A CdSe/CdS core/shell (dot in a rod) QR with maximal emission at EM ~560 nm (denoted as QR560 hereafter) was chosen to construct the QR-DiMan via our previously described ligand conjugation method. QR560 was chosen due to its similar core diameter to the QD scaffolds used in our previous studies, allowing for direct comparison of results. A dihydrolipoic acid-undeca(ethylene glycol)--1,2manno-biose (DHLA-EG11-DiMan, Figure , SI Section 2.1) based multi-functional ligand was synthesized as described previously. Each ligand contains three unique functional domains: a DHLA group for strong QR surface anchoring; 48 a flexible EG11 linker for imposing high water-solubility, stability and resisting non-specific interactions, and a terminal DiMan group for specific lectin binding. QR-DiMan was produced by performing cap-exchange using deprotonated DHLA-EG11-DiMan ligand in a homogeneous CHCl3/MeOH/EtOH solution, giving rise to high capexchange efficiency as described previously for QD-DiMan. QR-DiMan was found to be relatively compact, with a mean hydrodynamic diameter (𝐷 h ) of 20.8 ± 4.8 nm (𝐷 h ± ½ full width half maximum (FWHM)) and the first excitonic absorption and emission peaks at 541 nm and 560 nm, respectively. Its fluorescence quantum yield (QY) was determined as 6.2%, this represents a significant reduction of its nominal QY (~68%) prior to cap-exchange. This result agrees with other literature results, where CdSe/CdS based QDs and QRs have shown to display significantly reduced fluorescence QY after cap-exchange. By calculating the difference between the amount of ligand added and that remained in supernatant post cap-exchange via the phenolsulphuric acid carbohydrate quantification, the number of DHLA-EG11-DiMan ligands capped on each QR was estimated as 370 ± 30 (see SI Section 2.2). Binding Affinity Determination via FRET. The QR's strong fluorescence was exploited as a ratiometric FRET readout to quantify the binding affinity between QR-DiMan and acceptor fluorophore labeled DC-SIGN/R. Lectin labeling was achieved by coupling a maleimide-Atto-594 dye via the site-specifically introduced Q274C and R287C mutations in DC-SIGN and DC-SIGNR, respectively. These labeling sites lie close to, but not in, the glycan binding pockets, enabling us to obtain sufficient FRET signals without inhibiting the lectins' glycan binding properties, as confirmed previously. The QR-Atto-594 FRET pair has good spectral overlap and a respectable Förster radius 𝑅 0 of 4.8 nm (see SI, Section 2.3), ensuring that efficient FRET between QR-DiMan and labeled lectins can happen upon binding. Meanwhile, there is little overlap of their emission spectra, allowing for straightforward separation of the donor and acceptor fluorescence without the need of spectral deconvolution. The affinity assays were carried out by mixing QR-DiMan with labeled lectins under different concentrations (𝐶) but under a fixed protein : QR molar ratio (PQR) of 1:1 in a binding buffer (20 mM HEPES, 100 mM NaCl, 10 mM CaCl2 pH 7.8, containing 1 mg/mL of bovine serum albumin to reduce non-specific adsorption). The resulting fluorescence spectra were recorded using a fixed excitation wavelength (EX) of 450 nm, corresponding to the absorption minimum of the Atto-594 acceptor to reduce the dye direct excitation background. Exciting an equilibrated ; R 2 ≥ 0.99). ), 𝐾 d mono = 0.9 mM for CRD-DiMan binding. mixture of QR-DiMan with labeled lectins resulted in fluorescence of unbound QR (peaking at ~559 nm) or, if binding occurred, energy transfer via FRET from the excited QR donor to the Atto-594 acceptor, giving rise to simultaneously quenched QR and enhanced Atto-594 fluorescence (at ~627 nm, Figure ). These fluorescence spectra were corrected by subtracting dye direct excitation background spectra of the labelled lectins, without QR-DiMan, under identical conditions (Figure ). The resulting dye to QR fluorescence intensity ratio (apparent FRET ratio)-C relationship was then fitted by the Hill equation to extract the apparent binding 𝐾 d values (eq 1; where, 𝐹 is the FRET ratio, 𝐼 is the peak emission intensity, [P] is the protein concentration, 𝑛 is the Hill coefficient and 𝐾 d is the apparent equilibrium binding dissociation constant). Here, 𝑛 = 1 was assumed because most binding assays were measured under a PQR of 1:1, under which most QRs should be bound by just a single lectin, and hence there should be no positive or negative influence of QR-bound lectins toward further binding of lectins to the same QR-DiMan. As the FRET ratio is proportional to the fraction of protein bound to the QR under a fixed PQR of 1:1, this method is robust and can provide accurate affinity measurement of both strong and weak MLGIs.