The Application Of One Dimensional Conductive Polyaniline Nanoarray On The DNA Electrochemical Biosensors

IntroductionDNA biosensing technique can analyse and detect DNA fragments on the basis of complementary hybridization between the single-stranded DNA fragments with DNA probes acting as the sensing element.The measurement of DNA hybridization using electrochemical method possesses several advantages such as high analysing speed, simple operation procedure and high performance/price ratio and so on,making DNA electrochemical biosensing become a practical technique.However,this technique still possesses some technical challenges in target selectivity,detection sensitivity and long-term stability.It is the hope that this emerging nanotechnology can improve the performance of DNA electrochemical biosensing.Nanostructure conductive polyaniline is widely studied because of its high conductance,good chemical and physical properties and potential applications in many areas.In this study,systematic studies have been conducted to characterize the DNA electrochemical biosensor prepared with one dimensional conductive polyaniline nanoarray.Materials and Methods1.MaterialsAll of oligonucleic acids in this study were purchased from Takara Biotechnology (Dalian)Co.,Ltd.All of chemicals in this study are analytical grades.The electrodes were purchased from Shanghai Precision & Scientific Instrument Co.,Ltd. 2.Preparation of nano-structured DNA electrochemical biosensorsIn this study,one dimensional conductive polyaniline nanoarray was synthesized on graphite electrodes using template-assistant synthesis and electrochemical self-assembling approaches,respectively.Then the oligonucleotide probes were covalently immobilized on the nano-structured electrodes to prepare DNA electrochemical biosensors.3.Electrochemical measurements of DNA electrochemical biosensorIn this study,the nucleic acid hybridization conditions,including hybridization temperature and hybridization period,for the electrochemical measurements were optimized.The effect of electrochemical indicators(daunomycin and ferrocene)on the hybridization signals has been investigated,and two scanning methods,CV and DPV, were used to evaluate the measurements.The reproducibility of the two types of DNA electrochemical biosensors was also studied.Results1.Preparation and characterization of nano-structured DNA electrochemical biosensorsThe nano-structure electrodes were characterized using electrochemical and microscopical methods.The experimental results show that one dimensional polyaniline nanoarray synthezied on graphite electrodes are of highly-ordered structure with well-spaced array and well-aligned morphology.The immobilization of oligonucleicotide probes on the polyaniline nanofibers was also identified with fluorescence microscope.2.Hybridization reactionThe results showed that the 53.7℃and one hour were the optimal hybridization temperature and period.3.Electrochemical detection of DNA electrochemical biosensorsThe sensitivity and the selectivity of the DNA electrochemical biosensor were evaluated.The measurements using the biosensor prepared with the template-assistant synthesis method showed that the electrochemical signals of daunomycin were highly related with the target concentration in the range of 7.557fmol/L to 755.7fmol/L with a good linearity of R~2=0.9817.The detection limit of the polyaniline nanoarray DNA electrochemical biosensor is about 1fmol/L.The measurement using the biosensor prepared with the electrochemical self-assembling method showed that the electrochemical signals of daunomycin were also in a good relationship with the target concentration in the range of 0.7557nmol/L to 6.0456nmol/L(R~2=0.9858),and the detection limit was measured 75.5pmol/L.When ferrocene was used as the electrochemical indicator,the linear relationship was found in the target concentration of 15.83nmol/L to 126.64nmol/L(R~2=0.9835).4.Measurements of reproducibilityThe DNA electrochemical biosensors demonstrated a good measurement reproducibility.Discussions and ConclusionsIn this study,two types of nano-structured DNA electrochemical biosensors were prepared.One dimensional conductive polyaniline nanoarray was constructured on graphite electrodes using the template-assistant synthesis and electrochemical self-assembling approaches.The electrochemical and microscopic characterization show that one dimensional polyaniline nanoarray constructed on graphite electrodes was of highly-ordered structure with well-spaced array and well-aligned morphology. Due to the higher surface area ratio,every polyaniline nanofiber functions as a collector for large quantity of oligonucleotide probes.The highly-ordered polyaniline nanoarray structure provided an excellent micro-environment for DNA hybridization. Thus,the sensitivity of the DNA electrochemical detection was which improved significantly.The experimental results showed that the biosensor prepared with electrochemical self-assembling approaches is of lower sensitivity than that prepared with the method of template-assistant synthesis.It is interpreted as the results of the differences on the morphology of these two types of nanoarray structure.The data also demonstrated that the sensitivity of DNA electrochemical biosensors directly depended on the selectivity of electrochemical hybridization indicators to ssDNA and dsDNA.We measured the target oligonucleotide at different concentrations using daunomycin and ferrocene as indicators separately.The results showed ferrocene is less sensitive than daunomycin,which could be due to their different interaction modes with DNA.