Blue-to-Red Chromatic Sensor Composed of Gold Nanoparticles

The gold nanocomposites, which are used as a chromatic sensor, reveal chromatic change from blue to red with thermal stimuli, heating followed by cool...
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Langmuir 2007, 23, 11225-11232

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Blue-to-Red Chromatic Sensor Composed of Gold Nanoparticles Conjugated with Thermoresponsive Copolymer for Thiol Sensing Takeshi Shimada, Kouki Ookubo, Naoya Komuro, Tokuo Shimizu, and Nobuo Uehara* Graduate School of Engineering, Utsunomiya UniVersity, 7-1-2 Yoto, Utsunomiya, Tochigi, 321-8585, Japan ReceiVed March 7, 2007. In Final Form: June 9, 2007 We describe the first determination of thiol compounds with gold nanocomposites composed of gold nanoparticles and thermoresponsive copolymers having polyamino groups. The gold nanocomposites, which are used as a chromatic sensor, reveal chromatic change from blue to red with thermal stimuli, heating followed by cooling the solution. The blue-to-red chromatic change results from disassembly of the gold nanocomposites, which arises from shrinkage of the thermoresponsive copolymers bound to the gold nanoparticle surfaces due to the phase transition induced by thermal stimuli. The disassembly is inhibited by addition of thiol compounds through displacement of the adhered thermoresponsive copolymers. The detached copolymers no longer influence morphological change of the gold nanocomposites. Corresponding with increase of concentration of the thiol compounds, a solution of the gold nanocomposites after the thermal stimuli shows chromatic change, which was quantified with the a* value in L*a*b* chromatic coordinates. A linear relationship between the a* value and concentration of cysteine, examined as a bio-important thiol, is obtained below 7 × 10-6 mol dm-3, estimating a detection limit defined as 3σ of the blank to be 2.8 × 10-7 mol dm-3. The chromatic sensor of the gold nanocomposites is applied to the determination of cysteine in commercial supplements containing ascorbic acid, which seriously interferes with redox-based determination of cysteine. Analytical results obtained with the chromatic sensor are identical to those obtained with HPLC.

Introduction Gold nanoparticles (AuNPs) display plasmon absorption bands that depend on their shape and size.1 Typically, discrete AuNPs possess an absorption band around 520 nm, corresponding to red solution color, while aggregated AuNPs have it at longer wavelengths, corresponding to blue-purple solution color. Because discrete AuNPs tend to easily aggregate in response to change in their neighboring environment, and because aggregation causes rapid chromatic change from red to blue-purple, chromatic sensors using AuNPs have been widely explored.2-5 One effective strategy to develop chromatic sensors with AuNPs is to introduce probe molecules onto the surface of the AuNPs through chemical bonds. When the introduced probe molecules respond to guest * To whom correspondence should be addressed. E-mail: ueharan@ cc.utsunomiya-u.ac.jp. TEL, FAX: +81-28-689-6166. (1) (a) Schmid, G. Clusters and Colloids: From Theory to Applications; VCH: New York, 1994. (b) Henglein, A. J. Phys. Chem. 1993, 97, 5457-5471. (c) Kreibig, U.; Genzel, L. Surf. Sci. 1985, 156, 678-700. (d) Marie L. S.; Colby A. F., Jr. J. Phys. Chem. B 1999, 103, 11398-11406. (e) Christian D. G.; Adam M. S.; Thaddeus J. N., Jr. Jin, Z. Z. J. Am. Chem. Soc. 2003, 125, 549-553. (f) Takeuchi, Y.; Ida, T.; Kimura, K. Surf. ReV. Lett. 1996, 3, 1205-1208. (g) Kelly, K. L.; Coronado, E.; Zhao, L. L.; Schatz, G. C. J. Phys. Chem. B 2003, 107, 668-677. (h) Link, S.; Mohamed, M. B.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 3073-3077. (i) Zhong, Z.; Patskovskyy, S.; Bouvrette, P.; Luong, J. H. T.; Gedanken A. J. Phys. Chem. B 2004, 108, 4046-4052. (j) Link, S.; El-Sayed, M. A. J. Phys. Chem. B 1999, 103, 8410-8426. (k) Mulvaney, P. Langmuir 1996, 12, 788-800. (2) (a) Kim, Y.; Johnson, R. C.; Hupp, J. T. Nano Lett. 2001, 1, 165 -167. (b) Norsten, T. B.; Frankamp, B. L.; Rotello, V. M. Nano Lett. 2002, 2, 13451348. (c) Obare, S. O.; Hollowell, R. E.; Murphy, C. J. Langmuir 2002, 18, 10407-10410. (d) Lin, S.-Y.; Liu, S.-W.; Lin, C.-M.; Chen, C.-H. Anal. Chem. 2002, 74, 330-335. (e) Sugunan, A.; Thanachayanont, C.; Dutta, J.; Hilborn, J. G. Sci. Technol. AdV. Mater. 2005, 6, 335-340. (3) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature (London) 1996, 382, 607-609. (b) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L. Mirkin, C. A. Science 1997, 277, 1078-1081. (c) Reynolds, R. A., III; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-3796. (d) Sato, K.; Onoguchi, M.; Sato, Y.; Hosokawa, K.; Maeda, M. Anal. Biochem. 2006, 350, 162-164. (e) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 8012-8103. (f) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (g) 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.

molecules, i.e., analytes, bridging structures form between AuNPs via analytes, leading to the aggregation of the AuNPs. Sensing metal ions,2 DNA,3 proteins,4 and amino acids1i,5 have been accomplished with the types of AuNP color sensors. The chromatic change from red to blue-purple was observed corresponding to concentration of the analytes. Contrarily, there have been a few blue-to-red chromatic sensors of AuNPs for which the morphologic change is based on disassembly of gold nanocomposites, since discrete AuNPs are less stable than aggregated AuNPs. Liu et al.6 reported blueto-red chromatic sensors using AuNPs assemblies linked through DNA strands. The gold nanocomposites were disassembled by lead ions, cocaine, and adenosine, resulting in liberation of discrete AuNPs, which causes increased reddish color in the solution. The chromatic change was quantified with an absorbance ratio obtained at 522 and 700 nm. Lin et al.7 described another blueto-red chromatic sensor using gold nanocomposites, in which bridging structures formed through hydrogen bonds between crown ethers introduced on the gold surface. The gold nanocomposites were disassembled with lead ions, leading to a chromatic change to red. In this work, we describe a unique blue-to-red chromatic sensor based on inhibition of the disassembly of nanocomposites (4) (a) Park, S.-J.; Lazarides, A. A.; Mirkin, C. A.; Letsinger, R. L. Angew. Chem., Int. Ed. 2001, 40, 2909-2912. (b) Nam, J.-M.; Park, S.-J.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124 3820-3821. (c) Cobbe, S.; Connolly, S.; Ryan, D.; Nagle, L.; Eritja, R.; Fitzmaurice, D. J. Phys. Chem. B 2003, 107, 470-477. (d) Costanzo, P. J.; Patten, T. E.; Seery, T. A. P. Chem. Mater. 2004, 16, 1775-1785. (e) Otsuka, H., Akiyama, Y., Nagasaki, Y., Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226-8230. (f) Huang, C.-C.; Huang, Y.-F.; Cao, Z.; Tan, W.; Chang, H-T. Anal. Chem. 2005, 77, 5735-5741. (5) Yao, H.; Miki, K.; Nishida, N.; Sasaki, A.; Kimura, K. J. Am. Chem. Soc. 2005, 127, 15536-15543. (6) (a) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642-6643. (b) Liu, J.; Lu, Y. Chem. Mater. 2004, 16, 3231-3238. (c) Liu, J.; Lu, Y. Anal. Chem. 2004, 76, 1627-1632. (d) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2004, 126, 12298-12305. (e) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2005, 127, 12677-12683. (f) Liu, J.; Lu, Y. Angew. Chem., Int. Ed. 2006, 45, 90-94. (7) Lin, S.-Y.; Wu, S-H.; Chen, C-H. Angew. Chem., Int. Ed. 2006, 45, 49484951.

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consisting of AuNPs and thermoresponsive copolymers. We already reported a monochromatic sensor of nanocomposites composed of AuNPs conjugated with thermoresponsive copolymers having carboxyl groups.8 The monochromatic sensor is based on sedimentation of the nanocomposites resulting from displacement of the conjugated thermoresponsive copolymers with cysteine. In the exploration of thermoresponsive copolymers to be conjugated with AuNPs, the disassembly of gold nanocomposites with thermoresponsive copolymers possessing poly(ethyleneamine) was unexpectedly found. Nanocomposites consisting of AuNPs and thermoresponsive copolymers have been investigated for the exploitation of functional materials9-11 or morphologic control of gold nanocomposites.12 Jones et al.10 and other researchers11 have fabricated a thermoresponsive polymer gel containing AuNPs, which works as a heat converter of light, and they controlled the phase transition of the gels by light irradiation. As for the morphologic control of nanocomposites composed of AuNPs and thermoresponsive copolymers, reversible morphologic changes between dispersion and aggregation have been attained through the phase transition of thermoresponsive copolymer induced by thermal stimuli. Assemblies composed of AuNPs and thermoresponsive copolymers, however, have not been applied to chromatic sensors. The blue-to-red chromatic sensor exploited in this work is inhibited by thiol compounds. On the basis of the inhibition, the chromatic sensor is applied to the determination of cysteine. Cysteine, which is an important amino acid containing a thiol group, is not only a constituent of protein and an enzyme like cysteine protease, but is also an essential compound in several processes, such as redox, methyl transfer, and carbon fixation reactions in which CoA participates. Cysteine and its analogues are also related to metabolic disorder diseases.13 Therefore, much research has been focused on the determination of cysteine in pharmaceuticals, urine, serum, and plasma. Although HPLC14 and electrochemical analyses15 are versatile ways to determine cysteine content, they require expensive equipment. Spectrophotometry,16 fluorimetry,17 and chemical luminescent methods,18 which are rather ubiquitous techniques, are alternatives, but their (8) Okubo, K; Shimada, T.; Shimizu, T; Uehara, N. Anal. Sci. 2007, 85-90. (9) Lyon, L. A.; Debord, J. D.; Debord, S. B.; Jones, C. D.; McGrath, J. G.; Serpe, M. J. J. Phys. Chem. B 2004, 108, 19099-19108. (10) (a) Jones, C. D.; Lyon, L. A. J. Am. Chem. Soc. 2003, 125, 460-465. (b) Jones, C. D.; Serpe, M. J.; Schroeder, L.; Lyon, L. A. J. Am. Chem. Soc. 2003, 125, 5292-5293. (11) Gorelikov, I.; Field, L. M.; Kumacheva, E. J. Am. Chem. Soc. 2004, 126, 15938-15939. (12) (a) Zhu, M.-Q.; Wang, L.-Q.; Exarhos, G. J.; Alexander, D. Q. L. J. Am. Chem. Soc. 2004, 126, 2656-2657. (b) Kim, D. J.; Kang, S. M.; Kong, B.; Kim, W-J.; Paik, H-J.; Choi, H.; Choi, I. S. Macromol. Chem. Phys. 2005, 206, 19411946. (c) Bhattacharjee, R. R.; Chakraborty, M.; Mandal, T. K. J. Phys. Chem. B 2006, 110, 6768-6775. (d) Raula, J.; Shan, J.; Nuopponen, M.; Niskanen, A.; Jiang, H.; Kauppinen, E. I.; Tenhu, H. Langmuir 2003, 19, 3499-3504. (13) Slusser, S. O.; Grotyohann, L. W.; Martin, L. F.; Scaduto, R. C., Jr. Am. J. Phys. 1990, 258, F1547-F1553. (14) (a) Amarnath, V.; Amarnath, K. Talanta 2002, 56, 745-751. (b) Xu, H.; Zhang, W.; Zhu, W.; Wang, D.; Ye, J.; Yamamoto, K.; Jin, L. Anal. Chim. Acta. 2005, 545, 182-188. (c) Chwatko, G.; Bald, E. Talanta 2000, 52, 509-515. (d) Potesil, D.; Petrlova, J.; Adam, V.; Vacek, J.; Klejdus, B.; Zehnalek, J.; Trnkova, L.; Havel, L.; Kizek, R. J. Chromatogr., A 2005, 1084, 134-144. (e) Amarnath, K.; Amarnath, V.; Amarnath, K.; Valentine, H. L.; Valentine, W. M. Talanta 2003, 60, 1229-1238. (f) Amini, M. K.; Khorasani, J. H.; Khaloo, S. S.; Tangestaninejad, S. Anal. Biochem. 2003, 320, 32-38. (g) Shahrokhian, S.; Karimi, M. Electrochim. Acta 2004, 50, 77-84. (15) (a) Hignett, G.; Threlfell, S.; Wain, A. J.; Lawrence, N. S.; Wilkins, S. J.; Davis, J.; Compton, R. G.; Cardosi, M. F. Analyst 2001, 126, 353-357. (b) Salimi, A.; Pourbeyram, S. Talanta 2003, 60, 205-214. (c) Teixeiraa, M. F. S.; Dockalb, E. R.; Cavalheiro, E. T. G. Sens. Actuators, B 2005, 106, 619-625. (d) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Methods Enzymol. 1983, 91, 49-60. (e) Aitken, A.; Learmonth M. Protein Protocols Handbook, 2nd ed.; Walker, J. M., Ed.; Humana Press: Totowa, NJ, 2002; pp 595-596. (16) (a) Lee, S.-H.; Sohn, O.-J.; Yim, Y.-S.; Han, K.-A.; Hyung, G. W.; Chough, S. H.; Rhee, J. Talanta 2005, 68, 187-192. (b) Zaia, D. A. M.; Ribas, K. C. L.; Zaia, C. T. B. V. Talanta 1999, 50, 1003-1010.

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poor selectivity and/or laborious procedures have limited their application. Since the exploited method described here is highly selective toward thiol groups, it has become a promising technique to determine cysteine. Experimental Section Chemicals. Hydrogen tetrachloroaurate(III) tetrahydrate and N-isopropylacrylamide were obtained from Kanto Chemical (Tokyo, Japan). Poly(ethyleneamine)s, such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, and pentaethlenehexamine, were obtained from Tokyo Kasei (Tokyo, Japan). Amino acids (alanine, valine, leucine, isoleusine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, glutamic acid, lysine, arginine, histidine, and aspartic acid) and sulfur-containing compounds (homocysteine, penicillamine, 3-mercaptopropionic acid, ethanethiol, 2,3-dimercapto-1-propanol, 2-aminoethanethiol, 1,3-propanedithiol, 1,2ethanedithiol, 2-mercaptoethanol, S-methylcysteine, and cystine) were purchased from Wako Pure Chemical Industried (Osaka, Japan). All other reagents and solvents were obtained from commercial sources. N-Isopropylacrylamide was recrystallized with hexane before use. Methanol was distilled before use. Preparation of Gold Nanoparticles. All glassware used in these preparations was thoroughly cleaned in aqua regia (3 parts HCl, 1 part HNO3), rinsed in deionized and doubly distilled water, and oven-dried prior to use. AuNPs were prepared according to Grabar19 and Sutherland20 with slight modifications. The following stock solutions were prepared from deionized and doubly distilled water that had been filtered through a 0.4 µm membrane filter: 1% hydrogen tetrachloroaurate(III) tetrahydrate, 38.8 mmol dm-3 sodium citrate, and 1% sodium citrate. Other solutions were made fresh as needed using deionized and doubly distilled water. In a 1 dm3 round-bottom flask equipped with a condenser, 500 cm3 of 1 mmol dm-3 hydrogen tetrachloroaurate(III) tetrahydrate was brought to a rolling boil with vigorous stirring. Rapid addition of 50 cm3 of 38.8 mmol dm-3 sodium citrate to the vortex of the solution results 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 filtered through a membrane filter with 0.4 µm pore size. The resulting solution of colloidal particles was characterized by absorption maximum at 520 nm. Transmission electron microscopy (model JEM-2010, JEOL, Tokyo, Japan) indicated monodispersity with average particle size of 13 ( 1.7 nm (100 particles sampled). Preparation of Thermoresponsive Copolymer. We mainly used poly(N-isopropylacrylamide (90 mol %)-co- N-acryloyltryethylenetetramine (10 mol %)) (following poly(NIP0.9-TETA0.1) as a representative thermoresponsive copolymer having poly(ethyleneamine) units as functional groups to interact with AuNPs, since its nanocomposites exhibit typical disassembly. Furthermore, poly(NIP0.9-TETA0.1) is superior to other copolymers having poly(ethyleneamine) in terms of ease of synthesis and high yield. Poly(NIP0.9-TETA0.1) was synthesized with a radical copolymerization reported earlier21 with slight modification. The other thermoresponsive copolymers having different lengths of the poly(ethyleneamine) were synthesized by using a similar method to poly(NIP0.9-TETA0.1) with the corresponding poly(ethyleneamine)s. Prior to the radical copolymerization, a precursor, N-acryloyl triethylanetetramine, was synthesized from acryloyl chloride and (17) (a) Liang, S-C.; Wang, H.; Zhang, Z-M.; Zhang, X.; Zhang, H-S. Spectrochim. Acta, Part A 2002, 58, 2605-2611. (b) Wang, H.; Wang, W-S.; Zhang, H.-S. Talanta 2001, 53, 1015-1019. (18) (a) Nie, L.; Ma, H.; Sun, M.; Li, X.; Su, M.; Liang, S. Talanta 2003, 59, 959-964. (b) Lau, C.; Qin, X.; Liang, J.; Lu, J. Anal. Chim. Acta 2004, 514, 45-49. (c) Kamidate, T.; Tani, T.; Watanabe, H. Anal. Sci. 1998, 14, 725. (d) Sano, A.; Nakamura, H. Anal. Sci. 1998, 14, 731. (19) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735. (20) Sutherland, W. S. J. Colloid. Interface Sci. 1992, 148, 129. (21) Shimada, T.; Shimizu, T.; Uehara, N. Bunseki Kagaku 2002, 51, 689695.

Au Nanocomposite Chromatic Sensors triethylenetetramine (following TETA). 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 (14.6 g) of TETA. The white precipitate was filtered, followed by suspension in 100 cm3 of methanol containing 0.01 mol (0.59 g) of potassium hydroxide. After filtration of the precipitated potassium chloride, the filtrate, which contained N-acryloyl triethylanetetramine, was led to the following copolymerization immediately without purification. After the methanol solution of N-acryloyl triethylanetetramine was transferred into a 500 cm3 round-bottom separable 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 azobisisobutyronitrile were added into the flask. The mixture was kept at 60 °C under nitrogen atmosphere. After cooling, the solution was poured into the same volume of cooled diethyl ether. The crude precipitation of the copolymer was recrystallized with methanol and diethyl ether. The other thermoresponsive copolymers having poly(ethyleneamine) were synthesized in a similar manner as described above. Gel permeation chromatography with tetrahydrofuran used as eluent was used for determination of number average molecular weight (Mn) of the obtained copolymer, which measured ca. 1 × 104 by comparison with polystyrene calibration standards. Introduction of a triethylenetetramine group was confirmed with NMR and IR. Elemental analysis was used for determination of percentage of the triethylenetetramine group introduced into the copolymer. A practical introduction ratio of TETA groups in the copolymer was 6.5 mol % estimated form results of elemental analysis. A 20 g dm-3 of poly(NIP0.9-TETA0.1) solution was prepared and stored at 4 °C in the dark for at least 1 week before use. Determination of phase transition temperatures of the synthesized thermoresponsive copolymer, defined as the temperature giving 50% of transmittance of a 1 cm optical cuvette, was carried out with a light beam of 660 nm. Typical profiles of transmittance of copolymer solutions vs temperature are summarized in the Supporting Information (Figure S1). Determination of Cysteine. Prior to the determination, a solution of a chromatic sensor, gold nanocomposites composed of gold nanoparticles and thermoresponsive copolymer, was prepared as follows: 1.0 g of 20 g dm-3 poly(NIP0.9-TETA0.1) and 3.0 cm3 of a 0.18 g dm-3 AuNPs solution were taken into a 10 cm3 volumetric flask, followed by adjustment of the final volume. To determine cysteine, the resulting solution and 1 cm3 of sample solution were transferred into a centrifugal testing tube, and then the mixture was subjected to heating at 98 °C for 30 min in a water bath followed by cooling to 4 °C over 2 h, so that the gold nanocomposites disassembled. After being shaken vigorously for several seconds, visible spectra and a* and b* values in L*a*b* chromatic coordinate parameters of the solution were measured. Analysis of Supplements. After removal of the sugar-coated surface, supplements containing cysteine were ground with a mortar. 0.05 g of the powdered supplements was dissolved in 100 cm3 of water and subjected to vigorous stirring for 10 min. After filtration, the residue on the filter was dissolved in another 100 cm3 of water. The resulting solution was subjected to another filtration. The two filtrates were merged and the final volume of the solution was adjusted to 250 cm3 with water. After appropriate dilution with water, the sample solution was subjected to both determination protocols for cysteine described above and HPLC mentioned below. Determination of cysteine by HPLC was carried out with a 100 mm3 of a sample injection. The injected sample was separated with a Capcellpak C18SG120 column (4.6 mm i.d. × 250 mm length, Shiseido, Tokyo Japan) and an eluent composed of 5 wt % of acetonitrile and 95 wt % of water containing 3.5 × 10-3 mol dm-3 sodium 1-heptanesulfonate, 0.1% phosphoric acid. The eluent was pumped at a flow rate of 1.0 cm3 min-1, and the effluent was monitored at 210 nm spectrophotometrically. Mass Measurement of Gold Nanocomposites. The solution prepared by the method described in the Determination of Cysteine section was centrifuged for 10 min to sediment gold nanocomposites. After the supernatant was discarded, the precipitated gold nanocomposites were dispersed with 0.5 cm3 of water by sonication. One

Langmuir, Vol. 23, No. 22, 2007 11227 drop of the suspended solution was transferred onto an MS plate, followed by drying. MS spectra were measured with a MALDITOF mass spectrometer (Autoflex TOF/TOF, Bruker Daltonics) with a cation mode.

Results and Discussion Disassembly of Gold Nanocomposites. A chromatic sensor is composed of aggregated gold nanocomposites in which the thermoresponsive copolymer, poly(NIP0.9-TETA0.1), is noncovalently bound to the gold nanoparticle surface. The sensing mechanism is based on both disassembly of the gold nanocomposites and inhibition of the disassembly with thiol compounds, as shown in Figure 1. First, the aggregated gold nanocomposites are prepared by mixing a solution of thermoresponsive copolymer with a discrete gold nanoparticle solution. Interaction of the triethylenetetramine group incorporated in the thermoresponsive copolymer with gold surface causes aggregation of the gold nanoparticles, resulting in a chromatic change of the solution to blue-purple (Figure 1b). Although aggregated gold nanoparticles generally tend to sediment, sedimentation of the copolymer-conjugated gold nanocomposites is not observed, since the copolymers adhered on the nanocomposites work as stabilizer. This is favorable for exploitation of a highly reproducible chromatic sensor. Next, when the gold nanocomposite solution is heated at 98 °C, the phase transition of poly(NIP0.9-TETA0.1) arises, resulting in an educt of excess poly(NIP0.9-TETA0.1), which contains the Au nanocomposites. Subsequently, cooling the solution to below the phase transition temperature leads to dissolution of the educt, developing the red color of the solution (Figure 1c). The TEM images support the morphological change from an aggregated state to the dispersed state of the gold nanocomposites. As shown in Figure 1d, thiol compounds such as cysteine inhibit the disassembly. Development of the red color is caused by expansion of the interparticle distance of AuNPs due to the shrinkage of the conjugated thermoresponsive copolymer by heating. A model of the disassembly will be discussed in a later section. Effect of Heating Temperature on Disassembly. Heating temperature is also a dominant parameter in disassembly, since it governs the phase transition of the thermoresponsive copolymer. Figure 2 shows the influence of heating temperature on vis spectra and colors of the solution containing gold nanocomposites conjugated with poly(NIP0.9-TETA0.1) as a typical example. Spectral measurement was conducted after heating for 30 min followed by cooling to 4 °C. With increase in the heating temperature, the solution color changes from blue to red and absorption bands shift hypsochromically. The solution spectra obtained at 98 °C are almost identical to those of unmodified discrete gold nanoparticles. According to the MIE theory1f-k, absorption bands around 520 nm and over 600 nm are ascribed to independent discrete gold nanoparticles and aggregated ones, respectively. The hypsochromic shift in the absorption bands implies the process of disassembly of gold nanocomposites in the heating process. Considering that the phase transition temperature of poly(NIP0.9TETA0.1) is ca. 34 °C, it is concluded from Figure 2 that sufficient disassembly requires a higher heating temperature than the phase transition temperature. The requirement of the higher heating temperature results from restraint of the thermoresponsive copolymers bound to surfaces of gold nanoparticles. The restraint hinders the copolymer from shrinking during the phase transition process. The effect of solution temperature on the phase transition behavior of the thermoresponsive polymers themselves is summarized in the Supporting Information (Figure S1).

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Figure 1. Disassembly of copolymer-conjugated gold nanoparticles by thermal stimuli and its inhibition with thiol compounds. Copolymer to be conjugated is poly(n-isopropylacrylamide-co-acryloyltryethylenetetramine). Dispersed gold nanoparticles (a) are assembled through modification with the copolymer (b). Thermal stimuli attain disassembly of the aggregated nanoparticles (c). Cysteine inhibits the disassembly (d).

Figure 2. Temperature dependence of visible absorption spectra and color of solution of gold nanoparticle solution conjugated with poly(NIP0.9-TETA0.1) after heating followed by cooling. The solutions containing 0.054 g dm-3 gold and 2 g dm-3 poly(NIP0.9-TETA0.1) were heated between 40 and 98 °C for 30 min followed by cooling at 4 °C.

Roles of Functional Groups in Disassembly. In addition to the heating temperature, functional groups in thermoresponsive copolymers are also an important parameter in the disassembly, because the interaction between AuNPs and the copolymer depends on its functional groups. It was found by exploration of functional groups that poly(ethyleneamine) groups caused disassembly of the gold nanocomposites, and that no disassembly was observed with poly(n-isopropylacrylamide) incorporating methacrylate (10 mol %) and with poly(n-isopropylacrylamide) having no functional groups. The combination of carboxylates and poly(ethyleneamine) groups in the thermoresponsive copolymer caused no disassembly, either. These results are summarized in the Supporting Information (Figure S2). Figure 3 shows photos of the solution containing the gold nanocomposites with thermoresponsive copolymers, the chain length of functional groups, poly(ethyleneamine)s, of which is different after the thermal stimulation process. With increase of

Figure 3. Temperature dependence of solution color of gold nanoparticle conjugated with thermoresponsive copolymers after heating followed by cooling. The solutions containing 0.054 g dm-3 gold and 2 g dm-3 thermoresponsive copolymer were heated between 40 and 98 °C for 30 min followed by cooling at 4 °C. Conjugated thermoresponsive copolymers are (a) poly(N-isopropylacrylamide(90mol%)-co-N-acryloyldiethylenetriamine(10mol%), (b) poly(NIP0.9-TETA0.1), (c) poly(N-isopropylacrylamide(90mol%)co-N-acryloyl tetraethylenepentamine(10mol%), and (d) poly(N-isopropylacrylamide(90mol%)-co-N-acryloyl pentaethlenehexamine(10mol%).

the chain length of poly(ethyleneamine), heating temperatures provoking the disassembly decrease. Note that the thermoresponsive copolymer with diethylenetriamine (DETA) causes insufficient disassembly at any heating temperature. Increase of the chain length of poly(ethyleneamine), which means increase

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Figure 4. pH dependence of solution color (a) and visible spectra (b) of 0.054 g dm-3 gold nanoparticle conjugated with 2 g dm-3 poly(n-isopropylacrylamide) incorporating triethylenetetramine after heating followed by cooling.

of nitrogen atoms to interact with the AuNP surface, strengthens the interaction between the AuNP surface and the copolymer, facilitating the expansion of interparticle distances of AuNPs and shrinking of the copolymer with phase transition. Consequently, thermoresponsive copolymers with longer poly(ethyleneamine) chains cause the disassembly of the gold nanoparticles at lower heating temperatures. Effect of pH on Disassembly. Solution pH is another crucial parameter in the disassembly, because it controls not only the stability of unmodified gold nanoparticles, but also protonation of poly(ethyleneamine) groups in copolymers. Unmodified gold nanoparticles are unstable in an acidic media because of protonation of citrates on AuNPs, and they tend to aggregate spontaneously. As shown in Figure 4, the disassembly appears in the pH range from 6.5 to 8.0. Hindrance of disassembly below 6.0 seems to be due to the instability of gold nanoparticles themselves at lower pH, as mentioned above. Although an increase of the ionic strength of the solution due to the pH adjustment above 8.5 might cause the disassembly, it is difficult to explain the cause of the hindrance plausibly at this stage. The pH of the gold nanocomposite solution without a pH adjustment is 7.48, which lies within the pH range where the satisfied disassembly arises, and which can be buffered with TETA groups, the pKa value of which is 6.67.22 These are favorable for the practical application of polymer-modified gold nanocomposites as a chromatic sensor, because there are no requirements of pH adjustment or of the addition of an appropriate buffering agent. Mechanism of Disassembly. Considering the above results, schematic models of the disassembly and its inhibition are illustrated in Figure 5. Conjugation of thermoresponsive copolymers with AuNPs leads to aggregation of AuNPs due to their poly(ethyleneamine) groups. Heating the solution above the LCST of thermoresponsive copolymers induces phase transition, resulting in shrinkage of the copolymer bound on (22) Schwarzenbach, G. HelV. Chim. Acta 1950, 33, 974-985.

AuNPs surfaces. The shrunk copolymers expand interparticle distances of the AuNPs as if they are wedges, causing a blueshift of the absorption spectra of the gold nanocomposite solution. This consideration is supported by Figure S2 in the Supporting Information, which shows dark red educts of copolymer above the phase transition temperature. The dark red color implies a certain extent of expansion of interparticle distances of gold nanoparticle in the educts. Cooling the solution below the phase transition temperature leads to swelling of the copolymer, promoting further expansion of the distance between AuNPs.12a Repetition of thermal stimuli ascertains the complete disassembly, as shown in the Supporting Information (Figure S3). The above model is also verified with the following experiment. Aggregated gold nanocomposites without a thermoresponsive copolymer were prepared by mixing 1.0 mol dm-3 sodium chloride solution with an unmodified AuNP solution in a test tube, followed by centrifugation in such a way that the aggregated AuNPs sedimented. After removal of the supernatant in the test tube, a thermoresponsive copolymer, poly(NIP0.9-TETA0.1), solution was added. After vigorously shaking the test tube with ultrasonic irradiation for suspension of AuNPs, the test tube was heated above the phase transition temperature, so as to induce the phase transition. As a result, no disassembly arose, and the aggregated AuNPs sedimented at the bottom, indicating essentials of conjugation of thermoresponsive copolymer with AuNPs for disassembly. Inhibitors for Disassembly. Inhibition of disassembly with 21 common amino acids (alanine, valine, leucine, isoleusine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, glutamic acid, lysine, arginine, histidine, and aspartic acid) which play important roles in metabolism is explored for practical application of the gold nanocomposites. Only cysteine, a sulfur-containing amino acid, inhibits disassembly, suggesting the important role of thiol groups in disassembly and a potential to sense cysteine selectively. To confirm the role of thiol groups in disassembly, thiol compounds, such as homocysteine, penicillamine, 3-mer-

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Figure 5. Schematic model of redispersion with thermal stimuli.

Figure 7. Plots of chromaticity parameter a* of 0.054 g dm-3 gold nanoparticles conjugated with 2 g dm-3 poly(NIP0.9-TETA0.1) after heating followed by cooling vs concentration of thiol compounds such as O, 2,3-dimercaptopropanol; 4, 2-aminoethanethiol; b, 1,2ethanedithol; 2, 2-mercaptoethanol; 3, homocysteine; 1, butanethiol; 0, penicillamine; and 9, 3-mercaptopropionic acid.

Figure 6. (a) Change in solution color and (b) chromaticity parameters a* (solid circle) and b* (solid square) of 0.054 g dm-3 gold nanoparticles conjugated with 2 g dm-3 poly(n-isopropylacrylamide) incorporating triethylenetetramine after heating followed by cooling as a function of cysteine concentration.

captopropionic acid, ethanethiol, 1,3-propanedithiol, 2-aminoethanethiol, 1,3-propanedithiol, and 1,2-ethanedithiol, and other sulfur-containing amino acids such as S-methylcysteine and cystine are examined. Although every thiol compound inhibits the disassembly, S-methylcysteine, a monosulfide, and cystine, a disulfide, do not, indicating the crucial role of thiol groups in the inhibition. Other organic compounds without thiol compounds, such as alcohol, sugars, amines, and carboxylates, do not inhibit the disassembly, either. Concentration of thiol compounds in the aggregated AuNP solution also influenced the inhibition of the disassembly. Figure 6a,b shows a color gradation of the solution and plots of chromaticity parameters of a* and b* coordinates vs cysteine

concentration, as typical results of inhibition, respectively. Both the color gradation and the plots clearly respond to the cystine concentration. Since the a* value represents chromatic change between green and red, it gives a better response to the gradation of the solution than the b* value, which represents a chromatic change between blue and yellow.23 Therefore, the a* value is used in the following investigations for the sake of quantitative discussion. Inhibition of disassembly by other thiol compounds is explored to investigate auxiliary roles of other functional groups in the thiol compounds. The influence of concentration of thiol compounds on disassembly is shown in Figure 7. Remarkable inhibition is observed with 2,3-dimercapto-1-propanol, 1,2ethanedithiol, and 2-aminoethanethiol, which possess two thiols or one thiol and one amino group. Moderate inhibition is observed with 2-mercaptoethanol, butanethiol, and thiol amino acids, such as cysteine, homocysteine, and penicillamine. Contrarily, 3-mercaptopropionic acid gave weak inhibition. Note that on the basis of the order of inhibition degree of thiol compounds, thiol groups (23) (a) Mizuguchi, H.; Atsumia, H.; Hashimotoa, K.; Shimada, Y.; Kudo, Y.; Endo, M.; Yokota, F.; Shida, J.; Yotsuyanagi, T. Anal. Chim. Acta 2004, 527, 131-138. (b) Yokota, F.; Abe, S. Anal. Commun. 1997, 34, 111-112.

Au Nanocomposite Chromatic Sensors

Langmuir, Vol. 23, No. 22, 2007 11231 Table 1. Analytical Results of Cysteine in Supplement (/mg)

sample

this methoda (n ) 4) (mg g-1)

HPLCa (n ) 3) (mg g-1)

labeled value (mg g-1)

1 2 3 4

113 ( 3b 114 (1 182 ( 3 70.9 ( 1.1

113 ( 2 111 ( 2 182 ( 2 68.9 ( 0.9

119.6 108.6 199.5 66.2

a

Figure 8. Mass spectra of (a) gold nanocomposites conjugated with poly(NIP0.9-TETA0.1) and (b) gold nanocomposites conjugated with poly(NIP0.9-TETA0.1) and cysteine. Differences of mass number 56 in (a) and 32 in (b) are ascribed to a residue of ethyleneamine (NCH2CH2N) and a sulfur atom, respectively.

give the strongest effect on the inhibition. As to other functional groups, amino groups facilitate the inhibition, but carboxyl groups suppress it. The results are consistent with the fact that carboxylate in citric acid helps the dispersity of gold nanoparticles, and that addition of amine promotes aggregation of gold nanoparticles.1l,24 To study the role of thiol compounds in the inhibition, MALDITOF-MS was conducted for analysis of adhered substances on the gold nanocomposites. Two types of gold nanocomposites were prepared and subjected to MALDI-TOF-MS analysis. One is aggregated gold nanocomposites consisted of AuNPs and poly(NIP0.9-TETA0.1), and the other is that treated with a cysteine solution. Typical MS spectra obtained with both gold nanocomposites are illustrated in Figure 8. Some frequent peaks (z ) 197 × n) ascribed to gold clusters are observed in both of the mass spectra. Accompanying the peaks of gold clusters, some distinct peaks, mass numbers of which are 56 greater than the respective gold clusters peaks, are observed on the mass spectra (Figure 8a). Judging from the difference in the mass number, the peaks are ascribed to gold clusters with accompanying ethleneamine residue (NCH2CH2N, mass number ) 56). On the mass spectra of the solution with cysteine treatment, on the other hand (Figure 8b), some definite peaks, mass numbers of which are 32 (ascribed to sulfur atom) greater than respective gold clusters peaks, are observed, but the peaks of gold clusters with accompanying ethleneamine residue are not. The presence of sulfur residue and the absence of ethleneamine residue on AuNPs shown in Figure 8b indicate that thiol groups in cysteine displace the TETA group on the surface of gold nanocomposites. Displacement of oligonucleotides25 or amino compounds24 on (24) Chen, S-J.; Chang, H-T. Anal. Chem. 2004, 76, 3727-3734. (25) (a) Thaxton, C. S.; Hill, H. D.; Georganopoulou, D. G.; Stoeva, S. I.; Mirkin, C. A. Anal. Chem. 2005, 77, 8174-8178. (b) Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem. 2006, 78, 8313-8318.

n ) number of replicates. b average ( standard deviation.

the surface of gold nanoparticles with thiol compounds was reported by several researchers. We consider that the displacement of poly(ethyleneamine) in the copolymers causes detachment of the copolymer, and that the detached copolymers no longer lead to morphological change, i.e., disassembly, in the gold nanocomposites. Determination of Cysteine. Since the profile of the a* value vs thiol compound concentration depicted in Figure 6b is highly reproducible, the profile can be used as a calibration curve to determine cysteine. Therefore, the applicability of the aggregated gold nanocomposites as a chromatic sensor of cysteine was validated. The calibration range of the nanocomposite sensor can be tuned by controlling the concentration of the thermoresponsive copolymer in the AuNPs solution. Increase of the polymer concentration from 1 to 5 g dm-3 shifts the working range of the AuNPs sensor toward to the low concentration side, but an increase of the polymer concentration narrows the working range of cysteine. A 2 g dm-3 polymer concentration, which was the condition used in Figure 6, was optimum for reproducibility and for sensitivity in sensing cysteine. Increase of the polymer concentration above 5 g dm-3 raises the solution pH, leading to a shift out of the optimum pH range of the disassembly. Although the plots depicted in Figure 6 are curved, they can be approximated by a line below 7 × 10-6 mol dm-3 of cysteine. The detection limit of cysteine defined as 3 times the standard deviation of the blank value can be estimated with the slope of the approximated line, which is 2.8 × 10-7 mol dm-3. The estimated detection limit of the AuNPs sensor provides a comparable sensitivity to that of HPLC methods,14 and the sensitivity of the AuNPs sensor is greater that that of spectrophotometric methods based on redox reactions16a-c and with DTNB.16d,e Considering the inhibition studies of disassembly, the AuNPs sensor exhibits a certain extent of selectivity for cysteine, while DTNB reacts with sulfhydryls unselectively. The applicability of polymer-conjugated gold nanocomposite is validated by determining cysteine in commercial supplements, which also contain ascorbic acid as one of the main constituents. As ascorbic acid was added as an antioxidizing agent of cysteine, it caused serious interference in redox-based determinations of cysteine. However, since ascorbic acid does not inhibit the disassembly, it does not interfere with the determination of cysteine with the gold nanocomposites sensor. Table 1 summarizes the results obtained with the proposed method using disassembly of AuNPs and the HPLC method used as a crosscheck method. The labeled values given by manufacturers are also summarized in Table 1. Good agreements between the results obtained using both the proposed method and HPLC are observed, indicating the accuracy and reliability of the method based on disassembly of AuNPs. Although a slight difference is observed between the analytical results and labeled values in Table 1, the difference is because the labeled values are calculated with the feed content of cysteine and are not analytical results obtained with appropriate methods.

11232 Langmuir, Vol. 23, No. 22, 2007

Conclusion Conjugation of dispersed gold nanoparticles with a thermoresponsive copolymer possessing poly(ethyleneamine) groups caused abrupt aggregation of gold nanoparticles, resulting in the chromatic change from red to blue. Using the aggregated nanocomposites, a novel chromatic sensor was exploited. Thermal stimuli, heating above the phase transition temperature followed by cooling, induced disassembly of the gold nanocomposites, resulting in a return of the solution color to red. The disassembly resulted from expansion of interparticles of AuNPs due to the shrinkage of thermoresponsive copolymers. The disassembly was inhibited by thiol compounds through displacement of adhered poly(ethyleneamine) groups in the thermoresponsive copolymer. Amino acid containing thiol groups, such as cysteine, inhibited the disassembly, but other major amino acids without thiol groups did not, indicating high selectivity of the sensor. A series of sample solutions with different cysteine concentrations which developed the gradation of the solution after treatment

Shimada et al.

with thermal stimuli allowed us to sense cysteine. The quantitative analysis using the color sensor was achieved with an a* chromaticity coordinate. The color sensor was applied to the determination of cysteine in commercial tablets without any interference from ascorbic acid in the tablets. Conjugation of gold nanoparticles through nonchemical bonds provides us with a new strategy to exploit novel chromatic sensors of gold nanoparticles. We are continuing the research in developing other sensing systems with the disassembly. Acknowledgment. This research was supported by a Grantin-Aid for Science Research Japan B (16310059) and 21st Century COE Program “Nature-guided Material Processing” of Ministry of Education, Japan. Supporting Information Available: Three figures. This material is available free of charge via the Internet at http://pub.acs.org. LA700664U