Department of Biomedical Engineering, University of California, Davis, Davis, CA 95616, United States.
Biomaterials. 2012 Oct;33(30):7347-55. doi: 10.1016/j.biomaterials.2012.06.089. Epub 2012 Jul 17.
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.
细胞产生干扰素 (IFN)-γ 和肿瘤坏死因子 (TNF)-α 等细胞因子可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，我们描述了用于对免疫细胞产生的细胞因子进行多重检测的功能化微图案化电极的开发。通过一系列电极去保护和适体孵育步骤，将抗 IFN-γ DNA 适体和抗 TNF-α RNA 适体组装到可单独寻址的半环形电极上。适体分子经过巯基化处理以组装在金上，并通过亚甲基蓝氧化还原报告分子进行功能化，用于电化学生物信号转导。通过对重组细胞因子进行暴露，证实了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，将电极阵列集成到微流控设备中，并与免疫细胞孵育。表面的设计使得一小群~400 个细胞附着在由感应 IFN-γ 和 TNF-α 的半环形电极环绕的圆形附着点上。微器件由两个平行的微流道组成，每个通道包含四个细胞捕获/感应点。在有丝分裂原激活后，通过在可单独寻址的电极上进行方波伏安法 (SWV)，在不同时间点监测分泌的 IFN-γ 和 TNF-α 分子。该生物传感平台用于分析原代 T 细胞和单核细胞系中细胞因子的释放量和释放速率。进一步开发此平台可能会增强其功能，以实现对更多细胞因子的检测，并将免疫细胞中细胞因子释放的水平和动力学与传染病的诊断和治疗相关联。
