To investigate how GNP size and glycan density affect their MLGI with DC-SIGN, we quantified their binding affinities using GNP's strong fluorescence quenching properties. Here, varying concentrations of labelled DC-SIGN and Gx-psDiMan were mixed under a fixed mole ratio of 1:1 in a binding buffer (20 mM HEPES, 100 mM NaCl, 10 mM CaCl2, pH 7.8) containing large excess of a non-target serum protein, bovine serum albumin (BSA, 1 mg/mL), which serves to minimize any nonspecific interactions. It can also reduce nonspecific adsorption of proteins and/or Gx-psDiMans on surfaces, which can be a major source of experimental errors for assays performed at low concentrations (10 nM or below). Moreover, serum proteins are of high abundance in vivo, therefore, this also makes the binding environments resemble more closely to real biological situations. The Gx-psDiMan and labelled DC-SIGN samples were incubated for 20 min at room temperature before their fluorescence spectra (from 650 to 800 nm) were recorded under a fixed λEX of 630 nm. Labelled DC-SIGN only samples (without Gx-psDiMan) were also recorded under identical conditions, which serve as controls to determine the quenching efficiency (QE) at each concentration (C) via equation ( ): 14