Abstract 3676: Ultrasensitive protein detection using proximity initiated nucleic acid target amplification with digital biosensing

Abstract Proteins are vital biomarkers and provide a real-time dynamic look into cancer progression and disease states. Point-of-care simple diagnostics for rapid, ultrasensitive testing is necessary to study longitudinal changes of protein biomarkers for treatment monitoring, prognosis, or early detection. Proximity assays are a solution-based protein assay using antibody pairs that can achieve high sensitivity, specificity, and be used for high-throughput or multiplexing applications. However, standard proximity assays require complicated design, enzymatic amplification such as polymerase chain reaction (PCR), and thermocycling with expensive laboratory equipment and specialized laboratories. This limits the use for longitudinal monitoring or liquid biopsies. We demonstrate here the Proximity Initiated Nucleic Acid Target Amplification (PINATA) assay, a novel proximity immunoassay for protein detection that can be performed completely at room temperature in a single step using a small, portable, point-of-care benchtop detection instrument. The PINATA assay implements linear amplification using toehold-mediated strand displacement reactions of antibody-oligonucleotide conjugates along with digital detection of individual gold nanoparticle tags for ultrasensitive, rapid protein quantification in the femtomolar range, allowing detection in less than two hours for sub picogram per milliliter concentrations of human interleukin 6, a cytokine biomarker of inflammation that has been linked to certain cancers. These results showcase the promise of entropy-driven strand displacement proximity assays for fast and ultrasensitive point-of-care applications and disease diagnostics. Citation Format: Skye Shepherd, Weinan Liu, Brian T. Cunningham. Ultrasensitive protein detection using proximity initiated nucleic acid target amplification with digital biosensing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3676.