Electrochemiluminescence Detection Of Oxidative DNA Damage

The oxidative stress response in living organisms is due to the imbalance of reactive oxygen and antioxidant defense pathways.DNA,lipids and proteins in organisms are often exposed to reactive oxygen species such as hydroxyl radicals?OH·?,superoxide anions(O₂^·-),singlet oxygen?¹O₂?and peroxides?HOO^-?and so on,which can oxidize DNA to cause a variety of damage,including nucleotide deletion,oxidation,conversion and single/double-strand breaks.8-oxoguanoside?8-oxodGuo?is the most important DNA base oxidative damage product in the presence of hydroxyl radicals and singlet oxygen.When DNA is replicated,8-oxodGuo can cause mutations in the base G:C?T:A,resulting in genetic or genotoxicity.8-oxodGuo has been studied and applied in many fields as an effective biomarker for DNA oxidative damage.For example,in the field of medical diagnosis,8-oxodGuo is used to evaluate the risk of cancer in individuals and to diagnose diseases associated with free radicals.In the study of aging mechanisms or degenerative diseases,8-oxodGuo is also the main target.Therefore,it is necessary to construct an analytical method that can detect DNA damage quickly and sensitively.Until now,many methods for detecting DNA oxidative damage have been reported,in which the electrochemistry basedsensingtechniques?photoelectrochemistry,electrochemistry,electrochemiluminescence?have attracted the attention of many scientific researchers due to its rapid detection,simple equipment,and easy realization of high throughput.This thesis mainly includes two parts:The first part reviews the generation of oxidative DNA damage and its detection methods.On the other hand the principle,characteristics and application of electrochemiluminescence analysis are also summarized in detail.The second part is the research work.A"light switch"molecule was synthesized and introduced as an electrochemiluminescent signal probe,and a specific DNA glycosylase was introduced to amplify DNA damage.Based on which,a rapid,simple and sensitive electrochemiluminescence method for the detection of DNA oxidative damage was proposed.It mainly includes the following three parts:1.Synthesis and characterization of[Ru?bpy?₂dppz]²⁺?denoted as,Ru-dppz?and investigation of its interaction with DNA:Firstly,the bipyridyl complex dipyridine[3,2-a;2',3'-c]phenazine?Dppz?was prepared by the reaction of 1,10-phenanthroline 5,6-dione with o-phenylenediamine,and cis-bis?2,2'-dipyridyl?ruthenium dichloride?II?Dihydrate?Ru?bpy?₃Cl₂?react with dppz to produce bipyridine dipyridine[3,2-a;2',3'-c]phenazinium,?Ru-dppz?.Next,the structure of Ru-dppz was characterized by UV-vis spectroscopy,nuclear magnetic resonance spectroscopy and mass spectrometry.The interaction between the Ru-dppz and DNA were studied in by fluorescence method.The results showed that the fluorescence intensity of Ru-dppz gradually increased with the addition of DNA,indicating that Ru-dppz can be used as a DNA molecule"light switch".The binding constant of Ru-dppz to DNA was1.5×10⁷L/mol by fluorescence titration experiment,and the binding ratio was 0.14,which was basically consistent with the literature.2.The Ru-dppz prepared in the first step was used as the ECL signal probe to establish a label-free electrochemiluminescence method to detect DNA oxidative damage caused by Fenton reagent.As we know,the DNA glycosylation enzyme,Fpg?E.coli formamidopyrimidine?can recognize and excise 8-oxodGuo,hydrolyze phosphodiester bond to form a nick,which was introduced to to amplify oxidative damage of DNA and produce more DNA strand breaks.Firstly,considering the electrochemical and electrochemiluminescence behaviors of Ru-dppz,the indium oxide tin oxide?ITO?electrode modified with reduced graphene oxide?r-GO?and AuNPs?Au?composite nanomaterial is selected as the substrate electrode.A dsDNA film is firstly assembled on the ITO/r-GO@Au electrode.As a DNA intercalator with“light-switch”behavior,Ru-dppz is employed as the ECL signal reporter,and ECL emission is measured and the decrease of ECL intensity reflect the DNA damage,which were also confirmed by gel electrophoresis and fluorescence measurements.Under optimal conditions,the proposed method can detect DNA damage caused by 10nM Fe²⁺/40 nM H₂O₂ Fenton reagent,and the sensitivity is 1000 times higher than that of the similar photoelectrochemistry methods.This method provides a simple protocol for the highly sensitive detection of DNA oxidative damage.3.To further verify the universality of the proposed method,we studied DNA damage caused by another environmental pollutant,bengal rose.The mechanism of DNA oxidative damage caused by reaction with bengal red can produce singlet oxygen through energy transfer to damage DNA under irradiation.It is now well established that only guanine is the substrate of the ¹O₂ oxidation,and 8-oxodGuo is found to be the main¹O₂ oxidation product of DNA.As showing much higher specificity than hydroxyl radical,other?tandem?lesions are not produced,benefiting the detection of DNA damage with the proposed method.With the help of DNA glycosylation enzyme Fpg to specifically recognize and cleave 8-oxodGuo,the DNA damage caused by bengal rose was detected.The results showed that the method can detect the DNA damage caused by as low concentration as 0.1?g/mL of bengal rose red.