Gold Nanoparticles Amplified Ultrasensitive Quantification of Human Urinary Protein by Capillary Electrophoresis with On-Line Inductively Coupled Plasma Mass Spectroscopic Detection Jing-Min Liu, Yan Li, Yan Jiang, and Xiu-Ping Yan* Research Center for Analytical Sciences, College of Chemistry, Nankai University, 94 Weijin Road, Tianjin 300071, China Received January 21, 2010
Quantitative analysis of proteins play pivotal roles in basic discovery research and clinical applications, and the analytical challenge is to provide sufficient sensitivity to determine the proteins at endogenous levels. Here, we report a strategy for ultrasensitive quantification of human urinary protein by capillary electrophoresis with on-line inductively coupled plasma mass spectroscopic detection (CE-ICPMS) in conjunction with gold nanoparticles (AuNPs) amplification. The albumin in the sample solution was incubated with excess AuNPs to form the AuNP-albumin adduct. The excess AuNPs and the AuNP-albumin adduct were then effectively separated by CE for on-line ICPMS detection. As a result of AuNPs-tagging, more than 2000 gold atoms on average were attached to each albumin molecule to successfully achieve a significant amplification of ICPMS signal with extremely low limit of detection (0.5 pM for 280 nL of sample injection, corresponding to 0.1 amol) and a wide linear response over 4 orders of magnitude. The relative standard deviations of the migration time, peak area, and peak height for seven replicate injections of a mixture of 0.4 pM AuNPs and 9.0 pM albumin ranged from 1.8% to 4.4%. The developed method was successfully applied for detecting albumin in human urine samples with quantitative recoveries in the range of 93.0-99.7%. The methodology demonstrated here has potential for simultaneous determination of low-abundance multiple biomarkers of interest via multiple nanomaterials tags because of high-resolution CE separation and ultrasensitive ICPMS detection. Keywords: gold nanoparticles • CE-ICPMS • protein detection • albumin • proteomics
Introduction Ultrasensitive detection of proteins has received increasing interest because many important protein biomarkers are present at ultralow levels and ultrasensitive quantification is crucial for the diagnosis of specific diseases in clinical research.1-4 A recent review provides an excellent summary of tremendous advances in the development of analytical techniques for protein detection.3 Some of the commonly used techniques for protein detection, such as radioimmunoassay,5 enzyme immunoassay,6 fluorescence,7 quartz crystal microbalance,8 and surface-enhanced Raman spectroscopy,9 are able to provide relatively high sensitivity and low limits of detection, but most of them are only capable of detecting abundant proteins. In addition, some of these methods suffer drawbacks such as utilization of radioactive substances, and/or timeconsuming sample preparation steps before analysis. Recently, the combination of mass spectrometry (MS) and elemental tagging for sensitive detection of biomolecules has emerged as a powerful means for quantitative proteomics.10-19 Inductively coupled plasma mass spectroscopy (ICPMS) has gained a very wide acceptance due to its extremely high sensitivity, element specificity, robustness, and wide linearity * Corresponding nankai.edu.cn. 10.1021/pr100056w
author.
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(86)22-23506075.
2010 American Chemical Society
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of the response.20,21 However, proteins, as well as almost all biomolecules, are not efficiently detected by ICPMS. The atoms of hydrogen, carbon, oxygen, and nitrogen, which are the main components of proteins, are present in both the solvents used during sample introduction and the atmospheric air entrained into the ICPMS, and they cause extremely high background signals. In principle, by selecting proper nanomaterials of elements and labeling them to the biomolecules, ICPMS can indirectly detect biomolecules of interest via element-tag with extremely high sensitivity. Besides isotope-coded affinity tag (ICAT)22 and metal element chelate tag (MECT),23,24 nanomaterials labeling is an alternative method for quantification. On the basis of fundamental chemistry, nanomaterials science has developed over the past three decades into today’s powerful disciplines for biological and biomedical applications.25-34 Among those advanced nanomaterials, gold nanoparticles (AuNPs) have attracted the most attention because of their promising applications in biological fields, such as labeling, delivering, heating, and sensing.35-38 AuNPs are biocompatible; bind readily to a range of biomolecules such as amino acids,39 proteins/enzymes,40 and DNA;41 and expose large surface areas for immobilization of biomolecules. Thus, AuNPs are excellent candidates for bioconjugation, and the ideal labels for detecting Journal of Proteome Research 2010, 9, 3545–3550 3545 Published on Web 05/08/2010
research articles important biomolecules at low concentrations due to their uniform size and significant number of atoms per conjugate.14,15 Human serum albumin (HSA) is a major constituent of plasma proteins. During normal renal processing of the blood, a small amount of albumin is excreted with the urine. Monitoring the concentration change of albumin in urine with high precision and sensitivity can help to diagnose nephropathy in patients suffering from diabetes and hypertension.42-44 Here, we report AuNPs tagging for capillary electrophoresis (CE) with on-line ICPMS detection for ultrasensitive quantification of protein. Albumin was used as a model analyte in human urine samples to demonstrate the proof-of-concept. First, albumin was incubated with excess AuNPs to form the AuNP-albumin adduct. The excess AuNPs were separated from the AuNP-albumin adduct by CE. Both the AuNP-albumin adduct and AuNPs were on-line detected with ICPMS.
Experimental Section Reagents. All reagents were of the highest available purity and at least of analytical grade. Ultrapure water (18.2 MΩ cm-1) obtained from a Water Pro water purification system (Labconco Corporation, Kansas City, MO) was used throughout this work. Chloroauric acid (HAuCl4 · xH2O) was purchased from Aldrich. HSA (>96%; fraction V, lot no. A1653) was purchased from Sigma (St. Louis, MO). A protein stock solution of 1000 mg L-1 was prepared by dissolving 10 mg of HSA in 10 mL of ultrapure water, and was stored at 4 °C in the dark. Working solutions were prepared by serial dilution of the stock solution with 10 mM phosphate buffer solutions (PBS). The make-up solution was prepared from electronic-pure nitric acid (Beijing Institute of Chemicals, Beijing, China). The electrophoretic buffer solution was prepared from ammonium acetate (NH4Ac) (Beijing Chemicals, Beijing, China) and tris(hydroxymethyl)aminomethane (Tris) (Beijing Chemicals, Beijing, China), filtered through a 0.45-µm filter, and degassed in an ultrasonic bath prior to use. Instrumentation. The absorption and fluorescence spectra were recorded on a UV-3600 UV-vis-NIR spectrophotometer (Shimadzu, Japan) and an F-4500 fluorescence spectrophotometer (Hitachi, Japan), respectively. The fluorescence spectra of HSA were recorded under the excitation wavelength of 270 nm. The slit width of excitation and emission was 10 and 10 nm, respectively. Transmission electron microscopic (TEM) characterization was performed on a Tecnai G2 F20 (Philips, Holland) at 200 kV. The samples for TEM were obtained by drying sample droplets from water dispersion onto a 300-mesh Cu grid coated with a lacey carbon film, which was then allowed to dry prior to imaging. An X Series quadrupole ICPMS instrument (Thermo Elemental, Cheshire, U.K.) was used for all measurements. The Micromist nebulizer (GE, Australia) was mounted on the standard spray chamber without any modification. The experimental conditions for ICPMS measurements were optimized to give maximum signal for Au from AuNPs (Table 1). The separations were conducted on a laboratory-built CE system, which was composed of a 0-30 kV high-voltage power supply (Model DW 300-1, Tianjin Dongwen High-Voltage Power Supply Factory, Tianjin, China), a hydrodynamic sampling unit, and a 75-µm i.d. × 375-µm o.d. fused-silica capillary (Yongnian Optical Fiber Factory, Hebei, China). The power supply was operated in voltage-controlled mode. The inlet end of the capillary was held at a positive potential while the outlet end was grounded. The sample solution was introduced into the 3546
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Liu et al. Table 1. Optimized Operating Conditions for CE-ICPMS ICPMS instrument
RF power Plasma gas flow Auxiliary gas flow Nebulizer gas flow Nebulizer
Thermo Elemental X7 Series
Plasma Conditions 1340 W 15.0 L min-1 1.08 L min-1 0.98 L min-1 Micromist
Mass Spectrometer Settings Resolution Standard Dwell time 600 ms 197 Isotopes monitored Au Internal standard used 115In CE-ICPMS Interface 100 µL min-1 Homemade Fused silica, 75 µm i.d., 375 µm o.d., 50 cm length Electrophoretic buffer 20 mM NH4Ac + 20 mM Tris (pH 8.0) Electrophoretic voltage 17 kV Sample injection Hydrodynamic mode, 280 nL per injection Liquid uptake rate CE system Capillary
separation capillary using a hydrodynamic method. A positive voltage of 15-25 kV was used for the separations. The homemade CE-ICPMS interface used in this work (Figure S1 in Supporting Information) was constructed on the basis of a cross design for introducing a sheath flow around the CE capillary and a Pt electrode, which provided an electrical connection for stable electrophoretic separations.45 A 1/16-in. polyether ether ketone (PEEK) cross fitting was used to connect the CE capillary and the PTFE tubing for make-up solution delivery, the Pt electrode, and the PTFE tubing to the ICPMS system. To reduce the diffusion of sample zone, the capillary was inserted through the PEEK cross and went to the end of PTFE tubing to the ICPMS system. The CE capillary outlet was grounded through a Pt electrode. The mixture of CE effluent and HNO3 solution was nebulized by the Micromist nebulizer of ICPMS system, and introduced into the ICPMS by an Ar carrier. The signal of 115In, added to the make-up solution at 1 µg L-1 as internal standard, was monitored simultaneously and served to compensate for signal error due to possible instability of the make-up solution flow. The experimental conditions of the CE-ICPMS system used throughout this work are listed in Table 1. Before CE separation, the capillary was flushed sequentially with 1 M NaOH and 0.1 M NaOH for 5 min, rinsed with ultrapure water for 3 min, and then flushed with running electrolyte solution for 5 min. The capillary was also rinsed with running electrolyte solution for 5 min between each run. All CE experiments were performed at ambient temperature. Preparation of Colloidal AuNPs. Colloidal AuNPs were prepared by reduction of HAuCl4 solution with trisodium citrate according to the literature.46-48 Before preparation, the glassware was thoroughly cleaned with freshly prepared aqua regia, rinsed with ultrapure water, and oven-dried at 100-110 °C for 3 h. Briefly, 20 mL of HAuCl4 solution (0.1 mM) was brought to rolling boil with vigorous magnetic stirring. Rapid addition of 100 µL of trisodium citrate solution (0.1 M) to the vortex of the solution resulted in a color change from pale yellow into a brilliant red. The reaction was allowed to proceed for another 30 min with continuous stirring. The resulting colloidal suspension was cooled and centrifuged at 12 000 rpm for 20 min.
AuNPs Amplified Quantification of Human Urinary Protein
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Following removal of the supernatants, the oily precipitates were redispersed in 4 mM trisodium citrate solution and filtered through a 0.45 µm membrane. The prepared AuNPs was stored at 4 °C in dark prior to use. Procedures. AuNPs were made homodisperse in an ultrasonic bath for 30 min. Standard albumin solutions or urine samples were then incubated with excess colloidal AuNPs in 10 mM PBS (pH ) 4.5) for 1 h to ensure a thorough reaction of the albumin with the AuNPs. Finally, the incubated solution was analyzed by CE-ICPMS under the conditions listed in Table 1. Urine Samples. The urine samples were collected from healthy volunteers with informed and signed consent. After centrifugation at 10 000 rpm for 10 min, the supernatants were diluted with 10 mM PBS (pH ) 4.5) to reduce the interference from matrix and other biomolecules in urine and to ensure the albumin concentration involved in the linear range. No further complex pretreatment and deproteinization procedures were needed in the sample preparation before subsequent analysis. Then, AuNPs were added to make the final concentration of 100 pM in the pH-adjusted urine samples, and the incubation proceeded for 1 h.
Results and Discussion Characterization of Colloidal AuNPs. TEM measurement was performed on the prepared AuNPs, and at least 300 particles were selected at random to characterize the size distribution of the AuNPs. Figure 1a shows the size distribution of the prepared AuNPs with an average size of 16.3 ( 1.5 nm together with a typical TEM image as an inset. The narrow size distribution of the prepared AuNPs is favorable for reproducible quantification of albumin at low concentrations by CE-ICPMS assay. The maximal optical absorbance of the AuNPs was found at λmax ) 520 nm and the concentration of the AuNPs was determined by ultraviolet-visible (UV) spectroscopy using the molar extinction coefficient value and the absorbance intensity of AuNPs at the wavelength of 450 nm according to literature.49 Formation of the AuNP-Albumin Adduct. The citrate stabilized AuNPs have been proved to have the ability to adsorb proteins and the number of protein molecules adsorbed onto the AuNPs surface and the adsorption strength are mainly dependent on pH value of the buffer solution for incubation.50 To achieve the effective formation of the AuNP-albumin adduct, we investigated the effect of pH value on the labeling process by observing fluorescence quenching of albumin upon addition of AuNPs. PBS was chosen as a universal buffer to measure the influence of pH in order to minimize the effect of the different buffer components to the adsorption process. Since the fluorescence of tryptophan residues in proteins is very sensitive to their local environment, it can be used to study the interaction between AuNPs and proteins.51 The decrease of the fluorescence intensity was the most marked change in the fluorescence spectra observed upon addition of AuNPs (Figure 2a). As shown in Figure 2b, the highest quenching efficiency was obtained when pH value of PBS was 4.5, which was chosen as the best pH condition for the effective formation of the AuNP-albumin adduct. The interaction between AuNPs and albumin was reported to be driven by electrostatic force and albumin adsorption to AuNPs was mainly determined by surface charge characteristics.50 Thus, the pH effect on adsorption can be explained by the properties of albumin and the nanoparticle surface. The nanoparticle surface carries a negative charge introduced by
Figure 1. (a) Size distribution of AuNPs with an average diameter of 16.3 ( 1.5 nm. The inset shows a typical TEM image. The scale bar in the inset corresponds to 100 nm. (b) UV spectra of AuNPs and AuNP-albumin adduct.
citrate ions giving necessary colloidal stability to the nanoparticles and preventing aggregation.52 At pH lower than the isoelectric point of albumin (4.7), albumin adsorbs efficiently to the negatively charged nanoparticles because albumin and nanoparticles possess opposite charges. The adsorption is evidently reduced at higher pH. At pH lower than the pKa value of citric acid (3.15), the particle surface charge approaches zero, evidently lowering the electrostatic interaction and reducing protein adsorption. As shown in Figure 2c, particle aggregation (blue color) was observed at pH lower than 3.0. Thus, the final incubation proceeded in 10 mM PBS with pH 4.5. All protein solutions and urine samples were adjusted to pH 4.5 and then incubated with 100 pM AuNPs for 1 h before subsequent CEICPMS analysis. CE Separation of AuNPs and AuNP-Albumin Adduct. To examine the suitability of the interface of CE-ICPMS hybrid technique for AuNPs, we investigated the electrophoretic patterns of AuNPs with the buffer composed of 20 mM NH4Ac and 20 mM Tris and introduced two different methods for quantifying the AuNPs sample into the capillary. One is normally to promote the analyte into the detector by electrophoretic method with a 17 kV voltage across the two ends of capillary, while the other is to push the analyte by gas pressure of 120 kPa directly into the detector through the capillary. A good correlation was obtained (R2 ) 0.9999) between these methods, indicating the interface used is compatible for operating the on-line hyphenation of CE separation and ICPJournal of Proteome Research • Vol. 9, No. 7, 2010 3547
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Figure 2. (a) Effect of AuNPs on the fluorescence spectra of albumin (pH 4.5) with AuNPs concentration increased from top to bottom: 0, 0.075, 0.150, 0.225, 0.300, 0.375, 0.450, 0.525, 0.600 nM. (b) Effect of pH on the quenched fluorescence intensity of HSA (1 µM) at 1 nM AuNPs. (c) Effect of pH on the stability of AuNPs.
Figure 3. Correlation of electrophoretic and gas-push methods for the quantification of AuNPs injected into the capillary.
MS detection and that AuNPs running in the capillary show negligible adsorption onto the wall of capillary (Figure 3). To achieve an effective CE separation of the AuNP-albumin adduct from excess AuNPs, we tested several types of buffers and investigated the effects of pH and ionic strength on the 3548
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Figure 4. (a) Electropherograms obtained by CE-ICPMS assay for the interactions of 6 pM AuNPs with albumin concentration increased from top to bottom: 0, 100, 150, 200, 250 pM. CE separation was carried out using a fused-silica capillary (50-cm long, 75-µm i.d., and 375-µm o.d.) at 17 kV by using the mixture of 20 mM NH4Ac and 20 mM Tris as the running electrolyte (pH 8.0). (b) Peak intensity of AuNP-albumin adduct at variable albumin concentrations with fixed AuNPs concentration of 6 pM.
separation efficiency (Figures S2-S4 in Supporting Information). Tris-NH4Ac buffer was chosen as the most suitable buffer solution because it offered an effective separation of AuNPs and AuNP-albumin adduct by CE. The pH of the buffer solution used for CE separation is one of the most important factors. We found that the AuNP-albumin adduct was baseline separated from excess AuNPs in a pH range of 7.0-9.0 of the buffer solution (20 mM NH4Ac + 20 mM Tris). For the best separation efficiency and reproducibility, a pH of 8.0 of the buffer solution was chosen. The effect of the concentration of NH4Ac and Tris on the separation was examined in the range of 5-100 mM. The concentration of Tris showed little effect on the separation efficiency. As the concentration of NH4Ac decreased in the range of 20-100 mM, the resolution of the two AuNPs species increased, but the migration time and electric current also decreased. When the concentration of NH4Ac decreased to 5 mM, the separation efficiency reduced. Accordingly, 20 mM NH4Ac and 20 mM Tris were chosen for baseline separation of the AuNP-albumin adduct and AuNPs in a minimal period. Figure 4a shows albumin concentration dependent CEICPMS signals under optimal conditions (Table 1). As the
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AuNPs Amplified Quantification of Human Urinary Protein
Table 2. Analytical Figures of Merit of the Developed AuNPs Amplification CE-ICPMS for Albumin Determination precision (RSD, n ) 7) at 9.0 pM level/%
Migration time Peak area Peak height
1.8 3.6 4.4
detection limit (S/N ) 3)
/pM /amol Calibration function (A, peak area/ 103 counts; C, conc./pM) Correlation coefficient (R2)
Figure 5. Dependence of ICPMS intensity of AuNP-albumin adduct on albumin concentration.
concentration of albumin increased, the peaks of AuNP-albumin adduct gradually grew, while those of AuNPs decreased. As shown in Figure 4b, when the concentration of albumin increased upon 270 pM with that of AuNPs fixed as 6 pM, the peak intensity of AuNP-albumin adduct increased to top, which demonstrated the albumin was in excess relative to initial AuNPs. The number of albumin molecules bound per nanoparticle (n) was calculated from the change in the peak areas of AuNPs and AuNP-albumin adduct based on eq 1:53,54
n)
0 f b CAuNPs SAuNPs + SAuNPs 0 Calbumin
b SAuNPs
(1)
0 0 where CAuNPs and Calbumin are the initial molar concentrations b of AuNPs and albumin, respectively, SAuNPs is the peak area f measured for the AuNP-albumin adduct, and SAuNPs corresponds to the peak area belonging to the free (unbound) AuNPs. Thus, we conclude that each nanoparticle reacted with about 45 albumin molecules, that meant more than 2000 gold atoms on average were attached to each albumin molecule for signal amplification as the AuNPs with size of 16 nm contain ∼105 gold atoms. The dependence of 197Au peak area intensity of the AuNP-albumin adducts on albumin concentration showed a good linearity over 4 orders of magnitude in the concentration of albumin from 1.8 pM to 18 nM (R2 ) 0.9878) (Figure 5). Analytical Performance of the Developed AuNPs Amplification CE-ICPMS Hybrid Technique for Determination of Albumin. The analytical figures of merit of the developed AuNPs amplification CE-ICPMS for the determination of albumin are summarized in Table 2. The precisions (relative standard deviation, RSD) of the migration time, peak area, and peak height for seven replicate injections of a mixture of 0.4 pM AuNPs and 9.0 pM albumin were 1.8%, 3.6% and 4.4%, respectively. The detection limit (S/N ) 3) of the developed method was 0.1 amol of albumin, corresponding to 0.5 pM for 280 nL of sample injection. The developed technique is also compared with element-tagged techniques reported in the literature for highly sensitive detection of biomolecules with respect to detection limit (Table S1 in Supporting Information). We further tested the specificity of our AuNPs amplification CE-ICPMS assay for albumin. Human IgG, transferrin, glutathione, and cysteine were selected as interfering substances. Incubating any of the above interfering biomolecules with
0.5 0.1 A ) 0.32C - 0.06 0.9878
AuNPs under the same condition as albumin (10 mM PBS, pH ) 4.5) only gave the AuNP peak in the electropherograms. Furthermore, adding any of the above interfering biomolecules into the mixture of the AuNPs and albumin did not result in significant change of the electropherograms (see Figure S5 in Supporting Information). The results indicate that, under the chosen incubation conditions (10 mM PBS, pH ) 4.5), the selected interfering biomolecules gave no significant interference with the albumin determination. Application of the Developed AuNPs Amplification CE-ICPMS Assay to the Quantification of Albumin in Human Urine Samples. Monitoring the concentration change of albumin in urine with high precision and sensitivity is helpful to diagnose nephropathy in patients suffering from diabetes and hypertension. To demonstrate the applicability of the developed AuNPs amplification CE-ICPMS assay for real sample analysis, we applied it to the determination of albumin in human urine samples. Figure 6 shows the electropherograms for a human urine sample after 100-fold dilution with 10 mM PBS spiked with 100 pM AuNPs only (Figure 6a), and spiked with 100 pM AuNPs and 300 pM albumin together (Figure 6b). Addition of AuNPs to the urine sample gave the peak of the AuNP-albumin adduct and that of the AuNPs, indicating the binding of AuNPs to the original albumin in the urine sample. The addition of albumin to the sample solution led to a decrease of the peak of AuNPs, but an increase of the peak of the AuNP-albumin adduct, showing the binding of added albumin with excess AuNPs in the sample solution. The
Figure 6. Electropherograms obtained by the developed AuNPstagging CE-ICPMS assay under optimal conditions (Table 1) with injection of 280 nL of (a) human urine (diluted 100-fold with 10 mM PBS) spiked with 100 pM AuNPs, (b) the diluted human urine spiked with 100 pM AuNPs and 300 pM albumin. Journal of Proteome Research • Vol. 9, No. 7, 2010 3549
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Table 3. Analytical Results for Determination of Albumin in Urine Samples
samples
human human human human human
urine-1 urine-2 urine-3 urine-4 urine-4
dilution factor
determined albumin (mean ( s, n ) 3; nM)a
recovery (mean ( s, n ) 3) (%)
100 100 100 200 500
322.6 ( 0.9 270.4 ( 3.8 330.6 ( 4.3 301.6 ( 22 308.5 ( 4.9
97.9 ( 2.1 93.0 ( 1.2 95.3 ( 0.7 97.2 ( 2.3 99.7 ( 4.9
a The measured values were already adjusted for the dilution factor, thereby representing the concentrations of albumin in the original urine samples.
analytical results were consistent for various dilution factors used (100-, 200-, and 500-fold dilution) (Table 3). The recoveries of spiked 300 pM albumin in the urine samples ranged from 93.0% to 99.7% (Table 3), suggesting no interferences from the urine matrixes encountered. The concentrations of albumin in the urine samples determined by the developed method based on peak area measurements ranged from 270.4 to 330.6 nM (Table 3). The above results demonstrated the applicability of the developed AuNPs amplification CE-ICPMS hybrid technique for the quantification of albumin in real urine samples.
Conclusions In summary, we have developed an ultrasensitive method for the determination of human urinary protein based on AuNPs amplification CE-ICPMS. The extremely low detection limit for protein achieved by the present method provides a new possibility for biological assays and clinical diagnoses. The methodology demonstrated here has potential for simultaneous determination of low-abundance multiple biomarkers of interest via multiple nanomaterials tags because of high-resolution CE separation and ultrasensitive ICPMS detection.
Acknowledgment. This work was supported by the National Natural Science Foundation of China (Grant No. 20935001, 20975054), the National Basic Research Program of China (Grant No. 2006CB705703), and the Tianjin Natural Science Foundation (Grant No. 10JCZDJC16300). Supporting Information Available: Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Prange, A.; Pro¨frock, D. Anal. Bioanal. Chem. 2005, 383, 372–389. (2) Szpunar, J. Analyst 2005, 130, 442–465. (3) Zhang, H. Q.; Zhao, Q.; Li, X.-F.; Le, X. C. Analyst 2007, 132, 724– 737. (4) Zhang, H. Q.; Wang, Z. W.; Li, X.-F.; Le, X. C. Angew. Chem., Int. Ed. 2006, 45, 1576–1580. (5) Thomson, D. M. P.; Krupey, J.; Freedman, S. O.; Gold, P. Proc. Natl. Acad. Sci. U.S.A. 1969, 64, 161–167. (6) Pradelles, P.; Grassi, J.; Maclouf, J. Anal. Chem. 1985, 57, 1170– 1173. (7) Potyrailo, R. A.; Conrad, R. C.; Ellington, A. D.; Hieftje, G. M. Anal. Chem. 1998, 70, 3419–3425. (8) Furtado, L. M.; Su, H. B.; Thompson, M.; Mack, D. P.; Hayward, G. L. Anal. Chem. 1999, 71, 1167–1175. (9) Bizzarri, A. R.; Cannistraro, S. Nanomedicine 2007, 3, 306–310. (10) Bettmer, J.; Jakubowski, N.; Prange, A. Anal. Bioanal. Chem. 2006, 386, 7–11. (11) Prange, A.; Pro¨frock, D. J. Anal. At. Spectrom. 2008, 23, 432–459. (12) Sanz-Medel, A.; Montes-Bayo´n, M.; Ferna´ndez de la Campa, M. R.; Encinar, J. R.; Bettmer, J. Anal. Bioanal. Chem. 2008, 390, 3–16.
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PR100056W
