Highly sensitive detection of Escherichia coli O157:H7 via β-galactosidase-PNPG/pNP signal amplification assisted bipolar electrode-electrochemiluminescence

Herein, a novel bipolar electrode-electrochemiluminescence (BPE-ECL) biosensing strategy was developed, engineered to achieve highly and pathogen-specific detection of Escherichia coli O157:H7 ( E. coli O157:H7) through enzymatic cascade signal transduction. Crucially, this strategy employs β-galactosidase (β-gal) activity as a biomarker signature, wherein the enzymatic hydrolysis of p -nitrophenyl-β-D-galactopyranoside (PNPG) generates electroactive intermediates, p-nitrophenol (pNP). The pNP undergoes a six-electron reduction reaction at the closed BPE cathode electrode and simultaneously oxidizes phosphorus Ru(bpy) 3 2+ at the anode electrode to trigger obvious ECL signal. The ECL signal amplification of enzymatic substrates with mono-, bi-, and tetra-electronic systems is inherently constrained by inefficient electron transfer, resulting in suboptimal sensing performance. In contrast, the PNPG/pNP-based detection platform demonstrates superior sensing capabilities. For indirect quantification of E. coli O157:H7, the ECL intensity was linearly correlated with bacterial concentration. The optimized biosensor, through rigorous tuning of enzymatic catalysis and ECL signal amplification strategies, delivers a wide dynamic range (10(Zhang et al., 2023a 1) –10 6  CFU) and a low detection limit (LOD = 2 CFU) in 200 μL of bacterial samples. The real sample effect was evaluated, and the assay successfully detected E. coli O157:H7 in water, juice, and pure milk. This label-free platform integrates operational simplicity and high specificity, offering a promising solution for on-site monitoring of foodborne pathogens and clinical diagnostics.