Raman Signal Enhancement Dependence on the Gel Strength of Ag

Article pubs.acs.org/JPCC

Raman Signal Enhancement Dependence on the Gel Strength of Ag/ Hydrogels Used as SERS Substrates Sara Fateixa, Ana L. Daniel-da-Silva, Helena I. S. Nogueira, and Tito Trindade* Department of Chemistry-CICECO, Aveiro Institute of Nanotechnology, University of Aveiro, 3810-193 Aveiro, Portugal S Supporting Information *

ABSTRACT: A series of hydrogel samples composed of Ag nanoparticles dispersed in carrageenan gels have been prepared and used in SERS studies. These studies demonstrate the dependence of the enhancement of the SERS signal on the strength of the Ag/polysaccharide hydrogel. 2,2′-Dithiodipyridine was used as the analyte probe. Several strategies were employed in order to vary the gel strength. These include the increase of the polysaccharide content in the gel, the addition of KCl as cross-linker, and the variation of the type of carrageenan (κ, ι, λ) network. An increase in the gel strength originates an increase in the SERS enhancement observed. The results have been interpreted considering hot spots increase due to the formation of Ag particles nanojunctions as the biopolymer matrix tends to rearrange into stronger gels. This is the first report showing that there is a direct correlation between the gel strength of a hydrogel composite used as substrate and its analytical SERS sensitivity.

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INTRODUCTION

synergistic action of the plasmonic metal nanostructures and the external-stimuli responsive polymer. Carrageenans are linear sulfated polysaccharides obtained from red seaweeds (Eucheuma cottonii and Eucheuma spinosum).34,35 The different types of carrageenan (κ, ι, λ) differ in the position and number of ester sulfate groups which determine the physical properties (viscosity and gelation characteristics) of the biopolymer. For example, κ-carrageenan has a basic linear primary structure based on a repeating disaccharide unit of α(1−3)-D-galactose and β(1−4)-3,6anhydro-D-galactose and contains one sulfate group per disaccharide unit at carbon 2 of the 1,3 linked galactose unit34−37 (Figure 1). At temperatures above the gelling point, κ-carrageenan exists in solution as random coils, which undergo a double helix transition as the temperature decreases.36,37 The gel form appears when the double helices align to form quasi-crystalline regions, which happens in the presence of cations such as metal

Silver nanoparticles have been extensively used as SERS (surface-enhanced Raman scattering) substrates in the form of colloids,1−4 rough electrodes5,6 or as components in diverse types of composites that may include inorganic matrices7−9 and polymers.10−15 The advent of new techniques to control and monitor the formation of silver nanostructures has further advanced the widespread application of SERS in diverse fields.16−18 A significant advance in the development of highly effective SERS substrates has been the association of the SERS signal of an active molecule to its adsorption in specific metal nanojunctions.19,20 In this case, chemisorbed molecules show a plasmonic response to a local enhanced electromagnetic field that result in so-called “hot spots”.21−24 One approach to induce the formation of such metal nanojunctions has been the control of the morphology and aggregation of metal nanoparticles (e.g., Ag and Au) in polymer matrices. Illustrative examples include the synthesis of Ag/poly(butyl acrylate) composites using miniemulsions,25,26 the preparation of poly(methyl methacrylate) durable plastic films using an Ag/ PVP colloid,27 incorporation of Ag NPs in soft polymers to produce active SERS substrates,28 and the preparation of Ag/ bionanocomposites using cellulose,29 chitosan,30 or sporopollenin.31 In particular, polymers whose behavior is sensitive to external stimuli are attracting great interest as they might add complementary functionalities to the composite of the SERS active metal. Accordingly, Au/Ag nanorods in thermoresponsive hydrogel networks32 or mechanical responsive Ag-loaded agarose gels33 have been reported as substrates that show a SERS behavior dependent on “hot spots” induced by the © 2014 American Chemical Society

Figure 1. Structural disaccharide unit of κ-carrageenan. Received: January 10, 2014 Revised: April 14, 2014 Published: April 16, 2014 10384

dx.doi.org/10.1021/jp500301q | J. Phys. Chem. C 2014, 118, 10384−10392

The Journal of Physical Chemistry C

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SERS Experiments. The bionanocomposites with distinct κ-carrageenan content were used in the form of gels as new SERS substrates. Initial solutions of 2-dtpy in methanol with distinct concentrations (from 10−1 to 10−5 M) were prepared to establish the lower detection limit for the substrates used in SERS. These solutions were used to prepare the samples for SERS analysis by injecting 20 μL of the analyte solution into 0.98 mL of the silver nanocomposite gels. Prior to SERS analysis, and in order to ensure sample homogeneity, the gels were kept over 30 min at 80 °C, i.e., above sol−gel transition temperature, and then allowed to cool to room temperature. For all of the SERS measurements the pure biopolymer was used as control sample. Adenine SERS analysis was also performed in order to evaluate the SERS sensitivity with a bioanalyte. In this case, a solution of adenine (10−3 M) in a phosphate buffer at pH 4.5 was added to 1 mL of the Ag/κcarrageenan composites. Analytical enhancement factors (AEF) were determined using the following expression:48−50

alkali ions that act as cross-linkers via electrostatic interactions with the sulfate groups.38−40 The gel strength of a carrageenan gel can be increased either by adding a cross-linking agent to the gel or by increasing the polysaccharide concentration. Our previous studies have shown that κ-carrageenan can incorporate diverse types of inorganic nanoparticles via encapsulation in the biopolymer.41−46 In the present work, blended nanocomposites of Ag and κ-carrageenan have been prepared in order to obtain new analytical SERS platforms. Because of their tunable colloidal behavior between the sol−gel state, carrageenans are foreseen as interesting matrices to develop thermosensitive SERS substrates provided that there is the ability to create dynamic hot spots by varying the gelation conditions. As such, this research focuses on the effect of temperature and hydrogel strength on the performance of these new bionanocomposites as SERS substrates using 2,2′dithiodipyridine (2-dtpy) as the probe analyte. Furthermore, we show for the first time a strong correlation between the gel strength of a hydrogel composite used as substrate and SERS sensitivity, thus opening up new possibilities to develop new analytical platforms.

AEF = (ISERS/IRS)(C RS/CSERS)

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where CRS is the concentration of the analyte in bulk solution (1 M), CSERS is the concentration of the analyte in the colloid (2 × 10−3 M) that gives a SERS signal of ISERS intensity for a band assigned to a specific vibrational mode in the SERS spectrum, and IRS is the intensity of the band assigned to the same mode in the Raman spectrum of the bulk solution. Equation 1 was employed here to evaluate the variation in the SERS sensitivity for a series of Ag/κ-carrageenan hydrogels, in which all spectra were recorded with the same scan time and laser power. However, the AEF values obtained from this expression should be critically analyzed when comparing with values obtained in other experimental conditions, namely because parameters such as substrate coverage extension and chemisorption behavior of the analyte influence the final outputs.50 Rheological Measurements. The rheological behavior of the gels was evaluated via small amplitude oscillatory shear measurements. The nanocomposites were prepared by dispersing the Ag nanoparticles in κ-carrageenan solutions of variable concentration that have been kept at 80 °C; this aimed to guarantee homogeneous mixing in the sol state. Then, 2 mL of the resultant mixture was transferred to the rheometer measuring system set at 80 °C. The system was closed with metal plates to minimize evaporation losses. The gelation was monitored by measuring the storage (G′) and loss (G″) moduli as a function of temperature from 80 to 20 °C at 1 °C/min, at a frequency of 2 rad/s. The next step was to observe the time dependence of moduli by performing a time sweep test (20 °C, 2 rad/s) for 10 min. Finally, a frequency sweep test was done at 20 °C, from 0.01 to 100 rad/s, in order to assess the frequency dependence of the viscoelastic properties of the gel and using strain amplitude of 0.01. For all samples analyzed, the rheological properties of the nanocomposites were compared to those of the biopolymer matrix prepared under the same experimental conditions but without Ag nanoparticles. Instrumentation. A Jasco V 560 Ultra-Violet/Visible (UV/ vis) spectrophotometer was used for recording the UV/vis absorption. Fourier transform infrared spectroscopy analyses coupled to a horizontal attenuated total reflectance accessory (ATR-FTIR) were recorded using a Matson 700 FTIR spectrophotometer, using 128 scans at a resolution of 4 cm−1. Raman spectra were recorded using a Bruker RFS100/S FTRaman spectrometer (Nd:YAG laser, 1064 nm excitation), at a

EXPERIMENTAL SECTION Materials. The following chemicals were used as purchased: κ-carrageenan (Fluka), ι-carrageenan (Fluka), λ-carrageenan (Fluka), silver nitrate (AgNO3, 99.9%, J. M. Vaz Pereira), sodium citrate tribasic dihydrate (Na3C6H5O7·2H2O, 99%, Sigma-Aldrich), potassium chloride (KCl, 99%, Sigma-Aldrich), 2,2′-dithiodipyridine (C10H8N2S2, 98%, Sigma-Aldrich), and adenine (C5H5N5, 98%, Merck). Water was purified using a Sation 8000/Sation 9000 purification unit. SERS Substrates. The silver colloids were prepared by reduction of silver nitrate with sodium citrate, using the LeeMeisel method.47 An aqueous solution (250 mL) of silver nitrate (1 mM) was boiled under reflux for 10 min. Three mL of sodium citrate solution (1% w/v) was added dropwise to the boiling silver nitrate solution, under vigorous stirring. The mixture was then refluxed for 45 min and then slowly cooled to room temperature. A stable Ag hydrosol was obtained with particle average diameter of 30 ± 2.5 nm, as determined by DLS. κ-Carrageenan aqueous solutions were then prepared by dissolving κ-carrageenan powder into 10 mL of boiled distilled water. The amount of κ-carrageenan was varied between 5 and 30 g/L. Table 1 summarizes the conditions for the preparation Table 1. Preparation of Silver Nanocomposite Hydrogels sample Ag/ Ag/ Ag/ Ag/ Ag/

κ-carr_1 κ-carr_2 κ-carr_3 κ-carr_4 κ-carr_5

conc. of κ-carrageenan (g/L) 30 20 10 5 5

silver content (mL of Ag colloid) 10 10 10 10 10

(1)

KCl (1 M) no no no no yes

of the nanocomposites used as SERS substrates obtained as follows. The Ag/κ-carrageenan nanocomposites were prepared by dispersing Ag nanoparticles (10 mL) that have been previously isolated by centrifugation (1400 rpm, 10 min) from the above Ag colloid above, to the boiling κ-carrageenan solution. To promote the gelation of the Ag/κ-carrageenan bionanocomposite with 5 g/L, a KCl solution (2.5 mL, 1 M) was added. 10385



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power of 350 mV, with 250 scans at a resolution of 2 cm−1. Scanning electron microscopy (SEM) images were obtained using a Hitachi SU-70 SEM and EDX analysis was performed using an EDX Bruker Quantax 400. Rheology studies were performed via small amplitude oscillatory shear measurements using a Bohlin CVO HR 120 rheometer with parallel plate geometry (corrugated plate, 40 mm diameter, 1 mm gap).

show very weak bands in the Raman spectra which indicates that interference from the Ag/κ-carrageenan substrate in the SERS analysis is not expected, thus also favoring the use of these gels as SERS substrates for analytical detection.

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RESULTS AND DISCUSSION Silver Nanocomposite Hydrogels Characterization. Bionanocomposites, using hydrogels as matrices, are very often prepared as blends by homogeneous mixing inorganic nanofillers into the liquid dispersion medium (aqueous solution of the gel precursor) used as the continuous phase.41−46 In this work, the citrate method was used to obtain Ag nanoparticles that once isolated were added into boiled aqueous solutions of κ-carrageenan in order to form stable sols. All mixtures have formed gels after cooling to room temperature (ca. 20 °C) except the blends containing 5 and 10 g/L of κ-carrageenan, which have resulted in viscous liquids. Figure 2 shows the visible absorption spectra of hydrogels for increasing amounts of the polysaccharide and the same content of Ag nanoparticles. As expected, the visible spectra of the composite samples show the characteristic surface plasmon resonance (SPR) band of the silver nanoparticles around 420 nm. In comparison to the visible spectrum of the Ag hydrosol used in the preparation of the nanocomposites (Figure 2-b), band broadening was observed and a slight red shift of the absorption maximum occurred for samples with higher κ-carrageenan load (spectra df in Figure 2). Moreover, as the amount of polysaccharide is increased the spectra show a more pronounced absorption tail that extends to higher wavelength. These optical features are probably associated with the combination of two effects: (i) light dispersion due to formation of rigid gels and (ii) presence of Ag aggregates that are stabilized and confined in cages formed by the polymer chains as the gel network becomes more rigid. SEM analysis of the lyophilized samples have shown similarities in terms of surface texture to the pure κ‑carrageenan, though in some regions these aggregates have been clearly detected (Figure S1, Supporting Information). The ATR-FTIR spectra of the nanocomposites and of the pure biopolymer are very similar (Figure S2, Supporting Information). These spectra show the typical vibrational bands of κ-carrageenan, namely the SO stretching mode of sulfate ester at 1230 cm−1 and the C−O and C−OH stretching modes at 1160 and 1124 cm−1, respectively. The glycosidic linkage vibration is shown at 1060 cm−1, the presence of 3,6-anhydro-Dglactose is shown at 935 cm−1 and the presence of D-galactose4-sulfate at 844 cm−1.43,51,52 Therefore, the κ-carrageenan matrix retained its chemical identity when treated with the Ag nanoparticles to form the nanocomposites. SERS Studies Using κ-Carrageenan Hydrogels Containing Ag Nanoparticles. In this research, the compound 2,2′-dithiodipyridine (2-dtpy) (Figure S3, Supporting Information) was selected as molecular probe due to its SERS sensitivity.29,53,54 First, the Raman spectra for samples containing 2-dtpy in Ag/ κ-carr_2 and in the biopolymer matrix (2-dtpy final concentration of 2 × 10−3 M) have been recorded using an excitation line at 1064 nm (Figure 3). For comparative purposes, the Raman spectrum of a powdered sample of 2-dtpy is also shown in Figure 3 and the Raman spectra of κ-carrageenan (20 g/L) hydrogel and the respective silver nanocomposite are shown in SI (Figure S4). The latter

Figure 2. (1) Visible spectra of gels: (a) κ-carrageenan 20 g/L (for comparative purposes); (b) Ag aqueous colloid; (c) Ag/κ-carr_4; (d) Ag/κ-carr_3; (e) Ag/κ-carr_2; and (f) Ag/κ-carr_1 and (2) corresponding digital photographs.

Figure 3. (a) Conventional Raman spectrum for 2-dtpy powder; (b) SERS spectrum of 2-dtpy methanol solution (2 × 10−3 M final) using Ag/κ-carr_2 gel as substrate; (c) Raman spectrum of κ-carrageenan (20 g/L) with 2-dtpy methanol solution (2 × 10−3 M).

Figure 3 shows that Raman signals of 2-dtpy have been enhanced when the analyte was contacted with the Ag-κ-carr_2 nanocomposite but not in the presence of pure κ-carrageenan gel (20 g/L). Also, the spectrum shown in Figure 3b presents similarities to the SERS spectrum reported previously for this adsorbate but using the Ag colloid as substrate.29,53,54 In particular, band enhancement is clearly observed in the 1050− 1160 cm−1 region assigned to the aromatic ring in-plane vibrational modes: δ(CCC) + δ(C−H). These Raman signal enhancements have been interpreted by considering that adsorption of the analyte molecules to the surface of the silver 10386



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Figure 5. SERS spectrum 20 μL of 2-dtpy methanol solution (2 × 10−3 M final) on silver nanocomposites gels with increasing concentrations of biopolymer (a) 5 g/L; (b) 10 g/L; (c) 20 g/L; (d) 30 g/L.

Figure 4. SERS spectra of 2-dtpy with decreasing concentrations in the Ag/κ-carr_2 gel: (a) 2 × 10−3 M; (b) 2 × 10−4 M; (c) 2 × 10−5 M; (d) 2 × 10−6 M; (e) 2 × 10−7 M.

nanoparticles occurs with the aromatic ring in a tilted or perpendicular orientation.54 Note that the enhancement observed for the band assigned to the ν(S−S) at 547 cm−1 is consistent with chemisorption via the two sulfur atoms and with the two pyridine rings in a tilted position to the silver surfaces.54 This band was quite sensitive to experimental conditions and in some cases was not observed. The analysis of 2-dtpy methanol solutions (1 mL) with successive decreasing concentrations on the analyte was carried out in order to evaluate the limit of SERS detection using the Ag/κ-carr_2 nanocomposite. Figure 4 shows that the lowest concentration in the analyte for which a good signal could still be observed in these analytical conditions is 2 × 10−7 M. The above findings indicate that the composite hydrogels act as efficient SERS substrates due to the presence of the Ag nanoparticles dispersed within the matrix. It is well-known that the SERS signal is strongly dependent on the aggregation state of the metal nanoparticles used as substrate. Indeed, induced aggregation in aqueous Ag colloids by ionic strength increase has been a common practice to improve its SERS sensitivity. To clarify if a similar mechanism can be induced in the hydrogel nanocomposites, we have analyzed the dependence of the SERS signal with the gel strength of the hydrogel used as substrate. The strength of the polysaccharide gel was adjusted in this case by using different approaches that include, varying the amount of κ-carrageenan in the hydrogel, the addition of K+ as cross-linking agent and using distinct types of carrageenan (κ, ι, λ). Figure 5 shows the SERS spectra of 2-dtpy using silver nanocomposites prepared with variable concentration of κcarrageenan. It is clear that the intensity of the SERS signal increases with increasing the κ-carrageenan concentration in the substrate, i.e. as the gel network becomes more rigid. Therefore, these results are consistent with a mechanism in which the interaction of the analyte molecules and the Ag nanoparticles is favored by gelation of the matrix. Taking also into account the optical spectra shown in Figure 2, it is reasonable to assume that this mechanism involves the formation of Ag aggregates in the network cages in which the adsorbate molecules have been trapped. It is well-known that gelation of κ-carrageenan involves a coil to double-helix conformation transition, followed by helix aggregation to form an infinite three-dimensional network, that can be promoted by adding monovalent cations such as potassium.38−40,55 The SERS analysis with the Ag/κ-carra-

Figure 6. SERS spectra of 20 μL of 2-dtpy methanol solution (2 × 10−3 M final) in 1 mL of nanocomposite (a) Ag/κ-carr_4; (b) Ag/κcarr_5.

geenan nanocomposites with and without potassium cations demonstrates that the nanocomposite containing K+ (Ag/κcarr_5, higher strength) is more sensitivity to SERS (Figure 6). We explain these observations by considering the effect of these ions as cross-linkers in the gelation process, thus inducing the formation of regions with high SERS sensitivity due to clustering of the Ag nanoparticles in the cages formed within the rigid gel structure.38−40 The most common carrageenans are iota (ι), kappa (κ), and lambda (λ) carrageenan.51 Each natural carrageenan is a complex galactose-based polysaccharide that has different quantities of sulfate esters at different positions and with different distributions. The κ-, ι-, and λ-carrageenan dimers have one, two, and three sulfate ester groups, respectively.51,55 The solubility in water depends essentially on the number of sulfate groups and on their associated cations. Therefore, higher number of ester sulfate means lower temperature for complete solubility and lower gel strength. In order to have additional evidence of the SERS activity dependence on the strength of the polysaccharide, Ag nanocomposites have been prepared using as matrices the different types of carrageenan, for a selected concentration in the biopolymer (20 g/L) and then their SERS behavior was evaluated (Figure 7). These results confirm that by increasing the gel strength (λ < ι < κ) of the nanocomposites, the corresponding SERS sensitivity also increases. While λ-carrageenan appears as a liquid at room 10387



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temperature, in the same conditions the κ- and ι-carrageenan form thermo-reversible gels upon cooling, thus promoting the formation of a double helix structure that can trap the Ag nanoparticles in the cages, as explained above.51

the mixtures to room temperature. For all cases, the nanocomposites were kept at 80 °C for 30 min in the presence of the 2-dtpy analyte, and the SERS analysis was performed after cooling to room temperature. This procedure guarantees that the analyte is well dispersed within the matrix prior the SERS experiments, thus precluding effects on the Raman intensity signal due to local high concentration instead of a metal surface enhanced mechanism. On the other hand, it raises the question for the effect of the temperature treatment on the SERS sensitivity of the substrates. Therefore, the Ag/κ-carr_2 nanocomposites were used in the SERS analysis of 2-dtpy (2 × 10−3 M) over a temperature cycle treatment, starting from −40 °C and heating up to 80 °C, and then cooling back to −40 °C. Although several authors have reported that the SERS signal tends to increase by increasing the temperature of the sample under analysis,25,56−60 we have observed an opposite tendency as clearly shown in Figure 8. This is in line with the explanation above because at lower temperature we have a stronger gel in which the Ag aggregates have been trapped during the freezing of the solvent, causing the formation of regions with high SERS sensitivity (hot spots). Conversely, the intensity of the SERS signal of 2-dtpy decreases as the temperature has been increased because, at temperatures above Tg, the gel network collapses and the carrageenan chains appear as random coils and the silver particles are more dispersed in the matrix. Noteworthy this process is thermo-reversible and thereby further cooling of the liquid mixture restores a better SERS signal. In order to evaluate if the SERS sensitivity of the nanocomposites varies in function of the gel strength but for other type of analyte, experiments have been performed using adenine as the adsorbate. First, the activity of the Ag/ κ-carr_2 gel nanocomposite for SERS detection of adenine in phosphate buffer (pH 4.5) solutions was evaluated (S5-Supporting Information). The bands of adenine were clearly enhanced when using the Ag/κ-carr_2 gel substrate as compared to the same experiments but in which the pure hydrogel κ-carrageenan (i.e., without Ag NPs) was employed. Also, the effect of the gel strength on the SERS activity of Ag/κ-carrageenan for adenine detection was evaluated by using composites prepared with variable κ-carrageenan content. Figure 9 shows that as the gel strength increases, i.e., for increasing amounts of the biopolymer, there is a more pronounced enhancement of the bands assigned to the ring breathing mode at 731 cm−1, the C− N stretching at 1330 cm−1 and the NH3 scissoring at 1495 cm−1.26,61 Although this tendency is not so noticeable as compared to that one observed for 2-dtpy (Figure 5), it is still in line with an increase of the SERS sensitivity with the gel strength increase of the composite gels. Rheological Studies Using Ag/Carrageenan Hydrogels. The above spectroscopic studies suggest that as the strength of the Ag/carrageenan gel increased, either by increasing the concentration of the biopolymer or by adding a gelling promoter, there is an increase of the SERS sensitivity that can be related to the strength of the gel. A clear way to get a quantitative basis for this conjecture involves the assessment of the viscoelastic properties of the Ag/κ-carrageenan hydrogel nanocomposites by performing systematic rheological measurements. Figure 10 shows the temperature dependence of the dynamic moduli (G′ and G″) during gelation induced by cooling of pure κ-carrageenan and for the Ag/κ-carrageenan nanocomposites with variable concentration in carrageenan. The gelation process is characterized by an increase of both

Figure 7. SERS spectra of 2-dtpy methanol solution (2 × 10−3 M final) using silver nanocomposites with (a) λ-carrageenan (20 g/L); (b) ι-carrageenan (20 g/L); and (c) κ-carrageenan (20 g/L) as substrate.

Figure 8. SERS spectra of 2-dtpy (2 × 10−3 M) in 1 mL of Ag/κcarr_2 during a temperature cycle as also indicated.

Figure 9. SERS spectra of adenine phosphate buffer solution (2 × 10−5 M) in silver nanocomposite gels with increasing concentration of biopolymer (a) 5 g/L; (b) 10 g/L; (c) 20 g/L; (d) 30 g/L.

The results above show that the Ag/κ-carrageenan substrates are more SERS sensitive for increasing gel strength after cooling 10388



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Figure 11. Storage (G′ circles) and loss moduli (G″ triangles) as a function of frequency for pure κ-carrageenan (full symbols) and Ag/κcarrageenan (open symbols) at 20 °C: (a) 10 g/L; (b) 20 g/L; and (c) 30 g/L.

Figure 10. Storage (G′ circles) and loss moduli (G″ triangles) as a function of temperature for pure κ-carrageenan (full symbols) and Ag/ κ-carrageenan (open symbols) during gelation: (a) 10 g/L; (b) 20 g/ L; and (c) 30 g/L.

Table 2. Gelling Temperature (Tg) and Elastic Modulus (G′, 20°C, 2 rad/s) for κ-Carrageenan and Derived Ag Nanocomposites hydrogel κ-carrageenan (10 g/L) Ag/κ-carrageenan (10 g/L) κ-carrageenan (20 g/L) Ag/κ-carrageenan (20 g/L) κ-carrageenan (30 g/L) Ag/κ-carrageenan (30 g/L)

Tg (°C)

G′ (Pa)

± ± ± ± ± ±

138.8 165.6 2190 3704 5210 10 274

34.7 35.1 48.5 49.5 55.6 56.2

0.4 0.1 0.7 0.7 0.6 0.1

storage and loss moduli.62 As expected, before gelation the system behaves as a viscous fluid (G″ > G′) while elastic behavior predominates after gelation (G′>G″). The sudden increase of the elastic modulus (G′) during gelation is due to the increase of the connectivity between the basic structural units of the biopolymer and the development of the gel network.41,63

Figure 12. Representation of the analytical enhancement factors (AEF) in function of G′ modulus (20 °C) for Ag/κ-carrageenan hydrogels having variable biopolymer content. The AEF were calculated based on distinct Raman bands: 1571 cm−1 (square); 1558 cm−1 (circle); 1112 cm−1 (triangle); 1083 cm−1 (star). 10389



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Figure 13. Proposed mechanism for the formation of high sensitive SERS hydrogels due to the presence of Ag nanojunctions caused by κcarrageenan helices aggregation.

The gelling temperature (Tg) of the materials was calculated in each case as the onset temperature of the increasing ramp of the elastic modulus (G′) under cooling conditions (Table 2). For the pure hydrogel, the Tg increased from about 35 to 56 °C when the concentration of the polysaccharide increased from 10 to 30 g/L, which is in agreement with previous reports.35,36,41,63 The presence of Ag nanoparticles in the biopolymer did not cause significant changes in the Tg. Indeed, for all the cases analyzed, the Tg of the nanocomposites increased approximately 1 °C as compared to the corresponding κ-carrageenan pure matrices. Regarding the effect of Ag nanoparticles in the strength of the gel, it should be noted that the G′ of the hydrogels composites is higher than the corresponding G′ for pure carrageenan (Table 2). This effect is more pronounced for higher biopolymer concentrations. Thus, suggesting that the presence of Ag nanoparticles enhances the strength of the gel. Also, by increasing the biopolymer concentration, the G′ increases as expected for gels with higher strength. This trend was further confirmed in frequency sweep tests of the hydrogels (Figure 11). While for a 10 g/L carrageenan, the marked dependence of G′ with frequency indicates the formation of a weaker and less elastic structure, for higher biopolymer concentration, the G′ is independent of the frequency, which indicates few molecular rearrangements within the gel network over the frequency scale analyzed, which is a typical behavior of strong gels. Figure 12 gives a more complete picture of the SERS sensitivity dependence of the Ag/κ-carrageenan hydrogels for 2-dtpy with the corresponding gel strength. In Figure 12, the analytical enhancement factors (AEF) were estimated by applying eq 1 and by using distinct vibrational bands in order to confirm the general trend observed. Increasing the content of κ-carrageenan in the hydrogel causes tighter gels, as confirmed by higher G′ values, which correlates well with the increase of AEF estimated from the SERS spectra. All these experiments indicate that the SERS sensitivity of the composite hydrogels is dependent on the corresponding G′ values, which in turn take into account the number of κ-carrageenan chains per gel volume. Thus, an increase of G′ is associated with a more compact gel network in which probably clustering of the Ag particles is favored, as suggested by the corresponding optical spectra (Figure 2). As a consequence, the adsorbate molecules will be extensively trapped during the gelling process at the formed metal nanojunctions, which have been associated with the existence of hot spots for SERS detection. As discussed

above, the gel strength can be increased experimentally by adding a cross-linker agent, increasing the biopolymer content or lowering the temperature of the sample. Figure 13 illustrates this explanatory mechanism by using the effect of temperature on the κ-carrageenan helices aggregation.

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CONCLUSION In summary, this study demonstrates for the first time that the rheological behavior of Ag containing hydrogels influence their performance as SERS platforms. This research provided new insights into surface enhanced Raman scattering associated with changes on the rheological behavior of chemical environments comprising active metal surfaces such as Ag nanoparticles. Furthermore, a possible mechanism was formulated that correlates the gel strength of the biopolymer matrix with the SERS signal, as a consequence of the formation of metal nanojuntions caused by the closer proximity of Ag nanoparticles as the biopolymer chains interaction was promoted. Even though this tendency has been demonstrated for 2,2′dithiodipyridine and adenine as molecular adsorbates, it is reasonable to assume that a similar trend will be observed for other analytes. On the other hand, this research had focus on hydrogels composed of polysaccharides of the carrageenan family, which suggests caution in the anticipation of this SERS sensitive behavior for other types of polymer matrices. Although this has not been observed for the cases analyzed here, besides the rheological behavior also the chemical properties of the matrix can have a determinant effect on their use as thermosensitive SERS substrates. To conclude, we believe that this work can motivate research on the design of thermosensitive polymers that use rheological parameters for controlling the analytical sensitivity when used as SERS substrates.

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ASSOCIATED CONTENT

S Supporting Information *

SEM images and FTIR spectra of nanocomposites; Lewis structure of 2-dtpy; Raman spectra collected for control experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +351 234 370 726. Fax: +351 234 370 084. E-mail: [email protected]. 10390



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Author Contributions

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S.F. planned and performed the chemical and materials characterization tasks. H.I.S.N. and A.L.D.d.S. contributed respectively to SERS and rheology experiments and respective discussion. T.T. planned and supervised all the research. The manuscript was written through discussion and contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.F. thanks Fundaçaõ para a Ciência e Tecnologia (FCT/ FEDER) for the Grant SFRH/BPD/93547/2013. The authors acknowledge FCT/FEDER (PTDC/CTM-NAN/120668/ 2010, Pest-C/CTM/LA0011/2011), FSE and POPH for funding.

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