Differentiation of HSA and BSA and Instantaneous Detection of HSO₃^– Using Confined Space of Serum Albumins and Live Cell Imaging of Exogenous/Endogenous HSO₃^–

Abstract

The limitations of prevailing probes for the detection of human serum albumin (HSA) and HSO₃ ^- make it challenging to apprehend the cooperative effect of both HSA and HSO₃ ^- in biological systems. Herein, we present a multi-responsive fluorescent probe <b>MGTP</b>, which distinguishes HSA from bovine serum albumin (BSA) through an ∼104-fold fluorescence enhancement at an emission maximum of 595 nm with HSA and only an ∼10-fold increase at an emission maximum of 615 nm with a shoulder at 680 nm with BSA. The absorbance spectrum of <b>MGTP</b> also discriminates HSA and BSA with the respective absorption maxima at 543 nm and at 580 nm. <b>MGTP</b> in the confined space of HSA or BSA undergoes instantaneous conjugate addition of HSO₃ ^- and results in a ratiometric change in fluorescence intensity with diminishing of red fluorescence (600 nm) and emergence of green fluorescence (515 nm). <b>MGTP</b> in the absence of SAs does not react with HSO₃ ^- in phosphate-buffered saline buffer and reacts sluggishly in the dimethyl sulfoxide-water 1:1 mixture. The limit of detection values for the detection of HSA and HSO₃ ^- are 4 and 6.88 nM, respectively. The drug binding studies reveal that <b>MGTP</b> preferably confines itself at the bilirubin site of HSA. In MCF-7 cancer cells, <b>MGTP</b> is localized into mitochondria and reveals both exogenous and endogenous visualization of HSO₃ ^- through a change in fluorescence from the red to green channel.