Novel assay format for proteins based on magnetic molecularly imprinted polymer nanoparticles—detection of pepsin

Abstract

A novel, abiotic assay format for the quantification of pepsin has been developed featuring replacement of conventional antibody binders and enzyme reporters with entirely synthetic alternatives. The assay was based on the application of magnetic molecularly imprinted polymer nanoparticles (MIP NPs) in an enzyme-linked immunosorbent assay format, where colored or fluorescent polystyrene beads serve as a label instead of enzyme. Modification of the standard microtiter plate with magnetic inserts permits the instantaneous immobilization of magnetic MIPs, with a hole in the middle of each insert allowing unobstructed measurement of the sample. The magnetic MIP NPs were synthetized using a solid-phase synthesis method in which pepsin, which was chosen as a model protein target present in “gastric juice”, was used as a template. The results suggested that blue polystyrene particles with 10 μm diameter combined with magnetic MIP NPs and magnetic inserts can be used for reliable detection of pepsin in the concentration range between 10 and 50 µg mL⁻¹ in model solutions of pepsin. In order to measure pepsin in the “gastric juice” which constitutes a 100 mM solution of hydrochloric acid, the blue colored polystyrene beads were replaced with red fluorescent polystyrene beads that allowed precise measurement of clinical samples in nanomolar range following 10-fold dilution with buffer. This sensitivity, which was achieved using fluorescent polystyrene beads, permits quantifiable measurement of pepsin in the physiological range of concentrations between 5 and 50 μg mL⁻¹. Furthermore, the technology and protocols presented here are transferable and can be applied to identify proteins in a wide range of clinical, forensic and environmental samples, using stable synthetic binders and reporters.

Keywords:

• Pepsin
• molecularly imprinted polymer nanoparticles
• pseudo-ELISA
• gastric juice

Acknowledgments

The authors thank the BSc students Siu Kee Tam, Pratheepan Ramanathan, Tiffany Wu, Guoda Liepuoniute and Jacopo Piovesan from the Department of Chemistry at the University of Leicester for their participation in the project and Alexandra Piletska for her help with editing of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.