| dc.description.abstract | Cancer is a great worldwide public health concern with steadily rising incidence, especially in developing countries. According to GLOBOCAN 2020 data, colorectal cancer (CRC) ranks third in estimated number of incident cases and deaths worldwide, both sexes, all ages. The poor survival rate is largely because of lacking early diagnosis and precision medicine. Exosome is extracellular vesicle with diameter from 30 to 200 nm, secreted by all cells to body fluids, transporting functional biomolecules (nucleic acids, proteins, lipids, etc.) of normal physiology and acquired abnormalities. Namely, exosomal miRNA-21 and Carcinoembryonic antigen (CEA) have been revealed to regulate the pathogenesis of colorectal cancer and numerous diseases. However, non-invasive methods for early diagnoses and personalized treatments are hindered by challenges of isolating and profiling these circulating biomarkers.
Leveraging the capacity to obtain pure exosomes of immunoaffinity method and advantages of paper platform – an ideal candidate for point-of-care testing (POCT), this study presents a paper-based immunoaffinity device developed by fabricating Whatman paper surface with anti-CD63 antibodies to specifically target CD63 protein on exosome membrane. Number and morphology of captured exosomes were evaluated using paper-based enzyme-linked immunosorbent assays (P-ELISA) and scanning electron microscopy (SEM), respectively. Following initial successes, we designed further procedures for exosomal nucleic acids and proteins to be analyzed. First, we used high temperature double-distilled water, a highly purified and easily accessible laboratory water for biochemistry, to lyse the exosome and released nucleic acids were absorbed by silica particles before extracted miRNA-21 was evaluated by RT-qPCR. Otherwise, lysed solution of exosomes was directly performed ELISA for detection and quantification of exosomal CEA. At first, we used the standard exosomes from cell culture media of HCT116 (a human colorectal carcinoma cell line) to clarify the feasibility of the whole process from exosome isolation to extraction and quantification of exosomal nucleic acids and proteins. Afterward, biological samples (plasma, chronic wound fluids) were applied to demonstrate the clinical applicability of the designed methods. Finally, some properties of these approaches such as specificity, miRNA extraction efficiency compared with commercial kit as well as the stability of modified paper were also investigated.
The first success achieved was to demonstrate the feasibility of isolating and purifying exosomes from diverse samples and the ability to distinguish the number of captured exosomes. Thereafter, paper-based designed procedures for exosomal miRNA-21 extraction and exosomal CEA quantification were successfully experimented. Interestingly, the empirical results indicate that using high temperature double-distilled water as a lysis buffer was successfully applied for exosomal miRNA extraction, but not as effective as commercial lysis buffer in exosomal CEA study. Moreover, experimental results on wound fluids exposed an association between prolongation of healing and exosomal miRNA-21 reduction. Our designed procedure not only harvested more copious amount of exosomal miRNA but also consumed less sample volume than by commercial kit. These approaches feasibly adopt for different samples (plasma, chronic wound fluids, cell culture media) and manifest advantages for deeper inquiry on exosome-derived constituents. | en_US |