Studies on the Reversible Aggregation of Cysteine-Capped Colloidal

Saikat Mandal,† Anand Gole,† Neeta Lala,† Rajesh Gonnade,† Vivek Ganvir,‡ and. Murali Sastry*,†. Materials Chemistry Division, National Ch...
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Studies on the Reversible Aggregation of Cysteine-Capped Colloidal Silver Particles Interconnected via Hydrogen Bonds Saikat Mandal,† Anand Gole,† Neeta Lala,† Rajesh Gonnade,† Vivek Ganvir,‡ and Murali Sastry*,† Materials Chemistry Division, National Chemical Laboratory, Pune, 411 008, India, and Tata Research Development and Design Centre, Pune, 411 013, India Received April 10, 2001. In Final Form: July 10, 2001 The surface modification of aqueous silver colloidal particles with the amino acid cysteine and the cross-linking of the colloidal particles in solution is described. Capping of the silver particles with cysteine is accomplished by a thiolate bond between the amino acid and the nanoparticle surface. The silver colloidal particles are stabilized electrostatically by ionizing the carboxylic acid groups of cysteine. Aging of the cysteine-capped colloidal solution leads to aggregation of the particles via hydrogen bond formation between amino acid molecules located on neighboring silver particles. The aggregation is reversible upon heating the solution above 60 °C. The rate of cross-linking of the silver particles via hydrogen bond formation may be accelerated by screening the repulsive electrostatic interactions between the particles using salt. The process of aggregation and heat-induced dispersion of the particles has been studied by UV-vis spectroscopy, laser light scattering, and transmission electron microscopy measurements.

Introduction The area of nanotechnology is witnessing increased research activity due to its immense potential in various industrial applications such as optoelectronic devices,1 nonlinear optics,2 light-emitting diodes,3 and quantum dot lasers,4 to name a few. One of the goals in nanotechnology is the organization of nanoparticles in crystalline arrays with the ability to tailor the size and separation of the nanoparticles and thereby the optical and electronic properties of the assembly.5 While assembly of nanoparticles from solution into hexagonally close-packed monolayers and superlattice structures on solid surfaces has met with a fair degree of success,6-9 the controlled assembly of nanoparticles in solution remains a relatively unexplored area. The first steps in this direction leading to a rational nanoparticle assembly strategy were taken by the groups of Mirkin10 and Alivisatos11 who demonstrated that DNA-modified colloidal gold nanoparticles could be assembled into superstructures by hybridization * To whom all correspondence should be addressed. Phone: +91 20 5893044. Fax: +91 20 5893952/5893044. Ε-mail: sastry@ ems.ncl.res.in. † National Chemical Laboratory. ‡ Tata Research Development and Design Centre. (1) Henglein, A. Top. Curr. Chem. 1988, 143, 113. (2) Ghepremichael, F.; Kuzyk, M. G.; Lackritz, H. S. Prog. Polym. Sci. 1997, 22, 1147. (3) Colvin, V. L.; Schlamp, M. C.; Alivisatos, A. P. Nature 1994, 370, 354. (4) Alivisatos, A. P. Science 1996, 271, 933. (5) Collier, C. P.; Saykally, R. J.; Shiang, J. J.; Henrichs, S. E.; Heath, J. R. Science 1997, 277, 1978. (6) Examples of hexagonally ordered nanoparticle assemblies by solvent evaporation include: (a) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801. (b) Brust, M.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Adv. Mater. 1995, 7, 795. (c) Badia, A.; Gao, W.; Singh, S.; Demers, L.; Cuccia, L.; Reven, L. Langmuir 1996, 12, 1262. (d) Wang, Z. L.; Harfenist, S. A.; Whetten, R. L.; Bentley, J.; Evans, N. D. J. Phys. Chem. B 1998, 102, 3068. (e) Heath, J. R.; Knobler, C. M.; Leff, D. V. J. Phys. Chem. B 1997, 101, 189. (f) Wang, Z. L. Adv. Mater. 1998, 10, 13. (g) Connolly, S.; Fullam, S.; Korgel, B.; Fitzamurice, D. J. Am. Chem. Soc. 1998, 120, 2969. (h) Vijaya Sarathy, K.; Raina, G.; Yadav, R. T.; Kulkarni, G. U.; Rao, C. N. R. J. Phys. Chem. B 1997, 101, 9876.

of complementary base sequences in the surface-bound DNA molecules. From a fundamental point of view, Mirkin et al. have used this strategy to critically study the role of interparticle separation and aggregate size on the optical properties of DNA-modified colloidal gold solution.10c Other interactions such as the biotin-avidin molecular recognition process,12 hydrogen bonding between suitable terminal functional groups bound to the nanoparticle surface,13 electrostatic assembly on DNA templates,14 and control over electrostatic interactions stabilizing aqueous (7) Examples of nanoparticle immobilization on self-assembled monolayers include: (a) Chumanov, G.; Sokolov, K.; Gregory, B. W.; Cotton, T. M. J. Phys. Chem. 1995, 99, 9466. (b) Colvin, V. L.; Goldstein, A. N.; Alivisatos, A. P. J. Am. Chem. Soc. 1992, 114, 5221. (c) Grabar, K. C.; Smith, P. C.; Musick, M. D.; Davis, J. A.; Walter, D. G.; Jackson, M. A.; Guthrie, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1148. (d) Bandyopadhyay, K.; Patil, V.; Vijayamohanan, K.; Sastry, M. Langmuir 1997, 13, 5244. (e) Garcia, M. E.; Baker, L. A.; Crooks, R. M. Anal. Chem. 1999, 71, 256. (8) Examples of nanoparticle superlattices formed using dithiol linkers include: (a) Brust, M.; Bethell, D.; Kiely, C. J.; Schiffrin, D. J. Langmuir 1998, 14, 5425. (b) Musick, M. D.; Keating, C. D.; Keefe, M. H.; Natan, M. J. Chem. Mater. 1997, 9, 1499. (c) Sarathy, K. V.; Thomas, J. P.; Kulkarni, G. U.; Rao, C. N. R. J. Phys. Chem. B 1999, 103, 399. (9) Examples of nanoparticle superlattices formed by the LangmuirBlodgett method include: (a) Fendler, J. H.; Meldrum, F. Adv. Mater. 1995, 7, 607 and references therein. (b) Mayya, K. S.; Patil, V.; Sastry, M. Langmuir 1997, 13, 2575. (c) Sastry, M.; Mayya, K. S.; Patil, V.; Paranjape, D. V.; Hegde, S. G. J. Phys. Chem. B 1997, 101, 4954. (d) Patil, V.; Mayya, K. S.; Pradhan, S. D.; Sastry, M. J. Am. Chem. Soc. 1997, 119, 9281. (e) Mayya, K. S.; Sastry, M. J. Phys. Chem. B 1997, 101, 9790. (f) Mayya, K. S.; Patil, V.; Sastry, M. J. Chem. Soc., Faraday Trans. 1997, 93, 3377. (g) Mayya, K. S.; Sastry, M. Langmuir 1998, 14, 74. (h) Mayya, K. S.; Patil, V.; Kumar, M.; Sastry, M. Thin Solid Films 1998, 312, 308. (i) Sastry, M.; Mayya, K. S.; Patil, V. Langmuir 1998, 14, 5198. (j) Sastry, M.; Mayya, K. S. J. Nanopart. Res. 2000, 2, 183. (10) (a) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607. (b) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849. (c) 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. (d) Mirkin, C. A. Inorg. Chem. 2000, 39, 2258. (11) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609. (12) (a) Sastry, M.; Lala, N.; Patil, V.; Chavan, S. B.; Chittiboyina, A. G. Langmuir 1998, 14, 4138. (b) Shenton, W.; Davis, S. A.; Mann, S. Adv. Mater. 1999, 11, 449. (c) Fullam, S.; Rao, S. N.; Fitzmaurice, D. J. Phys. Chem. B 2000, 104, 6164.

10.1021/la010536d CCC: $20.00 © 2001 American Chemical Society Published on Web 08/31/2001

Reversible Aggregation of Silver Particles

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Scheme 1. Cartoon Showing the Assembly of Cysteine-Capped Silver Particles in Solution by Hydrogen Bonding of the Amino Acid Molecules on Different Silver Particles and Disaggregation of the Superclusters by Heating the Solution

nanoparticles15 have been used to assemble colloidal particles in solution. As part of our ongoing studies into the surface modification, stabilization, and optical properties of colloidal gold16 and silver17 hydrosols, we report herein an optical absorption, laser light scattering, and transmission electron microscopy (TEM) investigation of the reversible aggregation of aqueous silver colloids capped with the amino acid cysteine. The use of cysteine for stabilizing aqueous colloidal silver particles as opposed to 4-carboxythiophenol (4-CTP) as in the earlier studies16,17 was motivated by the following reasons. While 4-CTP was solubilized in water with ethanol prior to capping the gold and silver colloidal particles,16,17 cysteine is water soluble and would readily bind to the surface of the silver particles via a thiolate linkage. Furthermore, the surface layer of covalently bound cysteine molecules could cross-link the silver particles via hydrogen bond formation (as shown in Scheme 1), while such a process would not happen in silver particles derivatized with fully ionized carboxylic acid groups (as would be the case with 4-CTP capped silver particles).16 Presented below are details of the investigation. Experimental Section Chemicals. Silver sulfate, sodium borohydride, and L-cysteine were obtained from Aldrich Chemicals and used as received. Synthesis of Colloidal Silver and Capping with Cysteine. 100 mL of 10-4 M concentrated aqueous solution of silver sulfate (Ag2SO4) was reduced by 0.01 g of sodium borohydride (NaBH4) (13) (a) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763. (b) Johnson, S. R.; Evans, S. D.; Brydson, R. Langmuir 1998, 14, 6639. (c) Boal, A. K.; Ilhan, F.; DeRouchey, J. E.; ThurnAlbrecht, T.; Russell, T. P.; Rotello, V. M. Nature 2000, 404, 746. (14) Kumar, A.; Pattarkine, M.; Bhadbade, M.; Datar, S.; Dharmadhikari, C. V.; Ganesh, K. N.; Sastry, M. Adv. Mater. 2001, 13, 341. (15) (a) Shipway, A. N.; Lahav, M.; Gabai, R.; Willner, I. Langmuir 2000, 16, 8789. (b) Shipway, A. N.; Katz, E.; Willner, I. ChemPhysChem 2000, 1, 19. (c) Templeton, A. C.; Zamborini, F. P.; Wuelfing, W. R.; Murray, R. W. Langmuir 2000, 16, 6682. (d) Kim, Y.; Johnson, R. C.; Hupp, J. T. Nano Lett. 2001, 1, 165. (16) Mayya, K. S.; Patil, V.; Sastry, M. Langmuir 1997, 13, 3944. (17) Sastry, M.; Bandyopadhyay, K.; Mayya, K. S. Colloids Surf., A 1997, 127, 221.

at room temperature to yield colloidal silver particles as described elsewhere.17 The silver colloidal particles were stabilized by addition of 2 mL of aqueous solution of 10-3 M l-cysteine to 100 mL of the silver hydrosol. After addition of cysteine and aging the silver colloidal solution for 7 h, the solution was subjected to ultracentrifugation and the resulting pellet was washed with deionized water to remove any uncoordinated cysteine molecules. A film of the silver particles thus prepared was formed on a Si (111) wafer and analyzed by Fourier transform infrared (FTIR) spectroscopy. These measurements were carried out in the diffuse reflectance mode on a Shimadzu FTIR-8201 PC instrument at a resolution of 4 cm-1. The pH of the cysteine-capped silver colloidal solution was ca. 9. At this pH, the cysteine molecules on the surface of the silver particles would be negatively charged (pI of cysteine ) 5.02)18 and thus stabilized electrostatically. UV-Vis Spectroscopy Studies. The optical properties of the cysteine-capped silver colloidal solution were monitored as a function of time on a Hewlett-Packard diode array spectrophotometer (model HP-8452) operated at a resolution of 2 nm. To understand the nature of cross-linking of the cysteine-capped silver particles in solution, UV-vis spectra were recorded for a fully aged colloidal solution (silver hydrosol, 7 h after capping with cysteine) as a function of temperature in the range 20-90 °C at a heating rate of 0.5° min-1. These measurements were made on a Perkin-Elmer Lambda 15 UV-vis spectrophotometer employing a Julabo water circulator with programmed heating accessory. To understand the role of salt in screening the repulsive interactions between cysteine-capped silver particles, UV-vis measurements were carried out with time on the colloidal solutions to which NaCl was added in the concentration range (1-5) × 10-1 M. Appropriate control measurements were performed. Laser Light Scattering Measurements. The kinetics of aggregation of the cysteine-capped silver colloidal particles in the presence of varying concentrations of NaCl was studied on a Horiba model LA-910 laser light scattering particle size distribution analyzer. Prior to measurement, the silver solution was passed through a 0.22 µm membrane filter. The optical system consisted of a 1 mW He-Ne laser (632.8 nm) and a 50 W tungsten halogen lamp as the light source. An 18-division ring-shaped silicone photodiode served as the detector. The instrument was capable of accurately and rapidly measuring the size (and particle spread) of the aggregates in solution in the (18) Neal, A. L. Chemistry and Biochemistry: A Comprehensive Introduction; McGraw-Hill: New York, 1971; p 389.

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Figure 1. (A) FTIR spectra of the cysteine powder (curve 2) and the cysteine-capped silver particle film in the spectral window 2450-2650 cm-1 (curve 1). (B) FTIR spectra of the cysteine powder (curve 1) and the cysteine-capped silver particle film in the spectral window 1450-1750 cm-1. range 0.1-1000 µm. These measurements were carried out under steady stirring conditions. Transmission Electron Microscopy Measurements. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage of 120 kV. Samples for TEM studies were prepared by placing a drop of the cysteinecapped silver colloidal solution, aged for different time intervals, on carbon-coated TEM grids. A fully aged cysteine-capped colloidal silver solution (7 h of aging) was heated at 90 °C for 10 min, and this solution was also analyzed by TEM in a similar manner. The films on the TEM grids were allowed to dry for 1 min following which the extra solution was removed using a blotting paper. TEM analysis was also carried out on a cysteinecapped colloidal silver solution which was destabilized by addition of 3 × 10-1 M NaCl.

Results and Discussion The amino acid cysteine (formula, H2N-CH(CH2SH)COOH) plays an important role in defining the tertiary structure of proteins through disulfide (cystine) bridges. In this study, the free thiol groups in cysteine molecules have been used to bind to colloidal silver and thereby stabilize them electrostatically. The surface coordination of the silver particles with cysteine was accomplished as described in the Experimental Section. Figure 1A shows the FTIR spectrum recorded from the silver film in the spectral window 2450-2650 cm-1 (curve 1) along with the spectrum recorded from cysteine powder (curve 2). The prominent S-H vibrational band centered at ca. 2555 cm-1 is clearly seen in the free cysteine molecules (curve 2) and vanishes on coordination of these molecules with colloidal silver (curve 1). This is strong evidence of surface binding of cysteine to the silver particles via a thiolate linkage and agrees with earlier studies on alkanethiol modification of gold nanoparticles by Murray and coworkers.19 Further evidence for the presence of surfacebound cysteine is provided by FTIR measurements of the silver particle film in the spectral window 1450-1750 cm-1 (curve 1, Figure 1B). The carboxylate stretch vibration of the cysteine molecules is observed to occur at 1595 cm-1. It is well-known from studies on Langmuir-Blodgett films of metal salts of fatty acids that the position of this band is dependent on the nature of the bound metal cation.20 The position of the carboxylate stretch vibration in the silver cluster films suggests some interaction of the acid group with other cysteine molecules, possibly through (19) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66. (20) Pal, S. Ph.D. Thesis, Poona University, Pune, India, 1996.

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Figure 2. (A) UV-vis spectra of silver colloidal particles recorded as a function of time after capping with cysteine molecules. Curve 1: UV-vis spectrum of the as-prepared silver hydrosol immediately after capping with cysteine, t ) 0 h. Curve 2: UV-vis spectrum of the cysteine-capped silver sol aged for 7 h (curve 5) and heated at 90° C for 10 min. Curves 3, 4, and 5: UV-vis spectra recorded at time t ) 2, 5, and 7 h after capping the silver hydrosol with cysteine molecules (see text for details). (B) Absorbance at 550 nm monitored as a function of temperature of a cysteine-capped silver colloidal solution aged for 7 h (see text for details).

hydrogen bonding. This view is strengthened when one considers that the carbonyl stretch vibration of the acid group in free cysteine molecules occurs at 1650 cm-1 (curve 2, Figure 1B). UV-vis, TEM, and light scattering measurements indeed show significant cross-linking of the silver colloidal particles via hydrogen bonding between cysteine molecules located on different silver particles as shown in Scheme 1 and discussed below. Figure 2A shows the UV-vis spectra recorded from the colloidal silver solution as a function of time of capping with cysteine. Curves 1 and 3-5 in the figure correspond to the spectra recorded at time t ) 0, 2, 5, and 7 h after capping the colloidal solution with cysteine, while curve 2 is the spectrum recorded after aging the solution for 7 h (curve 5) and heating it to 90 °C for 10 min. The spectra have been shifted vertically for clarity. In all the spectra, a strong absorption at ca. 400 nm is observed that corresponds to excitation of surface plasmon vibrations in the silver particles.17 A comparison of curve 1 with curves 3-5 indicates that there is a broadening of the surface plasmon resonance with time of aging the cysteinecapped colloidal silver solution. The broadening is especially pronounced for the solution aged for 7 h with clear evidence of the growth of an additional peak at ca. 500 nm. No further changes were observed in the UV-vis spectra of the colloidal solution beyond 7 h of aging. The shoulder at 500 nm in the 7 h aged solution (curve 5) is due to the appearance of a longitudinal plasmon resonance and is a consequence of overlap of the dipole resonances between neighboring silver particles.17,21 Even though the silver colloidal particles are stabilized electrostatically by the negatively charged carboxylic acid groups of the surface-bound cysteine molecules, the broadening observed with time and the growth of the longitudinal plasmon resonance is clearly due to slow aggregation of the silver particles. We recollect that in our earlier studies on the optical properties of 4-CTP-capped silver colloidal particles, the solution was extremely stable over many weeks indicating that detectable cross-linking of the (21) Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci. 1982, 120, 435.

Reversible Aggregation of Silver Particles

particles had not occurred.17 The aggregation of the silver particles of this study could occur via hydrogen bonding between the cysteine molecules on the surface of the nanoparticles, a process that could not occur with 4-CTPcapped silver particles at high pH.17 That the bonding between the particles is fairly weak is indicated by the UV-vis spectrum of the aged colloidal solution heated at 90 °C for 10 min (curve 2). This spectrum is almost identical to that of the silver solution recorded immediately after capping with cysteine (curve 1) showing that particles may be disaggregated by this heating process. The reversibility of the aggregation of the silver particles upon heating the solution clearly highlights the role of hydrogen bonds in the aggregation process. We recollect that FTIR measurements (Figure 1) support the contention that hydrogen bonding is responsible for the silver particle aggregation. The presence of hydrogen bonds linking amine functional groups and carboxylate ions in cysteine molecules over neighboring particles is better studied by a more refined “melting analysis” as demonstrated by Mirkin and co-workers in their study of the optical properties of colloidal gold particles cross-linked by surface-bound DNA molecules.10c UV-vis spectra of the 7 h aged cysteinecapped silver colloidal solution were recorded as a function of temperature, and a plot of the absorbance at 550 nm with temperature is shown in Figure 2B. (The absorbance at 550 nm was chosen since the most rapid changes in the UV-vis spectra with temperature occurred at this wavelength.) Until 50 °C, there is little change in the absorbance at 550 nm, but above this temperature, a sharp fall in the absorbance occurs. Appropriate controls were performed wherein the as-prepared unaggregated sol was similarly heated. Variation in the absorbance band at 550 nm was not observed in this case. The decrease in the absorbance at 550 nm indicates dispersion of the particles in the aggregates, this process being almost complete by the time the solution is heated to 90 °C. This result is in agreement with the UV-vis results presented in Figure 2A (curves 1 and 2). The transition region of decreasing absorbance is fairly broad in comparison to that observed by Mirkin and co-workers for DNA-capped colloidal gold particles.10c This difference may be due to a spectrum of slightly differing bonding energies between the cysteine molecules and may be a consequence of disorder in the surface-bound cysteine molecules or variations in the size of neighboring silver particles. Direct evidence for the state of aggregation of the cysteine-capped silver particles is provided by TEM studies which are presented in Figure 3. The TEM picture of the silver colloidal solution immediately after capping with cysteine molecules is shown in Figure 3A. It is observed that the particles are well dispersed with no evidence of cross-linking. Figure 3B shows the particle size distribution histogram for the accompanying TEM micrograph. The solid line is a Gaussian fit to the data and results in a particle size of 4.4 ( 0.3 nm. Thus, the particle size distribution is reasonably monodisperse. The TEM micrographs recorded from the cysteine-capped silver colloidal solution at time t ) 2, 5, and 7 h are shown in Figure 3C-E, respectively. Open, stringlike structures form at t ) 2 h. Thereafter, the particles aggregate into larger structures until at t ) 7 h, the individual aggregates measure close to 80-100 nm. The inferences from the UV-vis measurements discussed above are thus borne out by the TEM studies presented in Figure 3. Figure 3F shows the TEM picture recorded from the cysteine-capped silver colloidal solution aged for 7 h and then heated to 90 °C for 10 min. The particles that had aggregated into

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Figure 3. (A) TEM micrograph of the silver colloidal particles immediately after capping with cysteine molecules (time t ) 0 h). (B) Particle size distribution histogram estimated from the micrograph shown in Figure 2A. The solid line is a Gaussian fit to the data and yields a silver particle size of 4.4 nm ( 0.3 nm. TEM micrographs of the silver hydrosol capped with cysteine as a function of time of aging: (C) t ) 2 h, (D) t ) 5 h, (E) t ) 7 h, and (F) after heating the cysteine-capped silver sol aged for 7 h at 90 °C for 10 min (see text for details).

large, ca. 100 nm structures (Figure 3E) have now dispersed to a very large extent. The structure of the aggregates is very similar to that observed for the cysteinecapped silver particle solution aged for 2 h (Figure 3C). This result is also in agreement with the UV-vis data presented earlier (curve 2, Figure 2A). Thus, the UV-vis and TEM results clearly show that significant cross-linking of the cysteine-capped silver particles occurs and furthermore that this process is reversible clearly pointing to hydrogen bonding between the silver particles. Screening of the repulsive interactions between the colloidal particles by addition of salt should enhance the rate of formation of aggregates and would thus provide

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Figure 4. (A) UV-vis spectra of the cysteine-capped silver colloidal solution recorded as a function of time after addition of 3 × 10-1 M NaCl. Curve 1: cysteine-capped silver solution before addition of NaCl. Curve 3: cysteine-capped silver solution immediately after addition of NaCl (time t ) 0 h). Curve 4: cysteine-capped silver solution 30 min after addition of NaCl. Curve 5: cysteine-capped silver solution 60 min after addition of NaCl. Curve 2: the spectrum recorded from the NaCldestabilized cysteine-capped silver colloidal solution shown as curve 5 after heating at 90 °C for 10 min. The inset shows a TEM micrograph of the cysteine-capped silver solution destabilized by addition of 3 × 10-1 M of NaCl (see text for details). (B) Mean particle diameter, d h , measured by light scattering measurements as a function of time after addition of 1 × 10-1 M NaCl (circles), 2 × 10-1 M NaCl (squares), and 3 × 10-1 M NaCl (triangles). The point represented by the diamond gives the size of the aggregates in the 3 × 10-1 M NaCl destabilized silver sol (triangles) after heating at 90° C for 10 min (see text for details).

further insight into the kinetics of the aggregation process. Figure 4A shows the UV-vis spectra recorded at different times after addition of 3 × 10-1 M NaCl to the cysteinecapped silver colloidal solution. These measurements were done on freshly capped silver colloidal solutions (and therefore not aged) to see the effect of added salt on the rate of change in the optical properties of the colloidal solution. The spectra recorded immediately after addition of salt (time t ) 0 min), 30 min after addition of salt, and 60 min after addition of salt are shown as curves 3-5, respectively, in Figure 4A. For comparison, the spectrum recorded from the cysteine-capped silver solution before addition of salt is also shown (curve 1, Figure 4A). A clearly resolved longitudinal plasmon resonance centered at ca. 540 nm is observed to grow immediately after addition of salt to the silver colloidal solution (curve 3). Furthermore, this resonance shifts to the red at larger time intervals and stabilizes at ca. 600 nm at 60 min after addition of salt. This result indicates that the size of the aggregates increases with time. It is clear that the salt accelerates the cross-linking of the colloidal particles by hydrogenbonding interactions by effectively screening the repulsive electrostatic interactions between the negatively charged silver particles. The inset of Figure 4A shows the TEM micrograph recorded from the [3 × 10-1 M NaCl]destabilized silver solution, 60 min after addition of salt. The presence of large aggregates of silver particles can clearly be seen and thus corroborates the UV-vis results presented above. As in the case of cysteine-capped silver colloidal particles aged at room temperature in the absence of salt, the salt-destabilized silver solution was heated at 90° C for 10 min and the UV-vis spectrum was recorded (curve 2, Figure 4A). It is clear that significant dispersion of the particles from the aggregates occurs on heating in this case as well. The changes in the UV-vis spectra both during aging of the cysteine-capped silver solution (Figure 2A) and after

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addition of NaCl (Figure 4A) provide (at best) indirect evidence for the aggregation of the silver particles in solution. Furthermore, the changes in the UV-vis spectra on addition of salt (Figure 4A) are too rapid to be followed by UV-vis spectroscopy alone. Consequently, the kinetics of aggregation of the cysteine-capped silver particles on addition of different amounts of NaCl was followed by laser light scattering measurements, this technique providing a rapid estimate of the mean aggregate size (and polydispersity) as a function of time in the different experiments. We also carried out light scattering measurements of the cysteine-capped silver solution as a function of time of aging to parallel the UV-vis study of Figure 2A. However, in this case the size of the aggregates in the fully aged solution was below the detection limits of the instrument used (100 nm). This result is in agreement with the TEM results that yielded an aggregate size in the range 80-100 nm for the fully aged sample (Figure 3E). Figure 4B shows a plot of the mean aggregate size as a function of time after addition of 1 × 10-1 M NaCl (circles), 2 × 10-1 M NaCl (squares), and 3 × 10-1 M NaCl (triangles) determined from light scattering measurements. While the aggregation is fairly slow in the case of low NaCl concentration (1 × 10-1 M) and continues up to 60 min after addition of salt, the particle size increases rapidly (within 8 min) for higher salt concentrations in solution and stabilizes soon thereafter. Further, the equilibrium size of the aggregates is considerably higher in the case of higher NaCl concentration in solution. These results clearly point to electrostatic stabilization of the silver particles by the surface-bound cysteine molecules which may be effectively screened by addition of suitable amounts of NaCl in solution. That the screening leads to hydrogen bonding (and consequently should be reversible) was tested by heating the [3 × 10-1 M NaCl]-silver solution (triangles) at 90 °C for 10 min and performing light scattering measurements on the heated sample. This experiment resulted in a decrease in the aggregate size from ca. 1.8 to ca. 1.44 µm (point shown as a diamond in Figure 4B). It is clear that while some disaggregation has occurred on heating, the process is not completely reversible. It would be instructive to briefly discuss the UV-vis spectroscopy (Figure 4A) and light scattering results for the salt-induced aggregation experiment (Figure 4B) in relation to the UV-vis and TEM studies of the aged cysteine-capped silver solution (Figures 2A and 3). In the case of the aged silver colloid, a weak peak at ca. 500 nm is observed which almost completely vanishes on heating at 90 °C for 10 min (Figure 2A, curves 5 and 2, respectively). The size of the aggregates formed on aging is smaller than 100 nm (below the detection limits of the light scattering instrument, also Figure 3E). Indeed, the spectrum from the heated solution is almost identical to the as-prepared cysteine-capped solution before aging (curve 1, Figure 2A). Another important point to note is that the position of the plasmon resonance remains at close to 400 nm even after complete aging of the silver solution. The above together with the TEM results (compare Figure 3A with Figure 3F) clearly indicate complete disaggregation of the superstructures formed in solution. In the case of the [3 × 10-1 M NaCl]-induced aggregated silver solution, the changes in the UV-vis spectra are rapid and the additional peak at longer wavelengths is much more pronounced (Figure 4A). Heating this solution at 90 °C for 10 min leads to some degree of disaggregation as evidenced by light scattering measurements (Figure 4B). Another interesting

Reversible Aggregation of Silver Particles

feature is that the surface plasmon resonance in the UVvis spectra shifts from 400 nm before addition of salt to 415 nm after NaCl addition (Figure 4A). Furthermore, heating the solution did not restore the position of the resonance which remained at 415 nm (curve 2, Figure 4A). This is a striking difference in the UV-vis behavior of the cysteine-capped silver solution aged for 7 h and that destabilized by addition of salt. The UV-vis results taken together with the light scattering measurements thus indicate that even though the asymmetry in the plasmon resonance disappears on heating the saltaggregated solution (and thus might lead one to erroneously conclude that complete disaggregation had occurred), the position of the resonance may be used as a measure of the extent of aggregation. The size of the aggregates in the case of the [3 × 10-1 M NaCl]-induced aggregated silver solution (ca. 1.8 µm, Figure 4B) is much larger than those observed for the 7 h aged silver solution (∼0.1 µm, Figure 3E), and consequently the energy required to destabilize the assembly would be much higher in the salt-destabilized solution. This would explain the partial disaggregation of the 1.8 µm superassemblies by heating at 90 °C which otherwise is sufficient to completely disaggregate the smaller structures. The rate of aggregation of the colloidal particles can be quantified in terms of a semiempirical flocculation parameter as first demonstrated by Whitesides and coworkers.13a The definition of this parameter has been modified by some of us and applied to the problem of aggregation of 4-CTP-capped gold particles16 as well as avidin-induced aggregation of biotinylated silver and gold particles in earlier studies.12a The basic idea behind this parameter is the following, the exact details of which may be obtained from our earlier work.12a,16 As shown by the UV-vis measurements, aggregation of the colloidal particles is accompanied by a broadening of the plasmon resonance feature. Therefore, the area under the UV-vis curve spanning a suitable integration range (encompassing the surface plasmon vibration) should be a suitable measure of the extent of aggregation and is termed the flocculation parameter.12a,16 The integration limits for the flocculation parameter were taken as 400-800 nm in this study. Figure 5A shows the variation of flocculation parameters as a function of time of aging of the silver colloidal nanoparticles derivatized by cysteine in the absence of salt. The parameter increases rapidly initially and stabilizes after 7 h of aging. On heating the 7 h aged cysteine-capped silver colloidal solution at 90 °C for 10 min, the flocculation parameter falls to nearly the starting value (at time t ) 0 h, point indicated by an arrow in Figure 5A). The use of the flocculation parameter in this case enables us to follow the aggregation behavior of the particles which was not detectable by light scattering measurements. The aggregation behavior of the silver colloidal particles after addition of different concentrations of salt and different aging times has also been studied in terms of the flocculation parameter, and the data obtained are shown in Figure 5B. The data corresponding to triangles, squares, and circles are the flocculation parameters calculated 60 min, 30 min, and immediately after addition of salt to the cysteine-capped silver solution, respectively. The inset of Figure 5B shows a plot of the variation in mean aggregate size, d h , with NaCl concentration in the cysteine-capped silver colloidal solution, taken from the light scattering data shown in Figure 4B at time t ) 0 min (circles), 4 min (squares), and 8 min (triangles). The trends in the aggregate size variation with salt concentration closely parallel that observed in the flocculation parameter

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Figure 5. (A) Flocculation parameters calculated for the cysteine-capped silver colloidal solution (from the UV-vis spectra shown in Figure 2A) plotted as a function of time (circles). After heating the aged sol, the reduction in the flocculation parameter is shown separately by a square (with an arrow, see text for details). (B) Flocculation parameters calculated from UV-vis spectra recorded as a function of time of addition of different concentrations of NaCl to cysteine-capped silver colloidal solution: (circles) immediately after addition of NaCl, (squares) 30 min after addition of NaCl, and (triangles) 60 min after addition of NaCl (see text for details). The inset shows a plot of the mean particle diameter d, estimated from light scattering measurements as a function of NaCl concentration at various times: (circles) 0 min, (squares) 4 min, and (triangles) 8 min.

calculations based on the UV-vis results (main part of Figure 5B) with the important difference being that the time scales are vastly different. While the flocculation parameter continues to change even 30-60 min after addition of salt, size stabilization as determined by light scattering measurements occurs within 10 min of addition of salt. This difference may be a consequence of the nature of definition of the flocculation parameter. It may also suggest that even though the size of the aggregates is invariant with time, small changes in the internanoparticle separation in the aggregates could occur due to a slower time scale associated with this process. It is known that both the interparticle separation and size of the aggregates contribute to changes in the UV-vis spectra of noble metal colloids10c and this process of comparison of light scattering data and flocculation parameters may be used to separate the two contributions. However, we hasten to add that this is purely speculative at the moment. It is observed that the flocculation parameter/mean aggregate size increases with increasing salt concentration for all the measurement times and is a result consistent with enhanced hydrogen bond formation due to better screening of the repulsive electrostatic interactions by the salt. Another interesting observation from the data presented in Figure 5B is that at small salt concentrations the flocculation parameters/mean aggregate size at the different times are nearly constant while at high salt concentrations there is significant deviation. This indicates that the rate of aggregation by hydrogen bond formation is critically determined by the extent of screening of the electrostatic repulsive forces acting across the colloidal particles. Conclusions The surface modification of colloidal silver particles with the amino acid cysteine has been described. The silver particles capped with cysteine are stabilized in an aqueous medium by charging of the carboxylic acid groups in the surface-bound cysteine molecules. The presence of amine

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and carboxylic acid functional groups on the surface of the colloidal particles leads to hydrogen bond formation and thereby cross-linking of the colloidal silver particles. This process is reversible, and the dispersion of the particles from the aggregates may be accomplished by heating the solution above 60 °C. The rate of hydrogen bond formation may be accelerated by screening the electrostatic repulsive forces between the particles with salt. The protocol based on amino acid derivatized colloidal

Mandal et al.

particles may be extended to controlled cross-linking using spacer molecules and is currently being pursued. Acknowledgment. S.M. and A.M.G. thank the Council of Scientific and Industrial Research (CSIR) and the University Grants Commission (UGC), Government of India, for research fellowships. LA010536D