Quantitative Detection of Cathepsin B Activity in Neutral pH Buffers Using Gold Microelectrode Arrays: Toward Direct Multiplex Analyses of Extracellular Proteases in Human Serum

Abstract

Proteases are critical signaling molecules and prognostic biomarkers for many diseases including cancer. There is a strong demand for multiplex bioanalytical techniques that can rapidly detect the activity of extracellular proteases with high sensitivity and specificity. This study demonstrates an activitybased electrochemical biosensor of a 3 × 3 gold microelectrode array for the detection of cathepsin B activity in human serum diluted in a neutral buffer. Proteolysis of ferrocene-labeled peptide substrates functionalized on 200 × 200 μm microelectrodes is measured simultaneously over the nine channels by AC voltammetry. The protease activity is represented by the inverse of the exponential decay time constant (1/τ), which equals to (k cat /K M )[CB] based on the Michaelis−Menten model. An enhanced activity of the recombinant human cathepsin B (rhCB) is observed in a low-ionic-strength phosphate buffer at pH = 7.4, giving a very low limit of detection of 8.49 × 10 −4 s −1 for activity and 57.1 pM for the active rhCB concentration that is comparable to affinity-based enzyme-linked immunosorbent assay (ELISA). The cathepsin B presented in the human serum sample is validated by ELISA, which mainly detects the inactive proenzyme, while the electrochemical biosensor specifically measures the active cathepsin B and shows significantly higher decay rates when rhCB and human serum are activated. Analyses of the kinetic electrochemical measurements with spiked active cathepsin B in human serum provide further assessment of the protease activity in the complex sample. This study lays the foundation to develop the gold microelectrode array into a multiplex biosensor for rapid detection of the activity of extracellular proteases toward cancer diagnosis and treatment assessment.

Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range

“…Cathepsin B is active at the neutral pH 7.2 , of the cytosol and other neutral pH compartments of biological systems, , as well as at the acidic pH 4.6 of lysosomes. , Importantly, cathepsin B is primarily a dipeptidyl carboxypeptidase (DPCP) type of exopeptidase and also functions as an endopeptidase shown by cleavage of peptide and protein substrates at sites distal from the C-terminus of peptide substrates. ,, …”

Electrochemical Biosensors for Whole Blood Analysis: Recent Progress, Challenges, and Future Perspectives

“…In comparison to the dedicated fabrication process of antibodies-based sensors, peptides feature advantageous properties including chemical versatility, better stability against denaturation and high specificity, and thus they have been widely employed as recognition elements for electrochemical biosensors. − Peptide, as biorecognition elements, can be selected from random libraries as for DNA aptamers. Alternatively, the epitope of specific antibody can be also engineered into electrochemical biosensor platform.…”

FRET‐Based Cathepsin Probes for Simultaneous Detection of Cathepsin B and D Activities

“…For detecting the activity of CTSB, various types of fluorescence‐based probes have been designed, such as fluorogenic [11,12] or Förster resonance energy transfer (FRET) probes, [13,14] and others [15] . However, there is a need for monitoring the physiological information associated with the action of CTSB, such as the local pH, concentrations of metabolites, and activities of other enzymes related to CTSB, simultaneously by other probes to further understand the interplay of CTSB in proteolytic processes revised [16–19] . For example, cathepsin D (CTSD), an aspartic protease universally found inside lysosomes, is an important protease and shows a complex relationship with CTSB [20–25] .…”

“…[15] However, there is a need for monitoring the physiological information associated with the action of CTSB, such as the local pH, concentrations of metabolites, and activities of other enzymes related to CTSB, simultaneously by other probes to further understand the interplay of CTSB in proteolytic processes revised. [16][17][18][19] For example, cathepsin D (CTSD), an aspartic protease universally found inside lysosomes, is an important protease and shows a complex relationship with CTSB. [20][21][22][23][24][25] CTSD is processed to be matured in cells by subsequent proteolytic cleavage involving CTSB and other cysteine proteases.…”