Coupling A Universal DNA Circuit With Graphene Sheets/Polyaniline/Aunps Nanocomposites For The Detection Of Bcr/abl Fusion Gene

Chronic myeloid leukemia(CML) is a myeloproliferative disease characterized by reciprocal translocation t(9; 22)(q34; q11) which lead to the generation of bcr/abl fusion gene. Thus, bcr/abl has been an important biomarker for early diagnosis, regular molecular assay, and therapeutic drug monitoring of CML. In addition, measurement of bcr/abl for diagnosis of minimal residual disease(MRD) is still a great challenge due to the sensitivity limit of current methods. Therefore, easy-to use, cost-effective and highly sensitive methods for bcr/abl detection are still greatly needed.Objective:In this work, we described a novel method by coupling a universal DNA circuit with graphene sheets/polyaniline/AuNPs nanocomposites(GS/PANI/Au NPs) for highly sensitive and specific detection of BCR/ABL fusion gene(bcr/abl) in chronic myeloid leukemia(CML). A transducer hairpin(HP) was designed for CHA to enable the reuse of DNA circuit for different inputs DNA. In addition, GS/PANI/AuNPs with excellent conductivity and enhanced effective area were coupled with CHA for further signal amplification, which may contribute to highly sensitive detection of bcr/abl. By combing the advantages of CHA and GS/PANI/AuNPs, a cost-effective, highly sensitive and specific DNA electrochemical sensor was fabricated.Methods:1. The pre-prepared GS/PANI solution was deposited on the pre-cleaned glass carbon electrode(GCE). Then electrodeposition of AuNPs was performed in 1% HAuCl4 solution. The obtained electrode was noted as GS/PANI/AuNPs/GCE.2. The capture probe(S) was immobilized on the surface of GS/PANI/AuNPs/GCE to capture signal probe.3. CHA was carried out by placing 10 μL of the the mixtures of target DNA, probe H1 and H2 on the prepared electrode. As a result, HP:C1 was relieved and available to trigger another cycle. Meanwhile, the S:H1:H2 complex was immobilized on the modified electrode surface. Streptavidin-alkaline phosphatase could bind H1 and catalyze 1-naphthyl phosphate(1-NP), resulting in electrochemical signal.4. The length of HP stem, concentration of HP, concentration of H2, and assembly time was optimized.5. Under optimized experimental conditions, the sensitivity of this method was evaluated by differential pulse voltammetry(DPV). The specificity of the biosensor was evaluated. The stability and reproducibility were investigated.6. To test real application of the fabricated DNA biosensor, bcr/abl amplified fragment from K562 cells, positive real sample(from CML patient), and negative real sample(from normal people) were obtained by PCR amplification.Results:1. The nanocomposites were characterized by a field emission scanning electron microscope(SEM) and Energy dispersive X-ray(EDX) spectra. Immobilization of S was analyzed by an atomic force microscope(AFM). The stepwise reactions on the surface of GCE were characterized by cyclic voltammetry(CV) and electrochemical impedance spectroscopy(EIS).2. The peak current varied linearly with logarithm of target DNA in the range from 10 pM to 20 nM. The correlation coefficient(R2) was 0.991. The detection limit was estimated to be 1.05 pM(S/N =3).3. It demonstrated that the DNA biosensor could retain about 92.6% of its initial current response after two-week storage at 4 ℃. Five modified electrodes prepared were used to detect target DNA(20 pM, 50 pM, and 100 pM), and the relative standard deviation was 5.26%, 4.59%, and 4.43%, respectively. Therefore, it could be concluded that the DNA biosensor provided satisfactory stability and reproducibility.4. The DPV signals of PCR products of K562 cells and positive real sample gave a mean average of 19.21 μA and 18.45 μA, which was much higher than that of negative real sample and blank.Conclusion:In summary, we proposed a sensing strategy by employing a universal DNA circuit and GS/PANI/AuNPs for highly sensitive and specific detection of target DNA. This strategy had several features. First, we introduced a transducer for CHA which make the DNA circuit be available for different kinds of target. Second, the stem, loop, and toehold of hairpins(HP, H1, H2) were rationally designed to improve signal-to-background ratio and specificity. Third, this protocol coupled an enzyme-free DNA circuit with easily synthesized GS/PANI/AuNPs, which was inexpensive and simple. By combining advantages of CHA and GS/PANI/AuNPs, this sensing system provided high sensitivity and specificity for the detection of bcr/abl. Moreover, this biosensor was applied for the detection of real sample with satisfactory results, demonstrating its great potential in clinical application.