An electrochemiluminescence biosensor for dopamine based on the recognition of fullerene-derivative and the quenching of cuprous oxide nanocrystals

Abstract

In this work, L-cysteine (L-Cys) and 3-aminophenylboronic acid (APBA) consecutively reacts with fullerene (C₆₀) to obtain a new fullerene-derivative (L-Cys–C₆₀–APBA) with better biocompatibility, conductivity and hydrophilicity. L-Cys-functionalized C₆₀ can act as an excellent co-reactant to the electrochemiluminescence (ECL) of peroxydisulfate–oxygen (S₂O₈²⁻–O₂) system and greatly amplify the luminescence signal. APBA-functionalized C₆₀ has specific recognition to dopamine (DA) with diol structure. Therefore, a sensitive sensing interface for DA can be constructed based on the obtained L-Cys–C₆₀–APBA and the ECL of S₂O₈²⁻–O₂ system. Furthermore, cuprous oxide (Cu₂O) nanocrystals, prepared with a modified reductive solution chemistry route, can promote the oxygen reduction reaction commendably. Thus, Cu₂O modified on the electrode has a strong quenching effect to the ECL of S₂O₈²⁻–O₂ system. And the quenching effect is positively correlated with concentrations of DA, by which DA can be selectively and sensitively detected. The linear range is from 0.01 μM to 40 μM with a relatively low detection limit of 0.003 μM (S/N = 3).

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1. Introduction

Dopamine (DA), as one of the most important neurotransmitters in the mammalian central nervous system, plays a very important role in regulating a variety of neuronal functions such as behavior, learning, attention, emotion, cognition and memory.^1,2 In addition, it is also regarded as a prognostic biomarker for several kinds of diseases, for instance Parkinson's disease, epilepsy and schizophrenia.^3,4 Therefore, the determination of DA has a great significance for biomedical diagnosis and treatment. Due to its excellent electro-activity, DA can be easily oxidized on the electrode, based on which a lot of electro-analysis methods are widely reported.^5,6 Nevertheless, some interference such as ascorbic acid (AA) and uric acid (UA), which coexisted in the biological samples and possessed similar oxidation potential, often greatly affected the electrochemical determination of DA. Thus, the fabrication of the specific, selective and sensitive methods for DA detection is imperative. It has been proved that boronic acid and its derivatives (such as 3-aminophenylboronic acid (APBA)) have specific affinity to diols.⁷ Therefore, based on this specific recognition, we consider constructing a sensitive interface for the specific and selective determination of DA.

Electrochemiluminescence (ECL), generated from an electrochemical reaction between electro-generated species, is a technique resulting from the combination of electrochemistry and chemiluminescence.^8–11 In recent decades, it has been widely applied for sensitive bioassays and biosensors owe to its apparent advantages, for instance simple instrumentation, low background, high detection sensitivity and excellent controllability.^12,13 Therefore, ECL is selected for the DA analysis. Up to now, several classic ECL reagents, such as luminol,¹⁴ quantum dots,¹⁵ tris (2,2′-bipyridyl) ruthenium(II) and their analogues,¹⁶ have been extensively studied. At the same time, some new substances with ECL properties are also gradually proposed. Peroxydisulfate–oxygen (S₂O₈²⁻–O₂) is just a newly discovered inorganic ECL system. Due to its potential advantages in simplicity, sensitivity and cheapness, the ECL system of S₂O₈²⁻–O₂ has been applied in much analytical detection since it was discovered.^17–19 For this system, the concentration of oxygen is a very important factor to the final signal. Based on this point, a series of analytical methods with “signal-on” or “signal-off” model can be established by adjusting the concentration of oxygen through some way.

For improving the detected sensitivity, some co-reactant and nanomaterials are applied to amplify the ECL signal. Fullerene (C₆₀) with a hollow spherical structure is an important member in the family of carbon-materials. Since being discovered, it has received much attention due to its highly symmetrical structure, unique chemical and physical properties.^20,21 It has been reported that C₆₀ treated with didodecyldimethyl ammonium bromide could greatly enhance the ECL signal of S₂O₈²⁻–O₂.²² However, the relatively poor water solubility of C₆₀ limits its further application in ECL. Owing to the existence of the rich conjugated π electrons, some chemicals with amino-group could be effectively decorated onto C₆₀ with good water solubility and some functional groups on the surface.²³ For example, L-cysteine (L-Cys), which is an α-amino acid with amplification effect to the ECL of S₂O₈²⁻–O₂ system, can react with C₆₀ to obtain L-Cys functionalized C₆₀ (L-Cys–C₆₀) which not only possesses better water solubility, but also acts as an more effective co-reactant to the ECL of S₂O₈²⁻–O₂.

Besides the signal increased ECL tape, quenching-type ECL is also an important mode which has less reported before, especially for the S₂O₈²⁻–O₂ ECL system. Recent decades, semi-conductor materials have attracted widely attention because of their excellent electronic and optical properties.^24,25 The applications of them cover many areas, such as electronics, optics, catalysis, chemical and biological sensing.²⁶ Cuprous oxide (Cu₂O), a common used semi-conductor material with a high optical absorption coefficient, has excellent potential in the applications of high-efficiency photocatalysis,²⁷ low-cost photovoltaics,²⁸ sensitive Raman spectroscopy,²⁹ and even high performance electrode materials.³⁰ Recently, it has been proved that Cu₂O nanoparticles dispersed on reduced graphene oxide could efficiently catalyze the reduction reaction of oxygen.³¹ Inspired by this, we try to synthesize Cu₂O nanocrystals with poly(ethylene glycol) (PEG) as capping agent and induct the Cu₂O nanocrystals into the ECL system of S₂O₈²⁻–O₂ to construct a quenching-type biosensor.

In this work, L-Cys–C₆₀–APBA, as a novel fullerene-derivative, was synthesized based on the reaction of amino group and the rich π electrons. The obtained L-Cys–C₆₀–APBA could not only act as co-reactant to the ECL system of S₂O₈²⁻–O₂, but also serve as a sensitive recognition element for DA. Moreover, Cu₂O nanocrystals were prepared with a chemical reduction method, which could promote the reduction of oxygen. Thus, Cu₂O labeled DA could greatly quench the ECL reaction of S₂O₈²⁻–O₂ system. Based on those factors mentioned above and AuNPs with good electrocatalytic ability and biocompatibility, a sensitive and selective biosensor for DA detection was constructed.

2. Experimental section

2.1. Reagents and apparatus

Gold chloride (HAuCl₄) was purchased from Kangda Amino Acid company (shanghai, china). Fullerene (C₆₀), 3-aminopropyltrimethoxysilane (AMPTS), glutaraldehyde (GA), L-cysteine (L-Cys) and 3-aminophenylboronic acid (APBA) were purchased from J&K Scientific Ltd (Beijing, China). K₂S₂O₈ was purchased from shanghai chemical Reagent company (Shanghai, China). Chloratum culture-medium (CuCl₂), sodium hydroxide (NaOH) and poly(ethylene glycol) (PEG) were obtained from Sigma Chemical (St. Louis, MO, USA). Ascorbic acid (AA) and dopamine (DA) were obtained From Chemical Reagent Co. (Chongqing, China). The serum specimens were obtained from local Hospital. Phosphate-buffered solution (PBS) (pH 6.5, 0.1 M) was prepared with 0.1 M Na₂HPO₄, 0.1 M KH₂PO₄ and 0.1 M KCl. Double distilled water was used throughout this study.

The ECL emission was monitored with a model MPI-A electroluminescence analyzer (Xi'an Remax Electronic science & Technology Co., Ltd, Xi'an, china) with the voltage of the photomultiplier tube (PTM) set at 800 V and the potential scan from 0 to −2.0 V in the process of detection. The experiment was performed with a conventional three-electrode system, in which the modified glassy carbon electrode (GCE) was the working electrode, a platinum wire was the counter electrode and an Ag/AgCl (sat. KCl) was the reference electrode. The morphologies of different nanocomposites were characterized by scanning electron microscopy (SEM, S-4800, Hitachi, Japan).

2.2. Synthesis of L-Cys and APBA functionalized C₆₀ (L-Cys–C₆₀–APBA)

The preparation of L-Cys–C₆₀–APBA is showed Scheme 1(A). Firstly, 1 g APBA, 2.0 g L-Cys and 1.7 g NaOH were dissolved in 5 mL distilled water, and then 40 mL ethanol was added. Subsequently, C₆₀ toluene solution (3 mg mL⁻¹) was added into the mixture solution mentioned above with stirring. After 5 days, the solution was filtered, centrifuged, and washed respectively with ethanol and distilled water for several times. The dark brown product of L-Cys–C₆₀–APBA was dried in vacuum for 24 h at 50 °C, and dispersed in distilled water when use.

Scheme 1 (A) The preparation of L-Cys–C₆₀–APBA, and (B) the preparation and reaction mechanism of ECL biosensor.

2.3. Synthesis of Cu₂O nanocrystals

Cu₂O nanocrystals were prepared according to the literature with some minor modifications.³² 0.5 g PEG was first dissolved in CuCl₂ aqueous solution (0.1 mM, 10 mL). Once PEG was completely dissolved, 0.06 g NaOH was added. Then, the solution immediately changed to blue color, indicating the formation of Cu(OH)₂. After 10 min, the AA solution (1 M, 0.2 mL) was dropwise added to the solution with the color slowly changed to orange. The Cu₂O nanocrystals were collected by centrifugation, washing and drying.

2.4. Fabrication of the ECL biosensor

Before modification, the glassy carbon electrode (GCE) (Φ = 4 mm) was polished with 0.3, 0.05 μm alumina slurry respectively, followed by rinsing thoroughly with bi-distilled water and sonicating in ethanol, bi-distilled water separately. As shown in Scheme 1(B), the GCE was firstly immersed in the HAuCl₄ solution (1%) and electrodeposited at −0.2 V for 30 s to get a gold nanoparticles (AuNPs) layer. Then, 15 μL L-Cys–C₆₀–APBA solution was dropped onto the electrode, which could be modified on the electrode through the Au–S bond. After washing, 15 μL hexanethiol (96%, HT) was used to block the remaining active groups. Subsequently, DA solutions with varying concentrations were added and incubated for 3 h. Finally, with glutaraldehyde (GA) as crosslinking reagent, amino group functionalized Cu₂O nanocrystals were modified onto the electrode surface. The Cu₂O nanocrystals had a strong quenching effect to the ECL of S₂O₈²⁻–O₂ system. And the ECL quenching effect became stronger with increasing the concentration of DA, based on which the DA could be sensitively detected.

3. Results and discussion

3.1. The characterization of L-Cys–C₆₀–APBA and Cu₂O nanocrystals

As shown in Fig. 1, the morphologies of different nanocomposites were monitored using scanning electron microscopy (SEM). C₆₀ was a hollow carbon sphere with a diameter of 20 ± 3 nm (Fig. 1A). L-Cys–C₆₀–APBA, had hollow sphere-like shape with bright border since the conductivity and hydrophilicity of C₆₀ were greatly enhanced after the modification of L-Cys and APBA (Fig. 1B). Meanwhile, the Cu₂O nanocrystals possessed a sphere shape with a concave-convex surface. According to Fig. 1C, the diameter of Cu₂O was about 50 ± 5 nm.

Fig. 1 SEM images of C₆₀ (A), L-Cys–C₆₀–APBA (B) and Cu₂O nanocrystals (C).

3.2. ECL characterization of the biosensor fabrication

The fabrication process of the ECL biosensor was stepwise characterized by ECL in 0.1 M peroxydisulfate solution. As shown in Fig. 2, the bare GCE in peroxydisulfate solution produced a low ECL (curve a). The ECL intensity was enhanced when AuNPs were electrodeposited onto the electrode (curve b), because AuNPs played an important role similar to a conducting wire and made it easier for the electron transfer. Furthermore, the ECL signal was greatly enhanced when L-Cys–C₆₀–APBA, a novel co-reactant to the ECL of S₂O₈²⁻–O₂ system, was modified onto the electrode (curve c). However, when non-electroactive HT was used to block nonspecific sites, the ECL response decreased (curve d). Then, the ECL signal further decreased obviously after the modification of Cu₂O through the cross linking with DA since Cu₂O could effectively quench the ECL reaction of S₂O₈²⁻–O₂ system (curve e).

Fig. 2 ECL profiles of (a) bare GCE, (b) GCE/AuNPs, (c) GCE/AuNPs/L-Cys–C₆₀–APBA, (d) GCE/AuNPs/L-Cys–C₆₀–APBA/HT, (e) GCE/AuNPs/L-Cys–C₆₀–APBA/HT/DA–Cu₂O (1 μM DA) in PBS (0.1 M, pH 6.5).

3.3. The possible luminescence mechanisms

The possible mechanisms to the ECL of S₂O₈²⁻–O₂ were described as follows:³³

S₂O₈²⁻ + e → SO₄˙⁻ + SO₄²⁻ (1)

SO₄˙⁻ + H₂O → HO˙ + HSO₄⁻ (2)

HO˙ + H₂O → HOO˙ + H₂ (3)

O₂ + H₂O + e → HOO˙ + HO⁻ (4)

SO₄˙⁻ + HOO˙ → HSO₄⁻ + ¹(O₂)^*₂ (5)

¹(O₂)^*₂ → 2³O₂ + hv (6)

It had been proved that the sole C₆₀ or L-Cys was an effective co-reactant to the ECL of S₂O₈²⁻–O₂.¹⁸ Therefore, L-Cys–C₆₀–APBA, synthesized in this study, not only served as a recognized element for DA but also acted as a more excellent co-reactant to the ECL of S₂O₈²⁻–O₂. The co-reactive mode was described as follows (eqn (7)–(9)): L-Cys–C₆₀–APBA losted electrons to form the strong oxidant intermediate (L-Cys–C₆₀˙⁺–APBA) which further reacted with HOO˙ generated in the change process of eqn (3) and (4) above to form ¹(O₂)^*₂. By this way, the ECL emission was enhanced with more ¹(O₂)^*₂ reacted back to ³O₂.

L-Cys–C₆₀–APBA − e → L-Cys–C₆₀˙⁺–APBA (7)

L-Cys–C₆₀˙⁺–APBA → L-Cys–C₆₀˙−APBA + H⁺ (8)

L-Cys–C₆₀˙–APBA + HOO˙ → ¹(O₂)^*₂ (9)

However, Cu₂O nanocrystals could effectively promote the reduction reaction of oxygen, by which the ECL signal of S₂O₈²⁻–O₂ was quenched.

Cu₂O + O₂ + H⁺ → Cu²⁺ + H₂O (10)

3.4. The detection of the ECL biosensor to DA

In order to evaluate its performance, the ECL biosensor was used to detect DA with different concentrations. As shown in Fig. 3, the ECL intensity decreased with increasing concentration of DA (curve a–g). Fig. 3 (insert) showed the calibration curve of proposed biosensor, where the ECL intensity was linear with the logarithm of DA concentrations. The linear equation was I = 8363.3–2304.7 log c (where I was the ECL intensity and c was the concentration of DA), and the correlation coefficient was 0.9981. The linear range for DA of the standard calibration curve shown in the inset figure was from 0.01 μM to 40 μM with a detection limit of 0.003 μM (S/N = 3). Table 1 shows the detection results of the biosensor with previous reports.^34–36 Compared to other methods, the biosensor have a relative low detection limit, which might hold a new promise for highly sensitive bioassays applied in clinical detection.

Fig. 3 ECL calibration curve of the immunosensor for CEA determination; the concentrations of DA: 0.01 μM (a), 0.1 μM (b), 0.5 μM (c), 1 μM (d), 10 μM (e), 20 μM (f), 40 μM (g).

Table 1 Comparison of our research with other methods for DA detection

Method	Linear range (μM)	Detection limit (μM)	References
Electrochemical sensor	0.05–10	0.033	34
Electrochemical sensor	0.99–103.1	0.26	35
Quartz glass capillaries	0.02–5.6	0.01	36
ECL	0.01–40	0.003	Present work

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3.5. Selectivity, reproducibility and stability of the biosensor

Several interfering agents were used to assess the selectivity of the proposed biosensor, such as uric acid (UA, 10 μM), ascorbic acid (AA, 10 μM), tyrosine (Tyr, 10 μM) and their mixture with DA (1 μM). According to Fig. 4, no remarkable changes were observed compared the ECL responses of each sole interfering agent to that of blank sample. And the ECL signal of the mixture was similar with that of the pure DA. The experimental results above indicated that the biosensor displayed good selectivity for the determination of DA. Relative standard deviations (ECL response) of intra- and inter-assays were used to evaluate the reproducibility. The relative standard deviations (R.S.D.) both of the intra- and inter-assay were not more than 5%, which suggested that the reproducibility of the proposed immunoassay was acceptable. Simultaneously, the stability of the ECL biosensor was evaluated under consecutive cyclic potential scans for 13 cycles. As shown in Fig. 5, the ECL intensity did not show obvious changes, which illustrated the good stability of the proposed biosensor.

Fig. 4 Comparison of ECL responses with different interfering agents: blank; DA (1 μM); AA (10 μM); UA (10 μM); Tyr (10 μM); a mixture containing DA (1 μM), AA (10 μM), UA (10 μM) and Tyr (10 μM).

Fig. 5 The ECL stability of proposed immunosensor under consecutive cyclic potential scans.

3.6. Preliminary analysis of real samples

The applicability of the proposed ECL biosensor was monitored by recovery experiments which were performed by standard addition methods in human serum. As shown in Table 2, the recovery (between 94.0% and 106.9%) was acceptable, which provided a promising tool for clinical determination of DA.

Table 2 Determination of DA added in normal human serum with the proposed biosensor

Sample number	Added/μM	Found/μM	Recovery/%
1	0.1	0.0940	94.0
2	0.5	0.492	98.4
3	1	0.982	98.2
4	10	10.69	106.9

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4. Conclusion

In conclusion, a quenching-type ECL biosensor based on S₂O₈²⁻–O₂ system was constructed for the detection of DA. L-Cys–C₆₀–APBA, as a novel fullerene-derivative, was synthesized to serve as a new co-reactant for signal amplification and a sensitive recognition element for DA. Meanwhile, Cu₂O nanocrystals were prepared with a modified chemical reduction method, which could promote the reduction of oxygen. Thus, Cu₂O labeled DA could effectively quench the ECL reaction of S₂O₈²⁻–O₂ system. The proposed biosensor exhibited high sensitivity and stability, good reproducibility and repeatability.

Acknowledgements

This work was financially supported by the NNSF of China (51473136, 21275119), Fundamental Research Funds for the Central Universities (XDJK2014A012), and the Chongqing Postdoctoral Research Project (xm2014022).

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