Photoluminescence and Electrochemiluminescence Dual-Signaling Sensors for Selective Detection of Cysteine Based on Iridium(III) Complexes

Abstract

Cysteine (Cys) is important in biosynthesis, detoxification, and metabolism. The selective detection of Cys over structurally similar homocysteine (Hcy) or glutathione (GSH) remains an immense challenge. Although there are many methods for detecting Cys, photoluminescence (PL) and electrochemiluminescence (ECL) techniques are well-suited for clinical diagnostics and analytical technology because of their high sensitivities. Herein, we report PL and ECL dual-channel sensors using cyclometalated iridium(III) complexes for the discrimination of Cys from Hcy and GSH. The sensors react with cysteine preferentially because of kinetic differences in intramolecular conjugate addition/cyclization, enabling phosphorescence enhancement and ECL decrease in the blue-shifted region. Sensor 1 shows ratiometric PL turn-on and ECL turn-off for Cys. In addition, unique ECL-enhancing behavior of sensor 1 toward GSH enables discrimination between Cys and GSH. Sensor 1 was successfully applied to the detection of Cys in human serum by the ECL method. We demonstrate the first case of a Cys-selective PL and ECL dual-channel chemodosimetric sensor based on cyclometalated iridium(III) complexes and expect that the rational design of efficient PL and ECL dual-channel sensors will be useful in diagnostic technology.

Scheme 1. Schematic Illustration for Detection of Cysteine (Cys) Using 1

Figure 1

Molecular structure of 1 showing displacement ellipsoids at 50%.

Scheme 2. Synthetic Scheme of Ir(III) Complexes (a) 1, 7, and (b) 12 (DCM = Dichloromethane, TEA = Trimethylamine)

Figure 2

(a) Time-dependent phosphorescence spectra of 1 (10 μM) in the presence of 100 equiv of Cys. (b) Time-dependent phosphorescence intensity changes of 1 (10 μM) in the presence of 100 equiv of Cys. (c) Phosphorescence titration curve of 1 (10 μM) upon the addition of Cys (0–1 mM). Limit of detection (LOD) = 3.16 μM. Inset: Linear plot of the PL intensity of 1 upon the addition of varying concentrations of Cys. Conditions: CH₃CN/ H₂O (1:1, v/v, pH 7.4, 10 mM HEPES, 25 °C) (λ_ex = 400 nm, bandwidth: 5/5 nm).

Figure 3

(a) Phosphorescence spectra of 1 (10 μM) at 1 h after addition of 100 equiv of various amino acids (Cys, Hcy, GSH, leucine, arginine, valine, methionine, threonine, isoleucine, lysine). (b) Corresponding PL intensity changes of 1 for various amino acids. (c) PL competition assays carried out by addition of 1 mM Cys to 10 μM 1 in the presence of 1 mM various amino acids. Conditions: CH₃CN/ H₂O (1:1, v/v, pH 7.4, 10 mM HEPES, 25 °C) (λ_ex = 400 nm, bandwidth: 5/5 nm).

Figure 4

(a) ECL intensity of 10 μM 1 upon addition of Cys (1 mM) as the potential is swept at a working electrode (Pt disk, diameter: 2 mm) over the range of 0–2.0 V vs Ag/Ag⁺ (scan rate: 0.1 V/s). (b) ECL titration curve of 1 (10 μM) upon addition of Cys. LOD = 0.23 μM. Conditions: CH₃CN/H₂O (1:1, v/v, pH 7.4, 10 mM HEPES, 100 mM TPrA, and 0.1 M TBAP as the supporting electrolyte).

Figure 5

(a) Competitive ECL binding assays carried out with addition of 1 mM Cys to 10 μM 1 in the presence of 1 mM amino acid. (b) Comparison of ECL intensities of 1 and 7. All the ECL measurements were performed after reaction for 1 h. Conditions: CH₃CN/H₂O (1:1, v/v, pH 7.4, 10 mM HEPES, 100 mM TPrA, and 0.1 M TBAP as the supporting electrolyte).

Figure 6

(a) ECL titration curve of 1 (10 μM) upon addition of Cys in human serum. (b) Continuous cyclic ECL scans of 1 (10 μM) in human serum at 0–1.5 V. Conditions: diluted serum (1:10, v/v, 10 mM HEPES, deproteinized) and CH₃CN (1:1, v/v, 100 mM TPrA, and 0.1 M TBAP as the supporting electrolyte). CA parameters were selected as follows: initial E: 0 V, high E: 1.5 V, number of steps: 80 (40 cycles), pulse width: 0.5 s, sample interval: 1 ms.

Figure 7

(a) Cyclic voltammograms of 1 and 1-Cys adduct. (b) HOMO/LUMO distributions obtained by DFT calculations and energy levels calculated from CV analysis of 1 and 1-Cys adduct.