• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文学位 >Use of Nanoparticles and Tunable Resistive Pulse Sensing Technology for Biosensing and Nanoflowers for Transfection.
【24h】

Use of Nanoparticles and Tunable Resistive Pulse Sensing Technology for Biosensing and Nanoflowers for Transfection.

机译：纳米粒子和可调节的电阻脉冲传感技术在生物传感和纳米花中的转染应用。

获取原文
获取原文并翻译 | 示例
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Infection with pathogens such as influenza virus or methicillin-resistant Staphylococcus aureus (MRSA) is a threat of public heath and economic burden. Loop-mediated isothermal amplification (LAMP) is a powerful technique without using thermal cycles to amplify target DNA from pathogens. LAMP is a highly sensitive and rapid detection method working at one temperature at 60 - 65 °C. With appropriate primers, target genes can be amplified by LAMP within 30 minutes. The specificity of LAMP is very high since four primers are utilized to amplify one DNA template. Another advantage is that LAMP can also be used to measure cDNA or RNA quantitatively, similar to that of the real-time PCR (qPCR).;Nowadays, SYBR Green I (SGI) is a commonly used DNA binding probe to report qPCR signals. According to work from Gudnason et al (2007)., SGI has effect in increasing the Ct value in qPCR in a dose dependent manner. However, due to the short history of LAMP, no or not too many studies using SGI for real-time LAMP (RT-LAMP) is available in the literature. In the first part of this thesis, the inhibitory effect of SGI and another DNA binding dye, SYTO-9 (ST9) on RT-LAMP for the detection of the panton-valentine leukocidin (PVL) gene of MRSA was confirmed. According to the RT-LAMP results, SGI blocked the PVL DNA amplification by LAMP in a dose-dependent manner. Also, this inhibition was confirmed using the RT-PCR tools, the amplification efficiency and threshold cycle value, for the analysis. On the contrary, ST9 showed less inhibitory effect on LAMP when similar concentration was used. Results from our study indicated that ST9 is more compatible with the RT-LAMP for fast and sensitive biosensing if fluorescence-based system is the only choice.;To avoid this negative effect, we tried to combined LAMP and gold nanoparticles (AuNPs) for biosensing. Through funcationalization, DNA were conjugated to the AuNPs which acted as carriers or probes for biosensing. We reported here that AuNPs coated with DNA were used to detect the PVL gene by LAMP. After DNA amplification at single temperature (65 °C), the Lamplicons (amplicons from LAMP) were mixed with the DNA probes on the surface of AuNPs. As a result of DNA-DNA hybridization, AuNPs formed aggregations and the changes were measured by the tunable resistive pulse sensing (TRPS) technology. Using the TRPS technique, the agglomerated AuNPs were allowed to pass through a size-tunable fluidic nanopore to give blockade events which provided information of the size and dynamic of the agglomerated AuNPs. The TRPS technique was sensitive and could achieve a detection limited of 208 copies of MRSA templates and the detction could be completed within a short period of time (2 hours). Therefore, the LAMP-based AuNP TRSP is a sensitive yet simple platform for detecting different kinds of antigens.;Nanoflowers (NF) are the flower-like structure with the core in the middle and branches extended. We confirmed here the low cytotoxicity feature of the NFs by AlamarBlue assay in the huamn basophils (KU812 cells) and human embryonic kidney cells (HEK293T cells). NFs were functionalized with the pEGFP-N1 and pDsRed1-C1 vectors with the green fluorescent protein (GFP) or red fluorescent protein (RFP) gene, respectively, for transfection by incubating the KU812 cells or HEK293T cells with the functionalized NFs in culture medium at 37 °C for 48 hr. From the results of flow cytometry, the expression rate of GFR or RFP in the KU812 and HEK293T cells was found to be high, approximately 50 % and 78 % respectively. Our expreiments therefore indicated that NFs is a biocompatible nanomaterial which is safe and a good carrier of DNA transfection in human cell lines.;On the other hand, TRPS, originally used in the field of nanotechnology, was employed to determine cell death in human red blood cells (RBCs) for toxicity studies. RBCs were treated with a potential anti-cancer agent polyphyllin D (PD) or the silver nanoparticles (AgNPs) for 24 hr, followed by the cell death detection with the TRPS. The data in our study thus demonstrated that the TRPS technique was able to detect the number of assemblies of different size. After subtracted from the background populations, more and larger positive populations bigger than 85 fL were found in the PD (2.5 and 1.25 muM)-treated RBCs when compared with the negative control. The combination of the TRPS technique and modified beads can therefore be used as a good sensing tool for cell death analysis. Taken together, the work in this thesis shows a key how to integrate and refine different technologies to provide a good platform for biosensing and other biomedical applications. (Abstract shortened by UMI.).
机译：感染诸如流感病毒或耐甲氧西林金黄色葡萄球菌（MRSA）的病原体是对公共卫生和经济负担的威胁。环介导的等温扩增（LAMP）是一项强大的技术，无需使用热循环即可从病原体扩增靶DNA。 LAMP是一种高灵敏度且快速的检测方法，可在60-65°C的一个温度下工作。使用适当的引物，可以在30分钟内通过LAMP扩增靶基因。 LAMP的特异性非常高，因为使用了四种引物来扩增一个DNA模板。另一个优点是，与实时PCR（qPCR）相似，LAMP还可以用于定量测量cDNA或RNA。如今，SYBR Green I（SGI）是报告qPCR信号的常用DNA结合探针。根据Gudnason等人（2007）的工作，SGI具有以剂量依赖性方式增加qPCR中Ct值的作用。但是，由于LAMP的历史短，在文献中没有或没有太多使用SGI进行实时LAMP（RT-LAMP）的研究。在本文的第一部分中，证实了SGI和另一种DNA结合染料SYTO-9（ST9）对RT-LAMP的抑制作用，以检测MRSA的泛白蛋白白介素（PVL）基因。根据RT-LAMP结果，SGI以剂量依赖的方式阻断了LAMP对PVL DNA的扩增。同样，使用RT-PCR工具，扩增效率和阈值循环值对这种抑制作用进行了确认。相反，当使用相似的浓度时，ST9对LAMP的抑制作用较小。我们的研究结果表明，如果仅基于荧光的系统是唯一选择，则ST9与RT-LAMP在快速和灵敏的生物传感方面更兼容。 。通过功能化，将DNA偶联到AuNPs上，该AuNPs充当生物传感的载体或探针。我们在这里报道了用DNA包被的AuNPs被LAMP检测到PVL基因。在单温度（65°C）下扩增DNA后，将Lamplicons（来自LAMP的扩增子）与AuNPs表面上的DNA探针混合。 DNA-DNA杂交的结果是，AuNPs形成聚集体，并通过可调电阻脉冲传感（TRPS）技术测量了变化。使用TRPS技术，使团聚的AuNPs通过大小可调的流体纳米孔，以产生封锁事件，从而提供了团聚的AuNPs的大小和动力学信息。 TRPS技术非常灵敏，可以检测到208个MRSA模板副本，并且可以在短时间内（2小时）完成检测。因此，基于LAMP的AuNP TRSP是检测不同种类抗原的灵敏而简单的平台。纳米（NF）是花状结构，其核心在中间，分支延伸。我们在这里通过AlamarBlue分析证实了在嗜碱性粒细胞（KU812细胞）和人类胚胎肾细胞（HEK293T细胞）中NF的低细胞毒性特征。通过分别用绿色荧光蛋白（GFP）或红色荧光蛋白（RFP）基因的pEGFP-N1和pDsRed1-C1载体对NFs进行功能化，通过将KU812细胞或HEK293T细胞与功能化的NFs在培养基中于37°C 48小时。根据流式细胞仪的结果，发现KU812和HEK293T细胞中GFR或RFP的表达率很高，分别约为50％和78％。因此，我们的实验表明，NFs是一种安全的生物相容性纳米材料，是人类细胞系中DNA转染的良好载体。另一方面，最初用于纳米技术领域的TRPS被用于确定人类红细胞死亡。血细胞（RBC）用于毒性研究。用潜在的抗癌药多聚叶绿素D（PD）或银纳米颗粒（AgNPs）处理RBC 24小时，然后用TRPS检测细胞死亡。因此，我们研究中的数据表明TRPS技术能够检测到不同尺寸的组件数量。从背景种群中减去后，与阴性对照相比，在PD（2.5和1.25μM）处理的RBC中发现了越来越多的大于85 fL的阳性种群。因此，将TRPS技术和修饰的磁珠结合起来可以用作细胞死亡分析的良好传感工具。综上所述，本文的工作表明了如何集成和完善不同技术以为生物传感和其他生物医学应用提供良好平台的关键。 （摘要由UMI缩短。）。

著录项

  • 作者

    Yang, Kar Lai Alice.;

  • 作者单位

    The Chinese University of Hong Kong (Hong Kong).;

  • 授予单位 The Chinese University of Hong Kong (Hong Kong).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 220 p.
  • 总页数 220
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号