| Bleomycin is an important antitumor drug in clinic. It exhibits significant anti-cancer effects, low bone marrow suppression, and low immune inhibition. It is used as the first line antitumor drug in the treatment of various cancers, such as squamous cell carcinoma, germ cell tumor, kaposi’s sarcoma, cervical cancer and malignant lymphoma and so on. Since bleomycin has narrow therapeutic window and different dosage of drug devotes to different plasma concentration, it is significant to monitor plasma concentration for better therapeutic effect, lower side effect and rational application of drug.It has been reported that bleomycin could oxidatively cleave DNA molecules. Therefore, utilizing DNA molecule as a substrate to recognize bleomycin, the DNA nanomaterial-based biosensing technology can be introduced into the work of bleomycin analysis. Compared with other detection technologies, biological sensing technology is sensitive, selective and programmed. This work constructs a novel DNA molecule recognition probe and takes full use of enzyme-mediated amplified strategy to develop novel biosensors, realizing simple, sensitive, specific and label-free detection of bleomycin.In this paper, the first chapter is the part of introduction. Firstly, this part sketches out discovery and clinical application of bleomycin, mechanism of bleomycin-induced DNA scission and development of detection methods. Secondly, DNA molecular recognition probes of non-nucleic acid targets are summarized. Finally, the enzyme-mediated amplification strategies are introduced.In the second chapter, a novel recognition probe named bicyclo-hairpin probe for bleomycin is constructed. And with this recognition probe, a fluorescent biosensor is developed for label-free detection of bleomycin. In this method, the bicyclo-hairpin probe contains two functional regions:5’-GTGC-3’recognition site for bleomycin and a length of DNA trigger sequence. This probe could recognizes bleomycin, releases the trigger sequence to initiate subsequent amplification reaction, and finally realizes signal amplification. Excess probe that is uncut could silence DNA trigger sequence to reduce non-specific hybridization. Thus, this method had low background. Based on bicyclo-hairpin probe and signal amplification, this strategy constructs a label-free and sensitive amplification strategy for bleomycin detection for the first time. The linear range is from 2 nM to 220 nM, and the detection limit of this method is 0.34 nM. Selectivity experiments toward four different antitumor drugs prove that this method has good selectivity. Recovery experiments (95%-99%) prove a good accuracy. However, it is reported that plasma concentration of bleomycin (24 h) ranges from 1.4 nM to 2.7 μM under intravenous injection. The sensitivity of this strategy is not enough for clinical plasma concentration monitoring.To improve the sensitivity, in the third chapter, a simple, highly sensitive and label-free fluorescent biosensor based on enzyme-mediated cascade amplification reaction is developed for bleomycin detection. In this method, with the presence of bleomycin, bicyclo-hairpin probe releases a trigger sequence. Then, the trigger sequence initiates strand displacement amplification reaction and exponential amplification reaction, realizing cascade signal amplification. The linear range of this strategy was from 40 pM-10 nM with the detection limit of 35 pM, which is lower than previous method. The sensitivity could meet clinical mornitoring of plasma concentration. This strategy has good selectivity toward bleomycin and three other antitumor drugs. Recovery tests (Results:95%-97%) prove that this strategy was accurate in complicated matrix. This strategy could be used in real sample analysis. |
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