Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Biophys J. 2012 May 2;102(9):2003-11. doi: 10.1016/j.bpj.2012.03.055.
Engineered protein biosensors, such as those based on Förster resonance energy transfer, membrane translocation, or solvatochromic shift, are being used in combination with live-cell fluorescence microscopy to reveal kinetics and spatial localization of intracellular processes as they occur. Progress in the application of this approach has been steady, yet its general suitability for quantitative measurements remains unclear. To address the pitfalls of the biosensor approach in quantitative terms, simple reaction-diffusion models were analyzed. The analysis shows that although high-affinity molecular recognition allows robust detection of the fluorescence readout, either of two detrimental effects is fostered. Binding of an intramolecular biosensor or of a relatively abundant intermolecular biosensor introduces observer effects in which the dynamics of the target molecule under study are significantly perturbed, whereas binding of a sparingly expressed intermolecular biosensor is subject to a saturation effect, where the pool of unbound biosensor is significantly depleted. The analysis explores how these effects are manifest in the kinetics and spatial gradients of the biosensor-target complex. A sobering insight emerges: the observer or saturation effect is always significant; the question is whether or not it can be tolerated or accounted for. The challenge in managing the adverse effects is that specification of the biosensor-target affinity to within a certain order of magnitude is required.
工程蛋白生物传感器，如基于Förster 共振能量转移、膜易位或溶剂化变色的生物传感器，与活细胞荧光显微镜结合使用，以揭示细胞内过程的动力学和空间定位。这种方法的应用进展是稳定的，但它是否适合定量测量仍然不清楚。为了从定量的角度解决生物传感器方法的缺陷，我们分析了简单的反应-扩散模型。分析表明，尽管高亲和力的分子识别允许对荧光读出进行稳健的检测，但两种有害影响中的任何一种都会受到促进。分子内生物传感器或相对丰富的分子间生物传感器的结合会引入观测者效应，在这种效应中，所研究的靶分子的动力学受到显著干扰，而表达不足的分子间生物传感器的结合则受到饱和效应的影响，其中未结合的生物传感器的池显著耗尽。该分析探讨了这些效应如何在生物传感器-靶标复合物的动力学和空间梯度中表现出来。一个令人清醒的认识出现了：观测者或饱和效应总是显著的；问题是是否可以容忍或解释它。管理不利影响的挑战在于需要将生物传感器-靶标亲和力指定到一定的数量级内。
