苯系物细胞传感器固定化检测条件的优化及其与降解相结合的研究
文献类型：学位论文
| 作者 | 唐阔 |
| 学位类别 | 硕士 |
| 答辩日期 | 2012-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 庄国强 |
| 关键词 | 苯系物细胞传感器 细胞与包埋剂的比例 固定化时间 固定化颗粒投加量 联合固定化 cell-based biosensor of benzene derivates cells and embedding agent ratio immobilized time immobilized beads dosing quantity co-immobilization |
| 其他题名 | Optimization of Immobilized Biosensor for Detection of the Benzene Derivates and Studies on its Detection Combined with Degradation |
| 学位专业 | 环境科学 |
| 中文摘要 | 细胞传感器的发展丰富了环境污染检测的方法和手段，但是由于自身使用条件的限制，难以在实验室以外的空间完成检测。相对于游离状态，经过固定化处理后的细胞传感器具备了更多优势：细胞浓度提高，效率也随之上升；固定化材料对内部的细胞起到保护作用，屏蔽或减少了外界环境中的毒性物质等不利因素的压力与干扰；此外，固定化细胞更加适合于实际环境中的应用。但是固定化过程中，由于外界条件的变化，细胞传感器自身的生理活性也发生一定的变化，而检测效果也随之受到了影响。因此，有必要对细胞传感器固定化过程中相关的重要因素进行研究，并对其进行优化，以便获得最佳的检测效果。 首先固定化细胞传感器与游离状态下的检测结果进行了比较，可以得到更高的单位体积信号强度。固定化过程对细胞的存活数产生了影响，固定化时间越长，颗粒中细胞传感器浓度越低。而在检测过程中，固定化颗粒中的细胞传感器数量没有出现明显增长，而且远小于游离状态下细胞传感器的数量。 以各个检测时间下的荧光信号强度为指标，对于细胞与包埋剂之间的比例、固定化时间、以及固定化颗粒的投加量等因素通过正交试验进行研究。结果表明：在较短的检测时间内，细胞与包埋剂之间的比例对荧光信号的影响程度最高，其次是固定化时间，最后是固定化颗粒投加量。经过优化后的固定化及检测条件为：细胞与包埋剂之间的比例为20 mL（菌液）/10 mL（包埋剂）；固定化为2 h，颗粒投加量为1.25 mL（颗粒）/50 mL（培养基）。 对不同浓度的苯进行检测，结果表明：在较短的检测时间内，固定化过程对于高浓度苯的检测信号，具有更高的提升效果。 将苯系物细胞传感器与降解菌进行联合固定化。降解菌的加入对于细胞传感器的激发-发射光谱未产生明显影响。加入降解菌后的联合固定化颗粒，其检测信号随降解菌加入的比例变化而变化。在较短的检测时间内，加入的降解菌比例越高，荧光信号越低。但是加入一定比例的联合固定化颗粒，经过长时间诱导后，获得的荧光信号比不含有降解菌的固定化细胞传感器更高。 联合固定化颗粒进行在线降解与检测，结果与单独降解和检测的结果进行比较。联合固定化颗粒与只具有降解功能的固定化颗粒都具有良好的降解能力。但是进行在线检测的联合固定化颗粒，和单独的固定化细胞传感器相比，获得的检测信号要低。同时，进水流量对降解效果也产生影响：流量越低，水力停留时间越长，降解效果越好；但是对在线监测的影响规律并不明显。 |
| 英文摘要 | The development of the cell-based biosensor enriches the environmental pollution detection methods and means, but it is difficult to complete detection outside of laboratory because of their own conditions of use restrictions. Immobilized cell-based biosensors have more advantages over the free ones: efficiency is enhanced via concentration of the cells; immobilization materials protect internal cells, and shield or decrease the pressure and interference of negative factors of the toxic substance in external environment; in addition, the immobilized cells are more suitable for application in a real setting. However, the physiological activities of cell-based biosensor will alter with the change of surrounding condition during immobilization, and time detection effects also be influenced at the same. Therefore, it is necessary to research and optimize the key factors in immobilization process of the cell-based biosensor, getting the best detection effect. First, we got higher signal unit per volume by immobilized biosensor compared with the free cell. The immobilization process affect the numbers of survival cells: the concentration of cell-based biosensor in the immobilization beads got lower as the immobilized time increased. In the process of testing, the number of cell-based biosensor in immobilized beads was much less than the free ones, and didn’t obviously increase. Taking the fluorescence signal intensity of each test time as the index, the factors of proportion between cells and embedding agent, the immobilized time and immobilized particles dosing quantity were taken into consideration through orthogonal test. The results show that in a relatively short period of short testing time, proportion between cells and embedding agent is most dominative factor, prior to immobilized time and immobilized particles dosing quantity. The immobilized and test conditions after optimized are as follows. The proportion between cells and embedding agent is 20 mL (bacteria liquid) / 10 mL (embedding agent); the immobilized time is 2h, beads dosing quantity is 1.25 mL (beads) / 50 mL (medium). Detecting different concentration of benzene, the results show that in a relatively short period of testing time, the immobilized process has higher promoting effect for high concentrations of benzene detection signal. The cell-based biosensor of benzenes and degrading bacteria were co-immobilized. Adding degrading bacteria didn’t obviously affect the stimulate-emission spectrum of cell-based biosensor of benzenes. The detection signal of co-immobilized beads alterd with the ratio of the degrading bacteria changed. In a relatively period of testing time, the fluorescent signal was lower with the proportion of degrading bacteria got higher. But to join a percentage of joint immobilized particles and after long time induction, the fluorescent signal of co-immobilized beads was higher than those without degrading bacteria. During online degradation and detection, co-immobilized beads turned out to have the same ability in degradation compared with immobilized beads of degrading bacteria only. But for the on-line detection, the detection signal of co-immobilized beads was lower compared with those of immobilized cells biosensor. At the mean time, the flow rate also had an impact on degradation: at lower flow rate and longer hydraulic retention time, the degradation effect got higher, but the influence on the on-line monitoring was not obvious. |
| 公开日期 | 2014-05-08 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/7200]  |
| 专题 | 生态环境研究中心_中国科学院环境生物技术重点实验室 |
| 推荐引用方式 GB/T 7714 | 唐阔. 苯系物细胞传感器固定化检测条件的优化及其与降解相结合的研究[D]. 北京. 中国科学院研究生院. 2012. |
入库方式： OAI收割
来源：生态环境研究中心
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