Development of a label-free SPR sensor for detection of matrixmetalloproteinase-9 by antibody immobilization on carboxymethyldextran chip

摘要

Surface plasmon resonance (SPR) immunosensor has been widely utilized for monitoring antigen-antibody interactions. The sensor measures changes of refractive index upon binding of analyte molecules to specific ligand immobilized on the sensor chip. This effort reports development of SPR immunosensor for real-time and label-free detection of recombinant human matrix metalloproteinases-9 (MMP-9), which has been associated with malignant tumor progression and metastasis by matrix degradation. MMP-9 was expressed in Escherichia coli BL21 and purified by Ni-NTA agarose column. CMD 50 D was activated by EDC/NHS for immobilization of monoclonal anti-MMP-9. Atomic force microscopy images showed uniform distribution of anti-MMP-9 over the sensor chip. Equilibrium constant (K-D), maximum binding capacity (R-max) and Delta G(b) values for interaction of MMP-9 and anti-MMP-9 were 0.4 nM, 680 mu RIU and -53.51 kJ/mol, respectively. Concentration of MMP-9 in saliva samples was determined, with linearity in the range of 10-200 ng/mol. The limit of detection was found to be 8 pg/mL, being lower than most of the previously reported techniques. (C) 2016 Elsevier B.V. All rights reserved.