Resonance Raman Based Cytochrome C Biosensor as a Tool for the Evaluation of the Redox Properties of Carbonaceus Particles

It is presently generally recognized that inhalation of diesel exhaust particles (DEP) is the cause of respiratory and cardiovascular diseases. So far, this effect has been ascribed to the fact that PM (particulate matter) and DEP consist of a carbonaceous/graphitic components mixed with organic molecules most of which belong to the class of polyaromatic molecules (PAH) . Among the PAH’s the class of quinones, formed during the combustion process, has been the target of many works which have shown that they are able to catalyze the generation of oxygen superoxide radicals (O2.-) producing reactive oxygen species (ROS), thus inducing oxidative stress in biological systems. This reaction needs to be activated by various reducing agents such as, for example, DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl) phosphine) 1,2 . The obvious request by the Science of the Environment has been that to exploit these experimental observations for the qualitative and quantitative evaluation (possibly with fast and in situ analysis) of the relationship between i) chemical composition and ii) size of PM and DEP and their capability of producing ROS. It has been shown that Cytochrome C (CytC) can be taken as a very suitable biosensor for the detection and evaluation of oxygen superoxide2. This fact has prompted the development of two independent analytical methods. An electrochemical/spectrophotometric sensor has been presented by Ciriello et al3. We present here an alternative method based on Resonance Raman Spectroscopy (RRS). Spiro et al. (1972) have provided a detailed understanding of the RRS of CytC and its changes with the oxidation state of the coordinated Fe (Fe3+ or Fe2+ )4. Thus, the use of RRS allows to monitor the reduction process of CytC in extremely small samples and even at very small concentrations (approx. 10-7 M of CytC)). In this research we have applied our method to study a variety of samples of carbonaceous particles namely DEP, graphite ( 99.99% purity) samples of ball milled HOPG for 10 and 20 hours, nanocarbon (99% purity). The commonly known Raman spectroscopic features (namely the G and D lines near 1300 and 1600 cm-1 respectively), observed with experiments and accounted for by theory, of carbonaceous particles are indeed observed in our samples, but show unquestionable differences. Multi-wavelength Raman experiments had been carried out on ball milled graphite to investigate the effect of varying crystallite size and defect concentration. The intensity ratio ID/IG has been shown to be related to the averagesize (or amount of edges) of the “graphitic” platelets, which make up most of a given sample of carbonaceous particle5. The experiments on these samples (pristine graphite, ball milled graphite (10 and 20 h), nanocarbon) lead to the following observations: a) The Raman intensities plotted vs. time allow to calculate the reaction time constants τ. b) We have measured the ID/IG intensity ratio and plotted τ vs ID/IG (Fig.1). From this figure we learn that superoxide formation is activated even by ball milled graphite. Furthermore, nanocarbon shows a value of τ well below the value obtained for 20h ball-milled graphite. c) From Fig.1 we observe a direct proportionality of these two parameters (τ,ID/IG) which highlights the role of the amount of edges of the carbon particles on the superoxide formation. In other words these experiments strongly suggest, that in the presence of an activator redox reactions probably involving the disordered edges or surfaces of the graphitic platelets, can contribute to the formation of oxygen superoxide.These results must be rationalized in terms of their biological relevance