Development and Application of Bioradical Detecting System for Oral Tissues : A Basic Research

近年来，活性氧被认为与多种疾病的发病机制有关。虽然药物代谢中活性氧中间体的存在可以通过最终产物分析或抗氧化剂或超氧化物歧化酶等酶的作用来推断，但只有电子自旋共振(ESR)技术才能直接检测这些高活性物种。然而，一些自由基物种由于其极短的半衰期而无法被ESR检测到，这导致了自由基的低稳态浓度或较短的自由基弛豫时间，这导致了非常宽的谱线。这些事实使得最近发展起来的自旋捕获和化学发光技术被广泛应用于自由基的检测。该项目的目标是开发各种可用于在生物模型中测量活性氧物种的分析方法。在这个研究项目中，我们希望建立(1)自旋捕获/电子自旋共振技术…(2)化学发光-光学生物传感器技术的应用，这是一种非常灵敏的方法，具有能够连续、在线、无损地监测活性氧物种的优点。1.去铁胺(DFX)是一种过渡金属离子螯合剂，已用于H_2O_2和过氧化物酶的检测。我们证明了DFX的单电子氧化产物DFX氮氧自由基是H_2O_2-过氧化物酶反应的中间产物，因此可以用ESR光谱(通过检测DFX自由基)来检测H_2O_2和过氧化物酶。2.使用NO捕捉剂Fe(DTCS)_2络合物的ESR光谱方法，可用于体外体系中NO的检测；然而，由于Fe(Dtcs)_2的毒性或L波段的ESR灵敏度较低，在Vevo中检测NO的条件并不理想。3.利用自旋陷阱2，2，6，6-四甲基哌啶(TEMP)研究了孟加拉玫瑰光活化产生单线态氧(^1O2)的ESR光谱。电子自旋共振研究结果表明，孟加拉玫瑰光活化可产生氧，并可检测到氧-TEMP产物(TEMPO；2，2，6，6-tetramethylpiperidine-N-oxyl).4.Optical)。该传感器是为次黄嘌呤的间歇分析系统研制的，其浓度范围为10^~(-4)~10^~(-4)M。这可以通过破坏基础释放的EDRF和/或内皮功能障碍来解释。较少

In recent years, reactive oxygen species have been implicated in the pathogenesis of a wide variety of disorders. Although the existence of reactive oxygen intermediates in drug metabolism can be inferred from end product analysis or from the effects of antioxidants or enzymes such as superoxide dismutase, only the technique of electron spin resonance (ESR) allows the direct detection of these highly reactive species. However, some free radical species cannot be detected by ESR due to their extremely short half-lives, which result in low steady-state concentrations of the radicals or to short radical relaxation times, which lead to a very broad line. These facts made recent development of spin-trapping and chemiluminescence techniques are widely used to detect free radicals. The goal of this project is to develop the various assays available for measurement of reactive oxygen species in biological models. In this research project, we wish to establish (1) the spin-trapping/ESR techniqu … More e in vitro and vivo and (2) the chemiluminescence-optical biosensor application of this technique, a very sensitive method that has the advantage of being able to provide continuous, online, nondestructive monitoring of reactive oxygen species.1.Desferioxamine (DFX), a transition metal ion chelator, has been used to detect H_2O_2 and peroxidase enzyme. We demonstrated that DFX's one-electron oxidation product, DFX nitroxide free radical, is an intermediate of H_2O_2-peroxidase reaction ; thus, H_2O_2 and peroxidase enzyme are detected (-muM) with ESR spectroscopy (by detecting DFX radical).2.ESR spectrometry method using NO-trapping reagent, Fe (DTCS) _2 complex, may be useful for detecting NO in vitro system ; however, the conditions used for NO detection in vevo are not optimal due to Fe (DTCS) _2 toxicity or to low L-band ESR sensitivity.3.Generation of singlet oxygen (^1O_2) from photoactivation of rose bengal was studied by ESR spectroscopy by use of the spin-trap 2,2,6,6-tetramethylpiperidine (TEMP). The result of ESR study showed that ^1O_2 was produced by photoactivation of rose bengal and was detected as ^1O_2-TEMP product (TEMPO ; 2,2,6,6-tetramethylpiperidine-N-oxyl).4.Optical biosensor was developed for the batch analysis system of hypoxanthine in the concentration range between 10^<-8> and 10^<-4> M.With this optical biosensor, it should be possible to measure oxygen-derived free radical generation in a variety of conditions and with multiple interventions.5.Oxygen-derived free radicals, possibly hydroxyl radical, directly produces vasoconstriction in the dog dental pulp of canine teeth. This could be explained by the destruction of basally released EDRF and/or endothelial dysfunction. Less