Abstract Biosensors based on the combination of semiconductor quantum dots (QDs) and Förster resonance energy transfer (FRET) have demonstrated many advantages for simple, fast, sensitive, and multiplexed diagnostics. However, the implementation of QDs as functional standard materials into homogeneous (single-step) FRET immunoassays has not yet been accomplished, because profound investigations of antibody-conjugation strategies concerning their influence on diagnostic performance for quantifying clinical biomarkers are lacking. Here, we report about a systematic study of size, type, orientation, specificity, nonspecific binding, and cross-reactivity of antibodies conjugated to QDs for single and duplexed EGFR and HER2 immunoassays. Time-gated terbium-to-quantum dot FRET detection on a clinical immunoassay fluorescence plate reader (KRYPTOR) enabled a direct comparison of matuzumab, cetuximab, trastuzumab, and pertuzumab monoclonal antibodies and EgA1, EgB4, 11A4, and 18A12 VHH nanobodies conjugated to 605 and 650 nm emitting QDs. Detection limits of 2.9 ng/mL EGFR, using cetuximab and matuzumab conjugates, and 8.0 ng/mL HER2, using oriented 11A4 and 18A12 conjugates, demonstrated the capability of detecting concentrations well below the clinical cutoff values. Multiplexed assays could quantify EGFR and HER2 at low nanomolar concentrations from the same sample. Our results show that careful optimization of QD-antibody conjugation is a prerequisite to implementing QDs into applied clinical diagnostics.
