Utilizing Constrained Bicyclic Peptides for <i>In Vitro</i> Diagnostics

Constrained bicyclic peptides (<i>Bicycle molecules</i>) with high affinity for biological targets have emerged as potentially powerful therapeutic agents, particularly for the <i>in vivo</i> targeting of cancer receptors. However, their antibody-mimetic properties have yet to be explored for use in diagnostic immunoassays. These synthetically derived compounds serve as biorecognition scaffolds that allow for facile site-selective modification and large-scale production. A phage display screen against various constructs of the SARS-CoV-2 nucleocapsid (N) protein identified several <i>Bicycle</i> molecules with binding affinities ranging from the micromolar to the low nanomolar range. These <i>Bicycle</i> molecules were validated in the development of enzyme- and nanozyme-linked immunosorbent assays, as well as enzymatic and colorimetric nanoparticle-based lateral flow immunoassays (LFIA) for the detection of ultralow concentrations of the SARS-CoV-2 N protein. We envision that these moieties enable robust, cost-effective, and large-scale development of ultrasensitive biosensors for a diverse range of biomarkers by leveraging their high binding affinity, minimalistic scaffold, and synthetic accessibility.