The library of 24 Lewis x trisaccharide analogues (LeX1 to LeX24) (Figure ) were converted into NGL probes to allow their non-covalent immobilization on nitrocellulose-coated microarray slides. The clustered and flexible presentation of glycans in the NGL-based microarray system is advantageous in revealing binding by a diverse range of glycan binding systems, including endogenous carbohydrate binding receptors, adhesins of microbes, virus particles or their adhesins. We used a new procedure for NGL preparation that takes advantage of the efficiency of the strain-promoted azide-alkyne cycloaddition (SPAAC, Figure ). The Lewis x analogues were conjugated to a new lipid reagent dibenzocycloctyne-functionalized DHPE (DBCO-DH) to generate the desired DBCO-DH-NGL probes in quantitative yields. A microarray was then constructed with the 24 Lewis x -based DBCO-DH-NGL probes (Figure ): unmodified Lewis X trisaccharide (LeX1); probes with modification of the GlcNAc residue only (LeX2-LeX6); modification of Gal only (LeX7-LeX12); mono-fluorination of Fuc (LeX13 and LeX14); difluorination on two monosaccharide residues (LeX15-LeX17); multi-fluorinated structures with modification on two or more sugar residues (LeX18-LeX24). Four conventional NGLs derived from natural oligosaccharides were included as references: Lewis x pentasaccharide lacto-N-fucopentaose III (LNFP III), its non-fucosylated analogue lacto-N-neotetraose (LNnT), pentasaccharide of GM1 ganglioside, and highmannose N-glycan Man9GN2 (Table ). The microarray was probed with nine glycan binding proteins (Table ): three recombinant Fc-tagged C-type immune lectin receptors, three anti-Lewis x monoclonal antibodies, and three closely related bacterial toxins, with the results of microarray analyses shown in Figure and SI (Figures and, Supplementary Dataset 1). Monoclonal anti-Lewis X antibodies. With the high specificity for Lewis X among the histo-blood group carbohydrate determinants, anti-Lewis X antibodies are widely used as research tools for identifying Lewis X expressed on different organisms, tissues, and cells. The monoclonal antibodies anti-SSEA-1 and anti-L5 together with a commercial antibody BG-7 were analysed in the microarray, and all gave binding to native Lewis X probes LeX1 and the LNFPIII standard. Distinct binding patterns were observed with the three antibodies: anti-SSEA-1 showed the most restricted and anti-L5 the broadest binding profiles (Figures and). Earlier study showed that anti-SSEA-1 recognizes Lewis X epitope on a longer carbohydrate backbone compared to anti-L5, and this is corroborated by the weaker binding detected to the LeX1 than LNFPIII. Nevertheless, a clear enhancement (almost 3-fold) of the binding of anti-SSEA-1 was observed with the 6F-Gal LeX9 compared to the native LeX1. The LeX9 was also the best ligand for BG-7 among all the Le X analogues. Interestingly, anti-L5 preferred for 6H-Gal (LeX10) compared to 6F-Gal (LeX9). The 3F-Gal, as in LeX7, abolished binding by all antibodies, indicating that the 3-OH group is likely to be a key hydrogen bond donor in the interactions of these antibodies. Most of the fluorine modifications on GlcNAc resulted in a reduction or abolition of antibody binding, except for C-6 di-fluorination as with LeX3, which was well tolerated by anti-L5 but not at all by anti-SSEA-1 and BG-7 (Figure and). The GlcNAc N-TFA modification alone was tolerated by all antibodies, whereas little binding was detected when this modification occurred in combination with other fluorination in the Lewis X trisaccharide. Modifications on Fuc had differential effects on antibody binding: LeX11 with 3F-Fuc was strongly bound by anti-L5 but only moderately bound by BG-7 and not at all by anti-SSEA-1. The LeX14 which has arabinose instead of Fuc in the trisaccharide structure gave no binding with anti-SSEA-1 and BG-7 and very weak binding with anti-L5, highlighting the importance of methyl group at the Fuc C-5 position for binding by the two antibodies.