title: Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins.
abstract: Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.
authors.affiliation: Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Uchiyama
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kuno
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Kubo
authors.forename: Mamoru
authors.initials: M
authors.lastname: Mizuno
authors.forename: Midori
authors.initials: M
authors.lastname: Noguchi
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
mesh_terms: D000818:Animals; D002645:Chickens; D005453:Fluorescence; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D009844:Oligosaccharides / Q000378:metabolism*; D037121:Plant Lectins / Q000737:chemistry / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D037121:Plant Lectins; C017707:Ricinus communis agglutinin-1