Sensitive electrochemiluminescence sensor based on ordered mesoporous carbon composite film for dopamine

Sensors and Actuators B 195 (2014) 22–27

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Sensitive electrochemiluminescence sensor based on ordered mesoporous carbon composite film for dopamine Beina Wu, Chongchong Miao, Lili Yu, Ziyi Wang, Chusen Huang, Nengqin Jia ∗ The Education Ministry Key Laboratory of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials, Department of Chemistry, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, PR China

a r t i c l e

i n f o

Article history: Received 27 September 2013 Received in revised form 20 December 2013 Accepted 3 January 2014 Available online 10 January 2014 Keywords: Electrochemiluminescence Biosensors Dopamine Ordered mesoporous carbon Medicine analysis

a b s t r a c t This work reports a sensitive electrochemiluminescence (ECL) detection strategy based on Ru(bpy)3 2+ /ordered mesoporous carbon/Nafion composite films modified glassy carbon electrode (GCE). Electrochemical and ECL behaviors of the prepared ECL sensors were studied with tri-n-propylamine (TPA) as coreactant. The proposed ECL sensor showed good reproducibility and high sensitivity to TPA with a wide linear range (4.75 × 10−9 to 6.25 × 10−4 M) and low detection limit (1.58 × 10−9 M). Under the optimized conditions, the ECL sensor with TPA as coreactant was employed to detect a neurotransmitter dopamine (DA), for estimating its practical application in the medicine analysis. The present sensor displayed a good response to the concentration of DA from 5.0 × 10−9 M to 5.0× 10−4 M. Moreover, the ECL sensor could be successfully applied to detect DA in real samples. The proposed signal amplification strategy for the preparation of ECL sensor could be easily attained and hold great promise for ultrasensitive medicine analysis. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Electrochemiluminescence (ECL), the production of light emission from electrochemically generated luminophore [1], is a noteworthy versatile and sensitive analytical method [2]. Due to the inherent advantages of electrochemistry and chemiluminescence, ECL has emerged in various research fields such as chemical sensing [3,4], imaging [5] and optical studies [6]. It also has a widespread application in environmental, clinical and medicine analysis [7]. Among various ECL compounds, Ru(bpy)3 2+ with the superior properties including good electrochemical stability, high sensitivity and efficiency [8] has received great attention. So far, extensive efforts have been made to create a solid-state ECL sensor [9] due to its low consumption, simple operation, reproducibility and stability [10,11]. And the approaches for immobilization of Ru(bpy)3 2+ onto electrode surfaces include Langmuir–Blodgett film (LB) [12], self-assembly monolayer (SAM) [13] and sol–gel technology [14]. Although a great progress of the ECL sensor has been made, those methods still cannot be applied widely as they suffer from instability at positively biased potentials and possible desorption in organic solvent [15]. To circumvent these disadvantages, ECL sensors based on various nano-materials which act as favorable electric conductors and excellent matrixes for immobilizing Ru(bpy)3 2+ are being developed [16,17].

∗ Corresponding author. Tel.: +86 21 64321045; fax: +86 21 64321833. E-mail address: [email protected] (N. Jia). 0925-4005/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2014.01.012

As one of novel carbon materials, ordered mesoporous carbons (OMC) have attracted increasing attention in many fields including adsorption, catalysis, capacitor, sensors, energy storage and etc [18,19], which greatly attribute to their excellent conductibility, high specific surface areas, large pore volume and remarkable ␲-conjunction [20–22]. Furthermore, when used as a platform for immobilizing organic and inorganic molecules onto electrode surfaces, OMC can effectively promote electron-transfer reactions of important molecules. This outstanding property promotes the increasing application of OMC-based sensors in the electrochemistry analysis [23–25]. Therefore, OMC will be a promising alternative candidate for ECL sensor material. In this work, the OMC@Nafion-based composite film was used to immobilize Ru(bpy)3 2+ onto a glass carbon electrode (GCE) surface. Due to the excellent ion-exchange ability of Nafion [26] and strong adsorption of OMC [27], Ru(bpy)3 2+ would be easily immobilized into the composite film. Moreover, this strategy can effectively prevent the immobilized ECL reagents from leaking into the solution, since the ion-exchange selectivity coefficient of Nafion for Ru(bpy)3 2+ is as high as 5.7 ×106 [28]. The presented Ru(bpy)3 2+ /OMC@Nafion composite film-modified GCE displayed good sensitivity for the ECL determination of coreactant tripropylamine (TPA). Under the optimal condition, the Ru(bpy)3 2+ –TPA system could be applied in the determination of dopamine (DA), which is an important neurotransmitter in the mammalian central nervous system and plays a significant role in the function of the renal, hormonal, and cardiovascular systems [29]. Some brain functions such as learning and memory formation are sensitive

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Scheme 1. Schematic illustration of the mechanism for ECL reaction of Ru(bpy)3 2+ with TPA and DA. PTPA is the oxidation product of TPA˙ ; PDA is the reduced product of DA+ ˙ .

to the change in the level of DA [30]. Additionally, it may lead to physiological and pathological process of Pakinson when DA concentration levels are extreme abnormalities [31]. Generally, a wide variety of conventional methods, including the capillary electrophoresis [32], high-performance liquid chromatography [33], liquid chromatography–electrospray tandem mass spectrometry [34] and radioimmunossay [35] have been carried out for quantitative analysis of DA. In spite of their high reliability, these assays still have some limitations such as complicated extraction or derivatization procedure, intensive labor and long analysis time. Therefore, it is necessary to develop a simple, rapid, and inexpensive method for quantitative detection of DA. As shown in Scheme 1, ECL response of the proposed Ru(bpy)3 2+ –TPA reaction system is decreased with the adding of DA. Therefore, the ECL sensor could be employed in quantitative determination of DA.

2. Experimental 2.1. Reagents and materials Tris (2,2-bipyridyl) dichlororuthenium(II) hexahydrate (Ru(bpy)3 Cl2 ·6H2 O) and TPA were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Dopamine hydrochloride (C8 H11 NO2 ·HCl) and potassium chloride (KCl) were purchased from Aladdin Chemistry Co. Ltd. Ordered mesoporous carbon (OMC) was obtained from XF Nano company (Nanjing, China). Nafion (5 wt%) was purchased from Fluka. Other reagents were of analytical grade and used as received. Freshly prepared solutions of DA and the ultrapure water were used throughout the work.

2.3. Preparation of Ru(bpy)3 2+ /OMC@Nafion modified electrodes Before modified, GCE was polished to a mirror-like with 1.0, 0.3, and 0.05 ␮m alumina slurry. The electrodes were successively sonicated in 1:1 anhydrous ethanol and doubly distilled water, then allowed to dry under N2 stream. The OMC@Nafion solution was firstly prepared by dispersing 2.0 mg of the OMC into 1 mL (0.5 wt%) Nafion mixture with ultrasonic oscillation to give a 2 mg mL−1 black suspension. Subsequently, eight microliter of as-prepared OMC@Nafion solution was cast on the surface of pretreated GCE and dried in a pressure desiccator to obtain the OMC@Nafion modified electrode. Finally, the modified electrode was immersed into 1.0 mM Ru(bpy)3 2+ solution for 2 h and then carefully washed to remove the loose Ru(bpy)3 2+ with distilled water. The mechanism of TPA and DA detection based on the prepared ECL sensor is shown in Scheme 1. 2.4. Determination of DA in real samples Since DA is stable in acidic media, the dopamine hydrochloride injection (Shanghai Harvest Pharmaceutical Co. Ltd) samples were freshly prepared by appropriately diluting with PBS (0.01 mol L−1 , pH 5.0) and subjected directly to the ECL detection. Noted that the prepared solutions should avoid exposure to light and air, which ensures their acidity and stability will not affect by external environment. 3. Results and discussion 3.1. Characterization of ordered mesoporous carbon and its composite film

2.2. Apparatus The electrochemical measurements were carried out on a CHI 660B electrochemical workstation (Shanghai CH Apparatus Inc., China). All electrochemical experiments were carried out with a conventional three-electrode system. The threecompartment electrochemical cell contains a platinum wire as auxiliary electrode, an Ag/AgCl (3 M KCl) as reference electrode and Ru(bpy)3 2+ /OMC@Nafion modified GCE (3 mm in diameter) as working electrode, respectively. The ECL emission was monitored with a model MPI-E electrochemiluminescence analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., Xi’An, China) at room temperature. The small-angle and wide-angle X-ray diffraction (XRD) measurements were performed on a Rigaku D/max B diffractometer using Cu K␣ radiation (40 kV, 30 and 40 mA). Transmission electron microscopy (TEM) images of the samples were taken with a JEOL 2011 instrument (Hitachi) operating at 200 kV.

TEM was first used to investigate the morphologies and structures of OMC materials. Fig. 1A displays TEM images of the carbon materials viewed perpendicular to the channel direction. It can be seen that the OMC consist of the ordered hexagonal array of carbon nanorods with an average size of about 5 nm in diameter. The hexagonally ordered arrangement leaded to the well-resolved XRD peaks, as evident from Fig. 1B, which can be indexed to (1 0 0), (1 1 0) and (2 0 0) in the range of 2 below 3.0◦ reflections of the 2D hexagonal symmetry with the space group p6 mm. In addition, the nitrogen adsorption/desorption isotherm curves of the OMC revealed a high specific surface area of 852 m2 g−1 with the average pore diameter of about 4.5 nm and the specific pore volume of 0.97 cm3 g−1 (curve a, Fig. 1C). After the formation of OMC@Nafion composite film, the relatively high BET surface area (380 m2 g−1 ) and the total pore volume (0.56 cm3 g−1 ) were retained (curve b, Fig. 1C), which may vastly amplify the active surface area for immobilization of ECL reagent.

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Fig. 1. (A) TEM image, (B) low-angle and wide-angle (inset) X-ray diffraction patterns, (C) N2 adsorption-desorption isotherms, pore size distributions (inset) of OMC materials (curve a) and OMC@Nafion composite film (curve b), (D) Raman spectra of OMC materials (curve a) and OMC@Nafion composite film (curve b).

Raman spectroscopy was carried out to obtain further information on the microstructure of OMC and its composite film. As shown in Fig. 1D, Raman spectrum of the order mesoporous carbon (curve a) displayed a smooth curve with the presence of D and G band, which located at 1325 and 1595 cm−1 , respectively. The relative intensity ratio of D and G bands (ID /IG ratio), which is a measurement of the number of defect sites in graphite carbon [36,37], was calculated to be 1.04, which approximate to that of pure mesoporous carbon (1.1; the carbon is calcined at 900◦ C in N2 ) [38]. Furthermore, Raman spectrum of the OMC@Nafion composite film (curve b) also showed the characteristic peaks of OMC as well as a weak vibrational band at 731 cm−1 , which was caused by the symmetric stretching vibrations of CF2 group in Nafion [39]. This result indicated that Nafion was successfully deposited on the OMC and the microstructure (e.g. defect sites) of OMC was less affected by the composite of Nafion film. 3.2. Electrochemical behaviors of different modified electrodes In order to investigate the influence of OMC on the proposed ECL sensor in this work, experiments were carried out with different modified GCE. As shown in Fig. 2, cyclic voltammograms of Ru(bpy)3 2+ /OMC@Nafion composite film modified GCE (curve b) manifested stable and well-defined redox peaks at 1.15 V and 1.02 V in 0.1 M PBS (pH 7.5) containing 0.25 mM TPA, and the peak current was much stronger than that of Ru(bpy)3 2+ /Nafion composite film modified GCE (curve a). As seen in the inset of Fig. 2, the ECL intensity–time curves also displayed an effect of OMC on performance of the prepared sensor. The Ru(bpy)3 2+ /OMC@Nafion composite film modified GCE exhibited a strong ECL signal (curve b), while the ECL intensity of Ru(bpy)3 2+ /Nafion composite film modified GCE was quite weaker (curve a) than the former. These results confirmed the excellent capacity of OMC as a nanoscale anchorage substrate to load a large amount of luminescent probes on the GCE surface, which could highly amplify the ECL response and generate an enhancement of the sensitivity.

3.3. Conditions optimization of ECL sensor for TPA sensing It is well known that Ru(bpy)3 2+ –TPA couple is considered to be its higher ECL signal compared to other generally used co-reactant. In order to improve the capacity of the ECL sensor for TPA sensing and obtain an enhanced sensitivity, several experimental parameters such as solution pH and photomultiplier tube (PMT) voltages interferences were optimized. As shown in Fig. 3A, the pH value of solution extremely affected the ECL system of Ru(bpy)3 2+ –TPA. The ECL response increased significantly along with increasing of pH value and then reached maximum at pH 7.5, a further increase of pH value resulted in a decline of ECL signal. Thus, pH 7.5 was selected to be the appropriate pH value for all subsequent work. The effect caused by PMT voltages on ECL signal of the sensor was also investigated. As seen in Fig. 3 B, the ECL intensities of the sensor in both blank buffer solution (curve a) and the solution containing TPA (curve b) increased as the voltages increasing. In order to obtain a high sensitivity and low background signal ECL sensor, 600 V was chosen as the optimal voltage. Both the influence of pH value and

Fig. 2. Cyclic voltammograms and ECL time curves (inset) of Ru(bpy)3 2+ /Nafion (a) and Ru(bpy)3 2+ /OMC@Nafion composite film (b) modified GCE in 0.1 M PBS (pH 7.5) containing 0.25 mM TPA at a scan rate of 100 mV s−1 .

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Fig. 3. (A) ECL signal of Ru(bpy)3 2+ /OMC@Nafion composite film modified GCE with different pH in 0.1 M PBS containing 0.25 mM TPA. (B) ECL signal of Ru(bpy)3 2+ /OMC @Nafion composite film modified GCE at different voltages in 0.1 M PBS (pH 7.5) (a) without and (b) with 0.25 mM TPA.

voltages on the ECL response of Ru(bpy)3 2+ /OMC@Nafion composite film modified GCE could be corresponding to the results gained at other modified GCE [40]. All of the above results demonstrated that the Ru(bpy)3 2+ /OMC@Nafion composite film was available to ECL sensor for analysis. 3.4. Analytical performance of the proposed ECL sensor Under optimal conditions, ECL response of the sensor to TPA was measured. As shown in Fig. 4A, the variation of amplified ECL signal gradually increased with increment in the concentrations of TPA (curves a–i). Fig. 4B depicts the calibration plot for the quantification of TPA from 0.00475 to 625 ␮M with a detection limit of 1.58 nM (S/N = 3). The linear regression equation was I = 12.285 × CTPA + 263.973 with a correlation coefficient of 0.9984, where I was the peak intensity of the ECL sensor, and C was the concentration of TPA. The proposed ECL sensor (Ru(bpy)3 2+ /OMC@Nafion/GCE) showed promising analytical

Table 1 Comparisons of the proposed protocol with other reported ECL sensors for the detection of TPA. No.

Linear range (nM)

Detection limit (nM)

Reference

1 2 3 4 5

50.00–1.00 × 106 8.50–8.10 × 104 1.00–10.00 × 104 250.00–1.50 × 105 4.75–6.25 × 105

10.00 2.80 0.50 5.00 1.58

[44] [45] [46] [47] This work

performances, which could be comparable with those reported TPA ECL sensors (Table 1). The ECL sensor also revealed excellent stability under continuous potential scanning over several cycles in the PBS (pH 7.5) containing 250 ␮M and 0.0025 ␮M TPA, respectively (Fig. 5A). After storing at 4 ◦ C for 3 weeks, the ECL sensor retained about 94% of its original ECL intensity (Fig. 5B). This result further confirmed the excellent repeatability and stability of Ru(bpy)3 2+ /OMC@Nafion modified electrodes. The outstanding

Fig. 4. (A) ECL intensity of the prepared sensor in 0.1 M PBS (pH 7.5) containing (a) 0.00475, (b) 0.3, (c) 0.6 (d) 2.4, (e) 4.8, (f) 19, (g) 78, (h) 313 and (i) 625 ␮M TPA. Scan rate: 100 mV s−1 . (B) Calibration curve of the sensor for TPA with a correlation coefficient of 0.9984 (n = 9). (C) ECL intensity of prepared sensor on the concentration of DA in 0.1 M PBS (pH 7.5) containing 0.25 mM TPA. Concentrations of DA are (from a to j): 0.005, 0.01, 0.05, 0.5, 1, 5, 10, 50, 100, 500 ␮M. (D) Calibration curve of the sensor for DA with a correlation coefficient of 0.9973 (n = 10) by sweeping the potential between 0.2 V and 1.25 V at a scan rate of 100 mV s−1 .

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Fig. 5. (A) ECL time curve of Ru(bpy)3 2+ /OMC@Nafion composite film modified GCE in 0.1 M PBS (pH 7.5) containing different TPA concentration at a scan rate of 100 mV s−1 . (B) ECL intensity of Ru(bpy)3 2+ /OMC@Nafion composite film modified GCE in 0.1 M PBS (pH 7.5) containing 0.25 mM TPA for a period of time. Table 2 Recoveries of the dopamine hydrochloride injection sample determined by the proposed assay protocol (n = 3). Sample no.

Original (␮M)

Amount added (␮M)

Amount found (␮M)

RSD (%)

Recovery (%)

1

0.0012 0.0012 31.30 31.30

0.0015 0.30 50.00 300.00

0.0029 0.31 75.97 328.37

4.88 6.19 6.02 6.80

107.41 103.57 93.44 99.12

2

Note: Each value is the mean of three experiments.

performance was largely attributed to the enhanced electron transfer rate from OMC@Nafion and amplified contacting surface area of electrode. In addition, with the excellent properties of the prepared ECL sensor, it can be used to perform the determination of biological molecule DA. As mentioned above, there was a rapid decline of the ECL response when DA was injected into the detection cell containing 0.25 mM TPA. The inhibition mechanism (Scheme 1) is outlined below: Ru(bpy)3 2+ − e− → Ru(bpy)3 3+

(1)

TPA − e− → [TPA• ]+

(2)

−

• +

DA − e → [DA ]

(3)

[TPA• ]+ → TPA• + H+

(4)

[DA• ]+ + TPA• → PDA + PTPA

(5)

Ru(bpy)3 3+ + TPA• → Ru(bpy)3 2+∗ + PTPA

(6)

Ru(bpy)3

2+∗

→ Ru(bpy)3

2+

+ h ( — — 620 nm)

(7)

During the ECL detection process, the concentration of TPA• decreased with the addition of DA, which resulted in quenching of the ECL intensity. With increasing concentration of DA, ECL signals of the proposed sensor decreased linearly with log(CDA ) in the range of 0.005–500 ␮M (Fig. 4C). And the calibration curve can be fitted with the equation I = 3386.7855–700 log(CDA /␮M), R2 = 0.9973 (Fig. 4D). The ECL sensor exhibited a detection limit of 1.7 nM (S/N = 3), which was much lower than those of 8.30 × 10−7 M at an Au nanoclusters–K2 S2 O8 system based ECL sensor for detection of DA, 7.1 × 10−9 M at an FI-ECL inhibition method for determination of DA [41,42]. As it is known that some biological small species such as ascorbic acid and uric acid possibly cause interference in the detection of DA [43], the anti-interference ability of the sensor was also investigated by detecting 5 ␮M DA solution in the presence of ascorbic acid and uric acid at the same concentration. It was found that compared with the ECL response obtained from pure DA, the ECL intensity of the mixed solution was changed less than 10%, suggesting the reliability of the ECL sensor for DA detection.

3.5. Real sample analysis The feasibility of the ECL sensor for practical application was investigated by detecting the concentration of DA in dopamine hydrochloride injection. As shown in Table 2, the recoveries and RSD were in the range of 93.44–107.41% and 4.88–6.80%, respectively. Furthermore, the recovery experiments were also carried out in human serum samples. 0.5, 1.0 and 1.5 ␮M standard DA solution was added to human serum samples, respectively. The average recoveries of the ECL sensor were between 94.11% and 108.06% (n = 3) with its RSD ranging from 4.25% to 7.42%. The results demonstrated that the proposed ECL sensor was applicable for the determination of DA in real sample with high accuracy. 4. Conclusions In summary, a simple, stable and sensitive Ru(bpy)3 2+ /OMC@Nafion based ECL sensor was constructed in the present work. The nanostructure of OMC aggrandized the electro-active surface of GCE, which promoted sensing performance of the ECL sensor. The proposed ECL sensor displayed a wide linear response and low detection limit toward TPA. Furthermore, the remarkable detection property of the prepared ECL sensor could benefit to its biosensing application. This Ru(bpy)3 2+ –TPA based ECL sensor provided a convenient, fast and sensitive way to detect DA with high accuracy in real sample analysis. Therefore, this preliminary strategy could promote the application of ordered mesoporous material-based ECL sensor in chemical and biochemical analysis. Acknowledgements This work was supported by the National Natural Science Foundation of China (21373138), National 973 Project (2010CB933901), Shanghai Sci. & Tech. Committee (12JC1407200), Program for Changjiang Scholars and Innovative Research Team in University (IRT1269). References

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Biographies Beina Wu is currently a M.S. candidate in the Department of Chemistry of Shanghai Normal University. Her current interests are biosensors. Chongchong Miao is currently a M.S. candidate in the Department of Chemistry of Shanghai Normal University. Her current interests are biosensors. Lili Yu is currently a M.S. candidate in the Department of Chemistry of Shanghai Normal University. Her current interests are biosensors. Ziyi Wang is currently a M.S. candidate in the Department of Chemistry of Shanghai Normal University. His current interests are biosensors. Chusen Huang is currently a lecturer in the Department of Chemistry of Shanghai Normal University. His main research interests are fluorescent dye and organic synthesis. Nengqin Jia is a professor in the Department of Chemistry, Shanghai Normal University. His main research interests are focused on bioelectrochemistry and nanobiotechnology.