Spectroscopic Real-Time Ellipsometry of Putidaredoxin Adsorption on Gold Electrodes

Abstract

Adsorption of putidaredoxin (Pdx) on gold electrodes was studied using dynamic spectroscopic ellipsometry and differential capacitance. A method to account for the metal surface optical perturbation during protein adsorption at constant potential is described. The method is based on the concept of the charged transition layer which develops between metal substrate and adsorbate. Transition-layer charge was determined from the differential capacitance and the ellipsometry measurements in buffer solutions. A three-phase (metal substrate/protein film/solution) reflecting surface model was amended with a charged metal transition layer as a fourth phase, which allowed the elucidation of the adsorbed Pdx complex index of refraction spectrum. On the basis of this spectrum, a time dependent protein surface concentration was calculated. For short times, this surface concentration was in good agreement with estimates based on mass transport limited adsorption.

Stark Spectroscopy of Tryptamine Immobilized on a Gold Electrode

“…A Å 01.84 e T G T hca (m* e 0 m* g )bDVa abs , [8] The ER response of the tryptamine layer was analyzed as a shift in the p-p* transition of the solvated indole-like where a abs is the amplitude of the measured molar absorptivring. The shift was ascribed to the interaction of the electrode ity line in m 2 /mol.…”

“…In principle, Eq. [8] should contain a sum over derivposition has been investigated in differential absorption mea-atives of at least two Gaussian functions, each one with its surements (15). There is a clear dependence on pH, ion own set of parameters.…”

“…The analysis of the ER difference between immobilized and solution measurements response starts with a least-squares fit to Eq. [8] to extract is that in isotropic solutions only second-order effects are the value of A which is then divided by a abs (obtained from observable while immobilized molecules possess a first-or-the measured solution absorbtivity) to yield the ER amplider response due to the inherent asymmetry of the environ-tude. The slope (mol/(cm m 2 V)) of the dependence of the ment at the interface.…”

Challenges in capturing oxygenase activity in vitro

“…In certain instances, a reversible electrochemical response from cyt c on bare gold or glassy carbon has been found just by careful choice of electrolyte solution (28). Rational electrode surface design for protein electrochemistry is increasingly being based on physical studies of the electrochemical interfaces (29)(30)(31)(32). Strategies with larger enzymes include use of electroactive groups in solution, or groups that are covalently attached to the electrode (22) or to the protein (19) surface.…”

Oriented immobilization of Pseudomonas putida putidaredoxin at a gold (111)–buffer interface: a real time scanning tunnelling microscopy study

“…Although both scanning tunnelling microscopy and atomic force microscopy (AFM) are being used to obtain direct information on biological molecules adsorbed at solid–liquid interfaces, STM has been preferred over AFM in most of these studies to obtain higher resolution and to lessen probe‐induced distortion in the imaged molecular shape. A study on adsorbed pdx layer on a gold electrode surface by ellipsometry has been reported (Reipa et al ., 1997). Here, we present STM investigations on pdx adsorption at the gold (111)–buffer interface and show that a thiolated mutant, C73S‐D58C, in which cysteine‐73 and aspartic acid‐58 are replaced by serine and cysteine, respectively, is necessary for oriented immobilization of the molecules on the gold surface.…”