Pre-coated Interface Proximity Extension Reaction assay enables trace protein detection with single-digit accuracy

Abstract Advances in trace protein detection contribute to the early diagnosis of diseases and exploration of stem cell development. The pre-coated interface proximity extension reaction (PIPER) assay enables target protein detection at trace levels and was developed based on protein biomarker recognition using sets of three specific antibodies and the extension of antibody-bound nucleic acid chains in proximity, accompanied by amplification and reading of protein signals via real-time quantitative polymerase chain reaction (qPCR). Noise generated in binding reactions and enzymatic steps was decreased by transferring the liquid-liquid reactions onto a liquid-solid interface in glutaraldehyde-treated tubes pre-coated with antibodies. Nucleic acid sequences of oligo-antibody-based probes were designed for extension and qPCR without pre-amplification when binding to a target molecule. As a proof of concept, the PIPER assay was used to profile slight variations in crucial biomarkers, high-sensitivity C-reactive protein, and cardiac troponin I. The detection sensitivity of the assay for the biomarkers was 0.05 pg/mL (1.25 fM) in 10% human serum. In phosphate-buffered saline, the PIPER assay detected fewer than 10 protein molecules per μL. The simple, widely applicable PIPER assay can detect trace protein biomarkers with single-digit accuracy, making it appropriate for the development of clinical hypersensitive protein detection and single-cell protein detection technology.