Surface plasma resonance biosensing of phosphorylated proteins via pH-adjusted specific binding of phosphate residues with UiO-66

Abstract

The abnormal protein phosphorylation is closely related to a series of diseases including cancers, neurodegeneration, and other immune/inflammatory disorders. However, a simple, accurate, and pragmatic methodology for the direct detection of phosphorylation levels of target proteins is largely unavailable. Herein, a surface plasma resonance (SPR) biosensor was developed to detect phosphorylated protein based on significant SPR enhancement using UiO-66 and its high affinity for phosphate residues. Furthermore, the pH of the incubation buffer and the NaOH concentration in the washing solution to eliminate nonspecific adsorption of nanoparticles on the chip surface were optimized to adjust the surface charges of UiO-66. This improved the specificity and sensitivity of the proposed method. The designed biosensor could distinguish 1% phosphorylated peptides from non-phosphorylated peptides background. Moreover, the method could directly analyze the phosphorylation level of target protein in complex samples such as cell or tissue extracts without requiring a purification step in a single workflow. This simple and robust biosensor is a sensitive, accurate, and pragmatic toolbox to detect phosphorylated proteins without destroying the protein structure and can significantly facilitate phosphoproteomics and clinical diagnosis research.