An electrochemical biosensor for the detection of pathogenic bacteria based on dual signal amplification of Cu3(PO4)2-mediated click chemistry and DNAzymes

Abstract

A novel electrochemical biosensor for detecting pathogenic bacteria was designed based on specific magnetic separation and highly sensitive click chemistry. Instead of enzyme–antibody conjugates, organic–inorganic hybrid nanoflowers [concanavalin A (Con A)–Cu₃(PO₄)₂] were used as the signal probe of the sandwich structure. The inorganic component, the copper ions of hybrid nanoflowers, was first used to amplify signal transduction for enzyme-free detection. Sodium ascorbate could dissolve Cu₃(PO₄)₂ of the signal probe to produce Cu²⁺, which was subsequently converted to Cu⁺, triggering the Cu⁺-catalyzed alkyne–azide cycloaddition (CuAAC) reaction between azide-functionalized ssDNA (a fragment of the DNAzyme-containing sequence) and alkyne-functionalized ssDNA immobilized onto the electrode surface. As a result, the DNAzyme was immobilized onto the gold electrode, which produced a positive and stable electrical signal. An exceptional linear relationship was observed between the electrical signal and the concentration of Salmonella typhimurium (10¹–10⁷ CFU mL⁻¹) with a detection limit of 10 CFU mL⁻¹. The developed electrochemical biosensor based on dual signal amplification of Cu₃(PO₄)₂-mediated click chemistry and DNAzymes exhibited good results in detecting S. typhimurium in milk samples.