DNAzyme-Immobilizing Microgel Magnetic Beads Enable Rapid, Specific, Culture-Free, and Wash-Free Electrochemical Quantification of Bacteria in Untreated Urine.

Rapid, ultrasensitive, and specific detection and identification of bacteria in unprocessed clinical specimens is critically needed to enable point-of-care diagnosis of infectious diseases. However, existing systems require sample processing and/or analyte enrichment for direct bacterial analysis in clinical samples, which significantly adds to the assay time and complexity. Herein, we integrate RNA-cleaving DNAzymes specific to Escherichia coli (E. coli) and programmed for electrochemical signal transduction, multifunctional microgel magnetic beads for immobilizing the DNAzyme into a hydrated and three-dimensional scaffold, and hierarchical electrodes for ultrasensitive electrochemical readout to achieve rapid bacterial analysis in undiluted and unprocessed urine collected from symptomatic patients suspected of having urinary tract infections (UTIs). The microgel magnetic bead assay enables highly efficient conjugation and hydration of the immobilized DNAzymes, resulting in low limits-of-detection of 6 CFU/mL in buffer and 138 CFU/mL in unprocessed urine with high specificity against multiple urinary pathogens within a 1 hour assay time. The assay successfully identifies which patients are infected with E. coli as the causative organism for their UTI symptoms, indicating the clinical relevance of this assay.