高灵敏双信标复合纳米探针电致化学发光比率型酶传感器研究

Ratiometric electrochemiluminescence (ECL) can effectively eliminate the interference of environmental factors and make the detection more convincing and more sensitive, thus exhibits promising future in the immunity, DNA and aptamer sensing analysis. The ratiometric detection of ECL enzymatic sensor, however, is hard to be constructed because of the unsuccessful step-by-step introduction of two signal probes. This project proposes to prepare highly sensitive dual-beacon combined nanoprobes using semiconductive nanocrystyallines as cathodic emitters and polyfluorene nanoparticles as anodic emitters. The dissolved oxygen and produced hydrogen peroxide are employed as cathodic and anodic co-reagents, respectively, to build enzymatic sensor. The ratiometric detections of small biologic molecules and enzymatic activity are achieved through the consumption of dissolved oxygen and the generation of hydrogen peroxide during the enzymatic reaction. The main research contents are as follows: synthesizing a series of dual-beacon combined nanoprobes with different semiconductive nanocrystyallines and polyfluorene nanoparticles; screening the highly sensitive dual-beacon combined nanoprobe by studying the effect of composition and structure on luminescence properties; constructing ECL ratiometric enzymatic senor to achieve the highly sensitive detection of hypoxanthine and acetylcholinesterase activities; studying the ECL mechanisms of dual-beacon combined nanoprobe and the response mechanisms of the enzymatic senor. This project would provide a new method for constructing novel ECL enzymatic sensors, presenting important theoretical value and practical significance meanwhile.

电致化学发光（ECL）比率法能有效避免环境因素干扰，提高分析灵敏度和可靠性，已在免疫、DNA和适体传感分析中显示出良好的应用前景。然而，ECL酶传感器难以分步引入两种信号探针，限制了其比率检测法的构建。本项目拟以半导体纳米晶和聚芴类纳米颗粒分别为阴极和阳极发光体，制备高灵敏双信标复合纳米探针；利用溶氧和生成物过氧化氢分别作为复合纳米探针的阴极和阳极共反应剂，构建酶传感器，借助酶促反应引起的溶氧消耗和过氧化氢增加，从而实现生物小分子及酶活性的比率检测。主要内容包括：利用不同半导体纳米晶和聚芴类纳米颗粒合成系列双信标复合纳米探针，研究其组成和结构等对发光特性的影响规律；以筛选出的高灵敏双信标复合纳米探针构建ECL比率型酶传感器，用于次黄嘌呤和乙酰胆碱酯酶活性的高灵敏检测；研究双信标复合纳米探针的ECL机理及传感器的响应机理。本项目可为新型ECL比率型酶传感器的构建提供新思路，具有重要价值。

电致化学发光（ECL）比率法能有效避免环境因素干扰，提高分析灵敏度和可靠性，已在免疫、DNA和适体传感分析中显示出良好的应用前景。然而，ECL酶传感器难以分步引入两种信号探针，限制了其比率检测法的构建。本项目以半导体纳米晶和聚芴类纳米颗粒分别为阴极和阳极发光体，制备了高灵敏双信标复合纳米探针；以酶促反应产物过氧化氢为双功能调控因子调控两信号的相反变化，构建酶传感器用于比率分析。主要研究成果如下：（1）合成了系列聚芴（PFO和PFBT）纳米颗粒和半导体（CdS和CdTe）纳米晶，并对其进行表征和ECL性能探究，发现聚芴类纳米颗粒在不同扫描电位下具有不同的阳极ECL发射，即扫描电位为0 ~ +1.25 V时，在+1.25 V能检测到一稳定的发射（ECL-1），扫描电位为0 ~ +2.0 V时，在+1.95 V能检测到一稳定的发射（ECL-2），ECL-1无需外加任何共反应试剂即可获得强而稳定的信号，且过氧化氢能高效猝灭ECL-1，增强ECL-2；（2）设计合成了以半导体纳米晶为阴极和聚芴类纳米颗粒为阳极发光体的系列双信标复合纳米探针，包括PFBT纳米颗粒和CdS纳米晶的复合材料，黑磷功能化的CdTe纳米晶，铱纳米棒与CdS纳米晶的复合纳米材料等，研究了组成、结构、尺寸等对复合纳米探针发光特性的影响规律，以调控制备出性能优越的双信标复合纳米探针；（3）构建了系列比率型ECL传感器和单信号的ECL传感器，实现了有机磷及生物小分子如次黄嘌呤和生物大分子如蛋白质和核酸的高灵敏检测；（4）借助光谱学和电化学手段，初步阐明了聚芴类的双阳极ECL发射机制和过氧化氢在双阳极发射中的作用机制，及双信标复合纳米探针的ECL机制和传感器ECL响应机理。本项目的研究成果为聚芴类材料在ECL领域的应用提供了参考，为新型ECL比率型酶传感器的构建提供了新思路，为环境及生物分析提供了新方法，具有重要价值。