Amplification by Polysaccharides for the Electrochemical Detection of Trypsin Activity

Although polysaccharides are ubiquitous in nature, biodegradable, and cheap, there is no report illustrating their use as functional materials for signal amplification in the biosensing of enzyme activities. Herein, we report the development of a polysaccharide-amplified electrochemical biosensor for the highly sensitive detection of trypsin activity. The substrate peptides without carboxyl sites are attached to the electrode surface via the N-terminus. Such that, the tryptic cleavage of the peptide layer gives rise to a free carboxyl site at the C-terminus of each truncated fragment, and the carboxyl-carrying polysaccharide chains are then recruited via the carboxylate-Zr(IV)-carboxylate linkage, followed by the decoration of redox labels via the cross-linking between the cis-diol sites of polysaccharide chains and the phenylboronic acid group. Through boronate cross-linking, each polysaccharide chain can be decorated with hundreds of redox labels, leading to the significantly amplified electrochemical detection of trypsin activity. The polysaccharide-based signal amplification is advantageous due to its simple operation, low cost, and mild reaction conditions. With alginic acid as the proof-of-concept polysaccharide, a detection limit of 103.5 μU/mL (∼0.41 ng/mL) has been obtained for trypsin detection. The cleavage-based recognition enabled the selective discrimination of trypsin against other proteases and proteins. In addition, the potential use of electrochemical biosensors in inhibitor screening, together with the interrogation of trypsin activity in urine samples, has been validated. Collectively, the results clearly show that the polysaccharide-amplified electrochemical biosensor is highly applicable to the highly sensitive detection of trypsin activity.