Michael Riepl

Capacitive monitoring of protein immobilization and antigen–antibody reactions on monomolecular alkylthiol films on gold electrodes

Self-assembled monolayers of omega-mercaptohexadecanoic acid and omega-mercaptohexadecylamine on gold electrodes are stable at neutral pH and display pure capacitive behavior at frequencies around 20 Hz. Different methods of covalent immobilization of proteins on these monolayers are compared. Various reagents including succinimides, thionylchloride, p-nitrophenol and carbodiimides were used to activate the carboxy groups of the adsorbed monolayer of omega-mercaptohexadecanoic acid. Glutaraldehyde, cyanuric chloride and phenylene diisocyanate were used to activate the amino groups of the monolayer of omega-mercaptohexadecylamine. The immobilization of albumin on the activated surface was studied by capacitive measurements. The N-hydroxysuccinimide and carbodiimide methods were identified as most suitable for protein immobilization in that they did not compromise the insulating properties of the alkylthiol layer and led to maximal increase of its dielectric thickness. These approaches were used for a layer-by-layer preparation of a capacitive immunosensor. Specifically, antibodies to human serum albumin were immobilized on the alkylthiol mono-layer. Binding of the antigen led to a decrease of the electrode capacitance. The detection limit of the immunosensor is as low as 15 nM (1 mg/l).

Optimization of capacitive affinity sensors: drift suppression and signal amplification

The detection limit of capacitive affinity sensors based on the gold–alkanethiol system can be improved by optimization of sensor preparation and by signal amplification. The dissociation of the gold–sulfur binding is often a critical point leading to operative errors of such sensors. The stability of self-assembled monolayers prepared with different thiols on gold electrodes in aqueous and organic solvents was studied by the capacitive technique. The results show that monolayers made of 16-mercaptohexadecanoic acid are stable in aqueous solution and can be hardly extracted from a gold surface by ethanol, methanol, or dioxane, while a considerable damage of self-assembled monolayers was observed due to incubation in chloroform or dimethylformamide. In contrast, self-assembled monolayers made from short-chain disulfides or thiols (such as 3,3′-dithio-bis(propionic acid N-hydroxysuccinimide ester) or 11-mercaptoundecanoic acid) displayed a poor stability in aqueous phase. Capacitive affinity sensors based on these short-chain thiols showed a considerable drift of the signal. The use of long-chain thiols resulted in a stable signal; it was applied to compare capacitive effects due to immobilization of different biological molecules and for preparation of different biosensors. The response of capacitive biosensors can be amplified by formation of a sandwich structure. This principle was illustrated by subsequent adsorption of polyclonal anti-HSA after binding of HSA with a sensor for HSA based on monoclonal antibodies.

Capacitive detection in thin film chemical sensors and biosensors

Thin film electrode chips on silicon substrate have been realized and utilized in development of different chemo- and biosensors. These affinity sensors are based on capacitive transducing and their general structure is thin film Au/alkanethiol/receptor. Several applications of thin film gold electrodes in capacitive sensors are presented.

Self-Assembled Monolayers with Molecular Gradients

In recent years, biosensors and sensor arrays have developed into very important analytical tools, which found applications in many fields such as pharmaceutical (high-throughput) screening, medical diagnosis, or industrial process control. One of the major challenges for material research is the preparation of appropriate sensor surfaces, providing an interface with a high sensitivity and selectivity toward a given analyte. This chapter discusses some straightforward and flexible approaches to study structure and/or composition-function relationships and response characteristics of polymeric and molecular sensor materials. The controlled continuous deposition of self-assembled monolayers (SAMs), e.g. of substituted thiols or silanes, paves the way for the generation of molecular gradients on solid surfaces. These are useful for the preparation of interfaces with spatially controlled chemical composition and/ or physical properties. These tools can help to improve the selectivity and specificity of surfaces for biosensors and biochips. They can also be utilized for the study of fundamental protein adsorption and exchange phenomena.