**5. Applications of SERS**

SERS is highly sensitive analytical spectroscopic technique available among the modern scientific communities and can contribute both to surface science and nanoscience. It is also associated with a broad range of other surface sensitive techniques to study various fundamental and applied research topics such as, corrosion, catalysis, advanced materials, diagnostics, biomedical applications, biological process, and sensing.

surface plasmons (LSP) of the metal nanoparticles. The localized surface plasmon resonanace (LSPR) arises as a result of resonance condition between the incident wavelength of light and the electrons in the nanoparticles. This facilitates a combined oscillation of the conduction electrons and it will give rise to two main consequences. The first consequence is the absorption of the wavelengths of light selectively by nanoparticles, which is responsible for this collective oscillation. The second consequence is the enhancement of electromagnetic fields that extend from the nanoparticle surfaces. These fields are mainly responsible for the large enhancement observed in SERS. The enhancement is approximately proportional to |*E*<sup>4</sup>

Localized surface plasmon resonance (LSPR), the lightning rod effect, and the image field effect, all these effects are responsible for the enhancement in SERS. Among them, LSPR contributes mainly to the electromagnetic field enhancement and SERS effect. Anisotropic metallic nanostructures have all of the characteristics of excellent SERS active substrates with good stability and high reproducibility. It has been demonstrated in the literature that anisotropic nanostructures such as nanorods, nanodisks, and nanoprisms will exhibit interesting size and shape-dependent properties. Anisotropic metal nanoparticles will exhibit "lightningrod effect" [24], which is a new kind of field enhancement refers to enhanced charge density localization at a tip or vertex of a nanoparticle. The theory based on "lightning-rod effect" was developed by Liao and Wokaun in the year 1982. The excitation of the free electrons of a metallic tip by an electromagnetic field (laser light) will generate an extremely localized, sturdy electric field at these sharp tips or vertex with large curvatures leading to large field enhancement in those regions. This effect gives rise to high SERS activity of the anisotropic nanostructures. Anisotropic metallic nanostructures have been extensively utilized as an effective SERS active substrate with high SERS activity [25, 26] as found in the literature.

The chemical enhancement (CM) mechanism corresponds to the enhancement effect arises as a result of the chemical interaction between the adsorbates and the metal surface. The CM mechanism can also be referred as the charge transfer (CT) mechanism, involving the photoinduced transfer of an electron from the Fermi level of the metal to an unoccupied molecular orbital of the adsorbate (LUMO) or vice versa. The enhancement factor of CM is generally in

as an SERS active substrate, while CM has a short-range effect taking place on the molecular scale. The two mechanisms of EM and CM are not reciprocally restricted, but these two effects work simultaneously to generate the overall SERS effect. However, it is very hard to differentiate CM from the EM effect. Several research groups all over the world have tried to solve

SERS is highly sensitive analytical spectroscopic technique available among the modern scientific communities and can contribute both to surface science and nanoscience. It is also associated with a broad range of other surface sensitive techniques to study various fundamental

. EM has a long-range effect, for which rough metallic surfaces can be used

or more, where *E* is the intensity of the electromagnetic field.

generally in the order of 108

304 Raman Spectroscopy and Applications

the order of 10–102

**5. Applications of SERS**

this problem, but the problem is unsolved so far.


The SERS-active Ag/AAO nanostructured system as mentioned earlier in this chapter has been used by Liu et al. to investigate antibiotic-induced chemical changes in bacterial cell wall [27]. They recorded high quality, highly reproducible SERS spectra, which are also sensitive and stable. The "chemical features" observed from the SERS spectra of bacterial cell wall facilitates fast identification of drug-resistant bacteria within an hour both qualitatively and quantitatively. Furthermore, the characteristic changes in the SERS spectra were clearly observed in the drug-sensitive bacteria at the near the beginning (i.e., 1 hour) of antibiotic exposure, which could be utilized to differentiate them from the drug-resistant ones. The rapid detection of pathogens such as bacteria and viruses using the SERS technique provides a novel approach for microbial diagnostics. The SERS-based novel technique was applied to a single bacterium. This rapid SERS detection of pathogens makes possible direct analysis of clinical specimen as an alternative to a pure culture specimen. Traditional diagnostic protocols for diagnosing bacterial infections are based on the isolation of pure culture of the bacterium, which is followed by the resolving of the nature of the isolate and an extensive assessment of the isolates responses to several antibiotics proliferation or viability. For such biological assays, an incubation period varying from days to weeks or even months is essential for the growth of bacteria with such a density that can be handled by the available diagnostic tools. Different PCR-based protocols have been inducted for the quantitative identification of bacteria. Mass spectrometry can also be alternatively utilized for culture-free bacterial diagnostics. Nevertheless, just like the PCR-based method, mass spectrometry-based protocol is dependent on the existing previous information in the literature about the pathogens. Finally, neither of the PCR or mass spectrometry-based protocols can be applied to a live bacterial sample to observe their responses to antibiotics or to carry out different functional tests. On the other hand, SERS-based spectroscopic technique can resolve the limitation of PCR-based identification methods for pathogens. The SERS active substrates based on the Ag/AAO system can be utilized for the fine changes observed in the bacterial cell wall during different stages of bacterium's growth and also for the bacterium's response to antibiotic treatment during the early period of antibiotic exposure.

In a recent development, Ankamwar et al. [28] fabricated a highly stable and almost homogeneous SERS active substrate from silver nanoparticles synthesized from the leaf extract of *Neolamarckia cadamba* for the rapid detection of two strains of bacteria, Gram-positive (*Staphylococcus aureus*) and Gram-negative (*Escherichia coli*) bacteria. **Figure 6** demonstrates the TEM image of the assynthesized silver nanoparticles along with their UV-visible spectrum, SAED pattern, and the resultant SERS spectra generated upon interaction with *S. aureus* and *E. coli*.

These silver nanoparticles upon interaction with bacteria can exhibit a large Raman enhancement factor ((3 ± 0.20) × 10<sup>7</sup> and (5 ± 0.40) × 10<sup>7</sup> for *S. aureus* and *E. coli* bacteria, respectively) with almost zero fluctuations. The SERS substrate developed by them is almost homogeneous with a relative standard deviation value of 6.32 calculated from 50 repeated measurements from various locations on the SERS substrate. In addition to this, the fabricated SERS substrates are extremely stable even after 3 months. Using this almost homogeneous, a stable SERS active substrate, Gram-positive bacteria can be differentiated from Gram-negative bacteria. The SERS data presented in the above study is highly stable, uniform, and reproducible, which shows the versatility of the biosynthesized SERS active substrate. This SERS active substrate is capable of detecting extremely low concentrations (103 CFU ml−1) of *E. coli* within a very short time of 1–5 s and also exhibits high sensitivity (see **Figure 7**). **Figure 7** demonstrates the SERS calibration curve obtained with SERS intensity of the peak at 1330 cm−1 (C–N stretching mode) as a function of concentration of bacteria *E. coli*. The 1330 cm−1 peak became detectable at 10<sup>3</sup> CFU/ml of *E. coli* concentration. The SERS intensity increases with the concentration of the bacterial solution, as it is exponentially correlated to the concentration of *E. coli* bacterial cells in the sample between 10<sup>3</sup> and 108 CFU/ml. Experiments were repeated five times with each bacterial concentration, and the standard errors of the mean for each concentration are also shown in **Figure 7**. The major intention of this SERS study using biosynthesized Ag nanoparticles was to develop a rapid fingerprinting method for the characterization of bacteria particularly *E. coli*, which is associated with urinary tract infection (UTI), a common disease among most people of all age groups in developed countries such as India and China.

**Figure 6.** The TEM image of the as-synthesized silver nanoparticles along with their UV-visible spectrum, SAED pattern, and the resultant SERS spectra generated upon interaction with bacteria *S. aureus* and *E. coli*. Reproduced with permission from Ankamwar et al. [28]. Copyright @ Royal Society of Chemistry, Inc.

The SERS-based pathogen detection method is especially useful for the analysis of slowgrowing bacteria, which typically may take few weeks during laboratory tests. The SERS spectra described above to detect and quantify bacteria lacks the molecular level specificity compared to other commonly used techniques. The SERS-based technique demonstrates a novel approach for rapid microbial diagnostics, where SERS can be directly applied on the clinical sample rather than pure cultured bacteria.

data presented in the above study is highly stable, uniform, and reproducible, which shows the versatility of the biosynthesized SERS active substrate. This SERS active substrate is capable of

s and also exhibits high sensitivity (see **Figure 7**). **Figure 7** demonstrates the SERS calibration curve obtained with SERS intensity of the peak at 1330 cm−1 (C–N stretching mode) as a func-

*E. coli* concentration. The SERS intensity increases with the concentration of the bacterial solution, as it is exponentially correlated to the concentration of *E. coli* bacterial cells in the sample

tration, and the standard errors of the mean for each concentration are also shown in **Figure 7**. The major intention of this SERS study using biosynthesized Ag nanoparticles was to develop a rapid fingerprinting method for the characterization of bacteria particularly *E. coli*, which is associated with urinary tract infection (UTI), a common disease among most people of all age

The SERS-based pathogen detection method is especially useful for the analysis of slowgrowing bacteria, which typically may take few weeks during laboratory tests. The SERS spectra described above to detect and quantify bacteria lacks the molecular level specificity

**Figure 6.** The TEM image of the as-synthesized silver nanoparticles along with their UV-visible spectrum, SAED pattern, and the resultant SERS spectra generated upon interaction with bacteria *S. aureus* and *E. coli*. Reproduced with

permission from Ankamwar et al. [28]. Copyright @ Royal Society of Chemistry, Inc.

CFU/ml. Experiments were repeated five times with each bacterial concen-

tion of concentration of bacteria *E. coli*. The 1330 cm−1 peak became detectable at 10<sup>3</sup>

CFU ml−1) of *E. coli* within a very short time of 1–5

CFU/ml of

detecting extremely low concentrations (103

groups in developed countries such as India and China.

between 10<sup>3</sup>

and 108

306 Raman Spectroscopy and Applications

**Figure 7.** The SERS calibration curve obtained with SERS peak area or SERS intensity of the peak at 1330 cm−1 (C–N stretching mode) as a function of concentration of bacteria *E. coli*. Reproduced with permission from Ankamwar et al. [28]. Copyright @ Royal Society of Chemistry, Inc.

Nie and Emory [15] carried out a single-molecule SERS experiment by employing the SERS technique along with the transmission electron microscopy (TEM) and scanning tunneling microscopy (STM) techniques. They observed surface Raman enhancement in the order of 1014–1015 for single rhodamine 6G (R6G) molecule adsorbed on selected Ag nanoparticles. For the single-molecule SERS study, a single event was observed rather than an ensemble averaged value usually attained for traditional SERS measurements. Advancement of the single molecule SERS technique has brought a new aspect in biomedical research, as it can act as a versatile probing tool to investigate various biological molecules such as virus, bacteria, and protein.

The limitation arises out of surface generality of the SERS effect has been resolved by the invention of tip-enhanced Raman spectroscopy (TERS) technique in 2004 [29], which is a modification of the conventional SERS technique. This new and novel technique is derived from the enhancement of the surface Raman scattering intensity (SERS signal) by merging Raman spectroscopy with a scanning probe microscopy technique such as atomic force microscopy (AFM) or scanning tunneling microscopy (STM). The experimental setup in the TERS technique consists of AFM or STM tip placed in a nearby surrounding area of an ultrasmooth substrate, generally single crystal metal surfaces and illuminated by an electromagnetic radiation of suitable wavelength. The contact or tunneling mode of AFM/STM can be used in the experimental setup of TERS. Irradiating with a laser beam of suitable wavelength, a localized surface plasmons are excited in the tip-substrate gap, generating a huge, local enhancement of electromagnetic field in comparison to the incident radiation. TERS was used to probe malachite green isothiocyanate, a dye molecule adsorbed on the Au(111) surface [29]. TERS has been used to study surface reactions on single crystal and smooth surfaces, as surface roughness of the substrate does not play any role in this enhancement.

Surface-enhanced Raman scattering spectroscopy (SERS) can be used for the identification short-live reaction intermediates such as radical and radical ions on the electrode surface and elucidation of the reaction mechanism in general. Tian and his research group at Xiamen University, China, carried out the first *in situ* electrochemical SERS (EC-SERS) investigation on the electrochemical reduction of PhCH<sup>2</sup> Cl in acetonitrile (CH3 CN) on the Ag electrode [30]. The benzyl radical anion as a reaction intermediate and 3-phenylpropanenitrile as the major reaction product were detected from the SERS study for the above surface reaction. The complete reaction mechanism enlightening the adsorption process of PhCH<sup>2</sup> Cl on the Ag surface and all other possible interactions including the solvent molecule have been determined from the systematic SERS study. The SERS results were further validated by quantum mechanical density functional theory (DFT) calculations, which confirm the detection of the reaction intermediate and products.

It was established by Mulvihill et al. that LB assemblies made of various polyhedral Ag nanocrystals can be utilized as high quality SERS active substrates for the high-sensitivity detection of arsenate and arsenite ions in aqueous solutions with a detection limit of 1 ppb [31]. The detection limit resulted from the analysis carried out by the SERS-based technique is an order of magnitude lower than the existing yardstick set by the World Health Organization (WHO). The SERS substrate can be used as a chemical sensor, which is simultaneously highly reproducible and portable, and therefore, this could be easily executed in field detection. The SERS technique can be further employed in environmental analysis. Pesticides, herbicides, pharmaceutical chemicals in water, banned food dyes, aromatic chemicals in regular aqueous solutions and in sea water, chlorophenol derivatives and amino acids, chemical warfare species, explosives, and various organic pollutants [32, 33] can qualitatively and quantitatively analyzed by the SERS-based detection technique. The partition property of SERS substrates as well as surface chemistry facilitates the complete separation of pollutants and analysis of complex environmental samples in real environmental analysis and monitoring.

Immobilized metal nanoparticles in the form of SERS substrates can be used for biomedical diagnostics. For instance, the SERS substrate can be used as a glucose sensor to detect glucose in human blood. Although glucose is most commonly monitored by electrochemicalbased sensors, a substitute protocol using SERS substrates fabricated by the NSL technique has been employed to detect glucose in blood [34]. In this new protocol, the SERS-based glucose sensor was developed by growing silver film over nanospheres (AgFON) surfaces prepared by the NSL technique. Nevertheless, glucose sensing on a bare AgFON surface was not successful and glucose was brought within the range of electromagnetic enhancement of the AgFON surface by formation of a self-assembled monolayer (SAM) on its surface to partition the analyte of interest, in a manner similar to the technique used to generate the stationary phase in high-performance liquid chromatography. Numerous SAMs were studied to partition glucose effectively to the AgFON surface and it was observed that both straight-chain alkanethiols and ethyleneglycol-terminated alkanethiols partitioned glucose most effectively. The key for the detection of glucose by the SERS-based technique was the surface chemistry of alkanethiol molecule on the AgFON surface. The SERS substrate was modified with an alkanethiol partition layer to facilitate the glucose adsorption to the metal surface. Real-time sensing and quantitative detection of glucose in bovine plasma by SERS has been reported in the literature [35].
