Electrochemical biosensing platform based on AuNWs/rGO-CMC-PEDOT:PSS composite for the detection of superoxide anion released from living cells.

Detection of superoxide anion (O2 ·- ) levels holds significant importance for the diagnosis and even clinical treatments of oxidative stress-related diseases. Herein, we prepared a composite electrode material to encapsulate copper-zinc superoxide dismutase (SOD1) for biosensing of O2 ·- . The sensing material consists of gold nanowires (AuNWs), reduced graphene oxide (rGO), carboxymethyl cellulose (CMC) and PEDOT:PSS. CMC provides abundant -COOH to bind SOD1, with a high adsorption coverage of 1.499 × 10-9  mol cm-2 on the sensor surface. rGO and PEDOT endow the composite with significant conductivity, whereas PSS has antifouling capability. Moreover, AuNWs exhibit excellent electrical conductivity and a high aspect ratio, which promotes electron transfer, and ultimately enhances the catalytic performance of the enzyme. Meanwhile, SOD1(Cu2+ ) catalyzes the dismutation of O2 ·- to O2 and H2 O2 , and H2 O2 is then electrochemically oxidized to generate amperometric signals for determination of O2 ·- . The sensor demonstrates outstanding detection performance for O2 ·- with a low detection limit of 2.52 nM, and two dynamic ranges (14.30 nM-1.34 μM and 1.34 μM-42.97 μM) with corresponding sensitivity of 0.479 and 0.052 μA μM-1 cm-2 , respectively. Additionally, the calculated apparent Michaelis constant (Km app ) of 1.804 μM for SOD1 demonstrates the outstanding catalytic activity and the surface-immobilized enzyme's substrate affinity. Furthermore, the sensor shows the capability to dynamically detect the level of O2 ·- released from living HepG2 cells. This study provides an inovative design to obtain a biocompatible electrochemical sensing platform with plenty of immobilization sites for biomolecules, large surface area, high conductivity and flexibility.