Conjugated polymer dots/oxalate anodic electrochemiluminescence system and its application for detecting melamine

Abstract

In this work, the anodic electrochemiluminescence (ECL) behavior of water-soluble poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), is firstly studied with Na₂C₂O₄ as a coreactant using cyclic voltammetry and ECL measurements. The possible anodic ECL mechanism of PFO–C₂O₄²⁻ system is proposed. Based on the fact that the melamine could efficiently quench the ECL signal of the PFO–C₂O₄²⁻ system, a new ECL sensing method for melamine was developed with a wide linear range from 9.0 × 10⁻¹¹ to 1.1 × 10⁻⁸ M. Furthermore, the sensor exhibited high sensitivity and good stability. Due to the excellent ECL behavior, the PFO–C₂O₄²⁻ system could be a promising platform for constructing ECL sensors.

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1. Introduction

The conjugated polymer poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO), an organic dye, has attracted a lot of attention because of its superior chemical stability and high electroluminescent (EL) quantum yields with blue light emission.^1–3 As early as 2001, Prieto and co-workers have investigated the ECL behavior of PFO in a benzene–MeCN solution.⁴ But so far there are still few reports concerning the application of PFO in the ECL field due to the poor water-solubility of PFO and the employment of poisonous organic solvents.⁴ Thus, it is urgent to develop a nontoxic aqueous solution system to further study the ECL behaviors of PFO, thereby expanding its potential application in the field of analytical chemistry.

Melamine (1,3,5-triazine-2,4,6-triamine), a component of plastic resin, is widely used in the chemical industry.⁵ Owing to containing a high nitrogen content (66% by mass), it has been added to foods and feeds illegally to distort the apparent protein content.⁶ Therefore, sensitive detection of melamine becomes increasingly important in the field of agriculture and food. Among various analysis methods for melamine, including surface enhanced Raman spectroscopy (SERS),⁷ fluorescence,⁸ electrochemical methods,⁹ liquid chromatography/mass spectrometry (LC-MS),¹⁰ enzyme-linked immunosorbent assay (ELISA),¹¹ and colorimetry,^12,13 electrochemiluminescence (ECL) is a better alternative since it not only could overcome obstacles such as being time-consuming, low sensitivity and complex manipulations, but also exhibit some outstanding features such as low background signal, good selectivity and high sensitivity.^14–17 However, the determination of melamine using the ECL method is in the initial stage, and only a few studies were reported. Recently, Guo et al. and Liu et al. have constructed an ECL method for melamine detection based on Ru(bpy)₃²⁺ system.^18,19 Due to the eximious ECL performance of Ru(bpy)₃²⁺, the sensors exhibited a low detection limit for melamine. However, in these studies, the strong base test condition and high-cost of luminophore would limit the application of the sensors in the determination of melamine in actual sample. Meanwhile, Hu et al. reported a sensor for the detection of melamine based on the fact that the melamine can inhibit the ECL response of CdTe quantum dots.²⁰ In this study, although the determination of melamine can be achieved in neutral test condition, the toxicity of CdTe quantum dots would limit the application of the sensor. Thus, it is still urgent and important to develop a low-cost, nontoxic, gentle and highly efficient ECL system to detect melamine.

Inspired by above observation, we prepared the water-solubility PFO dots, and investigated the anodic ECL behavior of PFO in gentle aqueous solution with Na₂C₂O₄ as a coreactant. It is found that the ECL signal of water-solubility PFO dots is relatively weak, whereas a much stronger ECL signal is measured with Na₂C₂O₄ as a coreactant. The ECL mechanism of PFO was investigated using cyclic voltammetry (CV) and ECL measurements, and the diagram is shown in Scheme 1. Meanwhile, the application of PFO in ECL field was discussed. Based on the fact that melamine could quench the ECL intensity of PFO–C₂O₄²⁻ system, the detection of melamine at the PFO dots modified electrode was achieved. Due to the excellent ECL performance of PFO–C₂O₄²⁻ system, the prepared sensor exhibited a high sensitivity and good selectivity for the detection of melamine. This strategy offers a promising luminophore for the construction of ECL sensing platforms based on PFO dots.

Scheme 1 Diagram of the ECL mechanism of PFO.

2. Experimental

2.1 Reagents and chemical

Melamine (2,4,6-triamino-1,3,5-trazine, 99%), tetrahydrofuran (THF) and sodium oxalate (Na₂C₂O₄) were purchased from Aladdin Ltd (Shanghai, China). Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO, MW 20 000) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Phosphate-buffered saline (PBS) solutions were prepared using 0.10 M KH₂PO₄ and 0.10 M Na₂HPO₄, and 0.10 M KCl was employed as a supporting electrolyte. Doubly distilled water was used throughout this work.

2.2 Apparatus

The ECL measurements were performed by a model MPI-A electrochemiluminescence analyzer (Xi'an Remax Electronic Science &Technology Co. Ltd, Xi'an, China). The voltage of photomultiplier tube (PMT) is set at 800 V and the potential scans from 0 to 2.0 V for this work. The Electrochemical signal was tested using a CHI660D electrochemical workstation (CH Instruments Co., China). Scanning electron micrograph was detected by a scanning electron microscope (SEM, Hitachi, Japan).

2.3 Synthesis of PFO dots

2 mg of poly(9,9-dioctylfluorenyl-2,7-diyl) was dissolved in 1 mL of THF by stirring overnight at room temperature. By filtering to remove any insoluble material, the solution was injected into 8 mL water. Then, the THF was removed through partial vacuum evaporation. After a small fraction of aggregates were removed through centrifugating at 6000 rpm for 5 min, the resultant solution was dried in air to obtain the PFO dots.¹

2.4 Preparation of the sensor

Firstly, a glassy carbon electrode (GCE, Φ = 4.0 mm) was polished with 0.3 μm and 0.05 μm alumina slurry and ultrasonically cleaned in ethanol and water, respectively. Then, 20 μL PFO dots solution was cast onto the surface of as-prepared GCE and dried in air to achieve a sensor (PFO/GCE).

2.5 Experimental measurements

The cyclic voltammetry and ECL measurements were performed at the PFO/GCE in 3.0 mL 0.10 M PBS containing 50 mM Na₂C₂O₄. In this work, we found that melamine could quench the ECL intensity of PFO–C₂O₄²⁻ system. The measurement was based on the change of ECL signal (ΔI = I₀ − I_t), here, I_t and I₀ are the ECL signals with and without melamine, respectively.

3. Results and discussion

3.1 Characterization of the PFO dots and the choice of coreactant

The surface topographies of the PFO dots were investigated by SEM. As seen in Fig. S1,† the uniform spherical structures of PFO dots with light border and dark center were observed and their sizes were about 30–60 nm.¹

It is important to select suitable coreactants for ECL systems, since coreactants could be transferred to reduced (or oxidized) species and help the emission of ECL light in the single direction potential sweep.²¹ In order to select the most suitable coreactant, four typical coreactants used in anodic ECL including H₂O₂, oxalate (C₂O₄²⁻), SO₃²⁻ and tripropylamine (TPA) were investigated in this study.^22,23 For every coreactant, we investigated the effect of the concentration on the ECL signal in 3.0 mL 0.10 M PBS solution (pH 6.5). The concentration in which the ECL signal arrived the maximum was chosen as the optimal concentration. As shown in Fig. 1, under the optimal concentration, the ECL activity of C₂O₄²⁻ is higher than those of SO₃²⁻, H₂O₂ and TPA. Thus, the C₂O₄²⁻ was chosen as the coreactant of PFO in this study. Although the ECL intensity of the PFO–H₂O₂ system is weaker than that of the PFO–C₂O₄²⁻ system, it also may have some potential applications in novel ECL biosensors since the PFO–H₂O₂ system is enough sensitive and H₂O₂ is a well known product in various enzymatic reactions.

Fig. 1 The ECL intensity change of PFO with the concentration of Na₂SO₃ (A), H₂O₂ (B), TPA (C), Na₂C₂O₄ (D).

3.2 ECL and electrochemical behavior of PFO/GCE

In order to study the ECL property of PFO, the PFO/GCE was measured in 0.10 M PBS without and with 50 mM Na₂C₂O₄, respectively. As seen from Fig. 2A curve a, PFO/GCE exhibited an obvious ECL response at the potential of approximately 1.95 V in absence of C₂O₄²⁻, which was due to the annihilation reaction of PFO, indicating that the PFO was a luminophore. Compared with curve a, a much higher ECL intensity is measured in the presence of Na₂C₂O₄ (curve b). This phenomenon conformed that the Na₂C₂O₄ could dramatically enhance the ECL signal of PFO, indicating that Na₂C₂O₄ plays an important role in the process of ECL. Fig. 2B displays the cyclic voltammetry curves of the PFO/GCE in 0.10 M PBS (pH 6.5) without (curve a) and with Na₂C₂O₄ (curve b). As seen from curve a, the oxidation current increases slightly when the scan potential is positive than ca. 1.00 V (vs. Ag/AgCl), which is owing to the annihilation reaction of PFO. Meanwhile, an oxidation peak at 1.64 V is observed in curve b, which is owing to the “oxidation–reduction” reaction of Na₂C₂O₄. The fact that the oxalate could act as the coreactant in ECL system has been reported in previous studies. For example, Rubinstein et al. used the oxalate as the coreactant for Ru(bpy)₃³⁺. In this system, the intermediate radical CO₂˙⁻, a strong reducing agent, can be generated from the oxidation of oxalate, and react with Ru(bpy)₃³⁺ to produce the excited state Ru(bpy)₃²⁺*. Then, light was emitted.²⁴ The oxalate was also used as the coreactant in quantum dots–C₂O₄²⁻ system. The CO₂˙⁻ was first generated from the oxidation of oxalate. Subsequently, CO₂˙⁻ would react with an ECL luminophore to produce an excited state by electron transfer and then generate light.^22,23 Meanwhile, the ECL mechanism of PFO has also been investigated by Bard's group. They have reported that the PFO can be oxidated to PFO˙⁺, and produce the ECL response owing to the annihilation reaction of PFO.⁴ Based on above reports, it is reasonable to expect that the C₂O₄²⁻ can act as coreactant in PFO ECL system. The possible ECL mechanisms are as follows. The C₂O₄²⁻ was oxidated to generate CO₂˙⁻. Meanwhile, PFO was oxidated to PFO˙⁺, which can react with CO₂˙⁻ to generate a excited state PFO* and light was emitted.

PFO − e → PFO˙⁺ (1)

C₂O₄²⁻ − e → C₂O₄˙⁻ (2)

C₂O₄˙⁻ → CO₂ + CO₂˙⁻ (3)

PFO˙⁺ + CO₂˙⁻ → PFO* + CO₂ (4)

PFO* → PFO + hv (5)

Fig. 2 (A) ECL behaviors of PFO/GCE without (a) and with 50 mM Na₂C₂O₄ (b). Inset: the enlarged picture of curve a; (B) CV curves of PFO/GCE without (a) and with 50 mM Na₂C₂O₄ (b).

3.3 Optimization of experimental conditions

The ECL responses of PFO/GCE were significantly affected by the concentration of coreactant C₂O₄²⁻ and the pH. The change in ECL intensity (ΔI) was detected at PFO/GCE in 0.10 M PBS with varied pH from 5.5 to 8.0. As seen from Fig. 3A, in the presence of 50.0 mM Na₂C₂O₄ and 3.5 nM melamine, the ΔI can be obviously influenced by the pH of PBS and achieve the maximum at pH 6.5. So, the optimal pH of 6.5 was chosen in the further study. Possible mechanism of the effect of pH on the ECL response of this system may be explained as following. For oxalic acid, pK_a1 and pK_a2 are 1.23 4.19, respectively, thus the predominant forms in acidic solution are H₂C₂O₄ and HC₂O₄⁻.²⁴ On the one hand, when the pH was higher than pK_a2, the predominant form in solution would be C₂O₄²⁻. With the increase of pH, the concentration of C₂O₄²⁻ in solution would increase, thereby, leading to an increase in the concentration of CO₂˙⁻. On the other hand, the potential at which oxalate oxidation occurs depends upon the pH and becomes less positive with an increase in pH.²⁴ Obviously, too high pH is unfavorable to the ECL response of PFO–C₂O₄²⁻ system. pH 6.5 was the optimal pH in our experiments. Fig. 3B displays the effect of the concentration of Na₂C₂O₄ on the ΔI in pH 6.5 PBS with 3.5 nM melamine. As displayed, when the concentration of Na₂C₂O₄ achieved 50 mM, the ΔI achieved the maximum value. Thus, 50 mM was chosen as the optimal concentration of Na₂C₂O₄ in the further study.

Fig. 3 Effects of (A) the pH of PBS and (B) the concentration of Na₂C₂O₄ on the ECL signal at the PFO/GCE.

3.4 ECL detection of melamine

Fig. 4A exhibits the ECL response of PFO/GCE towards various concentration of melamine. As seen, under optimum conditions, the ECL responses decreased with increasing concentration of melamine. The ECL intensity depicted linearity with the logarithm of the concentration of melamine in the range from 9.0 × 10⁻¹¹ to 1.1 × 10⁻⁸ M. The linear equation was ΔI = 2051 lg c + 20 698 (R = 0.991), and the detection limit (signal to noise = 3) was 2.7 × 10⁻¹¹ M. The quenching mechanism may be an energy-transfer process. First, the C₂O₄²⁻ was oxidated to generate CO₂˙⁻, which can react with PFO to generate an excited state PFO*. When melamine was added into solution, the active neutral free radical intermediate was produced through the oxidation of melamine at the positive potential.¹⁸ Then, The energy transfer was occurred owing to the collision between the active neutral free radical intermediate and CO₂˙⁻. The more melamine was added into solution, the more energy-transfer process was generated, resulting in a decrease of the quantity of CO₂˙⁻. Thereby, the ECL intensity of PFO–C₂O₄²⁻ system decreased.

Fig. 4 (A) ECL response of PFO/GCE towards different concentration melamine: 0.00 (a), 0.09 (b), 0.25 (c), 1.05 (d), 2.05 (e), 3.05 (f), 5.05 (g), 8.05 (h), and 11.05 (i) nM. (B) Calibration curve for melamine.

The analysis performances were compared between this method and other studies and the results are exhibited in Table S1.† As seen, in this study, the linear range achieved three orders of magnitude. Meanwhile, the detection limit of this work was achieved 10⁻¹¹ M. The results are better or comparable than those of previous studies, which is owing to the excellent performance of PFO. This work offers a gentle, simple and effective method for melamine analysis based on PFO–C₂O₄²⁻ system.

3.5 Stability and interference determination of the sensor

PFO/GCE was applied to detect melamine (2 nM) by ECL. It is found that the relative standard deviation (RSD) of 1.10% was obtained for 9 successive detections (Fig. 5). The long-term stability of the prepared sensor was measured in 0.10 M PBS with 50 mM Na₂C₂O₄ and 2 nM melamine. It is found that about 94.2% of the initial ECL intensity was noticed after ten days, and about 87.9% of the original ECL response was retained after twenty days, showing an acceptable stability.

Fig. 5 ECL emissions in 0.10 M PBS (pH 6.5) containing 50 mM Na₂C₂O₄ and 2 nM melamine under continuous cyclic scans between 0.0 and 2.0 V for 9 cycles.

The selectivity of the PFO/GCE was also measured using 300 nM Mg²⁺, Cl⁻, Na⁺, SO₄²⁻, ascorbic acid, uric acid, fructose, glucose, lactic acid, L-lysine, L-phenylalanine, L-isoleucine and 10 ng mL⁻¹ immunoglobulin as interfering substances. The results showed that above interfering substances do not cause obvious interference to the determination of 3 nM melamine.

3.6 Analytical application of the sensor in real samples

The PFO/GCE was used to detect melamine in commercial milk samples. It is found that no melamine was detected in chosen milk samples. Recovery experiments were also performed in milk samples using standard addition method and the results are exhibited in Table S2.† The recoveries ranged between 96.7% and 103%. Above fact indicates that PFO/GCE exhibits promising applications to detect melamine in milk samples.

4. Conclusion

The anodic ECL behavior of water-solubility PFO with Na₂C₂O₄ as coreactant was studied in this work. Then, a new ECL system for melamine sensing was developed based on the fact that the melamine could efficiently quench the ECL response of PFO–C₂O₄²⁻ system. Meanwhile, the PFO/GCE has been successfully used to determine melamine in commercial milk samples. Above facts confirmed that the PFO dots not only could develop a promising ECL sensing platform, but also open a new ECL system for the determination of melamine.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (21075100, 21275119), Ministry of Education of China (Project 708073), Research Fund for the Doctoral Program of Higher Education (RFDP) (20110182120010), Natural Science Foundation of Chongqing City (CSTC-2011BA7003, CSTC-2014JCYJA20005, CSTC-2010BB4121), Science and Technology Commission of Beibei (2012-27), Medical Scientific Research Projects of Health Bureau of Chongqing (2012-2-286), and the Fundamental Research Funds for the Central Universities (XDJK2012A004, XDJK2013C115), Specialized research fund for the doctoral program of higher education (swu113029) China.

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Footnote

† Electronic supplementary information (ESI) available: Fig. S1 SEM images of PFO dots. Table S1 comparison of this study with other sensor for the detection of melamine. See DOI: 10.1039/c5ra10809h

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