Ultrasensitive detection of CYN based on the cascade amplification strategy of RCA-DNAzyme and Cas12a

Cylindrospermopsin (CYN) is a class of cyanotoxins found widely across the globe, primarily in environments such as lake water, river water and drinking water. Ingestion can severely impair the function of organs such as the liver, kidneys and lungs. Consequently, the detection of CYN in the environment is crucial for safeguarding human health and public health safety. However, traditional detection methods are time-consuming and cumbersome to operate, and are prone to producing false-positive results. Consequently, there is an urgent need to establish a highly specific and accurate method for the ultra-sensitive detection of CYN. This study has developed a highly specific and ultra-sensitive detection method for CYN toxins based on a strategy combining rolling circle amplification DNAzyme with CRISPR/Cas12a cascade amplification. This technique utilises isothermal nucleic acid amplification technology and the trans-cleavage activity of CRISPR/Cas12a to amplify the signal. In the presence of the target algal toxin, the toxin binds to the arch-shaped probe, releasing an activator that triggers the amplified DNAzyme, thereby generating a large number of CRISPR/Cas12a activation sequences. This activates the trans-cleavage activity of CRISPR/Cas12a, which cleaves the reporter probe to produce a significantly enhanced fluorescent signal. The detection limit for CYN using this strategy is 2.4 nM. This method enables ultra-sensitive and highly specific detection of CYN and is suitable for the identification of CYN in real-world samples. The fluorescence detection of CYN achieved through the coupling of DNAzymes with CRISPR/Cas12a via rolling circle amplification provides an innovative solution for the ultra-sensitive detection of CYN.