High-throughput Screening of Proteins by Electrochemical Protein Chip

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By the near completion of the human genome sequencing project, attention is now shifting toward the analysis of proteins or proteoms. In this respect, protein chips are regarded as a high-throughput means of studying protein expression. Protein chips are made of various proteins immobilized on the surface of a substrate. The aim of this project is to develop a protein chip based on the knowledge obtained from our study of electrochemical DNA chips. To realize this purpose, we constructed a protein chip by immobilizing a protein carrying a cysteine residue on the gold electrode through the gold-sulfur linkage and tried to establish an electrochemical analysis system with it.First of all, we verified in a preliminary experiment with peptides that this system works as designed. Peptides carrying different charges immobilized on a gold electrode and their interaction with ferrocenecarboxylic acid was studied electrochemically. Ferrocenecarboxylic acid bound strongly to cationic peptides im … More mobilized on the electrode as proven by an increase in the redox current due to the ferrocene, whereas it failed to bind to anionic peptides because of electrostatic repulsion.In the next experiment, Rec A protein, known to discriminate single and double stranded DNA, and its functional domains, L2 and Helix G peptides, were synthesized by the peptide synthesizer with Fmoc chemistry and tested whether their interaction with single stranded DNA can be monitored electrochemically. These peptides were immobilized on the individual electrodes and were allowed to interact with single or double stranded DNA carrying an electrochemically active ferroncene moiety. This system, once established, should be useful for the screening of DNA binding peptides or proteins. Electrochemical as well as Quartz Crystal Microbalance experiments suggested that the L2 part of the protein is important for the interaction with nucleic bases of DNA and the Helix G part contributes to the discrimination between single and double stranded DNAs. These results opened up a new vista for the development of an electrochemical protein chip. Furthermore, high-throughput screening of various other proteins should be possible by using the scanning electrochemical microscope. Less