Effect of spacer length on the performance of peptide-based electrochemical biosensors for protease detection

Miniaturisation of a peptide-based electrochemical protease activity sensor using platinum microelectrodes

“…2A). Based on previous macroelectrode optimisation work, 20,21 the sensing layer consisted of a mixed SAM; the probe with the target peptide was tagged with MB and attached to a thiol-terminated PEG-6 spacer, backfilling MCH to minimise pinholes in the SAM layer and co-adsorbent PEG-based DT molecules to support the orientation, specificity and accessibility of the probe on the electrode surface. Two different MB-labelled peptides were used, the L-amino acid sequence for a trypsin-cleavable substrate or the D-amino acid sequence analogue as a trypsin-uncleavable control.…”

“…Two methylene blue-labelled peptides were synthesised inhouse following the synthetic experimental procedures described previously. 20,21 Briefly, the redox labelled peptides ( probe substrate and the control) (MB-Phe-Arg-Arg-PEG-6-Cys) were synthesised using Fmoc solid-phase chemistry on a polystyrene resin with a Rink-amide linker. First, cysteine was coupled to the resin, followed by a polyethylene glycol unit containing 6 ethylene glycol units (PEG-6) as a spacer, then a cleavable short peptide sequence (specific to trypsin) (Phe-Arg-Arg (FRR)) and finally the methylene blue redox label (MB).…”

“…After the addition of the target enzyme (trypsin), or the proteins, casein and BSA for characterisation of non-specific binding, the SWV signal was continuously monitored with time. Following the established analysis method for Au macroelectrodes, 20,21 this resulting signal is expressed as the percentage relative change in the SWV peak current with respect to the initial peak current (henceforth called the % signal change).…”

“…This is in addition to the uncleavable subset of the immobilised peptides at all [E] for both electrode types, previously attributed to electrode roughness and probe orientation variation as the sources of local site inaccessibility. 21,23 It is clear that this analysis method has now extended the trypsin measurement range to 100 nM, covering the clinically relevant range even for chronic pancreatitis conditions (60 ± 27 nM).…”

“…There is extensive literature reported on protease detection using this approach. [13][14][15][16][17][18][19][20][21][22] For example, Liu et al described a method that allowed detection of matrix metalloproteinases (MMPs), whose proteolytic activities have close association with cancer progression, at very low concentrations (with a limit of detection of 3.4 pM). 15 An alternative approach was developed by Lee et al who reported on the reference electrode-free sensing of MMP-9 using the self-gating effect on a concentric electrode system which consisted of an island and enclosing electrode.…”