Microreactor for electrochemical conversion: in drug screening and proteomics

The majority of marketed drugs are metabolized through oxidation by enzymes of the cytochrome P450 family, thereby producing phase I metabolites. For pharmaceutical companies it is essential to thoroughly screen candidate drugs for potentially toxic metabolites, in order to avoid high costs associated with their failure in late development stages. Generating phase I metabolites directly at an electrode in an electrochemical cell is a purely instrumental approach to metabolite analysis. Electrochemical cells can be coupled directly to analytical instrumentation such as liquid chromatography and mass spectrometry for rapid and sensitive detection.\ud Next to this, the identification and characterization of proteins is important to understand processes related to disease development. Standard routines in protein analysis involve enzymatic digestion using proteases such as trypsin, followed by mass spectrometric analysis of the resulting proteolytic peptides. Electrochemical protein cleavage is emerging as an instrumental alternative that enables specific cleavage at tyrosine and tryptophan peptide bonds, without the need for sample purification due to the absence of cleavage reagents.\ud Electrochemical cells designed for performing conversions in the often precious samples for proteomics and drug screening studies should be of low volume, efficient and either easy to clean or cheap and single-use. With microfabrication technology, structure dimensions can be controlled with micrometer precision and a variety of materials is available for chips and electrodes. We integrated an electrochemical cell with boron doped diamond electrodes and a mixer on a chip. This chip was used to mix electrogenerated products with (bio)molecules of interest. This allowed us to study combined phase I and phase II metabolism in a single device, as well as the investigation of potential toxicity of reactive metabolites due to protein binding.\ud Furthermore, site-specific oxidative peptide bond cleavage was demonstrated using these microreactors. It was shown that electrogenerated protein fragments can be used for protein identification with the use of a database searching algorithm. However, if no preventive measures are taken, protein fragments generated by peptide bond cleavage can be linked together by disulfide bridges, which hamper protein identification.\ud Our electrochemical microreactors can also offer a rapid and clean alternative to chemical reducing reagents. This work demonstrates the development and use of microreactors that are characterized by efficient electrochemical conversion and mixing of reagents. These chips are capable of driving combined electrochemical and chemical reactions for drug screening and proteomics studies in a rapid fashion and with minute amounts of sample