Role of Fluorosurfactant-Modified Gold Nanoparticles in Selective

Jul 9, 2008 - In this article, we report a simple approach for the selective sensing of homocysteine thiolactone (HTL) using fluorosurfactant (FSN)-ca...
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Anal. Chem. 2008, 80, 6345–6350

Role of Fluorosurfactant-Modified Gold Nanoparticles in Selective Detection of Homocysteine Thiolactone: Remover and Sensor Chia-Chi Huang† and Wei-Lung Tseng*,†,‡ Department of Chemistry and Medical University Joint Research Center, National Sun Yat-sen University, Kaohsiung, Taiwan In this article, we report a simple approach for the selective sensing of homocysteine thiolactone (HTL) using fluorosurfactant (FSN)-capped gold nanoparticles (AuNPs) as aminothiol removers and as sensors. We have shown that HTL did not bind to the surface of the FSN-AuNPs in the pH range of 4.0-10.0. In contrast, under these pH conditions, the FSN-AuNPs are aggregated upon the addition of homocysteine (HCys) and cysteine (Cys). On the basis of this feature, we have demonstrated that FSNAuNPs are effective sorbent materials for HCys and Cys, but not for HTL. It is found that the FSN-AuNPs can remove of >98% of HCys and >99% of Cys from an aqueous solution. Thus, after the centrifugation of a solution containing AuNPs, HTL, and other aminothiols, only HTL remains in the supernatant. When NaOH is added to the supernatant, HTL is hydrolyzed to HCys, leading to the aggregation of the FSN-AuNPs. As a result, the selectivity of the probe is significantly higher for HTL in aqueous solutions than for other aminthiols. The sensitivity of FSN-AuNPs toward HTL can be further improved by optimizing the AuNP concentrations. Under optimum conditions, the lowest detectable concentration of HTL through this approach is 100 nM. We have validated the applicability of our method through the analyses of HTL in urine samples. Elevated homocysteine (HCys) levels in plasma and urine are associated with many cardiovascular, metabolic, and neurodegenerative disorders.1–3 Although the mechanisms explaining the toxicity of HCys are not clear, one of them was found to be attributed to the metabolic conversion of HCys to a cyclic thioester, HCys thiolactone (HTL).4 Since the structure of HCys is similar to that of methionine, it participates in the initial step of protein synthesis and is misactivated by methionine aminoacyltRNA synthetase.5 The resulting product, homocysteinyl adeny* To whom correspondence should be addressed. E-mail: tsengwl@ mail.nsysu.edu.tw. Fax: 011-886-7-3684046. † Department of Chemistry. ‡ Medical University Joint Research Center. (1) Clarke, R.; Daly, L.; Robinson, K.; Naughten, E.; Cahalane, S.; Fowler, B.; Graham, I. N. Engl. J. Med 1991, 324, 1149–1155. (2) Taylor, L. M.; Defrang, R. D.; Harris, E. J.; Porter, J. M. J. Vasc. Surg. 1991, 13, 128–136. (3) Clarke, R.; Smith, A. D.; Jobst, K. A.; Refsum, H.; Sutton, L.; Ueland, P. M. Arch. Neurol. 1998, 55, 1449–1455. (4) Jakubowski, H.; Fersht, A. R. Nucleic Acids Res. 1981, 9, 3105–3117. 10.1021/ac8006973 CCC: $40.75  2008 American Chemical Society Published on Web 07/09/2008

late, is preferentially cleaved to yield HTL. The structure and isoelectric point of the protein can be modified by the reaction of the lysine residues of the protein with HTL, which yields free thiols.6 This modification can affect the function of the protein7 and induce immune responses.8 For example, the redox status of cytochrome c is very susceptible to modification by HTL.7a Additionally, cell damage might be caused by the homocyteinylation of cellular and extracellular proteins.9 Based on these observations, it can be reasonably stated that an increase in the concentration of HCys triggers the elevation of HTL levels in body fluids.10 Thus, HTL has been implicated in various HCys-related diseases.8a,10b,11 Although HTL is biologically and clinically significant, there are only a few reports on the determination of HTL concentration in body fluids and cells. Quantification of HTL can be directly detected by monitoring absorption peak at 240 nm.12 Thus, highperformance liquid chromatography coupled with UV detection has been successfully utilized for determination of HTL in biological samples.5 Thin-layer chromatography coupled with the scintillation counting of 35S offers high sensitivity in the detection of HTL, albeit at the expense of analysis time and convenience.6b Under alkaline conditions, the thiolactone ring of HTL is cleaved to yield HCys,13 which can be directly derivatized with ophthalaldehyde (OPA).14–16 By means of this derivatization step, the quantitative analysis of HTL in cell cultures,14 plasma,15 and urine16 has been accomplished using high-performance liquid (5) Jakubowski, H. Anal. Biochem. 2002, 308, 112–119. (6) (a) Jakubowski, H. FASEB J. 1999, 13, 2277–2283. (b) Jakubowski, H. J. Biol. Chem. 1997, 272, 1935–1942. (7) (a) Kajan, J. P.; Marczak, L.; Kajan, L.; Skowronek, P.; Twardowski, T.; Jakubowski, H. Biochemistry 2007, 46, 6225–6231. (b) Sauls, D. L.; Lockhart, E.; Warren, M. E.; Lenkowski, A.; Wilhelm, S. E.; Hoffman, M. Biochemistry 2006, 45, 2480–2487. (8) (a) Jakubowski, H. Cell. Mol. Life Sci. 2004, 61, 470–487. (b) Undas, A.; Perla, J.; Lacinski, M.; Trzeciak, W.; Kazmierski, R.; Jakubowski, H. Stroke 2004, 35, 1299–1304. (9) Jakubowski, H. J. Nutr. 2000, 130, 377S–381S. (10) (a) Chwatko, G.; Boers, G. H.; Strauss, K. A.; Shih, D. M.; Jakubowski, H. FASEB J. 2007, 21, 1707–1713. (b) Jakubowski, H. J. Nutr. 2006, 136, 1741S–1749S. (11) Jakubowski, H. Clin. Chem. Lab. Med. 2007, 45, 1704–1716. (12) (a) Gao, W.; Goldman, E.; Jakubowski, H. Biochemistry 1994, 33, 11528– 11535. (b) Jakubowski, H. J. Biol. Chem. 1995, 270, 17672–17673. (13) (a) du Vigneaud, V.; Patterson, W. I.; Hunt, M. J. Biol. Chem. 1938, 126, 217–231. (b) Jakubowski, H. Chem. Eur. J. 2006, 12, 8039–8043. (14) Mukai, Y.; Togawa, T.; Suzuki, T.; Ohata, K.; Tanabe, S. J. Chromatogr., B 2002, 767, 263–268. (15) Chwatko, G.; Jakubowski, H. Anal. Biochem. 2005, 337, 271–277. (16) Chwatko, G.; Jakubowski, H. Clin. Chem. 2005, 51, 408–415.

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Scheme 1. Procedure Associated with the Colorimetric Detection of HTL

chromatography with fluorescence detection. Moreover, the ringopening reaction of HTL can be catalyzed by HTL hydrolase under neutral conditions.17 The measurement of the HTL hydrolase activity has been successfully achieved by utilizing OPA as the derivatization agent and HTL as the substrate.18 Gas chromatography coupled with mass spectrometry is an alternative method for the determination of HTL concentration in human plasma samples.19 Recently, gold nanoparticles (AuNPs) have been demonstrated to have strong affinity toward aminothiols, such as cysteine (Cys), HCys, cysteinylglycine (Cys-Gly), glutathione (GSH), and γ-glutamycysteine (Glu-Cys).20 When these aminothiols are attached to the surface of the AuNPs through Au-S bonds, either hydrogen bonding or electrostatic interactions between two amino acid groups will engender the assembly of AuNPs.21 Thus, it is a simple process that uses AuNPs to selectively concentrate aminothiols from the starting solution. After centrifugation, the precipitates containing the aminothiols and AuNPs can be directly detected through surface-assisted laser desorption/ionization mass spectrometry.22 Based on this result, it is expected that the AuNPs (17) Jakubowski, H. J. Biol. Chem. 2000, 275, 3957–3962. (18) Togawa, T.; Mukai, Y.; Ohata, K.; Suzuki, T.; Tanabe, S. J. Chromatogr., B 2005, 819, 67–72. (19) Daneshvar, P.; Yazdanpanah, M.; Cuthbert, C.; Cole, D. E. Rapid Commun. Mass Spectrom. 2003, 17, 358–362. (20) (a) Zhang, F. X.; Han, L.; Israel, L. B.; Daras, J. G.; Maye, M. M.; Ly, N. K.; Zhong, C. J. Analyst 2002, 127, 462–465. (b) Chen, S. J.; Chang, H. T. Anal. Chem. 2004, 76, 3727–3734. (21) Lim, I. I.; Ip, W.; Crew, E.; Njoki, P. N.; Mott, D.; Zhong, C. J.; Pan, Y.; Zhou, S. Langmuir 2007, 23, 826–833.

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Figure 1. Visible absorption spectra of solutions of 0.48 nM FSNAuNPs upon the addition of (A) HCys and (B) HTL under acidic condition. Buffer: 200 mM phosphate solution at pH 4.0. The concentrations of aminothiols ranged from 0.5 to 25 µM. The aminothiols are incubated with the FSN-AuNPs for 20 min. (C) The effect of pH on the absorption ratio of a solution of FSN-AuNPs containing 10 µM HCys, Cys, and HTL, respectively. Buffer: 200 mM phosphate solution at pH ranged from 4.0 to 10.0.

can also serve as an effective sorbent nanomaterials for the removal of aminothiols from the biological fluids. In this study, we have developed a facile and selective method for the detection of HTL without involving chromatographic separations and derivatization steps. Under acidic conditions, the hydrolysis of HTL is so slow that it hardly binds to the surface of the fluorosurfactant-capped AuNPs (FSN-AuNPs). In contrast, under identical conditions, HCys and Cys can bind to the surface of the FSN-AuNPs.23 Subsequently, by centrifugation, HCys and Cys can be purged from the starting solution, whereas HTL still remains in the supernatant. Upon the addition of NaOH to the supernatant, HTL is hydrolyzed to HCys.13 The hydrolyzed HTL can induce the aggregation of the FSN-AuNPs. The entire procedure followed for the analysis of HTL is displayed in Scheme 1. To demonstrate its practicality, the present approach was applied to the determination of HTL in urine samples. EXPERIMENTAL SECTION Chemicals. HCys, Cys, GSH, Glu-Cys, Cys-Gly, and HTL were obtained from Sigma (St. Louis, MO). 3-Mercaptopropionate (MPA), hydrogen tetrachloroaurate(III) dehydrate, trisodium (22) Huang, Y. F.; Chang, H. T. Anal. Chem. 2006, 78, 1485–1493.

Figure 3. TEM images of a solution of FSN-AuNPs in the (A) absence and (B) presence of NaOH-treated supernatant. The supernatant, which was obtained by centrifugation of a solution of 25 µM HTL and 24.0 nM FSN-AuNPs, was reacted with 6.0 M NaOH (50 µL) for 5 min before addition to a solution of 0.48 nM FSN-AuNPs. Buffer: 200 mM phosphate solution at pH 4.0.

Figure 2. (A, B) Visible absorption spectra of 0.48 nM FSN-AuNPs upon addition of (a) aminothiol and (b) supernatant. The aminothiols include (A) HCys and (B) Cys. The supernatant was obtained by centrifugation of a solution of 25 µM aminothiol and 24.0 nM FSNAuNPs. After that, 0.48 nM FSN-AuNPs was utilized to detect the supernatant and the final concentration of aminothiol was 10 µM. (C) Visible absorption spectra of 0.48 nM FSN-AuNPs upon the addition of (a) HTL, (b) supernatant, and (c) NaOH-treated supernatant. The supernatant obtained from a solution of 25 µM HTL and 24.0 nM FSNAuNPs was reacted with 6.0 M NaOH (50 µL) for 5 min before the addition to a solution of 0.48 nM FSN-AuNPs. Buffer: 200 mM phosphate solution at pH 4.0. The aminothiols are incubated with the FSN-AuNPs for 20 min.

citrate, sodium hydroxide, Zonyl FSN-100, H3PO4, NaH2PO4, Na2HPO4, and Na3PO4 were purchased from Aldrich (Milwaukee, WI). The molecular formula of Zonyl FSN is F(CF2CF2)3-8CH2CH2O(CH2CH2O)xH. Milli-Q ultrapure water was used in all of the experiments. Apparatus. A double-beam UV-vis spectrophotometer (Cintra 10e; GBC, Victoria, Australia) was used to examine the absorbance of the FSN-AuNPs. A H7100 transmission electron microscopy (TEM) (Hitachi High-Technologies Corp., Tokyo, Japan) operating at 75 keV was used to collect TEM images of as-prepared AuNPs. Nanoparticle Synthesis. The citrate-capped AuNPs have been prepared by the chemical reduction of metal salt precursor (hydrogen tetrachloroaurate, HAuCl4) in a liquid phase.24 Briefly, 10% HAuCl4 (54 µL) was added rapidly to a solution of 0.075% sodium citrate (60 mL) that was heated under reflux. Heating (23) (a) Lu, C.; Zu, Y.; Yam, W. W. Anal. Chem. 2007, 79, 666–672. (b) Lu, C.; Zu, Y. Chem. Commun. 2007, 37, 3871–3873. (c) Wu, H.-P.; Huang, C.-C.; Cheng, T.-T.; Tseng, W.-L. Talanta 2008, 76, 347–352. (24) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391–3395.

under reflux was continued for an additional 15 min, during which time the color changed to deep red. The TEM images (not shown) confirmed that the size of the AuNPs is 13.1 nm with standard deviation of 0.3 nm. The maximum absorption wavelength of the AuNPs, which was examined by UV-vis spectrophotometer, was 520 nm. The particle concentration of the AuNP solution was 4.8 nM, which was determined by Beer’s law; the extinction coefficient of 13.1-nm AuNPs at 520 nm is ∼108 M-1 cm-1.25 The FSN-AuNPs were obtained when 240 µL of 10% FSN was added to a solution of 4.8 nM citrate-capped AuNPs (60 mL). The resulting mixture was stored at 4 °C until further use. Note that the FSN-AuNPs remain dispersed in the solution containing high concentration of salts. Sample Preparation and Detection. A stock solution of thiol (1 mM) was prepared in deionized water. We added a solution of 4.8 nM FSN-AuNPs (100 µL) to 900 µL of thiols (50.0-0.1 µM), which were prepared in 200 mM phosphate at pH 4.0-10.0. The resulting solutions were equilibrated at ambient temperature for 20 min and then examined by UV-vis spectrophotometer. To remove the aminthiols from the aqueous solution, 500 µL of 48.0 nM FSN-AuNPs was mixed with 500 µL of thiols (50 µM), which were prepared in 200 mM phosphate at pH 4.0. The resulting solutions were centrifuged at 18 000 rpm for 20 min to obtain the supernatant. We added a solution of 4.8 nM FSN-AuNPs (100 µL) and 400 mM phosphate (500 µL) to the supernatant (400 µL) for determining whether thiols were present in an aqueous solution. The final concentration of aminothiol was 10 µM. On the other hand, 50 µL of 6.0 M NaOH was mixed with the supernatant in order to hydrolyze HTL, which has been prepared in 200 mM phosphate at pH 4.0. After the reaction of 5 min, the resulting solution (950 µL) was blended with 100 µL of 4.8 nM FSN-AuNPs. Visible absorption spectra of the solution were recorded after 20min incubation. In the quantitative analysis, 50 µL of 6.0 M NaOH was mixed with 200 mM phosphate solution (pH 4.0) containing different concentrations of HTL. The resulting solution was incubated for 5 min, followed by the addition of 100 µL of 4.8 nM FSN-AuNPs; the final volume of the mixture was 1.0 mL, and the final concentration of HTL ranged from 100 to 500 nM. After 10-min (25) Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 12674–12675.

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Figure 4. Absorption ratio (A610nm/A520nm) of 0.48 nM FSN-AuNPs obtained after the addition of (A) thiols, (B) thiols reacted with 6.0 M NaOH, and (C) the supernatant from centrifugation of a solution of thiols and FSN-AuNPs. The supernatant was reacted with 6.0 M NaOH for 5 min before colorimetric assay. Buffer: 200 mM phosphate solution at pH 4.0. Thiols were incubated with the FSN-AuNPs for 20 min. The final concentration of each thiols was 10 µM. (D) Photographs of the FSN-AuNPs with added the supernatant, which has reacted with 6.0 M NaOH. From left to right: no analyte, HCys, Cys, HTL, GSH, Glu-Cys, Cys-Gly, and MPA.

incubation, the as-prepared solution was examined by UV-vis spectrophotometer. Detection of HTL in Urine. Urine samples were collected from a healthy female; the standard addition was carried out by spiking a certain amount of the HTL standard solution to urine samples. Note that HTL was prepared in 200 mM phosphate at pH 4.0. Subsequently, the as-prepared solution (900 µL) was treated with 240 nM FSN-AuNPs (100 µL) for the removal of the aminothiols from the starting solution at 4 °C. After centrifugation at 18 000 rpm for 20 min at 4 °C, 50 µL of 6.0 M NaOH was added to the supernatant. The resulting solution (900 µL) was incubated for 5 min, followed by the addition of 100 µL of 4.8 nM FSNAuNPs; the final volume of the mixture was 1.0 mL, and the final concentration of HTL ranged from 255 to 850 nM. Visible absorption spectra of the solution were recorded after 10-min incubation. RESULT AND DISCUSSION FSN-AuNPs as Removers. The hydrolysis rate of HTL is highly dependent on the pH of the solution.13 Under basic conditions, the hydroxide ion-catalyzed hydrolysis of HTL involves the ring opening of the thiolactone moiety, resulting in the liberation of a free sulfhydryl group. In contrast, HTL is relatively stable under acidic conditions. Moreover, previous studies have indicated that there is no significant loss of HTL in urine samples because most of the samples are slightly acidic.16 On the other hand, the FSN-AuNPs can be efficiently assembled in the presence of HCys and Cys. As indicated in the previous studies, the FSN molecules are adsorbed via their hydrophilic heads on the gold surface, while the hydrophobic chains are dispersed in the 6348

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solution.26 On the basis of this observation, it is proposed that both Cys and HCys diffuse smoothly into the FSN layers, which are attached to the nanoparticle surface. In comparison, the relatively large aminothiols including GSH, Cys-Gly, and Glu-Cys, cannot penetrate the FSN layer since they have strong hydrophobic interactions with the FSN molecules. Thus, only HCys and Cys can displace the FSN molecules from the Au surface, resulting in the aggregation of the AuNPs through hydrogen bonding, electrostatic interaction, or both. On the basis of this knowledge, it is expected that the FSN-AuNPs can not only be utilized as effective sorbent nanomaterials for the removal of aminothiols from an aqueous system but can also be aggregated upon the addition of hydrolyzed HTL. To prove this hypothesis, we first investigated the effect of solution pH and aminothiol concentration on the degree of aggregation of the FSN-AuNPs. At pH 4.0, it was not surprising that the HCys-induced aggregation of the FSNAuNPs resulted in significant broadening and a shift in the surface plasmon resonance (SPR) band, as shown in Figure 1A. However, under identical conditions, when different concentrations of HTL were added to a solution of the FSN-AuNPs, the observed absorption spectra did not show any shift in their SPR bands. This result clearly indicates that there is no interaction between the AuNPs and HTL (Figure 1B). Compared to the addition of HTL, the addition of HCys and Cys to a solution of FSN-AuNPs in the pH range of 4.0-10.0 resulted in a high ratio of absorption, from 610 to 520 nm (Figure 1C). The results provide clear evidence for the fact that HTL is not hydrolyzed in the pH range of 4.0-10.0. Moreover, the removal of HCys and Cys using the FSN-AuNPs (26) Li, F.; Zu, Y. Anal. Chem. 2004, 76, 1768–1722.

Figure 5. (A) Absorption spectral changes of 0.48 nM FSN-AuNPs upon addition of NaOH-treated HTL (0, 100, 200, 300, 400, and 500 nM). (B) Plot of A610 nm/A520 nm ratios of the FSN-AuNPs as a function of HTL concentration. HTL was reacted with 6.0 M NaOH (50 µL) for 5 min before colorimetric assay. Buffer: 200 mM phosphate solution at pH 4.0. HTL was incubated with FSN-AuNPs for 10 min.

could be performed in the pH range of 4.0-10.0 because they show strong interactions with the FSN-AuNPs. Next, the absorption spectra of the AuNPs were used to assess the efficiency of the removal of HCys and Cys under acidic conditions because the SPR band of the FSN-AuNPs was susceptible to the aminothiol concentration. We utilized 24.0 nM FSNAuNPs to efficiently remove the aminothiols from the aqueous solution and 0.48 nM FSN-AuNPs to detect the supernatant, which was isolated from the reacted nanoparticles. It is to be noted that the aggregation of 0.48 nM FSN-AuNPs did not occur when the concentrations of HCys and Cys were lower than 0.2 and 0.1 µM, respectively. A comparison with the absorption spectrum of a solution containing 0.48 nM FSN-AuNPs and 10 µM HCys (spectrum a in Figure 2A) showed that the SPR band of the AuNPs remained unchanged after the addition of the supernatant isolated from the reacted nanoparticles (spectrum b in Figure 2A). A similar phenomenon was discovered in the case of the removal of Cys (Figure 2B). The above results imply that 24.0 nM FSNAuNPs can remove >98% of HCys and >99% of Cys from the starting solution. However, the unchanged SPR band observed when 10 µM HTL (spectrum a in Figure 2C) and the supernatant (spectrum b in Figure 2C) were separately added to a solution of 0.48 nM FSN-AuNPs indicated that the FSN-AuNPs were welldispersed. It is to be noted that the supernatant was isolated from a solution of HTL and the FSN-AuNPs. This result further supported the assumption that the HTL molecules were not attached to the surface of the AuNPs. As soon as 6.0 M NaOH (50 µL) was added to the supernatant, the hydrolyzed HTL induced a rapid aggregation of the FSN-AuNPs (spectrum c in

Figure 6. Detection of HTL in urine samples. (A) Urine samples were spiked by standard solutions containing 0, 255, 425, 595, 765, and 850 nM HTL. Urine samples were treated with FSN-AuNPs to remove the aminothiols. HTL was dissolved in 200 mM phosphate solution at pH 4.0. The supernatant, which was isolated from centrifugation of a solution of FSN-AuNPs and urine, was reacted with 6.0 M NaOH in order to hydrolyze HTL. (B) Calibration curve for the detection of hydrolyzed HTL in urine samples. HTL was incubated with FSNAuNPs for 10 min.

Figure 2C). In addition, evidence for this HTL-induced aggregation can be seen in the TEM image (Figure 3). Hence, we believe that a solution containing HTL and aminothiols could be effectively separated by using the FSN-AuNPs as removers. Selectivity Improvement. Encouraged by these results, we attempted to extend the same method to the preparation of other thiols, including GSH, Glu-Cys, Cys-Gly, and MPA. As indicated in Figure 4A, the addition of HCys and Cys to a solution of 0.48 nM FSN-AuNPs resulted in significant increases in the absorption ratios (A610 nm/A520 nm), while the remaining thiols caused no such effect. The same results were observed upon the addition of 6.0 M NaOH to the sample solutions, except for the case of HTL, which was hydrolyzed to HCy (Figure 4B). It is obvious that low concentrations of the hydroxide ion did not affect the aggregation of the FSN-AuNPs. After the centrifugation of a solution containing 24.0 nM FSN-AuNPs and thiols, the supernatant was treated with 6.0 M NaOH for 5 min and subjected to colorimetric analysis (Figure 4C). The high absorption ratio (A610 nm/A520 nm) of the FSNAuNPs was observed only in the case of the hydrolyzed HTL, indicating the efficient removal of these thiols after the treatment of the FSN-AuNPs. Sensitivity and Application. We examined the effect of the FSN-AuNP concentration on the absorption ratio (A610 nm/A520 nm) upon the addition of hydrolyzed HTL (0.5 µM). The optimum concentration of the FSN-AuNPs was observed to be 0.48 nM Analytical Chemistry, Vol. 80, No. 16, August 15, 2008

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(Figure S1, Supporting Information). In addition, the aggregation kinetics of a solution of 0.48 nM FSN-AuNPs with different concentrations of hydrolyzed HTL was monitored by visible absorption spectra. After the addition of different concentrations of hydrolyzed HTL to a solution of FSN-AuNPs, the assembly was close to completion after 20 min; the optimum aggregation time for quantification was 10 min. As indicated in Figure 5A, the intensity of the absorbance signal at 610 nm increased with the concentration of the hydrolyzed HTL. Figure 5B shows a linear correlation between the absorption ratios (A610 nm/A520 nm) and the concentration of HTL over the range of 0-500 nM (R2 ) 0.9915). By applying this proposed method, the lowest detectable concentration of HTL was found to be 100 nM. We employed our proposed method, which has high sensitivity and selectivity, for the practical analyses of HTL in urine samples. The HTL concentrations in the urine samples of healthy individuals range from 11.0 to 473.7 nM.17 Although other aminothiols such as HCys and Cys also appear in the urine samples,27 their interference is not significant after the samples are pretreated with FSN-AuNPs. Figure 6A shows that an apparent increase in the absorbance signal at 610 nm was obtained after urine samples were spiked by standard solutions containing different concentrations of hydrolyzed HTL. A calibration curve derived from a solution containing HTL standards is shown in Figure 6B; this curve exhibits linearity in the range of 255-850 nM, which includes the maximum permissible limit of HTL in a healthy (27) Lochman, P.; Adam, T.; Friedecky´, D.; Hlı´dkova´, E.; Skopkova´, Z. Electrophoresis 2003, 24, 1200–1207. (28) Li, Z. P.; Duan, X. R.; Liu, C. H.; Du, B. A. Anal. Biochem. 2006, 351, 18–25.

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individual.10a,16 These results suggest that the proposed method will be suitable for routine urine assays in clinical studies. CONCLUSIONS We have demonstrated a highly selective method for detecting HTL based on the use of FSN-AuNPs as aminothiol removers and HTL sensors. A high absorption ratio of the FSN-AuNPs was obtained upon the addition of hydrolyzed HTL as compared to that obtained upon the addition of NaOH-untreated HTL. Moreover, the selectivity of the FSN-AuNPs toward HTL was apparently high after the removal of thiols. The present approach offers various advantages such as simplicity, cost efficiency, and homogeneous assays (separation free) over the conventional methods. Although the sensitivity of this approach for the detection of HTL is less than that of high-performance liquid chromatography coupled with fluorescence detection,14–16,18 we believe that it can be further improved by the extraction of HTL by solid-phase extraction5 and detection by resonance light-scattering techniques.28 ACKNOWLEDGMENT We thank National Science Council (NSC 96-2113-M-110-008-) and National Sun Yat-sen University-Kaohsiung Medical University Joint Research Center for financial support of this study. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 8, 2008. Accepted June 5, 2008. AC8006973