Construction Of Nanomaterial Mediated-ratiometric Fluorescence Biosensors For MicroRNA Detection And Imaging Of Living Cells

MicroRNAs(miRNAs)are a class of endogenous,non-coding,single-stranded RNA molecules consisting of approximately 18-24 bases.miRNA regulates various biological processes,including cell differentiation,cell proliferation and apoptosis,by regulating mRNA degradation or inhibiting the expression of translational regulatory genes.Numerous studies have displayed that the abnormal expression of miRNA is closely related to various diseases,such as cancer,cardiovascular disease and Alzheimer's disease.Thus,miRNA can be acted as potential marker for disease diagnosis and prognosis.Visualizing the detection of intracellular miRNAs is critical to better understand its role in the cell cycle and to further elucidate its biological function in clinical diagnosis.Based on the above analysis,a ratiometric fluorescence biosensor was constructed for sensitive,accurate detection of miRNA.In addition,this biosensor could also be used for cell imaging.This article is divided into three chapters:Chapter one is the introduction section,which mainly summarizes the production,formation mechanism,research significance of miRNA.Meanwhile,we introduce the traditional and new detection methods for miRNAs,and the scientific problems and research contents mainly solved in this paper.In chapter two,a MnO2 nanosheet-mediated ratiometric fluorescence biosensor was designed for miRNA detection and imaging in living cells.It contained MnO2 nanosheets acting as DNA carrier,and fluorescent donor(FAM)-labeled hairpin H1(recognition probe)and fluorescent acceptor(TAMRA)-labeled hairpin H2(amplification probe).When the biosensor entered cell,MnO2 nanosheets were reduced to Mn2+ via intracellular glutathione(GSH)and the adsorbed hairpins H1 and H2 were released.The intracellular target miRNA-21 hybridized with the recognition unit of H1 to initiate catalyzed hairpin assembly(CHA)and a large amount of H1-H2 duplexes were produced.This brought fluorescent donor FAM and fluorescent acceptor TAMRA into close proximity to produce fluorescence resonance energy transfer(FRET),inducing a ratiometric fluorescent response(donor signal decreased and acceptor signal enhanced)for miRNA-21 detection.Furthermore,this method could be applied to differentiate the expression levels of miRNA-21 in HeLa,HepG-2 and L02 cells.These results indicated that the proposed method possessed great potential in the early diagnosis of miRNA-related diseases.In chapter three,a biocoded fluorescence sensor was constructed based on FRET for multiplex detection and intracellular imaging of miRNAs.It consisted of MnO2 nanosheets and four single-stranded DNA probes(P1 AF 488,P2-Cy3,P3-AF 488 and P4-AF 594).As a DNA nanocarrier,MnO2 nanosheets could deliver probes into cells by endocytosis.Probe P1-AF 488 and P2-Cy3 sequences were partially complementary to miRNA-373,probe P3-AF 488 and P4-AF 594 sequences were partially complementary to miRNA-96.When the probe were delivered into the cells,in the presence of the corresponding target miRNA,they were form double-stranded structures.This caused the donor and acceptor to be close to each other to produce FRET,inducing the changes of fluorescence.In addition,the specific fluorescent color of the output could be used as a code to convert the presence of the miRNA into a different fluorescent signal,realizing multiple detection of miRNA.These results indicated that the biosensor provided a new tool for the simultaneous detection of intracellular miRNAs and was expected to provide more accurate information for the diagnosis and treatment of cancer.