Su Wei Wen, Liu Bo, Lu Wei-Bin, Xu Ning-Shou, Du Guo-Cheng, Tan Jing-Lu
Department of Molecular Biosciences and Bioengineering, University of Hawaii, Manoa, Hawaii 96822, USA.
Biotechnol Bioeng. 2005 Jul 20;91(2):213-26. doi: 10.1002/bit.20510.
The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.
从维多利亚多管水母中分离出的绿色荧光蛋白（GFP）是一种非常有用的实时生物过程传感报告基因。GFP培养物荧光是一种复合信号，会受到诸如培养物自发荧光、内滤光效应（IFE）和光漂白等因素的影响。这些因素使得从培养物荧光中准确估计GFP浓度变得复杂。在监测转基因植物细胞悬浮培养物中使用GFP时，IFE尤其成问题，因为这些培养系统中细胞具有聚集性且生物量浓度高。报道的用于在线补偿监测培养物NADH荧光或生物发光时的IFE的方法，除了荧光/生物发光测量外，还需要在线测量生物量密度或培养物浊度/光密度。在本研究中，培养物GFP荧光成功用于估计转基因烟草细胞生物反应器培养中的GFP浓度和其他重要状态，同时无需额外的生物量传感器即可校正IFE和培养物自发荧光的影响。这是通过建立一个基于模型的新型状态观测器实现的。首先，我们开发了一种改进的背散射荧光探针模型，该模型考虑了IFE和自发荧光对从在线荧光报告培养物GFP浓度的影响。然后基于荧光探针模型、植物细胞生物反应器的动态状态模型和在线GFP荧光测量，使用扩展卡尔曼滤波器（EKF）建立了状态观测器。引入了几种观测器版本以满足与监测植物细胞培养物GFP荧光相关的实际需求。所提出的方法为IFE的在线补偿提供了一种有效手段，能够对培养物荧光信号进行定量解释，以准确报告GFP或GFP融合蛋白的表达。
