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Novel DNA-Based Machine for Signal Amplification of DNA Hybridization

Author: FangCongCong
Tutor: ZhangShuSheng；ZhangHui
School: Qingdao University of Science and Technology
Course: Applied Chemistry
Keywords: DNA nanoparticles ATP circular amplification voltammetry
CLC: TP212.3
Type: Master's thesis
Year: 2011
Downloads: 75
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Abstract


With the development of molecular biology, the study on DNA has been a hot spot of the life science and materials science. In summary, the high sensitivity, ease of fabrication and a low cost make methods in this paper may provide an interesting alternative tool for detection protein in clinical laboratory. And it has a good developmental prospect.The main jobs of this paper can be concluded as follows:1. A Novel Electrochemical DNA Biosensor Based on One-to-One Recognition Tri-Au/CuS NPs Signal DNA ProbeThis new nano-structures due to the space terminals can be applied to the detection of biological samples by transmission electron microscopy （TEM） characterization. We confirm the truth of the formation of uniform bridge-type Tri–Au/CuS NPs structure. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands which were modified with CuS NPs. A preconcentration process of cupric ions performed by anodic stripping voltammetry （ASV） technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the DPASV intensity was increased with the increase of the concentration of target DNA in the range of 1.0×10-14 to 1.0×10-12 mol/L. A detection limit of 3.8×10–15 mol/L（3σ） of target DNA was achieved.2. An Autonomous Bio-barcode DNA Machine for Exponential DNA Amplification and Its Application to the Electrochemical Determination of Adenosine Triphosphate In the present study, a novel electrochemical method for exponentially amplified detection of ATP （as a model analyte） in cancer cells is developed by using an autonomous bio-bar-code DNA machine. Firstly, proportional relationship of ATP analyte and DNA （acted as signal conduction medium） is constructed on the basis of competition between ATP and DNA for ATP-binding aptamers. Secondly, signal conversion and exponential amplification of DNA is achieved in the DNA machine system that is driven by template–dependent DNA replication manipulated by Klenow fragment （KF） of Escherichia coli DNA polymeraseⅠ. Thirdly, a one–to–one recognition triple Au nanoparticles DNA probe labeled with CuS tags （tri–Au/CuS NPs probe） is used in a“sandwich–type”strategy for the detection of DNA exported from DNA machine system. CuS tags are dissolved in strong acidic solution and detected using a sensitive differential pulse anodic stripping voltammetry （DPASV） method. The method is sensitive to relevant changes in ATP concentrations (2×10-8 mol/L 2×10-6 mol/L) and shows a high selectivity for ATP when compared to other nucleoside triphosphate analogs. Furthermore, successful determination of ATP in cancer cells proves practicability of the DNA machine. In conclusion, the DNA machine provides not only a general protocol for the amplified detection of aptamer-based small molecule analyte, but also a model for applications of biomolecular devices.3. Ultrasensitive electrochemical analysis of Target DNA by using an autonomous DNA machine that works in a two-cycle modeWe reported a novel autonomous DNA machine for amplified electrochemical analysis of DNA. The DNA machine carries out a two–cycle working mode to amplify DNAs recognition events, which is assisted by two different nicking endonucleases （NEases）. A bio-barcode CdS NPs–DNA probe is used for tracing target DNA. The detection system is based on a sensitive differential pulse anodic stripping voltammetry （DPASV） method for the detection of Cd2+ tracers. It is proved that the DNA machine system can simultaneously amplify target DNA more than 4 orders of magnitude within 30 min at room temperature. Under the optimized conditions, detection limit as low as 4.1×10-17 mol/L （3σ） for target DNA is achieved.

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CLC: > Industrial Technology > Automation technology,computer technology > Automation technology and equipment > Automation components,parts > Transmitter ( converter),the sensor > Biological sensors,medical sensors
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