基于分子印迹有机磷水解模拟酶的仿生传感器

We propose a molecularly imprinted biomimetic enzyme with organophosphorus hydrolase (OPH)-like activity in order to develop the biomimetic sensors for neurotoxic organophosphate with enhanced stability and practicality. In this proposal, not only will the shape of the transition state of organophosphate be mimicked by imprinting but also at the same time appropriate catalytic groups and binding sites of natural OPH will be integrated into the active site in a predetermined orientation of the biomimetic OPH. The stable biomimetic OPH will be designed in accordance with the activity center of fragile natural OPH, prepared by the molecular imprinting technique and employed as a molecular recognition element. A binuclear metal ions complex will be designed to mimic the recognition, binding and catalytic functional groups of the activity center of natural OPH. Then, the designed complex will be synthesized and used as functional monomers. Meanwhile, the auxiliary monomer will be also designed to mimic the cooperative catalytic effect and synthesized. After that, the transition-state analogues of the organophosphate hydrolysis will be selected or designed as templates to incorporate the main catalytic elements. Thus, the biomimetic enzyme with organophosphorus hydrolase-like activity will be imprinted in term of stoichiometric noncovalent interaction. The characteristics of the biomimetic OPH will be examined as catalysts in the degradation of organophosphate. The interaction between OPH and its ligand will be also investigated using flexible molecular docking method. The influence of various functional groups on the activity of the biomimetic OPH will be studied to explore the crucial functional groups on the mimic of natural OPH. Finally, the biomimetic OPH will be covalently immobilized onto the disposable screen-printed electrodes and polystyrene microplates, respectively. Therefore, various biomimetic sensing modes (amperometric and fluorimetric,etc.)are expected to be established and examined to develop new methods for detection of organophosphate using the biomimetic OPH as molecular recognition element.

本项目提出：既利用分子印迹技术稳定有机磷水解的过渡态分子，又整体仿生模拟天然有机磷水解酶（OPH）的活性中心，合成稳定性优于天然OPH的仿生OPH，并作为分子识别元件构建有机磷仿生传感器，以提高有机磷传感器的稳定性和实用性。首先根据天然OPH各功能团间协同催化水解机制，设计合成含识别、结合和催化等功能团的双核金属离子配合物，并作为功能单体。同时，设计合成含起协同催化作用功能团的辅助单体。然后，以过渡态类似物为印迹模板，采用基于化学计量非共价作用的分子印迹法，整体仿生印迹天然OPH起识别、结合和催化作用的各功能团。研究仿生酶对有机磷底物的催化水解活性，并借助柔性分子对接方法揭示OPH与其配体的相互作用，探索功能团对仿生酶活性影响的规律。最后，研究将仿生酶共价固定于一次性印刷电极、聚苯乙烯微孔板上的方法，分别研究并完善有机磷仿生传感的电化学及光化学检测模式，建立有机磷仿生检测的新方法。

仿生模拟天然有机磷水解酶（OPH）活性位点是创制高催化性能人工OPH酶的有效策略，而使用人工OPH仿生酶进行有机磷神经毒剂降解和检测具有稳定性好的优势。根据项目合同书所规定的研究任务，设计并合成了分子印迹功能单体1-乙烯基咪唑和5-乙烯基-1,3-双（二-2-咪唑）羟甲基苯。利用沉淀聚合的方法，以对氧磷水解过渡态类似物4-硝基苄基磷酸二乙酯为模板分子，合成了两类具有OPH活性的分子印迹仿生酶，整体仿生模拟了天然OPH各催化官能团之间的协同催化水解作用。通过对这两类一系列具有OPH活性的仿生酶催化性能的研究，得到了稳定性好、可以反复使用的OPH仿生酶。与对氧磷自发水解相比，合成的OPH仿生酶催化水解对氧磷的效率提高了160000倍，催化水解对氧磷的最大速率Vm为0.38 mM/min，米氏常数Km为0.025 mM。将合成的分子印迹仿生OPH固定于一次性印刷电极上，构建了有机磷仿生传感器，建立了简单、快速、灵敏检测有机磷农药残留的新方法，检测限为5 nM，并对复杂基质（如牛奶）中有机磷农药残留进行了检测。另外，为了减小传质阻力对仿生酶活性的影响，将1-乙烯基咪唑共价接枝在甲基丙烯酸羟乙酯功能化的氧化石墨烯(GO)表面，成功合成了聚合物微球-GO复合物，合成了具有催化转化数（TOF）的仿生酶。在项目的支持下，通过功能团对仿生酶活性影响的研究，建立了提高仿生酶催化性能的设计策略，即充分考虑功能团的协同作用和底物传质阻力的设计原则，为进一步设计合成高催化活性的仿生酶奠定了基础。在项目执行期间，申报合成仿生OPH的发明专利3件，目前已授权1件，另外2件也已经公告。在本学科主流学术期刊上已发表学术论文8篇，其中影响因子大于5.0的学术论文5篇（包括Anal. Chem.：1篇；Biosens. Bioelectron.： 3篇； ACS Appl. Mater. Interfaces：1篇）。