Develop multiplex electrochemical biosensor arrays for cancer diagnosis based on protease activity profiling

Abstract

Proteases are critically involved in the progression of cancer, cardiovascular disease, and neurodegeneration, where dysregulated enzyme activity serves as an important biomarker for early diagnosis and therapeutic monitoring. Electrochemical detection platforms offer significant advantages for protease sensing due to their high sensitivity, compatibility with miniaturization, and multiplexing capability. This dissertation presents the development, optimization, and mechanistic evaluation of a 3×3 gold (Au) microelectrode array (MEA) functionalized with ferrocene (Fc) tagged peptide substrates for sensitive and selective electrochemical detection of cancer-associated proteases. Proteolytic cleavage of surface-immobilized peptide substrates was monitored using AC voltammetry (ACV), where enzymatic activity induces an exponential decay in redox current. The inverse decay time constant (1/[tau]) was shown to follow heterogeneous Michaelis-Menten kinetics, with 1/[tau] = (kcat/KM)[Protease], enabling quantitative assessment of catalytic efficiency. During early investigations with cathepsin B (CB), a critical interference mechanism was identified. The activation reagent dithiothreitol (DTT) that is required to maintain CB in its active form was found to disrupt the Au-thiol binding in the self-assembled monolayer (SAM) linking the peptide substrate to the Au electrode. Residual DTT caused the peptide to desorb from the Au electrode during continuous ACV measurements, generating a false signal decay and masking the true proteolytic activity. By removing DTT via centrifugal filtration and incorporating EDTA to preserve the enzyme activity, accurate CB kinetics were obtained. These findings clarified the roles of DTT and EDTA in enzyme activation and revealed a fundamental limitation of Au-thiol based electrochemical biosensors when exposed to DTT-like protease activating agents at the operating potentials used for Fc redox reaction.

To improve detection under physiologically relevant neutral pH conditions, rational peptide engineering was employed. Guided by systematic fluorogenic assays, incorporation of norleucine (Nle) at the P3 position of the substrate significantly enhanced CB activity at pH 7.3. The optimized hexapeptide substrate, H2N-(CH2)4-CO-Nle-Leu-Gly-Phe-Val-Ala-NH-CH2-CH2-Fc, demonstrated markedly improved electrochemical response on the Au MEA platform, achieving a limit of detection of 0.43 ± 0.08 nM for active CB at pH 7.3. This minimal site-directed modification strategy validated sequence-dependent cleavage behavior and demonstrated that targeted amino acid substitution can substantially enhance sensitivity and specificity, establishing a generalizable framework for substrate optimization in multiplex protease sensing.

The platform was further evaluated under complex biological conditions using 3% human serum to assess matrix effects and cross-reactivity from other clinically relevant proteases, including MMP-9 and ADAM-17. Introduction of human serum resulted in a pronounced initial signal decay, attributed to surface heterogeneity and nonspecific proteolytic activity. Kinetic analysis revealed that a double exponential model more accurately described the decay behavior, distinguishing a rapid matrix-induced component from a slower proteolytic process. The slower decay component exhibited a linear dependence on serum concentration, while the rapid component saturated at higher concentrations. Investigation of metalloprotease cross reactivity revealed that the activation agent APMA, used for MMP-9 activation, also disrupted the thiol-Au binding in the SAM and introduced electrochemical interference. Similar to the earlier CB studies, removing APMA after activation by centrifugal filtration preserved the enzymatic activity and eliminated surface disruption. In contrast to CB studies, EDTA was found to suppress metalloprotease activity. For multiplex assays, a better unified buffer system still needs to be developed. In general, the electrochemical proteolysis experiments demonstrated the highest CB activity, measurable MMP-9 activity (even in an EDTA-containing buffer), and the lowest ADAM-17 activity. Importantly, the Nle modified peptide showed no detectable cleavage by MMP-9 or ADAM-17, confirming enhanced specificity toward CB and validating rational substrate engineering as an effective strategy for minimizing the cross reactivity.

Overall, this dissertation establishes critical design principles for Au-S based electrochemical protease biosensors, including careful control of the interference by the activation agents, optimization of operating potential windows, rational peptide sequence engineering for detection in neutral pH buffers, and initial evaluation of matrix effects in complex biological samples. The Au MEA platform demonstrated here provides a scalable, multiplexing framework for profiling extracellular protease activity, representing an advancement in developing an interference-resistant electrochemical technique for early cancer diagnosis and therapeutic monitoring.