【作者】 张磊；

【导师】 雷建平；

【作者基本信息】 南京大学 ， 分析化学， 2015， 博士

【摘要】 光学探针由于其灵敏以及成像的优越性越来越引起分析化学研究者的兴趣。其原理一般是通过具有优良的光学(一般是荧光)性质的物质与目标物发生作用,引起探针发光性质的改变,通过光学检测手段获得被测组分定性或定量的信息,甚至还能用于细胞或活体里的可视化成像分析。结合纳米材料结构和性质的多样性,本文致力于生物功能化的光学纳米探针在生物传感和细胞分析中的应用。1.基于逐步化学反应策略的高灵敏电致化学发光生物传感利用逐步化学反应构建量子点电致化学发光(ECL)的高灵敏平台检测多巴胺。为了构建这个平台,合成一种硼酸功能化的芘识别探针,通过π-π堆积作用自组装固定在碳纳米管上。另外,合成3,3’-二硫代二丙酸-二(N-羟基丁二酰亚胺酯)(DSP)功能化的CdTe量子点(QDs)作为信号输出探针,并用显微镜和光谱仪进行表征。在目标物多巴胺存在下,发生逐步化学反应,多巴胺先被硼酸功能化的芘探针识别,然后又能与DSP-QDs偶联,使得DSP-QDs能够被固定到电极上作为ECL发光剂产生信号,这种方法的优势是在没有目标物的情况下背景信号很低。利用O2作为内源性共反应剂,这种ECL信号从无到有的方法能够实现对多巴胺定量分析,线性范围是50pM到10nM,检测限是26pM。该基于逐步化学反应的生物传感器还具有很高的抗干扰能力,能够用于检测人脑脊液样品中的多巴胺。该工作构建的逐步化学反应方法为高选择性的分析检测生物小分子开辟了新方向。2.自组装DNA凝胶作为开关用于蛋白质的荧光传感目标物诱导引起结构转变的材料由于高可控性和灵敏性在生命分析中具有重要意义。在本工作中,设计了一种适配体功能化的DNA水凝胶作为目标物诱导的可转换材料用于蛋白质的分析。这种DNA凝胶是用纯DNA自组装而成,不需要任何合成聚合物作为凝胶的骨架。它是用由一种Y-形状的DNA作为基本单元和含有适配体的L-DNA作为连接DNA构成,经过DNA杂交自组装形成。凝血酶作为检测的典型蛋白质,DNA水凝胶首先能通过包在凝胶里的金纳米颗粒(AuNPs)作为指示剂用于比色型检测。另外,合成一种带正电荷的聚乙酰亚胺包裹的量子点(PEI-QDs)作为荧光信号单元。目标物存在时,由于含有适配体的DNA与目标物具有更强的结合作用力,DNA凝胶发生溶液态的转变,从而释放出包夹在里面的AuNPs.释放的AuNPs与凝胶上层溶液中带正电荷的PEI-QDs通过静电作用力结合,发生荧光共振能量转移猝灭量子点的荧光,产生了一种灵敏检测目标蛋白的方法。这种方法能检测凝血酶的浓度范围在75 nM到12.5 gM,在3倍信噪比的情况下,检测限达到67 nM,并且能在复杂的血清样品中检测凝血酶。这种生物兼容的DNA凝胶为信号转变提供一种通用性的开关型材料,并且也为蛋白质的检测开辟新方向。3.基于可持续发光纳米探针的时间分辨荧光共振能量转移的生物传感和细胞凋亡寿命成像时间分辨荧光是一种能够有效减少短寿命的背景荧光和来自细胞和组织的自发荧光干扰的技术。本工作中,我们合成一种可持续发光纳米(PLNP)探针,构建通用性的时间分辨-荧光共振能量转移(TR-FRET)平台用于生物传感、细胞凋亡寿命成像和寿命定量细胞内的caspase-3。制备三种PLNP生物功能化的纳米探针用于检测半胱氨酸天冬酶caspase-3、microRNA和血小板源生长因子(PDGF),这些探针都是通过羧基功能化的PLNPs共价偶联染料标记的多肽或者DNA。多肽功能化的纳米探针也用于荧光寿命成像来监测细胞凋亡,并且研究细胞内荧光寿命和caspase-3活性的定量关系,从而构建一种原位的荧光寿命定量监测的方法。基于PLNPs的TR-FRET概念和分析方法对于探索生命过程中一些动力学信息具有很大的潜力。4.卟啉光敏化的金属有机框架用于凋亡酶响应的癌细胞治疗诊断设计一种多功能化的金属有机架构(MOF)纳米探针,用于癌细胞光动力治疗和细胞凋亡蛋白酶响应的治疗诊断。光敏剂卟啉衍生物(TMPyP)在MOF笼中的激发态能级改变,引起包夹卟啉的MOF纳米结构的单线态氧量子产率增大6.2倍。纳米探针通过MOF表面共价偶联H2N-PEG-叶酸和Cy3标记的多肽得到。探针选择性地进入癌细胞线粒体,激光辐照产生单线态氧,导致细胞凋亡和激活细胞质里caspase-3活性,释放Cy3,利用增强的荧光信号监测治疗效果。该方案实现一个纳米载体中治疗和诊断功能的集合,为癌症诊断和治疗提供新技术与新方法。5.原位激活和监控细胞里的半胱氨酸天冬酶家族演变细胞里半胱氨酸天冬酶(caspase)家族演变在细胞凋亡过程中起着重要的作用。为了评估这个过程,设计一个普适性的平台可以用来原位激活和监控细胞里caspase的演变。利用常见的金纳米材料作为载体和诱导细胞凋亡的光热材料,金纳米载体上组装两种染料标记的多肽分别是酶家族上游caspase-9底物和下游caspase-3底物作为信号开关,并修饰叶酸作为靶向部分,最后形成染料标记的多肽和叶酸修饰的金纳米探针。由于两种染料分别与金纳米材料的两个表面等离子体共振吸收发生能量转移,探针的荧光最初是被猝灭的。一旦探针被癌细胞内吞后对其进行光热治疗诱导细胞凋亡,探针上的多肽随着caspase家族从上游到下游的演变而顺序被剪切,荧光也逐渐被点亮,点亮的荧光信号可以用作检测癌细胞里caspase-9和caspase-3两种酶的活性和监控活体里的演变。恢复的荧光信号同样可以用来评估金纳米材料光热治疗的效果。这个工作为研究细胞里caspase家族提供有力的工具,也阐释细胞凋亡过程中caspase家族的生物学意义。6.多步组装的超级催化剂应用于超灵敏检测和细胞传感金属卟啉具有类似于过氧化酶的催化性质,但是由于他们溶解性差或者易聚集常常导致催化性能降低。本章设计了一种新型的多步自组装超级催化剂,该超级催化剂显著增强血红素(Hemin)的催化性质。多步自组装的方法如下：通过帕米磷酸二钠还原氯金酸合成了一种本身表面含有氨基的金纳米粒子(N-AuNPs),经过EDC反应将Hemin偶联到金纳米材料的表面形成Au-Hemin;合成的咪唑类有机化合物(MPT)能够通过Fe-N的配位作用轴向配位到Hemin形成Au-Hem-MPT;在Hg2+的存在下,这种轴向配体能诱导配位物聚合形成Au-Hem-MPT-Hg超级催化剂。构建的超级催化剂在催化效率和动力学上都显著提高了Hemin的催化性能。此外,超级催化剂能达到对Hg2+低至aM浓度的超灵敏检测以及能实现对细胞中活性氧的成像。这种自组装的高性能催化剂为催化机理研究和生物分子灵敏检测奠定了坚实的基础。更多还原

【Abstract】 Optical probes attract more and more interests because of their superiority of sensitivity and in situ imaging. The principle is generally elaborated as follows:upon the interaction with the target, the optical properties (especially the fluorescence) of the probe is usually changed, which can be measured through proper instrument for the quantitative or qualitative information, and even can be used for visual imaging analysis in living cells or in vivo. Combining with the nanomaterials of diverse structure and property, this project is dedicated to bio-functionalized optical nanoprobes in biosensing and celluar analysis.1. Stepwise chemical reaction strategy for highly sensitive electrochemiluminescent detection of dopamineA stepwise chemical reaction strategy based on the specific recognition of boronic acid to diol, and N-hydroxysuccinimide (NHS) ester to amine group, was designed to construct a "signal on’" electrochemiluminescence (ECL) platform for highly sensitive detection of dopamine. A boronic acid-functionalized pyrene probe was synthesized and was self-assembled on the sidewalls of carbon nanotubes via π-π stacking interactions as capture probes on a glassy carbon electrode. Meanwhile, 3,3’-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP)-functionalized CdTe quantum dots (QDs) were designed as signal probes and characterized with transmission electron microscopy and spectroscopic techniques. Upon stepwise chemical reaction of dopamine with boronic acid and then DSP-QDs, the QDs were captured on the electrode as ECL emitters for signal readout, leading to an ultralow background signal. By using O2 as an endogenous coreactant, the "signal on" ECL method was employed to quantify the concentration of dopamine from 50 pM to 10 nM with a detection limit of 26 pM. Moreover, the stepwise chemical reaction-based biosensor showed high specificity against cerebral interference and was successfully applied in the detection of dopamine in cerebrospinal fluid samples. The stepwise chemical reaction strategy should be a new concept for the design of highly selective analytical methods for the detection of small biomolecules.2. Self-assembled DNA hydrogel as switchable material for aptamer-based fluorescent detection of proteinThe methodology based on target-responsive structural switching is powerful in bioanalysis with the controllability and sensitivity. In this paper, an aptamer-functionalized DNA hydrogel was designed as a specifically target-responsive switchable material for protein detection. This pure DNA hydrogel was constructed by using a Y-shaped DNA and an aptamer linker through a DNA self-assembly without synthetic polymer backbone. With use of thrombin as the model analyte, the DNA hydrogel was first applied to visual detection with the entrapped Au nanoparticles (AuNPs) as indicating agent. Furthermore, the positively charged quantum dots (QDs) as the fluorophore were synthesized by using polyethyleneimine (PEI) as wrapper and characterized with spectroscopy, transmission electron micrograph, ζ potential, and dynamic laser scattering techniques. Along with a gel-to-sol transition in the presence of the target, the released negatively charged AuNPs from the hydrogel could approach the positively charged QDs. Due to the electrostatic interaction, fluorescence resonance energy transfer between PEI-QDs and AuNPs therefore occurred and quenched the fluorescence signal for the sensitive detection of thrombin. This assay for the detection of thrombin showed a good linear relationship in a range of 0.075 to 12.5 μM with a detection limit of 67 nM at 3σ, and demonstrated excellent feasibility in complex serum matrixes. The biocompatible DNA hydrogel provides a universal switchable material for signal transduction and significantly demonstrates proof-of-concept for the detection of proteins.3. Persistent luminescence nanoprobe for biosensing and lifetime imaging of cell apoptosis via time-resolved fluorescence resonance energy transferTime-resolved fluorescence technique can reduce the short-lived background luminescence and auto-fluorescence interference from cells and tissues by exerting the delay time between pulsed excitation light and signal acquisition. Here, we prepared PLNP probes to design a universal TR-FRET platform for biosensing, lifetime imaging of cell apoptosis and in situ lifetime quantification of intracellular caspase-3. Three kinds of PLNP-based nanoprobes are assembled by covalently binding dye-labeled peptides or DNA to carboxyl-functionalized PLNPs for the efficient detection of caspase-3, microRNA and protein. The peptides-functionalized nanoprobe is also employed for fluorescence lifetime imaging to monitor cell apoptosis, which shows a dependence of cellular fluorescence lifetime on caspase-3 activity and thus leads to an in situ quantification method. This work provides a proof-of-concept for PLNPs-based TR-FRET bioanalysis and demonstrates its potential in exploring dynamical information of life process.4. Porphyrin photosensitized metal-organic framework for cancer cell apoptosis and caspase responsive theranosticsA multifunctional metal-organic framework (MOF) is designed as an efficient nanoprobe for selective photodynamic therapy against cancer cells and caspase responsive theranostics. The porphyrin derivative (TMPyP) as a photosensitizer is incorporated in the cage of a variant MOF by one-pot synthesis, which significantly increases the singlet oxygen quantum yield of TMPyP by 6.2 times due to the change of its excited level in MOF cage. By covalent co-assembly of H2N-PEG-folate and Cy3-labelled peptide on the MOF surface, the obtained nanoprobe can be selectively internalized into cancer cells to produce singlet oxygen in mitochondria under laser irradiation, which induces cell apoptosis and activates caspase-3 in cytoplasm to specifically cleave the peptide. The released Cy3 produces bright fluorescence for in situ monitoring of therapeutic effectiveness. The integration of theranostic functions in a single nanocarrier holds great promise in precision cancer diagnosis and treatment.5. In situ activating and monitoring the evolution of intracellular caspase familyThe evolution of intracellular caspase family is crucial in cell apoptosis. To evaluate this process, a universal platform of in situ activating and monitoring the evolution of intracellular caspase is designed. Using well-known gold nanostructure as a model of both nanocarrier and matter inducing the cell apoptosis for photothermal therapy, a nanoprobe is prepared by assembly of two kinds of dye-labelled peptides specific to upstream caspase-9 and downstream caspase-3 as the signal switch and folic acid as a targeting moiety. The energy transfer from dyes to the gold nanocarrier at two surface plasmon resonance absorption wavelengths leads to their fluorescence quenching. Upon endocytosis of the nanoprobe to perform the thearapy agnasit cancer cells, the peptides are successively cleaved by intracellular caspase activation with the evolution from upstream to downstream, which lights up the fluorescence of the dyes sequentially, and can be used to quantify both caspase-9 and caspase-3 activities in cancer cells and to monitor their evolution in living mice. The recovered fluorescence could also be used to assesse the therapeutic efficiency. This work provides a novel powerful tool for studying the evolution of intracellular caspase family and elucidating the biological roles of caspases in cancer cell apoptosis.6. Multi-assembled supercatalyst for ultrasensitive detection and cytosensingMetal porphyrins have catalytic properties of peroxidase, however, the catalytic capabilities are often limited by their poor solubility, the bridge μ-oxide dimers (hindered access to catalytic sites) and oxidative self-degradation. In this work, we design a new multi-assembled supercatalyst, which significantly enhance the catalytic performance of Hemin. The multi-assembly method is as follows:gold nanoparticle with amino group (N-AuNP) is firstly synthesized through pamidronic acid disodium as a new reducing agent, and covalently bond with Hemin as Au-Hemin via the EDC reaction. Then, a prepared imidazole derivative named as MPT can be axially coordinated to the metal center though the Fe-N interaction, leading to the formation of Au-Hem-MPT. In the presence of Hg2+, a supercatalyst is finally formed by the Hg2+-induced polymerization of MPT. The designed supercatalyst significantly improves the catalytic performance of Hemin on both the catalytic efficiency and the dynamics. In addition, the catalyst can achieve the ultrasensitive detection of Hg2+ down aM and the imaging of reactive oxygen species in live cells. The multi-assembled highly-efficient catalyst provides a new avenue for the catalytic mechanism research and ultrasensitive detection.更多还原