Universal DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a bioassay for the synchronous detection of two exosomal proteins and its application in intelligent diagnosis of cancer

Exosomal proteins are considered to be promising indicators of cancer. Herein, a novel DNAzyme walkers-triggered CRISPR-Cas12a/Cas13a strategy was proposed for the synchronous determination of exosomal proteins: serum amyloid A-1 protein (SAA1) and coagulation factor V (FV). In this design, the paired antibodies were used to recognize targets, thereby ensuring the specificity. DNAzyme walkers were employed to convert the contents of SAA1 and FV into activators (P1 and P2), and one target can produce abundant activators, thus achieving an initial amplification of signal. Furthermore, the P1 and P2 can activate CRISPR-Cas12a/Cas13a system, which in turn trans-cleaves the reporters, enabling a second amplification and generating two fluorescent signals. The assay is highly sensitive (limits of detection as low as 30.00 pg/mL for SAA1 and 200.00 pg/mL for FV), highly specific and ideally accurate. More importantly, it is universal and can be used to detect both non-membrane and membrane proteins in exosome. Besides, the method can be successfully applied to detect SAA1 and FV in plasma exosomes to differentiate between lung cancer patients and healthy individuals. To explore the application of the developed method in tumor diagnosis, a deep learning model based on the expressions of SAA1 and FV was developed. The accuracy of this model can achieve 86.96%, which proves that it has a promising practical application capacity. Thus, this study does not only provide a new tool for the detection of exosomal proteins and cancer diagnosis, but also propose a new strategy to detect non-nucleic acid analytes for CRISPR-Cas system.