Highly Sensitive Immunoassay Based on ImmunogoldâSilver

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Highly Sensitive Immunoassay Based on Immunogold-Silver Amplification and Inductively Coupled Plasma Mass Spectrometric Detection Rui Liu,† Xing Liu,† Yurong Tang,† Li Wu,‡ Xiandeng Hou,†,‡ and Yi Lv*,† †

College of Chemistry and ‡Analytical and Testing Center, Sichuan University, Chengdu, Sichuan 610064, China ABSTRACT: In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL-1 with a limit of detection (LOD, 3σ) of 0.03 ng mL-1 (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay.

A

highly sensitive technique for protein quantification plays an important role in the early diagnosis and elucidation of molecular mechanisms for many diseases, because even a few molecules of proteins are sufficient to affect the biological functions of cells and trigger pathophysiological processes.1 Unfortunately, in many cases, it is still hard to use conventional methods to detect some important protein biomarkers, due to their low abundance in body fluids or tissues. Hence, it is extremely urgent to develop highly sensitive methods for detection of low abundant proteins. Generally, high sensitivity can often be obtained using a signal amplification procedure. Many signal amplification schemes have been reported for sensitive bioanalyte quantification, such as rolling circle amplification,2-5 avidin-biotin amplification,6,7 inline atom transfer radical polymerization,8 and exponential isothermal amplification.9 However, they usually require rather complex reagents or sophisticate protocols. Due to its easy handling, cheap reagents, robustness, and high sensitivity,10,11 the immunogold-silver amplification technique has been widely used and even successfully commercialized for the past 30 years since it was first developed in 1983 by Holgate et al.12 and Danscher et al.13 It is initially developed and commonly applied as a sensitive and specific immunohistochemical visualization technique.10 Later, Mirkin and co-workers developed a scanometric DNA array,14 an electrical detection-based DNA array,15 and a Raman spectroscopic fingerprints scheme for DNA and RNA detection16 using this technique for sensitivity improvement. Immunogold-silver amplification has also been r 2011 American Chemical Society

explored for bioanalyte quantification by coupling to microgravimetric biosensor,17 electrochemical stripping analysis,18,19 chemiluminescent analysis,20 dot-blot immunoassay,21,22 and microfluidic chips.23,24 Inductively coupled plasma mass spectrometry (ICPMS) is unarguably the predominant and most sensitive commercial instrument for the determination of a wide range of metals and several nonmetals. The advantages of ICPMS include low detection limits, low matrix effects, large dynamic ranges, and high spectral resolution for elements and isotopes.25,26 Therefore, element tagged immunoassay with ICPMS detection has become an emerging technique in immunoassay research27-30 since it was first reported by Zhang et al.31,32 ICPMS as a readout tool does not require nanoparticle reporters to possess the optical, electric, electrochemical, magnetic, or any other special properties since atomic ions from the nanoparticle are directly detected. A distinguished feature of this method is the great multiplexing potential for biological analytes endowed by the excellent element isotopic spectral resolution of the mass spectrometer.33-36 Another feature is that high sensitivity could be easily obtained by the use of the nanoparticle tag instead of metal ions, due to large quantities of detectable atoms in each nanoparticle tag.32,37 Methods for the detection of biological analytes such as small Received: December 15, 2010 Accepted: February 2, 2011 Published: February 24, 2011 2330

dx.doi.org/10.1021/ac103265z | Anal. Chem. 2011, 83, 2330–2336

Analytical Chemistry

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Table 1. Working Parameters of ICPMS parameters

values

RF power/W

1200

cool gas flow/L min-1

13

auxiliary gas flow/L min-1

0.8

nebulizer gas flow/L min-1

0.85

sample uptake rate/mL min-1

1

torch

shield torch

resolution

standard

dwell time/ms channels

10 3

sweeps

100 38

31,32,34,35,37,39-44

biomolecules, proteins, nucleic acids,45,46 and 47,48 have been proved to be successful. Additionally, it even cells is believed to be the ideal technique for the next generation of flow cytometers.47,49 It should be mentioned that Mcleod et al. combined immunohistochemistry with laser ablation ICPMS using the gold nanoparticles (Au-NPs) label and subsequent silver amplification for imaging of breast cancer-associated proteins.50 The method showed higher sensitivity and good resolution as compared to optical microscopy. Apparently, it is worthwhile to further explore quantitative application after silver amplification. In this work, therefore, a highly sensitive immunogold-silver amplified ICPMS immunoassay is presented through the catalytic precipitation of silver on the immunogold nanoparticles, which combined the intrinsically high sensitivity of ICPMS with the signal enhancement of immunogold-silver amplification. Carcinoembryonic antigen (CEA), a glycoprotein of 200 kDa that has been extensively studied as a tumor marker for clinical diagnosis and the individual’s annual medical checkup, was chosen as the model analyte. It is used as part of a panel of cancer markers for different cancers and even an independent prognostic factor, since it is present in about 95% of all colon tumors and 50% of breast tumors and is also associated with ovarian carcinoma, lung cancer, and other cancers.1

’ EXPERIMENTAL SECTION Instrumentation. An X Series ICPMS (Thermo Electron Co., Winsford, Cheshire, UK) was used throughout this work. A standard glass concentric nebulizer and a standard glass conical impact bead spray chamber were employed with an uptake rate of around 1 mL min-1. Single-channel and multichannel (8-channel) pipettes (Finnpipette Color, Thermo Scientific, USA) were used for convenient and rapid transfer of the solutions. Prior to analysis, the instrumental parameters were tuned using a 1% HNO3 solution containing 10 ng mL-1 In and Pb. The optimized parameters are listed in Table 1. The morphologies of the samples were observed with a transmission electron microscope (TEM, JEM-100CXII). Reagents and Immunoreaction Buffers. Deionized water with conductivity of 18.2 MΩ cm-1 from a water purification system (ULUPURE, Chengdu, China) was used in this work. Polystyrene 96-well microtiter plates (468667, NUNC, Denmark) were used to perform the immunoreactions. CEA antigen, primary CEA antibody, secondary CEA antibody, and bovine serum albumin (BSA) were purchased from Bejing Biosynthesis Biothechnology Co. (Beijing, China). Colloidal gold nanoparticles

Figure 1. TEM images of colloidal gold before (a) and after (b) 15 min silver amplification.

(Au-NPs) and secondary CEA antibody-colloidal Au conjugates were synthesized in our laboratory. Unless otherwise stated, all the reagents used in this study were at least of analytical grade and obtained from Changzheng Chemical Reagent Co. Ltd. (Chengdu, China). The buffers used were as follows: (A) coating buffer, 0.05 M carbonate/bicarbonate buffer solution, pH 9.6 (dissolve 2.601 g of Na2CO3 and 3.437 g of NaHCO3 in 1 L of deionized water); (B) blocking buffer, 1% (w/v, g mL-1) BSA in 0.01 M sodium phosphate buffered saline (PBS, dissolve 2.204 g of Na2HPO4 3 12H2O, 0.600 g of NaH2PO4 3 2H2O, and 8.766 g of NaCl in 1 L deionized water), pH 7.4. The blocking buffer was stored at 4 °C and used within a week; (C) assay buffer, 0.01 M PBS containing 1% BSA (w/v), pH 7.4; (D) washing buffer, 0.01 M PBS with 0.05% (v/v) Tween 20, pH 7.4; and (E) citrate buffer, pH 3.5 (dissolve 23.5 g of trisodium citrate dihydrate and 25.5 g of citric acid monohydrate in 850 mL of deionized water). This buffer can be kept at 4 °C for at least 2 to 3 weeks. Before use, it is adjusted to pH 3.8 with citric acid solution. Silver amplification solutions (A and B) are prepared freshly: Solution A, dissolve 80 mg of silver acetate in 40 mL of deionized water (silver acetate crystals can be dissolved by continuous stirring within about 15 min) and solution B, dissolve 200 mg of hydroquinone in 40 mL of citrate buffer. Solution A and solution B were mixed immediately with an equal volume before use. Preparation of Colloidal Gold Nanoparticles. Briefly, after boiling 100 mL of 0.01% (m/v) HAuCl4 3 4H2O with 4 mL of 1% (m/v) trisodium citrate in aqueous solution for 30 min, the resultant colloidal suspension was cooled and stored at 4 °C. The 2331

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Figure 3. Calibration curves for Ag and Au by ICPMS.

Figure 2. Schematic diagram of the sandwich immunoassay and silver amplification for human CEA with Au-NP tags.

average diameters of Au-NPs were 12 nm, as confirmed by TEM (Figure 1a). Preparation of Colloidal Gold-Antibody Conjugates. The Au-NP suspension was subjected to 5 min ultrasonication before conjugation. The secondary CEA antibody (10% more than the minimum amount, which was determined using a flocculation test) was added to 1 mL of pH-adjusted colloidal Au suspension followed by incubation at room temperature for 10 min. The conjugates were centrifuged at 14 000 rpm for 10 min, and the soft sediment was resuspended in 0.01 M PBS. A 1 mL suspension of Au-NPs was tagged with 40 μg of secondary CEA antibody. Addition of glycerol to a final concentration of 50% and BSA to a final concentration of 1% allows the storage of the colloidal Au-antibody conjugates at -20 °C for several months. Immunoassay Principles. The assay was performed in a polystyrene 96-well microtiter plate. The schematic diagram of the sandwich immunoassay and silver amplification for human CEA with Au-NP tags was shown in Figure 2. Initially, 200 μL of 10 μg mL-1 primary CEA antibodies in coating buffer was steadily attached on the solid polystyrene substrate via physical adsorption between hydrophobic groups of antibody molecule and polystyrene (step 1). The glass slide was washed off three times with 350 μL of washing buffer to remove any unbound antibodies, and the uncoated active sites of polystyrene substrate were saturated with 300 μL of blocking buffer, in which BSA was used as a blocking agent to prevent nonspecific adsorption of the antigens in the next step. BSA was also added to the antigen and Au-NPs conjugates solutions to minimize nonspecific binding and aggregation. Diluted CEA standard solutions (in assay buffer) or serum samples of 100 μL and diluted secondary CEA antibodies-colloidal gold conjugates of 100 μL were incubated over the polystyrene substrate to allow the formation of sandwich

complexes in the microtiter plate (step 2). Unbound antigens and gold conjugates were removed from the glass slide with 350 μL of washing buffer (three times) and 400 μL of pure water (three times). The attached Au-NPs were enlarged by 200 μL of silver amplification A and B mixture solution for silver metal deposition (step 3). Silver amplification A and B mixture solution was rapidly transferred to microtiter plates by use of a multichannel pipet, and a stopwatch was used to control exact reaction time. Silver amplification proceeds as an autocatalytic reaction: the Au-NPs serve as nucleation sites to catalyze the reduction of silver ions to metallic silver. Immediately after a 15 min amplification, the reaction was stopped by pouring out all the solutions from the microtiter plate. The metallic silver on the enhanced plates were washed with 400 μL of pure water (three times), dried, dissolved with 200 μL of 50% (v/v) high purity nitric acid solution for 10 min, and diluted to 4 mL with pure water. The atomic mass spectrometric signals obtained from the diluted solution were recorded by ICPMS for analyte quantification (step 4).

’ RESULTS AND DISCUSSION The present work aims at developing a highly sensitive ICPMS immunoassay by detecting the silver deposited on the Au-NPs. The proposed silver amplified immunogold ICPMS immunoassay combines the inherent high sensitivity of ICPMS detection with the signal amplification resulting from the catalytic precipitation of silver on the Au-NPs tags, pushing the sensitivity of the immunoassay to the low picomolar domain. The analytical procedure consists of the immunoreaction of the antigen with the primary antibody adsorbed on the walls of a polystyrene microtiter plate and a secondary colloidal gold-labeled antibody to form a sandwich complex, silver amplification, acid dissolution, and ICPMS detection of the silver (Figure 2). ICPMS was used earlier for determining the immunogold tags after immunoassay; thus, the comparison of analytical performance before and after silver amplification was also investigated. The detailed optimization and performance characteristics of the developed silver amplified ICPMS immunoassay are reported in the following sections. Detection of Gold and Silver by ICPMS. Au and Ag were detected using the 197Auþ and 107Agþ signal whose isotopic abundances are 100% and 51.35%, respectively. Theoretically, high interferences may be produced from 197TaOþ (99.7%) and 107 NbNþ (99.6%) to 197Auþ and 107Agþ. However, under the operating conditions of the proposed method, Ta and Nb atoms can be controlled at very low concentration levels using 2332



Figure 4. Dependence of the ICPMS Ag signal upon immunoassay parameters. (a) The antigen-antibody immunoreaction time. Concentration of human CEA, 1 μg mL-1; dilution ratio of the antibodycolloidal gold conjugate, 1:20; and silver amplification time, 15 min. (b) The dilution ratio of antibody-colloidal gold conjugate. Concentration of human CEA, 1 μg mL-1; antigen-antibody immunoreaction time, 60 min; and silver amplification time, 15 min.

Figure 5. Dependence of the ICPMS signal upon silver sources and amplification time. Concentration of human CEA, 10 ng mL-1; antigen-antibody immunoreaction time, 60 min; and dilution ratio of the antibody-colloidal gold conjugate, 1:20.

high purity reagents and deionized water. Therefore, the effects of 197TaOþ and 107NbNþ overlapping the 197Auþ and 107Agþ signals are negligible. Both 197Auþ and 107Agþ are suitable for our application. The optimizations of ICPMS parameters were carried out to obtain the maximum sensitivity. Parameters such as radio frequency (rf) power, cool gas flow rate, and nebulizer gas flow in ICPMS were optimized and listed in Table 1. Under the optimum conditions, the ICPMS calibration curves for Au and Ag were constructed with a serial of elemental standard solutions (Figure 3). The limits of detection (LOD, 3σ) for Au and Ag elements were measured as 0.001 and 0.002 ng mL-1,

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respectively. Linear ranges of more than 5 orders of magnitude were routinely obtained, with R2 > 0.999. Optimization of Immunoassay Conditions. The proposed ICPMS immunoassay of human CEA was performed as depicted in Figure 2. Immunoassay parameters including immunoreaction time and the dilution rate of antibody-immunogold conjugates were optimized using 1 μg mL-1 CEA solution. The dependence of the antigen-antibody reaction time upon the Ag signal was shown in Figure 4a. The response increased rapidly with the reaction time between 20 and 60 min and then leveled off above 60 min. This indicates that the interaction of antigen with antibody has reached equilibrium after 60 min, and hence, a reaction time of 60 min was selected for further investigations. During the colloidal gold conjugate-based immunoassay, the dilution rate of antibody-colloidal gold conjugates is a key factor affecting the detection sensitivity of the method, as was systematically studied in a previous report.51 In our study, the influence of the concentration of the antibody-colloidal gold conjugates upon the response of the ICPMS Ag signal was surveyed in the range of 1:10 to 1:200 dilutions. As shown in Figure 4b, the ICPMS Ag signal intensity remained almost a constant value while increasing the dilution rate (i.e., decreasing the concentration) of the colloidal gold-labeled antibody from 1:10 to 1:20 and then decreased in a nearly linear fashion by increasing the dilution rate. A dilution ratio of 1:20 was consequently selected for further studies. Silver Amplification. Immunogold silver amplification has been extensively studied and widely used in histochemical microscopy studies for 30 years,10 where functional immunogold acts as a catalyst to reduce silver ions to metallic silver in the presence of a reducing agent (such as hydroquinone). The autometallographic silver deposition procedure enlarges the size and darkens the color of the gold particles, such that protein-, antibody-, or DNA-conjugated Au-NPs become visible under electron or optic microscopy. In the subsequent quantitative applications of this technique, the sensitivity of detection in the case of Au-NP probes can be increased drastically by up to 5 orders of magnitude.11,52 The ingredient of silver amplification solution was adopted from Danscher et al.13,53 and Hacker et al.54 Apparently, when the molar concentration of each component of the silver amplification solution is fixed, the performance of silver amplification by catalytic precipitation on the Au-NPs tags would be strongly influenced by the silver source and silver amplification time. Silver Source. A number of silver salts, i.e., silver nitrate,19,20 silver lactate,13,53 and silver acetate,54,55 have been used as the silver amplification sources. The silver nitrate and silver lactate were light sensitive, and the amplification procedure consequently needed to be carried out in darkroom conditions or in a dark cupboard, while silver acetate was considered to be light insensitive during silver amplification. We have studied the performance of silver nitrate and silver acetate as silver sources, since they are widely used and commercially available. The silver amplifications by silver nitrate and silver acetate were compared in a dark room (Figure 5). As shown in Figure 5, a comparable performance of these two silver sources was obtained. However, silver amplification by silver nitrate gave rather a low signal-tonoise ratio under the light, while the performance of silver acetate remained almost invariable, which was in good accordance with the previous research.54 The light-insensitive silver acetate source was selected for the further study for the convenience of operation. 2333



Figure 6. Relationship between the concentration of CEA and visible or ICPMS signal. (a) The photograph of microtiter plate after immunogold labeled sandwich immunoassay; (b) The photograph of microtiter plate after immunogold labeled sandwich immunoassay and silver amplification; and (c) The ICPMS Ag and Au signal after dissolving the metallic elements in the microtiter plate.

Figure 7. Specificity for the determination of human CEA using the proposed immunoassay. Concentration of human CEA, 5 ng mL-1; and concentration of the other proteins, 100 ng mL-1.

Silver Amplification Time. We studied the appropriate time duration of silver amplification. As shown in Figure 5, the ICPMS signal of deposited silver increased nearly linearly with the silver amplification time. However, longer silver amplification time, while offering very favorable signal enhancement, led to an increase in the background. In contrast to the analytical signal generated by the silver deposited exclusively on the Au-NPs tags, such a background response might result from nonspecific binding of silver ions onto the walls of the polystyrene microtiter plate or the immobilized proteins,

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which also increases with the silver amplification time. Considering both signal sensitivity and background noise, we chose a 15 min silver amplification time for the further experiments. Figure 1 displays TEM images of colloidal gold before (a) and after (b) silver amplification. It can be seen that the diameters of the 12 nm Au-NPs increased dramatically to over 100 nm after 15 min of silver deposition/amplification. Analytical Performance. Under these optimal conditions described above, a negligible red color of the antibody-colloidal gold conjugate can be observed on the walls of a polystyrene microtiter plate after sandwich immunoassay (Figure 6a), which indicated that the immunogold labeled antibody has been adsorbed on the walls of the polystyrene microtiter plate. After 15 min of silver amplification, a naked-eye visible black color evidently appeared (Figure 6b), which visually confirmed the formation of metallic silver shell over Au-NPs labels. As low as 10 ng mL-1 CEA can be directly visualized, demonstrating a potential application for visual detection. Subsequently, the relationship between the concentration of CEA and ICPMS signal was investigated. As shown in Figure 6c, the dynamic range of the CEA concentration from 0.07 to 1000 ng mL-1 was obtained by the use of Ag ICPMS signal. The correlation equation was Y = 8.3 105 lg[CEA] þ 1.4 106, with the correlation coefficient r = 0.9917. The sensitivity enhancement of Ag over Au signal was up to 60-fold, i.e., the ratio of the slopes of the two calibration curves using the Ag and Au ICPMS signal, and the limit of detection (LOD, 3σ) was 0.6 and 0.03 ng mL-1 before and after silver amplification (calculated with Au and Ag signal intensity), respectively. The deviation from linearity was observed when the concentration of CEA was higher than 1000 ng mL-1. The reproducibility expressed as relative standard deviation (RSD) of 10 ng mL-1 CEA was 3.8% for within-batch (intra-assay) or 5.3% for between-batch (inter-assay). The specific recognition of antigenic species for the proposed ICPMS immunoassay was also investigated, with results shown in Figure 7. It can be seen that only the CEA can be recognized in the sandwich-type immunoreaction. Human IgG, rabbit IgG, goat IgG, or human AFP does not significantly interfere with the determination of CEA. A comparison of analytical performance of the present method with those of the other widely used immunoassay methods for the determination of CEA is given in Table 2. The LOD of the present method has more than 1 order of magnitude improvement compared to that of conventional Au-NP labeled ICPMS immunoassays and is even better than Eu3þ labeled laser ablation ICPMS immunoassay.35 It is also around 2 orders of magnitude lower than that of the widely used enzyme-linked immunosorbent assay (ELISA) or 1 order of magnitude lower than the clinical routine chemiluminescence immunoassay (CLIA) and time-resolved fluoroimmunoassay (TRFIA) for the determination of human CEA. Clinical Serum Sample Analysis. Sensitive detection of CEA is essential in clinical laboratories because increased correlation levels are found with a number of cancers including early colorectal, lung, breast, pancreatic, and bladder cancers.1 Clinical human serum samples were donated by Chengdu 7th People’s Hospital (Chengdu, China). As shown in Table 3, analytical results of the proposed method agree well with those by clinical chemiluminescent (CL) immunoassay, indicating that the present method could be applied to real clinical samples. 2334



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Table 2. Comparison of Various Immunoassay Methods for CEA Determinationa tags

immunoassay format

analytical method

limits of detection/ng mL-1

references

Au

microwells/sandwich-type

ICPMS immunoassay after silver amplification

0.03 (0.15 pM)

this work

Au


ICPMS immunoassay

0.6 (3 pM)

this work

Eu3þ


laser ablation ICPMS immunoassay

0.14

35

HRP


enzyme-linked immunosorbent assay

2

43

BHHCT-Sm3þ


time-resolved fluoroimmunoassay

0.30

56

BPTA-Tb3þ


time-resolved fluoroimmunoassay

0.20

57

HRP


chemiluminescence immunoassay

0.61

58

DMDSBA


chemiluminescence immunoassay

0.53

59

Au, Au-NPs; BHHCT-Sm3þ, Sm3þ chelate of 4,40 -bis (100 ,100 ,100 ,200 ,200 ,300 ,300 -heptafluoro-400 ,600 -exanedion-600 -yl)-chlorosulfo-o-terphenyl; BPTATb3þ, Tb3þ chelate of N,N,N1,N1-(2,6-bis(30 -aminomethyl-10 -pyrazolyl)-4-phenyl pyridine) tetrakis (acetic acid); HRP, horseradish peroxidase; DMDSBA, 10,100 -dimethyl-3,30 -disulfo-9,90 -biacridine. a

’ REFERENCES

Table 3. Analytical Results of Human Serum Samples sample

a

-1

this method /ng mL

-1

b

CL /ng mL

human serum 1

1.2 ( 0.1

1.4

human serum 2

2.5 ( 0.3

2.7

human serum 3 human serum 4

3.4 ( 0.4 35.8 ( 2.0

3.5 35.6

human serum 5

159.5 ( 10.3

167.7

Obtained value ( standard deviation. b CL, chemiluminescence immunoassay was performed by Chengdu 7th People’s Hospital. a

’ CONCLUSIONS The feasibility of a highly sensitive ICPMS immunoassay based on the quantitative precipitation of silver onto immunogold tags has been demonstrated. The great signal enhancement by silver amplification is successfully combined with the sensitive ICPMS detection. The silver amplification procedure is simple, low cost, and rapid. The proposed method is competitive to some conventional methods such as ELISA, CLIA, and TRFIA in clinical analysis for CEA, which also showed great potential for numerous applications in sensitive immunoassay, DNA hybridization, and other biological analysis. Other signal amplification schemes, such as rolling circle amplification, avidin-biotin amplification, and exponential isothermal amplification, may also exhibit some intriguing advantages for biosample analysis by coupling the sensitive ICPMS immunoassay. Furthermore, the ICPMS immunoassay has a wide choice of metal nanoparticle labels and the multielement detection power; thus, highly sensitive simultaneously multiplex immunoassay may be realized by incorporation of different signal amplification methods. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel./Fax: þ86-28-8541-2798.

’ ACKNOWLEDGMENT Authors acknowledge the financial support for this project from the National Natural Science Foundation of China (Nos. 20835003 and 21075084) and the Sichuan Youth Science and Technology Foundation (No. 2009-18-409). Mr. Hao Lv from Chinese Academy of Measurement and Testing Technology is thanked for helping with handling ICPMS. The authors also thank Mr. Guanglei Cheng of the Analytical and Testing Center for his assistance with analytical atomic spectrometric measurements.

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