Long-term stability of biosensors during storage and use is critical to assure reproducibility of measurements. To enable long-term stability tests, a model biosensor was developed consisting of a self-assembled monolayer (SAM) of L-cysteine on polycrystalline polished gold surface to which cytochrome-c (Cyt-c) was covalently attached using 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC) chemistry. To accomplish stability improvements, PEG-cyt-c neoconjugates were constructed and 4 and 7 PEG molecules (M(w) = 2000) attached to Cyt-c. The heme group electrochemistry was followed by cyclic and square wave voltammetry. It was found that all constructs were functional and that PEG modification of Cyt-c did not alter function and structure of the protein. Electrochemical results also show electron transfer to be a surface confined process which demonstrates that even when Cyt-c was PEG-modified it was covalently attached to the L-cysteine SAM. To assess the stability of the constructs electrodes were exposed to accelerated storage conditions (40 degrees C warm buffer) and current density determined for the reaction with H(2)O(2). A plot of current density versus storage time demonstrates that PEG-modified Cyt-c shows a much higher electrochemical response even after 2 months of exposure to accelerated storage conditions in comparison with Cyt-c modified electrodes. In summary, the present study demonstrates that covalent PEGylation of Cyt-c increases long term biosensor stability even under accelerated conditions. (C) 2011 Elsevier B.V. All rights reserved.