Allosteric DNAzyme‐Enabled Sensitive and Multiplex Detection of Biomarkers for Rapid Diagnosis of Urinary Tract Infections

ABSTRACT Urinary tract infections (UTI) are among the most prevalent infectious diseases, demanding rapid diagnosis to provide timely therapeutic interventions. Urinary molecules can indicate the responses of the host cells to pathogenic infections, which may serve as markers for UTI diagnosis. Sensitive and multiplex detection of urinary markers in one‐pot has remained challenging. Herein, we developed an allosteric DNAzyme‐based biosensor, denoted as SMART ( s ensitive and m ultiplex detection of A TP and mi R NAs for U T I diagnosis), to enable rapid UTI diagnosis. Multiple sensing modules were integrated into a unimolecular DNA strand of perfect stoichiometry and excellent thermodynamic stability that collectively led to enhanced detection capability. The binding of targets to the detection module induces conformational reconfiguration of SMART to activate sequence‐specific catalytic cleavage against fluorescent RNA reporters. SMART demonstrated remarkable sensitivity (ATP: ∼pM; miRNAs: ∼fM) and multiplexing capability (∼4 markers) to realize extraction‐free, preamplification‐free, and rapid (∼2.5 hr) detection of UTI markers. SMART‐based UTI diagnosis model yielded a detection accuracy of 95.5% in a cohort of 164 patients. SMART may serve as a technical platform for detecting various markers that could be applied to the diagnosis of UTI and many other diseases in the future.