A novel strategy for immobilising enzymes through the electrochemical control of self-assembled monolayers

The performances of enzymatic fuel cells (EFCs) are primarily affected by limitations in stability and power output. In order to improve the efficiency of the current EFC technology, researchers are striving towards an attempt to obtain devices which can resemble the biological function of enzymes in living matter. The goal is to mimic the ability to perform sequential transformation of a substrate to a final product using a sequence of enzymes in which the product of one enzyme serves as the substrate for an adjacent enzyme. The rate of reaction is controlled by the concentrations of substrates and cofactors and the activity of the enzymes involved: The performances of enzymatic fuel cells (EFCs) are primarily affected by limitations in stability and power output. In order to improve the efficiency of the current EFC technology, researchers are striving towards an attempt to obtain devices which can resemble the biological function of enzymes in living matter. The goal is to mimic the ability to perform sequential transformation of a substrate to a final product using a sequence of enzymes in which the product of one enzyme serves as the substrate for an adjacent enzyme. The rate of reaction is controlled by the concentrations of substrates and cofactors and the activity of the enzymes involved: the enzymatic cascade reaction (ECR). 

ECR can alleviate the limitation of a single substrate transformation which is typical of traditional EFCs, by enabling the sequential and more complete oxidation of the fuel. Such sequential reactions mimic cascade reactions in cells. 

Exploiting some intrinsic electrochemical properties, self-assembled monolayers (SAM) made of alkyl thiols on gold supports, can be used to sequentially immobilise a series of enzymes over different electrode areas which are determined by the characteristics of the electrodes with the application of specific voltages.

In the first work of the thesis, the controlled immobilisation of Cytochrome c has been used to demonstrate the ability to independently address and pattern the surfaces of two adjacent electrodes under conditions of neutral pH, without affecting the neighbouring bio-modified surface with SAM and the immobilised protein.

Subsequently, inspired by a prototype biofuel cell using a synthetic enzymatic cascade reaction for the conversion of methanol to carbon dioxide employing three NAD+dependent enzymes, alcohol (ADH), formaldehyde (FLDH) and formate (FoDH) dehydrogenases, the same approach and methodology used for cytochrome c proved to be effective for their immobilisation on three vicinal, and separate electrode surfaces. This strategy has the potential to specifically pattern and immobilise enzymes in a rapid manner, in benign and biocompatible conditions. The immobilisation of ADH, FLDH and FoDH at three adjacent gold electrodes has been proven using three thiols that were selected after a screening for optimal conditions of pH, buffer type and thiol. After screening, mercapto-1-hexanol was selected as thiol for the SAM to be used for physical adsorption of ADH, 1-methyl-5-mercapto-1,2,3,4-tetrazole (MMT) for the SAM to be used for physical adsorption of FLDH and mercaptoundecanoic acid for the SAM to be used for physical adsorption of FoDH. These thiols provided the optimal systems and working conditions for enzymatic activity after immobilisation. The selected thiols were successfully deposited using an electrochemical plug and play procedure on a three working electrode configuration, and the activity of the three enzymes was observed by measuring the production of NADH using UV-Vis spectrophotometry.