| Biosensors, which were based on analytical chemistry, biochemistry, biomedicine and biotechnology, have been an attractive researching topic in recent years. Biosensors have been paid attention to for the merits of simple device, easy to carry, low cost for testing, fast and convenient, sensitive and reliable, easy to implement in situ living detection. DN A florescence biosensors are one kind of the biosensors. DNA florescence biosensors translate and then, put out the signal of the target into florescence signal, thus to analysis the analyte qualitatively and quantitatively. This method combines the merits of the biosensors of specificity and easy to realize cycle amplification in signal and the specialty of sensitive, reliable, specific and widely used fluorescence biosensors. This combination has greatly enhanced the sensitivity, accuracy, practicality, and won this technology further reputation and thus, pushed this method to develop.However, with the development of gene sequencing, pathologic diagnosis, food and drug supervision, environmental protection, the request for DNA florescence biosensors is becoming higher and higher. In this thesis, DNA florescence biosensors are researched and experimentally used to detect the concentration of human p53 gene, thrombin and L-histidine.The specific contents of the study are as follows:Part I Sensitively and specifically detection of p53 gene based on the recycle amplification of DNA and the hydrolysis reaction of Exonuclease Ⅲ.Two partially complementary hairpins(HP1 and HP2) were designed as a probe pair. Since these two kinds of DNA strands had been annealed and formed hairpin structures separately to the utmost before being mixed, their spontaneous hybridization with each other was kinetically prevented for the complementary domains were caged in the stems of the hairpin structures. When target DNA was added to the system, it opened the stem region of HP1 in line with the principle of the toehold- mediated SDR. The freshly exposed sticky end of HP1 triggers a second SDR by associating with the sticky end of HP2. Then, the target molecule was released and could trigger more hairpin pairs into unfolding and forming duplex complex. It’s worth noting that HP1 and HP2 both had stretching 5’-hydroxyl termini in the end of their stems, and the duplex-structure formed between them was designed with extending 3’ ends, on the contrary. Thus, when Exo Ш, which can specifically hydrolyze double strand DNA from blunt or recessed 3’ end, was introduced into the mixture, the extra hairpins with recessed 3’ end were digested whereas the duplex formed of HP1 and HP2(HP1-HP2) remained for their extended 3’ end. Subsequently, when the fluorescent dye SYBR Green I was added, it intercalated into the as- formed dsDNA, and emitted significantly enhanced fluorescence signal. In the absence of the target DNA, the hairpin pair can not be unfolded, and are supposed to be digested by the enzyme. The fluorescence intensity varied positively with the concentration of the HP1-HP2 complex, and thereby reflected the amount of target DNA. The detection limit of this method for p53 gene is 5.34 pM with good selectivity and repeatability.Part II Toehold strand displace ment-driven assembly of duplex DNA for sensitive detection of thrombin.In this part, a principle for label- free detection of thrombin on the base of target- induced, toehold strand displacement-driven assembly of double-stranded DNA has been proposed. The DNA duplex forming sequences are originally locked in the hairpin structures of HP1 and HP2, and the inducing strand is partially hybridized with the thrombin binding aptamer strand. When there is no thrombin, no free inducing strand can be released and the DNA hairpins sta y locked. In this occasion, low fluorescence signal can be detected. In the presence of thrombin, the high binding affinity between the aptamer and thrombin cause the binding and thus, the inducing strand is released. The liberated inducing strand further hybridizes with HP1 and unfolds the locked hairpin of HP1, leading to the exposure of toehold for strand displacement by HP2. HP2 hybridizes with the newly exposed toehold and replaces the inducing strand through the toehold strand displacement principle. The replaced inducing strand again hybridizes with the hairpin HP1 and initiates the toehold strand displacement cycles, resulting in the generation of a great amount of DNA duplex. Subsequently, when the fluorescent dye SYBR Green I was added, it intercalated into the as- formed DNA duplex, significantly enhanced fluorescent emission can be emitted. The fluorescence intensity varied positively with the concentration of the HP1-HP2 complex, and thereby reflected the amount of target of thrombin molecules. The detection limit for thrombin of the proposed method is 0.42 pM and the selectivity is good.Part III A target recycling cascade amplification strategy for L-histidine detection based on L-histidine-cofactored DNAzyme.The unmodified DNAzyme- inducing strand nicking system serves as a molecular recognition machine for L- histidine. The signal reporting part is consisted of L-histidine, hairpin DNA, primer DNA, fluorescent dye SYBR Green I and polymerase. The DNAzyme consists of an enzyme strand, a substrate strand and a poly-T linking strand. The intramolecular linkage of the two strands can enhance the hybridization efficiency to provide high catalytic activity. The loop of the hairpin DNA is designed to be complementary to the released strand. The primer strand is complementary to the 3’-segment strand of the DNAzyme.In the presence of the target molecule, a DNA-histidine complex is formed. The complex can execute a catalytic reaction resulting in a cleavaged substrate strand at the scissile ribonucleic acid adenosine. Thus, the cleaved duplex regions lack thermal stability and the two parts of one DN Azyme separate. When the 5’-end shorter strand is released from the DNAzyme, it can hybridize with the loop of the hairpin. Under the assist of the polymerase, the released strand extends, forming new duplex with the hairpin DNA. And the hairpin DNA is unfolded, allowing the trigger DNA to attach and triggers a target-displacement polymerization and produces plenty of duplex DNA, which can exert strong fluoresce nce emission when SYBR Green I is added. While the 3’-end segment of the DNAzyme is exposed as an overhang, where the primer strand can attach and initiate another target-displacement polymerization which enhances the fluorescence signal further. At the sa me time, L- histidine is released and then binds to another DNAzyme to trigger another bout of target recycling reaction. So, the final fluorescence intensity can reflect the amount of the L- histidine. The detection limit of this method is calculated to be 1.73 p M with high specificity due to the specific recognition of the DNAzyme of L- histidine. |
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