Shape and Color of Au Nanoparticles Follow Biocatalytic Processes

The shaped Au NPs could be generated by a biocatalytic process using alcohol dehydrogenase, NAD+, and ethanol, and the resulting blue color provides a...
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Langmuir 2005, 21, 5659-5662

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Shape and Color of Au Nanoparticles Follow Biocatalytic Processes Yi Xiao,† Bella Shlyahovsky,† Inna Popov,‡ Valeri Pavlov,† and Itamar Willner*,†,‡ Institute of Chemistry and The Unit for Nanoscopic Characterization, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel Received February 3, 2005. In Final Form: April 6, 2005 The NAD(P)H-mediated growth of Au nanoparticles (NPs) in the presence of ascorbic acid, AuCl4-, and cetyltrimethylammonium bromide leads to the formation of shaped NP structures consisting of dipods, tripods, and tetrapods. The shaped particles exhibit a red-shifted plasmon absorbance at λ ) 680 nm, consistent with the existence of a longitudinal plasmon exciton. High-resolution transmission electron microscopy analysis of the tripod and tetrapod structures reveals directional growth along the 〈211〉 and 〈010〉 directions, respectively. The shaped Au NPs could be generated by a biocatalytic process using alcohol dehydrogenase, NAD+, and ethanol, and the resulting blue color provides a colorimetric test for ethanol.

Recent advances in nanotechnology use metal nanoparticles (NPs) as labels for detecting biorecognition events.1-3 Nucleic acid-functionalized Au NPs were used as labels for the detection of DNA hybridization or single base mismatches,4-6 and Au NPs modified with nucleic acids were employed as “weight labels” for the microgravimetric detection of DNA on piezoelectric crystals.7-9 The dissolution of metal NP labels was also used for the electrochemical detection of the dissolved ions.10-12 The catalytic properties of metal NPs were used for amplified biosensing, and the catalytic deposition of the metals on metal NP seeds was used to read-out biorecognition processes by electrical conductivity,13-15 optical means,16 or electrochemical methods.17 The integration of metal NPs with enzyme systems is, however, an unexplored field. Au NPs implanted into redox proteins were used to * Corresponding author. E-mail: [email protected]. † Institute of Chemistry, The Hebrew University of Jerusalem. ‡ The Unit for Nanoscopic Characterization, The Hebrew University of Jerusalem. (1) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (2) (a) Katz, E.; Willner, I.; Wang, J. Electroanalysis 2004, 16, 1944. (b) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 60426108. (3) Katz, E.; Shipway, A. N.; Willner, I. In Nanoparticles-from theory to applications; Schmid, G., Ed.; Wiley-VCH: Weinheim, 2003; Chapter 6, pp 368-421. (4) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (5) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (6) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (7) Weizmann, Y.; Patolsky, F.; Willner, I. Analyst 2001, 126, 15021504. (8) Zhou, X. C.; O’Shea, S. J.; Li, S. F. Y. Chem. Commun. 2000, 953-954. (9) Patolsky, F.; Ranjit, K. T.; Lichtenstein, A.; Willner, I. Chem. Commun. 2000, 1025-1026. (10) Wang, J.; Liu, G. D.; Merkoc¸ i, A. J. Am. Chem. Soc. 2003, 125, 3214-3215. (11) Wang, J.; Liu, G. D.; Zhu, Q. Y. Anal. Chem. 2003, 75, 62186222. (12) Wang, J.; Polsky, R.; Xu, D. K. Langmuir 2001, 17, 5739-5741. (13) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 15031506. (14) Velev, O. D.; Kaler, E. W. Langmuir 1999, 15, 3693-3698. (15) Urban, M.; Mo¨ller, R.; Fritzsche, W. Rev. Sci. Instrum. 2003, 74, 1077-1081. (16) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 90719077. (17) Wang, J.; Xu, D. K.; Polsky, R. J. Am. Chem. Soc. 2002, 124, 4208-4209.

electrically contact the biocatalyst and electrode supports.18 Recently, NADH-induced enlargement of Au NPs was employed for the colorimetric detection of NAD+dependent biocatalytic processes.19 The shape-controlled synthesis of metal NPs attracts substantial efforts, and NPs revealing rod,20 triangle,21 prism,22 tripod, and tetrapod23,24 structures were prepared. Although the mechanism of growth in these systems is not fully understood, it is believed that the added surfactant to the systems favorably binds to certain nanocrystalline phases, thus, inducing the particle enlargement in dictated directions that form the respective shapes. The use of biocatalysts for the synthesis of shaped particles is, however, scarce, and very recently the RNA-mediated growth of hexagonal Pd NPs was reported.25 Here we show that dihydronicotinamide adenenine dinucleotide (phosphate), NAD(P)H, cofactors mediate the growth of Au NP dipods, tripods, and tetrapods. The shape-controlled growth of the Au NPs is also accomplished by the biocatalyzed oxidation of ethanol in the presence of the NAD+-dependent alcohol dehydrogenase (AlcDH). As far as we are aware, this is the first example that demonstrates the biocatalytic shape-controlled synthesis of metal NPs. Besides the fundamental interest in this process, the structural features of the NPs allow the colorimetric analysis of enzyme processes. The present system consists of an aqueous solution that includes AuCl4-, 2.4 × 10-4 M, L-ascorbic acid (AA), 6.9 × 10-4 M, cetyltrimethylammonium bromide (CTAB), 4.8 ×10-2 M, and variable concentrations of NADH. Upon the adjustment of the pH of the system to about pH ) 11 the rapid formation of Au NPs is observed. Figure 1A shows the spectra formed in the presence of different (18) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2004, 299, 1877-1881. (19) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519-4522. (20) Jana, N. R.; Gearheart, L.; Murphy, C. J. Adv. Mater. 2001, 13, 1389-1393. (21) Jun, Y. W.; Lee, S. M.; Kang, N. J.; Cheon, J. J. Am. Chem. Soc. 2001, 123, 5150-5151. (22) Lee, S. M.; Jun, Y, W.; Cho, S. N.; Cheon, J. J. Am. Chem. Soc. 2002, 124, 11244-11245. (23) Chen, S. H.; Wang, Z. L.; Ballato, J.; Foulger, S. H.; Carroll, D. L. J. Am. Chem. Soc. 2003, 125, 16186-16187. (24) Sau, T. K.; Murphy, C. J. J. Am. Chem. Soc. 2004, 126, 86488649. (25) Gugliotti, L. A.; Feldheim, D. L.; Eaton, B. E. Science 2004, 304, 850-852.

10.1021/la050308+ CCC: $30.25 © 2005 American Chemical Society Published on Web 05/19/2005

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Figure 1. (A) Absorbance spectra of the Au NPs formed in the presence of different concentrations of NADH: (a) 0 M; (b) 1.3 × 10-7 M; (c) 2.7 × 10-7 M; (d) 5.4 × 10-7 M; (e) 8.0 × 10-7 M; (f) 1.3 × 10-6 M; (g) 2.0 × 10-6 M; and (h) 4.0 × 10-6 M. (B) Time-dependent evolution of the absorbance at λ ) 680 nm: (a) in the absence of NADH and (b and c) in the presence of NADH, 5.4 × 10-7 M and 4.0 × 10-6 M, respectively.

NADH concentrations. In the absence of NADH a single plasmon absorbance is observed at λ ) 530 nm (curve a). The resulting Au NPs are spherical (5-8 nm), and the absorbance of Au NPs reaches a constant value after a time interval of 2 min. In the presence of NADH and ascorbate and in the absence of any pre-synthesized Au NPs, the color of the system changes from red to dark blue, and at high concentrations of NADH the spectra include two bands at λ ) 530 nm and at λ ) 680 nm, Figure 1A, curves b-h. The plasmon band corresponding to the spherical Au NPs decreases while the band at λ ) 680 nm is intensified as the concentration of NADH increases. Control experiments reveal that in the absence of NADH only particles absorbing at λ ) 530 nm are formed, and these NPs originate from the reduction of the Au(III) salt by ascorbate. In the absence of ascorbate NADH is not reducing AuCl4-, at pH 11, to any particles exhibiting plasmon absorbance bands. Interestingly, the addition of NADH to a mixture that includes the ascorbate pre-synthesized Au NPs does not lead to the formation of the blue-colored NPs, and the plasmon band at λ ) 530 nm is unchanged. This suggests that the pre-synthesized Au NPs are protected by CTAB ligands that turn the NPs inaccessible to the catalytic growth by NADH (vide infra a discussion on the function of NADH in growing shaped Au NPs). Also, mixing the AuCl4-/AA/CTAB/NADH system at neutral pH does not lead to the formation of any NPs. The formation of the blue-colored NPs is timedependent, Figure 1B. The absorbance at λ ) 680 nm increases with time and reaches a saturation value. As the concentration of NADH in the system increases the level of saturated absorbance is higher. These results allow us to formulate the mechanism for the controlled growth of Au NPs in the presence of NADH. The reduced cofactor

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Figure 2. (A) Typical TEM image of shaped Au NPs generated in the presence of NADH, 4.0 × 10-6 M. (B-D) Typical images of dipod-, tripod-, and tetrapod-shaped Au NPs formed in the presence of NADH, 4.0 × 10-6 M, respectively. (E-G) “Embryonic-type” images of dipod-, tripod-, and tetrapod-shaped Au-NPs formed in the presence of NADH, 5.4 × 10-7 M, respectively.

(NADH) is kinetically prohibited to reduce AuCl4- to metal particles. The in situ formation of Au NP seeds by the reduction of AuCl4- by ascorbate (pH 11) yields catalytic sites for the reduction of AuCl4- by NADH, thus, allowing the enlargement of the Au NPs. We demonstrate that the growth of the particles leads to shaped Au NPs exhibiting the red-shifted plasmon band (λ ) 680 nm). We will see that the shaped particles originate from selective blocking of crystalline faces by catalytically generated NAD+ and due to the directional growth of Au by NADH as a result of the association of CTAB to growing gold faces. The structural features of the Au NPs generated in the absence and presence of NADH were analyzed by highresolution transmission electron microscopy (HRTEM). In the absence of NADH only spherical Au NPs with diameters ranging from 5 to 8 nm are observed. Figure 2A shows the TEM image of the Au NPs formed in the system that includes a high concentration of NADH, 4.0 × 10-6 M. We observe Au NPs of variable shapes, and by a statistical analysis of many images we find that in addition to spherical NPs (30%) most particles are composed of distinct structures and consist of L-shaped dipods (12%), tripods (45%), and tetrapods (13%), Figure 2B-D, respectively. HRTEM analysis of the tripod structure is depicted in Figure 3A. As shown in Figure 3B, a lattice of planes exhibiting an interplanar distance of 0.235 nm (corresponding to {111} type planes of crystalline gold) is observed. Analysis of the growth directions for three pods that are separated by about 120° one from another showed that the orientation of the crystallite is [011] and possible growth directions for the pods are of 〈211〉 type; namely, the pods extend in the directions [11 h2 h ], [2 h1 h 1], and [121], Figure 3A, inset. A similar HRTEM analysis of the tetrapod indicates that the crystallite is oriented along the [010] direction (only {220} type interplanar distances of 0.144 nm are observed).

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Langmuir, Vol. 21, No. 13, 2005 5661 Scheme 1. Colorimetric Detection of Ethanol Based on the Shape-Controlled Growth of the Au NPs

Figure 3. (A) TEM image of a representative tripod-shaped Au NP formed in the presence of NADH, 4.0 × 10-6 M. (B) HRTEM analysis of the tripod Au NP. The inset in part A shows the crystal planes and the tripod growth directions extracted from the HRTEM analysis.

The growth direction for these pods is of 〈110〉 type; namely, the pods extend in the directions [101], [1 h 01], [101 h ], and [1 h 01 h ]. At lower concentrations of NADH, 5.4 × 10-7 M, the stepwise formation of Au NP shapes is observed. Figure 2E-G shows images of typical “embryonic-type” L-shaped dipods, tripods, and tetrapods. It is clearly visible that while at high NADH concentrations NP shapes consisting of “arms” that are about 20-nm long (width 2-5 nm) are formed, substantially shorter pods (ca. 6-8 nm long) are observed for the “embryonic-type” NPs generated at lower NADH concentrations. Statistical analysis of many images indicates that the resulting Au NP mixture includes about 60% spherical particles, and about 10, 25, and 5% of the NPs consist of “embryonic-type” dipod, tripod, and tetrapod shapes as shown in Figure 2E-G, respectively. The structural features of the NADH-grown shaped NPs correlate nicely with the absorbance spectra of the systems. The blue color and the absorbance at 680 nm of the system consisting of the well-developed dipods, tripods, and tetrapods are attributed to a longitudinal plasmon exciton that exists in rodlike Au NPs. Indeed, such absorbance features were observed at λ ) 680-700 nm for tripod/ tetrapod structures that were prepared by chemical methods.23,26 The less intensive band at λ ) 530 nm observed in the spectra of the Au NPs generated at high NADH concentrations is attributed to residual spherical Au NPs. The spectrum of the system that includes less developed shaped Au NPs, generated at a lower concentration of NADH, consists of a significant band at λ ) 530 nm, corresponding to the spherical NPs, and a broad redshifted shoulder that indicates the formation of longitudinal-shaped structures. From the spectral and structural features of the Au NPs and the control experiments employed to study the formation of the shaped nanocrystals, we suggest the following mechanism for the growth of the shaped NPs. The reduced cofactor NADH is unable to reduce AuCl4to Au(0) NP (or to Au(I)) due to its low concentration in the system. Ascorbate reduces, however, AuCl4- to Au(0) nanocrystals. Formation of the Au(0) nanocrystals activates, however, the NADH-mediated reduction of the shaped Au NP. It is established that the electrocatalytic oxidation product of NADH bound to single-crystalline Au surfaces (NAD+) binds with different affinities to the faces, with a predominantly favored affinity to the (011) face.27 That is, the affinity association of NADH to the particles increased the local concentration of the reduced (26) Hao, E.; Bailey, R. C.; Schatz, G. C.; Hupp, J. T.; Li, S. Nano Lett. 2004, 4, 327-330.

cofactor at the Au NP, thus, facilitating the catalyzed reduction of AuCl4- to Au(0) and the enlargement of the Au NP. The resulting NAD+ binds, however, effectively to the (011) face, thus, blocking the further NADHmediated growth of the gold crystals on that face. Adsorption of CTAB to the Au NP controls the direction of growth of the particles in the 〈211〉 directions. The further NADH-mediated growth along these directions yields the tripod structures.28 It should be noted that in a previous report NAD(P)H cofactors were employed as reducing agents that generate spherical Au NPs of variable dimensions that lack any defined shapes.19 The plasmon absorbance of these spherical Au NPs was detected at λ ) 524 nm. Nonetheless, one should note the difference in the conditions for the synthesis of the particles in the present study and the previously reported system.19 While previously large Au NP seeds (13 nm) were employed to grow the particles, in the present system the in situ generation of small clusters allows the specific blockage of crystalline surfaces. Furthermore, in the previous report19 high concentrations of NADH (∼10-5 M) were employed, and this enabled the direct reduction of AuCl4- by NADH in solution. In the present study, a 100-fold lower concentration of NADH is used, thus, confining the reduction of AuCl4- to the surface of the Au NPs and specifically to the unblocked surfaces of the nanocrystal. Similar spectral changes are observed in the AuCl4-/AA/CTAB system upon the addition of NADPH (see Supporting Information). As the concentration of NADPH is higher the red-shifted absorbance at λ ) 680 nm is intensified, and the absorbance at λ ) 530 nm is reduced. Analogous TEM experiments confirm the formation of the shaped NPs (see Supporting Information). The NADH-mediated growth of structurally shaped, blue-colored, Au NPs provides a means to follow biocatalytic processes and to develop colorimetric biosensors based on the shape-controlled growth of the NPs. Because numerous redox enzymes catalyze the reduction of NAD(P)+ to NAD(P)H in the presence of the respective substrates, one may use the substrate-driven biocatalytic generation of NAD(P)H and the subsequent formation of the shaped Au NPs as a colorimetric test for the respective substrates (Scheme 1). Toward this goal, the NAD+dependent enzyme, AlcDH, was reacted in the presence of NAD+, 1 × 10-3 M, and different concentrations of ethanol at pH ) 9 for a time interval of 15 min, to generate NADH. From the resulting mixture a volume of 1 µL was introduced into an ascorbate aqueous solution, that included CTAB, 4.8 × 10-2 M, HAuCl4, 2.4 × 10-4 M, and AA, 6.9 × 10-4 M, at pH ) 6.0. At these conditions no Au NP seeds are generated. To the resulting mixture was added a NaOH solution, 1.3 × 10-2 M, to adjust the pH of the solution to 11. At these conditions, the NADH(27) Xing, X.; Shao, M.; Liu, C.-C. J. Electroanal. Chem. 1996, 406, 83-90. (28) One of the reviewers raised the possibility that the shaped Au NPs originate from the fusion of spherical precursor particles that is followed by the reductive growth of the pods (cf. Penn, R. L. J. Phys. Chem. B 2004, 108, 12707-12712). This possibility is, however, excluded because the single crystalline domains extend along the entire pod lengths.

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of enzymes to grow particles of pre-designed shapes. Such particles may be, in the future, building elements of nanodevices. Experimental Section

Figure 4. Absorbance spectra corresponding to the Au NPs formed by the biocatalytic generation of NADH in the presence of AlcDH and variable concentrations of ethanol: (a) 0 M; (b) 7.3 × 10-5 M; (c) 9.4 × 10-5 M; (d) 1.5 × 10-4 M; (e) 2.0 × 10-4 M; and (f) 2.9 × 10-4 M. Inset: calibration curve corresponding to the absorbance of the shaped Au NPs at λ ) 680 nm formed in the presence of different concentrations of ethanol.

mediated growth of the shaped Au NPs proceeds, Figure 4. In the absence of alcohol, no NADH is formed and only spherical Au NPs, λ ) 530 nm, are formed, curve a. As the concentration of ethanol increases, higher degrees of shaped particles are observed, curves b-f. Figure 4, inset, shows the derived calibration curve corresponding to the absorbance of the shaped particles at λ ) 680 nm formed in the presence of different concentrations of ethanol. The sensitivity limit for the detection of ethanol is 4.9 × 10-5 M, and the NP-based sensing of ethanol is easily visually qualitatively detected. TEM analyses of the NPs formed at high concentrations of ethanol reveal the dipod, tripod, and tetrapod structures as discussed before. To summarize, the present study has introduced a new concept to probe biocatalytic processes by following the shape-controlled synthesis of Au NPs of unique structural and spectral properties. Besides the versatile applicability of the shaped NPs for biosensing, we demonstrate the use

All chemicals, including AA, nicotinamide adenine dinucleotide (NAD+), 1,4-dihydro-nicotinamide adenine dinucleotide (NADH), 1,4-dihydro-nicotinamide adenine dinucleotide phosphate (NADPH), AlcDH (from bakers yeast, EC 1.1.1.1), CTAB, and HAuCl4‚3H2O, were purchased from Sigma-Aldrich and used as supplied. Ultrapure water from NANOpure Diamond Barnstead source was used in all experiments. For the generation of shaped Au NPs, solutions consisting of CTAB, 4.8 × 10-2 M, AuCl4-, 2.4 × 10-4 M, AA, 6.9 × 10-4 M, and different concentrations of NAD(P)H were prepared, and NaOH, 1.3 × 10-2 M, was rapidly added to the mixture to generate the shaped gold NPs. The spectra of the particles and the TEM images of the developed shaped Au NPs were recorded after a time interval of 5 min of reaction at room temperature. The growth of the Au NPs by the AlcDH-mediated oxidation of ethanol in the presence of NAD+ was performed in two steps: (i) In the first step, a Tris buffer solution, 50 mM, pH ) 9.0, that included NAD+, 1 × 10-3 M, AlcDH, 2.4 mg mL-1, and different concentrations of ethanol was allowed to react for 20 min at 30 °C. (ii) In the second step, 1 µL of the mixture prepared in (i) was added to 1 mL of the reaction solution consisting of CTAB, 4.8 × 10-2 M, AuCl4-, 2.4 × 10-4 M, and AA, 6.9 × 10-4 M at pH ) 6.0. Then, NaOH, 1.3 × 10-2 M, was added to the reaction solution. The absorbance spectra of the solutions were recorded after 5 min of reaction at room temperature. TEM images were obtained with a Tecnai F20 G2 transmission electron microscope operated at 200 kV. The Au NP solution, 8 µL, was drop-cast on a 400-mesh carbon-coated copper grid and allowed to dry followed by the introduction of the sample into the chamber rotary pumped down to 10-3 bar.

Acknowledgment. This research is supported by the German-Israeli Foundation (GIF). Supporting Information Available: Absorbance spectra of the Au NPs formed in the presence of different concentrations of NADPH and TEM images of representative shaped Au NPs formed in the presence of NADPH. This material is available free of charge via the Internet at http://pubs.asc.org. LA050308+