Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.
Fabrication of oligonucleotide and protein arrays on rigid and flexible substrates coated with reactive polymer multilayers.
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DOI：
10.1021/am302285n
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发表时间：
2013-01-23
影响因子：
9.5
通讯作者：
Lynn, David M.
中科院分区：
文献类型：
--
作者：
Broderick, Adam H.;Carter, Matthew C. D.;Lockett, Matthew R.;Smith, Lloyd M.;Lynn, David M.
关键词：
我们报道了一种自上而下的方法，在表面涂有超薄的胺反应性聚合物多层膜的表面上制备寡核苷酸和蛋白质阵列，这些多层膜是由共价的聚乙烯亚胺（PEI）和胺反应性的氮杂内酯功能化聚合物聚（2-乙烯基-4,4-二甲基内酯）（PVDMA）组成的。人工在涂有PEI/PVDMA多层膜（~35 nm厚）的平面玻片上定位胺端寡核苷酸探针序列，得到的固定探针阵列杂交荧光标记的互补序列具有高信号强度、高信噪比和高序列特异性。用无污染的小分子胺d-葡萄糖胺处理残留的氮唑内酯功能，导致这些阵列特征之间的区域抵抗蛋白质的吸附，并允许在含有高达10 mg/mL蛋白质的复杂介质中杂交。这些薄膜中残留的氮唑内酯基团也被用来固定蛋白质在薄膜表面，并制造蛋白质和酶的功能阵列。PEI/PVDMA多层膜沉积在任意尺寸、形状和组成的基底上的能力允许在多层涂覆的聚对苯二甲酸乙酯和热收缩聚合物薄膜表面上制造寡核苷酸阵列。在这些柔性塑料基板上制造的阵列可以弯曲，切割，调整尺寸和物理操作，这些方法很难使用更传统的刚性基板。因此，这种方法可能有助于开发新的分析格式和生物分子阵列的新应用。本文描述的方法易于实现，不需要使用专门的设备，并且还应与用于在更传统的表面上制造高密度寡核苷酸和蛋白质阵列的自动化液体处理方法兼容。
We report a top-down approach to the fabrication of oligonucleotide and protein arrays on surfaces coated with ultrathin, amine-reactive polymer multilayers fabricated by the covalent ‘layer-by-layer’ (LbL) assembly of polyethyleneimine (PEI) and the amine-reactive, azlactone-functionalized polymer poly(2-vinyl-4,4-dimethylazlactone) (PVDMA). Manual spotting of amine-terminated oligonucleotide probe sequences on planar glass slides coated with PEI/PVDMA multilayers (~35 nm thick) yielded arrays of immobilized probes that hybridized fluorescently labeled complementary sequences with high signal intensities, high signal-to-noise ratios, and high sequence specificity. Treatment of residual azlactone functionality with the nonfouling small-molecule amine d-glucamine resulted in regions between the features of these arrays that resisted adsorption of protein and permitted hybridization in complex media containing up to 10 mg/mL protein. The residual azlactone groups in these films were also exploited to immobilize proteins on film-coated surfaces and fabricate functional arrays of proteins and enzymes. The ability to deposit PEI/PVDMA multilayers on substrates of arbitrary size, shape, and composition permitted the fabrication of arrays of oligonucleotides on the surfaces of multilayer-coated sheets of poly(ethylene terephthalate) and heat-shrinkable polymer film. Arrays fabricated on these flexible plastic substrates can be bent, cut, resized, and manipulated physically in ways that are difficult using more conventional rigid substrates. This approach could thus contribute to the development of new assay formats and new applications of biomolecule arrays. The methods described here are straightforward to implement and do not require access to specialized equipment, and should also be compatible with automated liquidhandling methods used to fabricate higher-density arrays of oligonucleotides and proteins on more traditional surfaces.
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