**3.2. Blending or "grafting to" approach**

simplicity, although the resulting nanocomposites may exhibit inhomogeneity in terms of

In situ preparation is commonly used to prepare biopolymers based nanocomposites because natural polymers might be used to induce control over NPs size and aggregation state [63, 70], promoting the required biocompatibility for specific applications namely medical diagnostic and target detection in SERS [60, 64]. Polysaccharides such as chitosan [64, 65, 71, 72, 74], agarose [60, 63, 70], glucose [102], hyaluronic acid potassium salt [75] and cellulose [67–69] have been used as polymeric matrices for the one-step preparation of such polymer based nanocomposites. The preparation of composites containing polysaccharides in which in situ chemical or UV light reduction of metal ions occurred has been subject of great interest. Indeed, these nanocomposites can be applied in a variety of domains such as antimicrobial agents [66, 80], platforms for chemical detection [68, 69], textile dyeing monitoring process [67] and electronic paper [79]. For example, bacterial cellulose has been investigated as an alternative host matrix to vegetable cellulose in the preparation of electronic paper, SERS substrates and antimicrobial agents by using metal NPs as fillers. More recently, we have demonstrated that Raman imaging is a useful technique to characterize and monitor the textile dyeing process of antimicrobial fabrics [67]. **Figure 2** provides examples of cellulose composites for diverse applications.

The use of polysaccharides in the form of hydrogels have also been reported as matrices for dispersing metallic NPs, in certain cases showing capacity to collapse or be lyophilized upon drying and recover their structure by rehydration and subsequent use in SERS analysis [60, 63, 65]. For

**Figure 2.** Examples of applications for cellulose based nanocomposites loaded with metal NPs: a) Raman image obtained using the integrated intensity of the Raman band at 1620 cm−1 in the SERS spectra of methylene blue 10−4 M using a linen based composite containing Ag NPs as substrate (excitation at 633 nm); [67] Ag/bacterial cellulose nanocomposites for SERS biodetection of phenylalanine; [69] c) Nanocomposites containing copper based NPs and cellulose to be used as

antibacterial agents against *Staphylococcus aureus* [80].

morphology and fillers distribution in the polymer matrix [10, 129].

96 Raman Spectroscopy

Blending metallic nanofillers with a polymeric matrix is a simple methodology to fabricate nanocomposites with efficient SERS activity [84, 104, 106, 107, 124]. Besides its simplicity, this is a cost-effective and easily scalable method over a large area [90, 130]. In the literature, there are some reports on the use of synthetic polymers such as poly(methyl metacrylate) [105, 109], poly(*t*-butyl acrylate) [107], polystyrene-block-poly(acrylic acid) [110] and poly(vinyl alcohol) [128] as matrices for the incorporation of metallic NPs and subsequent use as SERS platforms. Although not so common in SERS research context, natural polymers can also be used as polymeric matrices for the preparation of polymer based composites, in which water compatible metal NPs are normally used. Marsch *et al.* have reported the development of a surface enhanced resonance Raman scattering (SERRS) substrate containing Ag NPs with a positive surface charge, due to a poly-L-lysine coating, which was then used in the analysis of organic anions [86]. In their work, these polymer based composites were successfully used as SERS substrates for the detection of bilirubin, a organic molecule of clinical interest formed as a metabolic waste product of heme breakdown. More recently, Chang *et al.* have reported flexible SERS substrates based on common filter paper loaded with gold nanorods, which exhibited more than two orders of magnitude SERS enhancement compared to silicon-based SERS substrates [92]. The authors have demonstrated that these platforms are excellent candidates for trace chemical and biological detection due to their efficient uptake, and transport of the analytes from the dispersing liquid to the surface of metal nanostructures.

Other examples of bionanocomposites containing metallic NPs have been reported including those based on chitosan [81], pullulan [83], cellulose [67, 79, 82], gelatin [85, 88] and carrageenan [84, 89]. **Figure 3** illustrates the use of biopolymers and Ag NPs in the fabrication of composites for antimicrobial and SERS applications. In certain situations, these applications can be complementary, such as in the case of producing a gel that due to the presence of Ag not only has the ability for SERS detection but also lasts longer periods of time due to the antimicrobial characteristics.

In ex situ methods, the polymer can be used either as a continuous phase or as an aqueous emulsion. For example, Lee *et al.* have reported the preparation of several metallic nanofillers

substrates. RAFT is a controlled radical polymerization approach, which involves multistep synthesis and sequential purification procedures. This polymeric approach is commonly used in the preparation of polymers with sulfur end groups, namely xanthates, dithioesters and thiocarbamates, that can be easily reduced to thiols [58, 133]. Besides the control of molecular weight and dispersity indexes of the polymers, several molecular structures can be achieved with the RAFT polymerization namely brush polymers, linear block copolymers, dendrimers and stars [58, 78, 116]. The polymer chains of the polymer based composites prepared by RAFT polymerization can be chemically functionalized with biomolecules that can be further used as SERS reports on the detection of specific analytes. In particular, metal loaded polymer composites functionalized with SERS reporters can be useful to conclude about the influence of the laser light source used in the target molecules or on the diffusion of the molecular probes through the polymer matrix. [58, 78, 134]. For example, Merican *et al.* have reported the surface modification of Au NPs with several SERS reporters, by using RAFT polymerization, in which a variety of polymers containing dithiocarbamate end groups were used. These polymer based composites were successfully

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used as SERS platforms for the detection of 2-naphthalenethiol and 2-quinolinethiol [58].

with specific selectivity for Cd2+ [122].

On the other hand, the ATRP approach controls the dispersity indexes and the molecular weight of the polymers by comprising an atom transfer step in the polymer chain growth phase in the polymerization [108, 120, 135, 136]. For example, core/shell structures prepared from surface functionalization of Au and Ag NPs by ATRP are often applied in a variety of fields such as catalysis, environmental monitoring, SERS detection and drug delivery [108, 120–122]. Mangeney and co-workers have fabricated Au–pNIPAM composites via ATRP method for the detection of methylene blue [121]. The authors have demonstrated that these hybrid materials are thermosensitive platforms, in which the Raman signal of the dye molecules increase with the increase of temperature. In addition, Yin *et al.* have reported the preparation of Au loaded polymer composites by ATRP approach to be used as SERS sensors

Our own investigation in this field has the focus on the preparation of such polymer based composites by in situ polymerization using distinct synthetic strategies, such as emulsion [137], suspension [138, 139] and (mini)emulsion polymerization [140, 141], a variant of emulsion polymerization. In the latter, the emulsions are nanosized and organically capped NPs can be allocated in the interior of the hydrophobic monomer droplets [142, 143]. The final properties of the polymer based composites can be tuned by varying several parameters namely the amount of monomer, surfactant, the size and surface organic capping of the inorganic NPs [143]. We have reported several organically capped NPs successfully coated with a series of polymers, using the (mini)emulsion polymerization, namely metallic NPs [113–115, 119], quantum dots [141, 144–146], ferromagnetic NPs [147] and lanthanide compounds [148, 149] Esteves *et al*. have reported pioneer research in this field by describing the encapsulation of TOPO capped CdS and CdSe quantum dots (QDs) using poly(styrene) and poly(*t*butylacrylate) as polymeric matrices [141]. They have demonstrated that by using the (mini) emulsion polymerization method, organically capped QDs NPs could be used as fillers leading to nanocomposites that still exhibit the typical photoluminescence of the dots at room temperature. Pereira and co-workers have synthesized oleylamine capped EuS nanocrystals from single-molecule precursors and investigated their use as fillers for polymer based

**Figure 3.** Blending of natural polymers and Ag NPs aiming at the fabrication of nanocomposites for SERS detection of analytes and antimicrobial applications.

with distinct sizes and shapes, which were used to decorated PS spheres stabilized in aqueous emulsion, producing active SERS platforms [104]. In addition, Fernández-López et al. have studied the thermoresponsive optical properties of poly(N-isopropylacrylamide) (pNIPAM) microgels doped with Au nanorods (Au NR) characterized by two different aspect ratios, observing a reversible behavior [112]. The thermoresponsive SERS sensitivity of the polymer composites was also analyzed using three different laser lines, demonstrating excitation wavelength-dependent efficiency, which can be controlled by either the aspect ratio (length/ width) of the assembled Au NR or by the Au NR payload per microgel.

#### **3.3. In situ polymerization and "grafting from" approach**

Current polymerization procedures can be fine-tuned to fabricate metal load composites to be used as SERS active platforms. These include (mini)emulsion polymerization, suspension polymerization, atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT). In in situ polymerization methods, the surface of the metallic nanofillers are modified with initiating species or chain transfer agents, allowing the growth of polymer chains from the metallic NPs surfaces [131]

By using the in situ emulsion polymerization, particles of the polymer based composite can be prepared with sizes ranging the micrometer and nanometer scale [117, 132]. In brief, this polymerization technique involves the formation of stable oil-in-water emulsions composed by small droplets of hydrophobic monomer dispersed in water that act as reactors after an initiator has been added. These micelles might contain surface modified inorganic NPs that at the end of the polymerization are coated or attached to the as prepared polymer.

Other polymerization strategies comprise the "grafting from" approach using reversible addition fragmentation chain transfer polymerization (RAFT) and atom-transfer radical polymerization (ATRP). There are a few reports on the use of ATRP and RAFT to produce metal/polymer SERS substrates. RAFT is a controlled radical polymerization approach, which involves multistep synthesis and sequential purification procedures. This polymeric approach is commonly used in the preparation of polymers with sulfur end groups, namely xanthates, dithioesters and thiocarbamates, that can be easily reduced to thiols [58, 133]. Besides the control of molecular weight and dispersity indexes of the polymers, several molecular structures can be achieved with the RAFT polymerization namely brush polymers, linear block copolymers, dendrimers and stars [58, 78, 116]. The polymer chains of the polymer based composites prepared by RAFT polymerization can be chemically functionalized with biomolecules that can be further used as SERS reports on the detection of specific analytes. In particular, metal loaded polymer composites functionalized with SERS reporters can be useful to conclude about the influence of the laser light source used in the target molecules or on the diffusion of the molecular probes through the polymer matrix. [58, 78, 134]. For example, Merican *et al.* have reported the surface modification of Au NPs with several SERS reporters, by using RAFT polymerization, in which a variety of polymers containing dithiocarbamate end groups were used. These polymer based composites were successfully used as SERS platforms for the detection of 2-naphthalenethiol and 2-quinolinethiol [58].

On the other hand, the ATRP approach controls the dispersity indexes and the molecular weight of the polymers by comprising an atom transfer step in the polymer chain growth phase in the polymerization [108, 120, 135, 136]. For example, core/shell structures prepared from surface functionalization of Au and Ag NPs by ATRP are often applied in a variety of fields such as catalysis, environmental monitoring, SERS detection and drug delivery [108, 120–122]. Mangeney and co-workers have fabricated Au–pNIPAM composites via ATRP method for the detection of methylene blue [121]. The authors have demonstrated that these hybrid materials are thermosensitive platforms, in which the Raman signal of the dye molecules increase with the increase of temperature. In addition, Yin *et al.* have reported the preparation of Au loaded polymer composites by ATRP approach to be used as SERS sensors with specific selectivity for Cd2+ [122].

with distinct sizes and shapes, which were used to decorated PS spheres stabilized in aqueous emulsion, producing active SERS platforms [104]. In addition, Fernández-López et al. have studied the thermoresponsive optical properties of poly(N-isopropylacrylamide) (pNIPAM) microgels doped with Au nanorods (Au NR) characterized by two different aspect ratios, observing a reversible behavior [112]. The thermoresponsive SERS sensitivity of the polymer composites was also analyzed using three different laser lines, demonstrating excitation wavelength-dependent efficiency, which can be controlled by either the aspect ratio (length/

**Figure 3.** Blending of natural polymers and Ag NPs aiming at the fabrication of nanocomposites for SERS detection of

Current polymerization procedures can be fine-tuned to fabricate metal load composites to be used as SERS active platforms. These include (mini)emulsion polymerization, suspension polymerization, atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT). In in situ polymerization methods, the surface of the metallic nanofillers are modified with initiating species or chain transfer agents, allowing the

By using the in situ emulsion polymerization, particles of the polymer based composite can be prepared with sizes ranging the micrometer and nanometer scale [117, 132]. In brief, this polymerization technique involves the formation of stable oil-in-water emulsions composed by small droplets of hydrophobic monomer dispersed in water that act as reactors after an initiator has been added. These micelles might contain surface modified inorganic NPs that at

Other polymerization strategies comprise the "grafting from" approach using reversible addition fragmentation chain transfer polymerization (RAFT) and atom-transfer radical polymerization (ATRP). There are a few reports on the use of ATRP and RAFT to produce metal/polymer SERS

the end of the polymerization are coated or attached to the as prepared polymer.

width) of the assembled Au NR or by the Au NR payload per microgel.

**3.3. In situ polymerization and "grafting from" approach**

analytes and antimicrobial applications.

98 Raman Spectroscopy

growth of polymer chains from the metallic NPs surfaces [131]

Our own investigation in this field has the focus on the preparation of such polymer based composites by in situ polymerization using distinct synthetic strategies, such as emulsion [137], suspension [138, 139] and (mini)emulsion polymerization [140, 141], a variant of emulsion polymerization. In the latter, the emulsions are nanosized and organically capped NPs can be allocated in the interior of the hydrophobic monomer droplets [142, 143]. The final properties of the polymer based composites can be tuned by varying several parameters namely the amount of monomer, surfactant, the size and surface organic capping of the inorganic NPs [143]. We have reported several organically capped NPs successfully coated with a series of polymers, using the (mini)emulsion polymerization, namely metallic NPs [113–115, 119], quantum dots [141, 144–146], ferromagnetic NPs [147] and lanthanide compounds [148, 149] Esteves *et al*. have reported pioneer research in this field by describing the encapsulation of TOPO capped CdS and CdSe quantum dots (QDs) using poly(styrene) and poly(*t*butylacrylate) as polymeric matrices [141]. They have demonstrated that by using the (mini) emulsion polymerization method, organically capped QDs NPs could be used as fillers leading to nanocomposites that still exhibit the typical photoluminescence of the dots at room temperature. Pereira and co-workers have synthesized oleylamine capped EuS nanocrystals from single-molecule precursors and investigated their use as fillers for polymer based 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 polymer based composites containing either Au NPs or CoPt3 NPs *via* (mini)emulsion polymerization 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 ferromagnetic polymer based composite composed by a magnetic core of CoPt3 NPs encapsulated by poly(*t*-butylacrylate) [147]. As a proof of concept, the chemical binding of bovine IgG antibodies to the hydrolyzed surfaces of CoPt3 /PtBA nanocomposites was described. 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 metal loaded polymer nanocomposites, there is also the presence of free polymer particles in the final emulsion. For certain applications this is not necessarily a limitation but nevertheless other options should be considered. For example, the use of magnetic NPs as fillers, such as in the case shown in **Figure 4d** for magnetite in poly(styrene), allows the application of magnetic

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

Environmental monitoring [85]

SERS mapping and imaging [60, 63]

SERS mapping and imaging [67, 68]

Ultradetection or single molecule detection [60]

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Medical diagnosis and target detection [157]

In situ molecular changes monitoring [65]

SERS mapping and imaging [73]

Cu Biomolecular detection [77]

Au Biomolecular detection [91]

Au Biomolecular detection [94] Au@Ag Biomolecular detection [94]

Au Biomolecular detection [74] Cu Substrate characterization [71] Au@Ag Biomolecular detection [72]

Chitosan Ag Medical diagnosis and target detection [64]

Poly-l-lysine Ag Medical diagnosis and target detection [86] Bovine serum albumin Ag Medical diagnosis and target detection [151] Natural rubber Au Ultradetection or single molecule detection [73]

Hyaluronic acid potassium salt Au Biomolecular detection [75]

separation in order to separate the nanocomposite from the free polymer beads.

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

Natural Gelatin Ag Biomolecular detection [61]

Gum Arabic Ag Biomolecular detection [62] Agarose Ag Biomolecular detection [63, 70]

Cellulose based materials Ag Biomolecular detection [69]

Carrageenan Ag Biomolecular detection [84] Paraffin Ag Biomolecular detection [90] Alginate Ag Biomolecular detection [94]

**Polymer matrix Metallic NPs Applications**

**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) (adapted with permission from [119]) and d) Fe3 O4 /poly(styrene) particles mixed with free PS beads (courtesy of P. C. Pinheiro).

metal loaded polymer nanocomposites, there is also the presence of free polymer particles in the final emulsion. For certain applications this is not necessarily a limitation but nevertheless other options should be considered. For example, the use of magnetic NPs as fillers, such as in the case shown in **Figure 4d** for magnetite in poly(styrene), allows the application of magnetic separation in order to separate the nanocomposite from the free polymer beads.
