基于微纳结构分离材料的微流控器件的构建及其在蛋白质分离中的应用

发布时间：2018-04-19 03:03

本文选题：微流控技术 + 蛋白质分离　；参考：《东华大学》2016年博士论文

【摘要】：在后基因组时代,蛋白质组学成为了生命科学的中心任务之一,而蛋白质分离则是蛋白质组学的基础。但传统的蛋白质分离技术不能满足当前高效高通量的蛋白质分离需求,因此需要发展一种新型的蛋白质分离技术。微流控技术具有集成化、微型化及自动化特点,若能将微流控技术应用于蛋白质分离,则有望实现蛋白质的高效高通量分离。目前过渡金属亲和色谱已广泛应用于多种蛋白质的分离,而将其与微流控技术相结合的研究也逐渐引起了广泛的关注。本文以构筑微纳结构的过渡金属化合物为主要研究对象,以调控其微观形貌为主要手段,将其与微流控技术相结合,深入研究微观形貌对于其蛋白质分离性能的影响,对其在高丰度蛋白分离及低丰度肽段富集等多个方面的应用进行了研究。利用微流体湿化学法在微通道内表面上构筑得到NiO阵列,通过调控反应液浓度构筑得到三种形貌的NiO阵列,并设计构建微流控器件。研究了p H和离子强度对器件的蛋白质吸附性能的影响,发现基于片状NiO阵列的微流控器件对牛血清蛋白(BSA)和牛血红蛋白(BHb)表现出最佳的吸附性能,当停留时间为120 s时,可分别对两种蛋白(0.5 mg mL-1)进行完全吸附;该微流控器件可在特定的pH和NaCl浓度下对混合蛋白质样品中的BHb进行选择性分离,并成功应用于人体血样中人血红蛋白(hhb)的分离。通过流体化学法在封闭微空间内部构筑得到了超长菱形zn(oh)f纳米棒阵列,其阵列长度达到了60μm,并构建得到微流控器件。研究了ph和离子强度对器件的蛋白质吸附性能的影响,发现器件可在一定的离子强度下表现出最佳吸附性能,其分别可在停留时间为14min和80s时完成对溶菌酶(lyz)和bhb(0.5mgml-1)的吸附,吸附量分别达到了3700和63094gm-3,与片状nio阵列相比提升了近10倍。该器件对lyz和bhb皆体现出了选择性吸附特性,并成功应用于蛋清中lyz以及人体血样中hhb的分离。在zn(oh)f阵列的基础上,研究了反应物中zn与f的混合摩尔比及氟源的不同对zno/zn(oh)f阵列的微观形貌的影响,构筑得到了纳米纤维状及三维网络结构状zno/zn(oh)f,并构建得到微流控器件。研究了微纳结构形貌对器件的光催化性能和蛋白质吸附性能的影响,发现基于纳米纤维状zno/zn(oh)f的微流控器件具有突出的光催化性能,其可在停留时间为50s时,将亚甲基蓝溶液(5ppm)完全降解,并对bhb(0.5mgml-1)进行全吸附。研究并验证了光催化特性应用于分离材料自身快速再生的可行性,当置于紫外光下照射50min时,器件即可恢复其蛋白质吸附性能。相较于pbs过夜浸泡的方法,光催化处理显著提升了器件的循环使用性能。当循环使用10次时,基于zno/zn(oh)f阵列的微流控器件仍可在停留时间为60s时吸附90%以上的bhb(0.5mgml-1),体现出了优异的循环使用性能。通过在原有三维ZnO/Zn(OH)F网络结构的基础上,进行Ni(OH)2的功能化修饰,通过调节反应液浓度及反应时间,成功构筑得到了三维Ni(OH)2网络结构,并构建得到微流控器件。该微流控器件表现出了突出的蛋白质吸附分离性能,可在25 s内对BHb(0.5 mg mL-1)进行完全吸附,其吸附容量达到了114450 g m-3,较ZnO/Zn(OH)F提升了30%。在循环使用10次时,该器件仍可在停留时间为25 s时,对90%的BHb(0.5 mg mL-1)进行全吸附,并可对100倍稀释的人体血样中HHb进行选择性分离。采用蒸发诱导自组装法和溶胶-凝胶法在玻片上制备得到了Ti O2-ZrO2反蛋白石结构薄膜,该薄膜具有比表面积大的特点,且其所含的TiO2与ZrO2成分在薄膜中均一分布。后设计构建了基于此薄膜的微流控器件,由于其结合了单多磷酸化肽富集材料的富集特性,研究表明其可在停留时间为30 s内完成对磷酸化肽的全富集。并且发现微流控器件的磷酸化肽富集特性取决于反蛋白石结构薄膜的微观结构,相较于其他两种薄膜,Ti O2-ZrO2-270表现出了较好的富集性能。
[Abstract]:In the post genomic era, proteomics has become one of the central tasks of life science, while the protein separation is based proteomics. But traditional protein separation techniques can not meet the current efficient high-throughput protein separation, protein separation technology, so it is necessary to develop a new type of microfluidic technology is integrated. Miniaturization and automation features, if the microfluidic technology applied to protein separation, is expected to achieve efficient high-throughput protein separation. The transition metal affinity chromatography has been widely used in a variety of proteins, and the study of combination with microfluidic technology has gradually aroused widespread concern. In this paper, in order to construct the transition the micro nano structure metal compounds as the main object of study, in order to control the morphology as the main method, combined with the microfluidic technology, in-depth research of micro The morphology of impact on the protein separation performance, the application in high abundance protein separation and enrichment of low abundance peptides and other aspects are studied. By using the micro fluid wet chemical method in the micro channel are constructed on the surface of the NiO array, by adjusting the reaction liquid concentration to build NiO array three kinds of morphologies. The design and construction of microfluidic devices. Effects of protein adsorption properties of P H and ionic strength on the device, found a microfluidic device for bovine serum albumin chip based on NiO array (BSA) and bovine hemoglobin (BHb) showed the best adsorption properties, when the residence time of 120 s, respectively in the two protein (0.5 mg mL-1) to complete the adsorption; microfluidic device for selective separation of protein mixture in BHb in particular pH and NaCl concentration, and successfully applied to human blood in human blood red protein (hHB) of the From. Through the fluid chemical method in a closed space within the building has been long micro diamond Zn (OH) f nanorod array, the array length reached 60 m, and the microfluidic device. The effects of protein adsorption properties of pH and ionic strength on the device, found that the device can exhibit the best adsorption performance in the ionic strength is certain, which are available in the residence time was 14min and 80s of lysozyme (Lyz) and BHb (0.5mgml-1) adsorption, the adsorption capacity reached 3700 and 63094gm-3, and the chip NiO array is increased by nearly 10 times. Compared with the device of Lyz and BHb are reflected in selective adsorption characteristics the separation, and successfully applied to the egg white in the Lyz and hHB in human blood. In Zn (OH) based on the f array, were studied in Zn and f mixed molar ratio and different sources of fluoride on zno/zn (OH) affects the morphology of the f array, by build The nano fibrous and three-dimensional network structure of zno/zn (OH) f, and to construct the microfluidic device. The effects on device photocatalytic properties and protein adsorption properties of micro nano structure morphology, found that the nano fibrous zno/zn (OH) based on microfluidic device f with photocatalytic performance is outstanding, which can when the retention time is 50s, the methylene blue (5ppm) and the complete degradation of all BHb (0.5mgml-1) adsorption. Research and verify the feasibility of photocatalytic properties of materials applied to the separation of its rapid regeneration, when placed in 50min under ultraviolet irradiation, devices can restore its adsorption protein. Compared with PBS. Immersion method, photocatalytic treatment significantly improves the device recycling performance. When recycling 10 times, based on zno/zn (OH) microfluidic devices still f array when the retention time is 60s adsorption more than 90% BHb (0.5mgml-1 ), reflects the excellent cycle performance. The original 3D ZnO/Zn (OH) based F network structure, Ni (OH) functionalized 2, by adjusting the reaction concentration and reaction time, the successful construction of the three Ni (OH) 2 network structure, and construct microfluidic the microfluidic device. The device exhibits the separation performance of the protein adsorption can be prominent, within 25 s of BHb (0.5 mg mL-1) were completely adsorption, the adsorption capacity reached 114450 g M-3, compared with ZnO/Zn (OH) F promoted 30%. in recycling 10 times, the device is still in the residence time 25 s, 90% BHb (0.5 mg mL-1) for adsorption, and the 100 times diluted in human blood HHb selective separation. By evaporation induced self-assembly method and sol-gel method on glass slides were prepared by the Ti O2-ZrO2 inverse opal film, the film has a large surface area the The characteristics of TiO2 and ZrO2, and the content of its content in the films uniform. After design and construct the microfluidic device based on this film, combined with the characteristics of single and multi enrichment of phosphopeptide enrichment materials, research shows that it can stay in time for 30 s to complete the full enrichment of phosphopeptides. And found that the microstructure in inverse opal films phosphorylation peptide enrichment depends on microfluidic device characteristics, compared with the other two films, Ti O2-ZrO2-270 showed enrichment of good performance.

【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q503

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