Study On The Preparation And Application Of An Electrical Active-inactive Switch Molecular Beacon

Study on the structure of gene and the mechanism of its functioning nowadays are a rather hot research field.And the most significant success of this field is the "Human Genome Project",which means genomic diagnose and treatment of human disease are in great prospect.The foundation of this kind of research is based on the recognition and determination of disease related DNA sequence.The traditional sequence detection methods,however,are usually time consuming and inconvenient. DNA biosensor,a new conception of genome detection which based on Watson-Crick base match principle offered an ideal option.And the DNA biosensor based on DNA hybridization technique,combining with different labeling methods such as fluorescent,chemical luminescent or electrochemical methods,demonstrate great advantages such as easy handling,quick detection,favorable selectivity,together with molecule recognition and DNA separation and purification abilities.Research on these biosensors has been one of the most interesting fields in genome study.The dissertation highlighted in two aspects.Firstly,molecule beacon and nanotech were introduced into the DNA biosensor model.Molecule beacon was used to optimize the DNA strand,which was the main part of biosensor.Its "hairpin" structure caused an additional "hairpin open" process in the biosensor detection,right before the DNA hybridization process.This made the hybridization more difficult to happen.Likewise,goldnanoparticles show affinity to single strand DNA,and the introduction of goldnanoparticles added another "peel off" process of the DNA strand, and increased the difficulty of hybridization again.Thus,to some extent,because of these two methods,the selectivity of the biosensor was greatly optimized.Secondly, in this dissertation,a totally new electrochemical molecule beacon model was settled. Different from the traditional electrochemical molecule beacon which depended on structural change of the one-end-immobilized molecule beacon induced distance variation between the signal molecule and the electrode surface and finally caused the difference of electrical signal,this new electrochemical molecule beacon is un-immobilized,and it conduct hybridization in the test solution instead of on the electrode surface.The signal variation came from an electrical active-inactive molecule switch.They were electrical inactive when the molecule beacon was in "hairpin" structure,and could become electrical active after the "hairpin" structure was opened.This new kind of electrochemical molecule beacon inherited advantages of fluorescent molecule beacon,such as quick,simple and easy to control,and had some unique predominance,such as low cost and the prospect of refit to portable devises.The dissertation includes four chapters:Chapter 1 PrefaceThe purpose and development of DNA electrochemical biosensors were introduced firstly,electrochemical determinations were the emphasis,molecular beacons and its especial detection methods were also included.Goldnanoparticles and its application in bioanalysis were introduced.At last the purpose and innovation point of the dissertation were pointed out.Chapter 2 Study on the preparation and application of a new electrical active-inactive switch molecular beaconWe developed a new electrical active-inactive switch molecular beacon and used carminic acid as signal molecule.Molecular beacon coupled with carminic acid was electrochemical inactivity,but after hybridized with complementary sequence, electrochemistry signal was greatly enhanced,and it was linear related with the concentration of complementary sequence in the range of 6.0×10^-8mol/L～1.4×10^-6 mol/L,with the detective limit of 1×10^-8mol/L.The signal of non-complementary sequence was almost the same as that of molecular beacon,and the responses of one base mismatched sequence,two bases mismatched sequence and three bases mismatched sequence were obviously distinguished from that of complementary sequence.With high sensibility and selectivity,this molecular beacon is suitable for quantitative detection of target DNA.Chapter 3 Study on the preparation and application of a high selective DNA electrical active-inactive switch molecular beacon based on goldnanoparticlsGoldnanoparticles were prepared by the reduction of chlorauric acid.An electrochemical molecular beacon based on goldnanoparticls with high selectivity was developed.The signal of non-complementary sequence was almost the same as that of molecular beacon,and the responses of one base mismatched sequence,two bases mismatched sequence and three bases mismatched sequence were obviously distinguished from that of complementary sequence.The signal of single-mismatched DNA was only 10%of that of the complementary sequence,which was much more super than any methods mentioned before,thus it could easily identify base mutation of DNA in practice.The electrochemical signal was linear related with the concentration of complementary sequence in the range of 8.39×10^-8mol/L～1.05×10^-6mol/L,with the detective limit of 2×10^-9mol/L.Chapter 4 The principle of a high selective DNA electrical active-inactive switch molecular beacon based on goldnanoparticlsElectrochemical methods were utilized for studying on the principle of the high selective molecular beacon based on goldnanoparticls.This molecular beacon could efficiently detect single-base mismatch of DNA.The reason was that ssDNA and dsDNA with goldnanoparticls had the different properties,ssDNA had strong attractive electrostatic interactions with negatively charged goldnanoparticls,and could interact with them through the coordination of nitrogen atoms,while dsDNA could not.Moreover,the selectivity of CAs-MB was related to the position and the type of mismatched base.This DNA electrochemical molecular beacon is suitable for single base mismatch detection,which provides a new way for developing simple, speedy and inexpensive bio-probe.