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A5.05 - Surface-Immobilized Cytochrome C Mutants for the Detection of ROS Species 
December 2, 2014   3:15pm - 3:30pm

The last years have witnessed a rapidly increased interest in the use of biomolecules as components of molecular bioelectronics devices. In most of the applications envisioned in the nano-biotechnology field, the biomolecule must be immobilized on the substrate. This can lead to a loss of the native functionality. Therefore, the design of strategies aiming at establishing a robust link between the surface and the biomolecule without affecting the activity of the latter is of paramount importance. The choice of the biomolecule should be based also on its stability in terms of temperature, pH and presence of organic solvent, as these experimental conditions can be extremely variable in the preparation of a bio-electronic device. Within this respect, electron transfer protein cytochrome c is an appealing candidate, displaying rather high stability and robustness. Nevertheless, this species lacks enzymatic activity which could be exploited in the design of biosensors.

We have designed, produced and characterized a set of yeast cytochrome c mutants, which endow the protein with novel peroxidase-like activity. These mutants can be adsorbed on gold electrodes functionalized with -COOH and/or -OH terminated Self Assembled Monolayers (SAMs) with retention of functionality. Both in solution and in the adsorbed phase, the investigated mutants are able to catalytically reduce O2, H2O2 and nitrite anion.

The non native pseudo-peroxidase activity was provided by modifying the heme coordination by means of site-directed mutagenesis. In the M80A (namely methionine at position 80 replaced by an alanine) set of mutants, the axial binding Met80 was permanently substituted with a non-coordinating alanine M80A mutation (1,2). This mutation leads to a dramatic change in the reduction potential and, most importantly, endows the protein with non-native, pseudo-peroxidase activity. The K79H (lys-to-his) mutant instead reversibly interconverts between the native-like, His-Met heme-ligated form and a His-His-ligated conformer with remarkably different redox and enzymatic properties, which can be reversibly switched on/off by slight adjustment of the solution pH in proximity of neutral values (3,4).

Moreover, these mutants maintain their natural ability to efficiently transfer electrodes to/from a conducting substrate, thus ensuring electrical communication, and the higher stability with respect to natural peroxidases.

We envision the possibility to integrate these mutants in electrolyte-gated organic transistors, either upon adsorption on Au gate electrode or following incorporation into a room temperature ionic liquid to pattern the active area of the OECT. This would allow for the design of (implantable) biosensors towards reactive oxygen species (ROS).


1. Casalini et al., J.Phys.Chem. B, 2008, 112, 15555.
2. Casalini et al., J.Phys.Chem. B, 2010, 114, 1698.
3. Bortolotti et al., Chem. Sci., 2012, 3, 807.
4. Ranieri et al., Metallomics, 2014, 4, 874.

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