Direct real-time measurements of superoxide release from skeletal muscles in rat limbs and human blood platelets using an implantable Cytochrome C microbiosensor

Oxidative stress and excessive accumulation of the superoxide (O-2(center dot-)) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O-2(center dot-)) 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-2(center dot-)) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of LCysteine 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-2(center dot-)) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/mu M and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O-2(center dot-)) 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-2(center dot-)) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies.

Automated summary

This study presents the design and characterization of a robust biosensor for real-time monitoring of superoxide (O-2(center dot-)) release. The biosensor is constructed by depositing a uniform layer of LCysteine on a gold wire electrode and conjugating it with Cytochrome C (Cyt c). The biosensor demonstrates stability and robustness in complex biological systems and shows potential for estimating oxidative injury in human pathologies.