Boosting Array‐based Diagnosis of Urinary Tract Infections via a Heterobifunctional Bridge‐Driven Strategy

Abstract Array‐based sensing platforms have witnessed significant progress in recent years, demonstrating considerable potential for high‐throughput and multiplexed analytes detection; however, their clinical translation remains hindered by inadequate sensitivity in complex biofluids. Herein, we introduce a heterobifunctional bridge‐driven (HB‐Bridge) strategy for constructing the sensor array, which incorporates asymmetric termini to enable dual‐mode sensing through concurrent covalent binding and electrostatic interactions. Upon addition of bacteria or lipopolysaccharide analogues, the array constructed by covalently complexing HB‐Bridge HB1 ‐ HB3 with perylene diimide fluorophores (PDIs) P1 ‐ P6 allow both fluorescence turn‐on through covalent competition and quenching through noncovalent aggregation. The optimized array enabled the simultaneous identification of 21 uropathogens in urine, achieving 84.52% blind‐test accuracy. Ultrasensitive detection was achieved for Escherichiacoli , the predominant cause of over 80% of clinical urinary tract infections (UTIs), with a limit of OD 600 = 0.0000417. Notably, in a double‐blind study of 150 clinical urine samples, the top‐performing multilayer perceptron (MLP), screened via multiple machine‐learning classifiers, achieved 98.50% accuracy in simultaneously distinguishing multiple bacterial types within 30 min using only 12.5 µL of urine, and remarkably maintained 97.92% accuracy even in clinically challenging polymicrobial cases. Overall, this study establishes a dual‐mode sensing platform enabling rapid and precise UTI diagnosis with strong potential for clinical translation.