**4. Metal loaded polymer nanocomposites as SERS substrates**

composites [148]. In this work, it was demonstrated that the magnetic properties observed for the EuS/poly(styrene) nanocomposites have varied in comparison to the starting EuS nanocrystals, which result from surface effects due to dispersion of the EuS nanocrystals within the polymer beads. Martins *et al*. have obtained stable aqueous emulsions of distinct

erization method. In the former, the optical properties of such composites depend not only on the Au NPs employed in their synthesis but also on the resulting morphology for the final composites [119]. In the later, they have demonstrated for the first time the preparation of a

sulated by poly(*t*-butylacrylate) [147]. As a proof of concept, the chemical binding of bovine

This strategy has established an interesting route for the development of nanocomposites materials for *in vitro* bioanalysis assays, which have been extended to other magnetic materials [150]. **Figure 4** presents TEM images of several polymer based composites prepared by (mini)emulsion polymerization. Although this method allows the preparation of diverse

**Figure 4.** TEM images of nanocomposites prepared by (mini)emulsion polymerization: a) Ag/poly(methyl metacrylate); [115] b) EuS/poly(styrene) ([148]–Reproduced by permission of The Royal Society of Chemistry); c) Au/poly(styrene)

/poly(styrene) particles mixed with free PS beads (courtesy of P. C.

O4

ferromagnetic polymer based composite composed by a magnetic core of CoPt3

NPs *via* (mini)emulsion polym-

/PtBA nanocomposites was described.

NPs encap-

polymer based composites containing either Au NPs or CoPt3

IgG antibodies to the hydrolyzed surfaces of CoPt3

100 Raman Spectroscopy

(adapted with permission from [119]) and d) Fe3

Pinheiro).

In the last years, a series of polymer-based nanocomposites containing plasmonic metal NPs have been investigated as a new class of SERS substrates. The preparation and properties of such



composites depends on the type of coating employed to stabilize the metal NPs, which were used as fillers. As discussed in the previous chapter, metal loaded polymer composites can be prepared by using different matrices such as synthetic [107, 113, 114, 123, 132] or natural polymers [61–63, 84, 87, 90, 151]. Polymer based composites containing metal NPs are of great interest due to their multifunctionality and potential for large-scale fabrication at low cost [23, 107, 152–154] In the context of SERS, these composites appear as a promising alternative for the development of efficient and scalable substrates for the detection of molecular species, such as dyes [68, 73, 95, 102, 103, 105, 121], biomolecules [64, 74, 75, 101, 113, 123, 155], and environmental pollutants [48, 60, 85]. For example, the application of polymer-based composites as stable and active smart devices or SERS chips has increased in the last years [126, 156–159]. Gao *et al.* have described the preparation of chips composed by metal NPs embedded in polymer nanofibers mats. [126]. The authors have demonstrated that such composites doped with distinct metal NPs are facile to store and to transport, and can be easily fixed in slides or in microfluidic channels for SERS detection of a variety of analytes. Long and co-workers have reported SERS assays loaded with Ag NPs that result from a low-cost production process by using screen printing techniques [156]. Chen *et al.* have decorated commercial tape with Au NPs to be used as SERS platforms for the directly extraction and trace detection of pesticide in vegetables and fruits by a "paste and peel

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Polymer nanocomposites containing metal NPs offer some advantages in SERS applications, namely by considering a judicious choice of the polymer used as the matrix. The polymer can be selected, for example, to provide a stimuli responsive platform or a porous polymeric matrix that facilitates the diffusion and entrapment of the biomolecules under analysis. In this context, an important objective in the application of SERS substrates has been the analysis of vestigial amounts of the target analyte for which minimal specimen preparation is required [69, 70, 72, 73, 107, 114, 115]. This is relevant to implement analytical protocols as many SERS applications are foreseen, such as environmental monitoring [48, 74], SERS mapping and imaging [67, 68, 93], medical diagnosis [109, 124, 126, 151, 160, 161] and substrate characterization [71, 96, 107]. **Table 2** lists polymer based composites mentioned in this chapter with the corresponding SERS applications by taking into account the type of polymer and the metal

In these nanocomposites, the polymer can also act as an active coating with influence in the interparticle distances of the dispersed plasmonic metal NPs and their close surroundings. These changes can endorse the formation of SERS active sites, the so called *hot spots*, that can be further accessed by molecular probes through diffusion in the polymeric matrix [113, 114, 152, 164]. Braun *et al.* have reported a general approach to create *hot spots* in SERS substrates via NPs linking, polymer coating and molecular permeation [152]. The polymer coating holds the analyte within the nanojunctions created by several Ag NPs trapped within the polymer. However, this approach requires the use of a linking agent prior substrate preparation and has been reported only for analyte detection in solution. More recently, Kim and co-workers have reported the preparation of microgels containing Ag nanocubes, providing molecular size-selective permeability and high SERS sensitivity for acetylsalicylic acid (aspirin) [164]. The size of the Ag aggregates in the microgel matrix allows selective diffusion of small molecules and also promotes the formation *hot spots* in the close vicinity of the Ag nanocubes. As

off" procedure [158]

nanofillers employed.

**Table 2.** SERS applications of metal/polymer nanocomposites.

composites depends on the type of coating employed to stabilize the metal NPs, which were used as fillers. As discussed in the previous chapter, metal loaded polymer composites can be prepared by using different matrices such as synthetic [107, 113, 114, 123, 132] or natural polymers [61–63, 84, 87, 90, 151]. Polymer based composites containing metal NPs are of great interest due to their multifunctionality and potential for large-scale fabrication at low cost [23, 107, 152–154]

**Polymer matrix Metallic NPs Applications**

Synthetic Poly(amide) Ag Biomolecular detection [87]

Poly(vinyl alcohol) Ag Biomolecular detection [95, 101]

Poly(styrene) Ag Biomolecular detection [98]

Poly(methyl methacrylate) Ag Biomolecular detection [115] Poly(acrylamide) Ag Biomolecular detection [118] Poly(acryloyl) Hydrazine Ag Biomolecular detection [162]

Poly(pyrrole) Ag Substrate characterization [96]

Poly(N-isopropylacrylamide) Au Biomolecular detection [121]

Poly(vinylidene fluoride) Ag Biomolecular detection [87]

Poly(acrylonitrile) Ag Biomolecular detection [127] Poly(sodium 4-styrenesulfonate) Au@Ag Biomolecular detection [155]

Fe3 O4

poly(ethylene glycol)

Poly(ethylene glycol dimethacrylate)

poly(ethylene glycol dimethacrylate-*co*-acrylonitrile)

Poly(styrene)-*block*-poly(acrylic

**Table 2.** SERS applications of metal/polymer nanocomposites.

diacrylate

102 Raman Spectroscopy

acid)

Poly(vinylpyrrolidone) Ag Biomolecular detection [99, 102, 105] Poly(aniline) Ag Biomolecular detection [100] Poly(*t*-butylacrylate) Ag Biomolecular detection [113, 114]

Substrate characterization [97]

Substrate characterization [104]

Heavy metal detection [122]

Ag Medical diagnosis and target detection [164]

Au Medical diagnosis and target detection [109]

Au Medical diagnosis and target detection [124] SERS mapping and imaging [124]

Ultradetection or single molecule detection [123]

Medical diagnosis and target detection [126]

Medical diagnosis and target detection [98, 117]

Au Biomolecular detection [126]

Au@Ag Biomolecular detection [128] Cu@Ag Biomolecular detection [103]

Au Biomolecular detection [107]

Au@Ag SERS mapping and imaging [93]

Au Biomolecular detection [110]

Au@Ag Biomolecular detection [125]

Au Biomolecular detection [165]

Poly(hexamethylene adipamide) Au Ultradetection or single molecule detection [161]

@Ag Environmental monitoring [48]

Poly(ethylene glycol) Au Medical diagnosis and target detection [11, 163]

In the context of SERS, these composites appear as a promising alternative for the development of efficient and scalable substrates for the detection of molecular species, such as dyes [68, 73, 95, 102, 103, 105, 121], biomolecules [64, 74, 75, 101, 113, 123, 155], and environmental pollutants [48, 60, 85]. For example, the application of polymer-based composites as stable and active smart devices or SERS chips has increased in the last years [126, 156–159]. Gao *et al.* have described the preparation of chips composed by metal NPs embedded in polymer nanofibers mats. [126]. The authors have demonstrated that such composites doped with distinct metal NPs are facile to store and to transport, and can be easily fixed in slides or in microfluidic channels for SERS detection of a variety of analytes. Long and co-workers have reported SERS assays loaded with Ag NPs that result from a low-cost production process by using screen printing techniques [156]. Chen *et al.* have decorated commercial tape with Au NPs to be used as SERS platforms for the directly extraction and trace detection of pesticide in vegetables and fruits by a "paste and peel off" procedure [158]

Polymer nanocomposites containing metal NPs offer some advantages in SERS applications, namely by considering a judicious choice of the polymer used as the matrix. The polymer can be selected, for example, to provide a stimuli responsive platform or a porous polymeric matrix that facilitates the diffusion and entrapment of the biomolecules under analysis. In this context, an important objective in the application of SERS substrates has been the analysis of vestigial amounts of the target analyte for which minimal specimen preparation is required [69, 70, 72, 73, 107, 114, 115]. This is relevant to implement analytical protocols as many SERS applications are foreseen, such as environmental monitoring [48, 74], SERS mapping and imaging [67, 68, 93], medical diagnosis [109, 124, 126, 151, 160, 161] and substrate characterization [71, 96, 107]. **Table 2** lists polymer based composites mentioned in this chapter with the corresponding SERS applications by taking into account the type of polymer and the metal nanofillers employed.

In these nanocomposites, the polymer can also act as an active coating with influence in the interparticle distances of the dispersed plasmonic metal NPs and their close surroundings. These changes can endorse the formation of SERS active sites, the so called *hot spots*, that can be further accessed by molecular probes through diffusion in the polymeric matrix [113, 114, 152, 164]. Braun *et al.* have reported a general approach to create *hot spots* in SERS substrates via NPs linking, polymer coating and molecular permeation [152]. The polymer coating holds the analyte within the nanojunctions created by several Ag NPs trapped within the polymer. However, this approach requires the use of a linking agent prior substrate preparation and has been reported only for analyte detection in solution. More recently, Kim and co-workers have reported the preparation of microgels containing Ag nanocubes, providing molecular size-selective permeability and high SERS sensitivity for acetylsalicylic acid (aspirin) [164]. The size of the Ag aggregates in the microgel matrix allows selective diffusion of small molecules and also promotes the formation *hot spots* in the close vicinity of the Ag nanocubes. As such, these nanocomposite enable the Raman analysis of small molecules dissolved in complex mixtures of proteins and cells without sample pre-treatment.

An interesting feature offered by certain polymer nanocomposites employed as SERS substrates is their chemical functionalization envisaging molecular recognition [106, 152, 167]. Nie *et al.* have reported a strategy to detect tumors in living animals, using SERS substrates based on *pegylated* Au NPs [111]. In this research, the surface of Au NPs were functionalized with organic dyes, namely diethylthiatricarbocyanine and malachite green, which acts as Raman reporters. The Au NPs were coated with a polyethylene glycol with a thiol group and then functionalized with antibody fragments, which would target the tumor. The tumor detection was successfully accomplished using the SERS technique, monitoring the signal of the corresponding Raman reporters. Additionally, Batt and co-workers have successfully prepared an apta-sensing SERS substrate composed by polymer-Au NP-aptamer composite microspheres that allows the detection of a target molecule such as malathion [109]. They have demonstrated that these nanocomposites when attached to an aptamer have extraction capabilities for a pesticide, whose Raman signal is strongly enhanced due to the presence of the Au NPs. Our research group has reported a series of polymer-based nanocomposites that enable the SERS detection of several analytes (**Figure 6**). In this context, synthetic polymers as well as polymers of natural origin have been investigated for these purposes. Examples include nanocomposites based on poly(*t*-butylacrylate), poly(methyl metacrylate), cellulose, linen,

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**Figure 6.** SERS analysis of biomolecules using polymer based composites as substrates. 1. detection of a DNA nucleobase using organically capped silver nanoparticles encapsulated with poly(*t*-butylacrylate) as SERS substrates; [113] 2. Au nanorods with poly(*t*-butylacrylate) for the detection of 2,2'-dithiodipyridine; [107] 3. Ag/gelatin nanocomposites as

active SERS platforms for pesticide detection and release [85].

Metal loaded polymer composites are also suitable for applying analyte trapping strategies [22, 118, 123, 152, 166]. For instance, the diffusion of the analyte molecules within the polymer towards the metal surface can be facilitated not only by existent porosity but also by external stimuli such as temperature, pH, ionic strength and dehydration [22, 84, 88, 123, 162, 163, 165]. Fateixa *el al.* have demonstrated that the gel strength of carrageenan hydrogel composites loaded with Ag NPs can be correlated with the signal enhancement observed in SERS studies [84]. This research has focused on the effect of hydrogel strength and temperature on the SERS behavior of these bionanocomposites for the detection of 2,2′- dithiodipyridine. In order to vary the gel strength of the biocomposite, several procedures were employed, such as the increase of the polysaccharide content in the gel, the addition of KCl as cross-linker, and varying the type of carrageenan (κ, ι, λ) gel. The authors have reported an increase in the SERS signal as the gel strength increased, which was attributed to the presence of sites with strong local electromagnetic field, that result from the formation of Ag particles nanojunctions as the carrageenan macromolecules tended to rearrange into stronger gels (**Figure 5**). Also, Contreras-Cáceres *et al.* have prepared core/shell microgel particles composed by a poly-(N-isopropylacrylamide) (pNIPAM) shell and a metallic core such as Au and Au@Ag nanospheres or nanorods [41, 123, 125]. In these systems, the molecules under analysis, which were trapped in the thermoresponsive polymer shells and at the vicinity of the metal surface, experience an enhancement of the Raman signal of about 10<sup>5</sup> times [41, 123, 125]. More recently, multifunctional substrates have been investigated by conferring magnetic properties to SERS substrates, thus allowing extraction of the molecular probes from liquid phase prior SERS analysis [48]. In this case, by encapsulating Fe3 O4 @Ag NPs in thermoresponsive pNIPAM shells, an increase of temperature promoted the formation of *hot pots* close to the Ag NPs. These new platforms were applied to monitor trace amounts of pentachlorophenol, a chlorinated environmental pollutant.

**Figure 5.** Illustration of biopolymer helices aggregation and consequently the creation of highly SERS sensitive Ag/kcarrageenan hydrogels and digital photographs of Ag/carrageenan hydrogels with variable carrageenan amount (left); SERS spectra of 2'2-dithiopyridine using Ag/k-carrageenan hydrogels as substrates with variable amount of biopolymer: (a) 5 g/dm3 ; (b) 10 g/dm3 ; (c) 20 g/dm3 ; (d) 30 g/dm3 (right) [84] (Reprinted with permission from ([84]). Copyright (2017) American Chemical Society.)

An interesting feature offered by certain polymer nanocomposites employed as SERS substrates is their chemical functionalization envisaging molecular recognition [106, 152, 167]. Nie *et al.* have reported a strategy to detect tumors in living animals, using SERS substrates based on *pegylated* Au NPs [111]. In this research, the surface of Au NPs were functionalized with organic dyes, namely diethylthiatricarbocyanine and malachite green, which acts as Raman reporters. The Au NPs were coated with a polyethylene glycol with a thiol group and then functionalized with antibody fragments, which would target the tumor. The tumor detection was successfully accomplished using the SERS technique, monitoring the signal of the corresponding Raman reporters. Additionally, Batt and co-workers have successfully prepared an apta-sensing SERS substrate composed by polymer-Au NP-aptamer composite microspheres that allows the detection of a target molecule such as malathion [109]. They have demonstrated that these nanocomposites when attached to an aptamer have extraction capabilities for a pesticide, whose Raman signal is strongly enhanced due to the presence of the Au NPs.

such, these nanocomposite enable the Raman analysis of small molecules dissolved in com-

Metal loaded polymer composites are also suitable for applying analyte trapping strategies [22, 118, 123, 152, 166]. For instance, the diffusion of the analyte molecules within the polymer towards the metal surface can be facilitated not only by existent porosity but also by external stimuli such as temperature, pH, ionic strength and dehydration [22, 84, 88, 123, 162, 163, 165]. Fateixa *el al.* have demonstrated that the gel strength of carrageenan hydrogel composites loaded with Ag NPs can be correlated with the signal enhancement observed in SERS studies [84]. This research has focused on the effect of hydrogel strength and temperature on the SERS behavior of these bionanocomposites for the detection of 2,2′- dithiodipyridine. In order to vary the gel strength of the biocomposite, several procedures were employed, such as the increase of the polysaccharide content in the gel, the addition of KCl as cross-linker, and varying the type of carrageenan (κ, ι, λ) gel. The authors have reported an increase in the SERS signal as the gel strength increased, which was attributed to the presence of sites with strong local electromagnetic field, that result from the formation of Ag particles nanojunctions as the carrageenan macromolecules tended to rearrange into stronger gels (**Figure 5**). Also, Contreras-Cáceres *et al.* have prepared core/shell microgel particles composed by a poly-(N-isopropylacrylamide) (pNIPAM) shell and a metallic core such as Au and Au@Ag nanospheres or nanorods [41, 123, 125]. In these systems, the molecules under analysis, which were trapped in the thermoresponsive polymer shells and at the vicinity of the metal sur-

recently, multifunctional substrates have been investigated by conferring magnetic properties to SERS substrates, thus allowing extraction of the molecular probes from liquid phase

pNIPAM shells, an increase of temperature promoted the formation of *hot pots* close to the Ag NPs. These new platforms were applied to monitor trace amounts of pentachlorophenol, a

**Figure 5.** Illustration of biopolymer helices aggregation and consequently the creation of highly SERS sensitive Ag/kcarrageenan hydrogels and digital photographs of Ag/carrageenan hydrogels with variable carrageenan amount (left); SERS spectra of 2'2-dithiopyridine using Ag/k-carrageenan hydrogels as substrates with variable amount of biopolymer:

O4

(right) [84] (Reprinted with permission from ([84]). Copyright (2017)

times [41, 123, 125]. More

@Ag NPs in thermoresponsive

plex mixtures of proteins and cells without sample pre-treatment.

face, experience an enhancement of the Raman signal of about 10<sup>5</sup>

prior SERS analysis [48]. In this case, by encapsulating Fe3

chlorinated environmental pollutant.

(a) 5 g/dm3

104 Raman Spectroscopy

; (b) 10 g/dm3

American Chemical Society.)

; (c) 20 g/dm3

; (d) 30 g/dm3

Our research group has reported a series of polymer-based nanocomposites that enable the SERS detection of several analytes (**Figure 6**). In this context, synthetic polymers as well as polymers of natural origin have been investigated for these purposes. Examples include nanocomposites based on poly(*t*-butylacrylate), poly(methyl metacrylate), cellulose, linen,

**Figure 6.** SERS analysis of biomolecules using polymer based composites as substrates. 1. detection of a DNA nucleobase using organically capped silver nanoparticles encapsulated with poly(*t*-butylacrylate) as SERS substrates; [113] 2. Au nanorods with poly(*t*-butylacrylate) for the detection of 2,2'-dithiodipyridine; [107] 3. Ag/gelatin nanocomposites as active SERS platforms for pesticide detection and release [85].

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.

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].

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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

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

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

chloramphenicol palmitate polymorphs [179]

(TERS) [170, 211–215].
