CRISPR/Cas12a regulated dual-channel ratiometric fluorescence biosensing for sensitive and accurate detection of kanamycin

• A CRISPR/Cas12a based ratiometric fluorescent biosensor is reported for Kana assay. • The biosensor was constructed on Cas12a-assisted dual-channel fluorescence outputs. • Telomerase extension and DNA recycling amplification led to its high sensitivity. • The sensor showed good accuracy and convenient operation for practical application. Antibiotic residues are regarded as a serious threat to public health and safety. Traditional fluorescence biosensors with single signal-output channel are susceptible to producing false results during practical applications. In this work, a novel ratiometric fluorescent biosensor with dual signal-output channels was developed for assaying kanamycin (Kana) antibiotic residues in complex matrices. Through highly specific aptamer recognition to trigger a nuclease-catalytic DNA recycling reaction, the CRISPR/Cas12a system was activated to realize the non-discriminatory cleavage of a short signal DNA and the G-quadruplexes produced from telomerase (TE)-catalytic extension reaction. These resulted in their “signal-on” and “signal-off” fluorescence outputs, respectively, to construct the ratiometric signal transduction strategy. Due to the TE-extension and DNA recycling-based signal amplification and the highly efficient reactivity of CRISPR/Cas12a, both the sensitivity and signal output efficiency of the sensing system were effectively improved. Meanwhile, the self-correcting advantage of the dual-channel reverse signal transduction mode ensured the superior detection accuracy of the method. Based on the synergistic action of the above functional units, this biosensor was able to accurately, rapidly and sensitively detect Kana residues in the concentration range from 1 pg mL −1 to 100 ng mL −1 with a very low detection limit of 0.28 pg mL −1 . In addition, the good agreement between the measurement results of the method and an ELISA kit with the relative errors less than 5.3 % indicates its favorable practicability.