Fig. 3: Structure-switchable DNA-based glycan chip combined with on-chip glycan biosynthesis.

a The workflow for the construction of a structure-switchable DNA-based glycan chip via on-chip complex glycan biosynthesis under optimized conditions. On-chip enzymatic glycosylation conditions were optimized by analyzing biosynthesized glycans isolated from the chip in acidic pH (4.5) through Bio-LC. To fabricate a DNA-based glycan chip composed of Globo H and its related structures, a lactose-oligonucleotide conjugate-immobilized surface was divided into five blocks, followed by treatment with glycosyltransferases under optimized conditions. For quality control, the DNA-based glycan chips were incubated with antibodies against starting materials and products. b Quantitative analyses of relative conversion efficiencies for on-chip enzymatic syntheses of Globo H hexasaccharide and related structures in Supplementary Figs. 6–10. Each value presents the mean ± SEM from forty-nine independent spots excluding the highest and lowest signals. Statistical significance was assessed using Student’s unpaired t test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Schematic illustrations and scanned raw images for on-chip enzymatic glycosylation of (c) SSEA-4 hexasaccharide and (d) Globo H hexasaccharide under optimized conditions (Scale bar: 800 μm). Synthesized complex glycans were detected by using DyLight 650-conjugated monoclonal antibodies or monoclonal antibodies with Alexa Fluor^® 647-conjugated polyclonal secondary antibodies. Anti-Gb5 Ab DyLight 650-conjugated anti-Gb5 monoclonal antibody, Anti-SSEA-4 Ab DyLight 650-conjugated anti-SSEA-4 monoclonal antibody, Anti-Globo H Ab Anti-Globo H monoclonal antibody (VK9), α2,3-SialT α-2,3-sialyltransferase, α1,2-FucT α-1,2-fucosyltransferase. Symbols: blue circle, Glc; yellow circle, Gal; yellow square, GalNAc; red triangle, Fuc; purple square, Neu5Ac. Source data are provided as a Source Data file.