Exploiting amplification byproducts to enable colorimetric ctDNA detection via nanozyme inhibition.

Sensitive circulating tumor DNA (ctDNA) analysis is increasingly important for mutation monitoring and molecular stratification in cancer management, yet most current approaches depend on complex signal transducers, fluorescent labels and sophisticated instrumentation. Here, we present an amplification byproduct-induced nanozyme suppression (ABINS) strategy that uses amplification byproducts as functional signals for colorimetric ctDNA detection. By coupling exponential amplification reaction (EXPAR) with Cu3(PO4)2 microflower nanozymes (Cu3(PO4)2 MFs), pyrophosphate (PPi) generated during amplification selectively inhibits the peroxidase-like activity of the nanozymes, resulting in a straightforward signal-off visual readout. This assay enables highly sensitive detection of KRAS G12D ctDNA at the femtomolar level and effectively discriminates target mutants from wild-type sequences. Furthermore, as a proof-of-concept, the sensing paradigm reliably discriminated KRAS-mutant patients from healthy donors in clinical plasma samples, showing good agreement with PCR-based analysis. The ABINS strategy provides a simple amplification-coupled and label-free colorimetric approach for mutation-associated ctDNA analysis, while broader validation in larger clinical cohorts and more representative ctDNA models will still be needed to further define its practical performance.