Ultrasensitive non-enzymatic protein detection using proximity immunoassay with photonic resonator absorption microscopy

Proximity assays have emerged as a leading technology for protein detection, yet broad adoption is currently limited by complex workflows that include enzymatic amplification, thermocycling, and expensive laboratory equipment for signal readout. In this report, we introduce a Proximity Initiated Nucleic Acid Target Amplification (PINATA) assay that is performed at room temperature with a simple two-step, 90-min protocol using a low-cost detection instrument. In contrast to alternative proximity assays, PINATA applies toehold-mediated strand displacement of nucleic acids for enzyme-free reactions that occur at room temperature to uniquely combine linear amplification and digital detection using Photonic Resonator Absorption Microscopy (PRAM). Utilizing human interleukin-6, we demonstrate a detection limit of 37 fg/ml with 6-log dynamic range, high selectivity against non-target cytokines, and assays were performed with complex sample matrices without significant loss of efficacy. We envision that PINATA can address a range of protein quantitation applications for life science research and diagnostics.