Experimentalstudies Of DNA Electrochemicalbiosensor Modified By Gold Nanoparticles In-situ Synthesized For Detection Of Fusion Gene In Chronic Myelogenous Leukemia

Objective：Chronic myelogenous leukemia （CML） is a clonal myeloproliferativedisorder, resulting from the neoplastic transformation of the primitivehemopoietic stem cells. CML is characterized by a reciprocal translocationbetween chromosome9and22with the formation of the so-calledPhiladelphia （Ph’） chromosome, which results in the formation ofBCR/ABL fusion gene finally. The abnormality usually occurs in more than95%of CML patients. Thus, the detection of BCR/ABL gene will be usefulfor an early diagnosis, a better prognosis of the disease, and animprovement for detecting minimal residual leukemia cells in the CMLpatients, especially after the bone marrow transplantation.In this study, we tried to construct a DNA electrochemical biosensor,based on the GNPs in situ synthesized, for the detection of BCR/ABLfusion gene. We characterised the electrochemical performance of the DNA biosensor. The aim is to explore a convenient, economy, fast and sensitivedetection method for the chronic myelogenous leukemia.Methods:1. Under certain conditions, we use the chitosan as reducing agent ofthe chlorine acid in situ synthesized of GNPs on the surface of theMWCNTs. The scanning electronmicroscope （SEM） was used tocharacterize the composite materials.2. An amperometric electrochemical biosensor for the detection ofhydrogen peroxide （H2O2） based on the GNPs/MWCNTs–Chits compositematerials. Then the thionine （Thi） was acted as the electrochemicalresponse indicator for the detection of H2O2. Besides, the performances ofthe H2O2were characterized.3. Using the simple method mentioned in the first step, we realized theGNPs in situ synthesized on the surface of CeO2and MWCNTs. Then thecapture probe was attached on the nanocomposite membrane modifiedglassy carbon electrode through anAu-S conjugated structure.4. After optimizing the experimental conditions, we characterized allthe performance of the DNAelectrochemical biosensor.5. The characteristics of the biosensor were depicted by cyclicvoltammogram （CV） and differential pulse voltammetry （DPV）. Thedecrease of the peak current upon hybridization of the probe with theBCR/ABL fusion gene were observed using the methylene blue as an electro-active indicator.Results:1. The SEM clearly illustrated that the GNPs were well-distributedalong the surface of the MWCNTs, which indicated that the GNPs werewell adsorbed on the surface of the MWCNTs and the size of the GNPswas approximately20nm.2. The fabricated hydrogen peroxide biosensor based on the GNPs insitu synthesized, showed good catalytic activity towards H2O2. The linearresponse range of the biosensor to H₂O₂concentration was from5×10^-7Mto1.5×10^-3M with a detection limit of3.75×10^-8M. With theamplification of gold nanoparticles in-situ synthesized, the sensitivity ofthe biosensor was greatly improved.3. Under the optimal condition, the experimental results indicated thatthe fabricated DNA electrochemical biosensor showed good specificity todistinguish the complementary sequence with different mismatchsequences and could detect BCR/ABL fusion gene quantitatively. Thelinear response range of the biosensor to DNA concentration was from1×10^-9M to1×10^-12M, with a detection limit of5×10^-13M.Conclusions:1. The SEM results indicated that the GNPs had successfully in situsynthesized on the surface of MWCNTs. The results indicated that thefabricated hydrogen peroxide biosensor showed better catalytic activity than some other methods.2. The in situ synthesized techolonology could make a variety ofnanomaterials became an organism. This method could make theconstructed biosensor have a better ability for the detection of the substrate.