A self-actuated CRISPR/Cas12a feedback amplification platform for ultrasensitive detection of exosome

The inherent heterogeneity of tumour-derived exosomes holds great promise for improving the accuracy of cancer diagnosis. Exosomes have emerged as valuable biomarkers for the early detection of cancer. Therefore, we need to develop a rapid, sensitive and highly specific technique for exosome detection. This manuscript presents an accurate and sensitive fluorescence strategy for detecting exosome surface proteins using allosteric Y-shaped structures (AYSs) combined with a dual CRISPR/Cas12a feedback amplification (DCFA) system. Our strategy relies on the revitalisation of the first CRISPR/Cas12a system by co-recognition of double-positive proteins and AYSs, which in turn induces the activation of the DCFA system, enabling amplified fluorescent detection of low abundance exosomes. The ingeniously designed AYSs that contain bi-specific aptamers and restrained activator enable to achieve precise and sensitive quantification of exosomal surface proteins owing to the dual-recognition mode, which effectively prevents the false-positive signals arising from the interfering proteins in the biological samples. Notably, the strategy demonstrated widen detection range across from 5 x 104 to 5 x 1010 particles/mL and improved sensitivity with the limit of detection as low as 1.15 x 104 particles/mL for exosome due to highlyefficient DCFA system. With its capability of accuracy, sensitivity, specificity and convenience, this AYSs and DCFA system-based strategy is anticipated to be a worthwhile tool for exosome identification, early diagnosis and therapeutic monitoring of cancer.