Theoretical and Experimental Study on the Functionalization Effect on the SERS Enhancement Factor of SiO2-Ag Composite Films

*Paulina De León Portilla, Ana Lilia González Ronquillo and Enrique Sánchez Mora*

## **Abstract**

Herein we addressed a study to determine the enhancement factor (EF) of the Raman signal reached by composite films with two main components, Ag nanoparticles and SiO2 spheres. The study involves the synthesis, structural composition and optical response by using experimental techniques and theoretical-numerical modeling. A colloid with single NPs and agglomerates of them, with a tannic acid layer on its surface, was produced. Separately, porous SiO2 spheres were obtained. A mixture of both, Ag NPs and SiO2 particles was used to produce the films by solvent evaporation method. It is shown that single or agglomerated Ag NPs are preferentially located at the interstices of the SiO2 spheres. Using discrete dipole approximation, the SERS EF has been estimated considering the agglomeration and tannic acid layer. Both, the dielectric spheres and tannic acid layer diminish the electric field intensity and therefore the SERS EF. When a Ag NP with/without a dielectric shell is touching a SiO2 sphere, the EF is as high as 1 103 , the zones where this value is reached are smaller when the dielectric layer is present. With a cluster of 3 nude Ag NPs surrounded by SiO2 spheres an EF of 2.4 103 is obtained.

**Keywords:** SiO2 spheres, silver nanoparticles, composite film, tannic acid, functionalization, near field intensity, discrete dipole approximation

### **1. Introduction**

Research around the optical properties of Ag nanoparticles (NPs) has been a topic of high importance for diverse applications as coating, plasmonic antennas, for drug delivery, as components of molecular detectors, among others [1–6]. The convenience of using Ag NPs lies in their intense plasmonic response with a surface plasmon resonance (SPR) in the visible optical range. The SPR is a phenomenon that occurs when a metal NP is excited by an electromagnetic wave, the conduction electrons respond to the electromagnetic field in such a way that oscillations of the electron charge distribution occur in the vicinity or on the surface of the particle. Ag NPs have their SPR in the range from 350 to 600 nm, depending on their size and morphology [7, 8].

To improve the colloidal stability and surface properties of Ag NPs the functionalization is a feasible option. This chemical process modifies the NP's surface charge using a specific chemical compound that successfully encapsulates the NP, attributing the surface with a neutral, negative or positive character [9]. Once NPs are functionalized can be used for different purposes, for example, to selectively adsorb drugs to later release them in a controlled way [3], to inhibit the growth of bacteria [5], to prevent agglomeration in a colloid [10], among others.

200 nm de diameter. The methodology describes different conditions to obtain a specific size. In essence, the required size is achieved from the number of growth steps. Here, Ag seeds of approximately 35 nm in diameter were obtained as follows. 50 ml of 0.1 mM tannic acid (Sigma Aldrich, 98%) and 50 ml of 5 mM sodium citrate (J. T. Baker, 96%) were mixed and then the solution was heated up to 90 ° C. Subsequently, 1 ml of 25 mM silver nitrate (Riedel-de Haën, 98%) was added and the solution was kept stirring for 30 minutes. At this point, to eliminate secondary products, the solution was centrifuged at 20,000 rpm, for 15 minutes. The supernatant solution was separated and subsequently the Ag seeds were redispersed in 20 ml of deionized water. The elimination of secondary products was repeated 3

*Theoretical and Experimental Study on the Functionalization Effect on the SERS…*

In order to obtain Ag NPs of 100 nm of diameter, a 4-stage growth procedure

A common method to synthesize SiO2 spheres is the Stöber method [20], which consists of the hydrolysis of tetraethylorthosilicate (TEOS) with an alcohol as solvent, and an hydrolysis catalyst. With this method polydisperse SiO2 colloids are obtained. On the other hand, Razo and collaborators [21] obtained monodispersed SiO2 microspheres with characteristic sizes between 150 and 600 nm in diameter. In their work they reported the relation between the sphere diameter of SiO2 as a function of the moles of TEOS. During all the study the number of moles of NH3/ H2O/C2H5OH was fixed. Using 0.46 moles of NH3, 2.89 moles of H2O and 2.15 moles of C2H5OH (relation A) they obtained microspheres with size from 150 nm to 600 nm in diameter. Whereas using 0.41 moles of NH3, 2.20 moles of H2O and 2.15 moles of C2H5OH (relation B) microspheres from 350 nm to 600 nm were produce. Herein we synthesize SiO2 spheres of 300 nm based on relation A. Separately,

two solutions were prepared: the first one consisted of 9 ml of TEOS (Sigma Aldrich, 98%) and 125 ml of ethanol (Sigma Aldrich, 80%). The second solution was formed from 52 ml of deionized water and 30 ml of ammonium hydroxide (J. T. Baker, 64%). Later, both solutions were mixed and the reaction was maintained for 3 hours, under constant stirring at room temperature. For the purification of the SiO2 spheres, the same washing procedure (elimination of the secondary products)

To obtain the films of the SiO2-Ag composites, the solvent evaporation method was used. For this, 25 ml of solution of SiO2 spheres (5 mM) dispersed in ethanol

On the other hand, the glass substrate was cleaned with neutral soap in a sonic bath, dried with pressurized air and immersed for 1 hour in 100 ml of piranha solution (3: 1 of sulfuric acid and hydrogen peroxide). After this time, the substrate was rinsed with deionized water and dried with compressed air. The substrate was

were mixed with 5 ml of Ag NPs solution (3 mM) dispersed in water.

was followed. The first stage consisted of diluting 20 ml of the seed solution (0.21 M) with 16 ml of deionized water and then heated up at 90 °C. Subsequently, 500 μl of 25 mM sodium citrate, 1.5 ml of 2.5 mM tannic acid and 1 ml of 25 mM silver nitrate were added. This stage ends after 30 minutes of reaction maintaining the temperature at 90 °C. As a second stage, the same amounts of sodium citrate, tannic acid and silver nitrate were added keeping the same reaction conditions. Third and fourth stages were alike than the second one. The elimination process of secondary products was carried out according to the one indicated above, with the

difference that a centrifugation velocity of 18,000 rpm was used.

times. The final solution is named the seed solution.

*DOI: http://dx.doi.org/10.5772/intechopen.97028*

**2.2 SiO2 spheres synthesis**

used for the Ag NPs was followed.

**173**

**2.3 SiO2-Ag composite film deposition**

Tannic acid is commonly used in synthesis methods as a stabilizer, it is a typical hydrolyzable tannin derived from plants with various biological properties such as antioxidant, antitumor, antimutagenic, antimicrobial and anti-inflammatory, besides its ability to interact with proteins [11]. The structure of tannic acid is made up of tannins that contain digalloyl ester groups connected to a central glucose nucleus. These groups can interact ionically with others, also can interact through hydrogen bonding with alike or different molecules, and moreover bond to a metal [12].

Silicon dioxide, SiO2, is considered a useful material for coating because of its physical and chemical properties such as chemical stability, optical transparency, biocompatibility, inert-material character and reactivity with various coupling agents [13, 14]. Specifically, amorphous SiO2 substrates have the advantage of a very high specific surface area. In addition, they promote the dispersion and homogeneous distribution of metallic NPs on it [14]. The last, seems promising for Surface Enhanced Raman Scattering (SERS) based-metal substrates purposes. The SERS enhancement factor (EF) that can be reached is directly related to the SPR of the used NPs through the j j *<sup>E</sup>* **<sup>4</sup>** approximation.

To determine the electric field intensity on the NP's surface and away from it, exist various options, some of the most common are based on finite elements methods, finite difference time domain or volume integral equations. In particular, Discrete Dipole Approximation (DDA) is a flexible integral equation method that allows to study the optical response of targets with a size smaller or similar to that of the incident wavelength. With this method is possible to study isolated or periodic targets with arbitrary shapes [15, 16]. On the other hand, this method has been widely used to study the absorbed and scattered light by several systems as nanoparticles, bacteria, films, red blood cells, among others [17, 18].

Along this chapter we present a study of films composed by Ag NPs and SiO2 spheres. The films are proposed as SERS substrates with the characteristic of Ag NPs very well located at the interstices of the silica spheres. It is worth mentioning that the NPs have a tannic acid layer on its surface. Through a combination of experimental techniques and theoretical-numerical modeling a study of structural and optical properties has been realized, in addition the SERS EF has been estimated. The content of the chapter is as follows. In section 2 the methodology followed for the synthesis of Ag NPs, SiO2 spheres and composite films is addressed. In sections 3 and 4, the determination of size and morphology of Ag NPs is presented, and the presence of a layer of tannic acid on its surface is also shown. Subsequently, in section 5 and 6 the structural composition of the SiO2 spheres and composite films is shown, respectively. Section 7 contains the details of the methodology followed to estimate the SERS EF of systems made of Ag NPs and SiO2 spheres. Finally, the conclusions are stated.

#### **2. Synthesis methodology**

#### **2.1 Ag NPs synthesis**

Ag NPs were synthesized using the seed growth method reported by Bastús et al. [19], they reported an experimental methodology para obtainer Ag NP de 4 a

*Theoretical and Experimental Study on the Functionalization Effect on the SERS… DOI: http://dx.doi.org/10.5772/intechopen.97028*

200 nm de diameter. The methodology describes different conditions to obtain a specific size. In essence, the required size is achieved from the number of growth steps. Here, Ag seeds of approximately 35 nm in diameter were obtained as follows. 50 ml of 0.1 mM tannic acid (Sigma Aldrich, 98%) and 50 ml of 5 mM sodium citrate (J. T. Baker, 96%) were mixed and then the solution was heated up to 90 ° C. Subsequently, 1 ml of 25 mM silver nitrate (Riedel-de Haën, 98%) was added and the solution was kept stirring for 30 minutes. At this point, to eliminate secondary products, the solution was centrifuged at 20,000 rpm, for 15 minutes. The supernatant solution was separated and subsequently the Ag seeds were redispersed in 20 ml of deionized water. The elimination of secondary products was repeated 3 times. The final solution is named the seed solution.

In order to obtain Ag NPs of 100 nm of diameter, a 4-stage growth procedure was followed. The first stage consisted of diluting 20 ml of the seed solution (0.21 M) with 16 ml of deionized water and then heated up at 90 °C. Subsequently, 500 μl of 25 mM sodium citrate, 1.5 ml of 2.5 mM tannic acid and 1 ml of 25 mM silver nitrate were added. This stage ends after 30 minutes of reaction maintaining the temperature at 90 °C. As a second stage, the same amounts of sodium citrate, tannic acid and silver nitrate were added keeping the same reaction conditions. Third and fourth stages were alike than the second one. The elimination process of secondary products was carried out according to the one indicated above, with the difference that a centrifugation velocity of 18,000 rpm was used.

#### **2.2 SiO2 spheres synthesis**

To improve the colloidal stability and surface properties of Ag NPs the functionalization is a feasible option. This chemical process modifies the NP's surface charge using a specific chemical compound that successfully encapsulates the NP, attributing the surface with a neutral, negative or positive character [9]. Once NPs are functionalized can be used for different purposes, for example, to selectively adsorb drugs to later release them in a controlled way [3], to inhibit the growth of bacteria [5], to prevent agglomeration in a colloid [10], among others. Tannic acid is commonly used in synthesis methods as a stabilizer, it is a typical hydrolyzable tannin derived from plants with various biological properties such as antioxidant, antitumor, antimutagenic, antimicrobial and anti-inflammatory, besides its ability to interact with proteins [11]. The structure of tannic acid is made up of tannins that contain digalloyl ester groups connected to a central glucose nucleus. These groups can interact ionically with others, also can interact through hydrogen bonding with alike or different molecules, and moreover bond to a metal [12].

*Silver Micro-Nanoparticles - Properties, Synthesis, Characterization, and Applications*

Silicon dioxide, SiO2, is considered a useful material for coating because of its physical and chemical properties such as chemical stability, optical transparency, biocompatibility, inert-material character and reactivity with various coupling agents [13, 14]. Specifically, amorphous SiO2 substrates have the advantage of a very high specific surface area. In addition, they promote the dispersion and homogeneous distribution of metallic NPs on it [14]. The last, seems promising for Surface Enhanced Raman Scattering (SERS) based-metal substrates purposes. The SERS enhancement factor (EF) that can be reached is directly related to the SPR of

To determine the electric field intensity on the NP's surface and away from it,

Along this chapter we present a study of films composed by Ag NPs and SiO2 spheres. The films are proposed as SERS substrates with the characteristic of Ag NPs very well located at the interstices of the silica spheres. It is worth mentioning that the NPs have a tannic acid layer on its surface. Through a combination of experimental techniques and theoretical-numerical modeling a study of structural and optical properties has been realized, in addition the SERS EF has been estimated. The content of the chapter is as follows. In section 2 the methodology followed for the synthesis of Ag NPs, SiO2 spheres and composite films is addressed. In sections 3 and 4, the determination of size and morphology of Ag NPs is presented, and the presence of a layer of tannic acid on its surface is also shown. Subsequently, in section 5 and 6 the structural composition of the SiO2 spheres and composite films is shown, respectively. Section 7 contains the details of the methodology followed to estimate the SERS EF of

systems made of Ag NPs and SiO2 spheres. Finally, the conclusions are stated.

[19], they reported an experimental methodology para obtainer Ag NP de 4 a

Ag NPs were synthesized using the seed growth method reported by Bastús et al.

exist various options, some of the most common are based on finite elements methods, finite difference time domain or volume integral equations. In particular, Discrete Dipole Approximation (DDA) is a flexible integral equation method that allows to study the optical response of targets with a size smaller or similar to that of the incident wavelength. With this method is possible to study isolated or periodic targets with arbitrary shapes [15, 16]. On the other hand, this method has been widely used to study the absorbed and scattered light by several systems as nanoparticles, bacteria, films, red blood cells, among others [17, 18].

the used NPs through the j j *<sup>E</sup>* **<sup>4</sup>** approximation.

**2. Synthesis methodology**

**2.1 Ag NPs synthesis**

**172**

A common method to synthesize SiO2 spheres is the Stöber method [20], which consists of the hydrolysis of tetraethylorthosilicate (TEOS) with an alcohol as solvent, and an hydrolysis catalyst. With this method polydisperse SiO2 colloids are obtained. On the other hand, Razo and collaborators [21] obtained monodispersed SiO2 microspheres with characteristic sizes between 150 and 600 nm in diameter. In their work they reported the relation between the sphere diameter of SiO2 as a function of the moles of TEOS. During all the study the number of moles of NH3/ H2O/C2H5OH was fixed. Using 0.46 moles of NH3, 2.89 moles of H2O and 2.15 moles of C2H5OH (relation A) they obtained microspheres with size from 150 nm to 600 nm in diameter. Whereas using 0.41 moles of NH3, 2.20 moles of H2O and 2.15 moles of C2H5OH (relation B) microspheres from 350 nm to 600 nm were produce.

Herein we synthesize SiO2 spheres of 300 nm based on relation A. Separately, two solutions were prepared: the first one consisted of 9 ml of TEOS (Sigma Aldrich, 98%) and 125 ml of ethanol (Sigma Aldrich, 80%). The second solution was formed from 52 ml of deionized water and 30 ml of ammonium hydroxide (J. T. Baker, 64%). Later, both solutions were mixed and the reaction was maintained for 3 hours, under constant stirring at room temperature. For the purification of the SiO2 spheres, the same washing procedure (elimination of the secondary products) used for the Ag NPs was followed.

#### **2.3 SiO2-Ag composite film deposition**

To obtain the films of the SiO2-Ag composites, the solvent evaporation method was used. For this, 25 ml of solution of SiO2 spheres (5 mM) dispersed in ethanol were mixed with 5 ml of Ag NPs solution (3 mM) dispersed in water.

On the other hand, the glass substrate was cleaned with neutral soap in a sonic bath, dried with pressurized air and immersed for 1 hour in 100 ml of piranha solution (3: 1 of sulfuric acid and hydrogen peroxide). After this time, the substrate was rinsed with deionized water and dried with compressed air. The substrate was

introduced into the colloid formed by microspheres of SiO2 and Ag NPs. Subsequently, it was heated in a muffle at 70 °C for 24 hours.

#### **3. Determination of the size and shape of Ag NPs**

**Figure 1** shows the Absorbance of the Ag NPs colloidal solution measured from the ultraviolet to the visible region. The maximum of the spectrum is located at 421 nm and the shape-line is asymmetrical, with a shoulder covering from 480 nm to 550 nm. To have an insight about the origin of the maximum and the shoulder we proceed to calculate the optical extinction efficiency (Qext) of a spherical Ag NP of various diameters. The Absorbance and extinction efficiency are directly related through the next expression:

$$A(\lambda) = \text{CQ}\_{\text{ext}}(\lambda) \pi r^2 L / \ln\left(10\right),\tag{1}$$

where *C* is the concentration of Ag NPs (in particles/cm<sup>3</sup> ), *L* is the length of the sample (1 cm), *r* the radius of the NP and ln is the natural logarithm function. The linear relation between *A* and *Qext* indicates that both of them have the same spectral shape-line but different intensities. The *Qext* was calculated using the well-known Mie theory [22].

As we can see in the **Figure 1**, the Qext spectra that better fit to the Absorbance are those of a Ag NP with a diameter of 63 nm and other of 100 nm. This indicates that in the colloidal solution predominates the presence of Ag NPs of 63 nm and 100 nm. According to the intensity of the spectrum one can expect that the number of NPs with a size of 63 nm is larger than that with a size of 100 nm.

Apart, the size distribution was also determined using Dynamic Light Scattering (DLS), a technique that provides information of the hydrodynamic diameter of the entities in the solution. Under optimal particle concentration, that is, sufficiently low to disesteem particle-particle interaction and sufficiently high to have a strong signal, the hydrodynamic diameter of spherical particles matches to the physical diameter. From the size distribution a main value around 66 nm is observed, also

the presence of NPs with a diameter of 100 nm is detected, see **Figure 2**. Therefore, there is a good agreement with the predicted calculations obtained with Mie theory

*Images by AFM showing single Ag NPs and agglomerates on a crystalline silicon wafer, the estimated mean*

Small and big Ag NPs were also observed by using an Atomic Force Microscope (AFM), see **Figure 3**. However, the "big NPs" are actually quasi spherical agglomerates of Ag NPs with an average size of 969 nm, the statistic was done over 166 particles. This fact clarifies the deductions made from DLS and spectroscopy techniques, and

In **Figure 4** the zeta potential profile of the Ag NPs is shown, there a negative superficial charge of 52.5 mV is detected. To expose the origin of the negative

Mie theory, the solution contains single Ag NPs and agglomerates of them.

*Size distribution of the Ag NPs in solution, measured with DLS technique.*

*Theoretical and Experimental Study on the Functionalization Effect on the SERS…*

*DOI: http://dx.doi.org/10.5772/intechopen.97028*

and shown in **Figure 1**.

**Figure 2.**

**Figure 3.**

**175**

*value is 96 9 nm.*

**4. Functionalization of Ag NPs**

#### **Figure 1.**

*Measured and calculated optical response of the Ag colloid. The calculated spectra were obtained using Mie theory for a spherical NP with a diameter D = 63 nm and other with a D = 100 nm.*

*Theoretical and Experimental Study on the Functionalization Effect on the SERS… DOI: http://dx.doi.org/10.5772/intechopen.97028*

**Figure 2.** *Size distribution of the Ag NPs in solution, measured with DLS technique.*

#### **Figure 3.**

introduced into the colloid formed by microspheres of SiO2 and Ag NPs.

*Silver Micro-Nanoparticles - Properties, Synthesis, Characterization, and Applications*

*A*ð Þ¼ *λ CQext*ð Þ*λ πr*

where *C* is the concentration of Ag NPs (in particles/cm<sup>3</sup>

of NPs with a size of 63 nm is larger than that with a size of 100 nm.

**Figure 1** shows the Absorbance of the Ag NPs colloidal solution measured from the ultraviolet to the visible region. The maximum of the spectrum is located at 421 nm and the shape-line is asymmetrical, with a shoulder covering from 480 nm to 550 nm. To have an insight about the origin of the maximum and the shoulder we proceed to calculate the optical extinction efficiency (Qext) of a spherical Ag NP of various diameters. The Absorbance and extinction efficiency are directly related

2

sample (1 cm), *r* the radius of the NP and ln is the natural logarithm function. The linear relation between *A* and *Qext* indicates that both of them have the same spectral shape-line but different intensities. The *Qext* was calculated using the

As we can see in the **Figure 1**, the Qext spectra that better fit to the Absorbance are those of a Ag NP with a diameter of 63 nm and other of 100 nm. This indicates that in the colloidal solution predominates the presence of Ag NPs of 63 nm and 100 nm. According to the intensity of the spectrum one can expect that the number

Apart, the size distribution was also determined using Dynamic Light Scattering (DLS), a technique that provides information of the hydrodynamic diameter of the entities in the solution. Under optimal particle concentration, that is, sufficiently low to disesteem particle-particle interaction and sufficiently high to have a strong signal, the hydrodynamic diameter of spherical particles matches to the physical diameter. From the size distribution a main value around 66 nm is observed, also

*Measured and calculated optical response of the Ag colloid. The calculated spectra were obtained using Mie*

*theory for a spherical NP with a diameter D = 63 nm and other with a D = 100 nm.*

*L=* ln 10 ð Þ, (1)

), *L* is the length of the

Subsequently, it was heated in a muffle at 70 °C for 24 hours.

**3. Determination of the size and shape of Ag NPs**

through the next expression:

well-known Mie theory [22].

**Figure 1.**

**174**

*Images by AFM showing single Ag NPs and agglomerates on a crystalline silicon wafer, the estimated mean value is 96 9 nm.*

the presence of NPs with a diameter of 100 nm is detected, see **Figure 2**. Therefore, there is a good agreement with the predicted calculations obtained with Mie theory and shown in **Figure 1**.

Small and big Ag NPs were also observed by using an Atomic Force Microscope (AFM), see **Figure 3**. However, the "big NPs" are actually quasi spherical agglomerates of Ag NPs with an average size of 969 nm, the statistic was done over 166 particles. This fact clarifies the deductions made from DLS and spectroscopy techniques, and Mie theory, the solution contains single Ag NPs and agglomerates of them.

#### **4. Functionalization of Ag NPs**

In **Figure 4** the zeta potential profile of the Ag NPs is shown, there a negative superficial charge of 52.5 mV is detected. To expose the origin of the negative

**5. Determination of size and shape of SiO2 particles**

*DOI: http://dx.doi.org/10.5772/intechopen.97028*

stay on the surface [13].

**Figure 6.**

**Figure 7.**

**177**

*Zeta potential of the SiO2 particles in solution.*

consequence of the defects mentioned above.

OH groups located on the surface or in the pores.

A transmission electron microscopy (TEM) image of the SiO2 spheres is shown

in **Figure 6A**. It is observed that the spheres are amorphous because of their porosity. In the inset of **Figure 6A** we illustrate the structure of one pore. The formation of the pores takes place when some periodic chains of Si-O were not carried out during the polymerization reaction, in addition, some OH groups may

*Theoretical and Experimental Study on the Functionalization Effect on the SERS…*

The optical UV–Vis spectrum is shown in **Figure 6B**. An absorption edge starting at approximately 300 nm and a maximum in 290 nm are observed. The width of absorbance spectrum corresponds to the band gap (Eg) energy of amorphous SiO2. The Eg was estimated by extrapolating the absorption edge to the photon energy axis through a linear fit, then the Eg value in the direct transition of SiO2 turned out to be 3.98 eV. This value is in agreement with the reported Eg of 3.8 eV in the case of SiO2 nanostructures [13, 24]. This value is lower than that reported for crystalline SiO2 (quartz), which is approximately 9 eV [25] and is a

The zeta potential of the SiO2 colloid is shown in **Figure 7**, a negative surface charge of 44.1 mV is detected. The minus sign is attributed to the oxygens of the

The spherical shape of the SiO2 particles is corroborated by the AFM images in

**Figure 8**. A large concentration of SiO2 spheres covering a glass substrate is

*(A) a TEM image of SiO2 spheres. (B) UV–vis spectrum of the colloidal solution of SiO2 particles.*

**Figure 4.** *Zeta potential of the Ag NPs in solution.*

**Figure 5.**

*Measured absorbance of the Ag NPs (black line), tannic acid (red line) and sodium citrate (blue line). To the right a scheme of the adsorption of tannic acid molecules on the NP surface is depicted.*

superficial charge, a comparison among the optical spectrum of the Ag NPs, the tannic acid and the sodium citrate is presented in **Figure 5**. The last two were used as reducing and stabilizing agents during the synthesis procedure of the NPs. The band of the sodium citrate is out of the measured interval, whereas the optical response of the tannic acid is characterized by one band located at 214 and other at 268 nm. These two bands are well superimposed to the shape line of the Ag NPs spectrum in the interval of ultraviolet light. Therefore, the tannic acid is present in the colloid even after the several washing times. The negative charge of the NPs is explained as follows. Assuming the tannic acid molecules are adsorbed on the surface of the NPs, the negative charge detected is probably because of a partial deprotonation of the OH groups bonded to aromatic ring (see scheme in **Figure 5**). It is worth mentioning the fact that the electronic interband transitions of Ag take place at wavelengths lower than 320 nm, therefore they also contribute to the Absorbance in the UV range [23].

*Theoretical and Experimental Study on the Functionalization Effect on the SERS… DOI: http://dx.doi.org/10.5772/intechopen.97028*
