Dual luminescent charge transfer probe for quantitative detection of serum albumin in aqueous samples

Abstract

In diagnostic medicine serum albumin is considered as an important biomarker for assessment of cardiovascular functions and diagnosis of renal diseases. Herein, we report a novel donor-π-π-acceptor fluorophore for selective detection of serum albumin in urine samples. In our design, a phenolic donor was conjugated with a tricyanofuran (TCF) acceptor through a dimethine bridge via a simple condensation reaction. The stereoelectronic effects of the incorporated methoxy (-OCH₃) groups and the TCF moiety-in conjunction with the extended π-electron conjugation-led to dual red and NIR-I absorption/emission in water. Moreover, due to superior electron transfer between a phenolate donor and the TCF acceptor and the subsequent energy decay from the charge transfer states, the fluorophore displayed negligible fluorescence emission in water and other polar solvents. Consequently, we have been able to utilize the fluorophore for quantitative estimation of serum albumin both in the red (<700 nm) and NIR-I (700-900 nm) regions of the electromagnetic spectrum with excellent reproducibility. The fluorophore selectively recognized human serum albumin over other proteins and enzymes with a limit of detection of 10 mg/L and 20 mg/L in simulated urine samples at red and NIR-I emission window of the spectrum, respectively. By molecular docking analysis and experimental displacement assays, we have shown that the selective response of the fluorophore toward human serum albumin is due to tighter supramolecular complexation between the fluorophore and the protein at subdomain IB, and the origin of the NIR-I (780 nm) emission was attributed to a twisted conformer of phenolate-π-π-TCF system in aqueous solution. These findings indicate that the fluorophore could be utilized for quantitative detection of human serum albumin in urine samples for clinical diagnosis of albuminuria.

Keywords: Dual emission probe; Human serum albumin; Microalbuminuria; Near infrared emission; Selective protein recognition.

Figure 1.

(a) Absorption spectra of probe 1 in various solvents. [1] = 1.0 × 10⁻⁵ M. Aqueous solution contains 1% DMSO. (b) Fluorescence spectra of probe 1 in various solvents. [1] = 1.0 × 10⁻⁵ M. Aqueous solution contains 1% DMSO. For emission spectra, all the samples were excited at the absorption maxima, except for in methanol solution (λ_ex = 485 nm in methanol).

Figure 2.

(a) Absorption spectra of 1 in water at different pH values. (b) Fluorescence spectra of 1 in water at different pH values. All the aqueous solutions contain 1% DMSO. For emission spectra, all the samples were excited at the absorption maxima. (c) Protonated, deprotonated, and probable twisted conformer of 1 in water. [1] = 1.0 × 10⁻⁵ M. Green circle indicates the isobestic point. Dotted ellipse shows absorption centered at 740 nm in higher pH solutions.

Figure 3.

(a) Molecular orbital plots (HOMO and LUMO) and the corresponding energies of the neutral form (figure 2c) in the ground state. (b) Molecular orbital plots (HOMO and LUMO) and the corresponding energies of the ionic form I (figure 2c) in the ground state.

Figure 4.

(a) Fluorescence spectra of 1 (10 μM) in different ratios of buffer (pH = 7.4)-ethylene glycol mixtures, λ_ex = 630 nm. (b) Fluorescence spectra of 1 (10 μM) in different ratios of buffer (pH = 7.4)-glycerol mixtures, λ_ex = 630 nm.

Figure 5.

(a) Plot of emission (λ_ex = 630 nm) of probe 1 in the presence of HSA. Inset: Showing fluorescence of 1 in absence (i) and presence (ii) of one equivalent of HSA when excited with a laser (source 630 nm) (b) Plot of emission (λ_ex = 740 nm) of probe 1 in the presence of HSA. (c) Linear relationship between the emission maxima of 1 (λ^max_em ~665 nm) and the amount of HSA in simulated urine. (d) Linear relationship between the emission maxima of 1 (λ^max_em ~780 nm) and the amount of HSA in simulated urine.

Figure 6.

(a) Fluorescence emission of 1 (10 μM) in presence of various ions and bio-analytes, λ_ex = 630 nm. (b) Fluorescence emission of 1 (10 μM) in presence of HSA and other biomacromolecules in phosphate buffer (pH = 7.4), λ_ex = 630 nm.

Figure 7.

(a) The change in fluorescence intensity at 665 nm (λ_ex = 630 nm) upon addition of increasing concentrations of site specific compounds warfarin and salicylic acid to the complexes of 1 (5 μM) and HSA (333 mg/L) (b) Docking conformation of 1@HSA complex showing predominant bindings at site IB. (c) The change in fluorescence intensity at 665 nm (λ_ex = 630 nm) upon addition of increasing concentrations of site specific compounds warfarin and salicylic acid to the complexes of 1 (5 μM) and BSA (333 mg/L).