In situ generation of a Zwitterionic fluorescent probe for detection of human serum albumin protein

Abstract

In this article, a new approach for human serum albumin selective fluorophore design has been reported. The fluorophore reported here comprises a substituted phenol donor and a cationic benzo[e]indolium acceptor connected with a π bond. Originally, the cationic fluorophore did not bind with human serum albumin. Upon deprotonation of the phenolic-OH by a water molecule the cationic form was transformed into an active zwitterionic form. Spectroscopic studies and theoretical calculations revealed that the new active form remained in a zwitterionic state in neutral aqueous solution, and it formed a strong supramolecular complex with human serum albumin. The spontaneous complexation resulted multi-fold increase of fluorescence intensity which increased linearly with the concentrations of the protein, thus giving an analytical tool to monitor human serum albumin in aqueous samples. We believe, this simple strategy applied on appropriate fluorogenic scaffolds would prove useful to develop new and improved turn-on fluorescent probes for pH regulated biological applications.

Keywords: Donor-acceptor fluorophore; Human serum albumin; Intramolecular charge transfer; Turn-on fluorescence.

Figure 1.

(a) Absorption spectra of 1 (10 μM) in different solvents. (b) Fluorescence spectra of 1 (10 μM) in different solvents. (c) Photograph of solutions of 1 in nonpolar and polar solvents.

Figure 2.

(a) Plot of λ_{max, abs} versus solvent polarity (using Reichardt’s E_T(30) scale). (b) Plot of λ_{max, FL} versus solvent polarity (using Reichardt’s E_T(30) scale).

Figure 3.

(a) UV-vis absorption spectra of 1 in solutions of different pH values; green circle indicates the isobestic point. (b) Steady state fluorescence of 1 in solutions of different pH values.

Figure 4.

(a) Fluorescence spectra of 1 in acidic solutions. (b) Deconvolution of fluorescence spectrum of 1 in pH 4.0 solution. Black line: experimental spectrum; Red line: fitted spectrum; Green lines: fluorescence identified at three different wavelengths.

Figure 5.

(a) Calculated frontier orbitals for 1 in vacuo. (b) Acid-base equilibrium between 1 and 2. (c) Calculated frontier orbitals for 2 in implicit water. (d) Electrostatic potential map of 1 and 2 as generated from DFT calculations.

Figure 6.

Plot of absorption of 2 (5 μM) in the presence of HSA in pH 7.4 buffer solution. (b) Denaturation of 2@HSA complex in the presence of guanidine hydrochloride in pH 7.4 buffer solution.

Figure 7.

(a) Fluorescence spectra of 2 (5 μM) upon addition of increasing concentration of HSA (0.033 μg/L to 1 mg/L) in physiological buffer (pH 7.4). (b) Determination of binding affinity from Benesi-Hildebrand equation.

Figure 8.

(a) The change in fluorescence intensity of 2@HSA (1:1) upon addition of phenylbutazone, ibuprofen, and salicylic acid. (b) Docking conformation of 2@HSA complex of the highest binding affinity. Hydrophobic interactions are shown as dotted grey lines and the cation-π interaction is shown as yellow dashed line.

Figure 9.

(a) Fluorescence intensity of 2 in presence of HSA and other biomolecules in physiological pH; average of two experiments is reported here. (b) Quantitative determination of HSA in deionized water (pH 6.23) (c) Qualitative determination of HSA with urine Chemstrip. The green color indicates albumin concentration greater than 0.90 μM.

Figure 10.

(a) Absorption spectra of 1 and 2 in presence of increasing amount of HSA in pH 5.0 buffer solutions. (b) Fluorescence spectra of 1 and 2 in presence of increasing amount of HSA in pH 5.0 buffer.

Scheme 1.

The synthetic route for the synthesis of compound 1.