A Highly Selective ECL DNA Sensor Based On Hairpin DNA Probe Labeled With Ruthenium Complex

Gene mutations and polymorphisms due to small genetic changes in DNA sequence, such as the replacement of one or several nucleic acid loss or increased, will lead to genetic change or the emergence of various diseases. Therefore, the analysis of human blood, body fluids and other specific DNA sequence, can be used to confirm the source of infection. DNA hairpins have been found to exhibit extraordinary stability, high selectivity and specificity compared to similar assays performed using single-stranded DNA. Wide-scale genetic testing requires the development of easy-to-use, fast, inexpensive, miniaturized analytical devices. Biosensors offer a promising alternative for nucleic acid assays duo to fast, cheap and simple.Electrogenerated chemiluninescence (ECL) is the luminescence generated by relaxation of exited state molecules that are produced during an electrochemically initiated reaction. The ECL method allows a highly sensitive detection and control the ECL the reaction through modulation of applied potential without expensive instrumentation. The generation of light in the vicinity of the electrode gives better spatial control for the sensitive detection of analytes. High sensitivity can be achieved by optimizing the material, size and position of the electrodes. Molecular beacon is a kind of new fluorescence nucleic acid probe with a high sensitivity and specificity. The molecular beacon technology by its operation simple, high sensitivity and specificity may be carried on the real-time quantitative determination to the nucleic acid, and living specimen analysis. It can be used not only in the widespread application in biology research, but also in clinical applications.This thesis includes review section and research section. In the review section, the principles and characteristics of different kinds of DNA biosensors are summarized. The basic principles, characteristics and systems of ECL, and characteristics of molecular beacon are introduced. The developments in applications of the molecular beacon in DNA sensors are reviewed, and the purpose and content of this thesis are presented. The aim of present work is to develop a high sensitivity and selectivity DNA biosensor. We combine the merit of hairpin DNA and the ECL technique with the nucleic acid hybridization technology to develop a new ECL sensor based on hairpin. It will be able to be used in the detection of DNA hybridization and mismatch rapidly and simply.The research section contains two subunits. In the first subunit, a novel sensitive ECL sensor for the detection DNA hybridization based on hairpin probes was fabricated. The ECL sensor - "switch off" mode - was fabricated by self-assembling the ECL probe on a gold electrode through thiol group at its 5' terminal. In the absence of target single strand DNA (ss-DNA), the ECL probe on the electrode was in the folded configuration and its termini were held in close proximity to the electrode, thus resulting in a strong ECL signal. In the presence of target ss-DNA, the loop of the ECL probe on the electrode was converted into a rigid and linear double helix configuration due to a hybridization with a complementary target ss-DNA, allowed to remove the tag of Ru(bpy)₂(dcbpy)NHS away from the electrode surface and thus the ECL signal was dropped off. The decreased ECL signal was found to be related to the concentration of complementary target ss-DNA in a range from 2.7×10^-10 mol L^-1 to 5.4×10^-6 mol L^-1 with a detection limit of 9×10^-11 mol L^-1.In the second subunit, the effect of the probe with different loops on the selectivity of ECL biosensors was investigated. It was found that mismatch target DNA can be discriminated from complementary one. This work demonstrated that the selectivity and specificity of ECL-based biosensor can be greatly improved using a hairpin DNA suitable of stem and loop length as a recognition element.