A Digital Single‐Molecule Nanocavity Assay to Monitor Phosphoprotein Circuits in Single‐Cell

ABSTRACT Advances in single‐cell and single‐molecule analytical technologies have improved our ability to uncover cellular heterogeneity and dynamic molecular signaling. However, direct quantification of post‐translational modifications such as protein phosphorylation at the single‐cell level remains technically challenging. Here, we develop a digital single‐cell and single‐molecule nanocavity platform designed for counting and multiplex detection of phosphoproteins obtained from single cells. The assay integrates anisotropic gold‐silver alloy nanoboxes as highly active SERS nanotags with immunocapture in poly (methyl methacrylate) nanocavity arrays, allowing for digital enumeration of individual phosphoprotein molecules from single‐cell lysates. To demonstrate the performance and clinical relevance of the platform, we employed circulating tumor cells (CTCs) from non‐small cell lung cancer (NSCLC) as a model system, given the established role of aberrant phosphorylation in NSCLC progression, immune evasion, and metastasis. Using this system, we successfully profiled key phosphoproteins—pEGFR, pPD‐L1, pBRAF, and pERK. Validation of the platform on NSCLC patient samples confirms its ability to accurately detect phosphoproteins in single CTCs and monitor CTC heterogeneity. This digital nanocavity assay holds promise for advancing our understanding of tumor biology, supporting personalized treatment strategies, and improving patient outcomes in NSCLC.