Article pubs.acs.org/ac
A Monochromatic Electrochemiluminescence Sensing Strategy for Dopamine with Dual-Stabilizers-Capped CdSe Quantum Dots as Emitters Shufeng Liu,† Xin Zhang,† Yanmin Yu,‡ and Guizheng Zou†,* †
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Department of Chemistry and Chemical Engineering, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China
‡
S Supporting Information *
ABSTRACT: A promising electrochemiluminescence (ECL) sensing strategy was proposed with dual-stabilizers-capped CdSe quantum dots (QDs) as ECL emitters. The dualstabilizers-capped CdSe QDs were covalently immobilized onto p-aminobenzoic acid modified glass carbon electrode with ethylenediamine as a link molecule. This strategy can preserve the completely passivated surface states of dual-stabilizerscapped CdSe QDs, so that the sensor demonstrated eye-visible greenish, band gap engineering and monochromatic ECL emission at 546 nm with a fwhm of 35 nm. Moreover, the proposed sensor could accurately quantify dopamine from 10.0 nM to 3.0 μM with a detection limit of 3.0 nM in practical drug, human urine, and cerebrospinal fluid samples without any signal amplification techniques. This strategy is promising for developing ECL sensors with high sensitivity and spectral selectivity.
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additional broad and red-shift peak on the ECL spectrum.19 Moreover, the limited monodispersity can also result in a broad ECL spectrum for completely passivated QDs. Recent research demonstrated that ECL spectra of QD-based ECL sensors were broad with the full width half-maximum (fwhm) around 100 nm,13,20 which lowered their spectral selectivity. Thus, efficient and monochromatic ECL emission is becoming more and more important for the QD-based ECL sensors. Our group developed a dual-stabilizers-capped synthetic strategy for preparing CdTe QDs with strong ECL emission in the near-infrared region.21,22 Recently, we found that this strategy can be utilized to prepare dual-stabilizers-capped CdSe QDs with strong, eye-visible and monochromatic ECL emission.23 The reason lies in two aspects: one is that the linkage of surface cadmium atoms of CdSe QDs to the dual stabilizers, mercaptopropionic acid (MPA) and modium hexametaphosphate (HMP), can effectively remove the nonradiative surface states and deep surface traps for enhanced ECL efficiency and monochromaticity; the other is that the dual-stabilizers-capped CdSe QDs are favorable for the electrochemical involved electron and hole injection processes. However, whether the dual-stabilizers-capped CdSe QDs can
lectrochemiluminescence (ECL) is the process in which electrogenerated radicals form excited species emitting light without the need of an external light source. ECL is capable of detecting a wide variety of analytes.1,2 Since the ECL emission of Si quantum dots (QDs) was reported in 2002,3 many ECL sensors have been developed with QDs as ECL emitters.4,5 Recent research also demonstrated that QDs are promising ECL tags for selective determination of macromolecules in real samples.6−9 However, there are still two significant barriers for configuring QD-based ECL sensors. One is the limited ECL efficiency of QDs. Because ECL intensity of QDs cannot come to that of conventional ECL reagents, such as Ru(bpy)32+,10 various signal amplification techniques have been utilized to improve the sensitivity of QD-based ECL sensors,11−13 including nanoparticle,6,14 enzyme,9 self-assembly,15 and multiple DNA cycle signal amplification.16 Bao presented an ECL sensor for dopamine (DA) by forming a CdSe/ZnS QDs assembly at a glassy carbon electrode (GCE) with 4-aminothiophenol as the linkage.15 Jie constructed a CdS QD-based ECL immunosensor for low-density lipoprotein with self-assembly and gold nanoparticle amplification techniques.14 The other is the broad ECL spectra of QDs,5,6 which lowered the spectral selectivity of the QDs-based ECL sensors. Previous research has demonstrated that ECL is more sensitive to the surface state energetics than PL.17 Completely passivated QDs should show an ECL spectrum that is very similar to the PL spectrum,18 while incompletely passivated QDs should show an © 2014 American Chemical Society
Received: January 5, 2014 Accepted: February 4, 2014 Published: February 4, 2014 2784
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Scheme 1. Schematic Illustration of ECL Sensor Configuration Strategy
(CV) and ECL of CdSe QDs were recorded with an MPI-A ECL analyzer (Xi’an Remex Analytical Instrument Co., Ltd. China) using a three-electrode system comprising a GCE work electrode (5.0 mm), a Pt counter electrode and a Ag/AgCl (saturated KCl) reference electrode. The voltage of the photomultiplier tube was biased at 500 V. ECL spectrum was obtained with a multichannel optical analyzer (SpectraPro300i, Acton Research Co., Acton, MA, U.S.A). The digital image was acquired with a Canon EOS 500D with exposure time of 0.01 s. Syntheses of Dual-Stabilizers-Capped CdSe QDs. CdSe QDs were synthesized according to the literature.23 Briefly, CdCl2 solution (0.20 M, 0.80 mL), HMP (72.5 mg), and MPA (34.6 μL) were dissolved in 50 mL of H2O successively. Then, pH was adjusted to 9.0, and Na2SeO3 solution (20.0 mM, 0.80 mL) was added to the mixture. After being refluxed at 100 °C for 10 min, the above mixture was added with 3.67 mL of N2H4·H2O and refluxed for another 10 h at 100 °C. The resultant was purified three times by isopropyl alcohol with centrifugation at 10 000 rpm and stored in the dark at 4 °C. The concentration of dual-stabilizers-capped CdSe QDs stock solution was estimated to be 7.10 μM with an empirical equation.24 Fabrication of the Sensor. As described in Scheme 1, a GCE was used as a substrate for immobilizing dual-stabilizerscapped CdSe QDs. The GCE was polished sequentially with slurries of 0.3 and 0.05 μm alumina and thoroughly rinsed with distilled water. For grafting QDs, an assembly of ABA was first formed on GCE (ABA-GCE) using a cyclic scanning electrode from 0.40 to 1.20 V in 10.0 mM, pH 7.4 PBS containing 1.0 mM ABA and 10.0 mM KCl at 10 mV s−1 for two cycles (Scheme 1a). The irreversible oxidation peak at ca. 0.82 V of ABA is ascribed to one-electron oxidation of the amino group turning into its corresponding cation radical, which can form a carbon−nitrogen linkage at the GCE surface (Supporting Information Figure S-1).25 After the ABA-GCE was washed with ethanol and dried with N2, a drop of 20.0 μL, 0.10 M, pH 7.0 PBS containing 100.0 mg·mL−1 EDC, 100.0 mg·mL−1 NHS, and 2% (v/v) En was placed onto its surface for 30 min to form En-ABA-GCE (Scheme 1b). Then, En-ABA-GCE was rinsed with water and dried with N2. Finally, a drop of 20.0 μL, 0.10 M, pH 7.0 MES containing 100.0 mg·mL−1 EDC, 100.0 mg· mL−1 NHS, and 2.36 μM CdSe QDs was placed onto the
preserve their unique ECL features or not is still crucial for their applications, especially when they are linked to some substrate or biomoleculars as ECL tags. Herein, we presented a promising sensing strategy with dual-stabilizers-capped CdSe QDs as ECL emitters (Scheme 1). We found that the dualstabilizers-capped CdSe QDs can be covalently immobilized onto the GCE surface with this strategy and preserve their efficient and monochromatic ECL features. More importantly, the proposed ECL sensor can accurately and sensitively determine DA in real samples without any signal amplification techniques.
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EXPERIMENTAL SECTION Materials and Reagents. All reagents were of analytical grade or better and used as received. Cadmium chloride was obtained from Hengxin Chemical Co., Ltd. (Shanghai, China). Sodium hexametaphosphate (HMP) was obtained from Guangcheng Chemical Reagent Co., Ltd. (Tianjin, China). Mercaptopropionic acid (MPA) was purchased from Aldrich Chemicals (St. Louis, MO, USA). Sodium selenite pentahydrate was obtained from Tianjin Chemical Reagent Research Institute (China). Ammonium persulfate, hydrazine hydrate, 2morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), uric acid (UA), and ascorbic acid (AA) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3 dimethyl-amino-propyl) carbodiimide hydrochloride (EDC) was obtained from Shanghai Medpep Co. Ltd. (China). p-Aminobenzoic acid (ABA) was obtained from Kermel Chemical Reagent Co., Ltd. (Shanghai, China). Potassium ferricyanide and ethylenediamine (En) was obtained from Xilong Chemical Co., Ltd. (Shantou, China). DA was purchased from J&K Chemical Co., Ltd. DA hydrochloride injection was purchased from Harvest Pharmaceutical Co., Ltd. (China). Real cerebrospinal fluid (CSF) samples were obtained from volunteer patients. The sample was centrifuged at 10 000 rpm for 10 min, and the supernates were collected for assay. EDC and NHS were dissolved in a 0.10 M pH 6.0 MES buffer solution containing 0.50 M KCl before use. All solutions were prepared with doubly distilled water. Apparatus. The absorption spectra were recorded on a TU1901 UV−vis spectrophotometer (Beijing, China). The photoluminescence (PL) was performed on a WGY-10 spectrofluorimeter (Tianjin, China). Cyclic voltammetry 2785
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increased peak-to-peak separation of 136 mV (curve d), which also proved the CdSe QDs were covalently grafted onto GCE. ECL Behaviors of the Proposed ECL Sensor. ECL and CV behaviors of the proposed sensor are shown in Figure 2. No
surface of En-ABA-GCE for 30 min to form QDs-En-ABAGCE (Scheme 1c).
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RESULTS AND DISCUSSION Characterization of Dual-Stabilizers-Capped CdSe QDs and Proposed ECL Sensor. As shown in Figure 1,
Figure 2. (A) ECL and (B) CV behaviors of (a) ABA-GCE, (b) EnABA-GCE and (c) CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8, and (d) CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS at a scan rate of 50 mV s−1. Inset A: ECL spectrum of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8. Insert B: digital image of the ECL emission of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8.
Figure 1. (a) Absorbance, (b) PL spectrum of dual-stabilizers-capped CdSe QDs. (c) ECL spectrum of dual-stabilizers-capped CdSe QDs in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 at 50 mV s−1. Inset: CV behaviors of (a) bare GCE, (b) ABA-GCE, (c) En-ABAGCE, and (d) CdSe QDs-En-ABA-GCE in solution containing 10.0 mM K3Fe(CN)6 and 0.50 M KCl at 50 mV s−1.
ECL emission was detected with ABA-GCE (cure a, Figure 2A) and En-ABA-GCE (curve b, Figure 2A) in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8. The efficient and eye-visible ECL emission of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 (curve c, Figure 2A) indicated a coreactant ECL route, as no ECL emission was detected with CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS (curve d, Figure 2A).30 The ECL of CdSe QDs-En-ABA-GCE was greenish and strong enough to be seen by the naked eye (inset B, Figure2), which further proved that the dualstabilizers-capped CdSe QDs were immobilized onto GCE by the proposed strategy. Compared with the CV curve of 0.10 M pH 7.4 PBS without (NH4)2S2O8 (curve d, Figure 2B), the wide cathodic peak at −1.05 V on the CV curve was observed for the CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing (NH4)2S2O8 (curves a, b, c, Figure 2B), which corresponded to the electrochemical reduction of (NH4)2S2O8.1 More importantly, the ECL spectrum of CdSe QDs-En-ABA-GCE displayed a single peak at 546 nm with a fwhm of 35 nm (inset A, Figure2), very similar to that of CdSe QDs dispersed in solution. No additional broad and red-shift peak was observed on the ECL spectrum, which indicated that the covalently immobilizing strategy can preserve the completely passivated surface states of dual-stabilizers-capped CdSe QDs. These results show that the strong and spectral selective ECL emission can be obtained with the proposed strategy. The ECL mechanism is proposed as follows: with the potential scanned negatively, the CdSe QDs were reduced (or electron-injected) to negatively charged species (CdSe•−). Meanwhile, the S2O82− at the surface of the electrode was reduced to oxidant SO4•−. Thereafter, the CdSe•− reacted with the SO4•− and produced excited state species (CdSe*) that emitted light. The equations corresponding to each step of the emission reactions are formulated as follows:
the PL spectrum of the dual-stabilizers-capped CdSe QDs centered at 545 nm with a fwhm of 39 nm. PL quantum yield (QY) of the dual-stabilizers-capped CdSe QDs was determined as around ∼15.8% by using rhodamine 6G in ethanol (QY = 95%) as a PL reference.23,26 The sharp excitonic peak in absorption (curve a) and the narrow PL spectrum (curve b) indicated their good absorption and light emission features. The ECL spectrum of the CdSe QDs demonstrated a single systemic peak at 541 nm (curve c), close to that of the PL spectrum (curve b). So, the dual-stabilizers-capped CdSe QDs should be completely passivated.23 The band gap engineered model ECL emission was highly monochromatic with a fwhm of 28 nm, indicating the dual-stabilizers-capped CdSe QDs were promising ECL emitter candidates. Thus, the dualstabilizers-capped CdSe QDs were chosen to fabricate QDbased monochromatic ECL sensor, as shown in Scheme 1. Potassium ferricyanide was chosen as a marker to investigate the changed surface states of GCE after each assembly step (inset of Figure 1). A well-shaped CV with a peak-to-peak separation of 70 mV was observed at bare GCE (curve a). Compared with bare GCE, the decreased peak current and enlarged peak-to-peak separation of 306 mV for ABA-GCE (curve b) might result from the electrostatic repulsion between ferricyanide anions and negative charged ABA-GCE surface (pKa‑COOH of ABA, 4.80).27 The increased peak current and diminished peak-to-peak separation (100 mV) of En-ABA-GCE (curve c) to that of ABA-GCE (curve b) should be due to the electrostatic attraction between ferricyanide anions and the positive charged En-ABA-GCE surface (pKa‑NH2 of En, 9.93),28 although the current was still smaller and the peak-to-peak separation was larger than those of bare GCE. Finally, because the CdSe QDs were negatively charged under the given conditions (pKa‑COOH of MPA, 4.32),29 the CdSe QDs-EnABA-GCE showed a decreased peak current as well as 2786
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CdSe + e− → CdSe•−
(1)
S2 O82 − + e− → SO4 2 − + SO4•− CdSe
•−
+ SO4
•−
→ CdSe* + SO4
CdSe* → CdSe + hv
(2) 2−
(3) (4)
Figure 3 demonstrated the effects of scan rate on ECL intensity of the proposed sensor. The ECL intensity increased
Figure 4. The quenching effects of DA on the ECL behaviors of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 and (a) 0.0, (b) 0.010, (c) 0.20, and (d) 3.0 μM DA at 50 mV s−1. Inset A: The linear relationship between the concentration of DA and ECL intensity quenching degree (I 0 /I). Inset B: Reproducibility of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 and 0.20 μM DA.
respectively. No obvious change of the ECL intensity was observed after the addition of interfering agents (a, b, c of Figure 5), which indicated that UA and AA cannot interfere
Figure 3. Effects of scan rate on the ECL intensity of CdSe QDs-EnABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8.
with the enhanced scan rate from 20 to 80 mV s−1, similar to that of dual-stabilizers-capped CdTe QDs.8,22 Because repeatability of the ECL emission decreased dramatically with scan rate up to 60 mV s−1, a scan rate of 50 mV s−1 with a relative standard deviation (RSD) of 3.7% (n = 5) was selected herein. Performance of the Proposed ECL Sensor. DA is an important neurotransmitter in the brain.31 Many efforts have been focused on simple and accurate methods for DA determination. In this case, DA was taken as a model molecular to evaluate the performance of the proposed ECL sensor.32 Figure 4 displayed the quenching effects of DA on ECL behaviors of CdSe QDs-En-ABA-GCE. The ECL intensity in the presence of DA (I) was lower than that in the absence of DA (I0), and the ECL intensity decreased gradually with increasing concentrations of DA. The reason might be that DA inhibited the ECL reaction between CdSe QDs and (NH4)2S2O8.10 The ECL intensity quenching degree (I0/I) linearly responds to the DA concentration from 10.0 nM to 3.0 μM with a detection limit of 3.0 nM (S/N = 3) (inset A, Figure 4), indicating the analytical performance of this sensor is better than that of the other existing ECL sensors for DA.5,10,11,15,33 Five proposed sensors exhibited similar ECL responses with DA at the 0.20 μM level, and the RSD was 4.3% (inset B, Figure 4), indicating acceptable reproducibility. After being stored in the refrigerator at 4 °C for three weeks, the average ECL intensity of five proposed sensors only decreased 4.9% in 0.10 M pH 7.4 PBS containing 0.10 M (NH 4 ) 2 S 2 O 8 (Supporting Information Figure S-2), which indicated good storage stability. This might due to no enzyme and mediator being involved in the sensing strategy.7 AA and UA were two common interfering agents for DA detection.32,34 An interference investigation was performed by using the solution containing 20.0 μM AA and 20.0 μM UA,
Figure 5. Interference of AA and UA with the ECL response of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 at 50 mV s−1. (a) Normalized initial ECL intensity of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 at 50 mV s−1, (b) a + 20.0 μM AA, (c) a + 20.0 μM UA, (d) a + 1.0 μM DA, (e) d + 10.0 μM AA + 10.0 μM UA.
with the ECL response of CdSe QDs-En-ABA-GCE at a level of 20.0 μM. When 10.0 μM AA and 10.0 μM UA were added to the PBS containing 1.0 μA DA, no obvious change of the ECL intensity for CdSe QDs-En-ABA-GCE (d, e of Figure 5) manifested that this ECL sensing strategy can be used to selectively detect DA. The applicability of the proposed ECL sensor was evaluatsed by detecting the DA in a DA hydrochloride injection, human urine from adult healthy volunteers, and CSF from volunteer patients with the standard addition method. The dopamine hydrochloride injection was diluted with 0.10 M phosphate buffer (pH 7.4) by 100 000 times as a real sample for analysis. The original DA concentration of dopamine hydrochloride injection was determined to be 0.055 M, close to the given value of 0.053 M. When 1.0 μM standard DA solution was added into the diluted injection, the average recovery was found 2787
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to be 106.9%. The human urine samples and CSF sample were diluted 10 times with a 0.10 M phosphate buffer (pH 7.4). The results are shown in Table 1, and the average recoveries ranged from 102% to 107.5%, showing a preliminary application of the sensor for the determining DA. Table 1. Results of Analysis of DA in Real Samples sample diluted injection CSF urine 1 urine 2
detected (μM)
added (μM)
found (μM)
recovery (%)
RSD (%) (n = 3)
0.55
1.00
1.069
106.9
3.0
----
0.20 0.20 0.20
0.215 0.204 0.209
107.5 102.0 104.5
2.5 4.2 4.3
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CONCLUSIONS An ECL sensor was designed for sensitive detection of small biomolecules by covalently immobilizing dual-stabilizerscapped CdSe QDs on GCE. The ECL sensor displayed eyevisible greenish and monochromatic ECL emissions without any signal amplification techniques and can be used to detect DA with high sensitivity, good precision, and acceptable storage stability. The strategy to form a dual-stabilizers-capped CdSe QD assembly on GCE is reliable for designing QD-based ECL sensors and devices.
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ASSOCIATED CONTENT
S Supporting Information *
CV of GCE in 10.0 mM pH 7.4 PBS containing 1.0 mM ABA and 10.0 mM KCl and storage stability of five proposed ECL sensors. This material is available free of charge via the Internet at http://pubs.acs.org
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AUTHOR INFORMATION
Corresponding Author
*Fax: +86-531-88564464. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project is supported by the National Natural Science Foundation of China (Grant Nos. 21375077 and 21376010), the Independent Innovation Foundation of Shandong University (Grant No. 2012TS002) and State Key Laboratory of Analytical Chemistry for Life Science (Grant No. SKLACLS1201).
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REFERENCES
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A Monochromatic Electrochemiluminescence Sensing Strategy for Dopamine with Dual-Stabilizers-Capped CdSe Quantum Dots as Emitters Shufeng Liu,† Xin Zhang,† Yanmin Yu,‡ and Guizheng Zou†,* †
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Department of Chemistry and Chemical Engineering, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China
‡
S Supporting Information *
ABSTRACT: A promising electrochemiluminescence (ECL) sensing strategy was proposed with dual-stabilizers-capped CdSe quantum dots (QDs) as ECL emitters. The dualstabilizers-capped CdSe QDs were covalently immobilized onto p-aminobenzoic acid modified glass carbon electrode with ethylenediamine as a link molecule. This strategy can preserve the completely passivated surface states of dual-stabilizerscapped CdSe QDs, so that the sensor demonstrated eye-visible greenish, band gap engineering and monochromatic ECL emission at 546 nm with a fwhm of 35 nm. Moreover, the proposed sensor could accurately quantify dopamine from 10.0 nM to 3.0 μM with a detection limit of 3.0 nM in practical drug, human urine, and cerebrospinal fluid samples without any signal amplification techniques. This strategy is promising for developing ECL sensors with high sensitivity and spectral selectivity.
E
additional broad and red-shift peak on the ECL spectrum.19 Moreover, the limited monodispersity can also result in a broad ECL spectrum for completely passivated QDs. Recent research demonstrated that ECL spectra of QD-based ECL sensors were broad with the full width half-maximum (fwhm) around 100 nm,13,20 which lowered their spectral selectivity. Thus, efficient and monochromatic ECL emission is becoming more and more important for the QD-based ECL sensors. Our group developed a dual-stabilizers-capped synthetic strategy for preparing CdTe QDs with strong ECL emission in the near-infrared region.21,22 Recently, we found that this strategy can be utilized to prepare dual-stabilizers-capped CdSe QDs with strong, eye-visible and monochromatic ECL emission.23 The reason lies in two aspects: one is that the linkage of surface cadmium atoms of CdSe QDs to the dual stabilizers, mercaptopropionic acid (MPA) and modium hexametaphosphate (HMP), can effectively remove the nonradiative surface states and deep surface traps for enhanced ECL efficiency and monochromaticity; the other is that the dual-stabilizers-capped CdSe QDs are favorable for the electrochemical involved electron and hole injection processes. However, whether the dual-stabilizers-capped CdSe QDs can
lectrochemiluminescence (ECL) is the process in which electrogenerated radicals form excited species emitting light without the need of an external light source. ECL is capable of detecting a wide variety of analytes.1,2 Since the ECL emission of Si quantum dots (QDs) was reported in 2002,3 many ECL sensors have been developed with QDs as ECL emitters.4,5 Recent research also demonstrated that QDs are promising ECL tags for selective determination of macromolecules in real samples.6−9 However, there are still two significant barriers for configuring QD-based ECL sensors. One is the limited ECL efficiency of QDs. Because ECL intensity of QDs cannot come to that of conventional ECL reagents, such as Ru(bpy)32+,10 various signal amplification techniques have been utilized to improve the sensitivity of QD-based ECL sensors,11−13 including nanoparticle,6,14 enzyme,9 self-assembly,15 and multiple DNA cycle signal amplification.16 Bao presented an ECL sensor for dopamine (DA) by forming a CdSe/ZnS QDs assembly at a glassy carbon electrode (GCE) with 4-aminothiophenol as the linkage.15 Jie constructed a CdS QD-based ECL immunosensor for low-density lipoprotein with self-assembly and gold nanoparticle amplification techniques.14 The other is the broad ECL spectra of QDs,5,6 which lowered the spectral selectivity of the QDs-based ECL sensors. Previous research has demonstrated that ECL is more sensitive to the surface state energetics than PL.17 Completely passivated QDs should show an ECL spectrum that is very similar to the PL spectrum,18 while incompletely passivated QDs should show an © 2014 American Chemical Society
Received: January 5, 2014 Accepted: February 4, 2014 Published: February 4, 2014 2784
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Scheme 1. Schematic Illustration of ECL Sensor Configuration Strategy
(CV) and ECL of CdSe QDs were recorded with an MPI-A ECL analyzer (Xi’an Remex Analytical Instrument Co., Ltd. China) using a three-electrode system comprising a GCE work electrode (5.0 mm), a Pt counter electrode and a Ag/AgCl (saturated KCl) reference electrode. The voltage of the photomultiplier tube was biased at 500 V. ECL spectrum was obtained with a multichannel optical analyzer (SpectraPro300i, Acton Research Co., Acton, MA, U.S.A). The digital image was acquired with a Canon EOS 500D with exposure time of 0.01 s. Syntheses of Dual-Stabilizers-Capped CdSe QDs. CdSe QDs were synthesized according to the literature.23 Briefly, CdCl2 solution (0.20 M, 0.80 mL), HMP (72.5 mg), and MPA (34.6 μL) were dissolved in 50 mL of H2O successively. Then, pH was adjusted to 9.0, and Na2SeO3 solution (20.0 mM, 0.80 mL) was added to the mixture. After being refluxed at 100 °C for 10 min, the above mixture was added with 3.67 mL of N2H4·H2O and refluxed for another 10 h at 100 °C. The resultant was purified three times by isopropyl alcohol with centrifugation at 10 000 rpm and stored in the dark at 4 °C. The concentration of dual-stabilizers-capped CdSe QDs stock solution was estimated to be 7.10 μM with an empirical equation.24 Fabrication of the Sensor. As described in Scheme 1, a GCE was used as a substrate for immobilizing dual-stabilizerscapped CdSe QDs. The GCE was polished sequentially with slurries of 0.3 and 0.05 μm alumina and thoroughly rinsed with distilled water. For grafting QDs, an assembly of ABA was first formed on GCE (ABA-GCE) using a cyclic scanning electrode from 0.40 to 1.20 V in 10.0 mM, pH 7.4 PBS containing 1.0 mM ABA and 10.0 mM KCl at 10 mV s−1 for two cycles (Scheme 1a). The irreversible oxidation peak at ca. 0.82 V of ABA is ascribed to one-electron oxidation of the amino group turning into its corresponding cation radical, which can form a carbon−nitrogen linkage at the GCE surface (Supporting Information Figure S-1).25 After the ABA-GCE was washed with ethanol and dried with N2, a drop of 20.0 μL, 0.10 M, pH 7.0 PBS containing 100.0 mg·mL−1 EDC, 100.0 mg·mL−1 NHS, and 2% (v/v) En was placed onto its surface for 30 min to form En-ABA-GCE (Scheme 1b). Then, En-ABA-GCE was rinsed with water and dried with N2. Finally, a drop of 20.0 μL, 0.10 M, pH 7.0 MES containing 100.0 mg·mL−1 EDC, 100.0 mg· mL−1 NHS, and 2.36 μM CdSe QDs was placed onto the
preserve their unique ECL features or not is still crucial for their applications, especially when they are linked to some substrate or biomoleculars as ECL tags. Herein, we presented a promising sensing strategy with dual-stabilizers-capped CdSe QDs as ECL emitters (Scheme 1). We found that the dualstabilizers-capped CdSe QDs can be covalently immobilized onto the GCE surface with this strategy and preserve their efficient and monochromatic ECL features. More importantly, the proposed ECL sensor can accurately and sensitively determine DA in real samples without any signal amplification techniques.
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EXPERIMENTAL SECTION Materials and Reagents. All reagents were of analytical grade or better and used as received. Cadmium chloride was obtained from Hengxin Chemical Co., Ltd. (Shanghai, China). Sodium hexametaphosphate (HMP) was obtained from Guangcheng Chemical Reagent Co., Ltd. (Tianjin, China). Mercaptopropionic acid (MPA) was purchased from Aldrich Chemicals (St. Louis, MO, USA). Sodium selenite pentahydrate was obtained from Tianjin Chemical Reagent Research Institute (China). Ammonium persulfate, hydrazine hydrate, 2morpholinoethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), uric acid (UA), and ascorbic acid (AA) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1-Ethyl-3-(3 dimethyl-amino-propyl) carbodiimide hydrochloride (EDC) was obtained from Shanghai Medpep Co. Ltd. (China). p-Aminobenzoic acid (ABA) was obtained from Kermel Chemical Reagent Co., Ltd. (Shanghai, China). Potassium ferricyanide and ethylenediamine (En) was obtained from Xilong Chemical Co., Ltd. (Shantou, China). DA was purchased from J&K Chemical Co., Ltd. DA hydrochloride injection was purchased from Harvest Pharmaceutical Co., Ltd. (China). Real cerebrospinal fluid (CSF) samples were obtained from volunteer patients. The sample was centrifuged at 10 000 rpm for 10 min, and the supernates were collected for assay. EDC and NHS were dissolved in a 0.10 M pH 6.0 MES buffer solution containing 0.50 M KCl before use. All solutions were prepared with doubly distilled water. Apparatus. The absorption spectra were recorded on a TU1901 UV−vis spectrophotometer (Beijing, China). The photoluminescence (PL) was performed on a WGY-10 spectrofluorimeter (Tianjin, China). Cyclic voltammetry 2785
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increased peak-to-peak separation of 136 mV (curve d), which also proved the CdSe QDs were covalently grafted onto GCE. ECL Behaviors of the Proposed ECL Sensor. ECL and CV behaviors of the proposed sensor are shown in Figure 2. No
surface of En-ABA-GCE for 30 min to form QDs-En-ABAGCE (Scheme 1c).
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RESULTS AND DISCUSSION Characterization of Dual-Stabilizers-Capped CdSe QDs and Proposed ECL Sensor. As shown in Figure 1,
Figure 2. (A) ECL and (B) CV behaviors of (a) ABA-GCE, (b) EnABA-GCE and (c) CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8, and (d) CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS at a scan rate of 50 mV s−1. Inset A: ECL spectrum of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8. Insert B: digital image of the ECL emission of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8.
Figure 1. (a) Absorbance, (b) PL spectrum of dual-stabilizers-capped CdSe QDs. (c) ECL spectrum of dual-stabilizers-capped CdSe QDs in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 at 50 mV s−1. Inset: CV behaviors of (a) bare GCE, (b) ABA-GCE, (c) En-ABAGCE, and (d) CdSe QDs-En-ABA-GCE in solution containing 10.0 mM K3Fe(CN)6 and 0.50 M KCl at 50 mV s−1.
ECL emission was detected with ABA-GCE (cure a, Figure 2A) and En-ABA-GCE (curve b, Figure 2A) in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8. The efficient and eye-visible ECL emission of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 (curve c, Figure 2A) indicated a coreactant ECL route, as no ECL emission was detected with CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS (curve d, Figure 2A).30 The ECL of CdSe QDs-En-ABA-GCE was greenish and strong enough to be seen by the naked eye (inset B, Figure2), which further proved that the dualstabilizers-capped CdSe QDs were immobilized onto GCE by the proposed strategy. Compared with the CV curve of 0.10 M pH 7.4 PBS without (NH4)2S2O8 (curve d, Figure 2B), the wide cathodic peak at −1.05 V on the CV curve was observed for the CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing (NH4)2S2O8 (curves a, b, c, Figure 2B), which corresponded to the electrochemical reduction of (NH4)2S2O8.1 More importantly, the ECL spectrum of CdSe QDs-En-ABA-GCE displayed a single peak at 546 nm with a fwhm of 35 nm (inset A, Figure2), very similar to that of CdSe QDs dispersed in solution. No additional broad and red-shift peak was observed on the ECL spectrum, which indicated that the covalently immobilizing strategy can preserve the completely passivated surface states of dual-stabilizers-capped CdSe QDs. These results show that the strong and spectral selective ECL emission can be obtained with the proposed strategy. The ECL mechanism is proposed as follows: with the potential scanned negatively, the CdSe QDs were reduced (or electron-injected) to negatively charged species (CdSe•−). Meanwhile, the S2O82− at the surface of the electrode was reduced to oxidant SO4•−. Thereafter, the CdSe•− reacted with the SO4•− and produced excited state species (CdSe*) that emitted light. The equations corresponding to each step of the emission reactions are formulated as follows:
the PL spectrum of the dual-stabilizers-capped CdSe QDs centered at 545 nm with a fwhm of 39 nm. PL quantum yield (QY) of the dual-stabilizers-capped CdSe QDs was determined as around ∼15.8% by using rhodamine 6G in ethanol (QY = 95%) as a PL reference.23,26 The sharp excitonic peak in absorption (curve a) and the narrow PL spectrum (curve b) indicated their good absorption and light emission features. The ECL spectrum of the CdSe QDs demonstrated a single systemic peak at 541 nm (curve c), close to that of the PL spectrum (curve b). So, the dual-stabilizers-capped CdSe QDs should be completely passivated.23 The band gap engineered model ECL emission was highly monochromatic with a fwhm of 28 nm, indicating the dual-stabilizers-capped CdSe QDs were promising ECL emitter candidates. Thus, the dualstabilizers-capped CdSe QDs were chosen to fabricate QDbased monochromatic ECL sensor, as shown in Scheme 1. Potassium ferricyanide was chosen as a marker to investigate the changed surface states of GCE after each assembly step (inset of Figure 1). A well-shaped CV with a peak-to-peak separation of 70 mV was observed at bare GCE (curve a). Compared with bare GCE, the decreased peak current and enlarged peak-to-peak separation of 306 mV for ABA-GCE (curve b) might result from the electrostatic repulsion between ferricyanide anions and negative charged ABA-GCE surface (pKa‑COOH of ABA, 4.80).27 The increased peak current and diminished peak-to-peak separation (100 mV) of En-ABA-GCE (curve c) to that of ABA-GCE (curve b) should be due to the electrostatic attraction between ferricyanide anions and the positive charged En-ABA-GCE surface (pKa‑NH2 of En, 9.93),28 although the current was still smaller and the peak-to-peak separation was larger than those of bare GCE. Finally, because the CdSe QDs were negatively charged under the given conditions (pKa‑COOH of MPA, 4.32),29 the CdSe QDs-EnABA-GCE showed a decreased peak current as well as 2786
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CdSe + e− → CdSe•−
(1)
S2 O82 − + e− → SO4 2 − + SO4•− CdSe
•−
+ SO4
•−
→ CdSe* + SO4
CdSe* → CdSe + hv
(2) 2−
(3) (4)
Figure 3 demonstrated the effects of scan rate on ECL intensity of the proposed sensor. The ECL intensity increased
Figure 4. The quenching effects of DA on the ECL behaviors of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 and (a) 0.0, (b) 0.010, (c) 0.20, and (d) 3.0 μM DA at 50 mV s−1. Inset A: The linear relationship between the concentration of DA and ECL intensity quenching degree (I 0 /I). Inset B: Reproducibility of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 and 0.20 μM DA.
respectively. No obvious change of the ECL intensity was observed after the addition of interfering agents (a, b, c of Figure 5), which indicated that UA and AA cannot interfere
Figure 3. Effects of scan rate on the ECL intensity of CdSe QDs-EnABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8.
with the enhanced scan rate from 20 to 80 mV s−1, similar to that of dual-stabilizers-capped CdTe QDs.8,22 Because repeatability of the ECL emission decreased dramatically with scan rate up to 60 mV s−1, a scan rate of 50 mV s−1 with a relative standard deviation (RSD) of 3.7% (n = 5) was selected herein. Performance of the Proposed ECL Sensor. DA is an important neurotransmitter in the brain.31 Many efforts have been focused on simple and accurate methods for DA determination. In this case, DA was taken as a model molecular to evaluate the performance of the proposed ECL sensor.32 Figure 4 displayed the quenching effects of DA on ECL behaviors of CdSe QDs-En-ABA-GCE. The ECL intensity in the presence of DA (I) was lower than that in the absence of DA (I0), and the ECL intensity decreased gradually with increasing concentrations of DA. The reason might be that DA inhibited the ECL reaction between CdSe QDs and (NH4)2S2O8.10 The ECL intensity quenching degree (I0/I) linearly responds to the DA concentration from 10.0 nM to 3.0 μM with a detection limit of 3.0 nM (S/N = 3) (inset A, Figure 4), indicating the analytical performance of this sensor is better than that of the other existing ECL sensors for DA.5,10,11,15,33 Five proposed sensors exhibited similar ECL responses with DA at the 0.20 μM level, and the RSD was 4.3% (inset B, Figure 4), indicating acceptable reproducibility. After being stored in the refrigerator at 4 °C for three weeks, the average ECL intensity of five proposed sensors only decreased 4.9% in 0.10 M pH 7.4 PBS containing 0.10 M (NH 4 ) 2 S 2 O 8 (Supporting Information Figure S-2), which indicated good storage stability. This might due to no enzyme and mediator being involved in the sensing strategy.7 AA and UA were two common interfering agents for DA detection.32,34 An interference investigation was performed by using the solution containing 20.0 μM AA and 20.0 μM UA,
Figure 5. Interference of AA and UA with the ECL response of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 at 50 mV s−1. (a) Normalized initial ECL intensity of CdSe QDs-En-ABA-GCE in 0.10 M pH 7.4 PBS containing 0.10 M (NH4)2S2O8 at 50 mV s−1, (b) a + 20.0 μM AA, (c) a + 20.0 μM UA, (d) a + 1.0 μM DA, (e) d + 10.0 μM AA + 10.0 μM UA.
with the ECL response of CdSe QDs-En-ABA-GCE at a level of 20.0 μM. When 10.0 μM AA and 10.0 μM UA were added to the PBS containing 1.0 μA DA, no obvious change of the ECL intensity for CdSe QDs-En-ABA-GCE (d, e of Figure 5) manifested that this ECL sensing strategy can be used to selectively detect DA. The applicability of the proposed ECL sensor was evaluatsed by detecting the DA in a DA hydrochloride injection, human urine from adult healthy volunteers, and CSF from volunteer patients with the standard addition method. The dopamine hydrochloride injection was diluted with 0.10 M phosphate buffer (pH 7.4) by 100 000 times as a real sample for analysis. The original DA concentration of dopamine hydrochloride injection was determined to be 0.055 M, close to the given value of 0.053 M. When 1.0 μM standard DA solution was added into the diluted injection, the average recovery was found 2787
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(5) Liu, X.; Jiang, H.; Lei, J.; Ju, H. Anal. Chem. 2007, 79, 8055− 8060. (6) Wang, J.; Han, H.; Jiang, X.; Huang, L.; Chen, L.; Li, N. Anal. Chem. 2012, 84, 4893−4899. (7) Liu, Q.; Han, M.; Bao, J. C.; Jiang, X. Q.; Dai, Z. H. Analyst 2011, 136, 5197−5203. (8) Liang, G.; Liu, S.; Zou, G.; Zhang, X. Anal. Chem. 2012, 84, 10645−10649. (9) Liu, X.; Zhang, Y.; Lei, J.; Xue, Y.; Cheng, L.; Ju, H. Anal. Chem. 2010, 82, 7351−7356. (10) Sun, F.; Chen, F.; Fei, W.; Sun, L.; Wu, Y. Sens. Actuators B 2012, 166−167, 702−707. (11) Yuan, D.; Chen, S.; Yuan, R.; Zhang, J.; Liu, X. Sens. Actuators B 2014, 191, 415−420. (12) (a) Niu, H.; Yuan, R.; Chai, Y.; Mao, L.; Liu, H.; Cao, Y. Biosens. Bioelectron. 2013, 39, 296−299. (b) Jie, G.; Yuan, J.; Zhang, J. Biosens. Bioelectron. 2012, 31, 69−76. (c) Jie, G.; Wang, L.; Zhang, S. Chem. Eur. J. 2011, 17, 641−648. (13) Wang, T.; Zhang, S. Y.; Mao, C. J.; Song, J. M.; Niu, H. L.; Jin, B. K.; Tian, Y. P. Biosens. Bioelectron. 2012, 31, 369−375. (14) Jie, G.; Liu, B.; Pan, H.; Zhu, J.-J.; Chen, H.-Y. Anal. Chem. 2007, 79, 5574−5581. (15) Bao, L.; Sun, L.; Zhang, Z.-L.; Jiang, P.; Wise, F. W.; Abruña, H. c. D.; Pang, D.-W. J. Phys. Chem. C 2011, 115, 18822−18828. (16) Jie, G.; Yuan, J. Anal. Chem. 2012, 84, 2811−2817. (17) Myung, N.; Lu, X.; Johnston, K. P.; Bard, A. J. Nano Lett. 2004, 4, 183−185. (18) Bae, Y.; Myung, N.; Bard, A. J. Nano Lett. 2004, 4, 1153−1161. (19) Myung, N.; Bae, Y.; Bard, A. J. Nano Lett. 2003, 3, 1053−1055. (20) Liu, X.; Guo, L.; Cheng, L.; Ju, H. Talanta 2009, 78, 691−694. (21) Liang, G.-d.; Shen, L.-p.; Zhang, X.-l.; Zou, G.-z. Eur. J. Inorg. Chem. 2011, 3726−3730. (22) Liang, G.; Shen, L.; Zou, G.; Zhang, X. Chem.Eur. J. 2011, 17, 10213−10215. (23) Liu, S.; Zhang, X.; Yu, Y.; Zou, G. Biosens. Bioelectron. 2014, 55, 203−208. (24) Yu, W. W.; Qu, L. H.; Guo, W. Z.; Peng, X. G. Chem. Mater. 2003, 15, 2854−2860. (25) Liu, S.; Wu, P.; Li, W.; Zhang, H.; Cai, C. Anal. Chem. 2011, 83, 4752−4758. (26) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991−1024. (27) Kazunori Yamada, T. T.; Sato, K.; Hirata, M. J. Appl. Polym. Sci. 2003, 89, 2535−2544. (28) Kroner, F.; Hubbuch, J. J. Chromatogr. A 2013, 1285, 78−87. (29) Ying, E.; Li, D.; Guo, S.; Dong, S.; Wang, J. PloS one 2008, 3, e2222. Zou, G. Z.; Liang, G. D.; Zhang, X. L. Chem. Commun. 2011, 47, 10115−10117. (30) Bertoncello, P.; Forster, R. J. Biosens. Bioelectron. 2009, 24, 3191−3200. (31) Swamy, B. E.; Venton, B. J. Analyst 2007, 132, 876−884. (32) Liu, X.; Cheng, L.; Lei, J.; Ju, H. Analyst 2008, 133, 1161−1163. (33) (a) Li, L.; Liu, H.; Shen, Y.; Zhang, J.; Zhu, J.-J. Anal. Chem. 2011, 83, 661−665. (b) Yan, Y.; Liu, Q.; Wang, K.; Jiang, L.; Yang, X.; Qian, J.; Dong, X.; Qiu, B. Analyst 2013, 138, 7101−7106. (c) Cui, R.; Gu, Y. P.; Bao, L.; Zhao, J. Y.; Qi, B. P.; Zhang, Z. L.; Xie, Z. X.; Pang, D. W. Anal. Chem. 2012, 84, 8932−8935. (34) (a) Liu, Q.; Zhu, X.; Huo, Z.; He, X.; Liang, Y.; Xu, M. Talanta 2012, 97, 557−562. (b) Liu, M.; Wang, L.; Deng, J.; Chen, Q.; Li, Y.; Zhang, Y.; Li, H.; Yao, S. Analyst 2012, 137, 4577−4583.
to be 106.9%. The human urine samples and CSF sample were diluted 10 times with a 0.10 M phosphate buffer (pH 7.4). The results are shown in Table 1, and the average recoveries ranged from 102% to 107.5%, showing a preliminary application of the sensor for the determining DA. Table 1. Results of Analysis of DA in Real Samples sample diluted injection CSF urine 1 urine 2
detected (μM)
added (μM)
found (μM)
recovery (%)
RSD (%) (n = 3)
0.55
1.00
1.069
106.9
3.0
----
0.20 0.20 0.20
0.215 0.204 0.209
107.5 102.0 104.5
2.5 4.2 4.3
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CONCLUSIONS An ECL sensor was designed for sensitive detection of small biomolecules by covalently immobilizing dual-stabilizerscapped CdSe QDs on GCE. The ECL sensor displayed eyevisible greenish and monochromatic ECL emissions without any signal amplification techniques and can be used to detect DA with high sensitivity, good precision, and acceptable storage stability. The strategy to form a dual-stabilizers-capped CdSe QD assembly on GCE is reliable for designing QD-based ECL sensors and devices.
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ASSOCIATED CONTENT
S Supporting Information *
CV of GCE in 10.0 mM pH 7.4 PBS containing 1.0 mM ABA and 10.0 mM KCl and storage stability of five proposed ECL sensors. This material is available free of charge via the Internet at http://pubs.acs.org
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AUTHOR INFORMATION
Corresponding Author
*Fax: +86-531-88564464. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This project is supported by the National Natural Science Foundation of China (Grant Nos. 21375077 and 21376010), the Independent Innovation Foundation of Shandong University (Grant No. 2012TS002) and State Key Laboratory of Analytical Chemistry for Life Science (Grant No. SKLACLS1201).
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REFERENCES
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