Specific Postcolumn Detection Method for HPLC Assay of

The new technique has been employed to determine total Hcy levels in ... Langmuir 2011 27 (1), 481-486 ... Analytical Chemistry 0 (proofing), .... Ana...
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Anal. Chem. 2007, 79, 666-672

Specific Postcolumn Detection Method for HPLC Assay of Homocysteine Based on Aggregation of Fluorosurfactant-Capped Gold Nanoparticles Chao Lu, Yanbing Zu,* and Vivian Wing-Wah Yam

Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China

Gold nanoparticles (GNPs) capped with nonionic fluorosurfactant molecules (Zonyl FSN) were synthesized, and with the colloidal solution as a probe reagent, a new postcolumn colorimetric detection method for HPLC assay of homocysteine (Hcy) has been developed. The FSN-capped GNPs exhibited excellent stability in aqueous solutions, even in the presence of high salt. The aggregation of the GNPs could be induced by either Hcy or cysteine, resulting in an absorption decrease of the colloidal solution at 525 nm and an absorption increase at longer wavelengths (600-700 nm); however, the GNPs did not respond to other amino acids and biomolecules such as glutathione, cysteinylglycine, and glucose. Under optimal conditions (i.e., high salt, neutral pH, and ∼70 °C), the color change of the GNP solution could almost complete (∼90%) within ∼30 s upon the addition of Hcy. The high selectivity and very fast kinetics of the reaction make it a promising system for HPLC postcolumn detection. The new technique has been employed to determine total Hcy levels in human urine and plasma samples, and the results are satisfactory. Homocysteine (Hcy), a naturally occurring thiol-containing amino acid, is a product of demethylation of methionine. The total Hcy (tHcy) in human plasma is normally lower than 15 µM, which includes protein-bound and free (most in disulfide form) Hcy.1 The elevation of tHcy level is a sensitive marker of folate and cobalamin (vitamin B12) deficiency and an independent risk factor for cardiovascular disease and is also related to birth defects, pregnancy complications, psychiatric disorders, and cognitive impairment in the elderly.1,2 Hcy is also excreted in urine. * To whom correspondence should be addressed. E-mail: [email protected]. (1) (a) Carmel, R., Jacobsen, D. W., Eds. Homocysteine in Health and Disease; Cambridge University Press: Cambridge, U.K., 2001. (b) Refsum, H.; Smith, A. D.; Ueland, P. M.; Nexo, E.; Clarke, R.; McPartlin, J.; Johnston, C.; Engbaek, F.; Schneede, J.; McPartlin, C.; Scott, J. M. Clin. Chem. 2004, 50, 3-32. (c) Rasmussen, K.; Moller, J. Ann. Clin. Biochem. 2000, 37, 627648. (2) (a) Ueland, P. M.; Vollset, S. E. Clin. Chem. 2004, 50, 1293-1295. (b) Cavalca, V.; Cighetti, G.; Bamonti, F.; Loaldi, A.; Bortone, L.; Novembrino, C.; De Franceschi, M.; Belardinelli, R.; Guazzi, M. D. Clin. Chem. 2001, 47, 887-892. (c) Nygard, O.; Vollset, S. E.; Refsum, H.; Stensvold, I.; Tverdal, A.; Nordrehaug, J. E.; Ueland, M.; Kvave, G. J. Am. Med. Assoc. 1995, 274, 1526-1533. (d) Refsum, H.; Ueland, P. M; Nygard, O.; Vollset S. E. Annu. Rev. Med. 1998, 49, 31-62. (e) Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D’Agostino, R. B.; Wilson, P.W. F.; Wolf, P. A. N. Engl. J. Med. 2002, 346, 476-483.

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Currently available methods for determining Hcy are generally based on chromatography and immunoassays. Chromatographic assays include high-performance liquid chromatography (HPLC) with colorimetric,3 fluorometric (FL),4 or electrochemical (EC) detection;5 capillary electrophoresis (CE) with FL6 or EC detection;7 gas chromatography-mass spectrometry;8 and liquid chromatography with tandem MS.9 During the latter half of 1990s, immunoassay-based commercial methods were developed. The fluorescence polarization immunoassay, run on Abbott’s IMx and AxSYM platforms, is now widely used.1b,10 However, these methods have their drawbacks. In immunoassays, relatively fragile and expensive biological reagents are utilized; while the chromatographic assays usually require time-consuming precolumn derivatization steps, and the selectivity of the commonly employed thiol-reactive reagents toward Hcy is low.11 It has been noted that the variations among methods and among laboratories are considerable, and there is a need for standardization of Hcy (3) (a) Andersson, A.; Isaksson, A.; Brattstrom, L.; Hultberg, B. Clin. Chem. 1993, 39, 1590-1597. (b) Kaniowska, E.; Chwatko, G.; Glowacki, R.; Kubalczyk, P.; Bald, E. J. Chromatogr., A 1998, 798, 27-35. (c) Chwatko, G.; Bald, E. J. Chromatogr., A 2002, 949, 141-151. (d) Kusmierek, K.; Glowacki, R.; Bald, E. Anal. Bioanal. Chem. 2006, 385, 855-860. (4) (a) Pastore, A.; Massoud, R.; Motti, C.; Russo, A. L.; Fucci, G.; Cortese, C.; Federici, G. Clin. Chem. 1998, 44, 825-832. (b) Mukai, Y.; Togawa, T.; Suzuki, T.; Ohata, K.; Tanabe, S. J. Chromatogr., B 2002, 767, 263-268. (c) Chou, S.-T.; Ko, L.-E.; Yang, C.-S. Anal. Chim. Acta 2001, 429, 331336. (5) (a) Melnyk, S.; Pogribna, M.; Pogribny, I.; Hine, R. J.; James, S. J. J. Nutr. Biochem. 1999, 10, 490-497. (b) Zhang, S.; Sun, W.-L.; Xian, Y.-Z.; Zhang, W.; Jin, L.-T.; Yamamoto, K.; Tao, S.-G.; Jin, J.-Y. Anal. Chim. Acta 1999, 399, 213-221. (6) (a) Causse, E.; Malatray, P.; Calaf, R.; Charpoit, P.; Candito, M.; Bayle, C.; Valdiquie, P.; Salvayre, R.; Couderc, F. Electrophoresis 2000, 21, 20742079. (b) Causse, E.; Siri, N.; Bellet, H.; Champagne, S.; Bayle, C.; Valdiguie, P.; Salvayre, R.; Couderc, F. Clin. Chem. 1999, 45, 412-414. (c) Causse, E.; Issac, C.; Malatray, P.; Bayle, C.; Valdguie, P.; Salvayre, R.; Couderc, F. J. Chromatogr., A 2000, 895, 173-178. (7) (a) Pasas, S. A.; Lacher N. A.; Davies, M. I.; Lunte, S. M. Electrophoresis 2002, 23, 759-766. (b) Inkayo, I.; Kirchhoff, J. R. Anal. Chem. 2002, 74, 1349-1354. (c) Zhong, M.; Lunte, S. M. Anal. Chem. 1999, 71, 251-255. (d) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307-311. (8) MacCoss, M. J.; Fukagawa, N. K.; Matthews, D. E. Anal. Chem. 1999, 71, 4527-4533. (9) Powers, H. J.; Moat, S. J. Curr. Opin. Clin. Nutr. Metab. Care 2000, 3, 391-397. (10) (a) Shipchandler, M. T.; Moore, E. G. Clin. Chem. 1995, 41, 991-994. (b) Frantzen, F.; Faaren, A. L.; Alfheim, I.; Nordhei, A. K. Clin. Chem. 1998, 44, 311-316. (11) Wang, W.-H.; Rusin, O.; Xu, X.-Y.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry, M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.; Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949-15958. 10.1021/ac061513c CCC: $37.00

© 2007 American Chemical Society Published on Web 11/23/2006

assays.1b Postcolumn techniques may minimize sample processing prior to assay and are generally influenced less by performance variation of the assay than precolumn derivatization techniques. However, only a few postcolumn reagents have been introduced for the detection of Hcy.3a,11 The development of specific postcolumn systems for the Hcy assay is attractive. Recently, the applications of gold nanoparticles (GNPs) in bioanalysis have drawn great interest because of their strongly distance-dependent optical properties and large surface areas. GNPs have extinction coefficients ∼3 orders of magnitude larger than those of organic dyes. The analyte-induced aggregation of GNPs shifts the surface plasmon resonance (SPR) absorption peak toward longer wavelength, which has been used for colorimetric sensing of duplex DNA formation,12 protein-ligand interactions,13 and metal ion ligand complexation.14 Some studies indicated that the aggregation of GNPs could be induced by amino acids possessing additional (besides the R-amine) functional groups such as amine, imidazole, thioether, or thiol.15 Under certain conditions, selective responses of GNPs toward aminothiols have been observed, based on which colorimetric detection of aminothiols was suggested.16 For example, Thomas and his coauthors utilized gold nanorods stabilized with cetyltrimethylammonium bromide (CTAB) to detect cysteine (Cys), cystine, and glutathione (Glu);16b Chen and Chang described the determination of thiols using Nile red-adsorbed GNPs.16c Although these methods can well sense aminothiols, it is difficult to determine a specific species. For the sake of selective detection, separation techniques such as HPLC and CE may be necessary prior to the sensing step. However, the above detection processes generally took several to tens of minutes, making it difficult to be conjugated with the separation methods. In this study, 12-nm GNPs capped with nonionic fluorosurfactant molecules, Zonyl FSN, were synthesized. The FSN-capped GNPs exhibited high stability in aqueous solutions, even in the presence of high salt. The aggregation of the GNPs could be induced by Hcy or Cys, but not by other amino acids and biomolecules such as Glu, cysteinylglycine, glucose, and bovine serum albumin (BSA). The high specificity was attributable to the unique feature of the FSN ligands. Under optimal conditions (i.e., high salt, neutral pH, and ∼70 °C), the colorimetric evolution (12) (a) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (b) Storhoff, J. J.; Lazarides, A. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L.; Schatz, G. C. J. Am. Chem. Soc. 2000, 122, 4640-4650. (c) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 8102-8103. (13) (a) Nam, J.-M.; Park, S.-J.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 38203821. (b) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (c) Mann, S.; Shenton, W.; Li, M.; Connolly, S.; Fitzmaurice, D. Adv. Mater. 2000, 12, 147-150. (14) (a) Liu, J.-W.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642-6643. (b) Obare, S. O.; Hollowell, R. E.; Murphy, C. J. Langmuir 2002, 18, 10407-10410. (c) Lin, S.-Y.; Liu, S.-W.; Lin, C.-M.; Chen, C.-H. Anal. Chem. 2002, 74, 330-335. (d) Lin, S.-Y.; Chen, C.-H.; Lin, M.-C.; Hsu, H.-F. Anal. Chem. 2005, 77, 4821-4828. (e) Kim, Y.; Johnson, R. C.; Hupp, J. T. Nano Lett. 2001, 1, 165-167. (15) (a) Zhong, Z.-Y.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gedanken, A. J. Phys. Chem. 2004, 108, 4046-4052. (b) Selvakannan, P. R.; Mandal, S.; Phadtare, S.; Pasricha, R.; Sastry, M. Langmuir 2003, 19, 3545-3549. (c) Naka, K.; Itoh, H.; Tampo, Y.; Chujo, Y. Langmuir 2003, 19, 55465549. (16) (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) Sudeep, P. K.; Joseph, S. T. S.; Thomas, K. G. J. Am. Chem. Soc. 2005, 127, 6516-6517. (c) Chen, S.J.; Chang, H.-T. Anal. Chem. 2004, 76, 3727-3734.

of the colloidal solution could be 90% complete within 30 s upon the addition of Hcy. The experimental results suggested that the rapid aggregation of the FSN-capped GNPs should be driven by the London-van der Waals attractive force, which proceeded much faster than those processes via a cross-linking mechanism. Therefore, a new postcolumn detection method for HPLC assay of Hcy has been developed based on the analyte-induced aggregation of the FSN-capped GNPs. This system offers fast reaction kinetics and high selectivity in responding to Hcy and can be successfully employed in the analysis of human urine and plasma samples. To the best of our knowledge, this is the first time that the GNP colloidal solution has been used as the HPLC postcolumn reagent. EXPERIMENTAL SECTION Chemicals. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4‚ 3H2O), trisodium citrate, 20 standard amino acids, cystine, homocysteine, homocystine, glutathione, cysteinylglycine, glucose, BSA, tris(2-carboxyethyl)phosphine (TCEP), ethylenediaminetetraacetic acid (EDTA), trifluoroacetic acid (TFA), Triton X-100 (4(C8H17)C6H4(OCH2CH2)nOH), and Zonyl FSN-100 (F(CF2CF2)17CH2CH2O(CH2CH2O)0-15H) were purchased from Sigma-Aldrich. All solutions were prepared with deionized water (Milli Q, Millipore). The solutions used for HPLC system were filtered through 0.2-µm membranes. The pH of the phosphate buffer solution (PBS) was adjusted with NaOH or HCl. Nanoparticle Synthesis. All glassware used for preparation of GNPs was thoroughly washed with freshly prepared aqua regia (HNO3:HCl ) 1:3), rinsed extensively with deionized and ultrahigh purity water sequentially, and then dried in an oven at 100 °C for 2-3 h. Colloidal GNPs with average diameters of ∼12 nm were prepared following the literature procedure.15a Briefly, a 60-mL solution of 0.075% sodium citrate was brought to a vigorous boil with stirring in a round-bottom flask fitted with a reflux condenser, and then 54 µL of 10% HAuCl4 was added to the stirring and refluxing HAuCl4 solution. The solution was maintained at the boiling point with continuous stirring for ∼15 min. After the solution was allowed to cool to room temperature with continued stirring, 240 µL of 10% FSN-100 was added. The suspension was stored at 4 °C until further use. Assuming spherical particles and density equivalent to that of bulk gold (19.30 g/cm3), the concentration of the GNPs was calculated (∼4.9 nM). The TEM specimens were prepared by depositing an appropriate amount of the FSN-capped GNPs onto the carbon-coated copper grids, and excess solution was wicked away by a filter paper. The grid was subsequently dried in air. Apparatus. Transmission electron microscopy (TEM) images were taken using a Philips microscope (Tecnai 20) operated at an acceleration voltage of 200 kV. UV-visible data were recorded on a Hewlett-Packard 8453 diode array UV-visible spectrophotometer using quartz cuvettes with an optical path length of 1 cm at room temperature. The HPLC separation system includes a Waters 600E pump, an inline degasser, a Rheodyne 7725i manual injector equipped with a 20-µL loop, a SunFire C18 guard column (4.6 × 20 mm, 5-µm particle size, Waters), a SunFire C18 analytical column column (4.6 × 150 mm, 5-µm particle size, Waters), and a UV-visible detector set at λ ) 680 nm (Waters 2487 Dual λ). Isocratic elution of a mobile phase containing 0.05% TFA was performed with a Waters Multisolvent Delivery System (model Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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600E). A Postcolumn Reaction Module (Waters) includes a reaction oven containing a RXN 1000 reaction coil (0.45-mm i.d., 1-mL volume) and a countercurrent heat exchanger (CHEX). A Reagent Manager (Waters), a single-piston and pulse-dampened pumping system, was used to deliver the postcolumn reagent, i.e., the solution of FSN-capped GNPs, to the Postcolumn Reaction Module. The temperature of the postcolumn reaction was controlled by a temperature control system (Waters). When the fluid stream left the oven, it was cooled to ambient temperature by passing through the CHEX unit and then passed out the Postcolumn Reaction Module and into the detector cell. The chromatographic system was connected with a personal computer via busSAT/IN Module, and the chromatograms were recorded and analyzed with the Millennium32 software (Waters). Pretreatment of Human Urine and Plasma Samples. Human urine sample was collected from a healthy male volunteer, and the analysis was conducted immediately after the sample collection. Commercial lyophilized human plasma sample was obtained from Sigma-Aldrich and simply reconstituted with deionized water. To 100 µL of plasma or urine sample in a centrifuge tube, 50 µL of 0.1 M EDTA and 5 µL of 0.2 M TCEP in pH 6 PBS were added. The mixture was kept at 60 °C for 30 min. After cooling to room temperature, the sample was gently vortex-mixed with 20 µL of 3.0 M perchloric acid solution (PCA), put aside at room temperature for 10 min, and then centrifuged at 13 000 rpm for 10 min. The clear supernate was filtered through a 0.22-µm filter and diluted when required, before injection (20 µL) into the HPLC. Calibration Standards. Stock solutions of 10 mM Hcy were daily prepared with water (deaerated with purified nitrogen). Working standard solutions with various Hcy concentrations were obtained by appropriate dilution with water and processed without delay. To prepare Hcy calibration standards in human urine or plasma samples, 100 µL of plasma or urine sample was placed in a centrifuge tube, which contains 50 µL of 0.1 M EDTA, 20 µL of 3.0 M PCA, and Hcy standard solutions of various concentrations. The mixture was kept at room temperature for 10 min and then centrifuged at 13 000 rpm for 10 min. The clear supernate was filtered through a 0.22-µm filter prior to HPLC analysis. The peak height was plotted against the final concentration of the added Hcy (the very small amounts of free reduced Hcy, usually accounting for less than 1-2% tHcy in the urine and plasma samples,1 can be ignored). The linear portion of the calibration curve was used for determining tHcy levels in the urine and plasma samples. RESULTS AND DISCUSSION Characterization of the FSN-Capped GNPs. Figure 1 shows the SPR absorption spectra of the colloidal solutions of 12-nm GNPs before and after FSN capping. The introduction of FSN ligands resulted in ∼4-nm red-shift of the SPR peak. This may be attributed to the increase of average refractive index of the environment surrounding the GNPs. A similar effect of the environmental change on the SPR spectra of GNPs induced by the binding of self-assembled monolayers of long-chain thiols was reported previously.17 The colloidal solution is wine-red in color, and the TEM image in Figure 1 shows that the GNPs are generally (17) Sun Y.-G.; Xia, Y.-N. Anal. Chem. 2002, 74, 5297-5305.

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Figure 1. UV-visible spectra of the GNPs with or without FSN capping. Inset, TEM image of the FSN-capped GNPs.

Figure 2. UV-visible spectra of the GNPs with or without FSN capping in the presence of NaCl.

spherical. The colloidal solution exhibited excellent stability in aqueous solutions with high ionic strength. The spectral bands were almost unchanged upon the addition of ∼500 mM NaCl, as shown in Figure 2. In contrast, the GNPs stabilized electrostatically by adsorbed citric anions (without FSN modification) were very sensitive to the increase of salt concentration; the addition of 50 mM NaCl led to immediate aggregation of the GNPs, and the colloidal solution color changed to blue rapidly. The results indicate that FSN ligands played a predominant role in preventing the GNPs from agglomerating in solutions with high ionic strength. Since salts are common components of biological samples, the high-salt tolerance of the FSN-capped GNPs is vital for their applications in bioanalysis. In addition, the FSN-capped GNPs are stable in a wide range of pH (from 1 to 13). Interaction of the FSN-Capped GNPs with Amino Acids and Other Biomolecules. We examined the spectral changes of the FSN-capped GNP colloids upon the addition of a variety of biomolecules including 20 standard amino acids, cystine, Hcy, homocystine, Glu, cysteinylglycine, glucose, and BSA. Figure 3 shows that, upon the addition of 4 µM Hcy, the absorption band at 525 nm decreased while new bands appeared at longer wavelengths, suggesting that the GNPs were ready to bind Hcy and aggregated subsequently. TEM images clearly show the aggregate formation. Similar results were also obtained upon the addition of 4 µM Cys. However, little spectral change occurred

Figure 3. UV-visible spectra for the FSN-capped GNP colloids in the absence and presence of Hcy. The solutions contain 100 mM PBS (pH ∼ 6). Inset, TEM image of the GNP aggregate induced by Hcy.

in the presence of other biomolecules, even at much higher concentrations (see Supporting Information). The highly selective responses of the FSN-capped GNPs toward Hcy and Cys are striking. Some previous studies indicated that amino acids may be attached to GNPs via their R-amine groups or additional functional groups such as amine, thioether, imidazole, or thiol.15 Therefore, the amino acid molecule possessing one of these additional functional groups could serve as a cross-linking agent for GNPs. However, the experimental results obtained in this study revealed the different behavior of Cys, Hcy, and other amino acids in interacting with the FSN-capped GNPs. It is known that the adsorption of FSN at the gold surface is quite strong and compact and could significantly inhibit many electrochemical reactions at a gold electrode.18 Similar adsorption behavior of FSN ligands on GNPs is expected, and the attachment of amino acids may be suppressed. The specific responses of the colloidal solution toward Hcy and Cys suggested that FSN ligands allowed for thiol-gold interaction while prohibiting the binding of other functional groups on the GNPs. The binding of amino acids may involve the replacement of the stabilizing ligands on the GNPs. Compared with other species, Hcy or Cys could be attached via strong Au-S bonds, which made the binding more favorable thermodynamically. It is a bit surprising that the FSN-capped GNPs did not respond to cystine, homocystine, cysteinylglycine, and Glu, although all of these species might be attached by thiol-gold linkages. This may probably be attributable to the steric effect, because these molecules are obviously larger than Cys and Hcy. Previous studies also revealed the slow kinetics of Glu-induced aggregation of GNPs.16a,b The effect was much more pronounced for the FSNcapped GNPs. It is noted that other surfactants, such as CTAB and nonionic hydrogenated surfactant, Triton X-100, could also be used to stabilize GNPs. The CTAB-capped gold nanorods have been used to detect Cys, but Glu and cystine could induce a similar response.16b We also examined the interaction between amino acids and the GNPs capped with Triton X-100, and no selectivity toward biothiols was observed. Clearly, the high selectivity of the FSN-capped GNPs in responding to Cys and Hcy is attributable (18) (a) Cha, C.-S.; Zu, Y. Langmuir 1998, 14, 6280-6286. (b) Li, F.; Zu, Y. Anal. Chem. 2004, 76, 1768-1772.

Figure 4. Salt effect on the Hcy-induced absorbance changes of the FSN-capped GNP colloids (pH ∼6). The absorbance values were measured at 5 min after the addition of 4 µM Hcy.

Figure 5. Time courses of extinction (at 680 nm) of the FSN-capped GNP colloids upon the addition of 4 µM Hcy at different temperatures (under gentle stirring). The solutions contain 100 mM PBS (pH ∼6).

to the unique feature of the FSN ligands. More detailed study on the interaction between the amino acids and the FSN-capped GNPs is currently underway. Hcy-Induced GNP Aggregation. The effects of solution ionic strength, pH, and temperature on Hcy-induced aggregation of the FSN-capped GNPs were examined. Figure 4 shows that the colorimetric evolution of the colloidal solution upon the addition of Hcy was affected significantly by PBS concentration (a similar effect was also found when other salts, such as NaCl and KCl, were used to alter the ionic strength). Under low-salt conditions, the aggregation of the GNPs might be hindered by the interparticle electrostatic repulsion force; as the solution ionic strength increased, however, the electrostatic interaction could be effectively screened, resulting in a much more rapid solution color change. The high-salt tolerance of the FSN-capped GNPs made it possible to utilize high ionic strength to achieve faster aggregation kinetics. It was found that the solution pH change in the range of 5-8 did not influence the rate of the GNP aggregation obviously, while lower or higher pH would slow down the kinetics. Our experiments were commonly carried out at pH 6. Figure 5 shows the influence of temperature on the Hcy-induced color change of the GNP colloids in 0.1 M PBS. At 25 °C, the time for attaining 90% colorimetric evolution was ∼60 s, which could be reduced to ∼30 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

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s as the solution temperature increased to 70 °C. Clearly, the GNPs aggregated much more rapidly at high temperatures. However, it was noted that, when the temperature was higher than ∼80 °C, the colloidal solution itself became unstable in the presence of high salt and a slow color change occurred. Therefore, the maximum temperature used in our experiments was 70 °C. Although the analyte-induced aggregation of GNPs has been intensively studied, the very rapid color change of the FSN-capped GNPs is unique. It has been proposed that the amino acid molecules bound to the GNPs may act as cross-linking agents to establish connection between the GNPs via either hydrogen bonding or electrostatic interaction.15a,b,16b However, the crosslinking mechanism cannot account for the remarkable salt effect as well as the very fast kinetics observed in our experiments. On the contrary, the rapid aggregation of the GNPs in high-salt solution suggests a non-cross-linking mechanism. Since FSN ligands should play the predominant role in stabilizing the GNPs under this condition, we believe that the replacement of the adsorbed FSN on the GNPs by Hcy molecules made the colloidal solution unstable, and the aggregation could be driven by the London-van der Waals attraction force, which was facilitated under high-salt conditions. This type of GNP aggregation has also been observed in a previous study,12c where the nanoparticles were stabilized by single-strand DNA molecules and rapid aggregation could be induced by DNA hybridization in high-salt solutions. Since the London-van der Waals attraction force takes effects from a distance while the cross-linking process depends on the random collisions between the GNPs, the non-cross-linking aggregation could proceed much faster. To further verify the non-cross-linking mechanism, the responses of the colloidal solution to some small thiol molecules, such as 3-mercapto-1-propanesulfonic acid, 2-mercaptoethanol, and 2-mercaptoethylamine, have also been examined. All these molecules (>10 µM) could induce rapid color change of the colloidal solution in the presence of 0.1 M NaCl. This suggests that hydrogen bonding or electrostatic interaction is not necessary for the GNP aggregation. Note that these small thiols are absent in most biosamples; therefore, they may not interfere with the detection of Hcy. Postcolumn HPLC Detection of Hcy. On the basis of the above results, the selective reaction between Hcy and the FSNcapped GNPs has been used for the HPLC postcolumn detection of Hcy. HPLC conditions, such as mobile-phase components, pH and flow rates of mobile-phase and postcolumn reagent, and postcolumn reactor temperature, were optimized. It has been found that the detection sensitivity became lower if the postcolumn reagent (4.9 nM FSN-capped GNPs) was diluted. Figure 6 shows a typical chromatogram of a mixture of Cys and Hcy (13 µM each). Two well-separated sharp chromatographic peaks appeared corresponding to Cys and Hcy with the retention times of 4.97 and 6.31 min, respectively. However, the injection of other amino acids and biomolecules, such as cysteinylglycine, Glu, glucose, etc., did not lead to the appearance of any peaks, demonstrating the high specificity of the detection method. The chromatographic peak height as a function of Hcy concentration is shown in Figure 7. The extinction change started to occur when Hcy concentration was higher than ∼3 µM. A linear increase of the peak height was observed in the concentration 670 Analytical Chemistry, Vol. 79, No. 2, January 15, 2007

Figure 6. Typical HPLC chromatogram of mixtures of standard Cys and Hcy (13 µM each). Chromatographic conditions: column, SunFire C18 (4.6 × 150 mm, 5-µm particle size, Waters); sample injection volume, 20 µL; mobile phase, 0.05% TFA at 0.5 mL/min; postcolumn reagent, colloidal solution of the FSN-capped GNPs (∼4.9 nM, containing 100 mM PBS, pH ∼6) at 0.5 mL/min; temperature of the reaction oven, 70 °C.

Figure 7. Chromatographic peak height (absorbance at 680 nm) versus Hcy concentration in the standard solution. Chromatographic conditions were the same as in Figure 6.

range of 5-43 µM. The linear relationship could be described by a regression equation: y ) 0.0024x - 0.0082 (R2 ) 0.9995). Further absorbance change was not obvious upon the addition of a larger amount of Hcy. The relative standard deviation of five replicates for each concentration was ∼1.5%. The lower limit of detection (LLD) for Hcy was ∼80 pmol with a signal-to-noise ratio of 3, per 20 µL of injection volume. Similar responses of GNPs to a variety of analytes have also been reported previously.14a,16b The dynamic region of the analyteinduced GNP aggregation is generally quite narrow. In addition, the GNPs respond only when the analyte concentration is higher than a certain threshold value. In the current case, the threshold Hcy concentration for the postcolumn detection system is ∼3 µM. As discussed above, the FSN-capped GNPs were made unstable when the FSN ligands were replaced by Hcy molecules. The GNPs started to aggregate as the amount of binding Hcy was large enough. Because of this feature, the proposed detection method offered a higher LLD as compared with the strategies based on FL or other colorimetric measurements.3,4 However, the analytical performance may be improved as long as the responding thresh-

Table 1. Results for Determination of tHcy in the Human Urine and Plasma Samples samplea (µM)

added (µM)

measureda (µM)

recovery (%)

urine tHcy

25.54 ( 0.63

plasma tHcy

3.68 ( 0.25

10.0 20.0 5.0 10.0

35.40 ( 0.27 46.06 ( 0.33 8.61 ( 0.24 13.65 ( 0.38

98.6 102.5 98.6 99.7

a

Figure 8. (A) HPLC chromatogram of a human urine sample. The pretreated urine sample solution was diluted three times with water before injection. (B) HPLC chromatogram of a human urine sample spiked with 8.0 µM Hcy. (C) HPLC chromatogram of a commercial human plasma sample spiked with 5.0 µM Hcy. Chromatographic conditions were the same as in Figure 6.

old value is determined in a certain sample, and thus, the nonresponding region of the calibration curve could probably be eliminated by spiking standard sample with a concentration of the threshold value. Assays of tHcy in Human Urine and Plasma Samples. The optimized postcolumn conditions were applied to the analysis of human urine and plasma samples. Figure 8A illustrates a typical chromatogram of a human urine sample. Figure 8B shows the identification of Hcy by adding a known amount of authentic Hcy standard into the same urine sample prior to the sample pretreatment. Hcy and Cys in the urine sample were well-separated, and the retention times were close to those of the standard Hcy and Cys. Note that the total concentration of Cys in human fluids is

Mean ( SD of three measurements.

usually ∼10-20 times larger than that of Hcy; therefore, the chromatographic peak of Cys was much higher. The background noises were relatively low because the high selectivity of the detection method greatly diminished the interferences from other species. Using the standard addition method described in the Experimental Section, the calibration curve in the urine sample was obtained and used to determine the urinary tHcy. After sample reduction and appropriate dilution (ensuring that the signal fell within the linear range), the tHcy concentration in the urine sample was measured and the result is shown in Table 1. A similar strategy was employed for the tHcy assay in a more complex matrix, commercial human plasma. The calibration curve was obtained by using the standard addition method. However, no chromatographic peak corresponding to Hcy was observed when a reduced plasma sample was analyzed. Because the tHcy level in the sample was relatively low, its concentration might fall within the nonresponding region of the postcolumn detection system. Therefore, a plasma sample spiked with an authentic Hcy standard (5.0 µM) was used for the analysis. The HPLC chromatogram for the spiked plasma sample is shown in Figure 8C. The peak height obtained was located in the linear responding region of the calibration curve, and the original tHcy in the commercial plasma sample has been determined to be ∼3.68 µM (see Table 1). This value is in good agreement with that reported previously.19 The validity of the postcolumn detection method was demonstrated by using urine and plasma samples spiked with authentic Hcy standards. The recoveries obtained are satisfactory (see Table 1). CONCLUSIONS Water-soluble FSN-capped GNPs (∼12 nm) have been synthesized and characterized. The GNPs exhibit excellent stability in aqueous solutions, even in the presence of high salt. Examination of their interaction with a variety of biomolecules revealed that rapid aggregation of the GNPs could be induced by Hcy and Cys, but not by other amino acids, glutathione, cysteinylglycine, glucose, and BSA. The kinetics of the Hcy-induced GNP aggregation could be significantly promoted by the increase of solution ionic strength. The experimental results suggested that, under high-salt conditions, the attachment of Hcy molecules resulted in the aggregation of the GNPs, which was driven by the Londonvan der Waals attraction force. The non-cross-linking aggregation (19) Wang, W.-H.; Escobedo, J. O.; Lawrence, C. M.; Strongin, R. M. J. Am. Chem. Soc. 2004, 126, 3400-3401.

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mechanism allowed for the very rapid color change of the GNP colloidal solution. The high selectivity and fast kinetics of the Hcyinduced aggregation of the FSN-capped GNPs led to the development of a novel postcolumn detection method for HPLC assay of Hcy. The results obtained from the analysis of a human urine sample and a commercial human plasma sample indicated that the new postcolumn detection method is promising for the determination of tHcy in the biological fluids. ACKNOWLEDGMENT This work has been supported by the University Development Fund on Luminescent Molecular Functional Materials of The

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University of Hong Kong. SUPPORTING INFORMATION AVAILABLE UV-visible spectra of the FSN-capped GNP colloids in the presence of Cys and a variety of biological molecules. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review August 15, 2006. Accepted October 20, 2006. AC061513C