The Construction Of DNA Biosensor Based On Carbon Dots@ Graphene Oxide Composite Toward The Detection Of PML/RARa Gene

C-dots have become one of the hotspot in the research of carbonnanomaterials, because of its excellent optical and electrical conductivity and goodbiocompatibility. Laser ablation, electrochemical oxidation, microwave-assistedand wet oxidation all can be used to synthesize C-dots. It was widely used in thefield of ion detection, biological imaging and so on. However, the reportedpreparation methods of CDs have some limitations in fluorescence quantum yieldand selection of carbon source. Therefore the more effective methods and suitablecarbon sources are urgently required. Graphene, as another research hot topic incarbon nanomaterials, owns superior physical and electrochemical properties dueto its layered structure leading to fine electronic transmission property. Meanwhilethe fluorescence energy transfer between graphene and fluorescent reagent caneasily take place. The combination of carbon dots and graphene has been a goodmethod in constructing a biosensor with high sensitivity and selectivity. Over 95%of cases are characterized by chromosome reciprocal translocation, t(15; 17)(q22;q12), resulting in the generation of fusion gene between promyelocytic leukemia(PML) and retinoic acid receptor alpha(RARα), which forms PML/RARα fusiongene. The detection of PML/RARα fusion gene has momentous significance for theearly diagnosis, disease monitoring and prognosis of APL. Thus, it is veryimportant to develop a new rapid and sensitive method to detect PML/RARα fusiongene for early diagnosis, disease monitoring and prognosis. The main contents ofthis paper are as follows:(1) The green fluorescent CDs have been synthesized by a one-stephydrothermal method of lotus root starch. The morphology, surface structure andoptical properties of the prepared CDs were characterized by TEM, XPS, UV-visand fluorescence spectrum method. It showed that C-dots exhibit excellentfluorescent quantum yield with 2.38% and fluorescent lifetime with 1.39 ns.Osteosarcoma cell was selected as the model for fluorescence labeling experiment.The results showed that the fluorescence labeling of osteosarcoma cell was carriedout by the prepared CDs. The fluorescence CDs was hopeful to be applied to cellmarking, bioimaging, biochemical analysis and photoelectrical research fields.(2) A fluorescent DNA biosensor for detecting acute promyelocytic leukemia ofPML/RARα fusion gene was constructed based on the fluorescence energy transferbetween carbon dots(CDs) and graphene oxide(GO) and the molecularhybridization technique. Firstly, the ss DNA-modified carbon dots(ss DNA-C-dots)was obtained by the modification of the single-strand DNA on the surface ofcarbon dots. Taking advantage of the electrostatic interaction between ss DNA andgraphene oxide with combination of electrostatic and π-π stacking interactionbetween carbon dots and graphene oxide, ss DNA-C-dots can been attached on thesurface of GO and then fluorescence signal of carbon dots ws quenched by theFRET between CDs an GO.. In the present of the target, ss DNA probe on thesurface of C-dots could hybride with the target to form the rigid ds DNA complex.Target binding allowed ss DNA-C-dots away from the surface of GO and resulted inan obvious increment in fluorescence intensity due to the decreased GO quenchingeffect. Based on the, the fluorescence DNA biosensor was constructed.The formation of ds DNA-C-dots breaks up electrostatic attraction and π-πstacking interaction between ss DNA-C-dots and GO, thus lead to liberation anddetachment of ds DNA-C-dots from GO, then fluorescence recovery from freeds DNA-C-dots, it was used for the detection of acute promyelocytic leukemia ofPML/RARα fusion gene. The experimental results revealed that the fluorescenceintensity increased with the concentration of target ss DNA in the range from 20nmol/L to150 nmol/L and its detection limit is 1.5 nmol/L. Furthermore, thebiosensors is able to identify complementary, single-base mismatch andpartial-base mismatch in hybridization solution. The proposed method wassensitive, specificity and quantify for the determination of PML/RARα fusiongene.(3) The using of electrostatic and π-π stacking interaction between carbon dotsand graphene oxide prepared carbon dots@graphene oxide(C-dots@GO)nanocomposites, combining the large surface area and rich carboxyl group of GO,the amino-modified ss DNA probes was fixed on the C-dots@GO electrode surfacevia covalent binding, increased the capacity of molecular probe, Then combinedwith strong conductivity C-dots, using methylene blue(MB) as a electrochemicalhybridization indicator, based on the difference between detect electrical signals,before and after hybridization, constructed an electrochemical DNA biosensors,used for the detection of acute promyelocytic leukemia PML/RARα gene fusion.The constructed DNA electrochemical biosensor through this method has goodspecificity and can be fine to distinguish ss DNA sequences of different mismatches.Under the optimized experimental conditions, it has a good linear relationshipbetween detect electrical signals and concentration of target DNA in the range of0.25 to 2.5 nmol/L, its detection limit was 0.083 nmol/L. Hence, it can realize thehigh sensitive and quantitative detection of target ss DNA sequences.