Enzymes are biomacromolecules for biological processes in a living being, assisting organisms in controlling physiological processes under a variety of environmental circumstances [1], [2], [3]. It is well recognized that numerous diseases such as Alzheimer’s disease, diabetes, Parkinson’s disease, and cancers, are closely correlated with the up- or down-regulation of particular enzyme activity [4], [5], [6], [7], [8]. Therefore, it is necessary to quantify and image the cellular enzymes for basic medical and biological research, as well as early disease diagnosis and drug discovery [9], [10], [11]. Small-molecule fluorescent probes are promising techniques that are employed for real-time imaging of living cells, monitoring cellular signaling pathway inside tissues and cells, identifying the binding with cellular and subcellular biomolecules and so on [12], [13], [14], [15], [16], [17], [18]. Because of the simplicity in synthetic procedure, fluorescent probes can selectively bind to a specific region or functional group of targeted biomolecules, along with changes in fluorescence signal (i.e. intensity or wavelength). Therefore, constructing fluorescence probes to track disease-related enzymes is pregnant and exciting work. In the last decades, substantial works were reported and results show large progress in this field [19], [20], [21], [22], [23], [24], [25], [26].

Human serum albumin (HSA) is an abundant protein in blood and tissue fluids with important physiological functions [27], [28]. The level of HSA in blood plasma is usually linked to variety of liver and kidney diseases such as diabetes, liver cirrhosis, chronic hepatitis, nephropathy and others [27], [28], [29], [30]. The sensitive and selective quantification of the HSA level in the biological fluids can thus serve as a diagnostic test for patients which suffer from the above-mentioned diseases. Indeed, urine albumin has long been accepted as diagnostic and prognostic marker of chronic kidney disease and diabetes [31], [32]. On the other hand, bovine serum albumin (BSA) is a globular protein with over 75 % similarity to HSA. because of its low cost and ready availability, BSA is widely used as a replacement of HSA in many biochemical and pharmacological applications [33]. However, BSA cannot replace the lost fluid in place of HSA and help restore the blood volume in surgery patients. In this way, it is important and urgent to distinguish HSA and BSA in therapeutic use.

Donor-π-acceptor (D-π-A) dipolar fluorescent probes are particularly interesting as their optical properties were strongly dependent to their environmental changes such as polarity or viscosity [34]. Many efficient fluorescent probes for HSA are known, which are mostly based on the fluorescence enhancement of dipolar probes caused by the environmental change from an aqueous medium to a binding pocket of HSA based on the probe-HSA chelation. However, they have been less exploited for the discrimination of HSA from BSA [35], [36], [37], [38], [39], [40], [41], [42], [43]. In this regard, the hydrophobic cavity of HSA is a key feature for designing fluorescent probes. Herein, we developed four lophine-derived D-π-A fluorescent probes by systematic variation of electron-donor and electron-acceptor groups. The selective experiment results demonstrated that dye M−H−SO3 with a hydrophilic sulfonate group at electron-acceptor part exhibited sufficient selectivity for discrimination of HSA from BSA. Dyes with substitutions at lophine group of electron-donor part, will increase steric hindrance in binding with HSA and give low selectivity. Analysis of the binding mechanism between dye M−H−SO3 and HSA by using both experimental and docking studies indicated that hydrogen-bond interactions were the major driving force for complexation of M−H−SO3 at site III in HSA, yielding a substantial fluorescent enhancement. Furthermore, the outstanding HSA detection capabilities of M−H−SO3 in urine allow us to develop a rapid and effective way to monitor the health status of people.