CRISPR/Cas12a-based biosensors for ultrasensitive tobramycin detection with single- and double-stranded DNA activators

In addition to its application in genome editing, the CRISPR/Cas system provides a new molecular tool for the construction of biosensor platforms due to its high target sequence specificity and programmability. To exploit the potential of the CRISPR/Cas system for detecting non-nucleic-acid targets, two ultrasensitive biosensors (sensor-ss and sensor-ds) consisting of allosteric aptamer probes integrated with the CRISPR/Cas12a system were designed and constructed for tobramycin detection. Both sensors utilized hairpin DNA containing a tobramycin aptamer sequence as the target recognition probe, which can respond to target binding and further produce signal transduction sequences. However, there are two differences between the sensors. First, in sensor-ss, single-stranded DNA was set to be the activator to trigger cutting activity (trans-cleavage) of CRISPR/Cas12a, leading to cleavage of the FQ-reporter (a short single-stranded DNA labeled with a fluorophore and quencher at each end) and increasing the fluorescent signal. However, double-stranded DNA containing a protospacer adjacent motif (PAM) site was produced in sensor-ds as the signal transduction sequence, which was recognized by CRISPR/Cas12a, resulting in the cleavage of the FQ-reporter. Second, sensor-ss involved a strand displacement amplification (SDA) reaction as the signal enhancement step, while no DNA amplification procedure was designed in sensor-ds. Through DNA sequence and reaction condition optimization, sensor-ds showed a linear relationship between the fluorescence response and tobramycin concentrations ranging from 10 to 300 pM with a limit of detection (LOD) as low as 3.719 pM. Although sensor-ss exhibited higher sensitivity (LOD = 1.542 pM) than sensor-ds, rapidly reaching the fluorescent signal saturation platform led to a narrow linear response range (5–30 pM). Moreover, our CRISPR/Cas12a-based assay can realize on-site detection by the naked eye readout under UV light. The constructed sensor-ds was also used to detect tobramycin in milk and lake water samples, and the results indicated that the detection system was reliable. The design strategy reported in this study could shed new light on CRISPR/Cas12a-based biosensor construction for small molecule detection.