Development of Fluorescent Reagent Based on Ligand Exchange Reaction for the Highly Sensitive and Selective Detection of Dopamine in the Serum

Abstract

A new fluorescent probe (BDP-Fe²⁺) was developed for targeting dopamine, with a boron-dipyrromethenyl (BDP) group as the fluorophore and a Fe²⁺ complex as the ligand exchange site. The free form of BDP-Fe²⁺ in solution displayed weak fluorescence emission, while it showed strong fluorescence emission after interaction with dopamine due to the release of Fe²⁺ from BDP-Fe²⁺, confirming the binding of Fe²⁺ to dopamine. The increase in fluorescence intensity was concentration-dependent, and a good linear relationship was observed between the fluorescence intensity and dopamine concentration. The detection limit of dopamine by BDP-Fe²⁺ was 1.1 nM, indicating a 20-fold higher sensitivity than that of previously reported compounds. The reaction of BDP-Fe²⁺ with dopamine was not affected by the presence of foreign substances, allowing the highly selective detection of dopamine in the human serum sample. The results of this study indicate that the novel compound BDP-Fe²⁺ is a reliable fluorescent molecular probe for the detection of dopamine and can be widely employed in diverse scientific areas.

Keywords: dopamine; fluorescence; high-throughput analysis; molecular probe; sensor; serum.

Figure 1

Chemical structures of the fluorescent reagents boron–dipyrromethenyl (BDP) and BDP-Fe²⁺.

Scheme 1

Method for the synthesis of the BDP-Fe²⁺.

Figure 2

Excitation (dotted line) and emission spectra (solid line) of BDP; [BDP] = 1.0 µM; solvent = 20.0 mM HEPES buffer (pH 7.0); excitation wavelength = 490 nm, monitored wavelength = 525 nm.

Figure 3

Fluorescence spectra of BDP (solid line) and its Fe²⁺ complex, BDP-Fe²⁺ (dotted line) recorded at room temperature (a), and the fluorescence intensity of BDP at 525 nm before and after the addition of various metal ions such as Fe²⁺, Ni²⁺, Co²⁺, Cu²⁺, Mn²⁺, Zn²⁺, Li⁺, Na⁺, K⁺, Cs⁺, Mg²⁺, Ca²⁺, Ba²⁺, and Pb²⁺ (b). [BDP] = 1.0 µM; solvent = HEPES buffer solution (pH 7.0); excitation wavelength = 490 nm.

Figure 4

Fluorescence spectra of BDP-Fe²⁺ before and after dopamine addition, recorded at room temperature (a), and fluorescence intensity of BDP-Fe²⁺ at 525 nm following dopamine addition at different concentrations (b); [BDP-Fe²⁺] = 1.0 µM; solvent = HEPES buffer solution (pH 7.0); excitation wavelength = 490 nm.

Figure 5

Variation of the fluorescence ratio of BDP-Fe²⁺ at 525 nm with time after dopamine addition. [BDP-Fe²⁺] = 1.0 µM; [Dopamine] = 5.0 µM; solvent = HEPES buffer solution (pH 7.0); excitation wavelength = 490 nm.

Figure 6

Fluorescence intensity of BDP-Fe²⁺ after the addition of dopamine in buffer at different pH values. [BDP-Fe²⁺] = 1.0 µM; [Dopamine] = 5.0 µM; excitation wavelength, 490 nm.

Figure 7

Fluorescence ratio of BDP and BDP-M²⁺ (M = Fe²⁺, Ni²⁺, Co²⁺, Cu²⁺, Mn²⁺, Zn²⁺) at 525 nm before and after the addition of dopamine. [BDP] = [BDP-M²⁺] = 1.0 µM; [dopamine] = 5.0 µM; solvent = HEPES buffer solution (pH 7.0); excitation wavelength = 490 nm. Y axis, I: fluorescence intensities of BDP or BDP-M²⁺ at 525 nm before and after the addition of dopamine, and I₀: fluorescence intensities of BDP or BDP-M²⁺ themselves at 525 nm.

Figure 8

Schematic representation of the reaction mechanism between BDP-Fe²⁺ and dopamine.

Figure 9

Fluorescence ratio of BDP-Fe²⁺ at 525 nm following the addition of dopamine and various related interference compounds; [BDP-Fe²⁺] = 1.0 µM; [interference compounds] = 5.0 µM; excitation wavelength = 490 nm. Y axis, I: fluorescence intensity of BDP-Fe²⁺ at 525 nm before and after addition of various amino compounds, and I₀: fluorescence intensity of BDP-Fe²⁺ at 525 nm.

Figure 10

Fluorescence ratios of BDP-Fe²⁺ and previously reported compounds, following the irradiation of excitation light for 24 h; [BDP-Fe²⁺] = [previous compound] = 1.0 µM; excitation wavelength: 490 nm for BDP-Fe²⁺, and 455 nm for previous compound.

Figure 11

Fluorescence ratio of BDP-Fe²⁺ at 525 nm following the addition of dopamine at different concentrations; [BDP-Fe²⁺] = 1.0 µM; solvent = HEPES buffer solution (pH 7.0) or human serum; excitation wavelength = 490 nm. Y axis, I: fluorescence intensity of BDP-Fe²⁺ at 525 nm before and after addition of various amino compounds, and I0: fluorescence intensity of BDP-Fe²⁺ at 525 nm.