基于稀土MOF的人工多酶体系构建及分析应用研究

It is of great importance to rationally fabricate artificial multi-enzyme system and further explore its synergistic catalysis effect, because of its roles in deeply understanding the complicated catalytic mechanism of multi-enzymatic reaction in cell and the fabrication of biosensors with high sensitivity. The key for successful fabrication of artificial multi-enzyme system lies on the selection of suitable carriers for enzyme immobilization. Due to its highly tunable nature, metal-organic framework (MOF) as carrier for enzyme immobilization has recently been attracted increasing attention. Nevertheless, the examples on the application of MOFs as enzymatic carriers for the fabrication of artificial multi-enzyme system are relatively rare, especially the development of biosensors based on such artificial multi-enzyme system. In this project, we attempt to employ Ce3+ as metal nodes to prepare a Ce-MOF with peroxidase-like activity, which was further used as a carrier for encapsulating natural oxidase. The integration of natural oxidase with Ce-MOF will lead to the formation of artificial multi-enzyme system with the ability of catalyzing multistep reaction in one-pot fashion, which is also called oxidase@Ce-MOF. After demonstrating the synergistic catalysis effect of oxidase and Ce-MOF, two kinds of biosensors based on cascade signal amplification from artificial multi-enzyme system, time-resolved fluorescent sensor and colorimetric immunosensor, would be developed upon the integration of Tb3+ or antibodies with oxidase@Ce-MOF. Because the high reaction efficiency of oxidase@Ce-MOF and the specific reorganization of its components to targets, these biosensors will display high sensitivity and excellent selectivity. We believe that the results of this project will not only provide a new insight for fabrication and analytical applications of artificial multi-enzyme system, but also provide new methods for the highly sensitive detection of important biochemical molecules and disease markers.

人工多酶体系的合理构建及其协同催化机理研究，对于深刻理解细胞内的复杂多酶反应和构筑高灵敏生物传感器等有着重要的指导意义。选择合适的载体进行酶固定化是其成功构建的关键。金属有机框架（MOF）作为酶固定化载体，因具有结构和性能高度可调的特性正备受关注，但是利用其构建人工多酶体系并用于生物传感的研究却有待进一步探索。在本项目中，我们拟利用稀土铈离子（Ce3+）构筑的Ce-MOF为载体来固定氧化酶，通过将Ce-MOF的过氧化物模拟酶活性与氧化酶进行偶联，构建可高效催化级联反应的人工多酶体系——氧化酶@Ce-MOF。在阐明氧化酶和Ce-MOF的协同催化机理的基础上，进一步将Tb3+或抗体整合到氧化酶@Ce-MOF中，发展基于人工多酶体系级联信号放大效应的高灵敏生物传感器。本项目的实施不仅将为人工多酶体系的构建及其分析应用提供一种新的研究思路，而且也将为重要生化分子和疾病标志物的高灵敏检测提供新方法。

在本课题中，我们围绕利用稀土MOF为酶固定化载体构建人工多酶体系并建立生物分析新方法这一既定目标，主要开展了以下几个方面的工作。首先，我们以Ce-MOF为模型，通过阐明它的催化机理，提出了有机小分子和DNA精准调控MOF类酶催化活性的新策略，不仅提高了Ce-MOF的传感特异性，而且也拓展了纳米酶在分析领域的应用范围。在此基础上，以胆固醇氧化酶为氧化酶代表，通过其与Ce-MOF自组装融合制备了具有高效级联催化活性的人工多酶体系，阐明了胆固醇氧化酶和Ce-MOF的协同催化机理，揭示了Ce-MOF的限域效应和保护作用。与此同时，通过Tb3+的共掺杂，验证了利用人工多酶体系建立高灵敏时间分辨荧光检测方法的可行性。研究发现，除氧化酶以外，利用共沉淀法还可实现稀土MOF与其他酶（如超氧化物歧化酶）的高效整合及其在高灵敏时间分辨荧光检测中的应用。在探明催化性MOF可以与抗体整合构建新型酶标抗体的基础上，我们利用MOF与抗体和酶的共融合，进一步构筑了基于人工多酶体系的新型酶标抗体，建立了具有级联信号放大功能的免疫传感反应，也实现了疾病标志物的高灵敏度和选择性检测。除此之外，我们还创新性提出了利用MOF的限域效应和自组装性能构建FRET荧光探针和多区室人工多酶体系的新策略。本课题的开展，不仅为人工多酶体系的构建及其生物分析应用提供了一种新的研究思路，而且也为重要生化分子和疾病标志物的高灵敏检测提供了新方法，极大地拓展了人工多酶体系在生物医学领域的应用。