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We report a lab-on-a-chip (LOC) that can separate and detect Escherichia coli (E. coli) in simulated urine samples for urinary tract infection (UTI) applications. The LOC consists of two (concentration and sensing) chambers connected in series and an integrated impedance detector. The two-chamber approach is designed to reduce the nonspecific absorption of a protein, e.g., albumin, that potentially coexists with E. coli in urine. We directly separate E. coli K-12 from cocktail urine in a concentration chamber containing microsized magnetic beads conjugated with anti-E. coli antibody. The immobilized E. coli is transferred to a sensing chamber for the impedance measurement. The measurement at the concentration chamber suffers from nonspecific absorption of albumin on the gold electrode, which may lead to false-positive response. By contrast, the measured impedance at the sensing chamber shows a ~ 60-kΩ impedance change. This is a clear distinction between 6.4 × 104 and 6.4 × 105 CFU/mL, covering the threshold of UTI (105 CFU/mL). The sensitivity of the LOC in detecting E. coli is characterized to be at least 3.4 × 104 CFU/mL. We also characterized the LOC for different age groups and white blood cell spiked samples. These preliminary data show promising potential for application in portable LOC devices for UTI detection.