Carbon-nanotube-supported POXA1b laccase and its hydrophobin chimera for oxygen reduction and picomolar phenol biosensing

Ilaria Sorrentino,Ilaria Stanzione,Alessandra Piscitelli,Paola Giardina,Alan Le Goff

doi:10.1016/j.biosx.2021.100074

Ilaria Sorrentino, Ilaria Stanzione + Show 3 more

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https://doi.org/10.1016/j.biosx.2021.100074

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Abstract

The immobilization of POXA1b laccase and its hydrophobin-fused chimera was performed at pristine Multiwalled Carbon Nanotube (MWCNT) and MWCNT-modified electrodes by electrografting of a 2-diazoniumanthraquinone salt. The influence of the hydrophobin domain and the MWCNT functionalization with anthraquinone groups towards immobilization of laccase was compared by direct electrochemistry under O2 and electrochemical biosensing of phenols. The hydrophobin domain affords the stable dispersion of MWCNT in water/ethanol, while being detrimental to the direct electron transfer between POXA1b and the electrode. On the contrary, the stronger hydrophobic interactions between anthraquinone and laccase affords direct electrochemistry of POXA1b and enabled the design of a highly sensitive phenol biosensor, reaching a limit-of-detection (LOD) of 2 pM and sensitivity of 23,600 mA L mmol−1 cm−2 for catechol, and a LOD of 15 nM and sensitivity of 0.053 mA L mmol−1 cm−2 for dopamine.

Highlights

The use of nanostructured carbons such as carbon nanotubes (CNTs) or graphene have tremendously improved the performance of biosensors (Holzinger et al, 2017; Sotiropoulou et al, 2003; Valentini et al, 2013)
Multi walled Carbon Nanotube (MWCNT) bioelectrodes were used as working electrodes, with a saturated calomel (SCE) and a platinum (Pt) wire acting as the reference and counter electrode, respectively
The addition of POXA1b-Vmh2 to a suspension of MWCNTs improved the dispersion stability after sonication for 10 min and centrifugation. This underlines the surfactant ability of POXA1b-Vmh2 owing to the adhesion properties of the hydrophobin domain towards hydrophobic surface such as MWCNT sidewalls

Summary

Introduction

The use of nanostructured carbons such as carbon nanotubes (CNTs) or graphene have tremendously improved the performance of biosensors (Holzinger et al, 2017; Sotiropoulou et al, 2003; Valentini et al, 2013). Hydro phobins strongly interact with several hydrophobic surfaces such as Teflon (Askolin et al, 2006; Portaccio et al, 2015; White, 2004), poly styrene (Wang et al, 2010), silicon (De Stefano et al, 2007, 2009), steel (Longobardi et al, 2015), and graphene (Gravagnuolo et al, 2015) This appealing ability has been further extended to laccases by genetic fusion of the hydrophobin to POXA1b(Sorrentino et al, 2019a,b), allowing achievement of simple and stable immobilization of the enzyme on different surfaces such as polystyrene or graphene sheets (Sorrentino et al, 2019, 2020). These biofunctionalized surfaces were employed in the design of laccase-based phenol biosensors(Sorrentino et al, 2020)

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