Terminal deoxynucleotidyl transferase-initiated molecule beacons arrayed aptamer probe for sensitive detection of metastatic colorectal cancer cells

摘要

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the world, which can lead to considerably high mortality rate. It was reported that the prognosis is extremely poor and survival is often measured in months once CRC metastases become clinically evident. Therefore, the development of effective approach for metastatic CRC cells detection and imaging may potentially be significant and helpful for CRC prognosis and treatment. Therefore, we proposed a sensitive and specific approach for high-metastatic CRC LoVo cells detection and imaging by using terminal deoxynucleotidyl transferase (TdT)-initiated molecule beacons (MBs) arrayed fluorescent aptamer probes (denoted as TMAP). In this approach, the aptamer W3 targeting high-metastatic CRC LoVo cells was elongated to form W3-poly A at the 3'-hydroxyl terminus with repeated A bases in the presence of TdT and dATP. The MBs designed with poly T sequence in the loop were then hybridized with the poly A in the aptamer W3. The TMAP was easily constructed without the need of aptamer modification. It was demonstrated that this approach could specifically detect and image the high-metastatic CRC LoVo cells from the mixture of high-metastatic CRC LoVo cells and non-metastatic HCT-8 cells. Compared with 6-carboxyfluorescein (6-FAM) labeled aptamer W3, the TMAP was demonstrated to have a much stronger fluorescence signal on the target cells, realizing a 4-fold increase in signal-to-background ratio (SBR). Determination by flow cytometry allowed for detection of as low as 23 CRC LoVo cells in 200 mu L cell culture medium. The high sensitivity and the capability for using in complicate biological samples imply that this approach holds considerable potential for metastatic CRC detection and therapy.