纳米氧化铜模拟髓过氧化物酶抗菌及清除生物膜的分子机制研究

Nanoscale forms of copper are particularly effective at inhibiting bacterial growth. The use of CuO nanoparticle deposited surface in hygiene-sensitive areas in food industry, or the application of metallic copper as the tap water pulp are good examples. The general mechanism of action for nanoscale CuO-based antimicrobial interfaces is largely unknown. Based on the knowledge that there are trace amount of hydrogen peroxide at mM orders of magnitude and relatively higher amount of the chlorine ion (more than 10 mM) in tap water and our daily foods. Therefore, we proposed at the first time that the mechanism behind might be that CuO nanoparticle mimicking myeloperoxidase catalyze hydrogen peroxide and chlorine to form reactive chlorine (RCS) such as hypochlorite, and small amount of reactive oxygen (ROS) as well, and the hypochlorite are largely responsible for antibacterial effects and elimination of biofilm. To elucidate the proposed content, firstly we are going to investigate the catalytic ability of CuO toward the oxidation of H2O2, Cl-1 and TMB, and the mechanism behind. Secondly, the influences such as the concentration of substrate, temperature, and pH on the antibacterial effects and removal of biofilm by CuO–H2O2–Cl-1 system are going to be verified. Thirdly, the mechanism will be elaborated by verifying the active components generated by CuO–H2O2–Cl-1 system including the possibility of the existence of HClO/ClO-1, chlorine radicals, and •OH. Simultaneously, by comparing the differences of reactive component generated by the CuO–H2O2–Cl-1, Fe3O4–H2O2–Cl-1 and CuO–H2O2 systems. Lastly, antibacterial performance of CuO deposited materials against microorganisms in bacterial suspensions, seawater, tap water and juice are going to be investigated to prove the proposed mechanism. The compliment of this proposal will help people better control the hygiene of food equipment, take advantage of CuO deposited materials for the design of food machinery. Furthermore, widen the understanding of Fenton chemistry and antifouling theory.

含铜材料（负载纳米CuO界面、空气中表面钝化成CuO的金属铜界面）具有抗细菌和清除生物被膜特性，其机理没有定论。我们提出CuO模拟髓过氧化物酶（myeloperoxidase, MPO），催化食品、水体中的H2O2和Cl-1产生活性氯（HClO/ClO-1，·Cl等）和极少量活性氧（·OH），而HClO/ClO-1等活性氯是抗菌和清除生物膜的主要成分。该观点尚属首次提出，拟从4点阐释。1.纳米CuO的催化活性与机制；2. CuO–H2O2–Cl-1杀灭食源性致病菌、清除生物被膜效果；3.重点探明CuO–H2O2–Cl-1活性成分组成及相关关系，对比Fe3O4–H2O2–Cl-1、CuO–H2O2，提出其杀菌、清除生物膜的分子机制；4.构建纳米氧化铜界面，在菌悬液、实际水体和食品中应用、验证该机制。本研究对于纳米抗菌材料的应用，食品加工运输接触界面安全卫生控制具有重要意义。

纳米模拟酶是一类既有纳米材料独特性能，又有催化功能的模拟酶。本研究首次发现纳米氧化铜（CuO NPs）具有模拟MPO酶（HPO酶的一种）特性，实验探究了在H2O2，Cl-，TMB三个底物存在时CuO NPs的催化反应机制，发现其符合乒乓机制，这与天然MPO 酶一致。即CuO NPs首先与H2O2反应产生•OH，然后Cl-被•OH氧化形成活性氯物质。研究发现CuO NPs对底物的Km值均大于天然V-CPO酶（HPO酶的一种），说明CuO NPs作为模拟酶对底物的亲和力不如天然酶；CuO NPs对底物的催化速率与CeO2-x和V2O5相近，但是也远远小于天然酶，结果表明虽然这些纳米颗粒具有类似天然HPO酶的性质，但催化效率比天然酶差，在一定程度上不足以代替天然的卤过氧化物酶。实验表明无论是氧化铜纳米颗粒还是负载氧化铜的界面，其促杀细菌的机理是通过氧化铜模拟MPO酶原位生成活性氯（氯自由基、次氯酸等），活性氮对游离的大肠杆菌和金黄色葡萄球菌具有显著的灭菌能力，对铜绿假单胞菌形成的生物被膜有较强的清除能力。活性氯通过降解细菌被膜中的多糖、DNA与蛋白质从而清除生物被膜，通过卤代化细菌用于信息交流的信号转导分子而抑制生物被膜的形成。同时，本课题拓展研究了增强氧化铜模拟MPO酶的方法与策略；拓展发现纳米氧化铜亦可以催化亚硝酸盐与双氧水生成二氧化氮自由基等活性氮，活性氮具有促杀细菌和抑制生物被膜生长的性能。本课题无论是对于阐释含铜材料的抗菌清除生物膜效果与机制，对于指导纳米抗菌材料的应用，对于食品药品加工、输送设备的设计以及卫生管理，以及加深加宽对经典芬顿反应的认识等多个领域均具有重要意义。