Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for many important biological events, such as infection, cell-cell communication, and the regulation of immune response. For example, pathogens often employ specific glycan patterns to target host cell lectin receptors (or vice versa) to initiate contact and infection, while immune cells often employ lectins to recognize specific pathogen-associated glycan patterns to differentiate pathogens and to instruct immune responses. Therefore, it is unsurprising that constructing glycan structures to target specific MLGIs has been a very active and attractive therapeutic approach against a wide range of viral infections, cancer, and other immune dysregulation diseases. Strategies employed often include the design of monovalent glycans against specific structures of individual carbohydraterecognition-domains (CRDs) and displaying glycans multi-/poly-valently onto various nanoscale scaffolds. This is mainly because most monovalent glycan-CRD interactions are too weak to produce high enough therapeutical effects. Displaying multiple glycans on a suitable scaffold to create a perfect spatial and orientation match to the target lectin's multiple CRDs will greatly enhance not only their binding affinity but also specificity. The latter is of great importance for potential applications in vivo due to the overlapping glycan specificity, at the monovalent levels, of various multimeric lectins. A wide variety of nanostructures, e.g., polymers, dendrimers, liposomes, polymersomes, proteins, and inorganic nanoparticles, have been employed as scaffolds to construct multi-/poly-valent glycoconjugates to enhance their MLGI affinity and specificity with target lectins. The biophysical parameters of binding to target lectins are mainly evaluated by conventional biophysical techniques, such as surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). While these traditional biophysical methods are powerful in obtaining quantitative binding affinity, kinetic, and thermodynamic data, they cannot provide key structural information, e.g., binding site organization, binding mode, and inter-binding site distances etc., which are of critical importance for designing spatial matched glycoconjugates against a particular MLGI for therapeutic interventions. In addition, each of these techniques also suffers from their own limitations. For example, while ITC can provide accurate measure of binding enthalpy changes (Hs), it cannot provide accurate binding affinities for very strong interactions with low-to sub-nM Kds. Whereas SPR measures binding interactions happening on surfaces, which is a very different environment from that happening in solution. As a result, the binding kinetic and thermodynamic data obtained in SPR may not reflect what happens in solution. Moreover, most previous studies have employed nanoparticles only as passive scaffolds to display polyvalent glycans to enhance MLGI affinity and/or specificity, however, the unique, size-dependent optical properties of nanoparticles, which are fundamental and critically important for nanomaterials, were not exploited as readout signals for MLGI affinity quantitation. To address the above stated limitations, we have recently demonstrated that small nanoparticles (e.g., ~4 nm CdSe/ ZnS quantum dots, QDs, and a ~5 nm gold nanoparticle, GNP, ) densely glycosylated with fragments of the natural high mannose structures are powerful probes for MLGIs. By harnessing the unique, size-dependent strong fluorescence (for QDs), or fluorescence quenching (for GNPs) properties, we have developed a robust, sensitive method for MLGI affinity quantitation based on the Förster resonance energy transfer (FRET, with QD) or fluorescence quenching (with GNP). We have further dissected the exact binding modes of the target MLGIs by analyzing the hydrodynamic size and capturing binding induced nanoparticle-lectin assemblies under their native dispersion state by exploiting the nanoparticle's size and high contrast under transmission electron microscopy (TEM) imaging. Using a pair of critically important, closely related tetrameric lectin viral receptors, DC-SIGN and DC-SIGNR , as model lectins, we have revealed that each DC-SIGN binds simultaneously to one nanoparticle-glycan via all four of its CRDs, giving rise to isolated nanoparticle-lectin assemblies and strong affinities (low to sub-nM Kds). In contrast, DC-SIGNR crosslinks with multiple nanoparticle-glycans, resulting in extended large-scale nanoparticle-lectin assemblies and markedly weaker affinities in binding to nanoparticle-glycans than DC-SIGN. Moreover, we have found that nanoparticle-glycans only potently and robustly block DC-SIGN-, but not DC-SIGNR-, mediated augmentation of Ebola virus cell entry, and thus demonstrating the critical role of the MLGI binding mode in the ability of glycoconjugates to block viral infections. Despite these advances, how nanoparticle scaffold size as well as glycan type and density control MLGI affinity and other key biophysical parameters remains to be explored. Here, a synthetic glycomimetic, a pseudo-α-1,2-mannobioside (psDiMan) that shows a different binding mode on DC-SIGN CRD from the natural high mannose fragment counterpart, -manno--1,2-biose (DiMan), has been displayed polyvalently onto two different sized GNP scaffolds (e.g., ~5 and ~13 nm in diameter, abbreviated as G5-psDiMan and G13-psDiMan, respectively) under systematically varying densities. psDiMan is designed by replacing the reducing end mannose of DiMan with a cyclohexanediol scaffold locked in a diaxial conformation by two carbomethoxy groups (Figure ). The cyclohexane framework was found to offer specific hydrophobic interactions with Val351 in DC-SIGN, resulting in moderate selectivity towards DC-SIGN CRD over that of langerin, despite their sharing glycan specificity. We have quantified Gx-psDiMan-DC-SIGN (x=5 or 13) binding affinities, via GNP's strong fluorescence quenching properties, and demonstrated that there is a minimal, GNP size-dependent, glycan density threshold in order to form strong MLGI with DC-SIGN. We have then applied the Van't Hoff analysis of their temperature dependent MLGI affinities and revealed that DC-SIGN binding with Gx-psDiMan (for both x = 5 and 13) is enthalpy driven and the binding thermodynamic parameters derived from fluorescence quenching match well to those obtained from ITC. Moreover, we have found that their MLGI enthalpy changes (Hs) are ~4 times that of monovalent binding, suggesting that all 4 CRDs in each DC-SIGN are engaged in binding. Finally, by employing particles pseudotyped with the glycoprotein (GP) of Ebola virus (EBOVpp), we have investigated the ability of Gx-psDiMan to block DC-SIGN-promoted cell entry of EBOV-GPpp. We reveal that Gx-psDiMan can potently block viral entry to host cells, and their antiviral potencies are positively correlated to their DC-SIGN binding affinity measured by fluorescence quenching. Together, these results have established Gx-psDiMan as a powerful new biophysical probe for MLGI affinity enhancing mechanisms, which can also serve as potent antiviral agents.