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

Abstract

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-visible 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 H 2 S in aqueous solutions over a two-decade concentration range and with a limit of detection of 0.35 M. Good analytical performance was also achieved in human plasma without significant interference from the biological matrix.

Electrografted Interfaces on Metal Oxide Electrodes for Enzyme Immobilization and Bioelectrocatalysis

“…Recent work demonstrated successful electrografting on high-surface area nanostructures. [38][39][40][41] Our strategy should be extendable also to these materials.…”

“…Diazonium salts have been successfully electrografted on a number of planar and porous oxide materials, where it is assumed that MÀ OÀ C interfacial bonds are formed, as has been proven for TiO 2 and ATO, [37,38] and indications of (extensive) polymerization are reported. [41] The first step was therefore to determine the effect of adding radical scavenger to the electrografting reaction and the thereby deposited interfaces. Highly conductive and reproducibly planar ITO electrodes were used to study the electrochemical interface, ruling out potential charge carrier transport limitations found in porous materials, as well as diffusion limitations and more complex surface chemistries within porous structures.…”

“…[38] Further studies demonstrated similarly high stabilities of such electrografted interfaces on tin-doped indium oxide (ITO), fluorine-doped tin oxide (FTO) and TiO 2 . [37,[39][40][41] The reactive aryl radicals, formed during electrografting, tend, however, to react with already-grafted species, resulting in the deposition of polymeric, branched interfaces that do not facilitate DET with, e. g., large redox enzymes. Unlike small molecules, enzymes cannot penetrate such structures.…”

“…A recent study described the immobilization of a redox enzyme on electrografted ITO, albeit in a non-electroactive manner. [41] The addition of radical scavengers during electrografting has been demonstrated as a simple and effective way of suppressing polymerization and favoring monolayer formation on carbon materials. [49][50][51][52] Similar effects in terms of grafting control towards optimized interfaces have been obtained by us for gold, enabling, e. g., tuning of the interfacial charge transfer resistance thereby allowing to enhance the reactivity of immobilized electrocatalytic molecular species.…”

“…Recently, we have demonstrated that electrografting aryl‐diazonium salts onto antimony‐doped tin oxide (ATO) electrodes resulted in robust, covalently‐bound interfaces that are stable in a very broad pH and potential range, and facilitated fast ET with an immobilized molecular electrocatalyst, such as an iron Hangman porphyrin [38] . Further studies demonstrated similarly high stabilities of such electrografted interfaces on tin‐doped indium oxide (ITO), fluorine‐doped tin oxide (FTO) and TiO 2 [37,39–41] . The reactive aryl radicals, formed during electrografting, tend, however, to react with already‐grafted species, resulting in the deposition of polymeric, branched interfaces that do not facilitate DET with, e. g., large redox enzymes.…”