Coupling Single Domain Antibodies to Catalytic Hairpin Assemblies for Homogeneous Assays

Abstract Immunoassays are widely used in various fields, including biomedical research, clinical diagnostics, and environmental monitoring. Single domain antibodies (sdAbs) provide small, tailorable, recognition elements that have been integrated into immunoassays for detecting a myriad of targets. Deoxyribonucleic acid (DNA) circuits are synthetic molecular devices composed of DNA strands that can perform logical or computational operations. They have a range of applications, including biosensing, diagnostics, and drug delivery. Recently, an immunoassay method was reported that used catalytic hairpin assemblies (CHA) with antibodies for homogeneous protein detection. The CHA process uses DNA hairpins that interact in the presence of a catalytic DNA sequence. This paper presents a new strategy to couple the recognition of sdAbs with CHA circuits using genetic fusions of sdAbs with rhizavidin (rz), a dimeric biotin binding protein. A pair of sdAb‐rz constructs is each functionalized with a biotinylated DNA sequence that represents half of the catalyst. When both sdAbs bind to the target protein, a signal is generated through the CHA circuit. The split catalyst approach amplifies signals through a DNA circuit without washing steps. Furthermore, this method distinctively utilizes programmable DNA circuits, which are highly modular and can accommodate new targets without disrupting the assay.