A microchip electrophoresis-based assay for ratiometric detection of kanamycin by R-shape probe and exonuclease-assisted signal amplification.

Excessive intake of kanamycin (KANA) can cause some serious drug-resistant diseases, so it is urgent to develop some accurate and rapid analytical methods for monitor KANA residues in foodstuffs with complex matrix. Recently, many ratiometric assays were reported to be capable of overcoming matrix interference. Herein, a ratiometric and homogeneous assay for KANA detection based on microchip electrophoresis (MCE) was developed. First, by one single strand DNA (S-DNA) and one hairpin DNA (H-DNA), a novel R shape DNA probe (R-DNA) was prepared. After the probe was incubated with KANA, the S-DNA-KANA complex was formed, and H-DNA was released. Moreover, in the presence of exonuclease I (Exo-I), S-DNA-KANA complex would be digested to release the captured KANA for triggering target recycling and signal amplification. With the reaction going on, the fluorescence intensity of H-DNA (I) increased and that of R-DNA (I) decreased. They can be separated at different voltage intensities and converted to fluorescent signals for signal readout by MCE. The signal ratio of I/I was found to be linear toward target from 0.5 pg mL to 10 ng mL, and the limit of detection was 150 fg mL. Moreover, it was successfully employed for KANA detection in milk and fish samples with consistent results of enzyme linked immune sorbent assay (ELISA). The R-DNA probe can quantitatively convert the amount of target to the intensity of DNA without label by MCE, and achieved exonuclease assisted signal amplification in homogenous solution. It was valuable to detect antibiotics residues in foodstuff with complex matrix. This approach broadened the application field of MCE to detect antibiotics without derivatization, which provided a promising platform for rapid screening of antibiotic residues in food.