Simultaneous detection of cell-secreted TNF-α and IFN-γ using micropatterned aptamer-modified electrodes.
Simultaneous detection of cell-secreted TNF-α and IFN-γ using micropatterned aptamer-modified electrodes.
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DOI：
10.1016/j.biomaterials.2012.06.089
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发表时间：
2012-10
期刊：
影响因子：
14
通讯作者：
Revzin, Alexander
中科院分区：
文献类型：
--
作者：
Liu, Ying;Kwa, Timothy;Revzin, Alexander
细胞产生干扰素 (IFN)-γ 和肿瘤坏死因子 (TNF)-α 等细胞因子，用于确定疾病特异性免疫反应，并可用于诊断结核病等传染病。在本文中，我们描述了用电活性适体功能化的微图案电极的开发，用于多重检测免疫细胞产生的细胞因子。使用一系列电极脱保护和适体孵育步骤在可单独寻址的半环电极上组装抗 IFN-γ DNA 适体和抗 TNF-α RNA 适体。适体分子被硫醇化以组装在金上，并用亚甲蓝氧化还原报告基因功能化以进行电化学信号转导。通过暴露于重组细胞因子来证实各个传感器对正确细胞因子种类的特异性。对于细胞检测实验，电极阵列被集成到微流体装置中并与免疫细胞一起孵育。表面的设计是这样的：一小群约 400 个细胞附着在由感应 IFN-γ 和 TNF-α 的半环电极包围的圆形粘附位点上。该微型装置由两个平行的微流体通道组成，每个通道包含四个细胞捕获/传感位点。有丝分裂激活后，通过在不同时间点在单独可寻址电极上进行方波伏安法（SWV）来监测分泌的 IFN-γ 和 TNF-α 分子。该生物传感平台用于分析原代 T 细胞和单核细胞系释放细胞因子的数量和速率。经过进一步开发，该平台可能会得到增强，以能够检测更多数量的细胞因子，并用于将免疫细胞中细胞因子释放的水平和动态与传染病的诊断和治疗相关联。
Cellular production of such cytokines as interferon (IFN)-γ and tumor necrosis factor (TNF)-α is used to determine disease-specific immune responses and may be used to diagnose infectious diseases such as tuberculosis. In this paper, we describe the development of micropatterned electrodes functionalized with electroactive aptamers for multiplexed detection of immune-cell-produced cytokines. A sequence of electrode deprotection and aptamer incubation steps were used to assemble anti–IFN–γ DNA aptamers and anti-TNF-α RNA aptamers on individually addressable half-ring electrodes. Aptamer molecules were thiolated for assembly on gold and were functionalized with methylene blue redox reporter for electrochemical signal transduction. Specificity of individual sensors to the correct cytokine species was confirmed by exposure to recombinant cytokines. For cell detection experiments, electrode arrays were integrated into microfluidic devices and incubated with immune cells. Design of the surface was such that a small group of ~400 cells attached in the circular adhesion sites surrounded by half-ring electrodes sensing IFN-γ and TNF-α. The microdevice consisted of two parallel microfluidic channels, each channel containing four cell capture/sensing sites. Upon mitogenic activation, secreted IFN-γ and TNF-α molecules were monitored by performing square wave voltammetry (SWV) at different time points at individually addressable electrodes. This biosensing platform was used to analyze the quantity and rate of cytokine release from primary T cells and a monocyte cell line. Upon further development of this platform may be enhanced to enable detection of larger number of cytokines and used to correlate the levels and dynamics of cytokine release in immune cells to diagnosis and treatment of infectious diseases.
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