A novel biosensor based on antibody controlled isothermal strand displacement amplification (ACISDA) system

Abstract

The overuse of antibiotics has aroused widespread concern in recent decades. Their residues in food and environment may pose potential risks to human health. Therefore, highly sensitive and rapid detection methods of antibiotics are urgently needed. Inspired by allosteric transcription factors (aTFs), we proposed a novel strategy for small molecules detection based on antibody controlled isothermal chain displacement amplification (ACISDA). A combination of nicking endonuclease, Klenow Fragment polymerase, specific antibody and a pair of antigen-labeled DNA regulate the synthesis of a G-quadruplex by isothermal chain displacement amplification. The presence of a target induces the antibody dissociation from the antigen-labeled DNA, which induces the synthesis of a G-quadruplex, and a fluorescent signal is produced by thioflavine T (ThT) binding to G-quadruplex. To test this notion, norfloxacin-conjugated DNA (named Primer-NOR) was prepared and ACISDA system was established combining with anti-norfloxacin antibody. This system could detect norfloxacin in a linear range of 0.1 ∼ 500 ng/mL with detection limit of 0.04 ng/mL, and this system could be applied to the detection of norfloxacin in real samples with good performance. Meanwhile, this system could also realize washing-free, immobilization-free and “ready-to-use”, and could be used for other small molecules quickly by replacing the antigen-labeled DNA and specific antibody.