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

Scheme1.Schematic illustration of (A) UiO-66 synthesis; (B) UiO-66-based SPR biosensor for the detection of phosphorylated protein. (a) The protein extract was injected into the flow chamber and then (b) UiO-66 was added. (c) NaOH solution was used to remove the non-specifically adsorbed UiO-66. • A SPR biosensor was proposed for the detection of phosphorylated protein. • UiO-66 can specifically bind of phosphate residues and enhance SPR effect. • pH was optimized to adjust the surface charges of UiO-66 for eliminating nonspecific adsorption. • This biosensor shows high sensitivity and directly analyzes phosphoprotein in complex samples. 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.