| Objective:MicroRNAs(mi RNAs)play a considerable role in cancer occurrence and development,which have been identified as promising noninvasive biomarkers.Studies have shown,the circulating level of mi RNAs is considerably higher in breast cancer,bladder cancer,prostatic cancer and nervous system disease patients in comparison to healthy individuals.Thus,the early,rapid and specific detection of microRNAs has become an urgent problem to be solved by clinical and scientific researchers.In this study,we used DNA nanotechnology and carbon nanoparticles to construct electrochemical biosensor for the detection of mi RNAs,which has provided a promising tool used in clinical screening and diagnosis cancer at an early stage.Methods:1.Construction of electrochemical biosensor: constructed a universal electrochemical biosensor for highly sensitive determination of mi RNAs based on isothermal target recycling amplification and DNA signal transducer triggered reaction;constructed an electrochemical biosensor platform for ultrasensitive detection of nucleic acids based on click chemistry-mediated enzyme-assisted target recycling(EATR)and multi-labeled functionalized fullerene nanoparticles(MFC60)dual-amplified strategy.To characterize the process of stepwise modification of electrode and the preparation of nanomaterials,we applied real-time fluorescence,agarose gel electrophoresis,polyacrylamide gel electrophoresis(PAGE),transmission electronmicroscopy(TEM),X-ray photoelectron spectroscopy(XPS)and electrochemical technology including cyclic voltammetry(CV),electrochemical impedance spectroscopy(EIS),differential pulse voltammograms(DPV)and chronocoulometry.2.Synthesis of MFC60: the fullerene(C60)aqueous suspension was prepared by solvent-exchange method;The synthesis of amino and thiol groups functionalized C60(FC60)was performed in supramolecular chemistry approach.3.Feasibility verification and experimental conditions optimization of this electrochemical biosensor: we used electrochemical technology to verify the feasibility of this biosensor;we investigated the sequence of capture probe,the immobilization time of capture probe,the ratio of nucleic acids strands and the sequences of azide-strand and alkyne-strand and so on.4.Evaluation of the biosensor and analysis of targets: evaluated the performance of these two kinds of biosensors reasonably,for instance,detection linear,detection limit,specificity,repeatability and stability.The target signal was converted into the corresponding electrochemical signal for detection.Results:1.The process of fabrication and modification of these two electrochemical biosensors and the synthesis of multi-labeled functionalized carbon nanomaterials were characterized by several methods.The results indicated that the biosensors were successful fabrication and the nanomaterials were successful synthesis.2.The performance of biosensor based on isothermal target recycling amplification and DNA signal transducer triggered reaction: detected mi RNA-21 highly sensitive;linear range was 0.5-2000 p M;detection limit was 56 f M;has good specificity,repeatability and stability.3.The performance of biosensor based on click chemistry-mediated EATR and MFC60 dual-amplified strategy: through dual-amplified strategy,this method could ultrasensitive detection of mi RNA-141;the linear ranges from 0.1 p M to 100 n M was achieved for mi RNA-141 with detection limit of 7.78 f M;has good specificity,repeatability and stability.Conclusions:This study proposed two novel sensing strategies,isothermal target recycling amplification combined with DNA signal transducer triggered reaction and click chemistry-mediated EATR combined with MFC60,to construct two electrochemical biosensors for detection of mi RNAs.These two biosensors were used to analyze mi RNA-21 and mi RNA-141,respectively.Under different signal amplification technologies,they were both displayed highly sensitive performance analysis for mi RNAs detection.Simultaneously,various methods have been used to characterize the synthesis of nanomaterials and the fabrication of the biosensors,the results indicated that the successful synthesis of nanomaterials and the successful fabrication of the biosensors.Signigicantly,these two biosensor platforms could be conveniently applied to other nucleic acids analysis by altering the corresponding sequence of capture probe;the sensoring platform based on click chemical also could be applied to detect the concentration of Cu2+.Therefore,the proposed electrochemical biosensors have good analysis performance and have great application prospect in the detection of nucleic acids. |
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