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

Abstract

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 alpha-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 alpha-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 Fc-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 Fc-peptide substrates is very high (i.e., low K(M) values) whereas that for the cleavage step itself is relatively low (low k(cat) 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 alpha-thrombin to cleave its Fc-peptide substrate whereas trypsin activity is unaffected by the presence of PEG chains.

Using G-Quadruplex/Hemin To “Switch-On” the Cathodic Photocurrent of p-Type PbS Quantum Dots: Toward a Versatile Platform for Photoelectrochemical Aptasensing

“…As a specific serine protease, TB is involved in the coagulation cascade, which catalyzes many coagulation-related reactions. 53 On one hand, it plays crucial role in physiological and pathological coagulation; on the other hand, it regulates many processes in inflammation and tissue repair at the vessel wall. 53 As shown in Scheme 2B, the TB aptamer (TBA) possessed the structure composed of the G-quadruplex sequence and the aptamer sequence specific for target TB.…”

“…53 On one hand, it plays crucial role in physiological and pathological coagulation; on the other hand, it regulates many processes in inflammation and tissue repair at the vessel wall. 53 As shown in Scheme 2B, the TB aptamer (TBA) possessed the structure composed of the G-quadruplex sequence and the aptamer sequence specific for target TB. The TBA sequence is 5'-G 1 G 2 T 3 T 4 G 5 G 6 T 7 G 8 T 9 G 10 G 11 T 12 T 13 G 14 G 15 -3' which binds to the TB using residues 4 through 12, while residues 1, 2, 3, 13, 14, and 15 locate at the exposed side of the aptamer.…”

A modular electrochemical peptide-based sensor for antibody detection

“…Recently, Plaxco, Lai and other groups have reported the development of electrochemical peptide-based (E-PB) sensors that use short antigenic peptides as recognition elements for the detection of diagnostically relevant antibodies. 2,6,7 In these platforms the recognition peptide is usually immobilized either by direct chemisorption of a thiol-end labelled peptide onto gold surfaces 8 or by attachment on alkylthiol self-assembled monolayers (SAMs) with terminal reactive groups via a ''clickchemistry'' approach; 9 the peptides are also labelled at one end with a redox reporter, most frequently ferrocene, 4,[10][11][12] or methylene blue (MB). 2,6 Once fabricated, these E-PB sensors allow the monitoring of the target antibody in a reagentless fashion and thus appear suitable for the development of portable and easy-to-use sensors for antibody detection.…”

Solid-Phase Synthesis of Aza-Lysine Peptide Analogue of Trypsin Substrate

“…The tetrapeptide Fmoc-Ser-Ala-Lys-Gly-NH2 was studied, because of its relationship to the previously synthesized substrate Fmoc-Ser-Pro-Arg-Gly [7] that could be monitored effectively in trypsin digestion experiments. To study the influence of aza-residues on trypsin cleavage, we have now synthesized Fmoc-Ser-Ala-azaLys-Gly (1) on Rink amide resin (Scheme 1).…”