工农业生产的快速发展导致环境中有毒有害物质的种类急剧增加，严重威胁到生态系统和人类健康，迫切需要建立快速有效的方法对环境质量进行评价。微生物传感器因其具有灵敏度高、成本低、可实现原位在线监测等特点，在环境质量监测中备受关注。欧洲亚硝化单胞菌（Nitrosomonas europaea）是一种化能自养菌，通过将环境中的氨氧化成亚硝酸根来获取生长所需的能量。该细菌对环境变化敏感，易受到诸如含硫化合物、芳香族化合物、卤代化合物等污染物的影响，因而常用于环境监测。本文依据欧洲亚硝化单胞菌受到污染物抑制后，氨的消耗速率或亚硝酸根的生成速率减小的原理，以欧洲亚硝化单胞菌为生物敏感元件、聚合物膜离子选择性电极为换能器，并结合流动分析技术构建微生物传感器系统，用于水体污染物毒性的快速测定。具体研究内容如下：1.换能器的构建：细菌代谢涉及亚硝酸根离子和铵离子浓度的变化，因而可以选用亚硝酸根离子选择性电极或铵离子选择性电极为换能器。本文合成了双水杨醛缩邻苯二胺合钴(Ⅱ)和四苯基卟啉合钴(Ⅱ)两种金属配合物，并将其作为离子载体构建聚合物膜亚硝酸根离子选择性电极。结果显示，基于所合成的金属配合物的亚硝酸根离子选择性电极的线性响应范围均为1.0×10-1～1.0×10-5 M，响应斜率为60 mV/decade。采用无活菌素为铵离子载体制备了铵离子选择性电极，结果显示，其线性响应范围为1.0×10-3～5.0×10-6 M，响应斜率为56 mV/decade。相比之下，铵离子选择性电极的灵敏度较高，因而最终选用铵离子选择性电极作为换能器。2.微生物膜的制备：将欧洲亚硝化单胞菌菌种进行活化和扩大培养，并测定其生长曲线，选取处于对数期末期的细菌为生物材料。采用夹心法将细菌固定于两层孔径为0.2μm的聚醚砜滤膜中间。本制备方法操作简单，条件温和，可防止细胞泄露。3.流动分析微生物传感器系统的集成及污染物毒性的测定：大多数生物传感器将生物活性材料直接固定到换能器表面，待测物质与生物活性材料作用所产生的物质需经扩散作用到达换能器表面，致使传感器响应时间长、灵敏度低。基于此，本研究结合流动分析方法，将固定化的细菌和电极分别放置于流动分析系统中的滤膜支架和电化学检测池中，使分子识别和信号转换分开进行。考察了背景溶液浓度、pH、温度、流速和细菌固定量对系统响应的影响，并在最优条件下测定了烯丙基硫脲、硫代乙酰胺和苯酚的抑制作用，测得其半抑制浓度（IC50）分别为0.17 μM、0.46 μM和49.5 μM。测定结果与光度法一致。与传统的生物传感器相比，该方法的优点在于：产物不需经过敏感层扩散到转换器表面，因而缩短了电极响应时间；响应灵敏度可通过增加活性材料的固定量得到提高；生物活性材料和换能器互不影响，可以各自处于最佳工作状态。该方法可用于水体污染物的灵敏快速测定。; A wide rang of toxic compounds have been released into the environment, causing potential risks to the ecosystem and human health. Hence rapid and efficient analytical methods are required for the assessment of environmental quality. Microbial biosensors have been widely used for environmental monitoring owing to their high sensitivity, low cost and versatility for online measurements.Nitrosomonas europaea (N. europaea)is chemoautotrophic and derives its energy for growth from the oxidation of ammonia to nitrite. The metabolism of N. europaea can be inhibited by a wide range of substrates including sulfur, aromatic, and halogenated compounds, and for this reason it is a well accepted target microorganism used for environmental detection. In this dissertation, a flow biosensing system using N. europaeaas a bioreceptor and a polymeric membrane ion-selective electrode as a transducer is proposed for the detection of toxicity in water. The system is based on the inhibition on the activity of N. europaea, which can be evaluated by measuring the ammonium consumption or the nitrite formation rate. The contents are as follows:1.Construction of the transducerCo(II)-salophen and Co(II)-tetraphenylporphyrin were synthesized as ionophores to prepare nitrite selective polymeric membrane electrodes. Both electrodes show a linear response towards nitrite in the range 1.0×10-1-1.0×10-5 M with a slope of 60 mV/decade. The response of ammonium selective electrode using nonactin as ionophore was also examined. The electrode shows a linear range of 1.0×10-3～5.0×10-6 M with a slope of 56 mV/decade. The ammonium selective electrode was chosen as the transducer for its lower detection limit.2. Preparation of the microbial filmN. europaea cells were activated and cultured in a growth media. The growth curve and morphology of the cells were examined. Cells were harvested during late-exponential growth and physically immobilized between two polyethersulfone filter membranes. The immobilization method offers simple procedures and mild conditions, and can avoid cell leakage. 3. Integration of the flow biosensing system for toxicity detectionFor traditional biosensors, the bioreceptor is usually immobilized on the transducer surface and the analyte is sensed by diffusing through the test solution to the inner detector surface. These sensors often suffer from problems of long response time and low sensitivity. In this work, a new type of biosensor which allows the molecular recognition and transduction processes to be done individually is proposed. Cells are immobilized on the polyethersulfone membranes packed upstream in a holder, while the membrane electrode is placed downstream in the flow cell. The flow biosensing mode simplifies the sensor construction and permits one to execute the individual unit operations under optimum conditions rather than to operate them concurrently under compromise conditions. Three inhibitors of ammonia oxidation including allythiourea, thioacetamid, and phenol have been tested. The IC50 values have been measured as 0.17, 0.46 and 49.5 μM for allythiourea, thioacetamid, and phenol, respectively. The proposed sensor offers advantages of simplicity, rapidity and high sensitivity for measuring toxicity in water.
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