PAM-assembled CRISPR-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for detecting prostate cancer exosomes

Prostate specific membrane antigen (PSMA)-positive exosomes hold significant potential for the diagnosis and risk assessment of prostate cancer. However, accurate detection is severely hindered by their low abundance in blood and interference from similarly sized particles. In this study, we have developed a protospacer adjacent motif (PAM)-assembled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for the highly sensitive detection of PSMA-positive exosomes. In this work, two split strands respectively containing CD63 and PSMA aptamers are utilized to bind CD63 and PSMA on the exosome surface, forming a template that induces the opening of a hairpin DNA (HP DNA). A PAM site forms via hairpin-to-double-stranded structure transition. CRISPR-Cas12a recognizes PAM, activates to cleave FAM-labeled probes for fluorescence, while cerium dioxide nanozyme (CeO 2 NZ) (with phosphatase-mimicking activity) hydrolyzes cleavage products. Hydroxyl radicals from hydrolysis oxidize 3,3′,5,5′-tetramethylbenzidine (TMB) to induce colorimetry. CRISPR-Cas12a-nanozyme dual recognition significantly improves prostate cancer exosome detection selectivity and sensitivity. Under optimized conditions, the limits of detection for the fluorescence and colorimetric modes reach 49 particles/μL and 63 particles/μL, respectively. By mutually validating dual detection modes, this biosensing technology accurately distinguishes prostate cancer patients from healthy individuals, holding great promise for early diagnosis.