Ultrasmall Affinity Proteins for Highly Sensitive Quantum Dot FRET Immunoassays

Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Förster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, we show that very small (~6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPTQD conjugates are significantly smaller than QD-conjugates based on IgG, Fab’, or single-domain antibodies. We demonstrate immediate applicability by the quantification of HER2 in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR. Limits of detection down to 40 pM (~8 ng/mL) were in a relevant clinical concentration range and outperformed previously tested assays with antibodies, antibody fragments,