A new method for enzyme substrate assembly and its use
in proteolytic
enzyme assays with colorimetric and electrochemical detection is presented.
The novelty of the method is the use of dual-function synthetic peptide
containing both gold clustering and protease-sensitive moieties, which
not only induces the simple formation of the peptide-decorated gold
nanoparticle test substrates but also allows for the detection of
proteolysis in the same batch. Protease-treated nanoparticles with
a destabilized peptide shell became more prone to electroactivity,
and thus, the model enzyme plasmin activity could be quantified with
stripping square wave voltammetry analysis as well, giving an alternative
method to conduct aggregation-based assays. Spectrophotometric and
electrochemical calibration data proved to be linear within the 40–100
nM active enzyme concentration range, with possible extensions of
the dynamic range by varying substrate concentration. The simple initial
components and the ease of synthesis make the assay substrate preparation
economic and easy to implement. The possibility of cross-check analytical
results with two independent measurement techniques in the same batch
greatly increases the applicability of the proposed system.