Microfluidic chip toward cellular ATP and ATP-conjugated metabolic analysis with bioluminescence detection
Microfluidic chip toward cellular ATP and ATP-conjugated metabolic analysis with bioluminescence detection
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DOI：
10.1021/ac0490447
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发表时间：
2005-01-15
影响因子：
7.4
通讯作者：
Terabe, S
中科院分区：
文献类型：
--
作者：
Liu, BF;Ozaki, M;Terabe, S
在本文中，展示了一种采用聚二甲基硅氧烷（PDMS）制造的集成毛细管电泳和生物发光（BL）检测的微流控平台，并采用芯片实验室技术进行细胞代谢分析。设计了“与 Y 交叉结合”的微通道网络，使用萤火虫荧光素-荧光素酶 BL 系统对 ATP 和 ATP 结合的代谢物进行芯片上样品制备、分离和 BL 检测。事实证明，PDMS 通道壁的动态修饰对于反转电渗流 (EOF) 的方向至关重要，这是通过使用阳离子表面活性剂双十二烷基二甲基溴化铵的预洗循环独特实现的。还研究了表面活性剂对EOF和BL反应的影响。定量分析显示 ATP 的动态线性范围超过 2 个数量级，检测限低至亚微摩尔（中阿托摩尔）。该方法通过测量大肠杆菌的细胞 ATP 进行了验证。直接进行芯片上细胞裂解。以半乳糖为例，进一步的工作重点是 ATP 结合的代谢物分析。人类尿液样本中半乳糖的测定证实了该方案的可靠性，这揭示了该平台用于 ATP 结合的亚代谢组分析的良好前景。
In this article, a microfluidic platform integrating capillary electrophoresis and bioluminescence (BL) detection that was fabricated in poly(dimethylsiloxane) (PDMS),with lab-on-a-chip technology was demonstrated for cellular metabolic analyses. A microchannels network, "cross combining with Y", was designed to perform on-chip sample preparation, separation, and BL detection of ATP and ATP-conjugated metabolites, using firefly luciferin-luciferase BL system. A dynamic modification of the channel wall of PDMS proved to be crucial to reverse the direction of electroosmotic flow (EOF), which was uniquely achieved by a prewash cycle with a cationic surfactant didodecyldimethylammonium bromide. The influences of surfactant on the EOF and BL reaction were also investigated. Quantitative analyses revealed a dynamic linear range over 2 orders of magnitude for ATP, with a detection limit down to submicromolar (midattomole). The method was validated by measuring cellular ATP of E. coli. with direct on-chip cell lysis. Further work was emphasized on ATP-conjugated metabolite analysis, using galactose as an example. Assays of galactose in human urine samples confirmed the reliability of the protocol, which revealed good prospect of this platform for ATP-conjugated submetabolomic profiling.