Biomedical Engineering Reference
In-Depth Information
challenge of developing effective label-free methods for protein detection, this
section highlights some emerging sensor architectures that utilize ferrocene-peptide
conjugates for this distinct purpose.
One class of proteins that has recently become an attractive target for biosensing
is proteases. Proteolysis, the reaction catalyzed by these enzymes, is a simple
hydrolytic process that cleaves the amide bond connecting adjacent amino acid
residues in proteins. While certain proteases mediate nonspecific hydrolysis, many
perform vital functions through selective and efficient cleavage of specific peptide
substrates. In healthy individuals, the activity of proteases is tightly regulated and
they are ubiquitous participants in maintaining fidelity in many critical biological
processes such as apoptosis, matrix remodeling, and blood clotting. However,
misregulation of protease activity has been implicated in an array of major life-
threatening diseases such as cancer, AIDS, and infectious and neurodegenerative
disorders. Thus, robust assay methods capable of quantifying target protease activ-
ity are in high demand.
Ferrocene-peptide conjugates are ideally suited as receptors for assaying prote-
ase activity when linked to interrogating electrodes. An example electrochemical
proteolytic beacon for detecting matrix metalloproteinase-7 (MMP-7) activity is
shown in Fig.
8
[
53
]. MMPs are a family of extracellular zinc proteases that have
long been associated with tumor invasion and metastasis [
54
] and thus may serve as
important cancer biomarkers. In this study, a known helix peptide substrate for
MMP-7 (Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser) was modified with an
N
-terminal
ferrocene and a Cys residue at the
C
-terminus to enable gold anchoring. The peptide
was synthesized via traditional solid-phase synthesis and ferrocene acetic acid was
conjugated to the
N
-terminus prior to cleavage from the resin. SAMs of the
ferrocene-peptide conjugate were grown by immersing clean gold electrodes in
ethanol solutions of the probe. In the initial “signal-on” state of the sensor,
voltammetric scanning yields a well-defined peak with quantifiable charge due to
efficient electron transfer between ferrocene and the underlying electrode. Upon
exposure to MMP-7, the peptide probe is cleaved between the Ala-Leu residues and
ferrocene is separated from the electrode. The removal of ferrocene by target
protease produces a measurable “signal-off” decrease in current. At a constant
incubation time, the amplitude of this signal decrease scaled with increasing
concentrations of MMP-7 resulting in a detection limit of 3.4 pM. Specificity was
confirmed in control experiments with different MMPs and BSA.
Importantly, similarly designed electrochemical protease biosensors have been
employed for the detection of alternate disease markers such as caspase-3 [
55
],
prostate-specific antigen (PSA) [
56
], plasmin [
57
], and trypsin and
-thrombin [
58
].
Hence, it is becoming increasingly more apparent that ferrocene-peptide conjugates
may be used as effective alternatives to fluorescent-labeled peptide substrates for
developing protease assays. This is significant as fluorescence-based assays are often
complicated by intricate excitation and detection schemes to reduce background
interference. It should be noted, however, that the electrochemical assays also require
some degree of optimization as the signal response is influenced by the monolayer
composition and probe densities. Looking ahead, one can easily envision multiplexing
a
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