Quantitative Imaging of Glutathione in Live Cells Using a Reversible Reaction-Based Ratiometric Fluorescent Probe

Abstract

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe—ThiolQuant Green (TQ Green)—for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies.

Fluorogenic diazaborine formation of semicarbazide with designed coumarin derivatives

“…The coumarin core structure has been widely used in the design of fluorescent molecules as its fluorescence can be easily tuned with electron donating and withdrawing groups. 14–18 The synthetic details of the coumarins are given in the Supporting Information. With the compounds in hand, we first characterized their absorption and fluorescence properties.…”

Theoretical and Experimental Investigation of Thermodynamics and Kinetics of Thiol-Michael Addition Reactions: A Case Study of Reversible Fluorescent Probes for Glutathione Imaging in Single Cells

“…1-3 An example of a Michael addition commonly used for bioconjugation is the thiol-maleimide reaction. This reaction is considered one of the “click” reactions due to its fast reaction rate and aqueous compatibility.…”

“…2 In addition, taking advantage of the reversibility of thiol Michael addition reactions, our group recently reported the first fluorescent probe for quantitative glutathione (GSH) imaging in living cells. 3 …”

Regenerative fluorescence “turn-on” probe for biothiols through Cu(II)/Cu(I) redox conversion

“…This was attributable to the reduction of Cu(II) ion of Cu II L to Cu(I) in the binding complex. Interestingly, the redox-based mechanism presents the possibility for regenerative sensing of biothiols [41,42] in which the fluorescence emission could be recovered repeatedly by not only addition of NEM, the thiol scavenger, but also application of the oxidative potential. Additionally, Cu II L was used successfully for the fluorescence imaging of the cellular GSH.…”