【作者】 杨丽敏；

【导师】 霍丹群；

【作者基本信息】 重庆大学 ， 细胞/组织工程与生物材料， 2010， 博士

【摘要】 有机磷化合物（organophosphorus compounds, OPs）不仅在农业生产中,被广泛应用于杀虫剂和除草剂,还可在军事中用作化学战剂,对生态环境、人身和社会安全构成了严重威胁。传统的检测手段虽然定量准确、灵敏度高,但分析设备多属于大型仪器,价格昂贵,需要专业人员操作,难于满足预防和控制突发事故的需要。因此,农残检测、现实反恐等迫切需要一种灵敏度高、选择性好、小巧、轻便、价格低廉、便于大规模使用的检测手段实现对微痕量OPs快速、灵敏、准确的检测,才能切实保障环境、社会和人身安全。鉴于此,本论文着重研究了植物酯酶-卟啉新型复合敏感材料的制备与筛选,深入研究了其与OPs的作用机理,为最终构建出一种新型的酶-卟啉复合膜阵列式传感系统奠定基础。具体开展的研究工作如下:（1）在传统双水相萃取技术的基础上,系统性研究了萃取过程参数（如双水相类型、成相盐、PEG分子量及浓度、系统pH值、中性盐的选择等）对植物酯酶分配行为的影响,开发出一种适于植物酯酶纯化的两步萃取法。第一步萃取中,采用27% PEG1000/ 13% NaH2PO4双水相体系,植物酯酶的纯化因子（PF）和产率（Y）分别为5.26和85.08%。再进行第二步萃取,其体系组成为27% PEG1000/ 13% NaH2PO4/ 6.0% （NH4）2SO4。经过两步萃取后,PF和Y分别达到18.46和83.16%。经过这种方法所得的植物酯酶的纯化倍数是传统盐析法的4.8倍。通过透析法将纯化酶中的（NH4）2SO4去除后,经冷冻干燥最终得到粉末状的植物酯酶纯酶制品。（2）以毒性较低的有机磷毒剂模拟剂甲基磷酸二甲酯（dimethyl methyl phosphonate,DMMP）为研究对象,利用紫外-可见吸收光谱,筛选了具有DMMP敏感性的植物酯酶-卟啉复合材料。结果表明,在所研究的复合材料中,对DMMP灵敏度最高的是植物酯酶-H₂TPPS₁（22380.95）,其灵敏度远远高于其他任何材料。其次为植物酯酶-CoTPPCl（1318.30）,最差的是植物酯酶-CuTPP(0.94×10^-6)。将植物酯酶-卟啉复合材料所得的灵敏度与卟啉单独检测时的灵敏度进行对比知,将卟啉与植物酯酶相复合后,除了CuTPP外,其他材料的灵敏度均有很大幅度的提高,其中效果最为突出的是H₂TPPS₁,其灵敏度提高了3.42×10⁶倍。因此,经过筛选,植物酯酶-H₂TPPS₁、植物酯酶-CoTPPCl、植物酯酶-ZnTPP、植物酯酶-FeTPPCl和植物酯酶-EuTPPCl等五种复合材料可以作为候选材料,用于构建对有机磷毒剂具有高灵敏度的生物传感器。（3）为阐明植物酯酶-卟啉复合敏感材料的检测机理,结合酶反应动力学、荧光光谱、UV-Vis吸收光谱等手段,进一步深入研究了卟啉-植物酯酶-OPs相互作用关系。结果表明,H₂TPPS₁和DMMP都是植物酯酶的竞争性抑制剂。它们与底物竞争结合酶的活性部位,影响底物与酶的正常结合。但H₂TPPS₁和DMMP对植物酯酶的抑制常数ki不同,k_{i（H2TPPS1）} > k_i（DMMP）。DMMP与植物酯酶之间的亲和力要大于H₂TPPS₁。因此,当用H₂TPPS₁-植物酯酶复合材料检测DMMP时,DMMP会将H₂TPPS₁从卟啉-植物酯酶复合物上置换下来,引起420 nm处吸收峰的降低,从而实现DMMP的光谱检测。（4）采用戊二醛交联法,在玻片基材上制备了固定化植物酯酶-H₂TPPS₁纳米传感膜。结果表明,H₂TPPS₁与固定化植物酯酶作用后会在422 nm处形成一特征峰。把固定化植物酯酶-H₂TPPS₁表面暴露于DMMP中,由于DMMP可以把卟啉从酶的活性位点上置换下来,引起422 nm处吸光度的降低。并且,在DMMP浓度低于9×10^-7 M的范围内,422 nm处吸光度的变化与DMMP的浓度线性相关。该膜材的纳米结构增加了其对DMMP的敏感性,降低了检测限,使其可以检测浓度低至的4.5×10^-10 M DMMP。更多还原

【Abstract】 Organophosphorus compounds （OPs） have been not only used as pesticides and herbicides in current agriculture, but also used as chemical warfare agents, becoming a serious threat to ecological environment, personal and social security. While traditional detecting methods have very high sensitivity, reliability and precision, they require expensive laboratory instrumentation, highly trained personnel and time-consuming analysis. These techniques are unsuitable for rapid warning and field analysis. Therefore, rapid, sensitive, and precise detection of OPs in the environment, public places or workplaces become increasingly important for homeland security and environment monitoring. In this thesis, the study was focused on the preparation and selection of plant-esterase-porphyrin complex. The interaction of the complex with OPs was also investigated to construct a new sensor array system integrating plant-esterase-porphyrin film array finally. Researches were carried out as follows:（1） Purification of plant-esterase in an aqueous two-phase system （ATPS） was investigated. The effect of various process parameters such as the type of ATPS, the phase-forming salt, the molecular weight and concentration of PEG, the system pH, and the types and concentrations of neutral salts on partitioning of plant-esterase were evaluated. Using 27% PEG1000/ 13% NaH2PO4, and 27% PEG1000/ 13% NaH2PO4/ 6.0% （NH4）2SO4, plant-esterase was purified by a two-step extraction. Compared to the results obtained with the conventional salting-out method, this method had a comparable yield （83.16% versus the original yield of 80%）, but produced plant-esterase that was 4.8 times as pure （18.46-fold）. Integrating dialysis into the aqueous two-phase extraction removed （NH4）2SO4 from the purified plant-esterase. Finally, plant-esterase was freeze-dried to convert the product to powder.（2） Plant-esterase-porphyrin complexes sensitive for dimethyl methylphosphonate （DMMP）, a simulant for organophosphorus agents, were selected with UV-Vis spectroscopy. The results showed that plant-esterase-H₂TPPS₁ had the highest sensitivity of 22380.95. The sensitivity of the plant-esterase-CoTPPCl, EuTPPCl, ZnTPP and FeTPPCl complex was 1318.3, 0.61, 0.17 and 0.05, respectively. Plant-esterase-CuTPP complex has the lowest sensitivity (0.94×10^-6). Compared to that got with the porphyrins only, the sensitivity was increased except plant-esterase-CuTPP. Especially for plant-esterase-H₂TPPS₁, the sensitivity was significantly increased by 3.42×10⁶. Therefore, five complexes, plant-esterase- H₂TPPS₁, CoTPPCl, EuTPPCl, ZnTPP and FeTPPCl, were selected as sensing material of biosensor for detection of organophosphorus agents.（3） To evaluate the mechanism of detection with plant-esterase-porphyrin complexes, the interaction of porphyrin-plant-esterase-OPs were investigated by the combination of UV-Vis absorption spectroscopy, fluorescence spectroscopy and enzyme kinetics methods. It was found that both H₂TPPS₁ and DMMP were competitive inhibitors of plant-esterase. They bind to plant-esterase at the active site, just like the substrate. However, DMMP exhibites higher inhibition potency than H₂TPPS₁ (k_{i（H2TPPS1）} > k_i（DMMP）). Therefore, the addition of DMMP to the performed plant-esterase-H₂TPPS₁ complex resulted in a loss of the 420 nm absorbance band as the porphyrin was displaced from plant-esterase.（4） A novel sensing nano-film immobilized plant-esterase-H₂TPPS₁ was prepared for the detection of DMMP. The results showed that the interaction of H₂TPPS₁ with immobilized plant-esterase yielded a characteristic peak at 422 nm. Exposure of the immobilized plant-esterase-H₂TPPS₁ surface to DMMP resulted in a decrease in absorbance intensity at 422 nm due to the displacement of the porphyrin from the active site by DMMP. The nano-structure of the film had a good effect on its sensitivity to DMMP and lowered the detection limit. The loss in absorbance intensity at 422 nm was linearly dependent on DMMP concentration below 9×10-7 M. DMMP concentration as low as 4.5×10^-10 M can be detected by the sensing nano-film.更多还原