Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography.
Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography.
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DOI：
10.1021/acsnano.5b05781
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发表时间：
2016-01-26
期刊：
影响因子：
17.1
通讯作者：
Maynard HD
中科院分区：
文献类型：
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作者：
Lau UY;Saxer SS;Lee J;Bat E;Maynard HD
同时检测多种生物标志物，如细胞外信号分子，是疾病分析和诊断的关键方面。抗体在表面上的精确定位，特别是在微纳米尺度上，对于在分子水平上改进检测、生物传感器和诊断非常重要，因此，追求设备小型化以实现并行、快速、小体积的检测是一个持续的挑战。在这里，我们描述了一种利用电子束光刻和海藻糖共聚物作为抗蚀剂的多重细胞因子免疫测定，用于在硅底物上直接写入抗体，从而实现微纳米级蛋白质固定的精度。具体地说，抗白细胞介素6 （IL-6）和抗肿瘤坏死因子α (tnf - α)抗体直接模式化。通过捕获受刺激的RAW 264.7巨噬细胞分泌的细胞因子，显示了图案抗体的特异性结合特性的保留。使用金纳米粒子和银增强的三明治免疫分析法，通过暗场显微镜检测局部表面等离子体共振，检测和可视化结合的细胞因子。在相应的细胞培养条件和细胞刺激后的不同时间，IL-6和tnf - α在单个芯片上的多路复用也获得了高特异性的成功证明。直接制造捕获抗体模式的细胞因子检测描述这里可能是有用的生物传感应用。
Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nano- scale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates allowing for micro- and nano-scale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and anti-tumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications.