Ortho-FusionMetaboliprobe for Sensitive and SpecificIn Situ Detection of Metabolites in Tumor-Derived Exosomes

Abstract Small molecule metabolites carried by exosomes have emerged as promising biomarkers for cancer liquid biopsy. However, their clinical application is hindered by the complexity of blood components, the difficulty in distinguishing tumor-derived exosome subpopulations, and the low abundance of metabolites. Here, we develop an Ortho-Fusion Metaboliprobe for the in situ detection of small molecule metabolites in tumor-derived exosomes with high specificity and sensitivity. The Ortho-Fusion Metaboliprobe employs a dual allosteric aptamer system targeting CD63 and EpCAM to form an orthogonal labeling barcode on the surface of tumor-derived exosomes, enabling precise discrimination of tumor exosome subpopulations from complex biological backgrounds. Following orthogonal labeling, liposomes functionalized with complementary DNA tags recognize this barcode via zipper hybridization, triggering targeted membrane fusion between the liposome and the exosome. This fusion event delivers the encapsulated Au NFs-based metabolite detection probes into the exosomal lumen, where they react with target metabolites and generate amplified fluorescent signals for sensitive and specific in situ detection. Using this assay, we successfully detected ATP and spermine in exosomes derived from prostate cancer cell lines and clinical plasma samples. Both metabolite markers effectively distinguished prostate cancer patients from benign controls, demonstrating the clinical potential of this assay. This strategy offers a powerful tool for the analysis of small molecule metabolites in tumor-derived exosomes and holds significant promise for early cancer diagnosis and screening.