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As a result of the very attractive pleiotropic properties of the heme-enzymes, three P450 cytochrome isoforms (P4501A2, P4502B4, P450SCC) have been utilized to identify a general optimal procedure to biodevice assembly for sensing a wide range of organic substances. The Langmuir-Blodgett films appears to yield the best stable working conditions as shown by UV-vis spectrophotometry, nanogravimetry, circular dichroism, and electrochemical characterization, to identify the ordered nanostructures of P450 cytochromes optimal for clozapine, styrene, and cholesterol sensing. Only in the presence of low purity grade protein, as in the case of P4501A2, a gel-matrix was needed to warrant the optimal clozapine sensing. By the combination of proper immobilization, transducer and nanostructured mutants of high-grade stable and selective P450-based sensors appear capable to detect the interaction with a wide range of organic substrates such as fatty acids, drugs, and toxic compounds.

Cytochromes P450 are a large superfamily of heme-thiolate enzymes involved in the metabolism of many different organic substrates such as drugs, fatty acids and toxic compounds. The aim of this work is to analyse the binding between the cytochrome P4501A2, in solution and in gel-matrix, and its substrate (clozapine), utilising voltammetric tests. The interaction measurements were carried out using two different screen printed electrodes (rhodium-graphite and graphite-riboflavin), and the results were compared. It was demonstrated that it is possible to realise a biosensor prototype to detect the presence of clozapine indirectly by chronoamperometry.

Molecular modeling and protein engineering were synergically employed to improve the fabrication of cytochrome P450scc mutant nanostructures for biodevice assembly. The optimization of protein three-dimensional structure by molecular modeling was performed using two models: in vacuum and simulating the presence of a polar solvent. Calculations were performed on a model to predict a P450scc mutant which could improve the process of molecules' immobilization onto solid supports. Engineerized cytochrome P450scc thin films were prepared and characterized by various biophysical techniques such as pi-A isotherms, surface potential measurements, Brewster angle microscopy, UV-vis spectroscopy, circular dichroism, nanogravimetry, and electrochemical analysis. This paper takes into consideration biomolecules modified by protein engineering that represent a new and powerful approach for obtaining synthetic simpler artificial structures with new or improved properties (i.e., specificity, stability, sensitivity, etc.) useful for biosensors development.

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