Electrochemical Detection of Biological Catalysts as Signatures of Life

The enzyme trypsin is chosen as a detection target due to its ubiquity in Earth biology. Trypsin catalyzes the break down of proteins through a catalytic triad consisting of histidine, aspartate and serine. The proposed sensor works by the detection of proteolytic cleavage of a redox-active tag. It is comprised of two gold electrodes. The first gold electrode houses a sensor probe, in this case a substrate peptide. The second electrode is a collector electrode that will detect the product of peptide cleavage. A methylene-labeled substrate peptide is self-assembled on a gold electrode using a thiol linkage. Any remaining accessible area of the electrode is then "blocked" or backfilled with an alkanethiol, which acts as an insulator and forces the peptide to stand up and form a close-packed structure on the gold surface. In this close-packed configuration, the peptide is accessible to the catalytic reaction with trypsin. Trypsin cleaves the peptide and releases the methylene blue terminus. A characteristic redox signature of methylene blue is detected at the second electrode using cyclic voltammetry and/or square wave voltammetry. Electrodes will be fabricated as 2 gold band electrodes on an inert substrate using a shadow mask and gold evaporation. While not required for proof of concept sensing, a simple fluidic channel will be added over the electrodes to demonstrate future integration ease with the ARC Universal Sample Processor for Life on Icy Worlds (SPLIce). More »