基于细菌趋化性的第二代细菌印迹技术研究

Selective separation of bacteria with similar structures plays important roles in microbiological examination, however there is lack of efficient and selective separation approach worldwide. Theoretically, molecular imprinting provides high potential for selective isolation of target bacteria. Recently, the applicant reported that the tendency of bacteria to assemble at oil-water interface could be utilized to create microbial recognition sites on the surface of polymer beads. The prepared bacterial imprinted polymers (BIPs) were successfully used for the selective isolation of the target bacteria. Based on our previous experience, in this project we will introduce bacterial chemotaxis into molecular imprinting, which broaden the theory of traditional molecular imprinting, and thus this imprinting method can be named the next generation of bacterial imprinting. Pickering emulsion polymerization or “Stamp fabrication” method will be used to prepare porous BIPs. Polymerization of the monomer followed by removal of the bacterial template resulted in well-defined polymers bearing bacterial imprints. It is noted that, the bacterial chemoattractant and chemorepellent will be loaded into the porous structures of the BIPs before the separation. Besides the passive recognition between bacteria and BIPs imprinted cavities, the selectivity of BIPs will be enhanced by bacterial chemotaxis. The mechanism of this enhanced selective recognition will be futher investigated. Furthermore, this method will be used to selectively separate bacteria with similar structures (e.g. the normal bacteria and drug-resistant bacteria). We believe that the achievment of the next generation of bacterial imprinting has great potential in food safety, disease prevention and clinical diagnosis.

选择性分离结构相近的细菌是微生物检验领域的研究热点和难点。理论上，分子印迹技术可实现靶细菌的高选择性分离。申请人最近在国际上率先采用细菌界面自组装的方式制备了细菌印迹聚合物（BIPs），可用于不同靶细菌的有效分离。本申请拟利用前期研究基础，将细菌趋化性引入分子印迹技术，拓宽传统分子印迹技术理论范围，制备第二代细菌印迹聚合物。在保证细菌与BIPs印迹孔穴之间亲和识别的基础上，利用细菌趋化性产生的主动识别性能对BIPs的吸附选择性进行二次增强。项目拟采用Pickering乳液界面聚合或表面压印方式制备具有多孔结构的BIPs，研究趋化剂的装载量、趋化剂的缓释浓度梯度对细菌识别的影响规律。验证该技术分离结构特征相近细菌（如正常细菌和耐药细菌）的有效性。第二代细菌印迹技术的建立与研究对我国食品安全、疾病预防及临床诊疗等领域的发展具有重要的意义。

靶细菌的高选择性分离在食品安全、疾病预防以及临床诊疗中至关重要。针对微生物检验领域中结构相近细菌难分离的难题，本项目将传统的细菌被动识别与细菌趋化性诱导的主动识别相结合开发了第二代细菌印迹技术。实验研究表明，以大肠杆菌为模板制备的细菌印记薄膜（Coli-BIM）对靶细菌有良好的识别性能。在该细菌印迹技术中，引诱剂的装载能够显著增加Coli-BIM对大肠杆菌的吸附性能和吸附选择性，实现了有鞭毛型大肠杆菌与无鞭毛型细菌的高选择性分离。不同的印迹体系测试表明，选择铜绿假单胞菌为模板制备Pae-BIM能够实现有鞭毛型且形状相似细菌的分离，证实第二代细菌印迹技术具有普适性。项目进一步以耐药性大肠杆菌为模板制备AMP-BIM，与传统的BIM相比，AMP-BIM对靶细菌的结合能力增加了10.8倍。基于该印迹聚合物，项目实现了结构相近细菌（仅胞内物质组成和含量有差异）的分离，拓宽了传统的分子印迹技术理论，可以满足分子生物学领域对细菌分离越来越精细的要求。生物学机制研究表明，靶细菌趋化性诱导的主动识别是第二代细菌印迹材料识别选择性大大增强的原因。项目还通过对比研究阐明了印迹空穴对细菌被动吸附及靶细菌趋化性诱导主动识别之间的关联性，该研究对其它微生物及细胞选择性分离具有重要的指导意义。此外，为了展示基于细菌趋化性的第二代细菌印迹技术的应用范围，项目还制备了一种基于细菌趋化性的新型细菌印迹多孔材料，实现了污水中耐药菌的选择性分离与清除。该高选择性分离靶细菌的新方法在实际的污水处理中有极大的应用价值，具有重要的科学意义。