Department of Chemistry, KU Leuven, Leuven, Belgium.
Department of Chemistry, University of Alberta, Edmonton, Canada.
Nat Commun. 2022 Apr 6;13(1):1850. doi: 10.1038/s41467-022-29508-w.
Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca concentration in live cells. We also develop intermittent quantification, a technique that combines absolute aquisitions with fast fluorescence acquisitions to deliver fast but fully quantitative measurements. We also show how the photochromism-based measurements can be expanded to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.
基于单个荧光蛋白的基因编码生物传感器被广泛用于可视化细胞内分析物水平或酶活性，尽管通常用于监测相对变化而不是绝对值。我们报告了光致变色实现的绝对定量（PEAQ）生物传感，这是一种利用生物传感器的光致变色特性提供分析物浓度或活性的绝对测量的方法。我们开发了流行的 GCaMP 家族 Ca 生物传感器的光致变色概念验证变体，并表明这些可以用于解析活细胞中绝对 Ca 浓度的动态变化。我们还开发了间歇式定量技术，该技术将绝对获取与快速荧光获取相结合，提供快速但完全定量的测量。我们还展示了如何将基于光致变色的测量扩展到绝对照明强度未知的情况。原则上，PEAQ 生物传感可以应用于具有光致变色特性的其他生物传感器，从而为复杂和动态系统中的完全定量测量提供更多可能性。
