Assay Of DNA And Tumor Cells Based On Rolling Circle Amplification

Electrochemical biosensor possesses the advantages of specific recognition of biological molecular, biocompatibility and amplification of nanoparticals and response enhancement of electrochemical detection. It has become the main stream of modern biochemical analysis with high sensitivity, good selectivity as well as being easy to miniaturization and automate.This thesis studied a novel electrochemical biosensor based on rolling circle amplification (RCA) and bio bar code probe and its application in assay of DNA and tumor cells. Chemiluminesece was carried out based on Au-CuS-DNA nanoprobe to detect the single nucleotide polymorphisms (SNPs) and achieved satisfying results. This thesis mainly consists of the following issues:1 An electrochemical biosensor based on RCA for highly sensitive detection of DNA was established. The"sandwich-type"DNA complexes were fabricated with the thiol-functionalized capture DNA firstly immobilized on the Au electrode and hybridized with one end of target DNA, the other end of which was recognized with signal DNA labeled on the surface of Au NPs modified with 6-ferrocenylhexanethiol. Well-defined DPV peaks of high signal intensity were obtained, and the peak heights were found to be dependent on the amount of target DNA, producing a highly sensitive sensor for target DNA ranging from 1.0×10^-17 6.0×10^-15 mol/L with a limit of detection of 2.8×10^-18 mol/L.2 A novel electrochemical cytosensing strategy was constructed by using rolling circle amplification (RCA) and bio bar code DNA-conjugated CdS nanoparticles (CdS NPs) to combine the selectivity and affinity of aptamers to allow for the sensitive detection of cancer cells. The anodic stripping voltammetry (ASV) technology was employed for the elemental detection of CdS NPs labeled on the bio bar code DNA for further assay the amount of the target cells. With the use of Burkitt's lymphoma cells (Ramos) as a model, the electrochemical method could detect cancer cells with a range from 10 to 1.0×10⁴ cells mL-1. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamer used in the assay indicating the wide applicability of the assay for diseased cell detection.3 A novel and sensitive flow injection chemiluminescence assay for detection of single nucleotide polymorphisms (SNPs) based on Au-CuS-DNA nanoprobe. A preconcentration process of cupric ions was performed by anodic stripping voltammetry technology to improve the sensitivity of the biosensor. this method could respond down to 1.9×10^-17 mol/L SNPs by the amplification of Au NPs and CuS NPs, showing very high sensitivity.