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A new amine functionalized electrochemical immunosensor for label-free detection of Escherichia coli using modified thermoplastic electrodes is reported for the first time. The E. coli-specific antibody was immobilized on diazonium-modified TPEs. The modification process was monitored using cyclic voltammetry and electrochemical impedance spectroscopy using the hexacyanoferrate redox couple as the electrochemical probe. The linear range was from 1 × 10³ to 1 × 105 CFU ml–1 with a linear correlation of 0.9823 and limit of detection of 27 CFU ml–1 for E. coli (DH5α strain) in phosphate buffered saline solution as a model system. The specificity of the proposed immunosensor was demonstrated by showing selective detection in the presence of different bacteria. The biosensor was successfully applied to urine samples spiked with 0.5 × 105 CFU ml–1 and 1 × 105 CFU ml–1 E. coli and obtained good recoveries 99% and 110%, respectively. The proposed system should be well suited for selective and sensitive detection of different pathogenic bacteria. [ABSTRACT FROM AUTHOR]