A universal and programmable platform for fluorescent detection and profiling of exosomes based on bispecific aptamer-programmed DNAzyme-switched CRISPR/Cas12a†

Recent research highlights the pivotal roles of exosomal surface proteins as diagnostic biomarkers for distinguishing various types of cancer. Consequently, comprehensive detection of exosomal surface proteins is essential to early diagnosis of cancer and protein function research. Therefore, there is a clear need for a portable and rapid method for the early detection of cancerous exosomal surface proteins. In this research, we proposed an intelligent, flexible and ultrasensitive system based on the combination of a bispecific aptamer-programmed DNAzyme, a universal DNA activator and CRISPR/Cas12a (DDC system), which was employed to achieve signal transduction and amplified fluorescent detection of exosomal surface proteins. Owing to the simultaneous binding of double-positive target proteins and bispecific aptamers, the false-positive result arising from interfering proteins in biological samples could be effectively eliminated. Moreover, the generated universal activator via DNAzyme-catalyzed cleavage of the hairpin probe enabled the detection of multiple surface proteins on exosomes with high specificity. Under the optimal conditions, our platform demonstrated an excellent dynamic range spanning 6 orders of magnitude and high sensitivity with a detection limit as low as 3.5 × 10³ particles per mL. To the best of our knowledge, the protein profiling of exosomes derived across various cell lines is established to differentiate even subtle variations in expression levels for the foundation of protein function research. Meanwhile, the proposed platform may provide a useful and practical tool for exosome identification, together with the associated early diagnosis, recurrence monitoring of cancer and elucidation for the rational design of functional nanosensors.