studies on the reversible aggregation of cysteine-capped

7/23/2019 Studies on the Reversible Aggregation of Cysteine-Capped

1/7

Studies on the Reversible Aggregation of Cysteine-CappedColloidal Silver Particles Interconnected via Hydrogen

Bonds

S a i k a t M a n d a l , Anand Gole, N e e ta L al a , Rajesh Gonnade, Vivek G a nvir , a n dM u ra l i S a s t r y *,

Ma te r i a l s Ch e mi str y Di vi si o n , Na ti o n a l Ch e mi ca l L a b o r a to r y, P u n e , 4 1 1 0 0 8 , In d i a , a n d Tata Research Development and Design Centre, Pune, 411 013, India

Recei ved A p r i l 1 0 , 2 0 01 . In Fi n a l Fo r m: Ju l y 1 0 , 2 00 1

The surface modification of aqueous silver colloidal particles with the amino acid cysteine and thecross-linking of the colloidal par ticles in solution is described. Ca pping of th e silver pa rticles wit h cysteineis accomplished by a th iolate bond between th e a mino a cid a nd th e nanopa rt icle surfa ce. The silver colloidalpart icles are sta bilized electrostat ically by ionizing t he carboxylic acid groups of cysteine. Aging of t hecysteine-capped colloidal solution leads to a ggregat ion of the par ticles via hyd rogen bond format ion betw eenamino acid molecules located on neighboring silver particles. The aggregation is reversible upon heatingth e solution a bove 60 C. The ra te of cross-linking of the silver part icles via hy drogen bond forma tion ma ybe accelerated by screening th e repulsive electrostat ic interactions between t he par ticles using salt . Theprocess of aggrega tion an d heat -induced dispersion of the part icles has been studied by UV-vis spectroscopy,laser light scattering, and transmission electron microscopy measurements.

Introduction

The a rea of nan otechnology is w itnessing increa sedresearch activity due to its immense potential in variousindustrial applications such as optoelectronic devices, 1

nonlinear optics,2 light-emitting diodes,3 a n d q u a n t u mdot lasers,4 to nam e a few. One of the goals in na notech-nology is the organization of nanoparticles in crystallinea r r a y s w i t h t h e a b i li t y t o t a i lor t h e s i z e a n d s e pa r a t i onof the na nopart icles a nd thereby the optical and electronicproperties of the as sembly.5 While assembly of nanopar-ticles from solution into hexagonally close-packed mono-layers a nd superlatt ice structures on solid surfaces hasm et w i t h a f a ir d eg r ee of s u cce ss ,6-9 the controlled

as sembly of nan opa rticlesin solu tionremains a relativelyunexplored a rea . The first st eps in this direction leadingto a rat ional nanoparticle assembly strategy were takenby t he groups of Mirkin 10 an d Alivisatos11 who demon-stra ted tha t D NA-modif ied colloidal gold na nopar ticlescould be a ssembled int o superstru ctures by hybridiza tion

of complementary base sequences in the surface-boundDN Am olecules. From a funda ment a l point of view, Mirkinet a l . have used this stra tegy to cr i t ical ly study the roleof interparticle separa tion a nd a ggregate size on the opticalproperties ofD NA-modified colloida l gold solution. 10c Otherinteractions such a s th e biotin-avidin molecular recogni-tion process,12 hydrogen bonding between suitable ter-minal functional groups bound t o the n an opar ticle sur-face,13 electrostatic assembly on D NA templates,14 a n dcontr ol over electrosta tic intera ctions sta bilizing aq ueous

* To wh om all correspondence should be ad dressed. Ph one: +9120 5893044. Fa x: +91 20 5893952/5893044. -m a il : s a s t r y @ems.ncl.res.in.

N a t ion a l C h e m ic a l L a b or a t or y . Tat a Research Development a nd D esign Centre.(1) Henglein, A. Top. Curr. Chem. 1988, 143, 113.(2 ) Ghe pre mic hae l, F . ; Kuz y k, M. G . ; L a c kri tz , H. S. Prog. Polym.

Sci. 1997, 22, 1147.(3) Colvin, V. L.; Schlamp, M. C.; Alivisatos, A. P. N a t u r e 1994,3 70,

354.(4) Alivisatos, A. P. Science1996, 271, 933.(5) Collier, C. P.; S ayka lly, R. J .; Shia ng, J . J . ; Henrichs, S. E.; Heat h,

J . R. Science1997, 277, 1978.(6) Exa mples of hexagonally ordered na noparticle assemblies by

solve nt e vaporat i on i ncl ude: (a) B rust , M.; Wal ker, M.; B e the ll , D . ;Schiffrin, D. J .; Whyma n, R. J . Chem. Soc., Chem. Commun . 1994, 801.(b)B rust, M.; B ethell, D.; Schiffrin, D. J .; Kiely, C. J .Adv. Mater. 1995,7, 795. (c) Ba dia, A.; G ao, W.; Singh, S .; Demers, L .; Cuccia, L.; Reven,L. L a n g m u i r 1996,12, 1262. (d)Wang, Z. L.; Harfenist, S. A.; Whetten,R. L . ; B e ntl ey , J . ; E vans, N. D . J . Phys. Chem. B 1998, 1 02, 3068. (e)Heath, J . R.; Knobler, C. M.; Leff, D. V. J. Phys. Chem. B 1997, 10 1,189. (f) Wang, Z. L. Ad v . M a t er . 1998, 1 0, 13. (g) Connolly, S.; Fullam,S.; Korgel, B.; Fitzamurice, D. J . Am . Chem. Soc.1998, 1 20, 2969. (h)Vi jay a Sarathy , K. ; Rai na, G. ; Yadav, R. T.; Kul karni , G. U . ; Rao, C .N. R. J. Phys. Chem. B 1997, 101, 9876.

(7) Exa mples of na noparticle immobilization on self-ass embledmonolayers include: (a) Chuma nov, G.; Sokolov, K.; Gr egory, B . W.;

Cott on, T. M.J . Phys. Chem. 1995,99, 9466. (b)C olvin, V. L.; Goldst ein,A. N.; Alivisatos, A. P. J . Am. Chem. Soc.1992, 1 14, 5221. (c) Grabar,K. C.; S mith, P . C.; Musick, M. D.; Da vis, J . A.; Walter, D . G.; J a ckson,M. A.; Gut hrie, A. P.; Na ta n, M. J . J . Am. Ch em. Soc.1996, 1 18, 1148.( d ) B a n d y o p a d h y a y , K . ; P a t i l , V . ; V i j a y a m o h a n a n , K . ; S a s t r y , M .L a n g m u i r 1997,13, 5244. (e)G ar cia, M. E.; B aker, L. A.; Crooks, R. M.Anal. Chem. 1999, 71, 256.

(8 ) E xampl e s of nanopart i c le superl at t i c es forme d usi ng di thi ollinkers include: (a) Brust , M.; B ethell, D.; Kiely, C. J .; Schiffrin, D. J .L a n g m u i r 1998, 1 4, 5425. (b) Musick, M. D.; Keating, C. D.; Keefe, M.H.; Nata n, M. J .C h e m . M a t er . 1997,9, 1499. (c)S ar at hy, K. V.; Thoma s,J . P . ; Kul karni , G. U . ; Ra o, C . N. R.J . Phys. Chem. B1999, 1 03, 399.

(9) Examples of na noparticlesuperlatt ices formed by the Lan gmuir-Blodgett m ethod include: (a) Fendler, J . H.; Meldrum, F . Ad v . M a t er .1995, 7, 607 and references therein. (b) Mayy a, K . S.; P at il , V.; Sa stry,M. L a n g m u i r 1997, 13, 2 57 5. ( c) Sastry , M.; May y a , K. S. ; P at i l , V. ;Pa ranjape , D. V.; He g de, S. G . J. Phys. Chem. B 1997, 101, 4954. (d)P a t i l, V .; M a y y a , K . S . ; P r a d h a n , S . D . ; S a s t r y , M . J . Am . Chem. Soc.1997, 119, 9281. (e) Mayya, K. S.; Sastry, M. J . Phys. Chem. B 1997,

101, 9790. (f)Ma yya , K. S.; Pa til , V.; Sa stry, M. J. Chem. Soc., FaradayT r a n s . 1997,9 3, 3377. (g) Mayy a, K. S.; Sa stry, M. L a n g m u i r 1998,1 4,74. (h) Mayya , K. S.; Pa til , V.; Kumar , M.; Sastr y, M.T h i n S ol i d F i l m s 1998, 3 12, 308. (i) Sastry, M.; Mayya, K. S.; Patil , V. L a n g m u i r 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.; S torhoff, J . J .N a t u r e 1996, 38 2, 607. (b) Storhoff, J . J . ; Mirkin, C. A. Chem. Rev.1999,99, 1849. (c)S torh off, J . J .; Laza rides, A. A.; Mucic, R. C.; Mirkin,C. A.; Letsinger, R. L.; Schat z, G. C. J . Am. Ch em. Soc. 2000,12 2, 4640.(d) Mirkin, C. A. Inorg. Chem. 2000, 39, 2258.

(11) Alivisat os, A. P .; J ohnsson, K . P .; P eng, X.; Wilson, T. E.; Low eth,C . J . ; Bruc he z, M. P . , J r .; Sc hul tz , P. G. N a t u r e 1996, 382, 609.

(1 2) (a) Sa stry , M.; L al a , N.; Pa t i l , V. ; C ha van, S . B . ; C hi t t i boy i na,A. G. L a n g m u i r 1998, 14, 4138. (b) Shenton, W.; Da vis, S. A.; Ma nn,S . Ad v . M a t er . 1999, 11, 449. (c) Fullam, S .; Ra o, S. N.; F itzma urice,D . J. Phys. Chem. B 2000, 104, 6164.

6262 L a n g m u i r 2001, 17 , 6262-6268

10.1021/la010536d CCC: $20.00 2001 American C hemical SocietyP ubl ish ed on Web 08/31/2001


2/7

nanoparticles15 h a v e b ee n u s ed t o a s s e m b le col loi d a lparticles in solution.

As pa rt of our ongoing st udies into the surface modi-fication, stabilization, and optical properties of colloidalgold 16 an d si lver 17 hydrosols, we report herein an opticalabsorption, laser light scattering, and transmission elec-tron microscopy (TEM) investigation of the reversibleaggregation of aqueous si lver colloids capped with theamino acid cysteine. The use of cysteine for stabilizinga queous colloidal silver pa rt icles a s opposed to 4-car boxy-thiophenol (4-CTP ) a s in the earl ier st udies 16,17 w a smotivat ed by the fol lowing reasons. While 4-CTP wa s

solubilized in wa ter w ith etha nol prior to capping the golda nd silver colloida l part icles,16,17 cysteine is wa ter solubleand would readily bind to the surfaceof the silver part iclesvia a thiolat e l inkage. Furt hermore, the surface layer ofcovalently bound cysteine molecules could cross-link thesilver pa rticles via h ydrogen bond forma tion (a s shown inScheme 1), while such a process would not ha ppen in silverparticles derivatized with fully ionized carboxylic acidgroups (as would be the case with 4-CTP capped silverparticles). 16 P resented below are detai ls of the investiga-tion.

Experimental Section

Chemicals.Silver sulfate, sodium borohydride, a nd L -cysteinewere obtained from Aldrich Chemicals and used as received.

SynthesisofColloidal SilverandCappingwith Cysteine.100 mL of 10-4 M concentra ted a queous solution of silver sulfat e(Ag2S O4) w a s reduced by 0.01 g of sodium borohydr ide (Na B H 4)

at r oom temperat ure to yield colloida l silver part icles as describedelsewhere. 17 The silver colloidal particles were stabilized bya ddition of 2 mL of aq ueous solution of 10-3 M l-cyst eine t o 100mL of the silver hydrosol. After a ddition of cysteine an d a gingthe silver colloidal solution for 7 h, the solution w as subjectedto ultracentrifugat ion an d the resulting pellet wa s wa shed withdeionized wa ter tor emovea ny uncoordinat ed cysteine molecules.A film of the silver part icles thus prepared wa s formed on a Si(111) wa fer and a na lyzed by Fourier tr an sform infrared (FTIR)spectroscopy. These measur ements were carried out in the diffusereflectan ce mode on a Shima dzu FTIR-8201 PC instrument ata resolution of 4 cm-1. The pH of the cysteine-capped silvercolloidal solution w as ca. 9. At t his pH, the cysteine moleculeson the surfa ce of th e silver part icles would be nega tively char ged(pI of cysteine ) 5.02)18 and thus stabilized electrostatically.

UV-Vis Spectroscopy Studies. The optical properties ofthe cysteine-capped silver colloidal solution were monitored asa f u n ct i on of t i m e on a He wl e t t -P a c kar d d i od e ar r a y s p e ct r o-photometer (model HP-8452) operated at a resolution of 2 nm.To underst a nd th e nat ure of cross-linking of the cysteine-cappedsilver particles in solution, UV-vis spectra were recorded for afully a ged colloidal solution (silver hydr osol, 7 h af t er cappingwith cysteine) as a function of tempera ture in the ra nge 20-90 C a t a h e a t i ng r a t e o f 0. 5 m i n-1. These measurements werema de on a P erkin-Elmer La mbda 15 UV-vis spectr ophotometeremploying a J ulabo wa ter circulat or with programm ed heatingaccessory. To understa nd the roleof salt in screening the repulsiveintera ctions between cysteine-capped silver part icles, UV-vi sm e as u r e m en t s we r e c ar r i e d ou t w i t h t i m e on t h e c ol l oi d als ol u t i on s t o w h i ch N a C l w as ad d e d i n t h e c on c en t r a t i on r an g e

(1-5) 10-1 M . Ap pr opr i at e c on t r ol m e as u r e m en t s we r eperformed.

Laser Light Scattering Measurements. The kinetics ofaggrega tion of the cysteine-capped silver colloidal part icles inthe presence of varying concentra tions of NaCl w as studied ona Hor i ba m od el L A-910 l a s e r l i g h t s cat t e r i n g p ar t i cl e s i z edistribution ana lyzer. Prior to measurement, t he silver solutionwas p as s e d t h r ou g h a 0.22 m membrane f i l ter. The opticalsystem consisted of a 1 mW He-Ne laser (632.8 nm) and a 50W tungsten halogen lamp as the l ight source. An 18-divisionring-shaped sil icone photodiode served as the detector. Thei n s t r u m e n t was c ap ab l e of ac c u r at e l y an d r ap i d l y m e as u r i n gthe size (an d part icle spread) of the aggregat es in solution in the

(13) (a) Weisbecker, C. S .; Merrit t, M. V.; Whitesides, G . M.L a n g m u i r 1996, 1 2, 3763. (b) J ohnson, S . R.; E van s, S. D .; Br ydson, R. L a n g m u i r 1998, 14, 6639. (c) Boal, A. K.; I lhan, F.; DeRouchey, J . E.; Thurn-Albrecht, T.; Russell, T. P.; Rotello, V. M. N a t u r e 2000, 40 4, 746.

(1 4) Kumar, A.; Pa t ta rki ne, M.; Bhadba de , M.; Dat ar, S . ; Dharma -dhi kari , C . V.; Gane sh, K. N.; Sastry , M. Ad v . M a t e r . 2001, 13, 341.

(15) (a) Shipw ay , A. N.; Laha v, M.; G aba i, R.; Willner, I . L a n g m u i r 2000,16, 8789. (b)Sh ipwa y, A. N.; Ka tz, E .; Willner, I. Ch emPh ysChem2000, 1, 19. (c) Templeton, A. C.; Zamborini, F . P .; Wuelfing, W. R.;Murray, R. W. L a n g m u i r 2000, 1 6, 6682. (d) Kim, Y.; J ohnson, R. C.;Hupp, J . T. N a n o L e t t .2001, 1, 165.

(1 6) May y a, K. S. ; P at i l , V. ; Sastry , M. L a n g m u i r 1997, 13, 3944.(1 7) Sastry , M.; B andy opadhy ay , K . ; May y a, K . S. Colloids Surf., A

1997, 127, 221.(18) Neal, A. L. C h em i s t r y a n d Bi o ch em i s t r y : A Comprehensive

I n t r o d u c t i o n ; McGra w-Hill: New York, 1971; p 389.

Scheme 1. Cartoon Showing the Assembly of Cysteine-Capped Silver Particles in Solution by HydrogenBonding of the Amino Acid Molecules on Di fferent Silver Particles and Disaggregation of the Superclusters by

Heating the Solution

R ever si bl e A ggr egat i on of Si l ver Par ti cl es L an gm u i r , V ol . 17, N o. 20, 2001 6263


3/7

r an g e 0.1-1000 m. These measur ements were carried out understeady stirring conditions.

Transmission Electron MicroscopyMeasurements.TEMm e as u r em e n t s we r e p er f or m e d on a J E OL m od el 1200E X

instrument operat ed at an accelerating volta ge of120kV. Sa mplesfor TEM st udies were prepa red by placing a drop of th e cyst eine-capped silver colloida l solution, a ged for different tim e interva ls,on ca rbon-coat ed TEM grids. A fully aged cysteine-cappedcolloidal silver solution (7 h of a ging) wa s heat ed at 90 C for 10m i n , an d t h i s s ol u t i on w as a l s o an a l y z ed b y TE M i n a s i m il arma nner. The f i lms on the TEM gr ids were a llowed t o dry for 1m i n f oll owi n g wh i c h t h e e xt r a s ol u t i on wa s r e m oved u s i n g ablott ing paper. TEM a na lysis wa s also carried out on a cysteine-capped colloidal silver solution wh ich wa s desta bilized by add itionof 3 10-1 M N aC l .

Results and Discussion

The a mino a cid cyst eine (formula, H 2N-CH(CH 2S H )-COOH) plays a n importa nt role in def ining the t ert iarystru cture of proteins th rough disulfide (cyst ine) bridges.

In t his st udy, t he free thiol groups in cysteine moleculeshave been used to bind to colloidal si lver and therebysta bilize th em electrosta tically. The surfa ce coordinat ionof the silver particles with cysteine was accomplished asdescribed in th e Experimenta l Section. Figure 1A showsthe FTIR spectrum recorded from the silver film in thespectral window 2450-2650 cm-1 (curve 1) a long withthe spectrum recorded from cysteine powder (curve 2).The prominent S-H vibrat iona l band centered at ca. 2555cm-1 is clearly seen in the free cysteine molecules (curve2) and vanishes on coordination of these molecules withcolloidal s ilver (curve 1). This is s tr ong evidence of surfa cebinding of cysteine to th e si lver pa rticles via a thiolat el in k a g e a n d a g r e e s w i t h e a r l ie r s t u d i e s o n a l ka n e t h i olmodif ication of gold nanoparticles by Murray and co-

workers. 19 Further evidence for the presence of surface-bound cysteine is provided by FTIR m easur ements of thesilver particlefilm in the spectra l window 1450-1750cm-1

(curve 1, Figure 1B). The car boxylate st retch vibra tion ofthe cysteine molecules is observed to occur at 1595 cm -1.It is well-known from stud ies on Lang muir-Blodgett filmsof meta l sal ts of fat t y a cids tha t t he posit ion of this bandis dependent on the na ture of the bound metal cat ion.20

The posit ion of the carboxylat e st retch vibration in thesilver cluster films suggest s some intera ction of the a cidgroup with other cysteine molecules, possibly through

hydrogen bonding. This view is strengt hened w hen oneconsiders tha t the carbonyl stretch vibrat ion of the a cidgroup in free cysteine molecules occurs a t 1650cm-1 (curve2, Figure 1B). U V-vis, TEM, and l ight scattering mea-surements indeed show significant cross-linking of thesilver colloidal particles via hydrogen bonding betweencysteine molecules loca ted on d ifferent silver part icles a sshown in Scheme 1 and discussed below.

Figure 2A shows the UV-vis spectra recorded from thecolloidal silver solution as a function of time of cappingwit h cysteine. Cur ves 1 a nd 3-5 in the figur e correspondto the spectra recorded a t t ime t) 0, 2, 5, and 7 h a f ter

capping the colloidal solution with cysteine, while curve2 is the spectrum recorded after aging the solution for 7h (curve 5)a nd h eat ing it t o 90 C for 10 min. The spectraha ve been shifted vertically for clar ity. In a ll th e spectra ,a s t r on g a b s or pt i on a t ca . 400 n m i s o bs er v ed t h a tcorresponds to excitation of surface plasmon vibrationsin the si lver particles.17 A comparison of curve 1 withcurves 3-5 i n d ic a t e s t h a t t h e r e i s a b r oa d e n i n g of t h esurfaceplasmon resonancewith timeof aging thecysteine-capped colloida l silver solution. The br oadening is espe-cially pronounced for the solution a ged for 7 h wit h clearevidence of the growth of an addit ional peak at ca. 500n m . N o f u r t h e r c h a n g e s w e r e o b s er v ed i n t h e U V-vi sspectra of the colloida l solution beyond 7 h of a ging. Theshoulder at 500 nm in the 7 h aged solution (curve 5) is

due to the appeara nceof a longitudinal plasmon resonancea nd is a consequence of overlap of the d ipole resona ncesbetw een neighboring silver pa rticles.17,21 Even though thesilver colloidal particles ar e sta bil ized electrosta tical lyby t he negat ively charged carboxylic acid groups of thesurface-bound cysteine molecules, the broadening ob-s e r v e d w i t h t i m e a n d t h e g r o w t h o f t h e l o n g i t u d i n a lplasmon resona nce is clearly due to slow aggrega tion ofthe silver pa rticles. We recollect t ha t in our earlier studieson th e optical pr operties of 4-CTP-ca pped silver colloida lparticles, the solution was extremely stable over manyw e e ks i n d ica t i n g t h a t d e t ect a b l e c r os s -l in k in g of t h e

(19) Templeton, A. C.; C hen, S .; G ross, S. M .; Murr ay , R. W. L a n g m u i r 1999, 15, 66.

(20) Pal, S. Ph.D. Thesis, Poona University, Pune, India, 1996.(21) Blatchford, C. G .; Ca mpbell, J . R.; Creighton, J . A. Su r f . Sc i .

1982, 120, 435.

Figure 1. (A) FTIR spectra of the cysteine powder (curve 2)and the cysteine-capped si lver particle fi lm in the spectralwindow 2450-2650 cm-1 (curve 1). (B ) FTIR spectra of th ecysteinepowder (curve 1)a nd t he cysteine-capped silver particlefi lm in the spectral w indow 1450-1750 cm-1.

Figure 2. (A) UV-vis spectra of si lver colloidal part iclesr e cor de d a s a funct i on of t i m e af t e r c appi ng w i t h cy st e inemolecules. Curve 1: UV-vis spectru m of the a s-prepared silverhydrosol immediat ely after capping with cysteine,t)0 h. Curve2: UV-vis spectrum of the cyst eine-capped silver sol aged for7 h (curve 5) an d heat ed at 90 C for 10 min. Curves 3, 4, a nd5: U V-vis spectra recorded at time t)2, 5, and 7 h aft er cappingthe silver hydrosol wit h cysteine molecules (see text for details).(B) Absorbance at 550 nm monitored as a function of temper-

ature of a cysteine-capped silver colloidal solution aged for 7h (see text for details).

6264 L an gm u i r , V ol . 17, N o. 20, 2001 M an d al et al .


4/7

part icles ha d not occurred. 17 The a ggrega tion of the silverpart icles of this stu dy could occur via hydr ogen bondingbetween the cysteine molecules on the surface of thenanoparticles, a process that could not occur with 4-CTP-capped si lver particles at high pH. 17 That the bondingbetween the particles is fair ly weak is indicated by theU V-vis spectru m of the a ged colloidal solution hea ted a t90 C for 10m in (curve 2). This spectr um is almost identicalto that of the silver solution recorded immediately aftercapping with cysteine (curve 1) showing that particles

m a y b e d is a g g r eg a t e d b y t h i s h ea t i n g p roce ss . Th ereversibilit y of th e a ggreg a tion of th e silver part icles uponheat ing the solution clearly highlights th e role of hydrogenbonds in t he a ggrega tion process. We recollect t ha t F TIRmeasurements (Figure 1) support the contention thathydrogen bonding is responsible for t he si lver pa rticleaggregation.

The presence of hydrogen bonds linking amine func-tiona l groups and ca rboxylat e ions in cysteine moleculesover neighboring particles is better studied by a morerefined melting ana lysis a s demonst ra ted by Mirkin andco-workers in their study of the optical properties ofcolloidal gold particles cross-linked by surface-bound DNAmolecules.10c U V-vis spectra of the 7 h aged cysteine-capped silver colloida l solution were recorded a s a functionof temperature, a nd a plot of the a bsorbance at 550 nmwit h tempera ture is shown in Figure 2B. (The absorba ncea t 550 nm w a s chosen since the most ra pid cha nges in theU V-vis spectra with temperature occurred at this wave-length.)Unti l50C,thereis l i t t lechangein theabsorbancea t 550 nm, but a bove th is temperat ure, a sha rp fall in thea bsorban ce occurs. Appropriat e cont rols were performedwherein the a s-prepared una ggregated sol wa s similar lyheated. Variat ion in the absorbance band a t 550 nm wa snot observed in th is case. The decrease in the a bsorban cea t 5 50 n m i n d ica t e s d i s p er s io n o f t h e p a r t i cl es i n t h eagg regat es, this process being almost complete by the timethe solution is heated to 90 C. This result is in agreementw i t h t h e U V-vis results presented in Figure 2A (curves1 and 2). The tra nsition region of decrea sing a bsorban ceis fairly broad in comparison to that observed by Mirkina nd co-w orkers for DN A-capped colloida l gold par ticles.10c

This dif ference ma y be due t o a spectrum of sl ightlydiffering bonding energies between t he cysteine moleculesan d may be a consequence of disorder in the surfa ce-boundcyst eine molecules or va ria tions in th e size of neighboringsilver particles.

D i r e ct e vi d en ce f o r t h e s t a t e of a g g r e g a t i on of t h ecyst eine-capped silver pa rt icles is provided by TE M stud ieswh ich are present ed in F igure 3. The TEM pictur e of thesilver colloidal solution immediately after capping withcysteine molecules is show n in Figure 3A. It is observedtha t the pa rticles ar e well dispersed with no evidence ofcross-linking. Figure 3B shows t he par ticle size distribu-

tion histogram for the accompanying TEM micrograph.The solid line is a Ga ussian f i t to the da ta an d results ina particle size of 4.4 ( 0.3 nm. Thus, the part icle sizedist ribut ion is rea sona bly monodisperse. The TEM m icro-graphs recorded from the cysteine-capped silver colloidalsolution at t ime t ) 2, 5 , a n d 7 h a r e s h ow n i n F i g u r e3C-E, respectively. Open, stringlike structures form att ) 2 h. Thereaf ter , the particles aggregate into largers t r u c t u r e s u n t i l a t t ) 7 h , t h e i n d i v i d u a l a g g r e g a t e smeasure close to 80-100 nm. The inferences from theU V-vis measurements discussed above are thus borneout by t he TEM s tud ies presented in Figure 3. Figure 3Fshows t he TEM picture recorded from the cysteine-cappedsilver colloidal solution a ged for 7 h an d t hen hea ted t o90 C for 10 min. The particles tha t ha d a ggregated into

l a r g e, ca . 100 n m s t r u ct u r es (F i gu r e 3E ) h a v e n ow dispersed to a very large extent. The structure of theaggr egat es is very similar to tha t observed for the cysteine-capped silver particle solution aged for 2 h (Figure 3C).This result is also in agreement with the UV-v i s d a t apresent ed ea rlier (curve 2, Figur e 2A). Thus , the U V-vi sa nd TEM result s clear ly show th a t significant cross-linkingof the cysteine-capped si lver particles occurs and fur-thermore that this process is reversible clearly pointingto hydr ogen bonding betw een the si lver part icles.

Screening of the repulsive interactions between thecolloidal particles by addition of salt should enhance therate of formation of aggregates and would thus provide

Figure3. (A) TEM m icrogra ph of the silver colloidal pa rticlesimmediately after capping with cysteine molecules (time t) 0h). (B) P art icle size distribution histogra m estimat ed from themicrograph sh own in Figure 2A. The solid l ine is a Ga ussianfi t to the da ta an d yields a si lver part icle size of 4.4 nm ( 0.3nm . TE M m i cr ogr aphs of t he si lve r hy d r osol c appe d w i t hcysteine as a function of t ime of aging: (C) t) 2 h, (D) t) 5h, (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 detai ls).



5/7

further insight into the kinetics of the a ggregat ion process.Figure 4Ashows t he UV-vis spectra recorded at differentt imes af ter a ddit ion of 3 10-1 M Na Cl to the cysteine-capped silver colloida l solution. These mea surement s weredone on freshly capped si lver colloidal solutions (andtherefore not a ged) to see the ef fect of added sa l t on t herat e of change in t he optical properties of th e colloidal

solution. The spectra recorded immedia tely a fter a dditionof sa lt (time t) 0 min), 30 min a fter a ddition of sa lt, a nd6 0 m in a f t e r a d d i t i on of s a l t a r e s h ow n a s cu r v es 3-5,respectively, in Figure 4A. For comparison, the spectrumrecorded from the cysteine-capped silver solution beforea ddit ion of sa lt is a lso shown (curve 1, Figur e 4A). A clea rlyresolved longitudinal plasmon resonance centered at ca.540 nm is observed to grow i m m edi at el y a f ter addit ion ofsalt tothe silver colloidal solution (curve 3). Furthermore,this resonance shif ts to the red at larger t ime intervalsand stabil izes at ca. 600 nm at 60 min af ter addit ion ofsal t . This result indicates tha t t he size of the a ggregatesincreases with t ime. I t is clear that the sal t acceleratesthe cross-linking of the colloidal particles by hydrogen-bonding intera ctions by effectively screening the repulsive

electrostaticinteractions between the negatively chargedsilver particles. The inset of Figure 4A shows the TEMm i cr og r a p h r e cor d e d f r om t h e [3 10-1 M N a C l ]-desta bilized silver solution, 60 min aft er ad dition of sa lt.The presence of large aggregates of silver particles canclear ly be seen and t hus corroborat es the UV-vis resultspresented a bove. As in th e case of cyst eine-capped silvercolloidal part icles aged a t room temperature in the absenceof salt, the salt-destabilized silver solution was heated at90 C for 10 min and t he UV-vis spectru m wa s recorded(curve 2, Figure 4A). It is clear that significant dispersionof the particles from the aggregates occurs on heating int h i s c a s e a s w e l l.

The changes in t he U V-vis spectra both during a gingof th e cysteine-ca pped silver solution (Figur e 2A)a nd aft er

addit ion of Na Cl (Figure 4A) provide (at best) indirectevidence for the aggregation of the si lver particles insolution. Furt hermore, the changes in the UV-vis spectr aon ad dition of salt (Figure 4A) a re too rapid t o be followedby UV-vis spectroscopy a lone. Consequently, the kineticsof aggregation of the cysteine-capped silver particles onaddition of di f ferent amounts of NaCl was fol lowed byl a s e r l i g h t s ca t t e r in g m e a s u r em e n t s , t h i s t e ch n i q u ep r ov id i n g a r a p i d e s t im a t e o f t h e m e a n a g g r e g a t e s i z e(a nd polydispersity) as a function of time in t he different

experiments. We also carr ied out l ight scattering mea-surements of the cysteine-capped si lver solution as afunction of t ime of aging t o para l lel the U V-vis study ofFigure 2A. However, in this case the size of th e aggrega tesin the fully aged solution was below the detection limitsof t h e i n s t r u m en t u s ed (1 00 n m ). Th i s r e s ul t i s i nagr eement with the TEM results tha t yielded a n aggregat esize in the range 80-100 nm for the fully aged sample(Figure 3E).

Figure 4B shows a plot of the mean a ggregate size asa f u n c t i o n o f t i m e a f t e r a d d i t i o n o f 1 10-1 M N a C l(circles ), 2 10-1 M NaC l (squa res), and 3 10-1 M N a C l(tr iangles) determined from l ight scattering measure-ments . While the agg rega tion is fairly slow in the case of

low Na Cl concentra tion (1 10-1

M) a nd contin ues up to60 min a f ter a ddit ion of sa l t , the particle size increasesrapidly (within 8 min) for higher sa l t concentra tions ins ol u t ion a n d s t a b i l iz es s oon t h e r ea f t e r . F u r t h e r , t h eequilibrium size of the a ggrega tes is considera bly higherin the case of higher Na Cl concentra tion in solution. Theseresults clearly point to electrosta tic sta bilization of thesilver part icles by the surfa ce-bound cysteine m oleculeswh ich may be effectively screened by a ddition of suitableamounts of NaCl in solution. That the screening leads tohyd rogen bonding (a nd consequent ly should be reversible)w a s t e s t e d b y h e a t i n g t h e [ 3 10-1 M N a C l ]-silversolution (tr ia ngles) at 90 C for 10 min a nd performinglight sca tt ering measurements on the heat ed sample. Thisexperiment resulted in a decrease in t he aggrega te size

from ca. 1.8 to ca. 1.44m (point shown as a diamond inFigure 4B). It is clear t ha t wh ile some disa ggrega tion hasoccurred on heating, the process is not completely revers-ible.

It would be instructive to brief ly discuss t he U V-vi sspectroscopy (Figure 4A) and light sca tt ering result s forthe salt-induced aggregation experiment (Figure 4B) inr el a t i on t o t h e U V-v i s a n d T E M s t u d i e s o f t h e a g e dcysteine-capped silver solution (Figures 2A a nd 3). In th ecase of the aged silver colloid, a w eak peak a t ca. 500 nmis observed wh ich almost completely vanish es on hea tinga t 90 C for 10m in (Figure 2A, curves 5 a nd 2, respectively).The size of the a ggrega tes formed on a ging is smaller tha n100 nm (below t he detection limits of the light scat tering

instrument, a lso Figure 3E). In deed, the spectrum fromthe hea ted solution is almost ident ical to the as-preparedcysteine-capped solution before a ging (curve 1, Figur e 2A).Another importa nt point to note is tha t t he position of theplasmon resona nce remains a t close to 400 nm even aftercomplete a ging of th e silver solution. The a bove togetherwith the TEM results (compare Figure 3A with Figure3F)clear ly indicate complete disaggr egat ion of the super-stru ctures formed in solution. In the ca se of th e [3 10-1

M Na Cl]-induced aggr egat ed silver solution, the changesi n t h e U V-vis spectra are ra pid and the a ddit ional peaka t longer wa velengths is much more pronounced (Figure4A). Heating this solution at 90 C for 10 min leads tos om e d e gr e e o f d i sa g g r e g a t i on a s e vi d en ce d b y l ig h tscatt ering measurements (Figure 4B).Another interesting

Figure 4. (A) UV-vis spectra of the cysteine-capped silvercolloidal solution recorded as a function of time after additionof 3 10-1 M NaCl. Curve 1: cysteine-capped silver solutionbefore a ddit ion of Na Cl. Curve 3: cystein e-capped silver solutionimmediately after addition of NaCl (t ime t ) 0 h). Curve 4:cysteine-capped silver solution 30 min after addition of NaCl.Cur ve 5: cyst eine-capped silver solution 60 min a fter a dditionof NaC l . C ur ve 2: t he spe ct r um r e cor de d fr om t he NaC l -desta bilized cyst eine-capped silver colloida l solution shown a scurve 5 after heating at 90 C for 10 min. The inset shows aTEM m icrogra ph of the cyst eine-capped silver solution desta -

bilized by addit ion of 3 10-

1 M of NaC l (see text for deta ils).(B) Mean particle diameter, dh , measured by l ight scatteringmeasurements a s a fun ction of t ime after addition of 1 10-1

M NaCl (circles), 2 10-1 M NaCl (squares), and 3 10-1 MNaCl (triangles). The point represented by the diamond givesthe size of the aggrega tes in the 3 10-1 M NaC l destabil izedsilver sol (trian gles) aft er heat ing a t 90 C for 10 min (see textfor details).



6/7

feat ure is th a t the surfa ce plasmon resonan ce in the UV-vis spectra shifts from 400 nm before addition of salt to415 nm af t er Na Cl addit ion (Figure 4A). Furt hermore,heating the solution did not restore the posit ion of theresonance which remained at 415 nm (curve 2, Figure4A). This is a striking difference in the UV-vis beha viorof the cysteine-capped si lver solution aged for 7 h andthat destabilized by addition of salt. The UV-vis resultstaken together with the l ight scattering measurementst h u s i n d i c a t e t h a t e v e n t h o u g h t h e a s y m m e t r y i n t h e

p la s m on r es on a n ce d is a p pe a r s on h ea t i n g t h e s a l t -aggrega ted solution (and t hus might lead one to errone-ously conclude th a t complete disa ggrega tion ha d occurred),the posit ion of the resonan ce may be used a s a measureof th e extent of aggrega tion. The size of the a ggrega tes inthe case of the [3 10-1 M NaC l]-induced aggrega tedsilver solut ion (ca. 1.8 m, Figure 4B) is much larger tha nthose observed for t he 7 h a ged silver solution (0. 1 m ,F i g u r e 3 E ) , a n d c o n s e q u e n t l y t h e e n e r g y r e q u i r e d t odestabil ize the assembly would be much higher in thesa lt-desta bilized solution. This would explain th e part ialdisaggregation of the 1.8 m superassemblies by heatinga t 9 0 C w h i ch ot h e r w i s e i s s u ff ic ie n t t o co mp le t el ydisaggregate the smaller structures.

The ra te of aggr egat ion of the colloida l part icles can bequanti f ied in terms of a semiempirical f locculation pa-r a m e t e r a s f i r s t d e m o n s t r a t e d b y W h i t e s i d e s a n d c o -workers. 13a The def init ion of this parameter has beenmodif ied by some of us and applied to the problem ofa ggrega tion of 4-CTP -capped gold pa rticles16 a s w e ll a savidin-induced agg regat ion of biotinyla ted silver an d goldparticles in earlier studies. 12a The ba sic idea behind t hispara meter is the following , the exa ct deta ils of w hich maybe obtained from our earlier work.12a,16 As shown by t heU V-v i s m e a s u r em e n t s , a g g r e g a t i on o f t h e co ll oi d a lpart icles is accompa nied by a broa dening of the pla smonresonan ce feat ure. Therefore, the a rea un der the U V-vi scurve spanning a suitable integration range (encompass-ing the surfa ce plasmon vibrat ion) should be a suitablemeasure of the extent of aggregation and is termed theflocculat ion para meter. 12a,16 The integra tion limits for theflocculat ion par a meter were ta ken as 400-800 nm in th iss t u d y . F i g ur e 5 A s h ow s t h e v a r i a t i on of f loc cu l a t i onparameters as a function of t ime of aging of the si lvercolloidal nan opart icles derivat ized by cysteine in thea bsence of sa lt. The para meter increases ra pidly initiallyand sta bilizes a f ter 7 h of aging. On heating t he 7 h agedcysteine-capped silver colloidal solution at 90 C for 10min, the f locculat ion para meter fal ls to nearly the sta r t ingv a l u e (a t t i m e t ) 0 h , p o i n t i n d i c a t e d b y a n a r r o w i nFigure 5A). The use of th e floccula tion pa ra meter in thiscase enables us to follow the aggregation behavior of thep a r t i cl es w h i ch w a s n ot d e t ect a b le b y l ig h t s ca t t e r in gmeasurements.

The a ggrega tion beha vior of th e silver colloida l par ticlesa f t e r a d d i t i on of d i ff er e n t c on ce n t r a t i on s o f s a l t a n ddifferent aging times has a lso been stud ied in terms of th ef locculation para meter , and t he dat a obta ined are shownin Figure5B . The da ta corresponding to tria ngles, squa res,and circles are the flocculation parameters calculated 60min, 30 min, an d immediat ely after a ddition of salt to thecyst eine-capped silver s olution, respectively . The inset ofFigure 5B shows a plot of the var iat ion in mean a ggrega tesize, dh , w ith NaC l concentra tion in the cysteine-cappedsilver colloidal solution, taken from the light scatteringdat a shown in Figure 4B at t imet) 0 min (circles), 4 min(s q u a r e s ), a n d 8 m i n (t r i a n g l es ). Th e t r e n d s i n t h eaggrega te size varia t ion with sal t concentra tion closelyp a r a l l el t h a t ob s er v ed i n t h e f loc cu l a t i on p a r a m e t e r

calculations based on the UV-vis results (main part ofFigure 5B ) with the importa nt d i f ference being th at thetime scales are vastly di f ferent. While the f locculationparameter continues to change even 30-6 0 m i n a f t e raddit ion of sal t , size sta bilization as determined by l ightscatt ering measurements occurs within 10m in of additionof sa lt. This difference may be a consequen ce of th e nat ureof def init ion of the f locculation parameter . I t may alsos u g g es t t h a t e ve n t h o u g h t h e s i z e o f t h e a g g r e g a t e s i sinvarian t w ith t ime, small cha nges in the internanopar-

ticle separation in the aggregates could occur due to aslower time scale associated w ith t his process. It is knownt h a t b o t h t h e i n t e r p a r t i c l e s e p a r a t i o n a n d s i z e o f t h eaggrega tes contr ibute to changes in the U V-vis spectraof noble metal colloids 10c and this process of comparisonof l ight sca ttering dat a a nd f locculation para meters maybe used to separat e the t wo contr ibutions. However, weha sten toa dd that this is purely speculat ivea t the moment.I t i s ob s er v ed t h a t t h e f locc ul a t i on p a r a m e t e r /m e a naggrega te sizeincreases with increasing salt concentra tionfor a l l the measurement t imes a nd is a result consistentwith enhanced hydrogen bond formation due to betterscreening of the repulsive electrosta tic intera ctions by thes a l t . An ot h e r i n t er e st i n g o bs e rv a t i on f r om t h e d a t apresented in Figure 5B is tha t at sma ll sa lt concentra tionsthe f locculat ion pa ra meters/mean aggrega te size a t thed i f f e r e n t t i m e s a r e n e a r l y c o n s t a n t w h i l e a t h i g h s a l tconcentra tions th ere is significa nt deviat ion. This indicat estha t t he rat e of aggrega tion by hydrogen bond formationis critically determined by the extent of screening of theelectrostatic repulsive forces acting across the colloidalparticles.

Conclusions

The surfa ce modifica tion of colloida l silver par ticles w iththe am ino acid cysteine ha s been described. The si lverpart icles capped wit h cysteine ar e sta bilized in an aq ueousmedium by charging of the carboxylic acid groups in thesur fa ce-bound cyst eine molecules. The presence of amin e

Figure 5. (A) Flocculat ion para meters calculated for th ecysteine-capped si lver colloidal solution (from the UV-vi sspec t r a show n i n Fi g ur e 2A) plot t ed as a funct i on of t i m e(ci r cl es). Aft e r he at i ng t he ag e d sol , t he r e d uc t ion i n t heflocculation para meter is shown separat ely by a squa re (withan arrow, see text for detai ls). (B) Flocculation parameterscalculated from U V-vis spectra recorded a s a fun ction of timeofa ddition ofd ifferent concentra tions ofNa Cl to cysteine-cappedsilver colloidal solution: (circles) immediat ely after a ddition ofNaC l, (squa res) 30 min a fter a ddition of NaC l, and (trian gles)60 min after addition of NaCl (see text for details). The insetshows a plot of the mea n par ticle diameter d, estimated fromlight scattering mea surements a s a function of NaC l concen-tra tion at various t imes: (circles) 0 min, (squares) 4 min, an d(trian gles) 8 min.



7/7

and carboxylic acid functiona l groups on t he surfa ce ofthe colloidal particles leads to hydrogen bond formationa nd t hereby cross-linking of the colloida l silver par ticles.This process is reversible, a nd the dispersion of theparticles from the aggregates may be accomplished byheating the solution a bove 60 C . The ra te of hydrogenb on d f or m a t i o n m a y b e a cce le r a t e d b y s cr e en i n g t h eelectrostatic repulsive forces between the particles withsalt . The protocolba sed on amino acid derivat ized colloidal

par ticles ma y be extended to cont rolled cross-linking usingspacer molecules and is currently being pursued.

Acknowledgment. S .M. an d A.M.G. tha nk the Coun-cil of Scienti fic and I ndustr ia l Research (CS IR) and theUniversi ty Grants Commission (UGC), Government ofIndia, for research fellowships.

LA010536D


studies on the reversible aggregation of cysteine-capped

Documents