Article pubs.acs.org/ac
Ratiometric Electrochemical Sensor for Selective Monitoring of Cadmium Ions Using Biomolecular Recognition Xiaolan Chai,† Limin Zhang,‡ and Yang Tian*,‡ †
Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, People’s Republic of China Department of chemistry, East China Normal University, North Zhongshan Road 3663, Shanghai 200062, People’s Republic of China
‡
S Supporting Information *
ABSTRACT: A selective, accurate, and sensitive method for monitoring of cadmium ions (Cd2+) based on a ratiometric electrochemical sensor was developed, by simultaneously modifying with protoporphyrin IX and 6-(ferroceney) hexanethiol (FcHT) on Au particle-deposited glassy carbon electrode. On the basis of high affinity of biomolecular recognition between protoporphyrin IX and Cd2+, the functionalized electrode showed high selectivity toward Cd2+ over other metal ions such as Cu2+, Fe3+, Ca2+, and so on. Electroactive FcHT played the role as the inner reference element to provide a built-in correction, thus improving the accuracy for determination of Cd2+ in the complicated environments. The sensitivity of the electrochemical sensor for Cd2+ was enhanced by ∼3-fold through the signal amplification of electrodeposited gold nanoparticles. Accordingly, the present ratiometric method demonstrated high sensitivity, broad linear range from 100 nM to 10 μM, and low detection limit down to 10 nM (2.2 ppb), lower than EPA and WHO guidelines. Finally, the ratiometric electrochemical sensor was successfully applied in the determination of Cd2+ in water samples, and the obtained results agreed well with those obtained by the conventional ICP-MS method.
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detectability of analytical methods, the accuracy and selectivity of the analytical methods especially for the applications in complex system is still the bottleneck. In this work, a novel ratiometric electrochemical sensor was developed for determination of Cd2+ in water with high selectivity and accuracy. As shown in Scheme 1A, first of all, protoporphyrin IX was employed as a selective biomolecular recognition element for Cd2+ because protoporphyrin IX was reconstituted by Cd2+ with high selectivity. Second, electroactive 6-(ferroceney) hexanethiol (FcHT) was simultaneously modified with protoporphyrin IX onto the electrode and served as the inner reference molecule to provide a built-in correction for improving the accuracy. Meanwhile, Au nanoparticles were employed for enhancing the sensitivity of electrochemical detection, since they show unique electrochemical properties, such as large surface area and high catalysis activity.31−33 The developed ratiometric electrochemical sensor exhibited high selectivity for Cd2+ over other metal ions, because of the selective biomolecular recognition of protoporphyrin IX for Cd2+. Under the optimized conditions, a linear range from 100 nM to 10 μM was obtained, and detection limit was achieved to
eavy metals (i.e., cadmium, lead, mercury, copper, and etc.) can cause serious problems in the environment and to human health, because they are not biodegradable and remain indefinitely in ecological systems.1−9 Cadmium (Cd), one of the most hazard heavy metals, is well-known to affect various cellular processes through membrane damage, disruption of electron transport, enzyme inhibition/activation, and DNA alteration.10−12 In addition, experimental and epidemiological studies have provided substantial evidence that low level of long-term exposure to Cd can contribute to many serious diseases and several types of cancers.13−15 According to the Environmental Protection Agency (EPA, the United States) and the World Health Organization (WHO) guidelines, maximum contaminant level (MCL) for Cd in drinking water is 5 and 3 ppb, respectively.16,17 Therefore, it is very critical to develop a facile, selective, and sensitive approach to determination of Cd2+ in drinking water with low-cost and high accuracy. To date, several elegant methods have been employed for monitoring of Cd2+, e.g., atomic adsorption spectrometry (AAS),18,19 inductively coupled plasma mass spectroscopy (ICP-MS),20−22 electrochemical, and photoelectrochemical approaches,23−26 and so on.27−30 Electrochemical methods have attracted more attention, because of the low cost, simple instrumentation, and easy to real time and on-site detection. Despite continuous progress in proving the © 2014 American Chemical Society
Received: July 9, 2014 Accepted: October 1, 2014 Published: October 1, 2014 10668
dx.doi.org/10.1021/ac502521f | Anal. Chem. 2014, 86, 10668−10673
Analytical Chemistry
Article
Scheme 1. (A) Ratiometric Sensor for Monitoring of Cd2+; (B) Preparation Procedures for the Modified Electrodes
GC/Au/Cys electrode was immersed in solution of protoporphyrin IX (10 μM) for 15 h, by adding EDC and NHS as the catalysts. The protoporphyrin IX-functional electrode was denoted as GC/Au/Cys/PP. For the comparison of sensitivity, gold electrode (2 mm in diameter, Chenhua, Shanghai) was also employed as working electrode. First, gold electrode were polished with aqueous slurries of successively finer alumina powder (down to 0.05 μm) on a polishing microcloth and then sonicated for 10 min in water. The electrode was then pretreated electrochemically in 0.05 M H2SO4 by potential cycling in the potential range of −0.2 to +1.5 V at a potential scan rate of 10 V s−1 until the cyclic voltammogram characteristic for a clean gold electrode was obtained. Finally, gold electrode was immersed in the solution of cysteamine and FcHT (50:1) for 12 h. Followed that, the electrode was adsorped in protoporphyrin IX solution (10 μM) for 15 h, by adding EDC and NHS as the catalysts. This modified electrode was denoted as Au/FcHT-PP. For measurements of UV−vis absorption spectra, an indium tin oxide (ITO)-coated glass plate (∼10 Ω cm−2 in square resistance, Nanbo Display Technology Co. Ltd., Shenzhen, China) was thoroughly cleaned by 1 h sonication in the following solvents: soapy water, neat ethanol, and 1 M KOH, followed by a rinse with doubly distilled water. The ITO substrate was then dried with nitrogen gas and treated as the modification processes as GC electrode. The modified electrode was denoted as ITO/Au/FcHT-PP. Apparatus and Instruments. Electrochemical experiments were performed with CHI 832 and CHI 660 electrochemical workstation (Chenhua, Shanghai). GC, gold, ITO, and the modified electrodes were used as working electrodes. A Pt wire was the counter electrode, and all potentials were based versus Ag/AgCl electrode (saturated with KCl). All measurements were carried out at ambient temperature and the pH value was determined with a pH meter. X-ray photoelectron spectroscopy (XPS) investigation was conducted in PHI-5000C ESCA system (PerkinElmer) with Mg Kα radiation (hν = 1253.6 eV). All binding energies (BEs) were referred to the C 1s peak (284.6 eV) arising from surface hydrocarbons (or adventitious hydrocarbon). SEM image was taken using a field emission gun Hitachi S-4800 scanning electron microscope (SEM) (Japan) operated at 1.0 kV. X-ray diffraction (XRD) was carried out by a Shimadzu XRD-6000. Inductively coupled plasma atomic emission spectroscopy (ICP-MS) was conducted a Hitachi P-4010 system (Japan). UV−vis spectrum was carried out using an Agilent 8453 spectrometer.
10 nM (2.2 ppb). Finally, the designed ratiometric electrochemical sensor was successfully applied to determine Cd2+ in water samples.
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EXPERIMENTAL SECTION Reagents and Chemicals. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4·3H2O, 99%), 6-(ferrocenyl) hexanethiol (FcHT), cysteamine, protoporphyrin IX (PP), 1-Ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS), methanol, ethanol, benzaldehyde (BzH), benzoic acid (BzOH), o-aminophenol (OAP), aniline (AN), humic acid (HA) and fulvic acid (FA) were purchased from Sigma-Aldrich (U.S.A.) and used as received. All other regents were of analytical grade and purchased from Aladdin-reagent company (Shanghai, China). The solution of metal ions was all prepared from their chloride salts with doubly distilled water. The solution used for electrochemical measurements was deoxygenated with nitrogen at least for 30 min before experiments. Preparation and Modification of Electrodes. Glassy carbon (GC, 3 mm in diameter, Chenhua, Shanghai) electrode was first polished with emery paper, 1.0 and 0.05 μm alumina slurry on a polishing cloth. Then, the electrode was sonicated in acetone and distilled water each for 3 min, and finally rinsed with doubly distilled water. Next, GC electrode was electrodeposited in HAuCl4 (20 mM) at the potential of −0.2 V for 150 s to form gold particles (AuPs) on the electrode. This substrate was denoted as GC/Au. Next, the electrode was immersed in the mixed solution of cysteamine (50 mM) and FcHT (1 mM) for 12 h. After the attachment of cysteamine and FcHT, the electrode was denoted as GC/Au/FcHT-Cys. Finally, GC/Au/FcHT-Cys electrode was immersed in solution of protoporphyrin IX (10 μM) for 15 h, by adding EDC and NHS as the catalysts. The protoporphyrin IX-functional electrode was denoted as GC/Au/FcHT-PP. The modification processes are illustrated in Scheme 1B. The normal biosensor for Cd2+ with recognition element of PP and no reference element of FcHT was constructed as follows. The GC/Au electrode was immersed in the solution of cysteamine (50 mM) for 2 h. After the attachment of cysteamine, the electrode was denoted as GC/Au/Cys. Finally,
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RESULTS AND DISCUSSION Characterization of the Modified GC Electrode. The schematic process for electrode modification is demonstrated in Scheme 1B. Initially, AuPs were electrodeposited on GC electrode, which was denoted as GC/Au. From typical SEM images of the AuPs (Figure 1A), we can see that the AuPs were uniformly distributed on the surface of GC electrode with a diameter of ∼1−1.5 μm. The XRD pattern (Figure 1B) shows sharp peaks corresponding to the (111), (200), (220), (311), and (222) diffraction peaks of metallic Au, and thus reveals that the precipitate is composed of crystalline Au. Then, FcHT and cysteamine were simultaneously attached onto GC/Au electrode through AuS covalent bond (GC/Au/FcHTCys). Finally, protoporphyrin IX was assembled on the electrode surface by using EDC and NHS to form amide 10669
dx.doi.org/10.1021/ac502521f | Anal. Chem. 2014, 86, 10668−10673
Analytical Chemistry
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Figure 1. (A) SEM image and (B) XRD pattern of AuPs after electrodeposition on GC surface.
Figure 3. UV−vis absorption spectra of (a) ITO/Au, (b) ITO/Au/ FcHT-Cys, (c) ITO/Au/FcHT-PP, and (d) ITO/Au/FcHT-PP with the addition Cd2+. Inset: UV−vis absorption spectra of 10 μM protoporphyrin IX solution (I) in the absencence and (II) in the presence of a stoichiometric amount of Cd2+.
between COOH group of protoporphyrin IX and NH2 moiety of cysteamine (GC/Au/FcHT-PP). The modification processes were tracked by X-ray photoelectron spectroscopy (XPS). After electrodeposition of Au particles, Au 4f7/2 (83.8 eV) and Au 4f5/2 (87.6 eV) were clearly observed, as shown in Figure 2A (curve a). The observation of
Continuous increasing of pH led to decreasing ratiometric peak current, which is due to the hydrolysis of Cd2+ in basic solution (Supporting Information, SI, Figure S1). Therefore, pH 7.0 (0.1 M PBS) was selected as the most suitable supporting electrolyte. Under the conditions employed, the reaction between protoporphyrin IX and Cd2+ showed saturate within 5 min (SI Figure S2). Thus, the GC/Au/FcHT-PP electrode was incubated in Cd2+ solution for 5 min before measurements. Electrochemical Responses of GC/Au/FcHT-PP Electrode. Considering its high sensitivity, differential pulse voltammetry (DPV) was employed for the quantitative determination of Cd2+. As demonstrated in Figure 4, cathodic
Figure 2. XPS spectrum of (A) Au 4f7/2 and Au 4f5/2, (B) Fe 2p3/2 and Fe 2p1/2, (C) N 1s, and (D) S 2p for (a) GC/Au, (b) GC/Au/FcHTCys, and (c) GC/Au/FcHT-PP.
Fe 2p3/2 (709.2 eV) and Fe 2p1/2 (722 eV) in Figure 2B (curve b), N 1s (400.0 eV) in Figure 2C (curve b), and S 2p (163.0 eV) in Figure 2D (curve b) indicates the successful modification of FcHT and cysteamine onto the GC/Au electrode, since no corresponding signals were obtained in the former modification process (curve a in Figure 2B−D). After the attachment of protoporphyrin IX, an obvious increase of peak at 400.0 eV for N 1s in Figure 2C (curve c) was observed, suggesting the successful assembly of protoporphyrin IX on the electrode. Meanwhile, from UV−vis absorption spectrum of protoporphyrin IX immobilized on the Au/FcHTCys surface (curve c, Figure 3), an obvious peak at 405 nm and four-modal band were observed, which is in good accordance with that of protoporphyrin IX solution. The results confirmed that protoporphyrin IX was successfully modified on the electrode. The influence of pH on the determination of Cd2+ was also investigated. As the pH increased from 5.0 to 7.0, the ratiometric peak current increased because at low pH values nitrogen atoms of protoporphyrin IX attached on GC electrode are protonated, therefore the GC/Au/FcHT-PP surface is positively charged and lose its ability of complexing with Cd2+.
Figure 4. Differential pulse voltammograms (DPVs) obtained at (a) bare GC, (b) GC/Au, (c) GC/Au/FcHT-Cys, and (d) GC/Au/ FcHT-PP electrodes in 0.1 M PBS (pH = 7.0) with the addition of 5 μM Cd2+.
peak located at 260 mV vs Ag|AgCl was clearly observed at GC/Au/FcHT-Cys electrode attributed to the reduction of FcHT in 0.1 M PBS solution (pH 7.0). However, the cathodic peak located at −224 mV vs Ag|AgCl was only obtained at GC/ Au/FcHT-PP electrode after protoporphyrin IX was immobilized on GC/Au/FcHT-Cys electrode in PBS solution with the addition of Cd2+, while no obvious responses were obtained at other modified electrodes even with the addition of Cd2+. The results indicate that as expected, the protoporphyrin IXfunctionalized electrode demonstrated sensitive response toward Cd2+ due to the complexation reaction of protoporphyrin IX with Cd2+. This observation was evident by UV− vis spectroscopy shown in Figure 3 (curve d). UV−vis absorption peak of protoporphyrin IX on the electrode surface located at 406 nm red-shifted to 438 nm after the addition of 10670
dx.doi.org/10.1021/ac502521f | Anal. Chem. 2014, 86, 10668−10673
Analytical Chemistry
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Cd2+, and four-modal band also changed to two-modal spectra with maximum at 561 and 592 nm. This observation agrees with the changes that observed in protoporphyrin IX solution with the addition of Cd2+ shown in the inset of Figure 3, indicating complexation of protoporphyrin IX with Cd2+. The complexation reaction showed 1:1 molar ratio, which was in a good agreement with those reported in earlier papers.34,35 As shown in Figure 5A, the cathodic peak obtained at −224 mV gradually increased with the increasing concentration of Figure 6. Selectivity and competition tests of metal ions against Cd2+. The white bars mean the addition of potential interferences. The black bars mean the subsequent addition of 10 μM Cd2+. Concentration of metal ions is 10 μM except of Ca2+, Na+, and Mg2+ which are 1 mM.
demonstrated in Figure 6, negligible effects (
