Researches On The Detection Of Tumor Markers MicroRNA Based On Spherical Nucleic Acid Probes

Tumor markers are bioactivators that characterized by their presence in tumor cells.They are produced and released from tumor cells to host body fluids.Their generation and change are closely related to the occurrence and development of cancer.Tumor markers can be detected by biological,immunological and chemical methods.MicroRNAs（miRNAs）,which contain about 22 nucleotides,are a class of noncoding small-molecule RNAs.They participate in regulation of ontogeny,cell proliferation,differentiation,and apoptosis by means of incomplete hybridization with the target gene and inhibition of target gene translation.Several studies have shown that some miRNAs have carcinogenic or anti-carcinogenic effects and are closely related to the pathogenesis,metastasis and other pathological processes of tumor.Therefore,developing of high sensitive and accurate detection methods for miRNAs is important for early diagnosis of cancer.Spherical nucleic acid（SNA）is a new kind of nano-material which connects a large number of radial nucleic acids on the surface of nanoparticles.Compared with the simple nucleic acid probes,SNA probes can directly penetrate the cell membrane without the aid of transfection agents and have a strong ability of anti-nuclease hydrolysis.Gold nanoparticles（AuNPs）have good biocompatibility,low toxicity and can quench fluorescence effectively.The thiol labeled nucleic acids modified on the surface of AuNPs through Au-S bond are stable,exhibit higher specificity and binding ability when hybridized with other nucleic acids,and can significantly improve the salt resistance and stability of AuNPs.These advantages make the SNA probes with AuNPs core have low background,high sensitivity and selectivity in the detection of biomarkers.DNAzymes are DNA sequences that have high catalytic efficiency and structure identification activities.They can recognize and cleave specific DNA or RNA sequences.There are two constituent parts of DNAzymes:the binding site and the catalytic site.The binding site makes DNAzymes hybridize with the substrate and the catalytic site cleaves the substrate with the help of cofactors.DNAzymes have many advantages compared with traditional protein enzymes.They are low cost,stable,and have high selectivity and recognition ability for different substrates to adapt to different reaction systems because of sequence designable of the binding site.DNAzymes are widely used in bio-detection based on the above advantages.The SNA probes with AuNPs core are employed to construct fluorescence switch for miRNA 155high sensitivity detection in vitro and imaging in living cells based on DNAzyme amplification strategy in this paper,the main contents are as follows:（1）Novel fluorescence switch for microRNA imaging in living cells based on DNAzyme amplification strategy.First,the complementary strand of miRNA 155 named Locker was used to hybridize with the DNAzyme to plug it and prevent the DNAzyme to bind with its substrate.AuNPs were modified with a lot of cy5-labeled hairpin DNA named FL DNA and the fluorescence of cy5 was quenched because of the close range between cy5 and AuNPs.In the presence of miRNA 155,the Locker was took away from the DNAzyme by miRNA 155 through strand displacement reaction and the DNAzyme was activated.Then,the active DNAzyme combined with its substrate DNA.The substrate was cleaved to two single DNA strands by the active DNAzyme.One of them was partly complementary to FL DNA named Key.Key hybridized with the FL DNA to open its hairpin structure and kept cy5 away from AuNPs to turn on its fluorescence.The DNAzyme circular cleaved its substrate DNA.Plenty of substrate DNA was added so that lots of the Key was released to turn on the fluorescence of cy5.And the amplification strategy based on DNAzyme was built.The method has a wide linear range from 0.1 to 10 nM and high sensitivity with a low detection limit of 44 pM on in vitro detection.The recovery rate in standard addition recovery experiment proved that the method had high accuracy.In complex systems containing multiple similar sequences of miRNAs,this method had high specificity for miRNA 155.In living cells experiment,the plugged DNAzyme and its substrate was loaded on MnO₂ nanosheets because of the good adsorption of single stranded DNA on MnO₂ nanosheets.MnO₂ nanosheets were employed as a transport to deliver DNA modules into cells and the AuNPs SNA probes entered the cells directly.The glutathione in cells reduced MnO₂ nanosheets to Mn²⁺so that the DNA modules would be released and the high level of miRNA 155 in tumor cells turned on the fluorescence of cy5.Under confocal laser microscopy,the fluorescence was obviously observed in tumor cells but not in normal cells,indicated that this method can image miRNA 155 in living cells.The DNA modules were transported into cells by MnO₂ nanosheets,and the Mn²⁺which reduced from MnO₂nanosheets by glutathione could also be used as cofactors of DNAzyme.Cytotoxicity assay proved that Mn²⁺at the used concentration was harmless to cells.A novel fluorescence switch based on DNAzyme amplification strategy was built for miRNA 155 high sensitivity detection in vitro and imaging in living cells,and this method may play important roles in clinical diagnosis and therapy of cancer.（2）Synthesis of positively charged AIE molecule.The positively charged aggregation induced emission（AIE）molecule was synthesized by means of functionalized tetraphenylethene.In the following research,the positively charged AIE molecule will be further functionalized,and load it in nanoparticles to enter cells for detection of tumor markers in living cells.