Unprecedented Wiring Efficiency of Sulfonated Graphitic Carbon Nitride Materials: Toward High-Performance Amperometric Recombinant CotA Laccase Biosensors

Control of electron-transfer (ET) processes across electrochemically active biomaterials by tuning the surface properties of platform materials plays a key role in the design of highly efficient biosensors. In this work, ET rates of recombinant CotA laccases have been drastically improved by an immobilization process on sulfonic group-modified graphitic carbon nitride (Sg-CN) materials. Cyclic voltammetry (CV) and Fourier transform infrared (FTIR) spectroscopy revealed that the enzymes undergo striking conformational changes onto graphitic carbon nitride (g-CN), adopting an electrochemically inactive configuration while retain their nativelike structure with a superb ET efficiency on Sg-CN surfaces. In fact, the resulting CotA laccase/Sg-CN biomaterial displayed an ET rate constant of (12 ± 0.5) s–1, the highest value reported to date for a direct electron-transfer reaction of multicopper oxidases attached to carbon-based materials. Importantly, the combined parallel tempering Monte Carlo (PTMC) and all-atom molecular dynamics (AAMD) theoretical calculations proved CotA incorporation in a highly ordered array with an overall positive surface density composed of lysine and arginine domains in contact with net negatively charged Sg-CN surfaces, which promoted a 1200-fold improvement in the free enzyme ET rate constant. An ET pathway has been put forward that takes into account the orientation of CotA laccase on the Sg-CN surface. Additionally, CotA laccase/Sg-CN biomaterial was tested as an amperometric biosensor delivering outstanding bioelectrocatalytic activities in the oxidation of catechol and syringol, which are relevant emerging pollutants. In fact, the sensitivities of the CotA laccase/Sg-CN/ITO electrodes were 0.95 and 0.41 A·M–1·cm–2 for catechol and syringol, respectively, surpassing most of the laccase biosensors reported in the literature.