Yan Jun, Sun Yinghua, Zhu He, Marcu Laura, Revzin Alexander
Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences Drive #2519, Davis, CA 95616, United States.
Biosens Bioelectron. 2009 Apr 15;24(8):2604-10. doi: 10.1016/j.bios.2009.01.029. Epub 2009 Jan 31.
The integration of sensing elements with small groups of cells is a critical step towards miniaturization of cell cultivation and analysis. This paper describes the development of an optical, enzyme-based biosensor for local detection of hydrogen peroxide (H(2)O(2)) secreted by stimulated macrophages. Photolithographic patterning of horseradish peroxidase (HRP)-containing poly (ethylene glycol) (PEG) hydrogel microstructures was used to create sensing structures on the glass surface. Importantly, enzyme-entrapping hydrogel micropatterns did not support protein or cell deposition and allowed to guide attachment of macrophages next to the sensing elements. Amplex Red, an organic molecule that becomes fluorescent in the presence of H(2)O(2) and HRP, was either immobilized inside hydrogel elements alongside enzyme molecules or added into the cell culture media during cell activation. The production of H(2)O(2) after mitogenic stimulation of macrophages resulted in appearance of fluorescence in the HRP-containing hydrogel microstructures, with fluorescence intensity being a strong function of analyte concentration. The novel cell culture system with integrated sensing elements described here may be enhanced in the future by incorporating additional biorecognition elements to enable multi-metabolite detection at the site of a cell.
将传感元件与小细胞群整合是实现细胞培养与分析小型化的关键一步。本文描述了一种基于酶的光学生物传感器的开发，用于局部检测受刺激巨噬细胞分泌的过氧化氢（H₂O₂）。通过对含辣根过氧化物酶（HRP）的聚乙二醇（PEG）水凝胶微结构进行光刻图案化，在玻璃表面创建传感结构。重要的是，包埋酶的水凝胶微图案不支持蛋白质或细胞沉积，并能引导巨噬细胞附着在传感元件旁边。Amplex Red是一种在H₂O₂和HRP存在下会发出荧光的有机分子，它要么与酶分子一起固定在水凝胶元件内部，要么在细胞激活过程中添加到细胞培养基中。巨噬细胞有丝分裂原刺激后产生的H₂O₂导致含HRP的水凝胶微结构中出现荧光，荧光强度是分析物浓度的强函数。本文所述的具有集成传感元件的新型细胞培养系统未来可能通过加入额外的生物识别元件得到改进，从而能够在细胞位点进行多代谢物检测。
