The Application Of Sensory Analysis Based On Signal Amplification Strategy In Tumor Cell-associated Active Molecules

Adopted non-destructive fluorescence analysis and fluorescence imaging technology,this paper designed the biological detection of telomerase activity with core-shell polymers,constructed intracellular target imaging technique which based on nucleic acid molecular aggregates,and extended DNA rolling circle replication reaction which based on telomerase.Based on double target cross probe and rolling circle amplification strategy,this paper also achieved the high sensitivity and high specificity detection of glutathione(GSH),miRNA and other tumor markers.The main contents and research results of this paper include the following aspects:1.The S-S bond molecular probe was designed.The probe entered into tumor cells,and DNA hybridization chain reaction was initiated by Thiol-disulfide exchange reaction.The fluorescent enrichment was formed,and the high sensitivity detection of GSH in tumor cells was achieved.The real-time monitoring and imaging experiments on hepatoma cell line(HepG2)showed that GSH stimulated the thiol-disulfide exchange reaction in the cells,triggered the DNA hybridization chain reaction,and generated the ordered nucleic acid molecule rich mass.As an active molecule,GSH has a high content in the cytoplasm of malignant tumor cells,which can be used to distinguish tumor cells from normal cells.GSH can cut off the disulfide bond on the nucleic acid probe through Thiol-Disulfide exchange reaction,triggering the intracellular DNA hybridization chain reaction,which is hybridized with the signal molecular probe,the hybridization products formed ordered nucleic acid molecular aggregates in the surface of functionalized nano silica materials.Thus,the signal amplified and aggregated can be detected and the GSH in a single cell can be effectively monitored,and the recognition of tumor cells and normal cells can be preliminarily realized.In this single cell analysis method,the cervical ring probe(modified quenching group BHQ and fluorescence group FAM)is in a fluorescence quenching state,and when GSH initiates a DNA chain hybridization reaction,the quenching effect decreases,and the fluorescence recovers;HCR products are self-assembled on mesoporous silicon nanospheres to form ordered nucleic acidaggregates,which can be used for GSH detection and intracellular imaging analysis.Self-assembled nucleic acid molecular accumulations can aggregate in a single cell and produce rich fluorescent spots in a single cancer cell,so they can effectively distinguish cancer cells from normal cells.2.A functional gold nanoprobe was designed to induce intracellular replication and amplification reaction by telomere lengthening to form a biological aggregates with silica nanoscale as the core.Thus,in situ detection of telomerase in tumor cells,aggregation of signal molecules in single cell and targeted release of anticancer drugs were achieved.The results showed that the aggregation of FAM fluorescence and the release of DOX were observed in HepG2,breast(MCF-7)and cervical(Hela)cancer cells,while in normal hepatocytes(L02),there were almost no FAM spots,and the amount of drugs released was also very small.This method of targeting tumor cells and releasing drugs can reduce the damage to normal cells.And a bifunctional nanoparticle probe was constructed,which has the capability of targeted intracellular telomerase and drugs released.At the same time,Based on telomere lengthening triggered intracellular rolling circle amplification reaction,forming a silicon oxide nano flower as the core of the biological aggregates,accordingly,telomerase detection in situ and the aggregation of signal molecules in a single cell can be achieved,and the anti-cancer drugs can be released simultaneously.Human telomerase is a unique ribonucleoprotein,can extend the repeat sequence TTAGGG of 3 ’end.In normal cells,telomeres are gradually shortened after each replication cycle.However,due to the telomerase is up-regulated or activated in active tumor cells,telomere length can be maintained in human cancer cells,which allows these cells to proliferate and survive.This technique can be used to detect telomerase activity in tumor cells by means of aggregated fluorescence signal,and it can effectively reduce the damage to normal cells by targeting the release of anticancer drugs.3.In this work,a dual target function probe was designed: with the intracellular tumor marker miRNA as drone,the expression changes of two genomes in cells were monitored.This cross probe can stimulate bidirectional rolling circle replication reaction in tumor cells and form bidirectional-signal fluorescence nanoclusters byelectrostatic self-assembly.The extraction of genomic components in tumor cells was carried out.The data show that this method has good specificity and sensitivity for the detection of miR21 and let7 a.The imaging of different cells was compared,it was found that the three kinds of tumor cells could form double signal fluorescence clusters,but only single signal clusters could be formed in normal cells.The reason is that the expression of miR21 in normal cells is low and the expression of let7 a is high.This dual signal recognition method is helpful to further improve the early diagnosis of tumor diseases.