International Centre of Biodynamics, Intr. Portocalelor 1 B, 060101, Bucharest, Romania; Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, Bucharest, 050095, Romania.
International Centre of Biodynamics, Intr. Portocalelor 1 B, 060101, Bucharest, Romania; Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, Bucharest, 050095, Romania.
Biosens Bioelectron. 2020 Apr 15;154:112003. doi: 10.1016/j.bios.2019.112003. Epub 2019 Dec 31.
We demonstrate a new biosensing concept with impact on the development of rapid, point of need cell based sensing with boosted sensitivity and wide relevance for bioanalysis. It involves optogenetic stimulation of cells stably transfected to express light sensitive protein channels for optical control of membrane potential and of ion homeostasis. Time-lapse impedance measurements are used to reveal cell dynamics changes encompassing cellular responses to bioactive stimuli and optically induced homeostasis disturbances. We prove that light driven perturbations of cell membrane potential induce homeostatic reactions and modulate transduction mechanisms that amplify cellular response to bioactive compounds. This allows cell based biosensors to respond more rapidly and sensitively to low concentrations of bioactive/toxic analytes: statistically relevant impedance changes are recorded in less than 30 min, in comparison with >8 h in the best alternative reported tests for the same low concentration (e.g. a concentration of 25 μM CdCl, lower than the threshold concentration in classical cellular sensors). Comparative analysis of model bioactive/toxic compounds (ouabain and CdCl) demonstrates that cellular reactivity can be boosted by light driven perturbations of cellular homeostasis and that this biosensing concept is able to discriminate analytes with different modes of action (i.e. CdCl toxicity versus ion pump inhibition by ouabain), a significant advance against state of the art cell based sensors.
我们展示了一种新的生物传感概念，该概念对快速、即时的基于细胞的传感发展具有影响，具有增强的灵敏度和广泛的生物分析相关性。它涉及到对稳定转染表达光敏感蛋白通道的细胞进行光遗传学刺激，以光学控制膜电位和离子动态平衡。延时阻抗测量用于揭示细胞动力学变化，包括细胞对生物活性刺激和光诱导的动态平衡紊乱的反应。我们证明，细胞膜电位的光驱动扰动会引起动态平衡反应，并调节转导机制，从而放大细胞对生物活性化合物的反应。这使得基于细胞的生物传感器能够对低浓度的生物活性/毒性分析物更快、更敏感地做出反应：与相同低浓度（例如 25μM CdCl）的最佳替代报告测试相比，统计上相关的阻抗变化在不到 30 分钟内记录，而在最佳替代报告测试中则需要 >8 小时。对模型生物活性/毒性化合物（哇巴因和 CdCl）的比较分析表明，细胞内动态平衡的光驱动扰动可以增强细胞的反应性，并且这种生物传感概念能够区分具有不同作用模式的分析物（即 CdCl 毒性与哇巴因对离子泵的抑制作用），这是对基于细胞的现有传感器的重大进展。
