Boronate affinity monolith with a gold nanoparticle-modified hydrophilic polymer as a matrix for the highly specific capture of glycoproteins.

As low abundance is the great obstacle for glycoprotein analysis, the development of materials with high efficiency and selectivity for glycoprotein enrichment is a prerequisite in glycoproteome research. Herein, we report a new kind of hydrophilic boronate affinity monolith by attaching 4-mercaptophenylboronic acid (MPBA) with 2-mercaptoethylamine (MPA) on the gold nanoparticle-modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate)) monolith for glycoprotein enrichment. With poly(ethylene glycol) diacrylate as the cross-linker and the further modification of gold nanoparticles, the matrix has advantages of good hydrophilicity and enhanced surface area, which are beneficial to improve the enrichment selectivity and efficiency for glycoproteins. The attachment of MPBA and MPA provide intramolecular BN coordination, which could further enhance the specificity of glycoprotein capture. Such a boronate affinity monolith was applied to enrich horseradish peroxidase (HRP) from the mixture of HRP and bovine serum albumin (BSA), and high selectivity was obtained even at a mass ratio of 1:1000. In addition, the binding capacity of ovalbumin on such monolith reached 390 µg g(-1) . Furthermore, the average recovery of HRP on the prepared affinity monoliths was (84.8±1.9) %, obtained in three times enrichment with the same column. Finally, the boronate affinity monolith was successfully applied for the human-plasma glycoproteome analysis. As a result, 160 glycoproteins were credibly identified from 9 µg of human plasma, demonstrating the great potential of such a monolith for large-scale glycoproteome research.

Aptamer functionalized hydrophilic polymer monolith with gold nanoparticles modification for the sensitive detection of human α-thrombin.

Low abundant proteins of body fluids participate nearly all physiological processes and indicate various kinds of diseases. The development of specific enrichment techniques is the key to identify and quantify the low abundant proteins. Herein, a novel kind of aptamer functionalized hydrophilic polymer monolith was developed for the specific enrichment and detection of human α-thrombin from the human plasma. Human α-thrombin aptamer, with thiol group modified at the 5' terminal, was immobilized on the gold nanoparticles (AuNPs) modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate) monolithic column, with the binding capacity of 277.1µmol/L. Due to the hydrophilic poly(ethylene glycol) diacrylate) as the cross-linking monomer, the detection recovery of the aptamer-functionalized hydrophilic polymer monolithic column could reach to 92.6±5.2% (n=3) and the dynamic range could reach 0.5-300ng/µL (S/N>10) with on-line UV detection. Meanwhile, the column could run over 100 times, because the poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate) stability structure and the AuNPs improved the stability of the matrix material. Furthermore, this column could even capture the target α-thrombin, which was spiked in 1000 folds of original human plasma. All these results demonstrated the great potential of the prepared aptamer functionalized hydrophilic polymer monolith for the recognition of the trace proteins in the biological samples.