Study Of Indirectly Labeled Ultrasensitive DNA Electrochemical Biosensor

1 IntroductionThe completion of the Human Genome Project is not only the genetic basis for the occurrence and development of human understanding of the disease,but also for the effective prevention,diagnosis and treatment of diseases of the molecular basis.Therefore,it is very important to study the mechanism of regulation of gene expression,to identify specific single nucleotide polymorphisms?SNP?as well as to carry out the clinical diagnosis of the gene mutation.There are many advantages in electrochemical analysis and detection of DNA detection,such as high speed,simple method,cost-effective features.But the low sensitivity of the electrochemical biosensor has been a huge obstacle to the practical application of biosensors.The current study to improve the sensitivity of electrochemical DNA sensors are more concentrated in search of better electrode modification methods to obtain greater effective surface area of the electrode,synthetic higher specific hybridization indicator between ssDNA and dsDNA and design more specific probe.Few people concerned on improving the sensitivity of the electrochemical indicator.This study improved DNA electrochemical sensor sensitivity through two aspects.Firstly,we modified conducting PANI nanowires array on the surface of platinum electrode in order to improve the effective surface area of the platinum electrode,thereby the number of probe DNA bonding on each PANI nanowire would be increased.Highly ordered PANI nanowires array provides excellent dynamic conditions and the micro-environment for DNA hybridization.The mechanisms mentioned above substantially improve the detection sensitivity of the DNA hybridization and furthermore improve the sensitivity of the DNA electrochemical sensor.Secondly,we designed a new type of DNA electrochemical signal enhancer.We used the lysine-rich polypeptide?LRP?as the medium.We covalently enriched ferrocene molecules to LRP on basis of the reaction between?-NH₂ in lysine and-COOH in ferrocenecarboxylate.Meanwhile the aniline used as an intercalator was linked to LRP.Such a structure can be intercalated on the same sites of the DNA,which enriched multiple ferrocene molecules,increasing the number of ferrocene molecules combined in the same double-stranded DNA molecules to improve the ferrocene molecules in the vicinity of the double-stranded DNA molecule i.e.,the relative concentration in the vicinity of the electrode,thereby increasing the peak current of the same concentration measured DNA response,thereby improving the detection sensitivity.2 MethodsConstant current electrochemical polymerization was used to polymerize PANI.The current density of the first step were respectively 0.02,0.04,0.08,0.16,0.32mA/cm²,and the following current densities of the next two steps were decreased step by step.Aniline in perchloric acid solution used as monomer was polymerized on the surface of Pt electrode.Electrochemical polymerization conditions were optimized to determine that the current density of the first stage was between0.04-0.16 mA/cm²,which could maximize the improvement in the effective surface area of the electrode.We use genetic engineering methods to construct different truncated sequence of human histone H1 C terminal recombinant expression vector pGEX-4T-2-LRP,which were transferred into E.coli BL21?DE3?and induced expression.Induced conditions?IPTG concentration,induction time,induction temperature?were optimized,and we obtained five different GST fusion protein?GST-LRP?.We purified GST-LRP obtained.The GST-LRPs were digested by thrombin and we obtained five different LRPsWe covalently linked probe DNA to PANI on Pt electrode to prepare DNA electrochemical biosensor.We used the biosensor to detect target DNA perfectly matching to probe DNA in order to explore the characters of the biosensor.In that detection,daunomycin was used as indicator.We explored the optimal hybridization temperature between the probe DNA molecule and its completely complementary target DNA by the biosensor.We explored a study on the stability of such a sensor.We synthesized FLAA by enriching ferrocene molecules to LRP chain step by step.We carried out a DNA electrophesis to observe DNA retardant by FLAA.We detected different concentrates target DNA by the biosensor using FLAA as indicator.We compared the five different types FLAA.3 ResultsHighly ordered PANI nanowires array were observed by SEM.Cyclic voltammetry scan indicated that the effective surface area of the modified electrode was apparently larger compared with that of bare electrode.We successfully constructed recombinant expression vector pGEX-4T-2-LRP.The vectors were transferred into E.coli BL21?DE3?and induced to express GST-LRP fusion protein.GST-LRP were digested by thrombin,and five different lengths of LRP were obtained.Daunomycin was used as indicator to detect the target DNA.The results showed that the sensor can detect the target DNA molecules in our experiments.The results of DNA retardation assay showed that it was aniline rather than LRP intercalated into DNA.FLAA comparison results showed that there was significant difference between the five types LRP when FLAA used as DNA electrochemical hybridization indicator.This new electrochemical hybridization enhancer was used to detect different concentration target DNA.The results showed that the DNA concentration linear range of 0.02pM to 2pM.The optimal hybridization temperature experimental results show that the electrochemical response of FLAA was the higest when the hybridization temperature was 57??Tm-10??.The biosensor showed good stability after three measured repeatedly.4 DiscussionsIn the present study,we have explored from two different aspects to improve the detection performance of the DNA electrochemical sensor.Firstly,we prepared PANI nanowires array showing a good homogeneity and orientation characteristics.Its electrochemical characterization of the structure also shows the PANI modified electrode has a much larger effective surface area compared with that of bare electrode.The polyaniline array modified electrode provides a greater effective surface area,so that each of the polyaniline nanofiber seems as a hybridization signal collection.And the highly order PANI nanowires array provides good dynamic conditions and the micro-environment,therefore the detection sensitivity of the DNA hybridization were substantially improved.Secondly,we synthesised a new type of DNA electrochemical signal enhancer.We used the lysine-rich polypeptide?lysine-rich peptide,LRP?as the medium.We covalently enriched ferrocene molecules to LRP on basis of the reaction between?-NH2 in lysine and?-COOH in ferrocenecarboxylate.Meanwhile the aniline used as an intercalator was linked to LRP.Such a structure can be intercalated on the same sites of the DNA,which enriched multiple ferrocene molecules,increasing the number of ferrocene molecules combined in the same double-stranded DNA molecules to improve the ferrocene molecules in the vicinity of the double-stranded DNA molecule i.e.,the relative concentration in the vicinity of the electrode,thereby increasing the peak current of the same concentration measured DNA response,thereby improving the detection sensitivity.5 ConclusionsIn this study,we optimized the conditions of polymerizing PANI nanowires array,which was modified on platinum electrode to prepare DNA electrochemical biosensor.In the present study,we used lysine-rich polypeptide as medium to synthesis a new type of DNA hybridizing signal enhancerPANI nanowire arrays modified DNA electrochemical biosensor was used to detect different concentrations of DNA.When the DNA concentration was within the range of from 0.02 pM to 2 pM,the new electrochemical enhancer had a good linear response.