Arylazoimidazole-based photoswitchable probe allowing visible-light-triggered rapid detection and imaging of live-cell biothiols via fluorophore release

Abstract

Elevated levels of biothiols—cysteine, homocysteine, and glutathione—are found in Alzheimer’s, cancers, or cardiovascular diseases. The discovery of fluorescent probes that can selectively detect biothiols in living cells and organisms holds immense potential for early disease diagnosis. Herein, we report a water-soluble biothiol-sensing fluorescent probe that is composed of a 2-(2-amino-5-alkoxyphenyl)benzothiazole fluorophore and a 2-arylazoimidazole photoswitch. Being a photochromic molecule, it exhibits reversible trans-cis photoisomerisation with high yields under visible light irradiation in aqueous media. In the initial state, the probe is non-fluorescent, and after incubation with biothiol solutions, both trans and cis isomers emit green fluorescence (φf = 10-19%) via azo bond cleavage. The limit of detection of biothiols varies between 2 and 17 μM concentrations. Remarkably, the cleavage of the cis azo-bond occurs at a much faster rate (8-14 times) than that of the trans isomer, allowing rapid detection of biothiols with a t1/2 of 2.4-10 min. The water solubility of the probe permits live-cell fluorescence imaging of biothiols, where the cis isomer could produce adequate fluorescent cell images within 15 min of its post-treatment to the cell culture, but the trans isomer could not develop any images within this time frame. With the theoretical calculations and the estimation of pKa of the azo unit, we have elucidated the mechanism of thiol-mediated azo bond cleavage. This type of photoswitchable turn-on fluorescent probe may permit biothiol imaging with enhanced bioavailability of the probe at the target site, spatiotemporal resolution, and high image contrast via background fluorescent signal subtraction.