To optimize biodevice assembly and cholesterol sensing, recombinant and wild type P450scc cytochromes are extensively characterized both in solution and in thin solid films, using X-ray scattering, Brewster angle microscopy, quartz crystal nanobalance, ellipsometry, cyclic voltammetry, and circular dichroism. Efficient expression systems are implemented in microbial cells for the production of recombinant P450scc proteins, which are then purified. Modeling of the cholesterol interaction was also studied for the given application. Both types of P450 form monolayers at the air/water interface which can be transferred onto solid substrates, but only in the case of recombinant protein does the engineered monolayer turn out to be more dense and regular, and its thickness corresponds better to the native protein size. By surface pressure and surface potential measurements it is shown that at the air/water interface P450scc molecules orient themselves at the initial stage of the monolayer compression by self-assembly; increasing surface pressure yields high homogeneity, as confirmed by Brewster angle microscopy. CD measurements confirmed a significant increase in the stability of the protein secondary structure in the recombinant monolayers whose regular structure appears by X-ray measurements. The cyclic voltammetric study on LB films of cytochrome P450scc as a function of number of monolayers and of cholesterol concentration pointed to a possible electron transfer from the electrochemical process to cytochrome P450scc that in turn reacted with cholesterol, compatibly with the modeling showing that the nearest cholesterol atoms belonging to the main chain are only 4−5 Å from the heme.

The photovoltaic (PV) solar cell, converting incident solar radiation directly into electrical energy, today represents the most common power source for the earth-orbiting spacecraft, and the utilization of organic materials in this context is here explored in comparison with the present state of the art placing emphasis in organic nanotechnology. Poly[3-3'(vinylcarbazole)] (PVK) was synthesized by oxidative polymerization with ferric chloride of N-vinylcarbazole. The resulting polymer was then deposited on solid support by using the Langmuir-Schaefer (LS) technique. The pressure-area isotherm of PVK revealed the possibility of compact monolayer formation at the air-water interface. Different layers of PVK were doped with iodine vapors. The cyclic voltammetry investigation of PVK-doped I2 showed a distinctive electrochemical behavior. The photoinduced charge transfer across a donor/acceptor (D/A) hybrid interface provided an effective method to study the PV properties of the composite LS films. The results are compared with other approaches within the biological framework, such as bacteriorhodopsin (BR), and organic nanostructured materials.

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.

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