Lab‐in‐a‐Tube System Integrating Hierarchically Wrinkled Electrodes and DNAzyme‐Functionalized Beads for Diagnosis of Clostridioides difficile Infection

ABSTRACT The diagnosis of bacterial infections remains slow because many existing methods rely on enrichment steps such as nucleic acid amplification or growth culture. RNA‐cleaving DNAzymes offer a promising route to accelerate bacterial diagnosis because they can be selected and programmed to specifically recognize disease‐causing bacteria and generate reporter DNA strands for signal readout. However, integrating DNAzymes into biosensors capable of analyzing complex biological matrices remains challenging, as matrix‐associated interferents can destabilize DNAzymes, suppress catalytic activity, and compromise signal detection. Here, we integrate redox RNA‐cleaving DNAzymes housed on antifouling magnetic beads with a lab‐in‐a‐tube platform that incorporates hierarchically structured sensing electrodes within a tubular flow cell for enrichment‐free detection of Clostridioides difficile in stool samples. The combination of antifouling magnetic beads and fluidic replenishment of DNA reporters across the hierarchical electrodes reduces nonspecific binding and enhances reporter capture efficiency, achieving a limit of detection as low as 1.3 × 10 2 CFU mL −1 in buffer. Using an optimized stool‐processing procedure, the sensor enabled detection of C. difficile infection in 38 human stool samples, demonstrating 94.7% concordance with the current gold‐standard polymerase chain reaction method. These results demonstrate a promising platform for enrichment‐free, point‐of‐care diagnosis of infectious diseases in clinic settings.