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Abstract: Glycoproteins are a large family of proteins which have great biological and clinical importance including molecular recognition, signal transduction, cell adhesion and immune response. Many glycoproteins are important biomarkers for disease diagnosis and as therapeutic targets. However, glycoproteins are present in very low abundance in the body and coexist alongside interfering species present at much higher abundance. So, specific recognition of glycoproteins is of great significance. Although antibodies are commonly used to solve this issue, they are associated with some apparent drawbacks, such as, difficult to prepare, poor storage stability, high cost and requiring harsh conditions for target release. Therefore, novel alternatives are highly desirable. Molecularly imprinted polymers (MIPs), which are synthesized through polymerization in the presence of a template, exhibit specific binding towards the template molecules. A series of molecular imprinting approaches have been developed to produce glycoprotein-specific MIPs. Boronic acids can covalently bind cis-diol groups of cis-diol-containing compounds like glycoproteins and glycopeptides, to form stable five- or six-membered cyclic esters at relatively high pH values. The esters dissociate reversibly when the pH of the environment is made acidic. In recent years, our group has developed a series of boronate affinity molecular imprinting techniques for specific recognition of glycoproteins, especially boronate affinity controllable-oriented surface imprinting method. Based on silane hydrolysis in alcohol phase, this method permits getting the desired thickness of the imprinting layer according to the size of the template molecules. The method allows for easy and efficient preparation of MIPs specific to glycoproteins, glycans, and monosaccharides, enabling promising applications. Recently, we can even achieve precision imprinting of glycopeptides for the facile preparation of glycan-specific MIPs. However, the molecular imprinting of intact proteins in organic phase often results in the denature of the template proteins while imprinting approaches using glycans as the templates require relatively tedious glycan preparation procedure. Herein, we developed a new approach for imprinting in organic phase using an intact target glycoprotein as a semi-template. This method allowed for the imprinting of only the glycans on the target glycoprotein, without worrying about possible denature of the target protein in organic phase. Alkaline phosphatase (ALP), which is a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was used as the target. ALP-specific molecularly imprinted magnetic nanoparticles (MI-MNPs) were prepared using this method. By using boronic acid-functionalized MNPs as the nanocores, the target molecules were immobilized onto the nanocores via boronate affinity. Then, a thin-layer of silica was controllably formed to cover the templates to an appropriate thickness through polycondensation of tetraethyl orthosilicate (TEOS) in ethanol. After that, the templates are removed facilely by washing with an acidic solution to disrupt the boronate affinity interaction. The prepared MIPs exhibited good specificity towards ALP, and this characteristic was preserved even in a complicated real sample. The developed approach exhibited high imprinting efficiency. It overcame the limitations of previous imprinting approaches. Also, the MI-MIPs synthesized in this work were simple to prepare, stable, cost-efficient and easy to collect. Besides, the target release just needed gentle conditions. It can be a promising means to produce glycoprotein-specific MIPs for important applications such as affinity separation and disease diagnosis.
Template and target information: glycoprotein, alkaline phosphatase, ALP
Author keywords: glycoprotein, magnetic nanoparticle, molecular imprinting, boronate affinity, controllable imprinting