Moreover, we have developed a new format of GNP-fluorescence quenching assay to minimize the interference arising from GNP's strong inner filter effect, allowing us to robustly quantify MLGI affinities for both small and large GNP-glycans. We find that the MLGI affinities between DC-SIGN/R and GNP-glycans are enhanced significantly with the increasing GNP size, while reducing glycan density from 100% to 50% does not markedly affect their affinity, although reducing the glycan density further to 25% weakens their affinity. By quantifying temperature-dependent MLGI affinities via GNP fluorescence quenching and applying Van't Hoff analysis, we have quantified their MLGI biophysical parameters, revealing that DC-SIGN/R bindings with GNP-glycans are enthalpy driven, and the negative binding enthalpy changes (Hs) for DC-SIGN are ~4 times that of the monovalent binding, while that for DC-SIGNR is about twice that of the monovalent binding, under our assay conditions.