Quantum Dot-Based Fret Immunoassay for Her2 Using Ultrasmall Affinity Proteins
Journal Article - Open Access
NAVAL RESEARCH LAB WASHINGTON DC WASHINGTON United States
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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 Forster resonance energy transfer FRET, the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small approximate to 6.5 kDa histidine-tagged albumin-binding domain-derived affinity proteins ADAPTs can efficiently self-assemble to zwitterionic ligand-coated quantum dots QDs. These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab, or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 HER2 in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 x 10-12m approximate to 8 ng mL-1 are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies.
- Medicine and Medical Research