All the filtrate and washing-through liquids were collected, combined, freeze-dried, and re-dissolved in 1.40 mL pure water to determine the amount of unbound LA-EG4-DiMan ligand using the phenolsulphuric acid method described previously. 11, 25 μL of each solution was diluted with water to a final volume of 125 μL. This solution was then mixed with 125 μL of 5% phenol and 625 μL of H2SO4, and then allowed to incubate at RT for 30 mins. The absorbance of the solution was recorded at 490 nm, and the dilution factors were then corrected to calculate the total amount of unconjugated glycan ligand against a standard calibration curve obtained with pure LA-EG4-DiMan ligand. The difference in LA-EG4-DiMan ligand amount between that added and that remained in the supernatant was conserved to have conjugated onto the GNP surface. 11