focal point review
FACULTY
MUSTAFA CULHA
DEPARTMENT OF GENETICS AND BIOENGINEERING
OF ENGINEERING, YEDITEPE UNIVERSITY, ATASEHIR
ISTANBUL 34755 TURKEY
Surface-Enhanced Raman
Scattering: An Emerging LabelFree Detection and Identification
Technique for Proteins
The detection and identification of biologically
important molecules has critical importance in
several fields such as medicine, biotechnology,
and pharmacology. Surface-enhanced Raman
scattering (SERS) is a powerful emerging
vibrational spectroscopic technique that allows
not only for the characterization, but also for
the identification and detection of biomacromolecules in a very short time. In this review,
efforts to utilize SERS for label-free protein
detection and identification is summarized after
a short introduction of proteins and the
technique.
Index Headings: Surface-Enhanced Raman
Scattering; Proteins; Label-Free; Detection;
Identification.
INTRODUCTION
A
mong the biomacromolecules
proteins are the most versatile
group with a wide range of
functional and structural duties in living
systems. As an end product of gene
expression they undertake numerous
responsibilities from catalyzing biochemical reactions to transporting and
storing small molecules. Therefore, they
show incredible variation in their strucReceived 15 October 2012; accepted 9 January
2013.
E-mail: [email protected].
DOI: 10.1366/12-06895
ture and size. Countless proteins are
found in the living world. To identify all
proteins in nature with the current
technology and level of understanding
is inconceivable. It is an extremely
difficult task even to study only the
human proteome, which deals with the
proteins expressed by about 23 000
genes. When differential mRNA splicing, post-translational modifications, and
protein complexes are also considered,
the complexity of studying proteins can
easily be seen. In molecular terms, the
diversity in their functions can be
attributed to the construction of their
molecular structures. They are composed of 20 naturally occurring amino
acids extended through the peptide
bond. Their enormous structural diversity is the result of the combinatorial
assembly of the amino acids to varying
lengths. This generates limitless combinations of protein structures. They
compose the major part of all living
cells and dependent tissues. Proteins are
classified into two groups, structural and
functional, based on their responsibility
in living systems. Structural proteins
form support materials such as collagen
and elastin in connective tissue and actin
and tubulin in cellular cytoskeletons.
Functional proteins, which are the
subject of this review (also known as
globular proteins referring to their general shapes), are responsible for duties
such as acting as catalysts and transporting small molecules.
CURRENT PROTEIN
DETECTION AND
IDENTIFICATION
TECHNIQUES
Several physical, chemical, and immunoassay-based methods are routinely
used for the detection and identification
of proteins. Physical methods requiring
the use of a spectroscopic technique
such as mass spectroscopy are usually
applied after a chromatographic or
electrophoretic separation step. Chemical methods, on the other hand, use
staining with organic dyes, metal chelates, or colloidal silver after a twodimensional electrophoretic separation
step. The modes of immunoassay-based
methods are commonly employed for
applications from clinical testing to
proteomics research when the target
protein is already known. In an immunoassay-based approach, a separation
step may not be necessary because the
capture antibody provides the necessary
selectivity.
Although all these approaches are
very valuable and critical for ongoing
research and clinical testing, the effort to
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develop a fast, sensitive, cost effective,
and simple protein detection and identification technique is underway. With
recent insights into the presence of
protein biomarkers for disease diagnosis
and prognosis, the need for the development of a more satisfactory detection
technique has become apparent.
Spectroscopic techniques such as
fluorescence, UV/Vis, IR, Raman, and
mass spectroscopy (MS) are best suited
for the detection and identification of
molecules. However, each of these
techniques has certain disadvantages.
For example, although fluorescence is a
very sensitive technique and can provide
information about the concentrationdependent presence of a molecule in a
sample, it may not provide fingerprint
information for molecular identification.
Besides, due to the broad spectral
bandwidth, it may not allow the detection of more than one type of molecule
in a sample. The vibrational spectroscopic techniques, IR and Raman, and
MS can provide fingerprint information
that can be used for the identification of
molecular species. However, IR spectroscopy suffers from over-sensitivity to
water and it is also not suitable for
detection at low concentrations. Mass
spectroscopy is the most powerful
technique of protein detection, however
it is a destructive technique and it
requires expensive instrumentation and
skilled personnel. For proteomic research, a number of different modes of
MS are employed. When a separation
step such as liquid chromatography (LC)
or capillary electrophoresis (CE) is
required, electrospray or a mode of
electrospray is used as the ionization
technique. The mass analyzer is usually
a quadrupole or ion trap. Matrix assisted
laser desorption ionization (MALDI) or
fast atom bombardment (FAB) is used
as the ionization technique along with
time of flight (TOF) as an analyzer when
an online separation system is not used.
Due to the extraordinary power of mass
spectroscopic techniques, they are unarguably the most important components
of proteomic research and protein identification and detection. However, in
addition to the disadvantages mentioned
above, the difficulty of interpretation of
obtained spectra due to the complexity
of the molecular structure and reproduc-
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ibility issues with the ionization methods make the employment of these
techniques too complex for easy and
effective use.
WHY LABEL-FREE
DETECTION?
Label-free detection involves the direct use of a property, such as electrochemical, spectroscopic, or physical, of
a molecular species. In the indirect
detection approach, a reporter molecule
covalently attached to the target molecule is used and the concentration of the
target molecule is indirectly determined
through a signal obtained from the
target-label conjugate. Direct detection
is highly desired because in indirect
detection the labeling step introduces
additional uncertainties to the measurement. In a SERS experiment, the
requirement that the label molecule and
the noble metal structure should be in
close contact also introduces further
uncertainties. Therefore, label-free detection offers more reliable measurement
because the signal originates from the
molecular structure itself. In addition, it
is highly desirable to be able to identify
the components of a mixture without a
separation step. Raman spectroscopy has
the power of multiplexing due to very
narrow spectral bands and SERS can
quickly provide information about the
presence of specific molecules in a
mixture. Hence, there is a significant
effort to develop new approaches for
label-free detection of biomolecules
using SERS.
SURFACE-ENHANCED RAMAN
SCATTERING
Raman spectroscopy as a vibrational
technique can provide very specific
information about the chemical structure
of a molecule. In addition, narrow
spectral bandwidths are much narrower
than those of Fluorescence and IR
spectroscopy, which may allow for
multiplex detection and identification
of different molecular components in a
sample. However, Raman scattering is a
very inefficient process and only 10-6 of
the intensity of the exciting frequency
appears as Raman scattering. Due to the
inefficiency in scattering, wide acceptance of the technique was delayed until
the development of high power laser
sources and sensitive solid-state detectors. The real acceptance of the technique gained momentum in 1977 with
the discovery that Raman scattering
from a molecule brought close to a
noble metal surface, such as silver and
gold, is extraordinarily enhanced. Because the enhancement phenomenon
took place on the surface, the technique
was called surface-enhanced Raman
scattering (SERS).1,2 It is now commonly accepted that the mechanism of this
phenomenon has two components: electromagnetic and chemical. The electromagnetic component is related to the
excitation of the surface plasmons with
the incident light.3 The role of chemical
enhancement component depends on the
level of interaction between analyte and
noble metal surface. Additional enhancement in Raman scattering can be
achieved with the use of a laser
wavelength that the molecule of interest
can absorb, which is called resonance
enhanced Raman scattering (RRS). It is
also possible to combine this feature
with SERS, which is called surfaceenhanced resonance Raman scattering
(SERRS).
Since the discovery of the SERS
phenomenon, a remarkable effort has
been devoted to using the technique in a
range of fields from material science to
medicine and in a variety of applications. The possibility of achieving as
large as 1014 enhancement, which implies that the detection of single molecules is possible and rivals fluorescence,
has contributed to the excitement about
the technique in the scientific community.4-10
Although SERS may offer extraordinary sensitivity and may even compete
with fluorescence, it is a fact that several
parameters must be carefully considered
for optimal enhancement and spectral
reproducibility. Figure 1 summarizes the
components of a standard SERS experiment. As seen, there are a number of
parameters influencing the outcome and
optimization should be performed based
on the nature of the application. Note
that the parameters indicated on the
figure do not necessarily compete with
each other, which outlines the significance of the parameters playing a role in
a SERS experiment. Because the ulti-
FIG. 1. Components of a SERS experiment.
mate goal is to achieve the highest
enhancement in a reproducible manner,
selection of a substrate and laser wavelength should be based on the chemical
nature of the target analyte. The chemical enhancement component is strongly
related to the chemical structure of the
molecule and its adsorption level onto
the noble metal surface. Small molecules that are rich in electrons are good
scatterers while large molecules without
moieties carrying sufficient electron
density are poor scatterers. For large
molecules or molecular structures such
as proteins, the nature of the contact
with the noble metal surface becomes
more critical because only molecular
moieties in contact with or in close
proximity to the surface contribute to the
overall spectrum.
Shortly after the discovery of the
SERS phenomenon, the potential of the
technique for the characterization of
molecules and molecular structures of
biological origin began to be explored.
The detection, identification, and characterization of a number of biomolecules
and biomolecular structures and organizations such as DNA,11 RNA,12 viruses,13 bacteria,14 yeast,15 living cells,16
and tissues17 were reported. In all of
these studies, a range of substrates from
colloidal nanoparticles to rationally de-
signed nanostructured surfaces were
employed.
SERS AND PROTEINS
SERS has been utilized for protein
characterization since the early years of
its discovery. Proteins such as Cytochrome c (Cyt c), Hemoglobin (Hb),
glucose oxidase, lactate oxidase, and
flavoproteins have been thoroughly
studied.18-22 Perhaps due to the difficulties of obtaining a reasonable and rich
spectrum, most of the early reports
focused on proteins with a heme group
and employed SERRS to benefit from
additional resonance enhancement. Silver electrodes or colloidal silver nanoparticles (AgNPs) were used as
substrates in almost all of these studies.
The interactions and characterization of
amino acids, water-soluble peptides,
proteins such as lysozyme and bovine
serum albumin (BSA), and bacterial
membrane proteins such as bacteriorhodopsin have also been studied by SERS
on colloidal AgNPs and Ag electrodes.23,24 The major conclusions of
these early studies were that the SERS
spectra of peptides and proteins had a
short-range character, and that those
peptides and proteins were dependent
on the degree of adsorption to the
surface and the orientation of macromolecules on the surface to the noble
metal substrate. It was also noted that
the chemical component of the enhancement mechanism became significant
upon adsorption of peptides or proteins
to the metal surfaces. The studies
conducted with heme-containing proteins revealed that the heme group
should interact with the surface plasmons to contribute to the spectrum.
Because heme is buried within the
protein and performance of SERS depends on the distance of the analyte to
noble metal surface, a strategy to
sandwich proteins between colloidal
AgNPs and gold nanoparticles (AuNPs)
was developed by Natan et al.25,26 The
entrapment of proteins between Ag and
AuNPs remedied the drawback of distance dependent issues with the enhancement of the heme group.
There are numerous SERS substrates
following several synthesis and fabrication strategies reported in the literature
for a range of applications.27-31 In the
SERS studies of proteins and peptides,
colloidal AgNPs or AuNPs, engineered
nanoparticles, or surfaces are used as
substrates. It is now clear that the
substrate choice is application-dependent
and one needs to carefully consider
several parameters for an optimal outcome from the SERS measurement as
discussed earlier. For label-free detection
of proteins, the following points arising
from the nature of the proteins and SERS
should be taken into account; a) nature of
the sample containing the protein and
knowledge of protein concentration level
in the sample, b) size and surface charge
of the protein and the presence of
functional groups on the protein’s surface
that have strong affinity for the noble
metal surface, c) choice of SERS substrate (nanostructured surfaces versus
nanoparticles), d) molecular or ionic
species present on the noble metal
surface originating from the preparation
of the substrate, and e) detection scheme
(assay format). Since the goal is to
eliminate reporter molecules and use the
intrinsic protein spectrum for the detection, these parameters should be carefully
evaluated for a healthy interpretation of
the obtained SERS spectrum.
Due to the enormous diversity and
functions of proteins in living systems,
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any constituent of a living system can be
the source of proteins. For example, a
biotechnology product, a clinical sample, or a eukaryotic cell can be the
sample of interest. A colloidal or nanostructured nanoparticle can be more
suitable for living eukaryotic cells while
a nanostructured surface might be more
appropriate for an immunoassay. Therefore, the nature of the sample is an
important factor for the design of the
substrate. The size, surface charge, and
the presence of functional groups such
as –SH and –NH2 on the protein surface
possessing high affinity for the noble
metal surface play a defining role in the
interaction and conformation of the
protein with the noble metal surface.
Finally, depending on the nature of the
sample, i.e., a cell lysate or clinical
sample, it is important to develop an
approach that is applicable. The samples
containing proteins are mostly very
complex mixtures and they should be
either purified or separated before detection with SERS. Alternatively, their
detection can be attempted while in the
complex sample using the multiplexing
power of SERS if the protein provides a
distinguishable spectrum. An immunoassay-based approach also can be used
for the capture and detection of a protein
in a complex sample or a living cell.
The major problem with the use of
SERS spectra for protein detection and
identification is the structural similarity
of proteins. For example, in an immunoassay-based detection scheme the
antibody and target protein have similar
biochemical structures and this similarity may prevent obtaining a spectrum
with a distinguishable spectrum from the
target protein because the antibody,
sandwiched between the noble metal
surface and antigen, is closer to the
noble metal surface. Considering the
depth of the surface plasmons on the
surface of the nanostructure, which is in
the range of 1-3 nm,32 the protein on the
top of the antigen may not feel the
surface plasmons. However, the molecular stress exerted on the antibody may
result in observable changes in the
vibrational modes of the bonds in the
antibody. Another issue is very low
concentration levels that may fall under
the detection limit of the technique,
which is the case for protein biomarkers.
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Volume 67, Number 4, 2013
A pre-concentration approach can be
necessary before detection in such cases.
In an immunoassay, a label molecule
as a reporter is used to monitor the
molecular level interactions between
antibody and antigen. The SERS based
immunoassays using a reporter molecule
were extensively studied due to the high
sensitivity and multiplexing capability
of the technique and excellent reports
are available in the literature.33-36 Although it is rather difficult to monitor the
antigen binding to the antibody, the
ability to observe antibody-antigen interactions with SERS without using a
reporter molecule was shown by Ozaki
et al. 37 It was demonstrated that the
IgG-anti-IgG complexes on AuNPs
could be detected with SERS using a
NIR laser. They found that this complex
selectively adsorbed onto the AuNPs.
Later, Cullum et al. demonstrated that
the biding of an antigen to its antibody
immobilized on a nanostructured silver
surface on silica particles assembled on
a glass slide could be observed from the
SERS spectra.38 In that study, they used
a substrate constructed by depositing an
optimal Ag layer on silica particles in
the range of 100–430 nm assembled on
a glass slide and removing them from
the surface by gently scrapping with a
sharp object. The antibodies specific for
the target protein were immobilized onto
the Ag surface through a crosslinker.
The prepared silica-Ag-antibody structure was successfully used for the
detection of insulin from lysed hybridoma T-cells in the cell culture media.39
The authors claimed that the additional
bands observed on the SERS spectrum
upon binding of the antigen to the
antibody originated from conformational
changes in the antibody after antigen
binding. This finding is particularly
important because the detection of
proteins without a label can be achieved
from the spectral changes that occur
with antigen binding. In a recent report,
it was shown that the binding of an
antigen to its antibody could be detected
from the change in vibrational modes of
a linker molecule with the application of
molecular stress to the linker molecule.40
Au nanoshells constructed by coating
SiO2 nanoparticles with an Au layer
were used for the characterization of
four different peptides with SERS.41
The SERS spectra obtained from proteins were compared to their bulk
Raman spectra to identify the bands
observed on the SERS spectra of
peptides. It was concluded that the
bands on the SERS spectra of the
peptides predominantly originated from
the aromatic amino acid side residues
and this information could be used for
the band assignments on the SERS
spectra of proteins and peptides.
Due to the structural similarity of
proteins, the technique to use for
detection should have the capability of
distinguishing small structural differences. There are a number of supporting
studies demonstrating this capability. In
a report by Chumanov et al., a subtle
difference in protein structure was
successfully detected with the technique.42 In that study, the structural
stability and redox properties of yeast
iso-1-cyt c and its mutant were investigated on a roughened Ag electrode by
SERRS. In the mutant form, phenylalanine at position-82 is replaced by
histidine. It was shown that SERRS
was sufficiently powerful and sensitive
enough to observe such a small change
in a complex molecule. In another
important study, the difference between
two protein forms, human insulin and
insulin lispro, was detected at a submonolayer density on nanostructured
adaptive silver films.43 Insulin lispro is
a recombinant analog of human insulin
that is mutated (Lys(B28) Pro(B29)) to
prevent its oligomerization into a hexamer state. Because this process significantly reduces solubility and its
subsequent adsorption under physiological conditions as a solution lysine and
proline on the C-terminus of the B-chain
are interlaced. The detection of Cyt c
from Saccharomyces cerevisiae and
myoglobin at single molecule level
using colloidal AgNPs was also reported.44,45 Citrate reduced colloidal AgNPs
and a 514 nm laser were used for the
detection of the protein both in an AgNP
suspension and on AgNPs immobilized
on a glass substrate.
A pre-concentration step before the
detection of very low concentrations of
protein biomarkers will be extremely
useful. Such a technique for charged
molecules before SERS detection was
demonstrated by Lee et al. 46 The
charged analytes were exposed to an
electrical field between rod shaped and
flat gold electrodes. The negatively
charged analyte migrated to the positively charged electrode acting as a
SERS substrate. The authors claim that
this method increases the detection
sensitivity eight orders of magnitude
for adenine and this approach can be
used for the label-free detection of a
number of charged biomolecules including proteins.
The detection of human integrins
aVb3 and a5b1, found in vascular
smooth muscle cells, was performed
using citrate reduced colloidal AgNPs.47
Integrins are a group of proteins found
on the cell surface that have numerous
critical physiological functions such as
aiding in blood clotting, blood pressure
regulation, and vascular remodeling.
Therefore, their detection is diagnostically important in medical and clinical
research. The detection limits for a5b1
and aVb3 integrins were found to be 30
and 60 nM, respectively. In another
study, a mutant form of a 55-residue bsheet protein possessing cysteine residues at two separate sites and providing
a few nanometers of spacing upon the
adsorption of thiol moieties to two
separate AgNPs, was used to study the
label free-detection of proteins.48 Because the small protein acts as a
bifunctional molecular spacer, a ‘‘hotspot’’ between the AgNPs can be
formed, which allows the determination
of lower detection limits without using
an external label.
Although these early studies provide
valuable information about the potential
of the technique, the difficulties of using
the technique for protein detection and
identification remain. The major problem in the use of SERS for proteins is
the diversity of proteins in size, shape,
and surface charge. In a series of reports,
Ozaki et al. employed several strategies
to obtain rich and reproducible SERS
spectra for label-free detection of proteins.49-52 Western blotting is an analytical technique used to detect proteins in
a sample. 53 First, the proteins are
separated using gel electrophoresis and
then the separated proteins are transferred onto a membrane. In the final step
of the procedure, the proteins are stained
TABLE I.
Physicochemical properties of some proteins.
Protein
BSA
Cytochrome c
Avidin
Lysozyme
Isoelectric
Point (pI)
MW (kDa)
4.7
9.8
10.5
11.4
66
12.3
66
14.3
Zeta
Potential (mV)
-15.6
þ22.5
þ27.1
þ35.9
with labeled target antibodies. After the
final step of the Western blotting
technique, the membrane can be stained
with colloidal AgNPs, and SERS used to
identify the proteins on the blot.49 In the
study, myoglobin (Mb) and bovine
serum albumin (BSA) were used as
model proteins. The detection limit for
Mb was found to be 4 ng with the
application of SERRS.
Surfaces that have been prepared
employing different strategies have also
been used for label-free detection of
proteins. Arrays of gold structures sized
80 to 100 nm and possessing 10–30 nm
gap size were prepared with electroplating and electron-beam lithography
and used for the detection of proteins.54
An attomole level detection limit for
myoglobin was established with the
substrate. A temperature gradient study
ranging from -65 to 90 8C was
performed using lysozyme, ribonuclease-B, bovin serum albumin, and myoglobin. It was shown that the changes in
conformation of proteins could be observed during the temperature gradient.
Uniformly distributed AgNPs on a
nanostructured polymer were also used
as a substrate for label-free detection by
addressing the reproducibility of the
new substrate.55 The detection and
identification of a dipeptide, Cys–Gly,
cytochrome c, coenzyme A, and hemoglobin A0 were accomplished successfully. Detection limits of 1 ng/mL and 1
lg/mL for dipeptide and cytochrome c
respectively were claimed.
The development of a label-free
detection and identification method
based on SERS is a significant effort in
our laboratory. In our studies, we
employ citrate reduced AgNPs as SERS
substrates and explore the interactions of
several proteins with the AgNPs to
develop an assay. Some of the results
of our studies are briefly summarized
here to point out challenges and advan-
6
6
6
6
4.2
4.4
5.1
3.5
Hydrodynamic
Radius (nm)
Property
6
6
6
6
Acidic
Basic
Basic
Basic
3.63
2.01
3.53
2.1
0.08
0.05
0.07
0.06
tages of the technique for the protein
analysis.
To use the colloidal noble metal
nanoparticles, the protein sample is
simply added into the colloidal-NP
suspension. The interaction between
NPs and proteins in the suspension is
defined by their surface charges and
other ionic species present in the
suspension. For example, citrate reduced
AgNPs possess a negative surface
charge due to the adsorbed citrate ions
on the surface, and therefore proteins
with positive charges such as cyt c and
lysozyme strongly interact with the
AgNPs and form aggregates. Aggregation is desired for optimal SERS performance because it may help to obtain
improved SERS spectra. However, in
the case of negatively charged protein
such as HSA, the interaction between
the AgNPs and protein is very poor due
to the negative surface charge of the
protein. A strategy to initiate the aggregation of AgNPs using sodium sulfate in
the presence of proteins was employed.50 In that study, lysozyme, ribonuclease B, avidin, catalase, and
hemoglobin were used as model proteins
and the detection limits for lysozyme
and catalase were found to be 5 lg/mL
and 50 ng/mL, respectively.
When colloidal noble metal nanoparticles are used as SERS substrates, an
aliquot of noble metal nanoparticle and
protein containing colloidal suspension
is spotted onto a surface and SERS
spectrum is acquired from the dried
droplet. Although the procedure is quite
simple, the distribution of NPs and
protein on the droplet area is chaotic
and strongly depends on the degree of
interaction of protein with the NPs,
which depends on the surface charge
of NPs and size and charge of the
protein. Since the simplicity of sample
preparation protocol is crucial for routine analysis, we have been interested in
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focal point review
FIG. 2. Illustration of the convective assembly process (A), thin films (B) of lysozyme (a) and BSA (b) on glass slide, and white light images
of the thin film prepared with BSA under 5x objective (C) and 50x objective (D). [Adapted with permission from Ref. 59, copyright American
Chemical Society.]
FIG. 3. SERS spectra of A) avidin, B) BSA, C) cyt c, and D) lysozyme and with decreasing concentrations in AgNP colloidal suspension
[Adapted with permission from Ref. 59, copyright American Chemical Society.]
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Volume 67, Number 4, 2013
FIG. 4. Photograph (A), schematic illustration (B) and solvent flow projection (C) of AgNP and protein containing suspended drying droplet
on CaF2 surface. [Adapted with permission from Ref. 64, copyright Royal Society of Chemistry.]
employing colloidal AgNPs as SERS
substrates and mixing and spotting onto
a surface for sample preparation. However, a well known phenomenon, which
is called the ‘‘coffee-ring’’ formation,56
governs the dynamics in a drying droplet
suspension containing the AgNP-analyte. During this process, all species
suspended or dissolved in the liquid
phase are dragged and jammed at the
liquid-solid-air interface. In a droplet of
suspension composed of citrate reduced
AgNPs and negative charged analytes,
this phenomenon is more dominant due
to the electrostatic repulsion forces
between the AgNPs and the analyte.
When an analyte sufficiently interacts
with the AgNPs, some level of precip-
itation of AgNP-analyte on the droplet
area is observed. This precipitation is
due to the formation of a network-like
structure of the NPs and analyte in the
suspension. This network of NPs and
analyte resist the pulling of the flow
toward the droplet contact line. The
addition of negatively charged proteins
into the colloidal suspension containing
AgNPs with negative surface charge
results in jamming the majority of the
species in the suspension at the droplet
edges because the repulsion forces
between AgNPs and proteins dominate
the behavior in the suspension. Although a level of aggregation of AgNPs
is highly desired, their tight packing is
not preferred because it causes dampen-
ing of the electron system of the AgNPs,
which results in the quenching of the
excitation of surface plasmons. Therefore, it is also important to have a good
knowledge of the physicochemical properties of proteins in the system of
interest. Table 1 shows some of the
physicochemical properties of proteins
that may be useful to know for method
development.
In an effort to benefit from the
dynamics in a droplet for label-free
detection of proteins with SERS, we
have investigated how to affect the
dynamics in a drying droplet to influence or control the aggregation profile.
‘‘Convective-assembly’’ is a process that
is used to assemble nanometer and
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361
focal point review
FIG. 5. SERS spectra of A) avidin, B) lysozyme, and C) cytochrome c with their decreasing concentrations in AgNP colloidal suspension.
[Adapted with permission from Ref. 64, copyright Royal Society of Chemistry.]
micrometer size particles at interfaces
from a drying and moving droplet.57,58
A simple experimental set-up was used
to benefit from this process for protein
detection.59 This approach was used to
assemble a variety of particles including
yeast60 and bacteria.61 Figure 2 shows
the illustration of the convective assembly set-up and process in our work (A)
thin films formed from AgNP-protein (alysozyme, b-BSA) (B) white light images of the thin film prepared with BSA
under 5x objective (C) and 50x objective
(D). First, the reproducibility of the
SERS spectra obtained from the thin
film was investigated. Several spectra
from the strips on x and y axes with 0.5
lm steps were acquired and revealed
that the spectra were sufficiently reproducible to enable identification. The
main source of variation was the decreasing protein concentration toward
the end of the convective assembly.
Finally, the detection limit for a number
of proteins was predicted using the
approach. The SERS spectra with decreasing protein concentration are given
on Fig. 3 for Avidin (A), BSA (B), Cyt c
(C), and Lysozyme (D). As seen, a
detection limit to 0.5 lg/mL can easily
be achieved.
In real world applications, almost all
analytes are in the form of a mixture.
Two approaches can be followed for the
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Volume 67, Number 4, 2013
analysis of mixtures. Either a purification or separation step before detection
or a multiplex detection scheme can be
employed. Since SERS is capable of
multiplex detection and identification it
can be used for the latter. As mentioned
earlier, an electrophoretic separation was
employed before detection of proteins
with SERS53. In convective-assembly,
the assembly process is controlled by
convection during the drying of water
and sedimentation.60 In sedimentation,
the heavier particles tend to settle down
first. Although citrate reduced AgNPs
have wide size variation (20–200 nm),
we explored the possibility of separating
the AgNP-protein structures with the
convective-assembly prior to their detection with SERS.62
Other means of influencing the aggregation of AgNPs in a dried droplet
were also investigated. For example,
inserting a tip into a drying droplet of
suspension containing AgNPs and protein prevents the uncontrolled jamming
of all species at the droplet edge, which
is generally not a favorable region to
acquire SERS spectra from proteins.63 In
our most recent study, we demonstrated
the effectiveness of drying an AgNPprotein containing droplet suspended
from a hydrophobic surface.64 Figure 4
shows the photography of a droplet
suspended from CaF2 surface (A),
depiction of the position of AgNPprotein structures at the suspended
droplet position (B), and the flow profile
during the drying of the droplet (C). As
water evaporates from the droplet, the
AgNP-protein structures accumulate at
the apex of the droplet due to gravity as
and finally adhere to the surface as a
large aggregate. Figure 5 shows the
SERS spectra of A) avidin, B) cytochrome c, and C) lysozyme with decreasing protein concentrations.
The SERS spectra of proteins presented in Figs. 3 and 5 demonstrate the
importance of the protocol used for the
sample preparation. Typical for any
SERS measurement, spectral reproducibility is the major problem. When
spectra of the same protein are obtained
with the two sample preparation methods, the convective-assembly and suspended-droplet, the main spectral
difference is the band at around 1050
cm-1. This band is attributed to the
nitrate ions, and is likely due to the coadsorption of proteins and nitrate ions
onto the AgNPs.65 As Fig. 5 shows, as
the concentration of proteins increases,
the intensity of this band increases as
reported. Although this band is also
observed on the SERS spectra of
proteins prepared with convective-assembly, it is rather weak. The intensity
difference in this band is due to the
concentration of nitrate ions distributed
on the unit area. In the case of the
suspended droplet, all species are confined in a much smaller area than that in
the convective-assembly.
When the spectra of each protein
obtained with the same sample preparation method are inspected some spectral
variations with the change of protein
concentration are also observed. This is
due to the variations in the aggregation
status of the AgNPs with varying protein
concentrations. However, some of the
major bands were retained on the spectra
and can be used for quantification. Note
that the other experimental conditions,
laser wavelength and colloidal AgNPs,
were the same. This again reveals the
importance of the SERS experimental
protocol for comparable results. For
quantification, linearity in concentration
versus spectral intensity is highly desired. However, this relationship for
proteins is not always linear and is
strongly related to the structural properties of proteins such as size, surface
charge, and flexibility.
CONCLUSIONS
The utility of SERS in biological
applications has long been of interest in
the scientific community. As the understanding of interactions of biomacromolecules and biological structures with
noble metal surfaces and nanoparticles
is clarified, there is steady progress in the
application of the technique for the
solution of biomedical, medical, and
biotechnology related problems. Developments in the biological applications of
the technique have been reviewed periodically.66-68 In this review, the efforts to
utilize SERS for label-free protein detection and identification have been summarized. Most of the earlier studies
focused on understanding the interaction
of proteins with the AgNPs or AuNPs
and increasing the SERS sensitivity.
However, the reproducibility issue has
not been fully addressed yet. The superiority of the AgNPs as substrates
influences their use as a substrate in
SERS experiments but the major drawback of using colloidal AgNPs as
substrates is their reproducibility issues.
This problem must be fully addressed
before the broad use of the technique in
label-free protein detection will be prac-
tical. It is clear that the use of the
technique for biological applications is in
its infancy yet. Most of the studies are
exploratory in nature and are aimed at
trying to understand the potential of the
technique. As the number of studies
increases, the full potential of the technique will be realized. The combining of
the technique with microfluidics could be
an area for future studies because separation of proteins from their mixtures can
be accomplished with microfluidics before their detection with SERS.
ACKNOWLEDGMENTS
The author acknowledges the financial support
of the Scientific and Technological Council of
Turkey (TUBITAK) and Yeditepe University for
the data partially presented in this study. The
author also acknowledges Assistant Prof. Dr.
Andrew Harvey for his valuable comments during
the writing process of the review.
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