硫酸盐还原菌的快速检测技术研究
摘要
硫酸盐还原菌（SRB）是腐蚀性最强，也是研究最广泛的腐蚀微生物。本论文针对传统SRB检测方法周期长的问题，分别构建了基于生物识别材料伴刀豆球蛋白A、基于SRB生物印迹薄膜材料和基于SRB特征代谢的快速检测方法，实现对海洋环境中SRB的高灵敏度、高特异性检测，扩展了海洋腐蚀微生物快速检测方法，具有一定的应用前景。具体的研究结果如下：
（1）构建了基于生物识别材料快速检测SRB的生物传感器。以伴刀豆球蛋白A为生物识别材料，以电化学方波伏安法为检测手段，借助多巴胺分子增强聚合作用将伴刀豆球蛋白A识别分子以及羧基二茂铁电化学信号分子共价修饰到Fe3O4@MnO2纳米材料表面。使用该电化学传感器能够将检测时间缩短至2h以内，同时将检测灵敏度提升3.4倍，检测限降低至40cfu mL-1。
（2）构建了基于生物印迹薄膜快速检测SRB的传感器。通过细胞介导法制备SRB生物印迹薄膜实现SRB检测的选择性，以电化学阻抗法为检测手段，引入石墨烯纳米材料提升SRB的吸附平台的导电性，增强检测电流信号。基于生物印迹薄膜快速识别检测SRB克服了生物材料易失活、价格昂贵等缺点，能够在1h内获得SRB种群数量。
（3）构建了基于SRB特征代谢过程快速检测SRB的方法。以SRB能够代谢产生硫化物的特征行为切入，构建了基于ZnO/ZnS阵列转化过程、基于ZnS纳米材料光催化性质、基于Thiobacillus thioparus细胞的微生物传感器、基于对巯基蛋白酶活性抑制作用和基于石墨烯催化性质特异性检测SRB的方法。借助纳米材料和生物酶的信号增强作用提高了检测灵敏度，在保证检测选择性的基础上缩短了检测所需时间。
Sulfate-reducing bacteria (SRB) are the most widely studied marine corrosivemicroorganisms. Since common SRB detection methods are time-consuming, thispaper focus on development and fabrication of fast SRB detection methods. Detectionmethods based on concanavalin A, cell-mediated bioimprinted films, and SRBcharacteristic metabolic process as the recognizing strategies were established forselective, sensitive and fast SRB quantitative analysis. The specific results of thethesis are as follows:
1. A biosensor based on biomaterial recognition was established for rapid SRBdetection. Concanavalin A was chosen as bio-recognition element, and square wavevoltammetry was used for bacterial detection. Concanavalin A and signalingmolecules, ferrocene, were conjugated onto the surface of Fe3O4@MnO2nanomaterials with the help of dopamine enhanced polymerization process. Thiselectrochemical biosensor could shorten bacterial detection time to less than2h.Besides, the detection sensitivity enhancement was on the order of340%, and thelimit of detection would be decreased to40cfu mL-1.
2. A sensor based on bioimprinted films was developed for rapid SRB detection.Bioimprinted films were synthesized by bacteria-mediated approach for selectivebacteria recognition, and electrochemical impedance spectroscopy was used for fastSRB population quantitative analysis. Graphene was introuced into bacterialattachment platform to obtain good electrochemical signals for bacterial detection.This bacterial detection method based on cell-mediated bioimprinted films couldavoid the use of biological bio-recognition elements, which are easily lost specificbinding abilities, and the bacterial population could be obtained within1h.
3. Detection protocols based on SRB characteristic bacterial metabolic processwere constructed for rapid SRB quantitative analysis. Since sulfide is thecharacteristic metabolite of SRB, SRB detection methods based on chemical conversion from ZnO nanorods arrays to ZnS arrays, the photocatalytic property ofmicrobial synthesized ZnS nanoparticles, Thiobacillus thioparus microbial biosensor,inhibition of cysteine protease activity, and reduced graphene sheets were establishedby taking advantage of this SRB characteristic metabolite. The detection sensitivitywas greatly enhacned with the help of nanomaterials and enzymes, and the time forselective SRB detection was greatly shortened as a result.
（1）构建了基于生物识别材料快速检测SRB的生物传感器。以伴刀豆球蛋白A为生物识别材料，以电化学方波伏安法为检测手段，借助多巴胺分子增强聚合作用将伴刀豆球蛋白A识别分子以及羧基二茂铁电化学信号分子共价修饰到Fe3O4@MnO2纳米材料表面。使用该电化学传感器能够将检测时间缩短至2h以内，同时将检测灵敏度提升3.4倍，检测限降低至40cfu mL-1。
（2）构建了基于生物印迹薄膜快速检测SRB的传感器。通过细胞介导法制备SRB生物印迹薄膜实现SRB检测的选择性，以电化学阻抗法为检测手段，引入石墨烯纳米材料提升SRB的吸附平台的导电性，增强检测电流信号。基于生物印迹薄膜快速识别检测SRB克服了生物材料易失活、价格昂贵等缺点，能够在1h内获得SRB种群数量。
（3）构建了基于SRB特征代谢过程快速检测SRB的方法。以SRB能够代谢产生硫化物的特征行为切入，构建了基于ZnO/ZnS阵列转化过程、基于ZnS纳米材料光催化性质、基于Thiobacillus thioparus细胞的微生物传感器、基于对巯基蛋白酶活性抑制作用和基于石墨烯催化性质特异性检测SRB的方法。借助纳米材料和生物酶的信号增强作用提高了检测灵敏度，在保证检测选择性的基础上缩短了检测所需时间。
Sulfate-reducing bacteria (SRB) are the most widely studied marine corrosivemicroorganisms. Since common SRB detection methods are time-consuming, thispaper focus on development and fabrication of fast SRB detection methods. Detectionmethods based on concanavalin A, cell-mediated bioimprinted films, and SRBcharacteristic metabolic process as the recognizing strategies were established forselective, sensitive and fast SRB quantitative analysis. The specific results of thethesis are as follows:
1. A biosensor based on biomaterial recognition was established for rapid SRBdetection. Concanavalin A was chosen as bio-recognition element, and square wavevoltammetry was used for bacterial detection. Concanavalin A and signalingmolecules, ferrocene, were conjugated onto the surface of Fe3O4@MnO2nanomaterials with the help of dopamine enhanced polymerization process. Thiselectrochemical biosensor could shorten bacterial detection time to less than2h.Besides, the detection sensitivity enhancement was on the order of340%, and thelimit of detection would be decreased to40cfu mL-1.
2. A sensor based on bioimprinted films was developed for rapid SRB detection.Bioimprinted films were synthesized by bacteria-mediated approach for selectivebacteria recognition, and electrochemical impedance spectroscopy was used for fastSRB population quantitative analysis. Graphene was introuced into bacterialattachment platform to obtain good electrochemical signals for bacterial detection.This bacterial detection method based on cell-mediated bioimprinted films couldavoid the use of biological bio-recognition elements, which are easily lost specificbinding abilities, and the bacterial population could be obtained within1h.
3. Detection protocols based on SRB characteristic bacterial metabolic processwere constructed for rapid SRB quantitative analysis. Since sulfide is thecharacteristic metabolite of SRB, SRB detection methods based on chemical conversion from ZnO nanorods arrays to ZnS arrays, the photocatalytic property ofmicrobial synthesized ZnS nanoparticles, Thiobacillus thioparus microbial biosensor,inhibition of cysteine protease activity, and reduced graphene sheets were establishedby taking advantage of this SRB characteristic metabolite. The detection sensitivitywas greatly enhacned with the help of nanomaterials and enzymes, and the time forselective SRB detection was greatly shortened as a result.
引文
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