Application Of Rolling Circle Amplification In Cancer Cell Detection And Molecular Logic Gate

Cancer and metal ion pollution has imperiled human health seriously due to worsening of the environment. Optical analysis method has become a popular analytical technique owing to its simplicity, high sensitivity, practicality and easy operation. This paper developed two new aptamer biosensors, used for high sensitivity detection of cancer cells and detection of trace metal ions. The method has the advantages of high selectivity and high sensitivity, also demonstrate a broad application prospects in environmental protection, clinical studies and in vitro diagnostic. The mainly research contents of the thesis are as follows:1. Metal ions triggered ligase activity for rolling circle amplification and its application in molecular logic gate operations.Hg2+specifically and strongly binding to a thymine-thhy mine (T-T) mismatch in DNA duplexes to form stable T-Hg2+-T base pairs, while Ag+binds to two cytosine (C) bases to form C-Ag+-C complexes, these features provide a new and versatile platform for the construction of molecular machines for metal ions detection, and even the performance of logic gates. This precise hybridization of padlock to the primer makes the3’and5’ends of padlock adjacent, which induces the activity of the DNA ligase. As a consequence, the padlock probe is specifically ligated and circularized with the primer by DNA ligase. Subsequently, an RCA reaction is conducted in the presence of DNA polymerase/dNTPs. The resulting RCA products with thousands of repetitive DNA domains make the molecular beacons (MBs) open and restore their fluorescence due to the hybridization between the RCA product and MBs, enhance the fluorescence signal greatly, realized the high sensitivity and excellent selectivity detection of the Hg2+and Ag+.2. The application of rolling circle amplification in cancer cell detection.we present a sensitive and versatile chemiluminescence imaging platform for the detection of Ramos cells (human Burkitt’s lymphoma cells) by combining the cell aptamer recognition with the amplification of RCA and bio-bar-code nanotechnology. The RCA reaction was initiated on the AuNPs by hybridizing primers with circular DNA templates in the presence of Klenow DNA polymerase/dNTPs. The HRP-labeled bio-bar-code AuNP probes were prepared by attaching a large amount of streptavidin-HRP to biotinylated detection probes on RCA-amplifled concatamers in a periodic pattern. Meanwhile, the arrays were prepared by immobilizing biotinylated capture aptamer TE02on the surface of streptavidin-coated96-well microtiter plate. In such a case, Ramos cells could be easily captured on the well surface by aptamer TE02and sandwiched between aptamers TE02and TD05. In the presence of luminol (reaction substrate), H2O2(oxidizer), and PIP (enhancer), the CL readout was finally performed on a cooled low-light CCD.