Proximity ligation assay (PLA) achieves extremely low limits of detection but requires the synthesis of antibody-DNA conjugates recognizing multiple target epitopes with appropriate proximity. In this work, we introduce a more generally applicable method by replacing antibody-DNA conjugates with nanoparticles which create ultradetectable PCR templates by capturing biotinylated oligonucleotides and catalyzing ligation. A competitive PCR readout was used to make the assay quantitative. We have demonstrated that NP-PLA can detect and quantitate human chorionic gonadotropin (hCG) levels as low as 2.6 fM (∼0.1 pg/mL), nearly 1000 times more sensitive than the current state of the art ELISA.
Keywords: competitive polymerase chain reaction; immunoassay; nanoparticle; protein detection; proximity ligation assay.