金纳米粒子传感器用于检测DNA和金属离子及核壳纳米材料制备
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摘要
金纳米粒子由于其优异的光学性能和强的表面吸附性能,以及具有强的表面等离子体共振、表面等离子体共振荧光、表面增强拉曼、表面易功能化等性质,在生物标记、分子识别、生物检测、金属离子检测等众多方面有着广泛的应用。
DNA电化学生物传感器提供了一种全新的DNA(基因)检测技术,具有简单、可靠、价廉、灵敏和选择性好等优点,并且与目前的DNA生物芯片技术兼容,在分子生物学和生物医学工程领域具有很大的实际意义和应用价值。
比色法由于具有操作方便、检测快捷、成本低、不需要复杂的检测仪器广泛应用于检测金属离子。金纳米粒子由于其独特的性质,成为了比色法检测金属离子的优质材料。
核壳复合纳米粒子因其组成、大小和结构排列的不同而具有特殊的化学、光学和电学等方面的特性,广泛应用于电子、催化、分离、生物诊断等许多领域。
结合上述几个方面,本论文主要利用金纳米离子传感器对DNA和金属离子进行了检测,以及制备了Au@SiO2以及Fe3O4@SiO2核壳复合纳米粒子。
具体研究工作分为三章,具体工作如下：
第二章,发展了一种新的电化学检测DNA的方法。该方法先通过生物条形码法将目标DNA信号放大并转化为可修饰到电极表面的巯基DNA,由于电化学活性物质六氨合钌(RuHex)阳离子能通过静电相互作用吸附在DNA骨架上并作为电化学信号物质,利用DNA杂交作用采用DNA功能化金纳米粒子对电极表面的巯基DNA进行再组装,组装后将大大提高电极表面负载的DNA量,其DNA骨架吸附更多的RuHex阳离子。该方法先利用生物条形码法将DNA信号放大,再通过组装将信号进一步放大,可以很大程度上提高DNA的检测限。实验结果表明用该方法检测DNA的初步检测限已达到1pM。这种方法结合了生物条形码法和电化学法的双重优势,具有良好的应用前景。
第三章,制备了两种金纳米传感器并用于检测金属离子。两种传感器表现出良好的水溶性和稳定性。1-金纳米粒子对Hg2+的检测具有特别高的选择性,通过肉眼就可检测到1μM的浓度,通过紫外可见光谱可以推进最低检测限至400nM。对于2-金纳米粒子来说,在金属离子浓度为100μaM时,肉眼可以看到2-金纳米粒子对Hg2+, Fe3+, Al3+, Fe2+和Cr3+等离子有颜色响应。当金属离子浓度小于10μM时,2-金纳米粒子对Hg2+的检测也具有非常高的选择性,能通过紫外可见光谱检测到限至1μM的Hg2+。进一步研究传感器颜色响应的机理发现：传感器的颜色响应的主要原因是功能分子与金属离子的配位效应,而不是受pH值和离子强度影响。
第四章,成功制备了Fe3O4@SiO2和Au@SiO2核壳纳米粒子。用水热法制备Fe304磁性纳米粒子,通过调节底物浓度,反应温度,反应时间得到了170nm和220nm的两种尺寸均一的质量较好的磁性纳米粒子。用改进的Stober法在Fe304表面包裹Si02,通过优化水醇比例,pH, TEOS加入量,底物浓度,反应时间等调节包裹层的Si02厚度。通过化学法和种子法制备了不同大小的金纳米粒子,再通过TEOS的水解,在不同粒径的金纳米粒子表面包裹Si02。通过优化水醇比例,pH, TEOS加入量,底物浓度,反应时间等调控Si02的壳层厚度。
Gold nanoparticles(AuNPs) possess excellent optical properties and strong surface adsorption. They have many properties such as strong surface plasmon resonance, surface plasmon resonance fluorescence, surface enhanced Raman, and can be used for a wide range of Biomarkers, Molecular recognition, genetic detection and metal ions detection. Electrochemical biosensing of DNA hybridization is a novel technique with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and it can be compatible with DNA biochip, and they are expected to have a broad prospect of application in clinic examination of inherited diseases and drug screening.
Colorimetry methods for detection of metal ions were widely used because of its fast detection, ease of operation, and low cost. Gold nanoparticles have special properties, are idle materials in colorimetry methods for detection metal ions.
Core-shell alloy nanoparticles have special chemical, optical and electrical properties for its difference in composition, size and structure and are thus widely used in electronic, catalytic, isolation and biologic diagnosis areas.
Combining these aspects, this thesis use gold nanopartical based ion transducer to detect metal ions and DNA, and alloy nanoparticles of Au@SiO2and Fe3O4@SiO2was prepared.
The concrete research work of this dissertation can be divided into three chapters as follows:
Chapter2. A method employing electric-chemical sensor for detection DNA was developed. This method use bio-barcode system to amplify and transfer target DNA signal to mercapto-DNA that can be modified to electrodes. RuHex cations work as electrical-chemical active agent and can be absorbed to anionic phosphate of DNA strands through electrostatic effect. Through DNA hybridization and in the presence of DNA functionalized gold nanoparticles the mercapto-DNA was reconstructed which resulted in a great increase in the amount of DNA loaded on the electrode and thus more RuHex cations were absorbed. This method first amplify DNA signal through bio-barcode system and further amplify the signal through nano self-assemble, which substantially increase the DNA detection limit. The result showed that this method can detect DNA of1pM. This method combined the advantage of bio-barcode system and electrical-chemical technique and has potential for future applications.
Chapter3. We have developed two sensors for detecting metal ions. Both sensors showed good water-soluble and stability.1-Gold nanoparticles(AuNPs) exhibited high selectivity to Hg2+and can detect Hg2+with the concentration of1μM by naked eye, the limit of detection can be further enhanced to400nM by ultraviolet-visible(UV-vis) spectra. Under naked eye,2-AuNPs had a distinct colour change after metal ions (Hg2+, Fe3+, Al3+, Fe2+and Cr3+)(100μM) was added respectively. When the concentration of metal ion is less than10μM,2-AuNPs also exhibited high selectivity to Hg2+and can detect Hg2+with the concentration of1μM by UV-vis spectra. Thorough investigation indicated that the mechanism of the colorimetric responses was mainly coordination effect of the modified organic molecules with metal ions and had little relationship with the change of ionic strength and pH value.
Chapter4. Core-shell nanoparticles of Au@SiO2and Fe3O4@SiO2were prepared. Hydrothermal synthesis of Fe3O4magnetic nanoparticle through controlling the substrate concentration, reaction temperature and reaction time yields two kinds of uniform sized products of170nm and220nm. Using modified Stober method the Fe3O4surface was coated with SiO2, through optimization of water-ethanol ratio, pH, TEOS amount, substrate concentration and reaction time the thickness of SiO2layer was controlled. Different sized gold nanoparticles was prepared through chemical method and seeding method, hydrolyzed through TEOS, and coated with SiO2. The thickness of the SiO2layer was controlled through optimization of the water-ethanol ratio, pH, TEOS amount, substrate concentration and reaction time.
DNA电化学生物传感器提供了一种全新的DNA(基因)检测技术,具有简单、可靠、价廉、灵敏和选择性好等优点,并且与目前的DNA生物芯片技术兼容,在分子生物学和生物医学工程领域具有很大的实际意义和应用价值。
比色法由于具有操作方便、检测快捷、成本低、不需要复杂的检测仪器广泛应用于检测金属离子。金纳米粒子由于其独特的性质,成为了比色法检测金属离子的优质材料。
核壳复合纳米粒子因其组成、大小和结构排列的不同而具有特殊的化学、光学和电学等方面的特性,广泛应用于电子、催化、分离、生物诊断等许多领域。
结合上述几个方面,本论文主要利用金纳米离子传感器对DNA和金属离子进行了检测,以及制备了Au@SiO2以及Fe3O4@SiO2核壳复合纳米粒子。
具体研究工作分为三章,具体工作如下：
第二章,发展了一种新的电化学检测DNA的方法。该方法先通过生物条形码法将目标DNA信号放大并转化为可修饰到电极表面的巯基DNA,由于电化学活性物质六氨合钌(RuHex)阳离子能通过静电相互作用吸附在DNA骨架上并作为电化学信号物质,利用DNA杂交作用采用DNA功能化金纳米粒子对电极表面的巯基DNA进行再组装,组装后将大大提高电极表面负载的DNA量,其DNA骨架吸附更多的RuHex阳离子。该方法先利用生物条形码法将DNA信号放大,再通过组装将信号进一步放大,可以很大程度上提高DNA的检测限。实验结果表明用该方法检测DNA的初步检测限已达到1pM。这种方法结合了生物条形码法和电化学法的双重优势,具有良好的应用前景。
第三章,制备了两种金纳米传感器并用于检测金属离子。两种传感器表现出良好的水溶性和稳定性。1-金纳米粒子对Hg2+的检测具有特别高的选择性,通过肉眼就可检测到1μM的浓度,通过紫外可见光谱可以推进最低检测限至400nM。对于2-金纳米粒子来说,在金属离子浓度为100μaM时,肉眼可以看到2-金纳米粒子对Hg2+, Fe3+, Al3+, Fe2+和Cr3+等离子有颜色响应。当金属离子浓度小于10μM时,2-金纳米粒子对Hg2+的检测也具有非常高的选择性,能通过紫外可见光谱检测到限至1μM的Hg2+。进一步研究传感器颜色响应的机理发现：传感器的颜色响应的主要原因是功能分子与金属离子的配位效应,而不是受pH值和离子强度影响。
第四章,成功制备了Fe3O4@SiO2和Au@SiO2核壳纳米粒子。用水热法制备Fe304磁性纳米粒子,通过调节底物浓度,反应温度,反应时间得到了170nm和220nm的两种尺寸均一的质量较好的磁性纳米粒子。用改进的Stober法在Fe304表面包裹Si02,通过优化水醇比例,pH, TEOS加入量,底物浓度,反应时间等调节包裹层的Si02厚度。通过化学法和种子法制备了不同大小的金纳米粒子,再通过TEOS的水解,在不同粒径的金纳米粒子表面包裹Si02。通过优化水醇比例,pH, TEOS加入量,底物浓度,反应时间等调控Si02的壳层厚度。
Gold nanoparticles(AuNPs) possess excellent optical properties and strong surface adsorption. They have many properties such as strong surface plasmon resonance, surface plasmon resonance fluorescence, surface enhanced Raman, and can be used for a wide range of Biomarkers, Molecular recognition, genetic detection and metal ions detection. Electrochemical biosensing of DNA hybridization is a novel technique with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and it can be compatible with DNA biochip, and they are expected to have a broad prospect of application in clinic examination of inherited diseases and drug screening.
Colorimetry methods for detection of metal ions were widely used because of its fast detection, ease of operation, and low cost. Gold nanoparticles have special properties, are idle materials in colorimetry methods for detection metal ions.
Core-shell alloy nanoparticles have special chemical, optical and electrical properties for its difference in composition, size and structure and are thus widely used in electronic, catalytic, isolation and biologic diagnosis areas.
Combining these aspects, this thesis use gold nanopartical based ion transducer to detect metal ions and DNA, and alloy nanoparticles of Au@SiO2and Fe3O4@SiO2was prepared.
The concrete research work of this dissertation can be divided into three chapters as follows:
Chapter2. A method employing electric-chemical sensor for detection DNA was developed. This method use bio-barcode system to amplify and transfer target DNA signal to mercapto-DNA that can be modified to electrodes. RuHex cations work as electrical-chemical active agent and can be absorbed to anionic phosphate of DNA strands through electrostatic effect. Through DNA hybridization and in the presence of DNA functionalized gold nanoparticles the mercapto-DNA was reconstructed which resulted in a great increase in the amount of DNA loaded on the electrode and thus more RuHex cations were absorbed. This method first amplify DNA signal through bio-barcode system and further amplify the signal through nano self-assemble, which substantially increase the DNA detection limit. The result showed that this method can detect DNA of1pM. This method combined the advantage of bio-barcode system and electrical-chemical technique and has potential for future applications.
Chapter3. We have developed two sensors for detecting metal ions. Both sensors showed good water-soluble and stability.1-Gold nanoparticles(AuNPs) exhibited high selectivity to Hg2+and can detect Hg2+with the concentration of1μM by naked eye, the limit of detection can be further enhanced to400nM by ultraviolet-visible(UV-vis) spectra. Under naked eye,2-AuNPs had a distinct colour change after metal ions (Hg2+, Fe3+, Al3+, Fe2+and Cr3+)(100μM) was added respectively. When the concentration of metal ion is less than10μM,2-AuNPs also exhibited high selectivity to Hg2+and can detect Hg2+with the concentration of1μM by UV-vis spectra. Thorough investigation indicated that the mechanism of the colorimetric responses was mainly coordination effect of the modified organic molecules with metal ions and had little relationship with the change of ionic strength and pH value.
Chapter4. Core-shell nanoparticles of Au@SiO2and Fe3O4@SiO2were prepared. Hydrothermal synthesis of Fe3O4magnetic nanoparticle through controlling the substrate concentration, reaction temperature and reaction time yields two kinds of uniform sized products of170nm and220nm. Using modified Stober method the Fe3O4surface was coated with SiO2, through optimization of water-ethanol ratio, pH, TEOS amount, substrate concentration and reaction time the thickness of SiO2layer was controlled. Different sized gold nanoparticles was prepared through chemical method and seeding method, hydrolyzed through TEOS, and coated with SiO2. The thickness of the SiO2layer was controlled through optimization of the water-ethanol ratio, pH, TEOS amount, substrate concentration and reaction time.
引文
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