Construction Of Novel Peroxynitrite Anion Sensor And Research Of Antioxidants

Peroxynitrite anion（ONOO^-）is one of the most important molecules in biological systems,and it is usually maintained at a very low concentration under normal physiological condition.However,under oxidative stress the body will produce high concentrations of ONOO^-abnormally,which may damage many biomolecules seriously and affect the physiological functions of cells.Therefore,it is very important to construct a sensor that can sensitively and rapidly detect ONOO^-released by cells.Meanwhile,it is also equally significant to explore a method for efficient removal of ONOO^-.In this dissertation,we constructed two ONOO^–electrochemical sensors by simple methods,and used to detect ONOO^–released by living cells.Meanwhile,we also investigated the synergistic effect of different antioxidants on the removal of ONOO^–from cells.The details of the study are as follows:（1）First,microporous polymer nanospheres（MPNS）were successfully prepared by a simple aldol condensation reaction.Then,the novel electrochemical sensor was constructed on a glassy carbon electrode（GCE）decorated with a nanocomposite（CTS-MPNS）synthesized from chitosan（CTS）functionalized microporous polymeric nanospheres（MPNS）.The prepared CTS-MPNS/GCE sensor showed a supernormal manifestation in measuring ONOO^-in a wide range of concentrations from 3.83 nM to0.104 mM,and the detection limit is as low as 1.28 nM（S/N=3）.In addition,satisfactory results were obtained from the selectivity,reproducibility and stability of the ONOO^-sensor,which made it possible to detect trace amounts of ONOO^-released from U87 cells.Significantly,the synergistic effect of different antioxidants（alpha lipoic acid,reduced glutathione,and ascorbic acid）on scavenging ONOO^-in biological systems is further studied by an electrochemical method for the first time.It is found that the antioxidant（ALA+GSH）has the strongest antioxidant capacity and better protective effect on cells.In addition,the ability of different antioxidants to scavenge ONOO^-is further investigated under the stimulation of heavy metal ions and drugs.Compared the results of the experiment,one can see that the antioxidants have better effect under the stimulation of heavy metal ions,which provide an efficient strategy for protecting cells against oxidative stress.（2）Based on the previous part of the experiment,the prepared microporous polymer nanospheres（MPNS）were carbonized further to obtain carbon nanospheres（CNS）,and then combined with the synthesized platinum nanoparticles（PtNPs）.Next,a novel electrochemical sensor was constructed with PtNPs/CNS and chitosan acetate solution（CTS）.The CTS/PtNPs/CNS/GCE sensor showed excellent analytical performance for ONOO^-detection.It has a wide linear range of 0.615 nM to 0.139mM,and an extremely low detection limit of 0.205 nM（S/N=3）.In addition,it also showed good selectivity,stability and reproducibility,and was applied to detect ONOO^-released from cells.We further investigated cells to release ONOO^-induced by Cd²⁺,and found that more ONOO^-released from cells under higher Cd²⁺concentration.Finally,the protective effects of theα-lipoic acid（ALA）and glutathione（GSH）on cells were further investigated,and antioxidants mixture（ALA+GSH）has better protect cells due to their synergistic effect.The experimental results show that the developed sensing platform and efficient antioxidation strategy provide the possibility for the future application in the biomedical field and cancer diseases.