Electrochemiluminescence sensor based on cationic polythiophene derivative and NH₂–graphene for dopamine detection

Abstract

In this study, a novel electrochemiluminescence (ECL) sensor was fabricated based on cationic polythiophene derivative poly[3-(1,1′-dimethyl-4-piperidinemethylene)thiophene-2,5-diyl chloride] (PTh-D) and NH₂–graphene (NH₂–G) for detection of dopamine (DA). PTh-D, which has advantages of high electrochemical stability, easy excitation, good water-solubility and good conductivity, and was used as luminescent material in an ECL assay. In the presence of NH₂–G, the ECL signal of PTh-D was significantly improved due to its efficient electron transfer. To obtain a stable ECL signal, Nafion was dropped on the surface of the modified electrode. Under the optimum experimental conditions, the ECL signal linearly decreased with the increase of DA concentration in the range of 0.1–50.0 μM with a detection limit of 0.04 μM. This simple prepared ECL sensor exhibited high sensitivity, selectivity, good reproducibility and long-term stability. The applicability of the proposed ECL sensor was also evaluated by detecting DA in real samples. This proposed method not only expands the application of PTh-D, but also opens new doors toward the detection of DA.

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Introduction

Conductive polymers with a large π conjugation system, such as polythiophene (PTh), polypyrrole and polyaniline, have attracted great interest in recent years.^1–4 These polymers exhibit special properties, such as tailoring of electronic properties, controllable electrical conductivity, nanostructured shapes (films, nanoparticles and nanowires), low cost and high yield synthesis.⁵ As one kind of new cationic polythiophene derivative, poly[3-(1,1′-dimethyl-4-piperidinemethylene)thiophene-2,5-diyl chloride] (PTh-D) has attracted considerable attention recently due to its high stability, easy modification and controllable electrochemical behavior.⁶ In the present work, we report the fabrication of an electrogenerated chemiluminescence (ECL) sensor based on (PTh-D) and NH₂–graphene.

Graphene consists of a monolayer of sp²-hybridized carbon atoms and single-atomic layer thickness arranged in a two-dimensional lattice.⁷ It has been applied in the areas including electronics, sensors and composite reinforcement capacitors due to its excellent electronic transport, outstanding optical properties, good fracture strength and high aspect ratio.⁸ Inspired by this thought, functionalized graphene was modified on the electrode surface in this work to increase the ECL intensity of PTh-D. However, the solubility of the chemically reduced graphene oxide sheets in water and most organic solvents is limited.⁹ To further improve the water solubility of graphene, NH₂–graphene (NH₂–G) with good solubility and excellent electronic properties was synthesized.

As one of the most important catecholamine neurotransmitters, dopamine (DA) plays a significant role in brain mood regulation and motivation circuits.¹⁰ DA is implicated in pathologies such as Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder (ADHD) and addiction.¹¹ Obviously, the determination of DA plays an important role in the field of physiological function research and clinical disease diagnosis. Various methods have been explored for the determination of DA, such as high performance liquid chromatography (HPLC),¹² fluorescence,¹³ electrochemistry,¹⁴ photoelectrochemistry¹⁵ and ECL.¹⁶ Compared with these methods, ECL has remarkable features such as simplicity, rapidity, high sensitivity and easy controllability.¹⁷

ECL is probably produced by chemiluminescence (CL) directly or indirectly.¹⁸ Generally speaking, the ECL reaction procedure includes two steps. Firstly, reactive species are formed from the electrochemical reaction. Secondly, electrogenerated substances are diffused from the electrode surface and reacted with each other or co-existing ones in the solution to produce light near by the electrode.¹⁹ So ECL is also a commendable model for investigating the mechanism of electron transfer.²⁰ As a powerful analytical technique, ECL has been extensively used for different analytical purposes such as squamous cell carcinoma antigen,²¹ glucose,²² cell tumor²³ and so on. ECL sensors have also been used broadly for sensitive detection of various analytes.²⁴

In this work, a novel ECL sensor based on PTh-D and NH₂–G for sensitive detection of DA was constructed. PTh-D was used as the luminescent material with the coreactant of K₂S₂O₈. NH₂–G was used to increase the ECL intensity because of its good performance in increasing the electron transfer efficiency. As a cation exchange polymer, Nafion was used for improving stability of the sensor due to its good electrical conductivity, high chemical stability and good biocompatibility.²⁵ The ECL sensor showed linear response to DA in the concentration range from 0.1 to 50.0 μM with a detection limit of 0.04 μM. The easily fabricated sensor exhibited sensitive ECL responses to DA even in the presence of a high concentration of ascorbic acid, uric acid and glucose. The proposed sensing strategy may open new era towards the fabrication of ECL sensor for DA detection.

Experimental

Materials

DA, ascorbic acid and uric acid were purchased from Acros Organics Co., Ltd. (Beijing, China). Chitosan and thiophene were obtained from Sigma-Aldrich (Beijing, China). Nafion (5%) was purchased from Alfa Aesar. graphite powder (SP). Glacial acetic acid (HAc), hydrochloric acid (HCl) and ammonia water (NH₃·H₂O) were obtained from Sinopharm Chemical Reagent Co., Ltd. Chloroform, methyl alcohol, ethyl alcohol, diethyl ether, ethylene glycol, hydrogen peroxide (H₂O₂, 30%), anhydrous ferric chloride (FeCl₃), anhydrous trichloromethane (CHCl₃), potassium permanganate (KMnO₄), potassium nitrate (KNO₃), and potassium persulfate (K₂S₂O₈) and potassium ferricyanide (K₃[Fe(CN)₆]) were purchased from Fuyu Chemical Co., Ltd (Tianjin, China). All other chemicals were of analytical reagent grade and were used without further purification. 0.1 M phosphate buffered saline (PBS) was used as an electrolyte for all the electrochemistry measurements. Ultrapure water (18.25 MΩ cm, 24 °C) was used for all the experiments. Chitosan was dissolved in 1% HAc. PTh-D and NH₂–G (5 mg) were respectively dissolved in 500 μL 0.5% chitosan.

Apparatus

The ECL measurements were performed with a MPI-F flow-injection chemiluminescence detector (Xi'an Remax Electronic Science Tech. Co. Ltd., China) and electrochemical measurements were carried out on CHI760D electrochemical workstation (Chenhua Instrument Shanghai Co., Ltd, China) using a three-electrode system consisted of a platinum wire as an auxiliary electrode, an Ag/AgCl electrode as reference electrode, and glassy carbon electrode (GCE, 4 mm in diameter) as working electrode.

Preparation of PTh-D

PTh-D was prepared according to the literature.²⁶ Typically, 1.02 g FeCl₃ was dispersed in 20 mL chloroform and stirred for 30 min under nitrogen, and then a solution of 3-(1,1′-dimethyl-4-piperidine methylene) thiophene methyl sulfate (0.50 g) in 20 mL chloroform was added dropwise to the above solution. The mixture was stirred at room temperature for 24 h. After evaporation of the reaction mixture to dryness, the crude product was washed quickly with 100 mL methanol and filtered. The filter cake and 1 mL hydrazine were mixed in 50 mL methanol and stirred overnight. The solution was filtered and evaporated to dryness. The resulting polymer was washed five times with a saturated solution of tetrabutylammonium chloride in acetone. After Soxhlet extraction with ethanol to remove impurity and oligomer, the resulting PTh-D was dispersed in 0.5% chitosan and stored at 4 °C until use.

Preparation of NH₂–G

The GO were synthesized according to the improved method reported by Daniela C. Marcano.²⁷ In this method, the mixture at a molar ratio of H₂SO₄ : H₃PO₄ = 9 : 1 (36 : 4 mL) was added to the mixture of graphite powder (0.3 g) and KMnO₄ (1.8 g). The reaction mixture was heated to 50 °C with constant stirring for 12 h. Then the reaction was cooled to room temperature and poured onto ice (40 mL) with 30% H₂O₂ (0.3 mL). The product was collected by centrifugation (9000 rpm for 10 min). The remaining precipitate was washed two times with HCl (0.2 M), ethyl alcohol and diethyl ether, respectively. Then the product was dried in vacuum at 35 °C.

The prepared GO (200 mg) was added to ethylene glycol (80 mL) under ultrasonication for 30 min, followed by the addition of NH₃·H₂O (2 mL) under constant stirring for several minutes. Then, the dark brown solution was transferred into autoclave for solvothermal reaction at 180 °C for 10 h. The obtained NH₂–G precipitation was washed three times with distilled water, and dried in vacuum at 50 °C.²⁸

Fabrication of the ECL sensor

Fig. 1 showed the fabrication procedures of the ECL sensor. Firstly, GCE was polished to a mirror-like finish with 0.05, 0.3 and 1.0 μm alumina powder and then thoroughly cleaned with ultrapure water and dried naturally in air. PTh-D (3 μL) was dropped on the surface of a well-polished GCE and dried at room temperature. Following that, NH₂–G (5 μL) was sequentially dropped on the surface of GCE and dried, then Nafion (0.5%, 3 μL) was dropped on the modified electrode surface and dried at room temperature. The prepared electrode was stored at room temperature until use.

Fig. 1 Schematic diagram for fabrication of the ECL sensor.

ECL detection of DA

10 mL of pH 8.0 PBS with different concentrations of DA, 0.1 M KCl and 30 mM K₂S₂O₈ were added to the ECL cell. The scanning potential was from −1.6 to 0 V, and the photomultiplier tube (PMT) was set at 800 V, scan rate: 0.1 V s⁻¹. Then the modified electrode was placed in the ECL cell and the ECL signal was measured.

Results and discussion

ECL behavior

The ECL intensity–potential curves were obtained by the electrodes modified with different products (Fig. 2). Obviously, bare GCE has weak ECL emission (curve a). However, after PTh-D was modified onto the GCE, the electrode has a good ECL emission peak (curve b). When NH₂–G was assembled on GCE/PTh-D, ECL response further increased (curve c), indicating that excellent conductivity of NH₂–G facilitated the ECL reaction. When DA was added into substrate solution, ECL response obviously decreased (curve d). Accordingly, the ECL sensor could be used for detection of DA.

Fig. 2 ECL intensity profiles of the bare GCE (curve a), GCE/PTh-D (curve b), and GCE/PTh-D@NH₂–G (curve c); GCE/PTh-D@NH₂–G/Nafion with DA (curve d).

The possible ECL mechanism was described as follows:²⁹ when the electrode was scanned from −1.6 to 0 V, the PTh-D was reduced to PTh-D˙⁻ (eqn (1)). Meanwhile, when the potential was negative enough, S₂O₈²⁻ was reduced to SO₄˙⁻ and SO₄²⁻ (eqn (2)). The strong oxidant SO₄˙⁻ further reacted with PTh-D˙⁻ to generate the excited state PTh-D* (eqn (3)) through electron transfer in the aqueous solution. When PTh-D* fell from the excited state to the ground state (eqn (4)), light was emitted and detected.

PTh-D + e⁻ → PTh-D˙⁻ (1)

S₂O₈²⁻ + e⁻ → SO₄²⁻ + SO₄˙⁻ (2)

PTh-D˙⁻ + SO₄˙⁻ → PTh-D* + SO₄²⁻ (3)

PTh-D* → PTh-D + hν (4)

Optimization of experimental conditions

The concentration of NH₂–G is an important factor that would affect the performance of the ECL sensor. As shown in Fig. 3A, when concentrations of NH₂–G changed from 1.0 to 2.0 mg mL⁻¹, the ECL intensity increased gradually and reached the maximum at 2.0 mg mL⁻¹. Then, with further increasing concentrations of NH₂–G from 2.0 to 8.0 mg mL⁻¹, the ECL intensity decreased. This might be the reason that excessive NH₂–G may be stacked each other.³⁰ Therefore, 2 mg mL⁻¹ was chosen as the best concentration of NH₂–G for subsequent experiments. Fig. 3B shows the effect of coreactant K₂S₂O₈ concentration in substrate solution on ECL emission. When the concentration of K₂S₂O₈ was 30 mM, the sensor performed the best ECL response. To achieve an optimal ECL signal, pH value of substrate solution was also optimized (Fig. 3C). When the pH value of substrate solution was 8.0, the ECL sensor displayed the optimal signal intensity. Consequently, pH 8.0 was selected as the optimal acidity condition and used throughout this study. Finally, the effect of scan rate on the ECL intensity was investigated and displayed in Fig. 3D. The ECL intensity increases steadily with the increase in scan rate (0.02–0.10 V s⁻¹) and then decreases at scan rates of 0.10–0.30 V s⁻¹. These results are consistent with other ECL sensors.³¹ The ECL efficiency is governed by the formation rate of the excited-state species as well as the diffusion rate of the coreactant.³² At high scan rates, the consumption of coreactant on the electrode interface would be much faster than the diffusion of the coreactant from the bulk solution to the electrode surface, leading to a low transient concentration of coreactant near the surface of the electrode with a concomitant decrease in ECL. In summary, too high of a scan rate would decrease the ECL. Thus, 0.10 V s⁻¹ was selected as the optimal scan rate in this work.

Fig. 3 The effect of NH₂–G (A), K₂S₂O₈ (B), pH (C) and scan rate (D) on the response of ECL intensity from GCE/PTh-D@NH₂–G (error bar = RSD, n = 3).

ECL quenching by DA and ECL detection of DA

The possible ECL enhancement mechanism may be as follows:³³ SO₄˙⁻ radical, which is produced by the electroreduction of S₂O₈²⁻, is a strong oxidant. The dissolved oxygen is reduced to OOH˙. The OOH˙ could react with SO₄˙⁻ to product light-emitting species (O2)^*₂, then O₂ is produced with light emitted. However, the possible ECL quenching mechanism by dopamine was described as follows:³⁴ the quenching reaction may be a static quenching, dopamine was oxidized to form a compound that was not fluorescence substance in alkaline solution. Therefore, the following reaction was suggested to explain the reason of the fluorescence quenching.

ECL sensor that based on GCE/PTh-D@NH₂–G/Nafion was used to detect different concentrations of DA under the optimal conditions (Fig. 4A). And the equation of the calibration curve is I = 4674.385 − 644.327 ln[c] (R = 0.964). The ECL intensity decreased linearly with the logarithm of DA concentration within the range from 0.1 μM to 50.0 μM with the limit of detection (LOD) was 0.04 μM (signal to noise = 3). The lower LOD might be attributed that the conductivity of PTh-D was largely improved by adding NH₂–G. A comparison of the performance of the proposed and referenced sensors for DA was provided. The analytical parameters including detection limit and linear range are better or comparable to the results reported for determination of DA, as displayed in Table 1.^17,35–38 Although the electrochemistry sensor which was developed by Jin et al. had lower detection limit than our work, the ECL sensor we proposed was more easy-to-use and faster than others.³⁶ Meanwhile, this proposed method not only expands the application of PTh-D, but also opens new doors toward the detection of DA.

Fig. 4 (A) ECL response of the ECL sensor to different concentrations of DA, from (a) to (g): 0.1, 0.5, 1, 5, 10, 30, 50 μM. (B) Calibration curve of the ECL sensor for DA at different concentrations. (C) ECL intensity of the GCE/PTh-D@NH₂–G/Nafion in PBS (pH = 8.0) containing 0.1 M KCl and 0.5 μM DA under continuous scanning for 15 cycles; (D) ECL intensity of the ECL sensor to 0 μM DA (1), 20 μM DA (2), 20 μM DA + 200 μM ascorbic acid (3), 20 μM DA + 200 μM uric acid (4), 20 μM DA + 200 μM glucose (5), 20 μM ascorbic acid (6), 20 μM uric acid (7), 20 μM glucose (8) (error bar = RSD, n = 3).

Table 1 A comparison of the performance of the proposed and referenced sensors for DA

The material of sensors	Method	linear range (μM)	detection limit (μM)	References
Mn3O4 nanoparticles	DPV	10–70	0.1	35
MWNTs–PEI–AuNPs	DPV	0.05–4.0	0.0066	36
SWCNT/Fe2O3	SWV	3.2–31.8	0.36	37
CdSe quantum dots	ECL	0.5–70	0.5	38
rGO/MWCNTs/AuNPs	ECL	0.20–70	0.067	17
This work	ECL	0.10–50.00	0.04	—

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Stability, selectivity and reproducibility of the ECL sensor

Operational stability was one of the major concerns for practical application of ECL sensors. Fig. 4C displayed the ECL emission of the PTh-D@NH₂–G modified GCE under 15 cycles of continuous potential scans from −1.6 to 0 V in PBS (pH = 8.0) containing 0.1 M KCl and 30 mM K₂S₂O₈ at 0.1 V s⁻¹. Strong and stable ECL signals were observed with the relative standard deviation (RSD) of 2.91%. The results indicated that the stability of the proposed ECL sensor was excellent.

In order to monitor the selectivity of the ECL sensor, the interferences of ascorbic acid, uric acid, and glucose were investigated for the determination of DA. It could be found that ascorbic acid, uric acid and glucose at a concentration of 10 times higher than that of DA did not cause an obvious interference with the determination of DA (20 μM). The RSD of the measurements was below 5.0% (Fig. 4D), indicating the selectivity of the ECL sensor was acceptable.

The reproducibility of the ECL sensor was also investigated by prepared seven electrodes for detection 0.5 μM of DA. RSD of measurements was under 5.0%. The results showed that the ECL sensor had satisfied reproducibility.

Real sample analysis

The feasibility of the ECL sensor for practical application was investigated by detecting the concentration of DA in human urine samples. As shown in Table 2, different concentration (5.00, 10.0 and 15.0 μM) of standard DA solution was added to human urine samples by standard addition methods, the relative standard deviation (RSD) and recoveries were in the range of 2.25–3.58% and 92.6–100.7%, respectively. The results showed that the developed sensor might be preliminarily applied for the determination of DA in real samples.

Table 2 The recovery of the proposed ECL sensor in urine samples

Urine samples (μM)	The addition content (μM)	The detection content (μM)	Average value (μM)	RSD (%)	Recovery (%)
1.56	5.00	5.96, 6.18, 6.32, 6.27, 6.23	6.19	2.25	92.6
	10.0	11.39, 11.28, 12.15, 11.73, 11.58	11.63	2.93	100.7
	15.0	16.92, 17.16, 15.84, 15.92, 16.58	16.48	3.58	99.5

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Conclusion

In this paper, a new ECL sensor was easily constructed based on PTh-D. NH₂–G was used to increase the electron transfer efficiency and so as to improve the ECL signal. The proposed ECL sensor showed high sensitivity, long-term stability, wide linear range, good reproducibility, acceptable selectivity, precision and accuracy. Therefore, this proposed method not only expands the application of PTh-D, but also may provide an attractive way in other targets determination.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21175057, 21375047 and 21377046), the Science and Technology Plan Project of Jinan (no. 201307010) and QW thanks the Science and Technology Development Plan of Shandong Province (no. 2014GSF120004), the Special Foundation for Taishan Scholar Professorship of Shandong Province and University of Jinan (no. ts20130937).

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