Study The Interaction Between Bel-7404 Cell Line And Its Aptamer Using Single Molecular Force Spectroscopy

As a novel kind of cancer-targeted recognition probe, aptamer has been paid attention in the research fields of biosensor, diagnosis and treatment because of its high binding affinity and high specificity. As molecular probes, aptamers are usually immobilized on a solid surface in these researches. To maintain the binding affinity and specificity of immobilized aptamer to its target cell, the immobilization strategy of aptamer is of great importance. Therefore, it is essential to investigate the effect of different immobilization methods on the interaction between aptamer and its target cell at the living cell level. In this thesis, taking human hepatocarcinoma cell line Bel-7404 for example, the effect of aptamer immobilization strategies on the interaction between Bel-7404 cell and its aptamer?ls2n? was systematically investigated using single molecular force spectroscopy?SMFS?. The effect of heavy metal ions on the interaction of aptamer/Bel-7404 cell was further evaluated by SMFS. The contents are as follows:1. The effect of different immobilization methods on the interaction between Bel-7404 cell and its aptamer?ls2n? was investigated using SMFS. The effect of different immobilization orientations?3'-end or 5'-end?, spacers?oligo?d T? spacer or dodecyl spacer? and cross-linkers?poly?ethylene glycol??PEG? or glutaraldehyde? on the interaction of aptamer/Bel-7404 cell was systematically evaluated by SMFS under physiological condition. It was found that the binding probability of 5'-end amino-modified aptamer/Bel-7404 cell was higher than that of 3'-end amino-modified aptamer/Bel-7404 cell, thus implying that 5'-end immobilization facilitated the binding of aptamer/Bel-7404 cell. In comparison with the modification method without spacer, oligo?d T? spacer could obviously improve the affinity of aptamer to Bel-7404 cell. However, dodecyl spacer could slightly improve the binding probability of aptamer/Bel-7404 cell only for glutaraldehyde immobilization, and almost had no effect on that of aptamer/Bel-7404 cell when PEG was used as the cross-liker. Thus, an oligo?d T? spacer was much more effective than a dodecyl spacer. In comparison with glutaraldehyde immobilization, higher binding probability of aptamer/Bel-7404 cell was obtained when PEG was used as the cross-linker. Therefore, PEG immobilization was superior to glutaraldehyde immobilization. This study may provide valuable information for aptamer-based chemical biosensing design.2. The effect of heavy metal ions on the interaction between Bel-7404 cell and its aptamer?ls2n? was investigated using SMFS. After Bel-7404 cells were treated with cadmium ion(Cd²⁺) or mercury ion(Hg²⁺), the interaction between aptamer and Bel-7404 cell was determined. The results showed that the binding probability of aptamer/Bel-7404 cell obviously decreased along with the increasing concentration of Cd²⁺ or Hg²⁺. But the unbinding force between aptamer and Bel-7404 cell treated with Cd²⁺ or Hg²⁺ increased. The possible reason was that Cd²⁺ and Hg²⁺ affected the well-balanced metabolizability of Bel-7404 cells, thus interfering with normal expression of target receptor on Bel-7404 cell, or both heavy metal ions caused alteration in cell structure. These results may provide valuable reference for understanding biomolecular interaction under complex conditions.