Quantifying binding affinity and thermodynamics via QD-FRET. The principle of the QD-FRET readout for quantifying the DC-SIGN/R (Atto-594 labeled)-QD-DiMan binding is shown schematically in Fig. . Since FRET can only happen over a short distance (e.g. < 10 nm), any unbound lectins (acceptors) would be too far away to participate FRET interactions with the QD donor and hence cannot contribute the FRET signal. Thus the observed FRET signal is directly linked to the equilibrium of QD-DiManlectin binding, and more specifically, the amount of QDbound lectins. This is a distinct advantage of QD-FRET readout, allowing for binding assays to be performed in homogenous solutions without the need of purification or separation. The binding affinities between QD-DiMan and the three lectins, quantified by their equilibrium dissociation constant, 𝐾 𝑑 , were measured via our recently established QD-FRET readout strategy. Briefly, the fluorescence spectra of pre-mixed QD-DiMan and lectin samples with varying protein concentrations under a fixed protein:QD molar ratio (PQR) of 1:1 for DC-SIGN or 10:1 for DC-SIGNR, were recorded at Ex = 450 nm, corresponding to the minimal absorption of Atto-594, to minimize the dye direct excitation background. A higher PQR for DC-SIGNR was used to compensate for its relatively low FRET signal due to weak binding. The corresponding dye direct excitation corrected fluorescence spectra (Fig. ) revealed that while both the fluorescence intensities of the QD donor (𝐼 𝐷 , at ~550 nm) and Atto-594 acceptor (𝐼 𝐴 , at ~628 nm) were increased with increasing concentration, 𝐼 𝐴 increased more quickly than 𝐼 𝐷 , leading an increasing apparent FRET ratio (IA/ID) at higher concentrations before reaching saturation (Fig. ). In contrast, incubating the labeled lectins with the QD-OH control without the terminal glycan did not produce any noticeable FRET signals, confirming that the FRET signal observed here was due to specific lectin-glycan interactions (SI, Fig. ). The apparent FRET ratioconcentration relationships were then fitted with the Hill's equation (Eq. 1) to derive the apparent binding 𝐾 𝑑 values (Fig. , Table ). Where 𝑥 is the protein concentration, 𝐹 is the apparent FRET ratio, and n is the Hill coefficient, which indicates binding cooperativity (i.e. 𝑛 < 1, = 1, and > 1, indicate binding to be negatively-, none-and positively cooperative, respectively). Here, 𝑛 = 1 was assumed for fitting all assays, as no CRD-CRD interactions should occur to inhibit or promote binding.