An optical H2S biosensor based on the chemoselective Hb-I protein tethered to a transparent, high surface area nanocolumnar electrode

Sensitive and selective detection of analytes in complex biological fluids can be an extremely challenging issue. The constructive association of biomolecules and transparent mesoporous electrodes is of interest in this area, as it can lead to innovative biosensors combining optical and electrochemical detection modes. This concept, however, requires the development of appropriate surface functionalization methodologies that are robust enough for long-term operation in physiological environments. In the present work, the high-surface area of 3D transparent mesoporous indium-tin oxide (ITO) electrodes (prepared by glancing angle deposition or GLAD) has been chemically functionalized with recombinant hemoglobin I from Lucina pectinata according to a versatile 2-step process. First, 4-diazoniumbenzoic acid salt is covalently electrografted onto the ITO surface, followed by amide coupling of the protein. The resulting electrodes were quantitatively characterized by cyclic voltammetry and UV-vis absorption spectroscopy, demonstrating high surface coverages (up to 45% of a closed-packed monolayer for Hemoglobin-I) and homogeneous distribution across the entire thickness of the GLAD mesoporous structure. Good stability is also observed when the modified electrodes are immersed for prolonged times in a high ionic strength saline buffer. We also show that the hemoglobin I-modified electrode can be used as an optical biosensor for the selective, reversible, and fast detection of H2S in aqueous solutions over a similar to two-decade concentration range (i.e. up to 10 mu M) and with a limit of detection of 0.35 mu M. Good analytical performance was also achieved in human plasma without significant interference from the biological matrix.