Electrochemical, Spectroscopic, and Mass Spectrometric Studies of the Interaction of Silver Species with Polyamidoamine Dendrimers

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1 Anal. Chem. 2005, 77, Eletrohemial, Spetrosopi, and Mass Spetrometri Studies of the Interation of Silver Speies with Polyamidoamine Dendrimers Fu-Ren F. Fan, Carolyn L. Mazzitelli, Jennifer S. Brodbelt, and Allen J. Bard* Department of Chemistry and Biohemistry, The University of Texas at Austin, Austin, Texas Eletrohemial, spetrosopi, and mass spetrometri (MS) methods were used to probe the interation (omplexation) of silver ions and zerovalent silver speies with polyamidoamine generation 1 amine-terminated (PAMA- MG1NH 2 ) and generation 2 hydroxy-terminated (PAMA- MG2OH) dendrimers (DDMs). Stability onstants (K q + ) and stoihiometries (q) (i.e., the number of silver ions omplexed per DDM moleule) were determined from the voltammetri data, that is, shifts in potential and hanges in peak or limiting urrent with addition of DDM. When the mole ratio of DDM to Ag + is g1, Ag + binds with PAMAMG2OH to form a dominant 1:1 omplex with a K 1 + value of M -1. Under similar onditions, Ag + binds with PAMAMG1NH 2, yielding a 1:1 omplex with K 1 + ) M -1, whih is onsistent with the finding of the MS experiments. When the mole ratio is <1, q g 2. The E 0 of the Ag-PAMAMG1NH 2 +/0 ouple shifted to a more negative value than that of the Ag +/0 ouple. The negative shift in the halfwave potential also suggests that DDM binds more strongly with Ag + than with zerovalent silver speies. Spetrosopi results suggest that hydroxyl-terminated PAMAMG2OH favors the formation of small zerovalent silver lusters after redution while amine-terminated PAMAMG1NH 2 allows for simultaneous formation of both lusters and larger nanopartiles at similar onditions. Other quantities, suh as diffusion oeffiients of the omplexes and molar absorptivity of the Ag + DDMs, are also reported. We desribe here eletrohemial (EC), spetrosopi, and mass spetrometri (MS) studies of the interation of silver ions and zerovalent silver lusters with two dendrimers (PAMAM DDMs). We report the dependene of the EC behavior on the nature of the ligands oordinated to the metal enters and their onentrations. The appliation of EC methods to the study of oordination of omplexing agents to metal ions provides a useful omplement to the spetrosopi and mass spetrometri tehniques. 1 Equilibrium onstants, K, for the omplexation of the metal ions and * Corresponding author. ajbard@mail.utexas.edu. (1) (a) Bard, A. J.; Faulkner, L. R. Eletrohemial Methods, Fundamentals and Appliations; New York: John Wiley & Sons, (b) Determination of Stability Constants; Rossotti, F. J. C., Rossotti, H., Eds.; MGraw-Hill: New York, 1961, Chapter 8. () Zhou, L.; Russell, D. H.; Zhao, M.; Crooks, R. M. Maromoleules 2001, 34, zerovalent metalli speies with different omplexing agents, suh as PAMAM DDMs, an be obtained from shifts in the peak potential, E p, or halfwave potential, E 1/2, and the number, q, of metal ions per DDM involved in binding. They an also 1 be obtained from the dependene of the urrent during redution of the omplexed ions on the effetive mole ratio of metal ions to the DDM ligands added. 1a The diffusion oeffiient, D, and the number of eletrons involved in the eletron-transfer reation, n, an also be determined by EC methods. Currently, DDMs are of interest beause of their unusual hemial and physial properties and also their potential appliations in suh diverse fields as mediine, biology, hemistry, physis, and material siene. 2,3 For example, DDMs have served as templates for nanopartile syntheses, 4 and reently, very small DDM-enapsulated silver nanodots were reported to be highly luminesent 5 and also show antimirobial ativity. 6 PAMAM an stabilize small metal lusters of defined size while the surrounding polymeri matrix protets the developing lusters against agglomeration following redution. 7 By ontrolling the hemial struture and size of the DDMs, differently sized soluble lusters that are suitable for EC, spetrosopi, and mass spetrometri studies an be prepared. One reason for ontrolling the size of metal lusters or nanopartiles is to study the strong size dependene of their photohemial, eletroni, and optial properties, as reported earlier by Henglein 8 for polyphosphate-enapsulated lusters of less than 10 silver atoms. In the present ase, (2) (a) Newkome, G. R.; Vögtle, F. Dendrimers and Dendrons; Wiley-VCH: Weinheim, (b) Dendrimers and other Dendriti Polymers; Fréhet, J. M. J., Tomalia, D. A., Eds.; Wiley: New York, (3) For some reent reviews, see, for example: (a) Heht, S.; Fréhet, J. M. J. Angew. Chem., Int. Ed. 2001, 40, 75. (b) Gorman, C. B.; Smith, J. C. A. Chem. Res. 2001, 34, 60. () Balzani, V.; Ceroni, P.; Juris, A.; Venturi, M.; Campagna, S.; Puntoriero, F.; Serroni, S. Coord. Chem. Rev. 2001, , 545. (d) Grayson, S. M.; Fréhet, J. M. J. Chem. Rev. 2001, 101, (e) Majoral, J.-P.; Caminade, A.-M.; Maraval, V. Chem. Commun. 2002, (f) Diederih, F.; Felber, B. Pro. Natl. Aad. Si. U.S.A. 2002, 99, (g) Gittins, P. J.; Twyman, L. J. Supramol. Chem. 2003, 15, 5. (4) (a) Crooks, R. M.; Zhao, M.; Sun, L.; Chehik, V.; Yeung, L. K. A. Chem. Res. 2001, 34, 181. (b) Baars, M. W. P. L.; Meijer, E. W. Top. Curr. Chem. 2001, 210, 131. (5) Zheng, J.; Petty, J. T.; Dikson, R. M. J. Am. Chem. So. 2003, 125, (6) Balogh, L.; Swanson, D. R.; Tomalia, D. A.; Hagnauer, G. L.; MManus, A. T. Nano Lett. 2001, 1, (7) Crooks, R. M.; Lemon, B. I.; Sun, L.; Yeung, L. K.; Zhao, M. Q. Dendrimer- Enapsulated Metals and Semiondutors: Synthesis, Charaterization, and Appliations. In Dendrimers III: Design, Dimension Funtion; Vögtle, F., Ed.; Springer-Verlag: Berlin, 2001; Vol. 212, pp (8) Henglein, A. Chem. Rev. 1989, 89, /a048133g CCC: $ Amerian Chemial Soiety Analytial Chemistry, Vol. 77, No. 14, July 15, Published on Web 05/25/2005

2 the threshold size of a silver nanopartile is determined by the appearane of the plasmon peak in the UV-vis spetra, as disussed below. We are partiularly interested in the EC behavior of very small (a few atoms) silver lusters and their biologial properties. Figure 1. A series of yli voltammograms (CVs) of 0.12 mm Ag + in 0.2 M NaNO 3 at an inlaid Au disk eletrode (radius 0.2 m) in the absene and presene of various PAMAMG1NH 2 onentrations. Potential san rate, v ) 0.1 V/s. Curve 1, no DDM; urve 2, 0.01 mm DDM; urve 3, 0.03 mm DDM; urve 4, 0.05 mm DDM; urve 5, 0.10 mm DDM. Initial potential and san diretion are labeled as S and arrow. In this paper, we desribe the appliation of EC measurements to quantify the binding of silver ion and zerovalent silver to PAMAM DDMs. Binding is interpreted in terms of the omplexation of silver speies with PAMAM as the ligands. We hose to begin these investigations with the low-generation PAMAM (G1, G2) to demonstrate the feasibility, usefulness, and also the limitations of various tehniques for the haraterization of metal- DDM omplexes and nanoomposites. EXPERIMENTAL SECTION Materials. Amine-terminated generation 1 PAMAM (PAMA- MG1NH 2 ) and OH-terminated generation 2 PAMAM (PAMA- MG2OH) dendrimers were purhased from Dendriteh, In. (Midland, MI) or Aldrih Chemial Co. (Milwaukee, WI) and were used without further purifiation. Silver perhlorate (99.99% purity) and anhydrous hydrazine were purhased from Aldrih Chemial Co. and were used as reeived. All other hemials were reagent grade and were used without further purifiation. Solutions were prepared using high-purity water (F)18 MΩ-m) from a Millipore Milli-Q water purifiation system. Instrumentation and Proedures. All EC measurements were performed with a Prineton Applied Researh (PAR) model 173 potentiostat/175 universal programmer/176 urrent-to-voltage onverter (or 179 digital oulometer) interfaed with a ustomdesigned data aquisition system. Ultraviolet-visible (UV-vis) spetra were obtained on a Milton Roy Spetroni 3000 Array Spetrophotometer with a 1-m quartz uvette. Eletrospray ionization (ESI)-mass spetra were taken on a ThermoFinnigan LCQ Duo quadruple ion trap mass spetrometer (San Jose, CA). A Harvard syringe pump (Holliston, MA) set at a flow rate of 3 µl/min was used to diretly infuse the sample solutions into the mass spetrometer. The ESI soure was operated in the positive ion mode with an eletrospray voltage of 4.0 kv and a heated apillary temperature ranging from 150 to 170 C. Sheath and auxiliary gas flow rates of 30 and 10 arbitrary units, respetively, were used to aid in desolvation. The base pressure of the trap was Torr. Spetra were aquired by summing 20 sans, eah omposed of 15 mirosans, with an ionization time of 100 ms. Collisional ativated dissoiation (CAD) experiments were performed on seleted dendrimer omplexes by isolating the desired preursor ion in the trap using resonane ejetion, followed by inreasing the ollision energy applied to the trap to produe fragmentation. All EC experiments were performed in a three-ompartment ell. The working eletrodes were either an inlaid Au disk (Bioanalytial Systems, West Lafayette, IN) with a geometri area of 0.13 m 2 or an inlaid ustom-made Pt disk of a geometri area of m 2. The working eletrodes were polished prior to eah series of experiments with 0.25-µm diamond paste (Buehler, Lake Bluff, IL) on a nylon loth and then subjeted to ultrasoni leaning for 5 min in 95% ethanol. A saturated alomel eletrode (SCE) was used as the referene eletrode in all experiments. The SCE was onneted to the ell through a salt bridge ontaining 0.2 M NaNO 3, whih was also used as the supporting eletrolyte in all EC measurements. A Pt flag served as ounter eletrode. Prior to all EC measurements (unless otherwise stated), where appropriate, solutions were deoxygenated via purging with N 2 or Ar gas for min. During measurements, a stream of N 2 or Ar was passed over the solution. Solutions for spetrosopi measurements were not deoxygenated, unless otherwise mentioned. All experiments were arried out at ambient temperature (24.5 ( 0.5 C). RESULTS EC Measurements. Silver-PAMAMG1NH 2 System, Cyli Voltammetry. A series of yli voltammograms (CVs) of 0.12 mm Ag + at an inlaid Au disk eletrode (radius, 0.2 m) in the absene and presene of various PAMAMG1NH 2 onentrations are shown in Figure 1. CV of Ag + at san rate, v, of 100 mv/s, in the absene of DDM (urve 1 of Figure 1) showed redution of Ag + to Ag 0 at a athodi peak potential, E p,of V (or halfwave potential,,of V) vs SCE. In this system, Ag 0 deposits on the E 1/ Analytial Chemistry, Vol. 77, No. 14, July 15, 2005

3 Figure 3. San rate dependene of CV for a solution ontaining mm AgNO 3 and 0.12 mm PAMAMG1NH 2 in 0.2 M NaNO 3 at a 4-mm-diameter Au disk. San rate 10 (urve 1), 20 (urve 2), 50 (urve 3), 100 (urve 4), 200 (urve 5), and 500 mv/s (urve 6). Figure 2. E 1/2 (frames A and B) and i p (frames C and D) values as funtions of the onentration of DDMs at a fixed [Ag + ] ) 0.12 mm. Squares, PAMAMG1NH 2; diamonds, PAMAMG2OH. San rate v ) 0.1 V/s. Frames B and D are the expanded sales of frames A and frame C, respetively. eletrode and is stripped during reoxidation of Ag 0 upon san reversal with an anodi peak potential, E pa of V. In the presene of 0.01 mm PAMAMG1NH 2, with 0.12 mm Ag +, E p ) V and E pa ) V vs SCE. Thus, both the E p and E pa shift to more negative values as ompared to a solution without PAMAMG1NH 2. In this ase, the E p and E pa shifted to more negative potentials by 60 and 38 mv, respetively, indiating that PAMAMG1NH 2 -omplexed Ag + is more diffiult to redue than unomplexed Ag +, whereas omplexed Ag 0 is easier to oxidize than bulk silver metal. Note that in the presene of DDM, the oxidation peak is harateristi of a solution speies rather than a stripping peak, showing that Ag 0 does not form bulk metal on the eletrode, but rather is present as a diffusing speies in solution. In addition, the absene of any obvious Ag stripping peak suggests that at this ratio of analytial onentrations, C Ag + /CDDM ) 12, almost all of the Ag + is omplexed. Both the E p and E 1/2 depended on the mole ratio of DDM to total Ag + onentration. Sine E 1/2, as ompared with E p in the present ase, an be determined more easily and reliably, we summarize in Figure 2 the E 1/2 values and the athodi peak urrents, i p, as funtions of the onentration of DDMs at a fixed Ag + onentration of 0.12 mm. A limiting shift of 0.16 V in E 1/2 was obtained for the Ag + -PAMAMG1NH 2 system at C DDM ) 1.5 mm. Reversibility of an eletron-transfer reation hanged in the presene of a DDM. As shown in Figure 3, in a solution ontaining 0.12 mm Ag + and 0.1 mm PAMAMG1NH 2, the E p shifts slightly to more negative values with inreasing san rate, while the E pa was nearly independent of v in the range of 5 < v < 500 mv/s, indiating that the eletron-transfer reations are fast. For a Nernstian reation, 1a i p ) n 3/2 AC* b D 1/2 v 1/2 (1) in whih A is the surfae area of the eletrode, C b / is the bulk Figure 4. CVs in a 0.2 M NaNO 3 solution ontaining mm Ag + -PAMAMG1NH 2 (0.118 mm AgNO 3 and 0.12 mm PAMAMG1NH 2) after redution with addition of different onentrations of hydrazine. Curve 1, 0.05 mm hydrazine; urve 2, CV in a 0.2 M NaNO 3 solution ontaining only 0.05 mm hydrazine; urve 3, mm hydrazine with omplex. onentration of the metal omplex, and the other quantities are previously defined. For a solution in whih the ratio of DDM to Ag + was high, the i p was proportional to v 1/2 (not shown) with a slope of the (i p vs v 1/2 ) plot yielding a D value of m 2 /s for PAMAMG1NH 2 -omplexed Ag +, assuming the redution was a reversible, one eletron-transfer proess. As will be disussed later, an experiment on the titration of Ag + with PAMAMG1NH 2 DDM showed that a (1:1) (Ag + /PAMAMG1NH 2 ) omplex was the major silver speies existing in suh a solution. The 1:1 PAMAMG1NH 2 -omplexed Ag + an be further transformed into DDM-bound zerovalent silver speies by adding a suitable reduing agent. There are several frequently used reduing agents, suh as borohydride, gluose, itrate, or hydrazine; however, signifiant rates of redution of Ag + by itrate or gluose an only take plae at elevated temperatures. To arry out the redution at a reasonably high rate at room temperature with minimum amounts of unwanted produts and large silver nanopartiles (not small lusters), hydrazine was used as the reduing agent. As shown in urve 3 of Figure 4, the addition of hydrazine (e1/4 of [Ag + ]) into a 1:1 PAMAMG1NH 2 /Ag + solution redued the omplex partially, as shown by the enhaned oxidation urrent when the eletrode potential was sanned positive from the initial rest potential ( -0.2 V vs SCE). A broad urrent peak assoiated with a diffusion-limited eletron transfer of a solution speies, rather than a sharp stripping peak assoiated with a surfae film, was obtained. Notie that this oxidation peak ourred at a potential more positive than that for hydrazine oxidation (urve 2, Figure Analytial Chemistry, Vol. 77, No. 14, July 15,

4 Figure 5. A series of CVs of 0.12 mm Ag + in 0.2 M NaNO 3 at an inlaid Au disk eletrode (radius 0.2 m) in the absene and presene of various PAMAMG2OH onentrations. Potential san rate, v ) 0.1 V/s. Curve 1, no DDM; urve 2, 0.1 mm DDM; urve 3, 0.3 mm DDM; urve 4, 0.6 mm DDM; urve 5, 1.0 mm DDM. 4) but less positive than that for bulk silver oxidation. We attribute this peak to the oxidation of a zerovalent silver speies. The formation of zerovalent silver speies is onfirmed by UV-vis spetra of the solution, as disussed later. The anodi peak urrent, i pa, inreased and the E pa shifted to more negative values with an inreasing onentration of hydrazine (e.g., 1/2 of [Ag + ] for urve 1 of Figure 4) due to the formation of more Ag 0 speies and, finally, from a ontribution for the oxidation of both Ag 0 speies and exess hydrazine. Chronoamperometry. A typial urrent vs time (i vs t) urve for a potential step (+0.35 to 0VvsSCE) experiment at an Au disk (radius 0.2 m) in a 0.2 M NaNO 3 solution ontaining 0.12 mm Ag + is shown in the Supporting Information setion, Figure S1A. The orresponding i vs t -1/2 plot is shown in Figure S1B. The i vs t -1/2 plot showed good linearity over the time domain between 1 and 40 ms. A small offset of a few miroamperes, assoiated with the small bakground urrent, was observed in the interept of the linear fitting line. For a diffusion-limited eletron-transfer reation at a marodisk eletrode, the slope of the linear i vs t -1/2 plot is nfac*(d/π) 1/4, 1a where F is the Faraday onstant and the other symbols are as defined previously. From the slope of the i vs t -1/2 plot, we determine a D value of m 2 /s for free Ag +, with nc* ) mol/m 3. This value agrees reasonably well with reported values. 9 A similar potential step experiment was arried out at the same disk after the addition of 0.1 mm PAMAMG1NH 2 into the Ag + solution. Good linearity with a nearly zero interept of the orresponding i vs t -1/2 plot was also found over a time domain longer than 10 ms (Figure SI2). The slope of the i vs t -1/2 plot with nc* ) mol/m 3 yields a D value of m 2 /s for the silver-pamamg1nh 2 omplex, that is, 2/3 that of unomplexed Ag + and slightly smaller than that determined from CV. Silver-PAMAMG2OH System, Cyli Voltammetry. Figure 5 shows a series of CVs of 0.12 mm Ag + analogous to those in Figure 1, exept in solutions ontaining various onentrations of PAMAMG2OH. As with Ag + -PAMAMG1NH 2, the addition of DDM into an Ag + solution auses the E p to shift to more negative values. However, the limiting shift in E 1/2 is smaller and estimated to be only V (see Figure 2). Although signifiant (9) (a) Meites, L. J. Am. Chem. So. 1951, 73, 395. (b) Meites, L. J. Am. Chem. So. 1951, 73, () West, P. W.; Dean, J.; Breda, E. J. Collet. Czeh. Chem. Commun. 1948, 13, 1. Figure 6. (A) San rate dependene of CV for a solution ontaining mm AgNO 3 and 0.12 mm PAMAMG1NH 2 in 0.2 M NaNO3 at a 4-mm-diameter Au disk. San rate: 10 (urve 1), 20 (urve 2), 50 (urve 3), 100 (urve 4), 200 (urve 5), and 500 mv/s (urve 6). (B) The orresponding san rate dependene of i p. reoxidation of omplexed Ag 0, as ompared with that of bulk silver metal, is observed at more negative potentials, the anodi urrent peaks are at more positive potentials, suggesting slower heterogeneous oxidation kinetis, as ompared to the oxidation of bare silver. As shown in Figure 6A, in a solution ontaining 0.12 mm Ag + and 0.1 mm PAMAMG2OH, the E p was nearly independent of v in the range of 5 < v < 500 mv/s, while the E pa shifted to more positive values with inreasing v, indiating slow kinetis for oxidation. The redution urrent, i p, (taken at E ) 0.2 V), was proportional to v 1/2 (see Figure 6B) and like the E p, depended on the onentration of DDM for a fixed total [Ag + ]. The i p as a funtion of the onentration of PAMAMG2OH at a fixed total [Ag + ] ) 0.12 mm is given in Figure 2. Chronoamperometry. A potential step experiment was also arried out, as with the G1NH 2 DDM, on the silver- PANMAMG2OH system. The i vs t urve in a solution ontaining 0.12 mm Ag + and 0.10 mm PAMAMG2OH is shown in the Supporting Information setion, Figure SI3A. The linearity of the orresponding i vs t -1/2 plot was good over a reasonably wide time domain, as shown in Figure SI-3B. By taking nc* ) mol/m 3, the slope of the i vs t -1/2 plot resulted in a D value of m 2 /s for this omplex, 1/5-1/6 that of unomplexed Ag +, onsistent with the muh higher moleular weight of the omplex ( 32 times), as ompared with free Ag +. The diffusion oeffiients of Ag + and 1:1 Ag + /PAMAM omplexes obtained from hronoamperometry are summarized in Table 1. Ultraviolet-Visible Spetrosopi Measurements. Comparison of the UV-vis spetra of various silver solutions with or without DDMs and before or after redution learly demonstrates the silver-ddm interation. Figure 7A shows the UV-vis spetra of PAMAMG1NH 2 at different onentrations. The absorption 4416 Analytial Chemistry, Vol. 77, No. 14, July 15, 2005

5 Figure 7. (A) UV-vis spetra of PAMAMG1NH 2 at different onentrations in water without Ag + : 0.01 (urve 1), 0.02 (urve 2), 0.03 (urve 3), 0.04 (urve 4), 0.05 (urve 5), 0.07 (urve 6), 0.1 (urve 7), and 0.3 mm (urve 8). (B) UV-vis spetra of PAMAMG1NH 2 at different onentrations in water with 0.12 mm Ag + : 0 (urve 1), 0.01 (urve 2), 0.02 (urve 3), 0.03 (urve 4), 0.04 (urve 5), 0.05 (urve 6), 0.07 (urve 7), 0.1 (urve 8), 0.3 (urve 9), and 0.5 mm (urve 10). Table 1. Diffusion Coeffiients (D) and Moleular Weights (MW) of Ag + and 1:1 Ag + /PAMAM Complexes Obtained from Chronoamperometry ompound MW normalized (MW) -1/2 D 10-6 m 2 /s Ag Ag + -PAMAMG1NH Ag + -PAMAMG2OH shows a peak at 284 nm, and the peak absorbane is proportional to the onentration. Addition of mm AgNO 3 to these DDM solutions auses the absorbane at 284 nm to derease slightly (e.g., ompare urve 8 of Figure 7B with urve 7 of Figure 7A for [PAMAMG1NH 2 ] ) 0. 1 mm with or without mm AgNO 3, respetively), suggesting some interation ours between Ag + and DDM. No signifiant absorption was observed in the same wavelength region in a solution ontaining only mm AgNO 3 (see urve 1 of Figure 7B), with no signifiant hange in the shape of the spetra of DDM in the presene of mm AgNO 3.As shown in Figure 8, similar photometri behavior was observed for PAMAMG2OH DDM in the absene or presene of AgNO 3. The absorbane at 284 nm as a funtion of DDM onentration obtained from Figures 7 and 8A and B is summarized in Figure 8C. Both DDMs have rather small molar absorptivity, ɛ, at 284 nm (ɛ ) 280 M -1 m -1 for PAMAMG1NH 2 and 190 M -1 m -1 for PAMAMG2OH). After the addition of 0.03 mm hydrazine into a DDMomplexed silver solution ontaining mm Ag + and 0.1 mm PAMAMG1NH 2, a new shoulder ( 345 nm) and a new peak Figure 8. (A) UV-vis spetra of PAMAMG2OH at different onentrations in water without Ag + : (urve 1), 0.02 (urve 2), 0.04 (urve 3), (urve 4), 0.1 (urve 5), 0.2 (urve 6), 0.3 (urve 7), and 0.6 mm (urve 8). (B) UV-vis spetra of PAMAMG1NH 2 at different onentrations in water with 0.12 mm Ag + : 0 (urve 1), (urve 2), 0.01 (urve 3), 0.04 (urve 4), (urve 5), 0.1 (urve 6), 0.2 (urve 7), 0.3 (urve 8), and 0.6 mm (urve 9). (C) Summary of the absorbane at 284 nm as a funtion of DDM onentrations. Squares, PAMAMG1NH 2 only; diamonds, 0.12 mm AgNO 3 and varying [PAMAMG1NH 2]; triangles, PAMAMG2OH only; and irles, 0.12 mm AgNO 3 and varying [PAMAMG2OH]. ( 410 nm) developed due to the prodution of and absorption by silver nanopartiles. Note that the sale in Figure 9, urve 1 represents 10 times lower sensitivity than the other urves. Notie also that the absorbane at 284 nm was substantially enhaned, and the peak position was slightly blue-shifted. It merged into a strong absorption band at wavelengths shorter than 250 nm due to the formation of smaller silver lusters. The addition of hydrazine to a solution ontaining only DDM affets the spetrum in the visible region slightly (ompare urve 3 with urve 2 of Figure 9). Similar behavior was observed for a DDM-omplexed silver solution ontaining mm Ag + and 0.1 mm PAMA- MG2OH (not shown). To minimize the agglomeration of silver lusters into larger nanopartiles during the redution proess, we used a lower onentration of Ag + and studied the onentration dependene of the growth rate of different zerovalent silver speies upon redution as shown by their different absorption peaks. As shown Analytial Chemistry, Vol. 77, No. 14, July 15,

6 Figure 9. Change in UV-vis spetra of Ag + -PAMAMG1NH 2 system after redution: mm Ag + only (urve 5); 0.1 mm PAMAMG1NH 2 and 0.05 mm hydrazine (urve 3); 0.1 mm PAMA- MG1NH 2 only (urve 2); 0.1 mm PAMAMG1NH 2 and mm AgNO 3 (urve 4); the above solution after redution with 0.05 mm hydrazine (urve 1). Notie that the absorbane shown in urve 1 was redued 10 times as ompared to the other urves. Figure 11. Absorbane of various solutions as funtions of hydrazine onentration at λ max 283 nm (A) and 410 nm (B). Open squares, 0.1 mm PAMAMG1NH 2 and mm Ag + ; open diamonds, 0.1 mm PAMAMG2OH and mm Ag + ; solid squares and solid triangle, 0.1 mm PAMAM only; solid diamonds and solid irle, 0.1 mm PAMAMG2OH only. Figure 10. (A) UV-vis spetra of a solution ontaining 0.1 mm PAMAMG2OH with or without mm AgNO 3 added: urve 1, 0.1 mm PAMAMG2OH only; urve 2, 0.1 mm PAMAMG2OH with 0.1 mm hydrazine; urve 3, mm AgNO 3 and 0.1 mm PAMAMG2OH; urve 4, solution used in urve 3 after the addition of mm hydrazine; urve 5, solution used in urve 3 after the addition of a total of 0.01 mm hydrazine; urve 6, solution used in urve 3 after the addition of a total of 0.02 mm hydrazine. (B) UV-vis spetra of a solution ontaining 0.1 mm PAMAMG1NH 2 with or without mm AgNO 3 added: urve 1, 0.1 mm PAMAMG1NH 2 only; urve 2, 0.1 mm PAMAMG1NH 2 with 0.06 mm hydrazine; urve 3, mm AgNO 3 and 0.1 mm PAMAMG1NH 2; urve 4, solution used in urve 3 after the addition of mm hydrazine; urve 5, solution used in urve 3 after the addition of a total of mm hydrazine; urve 6, solution used in urve 3 after the addition of a total of 0.01 mm hydrazine. in Figure 10A for a solution initially ontaining 0.1 mm PAMAMG2OH, the absorbane at the peak of 284 nm is nearly proportional to the onentration of added hydrazine, whereas that at 410 nm is nearly independent of the amount of hydrazine added into the solution. The enhanement of the absorbane at λ e 284 nm, after the redution proess, is mainly assoiated with the formation of small silver lusters of less than 10 silver atoms, 8 and the absorption at 410 nm is due to Mie surfae plasma resonane (SPR) 10 of larger silver nanopartiles. These spetrophotometri results suggest that the redution of the Ag + - PAMAMG2OH omplex favors the formation of small lusters, as ompared to the growth of bigger nanopartiles. Figure 10B shows the results of spetrophotometri titration of a solution ontaining 0.1 mm PAMAMG1NH 2. As summarized in Figure 11, the absorbane at peaks near to 284 and 410 nm are both proportional to the onentration of hydrazine added and inrease at a similar rate, suggesting that at least two differently sized zerovalent silver speies grow in a similar pattern. These results are onsistent with those observed previously for the Cu 2+ -DDM systems, 11 suggesting that PAMAMG2OH (OHterminated) favors the formation of intra-ddm silver lusters, whereas PAMAMG1NH 2 allows for the formations of both interiorand exterior-ddm zerovalent silver speies. The latter was less stable and ould further ause the agglomeration of silver lusters to form larger nanopartiles, as manifested by the observation of the SPR peak near 410 nm. Mass Spetrometri Measurement. To learn more about the stoihiometry of the silver-ddm omplexes, we arried out eletrospray ionization mass spetrometry (ESI-MS) studies of the Ag + -PAMAMG1NH 2 system. Solutions were prepared in water/ methanol mixed solvents to enhane the ion abundane and signalto-noise ratio. The spetra are fairly ompliated, but most of the major peaks an be assigned on the basis of the m/z values and ollisional ativated dissoiation (CAD) 12 fragmentation patterns. Figure 12A shows the ESI mass spetrum of PAMAMG1NH 2 (10) Kreibig, U. In Handbook of Optial Properties; Hummel, R. E., Wibmann, P., Eds.; CRC Press: Boa Raton, FL, 1997; Vol. II, Chapter 7. (11) Zhao, M.; Sun, L.; Crooks, R. M. J. Am. Chem. So. 1998, 120, Analytial Chemistry, Vol. 77, No. 14, July 15, 2005

7 Figure 12. SI Quadruple ion trap mass spetra of solutions ontaining 10 µm PAMAMG1NH 2 (abbreviated by DDM) alone (A), and 10 µm AgNO 3 and 10 µm PAMAMG1NH 2 (B) in 3:1 methanol/water mixed solvent. Peaks labeled with an asterisk (*) orrespond to omplexes ontaining the intat dendrimer. prepared in a 3:1 methanol/water (volume/volume) mixed solvent. A series of prominent peaks are assigned to the multiply harged moleular ions of protonated PAMAMG1NH 2 or ions omplexed with Na +. The main soure of the Na + ontamination is the glass apparatus used in the syntheses of DDM and the glass ontainers used for storage of the solution. Some of the peaks interspersed between the moleular ion peaks orrespond to DDMs ontaining defets, suh as missing branhes. 1 For example, a peak at m/z is assoiated with a Na + omplex of the parent DDM with the loss of one amidoamine branh of mass Da, and another peak at m/z is assoiated with a Na + omplex of the DDM after loss of two amidoamine branhes. Figure 12B shows the mass spetrum of omplexes of PAMAMG1NH 2 with silver(i) prepared by mixing equimolar (10 µm eah) Ag + and DDM in 3:1 methanol/water. The binding stoihiometries of 1:1 (Ag + /PAMAMG1NH 2 ) (peaks at m/z for HAgDDM 2+, for NaAgDDM +2, for AgDDM + ), were dominant for omplexes observed under these onditions. A signifiant amount of the omplex with a 2:1 (Ag + /PAMA- MG1NH 2 ) ratio (m/z 822.8) also existed in the solution. The single-branh DDM defet (m/z for its Na + omplex as mentioned above) formed omplexes with Ag + to yield a signifiant amount of a singly harged Ag + omplex of 1:1 ratio (m/z ), and the two-branh DDM defet (m/z 768.0) omplexes with Ag + to yield a singly harged omplex of 1:1 ratio (m/z 851.6). DISCUSSION For a Nernstian eletron-transfer reation in whih the oxidized and redued forms assoiated with a omplexation agent in solution, a square sheme (as shown below) an be applied for a single stoihiometri omplex system. For the ase presented in Sheme 1, L ) DDM.Here, E f 0 and E b 0 are the formal potentials of the Ag + /Ag 0 ouple in the free and omplexed forms, respetively, and q is the number of Ag + omplexed with one DDM (12) Bush, K. L.; Glish, G. L.; MLukey, S. A. Mass Spetrometry/Mass Spetrometry: Tehniques and Appliations of Tandem Mass Spetrometry; VCH Publishers: New York, Sheme 1 moleule. For simpliity, we assume that q values for DDM omplexed silver ion and zerovalent silver speies are idential, at least in the initial stage of the redution proess. K q + and K q are the mirosopi formal equilibrium onstants for omplexation of Ag + and Ag 0 speies to DDM and are expressed as follows. K q + ) [Ag q L q+ ]/([Ag + ] q [L]); K q ) [Ag q L]/(a Ag q [L]) (2) in whih [Ag q L q+ ] and [Ag + ] represent the equilibrium onentrations of the Ag + -DDM omplex and free Ag +, [Ag q L] and [a Ag ] are the orresponding onentrations of the Ag 0 -DDM omplex and the ativity of free Ag 0, and C s is the onentration of free DDM ligand. At 25 C and assuming that n ) 1 and q ) 1, we have 1a E f ) E f log([ag + ]/a Ag ) (3) E b ) E b log([agl + ]/[AgL]) (4) At equilibrium, one an obtain the relation shown below from eqs 2-4. E b 0 - E f 0 ) log(k 0 /K 1 + ) (5) Thus, from the CV, a limiting shift of 0.16 V in E 1/2 for the Ag + - PAMAMG1NH 2 omplex yields a K + 1 /K 1 of 515 for the Ag + - PAMAMG2OH omplex and a limiting shift of V, K + 1 / K 1 ) Analytial Chemistry, Vol. 77, No. 14, July 15,

8 13. These results indiate that DDM binds more strongly to Ag + than it does to zerovalent silver and also suggests that the silver speies interats with DDM predominantly via amine groups, espeially those periphery primary amine groups in the amineterminated DDM. This is onsistent with the K q + values determined from the voltammetri titrations disussed below. This square sheme has been utilized to probe the interation of metal omplexes with DNA 13 and to examine eletron-transfer reations assoiated with host-guest omplexation 14 on the basis of voltammetri measurements. Under the assumption of diffusionontrolled eletron transfer, two limiting ases hold for the urrent in voltammetri measurements, depending on whether the interonversion of free and omplexed Ag +, in Sheme 1, is treated as stati (no interonversion takes plae during the time of the normal CV experiment, eq 6) or mobile (rapid interonversion during a san, eq 7). 13,15 i p ) BC t + [D f 1/2 + (D b 1/2 - D f 1/2 )x b + ] (6) i p ) BC t + [D f + (D b - D f )x b + ] 1/2 (7) In whih B ) n 3/2 Av 1/2 for a Nernstian reation, whih represents the appropriate onentration-independent terms in the voltammetri expression. D f and D b are the diffusion oeffiients of free and omplexed Ag +, respetively, x b + is the mole ratio of the omplexed Ag +, and C t + is the analytial onentration of Ag(I), that is, Figure 13. Molar fration of omplexed Ag + as a funtion of R + at C t + ) M for different K + values; q ) 1 (frame A) and q ) 2 (frame B). Curve 1, K q + ) ; urve 2, K q + ) ; urve 3, K q + ) ; urve 4, K q + ) The unit is in M -1 for q ) 1 and M -2 for q ) 2. x b + ) q[ag q L q+ ]/C t + (8) C t + ) [Ag + ] + q[ag q L q+ ] (9) Normalizing i p in eqs 6 and 7 with the i p value at x b + ) 0, that is, BC t + D f 1/2 and with some mathematial manipulation, yields expressions in terms of a dimensionless parameter, I p, I p ) 1 + [(D b /D f ) 1/2-1)x b + for the stati ase (10) I p 2 ) 1 + [(D b /D f ) - 1)]x b + for the mobile ase (11) where I p ) i p /BC t + D f 1/2. Taking R + ) C s / /C t + for total Ag + speies, in whih C s / is the total onentration of DDM, we obtain eq 12 for R + as a funtion of x b +, K q +, C t + and q. R + ) x b + {1 + [1/(K q + (C t + ) q (1 - x b + ) q )]} (12) The variation of x + b with R + over a wide range of K + q at onstant C + t is shown in Figure 13, and the voltammetri behavior, in terms of I p or I p2 vs x + b for the stati and mobile ase is shown in Figure 14. The four urves in Figure 13A represent different omplexation onstants, K + q,of , 1.0 (13) Carter, M. T.; Rodriguez, M.; Bard, A. J. J. Am. Chem. So. 1989, 111, (14) (a) Matsue, T.; Evans, D. H.; Osa, T.; Kobayashi, N. J. Am. Chem. So. 1985, 107, 341. (b) Rusling, J. F.; Shi, C.-N.; Kumosinski, T. F. Anal. Chem. 1988, 60, () Zana, R.; Makay, R. A. Langmuir 1986, 2, 109. (15) Evans, D. H. J. Eletroanal. Chem. 1989, 258, 451. Figure 14. A series of theoretial urves of I p (frame A) and I p 2 (frame B) vs R + based on eqs 10, 11, and 12 for different K 1 + values. In the alulation, we take C t + ) 0.12 mm and q ) 1asR + > 1 and q ) 2asR + < 1, and D b/d f ) 0.18 and 0.55, as determined from hronoamperometry, for Ag + -PAMAMG1NH 2 and Ag + -PAMAMG1OH systems, respetively. Lines are theoretial urves: K 1 + ) (urve 1), (urve 2), (urve 3), and M -1 (urve 4) for q ) 1, and (urve 1), (urve 2), (urve 3), M -2 (urve 4) for q ) 2. Symbols are experimental data: squares (Ag-PAMAMG2OH system), diamonds (Ag-PAMAMG1NH 2 system). 10 6, , and M -1, respetively, with C t + ) 0.12 mm and q ) 1. The steepness of the initial rise in x b + with inreasing R + inreases as K q + beomes larger. At the lowest K q + value onsidered here, that is, M -1 ((solid squares) urve), x b + an reah 0.8 at R + ) 1 and approahes unity when R Analytial Chemistry, Vol. 77, No. 14, July 15, 2005

9 is >5. At K + q values higher than M -1, almost all (>98%) of the Ag + speies are in the omplexed form at R + ) 1. Thus, under suh onditions frequently used in our EC and spetrosopi experiments (i.e., [Ag + ] ) 0.12 mm and [DDM] ) 0.1 mm), most of the Ag + speies in the solution are omplexed. When K + q is large, a signifiant hange in x + b with inreasing R + ours at R + near 1, and little hange ours at larger R +. This is onsistent with the observation that both i p and E 1/2 hange signifiantly at R + < 1 and muh less at R + > 1. As shown in Figure 13B, when q ) 2, the initial rise in x + b with inreasing R + beomes less steep, as ompared to the ase of q ) 1, at the same value of K + q. The end point of the titration urve beomes more diffiult to define. At K + q of < 10 5 M -2, the urve is barely distinguishable from a straight line nearly parallel to the R + axis (see solid squares in Figure 13B). Figure 14 shows a series of theoretial urves of I p and I p2 vs R + based on eqs 10, 11, and 12 for different K + q values. In the alulation, we take C + t ) 0.12 mm and q ) 1atR + > 1 and q ) 2atR + < 1, and D b /D f ) 0.18 and 0.55, as determined from hronoamperometry, for Ag + -PAMAMG1NH 2 and Ag + -PAMA- MG1OH systems, respetively. The fit of the theoretial urves to the experimental data an be ahieved at R + + > 1 and gives K 1 ) M -1 for the 1:1 Ag + /PAMAMG1NH 2 omplex and K + 1 ) M -1 for the 1:1 Ag + /PAMAMG2OH omplex. The latter is omparable to K + 1 for the diamine omplex of Ag + and ammonia. 16 This voltammetri titration result, that is, the formation of a 1:1 omplex as the major silver-ontaining speies for the Ag + -PAMAMG1NH 2 system with equimolar Ag + and DDM, is onsistent with the finding obtained from the MS measurements. Notie that as indiated by the results of the voltammetri titration, Ag + binds more strongly with the amineterminated DDM, as ompared with the hydroxyl-terminated DDM of the same generation. At R + < 1, the experimental I p and I p2 data deviate substantially from the theoretial urves if the same set of parameters is used. A better fit an be ahieved in this domain by assuming q g 2 with omparable K + 2 values. However, one has to realize that mixed stoihiometri silver omplexes very likely oexist in this domain, and a more ompliated reation sheme is required for data analyses. It is worthwhile to examine the behavior of the two limiting models, eqs 10 and 11. In the limit of no interonversion of free and omplexed Ag +, on the time sale of the normal CV experiment, for example, v ) 0.1 V/s, the total urrent measured is always smaller than in the ase of rapid interonversion when R + is >0. Thus, the assumption of rapid interonversion will yield K + q values that are larger than those given by the stati model with the same diffusion oeffiients and q values. For our systems, the K + 1 values obtained from the two limiting models differ by no more than a fator of 1.8 beause the K + 1 values are large, leading to a small ontribution of the total urrent from free Ag +. Within experimental error, it is diffiult to determine whih model is more appropriate to use for the determination of K + q and q. We summarize the results in Table 2 on the basis of the alulation obtained from the stati model, sine it gives a slightly better fit of the theoretial urves to the experimental results with the same (16) Shumilova, N. A.; Zhutaeva, G. V. In Enylopedia of Eletrohemistry of the Elements; Bard, A. J., Ed.; Marel Dekker: New York, 1978; Vol. VIII, p 32. Table 2. Stability Constants (K q + ) and q Values of Ag + and PAMAM Complexes ligand q K q + PAMAMG1NH M -1 PAMAMG2OH M -1 PAMAMG1NH a M -2 PAMAMG2OH a M -2 a As R + < 1, better fit is obtained by assuming q g 2, and K q + now is in M -2. set of parameters (see Figure 14), although both models an give a good fit if different K 1 + values for different models are used. In the UV-vis spetrophotometri measurements of redued PAMAMG2OH-hosted silver speies at low onentration (see Figures 10A and 11), the lak of absorbane near 410 nm, whih is harateristi of Mie SPR for silver nanopartiles, indiates that zerovalent silver lusters rather than larger nanopartiles are produed under the onditions used. As the K q + value indiates, the surfae hydroxyl groups do not bind Ag + as strongly as surfae amine groups. Thus, OH-terminated DDM favors the formation of an intradendrimer Ag + omplex through interior tertiary amine groups, and interior-ddm silver lusters an be formed after redution. As opposed to Ag(I) redution in PAMAMG2OH, the results for PAMAMG1NH 2, as shown in Figures 10B and 11, show that both small lusters and larger nanopartiles are formed at the same onentration of the Ag + -PAMAMG1NH 2 omplex an hour after redution, although long-term stabilities and the growth kinetis of the lusters or nanopartiles have not been arried out. These results are onsistent with the onlusion drawn from the voltammetri titration by onsidering that amine-terminated DDM, as ompared with hydroxyl-terminated DDM, favors the formation of exterior-ddm silver lusters after redution, whih an lead to agglomeration of the Ag lusters to form larger nanopartiles. A photometri titration is, in general, useful for the determination of the stability onstant of a omplex. 17 However, the end point of the photometri titration urve, as shown in Figure 8C, is diffiult to define beause there are no dramati hanges either in the shape of the spetra or in the absorbane at peak 284 nm for the Ag + -PAMAM systems studied here. The urves are barely distinguishable from straight lines at high R + values, although more signifiant urvature is observed at low R +. CONCLUSIONS Eletrohemial methods, omplementary to spetrosopi and mass spetrometri tehniques, have been used to probe the omplexation of silver with low-generation amine- or hydroxylterminated PAMAM dendrimers. Stability onstants and the stoihiometry of the omplexes an be obtained from the dependene of peak urrent on R + from the voltammetri titration experiments. The stoihiometry of the omplexes obtained from the EC method agrees well with that obtained from mass spetrometry. Ag + binds more strongly with amine-terminated (17) (a) Rossotti, F. J. C.; Rossotti, H. The Determination of Stability Constants, MGraw-Hill: New York, (b) Ramette, R. W. J. Chem. Edu. 1967, 44, 647. Analytial Chemistry, Vol. 77, No. 14, July 15,

10 G1PAMAM than with hydroxyl-terminated G2PAMAM. E 0 of the Ag-PAMAMG1NH 2 +/0 ouple (estimated from the average of E p and E pa ) shifted to more negative values than that of Ag +/0. Spetrosopi results suggest that the OH-terminated G2PAMAM favors the formation of small zerovalent silver lusters after redution, whereas with amine-terminated G1PAMAM, both lusters and larger nanopartiles are formed simultaneously under similar onditions. This might be attributed to the formation of an exterior Ag + -DDM omplex for amine-terminated DDM, as ompared to the formation of an interior Ag + -DDM omplex for OH-terminated DDM prior to the redution proess. The present results also suggest that nuleation and growth are important during the redution proess, sine lusters or nanopartiles an be produed from a 1:1 Ag + -DDM omplex preursor. ACKNOWLEDGMENT We aknowledge support of this researh from Nuryst Pharmaeutials and the Welh Foundation (F-1155). SUPPORTING INFORMATION AVAILABLE Typial urrent vs time i vs t urve for the potential step (+0.35 to0vvssce) experiment at an Au disk (radius 0.2 m) in a 0.2 M NaNO 3 solution ontaining 0.12 mm Ag +, a solution ontaining 0.12 mm Ag + and 0.1 mm PAMAMG1NH 2, and a solution ontaining 0.12 mm Ag + and 0.1 mm PAMAMG2OH. This material is available free of harge via the Internet at Reeived for review Deember 17, Aepted April 26, AC048133G 4422 Analytial Chemistry, Vol. 77, No. 14, July 15, 2005