New Sensing Technologies Based On Both-end Blocked Hairpin Type DNAzyme And DsDNA-SYBR Green I Photosensitization

Nucleic acid is one of the most important biological macromolecules that make up living organisms.It guides and regulates the synthesis of proteins and the related functions of the organism's cells,so it is considered to be the basic genetic material of organisms.So far,nucleic acids have been used as an important biomarker for biological research and medical diagnosis.There are many methods for detecting nucleic acids,including fluorescence,colorimetry,electrochemical,and chemiluminescence.Catalysis reaction is always an wonderful tool for fabrication of ultrasensitive sensing technologies,and is widely used in life analysis,food analysis,environmental analysis,and medical analysis.Here,we combine DNAzyme catalyzed reaction and photosensitization to chemiluminescence sensing technologies,aiming to establish ultrasensitive sensing systems for detection of miRNA/DNA.The thesis includes two research sections:???Both-end blocked hairpin type DNAzyme was suggested for target-catalyzed hairpin assembly?CHA?to develop high signal to background sensing technology.In reported work,single-end blocked DNAzymes were usually applied for monitoring CHA process.This type of DNAzyme can probably lead to formation of intermolecular DNAzymes,accompanying high background and preventing the activation of DNAzyme?low signal?.The both-end blocked DNAzyme was inactivated by fixing its one end in the stem and the other end in the stem with an block efficiency as high as 96%;more importantly,its activity can be easily recovered by the CHA process,and the recovery efficiency can achieve 94%.The mechanism of the system was verified by polyacrylamide gel electrophoresis,UV spectroscopy,and chemiluminescence.The chemiluminescence intensity was linearly dependent on the amount of miRNA-21 in the range of 0.50 pM-0.50 nM with a detection limit of approximate 0.20 pM after optimizing the concentrations of H1,H2,hemin,incubation temperature and incubation time.Subsequently,other such as miRNA let-7a,miRNA let-7c,miRNA let-7i,single-base mismatched miRNA-21 sequence and three-base mismatched miRNA-21 sequences were selected to investigate its selectivity.Finally,the practical application of this method in cancer cell lysates and normal human cell lysates was investigated.The results showed that the chemiluminescent signal of the target miRNA-21 in the cancer cell lysates was significantly higher than that of the normal human cell lysate.And the chemiluminescence signal increased linearly with the increase of lysates of cancer cells.Therefore,this method is expected to be appealing for determination of miRNA in biological samples.???dsDNA-SG photosensitization-based chemiluminescence system was developed for sensing of DNA.Here we found K4[Fe?CN?6]can be oxidized into K₃[Fe?CN?₆]via dsDNA-SG photosensitization,and the resulted K₃[Fe?CN?₆]futher oxidize luminol,generation strong CL signals.The phenomenon was futher evolved in to a lable-free and ultrasentive sensing technology for DNA detection.The mechanism of the system was verified by UV spectroscopy and chemiluminescence.The concentration of K₄[Fe?CN?₆],the type of nucleic acid dye,the concentration of SG,the excitation wavelength,the illumination time and the luminol pH were optimized.Under optimal conditions,the linear range of detection of BRCA1 by photocatalytic chemiluminescence system was 5.0 pM-5.0 nM,and the detection limit of BRCA1 was 3.0 pM;the linear range for detection of BRCA2 is 10 pM-5.0 nM,and the detection limit for BRCA2 is 6.0 pM;the linear range of p53 detection was 10pM-5.0 nM,and the detection limit of p53 was 5.0 pM.The sensitivity of this system can be compared with the method with signal amplification technology.Through examination of the selectivity,this photocatalytic chemiluminescence system has strong selectivity for target DNA.In addition,the method can select the target DNA randomly for detection,and has the advantages of strong practicability,simple operation and low cost,and is expected to be applied to the detection of actual biological samples.