A Comparative Study on Surface Treatments in the Immobilization Improvement of Hexahistidine-tagged Protein on the Indium Tin Oxide Surface

Biosensors are becoming increasingly important in various sectors. However, choosing transducer type and surface treatment still faces severe limitations. Indium-tin oxide is widely used in electrochemical sensing, though surface cleaning and functionalization are not always straightforward and often poorly controlled. Here, we have covalently immobilized a hexahistidine-tagged model protein on three different treated ITO surfaces using a Ni2+ chelator moiety. The study shows that compared to two other treatments (H2O/H2O2/NH4OH and KOH treatments), the ITO Piranha treatment gives satisfactory results in term of yields of EC12 protein immobilized on the surface. The study in detail of treatments effect was realized by applying a combination of local and global techniques such X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), Attenuated total reflectance Fourier transform infrared spectroscopy (FTIR-ATR), contact angle measurements, and electrochemical impedance spectroscopy (EIS). The biofunctionalization allowed proteins to move freely around their anchoring bond. As the polyhistidine tags are widely used in protein engineering, such substrates offer a large panel of applications. This covalent and oriented immobilization process of recombinant proteins applied to ITO could provide transparent electroactive surfaces of high quality for electrochemical detection of soluble compounds, as well as cells.This work is the fruitful effort of different collaborations. A special thanks to Dr.\udHélène Feracci and Dr. Jean-Paul Salvetat for their important contributions. The work\udwas supported by institutional funding from CNRS (France), a CNanoGSO grant\ud(Hélène Feracci, Jean-Paul Salvetat), and by the Ministry of Higher Education and\udResearch of Tunisia. The ICMAB experiments were financed by Science Ministry\udGrant (MAT2011-24363) and Marato TV3 Foundation Grant (reference 110130/31)\ud(Dr. Nieves Casan). MBI stay at the CRPP and ICMAB was funded by a Ministry of\udHigher Education and Research of Tunisia fellowship. We are grateful to Sébastien\udChevalier for his help in early experiments, to Elizabeth Hillard for editing checking the\udmanuscript, to Marwa Sahraoui, Andres Gomez, and Christine Labrugère.Peer reviewe