Fluorescence Imaging And In Situ Detection Of Telomerase And MicroRNA In Living Cancer Cells Based On AuNP/GO Probe

Cancer,also known as the "malignant tumor",is a common and frequently-occurring disease,which is a serious threat to human health and life.For cancer patients,if the cancer can be diagnosed early,and effective treatment,it can greatly improve the cure rate.In fact,due to the general growth in human malignant cells tissues and organs,not recognized by the immune system and we as a foreign object,so in the current level of technology,it is difficult to get early diagnosis of cancer.So,If it can be early detected and timely treated,the cure rate of cancer can be greatly improved.With the advance and development of science and technology.The discovery and application of more and more cancer biomarker provides a new way for early diagnosis of tumors.MicroRNA and telomerase,are two kinds of well-studied tumor markers currently.In addition,in order to monitor the endogenous tumor markers in the body in real time,and provide direct evidence of some of the biological tissue associated with tumor markers,people need an in situ detection method for the non-invasive,repeatable monitoring and real-time imaging of tumor marker.This paper consists of three parts: 1?Detection of telomerase activity based on in situ imaging of gold nanoparticle fluorescent probeWith the above scientific problems as the goal,we prepared the nanocomposite probes by combining gold nanoparticle-DNA nanoflare probes and graphene oxide loaded DNA probes.Similarly,in the present nanoflare probe,the HS-DNA/Primer-DNA/Flare-DNA(“hybridized-DNA”)was bound to AuNPs by the AuS bonds and the terminal fluorophore FAM of the Flare-DNA was quenched via fluorescence resonance energy transfer(FRET).When the AuNP probe acts on telomerase,the primer-DNA on the AuNP probe will be extended,thus Flare-DNA will releasing the fluorescence.Thus,the in situ imaging of cancer cells can be applied to detect telomerase activity in the cell.2?Detection of telomerase activity and its in situ imaging based on AuNP/GO nanoparticle probeAfter the quenched Flare-DNA is released on the AuNP probe,Then,the GO loaded mimic-HCR between two species of fluorescent hairpin sequences(H1 and H2)for a signal amplification and enrichment role.The mimic-HCR system was composed of two FAM-modified hairpin sequences(H1-DNA and H2-DNA or H1 and H2,respectively),which were designed to alternate hybridize to generate the nicked double-stranded DNA structures just as HCR upon the introduction of initiator(Trigger-DNA),but the leakage of the system will occur.Additionally,the H1+H2 system was absorbed on GO by ?-? stacking,and the fluorescence of the FAM labels was quenched by GO.DNA-modified AuNPs and GO can directly enter cells by endocytosis without any transfection agent.After the AuNP/GO probe enters into cancer cells,Flare-DNA dissociates from the AuNPs upon Primer-DNA extension,inspired by intracellular telomerase.The existence of telomerase can be demonstrated by the fluorescent signals.Furthermore,released Flare-DNA can efficiently initiate GO-loaded mimic-HCR between two species of fluorescent hairpin sequences(H1 and H2)for a signal amplification and enrichment role.By the use of the nanocomposite probe and combining graphene-based signal enrichment effect and hybridization chain reaction signal amplification,telomerase can be detected sensitively and fluorescence imaged in living cells according to the characteristics that telomerase can synthesize extend the telomeric repeat sequences,and can distinguish normal cells from cancer cells.3?Fluorescence imaging and in situ detection of miR-21 and Pre-miR-21 in cancer cells based on AuNP/GO probeIn addition,Pre-miRNA is widespread in normal cells.When the cells become cancerous,they are cut into mature miRNA by Dicer enzyme,Therefore,the accurate identification of mature miRNA and its precursor pre-miRNA,distinguishing tumor cells from normal cells is of great significance for the early diagnosis and treatment of cancer cells.So,motivated by the previous reports,here we designed an AuNP and GO based nanocomposite probe to achieve simultaneously imaging of intracellular pre-miRNA and miRNA.This proposed strategy includes two parts: AuNP-based nanoflare probe designed for the imaging of pre-miRNA and GO-based HCR/GO system designed for the imaging of mature miRNA,the nanoflare probe consists of gold nanoparticles(AuNPs)functionalized with a dense shell of Cy5-labeled oligonucleotide duplexes by Au-S bond formation.The oligonucleotide duplexes are composed of two segments: a long thiol-labeled sequence(HS-DNA)and a short Cy5-terminated reporter sequence(Flare-DNA)which is complementary to HS-DNA.The surface coverage of oligonucleotide duplex on the AuNP is determined by the mercaptoethanol competition experiments.Next,we used the previously reported HCR/GO probe as the signal transducer of miRNA-21.Here,GO was used not only as the quencher of the fluorophore groups modified HCR system but also as the carrier of the HCR system into cells.The concentration of GO was also optimized with fluorescence spectra.When the target molecule miRNA appearing.A simulated hybridization chain system on the GO probe led to the formation of double helix structure of two hairpin sequences of H1 and H2,releasing FAM fluorophores on quenched H1 and H2,thus realizing the detection of miRNAIt is worth mentioning,monitor the change in telomerase and microRNA activity in response to a telomerase and microRNA inhibitor in living cells,It is intuitive understanding for in-depth studies of the changes of cancer cells,and more crucial for cancer diagnosis,monitoring and targeted drug therapy.