Biosensors and Bioelectronics 63 (2015) 519–524
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A novel signal-off electrochemiluminescence biosensor for the determination of glucose based on double nanoparticles Linlin Liu 1, Qiang Ma 1, Yang Li, ZiPing Liu, Xingguang Su n Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 May 2014 Received in revised form 25 July 2014 Accepted 29 July 2014 Available online 7 August 2014
In this work, a novel facile signal-off electrochemiluminescence (ECL) biosensor has been developed for the determination of glucose based on the integration of chitosan (CHIT), CdTe quantum dots (CdTe QDs) and Au nanoparticles (Au NPs) on the glassy carbon electrode (GCE). Chitosan displays high water permeability, hydrophilic property, strong hydrogel ability and good adhesion to load the double nanoparticles to the glassy carbon electrode surfaces. Au NPs are efficient glucose oxidase (GOx)-mimickess to catalytically oxidize glucose, similar to the natural process. Upon the addition of glucose, the Au NPs catalyzed glucose to produce gluconic acid and hydrogen peroxide (H2O2) based on the consumption of dissolved oxygen (O2), which resulted in a quenching effect on the ECL emission. Therefore, the determination of glucose could be achieved by monitoring the signal-off ECL biosensor. Under the optimum conditions, the ECL intensity of CdTe QDs and the concentration of glucose have a good linear relationship in the range of 0.01–10 mmol L 1. The limit of detection for glucose was 5.28 μmol L 1 (S/N¼3). The biosensor showed good sensitivity, selectivity, reproducibility and stability. The proposed biosensor has been employed for the detection of glucose in human serum samples with satisfactory results. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Biosensor Chitosan CdTe QDs Au nanoparticles Glucose
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiment section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Materials and apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Preparation of CdTe QDs and Au NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Fabrication of ECL biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. ECL measurement procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Human serum samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Characterization of CdTe QDs and AuNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. EIS behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Optimization of reaction conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Electrochemiluminescence behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Stability and reproducibility of the biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Glucose determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
n
Corresponding author. Tel.: þ 86 431 85168352. E-mail address: [email protected] (X. Su). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.07.087 0956-5663/& 2014 Elsevier B.V. All rights reserved.
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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 for glucose monitoring in the case of diabetes patients, it is also essential for non-diabetic acute care patients in order to control glucose levels (Matz et al., 2006). Therefore, a huge amount of glucose biosensors have been reported, such as those based on optical techniques (Barone et al., 2005), color metric measurement (Morris et al., 1992) and capacitive detection (Cheng et al., 2001). In recent years, a tremendous amount of attention and effort has been paid to develop ultrasensitive glucose biosensors including electrochemical (Si et al., 2013), surface enhanced Raman scattering (Wu et al., 2006), chemiluminescence (Li et al., 2000), electrochemical transistor biosensor (Liu et al., 2008) and potentiometric biosensor (Liao et al., 2007). However, many approaches suffer from limitations of complicated pretreatment steps, equipment costs and expensive labor resources and time are consuming. Obviously, an effective analytical method for rapid determination of glucose is urgently needed. Electrochemiluminescence (ECL) is a versatile analytical technique that combines the simplicity of electrochemistry with inherent sensitivity and wide linear ranges of the chemiluminescence method. Most importantly, ECL biosensors have lots of advantages, including high sensitivity, low backgound, ease of control and simple equipments (Zheng et al., 2001; Chovin et al., 2004). Semiconductor quantum dots (QDs) have attracted great attention in the past decade, due to their remarkable optoelectronic properties, such as narrow, symmetrical and size-tunable emission spectrum, broad excitation spectrum, large Stokes shift, high photo-bleaching threshold and good chemical stability (Hanif et al., 2002; Ma and Su, 2011). Uptil now, numerous reviews about QDs-based ECL have been published (Lei and Ju, 2011; Huang et al., 2011; Li et al., 2012). Further, QDs have been widely used to detect various substances via ECL methods. At the same time, great effort has been focused on the QD-based ECL behavior and mechanism. The thin-film technique has provided an effective way for the construction of QD-based ECL biosensors in aqueous solutions (Jie et al., 2008). Chitosan is a polysaccharide biopolymer, which displays excellent film-forming ability, high water permeability and hydrophilic property, strong hydrogel ability and good adhesion with the functional nanoparticles to the electrode surfaces (Kumar et al., 2004; Zhang et al., 2004). Furthermore, the previous research works demonstrated that the CHIT had strong adsorbing ability (Luo et al., 2005). Due to the attractive electronic properties, Au nanoparticles (Au NPs) have a wide range of applications like catalysis and bioanalysis (Gong and Mullins, 2009; Lee et al., 2010; Corma and
Garcia, 2008; Hammer, 2006). Recently, Au NPs were found to be efficient GOx-mimickess; they can catalytically oxidize glucose and produce gluconates in a “green” approach, similar to that of the natural enzyme of GOx (Comotti et al., 2004). Au NPs also catalyzed the oxidation of glucose with the cosubstrate oxygen (O2), producing gluconate and hydrogen peroxide (Beltrame et al., 2006). Comparing with glucose oxidase, Au NPs as biomimetic enzymes possess more advantages, such as providing an easy approach for preparation and purification, having high stability, avoiding degeneration and inactivation. In this paper, we report a new and simple method for the fabrication of an ECL biosensor by using an Au NPs-CdTe QDsCHIT/GCE composite film. Scheme 1 describes the preparation of the composite film and the mechanism of the ECL system for analysis of glucose. For the composite films, CHIT can connect CdTe QDs and Au NPs to the glass carbon electrode via powerful adsorption ability. When glucose is added into the system, Au 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 cathodic CdTe QDs ECL emission. Due to the consumption of dissolved O2 , a quenching effect would appear on the CdTe QDs ECL emission. The main features of this biosensor are: (i) chitosan displays excellent film-forming ability, high water permeability, strong hydrogel ability and good adhesion to load the double nanoparticles onto the glassy carbon electrode surfaces; (ii) Au NPs have not only accelerated electrical conductivity but can also oxidize glucose as GOx-mimickers in this green noenzyme system; and (iii) to the best of our knowledge, the signaloff CdTe QDs ECL biosensor for glucose determination based on Au NPs as GOx-mimickess has not been reported before. The proposed Au NPs-CdTe QDs-CHIT/GCE system is a novel ECL biosensor with good selectivity and stability. Further, it was successfully applied to the determination of glucose in samples of human serum with good accuracy and precision.
2. Experiment section 2.1. Materials and apparatus All chemicals and reagents were analytical grade and used directly without further purification. 3-Mercaptopropyl acid (MPA) was purchased from J&K Chemical. Tellurium powder (∼200 mesh, 99.8%), CdCl2 (99%) and NaBH4 (99%) were purchased from Aldrich Chemical Co. HAuCl4 was purchased from Acros Organics. Glucose and other materials were obtained from Beijing Dingguo Biotechnology Co. Ltd. All reagents were prepared using ultrapure water
Scheme 1. The schematic illustration of the ECL biosensor for determination glucose based on Au NPs-CdTe QDs-CHIT/GCE composite film.
L. Liu et al. / Biosensors and Bioelectronics 63 (2015) 519–524
with a resistivity of greater than 18 MΩ cm 1. All experiments were carried out at room temperature. Photoluminescence (PL) spectra measurements were performed on a Shimadzu RF-5301 PC spectrofluorophotometer (Shimadzu Co., Kyoto, Japan). UV–vis absorption measurements were obtained using a Varian GBC Cintra 10e UV–vis spectrometer. The ECL detection system consisted of a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) and a CHI 660B electrochemical workstation (Shanghai Chenhua Instrument Co., China). The voltage of the photomultiplier tube (PMT) was set at 900 V during the whole processes. Electrochemical impedance spectroscopic (EIS) analysis was performed on a CHI 660B electrochemical workstation in 2.5 mmol L 1 K3[Fe(CN)6]/K4[Fe(CN)6] with 0.1 mol L 1 KCl as the supporting electrolyte. A conventional three-electrode system was used. A glassy carbon electrode (GCE) was used as the working electrode. A platinum wire was employed as the counter electrode and an Ag/AgCl (saturated KCl) electrode as the reference electrode. 2.2. Preparation of CdTe QDs and Au NPs The water-soluble CdTe QDs were synthesized by refluxing routes (Ma et al., 2010). In brief, sodium hydrogen telluride (NaHTe) was produced in an aqueous solution by the reaction of sodium borohydride (NaBH4) with tellurium powder at a molar ratio of 2:1 at first. Later, freshly synthesized oxygen-free NaHTe solution was mixed with nitrogen-saturated 1.25 mmol L-1 CdCl2 solution at pH 11.4, with MPA as a stabilizing agent. The molar ratio of Cd2 þ /MPA/HTe was charged at 1:2.4:0.5. Then the CdTe precursor solution was subjected to reflux at 100 °C under stirring and condenser conditions. CdTe QDs with desirable size were synthesized by controlling the heating time. Stable water-compatible MPA-capped CdTe QDs with emission maximum at about 607 nm were used in the present experiment. The concentration of CdTe QDs was 4.77 μmol L 1. Au NPs were synthesized by the citrate reduction of HAuC14 according to the previous methods (Gao et al., 2011). Typically, 1 mL of 1% (v/v) HAuCl4 solution and 99 mL ofdeionized water were added into a round-bottom flask. The reaction proceeded under reflux condenser conditions under vigorous stirring. When the solution was heated to a boiling state, 5 mL of 1% trisodium citrate solution was quickly introduced to the flask. Then, when the color of the solution changed from pale yellow to deep red, the solution was cooled to room temperature and then stored at 4 °C in the refrigerator for further use. The concentration of the Au NPs was 4 nmol L 1. 2.3. Fabrication of ECL biosensors Firstly, the basal GCE was polished to a mirror by using 1.0 and 0.05 mm α-Al2O3 powder on fine abrasive paper. After each polishing, the electrode was sonicated in double distilled water, ethanol and double distilled water for 5 min, successively, in order to remove any adsorbed substances. Finally, it was dried under nitrogen atmosphere ready for use. 10 μL of 0.5% (w/v) CHIT solution, which was prepared by dissolving 0.5 g of CHIT in 1% acetic acid (HAc) with ultrasonication, was dropped onto the surface of the cleaned GCE and was kept at room temperature till dry (labeled as CHIT/GCE). Then, 10 μL of 4.77 μmol L 1 CdTe QDs solution was dropped onto CHIT/GCE and dried in air (labeled as CdTe QDs-CHIT/GCE). Finally, CdTe QDs-CHIT/GCE was further coated with 10 μL of Au NP solution and dried in air (labeled as Au NPs-CdTe QDs-CHIT/GCE). The biosensor was stored in 0.1 mol L 1 pH 7.0 PBS in 4 °C refrigerator when not used.
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2.4. ECL measurement procedure All electrochemical measurements were carried out on the aforementioned workstation with the three-electrode configuration at room temperature. A potential from 0.0 to 2.5 V was applied to the working electrode and the ECL signal was recorded simultaneously. For the measurements of glucose, the obtained Au NPs-CdTe QDs-CHIT/GCE biosensor was first immersed in 0.1 mol L 1 PBS (pH 8.0) solution containing different concentrations of glucose to study the electrochemical response by ECL. ECL signals related to the glucose concentrations could be measured. 2.5. Human serum samples The human blood samples were supplied from healthy volunteers at the Hospital of Changchun China Japan Union Hospital. All the blood samples were obtained through venipuncture. The samples were segregated by centrifugation at 10,000 rpm for 10 min after being stored for 2 h at room temperature. A 1.0 mL aliquot of serum sample was mixed with 1.5 mL of acetonitrile. After vigorously shaking for 2 min, the mixture was centrifuged at 10,000 rpm for 10 min. The obtained supernatant was adjusted to pH 8.0 with PBS. Different concentrations of glucose were added to the diluted serum samples to prepare the spiked samples.
3. Results and discussion 3.1. Characterization of CdTe QDs and AuNPs The UV–vis absorption and PL spectra of the CdTe QDs are shown in Fig. S1. The PL emission peak was at 607 nm and the maximum absorption wavelength was at 560 nm. According to Peng's empirical equations (Yu et al., 2003), the size and the concentration of CdTe QDs were estimated to be 3.3 nm and 4.77 μmol L 1, respectively. Fig. S2 shows the TEM micrograph of CdTe QDs and Au NPs. The size and the concentration of Au NPs were estimated to be 10 nm and 4 nmol L 1, respectively. 3.2. EIS behavior Electrochemical impedance spectroscopy (EIS) was employed to demonstrate the modification procedures of the electrode, as shown in Fig. 1. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance (Ret), and the linear part at lower frequencies corresponds to the diffusion process. It was observed that the EIS of the bare GCE displayed an almost straight line, which was characteristic of a mass diffusion limiting process (Fig. 1a). After the electrode was modified with 0.5% (w/v) CHIT solution, the EIS showed a low electron-transfer resistance of about 655.8 Ω ( Fig. 1b). With the further coating of CdTe QDs onto CHIT/GCE, the electron-transfer resistance was increased to about 5886 Ω (Fig. 1c). The semicircle increased significantly, which suggested the hindrance effect of the semiconductor CdTe QDs to the electrical conductivity. After the modification of Au NPs on CdTe QDs-CHIT/GCE, the electron transfer resistance was about 5600 Ω (Fig. 1d). The decrease of semicircles indicated that the presence of Au NPs has accelerated electrical conductivity. 3.3. Optimization of reaction conditions To achieve a high-performance ECL signal, the effects of pH value and scan rate on the ECL intensity of the biosensor were investigated. As shown in Fig. S3, the ECL intensity increased with pH increasing from 5.0 to 8.0. The strongest ECL signal was achieved at pH 8.0, which indicated that lower alkaline
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Fig. 1. EIS of (a), bare GCE, (b) CHIT/GCE, (c) CdTe QDs-CHIT/GCE, and (d) Au NPsCdTe QDs-CHIT/GCE in 0.1 mol L 1 KCl solution containing 2.5 mmol L 1 K3[Fe (CN)6]/K4[Fe(CN)6]. The impedance spectra were recorded in the frequency range of 0.01 Hz–100 kHz with the amplitude of 5 mV.
surroundings were suitable for CdTe QDs ECL emission. So, PBS of pH 8.0 was used as the electrolyte solution. ECL efficiency significantly depended on the rate of generation/ annihilation of excited-state QDs (Jiang and Ju, 2007). As shown in Fig. S4, it can be seen that the ECL intensity increased with the increasing scan rate and reached a maximum value at 0.6 V s 1. It was due to the formation of more excited-state CdTe QDs within short time span. The high scan rate could enrich excited-state QDs within short time span and enhance ECL intensity. When the scan rate was higher than 0.6 V s 1, the electrochemical process was irreversible, and the ECL intensity tended to decrease. It indicated that high scan rates were unfavorable to the electrochemical reaction of CdTe QDs. In the following experiments, a scan rate of 0.6 V s 1 was chosen to obtain the maximum ECL intensity.
3.4. Electrochemiluminescence behavior
Fig. 2. ECL–time curves of CdTe QDs/GCE in (a) air-saturated, (b) 320 μmol L 1 H2O2 in N2-saturated and (c) O2-saturated 0.1 mol L 1 pH 8.0 PBS.
Fig. 3. ECL curves of (a) CdTe QDs-CHIT/GCE, (b) CdTe QDs-CHIT/GCE containing 3 mmol L 1 glucose, (c) Au NPs-CdTe QDs-CHIT/GCE, (d) Au NPs-CdTe QDs-CHIT/ GCE containing 3 mmol L 1 glucose, and (e) Au NPs-CdTe QDs-CHIT/GCE containing 10 mmol L 1 glucose.
R* → R + hv
Although O2 and H2O2 can act as the ECL coreactants, the efficiencies of the two coreactants are quite different. O2 can capture more electrons from the electrochemically reduced CdTe QDs than H2O2 (Zhang et al., 2011). As a result, the reaction rate with the reduced QDs is also different (Jiang and Ju, 2007). Fig. 2 shows the enhanced ECL signals of the CdTe QDs/GCE electrode in the airsaturated 0.1 mol L 1 PBS solution (curve a), deaerated solution containing 320 μmol L 1 H2O2 (curve b) and O2-saturated 0.1 mol L 1 PBS solution (curve c). We can see that the ECL peak intensity of the CdTe QDs/GCE in O2-saturated 0.1 mol L 1 PBS was two times higher than that in air-saturated 0.1 mol L 1 PBS solution. The ECL peak intensity of the CdTe QDs/GCE in O2-saturated 0.1 mol L 1 PBS was 1.4 times than that in N2-saturated PBS containing 320 μmol L 1 H2O2 solution. It confirmed that the dissolved O2 is a more efficient coreactant to enhance the ECL emission of CdTe QDs. According to the previous report (Jiang and Ju, 2007), the possible ECL route of CdTe QDs (R) using dissolved O2 as the coreactant was proposed as follows:
CdTeQDs + ne− → nR⋅−
(1)
O2 + 2R⋅− + 2H+ → 2R* + H2 O2
(2)
(3)
According to recent researches, Au NPs are efficient GOx-mimickess that catalytically oxidize glucose in a“green” approach (Beltrame et al., 2006; Comotti et al., 2004 ). Au NPs catalyze the oxidation of glucose with the cosubstrate O2, producing gluconate and hydrogen peroxide. The catalytic reaction is shown as the following:
glucose + O2 + AuNPs → gluconic acid + H2 O2
(4)
ECL signals at each immobilization step were recorded to monitor the fabrication of the ECL biosensors. It is well known that there was no obvious ECL emission at bare GCE or CHIT modified GCE. Therefore, the ECL signal of CdTe QDs-CHIT/GCE should be produced from CdTe QDs. The ECL peak intensity of CdTe QDs-CHIT/GCE was about 7069 (Fig. 3, curve a). With 3 mmol L 1 glucose, the ECL peak intensity of CdTe QDs-CHIT/GCE was about 7005 (curve b). It indicated that glucose had no effect on CdTe QDs-CHIT/GCE. When Au NPs were conjugated onto CdTe QDsCHIT/GCE, the ECL peak intensity was decreased to about 3421 due to the inner filter effect of Au NPs toward the electrode surface (curve c) (Zhang et al., 2012; Shota et al., 2011). Upon addition of glucose, Au NPs catalyzed glucose to produce gluconic acid (Luo et al., 2010). The consumption of dissolved O2 [shown in reactions (4)] resulted in a quenching effect on the ECL emission of QDs (curve d and e).
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3.5. Stability and reproducibility of the biosensor The stability of the Au NPs-CdTe QDs-CHIT/GCE biosensor was evaluated by monitoring the response of the ECL emission of the biosensor as a function of different time intervals and consecutive cyclic potential scans from 0 to 2.5 V. As shown in Fig. S5, after the biosensor had been stored in the PBS solution in a refrigerator at 4 °C for two days, the ECL intensity of the biosensor was kept constant; it remained approximately 97% of the original value after storage for 10 day, and dropped to 93% after 15 day. The results indicate that the biosensor has better stability. After storing the the biosensor in the PBS solution in a refrigerator at 4 °C for over 10 days, the ECL signal–time curve under continuous potential scanning for 10 cycles is shown in Fig. S6. The ECL responses did not show obvious change, also indicating that the ECL biosensor was very stable. The reproducibility of the biosensor for glucose was estimated with an intra-assay. The intra-assay precision of the biosensor was evaluated by assaying one glucose level for three replicate measurements. The intra-assay relative standard deviations (RSDs) obtained from 10 μmol L 1 glucose were 4.3%. Obviously, the low value of intra-assay RSD indicated that the biosensor showed a good fabrication reproducibility. The stable ECL signal indicated that the biosensor was suitable for ECL detection of glucose. 3.6. Glucose determination The ECL inhibitory capability of glucose on Au NPs-CdTe QDsCHIT/GCE system was studied under optimum conditions. We can see from Fig. 4 inset that the ECL intensity decreased with the increasing concentration of glucose. The ECL intensity was linearly proportional to the glucose concentration in the range of 0.01– 10 mmol L 1. The regression equation is I ¼(2864.45 713.65) (156.36 72.74)C (mmol L 1) (I being the ECL intensity and C being
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the glucose concentration), with the regression coefficient of 0.9984. The detection limit (LOD) for glucose was calculated to be 5.28 μmol L 1. The detection limit is defined by the equation LOD¼ (3s/s), where s is the standard deviation of blank signals (n ¼7) and s is the slope of the calibration curve. As shown in Fig. 4, the ECL time trace for the glucose calibration curve shows the good repeatability and stability of the detected signal. Selectivity is a very important parameter to evaluate the performance of a new ECL biosensor, especially for a biosensor with potential applications in biomedical samples. Table S1 shows the interference effect of some coexistent substances on the determination of glucose (10 μmol L 1); a relative error of 75.0% was considered to be tolerable. As shown in Table S1, the tolerable concentration ratios of coexistent substances was 1000fold for Na þ and K þ and 500-fold for Ca2 þ , Mg2 þ , Zn2 þ , Ba2 þ , ascorbic acid, tyrosine, glycine, cysteine, phenylalanine and alamine. The results in Table S1 indicate that there was little interference from commonly coexistent substances. Thus, the biosensor displays high selectivity for the determination of glucose. In order to evaluate the feasibility of the present method, the method was used to detect glucose in human serum samples; the results are listed in Table 1. As shown in Table 1, the found values of glucose by the proposed method are consistent with that of hospital method. The RSD was lower than 2.5% and the average recoveries of glucose in the real samples were in the range of 96– 106%. These results indicated that the proposed biosensor for the determination of glucose was accurate and can be applied for a real samples assay. A comparison between the present biosensor and other reported methods and biosensors for glucose determination regarding the linear range and detection limit is summed up in Table S2. We can see that the linearity range and the sensitivity of our method are comparable with most of the reported methods and biosensors.
4. Conclusion
Fig. 4. ECL time trace of AuNPs-CdTe QDs-CHIT/GCE to different concentrations of glucose (a–h). Glucose concentration (mmol L 1): (a) 0, (b) 0.01, (c) 0.1, (d) 0.5, (e) 1, (f) 3, (g) 8 and (h) 10; inset shows the linear calibration curve for glucose determination.
In summary, a novel and highly sensitive signal-off ECL biosensor has been developed for the determination of glucose based on GCE sequentially modified by CHIT, CdTe QDs and Au NPs. CHIT offers ideal, strong hydrogel adsorbing ability and good adhesion with the functional nanoparticles to the electrode surfaces. Due to the consumption of dissolved O2 via the Au NPs as efficient GOxmimickers to catalyze glucose, there is a quenching effect on the CdTe QDs' ECL emission on the electrode. Compared with glucose oxidase, Au NPs possess more advantages, such as an easy approach for preparation and purification, high stability, avoiding degeneration and inactivation. We have demonstrated that the ECL biosensor shows good sensitivity, reproducibility and stability, which successfully provides a new ECL method for determination of glucose in human serum samples. Therefore, the satisfactory performance of the biosensor would open a new pathway for a qualified alternative for glucose determination in practical and routine analyses.
Table 1 Results of glucose determination in human serum samples. Sample
1 2 3 4
Hospital method founded (mmol L 1) 4.50 4.90 5.10 5.50
Proposed method founded (mmol L 1) 4.47 4.81 5.15 5.31
Added (mmol L 1) 4.00 4.00 4.00 4.00
Total founded (mmol L 1) 8.74 8.77 9.03 9.16
Recovery (%)
106 99.0 97.0 96.3
RSD (%, n ¼3) 1.15 2.08 1.43 1.54
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21075050 and 21005029) and Youth Science Fund of Jilin Province (20140520081JH).
Appendix A. Supplementary information Supplementary information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014. 07.087.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel signal-off electrochemiluminescence biosensor for the determination of glucose based on double nanoparticles Linlin Liu 1, Qiang Ma 1, Yang Li, ZiPing Liu, Xingguang Su n Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun 130012, China
art ic l e i nf o
a b s t r a c t
Article history: Received 4 May 2014 Received in revised form 25 July 2014 Accepted 29 July 2014 Available online 7 August 2014
In this work, a novel facile signal-off electrochemiluminescence (ECL) biosensor has been developed for the determination of glucose based on the integration of chitosan (CHIT), CdTe quantum dots (CdTe QDs) and Au nanoparticles (Au NPs) on the glassy carbon electrode (GCE). Chitosan displays high water permeability, hydrophilic property, strong hydrogel ability and good adhesion to load the double nanoparticles to the glassy carbon electrode surfaces. Au NPs are efficient glucose oxidase (GOx)-mimickess to catalytically oxidize glucose, similar to the natural process. Upon the addition of glucose, the Au NPs catalyzed glucose to produce gluconic acid and hydrogen peroxide (H2O2) based on the consumption of dissolved oxygen (O2), which resulted in a quenching effect on the ECL emission. Therefore, the determination of glucose could be achieved by monitoring the signal-off ECL biosensor. Under the optimum conditions, the ECL intensity of CdTe QDs and the concentration of glucose have a good linear relationship in the range of 0.01–10 mmol L 1. The limit of detection for glucose was 5.28 μmol L 1 (S/N¼3). The biosensor showed good sensitivity, selectivity, reproducibility and stability. The proposed biosensor has been employed for the detection of glucose in human serum samples with satisfactory results. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Biosensor Chitosan CdTe QDs Au nanoparticles Glucose
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Experiment section . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Materials and apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Preparation of CdTe QDs and Au NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Fabrication of ECL biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. ECL measurement procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Human serum samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Characterization of CdTe QDs and AuNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. EIS behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Optimization of reaction conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Electrochemiluminescence behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Stability and reproducibility of the biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Glucose determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
n
Corresponding author. Tel.: þ 86 431 85168352. E-mail address: [email protected] (X. Su). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bios.2014.07.087 0956-5663/& 2014 Elsevier B.V. All rights reserved.
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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 for glucose monitoring in the case of diabetes patients, it is also essential for non-diabetic acute care patients in order to control glucose levels (Matz et al., 2006). Therefore, a huge amount of glucose biosensors have been reported, such as those based on optical techniques (Barone et al., 2005), color metric measurement (Morris et al., 1992) and capacitive detection (Cheng et al., 2001). In recent years, a tremendous amount of attention and effort has been paid to develop ultrasensitive glucose biosensors including electrochemical (Si et al., 2013), surface enhanced Raman scattering (Wu et al., 2006), chemiluminescence (Li et al., 2000), electrochemical transistor biosensor (Liu et al., 2008) and potentiometric biosensor (Liao et al., 2007). However, many approaches suffer from limitations of complicated pretreatment steps, equipment costs and expensive labor resources and time are consuming. Obviously, an effective analytical method for rapid determination of glucose is urgently needed. Electrochemiluminescence (ECL) is a versatile analytical technique that combines the simplicity of electrochemistry with inherent sensitivity and wide linear ranges of the chemiluminescence method. Most importantly, ECL biosensors have lots of advantages, including high sensitivity, low backgound, ease of control and simple equipments (Zheng et al., 2001; Chovin et al., 2004). Semiconductor quantum dots (QDs) have attracted great attention in the past decade, due to their remarkable optoelectronic properties, such as narrow, symmetrical and size-tunable emission spectrum, broad excitation spectrum, large Stokes shift, high photo-bleaching threshold and good chemical stability (Hanif et al., 2002; Ma and Su, 2011). Uptil now, numerous reviews about QDs-based ECL have been published (Lei and Ju, 2011; Huang et al., 2011; Li et al., 2012). Further, QDs have been widely used to detect various substances via ECL methods. At the same time, great effort has been focused on the QD-based ECL behavior and mechanism. The thin-film technique has provided an effective way for the construction of QD-based ECL biosensors in aqueous solutions (Jie et al., 2008). Chitosan is a polysaccharide biopolymer, which displays excellent film-forming ability, high water permeability and hydrophilic property, strong hydrogel ability and good adhesion with the functional nanoparticles to the electrode surfaces (Kumar et al., 2004; Zhang et al., 2004). Furthermore, the previous research works demonstrated that the CHIT had strong adsorbing ability (Luo et al., 2005). Due to the attractive electronic properties, Au nanoparticles (Au NPs) have a wide range of applications like catalysis and bioanalysis (Gong and Mullins, 2009; Lee et al., 2010; Corma and
Garcia, 2008; Hammer, 2006). Recently, Au NPs were found to be efficient GOx-mimickess; they can catalytically oxidize glucose and produce gluconates in a “green” approach, similar to that of the natural enzyme of GOx (Comotti et al., 2004). Au NPs also catalyzed the oxidation of glucose with the cosubstrate oxygen (O2), producing gluconate and hydrogen peroxide (Beltrame et al., 2006). Comparing with glucose oxidase, Au NPs as biomimetic enzymes possess more advantages, such as providing an easy approach for preparation and purification, having high stability, avoiding degeneration and inactivation. In this paper, we report a new and simple method for the fabrication of an ECL biosensor by using an Au NPs-CdTe QDsCHIT/GCE composite film. Scheme 1 describes the preparation of the composite film and the mechanism of the ECL system for analysis of glucose. For the composite films, CHIT can connect CdTe QDs and Au NPs to the glass carbon electrode via powerful adsorption ability. When glucose is added into the system, Au 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 cathodic CdTe QDs ECL emission. Due to the consumption of dissolved O2 , a quenching effect would appear on the CdTe QDs ECL emission. The main features of this biosensor are: (i) chitosan displays excellent film-forming ability, high water permeability, strong hydrogel ability and good adhesion to load the double nanoparticles onto the glassy carbon electrode surfaces; (ii) Au NPs have not only accelerated electrical conductivity but can also oxidize glucose as GOx-mimickers in this green noenzyme system; and (iii) to the best of our knowledge, the signaloff CdTe QDs ECL biosensor for glucose determination based on Au NPs as GOx-mimickess has not been reported before. The proposed Au NPs-CdTe QDs-CHIT/GCE system is a novel ECL biosensor with good selectivity and stability. Further, it was successfully applied to the determination of glucose in samples of human serum with good accuracy and precision.
2. Experiment section 2.1. Materials and apparatus All chemicals and reagents were analytical grade and used directly without further purification. 3-Mercaptopropyl acid (MPA) was purchased from J&K Chemical. Tellurium powder (∼200 mesh, 99.8%), CdCl2 (99%) and NaBH4 (99%) were purchased from Aldrich Chemical Co. HAuCl4 was purchased from Acros Organics. Glucose and other materials were obtained from Beijing Dingguo Biotechnology Co. Ltd. All reagents were prepared using ultrapure water
Scheme 1. The schematic illustration of the ECL biosensor for determination glucose based on Au NPs-CdTe QDs-CHIT/GCE composite film.
L. Liu et al. / Biosensors and Bioelectronics 63 (2015) 519–524
with a resistivity of greater than 18 MΩ cm 1. All experiments were carried out at room temperature. Photoluminescence (PL) spectra measurements were performed on a Shimadzu RF-5301 PC spectrofluorophotometer (Shimadzu Co., Kyoto, Japan). UV–vis absorption measurements were obtained using a Varian GBC Cintra 10e UV–vis spectrometer. The ECL detection system consisted of a BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China) and a CHI 660B electrochemical workstation (Shanghai Chenhua Instrument Co., China). The voltage of the photomultiplier tube (PMT) was set at 900 V during the whole processes. Electrochemical impedance spectroscopic (EIS) analysis was performed on a CHI 660B electrochemical workstation in 2.5 mmol L 1 K3[Fe(CN)6]/K4[Fe(CN)6] with 0.1 mol L 1 KCl as the supporting electrolyte. A conventional three-electrode system was used. A glassy carbon electrode (GCE) was used as the working electrode. A platinum wire was employed as the counter electrode and an Ag/AgCl (saturated KCl) electrode as the reference electrode. 2.2. Preparation of CdTe QDs and Au NPs The water-soluble CdTe QDs were synthesized by refluxing routes (Ma et al., 2010). In brief, sodium hydrogen telluride (NaHTe) was produced in an aqueous solution by the reaction of sodium borohydride (NaBH4) with tellurium powder at a molar ratio of 2:1 at first. Later, freshly synthesized oxygen-free NaHTe solution was mixed with nitrogen-saturated 1.25 mmol L-1 CdCl2 solution at pH 11.4, with MPA as a stabilizing agent. The molar ratio of Cd2 þ /MPA/HTe was charged at 1:2.4:0.5. Then the CdTe precursor solution was subjected to reflux at 100 °C under stirring and condenser conditions. CdTe QDs with desirable size were synthesized by controlling the heating time. Stable water-compatible MPA-capped CdTe QDs with emission maximum at about 607 nm were used in the present experiment. The concentration of CdTe QDs was 4.77 μmol L 1. Au NPs were synthesized by the citrate reduction of HAuC14 according to the previous methods (Gao et al., 2011). Typically, 1 mL of 1% (v/v) HAuCl4 solution and 99 mL ofdeionized water were added into a round-bottom flask. The reaction proceeded under reflux condenser conditions under vigorous stirring. When the solution was heated to a boiling state, 5 mL of 1% trisodium citrate solution was quickly introduced to the flask. Then, when the color of the solution changed from pale yellow to deep red, the solution was cooled to room temperature and then stored at 4 °C in the refrigerator for further use. The concentration of the Au NPs was 4 nmol L 1. 2.3. Fabrication of ECL biosensors Firstly, the basal GCE was polished to a mirror by using 1.0 and 0.05 mm α-Al2O3 powder on fine abrasive paper. After each polishing, the electrode was sonicated in double distilled water, ethanol and double distilled water for 5 min, successively, in order to remove any adsorbed substances. Finally, it was dried under nitrogen atmosphere ready for use. 10 μL of 0.5% (w/v) CHIT solution, which was prepared by dissolving 0.5 g of CHIT in 1% acetic acid (HAc) with ultrasonication, was dropped onto the surface of the cleaned GCE and was kept at room temperature till dry (labeled as CHIT/GCE). Then, 10 μL of 4.77 μmol L 1 CdTe QDs solution was dropped onto CHIT/GCE and dried in air (labeled as CdTe QDs-CHIT/GCE). Finally, CdTe QDs-CHIT/GCE was further coated with 10 μL of Au NP solution and dried in air (labeled as Au NPs-CdTe QDs-CHIT/GCE). The biosensor was stored in 0.1 mol L 1 pH 7.0 PBS in 4 °C refrigerator when not used.
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2.4. ECL measurement procedure All electrochemical measurements were carried out on the aforementioned workstation with the three-electrode configuration at room temperature. A potential from 0.0 to 2.5 V was applied to the working electrode and the ECL signal was recorded simultaneously. For the measurements of glucose, the obtained Au NPs-CdTe QDs-CHIT/GCE biosensor was first immersed in 0.1 mol L 1 PBS (pH 8.0) solution containing different concentrations of glucose to study the electrochemical response by ECL. ECL signals related to the glucose concentrations could be measured. 2.5. Human serum samples The human blood samples were supplied from healthy volunteers at the Hospital of Changchun China Japan Union Hospital. All the blood samples were obtained through venipuncture. The samples were segregated by centrifugation at 10,000 rpm for 10 min after being stored for 2 h at room temperature. A 1.0 mL aliquot of serum sample was mixed with 1.5 mL of acetonitrile. After vigorously shaking for 2 min, the mixture was centrifuged at 10,000 rpm for 10 min. The obtained supernatant was adjusted to pH 8.0 with PBS. Different concentrations of glucose were added to the diluted serum samples to prepare the spiked samples.
3. Results and discussion 3.1. Characterization of CdTe QDs and AuNPs The UV–vis absorption and PL spectra of the CdTe QDs are shown in Fig. S1. The PL emission peak was at 607 nm and the maximum absorption wavelength was at 560 nm. According to Peng's empirical equations (Yu et al., 2003), the size and the concentration of CdTe QDs were estimated to be 3.3 nm and 4.77 μmol L 1, respectively. Fig. S2 shows the TEM micrograph of CdTe QDs and Au NPs. The size and the concentration of Au NPs were estimated to be 10 nm and 4 nmol L 1, respectively. 3.2. EIS behavior Electrochemical impedance spectroscopy (EIS) was employed to demonstrate the modification procedures of the electrode, as shown in Fig. 1. The semicircle diameter at higher frequencies corresponds to the electron-transfer resistance (Ret), and the linear part at lower frequencies corresponds to the diffusion process. It was observed that the EIS of the bare GCE displayed an almost straight line, which was characteristic of a mass diffusion limiting process (Fig. 1a). After the electrode was modified with 0.5% (w/v) CHIT solution, the EIS showed a low electron-transfer resistance of about 655.8 Ω ( Fig. 1b). With the further coating of CdTe QDs onto CHIT/GCE, the electron-transfer resistance was increased to about 5886 Ω (Fig. 1c). The semicircle increased significantly, which suggested the hindrance effect of the semiconductor CdTe QDs to the electrical conductivity. After the modification of Au NPs on CdTe QDs-CHIT/GCE, the electron transfer resistance was about 5600 Ω (Fig. 1d). The decrease of semicircles indicated that the presence of Au NPs has accelerated electrical conductivity. 3.3. Optimization of reaction conditions To achieve a high-performance ECL signal, the effects of pH value and scan rate on the ECL intensity of the biosensor were investigated. As shown in Fig. S3, the ECL intensity increased with pH increasing from 5.0 to 8.0. The strongest ECL signal was achieved at pH 8.0, which indicated that lower alkaline
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Fig. 1. EIS of (a), bare GCE, (b) CHIT/GCE, (c) CdTe QDs-CHIT/GCE, and (d) Au NPsCdTe QDs-CHIT/GCE in 0.1 mol L 1 KCl solution containing 2.5 mmol L 1 K3[Fe (CN)6]/K4[Fe(CN)6]. The impedance spectra were recorded in the frequency range of 0.01 Hz–100 kHz with the amplitude of 5 mV.
surroundings were suitable for CdTe QDs ECL emission. So, PBS of pH 8.0 was used as the electrolyte solution. ECL efficiency significantly depended on the rate of generation/ annihilation of excited-state QDs (Jiang and Ju, 2007). As shown in Fig. S4, it can be seen that the ECL intensity increased with the increasing scan rate and reached a maximum value at 0.6 V s 1. It was due to the formation of more excited-state CdTe QDs within short time span. The high scan rate could enrich excited-state QDs within short time span and enhance ECL intensity. When the scan rate was higher than 0.6 V s 1, the electrochemical process was irreversible, and the ECL intensity tended to decrease. It indicated that high scan rates were unfavorable to the electrochemical reaction of CdTe QDs. In the following experiments, a scan rate of 0.6 V s 1 was chosen to obtain the maximum ECL intensity.
3.4. Electrochemiluminescence behavior
Fig. 2. ECL–time curves of CdTe QDs/GCE in (a) air-saturated, (b) 320 μmol L 1 H2O2 in N2-saturated and (c) O2-saturated 0.1 mol L 1 pH 8.0 PBS.
Fig. 3. ECL curves of (a) CdTe QDs-CHIT/GCE, (b) CdTe QDs-CHIT/GCE containing 3 mmol L 1 glucose, (c) Au NPs-CdTe QDs-CHIT/GCE, (d) Au NPs-CdTe QDs-CHIT/ GCE containing 3 mmol L 1 glucose, and (e) Au NPs-CdTe QDs-CHIT/GCE containing 10 mmol L 1 glucose.
R* → R + hv
Although O2 and H2O2 can act as the ECL coreactants, the efficiencies of the two coreactants are quite different. O2 can capture more electrons from the electrochemically reduced CdTe QDs than H2O2 (Zhang et al., 2011). As a result, the reaction rate with the reduced QDs is also different (Jiang and Ju, 2007). Fig. 2 shows the enhanced ECL signals of the CdTe QDs/GCE electrode in the airsaturated 0.1 mol L 1 PBS solution (curve a), deaerated solution containing 320 μmol L 1 H2O2 (curve b) and O2-saturated 0.1 mol L 1 PBS solution (curve c). We can see that the ECL peak intensity of the CdTe QDs/GCE in O2-saturated 0.1 mol L 1 PBS was two times higher than that in air-saturated 0.1 mol L 1 PBS solution. The ECL peak intensity of the CdTe QDs/GCE in O2-saturated 0.1 mol L 1 PBS was 1.4 times than that in N2-saturated PBS containing 320 μmol L 1 H2O2 solution. It confirmed that the dissolved O2 is a more efficient coreactant to enhance the ECL emission of CdTe QDs. According to the previous report (Jiang and Ju, 2007), the possible ECL route of CdTe QDs (R) using dissolved O2 as the coreactant was proposed as follows:
CdTeQDs + ne− → nR⋅−
(1)
O2 + 2R⋅− + 2H+ → 2R* + H2 O2
(2)
(3)
According to recent researches, Au NPs are efficient GOx-mimickess that catalytically oxidize glucose in a“green” approach (Beltrame et al., 2006; Comotti et al., 2004 ). Au NPs catalyze the oxidation of glucose with the cosubstrate O2, producing gluconate and hydrogen peroxide. The catalytic reaction is shown as the following:
glucose + O2 + AuNPs → gluconic acid + H2 O2
(4)
ECL signals at each immobilization step were recorded to monitor the fabrication of the ECL biosensors. It is well known that there was no obvious ECL emission at bare GCE or CHIT modified GCE. Therefore, the ECL signal of CdTe QDs-CHIT/GCE should be produced from CdTe QDs. The ECL peak intensity of CdTe QDs-CHIT/GCE was about 7069 (Fig. 3, curve a). With 3 mmol L 1 glucose, the ECL peak intensity of CdTe QDs-CHIT/GCE was about 7005 (curve b). It indicated that glucose had no effect on CdTe QDs-CHIT/GCE. When Au NPs were conjugated onto CdTe QDsCHIT/GCE, the ECL peak intensity was decreased to about 3421 due to the inner filter effect of Au NPs toward the electrode surface (curve c) (Zhang et al., 2012; Shota et al., 2011). Upon addition of glucose, Au NPs catalyzed glucose to produce gluconic acid (Luo et al., 2010). The consumption of dissolved O2 [shown in reactions (4)] resulted in a quenching effect on the ECL emission of QDs (curve d and e).
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3.5. Stability and reproducibility of the biosensor The stability of the Au NPs-CdTe QDs-CHIT/GCE biosensor was evaluated by monitoring the response of the ECL emission of the biosensor as a function of different time intervals and consecutive cyclic potential scans from 0 to 2.5 V. As shown in Fig. S5, after the biosensor had been stored in the PBS solution in a refrigerator at 4 °C for two days, the ECL intensity of the biosensor was kept constant; it remained approximately 97% of the original value after storage for 10 day, and dropped to 93% after 15 day. The results indicate that the biosensor has better stability. After storing the the biosensor in the PBS solution in a refrigerator at 4 °C for over 10 days, the ECL signal–time curve under continuous potential scanning for 10 cycles is shown in Fig. S6. The ECL responses did not show obvious change, also indicating that the ECL biosensor was very stable. The reproducibility of the biosensor for glucose was estimated with an intra-assay. The intra-assay precision of the biosensor was evaluated by assaying one glucose level for three replicate measurements. The intra-assay relative standard deviations (RSDs) obtained from 10 μmol L 1 glucose were 4.3%. Obviously, the low value of intra-assay RSD indicated that the biosensor showed a good fabrication reproducibility. The stable ECL signal indicated that the biosensor was suitable for ECL detection of glucose. 3.6. Glucose determination The ECL inhibitory capability of glucose on Au NPs-CdTe QDsCHIT/GCE system was studied under optimum conditions. We can see from Fig. 4 inset that the ECL intensity decreased with the increasing concentration of glucose. The ECL intensity was linearly proportional to the glucose concentration in the range of 0.01– 10 mmol L 1. The regression equation is I ¼(2864.45 713.65) (156.36 72.74)C (mmol L 1) (I being the ECL intensity and C being
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the glucose concentration), with the regression coefficient of 0.9984. The detection limit (LOD) for glucose was calculated to be 5.28 μmol L 1. The detection limit is defined by the equation LOD¼ (3s/s), where s is the standard deviation of blank signals (n ¼7) and s is the slope of the calibration curve. As shown in Fig. 4, the ECL time trace for the glucose calibration curve shows the good repeatability and stability of the detected signal. Selectivity is a very important parameter to evaluate the performance of a new ECL biosensor, especially for a biosensor with potential applications in biomedical samples. Table S1 shows the interference effect of some coexistent substances on the determination of glucose (10 μmol L 1); a relative error of 75.0% was considered to be tolerable. As shown in Table S1, the tolerable concentration ratios of coexistent substances was 1000fold for Na þ and K þ and 500-fold for Ca2 þ , Mg2 þ , Zn2 þ , Ba2 þ , ascorbic acid, tyrosine, glycine, cysteine, phenylalanine and alamine. The results in Table S1 indicate that there was little interference from commonly coexistent substances. Thus, the biosensor displays high selectivity for the determination of glucose. In order to evaluate the feasibility of the present method, the method was used to detect glucose in human serum samples; the results are listed in Table 1. As shown in Table 1, the found values of glucose by the proposed method are consistent with that of hospital method. The RSD was lower than 2.5% and the average recoveries of glucose in the real samples were in the range of 96– 106%. These results indicated that the proposed biosensor for the determination of glucose was accurate and can be applied for a real samples assay. A comparison between the present biosensor and other reported methods and biosensors for glucose determination regarding the linear range and detection limit is summed up in Table S2. We can see that the linearity range and the sensitivity of our method are comparable with most of the reported methods and biosensors.
4. Conclusion
Fig. 4. ECL time trace of AuNPs-CdTe QDs-CHIT/GCE to different concentrations of glucose (a–h). Glucose concentration (mmol L 1): (a) 0, (b) 0.01, (c) 0.1, (d) 0.5, (e) 1, (f) 3, (g) 8 and (h) 10; inset shows the linear calibration curve for glucose determination.
In summary, a novel and highly sensitive signal-off ECL biosensor has been developed for the determination of glucose based on GCE sequentially modified by CHIT, CdTe QDs and Au NPs. CHIT offers ideal, strong hydrogel adsorbing ability and good adhesion with the functional nanoparticles to the electrode surfaces. Due to the consumption of dissolved O2 via the Au NPs as efficient GOxmimickers to catalyze glucose, there is a quenching effect on the CdTe QDs' ECL emission on the electrode. Compared with glucose oxidase, Au NPs possess more advantages, such as an easy approach for preparation and purification, high stability, avoiding degeneration and inactivation. We have demonstrated that the ECL biosensor shows good sensitivity, reproducibility and stability, which successfully provides a new ECL method for determination of glucose in human serum samples. Therefore, the satisfactory performance of the biosensor would open a new pathway for a qualified alternative for glucose determination in practical and routine analyses.
Table 1 Results of glucose determination in human serum samples. Sample
1 2 3 4
Hospital method founded (mmol L 1) 4.50 4.90 5.10 5.50
Proposed method founded (mmol L 1) 4.47 4.81 5.15 5.31
Added (mmol L 1) 4.00 4.00 4.00 4.00
Total founded (mmol L 1) 8.74 8.77 9.03 9.16
Recovery (%)
106 99.0 97.0 96.3
RSD (%, n ¼3) 1.15 2.08 1.43 1.54
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21075050 and 21005029) and Youth Science Fund of Jilin Province (20140520081JH).
Appendix A. Supplementary information Supplementary information associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014. 07.087.
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