Homogeneous Electrochemical Enzyme‐Linked Immunosorbent Assay Strategy Based On pH‐Mediated Redox Potential Regulation

Enzyme‐linked immunosorbent assay (ELISA) is widely recognized as the gold standard for protein detection. However, its reliance on expensive and bulky optical instruments limits its use in point‐of‐care and resource‐limited settings. Electrochemical technique emerges as a promising alternative due to its low cost, portability, and simple instrumentation. However, conventional electrochemical methods often require complex surface modifications and suffer from variability between electrodes. To overcome these limitations, a novel homogeneous electrochemical ELISA platform has been developed, that simplifies and accelerates signal acquisition in field settings. This platform leverages pH changes caused by alkaline phosphatase‐catalyzed hydrolysis of ATP, resulting in solution acidification. These pH variations are sensitively detected through shifts in the redox potential of methylene blue (MB), a proton‐sensitive electrochemical probe. By quantitatively correlating the target protein concentration with MB redox potential shift, a sensitive, reproducible, and quantitative electrochemical detection of a model protein, C‐reactive protein, is achieved. This versatile and cost‐effective approach holds significant potential to expand the applicability of ELISA to point‐of‐care diagnostics.