**The Intricate Nature of SERS: Real‐Life Applications and Challenges** Provisional chapter The Intricate Nature of SERS: Real-Life Applications

Nicoleta Elena Dina and Alia Colniță

Nicoleta Elena Dina and Alia Colniță

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65478

and Challenges

#### Abstract

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Testing for the presence of microorganisms in biological samples in order to diagnose infections is very common at all levels of health care. There is a growing need to ensure appropriate diagnosis by also minimizing the analysis time, both being very important concerns related to the risk of developing an antimicrobial resistance. Moreover, there are important medical and financial implications associated with infections. In this chapter, we will discuss the latest ultrasensitive and selective, but simple, rapid and inexpensive bacteria detection and identification methods by using receptor-free and innovative immobilization principles of the biomass. Raman spectroscopy, which combines the selectivity of the method with the sensitivity of the surface-enhanced Raman scattering (SERS) effect, is used in correlation with chemometric techniques in order to develop biosensors for pathogenic microorganisms.

Keywords: surface-enhanced Raman scattering (SERS), single-cell detection, label-free, principal component analysis (PCA), biosensors

#### 1. Introduction

Lately, the pathogens can be individually identified by using surface-enhanced Raman scattering (SERS), without the need of labeling or specific receptor usage like antibodies, for instance. Colloidal metallic suspensions offer the advantage of ambient conditions, fast completion, and minimal number of reactants, being economical, and resulting in a ready-to-use product. However, despite the progress achieved, concerns and problems with the preparation of metal nanoparticles (NPs) remain, such as the byproducts from the reducing agent, the multiple steps

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

often required, and the high concentration of protective agents. Furthermore, it has been a major bottleneck to elucidate the key factors (other than surface roughness enhanced electromagnetic fields) that play important roles in the SERS process of adsorbed biomolecules. The understanding of the mechanisms involved in the interaction of biological systems with inorganic materials is of interest in both fundamental and applied disciplines. Herein, the decisive know-how in investigating biological samples by using several SERS-active platforms will be described.

#### 1.1. SERS effect

Raman spectroscopy requires the illumination of a sample with monochromatic light. The inelastic scattering of a small fraction (approximately one in a million) of the incident photons toward lower (Stokes scattering) or higher frequencies (anti-Stokes scattering) than the incident light is known as Raman scattering. A typical Raman spectrum plots the intensity of the scattered light versus the number of probed molecules. There are several noteworthy advantages of this technique, such as speed, versatility, and the functionality under ambient conditions in nonspecific environments (by using portable, miniaturized spectrometers); the simplicity of sample preparation; the possibility of remote detection of Raman signals by using optical fiber probes; the chance to examine transparent samples; and the obliviousness to water, ubiquitous element for biological samples. Probably the biggest disadvantage lies in the extremely small cross section, typically (10−30–10<sup>−</sup>25) cm2 /molecule, which can be translated into long acquisition times and considerable high sample concentrations.

Raman spectra can be used for the identification and classification of microorganisms once a procedure with good reproducibility and reliability is established [1–4]. However, the spontaneous Raman effect is so weak that fluorescence, when it occurs, obscures the Raman spectrum. SERS represents the enhancement of Raman-active vibrations associated with the intimate contact (within few nanometers) to a surface covered with plasmonic NPs. Moreover, additional modes not found in the traditional Raman spectrum can be present in the SERS spectrum, while other modes can disappear.

The surface-selection rules that apply to infrared and Raman spectroscopies are extended for surface-enhanced vibrational spectroscopy (SEVS) by taking into account the local field and/or the roughness of the surface. SEVS spectra are the expression of the analyte-radiation interaction when the molecule is in the close proximity or adsorbed on the metallic nanostructure, which supports the surface plasmons [5]. So the presence of the plasmon resonance, for instance, will define the observed spectral intensities. When electromagnetic radiation with the same frequency is incident upon the nanostructure, the electric field of the radiation drives the conduction electrons into collective oscillation. Electromagnetic enhancement, the major contribution in the SERS effect, relies on the Raman-active molecules being confined within large electromagnetic fields (EFs), generated by the excitation of the local surface plasmon resonance (LSPR). So the extreme sensitivity of SERS to small increases in the local field is easily seen since it scales roughly as ω<sup>4</sup> (where ω = frequency). Therefore, the fall-off in intensity of high frequency vibrations is also explained; the driving field and scattered field cannot simultaneously excite the particle resonance if they are of very different frequencies. This explains also the different excitation profiles for different bands; maxima for higher frequency vibrations occur at shorter wavelengths, as the scattered field is brought closer to resonance [6].

often required, and the high concentration of protective agents. Furthermore, it has been a major bottleneck to elucidate the key factors (other than surface roughness enhanced electromagnetic fields) that play important roles in the SERS process of adsorbed biomolecules. The understanding of the mechanisms involved in the interaction of biological systems with inorganic materials is of interest in both fundamental and applied disciplines. Herein, the decisive know-how in investigating biological samples by using several SERS-active platforms

Raman spectroscopy requires the illumination of a sample with monochromatic light. The inelastic scattering of a small fraction (approximately one in a million) of the incident photons toward lower (Stokes scattering) or higher frequencies (anti-Stokes scattering) than the incident light is known as Raman scattering. A typical Raman spectrum plots the intensity of the scattered light versus the number of probed molecules. There are several noteworthy advantages of this technique, such as speed, versatility, and the functionality under ambient conditions in nonspecific environments (by using portable, miniaturized spectrometers); the simplicity of sample preparation; the possibility of remote detection of Raman signals by using optical fiber probes; the chance to examine transparent samples; and the obliviousness to water, ubiquitous element for biological samples. Probably the biggest disadvantage lies in

Raman spectra can be used for the identification and classification of microorganisms once a procedure with good reproducibility and reliability is established [1–4]. However, the spontaneous Raman effect is so weak that fluorescence, when it occurs, obscures the Raman spectrum. SERS represents the enhancement of Raman-active vibrations associated with the intimate contact (within few nanometers) to a surface covered with plasmonic NPs. Moreover, additional modes not found in the traditional Raman spectrum can be present in the SERS

The surface-selection rules that apply to infrared and Raman spectroscopies are extended for surface-enhanced vibrational spectroscopy (SEVS) by taking into account the local field and/or the roughness of the surface. SEVS spectra are the expression of the analyte-radiation interaction when the molecule is in the close proximity or adsorbed on the metallic nanostructure, which supports the surface plasmons [5]. So the presence of the plasmon resonance, for instance, will define the observed spectral intensities. When electromagnetic radiation with the same frequency is incident upon the nanostructure, the electric field of the radiation drives the conduction electrons into collective oscillation. Electromagnetic enhancement, the major contribution in the SERS effect, relies on the Raman-active molecules being confined within large electromagnetic fields (EFs), generated by the excitation of the local surface plasmon resonance (LSPR). So the extreme sensitivity of SERS to small increases in the local field is easily seen since it scales roughly as ω<sup>4</sup> (where ω = frequency). Therefore, the fall-off in intensity of high frequency vibrations is also explained; the driving field and scattered field cannot simultaneously excite the particle resonance if they are of very different frequencies. This explains also

/molecule, which can be translated

the extremely small cross section, typically (10−30–10<sup>−</sup>25) cm2

spectrum, while other modes can disappear.

into long acquisition times and considerable high sample concentrations.

will be described.

314 Raman Spectroscopy and Applications

1.1. SERS effect

SERS represents a relatively inexpensive alternative, compared to the conventional detection methods that also meet the clinical tools' requirements: simplicity, reliability, uniformity (for testing various pathogens), and high specificity. It completely overcomes the shortcoming of Raman small cross sections. SERS is capable to characterize [7–10], identify [11, 12], and differentiate [13, 14] pathogenic microorganisms in synergy with chemometrics, based on the biochemical, chemical, and their structural properties.

Even though SERS is a highly specific and sensitive detection method, well suited for biological issues, SERS measurements still suffer from low reproducibility of spectra. Fluctuations of spectral characteristics are induced by variation between different colloid batches, colloid concentration dependence, and inconsistent enhancement even within one colloid batch mainly due to an inhomogeneous and a rather uncontrollable aggregation of NPs [15]. The main issues consist in the difficulty to generate uniform distributed EFs, large EFs occurring only at localized positions (hot-spots) and the polydispersity of colloidal clusters. As Nie and coworkers [16, 17] have already quite convincingly demonstrated, the enhancement factor depends on the wavelength of exciting radiation, or rather on the relation between the wavelength and the size of the Ag NPs.

Still, for real-world applications, reproducibility is considered in particular cases more important than enhancement factors. Background signal from the food and environmental matrices represents a real challenge. In addition, proper and simplified sample pretreatment is needed before conducting a SERS measurement. For instance, sample preparation for SERS detection of bacteria is quite inconsistent referring to colloids as SERS-active substrates. The NPs can be either coated on the outside of the bacterial cell wall or directed to the interior of the bacterial cells. Whereas the first preparation results in spectral information mainly derived from cell wall components, the second one contains additional cytoplasmic information [18, 19]. Figure 1 shows the SERS signal acquisition process from a microbiologic sample, when the silver coverage of the bacteria (in blue) is successful.

Conclusively, the SERS effect depends on a wide range of parameters, such as the particular features of the laser excitation (wavelength, polarization, and angle of incidence), the experimental setup (scattering configuration), substrate-related parameters (geometry, adsorption, orientation with respect to the incident beam direction, and polarization), and is distancedependent. However, readiness remains an important parameter in choosing the suitable, fast, and reliable tool for detection at trace level, for large-scale applications.

#### 1.2. Gold or silver NPs in biomedical applications?

Gold NPs (Au NPs) are promising SERS candidates in biomedicine and have already been successfully tested for various biomedical applications. They are easy to prepare, significantly more stable than other metallic NPs (not easily oxidized), and are highly biocompatible. They can act as artificial antibodies due to their simple surface chemistry, precise binding affinity, and possibility of tuning by varying the density of ligands on their surfaces. Lately, a continuous effort was made to develop new low-cost and easier synthesis strategies for increasing

Figure 1. SERS signal acquisition from bacteria while irradiated with the laser light in backscattering configuration.

their cellular biocompatibility, by varying their geometries, their physical dimensions, and functionality. The mixing rate of the reactants could greatly influence the physical properties of the Au NPs, their stability over long periods of time, and their SERS sensitivity. It is reported that when the gold salt solution is rapidly added to the reaction mixture, preponderant spherical short- and long-chain polyethylene glycol (PEG) Au NPs with a mean diameter of 15 nm are obtained, whereas a drop-wise addition of the gold salt leads to a seeding effect and to Au NPs with a mean diameter of 60 nm [20]. The most common surface ligand used in biomedical applications is thiolated PEG (PEG-SH), which ensures the desired hydrophilicity and increased circulatory half-life in vivo systems [21]. Proteins, such as bovine serum albumin or collagen, can also serve as capping and stabilizing agents in the one-step synthesis of gold colloidal nanoassemblies and spherical Au NPs with tunable shape and size [22, 23]. Furthermore, the in vitro uptake and toxicity effect of Au NPs grown with a native collagen shell exhibit a lower toxic effect on cervical carcinoma and lung adenocarcinoma cells than synthetic polymer-coated Au NPs [24]. Additionally, due to their ability to efficiently convert light into heat, gold NPs can specifically allow thermal ablation of the targeted biological region and by absorbing high amounts of X-ray radiation become enhancers in cancer radiation therapy or computed tomography [21].

However, silver NPs (Ag NPs) show stronger plasmon fields than Au NPs due to the simple fact that their plasmon band does not overlap with the interband electronic transitions, as in the case of Au NPs [25]. Figure 2 presents our recent results obtained by using different SERS-

Figure 2. SERS spectra of E. coli by using five distinct platforms of detection: AgPEG200NPs/AgPEG8000NPs—silver NPs with PEG200/PEG8000 layer, AuPEG200NPs/AuPEG8000NPs—gold NPs with PEG200/PEG8000 layer, and Ag Hya NPs– in situ silver synthesized NPs by reduction with hydroxylamine hydrochloride.

their cellular biocompatibility, by varying their geometries, their physical dimensions, and functionality. The mixing rate of the reactants could greatly influence the physical properties of the Au NPs, their stability over long periods of time, and their SERS sensitivity. It is reported that when the gold salt solution is rapidly added to the reaction mixture, preponderant spherical short- and long-chain polyethylene glycol (PEG) Au NPs with a mean diameter of 15 nm are obtained, whereas a drop-wise addition of the gold salt leads to a seeding effect and to Au NPs with a mean diameter of 60 nm [20]. The most common surface ligand used in biomedical applications is thiolated PEG (PEG-SH), which ensures the desired hydrophilicity and increased circulatory half-life in vivo systems [21]. Proteins, such as bovine serum albumin or collagen, can also serve as capping and stabilizing agents in the one-step synthesis of gold colloidal nanoassemblies and spherical Au NPs with tunable shape and size [22, 23]. Furthermore, the in vitro uptake and toxicity effect of Au NPs grown with a native collagen shell exhibit a lower toxic effect on cervical carcinoma and lung adenocarcinoma cells than synthetic polymer-coated Au NPs [24]. Additionally, due to their ability to efficiently convert light into heat, gold NPs can specifically allow thermal ablation of the targeted biological region and by absorbing high amounts of X-ray radiation become enhancers in cancer radiation therapy or

Figure 1. SERS signal acquisition from bacteria while irradiated with the laser light in backscattering configuration.

However, silver NPs (Ag NPs) show stronger plasmon fields than Au NPs due to the simple fact that their plasmon band does not overlap with the interband electronic transitions, as in the case of Au NPs [25]. Figure 2 presents our recent results obtained by using different SERS-

computed tomography [21].

316 Raman Spectroscopy and Applications

active substrates for the detection of E. coli. We synthesized several types of Ag and Au sols by using PEG with two chain lengths as reducing agent [20] and compared the obtained SERS signals with the in situ synthesized Ag NPs SERS signal of bacteria. The SERS spectra were recorded using a Raman microscope (Lab RAM HR, HORIBA Jobin Yvon, Japan). The 633-nm line of a HeNe laser was used as the excitation source. The excitation wavelength dependency of the SERS enhancement can be explained by the optical absorption of the silver colloidal suspensions. We already characterized the herein used NPs in our previous work; the Ag Hya NPs particles feature a plasmon resonance band of around 402 nm. Hence, excitation with the 532 nm laser line is favorable as compared to longer wavelengths (633 nm), being closer to the plasmonic band. However, in the case of the Au PEG NPs the plasmon resonance band is specific to gold and was determined around 540–560 nm, depending on the particles'diameter. Generally, we waited 30 min as incubation time in order to obtain the higher and more stable SERS signal from the irradiated sample. As an effect of incubation time we observed an increasing agglomeration of the SERS-active colloids, leading to a coupling of the plasmon resonances, which again leads to a red-shift of the main absorption. In consequence, we achieved in these earlier works the best SERS performance with the 633 nm excitation wavelength. As an indicator we selected the intensity of the marker band found at 732 cm−<sup>1</sup> which was about fivefold higher for the in situ approach. Even when we preconcentrated the Au PEG NPs by centrifugation (38× more concentrated Au colloidal suspension), the enhancement factor was not comparable with the one obtained for the hydroxylamine reduced in situ Ag colloid [12]. Moreover, the single-cell detection of bacteria was successfully obtained by using the in situ method, while the other tested colloids enabled us to detect bacteria only in rather high concentrations (>10<sup>3</sup> CFU/ml). For single-cell detection assays, this aspect could make a difference in selecting the SERS detection platform.
