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Bridging the Bio-Electronic Interface with Biofabrication
Published on: June 6, 2012
Lectin-directed enzymatic silver deposition for electrochemical assay of endoprotease activities
Xiyao Zhang1, Jingjing Xu1, Di Wu1
1Center for Advanced Analytical Science, Guangzhou Key Laboratory of Sensing Materials and Devices, Guangdong Engineering Technology Research Center for Sensing Materials and Devices, School of Chemistry and Chemical Engineering, Guangzhou University, Guangzhou, 510006, PR China.
Background:
The assay of endoprotease levels is of great value in health assessment and medical diagnosis. Due to its good biocompatibility and high efficiency, alkaline phosphatase (ALP)-catalyzed silver deposition is well-suited for the highly sensitive electrochemical assay of biotargets. However, there is no report yet illustrating the use of enzymatic silver deposition in the electrochemical assay of protease activities, while ALP enzymes in previous studies are often recruited via the biotin-avidin or antigen-antibody interactions.
Results:
Herein, we illustrate a lectin-directed enzymatic silver deposition (LecESD)-based method for the signal-on electrochemical assay of endoprotease activities at very low concentration levels. Specifically, the LecESD-based method involves the end-tethering of a carboxyl-free peptide substrate via the N-terminus for the cleavage-based recognition of an endoprotease, the tethering of a carbohydrate module to the cleavage-generated carboxyl site via the carboxylate-Zr(IV)-carboxylate (CZrC) chemistry, the recruitment of ALP enzymes via the carbohydrate-lectin interactions, and the enzymatic hydrolysis of ascorbic acid 2-phosphate into ascorbic acid to bring about the reduction of Tollens' reagents into silver deposits. The CZrC chemistry can enable an equimolar labeling of the resulting carboxyl sites with carbohydrate modules for the site-directed recruitment of ALP enzymes. The synergy of the endoproteolytic cleavage and enzymatic silver deposition can greatly improve the assay sensitivity, and a limit of detection (LoD) of 54.8 μU/mL has been achieved for the signal-on assay of trypsin activity. In addition, the LecESD-based method has high selectivity and its potential use in endoprotease inhibitor screening has been demonstrated, along with the potential use to endoprotease activity assay in biological samples.
Significance:
Given that the site-directed recruitment of enzymes via the carbohydrate-lectin interactions is robust and convenient to use, the LecESD-based method is suited for the signal-on electrochemical assay of endoprotease activities at very low concentration levels.
