Drastically Different Porphyrin Adsorption and Metalation Processes in

larger globular clusters (cluster-cluster aggregation). This process is only cluster diffusion limited.32,33 However, as the aggregates grow, again th...
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J. Phys. Chem. B 2002, 106, 1543-1549

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ARTICLES Drastically Different Porphyrin Adsorption and Metalation Processes in Chemically Prepared and Laser-Ablated SERS-Active Silver Colloidal Substrates Marek Procha´ zka,*,†,‡ Josef Sˇ teˇ pa´ nek,‡ Pierre-Yves Turpin,† and Jirˇ´ı Bok‡ UniVersite´ Pierre et Marie Curie, LPBC (CNRS UMR 7033), 4 Place Jussieu, Case 138, F-75252 Paris Cedex 05, France, and Institute of Physics, Charles UniVersity, Ke KarloVu 5, CZ-12116 Prague 2, Czech Republic ReceiVed: August 2, 2001

Processes of adsorption and metalation of a cationic water-soluble free base porphyrin, i.e., 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP) on Ag colloids have been monitored by surface enhanced resonance Raman spectroscopy (SERRS) as a function of time and porphyrin concentrations. Ag colloids employed were prepared either by laser ablation or by chemical reduction (by sodium borohydride and citrate) of Ag salts. SERRS intensities of TMPyP depend on the morphology of the colloidal aggregates in each system; aggregation, in turn, is governed by a balance between the so-called “diffusion limited aggregation” and “contact limited aggregation” processes, strongly influenced by the TMPyP concentration and the initial state of the colloidal particles (i.e. the residuals ions stuck at the substrate surface). The time evolution of the overall SERRS intensities is thus a function of the colloid preparation procedure. On the other hand, the kinetics of metalation reflects primarily the accessibility of the substrate surface for porphyrin adsorption. Consequently, it is highly sensitive to porphyrin concentration, the covering of the colloid surface by porphyrin molecules being the main qualitative limiting factor. Moreover, the process of metalation is controlled by residual ions at the substrate surface and therefore completely different for laser-ablated and chemically prepared Ag colloids. In the former, the amount of the metalated species is only limited by the adsorbate concentration while in the latter it is also mediated by the removal of residual ions from the colloidal surface by porphyrin molecules.

Introduction Potential applications of exogenous porphyrin molecules in life sciences include photodynamic therapy of cancer (PDT),1 antiviral treatments,2 molecular biology applications,3 specific sensing of DNA sequences, and selective cleavage of nucleic acids.4 Candidates for such applications can be selected among new synthetic porphyrins on the basis of their physical and chemical properties. However, most of them have a strong tendency to self-aggregate in solution. Investigation of new porphyrin derivatives should therefore be carried out at extremely low concentration, close to those encountered under physiological conditions: surface-enhanced resonance Raman scattering (SERRS), obtained by using an excitation wavelength which fulfils the resonance conditions of the proper porphyrin adsorbed on an adequate metal surface, is a promising spectroscopic approach for this kind of investigation. In porphyrin SERS-active systems, a metalation process (i.e. incorporation of a metal ion from the SERS-active surface into the porphyrin core) can be detected in the SERRS spectra as a consequence of the direct adsorption of the original free base * To whom correspondence should be addressed. Permanent address: Charles University. Tel.: +420 2 21 91 14 74. Fax: +420 2 24 92 27 97. E-mail: [email protected]. † Universite ´ Pierre et Marie Curie. ‡ Charles University.

porphyrin onto the metal substrate. This was first observed at the beginning of the 1980s5-7 and reported for various porphyrins in various SERS-active systems including layered structures,8 roughened electrodes,9 MELLFs (metal liquidlike films),10 and colloids.11-13 Since this is an undesirable effect for free base porphyrin studies through SERRS spectroscopy, a considerable interest developed in discovering how to prevent free base porphyrins from metalation.14-16 On the other hand, a quantitative analysis of this metalation process has been recently used for probing the stability of surface properties of Ag colloids,17 the porphyrin self-aggregation,18 and/or their interactions with large biomolecules such as nucleic acids.19 However, the factors influencing the proper metalation process of free base porphyrins in SERS-active systems are not well-known yet. A previous work reported a quantitative method, based on a factor analysis (FA) approach,20 to monitor the SERRS spectral changes caused by porphyrin metalation. Marker bands of metalated and free base porphyrin forms (mainly at ca. 395, 1340 cm-1 and 331, 1337 + 1360 cm-1, respectively) were monitored. FA also allowed SERRS spectra of the two pure porphyrin species (i.e. free base and metalated) to be isolated and the metalation kinetics to be determined in terms of time dependence of the proportions of both species in the experimental spectra.21 Standard application of the metalation kinetics for porphyrin studies at low concentration requires, however,

10.1021/jp013002u CCC: $22.00 © 2002 American Chemical Society Published on Web 01/23/2002

1544 J. Phys. Chem. B, Vol. 106, No. 7, 2002 the knowledge of other factors that control the process (such as porphyrin concentration and substrate preparation). Although some rough concentration studies had already been done,9,22 only a precise quantitative analysis of the kinetics of metalation can help to understand the process. The present work shows the influence of the surface properties of Ag colloids and the porphyrin concentration on the efficiency of adsorption of the molecule on the metal surface and on the kinetics of metalation of free base porphyrins. A water-soluble, cationic model molecule, i.e., free base 5,10,15,20-tetrakis(1methyl-4-pyridyl)porphyrin (TMPyP), was adsorbed on three different Ag colloids (laser-ablated, borohydride-reduced, and citrate-reduced) at concentrations ranging from 0.01 to 1 µM. The SERRS spectra were quantitatively analyzed to obtain the time dependence of overall SERRS intensities and the proper kinetics of metalation. While the latter only reflects the efficiency of porphyrin adsorption, the former also allows the colloid aggregation process to be monitored. This will be discussed for the various Ag colloid SERS-active systems.

Procha´zka et al. measured from ∼30 s at the minimum to ∼20 min after adding TMPyP aliquots to the colloid. The excitation beam was adjusted close to the exit wall of the cuvette to reduce reabsorption of the scattered light. Spectra were recorded in two separate regions (250-1050 and 1051-1700 cm-1) with a minimum time delay of ca. 10 s between the two regions. Each spectral range was treated separately and presented here as low- and highwavenumber regions. In addition, the stability of experimental conditions in each series of measurements was checked using the water stretching vibrations as internal standard of intensity.24 Analysis of the SERRS Spectra. The sets of SERRS spectra were treated by a factor analysis (FA) procedure,20 using a “singular value decomposition” algorithm which provides a set of orthonormal subspectra Sj and weights Wj and a set of scores Vij representing the relative presence of the Sj subspectrum in each experimental spectrum Yi. A particular experimental spectrum Yi(ν) can then be approximated as M

Yi(ν) )

Experimental Section Chemicals. Analytical grade chemicals and redistilled deionized water were used for all sample preparations. NaBH4 (Merck), AgNO3, and sodium citrate (Lachema) were used for the chemical preparation of silver colloids. The 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (tetra-p-tosylate salt) (TMPyP) was purchased from Aldrich. Preparation of Ag Colloids. Chemical Ag colloids were prepared by using standard procedures for the reduction of AgNO3 with NaBH4 (borohydride-reduced, b.r.)14 or sodium citrate (citrate-reduced, c.r.).23 The obtained colloidal solutions showed standard surface plasmon absorption (SPA) spectra: maximum at 395 nm (b.r.) and 440 nm (c.r.); maximum absorbance (for 0.2 cm optical length) 0.63 (b.r) and 0.45 (c.r.). Colloids were used as they came. Laser-ablated (l.a.) Ag colloids were prepared according to a procedure published in reference.17 A silver foil (99.99%, ∼1 mm thickness) cleaned in 30% HNO3 was immersed in a quartz cell filled with redistilled deionized water and irradiated by a focused beam of the 1064 nm line of a Nd:YAG pulsed laser (Quantel YG 58110, 10 Hz repetition rate, 20 ns laser pulse duration, energy 30 mJ/pulse) for about 15 min. The solution was continuously stirred by a magnetic bar, allowing a secondary fracture of primary ablated fragments and lowering the colloidal size dispersion. The resulting yellowish colloid yielded a SPA maximum at 398 nm and maximum absorbance (for 0.2 cm optical length) of 0.52. Preparation of Ag Colloid/Porphyrin Systems. Ag colloid/ TMPyP systems were prepared by the addition of appropriate amounts of a 10 µM stock solution of TMPyP to the Ag colloid; the pH of all samples was 6.5-7. Three different sets of the systems were studied, named A-C for laser-ablated, borohydride-reduced, and citrate-reduced colloids, respectively, each with TMPyP concentrations of 0.01, 0.03, 0.05, 0.1, 0.2, 0.3, 0.5, and 1 µM. Instrumentation. Absorption spectra of initial Ag colloids and Ag colloid/TMPyP systems were recorded on a Varian Cary 1E UV/vis spectrometer in 0.2 cm quartz cells. SERRS spectra of the Ag colloid/TMPyP systems placed in a quartz cell (0.5 mL) were recorded at room temperature with a Jobin-Yvon T 64000 CCD Raman spectrometer using the 441.6 nm excitation line of a He-Cd laser (Liconix 4050, power 3-4 mW at the sample) and a 90° scattering geometry. Owing to the necessary time for manipulation, SERRS spectra were

∑ WjVijSj(ν)

j)1

The factor dimension M represents the minimum number of independent components resolvable in the analyzed spectral set which are sufficient to reconstruct, with the best possible approximation, the experimental spectra Yi(ν). The M value can be derived from the residual error of the approximation.21b Prior to factor analysis a baseline adjustment procedure was applied to all spectra to suppress possible artifacts due to variations in the background signal. Results and Discussion 1. SPA Spectra of Ag Colloid/TMPyP Systems. Prior to the proper SERRS measurements, surface plasmon absorption (SPA) spectra of the Ag colloid/TMPyP systems were recorded (not shown). They yield a typical behavior as a function of porphyrin concentration: low TMPyP concentration does not change the colloid SPA spectrum, indicating only weak, if any, aggregation, while, in contrast, high TMPyP concentration strongly aggregates Ag colloids, resulting in a decrease of the colloid SPA band and the appearance of a new broad band at a longer wavelength.24 For nonaggregated systems, the c.r. colloid yields a SPA maximum at 440 nm: this is very convenient for obtaining good SERRS spectra under our experimental conditions (laser excitation at 441.6 nm). In contrast, nonaggregated b.r. colloid, with a narrow SPA spectrum peaking at 395 nm, only shows a low absorption at 441.6 nm; as a consequence, the SERRS spectra obtained at 0.01 and 0.03 µM of TMPyP yield a very poor signal-to-noise ratio that does not allow quantitative analyses to be made. Finally, the SPA spectrum of l.a. colloids (maximum at 398 nm) is broadened at longer wavelengths due to the presence of large particles,17 and thus, the conditions for plasmon resonance are also ensured. The SPA measurements also indicate that the concentration limit between aggregated and nonaggregated (and/or weakly aggregated) systems is not the same for the three colloids. This limit likely corresponds to the “covering concentration”, i.e., the porphyrin concentration just sufficient to cover all of the possible surface of the colloidal particles.24 The following concentration limits have been estimated: ca. 0.2, 0.5, and 0.3 µM for l.a., b.r., and c.r. colloids, respectively. This is in very good agreement with what has been previously obtained for the b.r. Ag colloid/copper(II) TMPyP system (∼0.4 µM) .24

Porphyrin Adsorption and Metalation Processes

Figure 1. Top: (A) Typical time evolution of SERRS spectra of 1 µM TMPyP in laser-ablated colloid with delays between the system preparation and spectral acquisition (from bottom to top): ∼27, 62, 92, 167, 272, 337, 527, 677, 1187 s. Bottom: Typical SERRS spectra of 1 µM TMPyP (B) in borohydride-reduced colloid at 887 s after preparation and (C) in citrate-reduced colloid at 1187 s after preparation. All spectra are normalized on the intensity of the 1095 cm-1 band.

2. SERRS Spectra of TMPyP and Their Quantitative Analyses. Figure 1A shows an example of a time-dependent series of SERRS spectra measured for the l.a. Ag colloid with 1 µM TMPyP concentration. For comparison, Figure 1B,C shows the last SERRS spectra of a similar series for b.r. and c.r. colloids. Each set of time-dependent SERRS spectra obtained for a particular TMPyP concentration has been treated separately by factor analysis (FA). It clearly indicates qualitatively different behaviors in two concentration regions: the limit between these two regions for a particular colloid corresponds well to the covering concentration estimated from SPA spectra. Thus the terms “high-concentration” (equal and above the covering limit) and “low-concentration” samples (below the covering limit) will further be used. The residual errors obtained for high- and low-concentration samples of sets A-C clearly show that the factor dimension of the problem is 2 (see Figure 2 as an example); i.e., the first and second subspectra are sufficient to account for most of the changes in the experimental spectra. The first subspectrum represents a weighted average of the experimental spectral intensities, while the second one bears most of the spectral differences between them. Important spectral changes are observed in the 300-400, 950-1020, 1330-1360, and 15401550 cm-1 regions (see Figure 1 and second subspectra in Figure 2). It has been previously assumed that most of these spectral changes are caused by the metalation process of TMPyP adsorbed onto the silver surface: 328, 965, 1000, 1334, 1359, and 1550 cm-1 bands belong to the free base species, and the 392, 1010, 1337 and 1541 cm-1 ones, to AgTMPyP.8,12,21b The bands located in the 950-1020 and 1540-1550 cm-1 regions are known to also be sensitive to Cβ-Cβ vibrational deformations,12,25 i.e., porphyrin ring conformation. In a previous paper21b some spectral changes in the same regions were also reported in the resonance Raman spectra of aged solutions, probably due to some self-stacking process. Since one cannot rule out the influence of these effects on the intensity and position of these bands, in particular for highly concentrated samples, only the bands located in the 300-400 cm-1 and

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Figure 2. Factor analysis results obtained from low- (250-1050 cm-1) and high- (1051-1680 cm-1) wavenumber regions of the timedependent SERRS spectra from borohydride-reduced Ag colloid/1 µM TMPyP system: residual errors (top); details of the first (S1) and second (S2) subspectra; time dependences of the corresponding columns of the coefficient matrix (V1 and V2).

1330-1360 cm-1 regions have been used as proper metalation markers for further quantitative analysis. For low-concentration samples of set A, the second subspectrum of FA results does not show metalation changes similar to those observed for high-concentration samples (not shown here; see ref 22). This is probably due to the fact that for set A at low TMPyP concentrations the metalation process is extremely rapid and cannot be monitored by our measurement, which starts at least ∼30 s after sample preparation. However, this second subspectrum yields a new band at ∼385 cm-1 (that can only be detected as a badly resolved shoulder at the lowfrequency limb of the 392 cm-1 band in the SERRS spectra) with an opposite sign with respect to the 392 cm-1 band of metalated porphyrin.22 For high concentration samples of set A, the same spectral feature is observed in the third subspectrum22 but its significance (i.e. weight) is negligible in comparison to those of the first and second subspectra. For systems B we did not observe these spectral changes. For highly concentrated samples in system C, the 392 cm-1 band in the SERRS spectra is substantially broadened, thus indicating the presence of this additional downshifted band (Figure 1C). This 385 cm-1 band in the SERRS spectra of free base porphyrins had been previously observed in various SERS-active systems. Kim et al. reported it in SERRS spectra of TMPyP at acidic pH (3.1) and interpreted it as a marker of conformational changes in the porphyrin macrocycle.11 Adsorption of TMPyP onto laser-ablated Ag colloid coated by a sodium mercaptoacetate spacer also induced the appearance of such a band in the corresponding SERRS spectra.16 Last, similar spectral features have been observed in the SERRS spectrum of 5,10,15,20-tetra(R-pyridine-p-tolyl)porphyrin in complex with polynucleotides.26 This leads us to propose that, in our systems, the adsorption of TMPyP can possibly induce some conforma-

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Figure 3. SERRS spectra of TMPyP pure forms (i.e. free base and metalated) obtained from quantitative analysis of time-dependent SERRS spectra of 1 µM TMPyP in borohydride-reduced colloids. Spectra are normalized on the intensity of the 1095 cm-1 band.

tional change of the porphyrin macrocycle but this change only concerns a negligible percentage of porphyrin molecules in comparison to that affected by the proper metalation. In further analysis, the normalized SERRS spectra of both pure TMPyP forms (free base and metalated) were constructed as proper linear combinations of the first and second subspectra,21b by taking the 327, 1334, and 1360 cm-1 bands as free base markers on one hand and the 390 and 1337 cm-1 bands as metalated porphyrin markers on the other. The main criterion to estimate the correct value of the multiplier in the combination is the presence of markers of one form only, with no sign of spectral subtraction of the other form. Moreover, the spectra of both pure forms have been normalized to the 1095 cm-1 band intensity, assigned to Cβ-Η bending deformation,25 i.e., not sensitive to metalation. For set A and high-concentration samples, only the metalation markers from the high wavenumber region (1334, 1337, 1360 cm-1) were used to avoid a possible influence of the third subspectrum in the low wavenumber region (see above). For low-concentration samples of set A, it has not been possible to generate an adequate “pure free base” spectrum because the metalation is extremely rapid: free-base contribution is missing even in the very first SERRS spectra of the series. Since both “pure form” spectra should not in principle depend on the concentration, those constructed from highconcentration data have been also used for further analysis of results obtained at low concentrations. The pure forms spectra obtained from the time dependent SERRS spectra of 1 µM TMPyP in b.r. colloid are presented in Figure 3 as an example. They yield features that correspond very well to those observed in the resonance Raman spectra of free base and synthesized AgTMPyP.10,21b The experimental spectra (Yj) were then expressed in terms of both normalized free base (P1) and metalated (P2) components:21b

Yj(i) ) ajP1(i) + bjP2(i) Here i is the index of the spectral point and j that of the spectrum corresponding to the time delay tj after sample preparation. To monitor the mechanisms of molecular adsorption as a function of substrate preparation and porphyrin concentration, the time evolution of the overall SERRS intensities and the kinetics of metalation were quantitatively estimated in terms

Figure 4. Time dependence of the overall SERRS intensities of TMPyP (1-0.01 µM) in (A) laser-ablated, (B) borohydride-reduced, and (C) citrate-reduced colloids.

of variations of aj + bj and bj/(aj + bj), respectively, i.e., the time dependencies of global efficiency of porphyrin adsorption and fraction of metalated TMPyP form. 3. Time Dependence of Overall SERRS Intensity of TMPyP. Time dependences of overall SERRS intensity of TMPyP adsorbed on l.a., b.r., and c.r. colloids (Figure 4) are in very good agreement with results previously published for CuIITMPyP,24 showing different behaviors at high- and lowconcentration samples. For the latter ones (i.e. below the covering limit), SERRS intensities rapidly reach a plateau or only slightly increase as time elapses, and their levels are somewhat proportional to the concentration in all three sets (AC). For the former ones, i.e., when the covering limit is reached and over, thus inducing the colloid aggregation, the average level of SERRS intensity jumps up but its dependence on time and TMPyP concentration is much more complicated. SERRS intensity in high-concentration samples yield not only important changes with time but different concentration dependence for laser-ablated compared to chemically prepared colloids. In both chemical colloids (sets B and C), intensities obtained for high-concentration samples show that the covering limit is ca. 0.5 µM for b.r. and 0.3 µM for c.r. colloids (as stated above). In contrast to low concentration, highly concentrated samples (those that induce colloid aggregation) show amazing variations as a function of time: (i) Overall SERRS intensities go through a maximum almost independent from the concentration (or at least not following a rule of proportionality to the concentration). (ii) After reaching this maximum, overall intensities decrease as time elapses (Figure 4: see the 0.3, 0.5, and 1 µM of set C

Porphyrin Adsorption and Metalation Processes and 1 µM of set B). This is evident in particular for set C, in which the covering limit is lower than for set B: the higher the concentration, the shorter the time between system preparation and a decrease in SERRS intensity after reaching the maximum. At 1 µM in set C SERRS intensity even starts to decrease from the very first spectrum (Figure 4C) (an almost parallel observation can be made for 1 µM in set B; see Figure 4B). For laser-ablated colloids (set A), some maximum of intensity is also observed at concentrations equal to or higher than 0.2 µM (i.e. the covering limit). Again this maximum is not proportional to the concentration, and in contrast to chemical colloids, its time of occurrence increases when concentration increases: one can even observe that at 0.5 and 1 µM the maximum is barely reached after 20 min of preparation (Figure 4A). Let us now try to interpret these amazing observations. Recently, several authors reported an unexpected SERS intensity decrease for high concentrations of molecules adsorbed on both b.r. and c.r. Ag colloids, including nucleic acids bases,27,28 aminonaphthalene dyes,29 and pefloxacin.30 Sa´nchez-Corte´s et al.28 observed a time evolution of SERS intensity of 1-methylcytosine and 1,5-dimethylcytosine in chemical Ag colloids similar to that we obtained here for porphyrins in sets B and C. They explained this observation by a time-dependent aggregation of the colloidal substrate that leads to variations in the morphology of the aggregates. It is indeed well-known that the crucial parameters influencing the SERRS intensities obtained from metal colloids are the size and morphology of the proper colloidal clusters.31 Since adsorbates must reduce an electrical repulsive barrier of isolated colloidal particles to induce their aggregation, the latter strongly depends on the nature and concentration of adsorbates and on the colloid surface potential as well.32,33 As concerns this process two models have been proposed: the so-called “diffusion-limited aggregation” and “contact limited aggregation” which depends on the residual charges that remain on the metal surface after preparation and/ or adsorption of the adsorbate.32 Since the electrical barrier is substantially stronger for chemical colloids covered by borate (for b.r.) or citrate (for c.r.) anions than for the “chemically pure” laser-ablated colloids stabilized only by residual water ions (at neutral pH), we propose that high TMPyP concentrations result in two different aggregate morphologies for chemical colloids, on one hand, and laser-ablated colloids on the other, and consequently into different concentration- and timedependence variations of the overall SERRS intensities. For both chemical colloids (sets B and C) aggregation is a two-steps process. At an adsorbate concentration close to the covering limit, the adsorbate reduces the repulsive barrier between particles only moderately. Not all of the collisions are productive for growth, and aggregates grow only by collisions of individual particles with the growing cluster.32 The first step of aggregation is thus mostly controlled by contacts between particles and clusters of fractal shape, a high content of linear chains being formed.33 Aggregates grow slowly: this can be monitored by a slow increase of SERRS intensity (for example see in Figure 4C the 0.3 µM profile). However, as the aggregates grow, their own diffusion constants decrease while the mean space between them increases. Thus, the time necessary for them to diffuse and collide starts to dominate.32 The second aggregation step is thus limited by diffusion and leads to the formation of globular aggregates, probably by crossing of linear parts of clusters.28 Electromagnetic theory predicts that the more favorable structures for SERS effects are fractals,31 and thus, the formation of globular aggregates results in some SERRS

J. Phys. Chem. B, Vol. 106, No. 7, 2002 1547 intensity decrease.28 At higher adsorbate concentrations, however, the lowering of the electrical barrier is readily more efficient, and consequently, the first aggregation step (“contact limited”) can be drastically shortened.28 This might account for the observations made on set C (and at 1 µM in set B, Figure 4B): the time elapsed between the sample preparation, the reaching of the maximum, and the beginning of the SERRS intensity decrease is ca. 700 s for 0.3 µM, 150 s for 0.5, µM and so short for 1 µM that after 30 s it is practically over (Figure 4C). In contrast, in laser-ablated colloids the particle residual charge is low, and even at concentration close to the covering limit, the adsorbate can efficiently reduce the particle repulsive barrier: all collisions between particles are thus efficient. Large aggregates can be formed also by sticking together smaller clusters.32 We propose that at the beginning all isolated colloidal particles rapidly come together to form small aggregates. This is accompanied by slight SERRS intensity increase (Figure 4A). Then, sticking of small aggregates leads to the formation of larger globular clusters (cluster-cluster aggregation). This process is only cluster diffusion limited.32,33 However, as the aggregates grow, again their diffusion constants decrease while the mean space between them increases. Thus, aggregation becomes slower at later times.32,33 The higher the initial TMPyP concentration, the larger the aggregates with a lower diffusion and, consequently, the slower the formation of large globular clusters. This is actually reflected in our experiments: the SERRS intensity maximum and subsequent decrease is observed after ca. 200 s at 0.2 M and ca. 400 s at 0.3 µM. For 0.5 and 1 µM the maximum is even not observed (and a fortiori the subsequent intensity decrease) before 20 min.; the necessary time is probably longer than that (Figure 4A). 4. Metalation Kinetics of TMPyP. The kinetics of metalation is represented by the time dependence of the spectral fractions of the metalated form in each experimental spectrum (Figure 5). Assuming a two-step process, it has been fitted by a twoexponential function of time with time constants τ1 and τ2:

bj/(aj + bj) ) F1 + F2 - F1 exp(-tj/τ1) - F2 exp(-tj/τ2) τ1 and τ2 are listed in Table 1 for l.a. and b.r. colloids as a function of the porphyrin concentration: in the framework of this two-steps process, the fast time constant, τ1, is at least 1 order of magnitude faster than the slow one, τ2. The other parameters obtained from the fits (fraction F1 of the fast metalated form and total metalated fraction F1 + F2) are shown in Figure 6 for the three sets A-C, as a function of log of concentration. Figures 5 and 6 show that the metalation process not only depends on concentration but also on Ag substrate preparation. For l.a. colloids the lower the TMPyP concentration, the faster and more complete the metalation. There are clearly two concentration domains, i.e., below and above the covering limit. In the former case (i.e. 0.01 and 0.05 µM of TMPyP) F2 is practically null; a single-exponential function of time well fits the experimental data with the shortest τ1 time constant values (Table 1) and coefficients F1 close to 1 showing that the fast metalation is nearly complete (Figure 6A). The surface of the colloidal particles is accessible to all of the porphyrin molecules. At the covering concentration (∼0.2 µM) and above, the fit needs a two-exponential function. Both steps of metalation slow (τ1 and τ2 increase), F1 decreases to ca. 65% and F2 increases to ca. 20% when TMPyP concentration increases (see Table 1 and Figure 6); however, their sum never exceeds ca. 85%, indicating an incomplete metalation. The surface of the l.a.

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Figure 6. Concentration dependence of final fractions of the metalated TMPyP form within the fast metalation (white filled symbols) and total (black filled symbols) versus the logarithm of TMPyP molar concentration: (A) laser-ablated (B) borohydride-reduced, and (C) citrate-reduced colloids. Dotted lines indicate the estimated covering concentration limits.

Figure 5. Metalation kinetics of TMPyP (1-0.01 µM) in (A) laserablated, (B) borohydride-reduced, and (C) citrate-reduced colloids presented as the time dependence of spectral fractions of the TMPyPmetalated form.

TABLE 1: τ1 and τ2 Time Constants Obtained for a Two-Exponential Fit of the Kinetics of Metalation at Various TMPyP Concentrations for (A) Laser-Ablated and (B) Borohydride-Reduced Colloids A

B

c (µM)

τ1 (s)

τ2 (s)

τ1 (s)

τ2 (s)

1 0.5 0.3 0.2 0.1 0.05

23.6 ( 1.2 11.6 ( 0.7 9.6 ( 0.3 9.8 ( 0.6 8.4 ( 0.5