**5. Raman imaging of functional polymer nanocomposites**

In the last two decades, the rapid development of SERS has been in line with the scientific advances in nanofabrication and Raman instrumentation such as confocal Raman microscopy. Confocal Raman microscopy combines digital imaging technology with Raman spectroscopy in order to evaluate the chemical composition, molecular structure and spatial distribution of molecular components in a certain material, giving information about its homogeneity at the microscale level [168–171]. Delhaye and Dhamelincour have demonstrated for the first time the possibility to combine Raman spectroscopy and mapping microscopy in 1975 on a paper entitled "Raman microprobe and microscope with laser excitation" [172]. The authors have described the technique in detail, giving applications of the system such as the study of various materials such as rocks, plastics, composite materials, phases and inclusions and defects in solids. They emphasized that this new technique could became a valuable tool for the study of chemical reactions in micro-samples and also extended to biological samples.

In Raman imaging, thousands of linearly independent and spatially resolved spectra of the compounds existent in the specimen, are collected and analyzed. Among these spectra, the intensities of diagnosis bands for each species can be analyzed to generate images that are true maps for the spatial distribution of the compounds without the use of strains, dyes or contrast agents. This is a great advantage for materials characterization because little or no sample preparation is needed to characterize heterogeneous matrices [168–171, 173]. In fact, this technique is so versatile that has been applied in several fields, including pharmaceuticals' analysis [174–179], biology [180–182], biomedicine [183–186], label-free cell imaging [187–191], food industry [192–194], threat detection [195–197] and more fundamental research [198–202].

carrageenan and gelatin, loaded with colloidal Ag NPs as SERS substrates. Ultimately, these nanocomposites might find use to fabricate analytical platforms for distinct end uses, such as paper products, smart textiles, thermosensitive materials and drug delivery [67, 69, 84, 85]. Fateixa *et al.* have described Ag/gelatin A hydrogel samples with distinct gel strength as platforms for SERS detection and release of EtDTC, a pesticide model [85]. In this work, SERS was investigated as a spectroscopic method to detect the presence of low amounts of EtDTC in gelatin hydrogels, following the gradual release of the pesticide into water used as the dispersing medium. Noteworthy, this methodology can be used as an alternative to monitor the performance of hydrogel vehicles in the controlled release of pesticides, namely during the formulation and optimization stages of fabrication. On the other hand, P*t*BA matrices coating organically capped Ag NPs can be used either as aqueous emulsions or as cast films for active SERS substrates [114]. In this research, Trindade and co-workers have reported metal loaded polymer based composites with sensitivity for the SERS detection of thiosalicylic acid, even after the nanocomposite has been submitted to a temperature cycle (−60 to 65°C). The observations were interpreted by considering that the thiosalicylic acid molecules were entrapped within the polymer network and close to the Ag NPs. Similarly, DNA nucleobases, such as adenine, have been reported and detected by SERS using metal loaded polymer nanocomposites obtained by (mini)emulsion polymerization, namely Ag/PtBA and Ag/poly(metametylacrylate) (PMMA) [113, 115]. In addition, other plasmonic NPs can be used for the preparation of such polymer composites via (mini)emulsion polymerization namely gold nanorods (AuNRs). Fateixa *et al.* have reported SERS substrates obtained by a blending method of colloidal Au nanorods and PtBA aqueous emulsions [107]. The use of the blending method in this case limits morphological modifications of the rods that otherwise could occur in more drastic conditions. The composite blends were evaluated as SERS substrates, showing stronger signal enhancement when compared to the original Au NRs colloid, and using 2,2-dithiodipyridine as the analytical probe. This strategy involves a low-cost process with potential for the up-scale fabrication of

SERS substrates, namely by using other types of polymers.

106 Raman Spectroscopy

**5. Raman imaging of functional polymer nanocomposites**

of chemical reactions in micro-samples and also extended to biological samples.

In the last two decades, the rapid development of SERS has been in line with the scientific advances in nanofabrication and Raman instrumentation such as confocal Raman microscopy. Confocal Raman microscopy combines digital imaging technology with Raman spectroscopy in order to evaluate the chemical composition, molecular structure and spatial distribution of molecular components in a certain material, giving information about its homogeneity at the microscale level [168–171]. Delhaye and Dhamelincour have demonstrated for the first time the possibility to combine Raman spectroscopy and mapping microscopy in 1975 on a paper entitled "Raman microprobe and microscope with laser excitation" [172]. The authors have described the technique in detail, giving applications of the system such as the study of various materials such as rocks, plastics, composite materials, phases and inclusions and defects in solids. They emphasized that this new technique could became a valuable tool for the study For pharmaceutical industry, the Raman imaging technique became an important analytical tool to trace the active pharmaceutical ingredient (API) heterogeneity in tablet or granulates, controlled release systems, and orally inhaled and nasal drug products [174–176]. Šašić has reported the use of Raman imaging for the spatial distribution of the API and major excipient (mannitol) on common pharmaceutical tablets and granulates [175, 176]. In the same year, Widjaja *et al.* have reported the combination of Raman imaging with advanced multivariate data analysis method, namely band-target entropy minimization (BTEM) for the identification of minor components of pharmaceutical drug tablets [177]. They have detected, in model pharmaceutical tablets, minor components level as low as 0.2% by weight. In addition, the identification and quantification of polymorph forms, unique crystal packing lattice forms of molecules, of API is another important issue for pharmaceutical analysis and nowadays is routinely performed using Raman imaging. For example, Henson and Zhang have reported the detection and spatial distribution of a polymorphic impurity (0.05% w/w) of active pharmaceutical ingredient in a tablet, using Raman imaging [178]. Lin *et al.* have demonstrated the use of Raman mapping for microscopic characterization of the surface of tablets containing chloramphenicol palmitate polymorphs [179]

Besides the chemical specificity, Raman spectroscopy coupled with microscopy maps may hold several other desirable properties for imaging applications, such as high spatial resolution, multiplexing capability, low background signal, and excellent photostability [170, 171]. Over the past years, Raman imaging techniques have been developed in step with the latest Raman fields, such as coherent anti-Stokes Raman spectroscopy (CARS) [203–207], surface enhanced Raman scattering (SERS) [67, 169, 173, 208–210] and tip-enhanced Raman scattering (TERS) [170, 211–215].

The current developments in Raman imaging have brought a new overview on SERS platforms. There are few reports on the use of Raman imaging together with SERS methods, which makes SERS imaging an unexploited resource to answer unsolved questions about the materials functionalities and NPs synthesis, and complement substrates characterization in SERS platforms [67, 169, 173, 208–210]. SERS imaging has been successfully applied in different areas such as cellular imaging [173, 208, 216, 217], in vivo biosensing [218–221], pharmaceutics and cosmetics [209, 210] and textile industries [67, 68]. Some examples can be cited, Chao et al. have reported the use of diamond nanoparticles with two different sizes (5 and 100 nm) as SERS probes to bio-label human lung epithelial cells [217]. The interaction of the nanoparticles and the cells were probed by SERS imaging, demonstrating that the nanoparticles are not toxic to the cells. Gambhir and co-workers have injected Au@SiO<sup>2</sup> nanoparticles into a live mouse and have studied the mouse's liver through the skin using SERS mapped images [221]. In addition, Firkala el al. have reported, for the first time, the application of surface enhanced Raman imaging on pharmaceutical tablets containing an API in very low concentrations, using Ag colloids as SERS probes [209].

imaging [160]. The spatial distribution of thiophenol and an organic dye were achieved by SERS

SERS Research Applied to Polymer Based Nanocomposites

http://dx.doi.org/10.5772/intechopen.72680

109

We have been particularly interested in exploring Raman imaging in the characterization of SERS substrates. An illustrative example comprises the development of SERS substrates based on P*t*BA polymer beads coated with organically capped Ag NPs, accomplishing information at the level of both the substrate surface and the molecular adsorbate distribution (**Figure 7-1**) [114]. By coupling SERS images with AFM, we observed a rearrange of the polymeric chains of the PtBA after the addition of the analyte dispersed in ethanol, allowing a better diffusion of the molecular probe through the matrix and closer to the Ag NPs. This proximity enhanced the Raman signal of the thiosalicylic acid and their spacial distribution can be observed in the SERS images. In addition, linen fibers loaded with Ag NPs and then stained with methylene blue (MB) were investigated using SERS imaging. MB was selected as the molecular probe not only because it is a common organic dye but also due to the formation of dimer or monomer species, each one with characteristic visible absorption and Raman spectra. We have demonstrated that the SERRS effect together with confocal Raman microscopy offer a new tool to map the local distribution of the MB dye in the fibers and consequently the distribution of Ag NPs over the fabrics, using Raman imaging [67]. In addition, it is also possible to assess the preferred adsorbate form of MB on distinct types of nanocomposite fibers and their local distribution (**Figure 7-2**) [68]. This investigation allows to foresee the use of this approach in terms of quality control of antimicrobial Ag containing fabrics, which is a market in great

The development of polymer based composites as active SERS substrates has contributed considerably to the rise of this methodology as an important and significantly tool in several analytical contexts. This improvement could only be achieved due to the development of more versatile and powerful instruments, including portable Raman equipment, and a deeper knowledge about the underlying mechanisms in the Raman effect that occur in molecules adsorbed at metal surfaces. It should be also emphasized the unprecedented progress observed in the past decades on the synthesis of nanostructures having controlled morphology. Interestingly, it has become clear that improvements in SERS have also impact on the knowledge that we have about the materials required as substrates for applying such spectroscopic technique. In this chapter, the most recent developments in metal loaded polymer nanocomposites for SERS studies were reviewed, showing their applicability into diverse areas due to their multifunctional properties. The vestigial SERS detection of specific molecules using Raman reporters, SERS tags and specific external stimuli are examples of further developments in SERS technologies dependent on materials development. This research has also shown that Raman imaging combined with other techniques such as SERS are valuable assets that complement or eventually provide unique characterization data, with particular

relevance in the use of polymer based composites as SERS platforms.

mapping using smart films composed by natural rubber containing Au NPs [73]

expansion.

**6. Conclusions and outlook**

It is of special interest to mention in the context of this chapter that SERS imaging has been a useful technique to identify the SERS active sites in polymer based nanocomposite substrates [60, 63, 67, 68, 73, 93, 160]. SERS imaging has been applied to Ag/agarose beads films, giving an idea of the distribution of the *hot spots* in the polymer matrix [63]. Highly sensitive biological imaging of HEK293 cells expressing PLCγ1 cancer markers were obtained, using Au/Ag coreshell NPs, conjugated with monoclonal antibodies [93]. A porphyrin−phospholipid conjugate with quenched fluorescence have been reported as a Raman reporter molecule for SERS based

**Figure 7.** (1) AFM topography image of Ag/P*t*BA composites prepared by (mini)emulsion polymerization (A) before and (B) after the addition of 10 μL of an ethanolic solution of thiosalicylic acid 10−3 M; C) optical photograph of the sample with the scanned area marked in red; D) Raman images obtained using the integrated intensity of the Raman band at 1035 cm−1 in the SERS spectra of thiosalicylic acid 10−3 M using the Ag/P*t*BA composite as substrates; E) SERS spectrum of thiosalicylic acid (10−3 M) using Ag/P*t*BA composites as substrate (laser source: 532 nm); (2) Optical photograph (left) and combined Raman image (right), using two different Raman spectra of methylene blue (100 μM) adsorbed on Ag/ linen composite Inset: Raman spectra of monomer form and a mixture of monomer and dimer of MB used to create the combined Raman image [68].

imaging [160]. The spatial distribution of thiophenol and an organic dye were achieved by SERS mapping using smart films composed by natural rubber containing Au NPs [73]

We have been particularly interested in exploring Raman imaging in the characterization of SERS substrates. An illustrative example comprises the development of SERS substrates based on P*t*BA polymer beads coated with organically capped Ag NPs, accomplishing information at the level of both the substrate surface and the molecular adsorbate distribution (**Figure 7-1**) [114]. By coupling SERS images with AFM, we observed a rearrange of the polymeric chains of the PtBA after the addition of the analyte dispersed in ethanol, allowing a better diffusion of the molecular probe through the matrix and closer to the Ag NPs. This proximity enhanced the Raman signal of the thiosalicylic acid and their spacial distribution can be observed in the SERS images. In addition, linen fibers loaded with Ag NPs and then stained with methylene blue (MB) were investigated using SERS imaging. MB was selected as the molecular probe not only because it is a common organic dye but also due to the formation of dimer or monomer species, each one with characteristic visible absorption and Raman spectra. We have demonstrated that the SERRS effect together with confocal Raman microscopy offer a new tool to map the local distribution of the MB dye in the fibers and consequently the distribution of Ag NPs over the fabrics, using Raman imaging [67]. In addition, it is also possible to assess the preferred adsorbate form of MB on distinct types of nanocomposite fibers and their local distribution (**Figure 7-2**) [68]. This investigation allows to foresee the use of this approach in terms of quality control of antimicrobial Ag containing fabrics, which is a market in great expansion.
