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  • Capillary monolithic columns obtained by in situ polymerizat


    Capillary monolithic columns obtained by in situ polymerization have been developed rapidly as an efficient chromatographic stationary phase, which exhibits excellent reproducibility, reliable stability and fast transfer kinetics [18]. Recently, it has been extensively applied in capillary liquid chromatography (CLC) [19], gas chromatography (GC) [20], capillary electrochromatography (CEC) [21], solid-phase microextraction (SPME) [22] and microfluidic analysis [23]. Polymer monolith microextraction (PMME) is a type of SPME technique using polymer monolithic column as extraction medium [27]. In particular, the combination of SPME and BAC has become a powerful enrichment tool for cis-diols compounds from complex samples [[24], [25], [26]] and been widely adopted in separation and extraction of proteins [28], medicine [29] and environmental pollutant [30]. In view of facts above, we first reported a strategy of preparing a hydrophilic BAC monolithic column for extraction of glycoproteins. The monolith was made using AAPBA as functional monomer, OEG as auxiliary hydrophilic functional monomer, ethylene glycol dimethacrylate (EDMA) as crosslinking monomer and n-propanol and 1,4-butanediol as binary porogens. With systematic optimization of preparation and extraction parameters, monolithic column with homogeneous structure was formed. The optimized poly(AAPBA-co-OEG-co-EDMA) monolithic column was characterized with infrared dihydrofolate reductase inhibitor spectroscopy, field emission scanning electron microscopy and N2 adsorption experiment. The extraction performance of the newly boronate affinity monolith was evaluated by PMME of glycoproteins horseradish peroxidase (HRP) and ovalbumin (OVA). Moreover, the specific selectivity of the monolithic column to glycoproteins was demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
    Results and discussion
    Conclusion In this wok, a novel OEG incorporated hydrophilic boronate affinity monolith was successfully prepared and demonstrated higher extraction performance for glycoproteins using PMME. Besides, the OEG incorporated affinity monolith also can be used for molecular recognition and selective separation of other substances containing cis-diols groups. As a conclusion, the poly(AAPBA-co-OEG-co-EDMA) capillary monolith is a promising boronate affinity material for the extraction and pretreatment of glycoproteins from complex samples. In the future, more efficient and sensitive online-SPME methods by combining other detection methods (HPLC, MS and etc.) using the OEG incorporated monolith is expected.
    Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 21775109)
    Introduction Glycosylation, one of the most common and complex posttranslational modifications, acts as a key regulator in all kinds of physiological functions and biological procedures [1], [2]. Aberrant glycosylation within the cell is dynamics associated with the onset and development of various diseases. In addition, many glycoproteins are regarded as the vital clinical biomarkers and therapeutic targets. Thus, the detection of protein glycosylation is great important in further understanding a variety of cancer mechanisms and cellular signaling pathways [2], [3], [4], [5]. Besides antibodies, lectin is another molecular tool to profile different types of protein glycoforms from complex biological samples. For example, Lens culinaris agglutinin (LCA), Aleuria aurantia lectin (AAL) and P. squar-rosalectin (PhoSL) were used to target aberrant fucosylation [6], [7], Sambucus nigra agglutinin (SNA) was reported to detect aberrant sialylation [8], while Concanavalin A (ConA) [9] was known to recognize the high-mannose core of N-glycans. However, lectin approach suffers from high cost, poor stability and weak affinities for glycoproteins [10], [11]. Unlike lectins, which recognize specific carbohydrates, boronic acid does not show selectivity towards different glycoforms. Instead, boronic acid can selectively bind 1,2- and 1,3-cis-vicinal diol-containing biomolecules without irreversible alterations of glycan structure [12], which is an ideal solution for effective formation of glycoprotein recognition sites [13], [14]. Recently, boronic acid functionalized materials, such as monoliths [15], [16], nanoparticles [17], [18], magnetic beads [19], [20], [21] and mesoporous materials [22], [23] have gained an increasing attention due to their capability in the facile and selective isolation of glycoproteins. However, almost all of these current glycoprotein detection protocols are based on solid phase extractions, which make them difficult to be introduced into the detection of glycoprotein on membrane. Soluble nanopolymers, such as dendrimers, are attractive nanomolecules with hyperbranched surface groups for derivatization, which could offer the synergistic effect of simultaneous multiple binding to mimic the natural antibodies. It is also very easy for straightforward surface functionalization with various functional groups via well-established surface chemistry [24], [25], [26], [27], [28]. More important, the highly soluble nature of the molecule allows for the chelation between the material and glycoprotein in the solution phase.