Constructure And Application Of Novel DNA Electrochemical Biosensors

Deoxyribonucleic acid?DNA?is a basic unit that forms the genetic function of the organism and maintains the normal functioning of its life.It has become a hot spot in the field of life science and bioanalysis.Moreover,the development of functional nucleic acids and the use of a variety of signal amplification technologies based on gene amplification have made DNA play an increasingly significant role in the field of sensors.Electrochemical sensors have developed rapidly in recent years due to their advantages such as low cost,fast response,good selectivity and easy miniaturization.In order to improve the detection sensitivity or detect the genotoxicity of substances easier,DNA is often used to construct various types of electrochemical sensing systems.In this paper,aiming at the key problems of the electrochemical sensing system related to DNA,namely how to improve the detection sensitivity and how to detect the effect of substance on DNA,a series of new electrochemical sensing systems are constructed with different materials,different modification methods and different reaction systems,and are applied into the analysis of many kinds of sbstance.Various electrochemical techniques,such as cyclic voltammetry?CV?,electrochemical impedance spectroscopy?EIS?,differential pulse voltammetry?DPV?,and other techniques,for example,scanning electron microscopy?SEM?,ultraviolet visible spectrophotometry?UV-vis?and X ray diffraction?XRD?were used to studied in detail the structure,properties and detection performance of electrochemical sensors and the modified materials.The main research work of this paper is as follows:?1?A novel DNA electrochemical biosensor was constructed by modifying Pb²⁺-dependent DNAzyme to gold electrode via self-assembly combining the dual signal amplification strategy of HCR and enzyme catalysis plus redox reaction.The electrical properties of electrodes modified to different degrees were characterized by CV and EIS.The effects of cleavage and HCR time on the detection of Pb²⁺ were explored.Under the optimal experimental conditions,the linear range?0.1-1000 n M?and detection limit?0.01 n M?of the sensor were measured.Compared with other sensors,the sensor exhibited wider linear range and lower detection limit.In addition,the sensor had good selectivity,stability and reproducibility,which indicated the excellent overall analysis performance of the sensor.Finally,the sensor was applied to the detection of Pb²⁺ in the real water sample,and the acceptable recovery is obtained,which proved the practicability and accuracy of the sensor.The sensor provides a highly sensitive and highly selective method for the determination of Pb²⁺ for environmental detection.?2?By drop-coating method,the calf thymus ds DNA and?poly?diallyldimethyl ammonium chloride?,PDDA?were adsorbed to the glassy carbon electrode surface,combining with SnO₂ nanopowder?or WS₂ micropowder?,HCl and H₂O₂ mixture system,a novel DNA damage electrochemical biosensor system was constructed.Futhermore,the genotoxicity of SnO₂ nanopowder?or WS₂ micropowder?was successful detected by the prepared sensor.The morphology and structure of SnO₂ nanopowder and WS₂ micropowder were characterized by TEM and XRD.Using Co?NH₃?₆³⁺ and Ru?NH₃?₆²⁺ as electrochemical indicators,the electrochemical signals at the modified electrode before and after immersed in SnO₂ nanopowder?or WS₂ micropowder?,HCl and H₂O₂ mixture system were recorded by CV and DPV,and the signal difference before and after soaking proved the occurrence of ds DNA damage.Next,the effect of soaking time and solution component on ds DNA damage was investigated.The damage mechanism was deduced,namely,Fenton-like reaction took place in SnO₂ nanopowder?or WS₂ micropowder?,HCl and H₂O₂ mixture system,and produced ·OH,resulting in the oxidization damage to ds DNA.In addition,the construction of the biosensor provided a research idea for the genotoxicity detection of other nanomaterials.?3?Poly?3,4-ethylenedioxythiophene??PEDOT?and platinum nanoparticles?Pt NPs?were modified to the glassy carbon electrode by electrochemical method,and they greatly increased the electrochemical signal of chlorogenic acid?an active component of the Chinese medicine?,based on this,a high sensitivity electrochemical sensor for the detection of chlorogenic acid was constructed.In order to obtain the maximum sensitivity,the optimum acidity and scanning speed in the detection of chlorogenic acid are studied by experiments.Under the optimum condition of 0.1 M ABS?p H 5.0?,the sensor showed the linear range of 0.2-100 ?M and the detection limit of 0.05 ?M.Then,the sensor was used to detect chlorogenic acid in real sample of Shuanghuanglian injection with satisfactory recoveries.Furthermore,the interaction of chlorogenic acid and salmon sperm DNA was studied,and the results showed that the binding process mainly occured in the first 25 min,and the binding mode mainly was electrostatic mode,the binding number was 3,and the binding parameter was 1.62×103 L mol^-1.The binding force was non-covalent bond and small,resulting in the small binding constant,which proved that the chlorogenic acid produced another form of damage to DNA.?4?Graphene oxide was modified to glassy carbon electrode by drop-coating method,and greatly increased the oxidation current of m-Trihydroxybenzene?m-THB?and p-Nitrophenol?p-NP?,based on this,an electrochemical sensor for the simultaneous determination of m-THB and p-NP was preparared.CV was used to characterize the electrochemical properties of electrode and compare the difference in detection of m-THB and p-NP before and after modification.DPV was used to separate and simultaneous determine of m-THB and p-NP.At last,the interaction of m-THB,p-NP and herring sperm DNA was studied with two methods of UV visible absorption and electrochemistry.The results showed that the two phenolic compounds both combined with DNA mainly by the intercalation mode,and the binding number both was 1,the binding parameter of m-THB was 1.58×105 L mol^-1,and p-NP was 1.66×106 L mol^-1,which were larger than that of chlorogenic acid combination with electrostatic modes,indicating that the intercalation mode was more firmly than the electrostatic mode,and produce greater effect on DNA.?5?DPV was carried out to investigate the electrochemical signals of the four kind bases of DNA?guanine,adenine,thymine and cytosine?in the phosphate buffer solution?PBS?,Tris-HCl and acetate buffer solution?ABS?.The results showed that only in ABS,the four kind bases could obtain the oxidation signal at the same time.Thus,an electrochemical analysis method for simultaneous determination of four kind bases of DNA on bare glassy carbon electrode was established.And when this method was applied to the base determination of calf thymus DNA and salmon sperm DNA,the satisfactory results were obtained.The establishment of this sensing system not only provided more simple and convenient method for the study of DNA damage,but also provided useful reference information for other research directions about DNA.The establishment of these electrochemical sensing systems based on DNA provides new research ideas for the determination of toxic substances or the analysis of genotoxicity.It also provides a high sensitivity and selectivity research platform for the detection of similar substances.