Abstract
In recent decades, microarray has now been emerged as a high throughput screening tool for expanding biomedical application, such as biomolecular interactions of genomics, proteomics, and more recently in glycomics. The well-known specific binding event can be further developed for clinical diagnostic, environmental monitor, and so on in microarray format. To fabricate various kinds of microarray, there are varieties of surface functional groups available for immobilization of bio-molecules. Even so, none of them could be suitable for immobilization of all kinds of bio-molecules so far. Therefore, a new surface tool, which can be easily constructed and provides a new strategy for probe immobilization that minimizes probe destruction and allows covalent bond formation, are urgently demanded. It is well known that phenyl boronic acid (BA) and its derivatives can form cyclic boronate esters with vicinal cis-diols and polyols, such as glycerol and carbohydrates, even under aqueous conditions. Accordingly, BA has been employed for achieving glyco-targeting such as carbohydrate biosensors, polysaccharide detection, separation or enrichment of glycoprotein and glycopeptide, and so on. However, to our knowledge, it has not been attempted to use as chips. In this thesis, we take advantage of BA to fabricate oriented protein microarray and covalent polysaccharide microarray to improve accessibility of proteins and retention of polysaccharide on slide surface. To improve the protein conjugation on solid support, BA was used to site-specifically assemble Fc-fused lectin and antibody from its Fc domain for maintaining the best activity on the solid surface. We demonstrated that the protein activity can be conserved when protein molecule is oriented in an optimal manner on the BA surface. In addition, m-aminophenylboronic acid was used to easily construct covalent microarray of oligosaccharide and polysaccharide of molecular weight at least 0.5 KDa. The triple BSA layers surface was successfully applied to generate hydrophilic BA surfaces to significantly alleviate nonspecific adsorption for elucidation of covalent effect upon the carbohydrate. We demonstrated that BA based oligosaccharide microarrays offer better retention of carbohydrates on supports over non-covalent attachment during more stringent washing conditions. Furthermore, owing to its potential application in glycoprotein microarrays, we employed antibody microarray to detect RCA120, a low-toxic “ricin” surrogate, by using galactose-gold nanoparticle (g-AuNP) and in combination with silver enhancement by the naked eye. To enhance the detection sensitivity, we developed a method to generate and amplify the signal of a antibody microarray using AuNP-based agglutination to give a new ultrasensitive assay with detection sensitivity at the attomolar (aM) level that doesn’t required any optical instrument for readout.