Rojas Daniel, Hernández-Rodríguez Juan F, Della Pelle Flavio, Del Carlo Michele, Compagnone Dario, Escarpa Alberto
Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Sciences University of Alcalá, E-28871, Alcalá de Henares, Madrid, Spain; Faculty of Bioscience and Technology for Food, Agriculture and Environment University of Teramo, 64023, Teramo, Italy.
Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, Faculty of Sciences University of Alcalá, E-28871, Alcalá de Henares, Madrid, Spain.
Biosens Bioelectron. 2020 Dec 15;170:112669. doi: 10.1016/j.bios.2020.112669. Epub 2020 Oct 1.
A Prussian blue-based electrode array (PBEA) constituted by eight stencil-printed electrodes on a flexible PET (polyethylene terephthalate) substrate is proposed for in-situ HeLa cell culturing and real-time detection of the released HO. The array was suitably interfaced with a poly- (methyl methacrylate) (PMMA) well-containing holders resulting in a low cost multichambered chip. PBEA fabrication was carried out employing a xurography-based cost-effective benchtop microfabrication technology using just a desktop cutting plotter and office grade thermal-laminator. The hydrophobicity of the PET isolating layer allows to constrain cell-containing drops directly on top of the electrochemical cells. HeLa cells growth in the very close vicinity of the working electrode ensures in-situ cell seeding, incubation, and further electrochemical detection of the HO released, enabling high-throughput analysis. Selective and sensitive electrochemical sensing of hydrogen peroxide was carried out at -100 mV vs Ag|AgCl; the resulting LOD was 1.9 μM. Remarkably, the analytical exploitability of the approach was demonstrated by detection of the hydrogen peroxide released from HeLa cells stimulated with N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and after pretreatment of the cells with cocoa polyphenols, that induced a decreased oxidative stress levels. These data make our approach a promising tool for oxidative stress evaluation in cell cultures and biological systems.
本文提出了一种基于普鲁士蓝的电极阵列（PBEA），该阵列由柔性聚对苯二甲酸乙二酯（PET）基板上的八个模板印刷电极组成，用于原位培养HeLa细胞并实时检测释放的过氧化氢（HO）。该阵列与含有聚甲基丙烯酸甲酯（PMMA）孔的支架适当地连接，形成了一种低成本的多腔芯片。PBEA的制造采用了基于刻字术的经济高效的台式微制造技术，仅使用桌面切割机和办公级热压覆膜机。PET隔离层的疏水性使得含有细胞的液滴能够直接限制在电化学池的顶部。HeLa细胞在工作电极非常近的位置生长，确保了原位细胞接种、孵育以及对释放的HO进行进一步的电化学检测，从而实现高通量分析。在相对于Ag|AgCl为 -100 mV的电位下对过氧化氢进行了选择性和灵敏的电化学传感；得到的检测限为1.9 μM。值得注意的是，通过检测用N-甲酰-L-蛋氨酰-L-亮氨酰-L-苯丙氨酸（fMLP）刺激的HeLa细胞释放的过氧化氢，以及在用可可多酚预处理细胞后（可可多酚可诱导氧化应激水平降低），证明了该方法的分析可利用性。这些数据使我们的方法成为细胞培养和生物系统中氧化应激评估的一种有前景的工具。
