基于糖基化导电聚合物的细菌生物传感器构建以及用于抗生素耐药性检测

Many bacteria species have been identified as significant food and waterborne pathogens that have profound effects on humans, and the incidence of pathogen-related disease among humans has not yet decreased. Early diagnosis of bacterial infections increases the possibility of successful medical treatment. Furthermore, homeland security threats escalate the need for point-of-care (PoC) detection and identification of potentially hazardous bacterial pathogens. The major requirements for PoC testing systems are real time sensing and miniaturization into a portable, autonomous and inexpensive platform so that the immediate results can be obtained without a laboratory setting. Antibiotics are either naturally occurring or human-made compounds, which are widely used for improving human, animal and plant health and for preventing and treating infections caused by pathogenic bacteria. The continued evolution of antibiotic resistance in pathogenic bacteria has reached a level of public concern that is highlighted by frequent references in the media to the so-called “super-bugs”. Extensive research has been directed towards synergistic combinations of antimicrobial agents. Such combinations are vital for treating infections that fail to respond to single therapeutic agent. However, injudicious, empiric use of antibiotics has contributed to the emergence of more resistant pathogens and has also caused septic shocks in patients. These concerns, coupled with homeland security threats, escalate the need for reliable and effective assays for antimicrobial susceptibility testing. Bacterial cells express both carbohydrate and lectin adhesin structures on their outer cell walls. Certain antibiotics can act on cell wall biosynthesis which can dramatically affect the cell surface carbohydrate and lectin expression in the cell envelope, i.e. cytoplasmic membrane and cell wall. These affects such as the alternation of lipopolysaccharide (LPS) chain length or the alteration in lectin expressions can significantly affect the bacterial binding with the substrates. In this work, Taking advantage of functionalized conducting polymers as a solid state redox probe and the well-established conducting polymers based coupling chemistry for incorporating carbohydrate functionalities; a new glycosylation polymer interface is fabricated to form a carbohydrate platform for bacterial detection. Very importantly, this interface can be used for label free and reagentless detection, both by electrochemical and Quartz Crystal Microbalance (QCM) transducers, and by using lectin mediated LPS-carbohydrate binding. This integrated and orthogonal approach provided an enhanced sensitivity and limits of detection. At the same time, a real time information of QCM regarding these alteration can not only provide a fast bacterial susceptibility testing, but also can explain the fundamental mechanisms of the differences in mode of action of antibiotics, their influence on cell surface morphology, and antibiotic efficacies. With these analytical performances, the described biosensor is envisaged for being capable of differentiating gram negative bacterial strain and species, for many different applications.

本项目拟开展糖基化导电聚合物细菌传感器的研究。合成含有信号物质和糖化合物的导电聚合物单体；电化学聚合法制备糖基化导电聚合物修饰电极及其表征；以凝集素为分子识别物质，石英晶体微天平和电化学为检测手段，发展简单、快速、灵敏、特异性检测细菌的非标记型生物传感新方法；针对抗生素能使细菌表面的脂多糖脱落，使得与固定化凝集素结合的细菌量减少，通过石英晶体微天平和电化学检测的细菌量的变化，研究抗生素种类和用量对细菌结构的影响；探讨抗生素对凝集素和细菌之间相互作用的影响；建立抗生素耐药性检测的新方法。这项研究不仅为抗生素性能评价提供新思路，而且将为环境检测，食品安全，临床诊断和药物筛选等提供性能优良的分析器件，对临床医学、生命科学和材料科学研究以及生物传感器的发展均具有重要意义。

本项目开展了基于糖基化导电聚合物的细菌生物传感器构建以及用于抗生素耐药性检测的研究。以凝集素、三叉DNA以及多肽为分子识别物质，在分子识别物质，固定化方法以及传感器设计和分析应用方面进行了研究。（1）发展了基于对氨基苯甲酸和二维金属有机骨架（MOFs）电极修饰材料的纳米信号增强策略；（2）研究了DNA三叉结构节点碱基种类和个数和DNA三叉结构在电极表面固定化方法不同对传感器信号输出的影响。（3）利用抗生素对细菌的相互作用、基质金属蛋白酶底物对其的聚合作用以及目标物与三叉DNA相互作用，发展了检测金属离子、肿瘤标示物、细菌、抗生素等的电化学和电化学发光生物传感新器件和新方法；发展了细菌、酶与药物作用模型和药物筛选新方法。这项研究成果不仅为抗生素性能评价提供新思路，而且将为环境检测，食品安全，临床诊断和药物筛选等提供性能优良的分析器件，对临床医学、生命科学和材料科学研究以及生物传感器的发展均具有重要意义。

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