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J. Phys. Chem. B 2005, 109, 3454-3459
Identification and Characterization of Active and Inactive Species for Surface-Enhanced Resonance Raman Scattering Imran Khan,*,†,§ Dale Cunningham,† Duncan Graham,† David W. McComb,‡ and W. Ewen Smith† Department of Pure and Applied Chemistry, UniVersity of Strathclyde, Glasgow G1 1XL, UK, and Department of Materials, Imperial College London, London SW7 2AZ, UK ReceiVed: September 24, 2004; In Final Form: NoVember 29, 2004
The surface-enhanced resonance Raman scattering (SERRS) activity of a statistically significant number of silver nanoparticles has been studied using a correlated SERRS mapping and transmission electron microscopy (TEM) method. TEM allowed the nature of each entity to be directly identified, and the SERRS activity was obtained from the corresponding SERRS map. Particles in various states of aggregation were analyzed to establish relative activities. It was established that SERRS activity is dependent on the specific batch of colloid tested. By averaging different colloid batches, it was shown that increasing SERRS activity is observed with increasing numbers of particles in the aggregates. By reducing the surface coverage of the particles to the extent that single moieties could be examined optically, the ratio of the relative activities of single particles, dimers, trimers, and larger aggregates was estimated. High-resolution TEM images of a number of active and inactive particles are reported. However, no clear correlation between microstructure and SERRS activity was observed.
Introduction Since the discovery of single molecule detection by SERRS in 1997,1,2 there has been a renewed interest in understanding the enhancement mechanism of Raman scattering from analyte molecules adsorbed to silver nanoparticles.1-9 To this end, the different systems that have been studied include single particles,1,3 dimers,4,5 and aggregated clusters of particles.2,6-8 Previously we have reported a method allowing statistically significant numbers of immobilized silver particles to be analyzed by both SERRS and TEM.9 The method is based on the correlation of a SERRS map with a TEM collage of the area analyzed and clearly allows both SERRS active and inactive particles to be identified. One key problem in surface studies is the ease with which photodegradation can occur. However, for this method, conditions have been identified in which all particles can be analyzed without photodegradation or a reduction in SERRS activity of the particle-dye complex, thus allowing all particles to be quantitatively compared.9 In this paper we report a study of the dependence of the SERRS activity on (a) the particle density, (b) particle-particle interactions, and (c) particle microstructure. For the analysis of individual species it was necessary to study surfaces of low particle coverage populated with spatially isolated species where the separation between each species is greater than the optical resolution of the Raman system. In this way the relative enhancements of single particles, dimers, trimers, and larger aggregates could be investigated, and high-resolution TEM images of active and inactive particles could be recorded. The results presented in this paper were generated using a Raman excitation wavelength †
University of Strathclyde. Imperial College London. § Now at Department of Materials, Imperial College London, London, SW7 2AZ, UK. * Corresponding author. E-mail:
[email protected]. ‡
of 514.5 nm. Use of a different excitation wavelength would result in different particles and aggregates being active.8 Experimental Section For all studies presented in this paper, Raman spectra were obtained using a Renishaw Raman microprobe 2000 system in a confocal setup, equipped with a Spectra Physics 514.5 nm Ar+ laser and Renishaw XYZ mapping stage. Electron microscopy was performed using JEOL 2010 and JEOL 1200 EX transmission electron microscopes (TEM). Both the preparation of citrate reduced silver colloid and the immobilization of these colloids onto silanized TEM grids have been described previously.9 In all, five different batches of colloid were used in the study, each prepared as described previously. Grid 1 and grids 6-10 were prepared by applying 1 drop of colloid to one side of the grid and allowing the grid to dry completely. Grids 1, 6, and 7 were prepared from colloids aged for 3 months while grids 8-10 were prepared from fresh colloids, i.e., 1 day after colloid preparation. Grids 2-5 were prepared by immersion in a colloidal suspension for 1 h. Grid 2 was prepared using an undiluted stock colloid while grids 3-5 were prepared from stock colloid diluted by a factor of 10, 20, and 100, respectively. The azo dye 3,5-dimethoxy-4(5′-azobenzotriazoyl)phenylamine (ABT-DMOPA) was used as the analyte dye. The benzotriazole group causes this dye to adhere strongly to the silver surface without subsequent desorption.10 The dye can be adsorbed onto the silver particles after particle immobilization without the need of any activation procedure and with all detectable excess dye being washed cleanly from the rest of the surface. At the concentration used here an approximate monolayer of the analyte dye is adsorbed onto the particles, and it is believed that all active sites of all particles studied were coated with dye.9 For all grids, the SERRS maps were acquired using a laser power of 20 µW. For grids
10.1021/jp045661n CCC: $30.25 © 2005 American Chemical Society Published on Web 01/29/2005
Active and Inactive Species for SERRS
J. Phys. Chem. B, Vol. 109, No. 8, 2005 3455 TABLE 1: Number of Species Analyzed and Detected from Aged Colloid on (a) Grid 6 (b) and Grid 7a (a) Grid 6 species
no. analyzed
no. active
single particles dimers trimers aggregates (5-26 particles)
34 11 3 4
0 1 0 2
SERRS intensity range 90 30-40
(b) Grid 7
Figure 1. (a) TEM collage of immobilized particles with SERRS map superimposed. Each grid square represents a dimension of 1 µm2. Highresolution TEM images show (b) an inactive cluster present in an inactive pixel and (c) an active cluster present in an active pixel.
1-5 an acquisition time of 10 s was employed, and for grids 6-10 the acquisition time was 15 s. Under these conditions no photodegradation or decrease in SERRS activity to the particledye complex occurs.9 Results and Discussion The correlated SERRS/TEM method we have developed allows exactly the same region of the surface to be probed by both techniques. A SERRS map is recorded from a region of the surface, the sample is then transferred to the TEM, and exactly the same region is mapped. In this way the nature of the SERRS active and inactive particles can be unambiguously identified and characterized. Figure 1 shows a collage of TEM images from grid 1 with the SERRS map superimposed. Although the majority of the pixels are inactive, there are 10 pixels that are SERRS active, and these are clearly identified. High-resolution TEM images of both an active cluster and an inactive cluster are shown, and the position of each of these clusters on the grid is identified. Figure 1 demonstrates the power of the correlated SERRS/TEM method where active and inactive particles can be clearly identified and high-resolution TEM images of the particles can be recorded. Grids 2-5 were prepared by immersion in a colloid. This method results in a more even distribution of particles on the surface than the application of a drop of colloid to the grid and produces samples suitable for a systematic study of the effect of surface coverage on the SERRS activity of the immobilized particles. 3-D SERRS intensity maps obtained from grids 2-5 are shown in Figure 2. The correlated SERRS/TEM method was used to study each grid. TEM examination of grids 2-5 allowed the approximate number of particles and the state of the aggregation of particles to be established and correlated with the SERRS maps shown in Figure 2. In the area mapped, grid 2 contained in excess of 15 000 particles, the majority of which were aggregated into clusters comprising at least 30 particles and often several hundred particles. Grid 3 contains approximately 1500 particles spread evenly over the surface. In most cases these particles are present in low states of aggregation. In the mapped area grids 4 and 5 contain approximately 750 and 150 particles, respectively. These were present in low
species
no. analyzed
no. active min-max
SERRS intensity range
single particles dimers trimers aggregates (4-14 particles)
197 60 9 9
13-33 5-13 3-4 3-4
10-158 93-2170 18-270 90-170
a The minimum number of active species indicates the number of active species sufficiently spaced such that each species was analyzed individually. The maximum number of active species indicates all active species present in active pixels even when two or more species are present. The values quoted for the intensity ranges are taken from isolated species only; the intensity of signals containing contributions from two or more species are omitted since the relative contributions could not be separated.
states of aggregation, including a high percentage of single particles. SERRS activity is detected over the entire area analyzed in grid 2 (Figure 2). This suggests that the vast majority of clusters present are active. The signal intensity varies by 2 orders of magnitude across the surface with regions containing large aggregates being most intense and regions with few particles being the least intense. Figure 2 illustrates that as the surface coverage is reduced, inactive regions of the surface appear and the intensity of the signals detected in the active regions decreases. In the case of grid 3, reduction of the particle concentration by a factor of 10, compared to grid 2, produces a surface of which roughly one-third is inactive, one-third yields low intensity signals, and one-third yields high-intensity signals. Reduction of the particle concentration by a factor of 20 yields a surface, shown in grid 4, of which approximately half is inactive. When the particle concentration is reduced by a factor of 100 in grid 5, SERRS is detected from only 1% of the surface mapped, and the intensity of signal detected from active species is very low. These results show that the SERRS intensity appears to be linked closely to particle density, and as particle density increases the SERRS activity increases rapidly. Surfaces of low particle coverage were prepared on grids 6-10. In each case a single drop of colloid was placed on the surface of the grid to ensure that all of the particles analyzed were immobilized on the top side of the grid. The correlated SERRS/TEM method was employed to analyze each grid. Two different batches of aged colloid were used to prepare grids 6 and 7. In the region of the surface analyzed on grid 6 there were 106 particles in various states of aggregation, sufficiently spaced that scattering could be collected from each species separately. Only three SERRS active species were detected. These were identified by TEM as a dimer, an aggregate of 5 particles, and an aggregate of 26 particles. Table 1a lists the number of species analyzed against the number of active species detected. TEM analysis of grid 7 revealed that, in a few cases, active pixels contained more than one species. While these species were well separated within a pixel, typically 100-300 nm apart, and consequently no interaction between the species is likely,5 the SERRS activity could be associated with either
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Khan et al.
Figure 2. 3-D SERRS intensity maps of grids 2-5 illustrating the effect of reducing surface coverage on SERRS intensity.
or both of the species present in such a pixel. This is reflected in the third column of Table 1b; “minimum” indicates the number of active species detected that were sufficiently spaced such that SERRS could be detected from each species individually while “maximum” includes all species present in active pixels including occasions where two or more species were present in a pixel. A 3-D SERRS intensity map for both grids 6 and 7 is presented in Figure 3. A comparison of the results from grids 6 and 7 shows that a higher percentage of active species are present on grid 7. Furthermore, the SERRS signals from these active species are of greater intensity than those from active species on grid 6. An important point to note is that grid 7 contained a number of active single particles while on grid 6 no active single particles were identified. On grid 7 a greater percentage of trimers and larger aggregates (4-14 particles in size) analyzed were active compared to monomers and dimers. While grids 6 and 7 were prepared from different batches of aged colloid, grids 8 and 9 were prepared from the same batch of fresh colloid. Table 2 shows that the results from both grids 8 and 9 follow similar trends. A greater percentage of dimers are active compared to monomers although the SERRS intensities detected are comparable for both species. However, the results do not suggest that larger clusters are more active than dimers. Although there is still some variation between grids 8 and 9, these results show that there is a measure of reproducibility between samples prepared from the same batch of colloid.
The variations between the activities of different batches of colloid, such as between grids 6 and 7, suggest that, although all colloid batches were prepared by the same procedure, minor differences in the conditions of preparation, such as reaction temperature and time, could have a significant influence on particle activity. There is no evidence to suggest that differences between aged and fresh colloids are significantly larger than those found between different batches. In Table 3, the SERRS activities for each species are presented averaged over all colloid batches studied. The results from another batch of colloid that are not presented in detail have also been included. Only cases where one isolated species was present within an active pixel are considered. Also, the aggregates are subdivided into smaller species containing 4-9 particles and large species containing 10-26 particles. Table 3 contains measurements for 605 species, of which less than 10% were SERRS active. Table 3 suggests that the percentage of active species increases as the state of aggregation increases. Based on Table 3 the ratio of the percentage of active single particles:dimers:trimers:small clusters (4-9 particles):large clusters (10-26 particles) is estimated to be 1:1.9:2.4:3.5:8.3. The range of SERRS intensities detected from each species does not reveal a clear trend. Therefore, the trend of decreasing SERRS activity with decreasing surface coverage observed in Figure 2 is not observed when comparing the SERRS intensities of single particles and small clusters.
Active and Inactive Species for SERRS
J. Phys. Chem. B, Vol. 109, No. 8, 2005 3457
Figure 4. TEM images of (a, b) two active and (c-f) four inactive dimers.
TABLE 3: Number of Species Analyzed and Detected from Colloid Batches 1-4. Only Singly Detected Isolated Species Are Considered species single particles dimers trimers small aggregates (4-9 particles) large aggregates (10-26 particles) total Figure 3. 3-D SERRS intensity maps of (a) grid 6 and (b) grid 7.
TABLE 2: Number of Species Analyzed and Detected from Fresh Colloid on (a) Grid 8 and (b) Grid 9 (a) Grid 8 species
no. analyzed
no. active min-max
SERRS intensity range
single particles dimers trimers aggregates (4-15 particles)
135 31 9 17
5-27 3-8 0-2 2-2
25-100 10-110 50-200
(b) Grid 9 species
no. analyzed
no. active min-max
SERRS intensity range
single particles dimers trimers aggregates (4-12 particles)
52 15 3 8
3-10 3-5 0 1-1
20-100 20-100 100
That a particular species can be active and exhibit a high SERRS intensity, or active and exhibit a low SERRS intensity, or inactive suggests that the state of aggregation is only one of many factors involved in SERRS enhancement. The exact arrangement of particles, interparticle distances, and orientation relative to the polarization of the incident excitation together with surface roughness features on the nanometer and atomic scale are expected to contribute to a greater or lesser extent.1-7,11,12 Computational studies on spherical particles predict the activity of dimers to exceed that of single particles by many orders of magnitude.5 Although, when surface defects were accounted for, the activity from a single particle was shown to increase significantly and approach that of dimers.5 The high relative activity of monomers, as determined from this study, suggests
no. no. SERRS % analyzed active intensity range active 422 119 24 33
22 12 3 6
10-158 10-2170 18-270 30-461
5.2 10.1 12.5 18.2
7
3
40-200
42.9
605
46
10-2170
7.6
significant surface roughness is present on the particles. When imaged at high magnification in the TEM the majority of the particles exhibit diffraction contrast that is associated with defects (e.g., stacking faults, dislocations, grain boundaries, etc.) in the particles.13-15 It is possible that such defects could result in high-energy sites at the particle surface and cause the formation of intense electromagnetic fields at these localities, i.e., “hot spots”. At this stage definitive conclusions regarding correlations between particle-particle interactions, particle microstructure and SERRS activity cannot be made. Consider the images of two active and four inactive dimers presented in Figure 4. The active dimer (a) might be considered to possess three potentially significant features, namely (1) diffraction contrast is present in both particles and the features are aligned, (2) there is a very small silver particle positioned close, approximately 3 nm, to one of the particles of the dimer, and (3) the separation of the two particles of the dimer is approximately 1 nm. One can speculate that the SERRS activity of the dimer in Figure 4 is associated with one or more of these features. The active dimer shown in Figure 4b exhibits neither feature 1 nor feature 2, and the separation between the two particles is less than 0.5 nm. This might lead one to suggest that it is the separation that is critical since the junction between two particles has been shown to exhibit an enhanced electromagnetic field, the strength of which is critically dependent on the distance between the particles.5 The experimental evidence does not support this conclusion: the dimers in Figure 4c,d exhibit a separation between the two particles of approximately 0.5 and 1 nm, respectively, but in contrast to Figure 4b these dimers are SERRS inactive. The remaining images of dimers in Figure 4 exhibit some of the features that might be considered important for SERRS activity, yet all of these dimers are inactive. Although the analyte molecules are adsorbed onto the silver particles after particle immobilization, it is proposed that the
3458 J. Phys. Chem. B, Vol. 109, No. 8, 2005
Khan et al. aggregates measuring approximately 1 nm. In the study presented here, however, the presence of interparticle sites of this dimension in aggregates was not correlated with effective SERRS activity. It is apparent that the SERRS effect is more complex in origin and may be dependent on many other factors besides the presence of interparticle junctions. Many more active and inactive particles must be studied before the correlations between SERRS activity and microstructure can be unambiguously determined. Conclusions
Figure 5. TEM images of (a, b) two active and (c, d) two inactive clusters.
interparticle sites are accessible to the dye molecules for adsorption. In cases where particles are in contact and there is insufficient space available, the analyte could still adsorb close to the contact site and experiences the intense electromagnetic fields between the particles. The results do not show any dependence of the SERRS activity on the direction of the polarization of the incident light. Although the surface plasmon resonance of each dimer is unknown, calculations by Kall5 and co-workers suggest that dimer resonances are sufficiently broad, and plasmon excitation of each of the dimers presented here would occur at the same excitation wavelength. Therefore, it is believed that differences in surface plasmon resonance would not account for the activity or inactivity of these dimers. A similar lack of correlation between microstructure and SERRS activity is observed when aggregates of particles are analyzed by high-resolution TEM (Figure 5). The structural features that might be considered significant in the images of the active clusters (a, b) are generally also observed in the inactive clusters (c, d). In particular, in all of the images of clusters presented in Figure 5 it is possible to identify points of separation that are in the range considered to give enhanced electromagnetic fields. The results of this study show that the percentage of active particles, and thus the magnitude of the enhancement per particle increases from single particles through dimers and trimers to clusters. These findings support the conclusions of Nie and coworkers1,3 that effective single particle SERRS is possible. Work by the groups of Kall4,5 and Brus6,7,16 indicate that the active sites responsible for the SERRS enhancements that enable detection of a single molecule may be interparticle junctions in
The correlated SERRS mapping/TEM collage method allowed the SERRS intensities of a large number of particles present in various states of aggregation to be measured. Both SERRS active and inactive species were clearly identified, and a number of high-resolution TEM images were presented of active and inactive particles. At high surface coverage, the vast majority of clusters analyzed were SERRS active. As the surface coverage is reduced, the signal intensities and the frequency of signals decrease significantly and inactive clusters and particles are apparent. When isolated species were analyzed, less than 10% of species were found to be SERRS active. A number of different colloids were tested and a wide range of SERRS intensities observed. The clear dependence of SERRS activity on the specific batch of colloid tested suggests minor changes in conditions of preparation have an influence on particle activity. From a number of studies of different colloids a statistically significant number of particles were analyzed, and a clear correlation between the probability of a species being active and the size of the species was observed. The ratio of the percentage of active single particles:dimers:trimers:aggregates of 4-9 particles:aggregates of 10-26 particles was estimated to be 1:1.9:2.4:3.5:8.3. For each species a wide range of intensities was observed, suggesting that other factors besides the state of particle aggregation play an important role in surface enhancement. The study of high-resolution TEM images of a number of active and inactive dimers and clusters did not reveal any clear correlation between particle microstructure and SERRS activity. Many more active and inactive particles must be studied in detail using analytical high-resolution TEM in order to investigate further the origins of SERRS activity. Acknowledgment. We thank the EPSRC (GR/S12722 and GR/S12739) for funding of this research. References and Notes (1) Nie, S.; Emory, S. R. Science 1997, 275, 1102. (2) Kniepp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkhan, I. Phys. ReV. Lett. 1997, 78, 1667. (3) Nie, S.; Doering, W. E. J. Phys. Chem. B 2002, 106, 311. (4) Xu, H.; Bjerneld, J.; Kall, M.; Borjesson, L. Phys. ReV. Lett. 1999, 83, 4357. (5) Xu, H.; Aizpurua, E.; Kall, M.; Apell, P. Phys. ReV. E 2000, 62, 4318. (6) Michaels, A. M.; Nirmal, M.; Brus, L. E. J. Am. Chem. Soc. 1999, 121, 9932. (7) Michaels, A. M.; Jiang, J.; Brus, L. E. J. Phys. Chem. B 2000, 104, 11965. (8) Markel, V. A.; Shalaev, V. M.; Zhnag, P.; Huynh, W.; Tay, L.; Haslett, T. L.; Moscovits, M. Phys. ReV. B 1999, 59, 10903. (9) Khan, I.; Polwart, E.; McComb, D. W.; Smith, W. E. Analyst 2004, 129, 950. (10) McLaughlin, C.; Graham, D.; Smith, W. E. J. Phys. Chem. B 2002, 106, 5408.
Active and Inactive Species for SERRS (11) Moscovits, M. ReV. Mod. Phys. 1985, 57, 783. (12) Otto, A.; Mrozek, I.; Grabhorn, H.; Akemann, W. J. Phys.: Condens. Matter 1992, 4, 1143. (13) Ino, S. J. Phys. Soc. Jpn. 1966, 21, 346.
J. Phys. Chem. B, Vol. 109, No. 8, 2005 3459 (14) Marks, L. D.; Smith, D. J. J. Cryst. Growth 1981, 54, 425. (15) Marks, L. D. Rep. Prog. Phys. 1994, 57, 603. (16) Jiang, J.; Bosnick, K.; Maillard, M.; Brus, L. E. J. Phys. Chem. B 2003, 107, 9964.
