Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.
Biomaterials. 2011 Aug;32(23):5478-88. doi: 10.1016/j.biomaterials.2011.04.026. Epub 2011 May 7.
It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning steps, acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ∼ 6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells. To create cytokine immunoassays, micropatterned surfaces were incubated with biotinylated IFN-γ or TNF-α antibodies (Abs). Micropatterned immunoassays prepared in this manner were sensitive down to 1 ng/ml or 60 pM IFN-γ. To further prove utility of this biointerface design, macrophages were seeded into 30 μm diameter microwells fabricated on either bi-functional (acryl/thiol) or mono-functional silane layers. Both types of microwells were coated with avidin and biotin-anti-TNF-α prior to cell seeding. Short mitogenic activation followed by immunostaining for TNF-α revealed that microwells created on bi-functional silane layer had 3 times higher signal due to macrophage-secreted TNF-α compared to microwells fabricated on mono-functional silane. The rational design of cytokine-sensing surfaces described here, will be leveraged in the future for rapid detection of multiple cytokines secreted by individual immune cells.
通常希望将细胞固定在表面的特定位置，并将传感元件与细胞相邻放置。在本研究中，制备了表面，以便将细胞因子传感结构域定位在非污染聚（乙二醇）（PEG）水凝胶微井内。我们的目的是通过共价连接生物识别分子来提高微图案细胞因子免疫测定的灵敏度。为了实现这一目标，玻璃基底用丙烯酰基和巯基封端的甲氧基硅烷的二元混合物进行功能化。在随后的水凝胶光图案化步骤中，丙烯酰基部分用于将水凝胶微井锚定到玻璃基底上。重要的是，微井内的玻璃附着点含有可以用杂双功能交联剂激活的巯基，用于蛋白质的共价固定化。用荧光标记的亲和素孵育后，在混合丙烯酰基/巯基硅烷层上制造的微井发射的荧光比仅在丙烯酰基硅烷上制造的微井多 6 倍。这一结果突出了在微井内共价连接亲和素的优势。为了创建细胞因子免疫测定，将微图案化表面与生物素化 IFN-γ或 TNF-α 抗体（Abs）孵育。以这种方式制备的微图案免疫测定法的灵敏度低至 1ng/ml 或 60pM IFN-γ。为了进一步证明这种生物界面设计的实用性，将巨噬细胞接种到在双功能（丙烯酰基/巯基）或单功能硅烷层上制造的 30μm 直径微井中。在接种细胞之前，这两种类型的微井都用亲和素和生物素化抗 TNF-α 进行涂层。短暂的促有丝分裂激活后，用 TNF-α 的免疫染色显示，与在单功能硅烷上制造的微井相比，由于巨噬细胞分泌的 TNF-α，在双功能硅烷层上制造的微井具有高 3 倍的信号。这里描述的细胞因子传感表面的合理设计，将在未来用于快速检测单个免疫细胞分泌的多种细胞因子。
