Multiplexed and highly sensitive FRET aptasensor for simultaneous assay of multiple antibiotics via DNAzyme and catalytic strand displacement amplification cascades

Background

The emergence of antibiotic-resistant microorganisms poses significant risks to public health. Therefore, the development of technologies capable of detecting antibiotics with high sensitivity and selectivity is essential for monitoring and controlling the spread of antibiotic resistance. Yet, current major available antibody-based antibiotic detection methods often face limitations in sensitivity, complexity, and cost, and commonly one target antibiotic can be detected in one assay.

Results

On the basis of a three-way DNA junction (3-WJ) signal construct, we describe a multiplexed fluorescence resonance energy transfer (FRET) aptasensor strategy for highly sensitive simultaneous detection of sarafloxacin (SAR) and enrofloxacin (ENR) through cyclic DNAzyme and catalytic strand displacement reaction (CSDR) signal amplification cascades. Target antibiotics are recognized separately by the aptamers in DNAzyme/apamer duplexes to release active DNAzyme sequences, which cleave the dumbbell substrate hairpins to free ssDNAs to trigger subsequent CSDR between the assistance hairpins and the 3-WJ constructs for formation of many fluorophores 5-carboxyfluorescein (FAM)- and 2′,7′-dimethoxy-4′, 5′-dichloro-6-carboxyfluorescein (JOE)/6-carboxy-X-rhodamine (ROX)-labeled DNA duplexes. This leads to the pulling of FAM dye donor in proximity to the ROX and JOE dye acceptors, facilitating the yield of considerably amplified FRET signals at 555 nm and 605 nm for the SAR and ENR assays, respectively, with detection limits of 1.95 pM (0.76 ng/L) and 5.01 pM (1.8 ng/L) within 2.5 h. Additionally, this sensing method can selectively discriminate SAR and ENR against non-target antibiotics and has been validated for the simultaneous detection of SAR and ENR in milk samples.

Significance

Featured with the advantages of convenient and significant signal amplification capability as well as single excitation for multiplexed detection, the successful demonstration of our method for sensitive and simultaneous detection of two antibiotics therefore shows its promising potential for constructing different multiplexed aptasensors for detecting various low levels of biomolecules.