DOI 10.
1515/nanoph-2013-0024      Nanophotonics 2014; 3(6): 383–411
Review article
Dana Ciallaa, Sibyll Polloka, Carolin Steinbrücker, Karina Weber and Jürgen Popp*
SERS-based detection of biomolecules
Abstract: In order to detect biomolecules, different
approaches using for instance biological, spectroscopic
or imaging techniques are established. Due to the broad
variety of these methods, this review is focused on surface
enhanced Raman spectroscopy (SERS) as an analytical
tool in biomolecule detection. Here, the molecular speci-
ficity of Raman spectroscopy is combined with metallic
nanoparticles as sensor platform, which enhances the sig-
nal intensity by several orders of magnitude. Within this
article, the characterization of diverse biomolecules by
means of SERS is explained and moreover current applica-
tion fields are presented. The SERS intensity and as a con-
sequence thereof the reliable detection of the biomolecule
of interest is effected by distance, orientation and affinity
of the molecule towards the metal surface. Furthermore,
the great capability of the SERS technique for cutting-edge
applications like pathogen detection and cancer diagnosis
is highlighted. We wish to motivate by this comprehensive
and critical summary researchers from various scientific
background to create their own ideas and schemes for a
SERS-based detection and analysis of biomolecules.
Keywords: biomolecules; Raman; SERS.
a
The authors contributed equally to this manuscript.
*Corresponding author: Jürgen Popp, Institute of Photonic
Technology (IPHT) Jena, Albert-Einstein-Straße 9, 07745 Jena,
Germany; and Institute of Physical Chemistry and Abbe Center
of Photonics, Friedrich-Schiller-University Jena, Helmholtzweg 4,
07743 Jena, Germany, e-mail: juergen.popp@uni-jena.de
Dana Cialla, Sibyll Pollok, Carolin Steinbrücker and Karina Weber:
Institute of Photonic Technology (IPHT) Jena, Albert-Einstein-
Straße 9, 07745 Jena, Germany; and Institute of Physical Chemistry
and Abbe Center of Photonics, Friedrich-Schiller-University Jena,
Helmholtzweg 4, 07743 Jena, Germany
Edited by Lev T. Perelman
1 Introduction
The detection of biomolecules is essential in various ana-
lytical, medical, biochemical and pharmaceutical appli-
cation fields. Since the term “biomolecule” is defined as
a chemical substance generated by a living organism and
© 2014 Science Wise Publishing & De Gruyter
includes amongst others nucleic acids, peptides/proteins
and their building blocks, a variety of reliable detection
schemes are established. In most cases, immunological
or molecular biological methods such as enzyme-linked
immunosorbent assay (ELISA) or polymerase chain reac-
tion (PCR) are applied to detect peptides and proteins as
well as to amplify nucleic acids for a reliable, sequence-
specific detection, respectively [1, 2]. Moreover, optical
methods like fluorescence microscopy [3–5], UV-VIS
absorption [6] and vibrational spectroscopy (IR absorp-
tion, Raman spectroscopy) [7–9] are intensively used for
bioanalytical purposes. Since every method needs a pro-
found discussion about the capability of the detection
strategy as well as presenting the current cutting-edge
applications, this review focusses on surface enhanced
Raman spectroscopy (SERS).
Nanostructured metal surfaces are the key element,
which allow enhancing of the inherent weak Raman
signals of different molecules. Thus, this technique com-
bines the fingerprint specificity of the Raman method with
an increased sensitivity by several orders of magnitude. A
detailed description of the SERS method is found within
the next chapter. When analyzing the SERS application
fields based on the published items each year, it became
evident, that SERS is preferentially applied in bioanalyt-
ics. This technique is used in recent years for studying
not only the building blocks of life (nucleobases, amino
acids) but also complex functional structures (nucleic
acids and proteins) [10, 11]. Furthermore, SERS is an
attractive tool for the investigation and characterization of
whole prokaryotic cells, such as bacteria, bacterial spores,
biofilm, viruses or even eukaryotic cells and tissues [12–
14]. Also the use of SERS in biomedical and pharmaceuti-
cal research is a growing field in the last years [15, 16].
In order to investigate catalytic processes on metal
surfaces like silver, gold or copper SERS is an excellent
method due to the efficient enhancement of Raman modes
involving in the rearrangement of atomic bonds directly
at the surface [17]. Moreover, the detection of single mol-
ecules shows the great potential of this technique in sen-
sitivity [18, 19]. To realize an increase of spatial resolution
of SERS down to the nanometer scale, the technique is
combined with scanning probe microscopy creating tip
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384      D. Cialla et al.: SERS-based detection of biomolecules
enhanced Raman spectroscopy [20, 21]. As an example,
the TERS technique is applied to investigate inorganic
samples like metalloid nanorods [22] or carbon nanotube
composites [23], as well as biological samples such as
insulin fibrils [24].
This review summarizes novel findings and trends in
terms of biomolecules investigated by SERS, their charac-
terization and detection introducing cutting-edge SERS-
based application fields from the last 5 years.
2 S
 urface-enhanced Raman
spectroscopy: a brief introduction
The optical properties of metallic nanoparticles were
already used centuries ago, even before a detailed under-
standing of the nanoscale was developed. Using the
example of the Late Roman Lycurgus Cup, gold nano-
particles were applied to color glass [25]. This phenom-
enon can be understood based on the interaction of
light with metallic nanoparticles, whereby the electron
cloud is oscillating with a frequency known as localized
surface plasmon resonance [26]. The spectral position of
the plasmon resonance is defined by the size, shape and
material of the nanoparticle as well as the surrounding
medium [27, 28] and is associated with the fulfillment of the
plasmon resonance condition (Re(εi)≈-2εa; Im(εi) < < 1; εi –
dielectric constant of the metal; εa – dielectric constant of
the surrounding medium) [29]. Finally a strong evanes-
cent electromagnetic field is generated on the metallic
surface of the nanoparticle [26]. Based on this strong field
enhancement, various applications in ultrasensitive (bio)
analytics are feasible.
To detect and investigate substances contactless
with a molecular specificity combined with a simple
sample preparation protocol, Raman spectroscopy is
the method of choice in various application fields [8,
30–33]. Raman spectroscopy detects the vibrational
modes of functional groups of molecules. This means
that a Raman spectrum is dominated by group frequen-
cies at distinct spectral positions and is therefore a
molecular specific analytical tool. However, to detect
analyt molecules in low concentrations, the application
of Raman spectroscopy is often hindered by the inherent
weak Raman intensity. Consequently, the effective field
enhancement on the surface of metallic nanostructures
is employed for the amplification of the Raman inten-
sity of molecules located in close vicinity to the meta­llic
surface (surface-enhanced Raman spectroscopy –
SERS) [34–40]. Due to its high sensitivity based on the
exploiting of the electromagnetic field enhancement on
metal surfaces and fingerprint specificity from Raman
spectroscopy, the SERS technique is proven as powerful
method in chemical and biochemical applications [12,
41–44].
The SERS effect was first observed by investigating
traces of pyridine adsorbed on roughened silver elec-
trodes [45–47]. In order to explain the SERS enhancement
two main mechanisms are discussed – the electromag-
netic and the chemical enhancement, the electromag-
netic being the main contribution with a maximum
contribution to the enhancement factor in the order of
1011 [48]. When the excitation wavelength is resonant
to the plasmon absorption profile of the nanoparti-
cles, localized surface plasmon modes are excited and
a strong evanescent electromagnetic field is induced
on the metallic surface [29]. For a molecule, which is in
close vicinity to the metallic surface, the Raman modes
get efficiently enhanced because the Raman intensity is
proportional to the squared local electromagnetic field
intensity. Due to the characteristics of a Hertzian dipole
the Raman-scattered light is – summarized for all orien-
tations of the molecule – radiated in all spatial directions
and finally collected by a microscope objective. Moreover,
the Raman-scattered light undergoes a further enhance-
ment when the Raman mode overlaps with the plasmonic
profile. This effect is described in the literature as emis-
sion enhancement or second electromagnetic mecha-
nism [49–51]. The chemical mechanism is understood
as sum of different contributions: (1) a signal enhance-
ment based on chemical interactions between molecule
and nanoparticle in the ground state which are not based
on an excitation within the system, (2) a SERS enhance-
ment due to the resonant excitation of a charge transfer
between nanoparticle and molecule, and (3) a resonance
Raman enhancement due to the excitation of an elec-
tronic transition within the molecule [52]. One phenom-
enon in SERS spectroscopy called field gradient Raman
effect is the detection of Raman modes under SERS condi-
tions which are forbidden according to the spectroscopic
selection rules, [53, 54]. Furthermore, variations in Raman
intensity of different modes within the SERS spectrum are
explainable by means of surface selection rules [55–57].
A molecule is characterized by its Raman tensor and ori-
entation relative to the metallic surface. The enhance-
ment of various Raman-active modes is dependent on
the field components parallel and perpendicular to the
surface. Only vibrations with a dynamic dipole normal
to the surface are observed. In general, Raman modes
with an orientation parallel to the surface are not or only
with very weak signal intensity detectable. In summary,
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based on the discussed properties of the SERS mecha-
nism several consequences for the reliable detection of
molecules are expected. First, due to the range of the eva-
nescent field on the surface of metallic nanostructures,
the Raman signature is enhanced only in close vicinity up
to roughly 10 nm by several orders of magnitude. Thus,
the SERS response of large molecules like peptide and
protein structures are dominated by the parts which are
orientated very close to the metallic surface. Second, as
already mentioned the orientation of a molecule relative
to the metallic surface plays an important role in SERS,
due to the surface selection rules. As a consequence,
Raman modes perpendicular to the surface are preferably
enhanced and thus, are dominating the SERS spectrum of
the investigated biomolecules. Finally, since sulfur, nitro-
gen and oxygen containing chemical groups show a high
affinity to silver and gold surface, these moieties bind
preferably to the metal. Accordingly, vibrational modes
of these surface-bound chemical groups are dominating
the spectral response.
The development, preparation and characterization
of SERS-active substrates are one of the most prominent
research areas in SERS [25, 35, 36, 58]. Since thus is out
of scope for this review article, this research field is dis-
cussed very briefly. Since the first observation of the
SERS effect was done by investigating pyridine adsorbed
on roughened silver electrodes [45–47, 59], historically
the oldest SERS substrates are represented by roughened
metal electrodes with nanostructured features gener-
ated by oxidation-reduction-cycles [60]. Today, metal-
lic electrodes and their application as SERS substrate
are of less importance. Most commonly, colloidal silver
or gold nanoparticles are used for SERS-based investi-
gations. Due to their simple and cost-efficient prepara-
tion protocol based on adding a reduction agent to an
aqueous solution of, e.g., silver nitrate or chloroauric
acid, colloidal metal nanoparticle suspensions are the
most common used SERS substrate [58, 60, 61]. For the
detection of single molecules by means of SERS, aggre-
gated silver nanoparticles are used due to their excel-
lent signal enhancement properties [36, 62, 63]. A third
group of SERS substrates contains all plasmonic surfaces
with randomly distributed or regular arranged metallic
nanostructures. These are prepared by the deposition of
metallic nanoparticles on a substrate, by the evapora-
tion of pre-structured surfaces or by the application of
lithographic methods [25, 35, 58, 60]. All here described
SERS substrates consist mainly of silver, gold and copper
structures, since for these materials the plasmon reso-
nance condition is fulfilled within the visible and NIR
spectral region.
D. Cialla et al.: SERS-based detection of biomolecules      385
3 S
 ERS-based approaches to
characterize and detect
biomolecules
Biomolecules can be defined as any molecules that are
produced by a living organism. From a chemical per-
spective the molecular precursors are carbon, hydrogen,
oxygen, nitrogen, phosphor and sulfur. These atoms
specifically associate to small biomolecules that are the
building blocks of life: nucleotides, amino acids, fatty
acids and monosaccharides. In general, the macromol-
ecules itself tend to form larger biopolymers. These
biopolymers belong to four major groups of essential
components of prokaryotic as well as eukaryotic life:
nucleic acids, proteins, lipids and carbohydrates. Besides,
there are additional molecules with biological relevance
that are synthesized within cells, like small or complex
metabolites.
The characterization and detection schemes of nucleic
acids and proteins and its building blocks using SERS as
analytical tool are mainly addressed within this chapter.
In literature, the band positions and the according assign-
ment of Raman modes dominating the SERS spectra are
in the focus of interest. As mentioned in the previous
chapter, various effects influence the SERS intensity of
vibrational modes and their intensity ratios and thus, play
an important role in SERS analysis. (1) The orientation of
a molecule relative to the metallic surface affects the SERS
intensity, due to the surface selection rules. Raman modes
perpendicular to the surface are preferably enhanced and
are thus dominating the SERS spectrum. (2) Due to the
evanescent field character of the electric field close to the
metal surface, only Raman modes in close vicinity up to
roughly 10  nm distance are enhanced by several orders
of magnitude. (3) Vibrations of chemical groups which
bind preferably to the metal – sulfur, nitrogen and oxygen
showing a high affinity to silver and gold – are dominat-
ing the SERS spectrum. Within this chapter, an overview
over the characterization and detection of biomolecules
is given. Thus, the interested reader might be supported
to evaluate the cutting-edge application fields of SERS
introduced within the next chapter in relation to the own
research area in terms of biomolecules.
3.1 Nucleotides and nucleic acids
Nucleotides are composed of a sugar, a phosphoryl group
and a nucleobase, which can be a purine or a pyrimidine.
There are two purine bases, adenine (A) and guanine (G)
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386      D. Cialla et al.: SERS-based detection of biomolecules
and three pyrimidine bases, cytosine (C), thymine (T) and
uracil (U). Ribonucleic acid (RNA) and Deoxyribonucleic
acid (DNA) are the polymers of nucleotides, which are
de­signated by the type of the sugar in the backbone. In the
case of RNA the sugar is D-ribose and for DNA it is 2-deoxy-
D-ribose. The intrinsic genetic information of the DNA is
translated via the messenger RNA (mRNA) into a defined
peptide or protein sequence. Thus, mRNA molecules func-
tion as intermediate information storage. The expression
of the DNA-coded information into a given amino acid
chain can be regulated at various stages. One important
regulation mechanism involves so-called non-coding
RNAs that trigger gene silencing in eukaryotic cells.
Since the SERS spectra of DNA and RNA are domi-
nated by adenine related modes [64, 65], the interpreta-
tion of adenine SERS spectra and the discussion of the
high diversity of adenine compounds within the SERS
fingerprints play an important role. Moreover, the attach-
ment sites to the metal surface of adenine are often
investigated and discussed in the literature. For clarifi-
cation, the molecular structure of adenine is depicted in
Figure 1. SERS spectra of adenine were recorded applying
low concentrations of the nucleotide [66]. Adenine shows
a strong Raman mode at 730 cm-1, which can be assigned
to a breathing vibration and is dominating the SERS spec-
trum. In general, it is found that the SERS spectrum is
dominated by in-plane vibrational modes. Thus, accord-
ing to the surface selection rules, it is concluded, that the
orientation of the adenine rings should be perpendicular
to the silver surface. Due to the decreased intensity of the
vibrational modes involving the nitrogen atoms N1, N3 and
N9 as well as the preferentially enhanced SERS signals of
Raman modes involving the external amino group and
the nitrogen atom N7, it is suggested that the interaction
between adenine and the silver surface takes place via the
external amino group and N7. Furthermore, out-of-plane
modes appear strongly weakened in the SERS spectrum
but do not disappear completely. Thus, a slightly tilted
orientation of the adenine molecule to the metal surface
is probable. In further studies, the orientation of adenine
and its derivatives polyadenine single stranded DNA
Figure 1 Illustration of the molecular structure of adenine.
(polyA) and adenosine monophosphate (AMP) were inves-
tigated at different pH values using SERS as well as surface
enhanced IR absorption (SEIRA) on gold nanoshell parti-
cles [67]. Due to the protonation of adenine at various pH
values, relative changes in the band intensity are found.
The recorded SERS spectra of adenine and its investi-
gated derivatives polyA and AMP are dominated by the
adenine ring breathing mode at 735 cm-1 which is in con-
sistency to other studies. Based on a detailed vibrational
analysis of the recorded SERS as well as SEIRA spectra,
the ‘end-on’ binding of adenine via the nitrogen atom N3
with the C-NH2 bond normal to the surface is suggested
as the presumed orientation relative to the gold nanoshell
surface. In the case of polyA and AMP, an adsorption via
N3 is sterically hindered due to substituents bound at N9
and a binding via the external amino group is more likely.
In order to increase the sensitivity for the detection of the
nucleobases cytosine, adenine, thymine and guanine,
the automated SERS detection at different pH values is
developed [68] since the pH-sensitive SERS enhance-
ment is known from various studies [65, 67, 69, 70]. As an
example, the influence of the experimental conditions,
like type of colloid, adenine concentration and pH value
on the recorded SERS spectra is investigated [65]. It was
found, that the SERS spectra are in agreement with the
N1-protonated form of adenine at acidic pH values apply-
ing both silver and gold colloids. In contrast to previous
studies, the recorded spectra at neutral and alkaline pH
are assigned to the N9-deprotonated form rather than the
neutral adenine molecule. Thus, the authors concluded
that the ‘normal’ spectrum of adenine is attributed to the
deprotonated form, similar to guanine and uracil. For low
adenine concentrations applying silver colloids under
neutral and alkaline conditions, the detected SERS spectra
are attributed to different silver complexes of adenine.
The high cross section of the silver complexes might be
an explanation for the observed and difficult to interpret
changes in intensity with varying the adenine concentra-
tions. In addition, the SERS spectra of desoxyadenosine
and 5′-desoxyadenosine monophosphate (5′-dAMP) are
also dependent on the experimental conditions and the
results indicate that both protonated and neutral forms of
desoxyadenosine and 5′-dAMP are detectable as function
of the pH value [70]. Since the ribose unit is substituted
via the position N9, a deprotonated analogue to adenine is
not detectable. Based on the weakened SERS intensity of
the in-plane breathing modes, a much flatter orientation
to the metal surface of desoxyadenosine is found for both
the neutral and protonated forms compared to that of the
corresponding forms of adenine. In the case of 5′-dAMP,
a reorientation of the molecule on the metallic surface
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 D. Cialla et al.: SERS-based detection of biomolecules      387

is observed as function of the concentration, pH and the For high DNA concentrations which correspond to a high
used metal colloid. In summary, all introduced studies packing density, the adenine peak is more intense in com-
show that the SERS spectrum of adenine is dominated parison to guanine. In the case of low DNA concentra-
by the breathing mode of the entire molecule at around tions and thus low packing densities, the guanine peak is
730 cm-1, that the attachment of adenine is at least via the increased with respect to the adenine vibrational mode.
external amino group and moreover, hints are found for a Based on these studies, higher adenine signal intensity
N9-deprotonated form of the free adenine under normal indicates a less tilted orientation of the DNA mole­cule
conditions at neutral pH values. relative to the metal surface. A reorientation of double-
In addition to the detailed investigation of nucleo­bases stranded DNA towards an upright position was further
– especially adenine, also DNA and RNA are characterized done by means of insertion of polythymine single stranded
by means of SERS. The here presented results are the start- DNA (polyT) which is detectable via an increased adenine
ing point for a SERS-based application in label-free DNA peak intensity. Guanine-rich nucleic acid sequences are
and RNA analytics. In order to record reliable SERS spectra known to form a four-stranded DNA structure. The forma-
of nucleic acids, the interaction time between the molecules tion of these so-called G quadruplexes was characterized
of interest and the metallic surface as well as the amount by means of SERS as a label-free analytical method [73].
of molecules within the sample volume should be chosen The fluctuation in SERS intensity is quantitatively inves-
with caution due to strong influences on the SERS signal tigated before and after the quadruplex formation. Since
[71]. A detailed analysis on the correlation of the molecu- the single DNA strands show a random orientation relative
lar orientation and packing density of double-stranded to the metal surface, the detailed analysis of the quadru-
DNA adsorbed on a metallic surface was performed [72]. plex SERS spectra suggests a perpendicular orientation
In Figure 2, the detection strategy as well as the recorded of the G quadruplexes with respect to the metal surface.
SERS spectra is depicted. The SERS spectra were ana- The characterization of the SERS signal as function of the
lyzed regarding their peak ratio of adenine and guanine numbers of G planes illustrates the improved stability of
vibrational modes as function of the DNA concentration. the quadruplex structure with an increasing number of
G planes. In addition, the SERS technique is applied for
the characterization of RNA. Adenine-containing micro-
A RNA chains adsorbed on a silver surface were investigated
regarding their spectral features and moreover the struc-
tural arrangement relative to the metal surface [74]. The
recorded SERS spectra, which are dominated by Raman
modes of adenine, were analyzed in comparison with the
spectral fingerprint of adenine, adenosine and the silver-
adenine complex structure. Based on SERS and DFT results
for adenine and adenosine, an interaction of microRNA via
B the N3 atom of adenine is proposed. Furthermore, the aro-
G matic character of the pyrimidinic ring of adenine might
favor a stronger metal-molecule interaction. Since the
A SERS signal is proportional to the amount of free residues
Intensity (a.u.)

5
4
interacting with the metallic surface, this technique was
applied to monitor the catalysis of ribozyme cleavages –
3
catalytic active hairpin structures found within RNA mol-
2 ecules [75]. In further studies, the interaction of small mol-
1
ecules containing three imidazole as well as three pyridine
500 550 600 650 700 750 800
rings with single-stranded RNA polynucleotides, double-
Raman shift (cm-1)
stranded DNA polynucleotides and calf thymus DNA was
Figure 2 Characterization of the DNA orientation towards the metal subject of research [76]. Due to the wavenumber downshift
surface. (A) Illustration of the SERS-based detection scheme on the of imidazole and pyridine ring vibrations, an aromatic
basis of the double-stranded DNA tilt angle. (B) SERS response of
stacking structure of imidazole and pyridine aromatic moi-
double-stranded DNA for various concentrations (spectra 1–5 are
related to the concentrations 40, 20, 10, 5 and 1.25 μm). Reprinted
eties and DNA base-pairs is concluded. In summary, the
with permission from Barhoumi et al. [72]. Copyright 2008 American results presented within this paragraph open the way to
Chemical Society. label-free DNA and RNA SERS-based detection schemes

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388      D. Cialla et al.: SERS-based detection of biomolecules
and moreover allow the investigation of interactions
between small molecules and nucleic acids adsorbed on
metallic surfaces. As a consequence of surface selection
rules, Raman modes perpendicular to the surface are dom-
inating the SERS spectrum. Due to the evanescent field on
the metal surface, only Raman modes in close vicinity are
observable under SERS conditions. Furthermore, vibra-
tional modes of chemical groups which bind preferably
to the metal are dominating the SERS spectrum. Thus, a
change in the structural arrangement (reorientation) of
nucleic acids with respect to the surface due to attach-
ment of other molecule will cause a significant change in
SERS intensity and band ratios of the Raman modes of the
molecular compounds.
Miscellaneous proof-of-principle assays demonstrated
the applicability of the SERS technique for the detection
of adenine and its derivative as well as DNA or even RNA.
As mentioned previously, adenine is one of the four RNA/
DNA bases and therefore a main constituent of the nucleic
acid metabolism in living organisms. Moreover adenine
derivatives have pivotal roles in the cellular respiratory
chain (e.g., adenosine triphosphate – ATP, nicotinamide
adenine dinucleotide – NAD, flavin adenine dinucleotide
– FAD) as well as second messengers in signal transduc-
tion processes (cyclic adenosine monophosphate – cAMP).
Thus, the SERS-based detection of adenine or derivatives
is addressed in recent articles. A planar chitosan-Ag SERS
substrate was used for the quantification of adenine [77],
whereas adenosine monophosphate (AMP) or deoxyaden-
osine monophosphate (dAMP) was detected by mean of
SERS with noble metal particles [67, 78]. Also, DNA-aptamer
biosensors were successfully used for analyzing adeno-
sine/adenosine triphosphate [79–82]. Various papers
introduced the applicability of the SERS-approach for the
label-free detection of RNA or DNA molecules [83–85]. The
successful use of SERS as a biosensor for DNA hybridiza-
tion events with synthetic DNA or peptide nucleic acids
(PNA) was described by several research groups [86–90].
Additionally, several sandwich hybridization assays con-
comitant with SERS detection were discussed in the liter-
ature [91–94]. The prominent spectral feature of adenine
can also serve as an endogenous marker for a label-free
SERS detection of DNA hybridization. As demonstrated in
Figure 3 a DNA capture probe that harbors 2-aminopurine
(2-AP) instead of adenine, which already preserved an
adenine-like hybridization characteristic but lacked the
736 cm-1 breathing mode, was immobilized on a gold surface
[64]. After the successful hybridization with the unla-
beled complementary target DNA, that brought adenine
within the sequence, the characteristic adenine peak was
detectable. Another interesting tool for monitoring DNA
Target DNA Probe DNA 3′
5′ A T
P
P
G C
P
P
C G
P
P
T 2-AP 5′
3′
Au NS surface
Intensity (a.u.)
A
B
400 800 1200 1600
Raman shift (cm-1)
Figure 3 Label-free DNA detection applying SERS for read-out.
Recorded SERS spectra of (A) adenine containing and (B) adenine
non-containing DNA sequence are shown. Within the inset, the
hybridization model between probe DNA having 2-aminopurine
substituted for adenine and unlabeled target DNA is illustrated.
Reprinted with permission from Barhoumi et al. [64]. Copyright
2010 American Chemical Society.
hybridization was emphasized by dendrimer formation as
a signal amplification strategy [95].
Damaged DNA, more precisely oxidized and UV-light
exposed genomic DNA isolated from human cell lines, is
detectable by label-free SERS [96]. The SERS community
also implemented novel trends from the field of nucleic
acid diagnostics, namely isothermal DNA amplification and
microarray-based detection schemes. Rolling circle amplifi-
cation, as one isothermal amplification strategy, allows the
accumulation of target DNA and significantly improves the
resulting SERS signal [97–99]. The creation of SERS sub-
strates in array format on planar surfaces opens the pos-
sibility for multiplex detection by a plethora of dye-labeled
oligonucleotides [100]. After the detailed discussion about
the characterization as well detection of nucleotides and
nucleic acids, innovative approaches related to the char-
acterization of amino acids and their macromolecules pep-
tides and proteins as well as promising detection schemes
will be introduced within the next paragraph.
3.2 Amino acids and peptides/proteins
Amino acids are molecules containing an amine and a
carboxylic group as well as a side-chain that specifies
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each amino acid. Amino acids itself are the most versatile
group of biomolecules. They possess a variety of impor-
tant biological function, which include precursors of hor-
mones or neurotransmitters and, in particular, they serve
as the building blocks of peptides and proteins. There
are 22 proteinogenic amino acids which can be linked
together to form a variety of smaller peptides or complex
proteins. Within this paragraph, the characterization of
amino acids, small peptides and proteins under SERS
conditions is introduced. The following chapter includes
the discussion about the preferred binding sites of amino
acids relative to the metal surface which are important in
spectral analysis, since the vibrational modes of these sur-
face-binding moieties are dominating the SERS response.
Moreover, the influences on the SERS signature of pep-
tides and proteins are discussed and innovative detection
schemes of these biomolecules are introduced.
In detailed studies by various research groups, the
characteristic Raman modes of amino acids under SERS
conditions and their dependencies as well as the adsorp-
tion behavior and orientation relative to the metallic
surface was investigated. Applying SERS as analytical
tool in combination with supporting DFT (density func-
tional theory) calculations, the adsorption of the zwitter­
ionic L-cysteine on the silver surface via the carboxylate,
ammonium and sulphydryl groups is concluded [101].
Furthermore, it is found, that cysteine shows the tendency
to form the corresponding dipeptide in solution. The char-
acterization of L-tryptophan by means of SERS on silver
nanoparticles shows the result that Raman modes of car-
boxylate and amino groups become strong under SERS
conditions [102]. A time-dependent SERS study shows a
unique spectral response, related with the most stable
configuration and orientation to the metal surface, after
a stabilization period [103]. Overall, it is concluded, that
the attachment of this amino acid is preferably via the
carboxylate and amino groups of L-tryptophan [102, 103].
A study on L-lysine attached to silver colloidal surfaces
indicates no unique conformation and orientation relative
to the metal surface [104]. Within the study, no identical
SERS spectra were recorded under neutral pH conditions
indicating various conformation of L-lysine with respect
to the metal surface. In general, L-lysine interacts via the
carboxylate and amino groups with the silver surface,
which is also found for other amino acids. In the case of
arginine, the preferably interaction between the amino
acid and the silver surface is verified to be conducted
through the guanidinium moiety and not via the carboxy-
late and amino groups [105]. Reorientation of amino acids
adsorbed on silver surfaces is achieved by adding cations
which becomes visible within the SERS response. It is
D. Cialla et al.: SERS-based detection of biomolecules      389
found, that Ce3+ affects the structure of N-acetylalanine
self-assembled at silver surfaces due to a binding reac-
tion between Ce3+ and the carbonyl as well as the amino
moieties [106]. The same is true for the effect of Pb2+ influ-
encing the structure of L-glutathione due to the binding
possibly occurring between the cation and the carboxyl
and amino groups of the amino acids [107]. The adsorp-
tion behavior of enantiomeric and racemic forms of an
amino acid was investigated by using methionine and
collecting SERS spectra as function of pH value and elec-
trode potential [108]. Non negligible spectral differences
are found by comparing the SERS response of D-methio-
nine and a racemic mixture of D- and L-methionine under
acidic conditions for different electrode potentials. In
comparison, in alkaline medium where both carboxylate
and amino groups are responsible for the interaction with
the electrode surface, no differences in spectral response
are found in contrast to the observations under acidic
conditions. Thus, indications are found for a chirality-
dependent interaction based on intermolecular hydro-
gen bonds between adjacent molecules at pH 3. A further
study shows the stereoselective interaction between phe-
nylalanine and monolayers of cysteine adsorbed on rough
silver surfaces [109]. Significant changes in SERS intensity
were established for the interaction of L-phenylalanine
with L-cysteine monolayer adsorbed on silver surfaces as
well as for D-phenylalanine interacting with D-cysteine
monolayers. This effect is attributed to variations in
hydrogen bonding between the ammonium groups of the
adsorbed cysteine molecule with the carboxylate groups
of the phenylalanine. In summary, applying SERS for
characterization of amino acids, a preferably binding via
the carboxylate and amino moieties is found through the
enhancement of the Raman intensity of vibrations which
are influenced by the nature of the binding to the metal
surface. A reorientation of amino acids is found for adding
cations. Finally, indications are found that SERS is sensi-
tive to the chirality of amino acids.
Within the following paragraph, the characteriza-
tion of peptides, which are defined as small assemblies
of amino acids, is introduced and the influences on the
SERS response of peptides is discussed. The investiga-
tions are focused on studies of the orientation of peptides
relative to the metallic surface and the therewith related
marker modes within the SERS spectra. For L-carnosine
an interaction primarily via the carboxylate group with
the imidazole ring oriented perpendicular to the silver
surface and the alanine moiety with parallel orienta-
tion having the amino group close to the silver surface
is found [110]. A comprehensive study of alafosfalin and
its analogs, which are phosphonodipeptides of alanine,
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390      D. Cialla et al.: SERS-based detection of biomolecules
was done by using colloidal silver particles as SERS plat-
form supported by DFT calculations [111]. It is found, that
these peptides adsorb as intact anionic species with the
P-terminal acid group onto the silver surface. The detected
SERS response indicate that both the constituent amino
acids and the amide bond linking the N- and P-terminal
residues interact with the metallic surface. Moreover, the
adsorption mechanism of phosphonodipeptides con-
taining N-terminal glycine on silver surface is subject of
research [112]. Since a similar adsorption behavior is not
found for all analogs, it is concluded, that the aliphatic
spacer group play an important role within the interaction
of these dipeptides with the silver surface. For dipeptides
containing dehydroalanine and dehydrophenylalanine a
strongly by dehydroresidues influenced SERS profile is
detected [113]. Here, the most prominent SERS signal for
the investigated dipeptides is attributed to the symmet-
ric stretching vibration of the carboxylate group, which
indicates the preferred adsorption via the deprotonated
carboxyl group. Time-dependent and pH-dependent SERS
studies were performed to characterize the binding char-
acteristics of the homodipeptide diglycine to the silver
surface [114]. A time-dependent increase in signal inten-
sity is found for Raman modes representing the amino and
amide moiety of the dipeptide. Furthermore, the pH value
influences the way of adsorption due to the protonation
as well as deprotonation of functional groups. Cysteine-
containing aromatic peptides were characterized and the
spectral response is compared with the SERS spectra of
the cell-penetrating peptide oligomer penetratin [115]. It is
concluded that, beside the protein backbone groups, aro-
matic amino acid residues provide the dominant features
within the SERS spectra of peptides and proteins when
present in the molecule of interest. In the case of phos-
phonate tripeptides, the adsorption on the metal surface is
realized also via the phosphonate moiety which results in
a reasonable SERS enhancement of the related vibrational
modes [116]. Various studies are dealing with the char-
acterization of bombesin, a 14-amino acid peptide, and
its related fragments and analogues [117–121]. As a result
of one of the comprehensive studies, the 8–14 bombesin
fragment takes almost a parallel orientation with respect
to the metal surface while the 1–5 peptide chain is orien-
tated in a way, that it remains out of the range of the eva-
nescent field caused by the metal surface [120]. Moreover,
an effect on the orientation and adsorption mechanism is
found when substituting amino acids within the chain.
The same is true for fragments of human neurotensin and
mutated analogues, where the SERS response indicates
a slight influence on their adsorption behavior on silver
surfaces due to the substitution of native amino acids in
the investigated peptides [122]. When characterizing the
bombesin subfamily peptides phyllolitorin and a peptide
derived from Pseudophryne guntheri the moieties phenyl­
alanine and tryptophan rings, the sulfur atom of methio-
nine and the amide bond are in contact as well as in close
vicinity to the metallic surface [123]. Comparative analy-
sis of bombesin and five related peptides, among others
a homolog in mammals known as neuromedin B, shows
that the interaction between peptides and the metal-
lic surface depends on the geometry of the tryptophan,
amide bond, and S-C fragments of these molecules [121].
In a more detailed study, neuromedin B was investigated
using different electrode materials and applied elec-
trode potentials [124]. The authors demonstrated that the
peptide adsorbs via the molecules backbone (including
the phenylalanine and tryptophan rings) onto the silver,
gold and copper electrode surfaces. Moreover, it is found,
that the amide III mode, a typical Raman marker band of
peptides and proteins, exhibit an electrochemical Stark
effect which is reflected in potential depended frequen-
cies. Furthermore, the carboxy terminal polypeptide of the
β-subunit of human chorionic gonadotropin without car-
bohydrates moieties (P37) is characterized concerning the
P37-metal interaction [125]. Here, the SERS analysis is per-
formed based on the prior investigated oligopeptides with
selected amino acid sequences MRKDV, ADEDRDA and
LGRGISL [126, 127]. The SERS spectra of the investigated
oligopeptides are dominated by the guanidinium moiety
of the amino acid arginine which illustrates the orienta-
tion of the peptide relative to the metal surface that is
driven by guanidinium [127]. In the case of ADEDRDA and
LGRGISL, an additional interaction with the metal surface
via other amino acids is found [126]. Based on these find-
ings, the authors proposed the P37 metal interaction to
be mediated via positively charged fragments of selected
amino acids, mainly threonine, lysine and arginine [125].
Finally, evidences were found by means of SERS that the
secondary structure of peptides is modified due to the
adsorption on metallic surfaces [128]. To summarize, the
SERS signature of peptides is not only depending on the
orientation of the molecules to the metallic surface as well
as on the chemical composition or more specifically the
amino acid sequence of the peptide. Also the metal, pH
value and electrode potential play a key role for the SERS
spectrum. Finally, the paragraph illustrates how SERS
spectra of peptides are influenced by various parameters.
Thus the findings open a way to detect peptides in low
concentrations and investigate interactions of peptides
with other molecules.
The characterization of proteins, biological mac-
romolecules consisting of one or more chains of amino
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acids, is in the focus of the next paragraph. Furthermore,
approaches for an application of SERS as analytical tool
for the detection of proteins are introduced. Fundamental
studies on protein detection are introduced by investiga-
tion on the interaction of bovine serum albumin (BSA)
with the surface of gold nanoparticles. The presence of
the S-S stretching vibration bands of disulfide bridges
indicates that the disulfide bonds are unbroken and the
protein is not denatured at room temperature due to the
attachment on the gold surface [129]. The adsorption of
BSA to the metal surface is realized mainly via the tryp-
tophan residue. Thus, BSA is specifically attached to
the surface and undergoes only minor conformational
changes when interacting with gold nanoparticles. In the
case of lysozyme, the main Raman modes within the SERS
spectrum are attributed to the amino acids tryptophan,
tyrosine, phenylalanine and histidine, indicating the
close vicinity of these moieties to the metal surface [130,
131]. In order to prevent spectral fluctuations due to reori-
entation of proteins during the analysis, an approach to
bind proteins reproducible on silver surfaces is developed
by fusing a silver-binding peptide to the C-terminus of
maltose-binding protein [132]. Moreover, enzymes, which
are selective catalysts and mostly proteins, were charac-
terized by means of SERS. As a result of the orientation
of coactivator-associated arginine methyltransferase 1
(CARM1) in relation to the nanostructured silver surface,
strong Raman modes in the SERS spectrum occur which
are attributed to amide vibrations and aromatic ring
amino acids [133]. In the case of tyrosinase, the molecule
is oriented in a way that the aromatic rings are parallel
to the silver surface [134]. Furthermore, the adsorption
behavior of the coenzyme Q10 radical intermediate as func-
tion of the potential is characterized [135]. For an applied
potential lower than -0.30 V vs. SCE, a perpendicular ori-
entation is found. The quinone ring of the reduced form
adopts a face-on configuration for more positive elec-
trode potentials than -0.30 V vs. SCE. The impact of the
metal surface on the adsorption behavior of the protein
bovine pancreatic trypsin inhibitor (BPTI) was investi-
gated by using cetyltrimethylammonium bromide (CTAB)-
protected gold nanoparticles in comparison to bare gold
substrates [136]. While the disulfide stretching vibration
of the protein deposited on the roughened gold substrate
shows a significant heterogeneity, the S-S stretching
signal remains unchanged for the application of CTAB-
protected gold nanoparticle arrays. Thus, the authors
concluded that the adsorption of BPTI on the protected
nanoparticle array increases the stability of the protein.
Cytochrome c, a small heme protein, was immobilized on
2-mercaptoethanesulfonate (MES) monolayers on silver
D. Cialla et al.: SERS-based detection of biomolecules      391
nanostructures based on electrostatic attraction between
the protein and the MES layer [137]. The linkage is char-
acterized by SERS studies and finally the change of heme
orientation relative to the metal surface deduced from the
SERS response allowed the authors to propose protein
dynamics. Furthermore, SERS demonstrated its benefit
for the investigation of proteins on the basis of the char-
acterization of myeloperoxidase (MPO), its correspond-
ing antibody and their immunocomplex [138]. Within the
SERS response variations in peak position and intensity
can be assigned to conformational changes due to the
immunocomplex formation. Another possible application
of SERS is demonstrated by means of the characteriza-
tion of amyloid beta peptide aggregates, which are related
to the formation of neutric plaque, one of the primary
pathological hallmarks of Alzheimer’s disease [139]. The
spectral analysis of samples prepared by various aggrega-
tion procedures shows, that the Amide III band provides
information about the secondary structure (α-helix and
β-sheet) which allows understanding the properties of the
protein aggregates at very low concentrations down to the
femtomolar range. Using silver colloids as SERS substrate
the cancer-promoting protein S100A4 was characterized
[140]. Here, the SERS response of the dimer, tetramer and
twelvemer aggregates demonstrates the possible discrimi-
nability of these species. Since dimeric forms are found in
benign tumors, whereas oligomeric species are related to
a late stage of cancer, this SERS study illustrates a possible
application field of SERS in the field of biomolecule detec-
tion. Finally, the conformational changes of the photoac-
tive yellow protein (PYP) are characterized on the single
molecule level illustrating the high potential of SERS
as analytical tool in detecting biomolecules [141]. In the
ground state of the molecule, PYP has a para-Coumaric
Acid (pCA) chromophore and its deprotonated phenolic
oxygen is stabilized by means of hydrogen bridges. When
absorbing a photon around 450 nm, the photocycle of
PYP is initiated and yields a cascade of conformational
changes. The detected spectral changes are attributed to
temporal single molecule spectra and were related with
photoisomerization, protonation of the chromophore
and the carbonyl flip in the context of breaking hydrogen
bridges.
Investigating protein detection with the SERS tech-
nique, the classical model thrombin is frequently chosen.
Within this context, the measurement of free thrombin
plasma levels represents a promising biomarker reflect-
ing a patient’s individual hemostatic status to guide suc-
cessful treatment decisions and to avoid bleeding or even
thromboembolic complications [142]. Furthermore, throm-
bin is a useful marker for the diagnosis of pulmonary
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392      D. Cialla et al.: SERS-based detection of biomolecules

metastasis [143]. Aptamers, an alternative for antibodies,
are artificial single-stranded oligonucleotides (ssRNA
or ssDNA) that can bind to target molecules due to their
specific three-dimensional structures [144]. Unsurpris-
ingly, diverse aptamer-based immuno-SERS approaches
for thrombin detection were published recently [145–151].
As an example, an aptamer-based detection scheme
applying crystal violet as reporter molecule is depicted in
Figure 4 [149]. The thrombin detection is performed
according to the different amount of the reporter molecule
crystal violet contributing to the overall SERS signal, based
on electrostatic effects due to the changed structure of the
thrombin-binding aptamer. A number of articles deal with
the imaging of surface proteins by applying on-cell SERS.
The purpose of one experimental setting was to elucidate
the putative co-localization between the β2-adrenergic
receptor and caveolin-3 on rat cardiomyoctes [152]. To
that end, 4-(mercaptomethyl)-benzonitrile and d7-mer-
captomethylbenzen are synthesized as Raman reporters
for silver nanoparticle functionalization. Subsequently,
17% of the receptor co-localize with surface-exposed
caveolin-3 protein. With the help of antibody-conjugated
fluorescent SERS-dots the extracellular and respectively
intracellular expression of CD34, Sca-1 and SP-C proteins
are verified on bronchioveolar stem cell of the murine
lung [153]. An immunolabeling process involving keratan-
specific primary antibodies and Au-conjugated secondary
antibodies that trigger silver particle localization is used
for imaging of keratan sulfate within a corneal epithelium
[154]. To understand mannoprotein membrane dynamics
in a single living yeast cell, a SERS approach with silver
nanoaggregates is employed [155].
Furthermore, the determination of enzymatic protein
activity by means of SERS is addressed in recent studies.
The monitoring of enzyme kinetics is an important issue
of biochemistry, especially in the context of metabolism.
With respect to this, SERS is a novel tool for determin-
ing the catalytic activity of an isolated succinate dehy-
drogenase by a color change of a redox dye. The artificial
electron acceptor 2,6-dichlorphenolindophenol becomes
SERS active when oxidized [156]. A study claims the
applicability of SERS to quantitatively determine the
proteolytic activity of a bovine protease on glass-immo-
bilized substrates and rule out a detection limit of 0.43
mU/ml [157]. To detect the cAMP-protein kinase activity
and further enzyme inhibition kinetics by diverse inhibi-
tors, a SERS approach using anti-phosphoserine antibod-
ies and gold nanoparticles is presented [158]. Moreover,
the feasibility of SERS-based detection of two pterin
entities is characterized by analyzing the serum from
pterin-spiked rats [159]. A cascade of signal amplifica-
tion reactions allows the SERS detection of lysozyme with
a limit of detection of 10-15 m [160]. These findings and
results, which are summarized within the chapter, illus-
trate the potential of SERS for the detection of biomol-
ecules. Since various detection schemes of biomolecules
are often based on the interaction of a capture molecule
with the biomolecule of interest, investigations regarding
the interaction of various biomolecules are highlighted
within the final section of this chapter.

Aptamer Thrombin
Crystal violet

Crystal violet

Crystal violet

A B C

Figure 4 Aptamer-based immuno-SERS detection scheme. (A) Using unmodified metallic nanostructured surfaces, a strong SERS signal of
the positively charged reporter crystal violet is detected. (B) When binding a monolayer of aptamers, the amount of crystal violet molecules
on the metal surface is decreased due to electrostatic effects. This results in a lower SERS signal. (C) In the case of thrombin binding to the
aptamer structure, more crystal violet molecules are bound to the surface (B). Thus, the SERS signal increases after the thrombin binding,
albeit to a lower level than in case (A). Reprinted with permission from Hu et al. [149]. Copyright 2009 American Chemical Society.

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3.3 C
 haracterization of the interaction
between biomolecules
The interaction of biomolecules due to the accumulation
to affine groups is the basis of various detection schemes
in bioanalytics. This section summarizes the recent find-
ings regarding this important topic. Some immuno-SERS
approaches, like the interaction of DNA-aptamers with
target proteins and antigen-antibody recognition as spe-
cialized protein-protein interactions are highlighted in
the relevant application chapters. The study of DNA-pro-
tein interaction in the background of transcription factor
binding is characterized on the example of Wilms tumor
1 (WT1) [161]. Here, dye-labeled double-stranded DNA
with a WT1 protein binding site is immobilized on metal
nanoparticles and shows a significant SERS response
dominated by the label molecule. The sequence-specific
binding of the transcription factor to the DNA prevent the
access of an exonuclease. Thus, the SERS signal of the
dye label is still detectable. If no DNA-binding protein is
present, the DNA gets accessible for the DNase, is exo-
nucleolytic digested and the label molecule is released
from the surface, which yields a lower SERS intensity. In a
proof-of-principle approach, protein-protein and protein-
small molecule interactions are detected on glass-surface
immobilized human immunoglobulin G or avidin by the
help of colloidal silver [162]. The combination of magnetic
separation and SERS allows the detection of biotin-avidin
interaction on Ag-coated magnetic particles [163]. Cell
membranes are composed, amongst other biomolecules,
of fatty acid chains that form a lipid bilayer. Since the
incorporation of small molecules into cell membranes is
of biological importance in terms of the characterization
of drug effects, the insertion of ibuprofen into lipid layers
is investigated [164]. Within these studies, a hybrid bilayer
formed of dodecanethiol and phospholipids is prepared on
the surface of gold nanoparticles. The intercalation of ibu-
profen in the lipid structure is related to the enhancement
of ibuprofen Raman marker modes showing a concen-
tration dependency. On the basis of using silver nano-
particles as SERS substrate, the interaction of hypericin
with low-density lipoproteins and phosphatidylcholine
is characterized [165]. The authors observe that at high
hypericin concentrations these molecules are placed in
the outer shell of low-density lipoproteins forming aggre-
gates. These results show the great potential of SERS for
the investigation and detection of interactions between
biomolecules. Within the next chapter which deals with
cutting-edge application fields in bioanalytics of the SERS
technique, the capability applying this method as analyti-
cal tool is demonstrated.
D. Cialla et al.: SERS-based detection of biomolecules      393
4 C
 utting-edge application fields of
SERS in bioanalytics
To reflect the publication activity in the field of SERS in
conjunction with the term “biomolecule”, the detection
of nucleic acids and proteins is mainly addressed within
this chapter. In the following, state-of-the-art SERS appli-
cations in the fields of biomedicine and analytical chem-
istry are highlighted. Within this paragraph we report the
use of the SERS technique for various biomedical applica-
tions, focusing, mainly on pathogen detection and cancer.
4.1 Pathogen sensing
The SERS technique is extensively studied since decades
for the sensing of bacteria and viruses. This chapter gives
an overview of the current SERS literature within this
important biomedical field. A major challenge of modern
medicine is a rapid and specific identification of bacte-
rial pathogens in human body fluids. Efforts towards so-
called point-of-care settings encompass the improvement
of SERS with portable Raman microscopes, robust SERS
substrates and even a simplification of sample prepara-
tion [166]. The current SERS publication activity points
into the direction of an early diagnosis of bacteremia as
well as urinary tract infections. Bacteremia, the presence
of bacteria in the patient’s blood, resulted from severe
infections at sites in the body, surgical wounds, or con-
taminated implanted devices and may lead to a sepsis.
Sepsis is a cause of considerable mortality, morbidity,
cost and health care utilization [167]. Urinary tract infec-
tions are common, with 50% of all women experiencing
at least one in their lifetime. The rate of infection leads
to high economic costs as well as patient morbidity [168].
The current gold standard for testing in hospitals is based
on culturing and normally takes up to three days [169].
Due to our scope, that covers the SERS-based identifica-
tion of biomolecules, the plethora of great publications
that address whole bacteria capturing and detection is
excluded from this review.
A whole bunch of articles reports a nucleic acid based
identification of pathogens in conjunction with SERS. The
advantages in this context are the capability of multiplex-
ing and the improved sensitivity. Some proof-of-principle
publications demonstrate the proper identification of
various pathogens by using synthetic oligonucleotides.
For example, the omp A gene of Clamydia trachomatis, a
sexually transmitted bacteria, is used within an elegant
exo-SERS assay [170]. Here, a sandwich-hybridization is
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394      D. Cialla et al.: SERS-based detection of biomolecules
performed with a magnetic bead immobilized capture
probe, the target DNA and terminal phosphate/TAMRA-
modified reporter probe. The 5′-phosphorylated reporter
probe allow the partial degradation after the sequence-spe-
cific hybridization by a lambda-exonuclease [171]. Even-
tually, the shortened reporter probe is coated with silver
nanoparticles and SERS spectra were recorded. Another
sandwich hybridization assay highlight the usage of oligo-
nucleotide-gold and oligonucleotide-silver nanoparticles
for the probing of Staphylococcus aureus [172]. Two studies
claim multiplexed detection of several E. coli strains by
using either different dye-labeled reporter probes or even
Ag/Au-colloids and multivariate data analysis [173, 174].
The diagnosis of pathogens is also presented in conjunc-
tion with the amplification of organism’s genomic DNA
and SERS measurements. A multiplexed gold particle-
on-wire sensor allowed the identification of Staphylococ-
cus aureus and Vibrio vulnificus, which are causatives for
severe human diseases like sepsis or gastroenteritis [175].
The detection limit of this sensor is depicted as 10 pM. The
usability of SERS for the multiplexed detection of hospi-
tal-acquired methicillin-resistant Staphylococcus aureus
is addressed [176]. To that end target genes are amplified
by PCR. In case of hybridization with the complementary
fluorescence-probe, the formed dsDNA did not adsorb on
the surface of silver nanoparticles which result in a SERS
signal reduction. Another assay based on SERS-primers
and silver nanoparticles for the identification of Staphylo-
coccus epidermis is depicted in Figure 5 [177]. In case of the
absence of target DNA the SERS primer is closed, thus the
DNA is predominantly double-stranded and has a lower
affinity to adsorb to the nanoparticles, resulting in a low
SERS response. The improved SERS assay using PCR and
A B
NO target present
low SERS signal
Counts (a.u)
Ag NP
300 600 900 1200 1500 1800
Raman shift (cm-1)
3′ 5′
Dye
5′ 3′
Figure 5 Illustration of the detection scheme of DNA based on the application of silver nanoparticles (labeled as Ag NP in the figure). (A)
When no target DNA is present, the SERS primer is closed, which results in a low SERS signal. (B) In the case of the presence of complemen-
tary DNA, the loop structure is opened. Thus, the dye label is free to adsorb on the silver nanoparticle surface producing a high SERS signal.
Reprinted with permission from van Lierop et al. [177]. Copyright 2011 American Chemical Society.
enzyme digestion to generate dye-labeled single-stranded
DNA to detect pathogens like Staphylococcus aureus is
highlighted in a recent article [178]. A specific detection
of Staphylococcus aureus DNA through the combination of
TaqMan assay with SERS is also realizable [179]. A success-
ful multiplexed SERS detection of Mycoplasma mycoides,
the causative agent of contagious bovine pleuropneumo-
nia, is realized via combining magnetic nanoparticles
and DNA hybridization [180]. SERS is able to provide a
vibrational spectrum of the diverse cell wall molecules
of a single bacterium within a few seconds. A fingerprint
bacterial identification via their O-antigens, a part of the
lipopolysaccharides that are exposed on the outer bac-
terial wall, is described [181]. In the proof-of-principle
paper the reliable differentiation between Salmonella
typhimurium and E. coli O16 is enabled by characteristic
vibrational modes in the spectral regions 1250–1130 cm-1,
1030–730  cm-1 and 650–430  cm-1 that represented carbo-
hydrates, most probably O-antigens. Using a liquid core
photonic crystal fiber a detection limit of 106 Shewanella
oneides bacteria is achieved [182]. For an immuno-SERS
approach metallic nanoparticles were functionalized
with ligands like antibodies or aptamers that recognize
target biomolecules on the surface of the pathogen [183].
The simultaneous detection of three different bacteria
is accomplished by using gold, silver and Ag-Au core
shell nanoparticles co-immobilized with anti-Salmonella
typhimurium aptamers, anti-Staphylococcus aureus and
anti-Escherichia coli O157:H7 antibodies respectively and
unique Raman reporter molecules [184]. Gold nanopar-
ticles with a coat of anti-protein A and DTNB as Raman
reporter could be used for identification of Staphylococ-
cus aureus via the bacterial surface antigen. A detection
Target present
high SERS signal
e
Dy
5′
Counts (a.u)
Ag NP
300 600 900 1200 1500 1800
3′ Raman shift (cm-1) 5′
3′
5′ 3′
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limit of 1 pg/ml of protein A is achieved [185]. Gold nano-
particles are coated with DSNB and antibodies, which are
generated against a surface antigen of Mycobacterium
avium subsp. paratuberculosis [186]. This bacillus is the
causative of a cattle disease, which is responsible for dev-
astating losses in the worldwide dairy production. The
sandwich immuno-SERS assay allows the quantification
of 500 mycobacteria/ml milk.
Similar to bacterial sensing, the fast diagnosis and
therapy of viral infections is of great importance. The
West Nile virus (WNV), a pathogen with a RNA genome,
is the causative of West Nile fever and encephalitis. WNV
is transmitted to its vertebrate hosts by an infected mos-
quito during blood feeding. The replication of the virus
was documented in human monocytes in vitro, which
could lead to transmission via blood transfusion [187].
Thus, the screening of donor blood is desirable and
SERS techniques were implemented to identify WNV.
Various sandwich hybridization assays, that utilize syn-
thetic target DNA sequence instead of the WNV-RNA, are
applied on either gold or paramagnetic iron nanoparti-
cles [188, 189]. These approaches enable a limit of detec-
tion for WNV target nucleic acid of 10 pM and the authors
claim a future adaption to a clinical detection or on-site
platform using low-cost, handheld Raman instrumenta-
tion. A multiplexed immuno-SERS detection addressing
envelope and capsid antigens of WNV is also realized
[190]. The identification of new infections with human
immunodeficiency virus (HIV) is crucial to prevent trans-
mission as well as slowing disease progression by early
medical intervention. Thus, HIV RNA-based tests may
allow a very early diagnosis even before a common anti-
body test may become positive. In clinical laboratories the
assessment of HI virus load is done by so-called branched
DNA technologies [191]. A SERS-approach use HIV-1 DNA
sequence for sandwich hybridization that induced the
formation of a molecular junction based biosensor [192].
With regard to this platform, a concentration as low as
10-19 M HIV-1 DNA is detectable. An interesting experimen-
tal setup was invented to detect dengue virus sequences
by on-chip SERS [193]. To date, the diagnosis of dengue,
the most common mosquito-borne global disease,
remains challenging. Infection by the RNA virus could
lead to dengue fever or severe dengue illness [194]. Apply-
ing a microfluidic SERS assay, synthetic DNA sequences
that mimic viral dengue serotypes are used as TAMRA-
labeled targets. The complementary capture probes are
immobilized on gold nanoparticles and the SERS meas-
urement is performed within the optofluidic SERS-chip.
Characteristic peaks are found at 1653 cm-1, 1360 cm-1 and
1219  cm-1 [193]. Of further clinical importance is the fast
D. Cialla et al.: SERS-based detection of biomolecules      395
and reliable detection of influenza viruses. Nowadays
an extensive menu of diagnostic tools is available [195].
Within this diagnostic topic a SERS immunosensor exper-
iment is conducted [196]. Gold binding polypetides with
hemagglutinin H1 antigen are immobilized on a nanofor-
est-structured substrate, followed by an immersion with
anti-H1 detection antibodies. The relevant SERS peaks are
found at 993 cm-1 and 1525 cm-1, which correspond to phe-
nylalanine and tryptophan peaks, respectively. Moreover,
within a short communication an aptamer-based detec-
tion of influenza nucleoproteins is presented on the basis
of silver nanorods [197]. A label-free detection of DNA
hybridization limited to short oligonucleotides is intro-
duced for the analysis of the respiratory syncytial virus
(RSV) [198]. This pathogen is the major causative of lower
respiratory tract infections prevalent among infants and
elderly [199]. The Epstein-Barr virus (EBV)-associated
expression of latent membrane protein 1 (LMP1), a tumor
marker for nasopharyngeal carcinoma, is addressed by
immuno-SERS [200]. Au/Ag core-shell nano­ particles
coated with LMP1-specific antibody and 4-MBA as a
Raman reporter are used for the detection of LMP1 in
paraffin-embedded nasopharyngeal carcinoma tissues of
patients, that were EBV-positive and healthy volunteers.
The authors claim that with the LMP1-SERS, 97.1% of the
cancer patients are tested positive for the virus-encoded
protein, whereas only in 64.7% the protein was detectable
when conventional immunohistochemical staining tech-
nique was applied.
To summarize, the SERS technique is successfully
applied to detect pathogen contamination on the basis
of their building blocks such as DNA or proteins. Various
detection schemes are introduced to open the way towards
point-of-care applications of the SERS technique.
4.2 Cancer diagnosis and prognosis
Nowadays, cancer belongs like every other disease to the
risks of life of every person. Everyone has to deal with a
cancer diagnosis or treatment either in the personal life,
via family members or within the circle of friends. Often
the person affected ask “why me” and moreover a late
diagnosis has the consequence of a high mortality. Thus,
a reliable method for cancer diagnosis and prognosis is of
great importance. Within this chapter we address impor-
tant SERS activities in the field of cancer diagnosis/prog-
nosis in conjunction with the terms non-invasive cancer
detection, splice variants and single nucleotide poly-
morphisms, epigenetics, chemotherapeutics as well as
theranostics.
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396      D. Cialla et al.: SERS-based detection of biomolecules
The chances of curing cancer often correlate with the
time of diagnosis. The earlier cancer is detected the better
is the patient’s chance to be cured by an early treatment.
In addition the risk for metastasis is reduced by early
detection. Much effort has been made to develop rapid
and non-invasive cancer screening tests. The preferable
material for such a non-invasive diagnosis is blood plasma
or serum and the sample preparation is simple during
initial screening as well as following the patient’s treat-
ment. This non-invasive cancer detection in conjunction
with SERS includes the analysis of circulating nucleic
acids, circulating tumor cells, immunophenotyping of
cancer cells as well as the detection of various tumor
markers and paves the way towards personalized medi-
cine. Recently, a combination of membrane electrophore-
sis and SERS is applied for the optical detection of gastric
and nasopharyngeal cancer [201, 202]. To that end, blood
plasma from cancer patients and healthy volunteers is col-
lected; the albumin and globulin protein fractions are
separated by membrane electrophoresis, eluted and
mixed with silver nanoparticles. A PCA reveals that the
cancer and healthy group form distinct non-overlapping
clusters. Again, silver nanoparticles are used as SERS sub-
strate and directly mixed with blood plasma of gastric
cancer patients or healthy subjects [203]. A subsequent
PCA-LDA multivariate analysis point out that distinctive
spectral SERS features and intensity differences between
the two groups could reflect cellular changes associated
with malignant transformation. The authors found for
instance that the SERS peak at 725 cm-1 corresponding to
the C-H in-plane bending mode of adenine exhibits higher
signal in the cancer panel, indicating that there is an
increase in the relative amount of nucleic acids in blood of
cancer patient. This finding is matching reports that so-
called circulating nucleic acids are found in higher con-
centrations when subjects are affected by a neoplastic
disease [204]. Further cancer entities, namely naso-
pharyngeal, colorectal, esophageal and cervical cancer
are analyzed using silver or gold colloids and blood
plasma samples [205–208]. An increase of distinct SERS
signals are observed for patients with pathologically con-
firmed carcinoma that indicate an abnormal nucleic acid
metabolism. The reasons for an increase in the relative
amount of cell-free nucleic acids in cancer patients’ blood
have been proposed in the literature by cell death-related
cell disintegration or release of intact cells in the blood
stream followed by their lysis [209]. On the other hand a
decrease of signal intensities for SERS-bands, which origi-
nate from tyrosine, serine, galactosamine and mannose
reflects a higher tumor metabolism [206–208]. An overall
decrease of the 1400  cm-1 peak intensity in the cancer
patient’s plasma, reflecting the CH2 bending mode of col-
lagen might account for elevated concentrations of matrix
metalloproteinases that cleave the structure protein in
cancer dysplasia [208]. A label-free serum RNA analysis
enabled the detection of colorectal cancer by using 3D
silver nanofilm as a SERS-active substrate [210]. The
authors describe that RNA-SERS combined with PCA-LDA
multivariate analysis was applicable for the differentia-
tion of colorectal cancer patients from healthy subjects
with a diagnostic sensitivity of 89.1% and a specificity of
95.6%. These results employing the SERS technique put
forward the possibility of detecting circulating nucleic
acids from blood plasma samples as biomarkers for moni-
toring cancer occurrence. Malignant cells can disseminate
from the solid tumor and float as so called circulating
tumor cells (CTCs) in the bloodstream of patients, poten-
tially manifesting a metastasis [211]. The capturing from
patient’s blood and the analysis of these rare cancer cells
is of great importance for treatment decisions and moni-
toring. SERS is successfully applied for the detection of
CTCs, even in complex matrices like blood [212, 213]. Mag-
netic beads functionalized with anti-epithelial cell adhe-
sion molecule (EpCAM) antibodies are used for the
capturing of SKBR3 cells, which mimic CTCs, spiked in
human blood. Subsequent, the captured EpCAM-overex-
pessing cancer cells are detected with Nanoplex SERS tags
[213]. Another experimental setting uses gold nanoparti-
cles coated with antibodies against epidermal growth
factor (EGF) to specifically capture CTCs in the blood of
patients with head-and-neck cancer [212]. Here, 1–720
CTCs per ml of white blood cells get enriched. The diag-
nostic immunophenotyping of cancer cells is an impor-
tant issue concerning the identification and of
subpopulations and has a great value in predicting the
invasiveness and metastatic potential of a tumor. In
general, the amount of surface marker proteins, the cluster
of differentiation (CD), is evaluated by flow cytometry.
Antibodies against CD24 and CD44 antigens immobilized
on gold nanoparticles enable the SERS-subtyping of three
breast cancer cell populations [214]. The non-invasive but
selective targeting of hematologic malignancies, e.g.,
chronic lymphocytic leukemia (CLL) using SERS gold
nano­particles is recently reported [215, 216]. To that end,
e.g. anti-CD19 antibodies are conjugated covalently to
gold nanoparticles. The antibodies recognize the CD19
surface antigen on CLL cells prepared from leukemia
patients. The growing amount of publications which deals
with cancer biomarker detection in patient’s blood/serum
or on target cells by means of SERS reflects the efforts that
are made within this field. Well characterized tumor
markers are two ErbB family members: epidermal growth
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factor receptor (EGFR, ErbB-1) and human epidermal
growth factor receptor 2 (HER2, ErbB-2). The EGFR is
­significantly overexpressed in aberrant crypt foci, which
represent putative colon cancer precursors [217]. Immuno-
SERS techniques using antibody- or affibody-conjugated
SERS nanoparticles are applied to monitor the presence of
EGFR in vivo [218, 219]. The authors conclude that the
approach is capable of targeting EGFR biomarker in
human cancer cells as well as in xenograft mouse tumor
models. The Olivo group uses photonic crystal fibers filled
with lysate of cancer cell lines to immobilize the target
protein EGFR [220]. Then anti-EGFR-antibody conjugated
SERS nanotags are flown through the hollow fiber and
specifically bind to their target protein. An antibody-based
SERS platform allows the multiplex detection of EGFR and
HER2 on cancer cell lines [221]. Various publications
report the identification of HER2-positive breast cancer
cell lines by means of immuno-SERS [222–224]. The
sequence-specific DNA analysis using sentinel nano-
probes is exemplarily demonstrated for the breast cancer
marker HER2 and the proliferation marker Ki-67 [225]. The
researchers use two different DNA-aptamer structures
conjugated to silver nanoparticles for sensing the pres-
ence of synthetic HER2/Ki-67 target DNA. A well-known
marker for tumor-associated angiogenesis is the vascular
endothelial growth factor (VEGF) [226]. The functionality
of a sandwich immunoassay is documented by the combi-
nation of the target VEGF immobilization on the gold array
via antibodies and the detection by VEGF-aptamer-nano-
particles [227]. VEGF is also detectable by an immunosen-
sor setting in blood plasma from breast cancer patients
[228]. Hybrid Ag-Au-aptamer structures labeled with Rho-
damine 6G are constructed that are capable of recognizing
mucin 1 (MUC1) [229]. The transmembrane protein MUC1
is overexpressed in primary and metastatic breast cancers
[230]. In their experimental section the authors demon-
strate that MUC1-positive MCF-7 cells show a characteristic
SERS signal, whereas MUC1-negative HepG2 or MCF-10A
cells did not show any signal of the reporter. The screen-
ing for the cancer biomarker mucin 4 (MUC4) in the blood
serum of patients can help to early diagnose pancreatic
cancer [231]. Within this project, an immuno-SERS princi-
ple is performed to monitor the presence of MUC4 in
various serum samples. The results indicate that sera from
patients with prostate cancer produce significantly higher
SERS response for MUC4 compared to sera from healthy
subjects. Also, an antibody-based recognition of two
important markers in routine cancer diagnosis, namely
carcinoembryonic antigen (CEA) and α-fetoprotein (AFP),
respectively, is demonstrated recently by means of SERS
[232]. Here, a duplex SERS nanoprobe is created by
D. Cialla et al.: SERS-based detection of biomolecules      397
coupling two antibodies against the cancer biomarkers on
magnetic beads that allow the simultaneous detection of
both markers in blood serum with a single exitation wave-
length. A gold patterned microarray is used as a matrix for
the immuno-based detection of AFP and angiogenin, the
latter one seems to have an important role in tumor angio-
genesis [233–235]. The multiplexed detection of the pros-
tate specific antigen (PSA) in paraffin prostate cancer
sections is investigated with antibody-conjugated nano-
particles or the antibody-fluorochrome-conjugate [236].
Both detection reagents possess similar performance in
the tissue, supporting the development of Raman probes
for PSA tumor marker screening in situ. Soluble PSA as a
target protein is also detectable by antibody-conjugated
hybrid Fe3O4/Au nanoparticles, emphasizing the advan-
tages of magnetic particles in the bioassays [237]. Two
further serum biomarkers, matrix metalloproteinase 7
(MMP-7) and carbohydrate antigen 19-9 (CA 19-9) are suit-
able for the diagnosis of early stage pancreatic cancer
[238]. To that end, human serum was spiked with the
recombinant proteins and the subsequent immuno-SERS
allowed the measurement of these biomarkers. A biorecog-
nition between the bacterial redox protein azurin and
human p53 is exploited to realize a SERS-based detection
of the tumor suppressor at concentrations of 500 fM in
human serum [239, 240]. It is well documented in the lit-
erature that wild-type as well as mutant p53 protein is
found in elevated levels in tumor cells concomitant with
altered levels in the blood serum [241–243]. The SERS
approaches for ultrasensitive detection of p53 involve a
coupling of the protein via the interaction with 4-amithio-
phenol to gold nanoparticles and the subsequent interac-
tion with immobilized azurin molecules on a glass surface
[239, 240]. Azurin exhibit strong association with both, the
wild-type as well as the mutated p53 protein and repre-
sents due its combination with SERS a promising approach
for future tumor marker screenings. Another member of
the p53 tumor suppressor family, p65, is addressed by an
immuno-SERS approach together with the cyclin-depend-
ent kinase inhibitor p21cip [244]. Here, the two proteins
are spiked into human blood serum and are detected in a
multiplexed fashion by antibody-functionalized Au-Ag
nanorods. Immuno-SERS microscopy is applied for the
fast detection of p63 in paraffin-embedded prostate biop-
sies [245, 246].
Single nucleotide polymorphisms (SNPs) are single
base pair differences in the DNA among individuals that
account for more than 90% of all sequence variations [247,
248]. Roughly one of every 100–300 bases in the human
genome is a SNP [248, 249]. Numerous SNPs are detected as
reliable diagnostic biomarkers for selection of appropriate
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398      D. Cialla et al.: SERS-based detection of biomolecules
drug therapies in the upcoming field of personalized
medicine. For instance, a single nucleotide substitution in
different codon positions of the proto-oncogenic Kirsten
rat sarcoma (KRAS) is responsible for an activating muta-
tion. Thus patients with this type of mutations will not
respond to certain therapies [250]. An expansion of the
SNP screening method repertoire is facilitated by SERS.
A midsequence SNP discrimination by using synthetic
oligonucleotides and gold nanoparticles is realized as
dye-label-free SERS [251]. Another interesting approach
utilizing sequence-specific hybridization and the ligase
detection reaction (LDR) is introduced for discriminating
KRAS SNPs [252, 253]. When the SNP sequence matches
the discriminating primer, two adjacent oligonucleotide
sequences get ligated and a SERS active product result.
With the help of this LDR-SERS method a multiplexed
SNP genotyping of the KRAS oncogene is possible down
to a limit of detection of 10 pM [252]. A further SNP-related
study depicts the application of gold nanowire/nanofilm-
hybrids and a S1 nuclease digestion for assaying Wilson
disease [254]. The SNP at codon 504 of the breast cancer
1 (BRCA1) gene is chosen to be detected by SERS [255].
The authors demonstrate the usability of a SERS molecu-
lar sentinel that comprises a silver nanoparticle plus the
associated DNA hairpin structure that gets opened when
the target DNA hybridizes to it. Thus the Raman label is
physically separated from the particle and quenches the
SERS signal.
In addition to SNP analysis, the detection of BRCA1
splice variants is of great importance. Changes in the
alternative mRNA splicing profile of the BRCA1 gene
is associated with malignant transformation in breast
cancer [256]. The identification of cell and tissue spe-
cific splicing patterns helps to unravel the role of such
genes in tumor development. In two proof-of-concept
papers a SERS approach for a multiplexed detection of
BRCA1 splice variants through nanoparticle-DNA-Raman-
tag setup is presented with synthetic DNA [257, 258]. An
advanced gene expression assay by SERS, involving the
isolation of RNA from breast cancer cell lines followed
by an translation into complementary DNA, sensitively
quantify the expression levels of splice junction, skipping
exon 9 and 10, of the BRCA1 gene [259, 260]. Moreover,
SERS experiments are conducted that allowed the screen-
ing for wild-type and mutated BRCA1 peptides involving
gold nanocuboids [261].
The SERS technique is also introduced by numerous
groups to detect epigenetic modifications in the context
of cancer diagnosis and prognosis. Epigenetic variations
within the human genome are realized by means of non-
coding RNAs or covalent DNA modifications. Micro RNAs
(miRNAs) are small non-coding RNA molecules that regu-
late eukaryotic gene expression by mRNA degradation or
inhibition of translation [262, 263]. It has been suggested
that up to 30% of the human genome may be regulated
by miRNAs [264]. Of further importance is the fact, that
miRNA expression profiling can serve as diagnostic/prog-
nostic biomarker of human cancers. Recent studies high-
light the frequent downregulation of miR-15/16 in chronic
lymphocytic leukemia [265, 266]. In addition, a reduced
expression of multiple let-7 miRNA members has been
found to be associated with human cancers [267]. Emerg-
ing techniques for the detection of miRNA alterations are
bead-based flow cytometry and RNA-primed array-based
Klenow extention (RAKE) that significantly reduced
assay time [268, 269]. Nevertheless, the requirement of a
hybridization step remained. To further accelerate analy-
sis time, SERS is introduced as a label-free method for
the detection and classification of miRNA. The applica-
bility of SERS for miRNA analysis is demonstrated using
silver nanorods and synthetic human miRNAs, like the
pathogenic relevant miR-16 or let-7f [270–273]. The find-
ings indicate that miRNA sequences could be accurately
discriminated and quantified even in multicomponent
mixtures. The fact, that let-7 miRNA family members pro-
duced SERS spectra very similar to one other in number
and location of peaks is remarkable. But the spectra
differed significantly in the relative intensities of some
bands [270]. The SERS-based approaches enable rapid
as well as quantitative miRNA detection using minimal
sample volumes concomitant with an elimination of
probe labeling and hybridization steps. Thus, the SERS
technique is successfully adopted for the growing field of
miRNA expression profiling. In eukaryotic cells methyl-
ated cytosine (mC) in so called CpG islands is the most
common epigenetic mark [274]. More recently, hydroxy-
methylcytosine (hmC) was discovered as a novel DNA
methylation tag [275, 276]. Methylated DNA represents
an important epigenetic modification that provides
information for altering the gene expression. Moreover,
aberrant DNA methylation has been found to strongly
correlate with human cancer. In that context the relation-
ship of hypermethylation of CpG islands in the promotor
regions of tumor-suppressor genes like p16INK4A and the
promotion of tumor progression is reported [277, 278].
New scientific findings point in the direction of using
methylation profiles as diagnostic or predictive biomark-
ers [278–280]. So far, a whole bunch of techniques have
been developed for DNA methylation screening [281].
Recently, an article claims the single base extension reac-
tion-based SERS for the detection of methylated p16INK4A-
DNA [282]. Additionally, a SERS-based analysis enables
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the differentiation of the post-replicative DNA modifica-
tions mC and hmC [283]. The spectral variations between
normal and hydroxymethylated DNA are significant, so
an increase in the 665 cm-1 peak is an obvious marker of
the presence of hmC. The acquired data point into the
direction that SERS is a reliable method for discrimina-
tion between mC and hmC.
Besides the usefullness of the SERS technique for
cancer diagnosis and prognosis, it can also be success-
fully exerted for monitoring the effect of anticancer drugs
on target cells. With the help of a gold nanoflower-array
the immobilization of living HepG2 liver cells is real-
ized and this cell-based chip is used for studying the
various effects of anticancer drugs on the cell behavior
[284]. The chosen drugs are 5-fluorouracil and hydroxy­
urea which both interfere with DNA synthesis and cyclo-
phosphamide, a DNA damaging agent. The recorded
Raman spectra reflect a significant change in the cellu-
lar composition of nucleic acids and even proteins after
the chemotherapeutical treatment. The measurement of
drug concentrations in patient saliva instead of blood is a
further non-invasive option to monitor drug metabolism
and regulate drug dosage administration. In a proof-of-
principle experiment disposable SERS-active capillaries
filled with silver layers are tested for evaluation of 5-fluo-
rouracil and its metabolite concentrations in saliva [285].
Furthermore, a DNA approach is performed to illumi-
nate the interaction with the chemotherapy agent cis-
platin [286]. After incubation of dsDNA with cisplatin
the resulting intrastrand and interstrand adducts evoke
spectral changes. These characteristic time-dependent
spectra variations are caused by conformational distor-
tions due to the ligand binding. The intracellular release
of the chemotherapeutic agent 6-mercaptopurine, which
is coupled to gold nanoparticles, can be triggered by glu-
tathione. The release monitoring is performed by using
SERS [287].
Within the emerging field of personalized medicine
the development of so called theranostics is of great
interest. Theranostics is referred to as a treatment com-
bining therapeutics and diagnostics [288]. The aim is to
integrate diagnostic approach and therapeutic interven-
tion, allowing a simultaneous diagnosis, treatment as
well as efficacy monitoring [289]. The earlier mentioned
report about the binding of SERS-active nanostructures to
MUC1-expressing cancer cell lines has also outlined the
capability of absorbing near-infrared irradiation. They
are therefore eligible for the photothermal therapy to
selectively induce cell death of the targeted cancer cells
without destroying surrounding tissues [230]. Further
reports on photodynamic tumor therapy implementing
D. Cialla et al.: SERS-based detection of biomolecules      399
SERS are presented in the literature [290, 291]. In general
the photodynamic therapy (PDT) involves the selective
uptake and accumulation of a photosensitizing agent in
the target cells and the subsequent irradiation with light.
The induced generation of reactive oxygen species (ROS)
causes damaging of intracellular biomolecules, leading
to cell death by apoptotic processes [292]. The intracel-
lular accumulation of the photosensitizer-coated nano-
particles in cancer cells is realizable by either the use
of cell-penetrating peptides [291] or enhanced perme-
ability and retention (EPR) [290]. The latter EPR effect
accounts for the uptake of gold nanorods by tumor cells
and a large area of dead cells could be clearly seen in
PDT-treated tumors. A non-invasive in situ release moni-
toring possibility is introduced very recently by the help
of SERS [293]. The authors demonstrate the controlled
release of the cargo doxorubicin from gold nanocages
by applying heating, pointing into the direction of con-
trolled drug delivery. The development of plasmonic vesi-
cles assembled from SERS-active gold nanoparticles for
targeted drug delivery, which can be tracked by Raman
spectroscopy is reported [294]. The gold particles contain
the Raman reporter BGLA, mixed brushes of hydrophilic
poly ethylene glycol (PEG) and pH-sensitive hydropho-
bic copolymers of methyl methacrylate and 4-vinylpyri-
dine (PMMAVP) to allow drug release, HER2 antibodies
for cancer cell targeting and encapsulated doxorubicin
as an anticancer drug. To verify the targeting properties
concomitant with pH-triggered payload release, studies
with HER2-overexpressing breast cancer cells were per-
formed. The vesicle-coated cancer cells display a strong
fingerprint Raman signal of the BGLA probe that gradu-
ally decrease with time caused by acidic driven disrup-
tion of the vesicles and a drug release. The entire strategy
of this approach is depicted in Figure 6.
This chapter summarizes innovative strategies
based on the SERS application in cancer diagnostics and
prognosis. It is shown, that the SERS technique is used
by various detection schemes based on silver and gold
nanoparticles to contribute to the research field of non-
invasive cancer detection, which includes the detection
of circulating nucleic acids and tumor markers. More­
over, the sequence specific DNA detection to investigate
splice variants and single nucleotide polymorphisms as
well as the contribution to the research on epigenetics is
highlighted by using SERS as analytical tool. The detec-
tion of chemotherapeutics is of great importance in terms
of personalized medicine and drug treatment. Finally, the
findings within the research field theranostics illustrate
the great capability of SERS to detect and to release drugs
within cancer cells.
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400      D. Cialla et al.: SERS-based detection of biomolecules
A Amphiphilic gold nanoparticle
Poly(ethylene glycol)
pH-sensitive
polymer brush
Raman reporter
Plasmonic vesicles
: Anticancer drug
: HER2 antibody
Figure 6 Theranostics strategy based on plasmonic vesicles built of gold nanoparticles. (A) Scheme of the applied gold nanoparticles
coated with mixed polymer brushes and a Raman reporter as well as the drug-loaded plasmonic vesicle coated with antibodies for cancer
cell targeting. (B) Illustration of the plasmonic vesicle’s attachment to the outer cell membrane, their uptake and finally the disruption
of the SERS-active, pH-sensitive vesicle structure. Reprinted with permission from Song et al. [294]. Copyright 2012 American Chemical
Society.
4.3 F urther SERS-based application strate-
gies in biomedicine and biomolecule
detection
As described in the previous paragraphs, SERS is an alter-
native method to detect pathogens and cancer alterations.
In addition, SERS can be applied in other important bio-
medical fields. Thus, the following overview summarizes
SERS applications concerning disease-related mutation
detection, cell death and differentiation verification,
forensics, drug and neurotransmitter screening, metabo-
lism monitoring, prion protein detection in neurons,
oxygen-binding protein sensing as well as targeting of
immunoglobulins in blood. By employing SERS, the dis-
crimination between single nucleotide and triplet deletion
mutations as well as wild type of cystic fibrosis trans-
membrane regulator (CFTR) gene is accomplished [295,
296]. CFTR mutations cause cystic fibrosis, which is one
of the most common life-threatening inherited diseases in
Caucasians [297]. The use of an electroactive label for so-
called SERS-E-melting experiments that enable the CTFR
gene discrimination at the attomole level is reported [295].
A further SERS-E-melting approach is introduced for the
discrimination of short tandem repeats [298]. These poly-
morphic short sequences are commonly used to determine
genetic profiles for forensic individual identifications.
Programmed cell death, alias apoptosis, is a highly impor-
tant cellular process in vivo and the selective induction of
B Endocytosis
SERS ON
Cytosol
Nucleus
H+
SERS OFF
apoptosis in cancer tissue is an established chemothera-
peutic strategy. A label-free detection of apoptosis at the
single-cell level is realized by silver nanoparticles [299].
After apoptosis induction by detergents only a weak SERS
signal of DNA is observed due to specific cleavage of DNA
strands as a late apoptotic event. The verification of cell
differentiation is also accomplished by SERS [300, 301].
The in vitro differentiation of neuronal progenitor cells
into terminal differentiated neurons is realized by stauro-
sporine. To record SERS spectra corresponding to nucleic
acids, the accumulation of gold nanoparticles within the
nucleus of the different cell types is done by using the
nuclear localization signal of the SV40-large T antigen
[301]. The tracking of differentiation of mouse embryonic
stem cells by intracellular nanoparticles show the fol-
lowing results: SERS peaks derived from undifferentiated
cells are mostly attributed to nucleic acids (high prolifera-
tion rate) whereas the SERS peak at 1604 cm-1 originated
from mitochondria of the terminally differentiated cardio-
myocytes (high metabolic activity) [300]. As mentioned
earlier, aptamers are a versatile tool for bioanalytical
purposes. In conjunction with SERS the conformational
changes in the aptamers induced by target molecule inter-
action are monitored on a label-free level. To that end the
drug cocaine is chosen as a model substance [302, 303]. An
antibody-based immuno-SERS approach use silver-coated
carbon nanotubes for the detection of the major cocaine
metabolite benzoylecgonine [304]. Serotonin, a ubiqui-
tous neurotransmitter is quantified in a background of
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other indoles using SERS [305]. Lastly, SERS assisted
ultra-fast peptide screening is described as a novel prom-
ising tool for drug discovery [306]. The authors claim the
usability of microbeads with Au-Ag nanoparticle coat as a
solid support for the synthesis of combinatorial peptide
libraries. The subsequent SERS analysis enables the iden-
tification of several tripeptide combinations as a conse-
quence of the unique vibrational signature of each amino
acid. A transcutaneous in vivo glucose measurement in a
rat model is realized by SERS [307]. This work represents a
significant step towards an implantable, real time glucose
SERS-sensor. Another example of metabolism monitoring
is the detection of lactic acid at physiological concentra-
tions in blood samples [308]. The study of prion proteins
is performed by several SERS approaches [309–312]. The
focus lay on the native cellular prion protein (PrPc), which
is mainly expressed on the cell surface of neurons. The
physiological function of PrPc remains elusive, although
it has been related to copper metabolism [313]. SERS allow
the tracking of PrPc-Cu2+ interaction on the cell membrane
of different neuronal cells [309, 312]. The detection of PrPc
either in bovine serum or in blood samples with silver
nanorods is displayed [310, 311]. A proof-of-concept paper
focuses on the combination of isotachophoretic flow
electrophoresis on a chip-device and SERS for the detec-
tion of myoglobin, an abundant oxygen-binding protein
in muscle cells [314]. To gain relevant information about
putative drug effects on different hemoglobin subpopula-
tions in intact erythrocytes, SERS based on silver colloids
is performed [315]. The group shows that submembrane
and cytosolic hemoglobin molecules display significant
conformation differences. Beyond that, an immuno-SERS
approach is applied to detect immunoglobulin E (IgE)
in blood serum of rabbits [316]. Gold nanoparticles are
coated with protein A/G and malachite green as a Raman
tag. The formed immuncomplex allow the monitoring of
rabbit serum IgEs by SERS and represents an alternative
to classical ELISA formats. Mouse or human IgG is chosen
as a model protein to demonstrate the feasibility of SERS
as a novel spectroscopic technique in clinical diagnostics
[317, 318].
Finally, novel strategies regarding SERS for toxin
monitoring in water and food, identification of geneti-
cally modified organisms, extraterrestrial life sensing and
even art evaluation are presented. As an example, ricin, a
potential bioterrorism agent, is successfully quantified by
either an aptamer-based SERS approach or a pre-immu-
nomagnetic separation, respectively [319, 320]. At least
100 ng/ml ricin B is detectable in complex matrices like
milk. This highlighted the promising potential of the SERS
technique for bioweapon screening and the feasibility of
D. Cialla et al.: SERS-based detection of biomolecules      401
using portable Raman instruments for on-site detection
[319]. The assembly of gold nanorods is used for the prin-
ciple detection of microcystin, an algae toxin pollution in
drinking water reservoirs [321]. The presence of the toxin
interfered with the antibody-antigen triggered assembly
of nanorod chains, thus significantly reducing the SERS
signal for 4-ATP. In a study, the screening for genetically
modified organisms is performed by combining magnetic
pre-concentration of the target DNA, which was the 35S
gene promoter, with further SERS measurements. A real
sample analysis with Bt-176 maize DNA demonstrates the
feasibility of the assay concept [322]. Surprisingly, SERS
was utilized for analyzing nucleobases that mimic micro-
bial life on pyroxene rocks [323]. The authors depict that
the experimental procedure could be easily adapted for in
situ recognition of life traces in extraterrestrial environ-
ments, mainly detecting the marker band of adenine at
735 cm-1. Moreover, SERS is introduced in artworks. Here,
the precise identification and localization of proteins,
such as ovalbumin, collagen or casein is demonstrated by
indirect immuno-SERS in multilayered paintings [324].
In addition to the application fields of SERS patho-
gen sensing and cancer diagnostics, this section shows
the potential of SERS in further research areas. The inno-
vative, here described approaches might be the starting
point for the establishment of new application schemes
employing the SERS technique.
5 Conclusion
This review article summarizes the recent results and
developments of using SERS for the detection of biomole-
cules. First, the characterization of biomolecules and their
detection schemes were discussed. In general, the SERS
intensity depends on various parameters like the distance
of the molecule from, its orientation relative to and its affin-
ity to the molecule towards the metal surface. The great
capability of the SERS technique as an analytical tool to
detect biomolecules is illustrated by cutting-edge applica-
tions in the fields of pathogen sensing and cancer diagno-
sis. In the case of pathogen sensing, the bacterial or viral
contamination is detected via their building blocks such
as DNA and proteins. Moreover, innovative strategies in
cancer diagnosis are introduced. As an example, the SERS
technique is applied for non-invasive cancer detection
(which includes the detection of circulating nucleic acids
and tumor markers), epigenetics, chemotherapeutic drug
monitoring and theranostics. This highlights the contribu-
tion of the SERS technique among others to personalized
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402      D. Cialla et al.: SERS-based detection of biomolecules
medicine, which means an inclusion of individual circum-
stances to the treatment of patients. Finally, novel strate-
gies in cell investigations, forensics, biomarker screening
as well as environment monitoring and even art are intro-
duced. This comprehensive summary in the field of SERS-
based biomolecule detection might be a starting point
for the interested reader from different research areas to
create own approaches for a SERS-based detection in bio-
chemical, biological or biomedical applications.
References
[1] Ma L-n, Zhang J, Chen H-t, Zhou J-h, Ding YZ, Liu YS. An overview
on ELISA techniques for FMD. Virology J 2011;8: Article no. 419,
9 pages.
[2] Malou N, Raoult D. Immuno-PCR: a promising ultrasensitive
diagnostic method to detect antigens and antibodies. Trends
in Microbiol 2011;19:295–302.
[3] Seibel J, Koenig S, Goehler A, Doose S, Memmel E, Bertleff N,
Sauer M. Investigating infection processes with a workflow
from organic chemistry to biophysics: the combination of
metabolic glycoengineering, super-resolution fluorescence
imaging and proteomics. Expert Rev Proteomics 2013;10:25–31.
[4] Petryayeva E, Algar WR, Medintz IL. Quantum dots in
bioanalysis: a review of applications across various platforms
for fluorescence spectroscopy and imaging. Appl Spectrosc
2013;67:215–52.
[5] Pampaloni F, Ansari N, Stelzer EHK. High-resolution deep
imaging of live cellular spheroids with light-sheet-based
fluorescence microscopy. Cell Tissue Res 2013;352:161–77.
[6] Nicklas JA, Buel E. Quantification of DNA in forensic samples.
Anal Bioanal Chem 2003;376:1160–7.
[7] Ataka K, Kottke T, Heberle J. Thinner, smaller, faster: IR
techniques to probe the functionality of biological and
biomimetic systems. Angew Chem-Int Edit 2010;49:5416–24.
[8] Brauchle E, Schenke-Layland K. Raman spectroscopy in
biomedicine - non-invasive in vitro analysis of cells and
extracellular matrix components in tissues. Biotechnol J
2013;8:288–97.
[9] Downes A, Elfick A. Raman spectroscopy and related
techniques in biomedicine. Sensors 2010;10:1871–89.
[10] Culha M. Surface-enhanced raman scattering: an emerging
label-free detection and identification technique for proteins.
Appl Spectrosc 2013;67:355–64.
[11] Bantz KC, Meyer AF, Wittenberg NJ, Im H, Kurtulus O, Lee SH,
Lindquist NC, Oh SH, Haynes CL. Recent progress in SERS
biosensing. Phys Chem Chem Phys 2011;13:11551–67.
[12] Pahlow S, Maerz A, Seise B, Hartmann K, Freitag I, Kämmer E,
Böhme R, Deckert V, Weber K, Cialla D, Popp J. Bioanalytical
application of surface- and tip-enhanced Raman spectroscopy.
Eng Life Sci 2012;12:131–43.
[13] Negri P, Dluhy RA. Ag nanorod based surface-enhanced Raman
spectroscopy applied to bioanalytical sensing. J Biophotonics
2013;6:20–35.
[14] Vitol EA, Orynbayeva Z, Friedman G, Gogotsi Y. Nanoprobes
for intracellular and single cell surface-enhanced Raman
spectroscopy (SERS). J Raman Spectrosc 2012;43:817–27.
Acknowledgement: Funding of the research projects
“QuantiSERS” and “Jenaer Biochip Initiative 2.0” within
the framework “Unternehmen Region – InnoProfile
Transfer” the Federal Ministry of Education and Research,
Germany (BMBF) is gratefully acknowledged.
Received June 19, 2013; accepted September 5, 2013; previously pub-
lished online October 5, 2013
[15] Vendrell M, Maiti KK, Dhaliwal K, Chang Y-T. Surface-enhanced
Raman scattering in cancer detection and imaging. Trends
Biotechnolo 2013;31:249–57.
[16] Kho KW, Fu CY, Dinish US, Olivo M. Clinical SERS: are we there
yet?. J Biophotonics 2011;4:667–84.
[17] Joseph V, Engelbrekt C, Zhang J, Gernert U, Ulstrup J, Kneipp J.
Characterizing the kinetics of nanoparticle-catalyzed reactions
by surface-enhanced Raman scattering. Angew Chem-Int Edit
2012;51:7592–6.
[18] Le Ru, EC, Etchegoin PG. Single-molecule surface-enhanced
Raman spectroscopy. In: Johnson MA, Martinez TJ, eds. Annu
Rev Phys Chem 2012;63:65–87.
[19] Kneipp J, Kneipp H, Kneipp K. SERS - a single-molecule and
nanoscale tool for bioanalytics. Chem Soc Rev 2008;37:
1052–60.
[20] Stadler J, Schmid T, Zenobi R. Developments in and practical
guidelines for tip-enhanced Raman spectroscopy. Nanoscale
2012;4:1856–70.
[21] Treffer R, Boehme R, Deckert-Gaudig T, Lau K, Tiede S, Lin X,
Deckert V. Advances in TERS (tip-enhanced Raman scattering)
for biochemical applications. Biochem Soc T 2012;40:609–14.
[22] Reparaz JS, Peica N, Kirste R, Goni AR, Wagner MR, Callsen G,
Alonso MI, Garriga M, Marcus IC, Ronda A, Berbezier I,
Maultzsch J, Thomsen C, Hoffmann A. Probing local strain and
composition in Ge nanowires by means of tip-enhanced Raman
scattering. Nanotechnology 2013;24:185704.
[23] Suzuki T, Yan X, Kitahama Y, Sato H, Itoh T, Miura T, Ozaki Y.
Tip-enhanced Raman spectroscopy study of local interactions at
the interface of styrene-butadiene rubber/multiwalled carbon
nanotube nanocomposites. J Phys Chem C 2013;117:1436–40.
[24] Kurouski D, Deckert-Gaudig T, Deckert V, Lednev IK. Structural
characterization of insulin fibril surfaces using Tip Enhanced
Raman Spectroscopy (TERS). Biophys J 2013;104:49A–49A.
[25] Love SA, Marquis BJ, Haynes CL. Recent advances in
nanomaterial plasmonics: fundamental studies and
applications. Appl Spectros 2008;62:346A–362A.
[26] Willets KA, Van Duyne RP. Localized surface plasmon
resonance spectroscopy and sensing. Annu Rev Phys Chem
2007;58:267–97.
[27] Xia Y, Halas NJ. Shape-controlled synthesis and surface
plasmonic properties of metallic nanostructures. MRS Bull
2005;30:338–48.
[28] Haes AJ, Haynes CL, McFarland AD, Schatz GC, Van Duyne RP,
Zou S. Plasmonic materials for surface-enhanced sensing and
spectroscopy. MRS Bull 2005;30:368–75.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM

[29] Schatz GC, Van Duyne RP. Electromagnetic mechanism of
surface-enhanced spectroscopy. Handbook of Vibrational
Spectroscopy (Wiley & Sons, Chichester) 2002;1:759–74.
[30] Schmitt M, Popp J. Raman spectroscopy at the beginning of the
twenty-first century. J Raman Spectros 2006;37:20–8.
[31] Kudelski A. Analytical applications of Raman spectroscopy.
Talanta 2008;76:1–8.
[32] Petry R, Schmitt M, Popp J. Raman spectroscopy-a prospective
tool in the life sciences. ChemPhysChem 2003;4:14–30.
[33] Ferrari AC, Basko DM. Raman spectroscopy as a versatile tool
for studying the properties of graphene. Nat Nanotechnol
2013;8:235–46.
[34] Hering K, Cialla D, Ackermann K, Dörfer T, Möller R,
Schneidewind H, Mattheis R, Fritzsche W, Rösch P, Popp J.
SERS: a versatile tool in chemical and biochemical diagnostics.
Anal Bioanal Chem 2008;390:113–24.
[35] Cialla D, Maerz A, Boehme R, Theil F, Weber K, Schmitt M,
Popp J. Surface-enhanced Raman spectroscopy (SERS):
progress and trends. Anal Bioanal Chem 2012;403:27–54.
[36] Kleinman SL, Frontiera RR, Henry A-I, Dieringer JA, Van Duyne RP.
Creating, characterizing, and controlling chemistry with SERS
hot spots. Phys Chem Chem Phys 2013;15:21–36.
[37] Wang Y, Schluecker S. Rational design and synthesis of SERS
labels. Analyst 2013;138:2224–38.
[38] Bell SEJ, Sirimuthu NMS. Quantitative surface-enhanced
Raman spectroscopy. Chem Soc Rev 2008;37:1012–24.
[39] Smith WE. Practical understanding and use of surface
enhanced Raman scattering/ surface enhanced resonance
Raman scattering in chemical and biological analysis. Chem
Soc Rev 2008;37:955–64.
[40] Stiles PL, Dieringer JA, Shah NC, Van Duyne RP. Surface-
enhanced Raman spectroscopy. Annu Rev Anal Chem
2008;1:601–26.
[41] Xie W, Schluecker S. Medical applications of surface-enhanced
Raman scattering. Phys Chem Chem Phys 2013;15:5329–44.
[42] Anker JN, Hall WP, Lyandres O, Shah NC, Zhao J, Van Duyne RP.
Biosensing with plasmonic nanosensors. Nature Mater
2008;7:442–53.
[43] Graham D, Goodacre R. Chemical and bioanalytical
applications of surface enhanced Raman scattering
spectroscopy. Chem Soc Rev 2008;37:883–4.
[44] Huh YS, Chung AJ, Erickson D. Surface enhanced Raman
spectroscopy and its application to molecular and cellular
analysis. Microfluidics and Nanofluidics 2009;6:285–97.
[45] Fleischmann M, Hendra PJ, McQuillan AJ. Raman spectra
of pyridine adsorbed at a silver electrode. Chem Phys Lett
1974;26:163–6.
[46] Albrecht MG, Creighton JA. Anomalously intense Raman
spectra of pyridine at a silver electrode. J Am Chem Soc
1977;99:5215–7.
[47] Jeanmaire DL, Van Duyne RP. Surface Raman spectroelectro-
chemistry. Part I. Heterocyclic, aromatic, and aliphatic amines
adsorbed on the anodized silver electrode. J Electroanal Chem
Interf Electr 1977;84:1–20.
[48] Xu H, Aizpurua J, Kaell M, Apell P. Electromagnetic contri-
butions to single-molecule sensitivity in surface-enhanced
Raman scattering. Phys Rev E: Stat, Nonlin Soft Matter Phys
2000;62:4318–24.
[49] Cialla D, Petschulat J, Huebner U, Schneidewind H,
Zeisberger M, Mattheis R, Pertsch T, Schmitt M, Möller R, Popp J.
D. Cialla et al.: SERS-based detection of biomolecules      403
Investigation on the second part of the electromagnetic SERS
enhancement and resulting fabrication strategies of anisotropic
plasmonic arrays. ChemPhysChem 2010;11:1918–24.
[50] Itoh T, Yoshida K, Biju V, Kikkawa Y, Ishikawa M, Ozaki Y.
Second enhancement in surface-enhanced resonance Raman
scattering revealed by an analysis of anti-Stokes and Stokes
Raman spectra. Phys Rev B 2007;76:085405/085401–
085405/085405.
[51] Zhang WH, Fischer H, Schmid T, Zenobi R, Martin OJF.
Mode-selective surface-enhanced Raman spectroscopy Using
nanofabricated plasmonic dipole antennas. J Phys Chem C
2009;113:14672–5.
[52] Jensen L, Aikens CM, Schatz GC. Electronic structure methods
for studying surface-enhanced Raman scattering. Chem Soc
Rev 2008;37:1061–73.
[53] Knoll P, Marchl M, Kiefer W. Raman-spectroscopy of
microparticles in laser-light traps. Indian J Pure Ap Phy
1988;26:268–77.
[54] Ayars EJ, Hallen HD. Electric field gradient effects in Raman
spectroscopy. Phys Rev Lett 2000;85:4180–3.
[55] Otto A, Mrozek I, Grabhorn H, Akemann W. Surface-enhanced
Raman scattering. J Phys: Condens Matter 1992;4:1143–212.
[56] Gao X, Davies JP, Weaver MJ. Test of surface selection rules
for surface-enhanced Raman scattering: the orientation of
adsorbed benzene and monosubstituted benzenes on gold.
J Phys Chem 1990;94:6858–64.
[57] Moskovits M, Suh JS. Surface selection rules for surface-
enhanced Raman spectroscopy: calculations and application
to the surface-enhanced Raman spectrum of phthalazine on
silver. J Phys Chem 1984;88:5526–30.
[58] Brown RJC, Milton MJT. Nanostructures and nanostructured
substrates for surface-enhanced Raman scattering (SERS).
J Raman Spectros 2008;39:1313–26.
[59] Wu D-Y, Li J-F, Ren B, Tian Z-Q. Electrochemical surface-
enhanced Raman spectroscopy of nanostructures. Chem Soc
Rev 2008;37:1025–41.
[60] Vo-Dinh T. Surface-enhanced Raman spectroscopy using
metallic nanostructures. TrAC, T Anal Chem 1998;17:557–82.
[61] Vo Dinh T, Stokes DL. SERS-based Raman Probes. Handbook
of Vibrational Spectroscopy (Wiley & Sons, Chichester)
2002;2:1302–17.
[62] Kneipp K, Wang Y, Kneipp H, Perelman LT, Itzkan I, Dasari RR,
Feld MS. Single molecule detection using surface-enhanced
Raman scattering (SERS). Phys Rev Lett 1997;78:1667–70.
[63] Kneipp K, Kneipp H, Itzkan I, Dasari RR, Feld MS. Single
molecule detection using near infrared surface-enhanced
Raman scattering. Springer Ser Chem Phys 2001;67:144–60.
[64] Barhoumi A, Halas NJ. Label-free detection of dna hybridization
using surface enhanced Raman spectroscopy. J Am Chem Soc
2010;132:12792–3.
[65] Papadopoulou E, Bell SEJ. Structure of adenine on metal
nanoparticles: pH equilibria and formation of Ag+ complexes
detected by surface-enhanced Raman spectroscopy. J Phys
Chem C 2010;114:22644–51.
[66] Feng F, Zhi G, Jia HS, Cheng L, Tian YT, Li XJ. SERS detection
of low-concentration adenine by a patterned silver structure
immersion plated on a silicon nanoporous pillar array.
Nanotechnology 2009;20: Article no. 295501, 6 pages.
[67] Kundu J, Neumann O, Janesko BG, Zhang D, Lal S, Barhoumi A,
Scuseria G, Halas NJ. Adenine- and Adenosine Monophosphate
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM
404      D. Cialla et al.: SERS-based detection of biomolecules
(AMP)-gold binding interactions studied by surface-
enhanced Raman and infrared spectroscopies. J Phys Chem C
2009;113:14390–7.
[68] Carrillo-Carrion C, Armenta S, Simonet BM, Valcarcel M,
Lendl B. Determination of pyrimidine and purine bases by
reversed-phase capillary liquid chromatography with at-line
surface-enhanced raman spectroscopic detection employing a
novel sers substrate based on zns/cdse silver-quantum dots.
Anal Chem 2011;83:9391–8.
[69] Primera-Pedrozo OM, Rodriguez GDM, Castellanos J,
Felix-Rivera H, Resto O, Hernández-Rivera SP. Increasing
surface enhanced Raman spectroscopy effect of RNA and
DNA components by changing the pH of silver colloidal
suspensions. SpectrochimActa A 2012;87:77–85.
[70] Papadopoulou E, Bell SEJ. Surface-enhanced Raman evidence
of protonation, reorientation, and Ag+ complexation of
Deoxyadenosine and Deoxyadenosine-5 ′-Monophosphate
(dAMP) on Ag and Au surfaces. J Phys Chem C 2011;115:14228–35.
[71] Muntean CM, Leopold N, Halmagyi A, Valimareanu S. Surface-
enhanced Raman spectroscopy of DNA from leaves of in vitro
grown apple plants. J Raman Spectros 2011;42:844–50.
[72] Barhoumi A, Zhang D, Halas NJ. Correlation of molecular
orientation and packing density in a dsDNA self-assembled
monolayer observable with surface-enhanced-Raman
spectroscopy. J Am Chem Soc 2008;130:14040–1.
[73] Rusciano G, De Luca AC, Pesce G, Sasso A, Oliviero G, Amato J,
Borbone N, D′Errico S, Piccialli V, Piccialli G, Mayol L. Label-free
probing of G-quadruplex formation by surface-enhanced
Raman scattering. Anal Chem 2011;83:6849–55.
[74] Muniz-Miranda M, Gellini C, Pagliai M, Innocenti M, Salvi PR,
Schettino V. SERS and computational studies on MicroRNA chains
adsorbed on silver surfaces. J Phys Chem C 2010;114:13730–5.
[75] Percot A, Lecomte S, Vergne J, Maurel M-C. Hairpin ribozyme
catalysis: a surface-enhanced Raman spectroscopy study.
Biopolymers 2009;91:384–90.
[76] Miljanic S, Dijanosic A, Piantanida I, Meic Z, Albelda MT,
Sornosa-Ten A, García-Espana E. Surface-enhanced Raman
study of the interactions between tripodal cationic polyamines
and polynucleotides. Analyst 2011;136:3185–93.
[77] Luo XL, Buckhout-White S, Bentley WE, Rubloff GW. Biofab-
rication of chitosan-silver composite SERS substrates enabling
quantification of adenine by a spectroscopic shift. Biofab-
rication 2011;3: Article no. 034108, 9 pages.
[78] Yin P-G, Jiang L, Lang X-F, Guo L, Yang S. Quantitative analysis of
mononucleotides by isotopic labeling surface-enhanced Raman
scattering spectroscopy. Biosens Bioelectron 2011;26:4828–31.
[79] Chen J-W, Liu X-P, Feng K-J, Liang Y, Jiang JH, Shen GL, Yu RQ.
Detection of adenosine using surface-enhanced Raman
scattering based on structure-switching signaling aptamer.
Biosens Bioelectron 2008;24:66–71.
[80] Kim NH, Lee SJ, Moskovits M. Aptamer-mediated surface-
enhanced Raman spectroscopy intensity amplification. Nano
Letters 2010;10:4181–5.
[81] Li M, Zhang J, Suri S, Sooter LJ, Ma D, Wu N. Detection
of adenosine triphosphate with an aptamer biosensor
based on surface-enhanced Raman scattering. Anal Chem
2012;84:2837–42.
[82] Ye S, Xiao J, Guo Y, Zhang S. Aptamer-based SERS assay of
ATP and lysozyme by using primer self-generation. Chemistry
(Weinheim an der Bergstrasse, Germany) 2013;19:8111–6.
[83] Li M, Zhang J, Suri S, Sooter LJ, Ma D, Wu N. Quantitative
label-free RNA detection using surface-enhanced Raman
spectroscopy. Chem Commun 2011;47:7425–7.
[84] Rao S, Raj S, Balint S, Bardina Fons C, Campoy S, Llagostera M,
Petrov D. Single DNA molecule detection in an optical trap
using surface-enhanced Raman scattering. Appl Phys Lett
2010;96:213701.
[85] Liu R, Zhu S, Si M, Liu Z, Zhang D. Surface-enhanced Raman
scattering-based approach for DNA detection at low concen-
trations via polyvinyl alcohol-protected silver grasslike
patterns. J Raman Spectros 2012;43:370–9.
[86] Yuan W, Ho HP, Lee RKY, Kong SK. Surface-enhanced Raman
scattering biosensor for DNA detection on nanoparticle island
substrates. Appl Optics 2009;48:4329–37.
[87] Fang C, Agarwal A, Buddharaju KD. Khalid NM, Salim SM,
Widjaja E, Garland MV, Balasubramanian N, Kwong DL.
DNA detection using nanostructured SERS substrates
with Rhodamine B as Raman label. Biosens Bioelectron
2008;24:216–21.
[88] Strelau KK, Kretschmer R, Moller R, Fritzsche W, Popp J. SERS
as tool for the analysis of DNA-chips in a microfluidic platform.
Anal Bioanal Chem 2010;396:1381–4.
[89] Papadopoulou E, Bell SEJ. Label-free detection of single-base
mismatches in DNA by surface-enhanced Raman spectroscopy.
Angew Chem-Int Edit 2011;50:9058–61.
[90] Li J-M, Ma W-F, You L-J, Guo J, Hu J, Wang CC. Highly sensitive
detection of target ssDNA based on SERS liquid chip using
suspended magnetic nanospheres as capturing substrates.
Langmuir 2013;29:6147–55.
[91] Zhang Z, Wen Y, Ma Y, Luo J, Jiang L, Song Y. Mixed
DNA-functionalized nanoparticle probes for surface-enhanced
Raman scattering-based multiplex DNA detection. Chem
Commun 2011;47:7407–9.
[92] Thompson DG, Faulds K, Smith WE, Graham D. Precise
control of the assembly of dye-coded oligonucleotide silver
nanoparticle conjugates with single base mismatch discrim-
ination using surface enhanced resonance Raman scattering.
J Phys Chem C 2010;114:7384–9.
[93] Thuy NTB, Yokogawa R, Yoshimura Y, Fujimoto K, Koyano M,
Maenosono S. Surface-enhanced Raman spectroscopy for
facile DNA detection using gold nanoparticle aggregates
formed via photoligation. Analyst 2010;135:595–602.
[94] He Y, Su S, Xu T, Zhong Y, Zapien JA, Li J, Fan C, Lee S.-T. Silicon
nanowires-based highly-efficient SERS-active platform for
ultrasensitive DNA detection. Nano Today 2011;6:122–30.
[95] Sun Y-H, Kong R-M, Lu D-Q, Zhang X-B, Meng HM, Tan M,
Shen GL, Yu RQ. A nanoscale DNA-Au dendrimer as a signal
amplifier for the universal design of functional DNA-based
SERS biosensors. Chem Commun 2011;47:3840–2.
[96] Panikkanvalappil SR, Mackey MA, El-Sayed MA. Probing the
unique dehydration-induced structural modifications in cancer
cell dna using surface enhanced raman spectroscopy. J Am
Chem Soc 2013;135:4815–21.
[97] Hu J, Zhang C-y. Sensitive detection of nucleic acids with rolling
circle amplification and surface-enhanced raman scattering
spectroscopy. Anal Chem 2010;82:8991–7.
[98] Ye S, Yang Y, Xiao J, Zhang S. Surface-enhanced Raman
scattering assay combined with autonomous DNA machine
for detection of specific DNA and cancer cells. Chem Commun
2012;48:8535–7.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM

[99] Yan J, Su S, He S, He Y, Zhao B, Wang D, Zhang H, Huang Q,
Song S, Fan C. Nano rolling-circle amplification for enhanced
SERS hot spots in protein microarray analysis. Anal Chem
2012;84:9139–45.
[100] Hering KK, Moller R, Fritzsche W, Popp J. Microarray-based
detection of dye-labeled DNA by SERRS using particles
formed by enzymatic silver deposition. ChemPhysChem
2008;9:867–72.
[101] Diaz Fleming G, Finnerty JJ, Campos-Vallette M, Celis F, Aliaga
AE, Fredes C, Koch R. Experimental and theoretical Raman and
surface-enhanced Raman scattering study of cysteine.
J Raman Spectros 2009;40:632–8.
[102] Chuang C-H, Chen Y-T. Raman scattering of L-tryptophan en-
hanced by surface plasmon of silver nanoparticles: vibration-
al assignment and structural determination. J Raman Spectros
2009;40:150–6.
[103] Aliaga AE, Osorio-Roman I, Leyton P, Garrido C, Carcamo J,
Caniulef C, Celis F, Diaz F, Clavijo E, Gomez-Jeria JS, Campos-
Vallete MM. Surface-enhanced Raman scattering study of
L-tryptophan. J Raman Spectros 2009;40:164–9.
[104] Aliaga AE, Osorio-Roman I, Garrido C, Leyton P, Cárcamo J,
Clavijo E, Gómez-Jeria JS, Díaz F, Campos-Vallette MM. Surface
enhanced Raman scattering study of L-lysine. Vib Spectros
2009;50:131–5.
[105] Aliaga AE, Garrido C, Leyton P, Diaz FG, Gomez-Jeria JS,
Aguayo T, Clavijo E, Campos-Vallette MM, Sanchez-Cortes S.
SERS and theoretical studies of arginine. Spectrochim Acta A
2010;76:458–63.
[106] Yang H, Zhu X, Song W, Sun Y, Duan G, Zhao X, Zhang Z. N-
acetylalanine monolayers at the silver surface investigated by
surface enhanced Raman scattering spectroscopy and X-ray
photoelectron spectroscopy: effect of metallic ions. J Phys
Chem C 2008;112:15022–7.
[107] Sheng C, Zhao H, Gu F, Yang H. Effect of Pb2+ on L-glutathione
monolayers on a silver surface investigated by surface-
enhanced Raman scattering spectroscopy. J Raman Spectros
2009;40:1274–8.
[108] Graff M, Bukowska J. Surface-enhanced Raman scattering
(SERS) spectroscopy of enantiomeric and racemic methionine
on a silver electrode-evidence for chiral discrimination in
interactions between adsorbed molecules. Chem Phys Lett
2011;509:58–61.
[109] Graff M, Bukowska J. Enantiomeric recognition of pheny-
lalanine by self-assembled monolayers of cysteine: Sur-
face enhanced Raman scattering evidence. Vib Spectros
2010;52:103–7.
[110] Thomas S, Biswas N, Malkar VV, Mukherjee T, Kapoor S.
Studies on adsorption of carnosine on silver nanoparticles by
SERS. Chem Phys Lett 2010;491:59–64.
[111] Podstawka E, Andrzejak M, Kafarski P, Proniewicz LM.
Comparison of adsorption mechanism on colloidal silver
surface of alafosfalin and its analogs. J Raman Spectros
2008;39:1238–49.
[112] Podstawka E, Kafarski P, Proniewicz LM. Effect of an aliphatic
spacer group on the adsorption mechanism of phosphonodi-
peptides containing N-terminal glycine on the colloidal silver
surface. J Raman Spectros 2008;39:1396–407.
[113] Malek K, Makowski M, Krolikowska A, Bukowska J. Compara-
tive studies on IR, Raman, and surface enhanced Raman
scattering spectroscopy of dipeptides containing Delta Ala
D. Cialla et al.: SERS-based detection of biomolecules      405
and Delta Phe. J Phys Chem B 2012;116:1414–25.
[114] Yuan X, Gu H, Wu J. Surface-enhanced Raman spectrum of
Gly-Gly adsorbed on the silver colloidal surface. J Mol Struct
2010;977:56–61.
[115] Wei F, Zhang D, Halas NJ, Hartgerink JD. Aromatic amino acids
providing characteristic motifs in the Raman and SERS spec-
troscopy of peptides. J Phys Chem B 2008;112:9158–64.
[116] Podstawka E, Kafarski P, Proniewicz LM. Structural properties
of L-X-L-Met-L-Ala phosphonate tripeptides: a combined FT-IR,
FT-RS, and SERS spectroscopy studies and DFT calculations.
J Phys Chem A 2008;112:11744–55.
[117] Podstawka E. Effect of amino acid modifications on the
molecular structure of adsorbed and nonadsorbed bombesin
6-14 fragments on an electrochemically roughened silver
surface. J Raman Spectros 2008;39:1290–1305.
[118] Podstawka E, Niaura G, Proniewicz LM. Potential-dependent
studies on the interaction between phenylalanine-substituted
bombesin fragments and roughened Ag, Au, and Cu electrode
surfaces. J Phys Chem B 2010;114:1010–29.
[119] Podstawka E, Ozaki Y, Proniewicz LM. Structures and bonding
on a colloidal silver surface of the various length carboxyl ter-
minal fragments of bombesin. Langmuir 2008;24:10807–16.
[120] Podstawka-Proniewicz E, Ozaki Y, Kim Y, Xu Y, Proniewicz LM.
Surface-enhanced Raman scattering studies on bombesin,
its selected fragments and related peptides adsorbed at the
silver colloidal surface. Appl Surf Sci 2011;257:8246–52.
[121] Podstawka E, Proniewicz LM. The orientation of BN-related
peptides adsorbed on SERS-active silver nanoparticles:
comparison with a silver electrode surface. J Phys Chem B
2009;113:4978–85.
[122] Podstawka-Proniewicz E, Kudelski A, Kim Y, Proniewicz LM.
Structure and binding of specifically mutated neurotensin
fragments on a silver substrate: vibrational studies. J Phys
Chem B 2011;115:7097–108.
[123] Podstawka-Proniewicz E, Ignatjev I, Niaura G, Proniewicz LM.
Phe-MetNH(2) terminal bombesin subfamily peptides: poten-
tial induced changes in adsorption on Ag, Au, and Cu elec-
trodes monitored by SERS. J Phys Chem C 2012;116:4189–200.
[124] Ignatjev I, Podstawka-Proniewicz E, Niaura G, Lombardi
JR, Proniewicz LM. Potential induced changes in neuro-
medin B adsorption on Ag, Au, and Cu electrodes monitored
by surface-enhanced Raman scattering. J Phys Chem B
2011;115:10525–36.
[125] Aliaga AE, Aguayo T, Garrido C, Clavijo E, Hevia E, Gómez-
Jeria JS, Leyton P, Campos-Vallette MM, Sanchez-Cortes S.
Surface-enhanced Raman scattering and theoretical studies
of the C-terminal peptide of the beta-subunit human chorionic
gonadotropin without linked carbohydrates. Biopolymers
2011;95:135–43.
[126] Garrido C, Aliaga AE, Gomez-Jeria JS, Clavijo RE, Campos-
Vallette MM, Sanchez-Cortes S. Adsorption of oligopeptides
on silver nanoparticles: surface-enhanced Raman scattering
and theoretical studies. J Raman Spectros 2010;41:1149–55.
[127] Aliaga AE, Ahumada H, Sepulveda K, Gomez-Jeria JS, Garrido C,
Weiss-Lopez BE, Campos-Vallette MM. SERS, molecular
dynamics and molecular orbital studies of the MRKDV
peptide on silver and membrane surfaces. J Phys Chem C
2011;115:3982–9.
[128] Podstawka-Proniewicz E, Kosior M, Kim Y, Rolka K,
Proniewicz LM. Nociceptin and Its natural and specifically-
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM
406      D. Cialla et al.: SERS-based detection of biomolecules
modified fragments: structural studies. Biopolymers
2010;93:1039–54.
[129] Iosin M, Toderas F, Baldeck PL, Astilean S. Study of protein-gold
nanoparticle conjugates by fluorescence and surface-enhanced
Raman scattering. J Mole Struct 2009;924–26:196–200.
[130] Das R, Jagannathan R, Sharan C, Kumar U, Poddar P. Mecha-
nistic study of surface functionalization of enzyme lysozyme
synthesized Ag and Au nanoparticles using surface enhanced
Raman spectroscopy. J Phys Chem C 2009;113:21493–500.
[131] Chandra G, Ghosh KS, Dasgupta S, Roy A. Evidence of confor-
mational changes in adsorbed lysozyme molecule on silver
colloids. Int J Biol Macromol 2010;47:361–5.
[132] Sengupta A, Thai CK, Sastry MSR, Matthaei JF, Schwartz DT,
Davis EJ, Baneyx F. A genetic approach for controlling the
binding and orientation of proteins on nanoparticles. Lang-
muir 2008;24:2000–8.
[133] Kumar GVP, Selvi R, Kishore AH, KunduTK, Narayana C.
Surface-enhanced Raman, spectroscopic studies of coactiva-
tor-associated arginine methyltransferase 1. J Phys Chem B
2008;112:6703–7.
[134] Kudelski A. In situ SERS studies on the adsorption of tyrosi-
nase on bare and alkanethiol-modified silver substrates. Vib
Spectros 2008;46:34–8.
[135] Li D, Li D-W, Fossey JS, Long Y-T. In situ surface-enhanced
Raman scattering and X-ray photoelectron spectroscopic in-
vestigation of coenzyme Q(10) on silver electrode. Phys Chem
Chem Phys 2011;13:2259–65.
[136] Kaminska A, Forster RJ, Keyes TE. The impact of adsorption of
bovine pancreatic trypsin inhibitor on CTAB-protected gold
nanoparticle arrays: a Raman spectroscopic comparison with
solution denaturation. J Raman Spectros 2010;41:130–5.
[137] Krolikowska A, Bukowska J. Surface-enhanced resonance
Raman spectroscopic characterization of cytochrome c immo-
bilized on 2-mercaptoethanesulfonate monolayers on silver.
J Raman Spectros 2010;41:1621–31.
[138] Papazoglou ES, Babu S, Hansberry DR, Mohapatra S, Patel C.
SERS study on myeloperoxidase and its immunocomplex: Identi-
fication of binding interactions. Spectros Int J 2010;24:183–90.
[139] Choi I, Huh YS, Erickson D. Ultra-sensitive, label-free probing
of the conformational characteristics of amyloid beta aggre-
gates with a SERS active nanofluidic device. Microfluidics and
Nanofluidics 2012;12:663–9.
[140] Abdali S, De Laere B, Poulsen M, Grigorian M, Lukanidin E,
Klingelhöfer J. Toward methodology for detection of cancer-
promoting S100A4 protein conformations in subnanomolar
concentrations using Raman and SERS. J Phys Chem C
2010;114:7274–9.
[141] Singhal K, Kalkan AK. Surface-enhanced raman scattering
captures conformational changes of single photoactive yellow
protein molecules under photoexcitation. J Am Chem Soc
2010;132:429–31.
[142] Mueller J, Becher T, Braunstein J, Berdel P, Gravius S, Rohr-
bach F, Oldenburg J, Mayer G, Pötzsch B. Profiling of active
thrombin in human blood by supramolecular complexes.
Angew Chem-Int Edit 2011;50:6075–8.
[143] Nierodzik ML, Karpatkin S. Thrombin induces tumor growth,
metastasis, and angiogenesis: Evidence for a thrombin-regu-
lated dormant tumor phenotype. Cancer Cell 2006;10:355–62.
[144] Song K-M, Lee S, Ban C. Aptamers and their biological ap-
plications. Sensors 2012;12:612–31.
[145] Wu Z, Liu Y, Zhou X, Shen A, Hu J. A "turn-off′ SERS-based
detection platform for ultrasensitive detection of throm-
bin based on, enzymatic assays. Biosens Bioelectron
2013;44:10–15.
[146] Ochsenkuehn MA, Campbell CJ. Probing biomolecular interac-
tions using surface enhanced Raman spectroscopy: label-free
protein detection using a G-quadruplex DNA aptamer. Chem
Commun 2010;46:2799–801.
[147] Pagba CV, Lane SM, Cho H, Wachsmann-Hogiu S. Direct
detection of aptamer-thrombin binding via surface-enhanced
Raman spectroscopy. J Biomed Optics 2010;15.
[148] Fabris L, Dante M, Nguyen TQ, Tok JBH, Bazan GC. SERS
aptatags: New responsive metallic nanostructures for het-
erogeneous protein detection by surface enhanced Raman
spectroscopy. Adv Funct Mater 2008;18:2518–25.
[149] Hu J, Zheng P-C, Jiang J-H, Shen G-L, Yu RQ, Liu GK. Elec-
trostatic interaction based approach to thrombin detec-
tion by surface-enhanced Raman spectroscopy. Anal Chem
2009;81:87–93.
[150] Wang YL, Lee K, Irudayaraj J. SERS aptasensor from nanorod-
nanoparticle junction for protein detection. Chem Commun
2010;46:613–5.
[151] Yoon J, Choi N, Ko J, Kim K, Lee S, Choo J. Highly sensitive de-
tection of thrombin using SERS-based magnetic aptasensors.
Biosens Bioelectron 2013;47:62–7.
[152] Kennedy DC, Hoop KA, Tay L-L, Pezacki JP. Development of na-
noparticle probes for multiplex SERS imaging of cell surface
proteins. Nanoscale 2010;2:1413–6.
[153] Woo MA, Lee S-M, Kim G, Baek J, Noh MS, Kim JE, Park SJ,
Minai-Tehrani A, Park SC, Seo YT, Kim YK, Lee YS, Jeong DH,
Cho MH. Multiplex immunoassay using fluorescent-surface
enhanced Raman spectroscopic dots for the detection of
bronchioalveolar stem Cells in murine lung. Anal Chem
2009;81:1008–15.
[154] Hodges MD, Kelly JG, Bentley AJ, Fogarty S, Patel II, Martin FL,
Fullwood NJ. Combining immunolabeling and surface-en-
hanced Raman spectroscopy on cell membranes. ACS Nano
2011;5:9535–41.
[155] Sujith A, Itoh T, Abe H, Yoshida K-i, Kiran MS, Biju V,
Ishikawa M. Imaging the cell wall of living single yeast cells
using surface-enhanced Raman spectroscopy. Anal Bioanal
Chem 2009;394:1803–9.
[156] Hollywood KA, Shadi IT, Goodacre R. Monitoring the succinate
dehydrogenase activity isolated from mitochondria by
surface enhanced Raman scattering. J Phys Chem C 2010;114:
7308–13.
[157] Yazgan NN, Boyaci IH, Temur E, Tamer U, Topcu A. A high
sensitive assay platform based on surface-enhanced Raman
scattering for quantification of protease activity. Talanta
2010;82:631–9.
[158] Li T, Liu D, Wang Z. Microarray-based Raman spectroscopic
assay for kinase inhibition by gold nanoparticle probes. Bios-
ens Bioelectron 2009;24:3335–9.
[159] Stevenson R, Stokes RJ, MacMillan D, Armstrong D, Faulds K,
Wadsworth R, Kunuthur S, Suckling CJ, Graham D. In situ
detection of pterins by SERS. Analyst 2009;134:1561–4.
[160] He P, Zhang Y, Liu L, Qiao W, Zhang S. Ultrasensitive SERS
detection of lysozyme by a target-triggering multiple cycle
amplification strategy based on a gold substrate. Chemistry
(Weinheim an der Bergstrasse, Germany) 2013;19:7452–60.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM

[161] Joshi B, Chakrabarty A, Bruot C, Ainsworth H, Fraizer G, Wei
QH. DNA-WT1 protein interaction studied by surface-enhanced
Raman spectroscopy. Anal Bioanal Chem 2010;396:1415–21.
[162] Han XX, Kitahama Y, Tanaka Y, Guo J, Xu WQ, Zhao B, Ozaki Y.
Simplified protocol for detection of protein-ligand interac-
tions via surface-enhanced resonance Raman scattering and
surface-enhanced fluorescence. Anal Chem 2008;80:6567–72.
[163] Chen L, Hong W, Guo Z, Sa Y, Wang X, Jung YM, Zhao B.
Magnetic assistance highly sensitive protein assay based
on surface-enhanced resonance Raman scattering. J Colloid
Interf Sci 2012;368:282–6.
[164] Levin CS, Kundu J, Janesko BG, Scuseria GE, Raphael RM,
Halas NJ. Interactions of Ibuprofen with hybrid lipid bilay-
ers probed by complementary surface-enhanced vibrational
spectroscopies. J Phys Chem B 2008;112:14168–75.
[165] Lajos G, Jancura D, Miskovsky P, Garcia-Ramos JV, Sanchez-
Cortes S. Interaction of the photosensitizer hypericin with
low-density lipoproteins and phosphatidylcholine: a surface-
enhanced raman scattering and surface-enhanced fluores-
cence study. J Phys Chem C 2009;113:7147–54.
[166] Premasiri WR, Sauer-Budge AF, Lee JC, Klapperich CM,
Ziegler LD. Rapid bacterial diagnostics via surface-en-
hanced Raman microscopy. Spectroscopy - Special Issues
2012;27:s8–s21.
[167] O’Brien JM, Jr, Ali NA, Aberegg SK, Abraham E. Sepsis. Am J
Med 2007;120:1012–22.
[168] Brumbaugh AR, Mobley HLT. Preventing urinary tract infec-
tion: progress toward an effective Escherichia coli vaccine.
Expert Rev Vaccines 2012;11:663–76.
[169] Wertheim H, Verbrugh HA, van Pelt C, de Man P,
van ­Belkum A, Vos MC. Improved detection of methicillin-
resistant Staphylococcus aureus using phenyl mannitol
broth containing aztreonam and ceftizoxime. J Clin Microbiol
2001;39:2660–2.
[170] Dougan JA, MacRae D, Graham D, Faulds K. DNA detection
using enzymatic signal production and SERS. Chem Commun
2011;47:4649–51.
[171] Kujau MJ, Wolfl S. Efficient preparation of single-stranded
DNA for in vitro selection. Mole Biotechnol 1997;7:333–5.
[172] Graham D, Stevenson R, Thompson DG, Barrett L, Dalton C,
Faulds K. Combining functionalised nanoparticles and SERS
for the detection of DNA relating to disease. Faraday Discuss
2011;149:291–9.
[173] Faulds K, Jarvis R, Smith WE, Graham D, Goodacre R. Multi-
plexed detection of six labelled oligonucleotides using sur-
face enhanced resonance Raman scattering (SERRS). Analyst
2008;133:1505–12.
[174] Papadopoulou E, Bell SEJ. Label-Free detection of nanomo-
lar unmodified single- and double-stranded DNA by using
surface-enhanced Raman spectroscopy on Ag and Au colloids.
Chem Eur J 2012;18:5394–400.
[175] Kang T, Yoo SM, Yoon I, Lee SY, Kim B. Patterned multiplex
pathogen DNA detection by Au particle-on-wire SERS sensor.
Nano Letters 2010;10:1189–93.
[176] MacAskill A, Crawford D, Graham D, Faulds K. DNA sequence
detection using surface-enhanced resonance Raman spec-
troscopy in a homogeneous multiplexed assay. Anal Chem
2009;81:8134–40.
[177] van Lierop D, Faulds K, Graham D. Separation free DNA detec-
tion using surface enhanced Raman scattering. Anal Chem
2011;83:5817–21.
D. Cialla et al.: SERS-based detection of biomolecules      407
[178] van Lierop D, Larmour IA, Faulds K, Graham D. SERS primers
and their mode of action for pathogen DNA detection. Anal
Chem 2013;85:1408–14.
[179] Harper MM, Robertson B, Ricketts A, Faulds K. Specific
detection of DNA through coupling of a TaqMan assay with
surface enhanced Raman scattering (SERS). Chem Commun
2012;48:9412–14.
[180] Strelau KK, Brinker A, Schnee C, Weber K, Möller R, Popp J.
Detection of PCR products amplified from DNA of epizootic
pathogens using magnetic nanoparticles and SERS. J Raman
Spectros 2011;42:243–50.
[181] Osorio-Roman IO, Aroca RF, Astudillo J, Matsuhiro B,
Vásquez C, Pérez JM. Characterization of bacteria using its
O-antigen with surface-enhanced Raman scattering. Analyst
2010;135:1997–2001.
[182] Yang X, Gu C, Qian F, Li Y, Zhang JZ. Highly sensitive detection
of proteins and bacteria in aqueous solution using surface-
enhanced Raman scattering and optical fibers. Anal Chem
2011;83:5888–94.
[183] Arruebo M, Valladares M, Gonzalez-Fernandez A. Antibody-
conjugated nanoparticles for biomedical applications.
J Nanomaterials 2009;2009: Article ID 439389, 24 pages
(doi: 10.1155/2009/439389).
[184] Ravindranath SP, Wang Y, Irudayaraj J. SERS driven cross-
platform based multiplex pathogen detection. Sensor Actuat
B-Chem 2011;152:183–90.
[185] Lin C-C, Yang Y-M, Chen Y-F, Yang T-S, Chang H-C. A new pro-
tein A assay based on Raman reporter labeled immunogold
nanoparticles. Biosens Bioelectron 2008;24:178–83.
[186] Yakes BJ, Lipert RJ, Bannantine JP, Porter MD. Detection of
Mycobacterium avium subsp paratuberculosis by a sonicate
immunoassay based on surface-enhanced Raman scattering.
Clin Vaccine Immunol 2008;15:227–34.
[187] Colpitts TM, Conway MJ, Montgomery RR, Fikrig E. West nile
virus: biology, transmission, and human infection. Clin micro-
biol rev 2012;25:635–48.
[188] Zhang H, Harpster MH, Park HJ, Johnson PA. Surface-en-
hanced Raman scattering detection of DNA derived from the
west nile virus genome using magnetic capture of Raman-
active gold nanoparticles. Anal Chem 2011;83:254–60.
[189] Zhang H, Harpster MH, Wilson WC, Johnson PA. Surface-
enhanced Raman scattering detection of DNAs derived from
virus genomes using Au-coated paramagnetic nanoparticles.
Langmuir 2012;28:4030–7.
[190] Neng J, Harpster MH, Wilson WC, Johnson PA. Surface-
enhanced Raman scattering (SERS) detection of multiple viral
antigens using magnetic capture of SERS-active nanoparti-
cles. Biosens Bioelectron 2013;41:316–21.
[191] Tsongalis GJ. Branched DNA technology in molecular diagnos-
tics. Am J Clin Pathol 2006;126:448–53.
[192] Hu J, Zheng P-C, Jiang J-H, Shen G-L, Yu RQ, Liu GK. Sub-at-
tomolar HIV-1 DNA detection using surface-enhanced Raman
spectroscopy. Analyst 2010;135:1084–9.
[193] Huh YS, Chung AJ, Cordovez B, Erickson D. Enhanced on-chip
SERS based biomolecular detection using electrokinetically
active microwells. Lab on a Chip 2009;9:433–9.
[194] Tang KF, Ooi EE. Diagnosis of dengue: an update. Expert Rev
Anti-Infective Therapy 2012;10:895–907.
[195] Kumar S, Henrickson KJ. Update on influenza diagnostics:
lessons from the novel H1N1 Influenza a pandemic. Clin
microbiol rev 2012;25:344–61.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM
408      D. Cialla et al.: SERS-based detection of biomolecules
[196] Seol M-L, Choi S-J, Baek DJ, Park TJ, Ahn J-H, Lee SY, Choi YK.
A nanoforest structure for practical surface-enhanced Raman
scattering substrates. Nanotechnology 2012;23: Article
no. 095301, 7 pages.
[197] Negri P, Kage A, Nitsche A, Naumann D, Dluhy RA. Detection
of viral nucleoprotein binding to anti-influenza aptamers via
SERS. Chem Commun 2011;47:8635–7.
[198] Marotta NE, Beavers KR, Bottomley LA. Limitations of surface
enhanced Raman scattering in sensing DNA hybridization
demonstrated by label-free DNA oligos as molecular rulers of
distance-dependent enhancement. Anal Chem 2013;85:1440–6.
[199] Chang J. Current progress on development of respiratory
syncytial virus vaccine. Bmb Reports 2011;44:232–7.
[200] Chen Y, Zheng X, Chen G, He C, Zhu W, Feng S, Xi G, Chen R,
Lan F, Zeng H. Immunoassay for LMP1 in nasopharyngeal
tissue based on surface-enhanced Raman scattering. Int J
Nanomed 2012;7:73–82.
[201] Lin J, Chen R, Feng S, Pan J, Li B, Chen G, Lin S, Li C, Sun L,
Huang Z, Zeng H. Surface-enhanced Raman scattering spec-
troscopy for potential noninvasive nasopharyngeal cancer
detection. J Raman Spectros 2012;43:497–502.
[202] Lin J, Chen R, Feng S, Pan J, Li Y, Chen G, Cheng M, Huang Z,
Yu Y, Zeng H. A novel blood plasma analysis technique
combining membrane electrophoresis with silver nanopar-
ticle-based SERS spectroscopy for potential applications in
noninvasive cancer detection. Nanomed-Nanotechnol Biol
Med 2011;7:655–63.
[203] Feng SY, Chen R, Lin JQ, Pan JJ, Wu Y, Li Y, Chen J, Zeng H.
Gastric cancer detection based on blood plasma surface-en-
hanced Raman spectroscopy excited by polarized laser light.
Biosens Bioelectron 2011;26:3167–74.
[204] Pinzani P, Salvianti F, Pazzagli M, Orlando C. Circulating nu-
cleic acids in cancer and pregnancy. Methods 2010;50:302–7.
[205] Li S-X, Zeng Q-Y, Li L-F, Zhang Y-J, Wan MM, Liu ZM, Xiong HL,
Guo ZY, Liu SH. Study of support vector machine and serum
surface-enhanced Raman spectroscopy for noninvasive
esophageal cancer detection. J Biomed Opt 2013;18:027008.
[206] Lin D, Feng S, Pan J, Chen Y, Lin J, Chen G, Xie S, Zeng H,
Chen R. Colorectal cancer detection by gold nanoparticle
based surface-enhanced Raman spectroscopy of blood serum
and statistical analysis. Opt Express 2011;19:13565–77.
[207] Feng S, Chen R, Lin J, Pan J, Chen G, Li Y, Cheng M, Huang Z,
Chen J, Zeng H. Nasopharyngeal cancer detection based on
blood plasma surface-enhanced Raman spectroscopy and
multivariate analysis. Biosens Bioelectron 2010;25:2414–9.
[208] Feng S, Lin D, Lin J, Li B, Huang Z, Chen G, Zhang W, Wang L,
Pan J, Chen R, Zeng H. Blood plasma surface-enhanced Raman
spectroscopy for non-invasive optical detection of cervical
cancer. Analyst 2013;138:3967–74.
[209] Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA
in plasma or serum as biomarker of carcinogenesis: practical
aspects and biological significance. Mutat Res-Rev Mutat
2007;635:105–117.
[210] Chen Y, Chen G, Feng S, Pan J, Zheng X, Su Y, Chen Y, Huang Z,
Lin X, Lan F, Chen R, Zeng H. Label-free serum ribonucleic acid
analysis for colorectal cancer detection by surface-enhanced
Raman spectroscopy and multivariate analysis. J Biomed Opt
2012;17:067003.
[211] Arya SK, Lim B, Rahman ARA. Enrichment, detection and
clinical significance of circulating tumor cells. Lab on a Chip
2013;13:1995–2027.
[212] Wang X, Qian X, Beitler JJ, Chen ZG, Khuri FR, Lewis MM,
Shin HJ, Nie S, Shin DM. Detection of circulating tumor
cells in human peripheral blood using surface-enhanced
­Raman scattering nanoparticles. Cancer Res 2011;71:
1526–32.
[213] Sha MY, Xu H, Natan MJ, Cromer R. Surface-enhanced Raman
scattering tags for rapid and homogeneous detection of circu-
lating tumor cells in the presence of human whole blood. J Am
Chem Soc 2008;130:17214–5.
[214] Lee K, Drachev VP, Irudayaraj J. DNA-Gold nanoparticle revers-
ible networks grown on cell surface marker sites: application
in diagnostics. Acs Nano 2011;5:2109–17.
[215] MacLaughlin CM, Mullaithilaga N, Yang G, Ip SY, Wang C,
Walker GC. Surface-enhanced Raman scattering dye-
labeled Au nanoparticles for triplexed detection of leukemia
and lymphoma cells and SERS flow cytometry. Langmuir
2013;29:1908–19.
[216] Nguyen CT, Nguyen JT, Rutledge S, Zhang J, Wang C,
Walker GC. Detection of chronic lymphocytic leukemia cell
surface markers using surface enhanced Raman scattering
gold nanoparticles. Cancer Lett 2010;292:91–7.
[217] Dougherty U, Sehdev A, Cerda S, Mustafi R, Little N, Yuan W,
Jagadeeswaran S, Chumsangsri A, Delgado J, Tretiakova M,
Joseph L, Hart J, Cohen EE, Aluri L, Fichera A, Bissonnette M.
Epidermal growth factor receptor controls flat dysplastic aber-
rant crypt foci development and colon cancer progression in
the rat azoxymethane model. Clin Cancer Res 2008;14:
2253–62.
[218] Qian X, Peng X-H, Ansari DO, Yin-Goen Q, Chen GZ, Shin DM,
Yang L, Young AN, Wang MD, Nie S. In vivo tumor targeting
and spectroscopic detection with surface-enhanced Raman
nanoparticle tags. Nat Biotechnol 2008;26, 83–90.
[219] Jokerst JV, Miao Z, Zavaleta C, Cheng Z, Gambhir SS. Affibody-
functionalized gold-silica nanoparticles for Raman molecu-
lar imaging of the epidermal growth factor receptor. Small
2011;7:625–33.
[220] Dinish US, Fu CY, Soh KS, Bhuvaneswari R, Kumar A, Olivo M.
Highly sensitive SERS detection of cancer proteins in low
sample volume using hollow core photonic crystal fiber. Bios-
ens Bioelectron 2012;33:293–8.
[221] Maiti KK, Samanta A, Vendrell M, Soh K-S, Olivo M, Chang YT.
Multiplex cancer cell detection by SERS nanotags with cya-
nine and triphenylmethine Raman reporters. Chem Commun
2011;47:3514–6.
[222] Yang J, Wang Z, Zong S, Song C, Zhang R, Cui Y. Distinguishing
breast cancer cells using surface-enhanced Raman scattering.
Anal Bioanal Chem 2012;402:1093–100.
[223] Lee S, Chon H, Lee M, Choo J, Shin SY, Lee YH, Rhyu IJ,
Son SW, Oh CH. Surface-enhanced Raman scattering imaging
of HER2 cancer markers overexpressed in single MCF7 cells
using antibody conjugated hollow gold nanospheres. Biosens
Bioelectron 2009;24:2260–3.
[224] Park H, Lee S, Chen L, Lee EK, Shin SY, Lee YH, Son SW,
Oh CH, Song JM, Kang SH, Choo J. SERS imaging of HER2-
overexpressed MCF7 cells using antibody-conjugated gold
nanorods. Phys Chem Chem Phys 2009;11:7444–9.
[225] Wang HN, Vo-Dinh T. Multiplex detection of breast cancer
biomarkers using plasmonic molecular sentinel nanoprobes.
Nanotechnology 2009;20:065101.
[226] Ferrara N. Vascular endothelial growth factor: basic science
and clinical progress. Endocr Rev 2004;25:581–611.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM

[227] Ko J, Lee S, Lee EK, Chang S-I, Chen L, Yoon SY, Choo J. SERS-
based immunoassay of tumor marker VEGF using DNA aptam-
ers and silica-encapsulated hollow gold nanospheres. Phys
Chem Chem Phys 2013;15:5379–85.
[228] Li M, Cushing SK, Zhang J, Suri S, Evans R, Petros WP, Gibson
LF, Ma D, Liu Y, Wu N. Three-dimensional hierarchical plas-
monic nano-architecture enhanced surface-enhanced Raman
scattering immunosensor for cancer biomarker detection in
blood plasma. ACS Nano 2013;7:4967–76.
[229] Wu P, Gao Y, Zhang H, Cai C. Aptamer-guided silver-gold bi-
metallic nanostructures with highly active surface-enhanced
Raman scattering for specific detection and near-infrared
photothermal therapy of human breast cancer cells. Anal
Chem 2012;84:7692–9.
[230] Yu C, Hu Y, Duan J, Yuan W, Wang C, Xu H, Yang X.-D. Novel
aptamer-nanoparticle bioconjugates enhances delivery of
anticancer drug to MUC1-positive cancer cells in vitro. Plos
One 2011;6:e24077.
[231] Wang G, Lipert RJ, Jain M, Kaur S, Chakraboty S, Torres MP,
Batra SK, Brand RE, Porter MD. Detection of the potential pan-
creatic cancer marker MUC4 in serum using surface-enhanced
Raman scattering. Anal Chem 2011;83:2554–61.
[232] Chon H, Lee S, Yoon S-Y, Chang S-I, Lim DW, Choo J. Simul-
taneous immunoassay for the detection of two lung cancer
markers using functionalized SERS nanoprobes. Chem Com-
mun 2011;47:12515–17.
[233] Lee M, Lee S, Lee JH, Lim HW, Seong GH, Lee EK, Chang SI,
Oh CH, Choo J. Highly reproducible immunoassay of cancer
markers on a gold-patterned microarray chip using surface-
enhanced Raman scattering imaging. Biosens Bioelectron
2011;26:2135–41.
[234] Lee M, Lee K, Kim KH, Oh KW, Choo J. SERS-based immunoas-
say using a gold array-embedded gradient microfluidic chip.
Lab on a Chip 2012;12:3720–7.
[235] Srisa-Art M, Kang D-K, Hong J, Park H, Leatherbarrow RJ,
Edel JB, Chang SI, deMello AJ. Analysis of protein-protein
interactions by using droplet-based microfluidics. Chembio-
chem 2009;10:1605–11.
[236] Lutz B, Dentinger C, Sun L, Nguyen L, Zhang J, Chmura A,
Allen A, Chan S, Knudsen B. Raman nanoparticle probes for
antibody-based protein detection in tissues. J Histochem
Cytochem 2008;56:371–9.
[237] Zhou X, Xu W, Wang Y, Kuang Q, Shi Y, Zhong L, Zhang Q.
Fabrication of cluster/shell Fe3O4/Au nanoparticles and ap-
plication in protein detection via a SERS method. J Phys Chem
C 2010;114:19607–13.
[238] Granger JH, Granger MC, Firpo MA, Mulvihill SJ, Porter MD.
Toward development of a surface-enhanced Raman scattering
(SERS)-based cancer diagnostic immunoassay panel. Analyst
2013;138:410–6.
[239] Domenici F, Bizzarri AR, Cannistraro S. SERS-based nanobio-
sensing for ultrasensitive detection of the p53 tumor suppres-
sor. Int J Nanomed 2011;6:2033–42.
[240] Domenici F, Bizzarri AR, Cannistraro S. Surface-enhanced Raman
scattering detection of wild-type and mutant p53 proteins at very
low concentration in human serum. Anal Biochem 2012;421:9–15.
[241] Toledo F, Wahl GM. Regulating the p53 pathway: in vitro
hypotheses, in vivo veritas. Nat Rev Cancer 2006;6:909–23.
[242] Wu M, Mao C, Chen Q, Cu X-W, Zhang W-S. Serum p53 protein
and anti-p53 antibodies are associated with increased cancer
D. Cialla et al.: SERS-based detection of biomolecules      409
risk: a case-control study of 569 patients and 879 healthy
controls. Mol Biol Rep 2010;37:339–43.
[243] Balogh GA, Mailo DA, Corte MM, Roncoroni P, Nardi H, Vincent
E, Martinez D, Cafasso ME, Frizza A, Ponce G, Vincent E,
Barutta E, Lizarraga P, Lizarraga G, Monti C, Paolillo E, Vincent
R, Quatroquio R, Grimi C, Maturi H, Aimale M, Spinsanti C,
Montero H, Santiago J, Shulman L, Rivadulla M, Machiavelli
M, Salum G, Cuevas MA, Picolini J, Gentili A, Gentili R,
Mordoh J. Mutant p53 protein in serum could be used as a
molecular marker in human breast cancer. Int J Oncol 2006;
28:995–1002.
[244] Wu L, Wang Z, Zong S, Chen H, Wang C, Xu S, Cui Y. Simul-
taneous evaluation of p53 and p21 expression level for
early cancer diagnosis using SERS technique. The Analyst
2013;138:3450–6.
[245] Schutz M, Steinigeweg D, Salehi M, Kompe K, Schlucker S.
Hydrophilically stabilized gold nanostars as SERS labels for
tissue imaging of the tumor suppressor p63 by immuno-SERS
microscopy. Chem Commun 2011;47:4216–8.
[246] Salehi M, Steinigeweg D, Ströbel P, Marx A, Packeisen J,
Schlücker S. Rapid immuno-SERS microscopy for tissue imag-
ing with single-nanoparticle sensitivity. J Biophotonics 2013,
DOI: 10.1002/jbio.201200148.
[247] Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R,
Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L,
Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E,
Mittmann M, Morris MS, Shen N, Kilburn D, Rioux J,
Nusbaum C, Rozen S, Hudson TJ, Lipshutz R, Chee M, Lander
ES. Large-scale identification, mapping, and genotyping of
single-nucleotide polymorphisms in the human genome.
Science 1998;280:1077–82.
[248] Sachidanandam R, Weissman D, Schmidt SC, Kakol JM,
Stein LD, Marth G, Sherry S, Mullikin JC, Mortimore BJ,
Willey DL, Hunt SE, Cole CG, Coggill PC, Rice CM, Ning Z,
Rogers J, Bentley DR, Kwok PY, Mardis ER, Yeh RT, Schultz B,
Cook L, Davenport R, Dante M, Fulton L, Hillier L, Waterston RH,
McPherson JD, Gilman B, Schaffner S, Van Etten WJ, Reich D,
Higgins J, Daly MJ, Blumenstiel B, Baldwin J, Stange-Thomann N,
Zody MC, Linton L, Lander ES, Altshuler D; International SNP
Map Working Group. A map of human genome sequence
variation containing 1.42 million single nucleotide polymor-
phisms. Nature 2001;409:928–33.
[249] Aouacheria A, Navratil V, Wen WY, Jiang M, Mouchiroud D,
Gautier C, Gouy M, Zhang M. In silico whole-genome scanning
of cancer-associated nonsynonymous SNPs and molecular
characterization of a dynein light chain tumour variant. Onco-
gene 2005;24:6133–42.
[250] Martini M, Vecchione L, Siena S, Tejpar S, Bardelli A. Targeted
therapies: how personal should we go? Nature Rev Clin Oncol-
ogy 2012;9:87–97.
[251] Moody B, McCarty G. Statistically significant Raman detection
of midsequence single nucleotide polymorphisms. Anal Chem
2009;81:2013–16.
[252] Lowe AJ, Huh YS, Strickland AD, Erickson D, Batt CA. Multiplex
single nucleotide polymorphism genotyping utilizing ligase
detection reaction coupled surface enhanced Raman spec-
troscopy. Anal Chem 2010;82:5810–4.
[253] Huh YS, Lowe AJ, Strickland AD, Batt CA, Erickson D. Surface-
enhanced Raman scattering based ligase detection reaction.
J Am Chem Soc 2009;131:2208–13.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM
410      D. Cialla et al.: SERS-based detection of biomolecules
[254] Yoo SM, Kang T, Kim B, Lee SY. Detection of single nucleotide
polymorphisms by a gold nanowire-on-film SERS sensor
coupled with S1 nuclease treatment. Chem Eur J 2011;17:
8657–62.
[255] Wabuyele MB, Yan F, Vo-Dinh T. Plasmonics nanoprobes:
detection of single-nucleotide polymorphisms in the breast
cancer BRCA1 gene. Anal Bioanal Chem 2010;398:729–36.
[256] Okumura N, Yoshida H, Kitagishi Y, Nishimura Y, Matsuda S.
Alternative splicings on p53, BRCA1 and PTEN genes
involved in breast cancer. Biochem Biophys Res Commun
2011;413:395–9.
[257] Choi N, Lee K, Lim DW, Lee EK, Chang SI, Oh KW, Choo J.
Simultaneous detection of duplex DNA oligonucleotides using
a SERS-based micro-network gradient chip. Lab on a Chip
2012;12:5160–7.
[258] Sun L, Yu CX, Irudayaraj J. Raman multiplexers for alternative
gene splicing. Anal Chem 2008;80:3342–9.
[259] Sun L, Irudayaraj J. PCR-free quantification of multiple splice
variants in a cancer gene by surface-enhanced Raman spec-
troscopy. J Phys Chem B 2009;113:14021–5.
[260] Sun L, Irudayaraj J. Quantitative surface-Enhanced raman for
gene expression estimation. Biophys J 2009;96:4709–16.
[261] Das G, Chirumamilla M, Toma A, Gopalakrishnan A,
Zaccaria RP, Alabastri A, Leoncini M, Di Fabrizio E. Plasmon
based biosensor for distinguishing different peptides muta-
tion states. Scientific reports 2013;3:1792.
[262] Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism,
and function. Cell 2004;116:281–97.
[263] Zhang B, Wang Q, Pan X. MicroRNAs and their regulatory roles
in animals and plants. J Cell Physiol 2007;210:279–89.
[264] Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often
flanked by adenosines, indicates that thousands of human
genes are microRNA targets. Cell 2005;120:15–20.
[265] Calin GA, Croce CM. Genomics of chronic lymphocytic leu-
kemia microRNAs as new players with clinical significance.
Semin Oncol 2006;33:167–73.
[266] Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M,
Shimizu M, Wojcik SE, Aqeilan RI, Zupo S, Dono M, Rassenti L,
Alder H, Volinia S, Liu CG, Kipps TJ, Negrini M, Croce CM.
miR-15 and miR-16 induce apoptosis by targeting BCL2. P Natl
Acad Sci USA 2005;102:13944–9.
[267] Esquela-Kerscher A, Slack FJ. Oncomirs - microRNAs with a
role in cancer. Nat Rev Cancer 2006;6:259–69.
[268] Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D,
Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR,
Jacks T, Horvitz HR, Golub TR. MicroRNA expression profiles
classify human cancers. Nature 2005;435:834–8.
[269] Nelson PT, BaldwinDA, Scearce LM, Oberholtzer JC, Tobias JW,
Mourelatos Z. Microarray-based, high-throughput gene ex-
pression profiling of microRNAs. Nat Method 2004;1:155–61.
[270] Driskell JD, Seto AG, Jones LP, Jokela S, Dluhy RA, Zhao YP,
Tripp RA. Rapid microRNA (miRNA) detection and classifica-
tion via surface-enhanced Raman spectroscopy (SERS).
Biosens Bioelectron 2008;24:917–22.
[271] Driskell JD, Primera-Pedrozo OM, Dluhy RA, Zhao Y, Tripp RA.
Quantitative surface-enhanced Raman spectroscopy based
analysis of MicroRNA mixtures. Appl Spectros 2009;63:
1107–14.
[272] Driskell JD, Tripp RA. Label-free SERS detection of microRNA
based on affinity for an unmodified silver nanorod array sub-
strate. Chem Commun 2010;46:3298–300.
[273] Abell JL, Garren JM, Driskell JD, Tripp RA, Zhao Y. Label-free
detection of Micro-RNA hybridization using surface-enhanced
Raman spectroscopy and least-squares analysis. J Am Chem
Soc 2012;134:12889–92.
[274] Esteller M. Molecular origins of cancer: epigenetics in cancer.
N Engl J Med 2008;358:1148–59.
[275] Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxym-
ethylcytosine is present in purkinje neurons and the brain.
Science 2009;324:929–30.
[276] Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brud-
no Y, Agarwal S, Iyer LM, Liu DR, Aravind L, Rao A. Conversion
of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mamma-
lian DNA by MLL Partner TET1. Science 2009;324:930–5.
[277] Esteller M. CpG island hypermethylation and tumor suppres-
sor genes: a booming present, a brighter future. Oncogene
2002;21:5427–40.
[278] Laird PW. The power and the promise of DNA methylation
markers. Nat Rev Cancer 2003;3:253–66.
[279] Muller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G,
Perkmann E, Marth C, Widschwendter M. DNA methylation in
serum of breast cancer patients: an independent prognostic
marker. Cancer Res 2003;63:7641–5.
[280] Yu J, Cheng YY, Tao Q, Cheung KF, Lam CN, Geng H, Tian LW,
Wong YP, Tong JH, Ying JM, Jin H, To KF, Chan FK, Sung JJ.
Methylation of protocadherin 10, a novel tumor suppressor,
is associated with poor prognosis in patients with gastric
cancer. Gastroenterology 2009;136:640–51.
[281] Shames DS, Minna JD, Gazdar AF. Methods for detecting DNA
methylation in tumors: From bench to bedside. Cancer Lett
2007;251:187–98.
[282] Hu J, Zhang C-y. Single base extension reaction-based surface
enhanced Raman spectroscopy for DNA methylation assay.
Biosens Bioelectron 2012;31:451–7.
[283] Barhoumi A, Halas NJ. Detecting chemically modified DNA
bases using surface-enhanced Raman spectroscopy. J Phys
Chem Lett 2011;2:3118–23.
[284] El-Said WA, Kim TH, Kim H, Choi JW. Detection of effect of
chemotherapeutic agents to cancer cells on gold nanoflower
patterned substrate using surface-enhanced Raman scattering
and cyclic voltammetry. Biosens Bioelectron 2010;26:1486–92.
[285] Farquharson S, Gift A, Shende C, Inscore F, Ordway B, Farquhar-
son C, Murren J. Surface-enhanced Raman spectral measure-
ments of 5-fluorouracil in saliva. Molecules 2008;13:2608–27.
[286] Barhoumi A, Zhang D, Tam F, Halas NJ. Surface-enhanced Ra-
man spectroscopy of DNA. J Am Chem Soc 2008;130:5523–9.
[287] Ock K, Jeon WI, Ganbold EO, Kim M, Park J, Seo JH, Cho K, Joo SW,
Lee SY. Real-time monitoring of glutathione-triggered thiopu-
rine anticancer drug release in live cells investigated by sur-
face-enhanced Raman scattering. Anal Chem 2012;84:2172–8.
[288] Wang L-S, Chuang M-C, Ho J-a A. Nanotheranostics - a review
of recent publications. Int J Nanomed 2012;7:4679–95.
[289] Lee JE, Lee N, Kim T, Kim J, Hyeon T. Multifunctional mes-
oporous silica nanocomposite nanoparticles for theranostic
applications. Accounts Chem Res 2011;44:893–902.
[290] Zhang Y, Qian J, Wang D, Wang Y, He S. Multifunctional gold
nanorods with ultrahigh stability and tunability for in vivo
fluorescence imaging, SERS detection, and photodynamic
therapy. Angew Chem-Int Edit 2013;52:1148–51.
[291] Fales AM, Yuan H, Vo-Dinh T. Cell-penetrating Peptide
enhanced intracellular Raman imaging and photodynamic
therapy. Mole pharmaceutics 2013;10:2291–8.
Brought to you by | Universidade Federal de Viçosa UFV
Authenticated
Download Date | 4/20/18 1:27 PM

[292] Plaetzer K, Kiesslich T, Oberdanner CB, Krammer B. Apoptosis
following photodynamic tumor therapy: Induction, mecha-
nisms and detection. Curr Pharm Design 2005;11:1151–65.
[293] Tian L, Gandra N, Singamaneni S. Monitoring controlled
release of payload from gold nanocages using surface en-
hanced Raman scattering. ACS Nano 2013;7:4252–60.
[294] Song J, Zhou J, Duan H. Self-assembled plasmonic vesicles of
SERS-encoded amphiphilic gold nanoparticles for cancer cell
targeting and traceable intracellular drug delivery. J Am Chem
Soc 2012;134:13458–69.
[295] Mahajan S, Richardson J, Brown T, Bartlett PN. SERS-melting:
a new method for discriminating mutations in DNA sequenc-
es. J Am Chem Soc 2008;130:15589–601.
[296] Mahajan S, Richardson J, Ben Gaied N, Zhao ZY, Brown T, Bar-
tlett PN. The use of an electroactive marker as a SERS label in
an e-melting mutation discrimination assay. Electroanalysis
2009;21:2190–7.
[297] Lubamba B, Dhooghe B, Noel S, Leal T. Cystic fibrosis: Insight
into CFTR pathophysiology and pharmacotherapy. Clin Bio-
chem 2012;45:1132–44.
[298] Corrigan DK, Gale N, Brown T, Bartlett PN. Analysis of short
tandem repeats by using SERS monitoring and electrochemi-
cal melting. Angew Chem-Int Edit 2010;49:5917–20.
[299] Jiang X, Jiang Z, Xu T, Su S, Zhong Y, Peng F, Su Y, He Y.
Surface-enhanced Raman scattering-based sensing in vitro:
facile and label-free detection of apoptotic cells at the single-
cell level. Anal Chem 2013;85:2809–16.
[300] Sathuluri RR, Yoshikawa H, Shimizu E, Saito M, Tamiya E. Gold
nanoparticle-based surface-enhanced Raman scattering for
noninvasive molecular probing of embryonic stem cell dif-
ferentiation. Plos One 2011;6:e22802.
[301] Huefner A, Kuan W-L, Barker RA, Mahajan S. Intracellular
SERS nanoprobes for distinction of different neuronal cell
types. Nano Lett 2013;13:2463–70.
[302] Neumann O, Zhang DM, Tam F, Lal S, Wittung-Stafshede P,
Halas NJ. Direct optical detection of aptamer conforma-
tional changes induced by target molecules. Anal Chem
2009;81:10002–6.
[303] Chen JW, Jiang JH, Gao X, Liu GK, Shen G, Yu R. A new apta-
meric biosensor for cocaine based on surface-enhanced Ra-
man scattering spectroscopy. Chem Eur J 2008;14:8374–82.
[304] Sanles-Sobrido M, Rodriguez-Lorenzo L, Lorenzo-Abalde S,
Gonzalez-Fernandez A, Correa-Duarte MA, Alvarez-Puebla RA,
Liz-Marzán LM. Label-free SERS detection of relevant bioana-
lytes on silver-coated carbon nanotubes: the case of cocaine.
Nanoscale 2009;1:153–8.
[305] Tu Q, Eisen J, Chang C. Surface-enhanced Raman spectros-
copy study of indolic molecules adsorbed on gold colloids.
J Biomed Opt 2010;15:020512.
[306] Perez-Pineiro R, Correa-Duarte MA, Salgueirino V, Alvarez-
Puebla RA. SERS assisted ultra-fast peptidic screening: a new
tool for drug discovery. Nanoscale 2012;4:113–6.
[307] Yuen JM, Shah NC, Walsh JT, Jr, Glucksberg MR, Van Duyne RP.
Transcutaneous glucose sensing by surface-enhanced spa-
tially offset Raman spectroscopy in a rat model. Anal Chem
2010;82:8382–5.
[308] Hsu P-H, Chiang HK. Surface-enhanced Raman spectroscopy for
quantitative measurement of lactic acid at physiological con-
centration in human serum. J Raman Spectros 2010;41:1610–4.
[309] Manno D, Filippo E, Fiore R, Serra A, Urso E, Rizzello A,
Maffia M. Monitoring prion protein expression in complex
D. Cialla et al.: SERS-based detection of biomolecules      411
biological samples by SERS for diagnostic applications. Nano-
technology 2010;21:165502.
[310] Alvarez-Puebla RA, Zubarev ER, Kotov NA, Liz-Marzan LM.
Self-assembled nanorod supercrystals for ultrasensitive SERS
diagnostics. Nano Today 2012;7:6–9.
[311] Alvarez-Puebla RA, Agarwal A, Manna P, Khanal BP, Al-
deanueva-Potel P, Carbó-Argibay E, Pazos-Pérez N, Vigder-
man L, Zubarev ER, Kotov NA, Liz-Marzán LM. Gold nanorods
3D-supercrystals as surface enhanced Raman scattering
spectroscopy substrates for the rapid detection of scrambled
prions. P Natl Acad Sci USA 2011;108:8157–61.
[312] Serra A, Manno D, Filippo E, Buccolieri A, Urso E, Rizzello A,
Maffia M. SERS based optical sensor to detect prion pro-
tein in neurodegenerate living cells. Sensor Actuat B-Chem
2011;156:479–85.
[313] Lorca RA, Varela-Nallar L, Inestrosa NC, Huidobro-Toro JP. The
cellular prion protein prevents copper-induced inhibition of
P2X(4) receptors. Int J Alzheimer’s Disease 2011;2011: Article
ID 706576, 6 pages (doi: 10.4061/2011/706576).
[314] Becker M, Budich C, Deckert V, Janasek D. Isotachophoretic
free-flow electrophoretic focusing and SERS detection of myo-
globin inside a miniaturized device. Analyst 2009;134:38–40.
[315] Brazhe NA, Abdali S, Brazhe AR, Luneva OG, Bryzgalova NY,
Parshina EY, Sosnovtseva OV, Maksimov GV. New insight into
erythrocyte through in vivo surface-enhanced raman spec-
troscopy. Biophys J 2009;97:3206–14.
[316] Neng J, Harpster MH, Zhang H, Mecham JO, Wilson WC,
Johnson PA. A versatile SERS-based immunoassay for im-
munoglobulin detection using antigen-coated gold nanopar-
ticles and malachite green-conjugated protein A/G. Biosens
Bioelectron 2010;26:1009–1015.
[317] Chen Y, Cheng H, Tram K, Zhang S, Zhao Y, Han L, Chen Z,
Huan S. A paper-based surface-enhanced resonance Raman
spectroscopic (SERRS) immunoassay using magnetic separation
and enzyme-catalyzed reaction. Analyst 2013;138:2624–31.
[318] Chen J-W, Lei Y, Liu X-J, Jiang J-H, Shen GL, Yu RQ. Immuno-
assay using surface-enhanced Raman scattering based on
aggregation of reporter-labeled immunogold nanoparticles.
Anal Bioanal Chem 2008;392:187–93.
[319] He L, Deen B, Rodda T, Ronningen I, Blasius T, Haynes C,
Diez-Gonzalez F, Labuza TP. Rapid detection of ricin in milk
using immunomagnetic separation combined with surface-en-
hanced Raman spectroscopy. J Food Science 2011;76:N49–53.
[320] He L, Lamont E, Veeregowda B, Sreevatsan S, Haynes CL,
Diez-Gonzalez F, Labuza TP. Aptamer-based surface-enhanced
Raman scattering detection of ricin in liquid foods. Chem Sci-
ence 2011;2:1579–82.
[321] Zhu Y, Kuang H, Xu L, Ma W, Peng C, Hua Y, Wang L, Xu C.
Gold nanorod assembly based approach to toxin detection by
SERS. J Mater Chem 2012;22:2387–91.
[322] Guven B, Boyaci IH, Tamer U, Calik P. A rapid method for de-
tection of genetically modified organisms based on magnetic
separation and surface-enhanced Raman scattering. Analyst
2012;137:202–8.
[323] Muniz-Miranda M, Gellini C, Salvi PR, Pagliai M. Surface-
enhanced Raman micro-spectroscopy of DNA/RNA bases
adsorbed on pyroxene rocks as a test of in situ search for life
traces on Mars. J Raman Spectros 2010;41:12–15.
[324] Arslanoglu J, Zaleski S, Loike J. An improved method of
protein localization in artworks through SERS nanotag-com-
plexed antibodies. Anal Bioanal Chem 2011;399:2997–3010.
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