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Nonenzymatic electrochemiluminescence glucose sensor based on quenching effect on luminol using attapulgite–TiO2
Sensors and Actuators B 230 (2016) 449–455
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Nonenzymatic electrochemiluminescence glucose sensor based on quenching effect on luminol using attapulgite–TiO2 Yin-Zhu Wang a,b , Hui Zhong a,b,∗ , Xiao-Rong Li a,∗ , Gen-Qing Liu a,b , Kai Yang a,b , Min Ma a , Li-Li Zhang a,∗∗ , Jing-Zhou Yin a , Zhi-Peng Cheng a , Ji-Kui Wang b a Jiangsu Key Laboratory for Chemistry of Low-Dimensional Materials, School of Chemistry & Chemical Engineering, Huaiyin Normal University, Huaian 223300, PR China b College of Sciences, Nanjing University of Technology, Nanjing 210009, PR China
a r t i c l e
i n f o
Article history: Received 13 September 2015 Received in revised form 1 February 2016 Accepted 6 February 2016 Available online 10 February 2016 Keywords: Nonenzymatic Electrochemiluminescence Glucose Att–TiO2 Luminol
a b s t r a c t A new nonenzymatic glucose ECL sensor based on attapulgite (Att) integrated with semiconductor material TiO2 was developed. The prepared material was characterized by transmission electron microscopy (TEM), X-ray diffractometer (XRD), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Based on ECL experimental results, Att–TiO2 composites were found to be able to improve the ECL properties of luminol. When glucose is added into the system, TiO2 NPs serve as a catalyst and dissolved O2 acts as a cosubstrate for the glucose oxidation reaction. Dissolved O2 also worked as coreactant for the luminol ECL emission. Due to the consumption of dissolved O2 , a quenching effect would appear on the luminol ECL emission. Under the optimized conditions, the linear logarithmic relationship between ECL intensity and the concentration of glucose was valid in the range from 1.0 mM to 1.0 nM (R = 0.9976) with a detection limit (S/N = 3) of 10.0 pM. In addition, the proposed sensor presented good reproducibility, stability, and sensitivity for glucose detection and can be successfully applied in the determination of glucose in real blood samples. © 2016 Published by Elsevier B.V.
1. Introduction Glucose is the primary energy source of the body. The level of glucose in blood has been used for diagnosis of diabetes or hypoglycemia. Besides the need in glucose monitoring in the case of diabetes patients, it is also essential for non-diabetic acute care patients in order to control glucose levels [1]. Therefore, the development of fast, sensitive, selective and reliable methods for glucose monitoring is important in clinical diagnostics, food industry and biotechnology [2]. Electrochemiluminescence (ECL), a special form of chemiluminescence (CL) in which light emission is generated by electrochemical reactions, has been receiving higher and higher attention because of its excellent sensitivity, low background signal and some additional advantages [3], since Haghighi and Bozorgzadeh reported ECL behaviors of Si for the first time
∗ Corresponding author at: Jiangsu Key Laboratory for Chemistry of LowDimensional Materials, School of Chemistry & Chemical Engineering, Huaiyin Normal University, Huaian 223300, PR China. ∗∗ Corresponding author. E-mail addresses: [email protected] (H. Zhong), [email protected] (X.-R. Li), [email protected] (L.-L. Zhang). http://dx.doi.org/10.1016/j.snb.2016.02.026 0925-4005/© 2016 Published by Elsevier B.V.
in 2002 [4]. Among various ECL systems, luminol is considered as one of the most popular ECL luminophor due to its low oxidation potential, inexpensive reagent consumption and the high emission yields [5,6]. So, special attention had focused on the ECL studies concerning luminol for glucose analysis [7]. Luminol-based ECL glucose biosensors on the oxidation of glucose catalyzed by glucose oxidase have the advantages of being a simplified and sensitive instrument. However, these so-called biosensors have poor stability because glucose oxidase (GOx) quickly loses its activity at below pH 2 and above pH 8 and when temperature is above 40 ◦ C. In addition, these compounds suffer from badly damage or time consuming due to their long-winded fabrication procedure for the fixing enzyme onto the electrode [8]. Therefore, researchers have focused on nonenzymatic glucose ECL sensors. Currently, only four literatures have reported the corresponding nonenzymatic ECL behavior of luminol to determine glucose [9–12]. It is desirable to discover more materials to develop effective enzyme-less luminol-based ECL biosensors of detecting glucose. As far as we know, the ECL intensity is greatly influenced by the property of the working electrode, especially the material’s surface property. Titania dioxide (TiO2 ), an important semiconductor material with a large surface area, has previously been researched for photo-assisted degradation of a variety of toxic chemicals.
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Titania is known to be an effective catalyst for the cleavage of diols when reactive oxygen species (ROS) is used as oxidant [13]. Therefore, it would be a good working electrode for ECL detection and has attracted increasing interest in recent years [14]. Because the modified TiO2 nanocrystals are easy to fall off from the electrode surfaces, the electrodes have poor stability and the ECL intensity is not strong enough [15]. Considering the utilization and conversion of abundant negatively charged product, attapulgite (Att), which helps to form relatively high surface area and moderate cation exchange capacity [16] due to its permanent negative charges on its surface, has been successfully used to make TiO2 more active. There are large reserves of Att in Huai’an, China. The specific property and large distribution of Att make it to be studied undoubtedly. In present paper, we combined the huge specific surface area of Att, which can fix TiO2 onto the electrode surface more stable and prevent the deposition of luminol oxidation products. In addition, we found that AttTiO2 nanohybrids could effectively enhance luminol’s ECL in experimental process. The obtained sensor exhibited very sensitive ECL quenching responses for detection of glucose. The analytical procedure of this ECL sensing is illustrated in Scheme 1. First, Att–TiO2 composites material was prepared, then anchor it onto the surface of GCE, this composite can improve the ECL properties. While the glucose was added into the solution, glucose was oxidized in the presence of TiO2 , and part of oxygen radicals (O2 • −) were consumed, so that the ECL signal was quenched. The intensity of quenching signal was linearly associated with the logarithm of the concentration of glucose, based on it, the detection of glucose could be achieve. The stable and strong ECL emission guarantees that Att–TiO2 composite works as a sensitive sensor. 2. Experimental 2.1. Reagents and chemicals Luminol was obtained from J&K Chemical. Glucose and KH2 PO4 were received from Sinopharm Chemical Reagent Co., LTD. (Shanghai, China). K3 [Fe(CN)6 ] was purchased from Guangdong Shantou West Long Chemical Plant. K4 [Fe(CN)6 ]·3H2 O was obtained from Wuxi City and Yasheng Chemical. NaOH was from Nanjing Chemical Reagent Co., LTD. Phosphated-buffered solution (PBS) (pH 7.4, 0.1 M) was prepared using 0.1 M KH2 PO4 and 0.2 M NaOH. A 1.0 mM luminol (3-aminophthalhydrazide) stock solution was prepared by dissolving it in a small amount of 0.1 M NaOH. A stock solution of glucose (1.0 mM) was prepared in PBS and stored at 4 ◦ C when it was not in use. The stock solution of glucose was allowed to mutarotate at room temperature for 24 h before use. Different concentrations of working solutions were diluted step by step with PBS. Human serum samples were kindly provided by Huai’an First People’s Hospital. All chemicals were of analytical reagent grade and were used without further purification. All solutions were prepared exclusively in double distilled water.
4 h, the suspension was filtered, washed with water, dried at 80 ◦ C and calcined at 300 ◦ C for 4 h. After grinding to about 200 meshes in a carnelian mortar, TiO2 oxides coated onto Att were obtained and labeled as Att–TiO2 . 2.3. Instrumentation and procedures The morphologies of the Att–TiO2 nanohybrids were characterized by transmission electron microscopy (TEM) (JEM-3011, JEOL, Japan). The crystal structures of the samples were examined by an X-ray diffractometer (XRD, BrukerD8, Advanee, Germany) with Cu K␣ radiation () 0.15406 nm. Electrochemical impedance spectroscopy (EIS) measurements were monitored with an Autolab PGSTAT 30 Analyzer (Metrohm Autolab B.V., Switzerland) in a solution containing 5 mM Fe(CN)6 3−/4− and 0.1 M KCl. The frequency was ranged from 0.1 to 100,000 Hz with an alternating current voltage of 10 mV. Cyclic voltammetry (CV) and ECL measurements were taken using a homemade ECL/EC system. Cyclic voltammetrys (CVs) were carried out using an electrochemical working station (CHI 660D, Chenhua Inc., China) in 0.1 M KCl solution containing 5.0 mM K3 Fe(CN)6 /K4 Fe(CN)6 and in PBS (pH 7.4) for the bare GCE and Att–TiO2 -midified GCE. The ECL signals were acquired by a MPI-M multifunctional analytical system (Xi’an Remax Electronic Science Tech. Co., Ltd., Xi’an, China) with the voltage of the photomultiplier tube (PMT), which is used for transforming ECL emission into electrical signals, set at 600 V in the process of detection and the ECL detector cell was placed in front of the PMT. The detector cell was made of a microbeaker (high: 35 mm, i.d.: 25 mm) and we performed the ECL measurements in 3 mL PBS (pH 7.4) with 0.35 mM luminol and 1.0 M glucose. In addition, all electrochemical experiments were performed with a conventional three-electrode set-up where glassy carbon electrode was used as working electrode, platinum wire as counter electrode and Ag/AgCl (saturated KCl solution) as reference electrode, respectively. All measurements were performed at room temperature. 2.4. Electrodes preparation and modification A glassy carbon electrode (GCE, 3 mm diameter) was used for the preparation of the nonenzymatic glucose ECL biosensor. Before electrode modification, the GCE was polished with 0.3 and 0.05 m alumina paste (Gao Shi Rui Group Technology Co., LTD., Wuhan, China) on chamois leather and carefully rinsed with 1:20 (v/v) nitric acid, ethanol and doubly distilled water in an ultrasonic bath to give a smooth and clean electrode surface. Next, 1.0 mg of Att–TiO2 was dispersed in 1.0 mL double distilled water with ultrasonic agitation for 1 h to achieve a well-dispersed suspension. Then, a 5.0 L of the prepared Att–TiO2 suspension was pipetted on the surface of GCE to construct Att–TiO2 -modified GCE and allowed to be dried in air at room temperature. 3. Results and discussion
2.2. Synthesis of Att–TiO2 nanohybrids
3.1. Characterization of Att–TiO2 nanocomposites
The nanomaterials of Att–TiO2 were prepared by a similar route to the technique reported before [17]. The first step is the simple purification of Att. 1.0 g of raw Att when it was dispersed in 100 mL distilled water and ultrasound for 0.5 h, then the nether sand and large stones were removed. This produced a homogeneous Att suspension which was used directly in the later modification section without further acid activation. Secondly, 3.4 g of Ti(OBu)4 was added to the as-prepared Att suspension, and then 100 mL distilled water was added drop by drop to let the Ti(OBu)4 hydrolyze in situ to deposit Ti(OH)4 onto the surface of Att. After vigorous stirring for
Fig. 1A shows the XRD patterns of Att (a), TiO2 (b) and Att–TiO2 (c). The reflections of Att (a) at 2 = 8.34◦ , 19.8◦ , 26.6◦ , 28.0◦ and 34.9◦ were consistent with the standard spectrogram Att (JCPDS No. 37-0783) structure. Their corresponding crystal face index are (1 1 0), (0 4 0), (2 3 1), (4 0 0) and (1 0 2) respectively. The reflections (b) at 2 = 25.3◦ , 37.8◦ and 48.0◦ corresponded to TiO2 that is consistent with the standard spectrogram TiO2 (anatase) (JCPDS No. 65-5714) structure. Their corresponding crystal face index are (1 0 1), (0 0 4) and (2 0 0). Montmorillonite and quartz were also found. The characteristic reflections of Att were observed in the
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Scheme 1. The analytical procedure of Att–TiO2 electrode for glucose detection.
Fig. 1. (A) XRD characterization of Att (a), TiO2 (b) and Att–TiO2 (c) (♦ Attapulgite TiO2 (Anatase). (B) The TEM image of Att–TiO2 . (C) Electrochemical impedance spectra and cyclic voltammograms (D) of (a) bare GCE, (b) Att-modified GCE, (c) Att–TiO2 -modified GCE in 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl solution. Inset: the equivalent circuit.
XRD pattern of Att–TiO2 composite (c). In Fig. 1A, the characteristic reflections of TiO2 as anatase were also confirmed. In addition,
the TEM image of Att–TiO2 compounds s shown in Fig. 1B. The Att samples have an average diameter of about 50 nm. The surface
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Fig. 2. (A) IECL /E curves of luminol at the GCE (a), Att-modified GCE (b), Att–TiO2 -modified GCE(c). Inset: CV curves of luminol at the GCE (a), Att-modified GCE (b), Att–TiO2 modified GCE(c); conditions: luminol, 0.35 mM; supporting electrolyte, PBS (pH 7.4); scan rate, 100 mV/s. (B) IECL /E curves of luminol in the absence (a) and in the presence (b) of 1.0 M glucose at Att–TiO2 -modified GCE. Inset: CV curves of luminol in the absence (a) and in the presence (b) of 1.0 M glucose at Att–TiO2 -modified GCE; conditions: luminol, 3.5 × 10−4 M; supporting electrolyte, PBS (pH 7.4); scan rate, 100 mV/ s.
of Att–TiO2 was fully covered with nanoparticles about 10 nm in average and uniformly distributed, without obvious aggregation.
area and provided the conducting bridges for the electron-transfer of Fe(CN)6 3−/4− , which is consistent with the result of EIS. All of these results demonstrated that Att–TiO2 hybrids provided high electron conduction pathways.
3.2. Electrochemical characterization of Att–TiO2 modified GCE In the experiment, we respectively fabricated the bare GCE, Att and Att–TiO2 -modified GCEs and employed electrochemical impedance spectrum (EIS) of Fe(CN)6 4−/3− to monitor the features of above modified electrodes in Fig. 1C, because it is one of the most powerful and effective techniques to investigate the modification process [18]. The electron-transfer resistance (Rct) at the electrode surface is equal to the semicircle diameter of EIS and can be used to describe the interface properties of the electrode. The Randles circuit (inset of Fig. 1C) was chosen to fit the impedance data obtained. The resistance to charge transfer and the diffusion impedance (W) were both in parallel with the interfacial capacitance (Cdl). The diameter of the semicircle corresponds to the interfacial electrontransfer resistance (Rct) [19]. By fitting the data, the redox process of the [Fe(CN)6 ]3−/4− probe showed a Rct of 439.1 Ohm at the bare GCE (a). This value of Rct remarkably increased to about 643.2 Ohm after Att droped onto the GCE surface (b), indicating Att on the electrode surface blocked the electron transfer between [Fe(CN)6 ]3−/4− probe and electrode. The remarkable increase of Rct also indicates that Att has been successfully immobilized on the GCE surface. However, when the Att–TiO2 assembled onto the GCE surface, the Rct values decrease to about 306.7 Ohm (c), which suggested that Att–TiO2 was excellent electronic conducting materials and could accelerate the electron transfer between [Fe(CN)6 ]3−/4− probe and electrode. Considering that Att itself is not a conductor, the superior conductivity of Att–TiO2 here is may be that TiO2 play an important role similarly to a conducting wire or electron-conducting tunnel, which makes it easier for the electron transfer to take place. On the other hand, cyclic voltammograms of 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl solution on the bare GCE (a), Att-modified GCE (b) and Att–TiO2 -modified GCE (c) are shown in Fig. 1D. The quasi-reversible one-electron redox behavior of ferricyanide ions was observed on the bare GCE with a peak separation (Ep) of 74 mV at the scan rate of 100 mV/s. After being modified with Att, the peak current of Fe(CN)6 3−/4− was reduced compared with that observed at the bare GCE. For Att–TiO2 -modified GCE, the peak current of Fe(CN)6 3−/4− was increased greatly instead compared with that of the Att-modified GCE and bare GCE, revealing that the introduction of the Att–TiO2 hybrid played a role in the increase of the electroactive surface
3.3. Enhanced ECL response of luminol on Att–TiO2 -modified GCE In order to verify whether indeed the ECL sensor works as expected, we recorded step by step the ECL signal intensity of the sensor in PBS (pH 7.4) containing 0.35 mM luminol with the scan rate of 100 mV/s at the GCE, Att and Att–TiO2 -modified GCEs. As shown in Fig. 2A, in the IECL /E curves, the ECL signal of the bare GCE was found to be very weak (a). After the modification with Att, the ECL intensity of the electrode kept constant (b). That was attributed to the hindrance of Att. But if we use the Att–TiO2 modified GCE, a clear ECL wave occurred near 0.4 V during the anodic scan (−0.4–0.8 V), and then the ECL intensity rose steeply until it reached a maximum near 0.61 V (c), which was consistent with the oxidation potential of luminol [20]. The experimental results indicate that the ECL signal of the luminol is strongly dependent upon the TiO2 nanoparticles, the interaction between given chemical species and the surface of TiO2 nanoparticles would result in changes in the ECL efficiency. A similar enhancement effect was also observed in their corresponding cyclic voltammograms (CVs) (inset of Fig. 2A). In the CV curves, it can be clearly seen that there is no enhancement effect at bare GCE (a) and Att-modified GCE (b). When the modified electrode was incubated with Att–TiO2 (c), the anodic peak current intensity of luminol (at about 0.55 V) enhanced remarkably compared with Att, indicating Att–TiO2 provided large surface area and active sites for luminol oxidation reaction and consequently showed excellent electrocatalytic activity and high current intensity. 3.4. Quenching ECL response of Att–TiO2 nanocomposites toward glucose and the corresponding mechanism We studied the IECL /E curves and CV curves with and without glucose solution at pH 7.4 (Fig. 2B). In the IECL /E curves, an ECL signal at 0.61 V could be observed in luminol–PBS solution (a). Comparatively, we discovered that the ECL peak obviously decreased even if the solution contained only a small amount of glucose (b). As shown in inset of Fig. 2B, Att–TiO2 -modified GCE appeared an oxidization peak toward glucose (a). In addition, the anodic peak was
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3.5. Performance of the ECL sensor for glucose detection
Fig. 3. ECL spectrum of the luminol/Att–TiO2 (a) and Att–TiO2 (b) system.
negatively shifted when a proper amount of glucose was added (b). This indicates that Att–TiO2 has quenching effect on the oxidation of glucose. To explain the mechanism for the quenching effect, the ECL emission spectrum of Att–TiO2 and luminol/Att–TiO2 system are depicted in Fig. 3. It can be seen from curve a that the maximum emission at 420 nm is corresponding to the ECL emission of luminol. In addition, a quite weaker emission at 520 nm is corresponding to the ECL emission of TiO2 , which is nearly the same as the photoluminescence spectrum of TiO2 powder. Without luminol, the 520 nm emission is very weak (curve b). These results suggest that the ECL intensity is the combination of the two above optical signals. However, the intensity of the latter is negligible compared with that of the former, suggesting the little effect on the glucose detection. The possible inhibition mechanism of glucose on the luminol ECL response is shown as follows: TiO2 nanoparticles are reduced to TiO2 • − radicals at the surface of Att–TiO2 -modified electrode (Reaction (1)). The TiO2 • − radicals react with the dissolved O2 to produce strong oxidant O2 • − radicals (Reaction (2)) which play a key role in the progress of luminol ECL, as reported in the literature [21]. The strong oxidant O2 • − radicals react with the intermediate oxidation state of luminol (L− ) which was the product of the reaction of TiO2 • −with L− , thus generating the excited state of 3-aminophthalate according to Reactions (3) and (4). While the excited state of 3-aminophthalate goes back to the ground state, emission happens at 425 nm. The oxidation of luminol by the O2 •− radicals was indirectly confirmed by the result that luminol can be electrooxidized at the Att–TiO2 electrode in the range of −0.4–0.8 V. On the other hand, the reaction of TiO2 • − with L− would also produce the excited state of TiO2 with emission at 520 nm while it goes back to the ground state according to Reactions (3) and (5). This speculation was confirmed by such a result that the emission at 520 nm was enhanced with the addition of luminol. Without luminol, the emission at 520 nm is very weak. After adding of glucose into the luminol/Att–TiO2 system, glucose reacts with O2 •− radicals to generate gluconic acid (Reaction (6)) resulting in a decrease in the luminol ECL intensity [22]. The possible ECL route of luminol/Att–TiO2 using dissolved O2 as the coreactant was proposed as follows:
Before the application of this sensor to the accurate analysis, the optimized conditions such as the amount of Att–TiO2 , scan rates, luminol concentrations and the optimum pH were described in Supporting information. ECL intensity was measured step by step with Att–TiO2 -modified GCE in 9.0 mL PBS (pH 7.4) containing luminol (0.35 mM) and glucose with different concentrations. Fig. 4A shows the glucose concentration-dependent ECL intensity and the corresponding calibration curve (Fig. 4B). When the glucose concentration increases, the ECL signal decreases accordingly, indicating that the glucose could quench the ECL intensity of luminol in the presence of catalyst Att–TiO2 [23]. The inhibition ratio is linear with the logarithm of glucose concentration in the range of 1.0 mM–1.0 nM with a regression equation: Log I = −0.17065Log C + 2.6874(R = 0.9976) where I is the ECL intensity and C is the glucose concentration, the detection limit (S/N = 3) was 10 pM and the sensitivity was 288.6 nM−1 cm2 . As seen from Table 1, our non-enzymatic ECL sensor in this work exhibited a better performance than that in most reported works in terms of linear range and detection limit [9–12]. This may be due to the Att–TiO2 nanocomposites with good film stability and high conductivity for enhanced electrocatalytic response, which accelerates the electron transfer. In addition, dissolved oxygen, as the co-reagent of luminol, was only a very small amount in PBS, but luminol could be emitted at Att–TiO2 -modified GCE. So, it leads to such a high sensitivity of Att–TiO2 in sensing applications and is very sensitive to glucose. On the other hand, in the process of the reaction, when the glucose concentration increases, the ECL signals decrease accordingly.
3.6. Application of biosensor for detecting glucose in normal human serum sample In order to gauge the applicability of the proposed ECL biosensor to the detection of glucose in real samples, this nonenzymatic ECL biosensor was applied for determination of glucose concentrations in human serum samples (other parameters were used as optimum conditions), which were obtained from Huai’an First People’s Hospital. It should be noted that, prior to use, the serum samples were diluted 100-fold with normal saline. Proper quantity of the human serum was mixed with 9.0 mL solution containing PBS (pH 7.4) and luminal (0.35 mM). The results from these determinations are presented in Table 2, which shows an excellent agreement between the results from the present method and the reference values (reference values were provided by the Huai’an First People’s Hospital). In addition, a series of recoveries were also performed to evaluate the accuracy and the recovery values ranged from 98.7% to 101.9%, indicating good accuracy of the Att–TiO2 -modified GCE. These proved that this method was reliable and suitable for determining glucose in serum samples.
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Fig. 4. (A) ECL responses of luminol at Att–TiO2 -modified GCE toward different concentration of glucose. Conditions: luminol, 3.5 × 10−4 M; supporting electrolyte, PBS (pH 7.4); scan rate: 100 mV/s. a: 1.0 mM, b: 0.1 mM, c: 10 M M, d: 1.0 M, e: 0.2 M, f: 50 nM, g: 10 nM, h: 2.0 nM, i: 1.0 nM. (B) Logarithmic calibration curve of the prepared glucose biosensor. (C) ECL signal specificity of sensor toward 1.0 M glucose, 30 nM AA, 30 nM DA and 30 nM UA at the Att–TiO2 -modified GCE in PBS (pH 7.4) containing 0.35 mM luminol. (D) The successively cyclic ECL responses of the biosensor toward 1.0 M glucose for 14 times; Conditions: supporting electrolyte, 0.1 M phosphate buffer (pH 7.4); scan rate, 100 mV/s.
Table 1 Comparison of different non-enzymatic ECL glucose sensor. Electrode
Methods
Linear range (M)
Detection limit (M)
Ref.
Au NPs-CdTe QDs-CHIT/GCE PdNPs-FCNTse/nafion-GCE LaTiO3 –Ag0.1 /GCE ConA/Tween20/DexP/g-C3 N4 -PTCA/GCE Att–TiO2 -modified GCE
ECL ECL ECL ECL ECL
1.0×10−5 −1.0×10−2 5.0×10−7 −4.0×10−5 1.0×10−8 −1.0×10−4 1.0×10−10 −5.2×10−5 1.0×10−9 −1.0×10−3
5.28×10−6 9.0×10−8 2.5×10−9 4.0×10−11 10−11
[9] [10] [11] [12] This work
Table 2 Determination of glucose in human blood serum samples. Serum samples
Hospital (mM)
Sensor (mM)
RSD (%)
Added (mM)
Found (mM)
Recovery (%)
1 2 3 4 5 6 7
12.16 5.56 6.71 5.9 4.57 4.49 6.04
12.34 5.63 6.84 6.00 4.48 4.54 5.98
0.813 0.822 0.653 1.205 1.981 2.008 3.106
0.1 0.1 0.1 0.1 0.1 0.1 0.1
12.441 5.7315 6.9417 6.1019 4.5787 4.6418 6.0793
101.2 101.5 101.7 101.9 98.7 101.8 99.3
3.7. Interferences, reproducibility and stability One of the most important analytical factors for a sensor is the ability of the sensor to discriminate the electroactive interfering species. The easy oxidizable compounds such as ascorbic acid (AA), dopamine (DA) and uric acid (UA) are normally co-existed with glucose in natural samples. Because the normal physiological level of glucose in the human blood is about 30 times of AA, DA and UA [23], we studied the interference effect of 30 nM AA, DA and UA on
the ECL response of 1.0 M glucose in the present work. The corresponding results are shown in Fig. 4C. The interference caused by these electroactive interfering species could be neglected, indicating a good selectivity of the prepared biosensors for glucose. We also investigated the reproducibility of ECL biosensor. As shown in Fig. 4D, the relative standard deviation (RSD) of the ECL response to 1.0 M glucose was 0.7% for 14 successive measurements, giving an acceptable assay reproducibility of the method due to the stable structure of Att–TiO2 composite. The stability
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was measured by evaluating the responses of ECL biosensor to the glucose using the Att–TiO2 composite particles stored at room temperature in one month. The results showed that the ECL responses were kept constant in the first week, remained approximately 95% of the original value in the second week, and dropped to 85% in the fourth week. The results indicate that Att–TiO2 -modified GCE has a better stability. 4. Conclusions In summary, we have developed a high-performance nonenzymatic glucose ECL sensor based on Att integrated with semiconductor material TiO2 . The ECL behavior of luminol has been investigated in detail at the Att–TiO2 modified electrode, and glucose was found to be able to inhibit this ECL system. Based on this, an inhibited ECL detection method has been developed for determination of glucose. In addition, this sensor exhibited good reproducibility, wide-range linearity, high sensitivity and stability. The proposed method provided a promising way to develop efficient sensors to detect glucose in real blood samples. These demonstrations pave an avenue for multiple functional electrochemical sensors. Acknowledgments This research is supported by the National Natural Science Foundation of China (21375044, 51106061, 21201072, 21405055, 51472101), Natural Science Foundation of Jiangsu Province (BK20131211, BK20131214, BK2012241) and Natural Science Foundation of Jiangsu Higher Education Institutions (14KJB150004, 12JB150006). Appendix A. Supplementary data
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[11] F.F. Jia, H. Zhong, W.G. Zhang, X.R. Li, G.Y. Wang, J. Song, Z.P. Cheng, J.Z. Yin, L.P. Guo, A novel nonenzymatic ECL glucose sensor based on perovskite LaTiO3 –Ag0.1 nanomaterials, Sens. Actuators B 212 (2015) 174–182. [12] Y. Fan, X.R. Tan, X.F. Liu, X. Ou, S.H. Chen, S.P. Wei, A novel non-enzymatic electrochemiluminescence sensor for the detection of glucose based on the competitive reaction between glucose and phenoxy dextran for concanavalin a binding sites, Electrochim. Acta 180 (2015) 471–478. [13] A.M. Velarde, P. Bartl, T.E.W. Nießen, W.F. Hoelderich, Hydrogen peroxide oxidation of d-glucose with titanium-containing zeolites as catalysts, J. Mol. Catal. A: Chem. 157 (2000) 225–236. [14] C. Li, Q. Kang, Y. Chen, J. Li, Q. Cai, S. Yao, Electrochemiluminescence of luminol on Ti/TiO2 NT electrode and its application for pentachlorophenol detection, Analyst 135 (2010) 2806–2810. [15] J. Li, L. Yang, S. Luo, B. Chen, J. Li, H. Lin, Q. Cai, S. Yao, Polycyclic aromatic hydrocarbon detection by electrochemiluminescence generating Ag/TiO2 nanotubes, Anal. Chem. 82 (2010) 7357–7361. [16] S. Miao, Z. Liu, Z. Zhang, B. Han, Z. Miao, K. Ding, Ionic liquid-assisted immobilization of Rh on attapulgite and its application in cyclohexene hydrogenation, J. Phys. Chem C 111 (2007) 2185–2190. [17] L. Zhang, F. Lv, W. Zhang, R. Li, H. Zhong, Y. Zhao, Y. Zhang, X. Wang, Photo degradation of methyl orange by attapulgite–SnO2 –TiO2 nanocomposites, J. Hazard. Mater. 171 (2009) 294–300. [18] X. Sun, Y. Zhang, H. Shen, N. Jia, Direct electrochemistry and electrocatalysis of horseradish peroxidase based on halloysite nanotubes/chitosan nanocomposite film, Electrochim. Acta 56 (2010) 700–705. [19] Y. Guo, S. Guo, Y. Fang, S. Dong, Gold nanoparticle/carbon nanotube hybrids as an enhanced material for sensitive amperometric determination of tryptophan, Electrochim Acta 55 (2010) 3927–3931. [20] H.R. Zhang, M.S. Wu, J.J. Xu, H.Y. Chen, Signal-on dual-potential electrochemiluminescence based on lumino–Au bifunctional nanoparticles for telomerase detection, Anal. Chem. 86 (2014) 3834–3840. [21] Z. Lin, Y. Liu, G. Chen, TiO2 /nafion film based electrochemiluminescence for detection of dissolved oxygen, Electrochem. Commun. 10 (2008) 1629–1632. [22] H. Dai, X. Wu, Y. Wang, W. Zhou, G. Chen, An electrochemiluminescent biosensor for vitamin C based on inhibition of luminol electrochemiluminescence on graphite/poly(methylmethacrylate) composite electrode, Electrochim. Acta 53 (2008) 5113–5117. [23] Y.Z. Wang, H. Zhong, X.M. Li, F.F. Jia, Y.X. Shi, W.G. Zhang, Z.P. Cheng, L.L. Zhang, J.K. Wang, Perovskite LaTiO3 –Ag0.2 nanomaterials for nonenzymatic glucose sensor with high performance, Biosens. Bioelectron. 48 (2013) 56–60.
Biographies
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.02.026.
Yin-Zhu Wang obtained her MS in applied chemistry from Nanjing tech university, China. She is currently a Ph.D. candiadate at Nanjing University. Her main research interests are biosensors.
References
Hui Zhong is a professor of Huaiyin Normal University, China. She received her Ph.D. degree in analytical chemistry from Nanjing University, China in 2005. The main research interests of professor Zhong include chemical sensors and biosensors.
[1] L. Liu, Q. Ma, Y. Li, Z. Liu, X. Su, A novel signal-off electrochemiluminescence biosensor for the determination of glucose based on double nanoparticles, Biosens. Bioelectron. 63 (2015) 519–524. [2] S. Zhang, L. Han, C. Hou, C. Li, Q. Lang, L. Han, A. Liu, Novel glucose sensor with Au@Ag heterogeneous nanorods based on electrocatalytic reduction of hydrogen peroxide at negative potential, J. Electroanal. Chem. 742 (2015) 84–89. [3] H. Wang, Y. Yuan, Y. Chai, R. Yuan, Self-enhanced electrochemiluminescence immunosensor based on nanowires obtained by a green approach, Biosens. Bioelectron. 68 (2015) 72–77. [4] B. Haghighi, S. Bozorgzadeh, Fabrication of a highly sensitive electrochemiluminescence lactate biosensor using ZnO nanoparticles decorated multiwalled carbon nanotubes, Talanta 85 (2011) 2189–2193. [5] H.R. Zhang, J.J. Xu, H.Y. Chen, Electrochemiluminescence ratiometry: a new approach to DNA biosensing, Anal. Chem. 85 (2013) 5321–5325. [6] X. Jiang, Y. Chai, H. Wang, R. Yuan, Electrochemiluminescence of luminol enhanced by the synergetic catalysis of hemin and silver nanoparticles for sensitive protein detection, Biosens. Bioelectron. 54 (2014) 20–26. [7] Y. Cheng, R. Yuan, Y. Chai, H. Niu, Y. Cao, H. Liu, L. Bai, Y. Yuan, Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection, Anal. Chim. Acta 745 (2012) 137–142. [8] S. Park, H. Boo, T.D. Chung, Electrochemical non-enzymatic glucose sensors, Anal. Chim. Acta 556 (2006) 46–57. [9] L.L. Liu, Q. Ma, Y. Li, Z.P. Liu, X.G. Su, A novel signal-off electrochemiluminescence biosensor for the determination of glucose based on double nanoparticles, Biosens. Bioelectron. 63 (2015) 519–524. [10] X.M. Chen, Z.M. Cai, Z.J. Lin, T.T. Jia, H.Z. Liu, Y. Jiang, X. Chen, A novel non-enzymatic ECL sensor for glucose using palladium nanoparticles supported on functional carbon nanotubes, Biosens. Bioelectron. 24 (2009) 3475–3480.
Xiao-Rong Li is a lecturer of Huaiyin Normal University, China. She received her Ph.D. degree in analytical chemical from Nanjing University, China. Her interests are mainly in the areas of the synthesis of functional nanomateials and biosensor. Gen-Qing Liu is a MS candidate in the Nanjing Tech University, China. His main research interests are biosensors. Kai Yang is a MS candidate in the Nanjing Tech University, China. His main research interests are biosensors and elctrochenmistry. Min Ma is a student of Huaiyin Normal University. Her main research interests are elctrochenmistry. Li-Li Zhang is a professor of Huaiyin Normal University, China. She received her Ph.D. degree from Nanjing University of Science and Technology, China. She interests are in the areas of functional materials and new energy materials. Jing-Zhou Yin is a lecture of Huaiyin Normal University, China. He received her Ph.D. degree in chemistry from Nanjing University, China in 2010. His research interests are in the areas of biosensor. Zhi-Peng Cheng is an associate professor of Huaiyin Normal University, China. He received his Ph.D. degree in chemistry from Nanjing University of Science and Technology in 2008, China. His research interests are in the areas of functional material and analytical chemical. Ji-Kui Wang is a professor of Nanjing Tech University, China. He received his Ph.D. degree in applied chemistry from Nanjing Tech University. His interests are in the areas of applied chemistry.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Nonenzymatic electrochemiluminescence glucose sensor based on quenching effect on luminol using attapulgite–TiO2 Yin-Zhu Wang a,b , Hui Zhong a,b,∗ , Xiao-Rong Li a,∗ , Gen-Qing Liu a,b , Kai Yang a,b , Min Ma a , Li-Li Zhang a,∗∗ , Jing-Zhou Yin a , Zhi-Peng Cheng a , Ji-Kui Wang b a Jiangsu Key Laboratory for Chemistry of Low-Dimensional Materials, School of Chemistry & Chemical Engineering, Huaiyin Normal University, Huaian 223300, PR China b College of Sciences, Nanjing University of Technology, Nanjing 210009, PR China
a r t i c l e
i n f o
Article history: Received 13 September 2015 Received in revised form 1 February 2016 Accepted 6 February 2016 Available online 10 February 2016 Keywords: Nonenzymatic Electrochemiluminescence Glucose Att–TiO2 Luminol
a b s t r a c t A new nonenzymatic glucose ECL sensor based on attapulgite (Att) integrated with semiconductor material TiO2 was developed. The prepared material was characterized by transmission electron microscopy (TEM), X-ray diffractometer (XRD), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Based on ECL experimental results, Att–TiO2 composites were found to be able to improve the ECL properties of luminol. When glucose is added into the system, TiO2 NPs serve as a catalyst and dissolved O2 acts as a cosubstrate for the glucose oxidation reaction. Dissolved O2 also worked as coreactant for the luminol ECL emission. Due to the consumption of dissolved O2 , a quenching effect would appear on the luminol ECL emission. Under the optimized conditions, the linear logarithmic relationship between ECL intensity and the concentration of glucose was valid in the range from 1.0 mM to 1.0 nM (R = 0.9976) with a detection limit (S/N = 3) of 10.0 pM. In addition, the proposed sensor presented good reproducibility, stability, and sensitivity for glucose detection and can be successfully applied in the determination of glucose in real blood samples. © 2016 Published by Elsevier B.V.
1. Introduction Glucose is the primary energy source of the body. The level of glucose in blood has been used for diagnosis of diabetes or hypoglycemia. Besides the need in glucose monitoring in the case of diabetes patients, it is also essential for non-diabetic acute care patients in order to control glucose levels [1]. Therefore, the development of fast, sensitive, selective and reliable methods for glucose monitoring is important in clinical diagnostics, food industry and biotechnology [2]. Electrochemiluminescence (ECL), a special form of chemiluminescence (CL) in which light emission is generated by electrochemical reactions, has been receiving higher and higher attention because of its excellent sensitivity, low background signal and some additional advantages [3], since Haghighi and Bozorgzadeh reported ECL behaviors of Si for the first time
∗ Corresponding author at: Jiangsu Key Laboratory for Chemistry of LowDimensional Materials, School of Chemistry & Chemical Engineering, Huaiyin Normal University, Huaian 223300, PR China. ∗∗ Corresponding author. E-mail addresses: [email protected] (H. Zhong), [email protected] (X.-R. Li), [email protected] (L.-L. Zhang). http://dx.doi.org/10.1016/j.snb.2016.02.026 0925-4005/© 2016 Published by Elsevier B.V.
in 2002 [4]. Among various ECL systems, luminol is considered as one of the most popular ECL luminophor due to its low oxidation potential, inexpensive reagent consumption and the high emission yields [5,6]. So, special attention had focused on the ECL studies concerning luminol for glucose analysis [7]. Luminol-based ECL glucose biosensors on the oxidation of glucose catalyzed by glucose oxidase have the advantages of being a simplified and sensitive instrument. However, these so-called biosensors have poor stability because glucose oxidase (GOx) quickly loses its activity at below pH 2 and above pH 8 and when temperature is above 40 ◦ C. In addition, these compounds suffer from badly damage or time consuming due to their long-winded fabrication procedure for the fixing enzyme onto the electrode [8]. Therefore, researchers have focused on nonenzymatic glucose ECL sensors. Currently, only four literatures have reported the corresponding nonenzymatic ECL behavior of luminol to determine glucose [9–12]. It is desirable to discover more materials to develop effective enzyme-less luminol-based ECL biosensors of detecting glucose. As far as we know, the ECL intensity is greatly influenced by the property of the working electrode, especially the material’s surface property. Titania dioxide (TiO2 ), an important semiconductor material with a large surface area, has previously been researched for photo-assisted degradation of a variety of toxic chemicals.
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Titania is known to be an effective catalyst for the cleavage of diols when reactive oxygen species (ROS) is used as oxidant [13]. Therefore, it would be a good working electrode for ECL detection and has attracted increasing interest in recent years [14]. Because the modified TiO2 nanocrystals are easy to fall off from the electrode surfaces, the electrodes have poor stability and the ECL intensity is not strong enough [15]. Considering the utilization and conversion of abundant negatively charged product, attapulgite (Att), which helps to form relatively high surface area and moderate cation exchange capacity [16] due to its permanent negative charges on its surface, has been successfully used to make TiO2 more active. There are large reserves of Att in Huai’an, China. The specific property and large distribution of Att make it to be studied undoubtedly. In present paper, we combined the huge specific surface area of Att, which can fix TiO2 onto the electrode surface more stable and prevent the deposition of luminol oxidation products. In addition, we found that AttTiO2 nanohybrids could effectively enhance luminol’s ECL in experimental process. The obtained sensor exhibited very sensitive ECL quenching responses for detection of glucose. The analytical procedure of this ECL sensing is illustrated in Scheme 1. First, Att–TiO2 composites material was prepared, then anchor it onto the surface of GCE, this composite can improve the ECL properties. While the glucose was added into the solution, glucose was oxidized in the presence of TiO2 , and part of oxygen radicals (O2 • −) were consumed, so that the ECL signal was quenched. The intensity of quenching signal was linearly associated with the logarithm of the concentration of glucose, based on it, the detection of glucose could be achieve. The stable and strong ECL emission guarantees that Att–TiO2 composite works as a sensitive sensor. 2. Experimental 2.1. Reagents and chemicals Luminol was obtained from J&K Chemical. Glucose and KH2 PO4 were received from Sinopharm Chemical Reagent Co., LTD. (Shanghai, China). K3 [Fe(CN)6 ] was purchased from Guangdong Shantou West Long Chemical Plant. K4 [Fe(CN)6 ]·3H2 O was obtained from Wuxi City and Yasheng Chemical. NaOH was from Nanjing Chemical Reagent Co., LTD. Phosphated-buffered solution (PBS) (pH 7.4, 0.1 M) was prepared using 0.1 M KH2 PO4 and 0.2 M NaOH. A 1.0 mM luminol (3-aminophthalhydrazide) stock solution was prepared by dissolving it in a small amount of 0.1 M NaOH. A stock solution of glucose (1.0 mM) was prepared in PBS and stored at 4 ◦ C when it was not in use. The stock solution of glucose was allowed to mutarotate at room temperature for 24 h before use. Different concentrations of working solutions were diluted step by step with PBS. Human serum samples were kindly provided by Huai’an First People’s Hospital. All chemicals were of analytical reagent grade and were used without further purification. All solutions were prepared exclusively in double distilled water.
4 h, the suspension was filtered, washed with water, dried at 80 ◦ C and calcined at 300 ◦ C for 4 h. After grinding to about 200 meshes in a carnelian mortar, TiO2 oxides coated onto Att were obtained and labeled as Att–TiO2 . 2.3. Instrumentation and procedures The morphologies of the Att–TiO2 nanohybrids were characterized by transmission electron microscopy (TEM) (JEM-3011, JEOL, Japan). The crystal structures of the samples were examined by an X-ray diffractometer (XRD, BrukerD8, Advanee, Germany) with Cu K␣ radiation () 0.15406 nm. Electrochemical impedance spectroscopy (EIS) measurements were monitored with an Autolab PGSTAT 30 Analyzer (Metrohm Autolab B.V., Switzerland) in a solution containing 5 mM Fe(CN)6 3−/4− and 0.1 M KCl. The frequency was ranged from 0.1 to 100,000 Hz with an alternating current voltage of 10 mV. Cyclic voltammetry (CV) and ECL measurements were taken using a homemade ECL/EC system. Cyclic voltammetrys (CVs) were carried out using an electrochemical working station (CHI 660D, Chenhua Inc., China) in 0.1 M KCl solution containing 5.0 mM K3 Fe(CN)6 /K4 Fe(CN)6 and in PBS (pH 7.4) for the bare GCE and Att–TiO2 -midified GCE. The ECL signals were acquired by a MPI-M multifunctional analytical system (Xi’an Remax Electronic Science Tech. Co., Ltd., Xi’an, China) with the voltage of the photomultiplier tube (PMT), which is used for transforming ECL emission into electrical signals, set at 600 V in the process of detection and the ECL detector cell was placed in front of the PMT. The detector cell was made of a microbeaker (high: 35 mm, i.d.: 25 mm) and we performed the ECL measurements in 3 mL PBS (pH 7.4) with 0.35 mM luminol and 1.0 M glucose. In addition, all electrochemical experiments were performed with a conventional three-electrode set-up where glassy carbon electrode was used as working electrode, platinum wire as counter electrode and Ag/AgCl (saturated KCl solution) as reference electrode, respectively. All measurements were performed at room temperature. 2.4. Electrodes preparation and modification A glassy carbon electrode (GCE, 3 mm diameter) was used for the preparation of the nonenzymatic glucose ECL biosensor. Before electrode modification, the GCE was polished with 0.3 and 0.05 m alumina paste (Gao Shi Rui Group Technology Co., LTD., Wuhan, China) on chamois leather and carefully rinsed with 1:20 (v/v) nitric acid, ethanol and doubly distilled water in an ultrasonic bath to give a smooth and clean electrode surface. Next, 1.0 mg of Att–TiO2 was dispersed in 1.0 mL double distilled water with ultrasonic agitation for 1 h to achieve a well-dispersed suspension. Then, a 5.0 L of the prepared Att–TiO2 suspension was pipetted on the surface of GCE to construct Att–TiO2 -modified GCE and allowed to be dried in air at room temperature. 3. Results and discussion
2.2. Synthesis of Att–TiO2 nanohybrids
3.1. Characterization of Att–TiO2 nanocomposites
The nanomaterials of Att–TiO2 were prepared by a similar route to the technique reported before [17]. The first step is the simple purification of Att. 1.0 g of raw Att when it was dispersed in 100 mL distilled water and ultrasound for 0.5 h, then the nether sand and large stones were removed. This produced a homogeneous Att suspension which was used directly in the later modification section without further acid activation. Secondly, 3.4 g of Ti(OBu)4 was added to the as-prepared Att suspension, and then 100 mL distilled water was added drop by drop to let the Ti(OBu)4 hydrolyze in situ to deposit Ti(OH)4 onto the surface of Att. After vigorous stirring for
Fig. 1A shows the XRD patterns of Att (a), TiO2 (b) and Att–TiO2 (c). The reflections of Att (a) at 2 = 8.34◦ , 19.8◦ , 26.6◦ , 28.0◦ and 34.9◦ were consistent with the standard spectrogram Att (JCPDS No. 37-0783) structure. Their corresponding crystal face index are (1 1 0), (0 4 0), (2 3 1), (4 0 0) and (1 0 2) respectively. The reflections (b) at 2 = 25.3◦ , 37.8◦ and 48.0◦ corresponded to TiO2 that is consistent with the standard spectrogram TiO2 (anatase) (JCPDS No. 65-5714) structure. Their corresponding crystal face index are (1 0 1), (0 0 4) and (2 0 0). Montmorillonite and quartz were also found. The characteristic reflections of Att were observed in the
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Scheme 1. The analytical procedure of Att–TiO2 electrode for glucose detection.
Fig. 1. (A) XRD characterization of Att (a), TiO2 (b) and Att–TiO2 (c) (♦ Attapulgite TiO2 (Anatase). (B) The TEM image of Att–TiO2 . (C) Electrochemical impedance spectra and cyclic voltammograms (D) of (a) bare GCE, (b) Att-modified GCE, (c) Att–TiO2 -modified GCE in 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl solution. Inset: the equivalent circuit.
XRD pattern of Att–TiO2 composite (c). In Fig. 1A, the characteristic reflections of TiO2 as anatase were also confirmed. In addition,
the TEM image of Att–TiO2 compounds s shown in Fig. 1B. The Att samples have an average diameter of about 50 nm. The surface
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Fig. 2. (A) IECL /E curves of luminol at the GCE (a), Att-modified GCE (b), Att–TiO2 -modified GCE(c). Inset: CV curves of luminol at the GCE (a), Att-modified GCE (b), Att–TiO2 modified GCE(c); conditions: luminol, 0.35 mM; supporting electrolyte, PBS (pH 7.4); scan rate, 100 mV/s. (B) IECL /E curves of luminol in the absence (a) and in the presence (b) of 1.0 M glucose at Att–TiO2 -modified GCE. Inset: CV curves of luminol in the absence (a) and in the presence (b) of 1.0 M glucose at Att–TiO2 -modified GCE; conditions: luminol, 3.5 × 10−4 M; supporting electrolyte, PBS (pH 7.4); scan rate, 100 mV/ s.
of Att–TiO2 was fully covered with nanoparticles about 10 nm in average and uniformly distributed, without obvious aggregation.
area and provided the conducting bridges for the electron-transfer of Fe(CN)6 3−/4− , which is consistent with the result of EIS. All of these results demonstrated that Att–TiO2 hybrids provided high electron conduction pathways.
3.2. Electrochemical characterization of Att–TiO2 modified GCE In the experiment, we respectively fabricated the bare GCE, Att and Att–TiO2 -modified GCEs and employed electrochemical impedance spectrum (EIS) of Fe(CN)6 4−/3− to monitor the features of above modified electrodes in Fig. 1C, because it is one of the most powerful and effective techniques to investigate the modification process [18]. The electron-transfer resistance (Rct) at the electrode surface is equal to the semicircle diameter of EIS and can be used to describe the interface properties of the electrode. The Randles circuit (inset of Fig. 1C) was chosen to fit the impedance data obtained. The resistance to charge transfer and the diffusion impedance (W) were both in parallel with the interfacial capacitance (Cdl). The diameter of the semicircle corresponds to the interfacial electrontransfer resistance (Rct) [19]. By fitting the data, the redox process of the [Fe(CN)6 ]3−/4− probe showed a Rct of 439.1 Ohm at the bare GCE (a). This value of Rct remarkably increased to about 643.2 Ohm after Att droped onto the GCE surface (b), indicating Att on the electrode surface blocked the electron transfer between [Fe(CN)6 ]3−/4− probe and electrode. The remarkable increase of Rct also indicates that Att has been successfully immobilized on the GCE surface. However, when the Att–TiO2 assembled onto the GCE surface, the Rct values decrease to about 306.7 Ohm (c), which suggested that Att–TiO2 was excellent electronic conducting materials and could accelerate the electron transfer between [Fe(CN)6 ]3−/4− probe and electrode. Considering that Att itself is not a conductor, the superior conductivity of Att–TiO2 here is may be that TiO2 play an important role similarly to a conducting wire or electron-conducting tunnel, which makes it easier for the electron transfer to take place. On the other hand, cyclic voltammograms of 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl solution on the bare GCE (a), Att-modified GCE (b) and Att–TiO2 -modified GCE (c) are shown in Fig. 1D. The quasi-reversible one-electron redox behavior of ferricyanide ions was observed on the bare GCE with a peak separation (Ep) of 74 mV at the scan rate of 100 mV/s. After being modified with Att, the peak current of Fe(CN)6 3−/4− was reduced compared with that observed at the bare GCE. For Att–TiO2 -modified GCE, the peak current of Fe(CN)6 3−/4− was increased greatly instead compared with that of the Att-modified GCE and bare GCE, revealing that the introduction of the Att–TiO2 hybrid played a role in the increase of the electroactive surface
3.3. Enhanced ECL response of luminol on Att–TiO2 -modified GCE In order to verify whether indeed the ECL sensor works as expected, we recorded step by step the ECL signal intensity of the sensor in PBS (pH 7.4) containing 0.35 mM luminol with the scan rate of 100 mV/s at the GCE, Att and Att–TiO2 -modified GCEs. As shown in Fig. 2A, in the IECL /E curves, the ECL signal of the bare GCE was found to be very weak (a). After the modification with Att, the ECL intensity of the electrode kept constant (b). That was attributed to the hindrance of Att. But if we use the Att–TiO2 modified GCE, a clear ECL wave occurred near 0.4 V during the anodic scan (−0.4–0.8 V), and then the ECL intensity rose steeply until it reached a maximum near 0.61 V (c), which was consistent with the oxidation potential of luminol [20]. The experimental results indicate that the ECL signal of the luminol is strongly dependent upon the TiO2 nanoparticles, the interaction between given chemical species and the surface of TiO2 nanoparticles would result in changes in the ECL efficiency. A similar enhancement effect was also observed in their corresponding cyclic voltammograms (CVs) (inset of Fig. 2A). In the CV curves, it can be clearly seen that there is no enhancement effect at bare GCE (a) and Att-modified GCE (b). When the modified electrode was incubated with Att–TiO2 (c), the anodic peak current intensity of luminol (at about 0.55 V) enhanced remarkably compared with Att, indicating Att–TiO2 provided large surface area and active sites for luminol oxidation reaction and consequently showed excellent electrocatalytic activity and high current intensity. 3.4. Quenching ECL response of Att–TiO2 nanocomposites toward glucose and the corresponding mechanism We studied the IECL /E curves and CV curves with and without glucose solution at pH 7.4 (Fig. 2B). In the IECL /E curves, an ECL signal at 0.61 V could be observed in luminol–PBS solution (a). Comparatively, we discovered that the ECL peak obviously decreased even if the solution contained only a small amount of glucose (b). As shown in inset of Fig. 2B, Att–TiO2 -modified GCE appeared an oxidization peak toward glucose (a). In addition, the anodic peak was
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3.5. Performance of the ECL sensor for glucose detection
Fig. 3. ECL spectrum of the luminol/Att–TiO2 (a) and Att–TiO2 (b) system.
negatively shifted when a proper amount of glucose was added (b). This indicates that Att–TiO2 has quenching effect on the oxidation of glucose. To explain the mechanism for the quenching effect, the ECL emission spectrum of Att–TiO2 and luminol/Att–TiO2 system are depicted in Fig. 3. It can be seen from curve a that the maximum emission at 420 nm is corresponding to the ECL emission of luminol. In addition, a quite weaker emission at 520 nm is corresponding to the ECL emission of TiO2 , which is nearly the same as the photoluminescence spectrum of TiO2 powder. Without luminol, the 520 nm emission is very weak (curve b). These results suggest that the ECL intensity is the combination of the two above optical signals. However, the intensity of the latter is negligible compared with that of the former, suggesting the little effect on the glucose detection. The possible inhibition mechanism of glucose on the luminol ECL response is shown as follows: TiO2 nanoparticles are reduced to TiO2 • − radicals at the surface of Att–TiO2 -modified electrode (Reaction (1)). The TiO2 • − radicals react with the dissolved O2 to produce strong oxidant O2 • − radicals (Reaction (2)) which play a key role in the progress of luminol ECL, as reported in the literature [21]. The strong oxidant O2 • − radicals react with the intermediate oxidation state of luminol (L− ) which was the product of the reaction of TiO2 • −with L− , thus generating the excited state of 3-aminophthalate according to Reactions (3) and (4). While the excited state of 3-aminophthalate goes back to the ground state, emission happens at 425 nm. The oxidation of luminol by the O2 •− radicals was indirectly confirmed by the result that luminol can be electrooxidized at the Att–TiO2 electrode in the range of −0.4–0.8 V. On the other hand, the reaction of TiO2 • − with L− would also produce the excited state of TiO2 with emission at 520 nm while it goes back to the ground state according to Reactions (3) and (5). This speculation was confirmed by such a result that the emission at 520 nm was enhanced with the addition of luminol. Without luminol, the emission at 520 nm is very weak. After adding of glucose into the luminol/Att–TiO2 system, glucose reacts with O2 •− radicals to generate gluconic acid (Reaction (6)) resulting in a decrease in the luminol ECL intensity [22]. The possible ECL route of luminol/Att–TiO2 using dissolved O2 as the coreactant was proposed as follows:
Before the application of this sensor to the accurate analysis, the optimized conditions such as the amount of Att–TiO2 , scan rates, luminol concentrations and the optimum pH were described in Supporting information. ECL intensity was measured step by step with Att–TiO2 -modified GCE in 9.0 mL PBS (pH 7.4) containing luminol (0.35 mM) and glucose with different concentrations. Fig. 4A shows the glucose concentration-dependent ECL intensity and the corresponding calibration curve (Fig. 4B). When the glucose concentration increases, the ECL signal decreases accordingly, indicating that the glucose could quench the ECL intensity of luminol in the presence of catalyst Att–TiO2 [23]. The inhibition ratio is linear with the logarithm of glucose concentration in the range of 1.0 mM–1.0 nM with a regression equation: Log I = −0.17065Log C + 2.6874(R = 0.9976) where I is the ECL intensity and C is the glucose concentration, the detection limit (S/N = 3) was 10 pM and the sensitivity was 288.6 nM−1 cm2 . As seen from Table 1, our non-enzymatic ECL sensor in this work exhibited a better performance than that in most reported works in terms of linear range and detection limit [9–12]. This may be due to the Att–TiO2 nanocomposites with good film stability and high conductivity for enhanced electrocatalytic response, which accelerates the electron transfer. In addition, dissolved oxygen, as the co-reagent of luminol, was only a very small amount in PBS, but luminol could be emitted at Att–TiO2 -modified GCE. So, it leads to such a high sensitivity of Att–TiO2 in sensing applications and is very sensitive to glucose. On the other hand, in the process of the reaction, when the glucose concentration increases, the ECL signals decrease accordingly.
3.6. Application of biosensor for detecting glucose in normal human serum sample In order to gauge the applicability of the proposed ECL biosensor to the detection of glucose in real samples, this nonenzymatic ECL biosensor was applied for determination of glucose concentrations in human serum samples (other parameters were used as optimum conditions), which were obtained from Huai’an First People’s Hospital. It should be noted that, prior to use, the serum samples were diluted 100-fold with normal saline. Proper quantity of the human serum was mixed with 9.0 mL solution containing PBS (pH 7.4) and luminal (0.35 mM). The results from these determinations are presented in Table 2, which shows an excellent agreement between the results from the present method and the reference values (reference values were provided by the Huai’an First People’s Hospital). In addition, a series of recoveries were also performed to evaluate the accuracy and the recovery values ranged from 98.7% to 101.9%, indicating good accuracy of the Att–TiO2 -modified GCE. These proved that this method was reliable and suitable for determining glucose in serum samples.
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Fig. 4. (A) ECL responses of luminol at Att–TiO2 -modified GCE toward different concentration of glucose. Conditions: luminol, 3.5 × 10−4 M; supporting electrolyte, PBS (pH 7.4); scan rate: 100 mV/s. a: 1.0 mM, b: 0.1 mM, c: 10 M M, d: 1.0 M, e: 0.2 M, f: 50 nM, g: 10 nM, h: 2.0 nM, i: 1.0 nM. (B) Logarithmic calibration curve of the prepared glucose biosensor. (C) ECL signal specificity of sensor toward 1.0 M glucose, 30 nM AA, 30 nM DA and 30 nM UA at the Att–TiO2 -modified GCE in PBS (pH 7.4) containing 0.35 mM luminol. (D) The successively cyclic ECL responses of the biosensor toward 1.0 M glucose for 14 times; Conditions: supporting electrolyte, 0.1 M phosphate buffer (pH 7.4); scan rate, 100 mV/s.
Table 1 Comparison of different non-enzymatic ECL glucose sensor. Electrode
Methods
Linear range (M)
Detection limit (M)
Ref.
Au NPs-CdTe QDs-CHIT/GCE PdNPs-FCNTse/nafion-GCE LaTiO3 –Ag0.1 /GCE ConA/Tween20/DexP/g-C3 N4 -PTCA/GCE Att–TiO2 -modified GCE
ECL ECL ECL ECL ECL
1.0×10−5 −1.0×10−2 5.0×10−7 −4.0×10−5 1.0×10−8 −1.0×10−4 1.0×10−10 −5.2×10−5 1.0×10−9 −1.0×10−3
5.28×10−6 9.0×10−8 2.5×10−9 4.0×10−11 10−11
[9] [10] [11] [12] This work
Table 2 Determination of glucose in human blood serum samples. Serum samples
Hospital (mM)
Sensor (mM)
RSD (%)
Added (mM)
Found (mM)
Recovery (%)
1 2 3 4 5 6 7
12.16 5.56 6.71 5.9 4.57 4.49 6.04
12.34 5.63 6.84 6.00 4.48 4.54 5.98
0.813 0.822 0.653 1.205 1.981 2.008 3.106
0.1 0.1 0.1 0.1 0.1 0.1 0.1
12.441 5.7315 6.9417 6.1019 4.5787 4.6418 6.0793
101.2 101.5 101.7 101.9 98.7 101.8 99.3
3.7. Interferences, reproducibility and stability One of the most important analytical factors for a sensor is the ability of the sensor to discriminate the electroactive interfering species. The easy oxidizable compounds such as ascorbic acid (AA), dopamine (DA) and uric acid (UA) are normally co-existed with glucose in natural samples. Because the normal physiological level of glucose in the human blood is about 30 times of AA, DA and UA [23], we studied the interference effect of 30 nM AA, DA and UA on
the ECL response of 1.0 M glucose in the present work. The corresponding results are shown in Fig. 4C. The interference caused by these electroactive interfering species could be neglected, indicating a good selectivity of the prepared biosensors for glucose. We also investigated the reproducibility of ECL biosensor. As shown in Fig. 4D, the relative standard deviation (RSD) of the ECL response to 1.0 M glucose was 0.7% for 14 successive measurements, giving an acceptable assay reproducibility of the method due to the stable structure of Att–TiO2 composite. The stability
Y.-Z. Wang et al. / Sensors and Actuators B 230 (2016) 449–455
was measured by evaluating the responses of ECL biosensor to the glucose using the Att–TiO2 composite particles stored at room temperature in one month. The results showed that the ECL responses were kept constant in the first week, remained approximately 95% of the original value in the second week, and dropped to 85% in the fourth week. The results indicate that Att–TiO2 -modified GCE has a better stability. 4. Conclusions In summary, we have developed a high-performance nonenzymatic glucose ECL sensor based on Att integrated with semiconductor material TiO2 . The ECL behavior of luminol has been investigated in detail at the Att–TiO2 modified electrode, and glucose was found to be able to inhibit this ECL system. Based on this, an inhibited ECL detection method has been developed for determination of glucose. In addition, this sensor exhibited good reproducibility, wide-range linearity, high sensitivity and stability. The proposed method provided a promising way to develop efficient sensors to detect glucose in real blood samples. These demonstrations pave an avenue for multiple functional electrochemical sensors. Acknowledgments This research is supported by the National Natural Science Foundation of China (21375044, 51106061, 21201072, 21405055, 51472101), Natural Science Foundation of Jiangsu Province (BK20131211, BK20131214, BK2012241) and Natural Science Foundation of Jiangsu Higher Education Institutions (14KJB150004, 12JB150006). Appendix A. Supplementary data
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[11] F.F. Jia, H. Zhong, W.G. Zhang, X.R. Li, G.Y. Wang, J. Song, Z.P. Cheng, J.Z. Yin, L.P. Guo, A novel nonenzymatic ECL glucose sensor based on perovskite LaTiO3 –Ag0.1 nanomaterials, Sens. Actuators B 212 (2015) 174–182. [12] Y. Fan, X.R. Tan, X.F. Liu, X. Ou, S.H. Chen, S.P. Wei, A novel non-enzymatic electrochemiluminescence sensor for the detection of glucose based on the competitive reaction between glucose and phenoxy dextran for concanavalin a binding sites, Electrochim. Acta 180 (2015) 471–478. [13] A.M. Velarde, P. Bartl, T.E.W. Nießen, W.F. Hoelderich, Hydrogen peroxide oxidation of d-glucose with titanium-containing zeolites as catalysts, J. Mol. Catal. A: Chem. 157 (2000) 225–236. [14] C. Li, Q. Kang, Y. Chen, J. Li, Q. Cai, S. Yao, Electrochemiluminescence of luminol on Ti/TiO2 NT electrode and its application for pentachlorophenol detection, Analyst 135 (2010) 2806–2810. [15] J. Li, L. Yang, S. Luo, B. Chen, J. Li, H. Lin, Q. Cai, S. Yao, Polycyclic aromatic hydrocarbon detection by electrochemiluminescence generating Ag/TiO2 nanotubes, Anal. Chem. 82 (2010) 7357–7361. [16] S. Miao, Z. Liu, Z. Zhang, B. Han, Z. Miao, K. Ding, Ionic liquid-assisted immobilization of Rh on attapulgite and its application in cyclohexene hydrogenation, J. Phys. Chem C 111 (2007) 2185–2190. [17] L. Zhang, F. Lv, W. Zhang, R. Li, H. Zhong, Y. Zhao, Y. Zhang, X. Wang, Photo degradation of methyl orange by attapulgite–SnO2 –TiO2 nanocomposites, J. Hazard. Mater. 171 (2009) 294–300. [18] X. Sun, Y. Zhang, H. Shen, N. Jia, Direct electrochemistry and electrocatalysis of horseradish peroxidase based on halloysite nanotubes/chitosan nanocomposite film, Electrochim. Acta 56 (2010) 700–705. [19] Y. Guo, S. Guo, Y. Fang, S. Dong, Gold nanoparticle/carbon nanotube hybrids as an enhanced material for sensitive amperometric determination of tryptophan, Electrochim Acta 55 (2010) 3927–3931. [20] H.R. Zhang, M.S. Wu, J.J. Xu, H.Y. Chen, Signal-on dual-potential electrochemiluminescence based on lumino–Au bifunctional nanoparticles for telomerase detection, Anal. Chem. 86 (2014) 3834–3840. [21] Z. Lin, Y. Liu, G. Chen, TiO2 /nafion film based electrochemiluminescence for detection of dissolved oxygen, Electrochem. Commun. 10 (2008) 1629–1632. [22] H. Dai, X. Wu, Y. Wang, W. Zhou, G. Chen, An electrochemiluminescent biosensor for vitamin C based on inhibition of luminol electrochemiluminescence on graphite/poly(methylmethacrylate) composite electrode, Electrochim. Acta 53 (2008) 5113–5117. [23] Y.Z. Wang, H. Zhong, X.M. Li, F.F. Jia, Y.X. Shi, W.G. Zhang, Z.P. Cheng, L.L. Zhang, J.K. Wang, Perovskite LaTiO3 –Ag0.2 nanomaterials for nonenzymatic glucose sensor with high performance, Biosens. Bioelectron. 48 (2013) 56–60.
Biographies
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.02.026.
Yin-Zhu Wang obtained her MS in applied chemistry from Nanjing tech university, China. She is currently a Ph.D. candiadate at Nanjing University. Her main research interests are biosensors.
References
Hui Zhong is a professor of Huaiyin Normal University, China. She received her Ph.D. degree in analytical chemistry from Nanjing University, China in 2005. The main research interests of professor Zhong include chemical sensors and biosensors.
[1] L. Liu, Q. Ma, Y. Li, Z. Liu, X. Su, A novel signal-off electrochemiluminescence biosensor for the determination of glucose based on double nanoparticles, Biosens. Bioelectron. 63 (2015) 519–524. [2] S. Zhang, L. Han, C. Hou, C. Li, Q. Lang, L. Han, A. Liu, Novel glucose sensor with Au@Ag heterogeneous nanorods based on electrocatalytic reduction of hydrogen peroxide at negative potential, J. Electroanal. Chem. 742 (2015) 84–89. [3] H. Wang, Y. Yuan, Y. Chai, R. Yuan, Self-enhanced electrochemiluminescence immunosensor based on nanowires obtained by a green approach, Biosens. Bioelectron. 68 (2015) 72–77. [4] B. Haghighi, S. Bozorgzadeh, Fabrication of a highly sensitive electrochemiluminescence lactate biosensor using ZnO nanoparticles decorated multiwalled carbon nanotubes, Talanta 85 (2011) 2189–2193. [5] H.R. Zhang, J.J. Xu, H.Y. Chen, Electrochemiluminescence ratiometry: a new approach to DNA biosensing, Anal. Chem. 85 (2013) 5321–5325. [6] X. Jiang, Y. Chai, H. Wang, R. Yuan, Electrochemiluminescence of luminol enhanced by the synergetic catalysis of hemin and silver nanoparticles for sensitive protein detection, Biosens. Bioelectron. 54 (2014) 20–26. [7] Y. Cheng, R. Yuan, Y. Chai, H. Niu, Y. Cao, H. Liu, L. Bai, Y. Yuan, Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection, Anal. Chim. Acta 745 (2012) 137–142. [8] S. Park, H. Boo, T.D. Chung, Electrochemical non-enzymatic glucose sensors, Anal. Chim. Acta 556 (2006) 46–57. [9] L.L. Liu, Q. Ma, Y. Li, Z.P. Liu, X.G. Su, A novel signal-off electrochemiluminescence biosensor for the determination of glucose based on double nanoparticles, Biosens. Bioelectron. 63 (2015) 519–524. [10] X.M. Chen, Z.M. Cai, Z.J. Lin, T.T. Jia, H.Z. Liu, Y. Jiang, X. Chen, A novel non-enzymatic ECL sensor for glucose using palladium nanoparticles supported on functional carbon nanotubes, Biosens. Bioelectron. 24 (2009) 3475–3480.
Xiao-Rong Li is a lecturer of Huaiyin Normal University, China. She received her Ph.D. degree in analytical chemical from Nanjing University, China. Her interests are mainly in the areas of the synthesis of functional nanomateials and biosensor. Gen-Qing Liu is a MS candidate in the Nanjing Tech University, China. His main research interests are biosensors. Kai Yang is a MS candidate in the Nanjing Tech University, China. His main research interests are biosensors and elctrochenmistry. Min Ma is a student of Huaiyin Normal University. Her main research interests are elctrochenmistry. Li-Li Zhang is a professor of Huaiyin Normal University, China. She received her Ph.D. degree from Nanjing University of Science and Technology, China. She interests are in the areas of functional materials and new energy materials. Jing-Zhou Yin is a lecture of Huaiyin Normal University, China. He received her Ph.D. degree in chemistry from Nanjing University, China in 2010. His research interests are in the areas of biosensor. Zhi-Peng Cheng is an associate professor of Huaiyin Normal University, China. He received his Ph.D. degree in chemistry from Nanjing University of Science and Technology in 2008, China. His research interests are in the areas of functional material and analytical chemical. Ji-Kui Wang is a professor of Nanjing Tech University, China. He received his Ph.D. degree in applied chemistry from Nanjing Tech University. His interests are in the areas of applied chemistry.