Escherichia coli O157:H7 makes a major threat to human health. Aiming to detect Escherichia coli O157:H7 sensitively, hybridization chain reaction signal amplified immunoassay (immuno-HCR) based on contact quenching (CQ) and fluorescence resonance energy transfer (FRET) was developed. The background of the new designed HCR hairpins (CQ-FRET hairpins) was reduced by contact-quenching fluorescein (FAM) and breaking FRET from donor (FAM) to acceptor (Cy5). The F/F ratio of CQ-FRET hairpins (37.02) was obviously higher than that of two other common HCR fluorescent hairpins (CQ hairpins, 21.45; FRET hairpins, 4.61). The limit of detection of the assay was 3.5 × 10 CFU/mL and obviously lower than that of CQ hairpins based immuno-HCR (3.28 × 10 CFU/mL) and FRET hairpins based immuno-HCR (6.49 × 10 CFU/mL). The proposed low fluorescent background immuno-HCR with high sensitivity which was verified in contaminated milk samples could be potentially used in the detection of various pathogens.
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