A quasi-solid state electrochemical cell for in situ EXAFS measurements on biological samples.

A new "quasi-solid state" spectroelectrochemical cell for in-situ XAS measurements is described and tested using microperoxidase as reference material. The cell substantially improves conventional thin layer cells used for solution XAS spectroelectrochemistry in terms of assembling time and, more important, equilibration of the redox system under study with the applied potential. Spectra can be, in fact recorded simultaneously during a slow scan rate cyclic voltammetric scan thus permitting correlation of the spectra and the electrochemical curve. Other advantages are the possibility to use very small quantities of material also with second-generation rings. With high intensity sources having focussed beams a further decrease of the specimen weight can be easily obtained and the acquisition time of spectra further reduced.

In situ X-ray absorption spectroelectrochemical study of hydroxocobalamin.

An in situ X-ray absorption spectroscopy (XAS) spectroelectrochemical study of aquocobalamin (system B12a-B12r-B12s) has been carried out in aqueous solutions buffered at different pH values. To the best of our knowledge, this is the first structural study of aquocobalamin at room temperature under controlled oxidation conditions. Most of the previous work was in fact performed using frozen samples chemically treated to produce the species. The spectroelectrochemical approach offers several advantages: (1) the reduction products may be studied without poisoning the system with chemical reductive reagents and (2) any possible variation of the oxidation state owing to the electrons produced by the incident beam is avoided as the electrode, under potentiostatic control, acts as a scavenger. The spectroelectrochemical approach, together with more careful data analysis, has led to an improved interpretation of the XAS data. These conditions were not met in previous works where the oxidation state was not controlled and multiple scattering contributions were not taken into account. The general shape of the XAS spectra of the different species is not greatly affected by pH. A signature for the base-off square-planar coordination has been evidenced for the Co(II) compound at basic pH. A new signature for Co(I), indicating square-planar coordination, has been identified on the experimental spectra and simulated in theoretical X-ray absorption near-edge structure (XANES) studies. The flexibility of the electrochemical approach, that permits to unambiguously establish the formal oxidation state, has led to very reliable values for energy shift and peak intensity variations. The experimental XANES and extended X-ray absorption fine structure (EXAFS) spectra with a very good signal-to-noise ratio have been processed using the GNXAS package that takes into account multiple scattering contributions. EXAFS and XANES independent analysis result in the same structural model. The reduction from Co(III) to Co(II) produces the most significant structural changes: the cobalt coordination number decreases from six to five, and the edge position shifts by 2.4 +/- 0.3 eV. In addition, the XANES spectra are strongly modified. The reduction from Co(II) to Co(I) produces mainly electronic effects with no apparent change of the coordination number. A discussion of the limits and potentialities of EXAFS in this type of study has also been included.

Urea solid-state biosensor suitable for continuous dialysis control.

A solid state potentiometric biosensor for urea determination was assembled coupling a nonactin-based ISE without an internal solution with immobilized urease. The system proved to be useful for continuous monitoring of urea during dialysis treatment using the ultrafiltrate of blood as sample. A noticeable shift of potential occurred during the dialysis (probably due to continuous extraction of ionophore by the ultrafiltrate). Therefore a flow injection analysis assembly was necessary to control the shift of electrode potential.

A spectroelectrochemical study of microperoxidase at bare and gold-plated RVC thin-layer electrodes.

Direct electrochemistry of microperoxidase (the heme-undecapeptide from cytochrome c) has been followed at a bare and a gold plated RVC thin-layer electrode, using the spectropotentiostatic method or voltabsorptometry. Both techniques yield 'clean' and undistorted signals; their analysis easily provides quantitative information for the electrochemical parameters of microperoxidase and shows that spectroelectrochemistry is a powerful method to study the redox behavior of metalloproteins or their active site fragments.