This affinity represents a massive, 8.5 million fold MLGI affinity enhancement (β = Kd mono /Kd MLGI ) over that of the corresponding monovalent psDiMan-DC-SIGN binding (Kd mono , determined as 1.1 mM by ITC, see SI, Fig. ) and per glycan normalized enhancement factor, β/N, of ~8,700. This MLGI affinity is significantly stronger, by ~28 fold, than that of G5 coated with 100% LA-EG2-EG2-DiMan (e.g., apparent Kd ~ 3.8 nM), its equivalent DC-SIGN natural DiMan ligand of the same overall EG linker length, despite the fact that its monovalent affinity is slightly weaker than that of the latter (e.g., Kd mono 1.1 mM vs. 0.9 mM, both obtained from ITC, SI, Fig. ). This result shows that displaying psDiMan polyvalently on a GNP surface is more effective in enhancing its MLGI affinity with DC-SIGN than that with DiMan, a natural glycan ligand for DC-SIGN, presumably due to their different binding motifs on DC-SIGN CRD. Therefore, we cannot directly use the relative strength of lectin-glycan monovalent affinity to predict their relative MLGI strengths involving polyvalent glycoconjugates. (4) The per psDiMan normalized enhancement factor, β/N, as a function of GNP surface psDiMan contents was found to depend strongly on the GNP size. For G5-psDiMan, its β/N generally increased with the increasing psDiMan content and reached the maximum at 100% psDiMan; under which it gave a highly impressive β/N value of ~10000. While for G13-psDiMan, its β/N broadly plateaued at ~8000 for psDiMan content between 12.5% and 50%, and further increasing the psDiMan content led to a markedly reduced β/N value (Fig. ). This result reveals a key role of surface curvature of glycoconjugate in their ability to form strong MLGI with DC-SIGN. The difference in psDiMan density threshold for G5 and G13 -psDiMan to achieve strong MLGI with DC-SIGN can be rationalized from the assumption that strong MLGIs are only formed when all four CRDs in DC-SIGN are engaged in binding. While the detailed crystal structure of DC-SIGN tetramer remains unknown, results from our group as well as others indicate that all 4 binding sites in DC-SIGN point upwardly in the same direction, allowing them to bind simultaneously to multiple glycans on the same Gx surface. This was also confirmed from the hydrodynamic diameter (Dh) measurement of Gx-psDiMan100% + DC-SIGN samples under a variety of DC-SIGN: Gx-psDiMan molar ratios, which revealed only a single Dh species for both G5-and G13-psDiMan binding with DC-SIGN. Their Dhs initially increased with the increasing DC-SIGN: Gx ratio, and then plateaued at a ratio of ~6:1 or ~32:1 for G5-or G13-psDiMan, respectively. This result indicates an increasing number of DC-SIGN molecules are bound to each Gx-psDiMan before surface binding saturation. Moreover, the saturated Dhs were found to be monodisperse and comparable to that expected for a central Gx-psDiMan particle coated with a monolayer of DC-SIGN molecules (~50-60 nm), implying that DC-SIGN must have bound to the central Gx-psDiMan particle with all four CRDs (SI, Section 7, Figs. ). Given that the terminal psDiMans are displayed on the Gx surface via a flexible EG4 linker, it is reasonable to assume that any psDiMan groups within the projected footprint of each CRD on the Gx surface (~7 nm 2 , based on a spherical CRD structure of ~3 nm in diameter), could adapt and bind to that CRD. Therefore, any Gx-psDiMan conjugates with a glycan footprint smaller than 7 nm 2 are expected to be able to bind to all four CRDs in DC-SIGN, giving rise to strong MLGI affinity. This result matches well to the drastic decrease of DC-SIGN MLGI affinity observed for G13-psDiMan as psDiMan content decreased from 12.5% to 6.3% (i.e., average glycan footprint increased from ~4.9 to 9.4 nm 2 , see SI, Table ). The latter psDiMan content is below the threshold required to allow all four CRDs in DC-SIGN to engage in binding. For G5-psDiMan, a higher glycan content threshold is required to form tetravalent binding, presumably because of its larger surface curvature, giving rise to a glycan deflection angel being twice as big as that of its G13-psDiMan counterpart (e.g., 29.7 ± 1.3 o vs. 14.6 ± 0.7 o for G5-vs. G13-psDiMan12.5%, see SI, Table ). Fig. . The QE%-C relationships for DC-SIGN binding with G5-psDiMan100% (A) or G13-psDiMan100% (E) under three different temperatures fitted by Hill's equation, and the corresponding Van't Hoff plots (lnKd vs. 1/T plots) for DC-SIGN binding with G5-psDiMan (B) or G13-psDiMan (F). ITC titration curves for DC-SIGN binding with G5-psDiMan100% (C) or G13-psDiMan100% (G) and their respective fitting curves (D, H).