The Construction Of Multifunctional Nanoprobe And Detection Of Intracellular Active Substances

After AIDS,cancer has become another major public health problem worldwide.There are an estimated 1.7 million cancer diagnoses and 600,000 cancer deaths,according to cancer mortality data collected by the national center for health statistics.Malignant cancer accounted for 23.91%of the death rate of citizens.Therefore,early cancer diagnosis and intervention has become the primary task to reduce the cancer mortality.In this paper,the detection of tumor markers such as telomerase,miRNAs and MUC1 protein was realized based on biocompatible nano-noble metal materials combined with fluorescence resonance energy transfer and surface-enhanced Raman scattering.This paper mainly includes the following three parts:(1)In the first Chapter,a new“see-saw”ratio probe(SR)was constructed based on fluorescence resonance energy transfer and surface enhanced Raman scattering(SERS)technology,which realized the imaging and detection of telomerase in a single living cell.When SR probe entered MCF-7 cells through endocytosis of cell membrane,Rox molecules were close to gold nanoparticles.Although Cy3molecules were far away from gold nanoparticles,the fluorescence of Rox was quenched due to fluorescence resonance energy transfer,resulting in Rox Raman signal increased,the fluorescence of Cy3 recovered and Raman signal disappeared.In the presence of telomerase and dNTPs,the telomerase repeated sequence(TTAGGG)_n from the primer strand triggered a competitive hybridization reaction.SR probes were broken down,hairpin DNA H2 was released,and H1 hairpin structure was restored.Due to the change in the distance between Rox,Cy3 and gold nanoparticles,the Rox molecule was far away from the gold nanoparticles,while the Cy3 molecule was close to the gold nanoparticles.Due to the fluorescence resonance energy transfer phenomenon,the fluorescence of Cy3 was quenched and the Raman signal appears,the fluorescence of Rox was restored,and the Raman signal disappears.The dynamic change of SR probe was detected by fluorescence imaging,and the ratio of Raman intensity of double signal was used to detect the activity of telomerase quantitatively.(2)In the second chapter,a double-labeled nano probe(DL)was designed to realize the recognition of MUC1 protein on the cell membrane and miRNA-21 in the cell.When the double-labeled Raman probes were contacted with the cell membrane,the excessive expression of MUC1 protein on the MCF-7 cell membrane were combined with aptamer strand on probe H2.The H2 strand was dissociated from double-labeled Raman probe,the distance between the gold nanoparticles and the Rox molecule was increases,and the Rox Raman signals disappear,fluorescent signal recovery due to the phenomenon of fluorescence resonance energy transfer.Rox fluorescence signals were appeared on the cell membrane(blue).After Rox fluorescence signals appeared on the cell membrane(blue),the hairpin structure of DNA H1 was restored.The distance between Cy3 molecule and gold nanoparticle was reduced,the Raman signal of Cy3 appeared,and the fluorescence signal was quenched.When the intracellular miRNA-21 is present,DNA H1 hairpin structure is opened by miRNA-21 and the Raman signal of Cy3 disappeared,the fluorescence signal resumes.The Cy3 fluorescence signal appeared in the cytoplasm(red).(3)In the third Chapter,DNA logic gates including OR and AND were designed,and Rox Raman probes were prepared too.The detection of target microRNAs was achieved based on hybridization chain reaction(HCR)and surface enhanced Raman scattering(SERS).The hairpin DNA H1,H2 and S1 strands were recognised by target microRNAs and the footholds of HCR reaction were also exposed.And six kinds of hairpin DNA H3 and H4,H5 and H6,S2 and S3 were reacted,leading to occurrence of the hybridization chain reaction.Double-stranded DNAs were formed.Furthermore,Raman probes labeled with Rox were added into the reaction system and combined with the formed double-stranded DNA to detect the target miRNA.