Where QEmax, Kd, C, and n are the maximum QE (fixed at 100%), apparent binding equilibrium dissociation constant, protein concentration, and Hill coefficient, respectively. The representative fluorescence spectra of labelled DC-SIGN before and after mixing with G13-psDiMan (100% glycan density) at 1:1 molar ratio under different Cs were shown in Fig. (fluorescence spectra showing the binding of G5-psDiMan with labelled DC-SIGN were given in the SI, Fig. ). It is apparent that labelled DC-SIGN fluorescence was greatly reduced in the presence of G13-psDiMan (or G5-psDiMan, SI. Fig. ), especially at elevated concentrations. A plot of the integrated fluorescence (IF) vs. C (Fig. ) further revealed that, in the absence of G13-psDiMan, the fluorescence of labelled DC-SIGN alone increased linearly (R 2 >0.995) with the increasing C; while the presence of G13-psDiMan significantly and progressively quenched protein fluorescence, leading to the IF-C relationship to deviated more and more from linear (Fig. ). This result is fully consistent with that expected from that an increasing proportion of DC-SIGN are bound to G13-psDiMan and get quenched. The resulting QE -C relationships for DC-SIGN binding with G5-psDiMan and G13-psDiMan (with a variety of psDiMan contents) were obtained and fitted by the Hill's equation (eq. 2) as shown in Figs. and, respectively. The detailed fitting parameters were summarized in Table . The relationships of the apparent Kd, MLGI enhancement factor ( where  = Kd mono /Kd, and Kd mono = 1.1 mM obtained from ITC, see SI, Fig. ) and per psDiMan normalized enhancement factor (/N, where N is the psDiMan valency on each GNP) as a function of psDiMan content (%) were shown in Fig. , 3A and 3C, respectively. Based on the results of Fig. and Table , four conclusions can be drawn. (1) There is a minimal, GNP size dependent, psDiMan content on its surface in order to form strong DC-SIGN binding (i.e., sub-to low-nM Kds). The thresholds are 25% and 12.5% for G5 and G13 (denoted as G5-psDiMan25% and G13-psDiMan12.5%), respectively. (2) Above this threshold, DC-SIGN binding affinity increased gradually with the increasing psDiMan content on G5 till reaching 100%. While that for G13-psDiMan, the trend was less clear: it gave the strongest DC-SIGN affinity with 50% psDiMan content (although the differences with those of 75 and 100% psDiMan are small and close to the assay detection limit), and further increasing or reducing psDiMan content led to slightly reduced affinity. (3) The highest DC-SIGN affinity was obtained with G13-psDiMan50%, which gave an impressively strong apparent binding Kd of ~0.13 nM.