Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies
Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies
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DOI：
10.1039/c2lc20744c
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发表时间：
2012-01-01
期刊：
影响因子：
6.1
通讯作者：
Duffy, David C.
中科院分区：
文献类型：
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作者：
Kan, Cheuk W.;Rivnak, Andrew J.;Duffy, David C.
我们报告了一种方法，用于分离单个顺磁珠的毫微微升大小的威尔斯阵列和检测单酶标记的蛋白质在这些珠子上使用连续的流体流动在微加工聚合物阵列组件。基于DVD制造，使用注射成型在环烯烃聚合物（COP）中制造飞升大小的威尔斯阵列。将这些阵列结合到互补的流体结构上，该流体结构也在COP中模制以产生封闭的装置，以允许将液体递送到阵列。将悬浮在酶底物的水溶液中的酶相关的顺磁珠流体地递送至阵列，使得每个孔一个珠通过重力加载。然后将氟碳油流入装置中以从阵列表面去除过量的珠，并密封和隔离含有珠和酶底物的飞升大小的威尔斯孔。然后使用标准荧光成像对该装置进行成像，以确定哪些威尔斯孔含有单酶分子。该装置作为数字ELISA检测器的分析性能优于标准方法，即，玻璃阵列用硅胶垫圈机械密封;前列腺特异性抗原（PSA）可在0.011 pg mL（-1）至100 pg mL（-1）范围内检测。使用封闭的流体装置来分离单分子阵列中的珠粒提供了低成本制造、易于自动化和仪器开发的许多优点，以实现生物标志物验证和医学诊断中的应用。
We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.