DNA Electrochemical Biosensor Assisted By Enzymatic Tools For Signal Amplification

Gene detection plays a key role in clinical medicine,which is of great significance in early diagnosis,prognosis evaluation and drug treatment.In future,gene detection will move toward the development of point of care system for improving the clinical efficiency and reducing the medical cost so that a large number of people could be benefited.DNA electrochemical biosensor,with advantages of easy microminiaturization,speediness,high throughput,high sensitivity,high selectivity and low cost,shows a great prospect in clinical application.In order to achieve the trace analysis in real sample,it’s particularly important to improve sensitivity by means of signal amplification.This paper established simple and highly sensitive DNA electrochemical biosensor to realize the detection of complex real sample by employing enzymatic tools which have been widely applied in molecular biology for its highly catalytic activity,high biocompatibility,high specificity and high-flex model.This research will laid solid foundation for wide application of DNA electrochemical biosensor in clinic.The main content of this paper is depicted as follows:In the first part,electrochemical DNA biosensor based on grafting-to mode of terminal deoxyribonucleotide transferase-mediated（TdT）extension was constructed.Herein,a specific polymerase which could catalyze the incorporation of deoxy-ribonucleoside triphosphate（dNTP）into the terminal of 3’-OH in DNA without template under isothermal condition was utilized.Different from the traditional DNA electrochemical biosensor based on surface initiated enzymatic polymerization（SIEP）by employing TdT,this experiment introduced multiple signaling probe through the way of homogeneous extension prior to hybridization on the electrode surface.Results indicated that the high background in the mode of SIEP was caused by the TdT’s action on the surface rather than the nonspecific adsorption of DNA probe.Still,grafting-to mode could avoid the generation of high background so that a higher amount of TdT could be employed to complete the overall reaction in short time reducing from 120 min to 20 min.The comparisons in the aspects among background,time cost and signal intensity significantly showed grafting-to mode was greatly better than SIEP’s in the electrochemical DNA biosensor.In the second part,a novel electrochemical DNA biosensor based on ligation chain reaction（LCR）amplification and double-strand DNA（dsDNA）assembling mode was presented.Firstly,a large quantity of dsDNAs modified with thiol and biotin at each end were obtained from homogeneous LCR,and then the rigid dsDNAs were uniformly inserted into the interval of bovine serum albumins（BSAs）assembled on the gold electrode surface through Au-S bond by group of thiols.After that,horseradish peroxidases（HRPs）were fixed onto the gold electrode surface through the specific binding between biotin and streptavidin-HRP.Finally,the electrochemistry reduction current was measured after HRP catalysis in TMB-H₂O₂substrate.A linear relationship between the amperometric signal intensity and the logarithmical concentration of target DNA was achieved over a widely dynamic range from 1.0×10^-16 M to 1.0×10^-11 M extending six orders of magnitude with a limit detection of 0.5 aM（>3σ）in which only 15 copies of DNA molecule exist in 50μL volume of solution.Besides,this method could significantly differentiate any kind of one-base mismatch,which could be used for analysis of single nucleotide polymorphism（SNP）.In real sample assay,the proposed biosensor could detect gene of C-type hepatitis B virus（HBV）from complex real sample containing plenty of genome DNA and RNA with different length while it had no response to B-type and normal type sample,which exhibited excellent detection performance,also the electrochemical results in concordance with results from gene sequencing and gel electrophoresis were proven to be precious and reliable.Most importantly,this method is universal by designing different primer sequences,and it is expected to qualitative and quantitative analysis of different DNA markers relevant to clinical diseases.On the ground of the second part,the third part proposed an electrochemical biosensor coupling SA-PolyHRP with LCR to achieve dual-signal amplification for detection of PML/RARαfusion gene in acute promyelocytic leukemia（APL）,and it was realized by the binding of multiple HRPs onto the end of each dsDNA uniformly distributed on the electrode surface,which we called it post-amplification strategy.Herein,we chose two enzyme complexes,SA-HRP-biotin complex and SA coated ploy-HRP,to carry out our experiment,because these two products were widely applied in enzyme-linked immunosorbent assay（ELISA）to introduce multiple HRPs through two different ways.Results showed that the latter,due to its higher affinity to biotin on the electrode surface for its external streptavidin layer,had remarkable signal amplification than the former.Although,PML/RARαgene confined near the fusion site has limited in design of primer sequence,the sensitivity of PML/RARαgene’s detection based on the proposed method could be guaranteed,which could also detect as low as 15 copies of target DNA and had a linear relationship between the amperometric signal intensity and the logarithmical concentration of target DNA ranging from 1.0×10^-16 M to 1.0×10^-13 M.Compared with SA-HRP,SA-PolyHRP could improve the sensitivity by nearly two orders.In addition,this method could significantly distinguish any kind of single base caused by the mismatch,and the PML/RAR fusion gene probe designed in this experiment is more significant in the ability to distinguish single base mismatch.