Fe³⁺-immobilized nanoparticle-modified capillary for capillary electrophoretic separation of phosphoproteins and non-phosphoproteins

Abstract

A fused-silica capillary modified with Fe³⁺-immobilized magnetic nanoparticles (Fe³⁺-IMAN) has been investigated for the capillary electrophoretic (CE) separation of phosphoproteins and non-phosphoproteins. The Fe³⁺-IMAN capillary was achieved by covalently immobilising epoxy-based magnetic silica nanoparticles (160 nm) on the prederivatized 3-aminopropyl-trimethoxysilane (APTMS) fused-silica capillary (75 μm id), followed by disodium iminodiacetate and Fe³⁺. The buildup process was examined by measuring the streaming potentials of the bare capillary, APTMS capillary, epoxy-based nanoparticle capillary and Fe³⁺-IMAN capillary by varying the buffer pH. An inverted fluorescence microscope was used to determine the surface features of the Fe³⁺-IMAN capillary derivatized with morin. Further experimental results confirmed that Fe³⁺-IMAN bonded on the inner wall of the APTMS capillary could provide sufficient solute-bonded phase interactions to allow for the CE separation of phosphoproteins and non-phosphoproteins at concentration levels down to 50 μg/mL. The highest number of theoretical plates obtained was about 233,000/m, and the relative standard deviation (RSD) for migration times was <2.57% for eight consecutive runs, respectively. Additionally, the Fe³⁺-IMAN modifing method was also applied to the analyses of bovine milk proteins. With simplicity, high resolving power, and high repeatability, the proposed method has shown great potential for phosphoproteomics applications.