Preparation of an epitope-imprinted polymer with antibody-like selectivity for beta2-microglobulin and application in serum sample analysis with a facile method of on-line solid-phase extraction coupling with high performance liquid chromatography.

ABSTRACT

Molecularly imprinted polymers (MIPs) for protein recognition have great application potential in the biological analysis. However, preparation of protein imprinted polymer is still facing challenge. Beta2-microglobulin (ß2m) is a protein biomarker that can be used in diagnosis of different diseases. In this research, a novel MIP with ability of ß2m recognition has been developed by epitope and surface-confined imprinting approaches. A peptide with sequence of MIQRTPKIQ was selected as template. A strategy of combination of hierarchical imprinting and template immobilization was employed in the ß2m-MIP synthesis. Imprinted binding sites with open-entrance have been created that have good accessibility for ß2m and facilitated fast reversible binding kinetics. The experimental results demonstrated that the MIP has good selectivity. It can differentiate the template from peptide with different sequence and distinguish the ß2m from other proteins with similar size and pI values. After binding property study of the ß2m-MIP, a method of ß2m determination in serum was established in which ß2m was on-line extracted by MIP and analyzed by HPLC process. The recoveries for spiked serum was ≥83% with RSD <1.1%, indicating that the method has good accuracy and precisions. The LOD and LOQ were 0.058 and 0.195mgL-1 respectively, which meet the requirements of the ß2m analysis. The successful application of the ß2m-MIP demonstrated that ß2m has reversible binding on the MIP with a kinetics that can meet the requirements of the HPLC analysis. It also indicated that the ß2m-MIP has good mechanical strength and reusability that can be applied reliably in the practical analysis. As a synthetic antibody, ß2m-MIP is advantageous compared to the biological molecules.