Peptide-based biosensor for real-time monitoring of protease biomarker activity using multi-parametric surface plasmon resonance spectroscopy.

Matrix metalloproteinase-9 (MMP-9) is a key biomarker targeted in biosensing applications due to its involvement not only in maintaining good health but also in triggering various diseases such as cancer. While quantitative detection of MMP-9 is widely performed using bioanalytical detection kits such as enzyme-linked immunosorbent assay (ELISA), faster, label-free and real-time monitoring of MMP-9 activity would lead to improved disease diagnosis with better understanding of its role in underlying disease progression and development of therapeutic strategies. In this work, multi-parametric surface plasmon resonance spectroscopy (MP-SPR) is used to develop a highly sensitive MMP-9 sensor using immobilized synthetic peptides as MMP-9 substrates. Upon binding to MMP-9, the MMP-9 specific peptide is hydrolyzed between two sites of the amino acid sequence (P1 Gly and P1' Met), resulting in a decrease in the SPR signal response. The sensor detects different concentrations of MMP-9 in buffer and cell culture medium (RPMI-1640), indicating that it can be used under physiological conditions. The limit of detection (LOD) for MMP-9 in buffer is 0.34 pM and the linear detection range is between 5 pM and 9 nM, covering the clinically relevant detection range of MMP-9. To our knowledge, this is the first short synthetic peptide-based MP-SPR biosensor for monitoring MMP-9 activity. The sensor is faster than ELISA (minutes vs. hours) and provides real-time detection with access to binding kinetics information. The use of MP-SPR provides information on surface coverage and peptide thickness before and after cleavage, which is unique compared to other detection methods.