金属纳米颗粒探针的设计及其在生物检测中的应用

发布时间：2018-03-11 16:49

本文选题：金属纳米微粒　切入点：局域表面等离子共振　出处：《南京邮电大学》2016年博士论文　论文类型：学位论文

【摘要】：纳米生物材料作为纳米科技领域中的一个主要方面,其技术的进步和发展引领着生物医学、药物运输等领域的蓬勃发展。利用生物分子对纳米材料进行修饰,可以结合生物分子的识别能力与纳米材料的优良特性,构建出多种新型的功能性纳米材料探针,在生物传感、靶向治疗以及光电材料等方面具有重要的意义。纳米生物材料领域的关键问题之一是如何构建功能性纳米材料探针分子以充分利用生物分子(如:DNA、RNA以及蛋白质等)和纳米材料结合的优势。本论文旨在基于金属纳米颗粒构建探针分子用于核酸分子的检测。论文的研究内容包括以下四部分:1.单颗粒银纳米立方体探针的制备及其对早期肺癌标志物miRNA的实时检测MicroRNA作为肿瘤标志物,调控着很多细胞内过程。然而,由于其序列短、含量低等缺陷,很难开发简单、快速、廉价的检测方法。我们设计了巯基单链DNA (ssDNA)修饰的单颗粒银纳米立方体(AgNC)探针分子,并基于此探针分子构建了等离子体纳米生物传感器用于microRNA-21 (miR-21)的检测。我们首次通过原位实验成功地实时观测到核酸分子在单个AgNC表面杂交的过程。实验结果及时域有限差分方法(FDTD)仿真结果都表明了 AgNCs局域表面等离子体共振(LSPR)峰的红移是由于ssDNA分子与靶分子miR-21杂交引起AgNCs表面折射率(RI)增大的结果。随着靶分子miR-21浓度的增大,AgNCs的LSPR峰红移量增加,对靶分子miR-21的检测灵敏度可达到1fM。此外,通过FDTD仿真模型,成功的解释了反应的机理,每一个AgNC都可以作为一个纳米级传感探针用于早期肺癌标志物miR-21的检测。2.单颗粒金银核壳纳米立方体探针的制备及其对miRNA单分子水平的检测和逻辑门的构建在许多生物系统中,单分子水平的研究可以揭示分子间的相互作用、动力学以及构象的细微变化。目前用于单分子水平检测的可行方法通常需要荧光分子作标记,然而荧光探针容易发生光漂白等现象且信噪比较低。因此,我们设计了四面体结构的DNA (tsDNA)分子修饰的单颗粒金银核壳纳米立方体(Au@AgNC)探针分子,并基于此探针分子构建了“智能”等离子体纳米生物传感器用于单分子水平microRNA-21 (miR-21)以及核酸内切酶(KpnI和StuI)的检测。单个miR-21与tsDNA的杂交过程可引起Au@Ag NC的局域表面等离子体共振(LSPR)峰发生约0.4nm的红移。这种“智能”等离子体纳米生物传感器不仅可以实现对miR-21的超灵敏检测,检测动态范围从1 aM到1 nM,而且成功地执行了以miR-21、KpnI和StuI响应为模型的基于tsDNA的逻辑运算以及生物存储器。3.基于单层过渡金属二硫化物纳米片探针的制备及其对DNA的双元检测快速、灵敏的同时实现对多种靶分子的检测是基因表达和分子诊断领域的挑战性问题之一。针对这一问题,我们设计了基于单层过渡金属二硫化物(TMD,包括MoS2、TiS2和TaS2)纳米片的探针分子用于DNA分子的检测。利用TMD纳米片的高荧光猝灭能力以及对于染料分子标记的单链DNA (ssDNA)和双链DNA (dsDNA)不同的亲和力的特性构建的新型荧光传感平台。通过比较发现基于单层TaS2纳米片的DNA传感器对染料分子标记的ssDNA具有最高的猝灭效率(99%)和最低的检测限(0.05 nM)。重要的是,利用基于单层TaS2纳米片的DNA传感器首次实现了同时对靶分子H1N1和H5N1的检测。4.基于DNA为合成模板的银纳米簇探针的制备及其对DNA的双元检测与有机染料相比,金属纳米颗粒具有稳定性强、荧光强度高以及荧光寿命长等优点。我们进一步地设计了一种基于DNA为模板合成的荧光银纳米簇(AgNCs)的新型免标记的分子信标(MB)探针,用于DNA的双元检测。以两条不同的DNA序列为模板合成的发射光谱峰分别位于507 nm的绿光Ag NCs和597 nm的橙光Ag NCs作为荧光信号,可以实现廉价、免标记的对靶分子H1N1和H5N1的同时检测,检测限为25nM。
[Abstract]:Nanometer biological materials as one of the main aspects in the field of nanotechnology, the progress and development of the technology leads the vigorous development of biomedicine, drug transport and other fields. The use of biological molecular modification of nano materials, excellent properties can be combined with the ability to identify biological molecules and nano materials, build a variety of new functional nanomaterials probe in the biosensing, targeted therapy and photoelectric materials has important significance. One of the key issues in the field of nano biomaterials is how to construct the molecular probe of functional nanomaterials to make full use of biological molecules (such as: DNA, RNA and proteins) and combined with nano materials. The advantages of this thesis metal nano particles construction of molecular probe for the detection of nucleic acid molecules. Based on the research contents of this paper include the following four parts: 1. single particle silver nanocubes probe system Preparation and real-time detection of MicroRNA in early lung cancer marker miRNA as a tumor marker, regulates many cellular processes. However, due to its short sequences, low content of defects, it is difficult to develop a simple, fast and cheap detection method. We design the mercapto single stranded DNA (ssDNA) single particle (modified silver nanocubes AgNC) and the probe molecule probe molecule is constructed based on plasma nano biosensor for detection of microRNA-21 (miR-21). For the first time through the process of in situ experiments we successfully observed real-time nucleic acid molecules in a single AgNC surface hybridization. The experimental results and the finite difference time domain method (FDTD) simulation results show that the localized surface plasmon AgNCs resonance (LSPR) peak red shift is due to the ssDNA molecule and the target molecule miR-21 hybridization induced AgNCs surface index (RI) increased. With the increase of the concentration of target molecules of miR-21 LSPR, peak redshift AgNCs increases, the detection sensitivity of the target molecules of miR-21 can reach 1fM. in addition, through the FDTD simulation model successfully explains the reaction mechanism, each AgNC can be used as a nano sensing probe for early detection of lung cancer marker.2. miR-21 single particle silver core-shell nano cube the preparation of miRNA probe and single molecule detection and logic gate construction in many biological systems, studies on single molecular level reveals the interaction between molecules, dynamics and conformational changes. At present for the feasible method of single molecule level detection usually requires a fluorescent molecular marking, but prone to fluorescent probe photobleaching phenomenon and the SNR is low. Therefore, we design the tetrahedral structure of DNA (tsDNA) single particle silver core-shell nano cube molecular modification (Au@AgNC) probe This molecule, and molecular probe was constructed based on the "smart" plasma nanobiosensors for single molecule level microRNA-21 (miR-21) and endonucleases (KpnI and StuI) in the detection of hybridization. The single miR-21 and tsDNA Au@Ag NC can cause local surface plasmon resonance (LSPR) peak has a red shift of about 0.4nm. This "smart" plasma nanobiosensors can not only realize the ultra sensitive detection of miR-21, detection of dynamic range from 1 aM to 1 nM, and the successful implementation of miR-21, KpnI and StuI in response to the model based on tsDNA logic and biological memory.3. monolayer transition metal sulfide two probes were prepared based on nano film and the dual DNA assay is rapid, sensitive and realize the detection of a variety of target molecules is one of the most challenging problems in the field of molecular diagnostics and gene expression. In view of this question Questions, we designed a single transition metal sulfide based on two (TMD, including MoS2, TiS2 and TaS2) molecular probe nanosheets for the detection of DNA molecules. The high fluorescence quenching ability by TMD nanosheets and dye molecular markers for single stranded DNA (ssDNA) and double stranded DNA (dsDNA) a new fluorescent sensing platform different affinity characteristics. By comparing the ssDNA DNA sensor single-layer TaS2 nanosheets of dye molecular markers based on the highest quenching efficiency (99%) and the lowest detection limit (0.05 nM). It is important that the use of DNA sensor based on single TaS2 nanosheets first realized the simultaneous detection of.4. of target molecules H1N1 and H5N1 silver nanoclusters probe synthesis template preparation and dual DNA detection compared with organic dyes for DNA based metal nanoparticles has strong stability, high fluorescence intensity and long fluorescence lifetime etc. We have further advantages. Based on the design of a DNA as a template for the synthesis of fluorescent silver nanoclusters (AgNCs) of the new label free molecular beacon (MB) dual probe for the detection of DNA. With two different DNA sequences as the template synthesis and emission peaks are located at 507 nm green light Ag NCs and 597 nm orange light Ag NCs as fluorescence signal, can realize the detection of cheap, label free target molecules of H1N1 and H5N1 at the same time, the detection limit is 25nM.

【学位授予单位】：南京邮电大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2;TB383.1

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