Ultrasensitive Enzyme-Free Detection of Proteins on Magnetic Beads

., plasma or urine) can enable detection of diseases and transform clinical diagnostics and treatment. Digital protein detection assays, such as Single Molecule Arrays (Simoa) and Molecular On-bead Signal Amplification for Individual Counting (MOSAIC), enable ultrasensitive protein quantification, achieving detection limits that are at least 1000-fold lower than those of conventional ELISA assays. Currently, these ultrasensitive assays are performed in centralized laboratories that have access to specialized instrumentation and stable supply chains, which are rarely available in low- and middle-income countries (LMIC) or at the point-of-care (POC) in resource-limited settings. To address the need for ultrasensitive, accessible diagnostic assays, we have developed an enzyme-free platform for the detection of protein biomarkers with simple assay workflows. This approach uses enzyme-free signal amplifiers generated using hybridization chain reaction to achieve detection limits comparable to Simoa for most proteins. The signal amplifier reagent retains full performance when stored in the dark at 21 °C for up to six months. To investigate the utility of efMOSAIC for clinical workflows, we validated the platform for the sensitive detection of cytokines from human plasma and demonstrated multiplexed biomarker detection. With simple assay workflows and temperature stable reagents, efMOSAIC is poised to transform ultrasensitive protein assays making these technologies more accessible for use in LMICs.