基于构象转化的免标记生物分子化学发光适配体传感新技术

Aptasensors are currently receiving considerable attention in the fields of analytical chemistry and clinical disease, etc., in which those label-free aptamer-based optical and electrochemical sensors have been a hot point in these fields. My group has been developing novel chemiluminescence (CL) technologies for many years and already published nine research papers in the prestigious journal of "Analytical Chemistry". Based on our previous aptamer-related research work, here we present novel hairpin probes fitted with one streptavidin-related aptamer as our designing unit and then develop several magnetic separation and conformational switch-based aptasensors as demonstrated by microRNA let-7a and PDGF as model targets. The focus of this project is to firstly design and optimize the above-described conformational switch-based hairpin probes fitted with several recognition units, and then develop and optimize their analytical methodologies. Secondly several amplification platforms including the hybridization chain reaction and strand displacement reaction-based will be used for the improvement of their sensitivity and selectivity. In addition, this novel CL technique described in this project is a general one with a broad range of application possibilities and can be easily extended to the determination of other biomolecules. We now envision that this CL technique holds great promise for improving current optical aptasensor technologies.

适配体传感器的研究在分析化学等领域占据着重要地位，其中免标记型适配体传感技术的研究一直吸引着科学家的持续关注。申请人研究小组多年来一直致力于化学发光新技术的研究，研究成果已9次发表在国际著名分析化学杂志《Analytical Chemistry》上。本课题拟在以往课题组适配体相关研究的研究上，提出以嵌合有链霉亲合素适配体的发夹探针构象转化为设计基础，并以microRNA let-7a和PDGF为研究对象，构建多种具有创新意义的基于磁性分离和构象转化的免标记生物分子化学发光适配体检测技术。本研究主要致力于设计和优化以上嵌合有多种识别单元的构象转化型发夹探针、建立和完善方法学研究，并借助链式杂交和链替换等温循环等放大平台，达到提高生物分子检测灵敏度和选择性的目的。以上拟构建的新型构象转换型传感模式，能够很容易拓宽至其它生物分子的检测，因而具有广泛的用途，将有助于光化学适配体传感技术的发展。

适配体传感器的研究在分析化学等领域占据着重要地位，其中免标记型适配体传感技术的研究一直吸引着科学家的持续关注。按照原定计划，本课题以嵌合有链霉亲合素适配体的探针构象转化为设计基础，发展了多种具有创新意义的生物分子化学发光检测技术。主要包括以下内容：（1）构建了基于构象转化的免标记microRNA化学发光适配体检测技术；（2）基于滚环转录扩增的适配体磁性微球富集分离化学发光检测技术；（3）基于碱基位点突变的滚环扩增let-7 miRNA家族单核苷酸多态性适配体磁性微球富集分离检测方法；（4）偶合构象转换的等温扩增miRNA-486化学发光适配体磁性微球富集分离检测技术；（5）探索了聚苯乙烯微球-杂交链式反应双重放大的无标记化学发光检测新技术；（6）构建了基于xMAP流式荧光的构象转化型四组分microRNA检测技术；（7）构建了固相表面目标序列启动捕获探针标记技术，应用于三组分microRNA的高灵敏度高选择性检测。在上述工作的基础上，拓展设计了一种新型的H2S化学发光探针，并应用于细胞内和活体动物中H2S的检测；拓展构建了一种哑铃型银纳米簇结构探针，偶合链替代反应，实现了NAD+的荧光检测；拓展设计了一种新型的发夹型铜纳米簇模板，研究了其增敏和叠加效应。.围绕本课题，在国际一流分析化学杂志<<Analytical Chemistry>>上发表论文3篇，<<Analyst>>、<<Talanta>>、《Analytica Chimica Acta》、《Analytical and Bioanalytical Chemistry》、《Luminescence》和《Journal of Analysis and Testing》各1篇。基于本课题，现已培养博士研究生5名，硕士研究生4名。