Highly Sensitive Detection of Escherichia Coli O157:H7 Via Β-Galactosidase-Pnpg/Pnp Signal Amplification Assisted Bipolar Electrode-Electrochemiluminescence

Abstract

A novel bipolar electrode-electrochemiluminescence (BPE-ECL) biosensing strategy was developed for Escherichia coli O157:H7 (E. coli O157:H7) detection 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 cathodic pole of a closed BPE, which simultaneously drives the oxidation of the luminophore [Ru(bpy)₃]²⁺ at the anodic pole, triggering an 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. The biosensor delivers a wide dynamic range (101-106 CFU) and a low detection limit (LOD = 8 CFU) in 200 μL sample. The assay successfully detected E. coli O157:H7 in water, juice, and pure milk.

Keywords: β-gal, BPE-ECL, enzymatic hydrolysis, Six-electron reduction reaction