多光谱区段荧光纳米粒子的设计制备及其在生物检测方面的应用

发布时间：2018-08-22 16:45

【摘要】：近些年来,随着纳米技术的不断进步及科研人员的深入探索,大批新型荧光纳米材料如雨后春笋般涌现出来,在化学催化、光电器件、太阳能电池等领域得到了广泛应用。在这些纳米材料中,具有生物相容性的荧光纳米材料以其多光谱区段的荧光发射、较高的荧光量子效率、优良的荧光稳定性,以及极低的生物毒性等特质,在医疗检测、临床诊断、生物成像等方面展现出了广阔的应用前景。不同波段的荧光纳米材料在多个领域都发挥着举足轻重的作用,极大地推动了科技进步。因此,设计与制备不同光谱区段、不同功能作用的生物相容性荧光纳米材料,并及早地应用于生物传感、生物成像、临床诊断等领域,无疑有着十分重要的现实意义。本文以光谱为主线,以生物应用为目标,旨在研究不同光谱区段的含金属荧光纳米粒子在生物检测等领域的应用:通过设计与制备不同光谱区段、不同特定功能的荧光纳米粒子,研究荧光材料合成条件、粒子尺寸、组成成分对荧光性质的影响,探讨制备出的荧光纳米粒子在生物检测中的可行性与实际效果,进一步为新型的荧光材料应用于生物检测提供了可能。首先,我们以胺还原法,在油相中制备了兼具蓝色荧光和顺磁性的新型EuS纳米晶。具体过程为:以铕的无机盐和正十二烷基硫醇为原料,油酸、油胺为稳定剂,油胺与硫醇为还原剂,采用简单的“一锅法”,制备出二价态的EuS纳米晶。该纳米晶可发射蓝色荧光,发射峰位为475 nm,半峰宽为32 nm,荧光量子效率为3.5%,在室温下呈现出顺磁性。我们还发现,通过改变实验条件,可以合成到形貌各异、结构规整的纳米线、纳米棒和纳米球等纳米晶,并仍然保持了磁性与荧光等性质。我们进一步以两亲性聚合物F127为表面活性剂,通过自组装的方式,将F127修饰在EuS纳米晶表面,可以将纳米晶从油相转移至水相。转移后,EuS纳米晶依旧保持了良好的荧光与磁性。我们还首次实现了将EuS纳米晶作为荧光/磁性双功能造影剂,应用于细胞荧光成像与活体核磁共振成像中,并展现出了很好的成像效果。其次,我们通过电化学交换法,以Ag纳米点为模板,制备了具有红色荧光的au纳米点。au纳米点平均尺寸小于2.5nm,发射峰位为608nm,量子效率为8.7%。通过mtt细胞毒性测试和器官组织学检查,证明au纳米点具有很低的生物毒性。与pc12神经细胞共培养后,au纳米点可以进入细胞质中,实现对细胞的荧光成像。实验证明,au纳米点主要存在于细胞的溶酶体中。将au纳米点注射入大鼠体内10天后,荧光依然良好保持。进一步以化学键的方式,将au纳米点与细胞膜特异性结合的蛋白ctb相连接,形成ctb-aunds复合物。这种复合物不但保持了au纳米点的荧光性质,而且能特异性结合神经,并在较宽的ph范围内及较长时间的紫外光照射下,其荧光拥有良好的ph稳定性与抗光漂白性,展现了这种复合物良好的荧光稳定性。将复合物注射入大鼠坐骨神经5天后,可以在坐骨神经、神经背根节和脊髓中分别观察到红色荧光,表明这种复合物可以作为一种良好的长效逆行神经示踪剂,应用于生物体内的神经示踪领域。第三,为了制备具有更长荧光发射波长的纳米粒子,我们设计了一种表面部分修饰巯基的支化聚合物sh-pei,并以这种聚合物为配体,以水合肼为还原剂,通过“自下而上”法制备出了正电性、近红外一区荧光功能的au纳米点。这种纳米点展现了尺寸依赖的荧光可调性质:随着粒子粒径从2.38nm增加至3.08nm,其荧光峰位会从可见光区的609nm红移至近红外一区的811nm,荧光量子效率从9.4%降低至1.9%。我们以sh-pei分子为配体制备au纳米点的设计思路为:其一,与传统pei相比,在sh-pei的端基部分修饰上巯基并减少了胺基含量,使获得的au纳米点的细胞毒性显著降低。其二,以配体的巯基能够锚定在au纳米点表面形成au(i)-巯基复合物,这是au纳米点获得较强和稳定荧光性质的重要因素。其三,当配体的巯基作用在aunds表面后,配体的胺基端则会朝向外侧。这种结构不仅赋予了aunds优异的稳定性,而且使其具有正电性。其四,也是最重要的一点,具有正电性、低毒性的sh-pei-aunds可作为一种优异的基因载体,与负电性的基因通过静电作用相互结合后形成复合物,进而被细胞摄取,能够实现细胞对绿色荧光蛋白质粒基因的转染。最后,我们在制备近红外一区au纳米点的基础上,为了增强生物荧光对比度,设计合成具有更长荧光发射波长的纳米材料。向au纳米点的体系中引入铜元素,以cucl2、haucl4为原料,巯基化聚合物sh-pei为配体,通过胺还原法,制备出具有近红外二区荧光功能的au/cu合金纳米点。这种纳米点的粒径为2.7nm左右,荧光发射峰位在1080 nm,荧光量子效率为2%,是商品化碳纳米管荧光效率的5倍。此外,我们发现,Au/Cu合金纳米点具有荧光可调性质,即随着体系中铜含量的增加,其荧光发射波长可以逐渐从近红外一区的811 nm红移至近红外二区的1130 nm。这种荧光可调性质,可归因于合金纳米点的配体-金属-金属电荷转移作用(LMMCT)的结果。此外,这种纳米点还兼具稳定性高、生物毒性低等特点。将合金纳米点注射入大鼠体内,观察大鼠活体近红外二区荧光成像情况。我们发现,在Au/Cu合金纳米点作用下,只需很短时间即可完成对大鼠的近红外二区成像,由于纳米点的二区近红外荧光性质与动物的自身生物荧光对比差异显著,成像中可清晰展现大鼠的组织器官等细微结构。表明这种长波长荧光功能纳米材料可以应用于生物检测和荧光示踪领域。
[Abstract]:In recent years, with the continuous progress of nanotechnology and the deep exploration of researchers, a large number of new fluorescent nanomaterials have sprung up like mushrooms. They have been widely used in chemical catalysis, photoelectric devices, solar cells and other fields. Among these nanomaterials, biocompatible fluorescent nanomaterials have their multi-spectral areas. Fluorescence emission, high fluorescence quantum efficiency, excellent fluorescence stability, and very low biological toxicity have shown broad application prospects in medical detection, clinical diagnosis, biological imaging and other aspects. Therefore, it is undoubtedly of great practical significance to design and prepare biocompatible fluorescent nanomaterials with different spectral regions and different functions, and to apply them to the fields of biosensors, bioimaging and clinical diagnosis as early as possible. Applications of metal fluorescent nanoparticles in biological detection and other fields: Through the design and preparation of fluorescent nanoparticles with different spectral regions and different specific functions, the effects of synthesis conditions, particle size and composition on fluorescence properties of fluorescent materials were studied, and the feasibility and practical effects of the prepared fluorescent nanoparticles in biological detection were discussed. Firstly, a new type of EuS nanocrystals with blue fluorescence and paramagnetism were prepared in the oil phase by amine reduction method. Divalent EuS nanocrystals were synthesized by a simple one-pot method. The nanocrystals emitted blue fluorescence with a peak position of 475 nm, a half-peak width of 32 nm and a fluorescence quantum efficiency of 3.5%. They exhibited paramagnetism at room temperature. We further modified the surface of EuS nanocrystals with the amphiphilic polymer F127 as the surfactant by self-assembly, which can transfer the nanocrystals from the oil phase to the water phase. EuS nanocrystals were used as fluorescent/magnetic dual-functional contrast agents for the first time in cell fluorescence imaging and in vivo nuclear magnetic resonance imaging, and the imaging effect was very good. Secondly, we prepared gold nanodots with red fluorescence by electrochemical exchange method using Ag nanodots as template. 2.5 nm, emission peak 608 nm, and quantum efficiency 8.7%. The results of MTT cytotoxicity test and organ histology showed that Au nanodots had very low biological toxicity. After co-culture with PC12 nerve cells, Au nanodots could enter the cytoplasm and achieve fluorescence imaging of cells. Experiments showed that Au nanodots mainly existed in lysozyme of cells. In vivo, the fluorescence of Au nanodots was maintained after 10 days of injection into rats. Further, the Au nanodots were linked to the membrane-specific protein CTB by chemical bonding to form a ctb-aunds complex. The complex not only maintained the fluorescence properties of Au nanodots, but also specifically bound to nerves, and in a wide range of P. The fluorescence showed good pH stability and photobleaching resistance in the range of H and longer UV irradiation, showing the good fluorescence stability of the complex. Five days after the compound was injected into the sciatic nerve of rats, red fluorescence was observed in the sciatic nerve, dorsal root ganglion and spinal cord, respectively, indicating that the complex was feasible. Thirdly, in order to prepare nanoparticles with longer fluorescence emission wavelengths, we designed a branched polymer sh-pei with thiol groups partially modified on its surface, which was used as a ligand and hydrazine hydrate as a reductant through self-assembly. Au nanodots with positive charge and near-infrared fluorescence function were fabricated by bottom-up method. The nanodots exhibited size-dependent fluorescence tunability: with the particle size increasing from 2.38 nm to 3.08 nm, the fluorescence peak shifted from 609 nm in the visible region to 811 nm in the near-infrared region, and the fluorescence quantum efficiency decreased from 9.4% to 1.9%. The design idea of preparing Au nanodots with sh-pei molecule as ligand is as follows: firstly, compared with traditional pei, the end group of sh-pei is partly modified with thiol group and the amino group content is reduced, so the cytotoxicity of the obtained Au nanodots is significantly reduced. secondly, the thiol group of ligand can anchor on the surface of Au nanodots to form Au (i) - thiol complex, which is auna Thirdly, when the thiol group of the ligand acts on the surface of the aunds, the amino end of the ligand will face to the outside. This structure not only gives the aunds excellent stability, but also makes them have positive electricity. Fourthly, it is also the most important point that the sh-pei-aunds with positive electricity and low toxicity can be used as the ligand. As an excellent gene carrier, it can combine with negatively charged genes by electrostatic interaction to form a complex, and then be uptake by cells. Finally, we designed and synthesized a near infrared region Au nanodots to enhance the bioluminescence contrast based on the preparation of near infrared region Au nanodots. Au / Cu alloy nanodots with near-infrared two-zone fluorescence function were prepared by amine reduction method with copper element introduced into Au nanodot system, cucl_2 and haucl_4 as raw materials and sh-pei as ligand. In addition, we found that Au/Cu alloy nanodots have fluorescence tunable properties, i.e. the fluorescence emission wavelength can be gradually shifted from 811 nm in the near-infrared region to 1130 nm in the near-infrared region with the increase of copper content in the system. The results of ligand-metal-metal charge transfer effect (LMMCT) of the alloy nanodots were obtained. In addition, the nanodots also exhibited high stability and low toxicity. The alloy nanodots were injected into rats to observe the near-infrared two-zone fluorescence imaging in vivo. The two-zone near-infrared imaging of rats can be completed. Because the two-zone near-infrared fluorescence properties of nanodots are significantly different from the biofluorescence of animals, the microscopic structures of rat tissues and organs can be clearly displayed in the imaging. It shows that the long-wavelength fluorescent functional nanomaterials can be used in the field of biological detection and fluorescence tracing.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TB383.1;O657.3

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