Electroactive Myoglobin Films Grown Layer-by-Layer with Poly(styrenesulfonate) on Pyrolytic Graphite Electrodes

Abstract

Stable multilayer electroactive films were grown on rough pyrolytic graphite (PG) electrodes by alternate adsorption of layers of polyanion poly(styrenesulfonate) (PSS) and positively charged myoglobin (Mb) from their aqueous solutions. Incorporation of large amounts of electroactive Mb was facilitated by high electrode surface areas and by adsorbing coiled PSS from 0.5 M NaCl solutions. Cyclic voltammetry of {PSS/Mb}n films showed a pair of well-defined, chemically reversible peaks at about -0.25 V vs SCE at pH 5.5, characteristic of the Mb heme Fe III /Fe II redox couple. Electroactivity was extended to 7 {PSS/Mb} bilayers on rough PG surfaces, in comparison to 2 electroactive layers on smooth gold coated with mercaptopropanesulfonic acid. Square wave voltammograms of {PSS/Mb}n films gave good fits by nonlinear regression analysis to a model featuring dispersion of formal potentials, providing average formal potentials and an apparent rate constant. Oxygen and trichloroacetic acid were catalytically reduced by Mb in {PSS/Mb}n films with significant decreases in the electrode potential required. Making such films on PG rather than on gold provides a larger number of electroactive layers with no need for chemical pretreatment of the electrode.

Evidence for heme release in layer-by-layer assemblies of myoglobin and polystyrenesulfonate on pyrolitic graphite

“…We base this on (1) the large difference in midpoint potential of a {Mb-PSS} n multilayer on pyrolitic graphite compared with that of Mb in solution, (2) the similar pH dependence of the midpoint potential of a {Mb-PSS} n multilayer on pyrolitic graphite and adsorbed heme, (3) the similar coverage of a {Mb-PSS} 6 multilayer on pyrolitic graphite and adsorbed heme (4) the similar peak separation of a {Mb-PSS} 6 multilayer and a PDDA-PSS-heme-PDDA-PSS film, (5) the high electron transfer rate constants of a {Mb-PSS} n multilayer, which is unlikely for electron transfer to protein molecules, but is in line with the electron transfer expected for a layer of adsorbed heme, (6) the fact that desorption of the multilayer induced by high pH does not result in the disappearance of the voltammetric peaks and (7) the fact that a large voltammetric response can be obtained by direct immersion in a Mb-PSS mixture, showing that the formation of multilayers is not necessary. Our conclusion is in apparent disagreement with previous studies on {Mb-PSS} n layer-by-layer films on pyrolitic graphite [22][23][24][25], in which it was concluded that Mb retains a near-native conformation in the layer-by-layer films. This was concluded mainly on the basis of spectroscopic data, which consistently show that Mb retains a near-native conformation.…”

“…The midpoint potential is -0.04 ± 0.01 V versus SHE and the peak separation is 120 ± 10 mV. All these properties are in reasonable agreement with results in the literature [24] and show that we have prepared similar films.…”

“…It has been claimed that there is fast direct electron transfer between the electrode and the protein molecules in these assemblies and therefore these assemblies are interesting for mechanistic studies of redox enzymes and for use as biosensors. They can be made by alternately adsorbing negatively charged PSS and positively charged Mb [21][22][23][24][25]. Our goal was to determine whether the observed electrochemical response of these assemblies is due to the native protein or to a partially or completely denatured protein.…”

Direct electrochemistry of horseradish peroxidase immobilized on a monolayer modified nanowire array electrode

“…The˛n was calculated to be 0.52. Given 0.3 <˛< 0.7 in general (Ma et al, 2000), it could be concluded that n = 1 and˛= 0.52. So, the redox reaction between HRP and the NAE was a single electron transfer process.…”

Electrodeposition CaCO3 Nanoparticles‐chitosan Composite Film on Carbon Ionic Liquid Electrode as a Platform for Hemoglobin Electrochemical Biosensor

“…The value of the slope (-61.5 mV·pH -1 ) was close to that expected theoretically -59 mV·pH -1 for a one-electron oneproton reaction, indicating that a single proton transfer accompanied in the electrochemical process of Hb. The electrochemical reduction of Hb could be simply expressed as follows: 38 Hb heme Fe(III) + H + + e -« Hb heme Fe(II)…”