Electrogenerated Chemiluminescence Biosensing Method For The Determination Of MicroRNA Based On Nucleic Acid Amplification And Nanomaterials

MicroRNA（miRNA）is an endogenous,non-coding ribonucleic acid molecule（approximately18-25 nucleotides in length）,which plays an important role in various physiological processes including cell proliferation,differentiation and apoptosis.Moreover,it has been indicated that the abnormal expression levels of miRNA are associated with disease and has been served as biomarkers in the process of disease diagnosis.Therefore,it is imperative to exploit a highly sensitive method for the detection of miRNAs.However,there are limitations due to the intrinsic characteristics of miRNAs,such as high sequence similarity among family members,small sizes,low concentrations in cancer cells,and susceptibility to degradation.Electrogerated chemiluminescence（ECL）is widely applied to the detection of various biomolecules owing to its high sensitivity,low background signal,wide dynamic response range,and easy operation.As a signal amplification strategy,nucleic acid amplification and nanomaterial have been gained much attention in the fabrication of biosensors.In view of these merits,the aim of the research work is to develop a cascade-amplified ECL biosensor for ultrasensitive detection of miRNA-21 by combining nucleic acid amplification strategy and functionalized nanomaterials.This work opened a new path for the biomedical research and and therapeutic monitoring.The full text is divided into three chapters.The specific work is as follows:In chapter one,firstly,the basic principle and composition of biosensors were introduced.Additionally,the principle and characteristics of ECL were summarized.At the same time,the application of nucleic acid signal amplification strategy and nanomaterial signal amplification strategy in the field of biosensor was reviewed.Moreover,the research content and significance of this paper were clarified.In chapter two,an ultrasensitive and highly specific ECL biosensor platform was constructed for the detection of miRNA-21 based on isothermal strand-displacement polymerase reaction（ISDPR）and bridge-DNA-AuNP nanocomposites.Firstly,the bridge-DNA-AuNPs nanocomposites were prepared as a primary signal amplification unit.Meanwhile,assistant DNA fragment was generated by ISDPR and miRNA-21 was replaced in the presence of phi-29 DNA polymerase.The released miRNA-21 could induce the next new ISDPR cycle.After multiple cycles,a small amount of miRNA-21 was used to generate a large number of assistant DNA fragments,which further magnified the sensitivity of the detection.This process can be regarded as a secondary signal amplification unit.The assistant DNA was then introduced into the sensing interface by hybridizing between the thiolated capture probe（SH-CP）immobilized on the gold electrode and the bridge-DNA-AuNPs nanocomposites.Finally,the ECL signal probe SA-Ru was bound to the bridge-DNA-AuNPs nanocomposites by the specific interaction between streptavidin and biotin to generate ECL signal.The results indicated that the ECL intensity was linearly proportional to the miRNA concentration in the range of 0.01-10000 fM with a detection limit of 3.2 aM.In chapter three,a novel dual-signal enzyme-free amplification ECL biosensor was established based on toehold-mediated strand displacement reaction（TSDR）and target-catalyzed hairpin assembly（CHA）signal amplification strategy for the detection of miRNA-21 by using metal-organic frameworks（MOFs）Ru@MIL-101（Al）-NH₂ as ECL signal labels.Here,the MOFs was used as a carrier for the ECL signal substance Ru（bpy）₃²⁺to synthesize a new ECL signal labels Ru@MIL-101（Al）-NH₂.The presence of the target miRNA-21 could initiate the TSDR cycle reaction and release a large number of reporter strands（RS）.Subsequently,the liberated RS could be used to further participate in the CHA process for signal amplification by hybridizing with the hairpin probe H1.Finally,the ECL signal labels Ru@MIL-101（Al）-NH₂ were introduced into the surface of the sensing electrode to generate an ECL signal through hybridization of hairpin probe（H2）and the signal probe on the surface of ECL signal labels,and the ECL intensity is related to the concentration of the miRNA-21.The ECL biosensor has a good linear response from 10 fM to 10 nM with a detection limit of 4 fM.In addition,the proposed strategy has good reproducibility,stability and selectivity and can be used for the determination of miRNA-21 in total RNA samples extracted from human breast cancer cells（MCF-7）.