A CdSe/CdS/ZnS core/shell/shell QD (EM ~ 550 nm) was employed to construct glycan-QDs. It also acted as the donor for developing the QD-FRET based binding assays. Cap-exchange using deprotonated DHLA-EG11-DiMan in a homogeneous solution was employed to make DHLA-EG11-DiMan capped QD (denoted as QD-DiMan, hereafter) as reported previously. Moreover, a QD capped with the DHLA-EG3-OH control ligand (denoted as QD-OH hereafter) was also prepared and used as a negative control of lectin binding. Both constructs were found to be monodisperse, relatively compact (with hydrodynamic diameter, Dh, of ~12 and ~9 nm for QD-DiMan and QD-OH, respectively, SI, Fig. ) and highly stable: no change of physical experience or precipitation was observed after storage for 1 month in a fridge. The average glycan valency per QD was estimated as 212 ± 69 by measuring the difference between the amount of ligand added and that remained unbound post capexchange (SI, Fig. ). By calculating the average deflection angle of the glycan ligands, and using the Dh value above, the average inter-glycan distance was estimated to be 1.7 ± 0.3 nm (SI, Table ). The soluble extracellular segment of DC-SIGN/R, which have shown to faithfully retain the tetrameric structure and glycan binding properties of full length proteins, were used in all glycan-QD binding studies. DC-SIGN with its Cterminal 16 amino acids truncated (denoted as DC-SIGN-C hereafter) was constructed using standard molecular biology technique and the construct was confirmed by DNA sequencing. All proteins were expressed, purified and labeled with maleimide-modified Atto-594 dye (EM ~628 nm), which acted as the FRET acceptor, via a site-specific cysteine mutant at Q274 in DC-SIGN and DC-SIGN-C or R287 in DC-SIGNR, as before. The QD-Atto-594 FRET pair have good spectral overlap with a respectable Förster radius (R0 ~ 5.7 nm, SI, Fig. ), ensuring that efficient FRET can occur. Moreover, there is little overlap between the QD and dye emission spectra, making it straightforward to differentiate donor and acceptor fluorescence without the need of spectral deconvolution. The purity of proteins and successful dye labeling were confirmed by high resolution mass spectrometry (HRMS) and UV-vis spectroscopy (SI, Figs. ). The average dye labeling efficiency was estimated as ~80% per monomer for all three lectins.