Previously, most QD-FRET binding assays were carried out by varying the amount of protein (or other binder) whilst maintaining a fixed QD concentration, to obtain fluorescence-concentration relationships from which apparent 𝐾 𝑑 values were derived. While such a method can provide accurate 𝐾 𝑑 values for weak binders (e.g. true 𝐾 𝑑 >> 50% of the QD's saturate binding capacity), it cannot provide accurate measurements for strong binders (e.g. true 𝐾 𝑑 < 50% of QD's saturate binding capacity) where the measured 𝐾 𝑑 values will simply equal 50% of the QD's saturate binding capacity. In contrast, our above method does not have such limitations and can provide robust 𝐾 d measurements for both strong and weak binding partners. This is because the 𝐼 𝐴 /𝐼 𝐷 ratio is linearly proportional to the amount (or percentage, under a fixed PQR) of lectins bound to the QD in the binding system, thus it is independent of the protein concentration or QD's binding capacity, making it a highly robust parameter for 𝐾 𝑑 quantification. Consistent with that observed previously, the binding affinity of DC-SIGN to QD-DiMan is very strong, with an apparent 𝐾 𝑑 of ~1.5 nM at 20 o C, which is >20 fold stronger than that of DC-SIGNR. In order to obtain the binding thermodynamic parameters, each binding assay was repeated at three different temperatures (20, 25 and 30 o C, see Fig. , and SI Fig. for detailed spectra) and their . Van't Hoff plots were then constructed to extract the binding enthalpy and entropy changes by combining two Gibbs free energy equations, ∆𝐺 = 𝑅𝑇 ln(𝐾 𝑑 ) and Eq. 2, via a linear fit of the natural log of the 𝐾 𝑑 against the reciprocal of temperature, 1/T (Eq. 3) as shown in Fig. , where, R is the gas constant.