Zhu et al. Nanoscale Research Letters 2012, 7:613 http://www.nanoscalereslett.com/content/7/1/613 In order to examine the enhancement capability of GNP thin films to Raman signal, three R6G solutions with dif- ferent concentrations of 10−5, 10−6, and 10−7 M (which cannot be detected on bare ITO glass substrates with no nanoparticles) have been applied at three different points on sample S1, the film with the highest GNP density. As shown in Figure 4, salient surface-enhanced characteristic peaks of R6G molecules on this film can be seen notice- ably, even if the concentration drops dramatically to 10−7 M. The band at 615 cm−1 is due to plane bending of the C-C-C ring, whereas the band at 776 cm−1 has been assigned to out-of-plane bending of the hydrogen atoms of the xanthene skeleton [25]. The peak at 1,195 cm−1 is associated with C-C stretching vibrations, and those at 1,319, 1,365, 1,512, and 1,650 cm−1 correspond to aro- matic stretching vibrations [26]. Thus, these results indi- cate that the R6G solution at the very low concentration of 10−7 M can be successfully identified using sample S1 as a sensitive SERS substrate. In previous works [9,11], a plasmon resonance theory is widely used to explain SERS enhancement. In this the- ory, the LSPR effect of metal nanoparticles plays an im- portant role in Raman signal amplification because the electromagnetic field strength on the metal particle sur- face becomes strongest when LSPR is excited. Metal films that serve as SERS substrates often need high par- ticle deposition density to form ‘hot spots’ in the narrow gap regions between neighboring particles, which can harvest high electromagnetic field to enhance the Raman signal [15]. From Figure 3d, one can find that many nar- row gaps with sub-10-nm distance are clearly formed between neighboring particles on the film. These regions Page 5 of 7 can produce a strong LSPR effect on the surface of the thin film when GNPs are excited by the incident light and enable the Raman characteristic peaks of R6G at a very low concentration to be easily detected. Compared with the previous works [17,27], Raman signal enhance- ment is also observed in similar GNP films because of the presence of narrow gaps. For instance, in [17], R6G at an extremely low concentration of 10−9 M was successfully identified using nanosphere arrays with sub-10-nm gaps as sensitive SERS substrates. We have showed remarkable SERS signal amplifica- tion effect using sample S1 with the highest nanoparti- cle deposition density. To confirm the influence of the GNP deposition density on Raman signal measure- ment, we have carried out SERS studies on the other two films, S2 and S3. For simplicity, an R6G solution with a concentration of 10−5 M was applied on the two films. From the resulting Raman spectra (Figure 5), one can see that although sample S2 still can generate the characteristic peaks of R6G, it is much weaker than sample S1. For sample S3, there is even no dis- cernable Raman peak of R6G shown on the Raman spectrum. This behavior of losing Raman signal ampli- fication capability implies that the LSPR effects on samples S2 and S3 are much weaker than that of sam- ple S1. Obviously, these results show an excellent agreement of the micromorphology of the three sam- ples because the densities of GNPs deposited on sam- ples S2 and S3 were much lower than that of sample S1. Thus, the narrow gaps located between neighbor- ing particles on samples S2 and S3 were relatively fewer and caused the weaker Raman signals. Figure 4 SERS spectra of the different concentrations of R6G adsorbed on the surface of sample S1.