Graphene Sheets, Polyaniline And Aunps Based DNA Sensor For Electrochemical Detemination Of Bcr/abl Fusion Gene With Functional Hairpin Probe

Chronic myelogenous leukemia (CML) is a clonal neoplastic disorderof hematopoietic stem cells caused by expression of the chimeric bcr/ablfusion oncogene, which is the product of the t (9;22) Philadelphia (Ph’)translocation and proved to be the most characteristic feature of CML(abnormality occurs in more than95%patients). With that consideration,determination of bcr/abl is of great importance for an early diagnosis, abetter prognosis and an improvement for detecting minimal residualleukemia cells in CML patients.Objective:In this work, we developed an electrochemical DNA sensor adoptingfunctional hairpin structure probe for the detection of bcr/abl fusion gene ofCML. Taking advantages of AuNPs/PANI/CS-GS, biotin-avidin signalingamplification and alkaline phosphatase, the DNA sensor showed a highdetection sensitivity. Methods:1. Graphene sheets (GS) suspension was prepared with the aid ofchitosan (CS) solution and then fabricated onto the glassy carbon electrode(GCE), followed by the electro-polymerization of aniline to form the PANIlayer, then, Au nanoparticles (AuNPs) were electro-deposited onto themodified GCE to immobilize the capture probes.2. The capture probe employed a hairpin structure and dually labeledwith a5’-SH and a3’-biotin. After hybridization with the target DNA, hairpinstructure was compelled to open and3’-biotin was forced to stay away fromthe electrode surface.3. Streptavidin-alkaline phosphatase (SA-AP) was covalently binded tothe capture probe via biotin-avidin system. Reduction currents were thengenerated after catalyzing the hydrolysis of the electroinactive1-naphthylphosphate (1-NP) to1-naphthol and monitored by differential pulsevoltammetry (DPV).4. Optimize the experimental conditions: the concentration of probe,the immobilizing time of probe, the concentration of SA-AP and thehybridization time.5. Under the optimized experimental condition, the DNA sensor wasincubated in different concentrations of the target DNA solution to preparethe standard curve. The selectivity, stability and repeatability were alsoanalyzed in detail. 6. The real samples were detected by the proposed DNA sensor: thePCR products of bcr/abl fusion gene in K562cells, positive sample andnegative sample of CML patients were determined, respectively.Results:1. The field-emission SEM was used to characterize the morphologiesof CS-GS, PANI/CS-GS and AuNPs/PANI/CS-GS, respectively. AFM wasalso utilized to probe the attachment of DNA to the modified electrode. Theresults indicated that capture probes were successfully immobilized ontoAuNPs/PANI/CS-GS/GCE.2. When the concentration of probe was800nM, immobilizing timewas17h, hybridization time was120min and concentration of SA-AP was0.5μg mL-1, the DNA sensor exhibited the best behavior. Under theoptimized experimental conditions, the sensor showed a good response totarget DNA in the range from10to1000pM (I=2.493logC–0.810，R2=0.992), and the detection limit was calculated as2.11pM at asignal-to-noise ratio of3(S/N=3).3. When the biosensor was stored in refrigerator at4℃for15d,92.2%of its initial current response was retained compared with the freshlyprepared electrode. Five DNA sensors prepared with the same process weretested at100pM,300pM,500pM and800pM, and the relative standarddeviation (RSD) of4.2%,4.8%,3.6%and5.4%were obtained, respectively.4. K562cells, positive real sample and negative real sample (from normal people) were collected as the detecting targets. Total RNA fromthose samples was firstly extracted, then cDNA was obtained by applying areverse transcription method, followed by a PCR amplification procedure.The DPV results indicating that the expression level of bcr/abl fusion genein K562and positive real sample were up-regulated compared with that innegative real sample.Conclusion:In summary, an AuNPs/PANI/CS-GS based electrochemical DNAsensor with functional hairpin probe for the detection of bcr/abl fusion genein CML was successfully developed. Graphene sheets, aniline and AuNPswere respectively assembled onto the electrode surface to greatly improvethe conductivity and electrochemical effective surface area, and throughwhich the immobilizing amounts of hairpin probe were enlarged. Next,SA-AP specifically bound onto the electrode through biotin and avidininteraction, which could availably amplify the electrochemical response of1-NP. Based on these amplifications, the detection limit of2.11pM (S/N=3) was achieved. This DNA sensor showed an excellent selectivity fordifferent DNA sequences and had been successfully used for real-timedetection of bcr/abl fusion gene in PCR products from real samples.