Langmuir 1988,4, 1021-1032
1021
Conformation and Orientation of the Haptens, 2,4-Dinitrophenyl Amino Acids, on Colloidal Silver from Surface-Enhanced Raman Scattering Diane Curley and Olavi Siiman* Department of Chemistry, Clarkson University, Potsdam, New York 13676 Received December 29, 1987 Surface-enhanced Raman scattering (SERS) from N-substituted 2,4-dinitrophenyl (DNP) amino acids adsorbed on colloidal silver has been observed. SERS spectra of N-(2,4-dinitrophenyl)-substitutedmethionine, cystine, aspartic acid, and a-lysine, taken with 488.0-nm excitation far from the electronic absorption band of the DNP group at 360 nm, were dominated by intense bands of the DNP group, which in normal Raman spectra showed a much greater scattering intensity than the amino acid alone. SERS spectra of the parent amino acids were used to establish binding of at least one c&boxylate group to the silver surface. A rigorous resonance Raman spectrum of DNP-Met in aqueous solution with 386.4-nm excitation was used to make band assignments for the ortho and para nitro group stretching modes, v,(0-NO2)and v,(p-NO,), at 1271 and 1330 cm-', respectively. Both absolute and relative intensities of SERS bands changed with the total hapten concentration in the range 0.1-100 pM in the silver particle suspension. Lower limits on SERS enhancements were placed in the 103-104range. Relative intensity changes in bands associated with v,(o-NOz), v,(p-NOz), and the DNP ring symmetrical breathing mode at 833 cm-' were used mainly to monitor the conformation and orientation of the haptens on the silver surface. A t high concentrations (10-100 pM) it is proposed that the DNP amino acids adopt a folded-over conformation to occupy less surface area per molecule. In this case the v,(p-NOz) band was intense, and the DNP ring is shown to lie end-on with respect to the surface. At low concentrations a more open or extended conformation is assumed. In this structure the DNP ring has two possible orientations, either flat or edge-on with respect to the surface. Relative SERS band intensity changes in the low (I1pM) and intermediate (1-10 pM) concentration ranges, which show either a complete loss of intensity in v,(p-N02),v,(o-NOz), and v,(ring breathing) or only intensity in the v,(o-NO,) band, support a flat orientation at low concentrations and an edge-on orientation at intermediate concentrations. Similar conclusions regarding orientation of the 5-(dimethylamino)naphthalene-l-sulfonyl(DANS) group in DANS-Asp on colloidal silver could be reached from the SERS spectrum of this hapten, whose fluorescence emission totally obscures its normal Raman spectrum in solution.
Introduction The 2,4-dinitrophenyl (DNP) chromophore is a commonly used hapten, a term coined by Landsteiner' for a substance that can be bound by a specific antibody but cannot by itself induce the actual immune response or production of the antibody. A hapten can be coupled to suitable macromolecules called carriers such as proteins in order to become antigenic. For this reason a variety of techniques,2-12including resonance Raman scattering11J2 (RRS), has been pursued to study DNP haptens as well as their binding to immunoglobulins. The aim of the present work is to use surface-enhanced Raman scattering (SERS)as an alternative to RRS for DNP haptens since it can enhance a different set of vibrational modes.13 Since SERS intensity in particular Raman bands depends crit(1)Landsteiner, K. The Specificity of Serological Reactions; Harvard University Press: Cambridge, MA, 1945 (rev. ed., Dover: New York, 1964). (2)Kabat, E. A. Structural Concepts in Immunology and Zmmunochemistry, 2nd ed.; Holt, Rinehart, and Winston: New York, 1976. (3)Yalow, R. S.Annu. Rev. Biophys. Bioeng. 1980,9,327. (4)Velick, S. F.; Parker, C. W.; Eisen, H. N. Proc. Natl. Acad. Sci. U.S.A. 1960,46,1470. (5)Eisen, H.N.; Siskind, G. W. Biochemistry 1964,3, 996. (6) Haselkom, D.; Friedman, S.; Givol, D.; Pecht, I. Biochemistry 1974, 13,2210. (7)Dower, S. K.;Wain-Hobson, S.; Gettins, P.; Givol, D.; Jackson, W. R. C.; Perkins, S. J.; Sunderland, C. A,; Sutton, B. J.; Wright, C. E.; Dwek, R. A. Biochem. J . 1977,165, 207. (8) Kooistra, D. A.; Richards, J. H. Biochemistry 1978,17,345. (9)Hardy, R. R.; Richards, J. H. Biochemistry 1978,17,3386. (10)Gettins, P.; Dwek, R. A.; Stenhouse, I. FEBS Lett. 1980,117,23. (11)Kumar, K.;Phelps, D. J.; Carey, P. R.; Young, N. M. Biochem. J. 1978,175,727. (12)Gettins, P.;Dwek, R. A.; Perutz, R. N. Biochem. J. 1981,197,119. (13)Moskovits, M. Rev. Mod. Phys. 1985,57, 783.
ically on the orientation14J5of molecules with respect to the surface and on the distance16J7of functional groups in large molecules from the surface, it can give additional information on the structure of chemisorbed DNP derivatives. In this initial study several Ne-substituted DNP amino acids with methionyl, cystinyl, lysyl, and aspartyl residues have been chemisorbed onto colloidal silver particles. Raman band intensities of the hapten will be thus enhanced significantly over the normal solution Raman scattering intensity, especially if excitation is also tuned into or near an electronic absorption band of the 2,4-DNP group, resulting in surface-enhanced resonance Raman scattering (SERRS). The proximity of the haptens to a heavy metal atom surface also serves to quench any fluorescence emission that might be exhibited by the DNP group. This further allows SERS spectra to be obtained for highly fluorescent h a p t e n ~ such ~ ~ J as ~ &(dimethylamin0)naphthalene-1-sulfonyl-L-asparticacid (DANS-Asp) adsorbed on colloidal silver. Thus, additional information about the structure and bonding of these haptens, that is not available from excitation and fluorescence emission spectra, can be obtained from the positions and relative intensities of SERS bands. (14)Creighton, J. A. Surf. Sci. 1983,124, 209. (15)Moskovits, M.; DiLella, D. P. In Surface Enhanced Raman Scattering; Chang, R. K., Furtak, T. E., Eds.; Plenum: New York, 1982; p 243.
(16)Murray, C. A.; Allara, D. L. J. Chem. Phys. 1982,76,1290. (17)Kovacs, G. J.; Loutfy, R. 0.;Vincett, P. S.; Jennings, C.; Aroca, R. Langmuir 1986,2, 689. (18)Voss: E. W. Fluorescein Hapten: An ZmmunotogicalProbe; CRC: Boca Raton, FL, 1984. (19)Steward, M. W.; Steensgaard, J. Antibody Affinity: Thermodynamic Aspects and Biological Significance;CRC: Boca Raton, FL, 1983.
0743-7463/88/2404-lO21$01.50/00 1988 American Chemical Society
1022 Langmuir, VoE. 4 , No. 4 , 1988
The Na-substituted DNP amino acids are expected to form their strongest chemisorption bonds to silver through the 0, S, or N donor atoms of the carboxylate and residual groups that possess an established high affinity for silver. Confirmation of this affinity for the silver surface by the amino acid portion of the DNP derivatives could be obtained by measuring SERS spectra of the respective amino acids. Thus, with the amino acid portion of the molecules firmly tied down to the silver surface, several conformations and orientations of the DNP group can be assumed on or near the surface. Previous electrochemical studies of the orientation of aromatic compounds, thyadenine,22benzene-m-disulfonate and p-toluenesulfonate ions,23 diphenols with surface-active side chain^,^^.^^ mercaptans,26and quinones,24on mercury or platinum electrodes provide precedents for orientational changes that can occur on metal surfaces. Each of the cited studies supports the premise that at low concentration aromatic molecules adsorb flat on the metal surface while at high concentration they reorient, perhaps irreversibly,% to allow parallel close packing of their planes perpendicular to the surface and result in increased surface coverage due to attractive forces between these molecules. Since the DNP amino acids are so large, they may also fold up in order to occupy less surface area. Analogous folding of bromoacetyl derivatives of DNP-Lys has been invoked for these molecules to fit into antibody binding sites.6
Experimental Section T h e silver hydrosol with adsorbed citrate was prepared as previously describedSn The stock silver sol was stored in the dark a t approximately 10 "C in 1 X lo-, M aqueous sodium citrate at a silver concentration of about 2 g/L. Before use, the sol was diluted 10-fold with deionized doubly distilled water and treated with ion-exchange resin (Bio-Rad analytical grade mixed bed (H', OH- form) resin, AG 501-XS) t o remove excess sodium citrate and to displace some adsorbed citrate and sodium ions. The decrease in conductivity was followed by measurements on a Cole-Parmer digital conductivity meter until the sol reached 3-4 fiQ-l cm-'. T h e p H of the suspension at this point was about 6.5. Mixtures of N'-(2,4-dinitrophenyl)-~-lysine (a-DNP-Lys), N(2,4-dinitrophenyl)-~~-methionine (DNP-Met), and N,N-bis(2,4-dinitrophenyl)-~-cystine (DNP-Cys), were each made with a 5/100 dilution of the original stock silver sol for a final silver concentration of 0.1 g/L. Mixtures with N-(2,4-dinitrophenyl)-L-aspartic acid (DNP-Asp) were made with a 31100 dilution of the original stock silver sol for a final silver concentration of 0.06 g/L. All D N P derivatives, except a-DNP-Lys (Chemical Dynamics Corp.), and DANS-L-AS~ were obtained from Sigma. D N P amino acids (1 mM) were added to aliquots of deionized silver sol to give final concentrations of 100, 50, 10,5, 3, and 1 fiM (0.5 and 0.1 fiM also for DNP-Met). After the samples were prepared with the four haptens, they were stored in the dark until sufficient aggregation had occurred to give detectable SERS signals. The progress of the aggregation, followed by absorption spectrophotometry, showed the appearance and growth of an absorption band a t approximately 500 nm due to aggregates of silver particles. This band then increased in intensity and shifted to longer wavelengths as the size of the aggregates became larger. (20)Brabec, V.;Christian, S. D.; Dryhurst, G. J.Electroanal. Chem. 1977,85,389. (21)Kinoahita, H.; Christian, S. D.; Dryhurst, G. J. Electroanal. Chem. 1977,85,377. (22)Kinoshita, H.; Christian, S. D.; Dryhurst, G. J. Electroanal. Chem. 1977,83,151. (23)Parry, J. M.;Parsons, R. J. Electrochem. SOC.1966, 113,992. (24) Soriaga, M.; Wilson, P.; Hubbard, A.; Benton, C. J. Electroanal. Chem. 1982,142,317. (25)Soriaga, M.;Hubbard, A. J. Am. Chem. SOC.1982, 104, 2735. (26)Soriaga, M.;Hubbard, A. J. Am. Chem. SOC.1982, 104, 3937. (27)Siiman, 0.; Bumm, L. A.; Callaghan, R.; Blatchford, C. G.; Kerker, M. J . Phys. Chem. 1983,87,1014.
Curley and Siiman
i
i U 0.0J
0
4 I
I
5
10
I
15 ELAPSED TIME , d
I
I
I
20
25
30
Figure. 1. Time dependence of surface concentration of DNP-Met on colloidal silver for 3 mixture.
X
M DNP-Met and 0.1 g / L silver
Eventually sedimentation of large aggregated silver particles occurred. Ultracentrifugations were performed on a Beckman Model L-2 ultracentrifuge for 60 min a t 47 000 rpm and a temperature of 10 "C. Tubes of 13.5-mL volume were used in a Type 65 rotor. Continuous-wave Raman spectral measurements were made with instrumentation already described.B A rotating cylindrical quartz cell (2-cm path length) a t ambient temperature was used for sampling. Scattered light was collected a t 90" to the incident laser beam. Nanosecond pulsed excitation was provided by pumping a Lambda Physik model FL2002 dye laser containing laser dye, QUI in dioxane, with XeCl excimer laser (Model EMG 53MSC) pulses of 50 mJ and -17-ns width at 308 nm. The average power output from the dye laser at 386.4 nm was 15 mW a t a repetition rate of 25 Hz, as measured with a Scientech Model 361 power indicator and 1-in. disk calorimeter. Part of the output (1 mW) was background emission from the dye laser. The Raman scattered signals were processed with an EG & G Model 162 boxcar averager and a Model 165gated integrator. A fast photodiode (EG & G FND-100Q) was used as an external trigger for the boxcar, and an appropriate delay line was used between the P M tube (Hamamatsu R928, thermionically cooled in a Products for Research housing) and the gated integrator. The Spex CD2A Compudrive for the Model 1403 double monochromator was run in the burst mode a t a 1-cm-I increment and 1-s dwell time so that Raman signals were integrated over 25 pulses. Sampling was carried out with a stationary fluorescence cell with one face obliquely inclined at an angle of 20-30" to the incident laser beam. Scattered radiation off this face was collected at 90' to the incident beam. A long focal length lens was inserted near the output of the pulsed dye laser t o slightly defocus the laser beam and, thus, avoid dielectric breakdown in the quartz cell windows or the medium as well as nonlinear scattering phenomena and any photodecomposition of the sample. Calibration of the incident wavelength and timing of scattered pulses with boxcar gated integration was first performed with carbon tetrachloride in a stationary cuvette.
Results and Discussion Adsorption of DNP-Met on Colloidal Silver. The adsorption behavior of DNP-Met on colloidal silver was investigated as a function of DNP-Met concentration and elapsed time for a fixed amount of silver. In each case the amount of DNP-met adsorbed on silver particles was determined indirectly by ultracentrifugation of the sols at 40 000 rpm for 55 min and analysis of the supernatant for DNP-Met by spectrophotometry (measuring absorbance at 360 nm). The results for the concentration of DNP-Met adsorbed on the silver surface in a time-dependent run (3 X M DNP-Met on 0.1 g/L silver sol) are shown in (28)Lepp, A.;Siiman, 0. J. Phys. Chem. 1985,89,3494.
SERS of 2,4-Dinitrophenyl Amino Acids
Langmuir, Vol. 4, No. 4, 1988 1023
200 - 5 0
05
IO
IO
Ctotal x 1O5M
Figure 2. Adsorption of DNP-Met on colloidal silver, Cd versus C, after 10 days of equilibration.
Figure 1. The surface concentration, Cad, peaked after -6-7 days at 1 X 10-5M and remained about constant thereafter for 25 days. Subsequent measurements of Raman spectra, thus, were optimally made after adsorption of DNP-Met had reached its peak. In another analysis of DNP-Met-colloidal silver mixtures the adsorption isotherm was determined by the same technique (ultracentrifugation-spectrophotometry) after the mixtures had equilibrated for 10 days. The results of a plot of Cad versus C, in Figure 2 show that the surface becomes saturated with 1 X M DNP-Met at a minimum total concentration of 3 X M. Thus, surface DNP-Met concentrations below 1 X 10" M obtained with total DNP-Met concentrations less than 3 X 10-5M represent submonolayer coverage of DNP-Met on silver particles. In the course of these adsorption measurements, visual examination of the sols as well as spectrophotometric measurements showed that the particles of silver were also aggregating into clusters of various sizes. Mixtures of ( D N P - C ~ S DNP-Lys, )~, and DNP-Asp with the silver sol behaved in a similar way. Preparation and Absorption Spectra of Silver Hydrosols with Adsorbed DNP Amino Acids. Extinction refers to the reduction of the irradiance of a sample through both scattering and absorption; however, in our case the primary silver particles are small enough (mean diameter 13 nm) so that absorption dominates over scattering, and we can refer to the spectra as absorption spectraam The latter were used to monitor the extent of aggregation. A time-dependent run of the absorption spectra of a mixture of 3 X 10-5M DNP-Met and 0.1 g/L silver was made to coincide with the time-dependent measurements of DNP-Met adsorption. The results are displayed in Figure 3 for the same sample within a 30-day period. Within 11days, the band maximum at 400 nm due to single particles of silver decreased steadily in absorbance while concurrently a low-energy band developed and shifted to 555 nm. Subsequently, the 400-nm band broadened and its maximum shifted to 425 nm while the 555-nm aggregate band decreased in intensity. Mixtures with DNP-Met were reddish in color at their higher concentrations and dark red at the lower concentration. In the two spectra at the longest elapsed time in Figure 3 a small contribution from the DNP group absorption at 360 nm was also detectable as a shoulder to shorter wavelength of the 425-nm band. These absorption spectral data indicate that adsorption of DNP-Met on silver is accompanied by aggregation of the silver particles. (29) Bohren, C. F.;Huffman, D. R. Absorption and Scattering of Light by Small Particles; Wiley-Interscience: New York, 1983.
320
440
560
6 80
8 00
WAVELENGTH .nm
Figure 3. Absorption spectra as a function of time for 3 X M DNP-Met on colloidal silver. Elapsed time (days): 1,4;2,7; 3, 11; 4, 18;5, 29.
In general, after equilibration of DNP amino acid and silver sol mixtures a single narrow band centered at 400 nm was observed. This band has been associated30with the dipolar surface plasmon of small single spherical particles of silver. The lowest energy electronic absorption band maximum of the DNP group in the amino acid derivatives occurs between 350 and 370 nm. It thus overlaps with the absorption band of the single silver particles at 400 nm, but there is little overlap with the band due to aggregates of silver particles at higher wavelength. As aggregation of the silver particles proceeded, the absorption maxima were shifted to longer wavelengths and the colloidal suspensions changed color from yellow to amber to olive green to bluish gray, at which point they began to sedimentate. Ideally, the color change corresponds to the gradual disappearance of the 400-nm band and the appearance of a shoulder at 500 nm that grows and moves to longer wavelengths until the sample sedimentates and its absorption band shifts to about 800 nm. This color change is due to the increasing size of the particles from single particles to doublets, triplets, and higher order multiplets. Samples with (DNP-Cy& showed more aggregation with a decreasing concentration of the amino acid derivative. One month after preparation the samples ranged in color from amber to dark red-brown to a very dark red. The initial yellow to red color of the mixtures with a-DNP-Lys turned to a blood red color that grew more intense with an increase in a-DNP-Lys concentration. Samples below 1yM in a-DNP-Lys turned cloudy, indicating the presence of very large aggregates, and could not be used. Aggregation of DNP-Asp silver sol mixtures was also induced by adding sodium perchlorate. In the absence of salt, only samples at low concentration (1-5 yM) turned a very dark red-green. With 1-5 mM NaC104and 3 yM DNP-Asp, the color of the samples ranged from brown to olive green. In each case, as there was an increase in agggregation induced by the DNP amino acid derivatives and thus an intensity increase in the absorption spectrum of aggregates, there was a concurrent increase in SERS band intensities. DNP-Asp was chosen as a potential adsorbate for its high formal negative charge (-2) at pH -6.5 and for the established chemisorptionnJ1 ability of carboxylate groups, which appeared to be ideal for bridging silver particles and inducing further aggregation in the suspension. However, it appears that at high DNP-Asp concentrations single particles of silver acquire an overall high negative charge and are prevented from clustering through repulsive interparticle electrostatic forces. This charge stabilization (30) Papavassiliou, G.C.B o g . Solid State Chem. 1979,12, 185. (31) Suh, J. S.;Moskovits, M . J. Am. Chem. SOC.1986, 108, 4711.
1024 Langmuir, Vol. 4, No. 4 , 1988
Curley and Siiman
of the silver sol with adsorbed DNP-Asp is evident at concentrations above 5 pM at which no aggregation was detected. In order to induce aggregation in the DNPAsp-silver sol mixture, submonolayer concentrations of DNP-Asp from 1 to 5 pM had to be used. The particles then acquired a charge close to zero by compensation for some positively charged silver ions on the particle surface,32-34giving a greater probability for collisions that result in adhesion. Similar behavior was observed in DANS-Asp-silver sol mixtures. Since a negatively charged adsorbate is needed for the particles of the deionized silver sol to reach the point of zero charge, a compound such as DNP-Met with a lower negative charge and DNP-Cys with two carboxylate groups to serve as a potential bridging ligand between silver particles was observed to induce aggregation more easily. Even species such as the zwitterions, a-DNP-Lys and methionine, which have an isoelectric point close to pH 6.5, were used to successfully induce aggregation in the silver sol. This probably occurs since surfacebound amino groups in glycine and alanine have been shown31to exist in the NH, rather than NH3+ form so that the formal charge of the adsorbed species is -1. In order to induce more rapid aggregation in the silver sol-DNP-Asp system, several steps were taken: (1)higher silver particle concentrations were used, (2) the pH of the suspension was lowered, or (3) the salt of a noncoordinating anion was added. The first silver sol mixtures that were used at silver concentrations of 0.02 g/L showed no aggregation at all. Thus, the silver concentration was increased 3-fold to obtain aggregation in the lower concentration range for added DNP-Asp derivative. Alternatively, the pH of the silver sol was adjusted from 6.5 to about 3.0 with aqueous perchloric acid (0.1 M). With a pK, of 2.09 and 3.86 for the two carboxylic acid groups, lowering the pH to about 3.0 will lower the formal negative charge of the compound and the silver particles to which it is adsorbed and thus induce aggregation. Lowering the pH with the dropwise addition of 0.1 M perchloric acid, however, proved to be uncontrollable, and most of the samples gave immediate sedimentation of large aggregates of silver particles. The third alternative for controlled aggregation of silver hydrosols with adsorbed DNP-Asp was the most productive. It has been shown35that the addition of a salt of a noncoordinating anion such as sodium perchlorate will induce aggregation of colloidal gold particles. A high concentration of perchlorate will break down the electrical double layer of ions on the surface by compression and allow the closer approach of colliding particles to make aggregation into clusters more probable. Even though some of the DNP amino acid-silver sol mixtures required several months of equilibration to induce aggregation, their resultant SERS spectra (vide infra) showed no evidence of degradation of the DNP derivatives. Resonance Raman Spectra of DNP Amino Acids in Solution. The resonance Raman (RR) spectrum of 0.01 M DNP-Met in 0.01 M aqueous NaOH, obtained with 488.0-nm Ar+ excitation, is shown in spectrum A, Figure 4. The electronic absorption spectrum (Figure 5) of 10 pM aqueous DNP-Met, typical of all DNP amino acids that were used, has a distinct band maximum at 360 nm and a weaker shoulder near 400 nm. Excitation with Xo = 488.0 or 457.9 nm is expected to provide a resonance or
preresonance Raman condition with the low-energy shoulder but no rigorous resonance with the 360-nm peak. The RR spectra with 488.0- or 457.9-nm excitation were very similar for all DNP amino acids that were examined. Spectra of 0.01 M aqueous DNP-Asp and a-DNP-Lys and 0.01 M (DNP-Cys), in 0.01 M aqueous NaOH are included with SERS spectra (vide infra) of the respective DNP amino acids. Excitation into the 360-nm electronic absorption band of DNP-Met with 386.4-nm excimer-pumped dye laser irradiation gave very selective RR enhancement (Figure 4, spectrum B) of bands at 1330 and 714 cm-'. The spectra showed no time-dependent effects that could be associated with photodecomposition of DNP-Met in the excimerpumped dye laser beam. The 1330-cm-l band is similar in position and intensity to the vs(N02) RR band ob~ e r v e d ~in" various ~~ para-substituted nitrobenzene derivatives (nitrobenzene, p-nitrotoluene, p-nitrophenol,
(32) Frens, G.; Overbeek, J. T. G. Kolloid 2.2. Polym. 1969,233,922. (33) Miller, W. I.; Herz, A. H. In Colloid and Interface Science IV; Kerker, M., Ed.; Academic: New York, 1976;p 315. (34) Blatchford, C. G.; Siiman, 0.;Kerker, M. J . Phys. Chem. 1983, 87,2503. (35)Enustun, B. V.;Turkevich, J. J . Am. Chem. SOC. 1963,85,3317.
(36)Shorygin, P.P.Dokl. Akad. N a u k SSSR 1952,87, 201. (37)Shorygin, P. P. Izv. Akad. Nauk SSSR, Ser. Fiz. 1953,17, 581. (38)Shorygin, P. P.J. Chim. Phys. 1953,50,D31. (39)Behringer, J. In Raman Spectroscopy; Szymanski, H.,Ed.; Plenum: New York, 1967.
1
1600
1
1
1
1
~
1
1
1
1200 1000 800 RAMAN SHIFT,cm-'
1400
1
600
1
1
1
400
Figure 4. RR spectra (A and B) of 0.01 M DNP-Met in 0.01 M aqueous NaOH. Experimental conditions are as follows. Excitation wavelength A, 488.0 nm, Ar+; B, 386.4-nm pulsed dye laser. Spectral slit width: A, 10.2 cm-'; B, 10.0 cm-I. Photon counting time interval (A) 0.05 s; scan speed (A) 1.0 cm-'/s; laser power (A) 25 mW. B, 250-mV sensitivity, 0.5-w~time constant, and 5-11s aperture on 165; 68.2% of 100-ns aperture delay, 5-ns aperture duration, and 1.0-s time constant on 162. Reference Raman spectrum (C) of quartz cell in air, same conditions as spectrum B. .3
I
I
I
i
200
400
600
WAVELENGTH.nm
Figure 5. Electronic absorption spectrum of 0.01 M D iP. Jet in 0.01 M aqueous NaOH. Excitation wavelengths of laser nes used for obtaining Raman spectra are indicated with arrows.
Langmuir, Vol. 4, No. 4, 1988 1025
SERS of 2,4-Dinitrophenyl Amino Acids p-nitroaniline, p-nitrodimethylaniline, and p-nitrodiethylaniline) in nonpolar (benzene and cyclohexane) and polar (methanol) solvents as the excitation wavelength (434.8 or 404.9 nm) was tuned into the lowest energy electronic absorption band between 400 and 260 nm. However, in these latter cases, the intensity of the totally symmetric aromatic ring-breathing mode at -850 cm-' was more enhanced on approaching resonance than that of the symmetric NOz stretching mode at 1300-1350 cm-'. Thus, in the DNP-Met spectrum with Xo = 386.4 nm the very high relative RR intensity in the band assignable to v,(pNO2) and low intensity in all in-plane phenyl ring vibrational bands including the ring breathing mode at 833 cm-' suggest that the 360-nm absorption band originates from an electronic transition localized mainly in the p-N02 group and polarized along the long axis ( x ) of the phenyl ring. Since the various substituents (CH3, OH, NH2, N(CH3)2,and N(C,H,),) in p-nitrobenzenes strongly affected the position of the lowest energy absorption band, it is not surprising that this band when located near 400 nm might originate from a phenyl ring-nitro group electronic transition and thus give rise to an intense v,(ring breathing) mode when excited. Local, in-plane NO2group modes in nitroaromatics include vas(N02),v,(N02), and 6(N02),generally located39near 1560,1370, and 650 cm-'. The asymmetric NO2 stretching mode has been observed intensely in infrared spectra of nitroaromatics between 1490 and 1530 cm-' but has not been characterized in Raman spectra. It is expected to be even less intense in RR spectra but may become intense in SERS spectra (vide infra). By analogy, the second most intense RR band at 714 cm-' in DNP-Met excited with A,, = 386.4 nm radiation is assigned to the in-plane NO2 bending mode. Previous band assignments40have been made for 2,4dinitroaniline by obtaining spectra of the three 15N-labeled derivatives substituted at NHz, o-NO2,or p-NO2. Four bands between 1250 and 1400 cm-' were assigned to vibrations involving the NO2 and NH2 groups. The band at 1337 cm-' was shown to arise from the p-N02 group, while the other three bands were assigned to the "ortho structure", consisting of the H2N-C-C-N02 unit. A similar set of bands was also observedl2 in various DNP-NHR compounds, E-DNP-L-LYS, DNP-Gly, and DNP-L-As~. These are assignable to symmetric, in-plane, stretching modes, u,(p-N02) and v,(H2N-C-C-N02). The effect of various solvents on the RR spectra of DNP-NH2 in the 1250-1400-cm-' region was also investigated to verify any tendency to form a planar, intramolecular H-bonded ortho structure
Table I. SERS Enhancements DNP-Asp 10 5 1 3 with
0.255 0.257 0.042 0.94 4.06 1.89 1.37 0.97
1" 2" 3" 4" 5"
250 510 420
250 1100 870
3 100 14 000 6 300 4 600 3 200
6 500 29 000 13 000 9 600 6 200
180 720 3 200 7 200 4 200 11000
1800 3500 5200
DNP-Met 100 50 10 5 3 1
1.83 3.58 3.17 3.62 1.27 1.12
100 50 10 5 1
0.42 29.5 8.4 4.9 0.39
100 50 10 5
3.17 2.11 0.97 0.19 0.20
(DNP-CYS)~ 42 5900 8400 9900 3900
CY-DNP-LYS
1
320 420 980 370 2000
" CNsC104, mM* Table 11. Resonance Raman Band Positions (cm-') and Vibrational Mode Assignments for DNP-Met in Aqueous Solution
b,nm 457.9
386.4 1612 (2)
1583 (1)' 1574 (1) 1522 (1) 1432 (1) 1363 (9) 1330 (9) 1271 (6) 1233 (4) 1147 (1) 833 (10) 714 (1) 395-490 (3)
mode assignment 4 ua
1574 (1.8) 1522 (1.4) 1432 (1.6) 1330 (10)
v(C-NH) u,(~-NOZ) u,(o-NOz)
1147 (1.5) 833 (1.7) 714 (3.5)
us (ring
breathing)
" Values in parentheses are relative peak intensities. o ; N q N < H 0
0/N-o"
Shifts in bands associated with the ortho structure were closely correlated with dielectric constant and H-bonding strength and showed that this structure interacts significantly with the solvent. DNP-Asp, DNP-Met, a-DNP-Lys, and ( D N P - C ~ Sall) ~ showed bands at 1271, 1330, and 1363 cm-l. The two higher frequency bands were usually about twice as intense as the one at 1271 cm-'. The 1330-cm-' band is assigned to v,(p-N02);the other two bands to v,(02N-C-C-NHR). Another intense, narrow RR band of the nitroaromatic ring was observed at 833 cm-' in all DNP amino acid spectra. (40) Kumar, K.; Carey, P. R. J . Chem. Phys. 1975,63, 3697.
This band has been assigned to a symmetric breathing mode of the 1,2,4-trisubstituted phenyl ring. RR band positions and mode assignments for DNP-Met are summarized in Table 11. SERS Spectra and Enhancements of DNP Amino Acids on Colloidal Silver. SERS spectra of DNP-Met, (DNP-C~S)~, a-DNP-Lys, and DNP-Asp on colloidal silver are shown in Figures 6-9, respectively, for a series of total DNP amino acid concentrations at a fixed silver concentration. The SERS band intensity relationships among spectra in the series for each DNP amino acid are not simple functions of the total DNP derivative concentration. In each run, SERS intensity depends primarily on the surface coverage and both the degree of aggregation and the structure of the clusters of primary silver particles that were induced to form by addition of the DNP amino acid. Since the latter is a complicated function of conditions such as surface charge, pH of the medium, salt concen-
Curley and Siiman
1026 Langmuir, Vol. 4, No. 4, 1988 l
l
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Figure 7. RR spectrum (A) of 0.01 M (DNP-C~S)~ in 0.01 M aqueous NaOH. SERS spectra (B-E) of (DNPCys), on colloidal
silver. Excitation wavelength, 488.0 nm; laser power, 25 mW; spectral slit width, 10.2 cm-'; scan rate, 1.0 cm-l/s. (DNP-Cys)* concentration,photon counting time interval (s): A, 0.01 M, 0.05; B, 10 pM, 0.015; C, 5 pM, 0.02; D, 1 pM, 0.07.
RAMAN SHIFT, cm-1
Figure 6. SERS spectra of DNP-Met on colloidal silver. Excitation wavelength, 488.0 nm; laser power, 25 mW; spectral slit width, 5.0 cm-l; scan rate, 1.0 cm-l/s. DNP-Met concentration (pM),photon counting time interval (8): A, 100,0.03;B, 10,0.02; C, 5, 0.024; D, 3, 0.05; E, 1, 0.05; F, 0.5, 0.3; G, 0.1, 0.3.
tration, etc., there is no linear relationship between SERS band intensity and adsorbate concentration in the spectra shown ih Figures 6-9. Surface Raman band enhancements over solution Raman band intensities for the DNP haptens were typical of those for other chromophore^^^*^^ on silver surfaces, ranging from a lo2- to 104-foldenhancement. Enhancements listed in Table I were estimated for the intense narrow band at 833 cm-' in each case. The relative Raman intensity (RRI), &/&$, was obtained from integrated band intensities (peak height X fwhm) for spectra obtained with 488.0-nm excitation. The value of RRI was then scaled by the total DNP amino acid concentration in sol and solution, C, and C,h, to get the Raman enhancement from the sol, enh;, = RRI(Csol,,/Csol).Where ultracentrifugation data were available, the enhkl values were further corrected for the actual concentration of DNP amino acid on the surface to calculate enhz,,. The values of the enhancements need to be further qualified. First, one cannot be sure that the surface Raman spectra were taken when all the samples were at the same stage of aggregation. In fact they were not, as evidenced by the wide range of colors and absorption spectra of the
Figure 8. RR spectrum (A) of 0.01 M a-DNP-Lysin aqueous solution. SERS spectra (B-F)of a-DNP-Lyson colloidal silver. Excitation wavelength, 488.0 nm; laser power, 25 mW; spectral slit width (A) 10.2 cm-', (B-F)5.0 cm-'; scan rate, 1.0 cm-'/s. a-DNP-Lysconcentration (pM),photon counting time interval (9): A, lo4,0.04; B, 100, 0.02; C, 50, 0.03; D, 10, 0.07; E, 5 , 0.1;
(41)Lepp, A.; Siiman, 0.J. Phys. Chen. 1985,89, 3494. C.C.; Creighton, J. A. J. Electroanal. Chem. 1982, 133, 183.
silver sol-DNP amino acid mixtures. A measure of the extent of aggregation is provided by the increase in in-
(42) Busby,
lLOO
1100 800 RAMAN SHIFT,rm-'
F, 1, 0.1.
Langmuir, Vol. 4, No. 4, 1988 1027
SERS of 2,4-Dinitrophenyl Amino Acids l
I
l
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- lL
1 1400
l
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"
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'
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Figure 9. RR spectrum (A) of 0.01 M DNP-Asp in aqueous solution. SERS spectrum (B) of 5 pM DNP-Aspon colloidal silver. SERS spectra (C and D) of 3 pM DNP-Asp and 1 mM NaC104 (C) and of 2 mM NaC104 (D) on colloidal silver. Excitation wavelength, 488.0 nm; scan rate, 1.0 cm-l/s. Laser power (mW), spectral slit width (cm-I),photon counting time interval (8): A, 25, 10.2,0.025;B, 30, 5.0,0.3; C, 20, 10.2,0.05;D, 20, 10.2,0.02.
tensity of a shoulder or separate absorption band at 500 nm and the decrease in intensity of the 400-nm peak, originating from single particles of silver. Second, the most accurate values of enhancements should be those for the low concentrations of DNP amino acids since it is in the submonolayer region of the adsorption isotherm that almost all of the adsorbate is located on the surface of silver particles and adsorption sites, which are SERS active. An additional feature is the change in the enhanced Rgman spectra that occurs at extreme concentrations of 1.0 pM or lower. It may not be justified to compare results from the low and high concentration ranges with any great accuracy. The enhancements for DNP-Asp were of the same order as those reported for the other DNP derivatives. The same trend was seen in that there was an increase in magnitude with decrease in adsorbate concentration. Enhancements for DNP-Asp (3 pM)-silver mixtures that were further treated with aqueous sodium perchlorate (1-5 mM) leveled out at about 104-foldsuch that a further increase in perchlorate concentration did not increase the SERS enhancement. Corrected enhancement values were obtained for DNP-Met- and DNP-Asp-colloidal silver mixtures from experimental determinations of the amount that was adsorbed on the silver surface. Values of Csd for DNPMet on colloidal silver were already discussed. Similar analyses showed that about 46-49% of the DNP-Asp was adsorbed on the surface in the 2-50 pM concentration range for a silver concentration of 0.06 g/L and samples centrifuged shortly after mixing. A parallel run with
1600
1400
1200
1000 800 RAMAN SHIFT.cm-l
600
LOO
200
Figure 10. SERS spectra of amino acids L-Met (A), L-ASP (B), L-LYS(C), L-C~S-HC~ (D), and L-cystine (E) on colloidal silver. Excitation wavelength (A-D) 488.0 nm, (E) 647.1 nm; laser power, 25 m W spectral slit width, 10.0 cm-'; scan rate, 1.0 cm-l/s. Amino acid concentration (pM), photon counting time interval (8): A, 5,0.040; B, 1,0.030;C, 10,0.025;D, 1000,0.070for 200-700 cm-' and 0.100 for 550-1700 cm-l; E, 10, 0.100.
samples containing 0.1 g/L silver gave 56-33% DNP-Asp on the surface in the same 2-50 pM range. The corrected enhancement figures, enbd, for DNP-Asp and DNP-Met on silver are also listed in Table I. Since ultracentrifugation probed all adsorption sites of the DNP amino acid on single and aggregated silver particles, which do not all contribute to enhanced Raman signals, the corrected enhancements still represent a lower limit. SERS Spectra of Amino Acids on Colloidal Silver. Before we attempted to make vibrational assignments for DNP amino acids on silver, the SERS spectra of each parent amino acid, L-methionine, L-cystine, L-lysine, and L-aspartic acid, as well as the spectrum of L-cysteine hydrochloride (L-CYPHC~) on colloidal silver were obtained and are shown in Figure 10. The total concentration of amino acid (1 mM for L-CYS~HC~ and 410 pM for the others) was well below the limit (-0.1 M) for observing normal Raman spectra from these amino acids in aqueous solution under the same instrumental conditions. In each case the Raman spectra were run 1-3 days after the mixture of amino acid and silver particles was prepared so that absorption spectra showed a resolvable shoulder or distinct band due to aggregates of silver particles in the 500-600nm range. Vibrational mode assignments for some of the bands in SERS spectra of the above amino acids can be made by reference to the recent SERS spectra31reported for glycine, a- and @-alanine,and 6-aminocaproic acid on colloidal
1028 Langmuir, Vol. 4, No. 4, 1988 silver. A common feature in these spectra as well as our spectra for L-Met, L-As~,L-LYS,and L-cystine on silver is a very strong band in the 1350-1450-cm-' region, assignable in all cases to the symmetric carboxylate group stretching mode, v,(C02-). Only in the SERS spectrum of L-CYS~HC~ is the vs(C02-)band absent, and all major bands between 400 and 800 cm-' appear to be associated with the carbon-sulfur stretching mode, v,(C-s), or vibrationally coupled with it. Bands in the 800-1000-cm-' range can be assigned to v(C-C) stretching modes. Two bands which appear with variable intensity a t about 927 and 890 cm-' can be related to previous assignments for v(C-C). Their absence for L-cystine is further evidence for the lack of carboxylate-silver surface binding in this case. Both bands occur with equal intensity for L-As~.The 890-cm-' band is weaker for L-Met, absent or very weak for L-LYS,and very strong for L-cystine. The 927-cm-' band is absent or very weak for L-cystine. This probably demonstrates different orientations and/or coordination geometry for the COz-groups on the surface. L-LYSon silver shows the lowest frequency, 1366 cm-', for v,(CO~)and does not exhibit a detectable band at 890 cm-', whereas L-cystine gives the highest frequency, 1417 cm-', for v,(COz-) but does not have a band a t ca. 927 cm-l. According to vibrational data that have been summarized43for unidentate, bidentate, and bridging metal acetate complexes, the low frequency for v,(CO,-) with L-LYSindicates unidentate coordination to the surface. It is also noted that for L-LYS on silver the band a t 1571 cm-l, assignable to the asymmetric C0,- stretching mode, v,(COz-), is stronger than in any of the other spectra in Figure 10. This may be expected for unidentate coordination in which the asymmetric orientation of the noncoordinated C=O bond may be favorable for SERS enhancement. On the other hand, the high frequency (1417 cm-l) for v,(COz-) in L-cystine is close to the value for an ionic carboxylate group in solution and, thus, may indicate ionic interaction of the carboxylate groups in L-cystine with the silver surface or a bridging structure between adjacent silver atoms on the surface. The necessarily symmetric environment for oxygen atoms of the carboxylate group on the surface gives a SERS spectrum in which the v,(COz-) band was not observed in Figure 10, spectrum E. Further, the adjacent C-C bond to the C02- group is expected to be orientated exactly normal to the surface in a symmetric geometry for the two oxygen atoms so that the very strong SERS band at 890 cm-' can be assigned to this v(C-C02-) mode. SERS bands in the 1050-1200-~m-~ range can be assigned to v(C-N) or v(NH2) group modes, p,(",) or pw(NH,). Since a weak band between 1610 and 1640 cm-l, assignable to 6(NH2),was observed in all spectra in Figure 10, it is reasonable to also associate other bands in the 1050-1200-crn-' region with the C-NH2 group. All spectra also showed bands in the 700-850-cm-' region, which may be assigned to either p,(NH2) or other v(C-C) modes. Both L-cysteine and L-cystine on silver show characteristic SERS bands of sulfur-related vibrational modes. The medium bands at 686 and 500 cm-' for L-cystine are assigned to v(C-S) and v(S-S) modes of the disulfide linkage. Previous Raman spectra of disulfides and their extensive analysis have s h o ~ n *that ~ >the ~ ~position of v(S-S) is dependent on the conformation of the carbon atoms in the disulfide bridge. Bands at 510, 525, and 540 (43) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds; 4th ed.; Wiley-Interscience: New York, 1986. (44) Tu, A. T. Raman Spectroscopy i n Biology: Principles and A p plications; Wiley-Interscience: New York, 1982. (45) Parker, F. S. Applications o f Infrared, Raman, and Resonance Raman Spectroscopy in Biochemistry; Plenum: New York, 1983.
Curley and Siiman cm-' were assigned to the gauche-gauche-gauche, gauchegauche-trans, and trans-gauchetrans conformers, respectively. Our position of 505 cm-' for L-cystine on silver agrees most closely with the gauche-gauche-gauche rotomer, which is also the one that is found preferably in naturally occurring proteins. The medium band assigned to v(C-S) at 686 cm-' in L-cystine is somewhat higher in frequency than the C-S stretch usually obseved at 660 cm-' for disulfides with a 510-cm-' v(S-S) band. These shifts may be due to interaction of sulfur with silver atoms on the surface. For L-cysteine on silver the strong band at 590 cm-' is assigned to the v(C-S) mode. Extensive silver-sulfur binding appears to have downshifted the frequency of this band from its usual position at 650 cm-l. Thus, the very strong broad band at 432 cm-' may be associated with the silver-sulfur-cysteine metal-sulfur stretching mode. This v(M-S) mode can occur at relatively high frequencies (350-450 cm-') when strong metal-sulfur bonding is present, as previously observed46in resonance Raman spectra of ferric and cupric thiolate complexes. There is also a low-frequency strong-medium SERS band for L-LYSand L-ASPon silver at 210 and 205 cm-', respectively. As in the SERS spectra3' of glycine on colloidal silver at 241 cm-l, these bands are assigned to silvermolecule stretching modes, either v(Ag-02C) or v(Ag"2).
Conspicuously absent in the L-Met SERS spectrum are three intense Raman bands that are found45at 655,701, and 724 cm-' in aqueous L-Met a t pH 1.0. These bands are similar to those in liquid 2-thiabutane4 that have been assigned to C-S stretching frequencies: 723 cm-l, trans form; 675 cm-', gauche form; and 650 cm-', both forms. We therefore surmise that the thioether sulfur atom of L-Met does not coordinate to the silver surface. Vibrational band positions and mode assignments are collected in Table 111. Vibrational Mode Assignments for SERS Bands of DNP Amino Acids on Colloidal Silver. The enhanced Raman spectra, shown in Figures 6-9 for 488.0-nm excitation, of each of the DNP haptensilver sol mixtures were in many respects similar to each other even though widely different amino acid residues, methionine, aspartic acid, lysine, and cystine, were present in the compounds. The relative intensity of bands in the SERS spectra also showed some wavelength dependence. For example, a mixture of silver sol with 30 pM DNP-Met excited either with 647.1-nm Kr+ or 586.0-nm pulsed, excimer-pumped R6G dye laser radiation gave the two most intense SERS bands at 1323 and 833 cm-', similar to the spectra in Figure 6, A and B, taken with 488.0-nm Ar+ excitation. Notable changes that were observed in the 647.1-nm SERS spectrum included the following: bands at 1363 and 1271 cm-' were weak, the latter being broadened and centered at 1286 cm-l; only a weak band occurred at 1612 cm-'; no 1583-cm-' band was detected; a broader band at 1133 cm-' replaced the 1141-cm-' band; and the 928-cm-' band was weak and accompanied by another band at 890 cm-' of comparable intensity. The minor changes that were observed with excitation in the red appear to indicate the absence of any chromophoric decomposition products of DNP amino acid, generated by either adsorption on silver or irradiation, and also show a structural homogeneity of adsorbed DNP amino acid in aggregates of silver particles. The large width of the absorption band in the 450-700-nm region in Figure 3 due to the dipolar resonance of aggregates of silver particles is consistent with a wide distribution of sizes and shapes of aggregates. Thus, SERS spectra could be ob(46) Siiman,
0.; Carey, P.R. J. Inorg. Biochem. 1980, 12, 353.
SERS of 2,4-Dinitrophenyl Amino Acids
Langmuir, Vol. 4, No. 4, 1988 1029
Table 111. SERS Band Positions (cm-'), Relative Intensities, and Vibrational Mode Assignments for Amino Acids on Colloidal Silver predominant A B C D E vibrational L-Met L-ASP L-LYS L-CYPHC~ L-cystine mode 1617 w 1615 vw 1627 w 1611 w 1636 1571 mw 1571 w 1568 w 1500 mw 1446 w 1388 vs 1366 vs 1405 vs 1417 vs u,(COz-) 1295 w 1293 w 1290 w 1293 vw 1244 vs, sh 1254 vw pt(NHd 1105 vw 1157 w 1161 m, br PW("2) 1071 w 1055 w 1068 vw 1050 m 1052 mw v(C-N) 1019 vw 971 vw 985 vw 971 vw 923 mw 927 mw 928 mw u(C-C) 881 mw 890 w 890 w 890 vs u(C-CO,) 820 vw 827 w 840 vw 795 w 788 s 800 vw 763 m 771 mw 762 mw PA",) 722 vw 686 m w 695 w 695 w u(OC0) 686 m u(C-S) 584 w 593 w 590 s u(C-S) u(S-S) 505 m 500 w 495 mw 505 w *(CO,-) 480 s 432 vs 4Ag-S) 396 w, br 376 w 210 vs 205 m 4%-0)
tained over a wide range of excitation wavelengths. The spectra are dominated by bands of the DNP moiety since it has a greater relative Raman scattering cross section than the amino acid portion. SERS intensity has been shown to have an (a/r)12 dependence on the distance16J7that the molecule resides from the surface (a = radius of particle, -6.5 for silver; r = a + distance). Thus, if the hapten is chemisorbed on the silver through the amino acid functional groups (carboxylateand residue) and the DNP group is about 8 A from the surface, the amino acid groups will experience a greater SERS enhancement. However, even though the DNP group experiences a 23-fold lower SERS enhancement due to distance, it typically gives a 10-100 times higher Raman scattering in solution than amino acids with nonaromatic residues at the same concentration. As a result, Raman bands of the DNP group are also the ones that are most intensely observed from the species adsorbed on silver, particularly at relatively high adsorbate concentrations above 1pM. Also being embedded in SERS active sites between silver particles in aggregates, some adsorbed DNP amino acids may show double-sided coordination, i.e., through the amino acid side of the molecule to one surface and through the DNP group to another surface. This structure for adsorbed DNP amino acids would again favor large SERS band enhancements for the DNP part of molecule. Another common feature in the structure of each Nsubstituted DNP amino acid derivative is the availability of at least one carboxylate group for coordination to the silver surface. Since the SERS spectra of the parent amino acids in Figure 10 showed that L-Met, L - A s ~L-LYS, , and L-cystine each showed a strong attraction for silver by chemisorption to one or more carboxylate groups, we expect their N-substituted DNP amino acid derivatives to behave in the same way. Thus, although strong nitrophenyl group bands dominate the SERS spectra at high surface concentrations, a strong u,(C02-) band near 1400 cm-' begins to appear in SERS spectra taken at a total adsorbate concentration of 1pM or less for DNP-Met and (DNP-CYS),.
The most prominent features of the spectra are the bands between 1200 and 1400 cm-'. The SERS spectra of DNP haptens on silver include bands at 1271, -1330, and 1363 cm-' originating primarily from vibrational modes of the ortho structure and the symmetric stretching modes of the two nitro groups. The 1330-cm-' SERS band is located in the range for the p-NO, group symmetrical stretching mode. The corresponding mode for the 0-NO2 group has been assigned to a band in a lower frequency range below 1300 cm-l. The 1271-cm-l SERS band can be assigned to u,(o-NO2). Although the p-NO2 group in the four position on the ring may be the group farthest from the silver surface, the separation (approximately 4 A) between the two nitro groups gives at most only a 2-fold lower SERS intensity for the more distant group. The p N 0 2 group also has the greatest potential to twist. Thus, its direct interaction with the silver surface may account for its shifted position to 1323 cm-l in DNP-Met on silver and to 1317 cm-' in (DNP-Cys), on silver and for its splitting into two bands at 1332 and 1310 cm-l and shifted position to 1320 cm-' in DNP-Asp on silver. Eventually, at very low concentrations, the original set of SERS bands of the in-plane NO2 symmetric stretch as well as the C-NH stretch disappears and a new set of bands at 1295,1373, and 1410 cm-' in ( D N P - C ~ Son ) ~silver and at 1288 cm-l in DNP-Lys on silver appears. These latter bands also occurred in the SERS spectra of the parent amino acids on silver. The sharpest intense band in the SERS spectra is located at 833 cm-'. This band has been assigned to the symmetric breathing mode of the phenyl ring. Its position and intensity are not affected by the various amino acid residues. A similar band at 840 cm-' was also found in the SERS spectrum of 2-amino-5-nitropyridine on a silver ele~trode.~, The general trend with the 833-cm-' band as well as with bands originating from the ortho structure is a decrease in their relative intensity, as compared to other bands in the spectrum, with a decrease in concentration, even though their overall SERS enhancement is increasing, until
1030 Langmuir, Vol. 4, No. 4,1988
Curley and Siiman
Table IV. SERS Band Positions (cm-'), Relative Intensities, and Assignments for DNP-Met on Colloidal Silver high concn intermed concn low concn mode assignment 1612 ( l ) a 1583 (1) 1522 (1) 1432 (1)
1612 (3) 1583 (3) 1522 (1) 1496 (3) 1432 (1)
1363 (4) 1323 (10)
1363 (3) 1323 (6)
1271 (6) 1233 (3) 1147 (1) 1056 928 (1)
1271 (9) 1233 (3) 1147 (3) 1056 (3) 928 (4)
1612 (6) 1583 (6)
714 (1)
395 (1)
833 (10) 781 (4) 714 (1) 684 (3) 589 (1) 500 (3) 395 (1)
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v,(o-NOz) 1147 (6) 1056 (6) 928 (6) 890 (3) 781 (10) 684 589 500 395
1600
"(C-NH) 4p-NOz)
1290 (6)
833 (8)
I
1400
1200
1000
800
600
400
RAMAN SHIFT,cm-l
Figure 11. SERS spectrum of 1 pM DANS-Asp on colloidal silver. Experimental conditions: excitation wavelength, 488.0 nm Ar+; incident laser power, 25 mW; spectral slit width, 10.2 cm-'; photon counting time interval, 0.1 s; scan speed, 1.0 cm-'/s.
vg (ring breathing) vg (ring breathing)
(6) (3) (6) (6)
Values in parentheses are relative peak intensities.
-
a certain low concentration of 1yM is reached. A t this latter point (3 yM for DNP-Met, 1 pM for (DNP-Cys),, 5 yM for a-DNP-Lys),there appears to be a discontinuity or at least a drastic change in the observed SERS spectra. The symmetric ring-breathing and nitro group modes have lost almost all their SERS band intensity, and a different set of intense SERS bands is observed. Clearly, this change is most visible in the series of DNP-Met SERS spectra which could be measured for the widest range of concentrations. The positions and relative intensities of SERS bands of DNP-Met on colloidal silver together with suggested vibrational mode assignments are summarized in Table IV. Although most features of the SERS spectra of DNP amino acids on silver are common to all spectra at comparable concentration levels, some notable differences did occur. For a-DNP-Lys in Figure 6 the band at 1330 cm-l due to the v,(p-NO,) mode is always very weak and unshifted from its position in the solution spectrum. This suggests that the p-NO, group does not interact strongly with the silver surface in this case. For DNP-Asp in spectra B and D, Figure 9, medium intensity bands are located at 1612,1467,1209,1111,and 1056 cm-l. Although the 1612- and 1056-cm-l bands are also detected with increasing intensity at low concentrations with other DNP derivatives, the 1467,1209,and 1111-cm-' bands were only observed intensely in DNP-Asp spectra. For (DNP-Cys), on silver in Figure 7, an 1141-cm-' band in its solution spectrum is first accompanied by another shifted band at 1124 cm-' at high concentrations on silver and, then, by a band at 1161 cm-' at low concentrations on silver. Other minor differences can be readily noted from the labeled peak positions of bands in Figures 6-9. SERS Spectrum and Vibrational Band Assignments of DANS-Asp on Colloidal Silver. A variety of fluorescent-labeled amino acids, including fluorescein,'* 7-nitrobenz-2-oxa-l,3-diazole (NBD):' and DANSlg derivatives, have been used as haptenic determinants in immune reactions. Since quenching of fluorescence from emitterse5, with a wide range of quantum yields has been (47) Lancet, D.; Pecht, I. Biochemistry 1977, 16,5150. (48) Siiman, 0.;Lepp, A. J . Phys. Chem. 1984,88,2641.
WAVELENGTH,nm
Figure 12. Electronic absorption spectrum (LHS) of 10 pM aqueous DANS-Asp and fluorescence emission spectra (RHS) of 100 pM aqueous DANS-Asp with excitation wavelengths (A) 250, (B) 266, and (C) 296 nm.
observed on various silver surfaces, it is possible to obtain SERS spectra of haptens, normally fluorescent in solution, on colloidal silver. To avoid the interference from the fluorescence due to the solution species, it was imperative to use a low, submonolayer concentration of hapten. This assured operation in the early, linear part of its adsorption isotherm, where the hapten is nearly entirely adsorbed on the colloidal silver particles. The SERS spectrum of 1yM DANS-Asp on colloidal silver (3/100 dilution of stock) is shown in Figure 11for 488.0-nm Ar+ excitation. It contains no sign of the intense fluorescence background that is normally observed for DANS-Asp in aqueous solution with 488.0-nm radiation and, consequently, eclipses the normal Raman scattering from the solution species. The electronic absorption spectrum of 10 pM aqueous DANS-Asp and the fluorescence emission spectra of 100 yM DANS-Asp with 250-, 266-, and 296-nm excitation are shown in Figure 12. The lowest energy absorption bands of DANS-Asp at 320, 250, and 218 nm are far away from the 488.0-nm excitation wavelength that was used to measure the SERS spectrum. However, its emission maximum at 550 nm in aqueous solution is sufficiently close to interfere strongly with normal Raman scattering measurements. As in SERS spectra of DNP amino acids on silver at comparable concentrations, DANS-Asp showed two medium-intensity (49)Hildebrandt, P.; Stockburger, M. J. Raman Spectrosc. 1986,17, 55. (50) Copeland, R.A.; Foder, S. P. A,; Spiro, T. G. J. Am. Chem. SOC. 1984. 106. 3872. (51) Lee, N.-S.; Hsieh, Y.-Z.; Morris, M. D.; Schopfer, L. M. J . Am. Chem. SOC.1987, 109, 1358. (52) Holt, R. E.; Cotton, T. M. J. Am. Chem. SOC.1987,109, 1841.
Langmuir, Vol. 4, No. 4, 1988 1031
SERS of 2,4-Dinitrophenyl Amino Acids 0
0 H
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Figure 13. Structural models for proposed DNP-Met orientations on colloidal silver: I, folded-over conformation with end-on orientation of DNP group; 11,extened structure with edge-on orientation of DNP group; and 111,extended structure with flat orientation of DNP
group. SERS bands at 815 and 782 cm-l in the symmetrical ring-breathing-mode region. The Raman and infrared spectra of naphthalene and its derivatives have been extensively i n ~ e s t i g a t e d . ~ Their ~ Raman spectra generally show only one band at -800 cm-' in this region. For example, the Raman spectrum54(A, = 488.0 nm) of 1naphthol-2,5-disulfonic acid, which has its lowest energy electronic adsorption band at 301.5 nm, contains bands at 1577 (2), 1460 (1.5),1358 (lo), 1242 (0.5),1070sh (0.5),1043 (4), and 805 (1)cm-l. Relative peak intensities are given in parentheses. The SERS spectrum of DANS-Asp on silver also shows a very intense in-plane ring mode at 1371 cm-l and a medium band at 1575 cm-'. The SERS spectrum of naphthalene adsorbed on a gold electrode in methanol (0.01 M naphthalene, 0.02 M LiC104,0.002 M H,SO,) has been reported55 between 1300 and 1600 cm-l. It, too, showed a very intense band at 1376 cm-l together with a medium-weak band at 1550 cm-l.
Conclusions Orientation and Conformation of DNP Amino Acids on Colloidal Silver. Two main factors determine the relative intensities of SERS bands of molecules adsorbed on the surface of aggregates of silver particles: (1) the distance between the silver surface and the functional group in the adsorbed molecule that produces a particular SERS band; (2) the orientation of the functional group with respect to the surface. A given functional group vibrational mode will in general be more intense in SERS spectra if the group in the molecule is oriented so that vibrational motion is normal to the surface rather than tangential to it. According to SERS selection rules that have recently been ~ u m m a r i z e d , ~the ~ Jmost ~ intense SEW bands will originate from vibrational modes that produce large changes in the polarizability component, a,,,with the z-axis directed normal to the surface. (53) Dollish, F. R.; Fateley, W. G.; Bentley, F. F. Characteristic Raman Frequencies of Organic Compounds; Wiley-Interscience: New York, 1974. (54)
2198. (55) 379.
Carey, P. R.; Froese, A.; Schneider, H. Biochemistry 1973, 12,
Two major surface structures for N-substituted DNP amino acids with at least one metal-binding group, the carboxylate oxygen atom(s), are proposed. They are based on the observation of two distinct types of SERS spectra at low ( I 1 pM where [monolayer] 20 pM) and high (>1 pM) concentrations of the adsorbing species. At monolayer and greater concentrations we expect the adsorbed molecules to adopt a conformation that maximizes their number on the surface and, thus, minimizes the surface area covered per molecule. A folded conformation of the DNP amino acids, i.e., the DNP group folded over the carboxylate group (which represents a good metal binding site), is therefore proposed at high concentrations. This compact structure (Figure 13, structure I) would occupy the least surface area. It also allows d o u b l e - ~ i d e interaction d~~ of the molecule with two silver particle surfaces such that the DNP amino acid can function as a bridging ligand in the aggregates of silver particles that are induced to form. In this conformation the DNP ring system would be orientated either end-on or close to end-on with respect to the surface($. The p-nitro group would be aligned such that v,(NO,) would have a substantial a,, component whereas the o-nitro group would have v,(N02) with virtually no a,, component. At low concentrations the DNP amino acids would tend to adopt more open or extended structures in order to occupy a greater surface area per molecule and cover as much of the available surface as possible. Two extended structures with carboxylate oxygen atoms chemisorbed to the surface are envisioned. Both structures require conformational changes in the DNP group by rotation about either the phenyl C-NH or N-CH(CO0-) bonds, starting from the folded, high concentration structure. The ortho structure of the 0-NO2group and NHR group is retained in each case. To obtain structure 11, rotation about the C-N bond is required so that the DNP ring assumes an edge-on orientation (Figure 13) in which the normal to the phenyl ring lies parallel to the surface. In this case, the p-nitro group is oriented so that v,(NO,) has a very small a,, component while the o-nitro group mode, v,(NO,), has a large a,, component. A third conformer, structure 111,
-
Busby, C. C.; Creighton, J. A. J.Electroanal. Chem. 1982, 140, (56) Siiman,
0.; Feilchenfeld, H. J. Phys. Chem. 1988, 92, 453.
1032
Langmuir 1988,4, 1032-1039
can arise by further rotation about the N-C bond. In this structure, the geometry of the planar DNP ring system is almost flat (Figure 13) with respect to the surface and the molecule is in its most extended form. Both p-nitro and o-nitro groups would then be oriented so that v,(NOZ) would have a very small cy,, component. By analogy to SERS spectra of benzene, pyridine, and other planar ring compounds adsorbed on silver, the large enhancement of the Raman band assigned to the symmetrical ring-breathing mode located a t 833 cm-' for the DNP ring system, which was observed at concentrations 21 pM, can be indicative of either flat, side-on, or end-on orientation of the ring with respect to the surface. However, simultaneous SERS enhancement of the in-plane, v,(NO,), bands can only take place when these modes have a significant a,, component, in either a side-on or an end-on orientation of the ring. The p-nitro group v,(NO,) band was observed intensely at 1330 cm-' a t high concentrations. This is taken as support for the folded-over structure of DNP amino acids under these conditions. In the intermediate concentration range of 1-10 pM the onitro group v,(NO,) band at 1271 cm-' generally occurred more intensely. This serves as evidence for the extended structure I1 in which the DNP ring takes on a side-on orientation. Other in-plane modes of the DNP ring represented by bands at 1612 and 1583 cm-' (ezgmodes of the phenyl group) are intensified at the same time. At low concentrations both u,(NO,) bands are usually very weak, suggesting that the DNP ring lies almost flat on the sur-
face. Also, the 833-cm-' symmetrical ring-breathing mode shows very little or no intensity in its SERS band. On the other hand, a series of otherwise weak SERS bands at 1147, 1056,928, 890, 781, 684, 589,490, and 370 cm-' have increased considerably in relative intensity. We associate these bands with out-of-plane modes of the two nitro groups (NOz twist, rock, wag) and the phenyl ring. Similar arguments for at least two distinct orientations of DANS-Asp on silver can be made. The additional lower frequency SERS band at 782 cm-' is associated in an analogous way with a ring-breathing motion that contains a substantial out-of-plane polarizability component, aZZ, such that it becomes SERS-active when the planar ring is orientated flat on the surface. However, a large population of DANS-Asp molecules with the ring system orientated in a side-on or end-on fashion is also indicated by the presence of intense in-plane modes at 1371 and 1575 cm-'.
Acknowledgment. This work was supported by Army Research Office Grant DAAG29-85-K-0102 and by NIH Grant GM-30904. We thank Dr. Lee Guterman for the fluorescence emission measurements and Professor J. A. Koningstein and Dr. L. Haley of Carleton University for their advice on the pulsed Raman measurements. Registry No. DNP-Met, 3950-28-5;PNP-Cys, 35749-09-8; DNP-ASP, 7683-81-0; CPDNP-LYS,24696-20-6; DANS-ASP, 1100-24-9;Met, 63-68-3;Cys, 56-89-3;Asp, 56-84-8;Lys, 56-87-1; Ag, 7440-22-4.
Correlation between SERS of Pyridine and Electrochemical Response of Silver Electrodes in Halide-Free Alkaline Solutions M. L. A. Temperini and D. Sala Instituto de Quimica de Uniuersidade de SBo Paulo, C P 20780, Sdo Paulo, Brazil
G. I. Lacconi, A. S. Gioda, and V. A. Macagno INFIQC, Depto. de Fisicoquimica, Fac. de Ciencias Quimicas, Uniuersidad Nacional de CGrdoba, Sucursal 16, casilla de correo 61, (5016) Cdrdoba, Argentina
A. J. Arvia* Instituto de Investigaciones Fisicoquimicas Te6ricas y Aplicadas ( I N I F T A ) , Sucursal 4, casilla de correo 16, (1900) L a Plata, Argentina Received J u l y 13, 1987. I n Final Form: February 25, 1988 Surface-enhanced Raman scattering (SERS) of pyridine (Py) on Ag electrodes in alkaline solutions free of halide ions was obtained at 25 OC as a function of the applied potential. The Ag surface was activated for SERS through repetitive oxidation-reduction cycles (ORC), the effect being dependent on the electrochemical electrode history. The SERS effect was correlated to the activation for the hydrogen evolution reaction (HER),which can be obtained by means of potentiodynamic as well as potentiostatic procedures. The maximum SERS activity was achieved at potentials near the potential of zero charge (pzc) of polycrystalline Ag and appeared to be related to the maximum observed in the roughness factor vs potential curve. These results can be interpreted through the formation of a new uniform globular overlayer structure on the electroreduced Ag surface, which apparently exhibits a certain degree of preferred crystallographic orientation. Three well-defined potential regions can be distinguished for the complex competitive interactions between H,O, OH- ion, and P y with the new Ag electrode surface.
Introduction Surface-enhanced Raman scattering is recognized as a very powerful and sensitive method for characterizing and investigating structural aspects of adsorbates at electrode surfaces',' and for learning more about the structure of the electrical double layer. 0743-7463/S8/2404-1032$01.50/0
Since the discovery of SERS,3p4a large number of works have focused the attention to SERS of PYon Ag electrodes (1) Surface Enhanced Raman Scattering, Chang, R. K., Furtak, T. E., Eds.; Plenum: New York, 1982. (2) Seki, H. J. Electron Spectrosc. Relat. Phenorn. 1986, 39, 289.
0 1988 American Chemical Society
