The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays.
The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays.
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使用具有双功能硅烷的玻璃基板来设计微图案细胞分泌的细胞因子免疫测定。
DOI：
10.1016/j.biomaterials.2011.04.026
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发表时间：
2011
期刊：
影响因子：
14
通讯作者：
Revzin,Alexander
中科院分区：
文献类型：
--
作者：
Seo,JeongHyun;Chen,Li-Jung;Verkhoturov,StanislavV;Schweikert,EmileA;Revzin,Alexander
通常需要将细胞隔离在表面的特定位置，并在细胞旁边集成传感元件。在本研究中，制备了表面，以便在无污染的聚乙二醇（PEG）水凝胶微孔中定位细胞因子感知域。我们的目的是通过生物识别分子的共价附着来增加微模式细胞因子免疫测定的敏感性。为了实现这一目标，玻璃基板被丙烯酸酯和巯基甲氧基硅烷的二元混合物功能化。在随后的水凝胶光刻步骤中，丙烯酸酯部分用于将水凝胶微孔锚定在玻璃基板上。重要的是，微孔内的玻璃附着位点含有巯基，可以用异双功能交联剂激活，用于蛋白质的共价固定。用荧光标记的亲和素孵育后，在混合丙烯基/硫醇硅烷层上制备的微孔比单独在丙烯基硅烷上制备的微孔发出的荧光多6倍。这一结果突出了亲和素在微孔内共价附着的优势。为了创建细胞因子免疫分析，微图案表面与生物素化的IFN-γ或TNF-α抗体（Abs）孵育。以这种方式制备的微图免疫测定对1ng /ml或60pm IFN-γ敏感。为了进一步证明这种生物界面设计的实用性，将巨噬细胞植入直径为30 μm的微孔中，微孔由双功能层（丙烯/硫醇）或单功能硅烷层制成。两种类型的微孔在细胞播种前都包被亲和素和生物素-抗tnf -α。短时间有丝分裂激活和TNF-α免疫染色表明，双功能硅烷层微孔与单功能硅烷层微孔相比，巨噬细胞分泌TNF-α的信号高3倍。这里描述的细胞因子感应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。
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.