Label-free detection of miRNA cancer markers based on terminal deoxynucleotidyl transferase-induced copper nanoclusters

• Published in: Analytical biochemistry Vol. 585; p. 113346
• Main Authors: Li, Yiting, Tang, Dihong, Zhu, Li, Cai, Jingting, Chu, Chaonan, Wang, Jing, Xia, Man, Cao, Zhenzhen, Zhu, Hong
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.11.2019
• Subjects: Cancer diagnosis; Copper nanoclusters; Label-free; miRNA-21; Terminal deoxynucleotidyl transferase; Copper nanoclusters; Label-free; Cancer diagnosis; Terminal deoxynucleotidyl transferase; miRNA-21
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2019.113346

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, a label-free and sensitive fluorescent method for detection of miRNA-21 is described based on duplex-specific nuclease (DSN) assist target recycling and terminal deoxynucleotidyl transferase (TdT) induced copper nanoclusters (CuNCs). In the absence of target, the 3′-phosphorylated probe DNA cannot be hydrolyzed by DSN and extended by TdT, and failed to synthesizing fluorescent CuNCs. However, the target miRNA-21 can caused the digestion of probe DNA with DSN, releasing primer DNA with 3′-OH. After that, the primer DNA can forms long poly T with the assistance of TdT, leading to synthesize high fluorescent CuNCs. The fluorescence change of CuNCs can be used to identify the concentration of target miRNA-21. Under optimal experimental conditions, this strategy could quantitatively detect miRNA-21 down to 18.7 pM. We have also demonstrated the practical application of our proposed method for monitoring miRNA-21 expression levels in cancer cells. Moreover, this method show good specificity for miRNA-21 detection due to the strong preference of DSN for cutting perfectly matched DNA/RNA duplex, which holds great potential for highly specific quantification of biomarkers in bioanalysis and clinical diagnosis. A label-free and sensitive fluorescent method for detection of miRNA-21 is described based on duplex-specific nuclease (DSN) assist target recycling and terminal deoxynucleotidyl transferase (TdT) induced copper nanoclusters (CuNCs). This strategy was also applied to detecting miRNA-21 in cancer cell samples. [Display omitted] •A label-free and sensitive fluorescent method for detection of miRNA-21 is described based on copper nanoclusters.•Duplex-specific nuclease is used to cut probe DNA and assist target recycling.•Terminal deoxynucleotidyl transferases can extent the released primer into poly T DNA.•This method can also be used for monitoring miRNA-21 expression levels in different cancer cells.