Dually Exo III-catalytic recycling amplification to construct a novel ratiometric fluorescence biosensing method for bleomycin assay

The development of methods for the convenient determination of the bleomycin (BLM) antibiotic is significantly important in the clinical therapy field. Herein we report a novel ratiometric fluorescence biosensing method for BLM assays by utilizing two target-sensitive fluorescent probes to produce reversible signal response. One signal arose from the target recognition-triggered and exonuclease III (Exo III)-catalytic release of a G-quadruplex sequence. This could cause the recombination of the G-quadruplex with thioflavin T (ThT) to realize the “signal-on” fluorescence output. The other signal arose from the target recognition and Exo III-catalytic reaction-induced DNA hybridization of a ROX fluorophore-labeled strand with its quencher-labeled strand. This proximity quenching effect could realize the “signal-off” fluorescence output of the method. Meanwhile, the dually Exo III-catalytic recycling greatly amplified the ThT and ROX-based fluorescence response. Through the measurement of the ratio of the two reversible responses, a novel ratiometric fluorescence biosensing method was successfully constructed for the homogeneous assay of BLM in a very wide linear range from 50 pM to 5 μM, with a very low detection limit of 15.8 pM. The excellent analytical performance, convenient manipulation, and low assay cost of the method determine its high potential for future applications. • We designed two target-sensitive fluorescence probes for ratiometric assays of BLM. • ThT and ROX-based fluorescence signals provided reversible response for biosensing. • Dually Exo III-catalytic recycling reactions greatly amplified the two responses. • The smart DNA design decided the highly selective homogeneous assay of the method. • This biosensing method exhibited excellent performances for the POCT application.