Screening and analysis of cyclooxygenase-2 inhibitors from the complex matrix: A case study to illustrate the important effect of immobilized enzyme activity in magnetic ligand fishing

Abstract

Magnetic ligand fishing is one of the most widely used methods to screen and separate potentially bioactive compounds from complex mixtures. However, it is poorly understood the ambiguous relations between immobilized enzyme activity and fishing results (accuracy and sensitivity). Therefore, to investigate the underlying relationship of them, we fabricated the immobilized enzyme with different activities by introducing a novel support material, nickel ions (Ni2+) functionalized magnetic mesoporous silica microspheres (MMSM@PDA-Ni2+). It possesses a higher activity than the previous report because of site-directed immobilization. Then, the immobilized COX-2 with different activities were prepared using different immobilization methods, including Ni2+ affinity-oriented immobilization, polydopamine (PDA) covalent immobilization, and conventional glutaraldehyde (GA) covalent immobilization. A standard mixture (COX-2 inhibitors and noninhibitors) and green tea extract were used to compare and evaluate the fishing results systematically. It displayed the fished inhibitors were dramatically reduced with the decreased activity of immobilized COX-2, indicating that immobilized enzyme activity played a critical role in a reliable magnetic ligand fishing analysis. This development questions some of the previous studies aimed at rapid screening bioactive compounds in natural products and opens new possibilities for accurate and sensitive magnetic ligand fishing analysis. Also, the introduced novel materials with mild immobilization strategy preserve enzyme activity and spatial conformational, therefore, provides a valuable tool in future applications.