Parameters Important in Fabricating Enzyme Electrodes Using Self-Assembled Monolayers of Alkanethiols

Abstract

The immobilization of enzymes on the surface of electrodes modified with self-assembled monolayers (SAMs) provides a number of advantages as a method for the fabrication of enzyme electrodes. Using SAMs has the potential to provide enzyme electrodes with a high degree of reproducibility, 1,2 molecular level control over the spatial distribution of the immobilized enzymes 3-14 and the immobilization of the enzyme close to the electrode thus allowing direct electron transfer to be achieved. [15][16][17][18][19][20][21][22][23][24] These advantages have resulted in a recent surge in research into self-assembled monolayers for biosensor applications in general, and enzyme electrodes in particular. [25][26][27] However, despite the plethora of biosensor research papers using self-assembled monolayers as the base onto which the biomolecule is immobilized, there has been very little fundamental research into what steps are important in the fabrication process or which parameters control the response of the resultant biosensor.Alkanethiols modifying gold are the most popular SAMs. 25,26 The interaction between the thiol groups and the gold results in a strong pseudocovalent bond 28 which is stable throughout the potential range of 0.8 V to -1.4 V versus Ag/AgCl before being oxidatively or reductively desorbed. 29,30 If the alkanethiol has appropriate chemical functionality, such as an amine or carboxylic acid moiety, then once the SAM is formed, enzymes and other biomolecules can be easily covalently bound to the SAM. In this way, a monolayer or submonolayer of enzyme is immobilized. For enzyme electrodes a short alkyl chain alkanethiol, usually three carbons long, is chosen so that the enzyme is as close as possible to the electrode. An additional advantage of the short alkyl chain is that a relatively disordered SAM is formed which means the underlying metal is still electrochemically accessible. In contrast, if a long chain alkanethiol is used the electrode is passivated unless defects are deliberately introduced into the SAM. 31 There are a number of steps in the fabrication of an enzyme electrode using an alkanethiol. First, the metal surface must be prepared and cleaned. The alkanethiol must self-assemble onto the metal, followed by activation of the chemical functionality of the SAM, leading finally to the exposure of the activated SAM to the enzyme which is when attachment occurs. There are however many unknowns in this fabrication process. Questions that needs answers are: what effect does the roughness of the gold surface have? How long should the metal be exposed to the alkanethiol solution to allow a stable SAM to be formed? What is the optimal procedure for activating the SAM for enzyme attachment? How long should the activated SAM be exposed to enzyme? What should the concentration of the enzyme solution be during enzyme immobilization? Does the buffer used have any effect on subsequent performance? Can the amount of enzyme immobilized be controlled and do different enzyme loadings lead to different electr...

Self‐Assembled Monolayers into the 21st Century: Recent Advances and Applications

“…The molecular level control has seen multiple functionality integrated into a single layer and quite complicated multilayer molecular constructions fabricated. As many of the important issues in fabricating the SAM based enzyme electrodes have been reviewed [4,5,99,100] this discussion will concentrate on some of the more interesting integrated molecular systems.…”

Mediated Electron Transfer at Redox Active Monolayers. Part 3: Bimolecular Outer-Sphere, First Order Koutecky-Levich and Adduct Formation Mechanisms

“…Organic electro-oxidation at conductive hydrated metal oxide surfaces provides another example [29] . In more recent publications biphasic kinetic data are reported for glucose oxidation at thin electropolymerized poly(phenol) films containing immobilized glucose oxidase [30] and at alkanethiol self assembled monolayers containing adsorbed glucose oxidase [31] , and for both NADH [32] and ascorbate [33] oxidation at poly(aniline)/poly(vinylsulfonate) composite modified electrodes and ascorbate oxidation at an electrode modified by a Tosflex ionomer film containing ferricyanide [34].…”

Enhancement of the Protein Loading Density by a Pre-Cleaning Process of a Gold Substrate: Confocal Laser Scanning Microscopic Study

“…[1][2][3][4][5][6][7][8] They not only provide a high degree of reproducibility, 9,10 but also make it possible to immobilize proteins close to the surface of an electrode for the purpose of obtaining a direct electron transfer. [11][12][13][14][15][16][17][18] When the protein immobilization process using SAMs is practically applied to a biosensor, the density of loaded proteins should be greatly enhanced to improve its detection sensitivity.…”

“…It was known that the ratio of alcohol-terminated (-OH) thiol to carboxylic acid-terminated (-COOH) thiol has an important effect on the loading density of the immobilized proteins. 1,4 However, it was also found that the pre-cleaning process of gold substrates strongly affects the loading density of GOx. In the present work, different cleaning protocols of gold surfaces were examined to find the optimal conditions for the maximum GOx loading density.…”