Microfluidic Patterning of Miniaturized DNA Arrays on Plastic Substrates
Microfluidic Patterning of Miniaturized DNA Arrays on Plastic Substrates
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DOI：
10.1021/am900285g
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发表时间：
2009-07-01
影响因子：
9.5
通讯作者：
Veres, Teodor
中科院分区：
文献类型：
--
作者：
Geissler, Matthias;Roy, Emmanuel;Veres, Teodor
本文介绍了图案或DNA阵列塑料表面上使用弹性体，二维微毛细管系统（μ CS）。使用Versaflex CL 30通过热压光刻实现了流体结构。像弹性体的基础上聚（二甲基硅氧烷），这种热塑性嵌段共聚物是能够密封的表面在一个可逆的方式，使之有可能限制DNA探针的控制水平是无与伦比的使用标准的微喷技术，我们专注于亩CS支持阵列，包括高达2 × 48点，每个是45 μ m的直径。基底由两种硬热塑性材料，聚（甲基丙烯酸甲酯）和多环烯烃（例如，Zeonor 1060 R），它们都用1-乙基-3-[3-（2-甲氧基苯基）-2-（三氟甲基）苯基]-2-甲基-N-（2-甲氧基苯基）-3-（三氟甲基）-2-（三氟甲基）苯甲酰胺活化。（二甲基氨基）丙基]碳二亚胺盐酸盐和N-羟基琥珀酰亚胺来介导DNA分子的共价连接，该方法通过使用0.25-32 μ M用Cy 3或Cy 5荧光染料标记的氨基修饰的寡核苷酸在磷酸盐缓冲盐水中的溶液来举例说明。允许直接和灵敏地表征印刷阵列。将溶液在22、30和40 ° C下孵育1至> 48小时的持续时间，以探测获得高荧光强度的均匀斑点的条件。微流体供应通道的长度（l）和深度（d）对于消耗以及蒸发或溶剂都是重要的。虽然选择性活化或底物被证明有助于限制寡核苷酸沿着轨迹的非生产性损失，但在潮湿环境中孵育溶液是必要的，以防止液体的不受控制的干燥，保持固定化过程在延长的时间段内完整。当结合使用时，这些策略有效地促进了高质量DNA阵列的形成，使得可以以高度的均匀性平行排列多个探针，此外，我们表明所得阵列与标准杂交方案兼容，这使得当暴露于特定的ssDNA靶分子时可以可靠地区分单个链。
This paper describes the patterning or DNA arrays on plastic surfaces using an elastomeric, two-dimensional microcapillary system (mu CS). Fluidic structures were realized through hot-embossing lithography using Versaflex CL30. Like elastomers based on poly(dimethylsiloxane), this thermoplastic block copolymer is able to seal a surface in a reversible manner, making it possible to confine DNA probes with a level of control that is unparalleled using standard microspouting techniques, We focus on mu CSs that support arrays comprising up to 2 x 48 spots, each being 45 mu m in diameter. Substrates were fabricated from two hard thermoplastic materials, poly(mechylmethacrylate) and a polycyclic olefin (e.g., Zeonor 1060R), which were both activated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride and N-hydroxysuccinimide to mediate covalent attachment of DNA molecules, The approach was exemplified by using 0.25-32 mu M solutions of amino-modified oligonucleotides labeled with either Cy3 or Cy5 fluorescent dye in phosphate-buffered saline, allowing for a direct and sensitive characterization of the printed arrays. Solutions were incubated for durations of 1 to > 48 h at 22, 30, and 40 degrees C to probe the conditions for obtaining uniform spots OF high fluorescence intensity. The length (l) and depth (d) of microfluidic supply channels were both important with respect to depletion as well as evaporation or the solvent. While selective activation or the substrate proved helpful to limit unproductive loss of oligonucleotides along trajectories, incubation of solution in a humid environment was necessary to prevent uncontrolled drying of the liquid, keeping the immobilization process intact over extended periods of time. When combined, these strategies effectively promoted the formation of high-quality DNA arrays, making it possible to arrange multiple probes in parallel with a high degree of uniformity, Moreover, we show that resultant arrays are compatible with standard hybridization protocols, which allowed for reliable discrimination of individual strands when exposed to a specific ssDNA target molecule.