Disposable superoxide dismutase biosensors based on gold covered polycaprolactone fibers for the detection of superoxide in cell culture media.

A novel and disposable biosensor based on superoxide dismutase (SOD) immobilized on gold metallized polycaprolactone electrospun polymeric fibers (PCl/Au) has been developed for the determination of superoxide (O) in cell culture media. SOD biosensors were constructed employing three immobilization methods: cross-linking with EDC/NHS at a cysteine self-assembled monolayer (PCl/Au/SOD), biopolymer encapsulation with chitosan (PCl/Au/SOD) and cross-linking with glutaraldehyde (PCl/Au/SOD). Scanning electron microscopy was performed at the three different biosensors to evaluate their surface morphologies. Biosensors were employed for the electrochemical detection of superoxide by fixed potential amperometry at different applied potentials, with two distinct enzymatic mechanisms being proposed: i) the reduction of the enzymatically generated peroxide, at -0.3 V, for which the PCl/Au/SOD biosensor presented the highest value of sensitivity of 40.1 μA mM cm, and ii) the regeneration of the enzyme catalytic copper centre, at +0.3 V, for which the PCl/Au/SOD biosensor had the highest sensitivity value of 16.1 μA mM cm. The proposed recognition mechanisms were further confirmed by cyclic voltammetric measurements, which enabled also to determine the amount of immobilized electroactive SOD, with highest value corresponding to the PCl/Au/SOD biosensor. The biosensors with best analytical performance, PCl/Au/SOD and PCl/Au/SOD, were further investigated for stability and selectivity, with best results for the PCl/Au/SOD, chosen for superoxide monitoring in cell culture media. The study is promising for future application of PCl/Au/SOD for the on-line superoxide monitoring of superoxide in cell cultures, grown directly on the biosensor itself.