Electrochemical aided adsorption

Two methods for the spatially controlled deposition of proteins on microelectrodes are described. The first technique involves the entrapment of glucose oxidase in photopolymerized polyHK. The second uses electrochemically aid adsorption to deposit urease and to co-deposit glucose oxidase with bovine serum albumin. Both techniques were found to lead to active deposits and the properties and optimisation of the deposition procedures will be described. Further, to facilitate glucose measurement in complex medi the depointion of a thin film of polypyrrole following that of the protdns is described. The properties of s film with respect to two model interferents and complex yeast extract medium will be reported. 

Two principle techniques for electrochemical enzyme deposition have been reported, entrapment in an electrochemically grown polymer and electrochemically aided absorption. A wide range of electrochemically grown polymers have been used. The polymer can function as both an entrapment matrix and as an anti-interference layer (7, 12-20), as a matrix for the immobilisation of the protein with an electron transfer mediator (21-23), and as an electron transfer matrix alone (24, 25), Electrochemically aided adsorption has received comparably less attention (26-30), However, in our experience (31) the latter technique results in larger responses and is more appropriate to microelectrodes. Here we will present results on the electrochemically aided adsorption of GOx and BSA, and also of urease. Furthermore to reduce the interferences at the GOx/ BSA electrode we will describe the deposition of an anti-interference layer of polypyrrole, which is grown on the electrode after the deposition of the proteins. 

Electrochemical Aided Adsorption. The 1000 x 100 pm working electrode of the microcell was principally used for the GOx/ BSA depositions. Before any deposition was made the working electrode was pretreated by cyclic voltammetry in 0.5 M H2SO4 as follows four sweeps at 50 mV.s" were made, with the first two and the final one being made over the range -0.25 to 1.2 V, whilst for the third sweep the anodic limit was increased to 2.0 V. The final sweep was halted at 0.2V. The protdns were deposited out of PBS containing between 5 and 10% protein (wt/vol). The proteins were deposited simultaneously at, typically an plied current dens of 5 mA.cm-2 for a period of 2 min. The modified electrode was carefully washed in water for 5 s and then cross-linked with glutaraldehyde (2.5 % in PBS for 30 min or 25 % in PBS for 4 min) at room temperature. Finally the electrode was rinsed and stored in 10 mM potassium phosphate bi er at 4 imtil used. 

Many dehydrogenase enzymes catalyze oxidation/reduction reactions with the aid of nicotinamide cofactors. The electrochemical oxidation of nicotinamide adeniiw dinucleotide, NADH, has been studied in depthThe direct oxidation of NADH has been used to determine concentration of ethanol i s-isv, i62) lactate 157,160,162,163) pyTuvate 1 ), glucose-6-phosphate lactate dehydrogenase 159,161) alanine The direct oxidation often entails such complications as electrode surface pretreatment, interferences due to electrode operation at very positive potentials, and electrode fouling due to adsorption. Subsequent reaction of the NADH with peroxidase allows quantitation via the well established Clark electrode. 

Reversible attachment of nanostructures at molecular printboards was exemplified by the adsorption and desorption of CD-functionalized nanoparticles onto and from stimuli-responsive pre-adsorbed ferrocenyl-dendrimers at a CD SAM (Fig. 13.7).65 Electrochemical oxidation of the ferrocenyl endgroups was employed to induce desorption of the nanostructure from the CD SAM. An in situ adsorption and desorption of ferrocenyl dendrimers and CD-functionalized Au nanoparticles (d 3 nm) onto and from the molecular printboard was observed by a combination of surface plasmon resonance spectroscopy (SPR) and electrochemistry. Similar behavior was observed when larger CD-functionalized silica nanoparticles (d 60 nm) were desorbed from the surface with the aid of ultrasonication. 

The hydrophobias are a case where protein nanofibers can play a dual role in creating a biosensor. They can aid in the immobilization of bioactive components within a biosensor and also add further functionality to the transducing element of a biosensor device. Hydrophobins are self-assembling [3-sheet structures observed on the hyphae of filamentous fungi. They are surface active and aid the adhesion of hyphae to hydrophobic surfaces (Corvis et al., 2005). These properties can be used to create hydrophobia layers on glass electrodes. These layers can then facilitate the adsorption of two model enzymes glucose oxidase (GOX) and hydrogen peroxidase (HRP) to the electrode surface. The hydrophobin layer also enhances the electrochemical properties of the electrodes. 

This is, of necessity, the case since EMIRS is a difference technique and, as such, does not allow any quantitative investigation into the adsorption process, i.e. it is not possible to compare any integrated band intensities since the separate "absolute spectra of the species present at the two potentials cannot be obtained. Nonetheless, the paper clearly does show the versatility of the technique in giving an insight into an electrochemical system, aided by the high degree of energetic control afforded by potential modulation. 

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