Study On Novel DNA ECL Probes And Their Applications To DNA Assay

Deoxyribonucleic Acid (DNA) is known as inherited substance in life body. With the increasing of knowledge about human beings diseases it is found that the mutations of base sequence in DNA are responsible for numerous inherited human disorders. In order to make early diagnosis and cure for disease, much effort is needed to detect the specific DNA sequences related to diseases, which is also helpful for the selection of genes and the development of drugs.Most of the conventional methods for the analysis of specific DNA sequences are based on the nucleic acid hybridization. Single-stranded ss- DNA probe binds to its complementary strand (target sequence) to form a double-stranded (ds) DNA with high efficiency. The signal provided by the probe is a measure of the degree of hybridization.Traditional DNA probes are labeled with radioisotopes i.e. 32P, I25I, 3H or 35S. Although this method gives high sensitivity, the radioactive labels present many problems such as a potential hazard to analyst and environment, which limited its application in DNA diagnostic laboratories. In order to overcome these problems a serious of non-radioactive DNA probes such as fluorescent, chemiluminescent and electrochemical probes have been developed. Although these new 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 other rapid and sensitive methods for the detection of specific DNA sequences, which can also be used easily in non-specialized laboratories. Electrochemiluminescence (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. In this dissertation we focused on the preparation of a new type of DNA probes which were labeled with ECL activated substances. Based on coupling with the DNA hybridization and immobilization techniques, we have developed new ECL methods for the determination of special DNA sequence. Compared with other methods the new methods are simple, rapid as well as sensitive and selective. The dissertation is composed of four chapters as folio wings:Chapter 1: Introduced the ECL and its application in analytical chemistry field, paid more attention to the types of ECL reaction and ECL sensors. Reviewed the DNA sensors, including their principles, advantage and disadvantage. At last pointed out the purpose of the dissertation.Chapter 2: Study on the ECL behaviors of several substances and establishment of ECL analytical systems1 The ECL behavior of N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was studied and found ABEI could produce emission light when oxidized at a +1.0 V (vs Ag/AgCl) potential in alkaline solution. The addition of H₂O₂ markedly improved the ECL sensitivity. The effected factors on the ECL were examined. Under optimal conditions, ABEI took a linear response to ECL intensity in the range of 1.3x10-6 6.5 xl0-12 mol/L. The detection limit is 2.2 xl0-12 mol/L at a signal to noise ratio of 3.2 The catalytic reaction of ferrocenecarboxylic acid (FCA) to Luminol and hydrogen peroxide was investigated. It was found the Chemiluminescent intensity ofLuminol with hydrogen peroxide can be greatly enhanced by electrochemically oxidizing of FCA at low potential in Tris buffer solution. Under optimal conditions the ECL intensity exhibits a good linear response with the concentration of FCA in the range of 5.0x10^-9.².0xl0-4 mol/L. The detection limit is 1.5xl0-9mol/L.3 A study on the ECL of acridinum esters...