DNA Hybridization Detection Based On Electrogenerated Chemiluminescence

The analysis of DNA sequence and DNA mutant detection plays fundamental roles in the rapid development of molecular diagnostics and in the anticancer drug screening. Sensitivity in DNA hybridization and other bioassays is important in clinical diagnostics, forensic chemistry, environmental investigations, pharmaceutical studies and biological warfare agent detection. A single strand DNA (ssDNA) probe binds to its complementary strand (target ssDNA) to form double strand DNA (dsDNA) with high efficiency. The signal provided by the probe is a measure of degree of hybridization. A variety of techniques are available for the detection of DNA hybridization, of which electrochemical, fluorescent, radiochemical, piezoelectronic have been developed. Although these methods display many advantages, they have not been used to take place completely the traditional method because of low sensitivity or complex equipment or other shortcomings. So it is necessary to develop rapid and sensitive methods for the detection of specific DNA sequences.Electrogenerated chemiluminescence (ECL) is a technique that combines chemiluminescence (CL) and electrochemistry (EC). The CL reaction was initiated by an electrochemical reaction at electrode surface. Such an electro-initiation reaction introduces a large number of additional advantages, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. Due to these inherent advantages, ECL method has attracted much attention from all analytical fields, especially from biochemical analysis. The aim of the present work is to develop highly sensitive and selective methods for the DNA hybridization detection based on electrogenerated chemiluminescence. Based on coupling with DNA hybridization, immobilization techniques and nano-technology, we have developed a series of ECL methods for the determination of special DNA sequence, compared with other methods, the developed methods are simple, rapid, as well as sensitive. A part of research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No. 20375025).This thesis includes a review and a research section. In the review, the basic principles, characteristics and systems of ECL, the principle of DNA detection are introduced. The principles and characteristics of various kinds of DNA detection are summarized. Trends of ECL in DNA hybridization and drug (filtration) are reviewed and the purpose and content of the thesis finally are presented. The research section contains three subunits. Subunit 1, the study of ECL detection of specific DNA sequence A novel ECL method for the determination of DNA sequence is developed using Ru(bpy)2(dcbpy)NHS as label and target single strand DNA(HS-ssDNA) immobilized on Au electrode by self-assembly technique. Ru(bpy)2(dcbpy)NHS was synthesized and used as ECL label to prepare Ru(bpy>2(dcbpy)NHS-DNA probe. The Ru(bpy)2(dcbpy)NHS -DNA probe was then used to hybridize with the target ssDNA immobilized on the electrode and the hybridization events were evaluated by the ECL signals from Ru(bpy)2(dcbpy)NHS. The results show that the Ru(bpy)2(dcbpy)NHS -DNA probe is able to recognize complementary sequence to form dsDNA. The linear response to the complementary sequence is from 3.4xlO'10 to 3.4xlO"7mol/L with a detection limit of 1.2xlO"10 mol/L.Subunit 2, the study of ECL detection of specific DNA sequence based on sandwich hybridizationIn subunit 1, the target single strand DNA (HS-ssDNA) was immobilized on Au electrode by self-assembled monolayers technique, the target ssDNA must be functionalized at the 5' end with a thiol group. So the application is limited. In order to demonstrate further the application possibilities, sandwich hybridization was developed to assay the specific DNA sequences. In sandwich hybridization, the target ssDNA would not need to be modified with thiol group in the analytical procedure. The specific DNA could not only be captured by the immobilized probe, but also hybridized with the labeled probe (Ru(bpy)2(dcbpy)NHS-DNA). The results showed that the Ru(bpy)2(dcbpy)NHS-DNA probe is able to recognize complementary sequence to form dsDNA. The linear range for complementary sequence is from 8.8xlO'10 to 8.8xlO'8 mol/L with a detection limit of 3.0X 10"10 mol/L.Subunit 3, the study of ECL detection of specific DNA sequence based on Au nanoparticle self-assembly Au electrodeWe use Au nanoparticle to enhance the DNA immobilization amount on gold...