Colorimetric Assay of Glutathione Based on the Spontaneous

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Colorimetric Assay of Glutathione Based on the Spontaneous Disassembly of Aggregated Gold Nanocomposites Conjugated with Water-Soluble Polymer Nobuo Uehara,* Kouki Ookubo, and Tokuo Shimizu Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi 321-8585, Japan Received April 3, 2009. Revised Manuscript Received March 16, 2010 This article describes the glutathione-triggered disassembly of gold nanocomposites composed of gold cores and water-soluble copolymers [poly(N-n-isopropylacrylamide-co-acryloyldiethyletriamine)] attached to the surfaces of gold cores. The gold nanocomposites exhibit a bluish purple color because of the assembled gold cores that are conjugated with the diethylenetriamine groups incorporated into the copolymers. Glutathione added to the gold nanocomposite solution adsorbs onto the surface of the gold cores to liberate diethylenetriamine groups, resulting in spontaneous disassembly that changes the color of the solution to a reddish shade. Increasing the glutathione concentration facilitates the spontaneous disassembly of the gold nanocomposites. For the determination of glutathione, the colorimetric change of the gold nanoparticles is quantified with the a* value of the L*a*b* color coordinates defined by the CIE  (Commission Internationale de l’Eclairage) chromaticity diagram. A linear relationship between the a* value and the glutathione concentration of up to 6  10-6 mol/L is obtained 15 min after the addition of glutathione that has a detection limit (defined as 3σ) of 2.9  10-8 mol/L. The colorimetric assay is successfully applied to the determination of glutathione in eye drops and health supplements.

1. Introduction Gold nanoparticles (AuNPs) in an aqueous solution exhibit a distinctive color according to their morphology because of their surface plasmon resonance. Whereas discrete AuNPs in a solution are red in color, assembled AuNPs are bluish purple in color.1 A promising approach to utilizing AuNPs for colorimetric sensing involves their conjugation with functional polymers to form gold nanocomposites. Many colorimetric assays using gold nanocomposites have been investigated after Mirkin and co-workers reported the first study of a DNA assay with gold nanocomposites.2 One potential strategy to developing a colorimetric sensor with gold nanocomposites is based on the formation of interparticle bridging structures composed of analytes and functional groups in the conjugated polymers. When analytes are added to a solution of gold nanocomposites, these analytes interact with the functional groups of the conjugated copolymers to form interparticle bridges between the nanocomposites, resulting in the assembly of the nanocomposites changing the solution color from red to blue-purple. The formation of supermolecule *To whom correspondence should be addressed. E-mail: ueharan@ cc.utsunomiya-u.ac.jp. Tel, Fax: þ81-28-689-6166.

(1) (a) Link, S.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 8410–8426. (b) Sandrock, M. L.; Foss, C. A., Jr. J. Phys. Chem. B 1999, 103, 11398–11406. (c) Kelly, K. L.; Coronado, E.; Zhao, L. L.; Schatz, G. C. J. Phys. Chem. B 2003, 107, 668–677. (d) Grant, C. D.; Schwartzberg, A. M.; Norman, T. J., Jr.; Zhang, J. Z. J. Am. Chem. Soc. 2003, 125, 549–553. (e) Zhong, Z.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gedanken, A. J. Phys. Chem. B 2004, 108, 4046–4052. (2) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609. (3) (a) Lin, S.-Y.; Liu, S.-W.; Lin, C.-M.; Chen, C-h. Anal. Chem. 2002, 74, 330– 335. (b) Lin, S-Y; Chen, C-h; Lin, M.-C.; Hsu, H.-F. Anal. Chem. 2005, 77, 4821–4828. (4) (a) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078–1081. (b) Jin, C. R.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 7961–7962. (c) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643–1654. (d) Li, H.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126, 10958–10961. (e) Lim, I. S.; Chandrachud, U.; Wang, L.; Gal, S.; Zhong, C.-J. Anal. Chem. 2008, 80, 6038–6044. (5) (a) Kim, Y.; Johnson, R. C.; Hupp, J. T. Nano Lett. 2001, 1, 165–167. (b) Sugunan, A.; Thanachayanont, C.; Dutta, J.; Hilborn, J. G. Sci. Technol. Adv. Mater. 2005, 6, 335–340. (c) Lin, S.-Y.; Wu, S.-H.; Chen, C-h. Angew. Chem., Int. Ed. 2006, 45, 4948–4951. (d) Huang, C.; Chang, H.-T. Chem. Commun. 2007, 1215–1217.

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structures,3 double strands,4 coordination bonds,5 and specific biochemical interactions6 has been investigated in order to understand the formation of the interparticle bridging structures. Recently, certain types of gold nanocomposite colorimetric sensors other than those involving the formation of interparticle bridging structures have been reported. Maeda and co-workers7 developed a single-nucleotide polymorphism (SNP) sensor with gold nanocomposites into which single-stranded DNAs (ssDNAs) were introduced. The solvation of ssDNAs introduced onto the AuNPs kept the AuNPs dispersed in a concentrated NaCl solution because the ssDNAs worked as water-soluble polymers with a high flexibility and anionic charges to stabilize the AuNPs. Once the complementary single strands were added to the SNP sensor solution, the strands interacted with the ssDNAs to form rigid double strands, causing the entropic destabilization of the nanocomposites. The destabilized nanocomposites assembled spontaneously with the aid of a salting-out effect of NaCl, resulting in a change in the solution color to bluish purple. However, SNPs formed no stable double strands with the ssDNAs bound to the AuNPs. Therefore, the nanocomposites retained stability even in the NaCl solution. By exploiting this difference, we developed a gold nanocomposite colorimetric sensor that distinguished complementary single strands from SNPs and provided the response as a colorimetric change. Hence, red-to-blue colorimetric sensors based on the morphological change of AuNPs from a discrete to an assembled state have been investigated. Compared to red-to-blue colorimetric sensors based on gold nanocomposites, very few blue-to-red colorimetric sensors have been reported because of the difficulty in spontaneously disassembling the assembled AuNPs. Liu et al. reported blue-to-red (6) (a) Thanh, N. T. K.; Rosenzweig, Z. Anal. Chem. 2002, 74, 1624–1628. (b) Hirsch, L. R.; Jackson, J. B.; Lee, A.; Halas, N. J.; West, J. L. Anal. Chem. 2003, 75, 2377–2381. (c) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226–8230. (d) Nath, S.; Kaittanis, C.; Tinkham, A.; Perez, J. M. Anal. Chem. 2008, 80, 1033–1038. (7) (a) Sato, K.; Onoguchi, M.; Sato, Y.; Hosokawa, K; Maeda, M. Anal. Biochem. 2006, 350, 162–164. (b) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 8102–8103.

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colorimetric sensors that are based on the disruption of doublestrand linkages connecting gold cores in nanocomposites.8 We have reported an alternative blue-to-red colorimetric sensor using gold nanocomposites conjugated with water-soluble copolymers having poly(ethyleneamine) groups.9 The gold nanocomposites, which aggregate as a result of the copolymer conjugation, disassemble to exhibit a change in the solution color from bluish purple to red when they are heated and then cooled. Because the disassembly is inhibited by cysteine, the gold nanocomposites exhibit a color gradation between red and bluish purple in accordance with the cysteine concentration. This colorimetric change has been applied to the determination of cysteine in health supplements. We have found from further research that glutathione promotes the disassembly of gold nanocomposites even without thermal stimuli. Glutathione;composed of glutamic acid, cysteine, and glycine;is a ubiquitous tripeptide and is the principal nonprotein thiol involved in antioxidant cellular defense.10 It detoxifies a number of reactive oxygen species as well as other hazardous materials.11 Because changes in the concentration of glutathione in plasma and erythrocytes are associated with HIV syndrome,12 Alzheimer’s disease,13 and Werner syndrome,14 a rapid, accurate analysis of glutathione is required for an appropriate diagnosis. Glutathione in biological fluid has been determined by several analytical methods, including high-performance liquid chromatography,15 electrophoresis-based methods,16 flow-injection analysis,17 spectrophotometry,18 voltammetry,19 and potentiometry.20 (8) (a) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2004, 126, 12298–12305. (b) Liu, J.; Lu, Y. Nat. Protoc. 2006, 1, 246–252. (c) Liu, J.; Lu, Y. Adv. Mater. 2006, 18, 1667–1671. (d) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90–94. (e) Lu, Y; Liu, J. Acc. Chem. Res. 2007, 40, 315–323. (f) Lee, J. H.; Wang, Z.; Liu, J.; Lu, Y. J. Am. Chem. Soc. 2008, 130, 14217–14226. (9) Shimada, T.; Ookubo, K.; Komuro, N.; Shimizu, T.; Uehara, N. Langmuir 2007, 23, 11225–11232. (10) (a) Mates, J. M.; Perez-Gomez, C.; Blanca, M. Clin. Chim. Acta 2000, 296, 1–15. (b) Huet, O.; Cherreau, C.; Nicco, C.; Dupic, L.; Conti, M.; Borderie, D.; Pene, F.; Vicaut, E.; Benhamou, D.; Mira, J.-P.; Duranteau, J.; Batteux, F. Crit. Care Med. 2008, 36, 2328–2334. (11) (a) Kawakami, S. K.; Gledhill, M.; Achterberg, E. P. J. Phycol. 2006, 42, 975–989. (b) Preveral, S.; Gayet, L.; Moldes, C.; Hoffmann, J.; Mounicou, S.; Gruet, A.; Reynaud, F.; Lobinski, R.; Verbavatz, J.-M.; Vavasseur, A.; Forestier, C. J. Biol. Chem. 2009, 284, 4936–4943. (12) (a) Droege, W.; Gross, A.; Hack, V.; Kinscherf, R.; Schykowski, M.; Bockstette, M.; Mihm, S.; Galter, D. Adv. Pharmacol. 1997, 38, 581–600. (b) Opalenik, S. R.; Ding, Q.; Mallery, S. R.; Thompson, J. A. Arch. Biochem. Biophys. 1998, 351, 17–26. (c) Sung, J. H.; Shin, S. A.; Park, H. K.; Montelaro, R. C.; Chong, Y. H. J. NeuroVirol. 2001, 7, 454–465. (13) (a) Cecchi, C.; Latorraca, S.; Sorbi, S.; Iantomasi, T.; Favilli, F.; Vincenzini, M. T.; Liguri, G. Neurosci. Lett. 1999, 275, 152–154. (b) Bijur, G. N.; Davis, R. E.; Jope, R. S. Mol. Brain Res. 1999, 71, 69–77. (14) Pagano, G.; Zatterale, A.; Degan, P.; D’Ischia, M.; Kelly, F. J.; Pallardo, F. V.; Calzone, R.; Castello, G.; Dunster, C.; Giudice, A.; Kilinc, Y.; Lloret, A.; Manini, P.; Masella, R.; Vuttariello, E.; Warnau, M. Free Radical Res. 2005, 39, 529–533. (15) (a) Gotti, R.; Andrisano, V.; Cavrini, V.; Bongini, A. Chromatographia 1994, 39, 23–28. (b) Nozal, M. J.; Bernal, J. L.; Toribio, L.; Marinero, P.; Moral, O.; Manzanas, L.; Rodriguez, E. J. Chromatogr., A 1997, 778, 347–353. (c) Klejdus, B.; Zehnalek, J.; Adam, V.; Petrek, J.; Kizek, R.; Vacek, J.; Trnkova, L.; Rozik, R.; Havel, L.; Kuban, V. Anal. Chim. Acta 2004, 520, 117–124. (d) Zhang, W.; Wan, F.; Zhu, W.; Xu, H.; Ye, X.; Cheng, R.; Jin, L.-T. J. Chromatogr., B 2005, 818, 227–232. (e) Vacek, J.; Klejdus, B.; Petrlova, J.; Lojkova, L.; Kuban, V. Analyst 2006, 131, 1167–1174. (16) (a) Panak, K. C.; Ruiz, O. A.; Giorgieri, S. A.; Diaz, L. E. Electrophoresis 1996, 17, 1613–1616. (b) Ling, Y.-Y.; Yin, X.-F.; Fang, Z.-L. Electrophoresis 2005, 26, 4759–4766. (c) Musenga, A.; Mandrioli, R.; Bonifazi, P.; Kenndler, E.; Pompei, A.; Raggi, M. A. Anal. Bioanal. Chem. 2007, 387, 917–924. (17) (a) Hawkes, W. C.; Craig, K. A. Anal. Biochem. 1990, 186, 46–52. (b) Satoh, I.; Arakawa, S.; Okamoto, A. Sens. Actuators, B 1991, B5, 245–247. (18) (a) Eyer, P.; Podhradsky, D. Anal. Biochem. 1986, 153, 57–66. (b) Matsumoto, S.; Teshigawara, M.; Tsuboi, S.; Ohmori, S. Anal. Sci. 1996, 12, 91–95. (c) Rahman, I.; Kode, A.; Biswas, S. K. Nat. Protoc. 2006, 1, 3159–3165. (19) (a) Kinoshita, H.; Mira, T.; Kamihira, S. Bunseki Kagaku 1999, 48, 117– 120. 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Although electrochemical analyses and high-performance liquid chromatography with an appropriate detection technique have been mainly used for glutathione determination because of their selectivity and sensitivity, they require expensive equipment and laborious procedures. 5,50 -Dithiobis(2-nitrobenzoate) (DTNB), which is the most common reagent used for spectrophotometrically determining sulfhydryl compounds, requires a suitable separation technique to determine glutathione selectively because DTNB reacts with all thiol groups in sulfhydryl compounds.15b,21 Gold nanocomposites are also candidate materials for the colorimetric assay of glutathione because glutathione reacts with the surfaces of the gold cores in the nanocomposites through its thiol group.22,23 Recently, several studies have been conducted with regard to the assay of glutathione with AuNPs. Thomas’s24 and Wang’s25 research groups independently claimed the detection of glutathione with gold nanorods on the basis of the evolution of a new spectral band at a longer wavelength. Chang and coworkers26 studied the MS determination of glutathione with laser desorption and ionization assisted by AuNPs. In this article, we describe the glutathione-triggered disassembly of assembled gold nanocomposites conjugated with thermoresponsive copolymers. We then describe a practical application of the phenomenon to determine glutathione in eye drops and health supplements.

2. Experimental Section 2.1. Chemicals. Hydrogen tetrachloroaurate(III) tetrahydrate and N-isopropylacrylamide were obtained from Kanto Chemical (Tokyo, Japan). Diethylenetriamine was obtained from Tokyo Kasei (Tokyo, Japan). Sulfhydryl compounds and other amino acids examined here were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without further purification. The chemical structures of sulfhydryl compounds studied here are shown in Figure S1 (Supporting Information). All other reagents and solvents were obtained from commercial sources. N-Isopropylacrylamide was recrystallized with hexane before use. Methanol was distilled before use. 2.2. Synthesis of a Thermoresponsive Copolymer. A water-soluble copolymer, poly(n-isopropylacrylamide-co-acryloyl-diethylenetriamine) (hereafter referred to as poly(NIP0.9DETA0.1)) was obtained by copolymerizing 0.1 mol of acryloyldiethylenetriamine with 0.9 mol of isopropylacrylamide by radical polymerization with some modification of a previous method.9 In brief, prior to copolymerization, a precursor, N-acryloyl-diethylenetriamine, was synthesized from acryloyl chloride and diethylenetriamine (hereafter referred to as DETA). Subsequently, 0.01 mol (1.63 cm3) of acryloyl chloride in 25 cm3 of 1,4-dioxane was added to 100 cm3 of 1,4-dioxane solution containing 0.1 mol (10.3 g) of DETA. The white precipitate that formed was filtered and then suspended in 100 cm3 of methanol containing 0.01 mol (0.59 g) of potassium hydroxide. After the filtration of the precipitated potassium chloride, the filtrate containing N-acryloyl-diethylenetriamine was not purified but was used immediately for the following copolymerization. After a methanol solution of N-acryloyl-diethylenetriamine was transferred to a 500 cm3 round-bottomed separations flask equipped with a condenser, 0.09 mol (10.2 g) of N-isopropylacrylamide, 0.5 cm3 of 3-mercaptopropionic acid, and 0.82 g of (21) Aitken, A.; Learmonth, M. In The Protein Protocols Handbook, 2nd ed.; Walker, J. M., Ed.; Humana Press: Totowa, NJ, 2002; pp 595-596. (22) He, X.; Zhong, Z.; Guo, Y.; Lv, J.; Xu, J.; Zhu, M.; Li, Y.; Liu, H.; Wang, S.; Zhu, Y.; Zhu, D. Langmuir 2007, 23, 8815–8819. (23) Lim, I. S.; Mott, D.; Ip, W.; Njoki, P. N.; Pan, Y.; Zhou, S.; Zhong, C.-J. Langmuir 2008, 24, 8857–8863. (24) Sudeep, P. K.; Joseph, S. T. S.; Thomas, K. G. J. Am. Chem. Soc. 2005, 127, 6516–6517. (25) Kou, X.; Zhang, S.; Yang, Z.; Tsung, C.-K.; Stucky, G. D.; Sun, L.; Wang, J.; Yan, C. J. Am. Chem. Soc. 2007, 129, 6402–6404. (26) Huang, Y.-F.; Chang, H.-T. Anal. Chem. 2007, 79, 4852–4859.

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Uehara et al. Scheme 1. Disassembly of Gold Nanocomposites Conjugated with Water-Soluble Copolymer Poly(N-n-isopropylacrylamide-coacryloyldiethyletriamine) [poly(NIP0.9-DETA0.1)]a

a (a) Conjugation of gold nanoparticles with water-soluble copolymer, (b) disassembly induced by glutathione, and (c) disassembly induced by thermal stimuli.

azobisisobutyronitrile were added to the flask. The mixture was kept at 60 °C under a nitrogen atmosphere. After cooling, the solution was poured into cooled diethylether; both the solution and the diethylether had the same volume. The crude precipitation of the copolymer was recrystallized with methanol and diethylether. The average molecular weight (Mn) of the synthesized copolymer was estimated to be ca. 1.1  104 by using gel permeation chromatography with calibrated polystyrene standards. The chemical structure of poly(NIP0.9-DETA0.1) is illustrated in Scheme 1. 2.3. Preparation of AuNPs. AuNPs were prepared by using a citrate reduction method reported by Grabar27 and Sutherland28 with slight modifications. Briefly, in a 1 dm3 roundbottomed flask equipped with a condenser, 500 cm3 of 1 mmol/L hydrogen tetrachloroaurate(III) tetrahydrate was brought to a rolling boil with vigorous stirring. The rapid addition of 50 cm3 of 38.8 mmol/L sodium citrate to the vortex of the solution resulted in a color change from pale yellow to burgundy. Boiling was continued for 10 min; the heating mantle was then removed, and stirring was continued for an additional 15 min. After the solution reached room temperature, it was passed through a membrane filter with a pore size of 0.4 μm. The resulting solution of gold nanoparticles was characterized by an extinction maximum at 520 nm. The transmission electron microscope (model JEM-2010, JEOL, Tokyo, Japan) indicated a monodispersity with an average particle size of 13 nm (1.7 nm (number of particles sampled = 100). 2.4. Determination of Glutathione. To prepare assembled gold nanocomposites to be used for colorimetric sensing, 1.0 g (27) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735–743. (28) Sutherland, W. S. J. Colloid Interface Sci. 1992, 148, 129–141.

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of a 20 g/L poly(NIP0.9-DETA0.1) solution and 3.0 cm3 of a 0.18 g dm-3 AuNP solution were placed in a volumetric flask. Subsequently, 1.0 cm3 of a sample solution containing glutathione was added, followed by an adjustment of the final volume to 10 cm3 with water. The resulting solution was transferred to a centrifugal test tube and was then kept at room temperature (25 °C) for 20 min so that the gold nanocomposites disassembled. After vigorous shaking of the solution for several seconds, visible spectra of the solution from 380 to 850 nm were measured with a V-560 UV-vis spectrophotometer (Jasco, Tokyo, Japan). The L*a*b* colorimetric coordinate parameters of CIE chromaticity were calculated from the extinction spectra. The determination of glutathione by HPLC was carried out with the injection of a sample of 100 mm3. The injected sample was separated with a Capcellpak C18SG120 column (4.6 mm i.d.  250 mm length, Shiseido, Tokyo, Japan) and an eluent (5 wt % acetonitrile-95 wt % water) containing 3.5  10-3 mol/L sodium 1-heptanesulfonate and 0.1% phosphoric acid. The eluent was pumped at a flow rate of 1.0 cm3/L, and the effluent was monitored spectrophotometrically at 210 nm.

3. Results and Discussion 3.1. Spontaneous Disassembly Triggered by Glutathione. The disassembly of gold nanocomposites triggered by glutathione is illustrated in Scheme 1, where thermal-stimuli-induced disassembly is also depicted for the sake of comparison. Prior to disassembly, gold nanocomposites are prepared by mixing a solution of AuNPs with a solution of poly(NIP0.9-DETA0.1), a water-soluble copolymer containing poly(ethylene amine). The interaction of the poly(ethylene amine) groups with AuNPs results in the assembly of AuNPs to form gold nanocomposites, inducing the solution color to turn from red to bluish purple Langmuir 2010, 26(9), 6818–6825

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because of a change in the plasmon resonance of the gold nanocomposites. (Process a) Although the gold nanocomposites assemble, they do not sediment because of the stabilization caused by the conjugated water-soluble copolymer. The stability of the gold nanocomposites provides for the reproducibility of colorimetric sensing. When glutathione is added to a solution of assembled gold nanocomposites, the composites disassemble spontaneously and the solution color returns to red. (Process b) The spontaneous disassembly triggered by glutathione was unexpectedly discovered while investigating the effect of sulfhydryl compounds on the disassembly induced by the thermal stimuli9 (process c). We touch briefly upon the disassembly induced by the thermal stimuli before discussing the spontaneous disassembly. A watersoluble copolymer, poly(NIP0.9-DETA0.1), exhibits a thermoresponsive phase transition caused by the shrinkage of the polymer chains because of dehydration; this dehydration is induced by the thermal stimuli. The copolymers bound to the gold cores in the gold nanocomposites also shrink when the gold nanocomposite solution is heated. The shrunken copolymers expand the interparticle distance between the gold cores in the nanocomposites as if they were wedges. Therefore, the color of the solution changes from bluish purple to red in accordance with the morphological change in the gold cores as a result of the disassembly induced by the thermal stimuli.9 In contrast,

Figure 1. Change in the extinction spectra of gold nanocomposites conjugated with water-soluble copolymer in the presence of glutathione as a function of standing time at 25 °C. [Au] = 0.054 g/L, [poly(NIP0.9-DETA0.1)] = 2 g/L, [glutathione] = 1.0  10-4 mol/L, and pH 8.3.

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glutathione-triggered disassembly requires no thermal stimuli. Glutathione adheres to the surface of the gold cores to replace the attached water-soluble polymer as well as other sulfhydryl compounds. The resulting gold nanocomposites are stabilized by adhered glutathione. Because glutathione possesses negative charges at pH 8.3 (the experimental condition used here), the spontaneous disassembly is facilitated mainly by the electrostatic repulsion between the gold cores attached to negatively charged glutathione. Meanwhile, because diethylenetriamine groups in water-soluble copolymers have positive charges under the same pH condition, the electrostatic attraction between the glutathione on the gold cores and the diethylenetriamine groups of the liberated polymer relaxes the negative charges near the surface of the gold cores to facilitate disassembly as well. Details of the effect of the solution pH on the spontaneous disassembly will be discussed in a later section. Figure 1 depicts a change in the extinction spectra of gold nanocomposites after the addition of glutathione. With an increase in the standing time, the extinction spectra shift toward a shorter wavelength and there is a decrease in the extinction band at around 680 nm; this extinction band is assigned to the plasmon extinction of the assembled gold cores. Meanwhile, an increase is observed in the extinction band at around 520 nm; this band is assigned to isolated gold cores. The spectral change indicates a change in the morphologies of gold nanocomposites from the assembled to the disassembled state with an increase in the standing time. The change in the extinction spectrum is saturated more than 120 min after the addition of glutathione, and the spectrum is similar but not identical to that of unmodified discrete AuNPs, as shown in Figure S2 (Supporting Information). The maximum extinction wavelength of the saturated spectrum of the gold nanocomposites and that of the unmodified discrete AuNPs are 525 and 522 nm, respectively. The difference is due to the incomplete disassembly of the nanocomposite gold cores and the difference in the substances attached to the gold cores.1 Because the nanocomposite solution contains some types of gold assemblies of various sizes and morphologies, no isosbestic points are observed in Figure 1. TEM images of the nanocomposites after the addition of glutathione (Figure 2) also support the glutathione-triggered spontaneous disassembly of gold cores. The incomplete disassembly of each gold core observed in Figure 2 is consistent with the spectral studies. The glutathione-triggered disassembly of the gold nanocomposites is also monitored by dynamic light scattering (DLS) measurements. Data acquisition is carried out three times to

Figure 2. TEM images of gold nanocomposites conjugated with water-soluble copolymer in the (a) presence and (b) absence of glutathione after an incubation time of 120 min. Langmuir 2010, 26(9), 6818–6825

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Figure 3. Histogram of intensity contribution vs diameter (Dh in nm) of a gold nanocomposite solution ([Au] = 0.054 g/L, [poly(NIP0.9-DETA0.1)] = 2 g/L, pH 8.3) (a) without glutathione and (b) with 1.0  10-4 mol dm-3 glutathione at 25 °C. Because a single measurement with three replicated acquisitions required 12 min, the histogram in plot b reflects the averaged result obtained with three replicated acquisitions from 12 to 24 min after the preparation of the sample solution.

obtain reliable data for one DLS measurement, which required ca.12 min per measurement. However, because the disassembly of the gold nanocomposites proceeds during a DLS measurement, the obtained DLS data reflect the averaged information about the transient state of the disassembly, reducing the quantitative precision. Additionally, because DLS measures hydrodynamic diameters of gold nanocomposites, it tends to overestimate the diameters of AuNPs measured by TEM images. For example, the diameter of unmodified discrete AuNP with a particle size of 13 nm as determined by TEM is estimated to be 33 nm according to the DLS measurement. Nevertheless, as shown in Figure 3, a pronounced difference in the population of the gold nanocomposites before and after the addition of glutathione is observed. Whereas the distribution peaks around 1000 nm are dominant in the case of the gold nanocomposite solution before the addition of glutathione (Figure 3a), the peaks at around 100 nm become dominant after the addition of glutathione (Figure 3b). As determined by DLS, the average diameter of the gold nanocomposites changes from 808 nm in Figure 3a to 482 nm in Figure 3b. Considering that the average diameter of the gold nanocomposites disassembled by the thermal stimuli (134 nm) is considerably smaller than that of the gold nanocomposites disassembled by glutathione (482 nm), we concluded that the disassembly triggered by glutathione is less sufficient than that induced by the thermal stimuli. Control experiments indicate that the diameters of unbound [poly(NIP0.9-DETA0.1)] and poly(N-isopropylacrylamide)-modified gold nanocomposites with discrete morphologies are 69 and 88 nm, respectively, thereby suggesting insufficient disassembly. The results of spectrophotometric studies, TEM measurements, and DLS measurements described above indicate that glutathione serves as an effective wedge to expand interparticle distances of the assembled gold cores in the nanocomposites. Because the mechanism of the glutathione-triggered disassembly is different from that of the disassembly of AuNPs as a result of the disruption of interparticle bridging structures,8 glutathionetriggered disassembly offers an alternative approach to the exploitation of gold nanocomposites for colorimetric sensing. 3.2. Factors Affecting Spontaneous Disassembly. 3.2.1. Exploration of Sulfhydryl Compounds. First, a comprehensive survey of compounds that affect the spontaneous disassembly of gold nanocomposites was performed. Figure 4 shows the solution color of gold nanocomposites after the addition of sulfhydryl 6822 DOI: 10.1021/la100460w

Figure 4. Effect of sulfhydryl compounds on the spontaneous disassembly of gold nanocomposites evidenced by the color of the final solution after incubation for 120 min at 25 °C. [Au] = 0.054 g dm-3, [poly(NIP0.9-DETA0.1)] = 2 g/L, [sulfhydryl compound] = 1.0  10-4 mol/L, and pH 8.3.

compounds or biorelated compounds. Any amines, amino acids, or alcohols without thiol groups do not trigger spontaneous disassembly because their interaction with AuNPs is prevented by a water-soluble polymer surrounding them. Even glutathione in an oxidized form, glycine, glutamic acid, aspartic acid, lysine, and methionine have no effects on spontaneous disassembly because they lack thiol groups. In contrast, sulfhydryl compounds interact with AuNPs through their thiol group and give various results in accordance with their chemical structures. Substantial disassembly is observed upon the addition of glutathione and cysteinylglycine, whereas cysteine, penicillamine, and 3-mercaptopropionic acid promote slight disassembly. However, 2-mercaptoethanol, 1-butanethiol, and 2-aminoethanethiol do not facilitate spontaneous disassembly. Judging from the relationship between the chemical structures of sulfhydryl compounds (Supporting Information S1), we deduce that carboxyl groups and the bulkiness of sulfhydryl compounds play important roles in spontaneous disassembly. As for carboxyl groups, a typical difference is observed between 3-mercaptopropionic acid and ethyl 3-mercaptopropionate. That is, 3-mercaptopropionic acid promotes disassembly whereas no disassembly is observed upon the addition of ethyl 3-mercaptopropionate in which the carboxylate is esterified. As for bulkiness, spontaneous disassembly is far more pronounced for bulkier peptides such as glutathione (tripeptide) and cysteinylglycine (dipeptide) than for cysteine and penicillamine (monopeptides). The roles of carboxyl groups and the effect of bulkiness will be discussed in the Mechanism of Spontaneous Disassembly section. We have previously observed the opposing effects (facilitation or inhibition) of sulfhydryl compounds on the disassembly induced by the thermal stimuli.9 Typically, whereas glutathione and cysteinylglycine facilitated the disassembly induced by thermal stimuli, other sulfhydryl compounds such as 2-mercaptoethanol, 1-butanthiol, and 2-aminoethanthiol inhibited it. Therefore, similar effects of sulfhydryl compounds on the disassembly induced by the thermal stimuli and on spontaneous disassembly suggest that the compounds play important roles in both forms of disassembly. We consider this similarity to be caused by the dispersiveness of AuNP composites modified with the sulfhydryl compounds. The addition of sulfhydryl compounds to the gold Langmuir 2010, 26(9), 6818–6825

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Figure 5. Plots of the absorbance of gold nanocomposites composed of 0.054 g/L Au and 2 g/L poly(NIP0.9-DETA0.1) at 520 nm vs solution pH at 25 °C in (left) the presence and (right) the absence of 1.0  10-4 mol/L glutathione. The absorbance was measured after it reached a constant value.

nanocomposites of water-soluble copolymers liberates the copolymer, inducing the formation of nanocomposites attached to the sulfhydryl compounds; subsequently, the dispersiveness of the gold cores is governed by the attached sulfhydryl compounds. The control experiment in which the water-soluble copolymer with poly(ethylene amine) groups was added to a solution of glutathione-stabilized AuNPs failed to show AuNP assembly because amino groups in the water-soluble polymer cannot replace glutathione on the AuNPs. 3.2.2. Effect of the Length of Poly(ethylene amine) in a Polymer. The length of poly(ethyleneamine) in a thermoresponsive copolymer is a crucial parameter for the spontaneous disassembly of gold nanocomposites because the interaction between the poly(ethylene amine) groups and the AuNP surface causes the adhesion of the former onto the latter. We synthesized four types of thermoresponsive copolymers whose poly(ethylene amine) lengths were varied from diethylenetriamine to pentaethylenehexamine in order to examine the effect of the poly(ethylene amine) length on the spontaneous disassembly of gold nanocomposites. The glutathione-triggered spontaneous disassembly of gold nanocomposites was observed only when we used poly(NIP0.9-DETA0.1), which possessed the shortest poly(ethylene amine) examined. The results suggest that a weak interaction between poly(ethylene amine) and the AuNPs surface is favorable for spontaneous disassembly. The results are also consistent with the fact that sulfhydryl compounds liberate amino compounds adhered to the surface of AuNPs when they adsorb onto the surface.9,29 The reproducible spontaneous disassembly of gold nanocomposites was observed at copolymer concentrations between 0.2 and 0.5 wt %. Although glutathione provoked spontaneous disassembly below a copolymer concentration of 0.2 wt %, the reproducibility became poor under this condition. However, spontaneous disassembly proceeded slowly above a copolymer concentration of 0.5 wt %, causing an inferior response to a colorimetric output. Hence, the copolymer concentration was fixed at 0.2 wt % throughout the experiment. 3.2.3. Effect of Solution pH. The solution pH is also an important parameter for spontaneous disassembly because it controls not only the protonation of glutathione and the poly(ethylene amine) groups in the thermoresponsive copolymer but also the dispersiveness of unmodified gold nanocomposites. Figure 5a,b shows the influence of solution pH on the disassembly of gold nanocomposites of poly(NIP0.9-DETA0.1) with and without glutathione. The degree of gold nanocomposite assembly was (29) Chen, S.-J.; Chang, H.-T. Anal. Chem. 2004, 76, 3727–3734.

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quantified with absorbance at 520 nm, which was assigned to a plasmon band of discrete AuNPs.1 The absorbance of the solution at 520 nm decreased when the AuNPs assembled. As shown in Figure 5a, an increase in the solution pH facilitates the disassembly of gold nanocomposites. The profile shown in Figure 5a is reversible with respect to the pH change, indicating that the AuNPs repeat their morphological change between the assembly and the disassembly reversibly according to the change in the solution pH. However, the gold nanocomposites without glutathione exhibit a considerably different profile as compared to those with glutathione, as shown in Figure 5b. Although they remain in a disassembled state under the first basic condition, they become assembled when the solution is acidified. Once they assembled, they did not disassemble again under the second basic condition. The difference between the profiles indicates an essential role of glutathione in the disassembly. This will be discussed in the section on the mechanism of spontaneous disassembly. The extinction spectra and extinction (at 520 nm) of the nanocomposite solution do not vary above pH 7.8, at which the extinction spectra are identical to those of the nanocomposite solution 120 min after the addition of glutathione at pH 8.3 (Figure 1). The solution pH can be controlled by the buffering ability of the DETA groups in the absence of any buffering agent because the pKa2 of DETA is 9.13.30 Cysteinylglycine, which also provokes the spontaneous disassembly of gold nanocomposites, shows a profile similar to that of glutathione, as shown in S3 (Supporting Information). 3.3. Mechanism of Spontaneous Disassembly. A schematic representation of the mechanism of spontaneous disassembly is illustrated in Scheme 1 (step c). We have previously reported that cysteine liberated poly(ethylene amine) groups in a watersoluble copolymer through a replacement reaction at the AuNP surface of the nanocomposites.9 The MS spectra shown in S4 (Supporting Information) indicate that glutathione also liberated poly(ethylene amine) in the thermoresponsive copolymer bound to the gold cores through the formation of Au-S bonds with its cysteine moiety. As stated in the Exploration of Sulfhydryl Compounds section, features of the chemical structure of sulfhydryl compounds such as the carboxyl groups and bulkiness are crucial factors in facilitating spontaneous disassembly. Carboxyl groups in sulfhydryl compounds dissociate to introduce negative charges under the neutral pH conditions used in the experiment, leading to electrostatic repulsion between the gold cores adhered to the sulfhydryl compounds. Meanwhile, poly(ethylene amine) groups in the water-soluble copolymer protonate partially in order to possess positive charges, causing electrostatic attraction between the deprotonated sulfhydryl compounds on AuNPs and the librated water-soluble copolymer. Localization of the librated water-soluble copolymers between AuNPs with dissociated sulfhydryl compounds reduces the electrostatic repulsion and stabilizes the resulting gold nanocomposites. In addition, the bulkiness of sulfhydryl compounds is important for disassembly because bulkier sulfhydryl compounds cover a wider area of the AuNP surface than do smaller ones, leading to better stabilization.23 To test our hypothesis, the pH dependence of the dispersiveness of AuNPs with sulfhydryl compounds was examined and compared; the results of this examination are shown in Figure 5. Typical results obtained with glutathione and cysteine are shown in Figure 6a,b, respectively, in which the extinction at 520 nm is also used to represent the morphology of the gold cores in the nanocomposites. As shown in Figure 6a, the AuNPs with (30) Prue, P. L.; Schwarzenbach, G. Helv. Chim. Acta 1950, 33, 985–995.

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Figure 6. Plots of absorbance of gold nanoparticles with (a) glutathione or (b) cysteine at 520 nm vs solution pH at 25 °C. [Au] = 0.054 g/L

and [sulfhydryl compound] = 1.0  10-5 mol/L. The solution pH was repeatedly changed between acidic and alkaline regions. The absorbance was measured after it reached a constant value.

glutathione change their morphology in accordance with the solution pH with high repeatability. Considering the comparison of the profiles shown in Figure 5a,b, we conclude that glutathione attached to the gold cores plays an important role in causing the repeatable change in the morphology of the gold nanocomposites. The results are consistent with the study reported by Lim et al.,23 which showed that glutathione facilitated the assembly of AuNPs under acidic conditions and their disassembly under alkaline conditions. The reversible dissociation of a carboxyl group in glutathione as a function of pH caused the repeatable change in the morphology of glutathione-modified AuNPs. The hysteresis shown in Figure 6a is apparently due to the partial fusion of the assembled AuNPs. A stronger repulsive force is required to assemble partially fused AuNPs so that sufficient negative charges that are produced from the dissociated carboxyl groups of glutathione in order to assemble the AuNPs are attained at a higher solution pH. However, the cysteine-modified AuNPs did not exhibit a repeatable profile (Figure 6b). Once the modified AuNPs assembled, they did not disassemble again, even though the solution pH was raised in order to dissociate a carboxyl group in cysteine. The sulfhydryl compounds other than cysteinylglycine and glutathione listed in Figure 4 gave similar results to that of cysteine. Hence, AuNPs modified with smaller sulfhydryl compounds tend to fuse with each other when they are assembled. Once assembled, these AuNPs can never be disassembled again. In contrast, the pH-absorbance profile of cysteinylglycine gives a result similar to that of glutathione. Bulkier sulfhydryl compounds such as glutathione and cysteinylglycine prevented the fusion of the assembled AuNPs under acidic conditions.23 Tseng and co-workers claimed that sulfhydryl peptides such as cysteine and homocysteine assembled AuNPs coated with a surfactant whereas sulfhydryl oligopeptides such as cysteinylglycine and glutathione kept the AuNPs disassembled.31 They concluded that the bulkiness of sulfhydryl compounds is an important factor that governs the assembly of AuNPs modified with a surfactant. In addition to bulkiness, carboxyl groups in sulfhydryl compounds play important roles in the disassembly because, as mentioned above, the disassociated carboxylate groups introduce negative charges onto the AuNPs to induce the electric repulsion required to facilitate disassembly. This consideration is consistent with the role of 3-mercaptopropionic acid in Tseng’s report,31 showing that 3-mercaptpropionic acid stabilized AuNPs in spite of not being a bulky compound. Protonated carboxyl groups in sulfhydryl compounds on AuNPs under acidic conditions would also play an important role in (31) Huang, C.-C.; Tseng, W.-L. Anal. Chem. 2008, 80, 6345–6350.

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spontaneous disassembly because hydrogen-bonding networks composed of protonated carboxyl groups and water-soluble copolymers inhibit the fusion of gold cores in the nanocomposites. Hence, bulkier sulfhydryl compounds possessing carboxyl groups effectively promote spontaneous disassembly. A more detailed mechanism concerned with hydrogen bonding in the vicinity of AuNPs is under investigation. 3.4. Assay of Glutathione. Previously reported colorimetric sensors based on AuNPs use absorbance at a prescribed wavelength,3a,b,6a-c,29,32 the ratio of absorbance at two prescribed wavelengths,5c,d,8a,c,d,33 or a shift of the maximum wavelength6d,34 as the output. However, we have introduced an a* value into the L*a*b* color coordinates as colorimetric output to quantify a change in the solution color from blue to red.9 Although a* values are calculated from integrated absorbance,35 they are superior to other outputs with respect to the quantification of the colorimetric change in gold nanocomposites because they are directly associated with the human visual response from green to red. Because only particular sulfhydryl compounds such as glutathione and cysteinylglycine induce spontaneous disassembly, a high selectivity toward glutathione would be expected in assays based on spontaneous disassembly. Together with copolymer concentration and solution pH, which have already been investigated, glutathione concentration and incubation time are important factors that govern spontaneous disassembly. Figures 7 and 8 show the effects of glutathione concentration and incubation time on the colors and a* values of the solution of gold nanocomposites conjugated with poly(NIP0.9-DETA0.1)), respectively. The disassembly of gold nanocomposites proceeds with an increase in both parameters. As shown in the inset of Figure 8, a linear relationship with high reproducibility is obtained between the glutathione concentration and a* values at an incubation time of 15 min. Although the linear relationship is not directly related (32) (a) Schofield, C. L.; Field, R. A.; Russell, D. A. Anal. Chem. 2007, 79, 1356– 1361. (b) Okubo, K.; Shimada, T.; Shimizu, T.; Uehara, N. Anal. Sci. 2007, 23, 85–90. (c) Gates, A. T.; Fakayode, S. O.; Lowry, M.; Ganea, G. M.; Murugeshu, A.; Robinson, J. W.; Strongin, R. M.; Warner, I. M. Langmuir 2008, 24, 4107–4113. (d) Wu, S.-H.; Wu, Y.-S.; Chen, C-h. Anal. Chem. 2008, 80, 6560–6566. (33) (a) Huang, C.-C.; Huang, Y.-F.; Cao, Z.; Tan, W.; Chang, H.-T. Anal. Chem. 2005, 77, 5735–5741. (b) Liu, C.-W.; Hsieh, Y.-T.; Huang, C.-C.; Lin, Z.-H.; Chang, H.-T. Chem. Commun. 2008, 2242–2244. (c) Yu, C.-J.; Tseng, W.-L. Langmuir 2008, 24, 12717–12722. (d) Li, D.; Wieckowska, A.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 3927–3931. (34) Obare, S. O.; Hollowell, R. E.; Murphy, C. J. Langmuir 2002, 18, 10407– 10410. (35) (a) ISO 11664-4/CIE, CIE 1976. (b) Mizuguchi, H.; Atsumi, H.; Hashimoto, K.; Shimada, Y.; Kudo, Y.; Endo, M.; Yokota, F.; Shida, J.; Yotsuyanagi, T. Anal. Chim. Acta 2004, 527, 131–138.

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Article Table 1. Analytical Results of Glutathione in Eye Drops and Health Supplementsa this method (mg g-1)

HPLC (mg g-1)

labeled value (mg g-1)

eye drops 99 ( 7 96 ( 2 100 health supplements 49 ( 3 50 ( 1 50 a Average ( standard deviation (three replicate analyses).

Figure 7. Effect of glutathione concentration and standing time on solution colors of gold nanocomposites at 25 °C. [Au] = 0.054 g/L, [poly(NIP0.9-DETA0.1)] = 2 g/L, and pH 8.3.

positive error. However, cysteine and homocysteine inhibited disassembly, causing a negative error. Therefore, an appropriate separation technique is required to apply the assay to complicated samples such as blood plasma. We have validated the applicability of the gold nanocomposite assay by determining glutathione in eye drops and health supplements that contain no other sulfhydryl compounds. Table 1 summarizes the analytical results obtained with the proposed gold nanocomposite colorimetric assay and the HPLC method. Labeled values reported by the manufacturer are also summarized in Table 1 for the sake of comparison. Good agreement between the analytical results obtained by both methods indicates the accuracy and reliability of the proposed assay.

4. Conclusions

Figure 8. Plots of a* values of a gold nanocomposite solution vs standing time at various concentration of glutathione: (O) 0  10-6, (0) 2.0  10-6, (4) 4.0  10-6, and (3) 6.0  10-6 mol/L. (Inset) Plots of a* values of a gold nanocomposite solution at 15 min vs glutathione concentration. The a* values of gold nanocomposite solutions are obtained by the conversion of the photographs shown in Figure 7.

to the physical or chemical properties of the gold nanocomposites as other colorimetric sensor outputs are not related, the linearity and high reproducibility allows us to use the relationship as a calibration curve to determine glutathione. The profile shown in the inset exhibits a linear range of up to 6  10-6 mol/L glutathione with a relation coefficient (R2) of 0.9998 whereas it decurves above 6  10-6 mol/L. The detection limit, defined as 3 times the standard deviation of a blank (3σ, n = 4), was 2.9  10-8 mol/L. The decurved profile is caused by a nonlinear relationship between the a* values and the degree of AuNP disassembly. The interference of diverse compounds was investigated in order to devise practical applications of the present colorimetric sensors. As already stated, no interference was observed upon the addition of amines, alcohols, and carboxylates without thiol groups. None of the sulfur-containing compounds except for sulfhydryl compounds interfered with the determination of glutathione. Note that the oxidized form of glutathione did not affect disassembly, indicating the high selectivity of this sensor toward the reduced form of glutathione. However, sulfhydryl compounds caused a certain amount of interference. Because cysteinylglycine and γ-glutamylcysteine facilitated disassembly, they led to a

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A novel colorimetric sensor for assaying glutathione was developed by exploiting the spontaneous disassembly of gold nanocomposites conjugated with water-soluble copolymers. The color of a gold nanocomposite solution changed from blue to reddish purple in accordance with an increase in glutathione concentration. The colorimetric change could be successfully quantified with the a* value in L*a*b* color coordinates. The a* values of solutions with an incubation time of 15 min exhibited a linear response to the glutathione concentration. On the basis of this linear response, the concentration of glutathione in eye drops and health supplements was determined with the assay. The adhesion of glutathione onto the AuNP surface increased the intermolecular distance between AuNPs, resulting in spontaneous disassembly. The glutathione-triggered spontaneous disassembly showed that thermal stimuli were no longer necessary for the disassembly of gold nanocomposites conjugated with a water-soluble copolymer with thermoresponsive properties and suggested that AuNP disassembly could become a more versatile technique used in colorimetric nanoparticle sensors. We are continuing our research to develop other assays involving the disassembly of gold nanocomposites. Acknowledgment. This study was supported by Grants-in-Aid for Scientific Research Japan (C) (20550071) and (B) (20310040) and by the Tokuyama Science Foundation. We thank Dr. S. Ito (Utsunomiya University) for his assistance with the supporting MS measurements. Supporting Information Available: Chemical structures of amino acids and sulfhydryl compounds. Extinction spectra of discrete AuNPs and glutathione-triggered gold nanocomposites after the addition of glutathione. Plot of the absorbance of gold nanocomposites versus solution pH. Mass spectra for gold nanocomposites conjugated with poly(NIP0.9-DETA0.1) and gold nanocomposites conjugated with poly(NIP0.9-DETA0.1) and glutathione. This material is available free of charge via the Internet at http:// pubs.acs.org.

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