterization of a robust (O) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of L-Cysteine on a gold wire electrode to which Cytochrome C (Cyt c) was conjugated. The immobilized layers, studied using conductive Atomic Force Microscopy (c-AFM) revealed a stable and uniformly distributed redox protein on the gold surface, visualized as conductivity and surface topographical plots. The biosensor enabled detection of (O) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/μM and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O) release in activated human blood platelets and skeletal rat limb muscles following ischemia reperfusion injury (IRI), confirming the biosensor's stability and robustness for measurements in complex biological systems. The results demonstrate the ability of these biosensors to monitor real time release of (O) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies.
氧化应激和超氧阴离子（O）的过度积累是许多病理状况和多种疾病发生的根源。实时监测（O）的释放对于评估这些情况下氧化应激的程度非常重要。在此，我们提出了一种使用简单直接的程序设计、制造和表征（O）生物传感器的方法，该程序涉及在金线上沉积均匀的 L-半胱氨酸层，该金线与细胞色素 C（Cyt c）缀合。使用导电原子力显微镜（c-AFM）研究了固定化层，结果表明金表面上的氧化还原蛋白稳定且均匀分布，可通过导电性和表面形貌图进行可视化。该生物传感器能够在 0.15 V 的施加电位下检测（O），灵敏度为 42.4 nA/μM，检测限为 2.4 nM。该生物传感器在缺血再灌注损伤（IRI）后激活的人血小板和大鼠肢体骨骼肌中实时（O）释放的测量中的实用性得到了证明，证实了该生物传感器在复杂生物系统中的测量中的稳定性和鲁棒性。这些结果表明，这些生物传感器能够监测（O）的实时释放，并估计在易于转化为人类病理学的模型中的氧化损伤程度。
