Rapid and accessible tumor necrosis factor alpha detection using digital nanoparticle sensors towards point-of-care cytokine monitoring.

Cytokine monitoring is essential for understanding immune response and evaluating therapeutic outcomes in inflammatory and autoimmune diseases. Here, Tumor Necrosis Factor-alpha (TNF-α) is employed as an exemplary cytokine marker to demonstrate a nanoparticle-supported, rapid, electronic detection (NasRED) assay as an one-pot, in-solution sensing platform for cytokine quantification and therapeutic monitoring. Gold nanoparticles (AuNPs) functionalized with TNF Receptor-2 (TNFR2) are designed to react with TNF-α in phosphate-buffered saline (PBS), human pooled serum (HPS), whole blood (WB), cerebrospinal fluid (CSF), and synovial fluid, producing accelerated AuNP precipitation for signal transduction and digitization. The NasRED TNF-α assay demonstrates high sensitivity (1.2 fM), a broad dynamic range of seven orders of magnitude, and a short assay time (~20 min). Importantly, the platform enables analysis of the competitive interactions among TNFR2, TNF-α, and the therapeutic antibody Adalimumab, enabling rapid and multiparametric cytokine assessment. Furthermore, the NasRED assay is explored for detecting TNF-α in the presence of both Adalimumab and anti-drug antibody (ADA), modeling drug resistance scenarios during long-term therapy. This proof of principle study establishes a rapid, digital, and in-solution assay framework for cytokine and therapeutic monitoring in various biofluids and complex competitive environments, paving the way for improved understanding and management of inflammatory, autoimmune, oncological, and neurodegenerative diseases.