微流控芯片—毛细管电泳—微液滴喷射雾化器等离子体质谱联机进行单细胞内金属形态分析的研究

Metal speciation in single cells has great significance for increasing the ability of discriminating abnormal cells from the healthy ones and studying metal transformation and metabolites in cells. However, up to now, reports on metal speciation in single cell are scarce. The aim of the research proposal is achieving rapid speciation analysis of metal in single cells by developing an inkjet-based micro droplet aerosol generator interfacing capillary electrophoresis (CE)- inductively coupled plasma mass spectrometry (ICP-MS) with microfluidic chip single cell injection. The inkjet-based micro droplrt aerosol generator can be operated with high nebulizing efficiency at the low sample uptake rate as the electroosmotic velocity in CE without the use of nebulizing gas. Consequently,the make-up solution used in conventional CE-ICP-MS and suction effect from the conventional pneumatic nebulizer were eliminated in the proposed CE-ICP-MS system. For this reason, the absolute detection limit of the proposed CE-ICP-MS system was significantly reduced with better sensitivity and resolution. On this basis, a hyphenated system, which consists of microfluidic chip single cell manipulation, capillary electrophoretic separation and inductively coupled plasma mass spectrometry detection, will be developed for rapid single-cell metal speciation analysis. The metal speciation method in single cells will be developed for discriminating abnormal cells from normal cells and studying metal metabolites in single cells. The developed hyphennated system and speciation method will be anticipated to be widely used in studying metallomics and metabolomics.

单细胞中金属元素的形态分析对提高从正常细胞中辨别不正常细胞的能力，研究金属在细胞中代谢和迁移转化规律等有重要意义。目前尚稀见单细胞中金属元素形态分析的报道。本申请首次提出用喷墨打印头研制微液滴喷射雾化器，以及进一步研制以微液滴喷射雾化器作为接口的CE-ICP-MS联机装置。使ICP-MS雾化器的液流速度与CE电渗流的流速相匹配，消除传统联机系统中补充液对样品的稀释作用和电泳谱带增宽，消除ICP-MS气动雾化器对电泳分离的自吸作用，显著降低CE-ICP-MS的绝对检测下限，提高CE-ICP-MS联机系统进行单细胞中金属形态分析的灵敏度和分辨率等分析性能。在此基础上，进一步通过用微流控芯片操纵单细胞，研制微流控芯片-CE-ICP-MS联机装置，建立快速进行单细胞内金属元素形态分析的方法，研究金属元素在细胞中的代谢规律和细胞个体中金属形态的差异。在金属组学和代谢组学的研究中有广泛的应用前景。

生物样品（血清、尿样等）特别是单细胞中金属元素的形态分析对研究金属在生物体特别是细胞中代谢和迁移转化规律、从正常细胞中辨别不正常细胞等有重要意义。为了进行生物样品和单细胞的元素形态分析，需要对高灵敏元素特异性检测器-等离子体质谱（ICP-MS）的进样系统进行改造，提供雾化效率并降低进样速度，并依此建立ICP-MS与电泳和色谱联用的形态方法。本项目从ICP-MS进样系统研制、电泳-等离子体质谱联用方法、HPLC-ICP-MS联用与富集方法、加压电泳与电色谱柱制备与方法等方面开展了大量研究工作。研制成功了纳流气动雾化器和直接注入高效雾化器和纳升试样引入装置，可实现纳流速至微流速样品的高效引入。应用了场增强样品堆积和阵列分离通道来提高电泳与ICP-MS的灵敏度，灵敏度的提高倍数正比于阵列通道数，在此基础上建立了电泳/电色谱/纳流色谱-ICP-MS的碘、溴、砷、汞形态分析方法，分析时间均在1 min以内。用反相柱、离子交换柱和氧化石墨烯改性柱建立了汞/碘的形态分析方法，分析时间极大缩短；用离子交换颗粒、巯基硅胶、氧化石墨烯等材料来富集汞形态，实现了1000倍左右的富集效果。开发了加压毛细管电泳装置及碘形态方法，发展了多颗粒柱塞的填充柱制备技术，基本实现了各种不同规格的纳流柱和电色谱柱的实验室自给，制备了一系列改性有机整体柱（氧化石墨烯、碳纳米角、β-环糊精等），建立了17种多环芳烃的快速分离与在线富集方法。