JOURNAL OF RAMAN SPECTROSCOPYJ. Raman Spectrosc. 2007; 38: 799–801Published online 14 May 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jrs.1752

Rapid Communication

SER(R)S of porphyrins on Ag nanoparticlesimmobilized by silane: a unique way to obtainfree-base porphyrin spectra

Peter Molnar and Marek Prochazka∗

Charles University, Faculty of Mathematics and Physics, Institute of Physics, Prague, Czech Republic

Received 31 January 2007; Accepted 17 March 2007

We measured the surface-enhanced resonance Raman scattering (SER(R)S) spectra of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP) by using solid SERS-active substrates: Ag nanoparticlesimmobilized by aminosilane on glass plates. We report the surprising result that by using such substratesit is possible to obtain SER(R)S spectra of porphyrins in the unperturbed free-base form, although byusing silver nanoparticles directly in solution, the porphyrin molecules are completely metalated. Wesuggest that silane used for nanoparticle immobilization modifies the surface properties and, therefore,makes porphyrin metalation impossible. Copyright 2007 John Wiley & Sons, Ltd.

KEYWORDS: SERS; porphyrins; metalation; immobilized Ag nanoparticles

Porphyrins are very important biomolecules with applica-tions in photodynamic therapy of cancer, antiviral treat-ments, specific sensing of DNA sequences, vectorization ofoligonucleotides, etc. Surface-enhanced (resonance) Ramanscattering (SER(R)S) spectroscopy is widely employed forporphyrin studies at low concentrations. In the case offree-base porphyrins studied using silver SERS-active sur-faces, metalation (incorporation of a silver atom from thesurface into the porphyrin core) is well known.1 In thecase of the cationic porphyrin 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP) in systems with Ag colloids,the measured SER(R)S spectra are spectral mixtures ofthe free-base and metalated porphyrin forms characterizedby spectral bands at ca 330, 965 C 1000, 1337 C 1360 and1550 cm�1, and 395, 1340, 1010 and 1545 cm�1, respectively,but only those at ca 330, 395 and 1330–1360 cm�1 are purefree-base/metalation markers.2 In some special cases, evenextra spectral features at ca 385, 1365 and 1565 cm�1 cor-responding to porphyrin metalated by neutral silver havebeen observed.3,4 A quantitative method, based on a factoranalysis approach, has been developed to analyze spectralchanges caused by porphyrin metalation.5 The metalation

ŁCorrespondence to: Marek Prochazka, Charles University, Facultyof Mathematics and Physics, Institute of Physics, Ke Karlovu 5,CZ-121 16 Prague 2, Czech Republic.E-mail: prochaz@karlov.mff.cuni.cz

substantially decreases reproducibility of porphyrin SER(R)Sspectra and even makes obtaining the SE(R)RS spectra of theunperturbed free-base porphyrin impossible. Although con-siderable attention has been paid to discovering methodsto prevent free-base porphyrins from metalation (e.g. byusing an efficient molecular spacer6 – 8 or gold as an SERS-active surface9,10), it seems to be extremely difficult in somecases.

In this communication, we measure spectra of TMPyPfrom silver colloidal nanoparticles immobilized on silanizedglass plates. Slight modifications of the published prepa-ration procedures for silver11 and gold12 substrates wereemployed. Glass slides cleaned using ‘piranha’ solutionand aqua regia were silanized by a 20% solution of 3-aminopropyltrimethoxysilane (APTMS, 97% Sigma-Aldrich)in methanol (99.8% Sigma-Aldrich) for 30 min. After silaniza-tion, the substrates were rinsed several times with methanoland then with water to remove any physisorbed silane,which could cause nanoparticle aggregation in the colloidalsuspension during the next step. Each silanized glass platewas dipped into a tube containing 1 ml of the colloidal sus-pension (borohydride-reduced Ag colloid, preparation inRef. 2) for 6 h. Then they were rinsed with water.

For SERS measurements in solution, TMPyP was mixedwith the Ag colloid to obtain a final TMPyP concentrationof 1 µM. In the case of immobilized nanoparticles, the SERS

Copyright 2007 John Wiley & Sons, Ltd.

800 P. Molnar and M. Prochazka

substrate was placed in TMPyP of concentration 1 µM for20 min and rinsed by deionised water, and the SER(R)Sspectrum was measured. SER(R)S spectra were recordedat room temperature with a Raman spectrometer usingthe 514.5 nm excitation line of an ArC laser INNOVA 300(Coherent, power ¾150 mW at the sample). Accumulationtime was 1 ð 60 s and 5 ð 30 s for measurements fromsolution and solid substrates, respectively. The scatteredradiation was collected at 90° relative to the excitation beamand detected with a nitrogen-cooled CCD detector.

Figure 1 compares the SER(R)S spectra of TMPyP mea-sured directly from Ag colloidal nanoparticles in solution(Fig. 1(A)) and from the solid substrate (Ag nanoparti-cles immobilized on silanized glass plates) (Fig. 1(B)). TheTMPyP concentration added to the Ag colloid and the soak-ing concentration in the case of solid substrates were thesame (1 µM). The SER(R)S spectra measured for varioustime delays between the system preparation and spec-tral acquisition (from 5 to 30 min) show very surprisingresults: whereas TMPyP is completely metalated in the Agcolloidal solution, in the case of immobilized Ag nanopar-ticles, TMPyP remained in the unperturbed free-base form

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Figure 1. Time dependence of TMPyP SER(R)S spectrameasured from: (A) Ag nanoparticles in solution, 1 µM TMPyPconcentration; and (B) from Ag nanoparticles immobilized onglass plates, 1 µM TMPyP soaking concentration and 20 minsoaking time. The time between system preparation andspectral acquisition for both spectral series (from top tobottom) was 5, 10, 15, 20 and 30 min.

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Figure 2. SER(R)S spectra of TMPyP measured from differentspots of the same sample: Ag nanoparticles were immobilizedon glass plates, with 1 µM TMPyP soaking concentration and20 min soaking time.

even after 30 min. The metalated AgTMPyP form is clearlycharacterized by spectral bands at 397 and 1015, 1343 and1546 cm�1 (Fig. 1(A)) and free-base H2TMPyP form by 333,1004, 1335 C 1365 and 1555 cm�1 (Fig. 1(B)). Factor analy-sis of the spectral set in Fig. 1(B) clearly shows that thereare no spectral traces of metalated AgTMPyP. The sameresults were obtained for tens of substrates prepared in thesame way and even for longer time delays between systempreparation and measurement (several hours). Moreover,SER(R)S spectra of TMPyP measured from different spots ofthe same substrate (Fig. 2) showed excellent reproducibilitynot only in absolute intensity but also in spectral featuresthat belong only to the free-base form. Thus, we concludethat Ag nanoparticles immobilized by aminosilane on glassplates provide a simple but unique way to obtain SER(R)Sspectra of the native free-base TMPyP form. We suggest thatsilane used for nanoparticle immobilization modifies surfaceproperties by picking up AgC adsorption sites suitable forTMPyP metalation. A detailed study of the effect of silane onporphyrin SER(R)S spectra is in progress.

AcknowledgementsFinancial support from the Ministry of Education, Youth andSports of the Czech Republic (grant number 1P05ME809 andMSM0021620835) is gratefully acknowledged.

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Copyright 2007 John Wiley & Sons, Ltd. J. Raman Spectrosc. 2007; 38: 799–801DOI: 10.1002/jrs