Novel Molecular Aptamer Beacon for the Specific Simultaneous Analysis of Circulating Tumor Cells and Exosomes of Colorectal Cancer Patients

Liquid biopsy provides non-invasive and real-time detection for cancer diagnosis, but the lack of specific markers targeted to liquid biopsy components, such as circulating tumor cells (CTCs) and exosomes, has impeded its effective utilization in clinical settings. W3 is an aptamer, and its target has been previously demonstrated to be a predictor of colorectal cancer (CRC) metastasis. Herein, we developed a W3-based molecular beacon (MAB-W3-3G) to specifically detect CTCs and exosomes derived from CRC patients by modifying the W3 sequence and adding a fluorescent group FAM at the 5' end and a quencher group BHQ1 at the 3' end, resulting in a detectable green fluorescence only in the presence of the target. MAB-W3-3G retained features similar to those of the original W3, including high specificity and affinity for metastatic CRC cells, as well as excellent plasma stability. Notably, W3 target-positive CTCs were visualized, positive exosomes were quantified in CRC patients' whole blood without any sample pretreatment, and both detections could be finished in one step without any routine washing procedures. For CRC, the W3 target-positive CTC enumeration in metastasis was higher than that in non-metastasis (p < 0.01), and the quantitation of positive exosomes was correlated with CRC patients (p < 0.0001). Moreover, the MAB-W3-3G-based simultaneous detection of CTCs and exosomes was proven to have the potential for more precise clinical diagnosis. In conclusion, MAB-W3-3G could detect CTCs and exosomes in the blood samples of tumor patients with simple manipulation, rapid analysis, and high specificity, providing an effective liquid biopsy tool for the prediction of CRC.