Fluorescence Sensing Platform Based On Ruthenium Complexes As Sensors For The Detection Of MiRNA Involved In Cancer

Cancer is one of the health-threatening diseases in the world.In the recent years,its incidence and mortality keeps the trend of rising,which make it become a major public health problem in the world.Although there are many methods for the diagnosis and treatment of cancer currently,most patients are diagnosed at late stage.Therefore,early diagnosis has great significance to the treatment and prognosis of cancer.Searching for rapid and efficient early diagnosis of cancer has gradually becoming the focus of the researchers.MicroRNAs are small noncoding RNAs of 18-25 nt that can play vital roles in regulating human fuction.And more importantly,the expression of miRNA is closely related to many human diseases,especially for cancer.The deregulation of miRNA can reveal the occurrence of cancer.MiRNA can be used as the biomarker of cancer at its early stage.At present,the methods for sensing miRNA in clinical trials(Northen blotting,RT-PCR,Genechip and so on)are often required complicated operation and long time.Recently,the use of fluorescence spectroscopy to sense miRNA is becoming a research hotspot due to its merits of easy operation,low cost,high sensitivity and selectivity.In recent years,our group has been engaged in using metal organic frameworks(MOFs)for DNA/RNA detection.MOFs contain aromatic rings and positively charged metal cation centers in their structures.Such backbones have been proven to promote interactions with fluorophore labeled probe DNA through ?-stacking and electrostatic interactions to quench the fluorescence of fluorophore.The formed system can be used as sensors for the detection of disease-related nucleic acid,such as Ebola virus RNA and HIV virus DNA sequences.The ruthenium complexes based on polypyridine exhibit excellent properties in the study of the interaction with nucleic acids for its advantages of rich conjugated ? system,water solubility and stability.According to the detection mechanism for the nucleic acid,herein,we synthesized a serial of ruthenium complexes as a new material for the detection of cancer-related miRNAs.The results are summarized as follows.Part 1,Four ruthenium complexes([Ru(bpy)2(amtp)]Cl2(Ru 1),[Ru(phen)2(amtp)]Cl2(Ru 2),[Ru(dmp)2(amtp)]Cl2(Ru 3)and[Ru(bpy)2(thtp)]Cl2(Ru 4))have been synthesized and characterized and used in the detection of cancer-related miR-185.The results indicate that Ru 1-4 can have electrostatic,?-stacking and hydrogen-bonding interactions with P-DNA to form the P-DNA@Ru sensing platform.All these four P-DNA@Ru platforms can detect miR-185 with high sensitivity,specificity and speed,giving the detection limits of 0.42 nM for Ru 1,0.28 nM for Ru 2,0.32 nM for Ru 3,0.85 nM for Ru 4,all with instant detection time in 1 minute.Part 2,miR-185 is associated with a variety of cancers and it is not specific for certain cancer detection.Herein,we screened out the biomarkers miR-221 and miR-222 as subjects,which were upregulation in breast cancer.Based on Ru 1,we designed and synthesized[Ru(bpy)2(hdppz)]C12(Ru 5)and[Ru(bpy-NH2)2(amtp)]Cl2(Ru 6)with more large conjugated system.The results indicated that both Ru 5 and Ru 6 have high affinity toward carboxyfluorescein(FAM)or 5(6)-carboxyrhodamine,triethylammonium salt(ROX)-tagged single stranded probe DNA(P-DNA)through electrostatic,?-stacking and hydrogen-bonding interactions,thus quenching the fluorescence of tag.The two formed P-DNAs@Ru systems can be applied for the detection of miR-221 and miR-222 with single and synchronous fluorescence detection methods.The detection effect of P-DNAs@Ru platforms formed by Ru 5 or Ru 6 is superior to that of Ru 1,giving the simultaneous detection limits of 0.20 nM and 0.52 nM for Ru 5,0.28 nM and 1.03 nM for Ru 6,both with instant detection time in 1 minute and 2 minutes,respectively.The two miRNAs do not interfere with each other during the detection process.Part 3,On basis of the above research,in order to apply the formed P-DNAs@Ru sensing platform to detect miRNA at cell level,we further designed complex[Ru(dip)2(hdppz)]Cl2(Ru 7)with better liposoluble.The results revealed that the detection limits of P-DNAs@Ru 7 platform for miR-221 and miR-222 were 0.13 nM and 0.58 nM with detection time in 2 minutes,which were similar to Ru 5.The formed P-DNAs@Ru 7 sensor can further enable the highly specific detection of miRNAs in living cancer cells.