内标型上转换酶纳米探针的构建及缺血性脑卒中疾病相关酶活性定量检测及成像分析应用研究

Cerebral ischemic stroke is a very complicated pathophysiologic process involved with metabolism related enzyme activity changes. At present, the cerebral ischemic stroke diagnosis relies on computer tomography imaging (CT) for estimating the degree of apoplexy patients, which is difficult to realize early diagnosis and pathological changes. The determination of the enzyme activity caused by cerebral ischemic stroke has not received enough research attention yet,while it actuallyplays a key role in the field of biomedical research toinvestigate the molecular mechanism of signal transmission, as well as enzyme targeted drug design and early diagnosis. The use of upconversion imaging technology can eliminate the interference of biological spontaneous fluorescence, which has attracted extensive research interests in the field of bioanalysis. However, most of the upconversionnano-probes are based on the wavelength change of a single peak, which is difficult for quantitative detection in biological systems. In this project, we propose tosynthesize internal standard typeupconversion enzymenanoprobes with dual emission wavelengths, in which the longer emission wavelength is used as internal standard and the quencher with the absorbance peak locates at the shorter wavelengthwas modified with the peptide or aptamer. A series of upconversion enzyme nano-probes will be developed with high sensitivity, good repeatability, strong specificity, and good biocompatibility. The ratio of the measured signal intensity to the internal standard signal intensity can eliminate the interference of environmental factors and achieve cerebral ischemic stroke related enzyme activity quantitative detection and analysis of intracellular imaging. This research can provide theoretical basis and technical support for the theranostics and mechanism studyof these diseases and early diagnosis and therapy.

缺血性脑卒中具有发病率高、死亡率高和致残率高的特点，其病理机制复杂。现有的医学诊疗中常通过电子计算机断层扫描成像对患病程度进行判断，难以实现早期诊断及病变机制研究。缺血性脑卒中往往伴随着和代谢相关的酶活性变化，检测其引起的酶活性的变化不仅在研究信号传递的分子机制方面起关键作用，同时在早期诊断和指导酶靶向的药物设计方面有重要意义，但目前尚未引起足够关注。上转换成像技术可消除生物自发荧光的干扰，已在生物分析领域引起广泛研究兴趣。但是，目前大部分上转换纳米探针基于单个峰波长改变进行检测，难以用于生物体系定量检测。本项目拟合成具有双发射峰的上转换纳米材料，利用其中长波长的峰作为内标，结合修饰了吸收峰位于短波长范围的猝灭基团的多肽序列或核酸适体，构建一系列内标型上转换酶纳米探针，利用报告峰信号与内标峰信号强度的比值消除环境因素的干扰，实现缺血性脑卒中类疾病相关酶活性的定量检测及细胞内成像分析。

针对疾病标志物、环境与食品监测的定量检测及细胞内成像分析，开发了一系列具有特定功能的新型过渡金属三维纳米阵列材料、金属有机框架和二维纳米材料用于电化学、光电化学传感和电催化领域，为构建电化学、光电化学和传感研究奠定了基础。通过调控纳米材料表界面构效提升分析检测灵敏度，结合纳米生物技术和荧光、光电分析方法，建立了多种新型传感界面。主要研究内容主要归纳为四个方面：（1）围绕恶性肿瘤、心脑血管疾病相关蛋白、miRNA的检测开展系统研究，建立了以二氧化锰、硫/氧化物纳米阵列、半导体纳米组装结构等纳米功能材料的光化学、电化学信号转换的生物传感新方法。开发了疾病相关生物酶快速、高灵敏检测传感器，为癌症、糖尿病、心血管等重大疾病筛查提供了新思路、新方法。（2）通过纳米材料的合成和表界面结构的功能化，增强材料表面的特异性吸附、聚集或催化等表界面行为，提高光、电催化性能。研究电催化活性位点和异质界面的高效调控，为建立低价、简便、高性能电催化剂提供可控合成方法。设计了三维纳米阵列，具有比表面积大、活性位点密度高、阻抗低、稳定性好、有利于电解液扩散等优点。实现了纳米阵列功能材料电活性位点和异质界面的高效调控，显著提高电化学活性面积和催化活性。（3）开发了一系列基于金属有机框架设计的二维、三维纳米材料，结合纳米生物技术和荧光、光电分析方法，建立了多种新型传感界面，发展了疾病标记物、环境污染物的高灵敏传感方法，为疾病诊断、环境与食品监测等相关重要问题提供了理论基础和解决方案。开发了一系列具有特定功能的热敏金属有机框架，探究其在荧光传感中的应用。（4）对诊断和治疗的功能性核酸适体修饰的纳米材料和DNA修饰的上转换材料分别发表综述类文章。