Synthesis of magnetic nanoparticles with immobilized aminophenylboronic acid for selective capture of glycoproteins Zi-An Lin, * a Jiang-Nan Zheng, a Fen Lin, b Lan Zhang, a Zongwei Cai c and Guo-Nan Chen * a Received 16th July 2010, Accepted 8th September 2010 DOI: 10.1039/c0jm02300k Aminophenylboronic acid (APBA)-functionalized magnetic iron oxide nanoparticles (Fe 3 O 4 MNPs) were synthesized for the selective capture of glycoproteins from unfractionated protein mixtures. The morphology, adsorption, and recognition properties of the resultant particles were investigated and uniform size APBA-coated MNPs with a mean diameter of 15 nm and high magnetic saturation value of 30.6 emu g 1 were obtained, which endued the adsorbent with a large surface area and convenience of isolation. The selectivity and binding capacity of APBA-coated MNPs were evaluated by using standard glycoproteins (cellulose and ovalbumin) and nonglycoproteins (bovine hemoglobin, bovine serum albumin and lysozyme) as model samples. Adsorption experiments and SDS-PAGE demonstrated that the APBA-coated MNPs had higher binding capacity and selectivity for glycoproteins compared to nonglycoproteins. In addition, the practicability of the as-prepared MNPs was further assessed by specific capture of ovalbumin from an egg white sample. Introduction Glycosylation of proteins is the most ubiquitous post-trans- lational modification, which plays crucial roles in various bio- logical processes, such as protein folding, cell division, signal transduction, inflammation, tumorigenesis, differentiation, as well as cell–cell recognition. 1–3 The isolation, discovery and subsequent identification of glycopeptides and glycoproteins are becoming increasingly important in diagnosis and glyco- proteomics. Although glycoprotein abundance in organisms is relatively high, it is difficult to analyze because of the presence of other highly abundant nonglycoproteins in complex biological samples. Consequently, isolation and enrichment of glycosylated proteins is generally required prior to analysis by liquid chro- matography coupled to mass spectrography (LC-MS). Several strategies have been developed for isolation and enrichment of glycoproteins based on different mechanisms, including a hydrazide chemical reaction, 4,5 lectin-based affinity chromatography, 6–9 hydrophilic interaction liquid chromatog- raphy 10,11 and so on. Among them, lectin-based affinity chro- matography is the most widely used due to its highly specific binding affinity for N-linked glycoproteins. Besides, phenyl- boronic acid-based affinity chromatography, as an effective means for the specific capture of cis-diol-containing compounds, 12–14 has also been used to attempt the isolation of glycol-structure biomolecules in recent researches, 15–17 where the recognition principle relies on reversible covalent complex formation/dissociation between boronic acids and cis-diols in a basic/acidic aqueous media. 18,19 The unique property of boronic acids makes it an ideal biomaterial for the selective capture of glycoproteins. Combining nanomaterials and biomaterials into an integrated system for applications in drug delivery, cell separation, protein recognition and bioanalysis has been a key research focus in nanobiotechnology. 20–22 Particularly, magnetic Fe 3 O 4 nano- particles are the most commonly used because of their good biocompatibility, superparamagnetic property, low toxicity and easy preparation. 23,24 Recently, functionalized magnetic beads applied for protein separation have received great interest due to their simple and fast procedure by applying an external magnet. 25–28 Sparbier et al. 29 employed commercial concanavalin A- (ConA) and boronic acid-functionalized magnetic beads to isolate and enrich glycosylated peptides and proteins prior to MALDI-TOF-MS analysis, where carboxyl-functionalized magnetic particles were used to couple ConA and 3-amino- phenyl-di-boronic acid onto the beads by using the carbodiimide method and polyglutaraldehyde method, respectively. The cor- responding magnetic ConA beads and boronic acid beads showed specific binding toward the model proteins containing N-glycans of different oligosaccharide types and containing cis- diol groups, respectively. Lately, Zhou et al. 26 developed a facile synthetic route for the preparation of boronic acid-coated magnetic microspheres by using a solvothermal reaction and a two-step amidation reaction, with which the glycopeptides and glycoproteins could be well isolated from the spiked protein mixtures and analyzed by LC-MS. Despite this attractive feature of magnetic nanoparticles, there are few studies to date on the isolation and enrichment of glycoproteins by using functional magnetic nanoparticles. 26,27,29–31 In this work, we report a synthesis of aminophenylboronic acid (APBA)-functionalized magnetic nanoparticles using chemical coprecipitation and a multi-step covalent modification. The properties of APBA-coated Fe 3 O 4 MNPs were characterized a Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, Fujian, 350002, China. E-mail: gnchen@fzu.edu.cn; zalin@fzu.edu.cn; Fax: +86-591-83713866 b Instrumentation Analysis and Measurement Center of Fuzhou University, Fuzhou, Fujian, 350002, China c Department of Chemistry, Hong Kong Baptist University, Hong Kong, SAR, P. R. China 518 | J. Mater. Chem., 2011, 21, 518–524 This journal is ª The Royal Society of Chemistry 2011 PAPER www.rsc.org/materials | Journal of Materials Chemistry Published on 27 October 2010. Downloaded by NORTHWEST UNIVERSITY LIBRARY on 11/13/2019 7:48:42 AM. View Article Online / Journal Homepage / Table of Contents for this issue