Grup de Sensors i Biosensors, Departament de Química, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
Biosensing and Bioanalysis Group, Institute of Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain.
Anal Chem. 2023 Jan 31;95(4):2487-2495. doi: 10.1021/acs.analchem.2c04773. Epub 2023 Jan 22.
Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 10 exosomes μL. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.
外泌体作为癌症检测的新型生物标志物受到了广泛关注，因为它们在不同的生物体液中大量由肿瘤细胞释放。在本文中，外泌体通过免疫磁分离（IMS）基于 CD326 受体进行预浓缩，CD326 受体是一种特定的上皮癌相关生物标志物，并通过甘油醛-3-磷酸脱氢酶（GAPDH）转录本进行检测。捕获的外泌体裂解后，通过逆转录聚合酶链反应（RT-PCR）用聚（dT）修饰的 MPs 上的双标记引物扩增释放的 GAPDH 转录本，以提高灵敏度。然后通过电化学生物传感定量双标记扩增子。IMS/双标记 RT-PCR/电化学生物传感方法首先用于敏感检测 MCF7 乳腺癌细胞来源的外泌体，并在分析性能方面与 CTCs 进行比较，显示出 4×10 个外泌体 μL 的检测限。该基因传感器通过直接从乳腺癌患者的血清中免疫捕获外泌体应用于人体样本，并显示出更高的电化学信号（3.3 倍， < 0.05），与健康对照组相比，表明乳腺癌患者血清来源的外泌体中 GAPDH 的过表达。仅使用特异性磁性颗粒从 1.0 mL 人血清中即可检测到 GAPDH 转录本，这提高了分析的简化程度并避免了超速离心步骤，证明了这是一种用于微创液体活检的很有前途的策略。
