Construction of a Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO-based electrochemiluminescence chiral sensor and its application for simultaneous recognition of menthol and penicillamine enantiomers†

Rui Kuang*^a and Bing Yang^b
aCollege of Traffic Civil Engineering, Shandong Jiaotong University, Jinan 250023, China. E-mail: kuangrui@sdjtu.edu.cn
^bSchool of Intelligence Engineering, Shandong Management University, Jinan 250357, China

First published on 15th August 2025

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Introduction

Rare earth metal complexes of ruthenium exhibit excellent luminescent capabilities. Since the report of the electrochemiluminescence (ECL) system of Ru(bpy)₃²⁺ (bpy = 2,2′-bipyridine) in tripropylamine (TPA) aqueous solution in 1990,¹ ruthenium-related compounds have been extensively studied and applied in ECL analysis due to their high ECL efficiency, good chemical stability, and outstanding electrochemical properties.^2,3

2-Isopropyl-5-methylcyclohexanol (Men) is one of the main components in mint and other mint leaves or oils, and it is widely used in fields such as food, pharmaceuticals, fragrances, tobacco, and insect repellents.⁴ Men with different configurations have different industrial application values. L-Men is an important flavoring agent and has a strong minty odor and an obvious cooling sensation,⁵ and thus it is used to make medications for treating contact urticaria, headache, or acute bronchitis. In contrast, D-Men has a woody aroma and a slight camphor odor.^6,7 Therefore, the application value of L-Men is higher than that of D-Men. Currently, the main methods for identifying D/L-Men enantiomers include optical methods,⁵ biological methods,⁴ etc. However, practical operation difficulties and the high cost of chiral selectors limit the application of the identification of D/L-Men. At present, there are relatively few relevant literature studies on the identification and detection of D/L-Men enantiomers. Therefore, it is of great practical significance to develop more green and efficient methods that can be applied to the detection of menthol enantiomers.

In recent years, metal–organic frameworks (MOFs) have been widely used as adsorbents and stationary phases due to their excellent crystalline structures and coordination modes, playing an important role in fields such as catalysis, chromatographic column separation, and chiral recognition.⁸ Solid-state ECL sensors based on semiconductor materials have emerged widely in recent years. Metal oxide semiconductor nano-ZnO has been extensively applied in ECL sensing because of its non-toxicity, low cost, and easy preparation.^9,10 There is a strategy of in situ transformation of ZnO nanorod arrays into chiral ECL sensors.

To achieve this goal, in this study, the potentiostatic electrolysis method was first adopted to electrodeposit uniformly distributed achiral ZnO nanorod arrays on fluorine-doped conductive glass. Using this template as a zinc source, the ZnO nanorod arrays were partially in situ transformed into a Zn-based chiral MOF, namely the Zn(L-tar)(4,4′-bipy) structure. Furthermore, an ECL chiral sensor of ZnO@Zn(L-tar)(4,4′-bipy)/FTO was constructed, realizing the identification of D/L-Men enantiomers.

Potential-resolved ECL systems can detect multiple analytes in a single scan without the need to use filters or beam splitters to distinguish signals, which greatly simplifies the operation procedure and shortens the analysis time.^11–13 To the best of our knowledge, there are currently no reports in the literature on ECL sensors that can achieve chiral detection of multiple samples in a single potential scan.

For simultaneous discrimination of multiple analytes, we strategically integrated the anodic luminophore Ru(phen)₂L-Asp with cathodic-emitting ZnO@Zn(L-tar)(4,4′-bipy)/FTO to construct a novel Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor. This configuration represents the first reported attempt at potential-resolved simultaneous recognition of D/L-menthol and D/L-penicillamine enantiomers through a dual-ECL approach.

Experimental section

Synthesis of Ru(phen)₂Cl₂

Ru(phen)₂Cl₂ was synthesized according to the following steps:¹⁴ 0.5000 g (2.5 mmol) of RuCl₃, 1.0000 g (5 mmol) of 1,10′-phenanthroline (phen) and 0.6400 g (14 mmol) of LiCl were dissolved in 10 mL of N,N-dimethylformamide (DMF) and transferred into a three-necked flask. The mixture was refluxed at 140 °C under N₂ protection for 8 h, during which the red solution turned dark purple. After cooling, 25 mL of cold acetone was added, and the mixture was allowed to stand at 4 °C for 12 h. The resulting black-purple precipitate was obtained by filtration. It was washed three times with cold deionized water and acetone and then dried in a vacuum drying oven for 24 h. The quantity of Ru(phen)₂Cl₂ obtained was 1.1690 g, with a yield of 91.6%.

Synthesis of Ru(phen)₂L-Asp

Ref. 15 describes the synthesis of the chiral complex Ru(phen)₂L-Asp. In this procedure, 0.1060 g of the precursor Ru(phen)₂Cl₂ and 0.0270 g (0.2 mmol) of L-aspartic acid (L-Asp) were dissolved in a mixture of 7 mL deionized water and anhydrous ethanol (v/v = 1:1). The solution was then placed in a high-pressure reactor and reacted at 100 °C for 2 h. The solution turned back to its original red color. Saturated NH₄PF₆ solution was dripped into 1 mmol of methanol, yielding a red precipitate. The precipitate was further recrystallized using acetone/methanol (1:1, v/v), resulting in purple crystals of Ru(phen)₂L-Asp. Excess L-Asp was removed by rinsing with ultrapure water and the product was dried at 55 °C under vacuum for 24 h, yielding the desired material, the chiral complex Ru(phen)₂L-Asp (Scheme 1).

Synthesis of conductive glass (FTO)

The FTO glass was placed in chloroform and acetone respectively, and ultrasonic treatment was performed for 30 minutes. Then, it was washed with ultrapure water and absolute ethanol, and ultrasonic treatment was conducted for 15 minutes. Finally, it was dried and kept for future use.

Synthesis of ZnO nanorod arrays

Synthesis was according to ref. 15, with the following steps: electrolyte solution containing 0.005 mol L⁻¹ zinc acetate and hexamethylenetetramine was prepared. A three-electrode system was assembled, consisting of a pretreated FTO substrate as the working electrode, a saturated calomel electrode (SCE) as the reference electrode, and a platinum sheet as the counter electrode. Electrodeposition was performed using an RST5000F electrochemical workstation via chronoamperometry (i–t curve method). The initial resting potential was set at −1.2 V for 10 s, followed by a constant potential of −1.0 V for 3000 s. After completion of the reaction, the sample was thoroughly rinsed and dried, yielding a uniformly distributed ZnO nanorod array.

Preparation of ZnO@Zn(L-tar)(4,4′-bipy) nanorod arrays

Synthesis was according to ref. 16 and 17: the prepared ZnO nanorod arrays were immersed in a reaction kettle containing 0.225 g (1.5 mM) of L-tartaric acid (L-tar) and 0.234 g (1.5 mM) of 4,4′-bipyridine (4,4′-bipy) in 16 mL of a methanol and N,N-dimethylformamide (DMF) mixed solvent (v/v = 1:1). The reaction was carried out at 120 °C for 48 h. After completion, the product was rinsed three times with ultrapure water and anhydrous ethanol and dried, and finally the FTO-supported ZnO@Zn(L-tar)(4,4′-bipy) nanorod arrays were obtained for the fabrication of the ECL chiral sensor.

Construction of the ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor

0.1 M phosphate buffer solutions were prepared with varying pH as electrolytes, using 0.05 M S₂O₈²⁻ as a co-reactant. D/L-Men was added to the electrolyte at a fixed molar concentration of 2.5 mM. A saturated Ag/AgCl electrode was employed as the reference electrode, a platinum sheet was used as the counter electrode, and a conductive glass coated with ZnO@Zn(L-tar)(4,4′-bipy) nanoarray was used as the working electrode. The ZnO@MOF/FTO ECL chiral sensor was constructed using an electrochemical workstation (scan rate: 50 mV s⁻¹) and an ultra-weak luminescence detector (sampling rate: 50 T s⁻¹) for the recognition of D/L-Men enantiomers. All experiments were conducted under dark conditions at 25 °C (Scheme 2).

Construction of the Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO dual-potential ECL chiral sensor

5 mg of the self-synthesized chiral complex Ru(phen)₂L-Asp was dissolved in a mixed solvent system containing 600 μL deionized water and 380 μL anhydrous ethanol, followed by addition of 20 μL Nafion to prepare a 1.000 mL homogeneous solution. The solution was ultrasonicated for 5 minutes to ensure uniform dispersion and then loaded onto the prepared ZnO@Zn(L-tar)(4,4′-bipy)/FTO to obtain Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO. Using an electrolyte containing 50 mM TPrA and 0.05 M S₂O₈²⁻ in a 0.1 M phosphate buffer solution, the fabricated Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO served as the working electrode to construct a potential-resolved dual-target ECL chiral sensor. This sensor achieved simultaneous recognition of Pen and Men enantiomers (Scheme 3).

Results and discussion

The morphological characterization

Fig. 1 shows the FE-SEM images of the precursor Ru(phen)₂Cl₂ and the target material Ru(phen)₂L-Asp. From Fig. 1a and b, it can be observed that Ru(phen)₂Cl₂ forms irregular and differently sized two-dimensional flakes. The formation of this morphology is attributed to the aromatic rigid structure of the ligand phenanthroline, which allows for the extension of zero-dimensional Ru(phen)₂Cl₂ complexes into two-dimensional layers through π–π interactions in the XY plane. These thin flakes stack on top of each other through layer-by-layer π–π interactions, resulting in a certain thickness in the Z-axis direction. Fig. 1c and d demonstrate that after replacing the Cl⁻ ions on Ru(phen)₂Cl₂ with the ligand L-Asp, the resulting Ru(phen)₂L-Asp exhibits irregular particle-like morphology, but the size is significantly smaller than that of sheet-like Ru(phen)₂Cl₂. This is also attributed to the structural rearrangement, where L-Asp restricts the simultaneous extension of Ru(phen)₂L-Asp in all three dimensions.

As shown in Fig. 2, the ZnO nanorod arrays exhibit smooth hexagonal prism morphology, with diameters ranging between 150 and 200 nm and lengths of approximately 600 nm, displaying a uniform and upright distribution (Fig. 2a and b). Fig. 2c and d show the morphology of the ZnO nanorod arrays after the solvothermal reaction with L-tartaric acid (L-tar) and 4,4′-bipyridine (4,4′-bipy) at 120 °C for 36 h. It can be observed that thin layers of material have formed on the originally smooth hexagonal prism surfaces, increasing the diameter by approximately 20 nm while maintaining a distinct hexagonal structure.

Fig. 2e and f present the morphology of the material obtained after extending the reaction time to 48 h under the same conditions. The surfaces of the nanorods become noticeably rough, and their diameters increase to 200–300 nm. Further prolonging the reaction to 60 h (Fig. 2g and h) leads to additional diameter expansion and a transformation of the hexagonal prism tips into irregular shapes. Due to excessive conversion of ZnO nanorods into MOF, the resulting composite exhibits diminished ECL performance. Therefore, in subsequent experiments, the optimal reaction time for material synthesis was determined to be 48 h.

Structural characterization

This study further characterizes the functional groups of the synthesized products using RT-IR spectra, as depicted in Fig. S1a.† The infrared spectrum of the precursor Ru(phen)₂Cl₂ exhibits distinctive peaks associated with phen (ν_CN = 1421 cm⁻¹, ν_CC = 1504 cm⁻¹, δ_CC–H = 852 cm⁻¹, 738 cm⁻¹). The infrared spectrum of the chiral complex Ru(phen)₂L-Asp, which is formed through substitution, displays characteristic peaks for both phen and L-Asp (ν_CO = 1690 cm⁻¹, ν_C–N = 1251 cm⁻¹). Notably, the carbonyl CO vibration frequency in Ru(phen)₂L-Asp, influenced by ring strain, shifts to a higher wavenumber of 1735 cm⁻¹, compared to the characteristic absorption peak of CO at 1690 cm⁻¹ observed for L-Asp.

Thermogravimetric analysis was employed to ascertain the stoichiometry of ligands within the complex Ru(phen)₂L-Asp. Fig. S1b† illustrates the thermogravimetric profile of the precursor Ru(phen)₂Cl₂, which displays a pronounced thermal step between 200 and 400 °C, accompanied by a weight loss of 13.2%. This loss aligns precisely with the theoretical weight percentage of Cl in the complex, which is 13.3%, suggesting that the observed weight loss at this temperature range is attributable to the detachment of the Cl⁻ ligand. For the complex Ru(phen)₂L-Asp, the thermogravimetric curve reveals a weight loss of 22.4% over the temperature range of 150 to 300 °C. This loss closely matches the theoretical weight percentage of L-Asp, which is also 22.4%, indicating the presence of a single L-Asp ligand within the complex.

Fig. S1c† depicts the fluorescence emission (FL) spectra of Ru(phen)₂L-Asp and its precursor Ru(phen)₂Cl₂; the concentration of the solution is 10⁻⁴ mol L⁻¹. Notably, the fluorescence spectra of the two materials diverge significantly. Ru(phen)₂Cl₂ achieves its peak emission at Ex = 593.4 nm, excited at a wavelength of 350 nm. In contrast, Ru(phen)₂L-Asp reaches its maximum emission at Ex = 589.8 nm, excited at 450 nm. Relative to Ru(phen)₂Cl₂, Ru(phen)₂L-Asp experiences a subtle blue shift in its maximum fluorescence emission towards shorter wavelengths, accompanied by a decrease in fluorescence intensity. This shift and the diminished absorption are attributed to the introduction of the auxiliary chromophore –COOH by the ligand.

Fig. S1d† illustrates the UV-vis absorption spectra of Ru(phen)₂L-Asp and its precursor Ru(phen)₂Cl₂, each at a concentration of 10⁻⁴ mol L⁻¹. The ethanolic solution of Ru(phen)₂Cl₂ reveals three broad and subdued absorption bands peaking at 445, 535, and 620 nm. These bands are ascribed to metal–ligand charge transfer (MLCT) transitions, specifically involving the electronic movement from dπ(Ru) → π*(phen), as well as metal-centered d–d electronic transitions from ²T_2g → ²T_1g. Ru(phen)₂L-Asp exhibits distinct UV absorption peaks at 410 and 500 nm, which are attributed to the n → π* transitions of the carbonyl group (CO) in L-Asp. These transitions result in the complex's absorption in the near-ultraviolet region, indicative of the influence of the L-Asp ligand on the electronic structure and absorption characteristics of the complex.

Fig. S2a† presents the XRD patterns of the synthesized ZnO@Zn(L-tar)(4,4′-bipy) nanocomposite compared with standard Zn(L-tar)(4,4′-bipy) (Fig. S2a† simulated, CCDC no: 906967)^16,17 and ZnO (Fig. S2a† ZnO). The experimental pattern of our nanoarray material (Fig. S2a† experimental) clearly exhibits characteristic diffraction peaks from both Zn(L-tar)(4,4′-bipy) and ZnO phases. This confirms the coexistence of the ZnO crystal structure and Zn(L-tar)(4,4′-bipy) crystalline framework in the composite, demonstrating the successful preparation of the ZnO@Zn(L-tar)(4,4′-bipy) nanocomposite.

A comparison of the FT-IR spectra of the ZnO@Zn(L-tar)(4,4′-bipy) nanoarray with those of L-tar and 4,4′-bipy (Fig. S2b†) reveals that the characteristic IR peaks of the carboxyl O–H stretching vibrations (ν = 3406 cm⁻¹, 3336 cm⁻¹, 1743 cm⁻¹) and in-plane bending vibrations (δ = 1190 cm⁻¹, 1255 cm⁻¹) of L-tar and the amine N–H stretching vibration (ν = 3028 cm⁻¹) and in-plane bending vibration (δ = 1531 cm⁻¹) of 4,4′-bipy, disappear in the ZnO@Zn(L-tar)(4,4′-bipy) nanoarray. This confirms the successful coordination of the ligands L-tar and 4,4′-bipy.

As shown in the UV-vis absorption spectra of the ZnO@Zn(L-tar)(4,4′-bipy) nanoarray and ZnO nanorod array (Fig. S2c†), the ZnO nanorod array exhibits a UV absorption peak at 370 nm, whereas the UV absorption peak of ZnO@Zn(L-tar)(4,4′-bipy) is significantly enhanced, with a strong absorption at 240 nm. The absorption peak exhibits a blue shift of 140 nm to lower wavelengths, which is attributed to the π → π* transition in the closed-ring conjugated double bonds of 4,4′-bipy. The extended conjugation system induces the blue shift and the high absorption intensity leads to a pronounced enhancement in UV absorption.

Fig. S2d† compares the FL spectra of the ZnO@Zn(L-tar)(4,4′-bipy) nanoarray and the ZnO nanorod array. At the maximum excitation wavelength (Ex = 350 nm), ZnO shows a fluorescence emission peak at 400 nm. In contrast, ZnO@Zn(L-tar)(4,4′-bipy), when excited at Ex = 330 nm, exhibits a strong fluorescence emission peak at 370 nm with increased emission intensity.

To substantiate the ligand stoichiometry within the synthesized materials, this study performed ¹H NMR analysis on the ligand phen, the precursor Ru(phen)₂Cl₂, and the target complex Ru(phen)₂L-Asp, with the findings depicted in Fig. 3. The ligand phen displays four distinct sets of peaks (A, B, C, and D) at chemical shifts δ 9.11, 8.50, 8.00, and 7.78, each comprising two protons, accounting for a total of eight protons. The precursor Ru(phen)₂Cl₂, which incorporates phen as the ligand, exhibits corresponding peaks at δ 9.23, 8.82, 8.66, 8.15, and 8.06, aligning with the protons on phen. In the synthesized Ru(phen)₂L-Asp, the chemical shift range δ 9.05–7.00 corresponds to the protons of phen, with a peak for the methylene group of L-Asp at δ 4.10 and methine group of L-Asp at δ 2.93. The integrated ratio of the proton peak areas is 16:1:2, which suggests that each Ru(phen)₂Cl₂ molecule is coordinated with a single L-Asp ligand.

Electrochemical performance testing of Ru(phen)₂L

To assess the electrochemical performance of the Ru(phen)₂L-Asp/GCE ECL sensor, this study employed CV and electrochemical impedance EIS to evaluate both the precursor and the synthesized chiral material-based sensors. These tests were conducted in a mixed solution containing 5 mM Fe(CN)₆^3+/4+ and 0.1 M KCl, with an initial frequency set at 10⁵ Hz, a final frequency at 0.1 Hz, and an amplitude of 0.01 V. The outcomes are presented in Fig. S3.†

As depicted in Fig. S3a,† the CV plot of Ru(phen)₂Cl₂/GCE reveals a pair of well-defined redox peaks. When compared to a bare glassy carbon electrode, there is a notable increase in redox peak currents, and the voltage difference between the peaks is significantly reduced, signifying the efficient electron transfer capability of Ru(phen)₂Cl₂/GCE. Upon substitution of the Cl⁻ ligands in Ru(phen)₂Cl₂ with the chiral ligand L-Asp to form chiral Ru(phen)₂L-Asp/GCE, the redox peak currents diminish and the voltage difference between the peaks decreases, suggesting that the coordination with the chiral organic ligand impedes electron transfer, thereby reducing conductivity.

Fig. S3b† provides additional evidence to support these findings. The impedance radius of the bare glassy carbon electrode is recorded at 2150 ohm, whereas that of Ru(phen)₂Cl₂/GCE is considerably lower at 450 ohm. This indicates that electron transfer within Ru(phen)₂Cl₂/GCE is far less hindered compared to that in the bare glassy carbon electrode, implying that Ru(phen)₂Cl₂ possesses excellent electrical conductivity. Following the replacement of Cl⁻ with the chiral organic ligand L-Asp to yield Ru(phen)₂L-Asp, the impedance radius increases to 1300 ohm and 1450 ohm, respectively. This increase in impedance suggests that the conductivity of Ru(phen)₂L-Asp is inferior to that of Ru(phen)₂Cl₂.

As shown in Fig. S4a,† the bare ZnO nanorod array exhibits the highest redox peaks, indicating that among the three materials, this semiconductor ZnO nanorod array possesses the strongest electron transport capability. After coating the ZnO nanorod array with the Zn(L-tar)(4,4′-bipy) MOF, the redox peak current decreases. This is attributed to the presence of poorly conductive organic ligands in the MOF, which reduce the overall conductivity of the composite compared to the semiconductor ZnO, leading to diminished electron transport and a lower peak current. However, the peak current remains significantly higher than that of the bare electrode, confirming that the composite still retains appreciable conductivity.

In Fig. S4b,† the EIS results show that the ZnO nanorod array has a semicircle radius of approximately 650 Ω. Upon coating with the Zn(L-tar)(4,4′-bipy) MOF, the impedance radius increases to 1050 Ω, further indicating reduced electron transfer efficiency in the composite. Nevertheless, this value remains lower than that of the bare electrode (2200 Ω), demonstrating that the composite retains higher conductivity than the bare electrode. These findings are consistent with the CV results.

Feasibility analysis of chiral recognition using the Ru(phen)₂L-Asp/GCE ECL sensor

To explore the enantiomeric discrimination ability of the synthesized chiral Ru(phen)₂L-Asp/GCE ECL sensor, this study first evaluated its ECL response toward a series of enantiomers, including D/L-Pen, D/L-Phe, D/L-His, D/L-Asp, D/L-Tyr, D/L-Met, D/L-Men, and D/L-Trp. The measurements were conducted in a phosphate buffer solution (pH = 6.98) containing each chiral isomer. The results are presented in Fig. 5. The Ru(phen)₂L-Asp/GCE sensor exhibited a significant ECL signal ratio I_D-/I_L- of 2.33 for D/L-Pen, while the ratios for the other chiral molecules ranged between 0.72 and 1.14, indicating superior chiral recognition efficiency toward D/L-Pen enantiomers.

Feasibility analysis of chiral recognition using the ZnO@Zn(L-tar)(4,4′-bipy) ECL sensor

Fig. 6a and b present the ECL response profiles of the constructed ZnO@Zn(L-tar)(4,4′-bipy)/FTO chiral ECL sensor toward different chiral compounds. Notably, the sensor exhibits distinct ECL signal variations in phosphate buffer solution (pH = 6.98) containing different chiral compounds. The most pronounced discrimination effect is observed for D/L-Men enantiomers, with an ECL intensity ratio I_D-/L- of 1.6, demonstrating excellent chiral recognition capability. In contrast, the sensor shows moderate responses to other chiral compounds (D/L-Phe, D/L-Trp, D/L-Met, D/L-His, D/L-Pen, D/L-Tyr, and D/L-Asp), with ECL intensity ratios ranging between 0.8 and 1.2.

Fig. 6c presents the calibration curves for quantitative analysis of D/L-Men using the ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor, while Fig. 6d displays the corresponding ECL measurement profiles. To investigate the concentration-dependent ECL response, the sensor demonstrated excellent linear relationships for both D- and L-Men enantiomers within the concentration range of 1–5 mM. The linear regression equations were determined as: ECL_D-Men = 2696 + 370C_D-Men; ECL_L-Men = 1971 + 152C_L-Men, with correlation coefficients (R²) exceeding 0.99 for both enantiomers. The detection limits were calculated to be 3.12 × 10⁻⁵ M and 6.68 × 10⁻⁵ M for D-Men and L-Men, respectively.

These experimental results confirm that the ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor not only exhibits excellent discrimination capability for D-/L-Men enantiomers but also serves as an effective platform for quantitative enantiomeric analysis.

ECL luminescence mechanism

Anodic process: Ru(bpy)₃²⁺ loses an electron to form Ru(bpy)₃³⁺. This typically occurs at an electrode surface at applied potential (eqn (1)). TPrA is oxidized by losing an electron, forming a radical cation TPrA·⁺. This species rapidly loses a proton (deprotonation) to generate a neutral radical TPrA^· (eqn (2)). The TPrA^· radical donates an electron to another Ru(bpy)₃²⁺ molecule, reducing it to Ru(bpy)₃⁺ while regenerating the oxidized TPrA^·+ radical cation (eqn (3)). Ru(bpy)₃³⁺ and Ru(bpy)₃⁺ species undergo an electron transfer reaction. This produces an excited-state Ru(bpy)₃²⁺* and regenerates the ground-state Ru(bpy)₃²⁺ (eqn (4)). Ru(bpy)₃²⁺* relaxes to its ground state, releasing energy as visible light (hv). This is the electrochemiluminescence signal used in analytical applications (eqn (5)). The reaction sequence is summarized as follows:^18,19

Ru(bpy)32+ → Ru(bpy)33+ + e−	(1)

TprA-e → TprA˙+ → TprA˙ + H+	(2)

Ru(bpy)32+ + TprA˙ → Ru(bpy)3+ + TprA˙+	(3)

Ru(bpy)33+ + Ru(bpy)3+ → Ru(bpy)32+* + Ru(bpy)32+	(4)

Ru(bpy)32+* → Ru(bpy)32+ + hν	(5)

Cathodic process: S₂O₈²⁻ are electrochemically reduced to form SO₄˙⁻ (eqn (6)), a critical oxidizing intermediate. Simultaneously, ZnO undergoes one-electron reduction to generate its radical anion ZnO˙⁻ (eqn (7)). These electrogenerated intermediates (SO₄˙⁻ and ZnO˙⁻) undergo redox annihilation, resulting in the formation of an excited state ZnO* accompanied by sulfate ion regeneration (eqn (8)). Radiative decay of ZnO* to its ground state produces emission (hν), which is quantitatively detected as the ECL signal (eqn (9)). The reaction cascade is summarized as follows:^18–20

S2O82− + e− → SO42− + SO4˙−	(6)

ZnO + e− → ZnO˙−	(7)

ZnO˙− + SO4˙− → ZnO* + SO42−	(8)

ZnO* → ZnO + hν	(9)

Optimization of experimental conditions and evaluation of sensor stability and reproducibility

To investigate the effect of experimental conditions on the sensor's recognition capability, ECL measurements for D/L-Men enantiomers were conducted in electrolytes with varying pH values (5.91, 6.34, 6.98, 7.38, and 8.04). As shown in Fig. 7a, the optimal chiral discrimination performance was achieved in phosphate buffer solution (pH = 6.98), yielding the highest intensity ratio (I_D-/L- = 1.61). Consequently, all subsequent experiments were performed under this optimized condition.

Fig. 7b demonstrates the time-dependent ECL response of the fabricated sensor towards D/L-Men enantiomers. The sensor maintained excellent chiral recognition capability for one week, as evidenced by relative standard deviations (RSDs) of 0.017 and 0.013 for D-Men and L-Men detection respectively, confirming the outstanding reproducibility of the ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor. To evaluate the operational stability of the ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor for D-/L-Men discrimination, continuous cyclic scanning was performed for 10 cycles using an electrochemical workstation. The corresponding ECL intensity profiles are presented in Fig. 7c and d, showing RSD values of 0.0255 and 0.0249 for D-Men and L-Men respectively. These results demonstrate the remarkable stability of the developed chiral sensing platform.

Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor for simultaneous discrimination of Pen and Men enantiomers

Using a phosphate buffer solution (0.1 M) with a pH of 6.98 containing enantiomers of D/L-Pen and D/L-Men at the same concentration (0.5–2.5 mM) as the electrolyte, an ECL test was carried out using the constructed Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor. The quantitative analysis results are shown in Fig. 8a. When the Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor simultaneously recognizes the enantiomers of D/L-Pen and D/L-Men, there is a linear relationship between the concentrations of D-Pen and D-Men and the values of the ECL signal response. The linear regression equations are I_D-Pen = 3973.4 + 252.6C_D-Pen and I_D-Men = 2580.7 + 213.9C_D-Men, and the ECL signal response diagram is shown in Fig. 8b. There is a linear relationship between the concentrations of L-Pen and L-Men and the values of the ECL signal response. The linear regression equations are I_L-Pen = 1546.4 + 200.0C_L-Pen and I_L-Men = 1403.6 + 198.4C_L-Men, and the ECL signal response diagram is shown in Fig. 8c. Moreover, the correlation indices are all above 0.99, indicating that this chiral sensor can simultaneously recognize the enantiomers of D/L-Pen and D/L-Men, and it can perform quantitative analysis of the enantiomers of D/L-Pen and D/L-Men within the linear range.

Comparison of ECL sensor performance in amino acid enantiomer detection

As shown in Table 1,we present a comprehensive performance comparison between our chiral sensor and previously reported systems. In this work, we successfully developed a ZnO@Zn(L-tar)(4,4′-bipy)/FTO-based chiral sensor that demonstrates remarkable performance advantages for D/L-Men enantiomer detection. Notably, compared with previous studies,^18–21 our approach exhibits superior enantiomeric recognition capability for amino acids, establishing an innovative platform for rapid and precise enantiomer discrimination and trace-level analysis of amino acids.

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Selective testing and practical application

To evaluate the practical efficacy of the established chiral detection sensor, Ru(phen)₂L-Asp/ZnO@Zn(L-tar)(4,4′-bipy)/FTO was employed for ECL detection of D/L-Men mixtures with a total concentration maintained at 3 mM and D/L-Phe mixtures with a total concentration of 2.5 mM. The experimental results demonstrated a linear correlation between the ECL signals and the percentage of D-Men and D-Phe in their respective mixtures. The corresponding linear regression equations were determined to be y = 2440.2 + 15.9483x (Fig. S5a†) and y = 5553.7 + 56.4058x (Fig. S5b†), respectively.

To evaluate the practical applicability of the sensor, predetermined amounts of D-Men and L-Phe were dissolved in authentic human urine samples for detection. As summarized in Table S1,† the recovery rates for D-Men and L-Phe ranged from 93.6% to 97.3% and 91.4% to 96.7%, respectively, demonstrating excellent practicality and reliability for the stereoselective detection of Men and Phe enantiomers.

Conclusions

This study constructed a ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor. CV and EIS analyses demonstrated that this material exhibits weaker electron transfer capability compared to ZnO nanorods, yet it displays satisfactory qualitative and quantitative ECL analysis performance for chiral D/L-Men enantiomers, with a good linear relationship observed in the concentration range of 1–5 mM. The developed ZnO@Zn(L-tar)(4,4′-bipy)/FTO ECL chiral sensor shows excellent stability and high reproducibility, providing a novel research direction for cathodic potential-based ECL recognition of Men enantiomers.

Based on the experimental results, the ZnO@Zn(4,4′-bipy)(L-tar)/FTO ECL chiral sensor effectively recognizes Men at −1.7 V, while the Ru(phen)₂L-Asp ECL chiral sensor successfully identifies Pen at +1.3 V. This inspired the design of a dual-color ECL chiral recognition strategy employing different luminophores. By immobilizing Ru(phen)₂L-Asp on the ZnO@Zn(4,4′-bipy)(L-tar)/FTO substrate, we constructed an integrated Ru(phen)₂L-Asp/ZnO@Zn(4,4′-bipy)(L-tar)/FTO ECL chiral sensor, achieving simultaneous detection of Pen and Men enantiomers.

Data availability

All relevant data are within the manuscript and its additional files.

Author contributions

R. Kuang: performed research, analyzed the data, and wrote the manuscript. B. Yang: analyzed the data and revised the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This work was supported by the Natural Science Foundation of Shandong Province, China (ZR2020QB065).

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Footnote

† Electronic supplementary information (ESI) available: Apparatus, electronic spectra, IR spectra, thermogravimetric analysis, X-ray diffraction spectrum, cyclic voltammograms, and the influence of the pH value. See DOI: https://doi.org/10.1039/d5ay00528k

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