电化学传感器检测堆肥微生物及几种典型环境污染物的研究
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摘要
电化学方法是现代环境污染物以及环境微生物检测技术中新兴起来的一种,它以其独特的优势吸引着众多研究者的关注和兴趣。随着各学科知识的不断融合以及各种各样化学修饰电极的出现,电化学传感器的应用领域越来越广泛。堆肥是固体废物处理方法中一种很有效的生物方法,而堆肥的主要影响因素就是微生物,因而研究堆肥过程中的微生物的有关参数对有效控制堆肥效率起着至关重要的作用。随着社会和经济的发展,进入环境中的污染物越来越多,给人类的生存发展乃至整个地球生态系统带来毁灭性的灾难；污染物的种类繁多,而其中的重金属和持久性有机污染物的危害尤其严重,因而迫切需要能够在环境污染现场快速且准确地对它们进行定性或定量的检测分析手段。
论文根据铜绿假单胞菌16S rRNA的特异性序列设计了一种发夹形DNA探针,两端分别标记荧光基团和淬灭基团,利用荧光共振能量转移的原理对与探针互补的人工合成的核苷酸链进行了检测。该探针显示出良好的特异性,在最佳实验条件下,发现在FAM最大发射波长处测得的荧光值与目标核酸的浓度呈一定线性关系,该探针的检测范围为0.1-50nM,最低检测限为0.02nM。从铜绿假单胞菌中用试剂盒提取总RNA,与探针反应,检测其中的16S rRNA特异性片段,得到较为满意的结果。
论文将源于铜绿假单胞菌16S rRNA的特异性序列设计的发夹形DNA探针通过自组装固定在金电极表面,并以链霉亲和素-辣根过氧化物酶(HRP-SA)做电化学活性指示剂构建电化学传感器。优化实验条件后,得到该电化学DNA传感器的检测范围为0.05～100nM,宽于荧光法的检测范围(0.1-50nM),最低检测限为0.002nM,比荧光法的检测限(0.02nM)要低。培养铜绿假单胞菌,提取其总RNA后预处理,用该传感器对RNA进行检测,检测结果与紫外分光光度法的检测结果基本一致。
论文在较低温度下(80℃)合成纳米二氧化钛/GO (TiO2/GO)复合材料,扫描电镜(SEM)的结果显示了该复合物的结构特点。把该复合材料分散在0.05%Nafion中,然后修饰玻碳电极来构建电化学传感器,通过方波伏安法(SWV)检测了有机污染物五氯酚(PCP),该传感器在2.5×10-9～1.0×10-7M范围内和在5.0×10-7-2.5×10-5M范围内分别得到线性关系,对PCP浓度的检出限为1.0×10-10M。用该电化学传感器对从堆肥中提取的样品进行了检测,采用标准加入法,得到较好的结果。
论文用天然鳞片石墨制备了氧化石墨,并通过超声作用得到氧化石墨烯胶体,采用电化学还原法对修饰在玻碳电极表面的氧化石墨烯胶体进行还原,得到石墨烯修饰的电极,再修饰Nafion膜,然后预镀一层铋膜,用微分脉冲阳极溶出伏安法(DPASV)检测了痕量重金属铅和镉。实验结果表明,铅和镉在石墨烯/Nafion铋膜电极上能显示出灵敏的溶出峰,在最佳实验条件下,铅和镉的检出限分别为0.02μg/L和0.01μg/L。对市售新鲜小白菜叶中含有的铅和镉进行了检测,将检测结果与原子吸收光谱法的结果进行了比对,二者表现出一致。
Electrochemical method is a modern kind of technology in environmental pollutants and environmental microbial detection, it have attracted growing interests owing to its inherent unique advantages. With the continued integration of academic knowledge and the emergence of a variety of chemically modified electrodes, electrochemical sensor became more and more common in many areas. Compost is an effective biological method of solid waste disposal, one of the main influencing factors is the microorganisms. Thus, if want to control of the composting effectively, it is very important to study the correlation parameter of microorganisms in the composting process. With the social and economic development, more and more pollutants enter in our environment, which created a devastating disaster to human survival and development, even the whole ecosystems in the earth. Heavy metals and persistent organic pollutants (POPs) are two of the typical in multitudinous environmental pollutants, therefore, it have an urgent call for methods to detect the pollutants qualitative or quantitative rapidly and accurately in the in situ measurement.
A hairpin-shaped DNA probe has been designed according to the special sequence of Pseudomonas aeruginosa16S rRNA gene. The probe modified a quencher (DABCYL) at its3'end and a carboxyfluorescein (FAM) at its5'end, then the probe detect its complementary strand based on the principle of fluorescence resonance energy transfer. The probe showed a good specificity, and also obtained a linear regression of fluorescence intensity vs. the logarithm of complementary strand concentration under the optimum condition. The detection range was0.1-50nM and detection limit was0.02nM. Total RNA isolated from Pseudomonas aeruginosa using the isolate kit, the16S rRNA was analyzed by this probe, showing a good result.
An electrochemical DNA biosensor based on immobilizing the hairpin-shaped DNA probe on a gold electrode for Pseudomonas aeruginosa detection was developed, streptavidin-horseradish peroxidase (HRP) was used as electrochemical activity indicator. The probe modified with a biotin at its3'end and a thiol at its5'end was immobilized onto the gold electrode surface through self-assembly. Under the optimum condition, the detection range of the electrochemical sensor was0.05～100nM, and the detection limit was0.002nM, it is obviously better than the fluorescence method. The16S rRNA extracted from Pseudomonas aeruginosa was analyzed by this proposed sensor. The results were in agreement with the reference values deduced from UV spectrometric data. The biosensor was indicative of good precision, stability, sensitivity, and selectivity.
TiO2-GO intercalated composite was prepared at a low temperature (80℃) with single and multi-layered graphite oxide mixture (GO) and titanium sulfate (Ti (SO4)2) as initial reactants. The surface morphology of TiO2-GO intercalated composite was characterized by Field Emission Scanning Electron Microscope (FE-SEM). TiO2-GO intercalated composite was dispersed in0.05%Nafion solution, for fabricating the electrochemical sensing platform to determine the pentachlorophenol (PCP) by square wave voltammetry (SWV). Under the optimum experiment conditions, the SWV oxidation peak current was linearly related to the concentration of PCP, ranging from2.5×10-9M to1.0×10-7M and from5.0×10-7M to2.5×10-5M, with a detection limit of1.0×10-10M. The practical application of the proposed methods was verified in the compost sample determination.
Graphite oxide was prepared from natural flake graphite, and graphene oxide was obtained via sonication. The graphene was prepared from electrochemical reduction of graphene oxide on the glassy carbon electrode, and then the graphene/GCE was modified with Nafion. Bismuth film modified graphene/Nafion/GCE was prepared by ex-situ plating for the simultaneous determination of a trace level of Pb2+and Cd2+by differential pulse anodic stripping voltammetry (DPASV). Results showed that sensitive potential stripping peak was obtained for Pb2+and Cd2+with the proposed electrochemical sensor, under the optimum experiment conditions, the detection limit of Pb2+and Cd2+was0.02μg/L and0.01μg/L, respectively. The sensor was applied to determination of Pb2+and Cd2+in fresh cabbage leaves, comparing the result with that of atomic absorption spectrometry (AAS), the concordance of results was satisfactory.
论文根据铜绿假单胞菌16S rRNA的特异性序列设计了一种发夹形DNA探针,两端分别标记荧光基团和淬灭基团,利用荧光共振能量转移的原理对与探针互补的人工合成的核苷酸链进行了检测。该探针显示出良好的特异性,在最佳实验条件下,发现在FAM最大发射波长处测得的荧光值与目标核酸的浓度呈一定线性关系,该探针的检测范围为0.1-50nM,最低检测限为0.02nM。从铜绿假单胞菌中用试剂盒提取总RNA,与探针反应,检测其中的16S rRNA特异性片段,得到较为满意的结果。
论文将源于铜绿假单胞菌16S rRNA的特异性序列设计的发夹形DNA探针通过自组装固定在金电极表面,并以链霉亲和素-辣根过氧化物酶(HRP-SA)做电化学活性指示剂构建电化学传感器。优化实验条件后,得到该电化学DNA传感器的检测范围为0.05～100nM,宽于荧光法的检测范围(0.1-50nM),最低检测限为0.002nM,比荧光法的检测限(0.02nM)要低。培养铜绿假单胞菌,提取其总RNA后预处理,用该传感器对RNA进行检测,检测结果与紫外分光光度法的检测结果基本一致。
论文在较低温度下(80℃)合成纳米二氧化钛/GO (TiO2/GO)复合材料,扫描电镜(SEM)的结果显示了该复合物的结构特点。把该复合材料分散在0.05%Nafion中,然后修饰玻碳电极来构建电化学传感器,通过方波伏安法(SWV)检测了有机污染物五氯酚(PCP),该传感器在2.5×10-9～1.0×10-7M范围内和在5.0×10-7-2.5×10-5M范围内分别得到线性关系,对PCP浓度的检出限为1.0×10-10M。用该电化学传感器对从堆肥中提取的样品进行了检测,采用标准加入法,得到较好的结果。
论文用天然鳞片石墨制备了氧化石墨,并通过超声作用得到氧化石墨烯胶体,采用电化学还原法对修饰在玻碳电极表面的氧化石墨烯胶体进行还原,得到石墨烯修饰的电极,再修饰Nafion膜,然后预镀一层铋膜,用微分脉冲阳极溶出伏安法(DPASV)检测了痕量重金属铅和镉。实验结果表明,铅和镉在石墨烯/Nafion铋膜电极上能显示出灵敏的溶出峰,在最佳实验条件下,铅和镉的检出限分别为0.02μg/L和0.01μg/L。对市售新鲜小白菜叶中含有的铅和镉进行了检测,将检测结果与原子吸收光谱法的结果进行了比对,二者表现出一致。
Electrochemical method is a modern kind of technology in environmental pollutants and environmental microbial detection, it have attracted growing interests owing to its inherent unique advantages. With the continued integration of academic knowledge and the emergence of a variety of chemically modified electrodes, electrochemical sensor became more and more common in many areas. Compost is an effective biological method of solid waste disposal, one of the main influencing factors is the microorganisms. Thus, if want to control of the composting effectively, it is very important to study the correlation parameter of microorganisms in the composting process. With the social and economic development, more and more pollutants enter in our environment, which created a devastating disaster to human survival and development, even the whole ecosystems in the earth. Heavy metals and persistent organic pollutants (POPs) are two of the typical in multitudinous environmental pollutants, therefore, it have an urgent call for methods to detect the pollutants qualitative or quantitative rapidly and accurately in the in situ measurement.
A hairpin-shaped DNA probe has been designed according to the special sequence of Pseudomonas aeruginosa16S rRNA gene. The probe modified a quencher (DABCYL) at its3'end and a carboxyfluorescein (FAM) at its5'end, then the probe detect its complementary strand based on the principle of fluorescence resonance energy transfer. The probe showed a good specificity, and also obtained a linear regression of fluorescence intensity vs. the logarithm of complementary strand concentration under the optimum condition. The detection range was0.1-50nM and detection limit was0.02nM. Total RNA isolated from Pseudomonas aeruginosa using the isolate kit, the16S rRNA was analyzed by this probe, showing a good result.
An electrochemical DNA biosensor based on immobilizing the hairpin-shaped DNA probe on a gold electrode for Pseudomonas aeruginosa detection was developed, streptavidin-horseradish peroxidase (HRP) was used as electrochemical activity indicator. The probe modified with a biotin at its3'end and a thiol at its5'end was immobilized onto the gold electrode surface through self-assembly. Under the optimum condition, the detection range of the electrochemical sensor was0.05～100nM, and the detection limit was0.002nM, it is obviously better than the fluorescence method. The16S rRNA extracted from Pseudomonas aeruginosa was analyzed by this proposed sensor. The results were in agreement with the reference values deduced from UV spectrometric data. The biosensor was indicative of good precision, stability, sensitivity, and selectivity.
TiO2-GO intercalated composite was prepared at a low temperature (80℃) with single and multi-layered graphite oxide mixture (GO) and titanium sulfate (Ti (SO4)2) as initial reactants. The surface morphology of TiO2-GO intercalated composite was characterized by Field Emission Scanning Electron Microscope (FE-SEM). TiO2-GO intercalated composite was dispersed in0.05%Nafion solution, for fabricating the electrochemical sensing platform to determine the pentachlorophenol (PCP) by square wave voltammetry (SWV). Under the optimum experiment conditions, the SWV oxidation peak current was linearly related to the concentration of PCP, ranging from2.5×10-9M to1.0×10-7M and from5.0×10-7M to2.5×10-5M, with a detection limit of1.0×10-10M. The practical application of the proposed methods was verified in the compost sample determination.
Graphite oxide was prepared from natural flake graphite, and graphene oxide was obtained via sonication. The graphene was prepared from electrochemical reduction of graphene oxide on the glassy carbon electrode, and then the graphene/GCE was modified with Nafion. Bismuth film modified graphene/Nafion/GCE was prepared by ex-situ plating for the simultaneous determination of a trace level of Pb2+and Cd2+by differential pulse anodic stripping voltammetry (DPASV). Results showed that sensitive potential stripping peak was obtained for Pb2+and Cd2+with the proposed electrochemical sensor, under the optimum experiment conditions, the detection limit of Pb2+and Cd2+was0.02μg/L and0.01μg/L, respectively. The sensor was applied to determination of Pb2+and Cd2+in fresh cabbage leaves, comparing the result with that of atomic absorption spectrometry (AAS), the concordance of results was satisfactory.
引文
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