Research Article

Received: 19 July 2011 Revised: 22 August 2011 Accepted: 27 September 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jrs.3092

Proline and hydroxyproline deposited on silver

nanoparticles. A Raman, SERS and theoretical study
J. J. Cárcamo, A. E. Aliaga, E. Clavijo, C. Garrido, J. S. Gómez-Jeria
and M. M. Campos-Vallette*
The Raman and surface-enhanced Raman scattering (SERS) spectra of L-proline (Pro) and trans-4-hydroxy-L-proline (Hyp) were
recorded. SERS spectra were obtained on colloidal Ag prepared by reduction with hydroxylamine. Allowing sufficient time for
Pro and Hyp to adjust in the colloidal solution resulted fundamentally in obtaining unique and reproducible SERS spectra. Hyp
stabilizes on the surface more rapidly than Pro. The spectral analysis indicates that Pro interacts with the Ag surface through
the carboxylate group. The interaction of Hyp with the metal surface occurs through the amino, methylene and carboxylate
moieties of the molecule. The spectroscopic results are supported by quantum chemical calculations, performed using
extended Hückel theory (EHT) of the title compounds interacting with an Ag cluster model. The assignment of the Raman
bands was supported by a normal coordinate analysis performed through Becke, three-parameter, Lee–Yang–Parr/6-311
G* + calculations. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: Raman; surface-enhanced Raman scattering; L-proline; trans-4-hydroxy-L-proline; theoretical calculations

Introduction Raman instrumentation

L-Proline (Pro) and trans-4-hydroxyproline (Hyp) are the only The Raman and SERS spectra were scanned by means of a Raman
naturally occurring amino acids with a secondary a-amino group, Renishaw Microscope System RM1000 apparatus, equipped with
which explains their more basic character with respect to many 514, 633, and 785 nm laser lines for excitation, a Leica microscope
other amino acids. Hyp, a major component of the protein colla- and an electrically cooled charge-coupled device camera. The
gen, is a common nonproteinogenic amino acid (see Fig. 1). Hyp best Raman and SERS spectra were obtained using the 514 nm
and Pro play key roles in the stability of collagen.[1] Vibrational laser line. Macro Raman measurements were obtained by using
spectral data on Pro and Hyp, recorded at different pH values, adequate accessories. The signals were calibrated using the
have been reported. The results, therefore, are diverse and there 520 cm–1 line of a Si wafer and a 50 objective. The resolution
is no coherent idea about how these molecules are organized or was set to 4 cm–1 and 5 to 20 scans of 10 s each were averaged.
oriented on a metal surface. In the present contribution, we pre- Spectra were recorded in the 200–1800 cm–1 region to observe
pared solutions of the samples at a controlled and fixed pH, addi- both the Raman and SERS spectra. The spectral scanning condi-
tionally allowing the analyte to evolve in time on the surface until tions were chosen to avoid sample degradation.
unique and reproducible surface-enhanced Raman scattering
(SERS) spectra were obtained. Following this approach we have Raman measurements
obtained optimal results in our studies on tryptophan[2] and
Raman spectra of Pro and Hyp were scanned directly from the
lysine.[3] However, lysine did not give a unique SERS spectrum;
samples using the 514 nm laser line.
different spectra resulted from the diverse conformational/
structural possibilities available to the aliphatic moiety. We have
now performed quantum chemical calculations using extended Preparation of silver nanoparticles – SERS measurements
Hückel theory (EHT) for models of Pro and Hyp interacting with Nanoparticles (NPs) were prepared by chemical reduction of
an Ag surface to assist the interpretation of our SERS spectra. A silver nitrate with hydroxylamine hydrochloride.[4] The resulting
normal coordinate analysis to support the assignment of the colloid displays a final pH close to 7. The aqueous solutions
Raman bands for the title compounds was achieved by using utilized for the NP formation were prepared using nanopure
density functional theory (DFT) calculations at the Becke, water. Metal surfaces of Ag nanoparticles for micro-SERS
three-parameter, Lee–Yang–Parr (B3LYP)/6-311 G* + level. measurements were prepared by immobilizing the colloidal
nanoparticles. Aqueous solutions of the samples were obtained

Experimental * Correspondence to: M. M. Campos-Vallette, University of Chile, Faculty of

Sciences, Department of Chemistry, POBox 653, Ñuñoa, Santiago, Chile. E-mail:
Amino acids [email protected]

Proline and hydroxyproline were obtained from Fluka; purity was Vibrational Spectroscopy Laboratory, Faculty of Sciences, University of Chile,
checked by routine infrared analysis. POBox 653, Santiago, Chile

J. Raman Spectrosc. (2011) Copyright © 2011 John Wiley & Sons, Ltd.
 J. J. Cárcamo et al.

obtained in the published spectral studies of the simple amino

acid molecules, e.g. tryptophan,[2] lysine,[3] cysteine,[14] Pro and
valine,[15] histidine,[16] Pro and Hyp,[17] related systems such as
oligopeptides containing Pro,[18] several other amino acids,[19]
including the aromatic phenylalanine and tyrosine[20] and ab
initio vibrational wavenumbers.[21] A normal coordinate analysis
was performed for Pro and Hyp to complete the vibrational spec-
tral assignments. The results of these calculations are available on
request. Calculated bands are listed in Table 1; for the sake of
Figure 1. Molecular models for proline and 4-hydroxyproline. simplicity we have discussed only the fingerprint spectral region
300–1800 cm–1. The Raman and SERS spectra now measured for
Pro differ from those published by Sheena Mary et al.,[22] who
added the aqueous solution of Pro to a citrate-reduced silver
by dissolving the materials at 10–3 M concentration. Samples for
colloid; their Raman spectrum displays several bands probably
SERS were prepared by adding 100 mL of the sample solution to
corresponding to impurities. The source of the amino acid used
500 mL of the silver colloid. The pH was adjusted to 7 by adding
by them is not indicated.
aqueous HCl and NaOH. Final solutions were allowed to stand
for 2, 24, 48 and 72 h. After completing each lapse of time, 10
mL of the final mixtures containing the Pro and Hyp samples were Raman spectra of Pro and Hyp
dropped on a slide and dried at room temperature. Laser powers The Raman spectra of Pro and Hyp in the solid state are displayed
in the 100 mW to 10 mW range and selected spectral scanning in Figs 2 and 3, respectively. Bands ascribed to nOH, nNH are
conditions guaranteed nondestructive sample treatment. active around 3400 cm–1 while the aliphatic nCH modes are
observed below 3000 cm–1. The dNH+2 deformation is seen at
Molecular models and calculations for Pro and Hyp about 1620 cm–1 while the nasCOO– mode is attributed to the
band close to 1550 cm–1. The nsCOO– mode corresponds to the
To help with the band assignment, the infrared and Raman medium-intensity band at 1373 cm–1. Bands ascribed to deforma-
spectra of Pro and Hyp were calculated using DFT at the B3LYP/ tions of the aliphatic CH2 groups are observed at about 1470 and
6-311 G* + level with full geometry optimization. The zwitterionic 1450 cm–1. The band at about 1350 cm–1 is a wagging CH2 mode.
form was selected because of the SERS experimental conditions. Coupled NH and CH deformation modes are expected between
GAUSSIAN software was used.[5] The silver surface was simulated as 1320 and 1210 cm–1. Rocking and twisting CH2 modes are nor-
follows: a large (a = 0.408 nm) Ag cube with a face-centered cubic mally observed in the range 800 to 1250 cm–1. Bands around
structure was built as in our previous studies.[2,3,6,7] The resulting 1170 cm–1 are probably due to coupled CH2 deformation modes.
structure was trimmed to get a planar double layer composed of In the series of amino acids only Pro and derivatives display char-
324 Ag atoms. Molecular mechanics was employed to optimize acteristic bands in the range 1100–980 cm–1, which are attributed
both the Pro-Ag and the Hyp-Ag surface geometries. The metallic to the nCN and dCCN modes. The band at 950 cm–1 is a nCC vibra-
layer geometry was kept constant. Proline was placed at different tion of the pyrrolidine ring. The strong band at 917 cm–1 is
distances and orientations from the center of the Ag layer. EHT ascribed to the nCC vibration of the C–COO– group. The strong
was used to calculate the wave function of proline as an isolated bands at 896 and 839 cm–1 are due to CC stretching and CH2
system and interacting with the metal surface. The HYPERCHEM twisting modes, respectively. At 790 cm–1 a weak band is
program was used.[8] EHT calculations produce qualitative or observed, probably associated with a deformation of the COO–
semiquantitative descriptions of molecular orbital and electronic group. Bands around 695 and 640 cm–1 are assigned to different
properties.[9] The combination of EHT with molecular mechanics COO– deformation modes. Bands around 570 cm–1 probably
was able to give, for example, a qualitative explanation of involve rCO modes. Pyrrolidine ring deformations are ascribed
our previous SERS work on nanotubes,[6] humic acids,[10] to bands at about 445 cm–1. Other vibrations related to deforma-
tryptophan,[2] lysine,[3] and arginine[11] interacting with Ag tion modes of the NCCO and CCCO fragments are expected
surfaces. EHT was chosen because it was shown, within the below 450 cm–1.
Hartree–Fock–Rüdenberg picture, to be compatible with the
nonempirical Hartree–Fock method in Roothaan’s form.
Hartree–Fock–Rüdenberg thus explains why EHT proved to be SERS spectra of Pro and Hyp
qualitatively successful.[12,13]
Time-dependent SERS measurements have been performed.
Results and Discussion This technique was successfully tested by us in the study of
tryptophan and by Podstawka et al.[23] to study the binding spec-
The Raman and SERS spectra were recorded for the analytes and ificity of Gly–Gly on a silver surface. Different spectra were
the surfaces plus the analytes. Spectral modifications (intensity obtained after 2 and 24 h of mixing Pro and Hyp solutions with
and wavenumber) of the analytes because of surface effects were the colloidal Ag suspension. The fact that no evident SERS spectra
evaluated from a structural viewpoint. of Pro and Hyp were obtained at shorter time periods suggests
that the nature of the substrate–adsorbate interaction is drasti-
cally modified. Reproducible spectra were obtained at 48 h for
Vibrational analysis – General information
Hyp. Only after 72 h was a batch-to-batch reproducible spectrum
The following proposed band assignment of the Pro and Hyp obtained for Pro. SERS spectra at 72 and 48 h along with the
samples is based on our Raman and SERS information and that Raman spectra of the solids are displayed in Figs 4 and 5 and

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Proline and hydroxyproline deposited on silver nanoparticles

Table 1. Experimental and calculated Raman spectra, SERS data, and the most likely band assignments for Pro and Hyp
Raman Pro exp. Raman Pro calc. SERS Pro 72 h Raman Hyp exp. Raman Hyp calc. SERS Hyp 48 h Assignment

1623 mw 1640 wm 1608 w 1642 vw 1640 wm 1585 m dNH+2

1548 m 1510 s 1572 m 1520 ms 1562 ms nasCOO–
1473 wsh. 1483 ms 1477 vw 1450 wm 1477 s dCH2
1449 md dCH2
1410 wm 1409 mb 1428 vwd oCH2, oNH+2
1373 m 1350 ms 1373 w 1389 wm 1390 w 1390 ms nsCOO–
1320 sh 1352 vw 1350 ms oCH2
1317 vw 1312 ms 1318 wmult. 1300 wm 1301 wsh. tCH2
1286 m 1290 sh 1279 w 1280 w 1273 md rCH2, tCH2
1263 m 1270 ms 1262 vw 1258 vw 1260 m dCH, dNH
1237 m 1210 m 1215 s as. 1223 w 1230 vw 1240 s dCH, dNH
1173 md 1170 w 1164 w 1183 w 1190 w oCH2
1100 w 1102 wb 1110 m 1111 vw dCNH
1081 m 1085 w 1070 vw nCN
1054 m 1049 wb 1047 wd 1065 m oCH2
1032 m 1030 m 1032 w 1030 m nCCN
983 m 990 w 1000 m 984 sh nCCN, nCC
950 m 962 wm 956 m 966 m nCC pyrim.
917 s 918 w 918 w nC-COO–
896 s 910 m 877 m 850 vs nCC
839 ms 850 m 831 w 850 vs 820 m 851 m tCH2
790 w 780 w 757 wm 756 wm 790 wb 793 m dCOO-
670 vw 697 wd 696 w 670 w d,o COO –
638 m 630 w 642 m 616 w 640 wm 654 mwb r,d,oCOO-
572 vw 580 vw 593 w 590 mb mult. rCO
527 vw 550 w dCCN
445 m 470 m 484 vw 464 m 480 m 461 w Pyrrolid. deform.
421 m
370 wb 350 m 350 m NCCO, CCCO def.
300 vw 302 wm 250 wm

Band description: w, weak; vw, very weak; wb, weak broad; mw, medium weak; md, medium double; ms, medium strong; sh, shoulder; s, strong;
mult. multiple; pyrrolid. pyrrolidine cycle.

Figure 2. Raman theoretical (a), experimental (b), and SERS (72 h) (c) spectra of proline.

J. Raman Spectrosc. (2011) Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jrs
 J. J. Cárcamo et al.

Figure 3. Raman theoretical (a), experimental (b), and SERS (48 h) (c) spectra of hydroxyproline.

but its intensity decreases. The SERS bands at 1409 cm–1 (oCH2,
oNH+2 ) and at 697 cm–1 (d,o COO–) appear as a consequence of
the surface effect. This spectral behaviour, according to the SERS
selection rules[24] indicating that modes having their Raman
polarizability z-component perpendicular to the surface are likely
to become more enhanced than the parallel ones, is consistent
with a CO bond vibrating perpendicularly to the metal surface.
In particular, the intensity increase suggests a close CO-metal
surface interaction. The interaction involving the NH moiety and
the metal surface is probably less intense than that of the carbox-
Figure 4. Predicted proline-Ag surface interaction. ylate group. This is verified by the wavenumber shifts, intensity
variations or disappearance of the Raman bands involving
the NH vibrations at 1623 cm–1 (dNH+2 ) and 1317, 1286, and
1263 cm–1 (CH and NH deformations). Thus, the band at
1623 cm–1 shifts to 1608 cm–1 keeping a similar relative intensity,
while the band at 1263 cm–1 decreases in intensity. The band at
1317 cm–1 shifts to 1312 cm–1 and increases its intensity drasti-
cally. The 1286 cm–1 band is not clearly identified in the SERS
spectrum. Thus, it is possible to infer that the molecular fragment
accounting for this band is not too far from the surface, or its
interaction with the metal surface is feeble. Other vibrations
concerning the ring fragment, in particular the CH2 modes of
the propyl moiety, are influenced by the surface effect. This sug-
gests that this moiety or some of the CH2 groups are probably
Figure 5. Predicted hydroxyproline-Ag surface interaction. close to the metal surface. This is supported by the spectral mod-
ifications of the CH2 mode at 1473 cm–1, which increases in inten-
Table 1, for Pro and Hyp, respectively. No SERS bands were sity while the band 1449 cm–1 disappears as a result of the
observed in the 3500–2500 cm–1 region for either compound. surface effect. A shoulder at 1320 cm–1 ascribed to a oCH2 mode
is observed only in the SERS spectrum.
Proline
4-Hydroxyproline
Several bands ascribed mainly to vibrational modes involving the
carboxylate fragment of Pro display modifications in their relative Several bands ascribed mainly to vibrational modes involving the
intensities and/or wavenumber shifting by the surface effect; carboxylate, amino, and methylene fragments of Hyp display
some bands become active, while others disappear (see Fig. 4 modifications in the relative intensity and/or wavenumber shift-
and Table 1). This is interpreted in terms of an analyte–substrate ing by the surface effect (see Fig. 5 and Table 1). This is inter-
interaction. In fact, the Raman nasCOO– band at 1548 cm–1 shifts preted in terms of a net analyte–substrate interaction. In fact,
to 1572 cm–1 while the nsCOO– mode at 1373 cm–1 does not, the very weak Raman band at 1642 cm–1 (dNH+2 ) increases its

wileyonlinelibrary.com/journal/jrs Copyright © 2011 John Wiley & Sons, Ltd. J. Raman Spectrosc. (2011)
Proline and hydroxyproline deposited on silver nanoparticles

intensity and shifts to lower wavenumbers because of the surface electrostatic interactions are also the main factor in the Hyp-
effect. Bands involving NH and CH deformation modes at 1279 Ag layer interaction.
and 1223 cm–1 are enhanced. The nasCOO– mode appears at
1562 cm–1 displaying a strong intensity. The symmetric mode at
1389 cm–1 also increases its intensity. The relative intensity of Conclusions
the dCOO– mode at 756 cm–1 increases, and its wavenumber
shifts to higher energy (793 cm–1). The band corresponding to The Raman spectra of proline (Pro) and hydroxyproline (Hyp)
other COO– deformations at 616 cm–1 changes its spectral have been recorded and a complete band assignment by using
behaviour because of the surface effect. The weak band at DFT at the B3LYP/6-311 G* + level of theory performed. SERS
918 cm–1 ascribed to nC–COO– is not observed in the SERS spectra of Pro and Hyp were recorded using colloidal Ag
spectrum. The Raman bands involving the pyrrolidine moiety, obtained by reduction with hydroxylamine. The time-dependent
in particular those ascribed to nCC at 877 cm–1, and the SERS spectra suggest a continued aggregation process. It is also
pyrrolidine deformation at 464 cm–1, are either not observed concluded that prolonged adjustment time of Pro and Hyp in
or decrease their intensity in the SERS spectrum; the strong the colloidal solution resulted fundamentally in obtaining a
tCH2 band at 850 cm–1 appears in the SERS spectrum at unique and reproducible SERS spectrum. Hyp stabilizes on the
851 cm–1 with a weak relative intensity. The medium-intensity metal surface more rapidly (48 h) than Pro (72 h). The spectral
nCC band at 956 cm–1 shifts to 966 cm–1. Another nCC band analysis suggests that Pro interacts with the metal surface
appears because of the surface effect at 984 cm–1. According through the carboxylate group. The interaction of Hyp is mainly
to the SERS selection rules, the observed spectral behaviour governed by the carboxylate, amino, and methylene moieties. It
of the pyrrolidine ring CC bands is interpreted in terms of a is known that Pro and Hyp in collagen restrict the orientational
nearly parallel orientation of the ring on the surface. The fact freedom of the chain and give rigidity and stability to the triple
that the very weak Raman dCH2 mode at 1477 cm–1 becomes helix; Hyp confers additional stability to collagen structure.[25,26]
very strong in the SERS spectrum suggests that the The additional stability is inferred from the present SERS experi-
corresponding molecular fragment is close to the metal sur- ments. The SERS spectroscopic results are supported by quantum
face. Thus, Hyp is inferred to interact with the metal surface chemical calculations, performed using EHT for a model of the
through its amino and carboxylate groups and part of the title amino acids interacting with an Ag cluster.
aliphatic moiety of the pyrrolidine ring.
Acknowledgements
This contribution was financially supported by FONDECYT grant
Theoretical aspects
Nº 1090074. JJC is grateful to Becas Chile 2010 and a CONICYT
Amino acid–metal interactions. For the case of the isolated Doctoral Fellowship. AEA acknowledges receipt of AT grant Nº
silver bilayer, the highest occupied molecular orbital (HOMO) 24090050 and a CONICYT Doctoral Fellowship.
and lowest unoccupied molecular orbital (LUMO) energies are
degenerate ( 6.14 eV) and the valence and conduction bands References
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