Catalytic hairpin assembly combined with graphene oxide for the detection of emetic Bacillus cereus in milk

Abstract

A fluorescence assay combined with PCR, catalytic hairpin assembly (CHA), and graphene oxide (GO) was established to detect emetic Bacillus cereus in milk samples. The processes of the assay are not new, but components of the processes make the assay useful. Two partially complementary hairpin probes (H1 and FAM-H2) were designed according to the target single-strand DNA (ssDNA). The CHA reaction could be initiated only by the target ssDNA, which was generated by the denaturation of PCR amplicons. In the absence of the target ssDNA, CHA reaction could not be triggered, which caused the H1 and FAM-H2 adsorbing on the surface of GO and exhibiting a low fluorescence intensity. Addition of the target ssDNA resulted in opening of the hairpin H1 that subsequently hybridized with H2. Then, target ssDNA would be replaced from the H1 and recycled to promote another CHA reaction. Through the CHA reaction, multiple H1-H2 duplexes were generated that could not adsorb on the surface of GO. Thus, a strong fluorescence signal would be obtained. The assay showed a limit of detection for emetic B. cereus of 6.2 × 10 1 cfu/mL in pure culture and 5.9 × 10 2 cfu/mL in spiked milk without enrichment. By changing the PCR primer, the assay developed in this study had potential to detect other bacteria.

Applications of Catalytic Hairpin Assembly Reaction in Biosensing

“…Multiple analytical readouts applied for CHA based bioanalysis methods are illustrated in Figure , including fluorescent, chemiluminescent, colorimetric, electrochemical, and SPR methods. The optical method is the most satisfactory and widely used technique for the detection of biomarkers.…”

“…Various readouts applied for CHA circuits including A) fluorescence, B) colorimetry, C) chemiluminescence, D) surface plasmon resonance, and E) electrochemistry …”