Binding Mode Determination via FRET and Hydrodynamic Size Analysis. In order to more empirically establish the binding modes between the two lectins and QR-DiMan, the effect of titrating protein against a fixed C of QR was analyzed using FRET and hydrodynamic size analysis. Binding of both lectins with QR-DiMan yielded an initial linear increase of FRET ratio with increasing PQR before reaching saturation (Figure ). This behavior is similar to that observed previously for DC-SIGN binding with QD-DiMan, but is very different from the binding of DC-SIGNR with QD-DiMan, which displays a sigmoidal relationship with very little binding occurring at the low PQRs. This difference agrees with the aforementioned observation that a significant amount of strong affinity, higher order multivalency complexes are established for QR-DiMan•DC-SIGNR complexation, allowing significant binding to occur even at these lower PQRs. By fitting the linear region of the FRET ratio-PQR relationship and taking the intersection with the maximum recorded FRET ratio (Figure ), the "apparent" PQRs required to achieve saturate QR binding (i.e. maximal FRET ratio) are estimated as ~6 for DC-SIGN and ~33 for DC-SIGNR. Please note that these values do not represent the actual number of lectins that are bound to each QR-DiMan but, rather, the number of lectins per QR required to achieve saturate binding because not all added lectins will be able to bind to the QR under the natural association/dissociation equilibrium. As the overall binding affinity of QR-DiMan with DC-SIGNR is significantly weaker than that with DC-SIGN, the proportion of added DC-SIGNR molecules that are bound to the QR would be considerably lower than that for DC-SIGN. In addition, it is also worth noting that these values are likely to be smaller than the "true" PQR required to achieve saturated protein coverage of QR. This is because, according to our previous EDX mapping, the fluorescent core is situated at the center of the rod, thus only lectins bound within proximity of the central region will be close enough to engage in FRET, due to the inverse sixth power dependency of the FRET efficiency to the distance between the donor and acceptor. The binding modes of the two proteins with QR-DiMan were further confirmed by analyzing the hydrodynamic diameters (𝐷 h ) of the resulting QR-lectin complexes. The apparent 𝐷 h values were obtained from Gaussian fits of the 𝐷 h distribution histograms over a PQR range of 0 to 20. For QR-DiMan•DC-SIGN complexation, only a single size distribution is observed, which plateaus at ~60 nm (Figure ) after a PQR of ~6. This value is approximately equal ). This size is therefore likely to be representative of a monolayer of lectin with all CRDs simultaneously specifically bound to a single QR-DiMan particle. QR-DiMan•DC-SIGNR complexation, on the other hand, demonstrates two distinct size distributions (Figure -H): one plateaus at ~60 nm and matches well with that of DC-SIGN complexation, while the other plateaus at ~140 nm. The similarity in 𝐷 h values between the smaller size peak in DC-SIGNR and that observed for DC-SIGN evidently confirms the presence of simultaneous binding in DC-SIGNR. Whereas, the larger size distribution with DC-SIGNR is indicative of the formation of crosslinked clusters, similar to that observed previously with QDs, although the size here is somewhat smaller. Fitting the average 𝐷 h (weighted with respect to their integrated areas) against PQR using an offset Hill function (see Figure ), it is observed that the average 𝐷 h plateaus at 62 ± 6 and 150 ± 10 nm for DC-SIGN and DC-SIGNR, respectively. This demonstrates that the number of DC-SIGNR clustered particles tends to increase with increasing PQR. This result provides further evidence that the high affinity interaction observed at low concentrations for DC-SIGNR does indeed correlate with the simultaneous binding mode, and the weaker affinity binding correlates with an increase of crosslinked binding at the higher PQRs.