金纳米与磁纳米颗粒及其复合物的生物传感和细胞成像研究

发布时间：2018-09-14 11:53

【摘要】：贵金属纳米颗粒具有独特的表面等离子体共振(surface plasmon resonance, SPR)吸收和散射性质,已被广泛用于生物传感、细胞成像及癌症治疗等生物化学领域。而磁纳米颗粒(MNPs),尤其是超顺磁性四氧化三铁(Fe304)纳米颗粒,也在磁分离、磁驰豫开关、磁治疗以及核磁共振成像(MRI)等生物医学领域引起了人们广泛关注。近年来,复合纳米材料,特别是核壳结构的纳米材料,由于其能克服单一材料存在的稳定性差、修饰较难、功能单一等缺陷,还能复合各种材料的性能,因而具有独特的优良性质。其中,金纳米包覆磁性纳米复合颗粒(Fe3O4@Au)合成简单、易修饰,且具有SPR和磁性双重性质,体现了巨大的应用潜力,已经逐渐成为人们研究的焦点。然而,这些纳米材料的应用仍存在一些问题,例如生物分子如何有效调控金属纳米颗粒SPR性质,使其能更好的应用于生物传感以及疾病诊断和治疗?纳米材料在细胞膜表面是否具有防御病毒功能?多功能纳米材料已有很多报道,但很少能集靶向、多模式成像及多治疗手段于一身。基于此,针对上述存在的问题,本文以金纳米颗粒(AuNPs)、MNPs、以及Fe3O4@Au复合纳米颗粒为代表,拓宽了其在生物传感的应用,分析了生物分子如何有效调控金属纳米颗粒等离子共振性质,以进一步用于癌细胞成像与治疗,并考察了纳米材料修饰的细胞膜是否具有防御病毒功能。具体的研究内容包括以下两部分：第一部分：基于AuNPs与MNPs在生物传感中的应用。利用AuNPs的SPR吸收性质,以正电AuNPs为探针实现了可视化检测三磷酸腺苷(ATP)与碱性磷酸酶(ALP)；将核酸适配体aptamer)的高选择性与MNPs磁性分离的性质结合,利用银纳米颗粒(AgNPs)的SPR散射性质,建立了高灵敏、高选择性地测定溶菌酶(lysozyme)的方法。考察了生物分子如何有效调控贵金属纳米颗粒SPR性质。具体工作如下： 1.ATP诱导正电AuNPs聚集的光学性质研究及定量检测ATP。实验发现,通过调节ATP浓度,可以有效调控AuNPs在可见区的SPR吸收性质。提出了ATP与AuNPs的作用主要通过两种方式：首先是ATP的磷酸根与AuNPs表面阳离子表面活性剂之间的静电作用,其次是金原子与ATP含N碱基之问的配位作用。基于AuNPs的SPR吸收用于定量检测ATP,方法简单、成本较低、耗时较短、选择性好,检测限达到0.82μM。进一步实验发现,ATP的类似物包括GTP, UTP, CTP, ADP,AMP等,诱导AuNPs聚集程度各不相同,由此可建立了一种简单的可视化分析方法区分ATP类似物。 2.ALP经过脱磷酸化反应使ATP转化为腺苷,建立了一种基于AuNPs的免标记可视化方法检测ALP。该方法的检测范围可以通过加入金属离子动态调节,加入Ca2+或Pb2+可以使其线性范围从100-600unit/L分别调节至5.0-100unit/L及0.2-20unit/L,灵敏度得到极大提高。该方法具有高选择性、高灵敏度、检测范围动态可调的优点,用于检测人血清样品中ALP含量,与临床结果一致。 3. Aptamer偶联MNPs用于lysozyme的富集与高灵敏分析检测。建立了一种基于MNPs与aptamer作为高选择性的分离富集载体,AgNPs作为散射信号探针的方法用于检测lysozyme。首先利用MNPs与aptamer偶联复合物特异性分离并富集lysozyme,再根据其正电荷性质,可与柠檬酸根包被的AgNPs通过静电作用结合,磁性分离上清液中AgNPs强烈的表面等离子共振散射光降低,可用于定量分析lysozyme。磁性分离下层吸附的AgNPs具有强烈的暗场散射光,可用暗场敞射成像对lysozyme进行半定量分析。方法结合了aptamer的高选择性以及lysozyme带正电荷的两个性质,为测定lysozyme设下双重开关,能提高方法的选择性及测定结果的准确性。以具有强散射信号的AgNPs作为信号探针,可以提高方法灵敏度,检测限可低至0.1nM。该方法设计具有普遍适用性,对于复杂样品的测定具有重要意义。第二部分：MNPs与Fe3O4@Au复合纳米颗粒在细胞成像中的应用。首先,以MNPs为例,考察了在细胞膜表而构建“铁笼子”是否具有防御病毒入侵的功能,拓宽了磁纳米材料在细胞成像及生物医学方而的应用。其次,我们结合了Au与Fe3O4各自优越的性能,制备成复合纳米颗粒,修饰靶向配体,构建了Fe3O4@Au多功能纳米材料用于癌细胞的靶向多模式成像与治疗。具体工作如下： 1.细胞膜表面构建“铁笼子”用于抗病毒入侵。本文使用人喉癌上皮细胞(HEp-2),探讨了细胞膜外形成网状“铁笼子”是否能抑制呼吸道合胞病毒(RSV)的感染。MNPs可通过链霉亲和素与生物素的特异性反应吸附到细胞膜表面,利用颗粒表面修饰DNA的多价效应,通过杂交形成网状“铁笼子”,期望达到抑制病毒入侵的效果。实验采用扫描电镜(SEM)证实了“铁笼子”在细胞膜表面形成。其具有良好的生物相容性,能在一定程度上防御病毒侵染细胞。当病毒侵染MOI值为2时,有“铁笼子”保护的细胞,其细胞存活率能从24.1士4.4%提高到49.0±10.0%。利用免疫荧光显微技术,表明“铁笼子”抑制病毒入侵可能通过以下两种方式：其空间位阻效应阻碍了病毒与细胞膜的结合；降低细胞膜柔韧性,抑制病毒出胞以及在细胞间的扩散。本文利用纳米材料在一定程度上实现抗病毒入侵,为开发新的抗病毒药物以及其它防治病毒性疾病的新方法研究提供了思路。 2.多功能Fe3O4@Au核壳纳米花靶向癌细胞双模式成像与治疗。发展能结合诊断与治疗于一身的多功能纳米材料在分子医学领域有极其重要的意义。本文将五个独特的功能有机地整合到了一个粒径为70nm的Fe3O4@Au纳米花上。Fe304核可作为MRI显影剂；表面包被金壳,使其具有近红外吸收,产生光热效应使细胞温度升高,可用于光热治疗。高温能促进负载的抗癌药物盐酸阿霉素(Dox)快速释放,产生红色荧光,可通过共聚焦荧光显微镜监测其释放过程。颗粒表面偶联aptamer可提高药物的靶向释放。该Fe3O4@Au多功能纳米材料结合了荧光成像与MRI双模式成像方式,可提高癌细胞诊断准确性,并有利于监测药物释放。当纳米材料负载0.8μM Dox时达到的治疗效果,可与单独使用2.0μM Dox时相当。因此,将化疗与光热治疗结合使用,可以减少药物用量,降低非特异性的毒副作用。总之,该多功能纳米材料同时具有靶向、双模式成像与双治疗手段结合的功能,对生物医学领域,尤其是癌症治疗具有潜在的意义。总之,本论文具有以下三个创新点：(一)利用生物分子实现了对金属纳米颗粒SPR性质的有效调节。(二)构建了与传统的小分子设计不同的病毒防御系统,以磁纳米颗粒在细胞膜表面形成“铁笼子”抗病毒侵染。(三)制备了同时具有靶向、双成像模式及双治疗手段五个功能的多功能纳米材料。该研究论文拓宽了金属纳米颗粒在化学与生物传感方面的应用,对于将金属纳米颗粒进一步应用于细胞成像及光热治疗领域提供了一定依据,在癌细胞诊断与治疗方面具有一定的临床价值。
[Abstract]:Noble metal nanoparticles have unique surface plasmon resonance (SPR) absorption and scattering properties, and have been widely used in biochemical fields such as biosensors, cell imaging and cancer therapy. In recent years, composite nanomaterials, especially core-shell nanomaterials, have attracted much attention due to their ability to overcome the shortcomings of single materials, such as poor stability, difficult modification, single function, and composite properties of various materials. Among them, gold nanoparticles coated with magnetic nanoparticles (Fe3O4@Au) are easy to modify, and have the dual properties of SPR and magnetism. It has become the focus of research. However, there are still some problems in the application of these nanomaterials, such as how biological molecules effectively regulate gold. As a kind of SPR nanoparticles, nanoparticles can be used in biosensors, disease diagnosis and treatment. Does nanomaterials have the ability to defend against viruses on the surface of cell membranes? Many reports have been reported on multifunctional nanomaterials, but few of them can be combined with targeting, multimodal imaging and multimodal therapy. The applications of AuNPs, MNPs and Fe3O4@Au composite nanoparticles in biosensors have been broadened. The effective regulation of plasma resonance properties of metal nanoparticles by biological molecules has been analyzed for further application in cancer imaging and treatment. The defense of cell membranes modified with nanomaterials has been investigated. The specific research contents include the following two parts:
The first part is based on the application of AuNPs and MNPs in biosensors.Using the SPR absorption property of AuNPs and the positively charged AuNPs as the probe, the visual detection of ATP and ALP was realized.The highly selective aptamer was combined with the magnetic separation property of MNPs and the SPR dispersion of AgNPs was utilized. A highly sensitive and selective method for the determination of lysozyme was developed. The effective regulation of SPR properties of noble metal nanoparticles by biological molecules was investigated.
1. Optical properties of ATP-induced aggregation of positive AuNPs and quantitative detection of ATP. It was found that the SPR absorption properties of AuNPs in the visible region could be effectively regulated by adjusting the ATP concentration. It was proposed that the interaction between ATP and AuNPs could be achieved by two ways: first, the electrostatic interaction between ATP phosphate and surface cationic surfactants of AuNPs. SPR absorption based on AuNPs can be used to quantitatively detect ATP. The method is simple, cost-effective, time-consuming and selective. The detection limit is 0.82 mu M. Further experiments show that the analogues of ATP, including GTP, UTP, CTP, ADP, AMP and so on, induce the aggregation degree of AuNPs to be different, which can be established. A simple visual analysis method is used to distinguish ATP analogues.
2. After dephosphorylation, ATP was converted into adenosine. A label-free visualization method based on AuNPs was established to detect ALP. The detection range of this method can be adjusted dynamically by adding metal ions. The linear range of the method can be adjusted from 100-600 unit/L to 5.0-100 unit/L and 0.2-20 unit/L by adding Ca2+ or Pb2+ respectively, and the sensitivity can be obtained. This method has the advantages of high selectivity, high sensitivity and dynamic adjustable detection range. It is used to detect the content of ALP in human serum samples, which is consistent with clinical results.
3. Aptamer-coupled MNPs were used for lysozyme enrichment and highly sensitive detection. A method based on MNPs and aptamer as highly selective separation and enrichment carriers and AgNPs as scattering signal probes was developed for lysozyme detection. The strong surface plasmon resonance scattering light of AgNPs in the supernatant of magnetic separation can be used for quantitative analysis of lysozyme. Considering the high selectivity of aptamer and the two properties of lysozyme with positive charge, a double switch for the determination of lysozyme can improve the selectivity of the method and the accuracy of the determination results. AgNPs with strong scattering signal can be used as a signal probe to improve the sensitivity of the method, and the detection limit can be as low as 0.1nM. The design of the method is generally applicable. It is important for the determination of complex samples.
The second part is the application of MNPs and Fe3O4@Au composite nanoparticles in cell imaging. Firstly, taking MNPs as an example, we investigated whether the "iron cage" constructed on the surface of cell membrane has the function of preventing virus invasion, which broadens the application of magnetic nanomaterials in cell imaging and biomedicine. Secondly, we combined the advantages of Au and Fe3O4. Fe3O4 @ Au multifunctional nanomaterials were prepared to be used in targeted multimodal imaging and therapy of cancer cells.
1. An iron cage was constructed on the surface of the cell membrane to resist virus invasion. In this paper, the human laryngeal carcinoma epithelial cells (HEp-2) were used to investigate whether the reticulated iron cage could inhibit the infection of respiratory syncytial virus (RSV). Surface-modified DNA has a multivalent effect, which is expected to inhibit viral invasion by hybridization. Scanning electron microscopy (SEM) was used to confirm the formation of "iron cage" on the surface of cell membrane. The survival rate of the cells protected by the cage was increased from 24.1 to 4.4% to 49.0 (+ 10.0%). Immunofluorescence microscopy showed that the cage could inhibit virus invasion in two ways: steric hindrance hindered the binding of the virus to the cell membrane, decreased the flexibility of the cell membrane, inhibited the emergence of the virus, and inhibited the viral invasion. In this paper, nano-materials are used to achieve anti-virus invasion to a certain extent, which provides ideas for the development of new antiviral drugs and other new methods for the prevention and treatment of viral diseases.
2. Multifunctional Fe3O4@Au core-shell nanoflowers are targeted at cancer cells by dual-mode imaging and therapy. The development of multifunctional nanomaterials combining diagnosis and therapy is of great significance in the field of molecular medicine. High temperature can promote the rapid release of doxorubicin hydrochloride (Dox), a loaded anticancer drug, and produce red fluorescence. The release process can be monitored by confocal fluorescence microscopy. Targeted drug release. The Fe3O4@Au multifunctional nano-material combines fluorescence imaging with dual-mode MRI imaging to improve the diagnostic accuracy of cancer cells and facilitate monitoring drug release. The therapeutic effect achieved when the nano-material is loaded with 0.8 mu mDox is comparable to that achieved when 2.0 mu mDox is used alone. Therefore, chemotherapy and photothermal therapy are combined. In a word, this multifunctional nano-material has the function of targeting, dual-mode imaging and dual-therapy, which has potential significance for biomedical field, especially cancer treatment.
In summary, this paper has three innovations: (1) The SPR properties of metal nanoparticles can be effectively regulated by using biological molecules. (2) A virus defense system different from traditional small molecule design was constructed to form "iron cage" on the surface of the cell membrane to resist virus infection. (3) Targeted metal nanoparticles were prepared. This paper extends the application of metal nanoparticles in chemical and biosensor fields, provides a certain basis for further application of metal nanoparticles in cell imaging and photothermal therapy, and has a certain extent in cancer cell diagnosis and treatment. Clinical value.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08;TB383.1

【共引文献】