Long-wavelength TCF-based fluorescence probes for the detection and intracellular imaging of biological thiols

Two ‘turn on’ TCF-based fluorescence probes were developed for the detection of biological thiols (TCF-GSH and TCFCl-GSH).


Reaction mechanism
As previously reported 1,2 the system has an internal charge transfer (ICT) donor-π-acceptor (D-π-A) structure. Before the addition of GSH, it is a pull-pull system. The addition of GSH leads to the deprotection of the phenol by reacting with the dinitrobenzenesulfonyl group. This produces the push-pull ICT system with a colorimetric and fluorescence response.

Stability of TCF fluorophores towards GSH
As shown in the manuscript (Fig. 2), at higher GSH concentrations the fluorescence intensity began to decrease. Therefore, we investigated the stability of the TCF phenols towards the addition of GSH. As illustrated below in Fig. S11, there is a clear colour change from purple to brown with the addition of 2 mM GSH. A clear decrease in fluorescence intensity was observed with the addition of GSH (Fig. S4) Despite only a small change in colour, a decrease in fluorescence intensity with the addition of GSH to TCFCl-OH - Fig. S14. However, in comparison to TCF-OH, there was smaller change in the fluorescence intensity indicating a greater stability to GSH.

Method
Cells were seeded in a 96-well plate with culture media. After overnight culture, cells were incubated with each concentration of sample for 24 h. To identify cell viability, reagents were removed and 0.5 mg/ml of MTT (Sigma) media was added to the cells and incubated for 4 hr at 37 ℃ CO2 incubator and the produced formazan was dissolved in 0.1 ml of dimethylsulfoxide (DMSO) and read at OD 650 nm with a Spectramax Microwell plate reader. Absorbance was determined and the mean cell viability was calculated as a percentage of the mean vehicle control and experiments were carried out in three independent tests. To reduce intracellular thiol concentrations, cells were treated with 500 μM H2O2 or 200 μM cisplatin for 6 hr and 2 mM NAC (N-acetylcysteine) was cotreated to recover this effect.

Photobleaching experiments
As shown below in Fig. S22 and S23, photobleaching experiments were carried out against the known biological dye 4',6-diamidino-2-phenylindole (DAPI). The cells were continuously irradiated and the fluorescence intensity was measured over time. Both TCF-based fluorophores displayed good photostability against DAPI with TCFCl-GSH having the best photostability.

Limit of detection
The limit of detection was calculated using the formula shown below: Limit of detection (LOD) = 3σ/slope σ = Standard deviation of the lowest concentration