| Considering the rapid development of bioscience, it is of great significance toprofile important biomolecules, especially in the aspects of desease early diagnosis,prediction and cure. Biosensing technology, as a novel analyzing tool in analyticalchemistry, has been widely used in different areas such as bioscience, clinical medicine,molecular biology and so forth, due to the advantages of high sensitivity, goodselectivity, low cost, fast analysis, continuous monitoring, etc. With the greatdevelopment of biochemistry, DNA is known as far more than just a carrier of geneticinformation. Its properties in chemical and bioelectronical have been researchedwidely and deeply, and DNA has become novel sensing materials, catalysts, and evenpharmaceuticals.On the basis of reviewing related reports, this thesis focuses on enhancingdetection sensitivity, broadening the linear response range, simplifying the process,reducing the cost, etc. Several novel, label-free DNA biosensing strategies have beenconstructed based on enzyme amplification technology, DNA biorecognition andcatalysis，and successfully applied to sensitive, specific detection of several importantbiomolecules. The details are summarized as follows:(1) Based on the PCR nucleic acid amplification, methylation-resistant restrictionendonuclease, and DNA in vitro expression, a novel sensitive sensing method wasdeveloped. The detection limit reached0.08U/mL, and the linear range betweenbioluminescence and DNA methyltransferase concentration was from0.2to100U/mL.What featured the method most was the PCR amplified linear double-stranded DNAproducts, which acted as the substrates of DNA methyltransferase andmethylation-resistant restriction endonuclease, as well as the in vitro expressiontemplate, since it contained the promoter sequence and luciferase-encoding sequence.Only all the methylation sites kept intact could the expression process undergosuccessfully. And then the expression products luciferase catalyzed the luciferinbioluminescent reaction.(Chapter2)(2) A label-free hairpin DNA probe was designed as a recognition platform for thedetection of T4Polynucleotide Kinase (PNK) based on the preferred digestion of5’phosphorylated double-stranded DNA by lamda exonuclease. The amount ofDNAzyme released from the hair DNA probe was controlled by the coaction of PNK and lamda exonuclease. The released DNAzyme then combined Hemin and catalyzedthe colorimetric reaction of ABTS2-. The method was successfully applied to thesimple, rapid, colorimetric and sensitive detection of PNK, and the screening ofrelative inhibitors. As low as0.06U/mL of PNK was detected, and a linear relationshipbetween UV-vis absorbance and PNK logarithm was obtained in the range of0.06to100U/mL of PNK.(Chapter3)(3) Making use of isothermal circular strand displacement amplification (ICSDA),target molecules and signal elements were amplified at the same time on the sametemplate for the simple sensitive detection of microRNA. The limit of detection was aslow as0.5fM, and the linear relationship between UV-vis absorbance and microRNAwas obtained ranging from0.5fM to1nM of target. Based on the conformation changeof DNA probe in the presence of target, a label-free DNA hairpin probe was designed.The opened DNA which was opened by the target acted as the template fo r thesubsequent ICSDA reaction. As the nicking enzyme nicked the polymerized strand,two kinds of DNA fragments were produced: one functioned in the same manner as thetarget, and the other one functioned as a signal element. The accumulated elementsthen bound Hemin, resulting in a DNAzyme-Hemin complex, and catalyzed theoxidation of ABTS2-. The UV-vis absorbance of the green oxidized products was usedto quantify the target.(Chapter4)... |
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