Article pubs.acs.org/ac
Detection of Arsenic(III) through Pulsed Laser-Induced Desorption/ Ionization of Gold Nanoparticles on Cellulose Membranes Cheng-I Weng,† Jin-Shun Cang,‡ Jia-Yaw Chang,§ Tung-Ming Hsiung,† Binesh Unnikrishnan,† Yu-Lun Hung,† Yu-Ting Tseng,† Yu-Jia Li,† Yu-Wei Shen,† and Chih-Ching Huang*,†,⊥,# †
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan Department of Chemistry, Yancheng Institute of Industry Technology, Yancheng, Jiangsu 224005, P. R. China § Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan ⊥ Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan # School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan ‡
S Supporting Information *
ABSTRACT: We have developed an assay based on gold nanoparticle-modified mixed cellulose ester membrane (Au NPs-MCEM) coupled with laser-induced desorption/ionization mass spectrometry (LDI-MS)-for the detection of arsenic(III) ions (arsenite, AsO2−) in aqueous solution. When the Au NPs reacted with lead ions (Pb2+) in alkaline solution (5 mM glycine−NaOH, pH 12), Au−Pb complexes, PbO, and Pb(OH) were formed immediately on the Au NP surfaces. The Pb species reacted rapidly with subsequently added AsO2− to form PbOAs2O3, (PbO)2As2O3, and/or (PbO)3As2O3 shells (2−5 nm) on the Au NPs’ surfaces. As a result, significant observable aggregation of the Au NPs occurred in the solution. This Pb2+/Au NP probe allowed the detection of AsO2− at concentrations as low as 0.6 μM with high selectivity (at least 100-fold over other anions and metal ions). To further improve the sensitivity, we prepared Au NPs-MCEM for the LDI-MS-based detection of AsO2− ions. The intensity of the signal for the [Pb]+ ions in the mass spectra increased when the Au NPs-MCEM reacted with AsO2−; in contrast, the intensity of the signal for [Au]+ ions decreased. Accordingly, the [Pb]+/[Au]+ peak ratio increased upon increasing the AsO2− concentration over the range from 10 nM to 10 μM. The limit of detection at a signal-to-noise ratio of 3 was 2.5 nM, far below the action level of As (133 nM, ca. 10 ppb) permitted by the US EPA for drinking water. Relative to other nanoparticle-based arsenic sensors, this approach is rapid, specific, and sensitive; in addition, it can be applied to the detection of AsO2− in natural water samples (in this case, streamwater, lake water, tap water, groundwater, and mineral water).
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rsenic is a toxic metalloid found widely in nature.1 The arsenic content of the Earth’s crust is 1.8 ppm; in the soil, it is 0.2−40 ppm with an average concentration of 5 ppm.2 Volcanic materials contain arsenic at 20 ppm; the average concentration of arsenic in seawater is approximately 3 ppm.3 Arsenic is released into the atmosphere mainly from the weathering of rocks, from the soil, and from plants; the estimated annual emission of arsenic into the atmosphere from the Earth’s surface is approximately 2.37 × 107 kg.4 Methylation of arsenic by soil microbes generates volatile methylated arsenic species, having the general formula (CH3)nAsH(3−n) (n = 1−3), that are released into the atmosphere.5 Plants intake arsenic through their roots; after conversion, they also discharge the methylated arsenic species (CH3)2AsH and (CH3)3As into the air.6 These compounds react with O2 to produce (CH3)2AsO and (CH3)2As(OH), which further react with ozone (O3) and N2O4 to generate As4O6, which reenters the soil and is then converted back to HAsO2.7 Arsenic can flow into lakes, rivers, © 2014 American Chemical Society
and groundwater through the action of rain or wastewater discharge.8 Arsenic can cause acute and chronic poisoning, including abdominal pain, bloody diarrhea, acute renal failure and neuropathy, muscle weakness, skin keratinization, pigmentation, and cancer (lung, liver, bladder, and skin cancer).9 In addition, arsine (AsH3) gas, which is used commonly in the electronics industry and has a garlic flavor, readily induces hemolysis and then death through acute renal failure.10 Trivalent arsenic [arsenic(III)] and pentavalent arsenic [arsenic(V)] enter the human body most readily through drinking water.11 Arsenic(III) species form bonds with the sulfhydryl groups of protein S (R−AsO + 2 RSH → R−As− (SR)2 + H2O) and can interfere with the reactions of other Received: January 6, 2014 Accepted: February 20, 2014 Published: February 20, 2014 3167
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Scheme 1. Cartoon Representations of (A) the Colorimetric Sensing Mechanism of the Pb2+/Au NPs Probe and (B) the Use of a Pb2+/Au NPs-MCEM for the Detection of AsO2− Ions through LDI-MS
AsO2− based on a specific AsO2−-binding aptamer and gold nanoparticles (Au NPs); it operated through AsO2−-mediated interaction of the aptamer and poly(diallyldimethylammonium) and aggregation of the Au NPs.19 A solution of arsenic-binding aptamer and crystal violet will form crystal violet/aptamer complex NPs; changes in the sizes of these NPs, induced by AsO2−, enable the detection of AsO2− based on resonance Rayleigh scattering spectrometry.20 Although these NP-based optical sensors all exhibit high sensitivity for the detection of arsenic ions, they have limited applicability because of matrix interference, high cost, poor stability, and the need for special agents to selectively detect arsenic ions. Therefore, the development of reliable and rapid routine methodologies for the selective detection of arsenic ions in water remains an attractive challenge. In this study, we developed a laser desorption/ionization mass spectrometry (LDI-MS)-based assay for the detection of AsO2− using Au NP-modified membranes. As displayed in Scheme 1A, the addition of Au NPs to a lead ion (Pb2+) solution in an alkaline medium can form AuPb alloys as well as PbO or Pb(OH)2 species on the surface of the Au NPs. When AsO2− is added to the alkaline solution of Pb/Au NPs, the AsO2− and PbO species will react to form As2O3 or Pb(AsO2)2, leading to aggregation of the Au NPs. To increase the sensitivity and linear range, we adsorbed the Au NPs onto a mixed cellulose ester membrane (MCEM) for use as a substrate for LDI-MS-based detection of AsO2−, that is, by measuring the signal for the [208Pb]+ ions, rather than detecting AsO2− directly (Scheme 1B). Upon pulsed laser irradiation, we detected the [208Pb]+ ions at m/z 208, with laser-induced desorption and ionization of [208Pb]+ being enhanced dramatically in the
enzymes or proteins.12 Pentavalent arsenic [arsenate(V)] can replace some phosphate units because of their similar structures; for example, it can weaken the bonding in adenosine triphosphate (ATP), thereby leading to the generation of less energy and disrupting normal biological or cellular energy supply.13 The US Environmental Protection Agency (EPA) has established the limit of arsenic in drinking water at 10 ppb (133 nM).14 Most of the arsenic in water that comes into contact with air (e.g., in rivers, lakes, or seawater) is of the form arsenic(V), while that in closed water (e.g., groundwater) is typically in the form of arsenic(III) [arsenite (AsO2−)].15 Thus, it is inevitable that AsO2− will enter living systems from water sources, potentially leading to severe poisoning. Therefore, determining the AsO2− contents of various water samples is a necessary and indispensable task. At present, the techniques employed most widely to detect arsenic in water samples are inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICPMS), graphite furnace atomic absorption spectrometry (GF-AAS), and hydride generation atomic absorption spectrometry (HG-AAS).16 These techniques often suffer, however, from complex and cumbersome sample handling and the inability to detect AsO2− selectively. Although the detection limit of ICPMS is very low relative to the other methods, interference caused by the formation of isobars in the plasma can be problematic. In recent years, several nanomaterial-based optical sensors have been developed for the detection of arsenic ions in water samples.17 For example, fluorescent gold nanoclusters have been used to sense AsO2− with limit of detection (LOD) of 28 ppb.18 Wu et al. reported a colorimetric sensor for the selective detection of 3168
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presence of AsO2−. It appears that the Pb(AsO2)2 species formed from the reaction of AsO2− ions with PbO allow for detection of [208Pb]+ ions with greater sensitivity and less noise than the detection of the AsO2− signals directly. This method exhibits low interference and can, therefore, be applied to the detection of AsO2− in natural water samples.
centrifugation/wash cycles to remove most of the free Pb2+ ions in solution. Centrifugation was performed at a relative centrifugation force (RCF) of 30,000g at 25 °C for 20 min, and ultrapure water (1 mL × 3) was used to wash the Au NPs pellets. The purified Pb/Au NPs were then collected and acidified with 2% HNO3 before the metal ion concentrations were determined by ICPMS (Agilent 7700 Series, Agilent Technologies, California, USA). Detecting Arsenite Using the Membrane-Based Probe. The as-prepared Au NPs-MCEM was immersed in 5 mM glycine−NaOH solution (pH 12, 1.0 mL) containing Pb2+ (100 nM) for 30 min and then immersed in AsO2− (0−10 μM) ions for 30 min at room temperature. The membranes were gently washed with DI water (5 mL) for 30 s, dried in air at room temperature, and then attached to a MALDI plate using adhesive polyimide film tape. Mass spectrometry experiments were performed in the reflectron positive-ion mode using an AutoflexIII MALDI time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). The samples were irradiated using a pulse laser (355-nm Nd:YAG, 100 Hz; pulse width: 6 ns). A delayed extraction period of 20 ns was set to energetically stabilize the positively charged ions produced during LDI, and then, the ions were accelerated through the TOF chamber in the reflection mode prior to entering the mass analyzer. The accelerating voltages were applied in the range from +20 to −20 kV. The instrument was calibrated to the signals for the Au clusters ([Aux]+; x = 1−5) prior to recording the mass spectra. To accumulate signals, 300 pulsed laser shots at a laser fluence of 4.9 × 104 W cm−2 were applied to five random positions on the MALDI target plate. Analysis of Real Samples. Water from a stream on the campus of National Taiwan Ocean University, lake water from National Taiwan University, local tap water and groundwater, and mineral water from Young Energy Source Co., Ltd. were filtered through a 0.2 μm membrane. For the detection of AsO2−, aliquots of the water samples (500 μL) were spiked with standard AsO2− ion solutions (from 10 nM to 10 μM). The spiked samples were diluted to 1.0 mL with 5 mM glycine−NaOH solution (pH 12, 1.0 mL), and then, the Pb2+/ Au NPs-MCEMs were immersed in the solution for 30 min and then washed prior to MS measurements. All spiked samples were also analyzed by ICPMS.
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EXPERIMENTAL METHODS Materials. Citric acid, trisodium citrate, and all metallic salts used in this study were purchased from Aldrich (Milwaukee, WI). Hydrogen tetrachloroaurate(III) trihydrate were obtained from Acros (Geel, Belgium). MCEM (pore size: 0.45 μm) was purchased from Advantec (Toyo Roshi Kaisha, Japan). Milli-Q ultrapure water was used in all experiments. Glycine obtained from J. T. Baker (Phillipsburg, NJ, USA) was used to prepare the glycine buffer (50 mM, pH 9−12, adjusted with 1.0 N NaOH). Au NPs and Au NPs-MCEMs. The 32 nm spherical Au NPs were prepared through citrate-mediated reduction of HAuCl4.21 Aqueous 0.01% HAuCl4 (50 mL) was brought to a vigorous boil while stirring in a round-bottom flask fitted with a reflux condenser; 1% trisodium citrate (0.5 mL) was added rapidly, and then heating was continued for another 8 min, during which time the color of the solution changed from pale yellow to red−purple. The solution was cooled to room temperature with continuous stirring. The sizes of the Au NPs were verified using transmission electron microscopy (TEM; H7100, Hitachi High-Technologies Corporation, Tokyo, Japan); they appeared to be nearly monodisperse, with an average size of 32.3 ± 2.8 nm. A double-beam UV−vis spectrophotometer (Cintra 10e, GBC, Victoria, Australia) was used to measure the absorption of the Au NPs solution. The particle concentration of the Au NPs (280 pM) was determined according to Beer’s law, using an extinction coefficient of 1.2 × 109 M−1 cm−1 at 526 nm for the 32 nm Au NPs. An MCEM having a diameter of 0.6 cm was used to prepare the Au NPs-MCEM substrate. The MCEM was immersed in a Au NP (140 pM)-containing citrate solution (0.25 mM, pH 5.8, 20 mL) and incubated for 2 h. The Au NPs-adsorbed MCEM (Au NPs-MCEM) was washed gently with ultrapure water (20 mL) for 2 min and then dried in air for 2 h at room temperature. Characterization of Au NPs. TEM images of Au NPs were recorded using a H7100 transmission electron microscope (operated at 125 kV). Samples for TEM measurements were prepared by placing 20 μL of the Au NPs solution on a carboncoated copper grid and then drying at room temperature. The zeta potentials of the Au NPs were measured using a Zetasizer 3000HS analyzer (Malvern Instruments, Malvern, UK). Pb2+/Au NP-Based Colorimetric Sensor for Arsenite. The Pb2+/Au NP system was prepared by equilibrating the Au NPs (140 pM) and Pb2+ ions (5.0 μM) in 5 mM glycine− NaOH (pH 12) solution for 30 min. For AsO2− sensing, aliquots (500 μL) of the as-prepared Pb2+/Au NPs and AsO2− ions (0−10 μM) were equilibrated at room temperature for 30 min. The mixtures were then transferred separately into 96-well microtiter plates, and their UV−vis absorption spectra were recorded using a μ-Quant monochromatic microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). Herein, the final concentrations of the species are provided. To determine the number of Pb atoms/ions on the Au NPs’ surface, the mixtures of Pb2+ and Au NPs solutions were maintained at 25 °C for 1 h and were then subjected to three
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RESULTS AND DISCUSSION Interaction of AsO2− and Pb2+/Au NPs. Scheme 1A outlines the colorimetric sensing mechanism for AsO2− in this study. When the Au NPs (32-nm, 140 pM) reacted with Pb ions (Pb2+, 5.0 μM) in alkaline solution (5 mM glycine− NaOH, pH 12), Au−Pb complexes and PbO and Pb(OH) species were formed immediately on the surfaces of the Au NPs (Figures S1 and S2, Supporting Information), leading to a slight increase in their surface plasmon resonance (SPR) absorption (curve B, Figure 1). The TEM images in Figure 2 indicate that the Au NPs in the presence of Pb2+ ions were almost the same size and shape as those in the absence of the Pb2+ ions, suggesting formation of only a monolayer or submonolayer of Pb complexes on the NP surfaces. In addition, using ICPMS to quantify the content of Pb species on the Au NPs, we obtained a value of 29,400 Pb atoms/ions per Au NP, close to the number of surface atoms of the 32 nm Au NPs (ca. 40,000 atoms per particle). After adding AsO2− (10 μM), the Pb species on the NPs’ surfaces react rapidly to form PbOAs2O3, (PbO)2As2O3, and/or (PbO)3As2O3 shells (2−5 nm) on the 3169
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induce aggregation of the citrate-capped Au NPs (curve D, Figure 1). Parameters for Detection of AsO2−. We investigated the effect of the concentration of Pb2+ ions (0−10 μM) on the aggregation of the Au NPs in the absence and presence of AsO2− ions (0−10 μM). The absorption (extinction) bands of the Au NPs at 750 and 526 nm were related to the quantities of aggregated and dispersed Au NPs, respectively. The extinction ratio (Ex750/526) could, therefore, be used as a measure of the degree of aggregation, with high values corresponding to high degrees of aggregation. The degree of aggregation of the Au NPs in the 5 mM glycine−NaOH (pH 12) solution increased upon increasing the concentration of Pb2+ ions in the absence of AsO2− ions (Figure S5A, Supporting Information). This phenomenon was due mainly to the formation of larger amounts of PbO and/or Pb(OH) on the NPs’ surfaces under higher concentrations of Pb2+ ions (>5.0 μM). In addition, the degree of aggregation increased upon increasing the concentration of AsO2− ions (0−10 μM) in the presence of a constant concentration of Pb2+ ions (within the range of 2.5−7.5 μM). In a subsequent study, we maintained the concentration of Pb2+ ions in the solution at 5.0 μM and detected the AsO2− ions in glycine buffers (5 mM) at values of pH from 9.0 to 12.0. The sensitivity of the Pb2+/Au NP probe for AsO2− ions increased upon increasing the pH of the solution (Figure S5B, Supporting Information). We presume that PbO and/or Pb(OH) formed readily, depositing on the Au NPs’ surfaces, at higher values of pH; this phenomenon favored the formation of Pb/As shells on the Au NPs, thereby inducing their aggregation. We noted that the Au NPs tended to aggregate in the absence of Pb2+ and AsO2− ions when the pH of the glycine−NaOH buffer was greater than 12 (data not shown). Figure S5C (Supporting Information) indicates that the aggregation of Pb2+ (5.0 μM)/ Au NPs (140 pM) induced by the AsO2− ions (10 μM) decreased when we increased the temperature from 25 to 80 °C, mainly because As−Pb composites [e.g., PbOAs2O3, (PbO)2As2O3, (PbO)3As2O3] are difficult to form at higher temperature. Under the optimal solution conditions [5 mM glycine−NaOH (pH 12), Pb2+ (5.0 μM), room temperature (ca. 25 °C)], the Pb2+/Au NP probe allowed the detection of AsO2− at concentrations as low as 0.6 μM (Figure S6A, Supporting Information) with high selectivity [at least 100-fold over other anions, metal ions, and amino thiol (cysteine); Figure S6B, Supporting Information]. The high selectivity of the Pb2+/Au NPs for AsO2− ions was presumably due to the specific reactions of Pb ions, PbO and Pb(OH) species with AsO2− ions on the Au NPs’ surfaces under alkaline conditions (pH 12). The response of the sensor toward AsO2− ions in the presence of various ion species was also tested and found that the tolerance concentrations of other metal ions, cysteine, and anions (within a relative error of ±5%) for the sensing of AsO2− using this Pb2+/Au NPs probe were at least 10 times the AsO2− concentrations (Figure S7A, Supporting Information). Our probe showed a high tolerance to high concentrations of amino thiol (cysteine) which is otherwise capable of forming complexes with the AsO2− ions and interferes with the detection of the AsO2−. These results suggest that the species we tested should not interfere with the determination of AsO2− when applying our developed probe. Membrane-Based Sensor for AsO2−. To further improve the sensitivity, we prepared a Au NPs-MCEM for LDI-MSbased detection of AsO2− ions (Scheme 1B). In previous reports, we demonstrated a Au NP-MCEM that provided very
Figure 1. UV−vis absorption spectra of solutions containing (A) Au NPs, (B) Au NPs and Pb2+, (C) Au NPs, Pb2+, and AsO2−, and (D) Au NPs and AsO2−. Inset: Photograph of the four Au NPs solutions. The concentrations of the Au NPs, PbNO3, and NaAsO2 were 140 pM, 5.0 μM, and 10 μM, respectively. Buffer: 5 mM glycine−NaOH solution (pH 12).
Figure 2. TEM images of Au NPs in the (A) absence and (B−D) presence of (B) Pb2+, (C) Pb2+ and AsO2−, and (D) AsO2− in 5 mM glycine−NaOH solution (pH 12). Other conditions were the same as those described in Figure 1.
Au NPs’ surfaces (see arrow in Figure 2C).22 Energy-dispersive X-ray spectroscopy (EDS) revealed an As-to-Pb atomic ratio of close to 2:3 (Figure S3, Supporting Information), suggesting that the major component in the shell was (PbO)3As2O3. Scanning transmission electron microscopy/energy-dispersive X-ray mapping (STEM−EDS; Figure S4, Supporting Information) indicated that the major components in the shell were Pb and As. We also observed that the zeta potential (ξ) of the Au NPs decreased, from −47.8 to −21.2 mV, after reaction with AsO2−. As a result, significant aggregation of the Au NPs occurred in the solution (Figure 2C). The aggregated Au NPs induced SPR absorption coupling, with the absorption band of the Au NPs at 526 nm undergoing a red-shift with decreased absorption, while the intensity of the absorption band at 750 nm increased (curve C in Figure 1). Dynamic light scattering (DLS) measurements revealed that the Pb2+/Au NPs in the absence and presence of AsO2− (1.0 μM) had diameters of 39.5 (±7.8; n = 4) and 254.5 (±29.5; n = 4) nm, respectively, further confirming that AsO2− induced aggregation of the Pb2+/ Au NPs. In a control experiment, the addition of AsO2− did not 3170
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homogeneous, clean mass spectra of [Aun]+ (n = 1−5) cluster ions, with the formation of the [Aun]+ cluster ions being very sensitive to the particle’s surface properties (composites), under pulsed laser irradiation.23 Figure 3A displays LDI mass spectra
because the boiling temperature is easy to reach under irradiation with a nanosecond-pulse laser, whereas a fragmentation temperature that exceeds the Rayleigh instability limit is not.25 We noted that almost all of the Au NPs had desorbed from the surface of the MCEM (Figure S9, Supporting Information) after 300 shots of pulsed laser irradiation at 4.9 × 104 W cm−2. Figure 3 reveals that the intensities of the signals for the [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ ions all increased upon increasing the concentration of AsO2− ions. We suggest that the formation of more Pb−As nanocomposites on the Au NPs upon increasing the concentration of AsO2− was the main reason for the increase in the intensity of the Pb-related peaks ([Pb]+, [AuPb]+, [Au2Pb]+). After formation of Pb−As nanocomposite shells on the Au NPs, however, the laserirradiated Au NPs transferred their energy to the surface Pb−As nanocomposites, thereby suppressing evaporation of the surface Au atoms into the gas phase and resulting in low abundances for the signals of the Au clusters in the mass spectra. We observed no signals for Pb−As hybrids [e.g., PbOAs2O3, (PbO)2As2O3, (PbO)3As2O3] in the mass spectra, presumably because these composites decompose under the pulsed laser irradiation and/or react with other ions or molecules in the gas phase. The average relative standard deviations (RSDs) for the signal fluctuations of the [208Pb]+, [Au208Pb]+, and [Au2208Pb]+ ions collected from 50 different mass spectra were approximately 15% (Figure S10, Supporting Information) when using the Pb2+/Au NPs/MCEM for detection of AsO2− ions (100 nM). In addition, each of the RSDs decreased to less than 5% when we normalized the signal intensity of the [208Pb]+ ions with respect to the signal intensity of the [Au1]+ ions ([208Pb]+/ [Au1]+) from each MS measurement. Quantitation of AsO2− ions in solution was possible after recording the ratio of the signal intensities of the [208Pb]+ and [Au1]+ ions from 300 different sample spots in membranes (Figure 4). The [208Pb]+/
Figure 3. (A) LDI mass spectra of the Pb2+/Au NPs-MCEM in the absence and presence of AsO2− (1.0 nM−10 μM). (B) Relative peak intensities (S/S0) of [Au1]+, [Au2]+, [Au3]+, [Au4]+, [Au5]+, [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ plotted against the concentration of AsO2− (1.0 nM−10 μM). The descriptors S0 and S represent the mass spectral signal intensities of the [Au1]+, [Au2]+, [Au3]+, [Au4]+, [Au5]+, [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ ions in the absence and presence of AsO2−, respectively. Error bars represent standard deviations derived from four repeated experiments. Figure 4. Relative signal intensity [208Pb]+/[Au1]+ plotted with respect to the concentration of AsO2− ions (1.0 nM−10 μM). Other conditions were the same as those described in Figure 3.
of the Pb2+/Au NPs-MCEMs in the absence and presence of AsO2− ions (0−10 μM). Under pulsed laser irradiation (355 nm Nd:YAG, 4.9 × 104 W cm−2; pulse width: 6 ns), we detected Au cluster ions ([Aun]+; n = 1−5) and [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ ions produced from the Au NPs’ surfaces (Figure 3A, panel a). Using the Au clusters ([Aun]+; n = 1−5) as the calibrating internal standard, Figure S8 (Supporting Information) reveals that all of the masses of the [Pb]+ ions in the mass spectra were accurate to within 28 ppm of their theoretical masses, with isotopic distributions close to the predicted isotope patterns. Photothermal surface evaporation, Coulomb explosion, and near-field ablation have all been proposed as mechanisms for the desorption and ionization of metallic NPs.24 The fragmentation of the Au NPs may favor the photothermal surface evaporation mechanism
[Au1]+ ratio increased upon increasing the AsO2− concentration over the range from 10 nM to 10 μM. The plot of [208Pb]+/ [Au1]+ against the logarithm of the concentration of AsO2− ions over the range from 10 nM to 10 μM was linear (y = 0.0915x + 0.8686; y: [ 208 Pb] +/[Au 1 ]+ ratio; x: logarithm of the concentration of AsO2− ions), with a correlation coefficient (R2) of 0.99. The LOD at a signal-to-noise (S/N) ratio of 3 was 2.5 nM, far below the action level of As (133 nM, ca. 10 ppb) permitted by the US EPA for drinking water. Figure S11 (Supporting Information) reveals the good reproducibility (RSD < 5%) of the mass spectra obtained from a solution of 3171
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Figure 5. Analyses of AsO2− ions in representative streamwater, lake water, tap water, groundwater, and mineral water samples, determined using Pb2+/Au NPs-MCEMs probes. Water samples (diluted 10-fold) were spiked with AsO2− ions (1.0 nM−10 μM). Other conditions were the same as those described in Figure 3.
100 nM AsO2− ions when using 10 different Au NP-MCEMs as substrates with three replicate recordings. The tolerance test for the membrane-based probe for different metal ions, anions, and amino thiol (Figure S7B, Supporting Information) suggests that the tolerance concentrations of these species for the sensing of AsO 2 − were at least 10 times greater than the AsO2 − concentrations. Since the Au NPs modified MCEM is treated with Pb2+ in alkaline solution (5 mM glycine−NaOH, pH 12), the Pb ions form compounds such as PbO or Pb(OH) species on the Au NPs’ surface, which does not easily react with S2− ions avoiding the possibility for interference. The selectivity and tolerance studies (Figure S6 and S7, Supporting Information) support this fact. Detection of AsO2− Ions in Real Samples. To validate the practicality of our proposed MS-based sensing approach for the analysis of AsO2− ions in environmental samples, we used our Pb2+/Au NPs-MCEMs to determine the concentrations of AsO2− ions spiked in samples of streamwater, lake water, tap water, groundwater, and mineral water. Prior to the analyses, we filtered the samples through a 0.2 μm membrane and diluted them 10-fold with 5 mM glycine−NaOH buffer (pH 12). Here, we obtained linear correlations (R2 = 0.99) between the ratio of peak intensities [208Pb]+/[Au1]+ and the logarithm of the spiked concentration (10 nM−10 μM) of AsO2− ions (Figure 5). In these measurements, the probe provided recoveries of 89.2−117.5% for AsO2− ions. The minimum concentration of AsO2− ions detectable by our Pb2+/Au NPsMCEMs probe in these water samples was approximately 10 nM. Neither an ICPMS-based system nor our sensing approach could detect the presence of any AsO2− ions in these original water samples. Therefore, our proposed approach appears to be applicable to the practical screening of AsO2− ions in environmental water samples.
formed Pb−As complexes when reacted with AsO2− ions, resulting in changes in the [208Pb]+/[Au]+ peak ratio in the mass spectrum. The increase in desorption and ionization efficiency of the [208Pb]+ ions upon increasing the concentration of AsO2− ions appears to be due to the ready decomposition of the composites formed between Pb and As atoms on the surfaces of the Au NPs. Our Au NP-modified MCEM probe, that can be fabricated as test strips, allows for the detection of AsO2− ions in the nanomolar range, suggesting practical utility for real-life applications. Further modification of such probes could extend their applicability to other analytes (e.g., DNA, proteins, heavy metal ions, etc.).
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ASSOCIATED CONTENT
S Supporting Information *
Additional information (Figures S1−S11) as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Address: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung 20224, Taiwan. Tel.: 011-886-2-2462-2192 ext. 5517. Fax: 011-886-22462-2320. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the National Science Council of Taiwan under contract NSC 101-2628-M-019-001-MY3, 1022113-M-019-001-MY3, and 102-2627-M-019-001-MY3.
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CONCLUSIONS We have demonstrated Au NPs-MCEMs are efficient substrates for the adsorption of AsO2− ions from dilute aqueous solutions. Under LDI-MS analysis, the [208Pb]+/[Au1]+ peak ratio in the mass spectra provided a highly amplified indicator for the concentration of AsO2− ions. Our study has revealed that Pb2+ ions formed a monolayer on the Au NPs’ surfaces and then
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(4) Yang, G.; Ma, L.; Xu, D.; Li, J.; He, T.; Liu, L.; Jia, H.; Zhang, Y.; Chen, Y.; Chai, Z. Chemosphere 2012, 87, 845−850. (5) Tsai, S.-L.; Singh, S.; Chen, W. Curr. Opin. Biotechnol. 2009, 20, 659−667. (6) Ali, W.; Isayenkov, S. V.; Zhao, F.-J.; Maathuis, F. J. M. Cell. Mol. Life Sci. 2009, 66, 2329−2339. (7) (a) Khan, M. A.; Ho, Y.-S. Asian J. Chem. 2011, 23, 1889−1901. (b) Zhao, F. J.; Ma, J. F.; Meharg, A. A.; McGrath, S. P. New Phytol. 2009, 181, 777−794. (8) Sullivan, C.; Tyrer, M.; Cheeseman, C. R.; Graham, N. J. D. Sci. Total Environ. 2010, 408, 1770−1778. (9) (a) Jomova, K.; Jenisova, Z.; Feszterova, M.; Baros, S.; Liska, J.; Hudecova, D.; Rhodes, C. J.; Valko, M. J. Appl. Toxicol. 2011, 31, 95− 107. (b) Rahman, M. M.; Ng, J. C.; Naidu, R. Environ. Geochem. Health 2009, 31, 189−200. (10) (a) Lee, J. Y.; Eom, M.; Yang, J. W.; Han, B. G.; Choi, S. O.; Kim, J. S. Renal Failure 2013, 35, 299−301. (b) Danielson, C.; Houseworth, J.; Skipworth, E.; Smith, D.; McCarthy, L.; Nanagas, K. Transfusion 2006, 46, 1576−1579. (11) (a) He, J.; Charlet, L. J. Hydrol. 2013, 492, 79−88. (b) Sorg, T. J.; Chen, A. S. C.; Wang, L. Water Res. 2014, 48, 156−169. (c) Mondal, P.; Bhowmick, S.; Chatterjee, D.; Figoli, A.; Van der Bruggen, B. Chemosphere 2013, 92, 157−170. (12) Kitchin, K. T.; Wallace, K. J. Inorg. Biochem. 2008, 102, 532− 539. (13) (a) Rosen, B. P.; Ajees, A. A.; McDermott, T. R. BioEssays 2011, 33, 350−357. (b) Rosen, B. P.; Liu, Z. Environ. Int. 2009, 35, 512−515. (14) http://water.epa.gov/lawsregs/rulesregs/sdwa/arsenic/ regulations.cfm (accessed November 2013). (15) (a) Gupta, V. K.; Nayak, A.; Agarwal, S.; Dobhal, R.; Uniyal, D. P.; Singh, P.; Sharma, B.; Tyagi, S.; Singh, R. Desalin. Water Treat. 2012, 40, 231−243. (b) Sharma, V. K.; Sohn, M. Environ. Int. 2009, 35, 743−759. (16) (a) Anawar, H. M. Talanta 2012, 88, 30−42. (b) Rajaković, L. V.; Marković, D. D.; Rajaković-Ognjanović, V. N.; Antanasijević, D. Z. Talanta 2012, 102, 79−87. (17) (a) Wu, Y.; Liu, L.; Zhan, S.; Wang, F.; Zhou, P. Analyst 2012, 137, 4171−4178. (b) Kalluri, J. R.; Arbneshi, T.; Khan, S. A.; Neely, A.; Candice, P.; Varisli, B.; Washington, M.; McAfee, S.; Robinson, B.; Banerjee, S.; Singh, A. K.; Senapati, D.; Ray, P. C. Angew. Chem., Int. Ed. 2009, 121, 9848−9851. (c) Domínguez-González, R.; González Varela, L.; Bermejo-Barrera, P. Talanta 2014, 118, 262−269. (d) Liang, R.-P.; Wang, Z.-X.; Zhang, L.; Qiu, J.-D. Chem.Eur. J. 2013, 19, 5029−5033. (e) Tang, M.; Wen, G.; Liang, A.; Jiang, Z. Luminescence 2013, DOI: 10.1002/bio.2589. (f) Li, J.; Chen, L.; Lou, T.; Wang, Y. ACS Appl. Mater. Interfaces 2011, 3, 3936−3941. (18) Roy, S.; Palui, G.; Banerjee, A. Nanoscale 2012, 4, 2734−2740. (19) Wu, Y.; Zhan, S.; Wang, F.; He, L.; Zhi, W.; Zhou, P. Chem. Commun. 2012, 48, 4459−4461. (20) Wu, Y.; Zhan, S.; Xing, H.; He, L.; Xu, L.; Zhou, P. Nanoscale 2012, 4, 6841−6849. (21) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss Faraday Soc. 1951, 11, 55−75. (22) (a) Sahai, R.; Prakash, G. J. Electroanal. Chem. 1961, 2, 242− 248. (b) Firoozi, S.; Harris, R. Metall. Mater. Trans. B 2007, 38, 425− 432. (23) (a) Tseng, Y.-T.; Chang, H.-Y.; Huang, C.-C. Chem. Commun. 2012, 48, 8712−8714. (b) Chiu, W.-C.; Huang, C.-C. Anal. Chem. 2013, 85, 6922−6929. (c) Li, Y.-J.; Tseng, Y.-T.; Unnikrishnan, B.; Huang, C.-C. ACS Appl. Mater. Interfaces 2013, 5, 9161−9166. (d) Liu, Y.-C.; Li, Y.-J.; Huang, C.-C. Anal. Chem. 2013, 85, 1021−1028. (e) Liu, Y.-C.; Chang, H.-T.; Chiang, C.-K.; Huang, C.-C. ACS Appl. Mater. Interfaces 2012, 4, 5241−5248. (24) Tan, D.; Zhou, S.; Qiu, J.; Khusro, N. J. Photochem. Photobiol., C 2013, 17, 50−68. (25) (a) Hashimoto, S.; Werner, D.; Uwada, T. J. Photochem. Photobiol., C 2012, 13, 28−54. (b) Werner, D.; Hashimoto, S. J. Phys. Chem. C 2011, 115, 5063−5072.
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Detection of Arsenic(III) through Pulsed Laser-Induced Desorption/ Ionization of Gold Nanoparticles on Cellulose Membranes Cheng-I Weng,† Jin-Shun Cang,‡ Jia-Yaw Chang,§ Tung-Ming Hsiung,† Binesh Unnikrishnan,† Yu-Lun Hung,† Yu-Ting Tseng,† Yu-Jia Li,† Yu-Wei Shen,† and Chih-Ching Huang*,†,⊥,# †
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 20224, Taiwan Department of Chemistry, Yancheng Institute of Industry Technology, Yancheng, Jiangsu 224005, P. R. China § Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, 10607, Taiwan ⊥ Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan # School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan ‡
S Supporting Information *
ABSTRACT: We have developed an assay based on gold nanoparticle-modified mixed cellulose ester membrane (Au NPs-MCEM) coupled with laser-induced desorption/ionization mass spectrometry (LDI-MS)-for the detection of arsenic(III) ions (arsenite, AsO2−) in aqueous solution. When the Au NPs reacted with lead ions (Pb2+) in alkaline solution (5 mM glycine−NaOH, pH 12), Au−Pb complexes, PbO, and Pb(OH) were formed immediately on the Au NP surfaces. The Pb species reacted rapidly with subsequently added AsO2− to form PbOAs2O3, (PbO)2As2O3, and/or (PbO)3As2O3 shells (2−5 nm) on the Au NPs’ surfaces. As a result, significant observable aggregation of the Au NPs occurred in the solution. This Pb2+/Au NP probe allowed the detection of AsO2− at concentrations as low as 0.6 μM with high selectivity (at least 100-fold over other anions and metal ions). To further improve the sensitivity, we prepared Au NPs-MCEM for the LDI-MS-based detection of AsO2− ions. The intensity of the signal for the [Pb]+ ions in the mass spectra increased when the Au NPs-MCEM reacted with AsO2−; in contrast, the intensity of the signal for [Au]+ ions decreased. Accordingly, the [Pb]+/[Au]+ peak ratio increased upon increasing the AsO2− concentration over the range from 10 nM to 10 μM. The limit of detection at a signal-to-noise ratio of 3 was 2.5 nM, far below the action level of As (133 nM, ca. 10 ppb) permitted by the US EPA for drinking water. Relative to other nanoparticle-based arsenic sensors, this approach is rapid, specific, and sensitive; in addition, it can be applied to the detection of AsO2− in natural water samples (in this case, streamwater, lake water, tap water, groundwater, and mineral water).
A
rsenic is a toxic metalloid found widely in nature.1 The arsenic content of the Earth’s crust is 1.8 ppm; in the soil, it is 0.2−40 ppm with an average concentration of 5 ppm.2 Volcanic materials contain arsenic at 20 ppm; the average concentration of arsenic in seawater is approximately 3 ppm.3 Arsenic is released into the atmosphere mainly from the weathering of rocks, from the soil, and from plants; the estimated annual emission of arsenic into the atmosphere from the Earth’s surface is approximately 2.37 × 107 kg.4 Methylation of arsenic by soil microbes generates volatile methylated arsenic species, having the general formula (CH3)nAsH(3−n) (n = 1−3), that are released into the atmosphere.5 Plants intake arsenic through their roots; after conversion, they also discharge the methylated arsenic species (CH3)2AsH and (CH3)3As into the air.6 These compounds react with O2 to produce (CH3)2AsO and (CH3)2As(OH), which further react with ozone (O3) and N2O4 to generate As4O6, which reenters the soil and is then converted back to HAsO2.7 Arsenic can flow into lakes, rivers, © 2014 American Chemical Society
and groundwater through the action of rain or wastewater discharge.8 Arsenic can cause acute and chronic poisoning, including abdominal pain, bloody diarrhea, acute renal failure and neuropathy, muscle weakness, skin keratinization, pigmentation, and cancer (lung, liver, bladder, and skin cancer).9 In addition, arsine (AsH3) gas, which is used commonly in the electronics industry and has a garlic flavor, readily induces hemolysis and then death through acute renal failure.10 Trivalent arsenic [arsenic(III)] and pentavalent arsenic [arsenic(V)] enter the human body most readily through drinking water.11 Arsenic(III) species form bonds with the sulfhydryl groups of protein S (R−AsO + 2 RSH → R−As− (SR)2 + H2O) and can interfere with the reactions of other Received: January 6, 2014 Accepted: February 20, 2014 Published: February 20, 2014 3167
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Scheme 1. Cartoon Representations of (A) the Colorimetric Sensing Mechanism of the Pb2+/Au NPs Probe and (B) the Use of a Pb2+/Au NPs-MCEM for the Detection of AsO2− Ions through LDI-MS
AsO2− based on a specific AsO2−-binding aptamer and gold nanoparticles (Au NPs); it operated through AsO2−-mediated interaction of the aptamer and poly(diallyldimethylammonium) and aggregation of the Au NPs.19 A solution of arsenic-binding aptamer and crystal violet will form crystal violet/aptamer complex NPs; changes in the sizes of these NPs, induced by AsO2−, enable the detection of AsO2− based on resonance Rayleigh scattering spectrometry.20 Although these NP-based optical sensors all exhibit high sensitivity for the detection of arsenic ions, they have limited applicability because of matrix interference, high cost, poor stability, and the need for special agents to selectively detect arsenic ions. Therefore, the development of reliable and rapid routine methodologies for the selective detection of arsenic ions in water remains an attractive challenge. In this study, we developed a laser desorption/ionization mass spectrometry (LDI-MS)-based assay for the detection of AsO2− using Au NP-modified membranes. As displayed in Scheme 1A, the addition of Au NPs to a lead ion (Pb2+) solution in an alkaline medium can form AuPb alloys as well as PbO or Pb(OH)2 species on the surface of the Au NPs. When AsO2− is added to the alkaline solution of Pb/Au NPs, the AsO2− and PbO species will react to form As2O3 or Pb(AsO2)2, leading to aggregation of the Au NPs. To increase the sensitivity and linear range, we adsorbed the Au NPs onto a mixed cellulose ester membrane (MCEM) for use as a substrate for LDI-MS-based detection of AsO2−, that is, by measuring the signal for the [208Pb]+ ions, rather than detecting AsO2− directly (Scheme 1B). Upon pulsed laser irradiation, we detected the [208Pb]+ ions at m/z 208, with laser-induced desorption and ionization of [208Pb]+ being enhanced dramatically in the
enzymes or proteins.12 Pentavalent arsenic [arsenate(V)] can replace some phosphate units because of their similar structures; for example, it can weaken the bonding in adenosine triphosphate (ATP), thereby leading to the generation of less energy and disrupting normal biological or cellular energy supply.13 The US Environmental Protection Agency (EPA) has established the limit of arsenic in drinking water at 10 ppb (133 nM).14 Most of the arsenic in water that comes into contact with air (e.g., in rivers, lakes, or seawater) is of the form arsenic(V), while that in closed water (e.g., groundwater) is typically in the form of arsenic(III) [arsenite (AsO2−)].15 Thus, it is inevitable that AsO2− will enter living systems from water sources, potentially leading to severe poisoning. Therefore, determining the AsO2− contents of various water samples is a necessary and indispensable task. At present, the techniques employed most widely to detect arsenic in water samples are inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICPMS), graphite furnace atomic absorption spectrometry (GF-AAS), and hydride generation atomic absorption spectrometry (HG-AAS).16 These techniques often suffer, however, from complex and cumbersome sample handling and the inability to detect AsO2− selectively. Although the detection limit of ICPMS is very low relative to the other methods, interference caused by the formation of isobars in the plasma can be problematic. In recent years, several nanomaterial-based optical sensors have been developed for the detection of arsenic ions in water samples.17 For example, fluorescent gold nanoclusters have been used to sense AsO2− with limit of detection (LOD) of 28 ppb.18 Wu et al. reported a colorimetric sensor for the selective detection of 3168
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presence of AsO2−. It appears that the Pb(AsO2)2 species formed from the reaction of AsO2− ions with PbO allow for detection of [208Pb]+ ions with greater sensitivity and less noise than the detection of the AsO2− signals directly. This method exhibits low interference and can, therefore, be applied to the detection of AsO2− in natural water samples.
centrifugation/wash cycles to remove most of the free Pb2+ ions in solution. Centrifugation was performed at a relative centrifugation force (RCF) of 30,000g at 25 °C for 20 min, and ultrapure water (1 mL × 3) was used to wash the Au NPs pellets. The purified Pb/Au NPs were then collected and acidified with 2% HNO3 before the metal ion concentrations were determined by ICPMS (Agilent 7700 Series, Agilent Technologies, California, USA). Detecting Arsenite Using the Membrane-Based Probe. The as-prepared Au NPs-MCEM was immersed in 5 mM glycine−NaOH solution (pH 12, 1.0 mL) containing Pb2+ (100 nM) for 30 min and then immersed in AsO2− (0−10 μM) ions for 30 min at room temperature. The membranes were gently washed with DI water (5 mL) for 30 s, dried in air at room temperature, and then attached to a MALDI plate using adhesive polyimide film tape. Mass spectrometry experiments were performed in the reflectron positive-ion mode using an AutoflexIII MALDI time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany). The samples were irradiated using a pulse laser (355-nm Nd:YAG, 100 Hz; pulse width: 6 ns). A delayed extraction period of 20 ns was set to energetically stabilize the positively charged ions produced during LDI, and then, the ions were accelerated through the TOF chamber in the reflection mode prior to entering the mass analyzer. The accelerating voltages were applied in the range from +20 to −20 kV. The instrument was calibrated to the signals for the Au clusters ([Aux]+; x = 1−5) prior to recording the mass spectra. To accumulate signals, 300 pulsed laser shots at a laser fluence of 4.9 × 104 W cm−2 were applied to five random positions on the MALDI target plate. Analysis of Real Samples. Water from a stream on the campus of National Taiwan Ocean University, lake water from National Taiwan University, local tap water and groundwater, and mineral water from Young Energy Source Co., Ltd. were filtered through a 0.2 μm membrane. For the detection of AsO2−, aliquots of the water samples (500 μL) were spiked with standard AsO2− ion solutions (from 10 nM to 10 μM). The spiked samples were diluted to 1.0 mL with 5 mM glycine−NaOH solution (pH 12, 1.0 mL), and then, the Pb2+/ Au NPs-MCEMs were immersed in the solution for 30 min and then washed prior to MS measurements. All spiked samples were also analyzed by ICPMS.
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EXPERIMENTAL METHODS Materials. Citric acid, trisodium citrate, and all metallic salts used in this study were purchased from Aldrich (Milwaukee, WI). Hydrogen tetrachloroaurate(III) trihydrate were obtained from Acros (Geel, Belgium). MCEM (pore size: 0.45 μm) was purchased from Advantec (Toyo Roshi Kaisha, Japan). Milli-Q ultrapure water was used in all experiments. Glycine obtained from J. T. Baker (Phillipsburg, NJ, USA) was used to prepare the glycine buffer (50 mM, pH 9−12, adjusted with 1.0 N NaOH). Au NPs and Au NPs-MCEMs. The 32 nm spherical Au NPs were prepared through citrate-mediated reduction of HAuCl4.21 Aqueous 0.01% HAuCl4 (50 mL) was brought to a vigorous boil while stirring in a round-bottom flask fitted with a reflux condenser; 1% trisodium citrate (0.5 mL) was added rapidly, and then heating was continued for another 8 min, during which time the color of the solution changed from pale yellow to red−purple. The solution was cooled to room temperature with continuous stirring. The sizes of the Au NPs were verified using transmission electron microscopy (TEM; H7100, Hitachi High-Technologies Corporation, Tokyo, Japan); they appeared to be nearly monodisperse, with an average size of 32.3 ± 2.8 nm. A double-beam UV−vis spectrophotometer (Cintra 10e, GBC, Victoria, Australia) was used to measure the absorption of the Au NPs solution. The particle concentration of the Au NPs (280 pM) was determined according to Beer’s law, using an extinction coefficient of 1.2 × 109 M−1 cm−1 at 526 nm for the 32 nm Au NPs. An MCEM having a diameter of 0.6 cm was used to prepare the Au NPs-MCEM substrate. The MCEM was immersed in a Au NP (140 pM)-containing citrate solution (0.25 mM, pH 5.8, 20 mL) and incubated for 2 h. The Au NPs-adsorbed MCEM (Au NPs-MCEM) was washed gently with ultrapure water (20 mL) for 2 min and then dried in air for 2 h at room temperature. Characterization of Au NPs. TEM images of Au NPs were recorded using a H7100 transmission electron microscope (operated at 125 kV). Samples for TEM measurements were prepared by placing 20 μL of the Au NPs solution on a carboncoated copper grid and then drying at room temperature. The zeta potentials of the Au NPs were measured using a Zetasizer 3000HS analyzer (Malvern Instruments, Malvern, UK). Pb2+/Au NP-Based Colorimetric Sensor for Arsenite. The Pb2+/Au NP system was prepared by equilibrating the Au NPs (140 pM) and Pb2+ ions (5.0 μM) in 5 mM glycine− NaOH (pH 12) solution for 30 min. For AsO2− sensing, aliquots (500 μL) of the as-prepared Pb2+/Au NPs and AsO2− ions (0−10 μM) were equilibrated at room temperature for 30 min. The mixtures were then transferred separately into 96-well microtiter plates, and their UV−vis absorption spectra were recorded using a μ-Quant monochromatic microplate spectrophotometer (Biotek Instruments, Winooski, VT, USA). Herein, the final concentrations of the species are provided. To determine the number of Pb atoms/ions on the Au NPs’ surface, the mixtures of Pb2+ and Au NPs solutions were maintained at 25 °C for 1 h and were then subjected to three
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RESULTS AND DISCUSSION Interaction of AsO2− and Pb2+/Au NPs. Scheme 1A outlines the colorimetric sensing mechanism for AsO2− in this study. When the Au NPs (32-nm, 140 pM) reacted with Pb ions (Pb2+, 5.0 μM) in alkaline solution (5 mM glycine− NaOH, pH 12), Au−Pb complexes and PbO and Pb(OH) species were formed immediately on the surfaces of the Au NPs (Figures S1 and S2, Supporting Information), leading to a slight increase in their surface plasmon resonance (SPR) absorption (curve B, Figure 1). The TEM images in Figure 2 indicate that the Au NPs in the presence of Pb2+ ions were almost the same size and shape as those in the absence of the Pb2+ ions, suggesting formation of only a monolayer or submonolayer of Pb complexes on the NP surfaces. In addition, using ICPMS to quantify the content of Pb species on the Au NPs, we obtained a value of 29,400 Pb atoms/ions per Au NP, close to the number of surface atoms of the 32 nm Au NPs (ca. 40,000 atoms per particle). After adding AsO2− (10 μM), the Pb species on the NPs’ surfaces react rapidly to form PbOAs2O3, (PbO)2As2O3, and/or (PbO)3As2O3 shells (2−5 nm) on the 3169
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induce aggregation of the citrate-capped Au NPs (curve D, Figure 1). Parameters for Detection of AsO2−. We investigated the effect of the concentration of Pb2+ ions (0−10 μM) on the aggregation of the Au NPs in the absence and presence of AsO2− ions (0−10 μM). The absorption (extinction) bands of the Au NPs at 750 and 526 nm were related to the quantities of aggregated and dispersed Au NPs, respectively. The extinction ratio (Ex750/526) could, therefore, be used as a measure of the degree of aggregation, with high values corresponding to high degrees of aggregation. The degree of aggregation of the Au NPs in the 5 mM glycine−NaOH (pH 12) solution increased upon increasing the concentration of Pb2+ ions in the absence of AsO2− ions (Figure S5A, Supporting Information). This phenomenon was due mainly to the formation of larger amounts of PbO and/or Pb(OH) on the NPs’ surfaces under higher concentrations of Pb2+ ions (>5.0 μM). In addition, the degree of aggregation increased upon increasing the concentration of AsO2− ions (0−10 μM) in the presence of a constant concentration of Pb2+ ions (within the range of 2.5−7.5 μM). In a subsequent study, we maintained the concentration of Pb2+ ions in the solution at 5.0 μM and detected the AsO2− ions in glycine buffers (5 mM) at values of pH from 9.0 to 12.0. The sensitivity of the Pb2+/Au NP probe for AsO2− ions increased upon increasing the pH of the solution (Figure S5B, Supporting Information). We presume that PbO and/or Pb(OH) formed readily, depositing on the Au NPs’ surfaces, at higher values of pH; this phenomenon favored the formation of Pb/As shells on the Au NPs, thereby inducing their aggregation. We noted that the Au NPs tended to aggregate in the absence of Pb2+ and AsO2− ions when the pH of the glycine−NaOH buffer was greater than 12 (data not shown). Figure S5C (Supporting Information) indicates that the aggregation of Pb2+ (5.0 μM)/ Au NPs (140 pM) induced by the AsO2− ions (10 μM) decreased when we increased the temperature from 25 to 80 °C, mainly because As−Pb composites [e.g., PbOAs2O3, (PbO)2As2O3, (PbO)3As2O3] are difficult to form at higher temperature. Under the optimal solution conditions [5 mM glycine−NaOH (pH 12), Pb2+ (5.0 μM), room temperature (ca. 25 °C)], the Pb2+/Au NP probe allowed the detection of AsO2− at concentrations as low as 0.6 μM (Figure S6A, Supporting Information) with high selectivity [at least 100-fold over other anions, metal ions, and amino thiol (cysteine); Figure S6B, Supporting Information]. The high selectivity of the Pb2+/Au NPs for AsO2− ions was presumably due to the specific reactions of Pb ions, PbO and Pb(OH) species with AsO2− ions on the Au NPs’ surfaces under alkaline conditions (pH 12). The response of the sensor toward AsO2− ions in the presence of various ion species was also tested and found that the tolerance concentrations of other metal ions, cysteine, and anions (within a relative error of ±5%) for the sensing of AsO2− using this Pb2+/Au NPs probe were at least 10 times the AsO2− concentrations (Figure S7A, Supporting Information). Our probe showed a high tolerance to high concentrations of amino thiol (cysteine) which is otherwise capable of forming complexes with the AsO2− ions and interferes with the detection of the AsO2−. These results suggest that the species we tested should not interfere with the determination of AsO2− when applying our developed probe. Membrane-Based Sensor for AsO2−. To further improve the sensitivity, we prepared a Au NPs-MCEM for LDI-MSbased detection of AsO2− ions (Scheme 1B). In previous reports, we demonstrated a Au NP-MCEM that provided very
Figure 1. UV−vis absorption spectra of solutions containing (A) Au NPs, (B) Au NPs and Pb2+, (C) Au NPs, Pb2+, and AsO2−, and (D) Au NPs and AsO2−. Inset: Photograph of the four Au NPs solutions. The concentrations of the Au NPs, PbNO3, and NaAsO2 were 140 pM, 5.0 μM, and 10 μM, respectively. Buffer: 5 mM glycine−NaOH solution (pH 12).
Figure 2. TEM images of Au NPs in the (A) absence and (B−D) presence of (B) Pb2+, (C) Pb2+ and AsO2−, and (D) AsO2− in 5 mM glycine−NaOH solution (pH 12). Other conditions were the same as those described in Figure 1.
Au NPs’ surfaces (see arrow in Figure 2C).22 Energy-dispersive X-ray spectroscopy (EDS) revealed an As-to-Pb atomic ratio of close to 2:3 (Figure S3, Supporting Information), suggesting that the major component in the shell was (PbO)3As2O3. Scanning transmission electron microscopy/energy-dispersive X-ray mapping (STEM−EDS; Figure S4, Supporting Information) indicated that the major components in the shell were Pb and As. We also observed that the zeta potential (ξ) of the Au NPs decreased, from −47.8 to −21.2 mV, after reaction with AsO2−. As a result, significant aggregation of the Au NPs occurred in the solution (Figure 2C). The aggregated Au NPs induced SPR absorption coupling, with the absorption band of the Au NPs at 526 nm undergoing a red-shift with decreased absorption, while the intensity of the absorption band at 750 nm increased (curve C in Figure 1). Dynamic light scattering (DLS) measurements revealed that the Pb2+/Au NPs in the absence and presence of AsO2− (1.0 μM) had diameters of 39.5 (±7.8; n = 4) and 254.5 (±29.5; n = 4) nm, respectively, further confirming that AsO2− induced aggregation of the Pb2+/ Au NPs. In a control experiment, the addition of AsO2− did not 3170
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homogeneous, clean mass spectra of [Aun]+ (n = 1−5) cluster ions, with the formation of the [Aun]+ cluster ions being very sensitive to the particle’s surface properties (composites), under pulsed laser irradiation.23 Figure 3A displays LDI mass spectra
because the boiling temperature is easy to reach under irradiation with a nanosecond-pulse laser, whereas a fragmentation temperature that exceeds the Rayleigh instability limit is not.25 We noted that almost all of the Au NPs had desorbed from the surface of the MCEM (Figure S9, Supporting Information) after 300 shots of pulsed laser irradiation at 4.9 × 104 W cm−2. Figure 3 reveals that the intensities of the signals for the [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ ions all increased upon increasing the concentration of AsO2− ions. We suggest that the formation of more Pb−As nanocomposites on the Au NPs upon increasing the concentration of AsO2− was the main reason for the increase in the intensity of the Pb-related peaks ([Pb]+, [AuPb]+, [Au2Pb]+). After formation of Pb−As nanocomposite shells on the Au NPs, however, the laserirradiated Au NPs transferred their energy to the surface Pb−As nanocomposites, thereby suppressing evaporation of the surface Au atoms into the gas phase and resulting in low abundances for the signals of the Au clusters in the mass spectra. We observed no signals for Pb−As hybrids [e.g., PbOAs2O3, (PbO)2As2O3, (PbO)3As2O3] in the mass spectra, presumably because these composites decompose under the pulsed laser irradiation and/or react with other ions or molecules in the gas phase. The average relative standard deviations (RSDs) for the signal fluctuations of the [208Pb]+, [Au208Pb]+, and [Au2208Pb]+ ions collected from 50 different mass spectra were approximately 15% (Figure S10, Supporting Information) when using the Pb2+/Au NPs/MCEM for detection of AsO2− ions (100 nM). In addition, each of the RSDs decreased to less than 5% when we normalized the signal intensity of the [208Pb]+ ions with respect to the signal intensity of the [Au1]+ ions ([208Pb]+/ [Au1]+) from each MS measurement. Quantitation of AsO2− ions in solution was possible after recording the ratio of the signal intensities of the [208Pb]+ and [Au1]+ ions from 300 different sample spots in membranes (Figure 4). The [208Pb]+/
Figure 3. (A) LDI mass spectra of the Pb2+/Au NPs-MCEM in the absence and presence of AsO2− (1.0 nM−10 μM). (B) Relative peak intensities (S/S0) of [Au1]+, [Au2]+, [Au3]+, [Au4]+, [Au5]+, [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ plotted against the concentration of AsO2− (1.0 nM−10 μM). The descriptors S0 and S represent the mass spectral signal intensities of the [Au1]+, [Au2]+, [Au3]+, [Au4]+, [Au5]+, [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ ions in the absence and presence of AsO2−, respectively. Error bars represent standard deviations derived from four repeated experiments. Figure 4. Relative signal intensity [208Pb]+/[Au1]+ plotted with respect to the concentration of AsO2− ions (1.0 nM−10 μM). Other conditions were the same as those described in Figure 3.
of the Pb2+/Au NPs-MCEMs in the absence and presence of AsO2− ions (0−10 μM). Under pulsed laser irradiation (355 nm Nd:YAG, 4.9 × 104 W cm−2; pulse width: 6 ns), we detected Au cluster ions ([Aun]+; n = 1−5) and [Pb]+, [PbOH]+, [AuPb]+, and [Au2Pb]+ ions produced from the Au NPs’ surfaces (Figure 3A, panel a). Using the Au clusters ([Aun]+; n = 1−5) as the calibrating internal standard, Figure S8 (Supporting Information) reveals that all of the masses of the [Pb]+ ions in the mass spectra were accurate to within 28 ppm of their theoretical masses, with isotopic distributions close to the predicted isotope patterns. Photothermal surface evaporation, Coulomb explosion, and near-field ablation have all been proposed as mechanisms for the desorption and ionization of metallic NPs.24 The fragmentation of the Au NPs may favor the photothermal surface evaporation mechanism
[Au1]+ ratio increased upon increasing the AsO2− concentration over the range from 10 nM to 10 μM. The plot of [208Pb]+/ [Au1]+ against the logarithm of the concentration of AsO2− ions over the range from 10 nM to 10 μM was linear (y = 0.0915x + 0.8686; y: [ 208 Pb] +/[Au 1 ]+ ratio; x: logarithm of the concentration of AsO2− ions), with a correlation coefficient (R2) of 0.99. The LOD at a signal-to-noise (S/N) ratio of 3 was 2.5 nM, far below the action level of As (133 nM, ca. 10 ppb) permitted by the US EPA for drinking water. Figure S11 (Supporting Information) reveals the good reproducibility (RSD < 5%) of the mass spectra obtained from a solution of 3171
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Figure 5. Analyses of AsO2− ions in representative streamwater, lake water, tap water, groundwater, and mineral water samples, determined using Pb2+/Au NPs-MCEMs probes. Water samples (diluted 10-fold) were spiked with AsO2− ions (1.0 nM−10 μM). Other conditions were the same as those described in Figure 3.
100 nM AsO2− ions when using 10 different Au NP-MCEMs as substrates with three replicate recordings. The tolerance test for the membrane-based probe for different metal ions, anions, and amino thiol (Figure S7B, Supporting Information) suggests that the tolerance concentrations of these species for the sensing of AsO 2 − were at least 10 times greater than the AsO2 − concentrations. Since the Au NPs modified MCEM is treated with Pb2+ in alkaline solution (5 mM glycine−NaOH, pH 12), the Pb ions form compounds such as PbO or Pb(OH) species on the Au NPs’ surface, which does not easily react with S2− ions avoiding the possibility for interference. The selectivity and tolerance studies (Figure S6 and S7, Supporting Information) support this fact. Detection of AsO2− Ions in Real Samples. To validate the practicality of our proposed MS-based sensing approach for the analysis of AsO2− ions in environmental samples, we used our Pb2+/Au NPs-MCEMs to determine the concentrations of AsO2− ions spiked in samples of streamwater, lake water, tap water, groundwater, and mineral water. Prior to the analyses, we filtered the samples through a 0.2 μm membrane and diluted them 10-fold with 5 mM glycine−NaOH buffer (pH 12). Here, we obtained linear correlations (R2 = 0.99) between the ratio of peak intensities [208Pb]+/[Au1]+ and the logarithm of the spiked concentration (10 nM−10 μM) of AsO2− ions (Figure 5). In these measurements, the probe provided recoveries of 89.2−117.5% for AsO2− ions. The minimum concentration of AsO2− ions detectable by our Pb2+/Au NPsMCEMs probe in these water samples was approximately 10 nM. Neither an ICPMS-based system nor our sensing approach could detect the presence of any AsO2− ions in these original water samples. Therefore, our proposed approach appears to be applicable to the practical screening of AsO2− ions in environmental water samples.
formed Pb−As complexes when reacted with AsO2− ions, resulting in changes in the [208Pb]+/[Au]+ peak ratio in the mass spectrum. The increase in desorption and ionization efficiency of the [208Pb]+ ions upon increasing the concentration of AsO2− ions appears to be due to the ready decomposition of the composites formed between Pb and As atoms on the surfaces of the Au NPs. Our Au NP-modified MCEM probe, that can be fabricated as test strips, allows for the detection of AsO2− ions in the nanomolar range, suggesting practical utility for real-life applications. Further modification of such probes could extend their applicability to other analytes (e.g., DNA, proteins, heavy metal ions, etc.).
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ASSOCIATED CONTENT
S Supporting Information *
Additional information (Figures S1−S11) as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Address: Institute of Bioscience and Biotechnology, National Taiwan Ocean University, 2, Pei-Ning Road, Keelung 20224, Taiwan. Tel.: 011-886-2-2462-2192 ext. 5517. Fax: 011-886-22462-2320. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported by the National Science Council of Taiwan under contract NSC 101-2628-M-019-001-MY3, 1022113-M-019-001-MY3, and 102-2627-M-019-001-MY3.
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CONCLUSIONS We have demonstrated Au NPs-MCEMs are efficient substrates for the adsorption of AsO2− ions from dilute aqueous solutions. Under LDI-MS analysis, the [208Pb]+/[Au1]+ peak ratio in the mass spectra provided a highly amplified indicator for the concentration of AsO2− ions. Our study has revealed that Pb2+ ions formed a monolayer on the Au NPs’ surfaces and then
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