B-PER tandem assembly CRISPR/Cas12a cascade amplification strategy based fluorescence/colorimetric dual-mode for...

Microcystins are the most common, most powerful and most toxic types of cyanobacteria, which seriously threaten the public health and ecological environment. Biosensor has been widely used in Microcystin-Leucine-Arginine (MC-LR) detection. In this study, we report an efficient single-hairpin with double primers (the trigger chain T hybridizes with the left end a, and the amplified product ab hybridizes with the right end a) for bidirectional PER (B-PER) amplification strategy for uniform visual and fluorescence detection of trace amounts of MC-LR in the environment using quantum dots (QDs) as signal reporters. The biosensor is triggered by a single stranded DNA of MC-LR specificity to initiate the amplification by B-PER. From the long product of the B-PER, the trans-cleavage activity of CRISPR/Cas12a is activated, and the Ag⁺ is released from the C-Ag⁺-C. The released Ag⁺ undergoes cation exchange reaction (CER) with CdTe QDs (QDs), quenching QDs fluorescence and generating visual and fluorescent dual signals. The biosensor can simultaneously complete the display of dual signals of naked eyes and fluorescence, and can successfully used for the detection of the actual environment sample. In addition, the biosensor has low detection limits, high sensitivity, good accuracy and high selectivity. The detection range under the best conditions was 0.05-500 nM, and the detection limit was 0.705 pM. In summary, this strategy provides a general detection platform for detecting trace pollutants in the environment by using biosensors.