【摘要】：近些年來(lái),隨著納米技術(shù)的不斷進(jìn)步及科研人員的深入探索,大批新型熒光納米材料如雨后春筍般涌現(xiàn)出來(lái),在化學(xué)催化、光電器件、太陽(yáng)能電池等領(lǐng)域得到了廣泛應(yīng)用。在這些納米材料中,具有生物相容性的熒光納米材料以其多光譜區(qū)段的熒光發(fā)射、較高的熒光量子效率、優(yōu)良的熒光穩(wěn)定性,以及極低的生物毒性等特質(zhì),在醫(yī)療檢測(cè)、臨床診斷、生物成像等方面展現(xiàn)出了廣闊的應(yīng)用前景。不同波段的熒光納米材料在多個(gè)領(lǐng)域都發(fā)揮著舉足輕重的作用,極大地推動(dòng)了科技進(jìn)步。因此,設(shè)計(jì)與制備不同光譜區(qū)段、不同功能作用的生物相容性熒光納米材料,并及早地應(yīng)用于生物傳感、生物成像、臨床診斷等領(lǐng)域,無(wú)疑有著十分重要的現(xiàn)實(shí)意義。本文以光譜為主線,以生物應(yīng)用為目標(biāo),旨在研究不同光譜區(qū)段的含金屬熒光納米粒子在生物檢測(cè)等領(lǐng)域的應(yīng)用:通過(guò)設(shè)計(jì)與制備不同光譜區(qū)段、不同特定功能的熒光納米粒子,研究熒光材料合成條件、粒子尺寸、組成成分對(duì)熒光性質(zhì)的影響,探討制備出的熒光納米粒子在生物檢測(cè)中的可行性與實(shí)際效果,進(jìn)一步為新型的熒光材料應(yīng)用于生物檢測(cè)提供了可能。首先,我們以胺還原法,在油相中制備了兼具藍(lán)色熒光和順磁性的新型EuS納米晶。具體過(guò)程為:以銪的無(wú)機(jī)鹽和正十二烷基硫醇為原料,油酸、油胺為穩(wěn)定劑,油胺與硫醇為還原劑,采用簡(jiǎn)單的“一鍋法”,制備出二價(jià)態(tài)的EuS納米晶。該納米晶可發(fā)射藍(lán)色熒光,發(fā)射峰位為475 nm,半峰寬為32 nm,熒光量子效率為3.5%,在室溫下呈現(xiàn)出順磁性。我們還發(fā)現(xiàn),通過(guò)改變實(shí)驗(yàn)條件,可以合成到形貌各異、結(jié)構(gòu)規(guī)整的納米線、納米棒和納米球等納米晶,并仍然保持了磁性與熒光等性質(zhì)。我們進(jìn)一步以?xún)捎H性聚合物F127為表面活性劑,通過(guò)自組裝的方式,將F127修飾在EuS納米晶表面,可以將納米晶從油相轉(zhuǎn)移至水相。轉(zhuǎn)移后,EuS納米晶依舊保持了良好的熒光與磁性。我們還首次實(shí)現(xiàn)了將EuS納米晶作為熒光/磁性雙功能造影劑,應(yīng)用于細(xì)胞熒光成像與活體核磁共振成像中,并展現(xiàn)出了很好的成像效果。其次,我們通過(guò)電化學(xué)交換法,以Ag納米點(diǎn)為模板,制備了具有紅色熒光的au納米點(diǎn)。au納米點(diǎn)平均尺寸小于2.5nm,發(fā)射峰位為608nm,量子效率為8.7%。通過(guò)mtt細(xì)胞毒性測(cè)試和器官組織學(xué)檢查,證明au納米點(diǎn)具有很低的生物毒性。與pc12神經(jīng)細(xì)胞共培養(yǎng)后,au納米點(diǎn)可以進(jìn)入細(xì)胞質(zhì)中,實(shí)現(xiàn)對(duì)細(xì)胞的熒光成像。實(shí)驗(yàn)證明,au納米點(diǎn)主要存在于細(xì)胞的溶酶體中。將au納米點(diǎn)注射入大鼠體內(nèi)10天后,熒光依然良好保持。進(jìn)一步以化學(xué)鍵的方式,將au納米點(diǎn)與細(xì)胞膜特異性結(jié)合的蛋白ctb相連接,形成ctb-aunds復(fù)合物。這種復(fù)合物不但保持了au納米點(diǎn)的熒光性質(zhì),而且能特異性結(jié)合神經(jīng),并在較寬的ph范圍內(nèi)及較長(zhǎng)時(shí)間的紫外光照射下,其熒光擁有良好的ph穩(wěn)定性與抗光漂白性,展現(xiàn)了這種復(fù)合物良好的熒光穩(wěn)定性。將復(fù)合物注射入大鼠坐骨神經(jīng)5天后,可以在坐骨神經(jīng)、神經(jīng)背根節(jié)和脊髓中分別觀察到紅色熒光,表明這種復(fù)合物可以作為一種良好的長(zhǎng)效逆行神經(jīng)示蹤劑,應(yīng)用于生物體內(nèi)的神經(jīng)示蹤領(lǐng)域。第三,為了制備具有更長(zhǎng)熒光發(fā)射波長(zhǎng)的納米粒子,我們?cè)O(shè)計(jì)了一種表面部分修飾巰基的支化聚合物sh-pei,并以這種聚合物為配體,以水合肼為還原劑,通過(guò)“自下而上”法制備出了正電性、近紅外一區(qū)熒光功能的au納米點(diǎn)。這種納米點(diǎn)展現(xiàn)了尺寸依賴(lài)的熒光可調(diào)性質(zhì):隨著粒子粒徑從2.38nm增加至3.08nm,其熒光峰位會(huì)從可見(jiàn)光區(qū)的609nm紅移至近紅外一區(qū)的811nm,熒光量子效率從9.4%降低至1.9%。我們以sh-pei分子為配體制備au納米點(diǎn)的設(shè)計(jì)思路為:其一,與傳統(tǒng)pei相比,在sh-pei的端基部分修飾上巰基并減少了胺基含量,使獲得的au納米點(diǎn)的細(xì)胞毒性顯著降低。其二,以配體的巰基能夠錨定在au納米點(diǎn)表面形成au(i)-巰基復(fù)合物,這是au納米點(diǎn)獲得較強(qiáng)和穩(wěn)定熒光性質(zhì)的重要因素。其三,當(dāng)配體的巰基作用在aunds表面后,配體的胺基端則會(huì)朝向外側(cè)。這種結(jié)構(gòu)不僅賦予了aunds優(yōu)異的穩(wěn)定性,而且使其具有正電性。其四,也是最重要的一點(diǎn),具有正電性、低毒性的sh-pei-aunds可作為一種優(yōu)異的基因載體,與負(fù)電性的基因通過(guò)靜電作用相互結(jié)合后形成復(fù)合物,進(jìn)而被細(xì)胞攝取,能夠?qū)崿F(xiàn)細(xì)胞對(duì)綠色熒光蛋白質(zhì)�；虻霓D(zhuǎn)染。最后,我們?cè)谥苽浣t外一區(qū)au納米點(diǎn)的基礎(chǔ)上,為了增強(qiáng)生物熒光對(duì)比度,設(shè)計(jì)合成具有更長(zhǎng)熒光發(fā)射波長(zhǎng)的納米材料。向au納米點(diǎn)的體系中引入銅元素,以cucl2、haucl4為原料,巰基化聚合物sh-pei為配體,通過(guò)胺還原法,制備出具有近紅外二區(qū)熒光功能的au/cu合金納米點(diǎn)。這種納米點(diǎn)的粒徑為2.7nm左右,熒光發(fā)射峰位在1080 nm,熒光量子效率為2%,是商品化碳納米管熒光效率的5倍。此外,我們發(fā)現(xiàn),Au/Cu合金納米點(diǎn)具有熒光可調(diào)性質(zhì),即隨著體系中銅含量的增加,其熒光發(fā)射波長(zhǎng)可以逐漸從近紅外一區(qū)的811 nm紅移至近紅外二區(qū)的1130 nm。這種熒光可調(diào)性質(zhì),可歸因于合金納米點(diǎn)的配體-金屬-金屬電荷轉(zhuǎn)移作用(LMMCT)的結(jié)果。此外,這種納米點(diǎn)還兼具穩(wěn)定性高、生物毒性低等特點(diǎn)。將合金納米點(diǎn)注射入大鼠體內(nèi),觀察大鼠活體近紅外二區(qū)熒光成像情況。我們發(fā)現(xiàn),在Au/Cu合金納米點(diǎn)作用下,只需很短時(shí)間即可完成對(duì)大鼠的近紅外二區(qū)成像,由于納米點(diǎn)的二區(qū)近紅外熒光性質(zhì)與動(dòng)物的自身生物熒光對(duì)比差異顯著,成像中可清晰展現(xiàn)大鼠的組織器官等細(xì)微結(jié)構(gòu)。表明這種長(zhǎng)波長(zhǎng)熒光功能納米材料可以應(yīng)用于生物檢測(cè)和熒光示蹤領(lǐng)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TB383.1;O657.3

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