Abstract: Disclosed herein are methods for preparing modified, functional conjugates that includes the steps as follows. First, biotinylated peptides, each having a His-tag sequence, are gradually added into avidin-like molecules in a molar ratio between 1:4 to 16:1 so as to form a plurality of avidin-biotin-peptide conjugates with different valence states. The avidin-biotin-peptide conjugates are then separated using a nickel affinity column eluted with an imidazole gradient to produce a first eluate including mostly first single-valent avidin-biotin-peptide conjugates each having a first valence. The first single-valent avidin-biotin-peptide conjugates are then mixed with functional molecules under conditions allowing the formation of functional molecules modified with the first single-valent biotin-binding peptides of the first valence. Methods for preparing single-valent, modified avidin-like molecules are also disclosed herein.