Rapid and Visual Detection of Urinary Pathogens by Employing Bifunctional Deoxyribonucleic Acid Sensors and Aggregation of Gold Nanoparticles

A simple, rapid, and visual approach is developed to perform diagnosis of urinary tract infection (UTI) and antimicrobial susceptibility testing (AST) by employing smart bifunctional DNA (bfDNA) sensors, exonuclease III, concatermers of CuO nanoparticles (CuONPs), and gold NPs (AuNPs) aggregation [AuNPs agglutination (AA)], namely, the bfDEC-AA method. The bfDNA sensors serve as probes for identifying 16S rRNA genes of bacterium or 18S rRNA of fungus and as mediators connecting the concatermers of CuONPs. The AA as a signal source is triggered by Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry. In this study, the urine samples are analyzed directly, eliminating the need for overnight incubation or bacterial isolation. A crucial color change from red to purple occurs at the UTI diagnostic threshold of 10⁴ CFU mL^-1; thus the results of UTI diagnosis can be qualitatively interpreted by the unaided eyes. Similarly, AST was performed visually under our optimal conditions. The color shift can also be quantified using a point-of-care (POC) device. UTI identification, bacteria strain identification, and AST were achieved within 55, 55, and 145 min for 96 samples, respectively. Here, 128 urine samples from suspected UTI patients were tested. Notably, the sensitivity and specificity of our method were 99% and 100% for Gram-negative bacteria (G^-) infection, 100% and 100% for Gram-positive bacteria (G⁺) infection, and 98% and 100% for AST, respectively. Furthermore, the low cost of our POC device (US$156) is friendly to underdeveloped regions.