Streptavidin–Biotin Binding Is Intrinsically Noncooperative, but Experimental Artifacts Can Induce Apparent Positive Cooperativity

Abstract The biotin–streptavidin interaction is a key component of nanobiotechnology, yet the nature of its cooperativity remains a debated question. Here, we introduce a label-free spectroscopy approach based on confocal UV autofluorescence microscopy to probe the binding cooperativity of this high-affinity system. By leveraging the intrinsic autofluorescence of tryptophan residues, we avoid artifacts associated with extrinsic fluorescent labels, enabling precise measurements of fully biotin-saturated streptavidin across a wide range of molar ratios. Our experimental results, supported by Monte Carlo simulations, provide conclusive evidence for the absence of cooperativity. However, we show that false signs of apparent positive cooperativity can emerge from local concentration inhomogeneities occurring when rapid reaction kinetics outpace mixing. By establishing the noncooperative nature of biotin–streptavidin binding and identifying the key conditions to avoid cooperativity artifacts, these insights resolve a longstanding debate and offer critical implications for streptavidin-based biotechnological applications.