A Novel DNA Biosensor Integrated With Polypyrrole/streptavidin And Au-pAMAM-CP Bionanocomposite Probes To Detect The Rs4839469 Locus Of The VANGL1 Gene For Dysontogenesis Prediction

ObjectiveWe first propose the use of a DNA biosensor to detect the missense single nucleotide polymorphism(rs4839469 c.346 G > A p.Ala116Thr)of the VANGL1 gene in this work.Methods(1)Polypyrrole(PPy)and streptavidin were integrated to modify a gold electrode.We took advantage of the PPy's good biocompatibility and excellent conductivity.Three-dimensional laser scanning,SEM,FT-IR spectroscopy,and CV techniques were performed to characterize the PPy/streptavidin film.To confirm that PPy has been successfully deposited with streptavidin.(2)To further accelerate the electron transfer process at the electrode surface,polyamidoamine dendrimer-encapsulated gold nanoparticles(Au-PAMAM)were used,because Au-PAMAM possess a large number of amino groups to load capture probes(CP).Capture probes was designed to detect the rs4839469 locus of the VANGL1 gene.Au-PAMAM and Au-PAMAM-CP were characterized by TEM,HRTEM,FT-IR spectroscopy and UV-vis spectroscopy.(3)When detect the target DNA,using the biotin-streptavidin system,the Au-PAMAM-CP bionanocomposite probe,which can detect the target DNA,was conjugated to the electrode surface.The successful coupling of the target DNA and CP was investigated by agarose gel electrophoresis.(4)CV and EIS were applied to characterize the modification steps of the DNA biosensor using [Fe(CN)6]3-/4-as a redox probe,and were performed in a 5 m M [Fe(CN)6]3-/4-solution containing 0.1 M KCl.(5)The electrochemical performance of the DNA biosensor may be influenced by many experimental conditions,such as the deposition time,hybridization temperature,hybridization time,and capture time.Therefore,a series of optimizations of these parameters was studied systematically.(6)The electrochemical responses of [Fe(CN)6]3-/4-toward different concentrations of target DNA(0.1,1,10,50,100 n M)were measured by DPV under the optimal conditions.(7)The specificity of the biosensor was investigated with four types of 10 n M ss DNA: Mixture,single-base mismatched DNA(SBM),three-base mismatched DNA(TBM),non-complementary DNA(nc DNA);Repeatability is another important criterion for DNA biosensors: five modified electrodes were used to detect c DNA(10 n M)for getting the relative standard deviation(RSD);Furthermore,the DNA biosensor was storaged at 4? for two weeks to test the stability;(8)To exclude the potential interfering species,the validity of the proposed signal amplification strategy to real clinical samples was investigated.We challenged our sensing strategy with the presence of target DNA in a complex sample matrix: 5% and 10% human serum.Target DNA(10 n M)was spiked into the human serum and was assayed using the developed DNA biosensor;To examine the applicability of the present DNA biosensor to biological samples,the biosensor was used to test the recoveries of three concentrations of the target DNA.In this study,the real samples were prepared by a standard addition method.Each concentration was measured three times using five different electrodes with DPV analysis.Results(1)The PPy has been successfully deposited with streptavidin.(2)The Au-PAMAM-CP bionanocomposite probes were successfully synthesized.(3)The formation of the stem-loop structure of the CP was demonstrated.(4)The DNA biosensor in this study was successful.(5)The optimal deposition time,hybridization time,capture time and hybridization temperature were 4 min,30 min,40 min 35?,respectively.(6)Under optimal conditions,the DNA biosensor exhibited a wide linear range of 0.1 n M to 100 n M with a low detection limit of 0.033 n M(S/N = 3).(7)we performed a comparison study between mismatched targets(SBM,TBM,nc DNA),a perfect complementary target(c DNA)and a blank control,and the biosensor made a good specialty;Five modified electrodes were used to detect c DNA(10 n M),and the relative standard deviation(RSD)was 3.1%,suggesting that the reproducibility of the DNA biosensor is acceptable;The DNA biosensor also remained bioactive after two weeks of storage at 4? and the current response showed no significant changes.The result demonstrates that the developed electrochemical DNA biosensor has satisfactory stability.(8)There is no obvious current change,which demonstrated the capacity of this assay for the analysis of real samples in which the influence of the matrix could be neglected;The validity of the proposed signal amplification strategy to real clinical samples was good,and the recovery was in the range of 103% to 105%.ConclusionThe results suggest that this approach has the advantages of simple,low cost,high sensitivity and good reproducibility,and can be used for detection of rs4839469 locus of VANGL1 gene We propose that this assay might be a promising approach to perform DNA-based diagnostics.