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 Chapter 15

The Intricate Nature of SERS: Real‐Life Applications and

Challenges

Nicoleta Elena Dina and Alia Colniță

Additional information is available at the end of the chapter

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

Abstract
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 com-
bines 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. How-
ever, 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.
314 Raman Spectroscopy and Applications

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 elec-
tromagnetic 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 inci-
dent 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 advan-
tages of this technique, such as speed, versatility, and the functionality under ambient condi-
tions 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 (10−30–10−25) 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 sponta-
neous Raman effect is so weak that fluorescence, when it occurs, obscures the Raman spec-
trum. 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 interac-
tion 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 contri-
bution 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 ω4 (where ω = frequency). Therefore, the fall-off in intensity of high
frequency vibrations is also explained; the driving field and scattered field cannot simulta-
neously excite the particle resonance if they are of very different frequencies. This explains also
 The Intricate Nature of SERS: Real‐Life Applications and Challenges 315
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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].
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 spec-
tral characteristics are induced by variation between different colloid batches, colloid concentra-
tion 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 impor-
tant 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 experi-
mental setup (scattering configuration), substrate-related parameters (geometry, adsorption,
orientation with respect to the incident beam direction, and polarization), and is distance-
dependent. 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 continu-
ous effort was made to develop new low-cost and easier synthesis strategies for increasing
316 Raman Spectroscopy and Applications

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]. Further-
more, 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-
 The Intricate Nature of SERS: Real‐Life Applications and Challenges 317
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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.

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
318 Raman Spectroscopy and Applications

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 wave-
length. As an indicator we selected the intensity of the marker band found at 732 cm−1 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 (>103 CFU/ml). For single-cell detection assays, this aspect could make a
difference in selecting the SERS detection platform.

2. Label-free SERS-based assays

The impact on the public health demands sensitive analytical tools for detecting pathogens.
Rapid, culture-free, ultrasensitive pathogens’ detection and identification are of paramount
importance, since there are infections caused by a single microorganism (mycobacteria) and
some pathogens need 20 days to proceed through one division cycle (while some E. coli strains
take only 20 min), making laboratory culture a slow process.

Conventional methods currently used for microorganisms’ identification are nucleic acid-
based polymerase chain reaction (PCR, qPCR, and real-time PCR), on-chip nucleic acid ampli-
fication [26], enzyme-linked immunosorbent assay (ELISA) [27], chemiluminescence-based
microarrays [28, 29], and matrix-assisted laser desorption/ionization (MALDI, MALDI-TOF)
[30, 31]. Major drawbacks of these culture-based detection techniques are the time required,
the high costs, the need of prelabeling, and/or use of antibodies or DNA sequencing, and also
the concerning increased rate of false negatives and false positives. In addition, biosensors for
bacteria detection still rely on the specific capture of the targeted pathogen by using antibodies
[9, 11, 32], aptamers [33], and substrates that contain metallic nanosculptured thin films [34], or
other different complex surface morphologies fabricated by using photolithography combined
with deposition techniques [35]. This approach leads to costly microarrays, which can only be
handled by trained personnel, in laboratory conditions. However, before any of these whole-
organism fingerprint techniques can be used to analyze the samples, the microorganisms must
be cultured in order to isolate the microorganism of interest from other sample constituents
and/or produce sufficient biomass for analysis.
Recently, spectroscopic techniques look more and more promising with the development of
low-cost, label-free, and ultrasensitive detection protocols enabling for the first time to be fast,
specific, and sensitive enough in vital issues as healthcare. Particularly, Raman spectroscopy is
a nonintrusive in situ analysis method, requiring small efforts for sample preparation and can
be easily used outdoors with portable, miniaturized, and even handheld Raman spectrome-
ters. Moreover, when using SERS, the spectral fingerprint reflects the physiological state of a
bacterial cell, e.g., when pathogenic bacteria were cultured under conditions known to affect
virulence, their SERS fingerprints changed significantly. It is also known that bacteria respond
to environmental triggers, such as temperature, pH, and nutrient concentrations, by switching
 The Intricate Nature of SERS: Real‐Life Applications and Challenges 319
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to different physiological changes in their biochemical profile, including the number and
composition of outer membrane proteins, lipopolysaccharides (LPS), and cellular fatty acids
[36].
For SERS detection of bacteria, several innovative approaches are reported. Sengupta and
coworkers [37–39] reported straightforward analysis of a colloidal-bacterial mixture in an
optical glass cuvette. The preferred excitation laser line is 514.5 nm in their study of the pH
influence and the time-dependent behavior of colloidal-bacterial suspensions, even if this
wavelength is too long to resonate with excitations of the aromatic ring breathing mode.
By using the same excitation wavelength, Kahraman et al. [40] developed a uniform bacterial
sample preparation method based on the convective assembly. Aggregation and clustering
was frequently applied for obtaining higher SERS signal from “hot-spots” [41]. Knauer and
others [9, 11, 42] optimized the microarray detection of single-bacterium by using different Ag
sols and aggregation with sodium chloride or sodium azide in low concentrations. However,
in these studies, the 633 nm laser line was selected for SERS-based detection on the antibody-
activated microarray and the substrate used for enhancement was an Ag colloid produced by
using a modified Leopold and Lendl method [43].

Efrima and Zeiri also proposed a novel approach, to use colloid produced in the presence of
the biomass [18, 19]. The authors used the 633 nm laser line as an excitation wavelength,
therefore they were able to report the ring breathing mode band observed at 1004 cm−1 and
assigned to the phenylalanine residue [10]. Excepting Knauer's group work [9, 11, 42] and
recent studies reported by Zhou et al. [12–14, 44], when applying the in situ approach, the
Leopold and Lendl SERS active substrate was not so exploited in the bacteria detection, as the
usage of the 633 nm laser line is mere. The hydroxylamine-reduced silver colloid was shown as
ideal for obtaining SERS structural information on biological molecules contained in the
bacterial cell wall, since it provides a high enhancement factor and shows almost no anomalies
in the spectral band position upon aggregation [45] in comparison to the citrate-reduced Ag
sols.

Another bacteria detection assay reported used crystal violet (CV) as Gram stain [46]: the
procedure involved staining bacterial samples with CV which binds to the peptidoglycan layer
of the Gram-positive and Gram-negative bacteria. Despite the simple and robust methodology
of staining, the detection relies on optical microscopy, which is often susceptible to user-
dependent sampling error. Therefore, by developing magneto-fluorescent NPs, the detection
was improved and was successfully tested for both Gram-positive and Gram-negative bacteria
(E. coli and S. aureus).
Label-free SERS-based detection is a very promising alternative for rapid monitoring real
samples, offering single-cell sensitivity [1, 47], providing spectra with no contribution from
the aqueous environment (prominent in the biological samples), and a high precision classifi-
cation of bacteria, at strain level [1–3]. Recently, innovative approaches for the rapid SERS
label-free detection of bacteria were developed:
(i). Simple, receptor-free immobilization of bacteria on the glass surface [14]. Mircescu et al.,
based on molecular-specific SERS spectra of uropathogens at single-cell level, discriminated
320 Raman Spectroscopy and Applications

between rough and smooth strains of E. coli and P. mirabilis. The innovative and effective
principle of bacteria immobilization through electrostatic forces, by inducing a positive charge
on the silanized microarray surface was demonstrated for Gram-negative bacteria. In addition,
the monitoring of single-cell SERS spectra of bacteria in different growth phases was assessed.
(ii). In situ silver NPs preparation in the presence of bacteria (Bacteria@ Ag NPs) [12]. Zhou
et al. developed the in situ Ag colloid synthesis in two steps, resulting in coating the bacterial
cell wall with a silver SERS-active layer. The assay requires about 10 min and only one sample
droplet of 3 µl. By using this novel strategy, SERS detection (about 30-fold higher enhanced
SERS signal) and hierarchy cluster analysis (HCA) discrimination of three strains of E. coli and
one strain of S. epidermidis was reported.

2.1. In situ Ag NPs synthesis: extended approach

Currently and also in the future, biosensors with integrated nanotechnology promise to
address the analytical needs in practical pathogen diagnosis. Recently, comprehensive reviews
concerning the bacteria detection by using Raman and SERS spectroscopies were reported [15,
48, 49]. The increased sensitivity and high information content of SERS is acknowledged,
mostly when this powerful tool is used in conjunction with advanced analysis and classifica-
tion techniques. The key advantages that SERS-based biosensors include are the easy-to-use
detection platforms and reduced testing time resulting in immediate diagnostic (within 5–15
min) [50, 51], superior sensitivity and multiplex capability [52], reduced sample volume, and
high sensitivity and specificity. Thus, a great deal of research has been invested into the
development of SERS-based biosensors for pathogenic microorganisms.
For instance, SERS mapping by using Ag dendrites [53] as SERS active substrate, both for
Gram-negative and Gram-positive bacteria was reported. Not so promising results were
obtained in case of the Gram-positive bacteria, probably due to their different membrane
structure, containing less outside proteins. Usually, the marker bands used for detection of
the pathogens are either the 1332 cm−1 band assigned to the CH deformations in proteins [53],
either the 730 cm−1 band assigned to adenine [14, 54].
In this section we will mainly focus on the latest studies involving in situ Ag NPs synthesis
approach for SERS detection in biomedical applications. In a more recent study, Zhou et al. [13]
applied the Bacteria@ Ag NPs approach for live and dead bacteria counting/discrimination.

Figure 3. Scheme describing the in situ AgNPs synthesis on the bacterial cell wall.
 The Intricate Nature of SERS: Real‐Life Applications and Challenges 321
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Moreover, by using antibiotics (ampicillin, polymyxin B, and chloramphenicol) with different

action mechanisms on bacteria and by monitoring the SERS signal decay in time, they were
able to determine which microorganism is or not developing a drug resistance in less than 4 h.
Lately, the same group [44] completed the previous work by applying the Bacteria@ Ag NPs
approach on a microarray (containing antibodies). Figure 3 exhibits the schematic protocol of
generating the Bacteria@ Ag NPs for SERS detection of microorganisms at single-cell level.
Since Efrima's group reported on producing in situ NPs through external (bacterial cell-wall) or
internal (interior components mode) synthesis on bacteria [18], the use of in situ synthesized
Ag colloids for bacteria detection was demonstrated in several assays only by Haisch's group
[12–14, 44, 54].
Recently, a label-free NIR-SERS detection and discrimination of bacteria after pretreatment of
bacterial cell membrane with disrupting agents was presented, featuring a sensitivity down to
103 CFU/ml and a measuring time of less than 5 min [55]. Latest studies underline the applica-
bility of the in situ synthesized Ag colloids also in environmental research, for instance, for the
detection of bacteria in plant roots [56] and for pesticide monitoring in spinach leaves [57].
These results demonstrate the applicability of SERS-based noninvasive detection approaches
for identification and characterization of pathogens and their secreted metabolites. The in situ
synthesis of Ag colloid ensures the structural integrity of the coated bacterial cells and thus
helps to rapidly generate spectral bacterial signatures with high sensitivity and specificity.
Figure 4 contains a collection of microscopic images illustrating the reproducible coverage of
E. coli cells with a silver layer by using the in situ synthesis approach.

Figure 4. Microscopic 100× images showing the Ag NPs coverage of bacteria (E. coli) when using the in situ synthesis
approach (Bacteria@ Ag NPs).
322 Raman Spectroscopy and Applications

The influence at strain level of the O-antigen presence was already demonstrated by using
unspecific surface chemistry as means of bacteria adsorption and the in situ synthesis approach
[14]. O-antigen is the terminal structural part of the Gram-negative bacterial outer membrane
that contains the significant variation between virulent strains and lab-designed strains. The
differences in the composition of the monosaccharide units and sugar linkages translate into
strain discrimination, by using molecular serotyping tests and chemometric analysis. Consid-
ering all this, O-antigen is the most variable cell constituent and of great importance when
dealing with bacteria identification at strain level.

Raw SERS spectra collected from single cells of four different strains of E. coli are shown in
Figure 5. For the accurate detection and discrimination between these strains, one has to make
sure that all experimental conditions are standardized for each measurement. In this context,
we established a constant timeline in the preparation steps of the samples and we used constant
experimental parameters in acquiring the SERS spectra. The used E. coli strains are rough (K12)–
MG1655, TOP 10 (Figure 5A and C) and smooth strains (B2)-536, UTI89 (Figure 5B and D).

Figure 5. Raw SERS spectra of rough (A and C) and smooth (B and D) E. coli strains collected by using the in situ
synthesis approach (Bacteria@ Ag NPs).
 The Intricate Nature of SERS: Real‐Life Applications and Challenges 323
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Specific SERS bands in each case (with or without the O-antigen) are discussed in our previous
study [14], where a clear discrimination between K12 and B2 strains is demonstrated by using
chemometrics (principal component analysis, PCA).

In the last decades, SERS was used to identify: DNA bases [58], a wide range of explosives and
trace materials [59], food additives [60], therapeutic agents [61], different species of pathogenic
and nonpathogenic bacteria [62–64], protozoa [65], fungi [66, 67], and their spores [68], respec-
tively. Furthermore, as previously described, vibrational spectroscopy can be used to study the
uniqueness of microorganisms. Consequently, we envision that the in situ synthesis approach
could be used to some extent in other microorganisms’ detection as well, not only bacteria.

Particularly, Raman and SERS spectroscopies were already applied in the detection, character-
ization, and monitoring of growth cycle for fungi. For example, various pathogens such as
Candida albicans, C. glabrata, and C. tropicalis isolated from blood cultures have been rapidly
identified [66]. Rapid diagnosis of infections caused by fungi from Candida genus is extremely
important, since intra-abdominal infections caused by these fungi lead to high mortality rates
[67]. Yang and Irudayaraj [69] were able to easily identify A. niger and F. verticillioides patho-
genic fungi from apple surface, using FT-Raman spectroscopy. Szeghalmi and coworkers [70]
studied the growth of A. nidulans strain A28 hyphae over the Au-coated Klarite SERS sub-
strate, and they detected a strong signal in the close proximity of the hyphal cell wall because
of the excretion of some extracellular components during growth.

Another field of interest in fungi studies using Raman spectroscopy and SERS is the character-
ization of various bioactive compounds extracted from different fungi. De Oliveira and
coworkers [71] successfully identified the chemical composition of the extracts obtained from
P. sanguineus fungus. The major bioactive components of P. sanguineus extracts are ergosterol
and cinnabarin, the last one being responsible for the antibiotic activity of the extract [72].

Zinc oxide nanoparticles (ZnO NPs) were tested for their antifungal activity against B. cinerea
and P. expansum fungi. He and coworkers [73] used traditional microbiological plating, along
with SEM and Raman spectroscopy and showed that a concentration higher than 3 mmol/l of
ZnO NPs can significantly inhibit the growth of B. cinerea and P. expansum. The last one is more
sensitive to the action of ZnO NPs because these inhibit the development of conidiophores and
conidia, resulting in the death of fungal hyphae. The detection of fungal infection in mice lungs
with P. brasiliensis and follow-up treatment with magnetic NPs functionalized with
amphotericin B can be achieved using SERS analysis [74].

SERS imaging and analysis have been effectively used for the characterization of in vitro
biosynthesis of NPs by different species of fungi. In this regard, Mukherjee and coworkers
[75] established a controlled biosynthetic route to obtain the nanocrystalline Ag particles using
T. asperellum. Using TEM and XRD, the obtained Ag NPs were found to be in a range of 13–18
nm. C. cladosporioides was also reported to be able of Ag NPs extracellular biosynthesis [76]. In
fact, fungi are not only able to biosynthesize Ag NPs, but also Au NPs. Extract from the
filamentous fungi A. nidulans was used in the formation of Au NPs within and adjacent to
hyphae. Also, the Neurospora crassa extract was tested by Quester and his coworkers [77] for
the formation of Au NPs under different experimental conditions. The authors were able to
324 Raman Spectroscopy and Applications

synthesize Au NPs with different shapes and sizes ranging from 3 to 200 nm by using
methylene blue as target molecule.
Concluding this chapter, it is a challenge to entrench how to use most effectively the SERS
effect in our favor. The simple reasoning is that SERS is still a not fully understood phenome-
non. However, the ongoing studies in the biomedical area show the huge potential of this
ultrasensitive technique to actually improve our life quality and the diagnosis procedures of
infections and to significantly prevail essential real-life issues. Apart from infections diagnos-
tics, cancer treatment or imaging, drug delivery, and personalized medicine or other health
care branches can greatly benefit from Raman/SERS detection and mapping in synergy with
functionalized NPs and high-performance support vector machines.

Acknowledgements

This chapter could not be written to its fullest without Dr. Nicolae Leopold, Prof. Dr. Vasile
Chis (Biomolecular Physics Department, Babes-Bolyai University, Cluj-Napoca, Romania), and
Dr. Christoph Haisch (Analytical Chemistry Chair, Technische Universität München, Ger-
many) who served as my supervisors, as well as partners who challenged and encouraged
me throughout my time spent as a PhD student. They would have never accepted anything
less than my best efforts, and for that, I sincerely thank them. This work was supported by a
grant of the Romanian National Authority for Scientific Research and Innovation, CNCS –
UEFISCDI, project number PN-II-RU TE-2014-4-0862.

Author details

Nicoleta Elena Dina* and Alia Colniță

*Address all correspondence to: [email protected]

National Institute of R & D of Isotopic and Molecular Technologies, Donat, Cluj-Napoca,

Romania

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