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Layer-by-layer Assembly of DNA Multilayer Films and Preparation Investigation on DNA Damage

Author: LiaoTianLu
Tutor: LuXiaoQuan
School: Northwest Normal University
Course: Analytical Chemistry
Keywords: dsDNA Layer-by-layer assembly Reversible loading Adriamycin Methylene blue Quercetin Fenton’s reagent Styrene oxide DNA damage
CLC: Q523
Type: Master's thesis
Year: 2008
Downloads: 12
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Abstract

DNA molecule immobilization is one of the most important research fields and is cared more and more. For example, the controlled adsorption of DNA is essential in the fields of investigating structure function relationship of DNA, designing the third-generation electrochemical sensor and disease diagnostics. Layer-by-layer assembled technique has brought a new opportunity for biofunctional material, owing to its easy preparation method and facile controlling every layer’s composition, thickness and orientation. Layer-by-layer assembly technique is frontal research direction in microcosmic physics and chemistry field, and supply new idea for developing next molecular instrument.The main work of this paper is focus on one of the most active field where assembled technique combines with electroanalytical chemistry, developing and fabrication novel modified electrodes based on electrochemical sensors.This paper is to find out and study the assembled electrodes modified by layer-by-layer assembled technique. Besides, the emphasis of the paper is to apply these novel modified electrodes to realizing the detection and analysis of DNA damage, which may provide many important analytical methods for the life science and relative fields. In this work, layer-by-layer {dsDNA/PEI}n films of polycationic polyethyleneimine (PEI) and negatively charged dsDNA were assembled onto glassy carbon electrode (GCE) surface. The electrochemical characteristics of the {dsDNA/PEI}n films have been studied by electrochemical impedance spectroscopy. The detection of natural dsDNA damage in {dsDNA/PEI}2 layer-by-layer films induced by model genotoxic agent SO or Fenton’s reagent is realized by using electroactive probe ADM and MB or QC. This DNA damage sensor may provide a novel and general approach for detecting DNA damage electrochemically. The details are listed below:The first Chapter: General Introduction and ConclusionThis part reviews the theoretical and practical significance of the research of DNA damage, the Layer-by-layer assembled technique’s preparation, characteristic, and studies on application in DNA molecular immobilization and detection DNA damage. The second Chapter: Characterization of dsDNA layer-by-layer films using electrochemical impedance spectroscopy A new dsDNA layer-by-layer films fabricated based on electrostatic attraction in this laboratory was firstly characterized by the electrochemical impedance spectroscopy (EIS). The relationship between the charge-transfer resistance and double-layer capacitance with the number of layers was obtained through analyzing the impedance data. It demonstrated that the multilayer film showed a unique structure with the film growth. Compared to other electrochemical methods, the electrochemical impedance spectroscopy was proved to be a very sensitive and useful technique for characterizing the dsDNA layer-by-layer films. The third Chapter: Detection of DNA damage induced by styrene oxide in dsDNA layer-by-layer films using Adriamycin as electroactive probeBy differential pulse voltammetry (DPV) measurement, two bilayers were selected as the optimum films in our experiments. The detection of natural dsDNA damage in {dsDNA/PEI}2 layer-by-layer films induced by model genotoxic agent SO is realized by using electroactive probe ADM. Based on the loading reversibility of ADM into the dsDNA films, the unique‘‘loading/release/reloading’’mode is used to highlight the difference of loading capability between intact and damaged dsDNA in the films and minimize the influence of ADM leaking from the films. The result was consistent with those done by Scanning electrochemical microscope (SECM). It may provide a general and sensitive approach to detect chemical DNA lesion. The fourth Chapter: Detection of DNA damage induced by styrene oxide in dsDNA layer-by-layer films using methylene blue as electroactive probeBy differential pulse voltammetry (DPV) measurement, two bilayers were selected as the optimum films in our experiments. The detection of natural dsDNA damage in {dsDNA/PEI}2 layer-by-layer films induced by model genotoxic agent SO is realized by using electroactive probe MB. Based on the loading reversibility of MB into the dsDNA films, the unique‘‘loading/release/reloading’’mode is used to highlight the difference of loading capability between intact and damaged dsDNA in the films and minimize the influence of MB leaking from the films. The result was consistent with those done by Scanning electrochemical microscope (SECM). This DNA damage sensor may provide a general and sensitive approach to detect chemical DNA lesion. The fifth Chapter: Detection of natural DNA damage induced by Fenton reagent with dsDNA layer-by-layer filmsThe layer-by-layer {dsDNA/PEI}2 films assembled by alternate adsorption of polycationic polyethyleneimine (PEI) and negatively charged natural DNA onto glassy carbon electrode (GCE) surface. Quercetin was utilized as the electroactive probe and loaded inside the DNA films to detect the chemically damaged DNA caused by Fenton’s reagent. Based on the loading reversibility of quercetin into the dsDNA films, the unique‘‘loading/release/reloading’’mode is used to highlight the difference of loading capability between intact and damaged dsDNA in the films. Cyclic voltammetry (CV) of loaded quercetin was used to detect dsDNA lesion at different time of DNA layer-by-layer films in solution of Fenton’s reagent. This DNA damage sensor may provide a novel and general approach for detecting DNA damage electrochemically.

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