Colorimetric Detection Of Proteins And Nucleic Acids Based On DNAzyme Amplification

Proteins, nucleic and other biological molecules are very important formaintaining life activities. Changs of these biological molecules will lead tophysiological disorders and dieases. Therefore, the detection of protein and nucleicacid has the realistic significance for the normal function research and diagnosis ofdiseases. Colorimetric methods, due to its excellent properties such as simplitity, lowcost and potential ability of on-line detection, will show wide applications in the fieldof biosensing assay for proteins and nucleic acids. However, one major limition ofcolorimetric approch is its low detection senstivity. Aming at the chllange incolorimetric assays,based on the metal ion-catalyzed cleavage property of8-17DNAzyme and HRP-miniking catalytic property of G-Quadruplex DNAzyme, thefllowing three works have been finished in this thesis by engineering8-17DNAzymewith G-Quadruplex DNAzyme:1. Cleavege of DNA sequence and fabrication of DNAzyme cascade signalamplification-based colorimetric sensing platform.By the help of denaturingpolyacrylamide gel electrophoresis, the digestion experiments of DNA or DNA/proteinconjugates were explored by the addtion of ExoI with the3’â†'5’ cleavage property.Morever, cleavage of the8-17DNAzyme was also investigated by using gelelectrophoresis. From gel results, we can conclude that the ExoI has the bestsingle-strand cleavage property. Based on these investigations, a DNAzyme-medidatedsignal amplification-basd colorimetric biosesing platform was proposed, offering areliable foundation for the next detection applications.2. DNAzyme-medidated signal amplification for colorimetric assay of protein. Inthis paper, one of the numerous growth factors that regulate cell growth and division,platelet-derived growth factor （PDGF）-BB，was chosen as the model biomarker. Theaptamer probe was first band to the target protein in simple solution, and then theaptamer probes that bind target proteins are protected, whereas free single-strands arecompletely degraded by Exo I. The protected aptamer probes are then hybridized withharpin-structured catalytic-beacon substrates, and the cleavage reaction will be carriedout and the HRP-miniking DNAzyme is continuously generated. Then the producedHRP-miniking DNAzyme is used to catalyze the H2O2-mediated ABTS colorimetricsystem. So, the detection of PDGF-BB can be realized via the absorbance change. The signal response of this sensing ensemble appear a good linear correlation in the targetconcentration range from0.02to5nM, with a detection limit of10pM. The selectivityof this sensing ensemble is very good.3. DNAzyme-medidated signal amplification for colorimetric assay of SNP. Inthis paper, thalassemia gene was chosen as the model biomarker. The capture probeswas first immobilized on the surface of magnetic nanoparticles. In the presence oftarget DNA sequence, the8-17DNAzyme enzyme sequence will be captured on to themagetic nanoparticles surface via the cross-linking between8-17sequence and thecapture probe under the precense of DNA ligase. After the magetic sepration,harpin-structured catalytic-beacon substrates was added. Then the cleavage reactionwill be carried out in the precense of Pb2+and the HRP-miniking DNAzyme iscontinuously generated. Last, the produced HRP-miniking DNAzyme is used tocatalyzed the H2O2-mediated ABTS colorimetric system to realized the SNP detection.The signal response of this sensing ensemble show a good liner correlation in thetarget concentration range from2to100nM, with a detection limit of0.97nM.