The Selection Of High Insulin Treament Related Aptamer And Insulin Detection Based On Aptamer Technology

Chapter 1 Investigation on the construction of Cell-based Systematic Evolution of Ligands by Exponential EnrichmentObjective:To constract the Cell-based systematic evolution of ligands by exponential enrichment(SELEX)and to obtain the aptamers for the membrane proteins on the liver cells.Methods:The DNA library is consisted by two primers which length are 20bp. Primer1 is labeled with fluorescein isothiocyanate(FITC), while Primer2 is labeled with biotin. There is 20bp random sequence in the middle. When the HepG2 in the cell culture dish(100mm)grows to 95% confluence, get rid of the cell culture medium, and wash it by 4℃washing buffer. Dillute 20nmole library by 1ml binding buffer, and incubate it with HepG2 in 4℃for 1h. Get rid of the suspension and wash the cell by washing buffer twice. Use cell brush collect the cells in 1.5ml centrifuge tube by 1ml DI water, and heat the tube in the heat block in 95℃for 15min. Then centrifuge the tube and get the supernatant. This is our first pool for selection. Our binding sequences was inside. Using PCR to amplify these sequences. After PCR, we have double strain DNA(dsDNA), which contain the target strain and the complemental strain. Incubate these dsDNA with streatavidin coated beads, and get rid of the suspension. The dsDNA is opened into ssDNA by 2%NaOH, then our target strain was dissolved in the NaOH solution. Desalt the solution by NAP-5 column and meansure the concentration of these ssDNA to get the gross.At last dry the final sollution by DNA dryer. That is our first pool. Culture our target cells in 60mm cell culture dish. Dissovle 100pmole pooll by 500ul binding buffer which contain 10%FBS. And then do the next round of SELEX follow the way hereinbefore. Test the bound between each pool and our target cells every 3 to 5 rounds. When HepG2 grows to 95% confluence, using non-enzymatic buffer digest the cells, and make the cells into single one. Wash the cells, and put dillute the cells by binding buffer. Every 5×105 cells incubate with 25pmole pool in 4℃for 30min, the concentration of the pool is 250nM. The cells incubate with DNA library is the negative control. Using flow cytometry to analyse the binding. When the binding signal stop increasing, take the best 3 binding pools to do the 454 sequencing. After we get the sequences, synthesize these sequences and test the binding one by one with target cells to determine our aptamers. Using trypsin and proteinase K digesting HepG2 cells respectively, the treating time range from 5min to 30min, and compare the binding before and after the enzyme treatment. On the other hand, we incubate each aptamer with other kinds of cells (Including LH86, Huh7, WT, IRS/KO, M7617, Ramos, CEM, H23, H69, A549, HBE, H661, TOV-21G, CAOV3) and find the binding specificity.Results:In this paper, we do SELEX on HepG2 cells, and finally got 4 binding aptamers. There are IR01, IR03, IR04 and IR06. The best binding aptamers are IR01 and IR04, whose binding dissociated constant (Kd) are 11.287±3.786nM and 88.849±22.339nM respectively; Although our IR03 only have weak binding with HepG2 cells (Kd=129.513±47.924nM), but seems it only binds with HepG2, and almost no cross link with other kinds of cells; IR01, IR04 and IR06 can not only bind with HepG2 but also can bind with other kinds of liver cells. IR01 is the strongest binding sequence we got. And the most interesting thing is the bound of IR01 can not be eliminated by the digestion of proteinase.Conclusion:By doing SELEX, we can get the aptamers for the target cells with high specificity and high affinity. These aptamers can bind with their target biomolecules which are on the surface of cells membrane. In that way, they can bind different kinds of cells which express same biomolecules. Chapter 2 Select one aptamer for high insulin treatment related membrane protein on the surface of liver cellObjective:Based on the first part experiment, to select one aptamer for high insulin treatment related membrane protein on the surface of liver cell from the aptamers we got from the Cell-based SELEX on HepG2.Methods:Let the HepG2 cells grow to 70% confluence, starve from FBS for 12h, then divide these cells into four groups. The first group is treated by OnM insulin. The second group incubate with 0.1 nM insulin. The third group was treated with 100nM insulin. The last group was treated by 500nM insulin. All the four groups were incubated 24h. We also treat the fetal mice hepatocyte wild type(WT) and insulin receptor substrate 2 gene knock out fetal mice hepatocytes(IRS/KO) with 100nM insulin in the same way as HepG2 cells. At the same time, we also treat the WT and IRS/KO cells with OnM insulinfor control. And all the above cells are incubated in the FBS free medium. After 24h, using non-enzymatic buffer digest the cells into single one. Wash them by washing buffer and dissolve the cells into binding buffer. Every 5×105 cells incubate with 25pmole aptamers for 30min in 4℃. The concentration of aptamers is 250nM. After incubation, washing the cells by washing buffer, and using the flow cytometry to analyse if the high insulin treatment will change the binding aptamers and cells.Results:The binding between IR04 and HepG2 cells was inhibited by 100nM insulin obviously. When the concentration of insulin increased to 500nM, the binding between IR04 and HepG2 almost disappeared completely. But those binding won’t affect by physiological dose insulin. Also the high insulin treatment can decrease the binding between IR04 and fetal mice hepatocyte (WT), but won’t affect the binding between IR04 and the insulin receptor substrate 2 gene knock out fetal mice hepatocyte (IRS/KO). But aptamers IR01, IR03 and IR06’s binding will not be influented by high insulin treatment.Conclusion:The binding between IR04 and target cells are inhibited by high insulin treatment. And the degree of the binding has positive correlation with insulin concentration. But the physiologic concentration of the insulin won’t affect IR04’s binding. So we guess the target of IR04 is one kind of insulin related membrane protein, which will be down-regulated by high insulin treatment. But the detail is not very clear. The target of IR04 are highly homologous in both human beings’ and mice hepatocyte. Also the mechanism under the high insulin treatment might be the same. Chapter 3 Aptamer Conjugated Graphene Oxide based Insulin DetectionObjective:Use insulin binding aptamer conjugated graphene oxide (GO) to do insulin detection. And add DNase to gain a output amplification.Methods:Ground the graphite powder with NaCl to reduce the particle size. Removing the salt, then graphite was added to the concentrated H2SO4 and stirring for 2 hours. After, KMnO4 was added slowly gradually under stirring.Finally, we add distilled water and H2O2 solution to end the reaction. At last, the mixture was washed again. Sonicate GO dispersion under ambient condition for 4 hours. The resulted sample was centrifuged, and collect the upper solution for future experiments.IBA will bind with insulin and escaped from the GO surface. In that case the quenching effect of GO to FITC-IBA will be decreased a lot. Then we can detect the fluorescence signal. Further more, we add DNase I into the GO-aptamer solution and allow insulin incubate with them for 2 hours. When the IBA bind with insulin, it escaped from the GO surface, so the GO can not protect the IBA from the digestion of DNAse any more, it will be digested into pieces, and can not bind with insulin anymore. But the FITC release into the solution and won’t quench by GO. The other IBA which still on the GO surface can be protect from the DNAse digestion. Because the binding IBA was destroyed by DNAse I, other IBA can continue release from the GO surface and bind with the insulin, and the above reaction go on and on. So one insulin can bind with more than one IBA, and the output fluorescence signal in the solution has been amplified. At the same time, we test biotin, streptavidin and BSA with the same concentration as insulin to determine the specificity of the above insulin detecion system.Results:The IBA conjugated GO is a good tool to do insulin selection. Depend on that, we can detect insulin in a simple and fast way, and our detection limit is 500nM. While with the amplification strategy, we added DNase I to amplify the output signal, the detection limit was decreased into 5nM. And the detection limit is improved 100 times.Conclusion:The IBA conjugated graphene oxide is a wonderful tool for insulin detection, but without singal amplification, the detection limit is not low enough. By adding DNase I, we can improve the detection limit over 100 times.