Cleavage-Sensing Redox Peptide Monolayers for the Rapid Measurement of the Proteolytic Activity of Trypsin and α-Thrombin Enzymes

Ferrocene (Fc)-labeled peptides are end-grafted onto gold electrodes via a flexible polyethylene glycol (PEG) linker, and their ability to act as substrates for proteolytic enzymes trypsin and a-thrombin is investigated by cyclic voltammetry. It is shown that whereas a short Fc-tetrapeptide substrate is rapidly cleaved by trypsin, a longer Fc-heptapeptide substrate is required for a-thrombin detection. However, in both cases it is observed that not all of the Fc-peptide chains present on the electrode surface are cleavable by the proteases and that the cleavage yield is actually controlled by the surface coverage in the Fc-peptide. Surface dilution of the Fc-peptide using a backfilling molecule such as MCH (6-mercapto-1-hexanol) was required to obtain a cleavage yield larger than 80%. The kinetics of Fe-peptide cleavage by trypsin or alpha-thrombin is then shown to be adequately described by Michaelis Menten kinetics, allowing enzymatic constants k(cat) and K-M to be determined. The obtained rate constant values showed that the affinity of the enzymes for their respective Fe-peptide substrates is very high (i.e., low K-M values) whereas that for the cleavage step itself is relatively low (low kcat values). Partial compensation of these parameters yields a fast response of the Fc-peptide electrodes to the proteases in solution in the 1-1000 nM range. The type of molecule used to backfill the Fc-peptide layers, either MCH or PEG(6) chains, is shown to modulate the activity of the proteases versus the Fc-peptide layers: in particular, the PEG(6) diluent is specifically shown to decrease the ability of a-thrombin to cleave its Fc-peptide substrate whereas trypsin activity is unaffected by the presence of PEG chains.