Review

pubs.acs.org/CR

SERS-Activated Platforms for Immunoassay: Probes, Encoding

Methods, and Applications
Zhuyuan Wang, Shenfei Zong, Lei Wu, Dan Zhu, and Yiping Cui*
Advanced Photonics Center, Southeast University, Nanjing 210096, Jiangsu, China

ABSTRACT: Owing to their excellent multiplexing ability, high sensitivity, and large
dynamic range, immunoassays using surface-enhanced Raman scattering (SERS) as the
readout signal have found prosperous applications in fields such as disease diagnosis,
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environmental surveillance, and food safety supervision. Various ever-increasing

demands have promoted SERS-based immunoassays from the classical sandwich-type
ones to those integrated with fascinating automatic platforms (e.g., test strips and
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microfluidic chips). As recent years have witnessed impressive progress in SERS

immunoassays, we try to comprehensively cover SERS-based immunoassays from their
basic working principles to specific applications. Focusing on several basic elements in
SERS immunoassays, typical structures of SERS nanoprobes, productive optical spectral
encoding strategies, and popular immunoassay platforms are highlighted, followed by their representative biological applications
in the last 5 years. Moreover, despite the vast advances achieved to date, SERS immunoassays still suffer from some annoying
shortcomings. Thus, proposals on how to improve the SERS immunoassay performance are also discussed, as well as future
challenges and perspectives, aiming to give brief and valid guidelines for choosing suitable platforms according to particular
applications.

CONTENTS 4. SERS Encoding for Multiplex Detection 7926

4.1. Design and Fabrication of SERS Encoders 7926
1. Introduction 7911 4.2. SERS Spectral Encoding Strategy 7928
1.1. Historical View of Immunoassay Protocols 7911 4.2.1. Raman-Frequency-Based Encoding 7928
1.2. SERS-Based Immunoassay (History, Design, 4.2.2. Signal-Intensity-Based Encoding 7928
and Advantages) 7912 4.3. SERS-Included Joint Encoding 7929
2. Principles of SERS-Based Immunoassay 7912 4.3.1. SERS−Fluorescence Joint Spectral En-
2.1. Construction of SERS-Based Immunoassays 7912 coding 7929
2.2. Design and Fabrication of SERS Probes 7915 4.3.2. Spectral−Spatial Joint Encoding 7930
2.2.1. Optical Label: Organic and Inorganic 5. Applications of SERS-Based Immunoassay 7930
Raman Reporters 7915 5.1. Analysis of IgGs 7931
2.2.2. Signal Amplifiers: Metal Nanoparticles 7916 5.2. Study of Disease Biomarkers 7931
2.2.3. Protection Layer: Silica, Polymers, Etc 7917 5.2.1. Detection of Protein Biomarkers 7932
2.2.4. Targeting Molecules: Antibodies and 5.2.2. Detection of miRNAs and Circulating
Aptamers 7917 DNAs 7934
3. Platforms for SERS-Based Immunoassay 7917 5.2.3. Detection of Telomerase Activity and
3.1. SERS-Based Immunoassay on Solid Sub- Telomere Length Measurement 7935
strate 7918 5.3. Identification of Bacteria and Viruses 7936
3.1.1. Nonmetallic Substrate 7918 5.4. Detection of Ions and Toxins 7936
3.1.2. Metallic Substrate 7918 5.5. Sorting of Cells 7938
3.2. SERS-Based Immunoassay in Liquid Phase 5.6. Multiplex Biochemical Analysis Using SERS
(Suspension Array) 7919 Encoders 7939
3.2.1. Nonmagnetic Substrate 7920 5.6.1. Multiplex Immuno-Detection of Biomo-
3.2.2. Magnetic Substrate 7921 lecules and Cells 7939
3.3. Immunoassays on a SERS−Microfluidic Chip 7922 5.6.2. Multiplex Ion Detection 7943
3.3.1. Microfluidic Channels 7922 6. Challenges and Perspectives 7943
3.3.2. Droplet Microfluidics 7923 6.1. Improving the Reproducibility of SERS-
3.4. SERS-Based Immunoassay on Optical Fibers 7924 Based Immunoassay 7943
3.5. Paper-Based SERS Platforms 7924 6.1.1. Uniformity of the Immune Substrates 7943
3.6. Hydrophobic SERS Substrates 7925
3.7. Comparison between Different Platforms for
SERS-Based Immunoassay 7926 Received: January 12, 2017
Published: May 23, 2017

© 2017 American Chemical Society 7910 DOI: 10.1021/acs.chemrev.7b00027

Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews Review

6.1.2. Stability of SERS Nanoprobes 7945 SERS-based immunoassays. The design and fabrication of SERS
6.2. Real Sample Detection 7945 nanoprobes are introduced in section 2. As this part has been
6.2.1. Serum and Plasma 7945 extensively reviewed previously,7,12−14 we briefly summarize the
6.2.2. Whole Blood 7946 typical design and structure of a SERS nanoprobe. In section 3,
6.2.3. Other 7947 different platforms in SERS-based immunoassay are summar-
6.3. Point-of-Care Testing (POCT) 7947 ized, ranging from solid substrates to liquid microspheres to
6.4. Practical SERS-Encoding Technique 7947 microfluidic chips, etc. The advantages and disadvantages of
6.5. Combining SERS with Other Techniques for each platform will be compared and discussed, which is
Multimodal Immunoassay 7948 expected to provide a brief guideline for choosing a suitable
6.5.1. SERS−SPR Immunoassay 7948 platform for specific applications. Aside from the nanoprobes
6.5.2. SERS−Fluorescence Immunoassay 7948 and platforms, spectral-encoding techniques are usally
6.5.3. SERS−Electrochemistry Immunoassay 7949 employed in an immunoassay system for multiplex detec-
6.5.4. Immunoassay with Hyperbolic Metama- tion.15−17 Therefore, in section 4, the typical SERS-encoding
terials 7949 techniques are summarized, including those based on Raman
7. Conclusion 7949 frequency or intensity, as well as several SERS-involved
Author Information 7949 multimodal-encoding strategies. We then provide considerable
Corresponding Author 7949 details on the biochemical applications of SERS-based
ORCID 7949 immunoassay in analyzing various targets in section 5. Finally,
Notes 7950 the challenges and future perspective of designing next-
Biographies 7950 gerneration SERS immunoassays are presented in sections 6
Acknowledgments 7950 and 7.
Abbreviations Used 7950
1.1. Historical View of Immunoassay Protocols
References 7950
An immunoassay is defined as a test for the quantitative
determination of chemical substances, such as hormones, drugs,
1. INTRODUCTION and specific proteins, that utilizes the highly specific binding
Immunoassay is one of the most famous tools for the between antibodies and antigens or haptens.18,19
quantitative detection of biochemical targets (e.g., proteins The development of immunoassay starts from the detection
and toxins). The fine accuracy and operability of immunoassays of insulin by Yalow and Berson.20 They found out that the
have promoted thorough investigations of stringent issues, such globulins bound to insulin in patients treated with exogenous
as disease diagnosis, food safety, and environmental protection. animal insulin were actually antibodies. Afterward, insulin
Recent years have witnessed significant progress in developing antibodies were purified and the first radio-immunoassay (RIA)
more efficient immunoassay protocols. Immunoassays have was established to detect insulin using a radioactive iodine
evolved from the traditional ones performed on microplates to label.21 Later, together with two other scientists, Yalow won the
those conducted with highly automized platforms using various Nobel Prize in Medicine/Physiology due to her pioneering
kinds of readout signals (e.g., colorimetry, electrochemistry, work on devising RIA. Since its discovery, RIA has paved the
surface plasmon resonance, fluorescence, and surface-enhanced way for the rapid development of immunoassays.
Raman scattering). To date, immunoassay is no longer In the late 1960s, immunoassay techniques were developed
regarded as just a simple test. It is more appropriate to for chemically linking enzymes to antibodies. Soon after that, in
consider immunoassay as a sophisticated analyte-detecting 1971, the enzyme immunoassay (EIA) and enzyme-linked
system composed of both biochemical and electrical or optical immunosorbent assay (ELISA) were developed by Engvall and
techniques. Since other immunoassay strategies have been well- Perlmann22 and van Weeman and Schuurs,23 respectively. The
reviewed elsewhere,1−3 here we only focus on optical replacement of radiolabels with enzymes made immunoassay
immunoassays, especially those using surface-enhanced much simpler and more popular. Today, most of the detection
Raman scattering (SERS) as the readout signal. SERS is well- of clinical protein biomarkers is performed with ELISA kits.
known for its high sensitivity, fine specificity, and excellent Since the 1970s, various detection techniques have been
multiplexing ability, leading to the blooming use of SERS-based employed to develop the formats of immunoassays. For
immunoassays.4−9 The generation of Raman enhancement example, immunoassays by electrochemical techniques can
needs a nanoscopic metallic surface;10,11 consequently, SERS- determine the level of analytes by the changes in electrical
based immunoassays have some special requirements on the signals such as potential, current, resistance, and capacitance.24
layout of the assay. SERS immunoassays usually contain several As a label-free method, electrochemical immune detection
basic elements (i.e., nanoprobes, SERS spectra, and assay offers the advantages of fast response, low cost, and simplicity,
platforms), each of which requires an elaborate design. Despite which has a wide range of applications in biosensing. On the
its rapid development, up to now, there is no review covering other hand, with the rapid development of optical materials and
SERS immunoassay in detail. methods, the optical detection technique has become one of
Hence, in this review, we try to comprehensively cover the most popular methods for immunoassays in recent years. In
SERS-based immunoassays from basic principles to abundant the beginning, chemiluminescence immunosensors have been
applications. Typical structures of SERS nanoprobes, produc- employed for routine clinical diagnosis or biomedical research
tive optical-encoding strategies, and popular immunoassay due to their advantages such as no radioactive waste, the
platforms are highlighted, followed by the introduction of relatively simple instrumentation, and high sensitivity. In
representative biological applications using SERS immuno- principle, they employ chemical reactions to produce optical
assays. Specifically, we start with the history, principles, and signals for immunoassays.48 Later, fluorescence labels, including
classifications of immunoassays in section 1. Then, we focus on fluorescent dyes and quantum dots, were used to develop
7911 DOI: 10.1021/acs.chemrev.7b00027
Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews
optical immunoassays.49,50 The advantages of fluorescence
immunoassays include simplicity of system design, improved
sensitivity, and fast readout process. More recently, immuno-
assays based on SERS, which utilize Raman reporters as the
optical labels, have attracted much attention due to their
ultrahigh sensitivity, excellent multiplexing ability, and high
photostability.8 Aside from these labeled optical techniques,
surface plasmon resonance (SPR) immunoassays provide a
label-free optical sensing platform,51 in which the specific
interactions between antibodies and antigens could be
determined by the changes in reflective index in real time.
Another kind of immunosensor employs the quartz crystal
microbalance (QCM) technique, which measures the mass
change through a quartz crystal resonator,52 to perform the
label-free, noninvasive, and real-time detection. Summarily, the
diversity of detection techniques makes it easy and flexible to
design efficient immunoassays. Table 1 shows some typical
examples of immunoassays using the above-mentioned
techniques, in which the comparisons among different
techniques are also summarized.
1.2. SERS-Based Immunoassay (History, Design, and
Advantages)
Among the various detection techniques employed for
immunoassays, SERS has gained increasing popularity in recent
decades. In this review, we specially focus on the development
of immunoassays based on SERS technique. SERS-based
immunoassays refer to those that using surface-enhanced
Raman scattering as the readout signal. As is well-known,
Raman scattering is the inelastic scattering of a photon, which
was discovered by Raman and Krishnan in liquids53 and by
Landsberg and Mandelstam in crystals,54 respectively. Raman
scattering can provide rich structural and quantitative
information on nearly all kinds of molecules and materials.
However, the weak intensity of Raman spectroscopy limits its
application in biomedical and chemical studies. In 1973,
Fleischmann et al. discovered that pyridine molecules adsorbed
onto the rough surface of silver exhibited a dramatically
enhanced Raman scattering light signal.55 The enhancement
factor could reach from 103 to 1010,56−58 which has then been
widely used for unltrasensitive biochemical analysis.
The first typical SERS-based immunoassay was developed in
1999 using a “sandwich” scheme (Figure 1).59 In that protocol,
capture antibodies are bound to a flat gold surface to form an
immune substrate. Gold nanoparticles were labeled with Raman
reporters and conjugated with detection antibodies to form
SERS probes. Then, the target antigens could be captured by
the antibodies on the substrate and subsequently be recognized
by the SERS probes. The presence and concentration of a
specific antigen is determined by the characteristic SERS
spectrum of the Raman reporters. Importantly, the authors also
demonstrated that the method could be used for multiplex
detection through the SERS-encoding method.
In the following decades, the platforms for SERS-based
immunoassay have experienced a rapid development, ranging
from solid substrates (metallic59 and nonmetallic60) to liquid
phase (magnetic61 and nonmagnetic62) to microfluidic chips,63
paper devices,64 and optical fibers.28 The diversity of SERS-
active platforms offers sufficient choices for analyzing proteins,
disease biomarkers, ions, toxins, bacteria, viruses, cells, etc. On
the other hand, there is a growing demand for multiplex, high-
throughput analysis of large quantities of analytes in a single
sample, especially in the fields of clinical diagnosis, drug
7912
Review
screening, and environmental monitoring.9 Additionally and
importantly, the SERS-based optical-encoding technique has
been well-employed for high-throughput detection, in which
multiple targets could be distinguished simultaneously by the
Raman frequency or intensity.65 Further, SERS has also been
combined with fluorescence17 or spatial coordinate66 for joint
encoding. The combination of SERS with other techniques
greatly improved the encoding capacity, which is of great
importance for achieving high-throughput analysis.
In brief, SERS-based immunoassays mainly have three
unique advantages. First, due to the large enhancement factor
up to ∼1010, SERS-based detection could reach a high
sensitivity to a single molecule level, making it extremely useful
for trace analysis.67−69 Second, the narrow Raman bands could
be employed for highly efficient spectroscopic encoding, which
can facilitate high-throughput detection.9,70 Third, SERS is
insensitive to photobleaching and quenching.71 The high
stability of SERS signals allows repetitive signal measurements
to reduce test errors. Owing to these advantages, SERS-based
immunoassays have found an enormous number of usages in
both research and practical applications.
2. PRINCIPLES OF SERS-BASED IMMUNOASSAY
Typically, the development of SERS-based immunoassay
requires two critical components: the immune substrate and
the SERS immunoprobe. Basically, the immune substrates are
modified with targeting molecules (antibodies, aptamers, etc.)
to capture the specific analytes from the samples. Afterward, the
SERS probes are employed to quantify the concentration of
analytes. There are two basic functions of SERS probes: (1)
specifically recognizing and binding to the analytes captured by
the immune substrate and (2) providing SERS signals for the
quantitative detection of analytes. In accordance with the
universal classification for immunoassays, generally, SERS-
based immunoassays could be classified into the heterogeneous
assays and homogeneous ones, noncompetitive assays and
competitive ones, and labeled and label-free ones. In this
section, we first introduce the basic principles of SERS-based
immunoassay in different formats. Then, the design and
fabrication of SERS probes are summarized. The discussion
on different immune substrates (platforms) is concretely
described in section 3.
2.1. Construction of SERS-Based Immunoassays
According to the different separation and detection processes,
SERS-based immunoassays could be classified into heteroge-
neous and homogeneous immunoassays (Figure 2). The
heterogeneous immunoassay requires the separation of bound
SERS probes from free ones and the concentration of antigens
is measured through the bound SERS probes. The separation
step can be fulfilled using solid substrates or liquid microbeads.
On the contrary, in homogeneous SERS immunoassays, the
binding between SERS nanoprobes and target molecules could
induce the changes in SERS intensity, which are used for the
qualitive and quantitative analysis. In general, the homogeneous
immunoassay is rapid, straightforward, and more adaptable to
automated systems. However, it is also more vulnerable toward
nonspecific antibody−antigen cross-reactions. In contrast,
heterogeneous immunoassay is excellent in detection perform-
ance, including sensitivity and reproducibility, although it
requires repetitive separation procedures.
When considering the binding manner of the immunocom-
plex, the SERS-based immunoassays can be classified into
DOI: 10.1021/acs.chemrev.7b00027
Chem. Rev. 2017, 117, 7910−7963
 Table 1. Some Typical Examples of Immunoassays Using Different Techniquesa
label- operation read-out
methods analyte dynamic range limit of detection free time read-out speed instrument multiplexing ability ref
electrochemistry CEA 10.0 pg/mL to 50.0 ng/mL 4.4 ± 0.1 pg/mL no real-time yes parallel multiplexing 25
PSA 500 pg/mL to ca. 100 ng/mL; in 0.5 ng/mL; in serum, 1 ng/mL yes <30 min yes 26
serum, 1−100 ng/mL
Chemical Reviews

H1N1 split influenza vaccine 5−500 ng/mL 5 ng/mL yes <30 min yes 26
simultaneous detection of 500 pg/mL to ca. 10 μg/mL; 1 ng/mL 500 pg/mL; 1 ng/mL yes <30 min yes 26
AFP/ PSA to ca. 10 μg/mL
CA 125 0−0.1 U/mL 0.0016 U/mL yes yes 27
AFP 10.0 pg/mL to 10.0 ng/mL 3 pg/mL yes yes 28

QCM HIgG 5.0 pg/mL to 20.0 ng/mL 5.0 ± 0.18 pg/mL no >1 h real-time yes parallel multiplexing 29
thrombomodulin 10−5000 ng/mL 2 ng/mL yes >1 h yes 30

chemiluminescence CRP 1−1000 ng/mL 0.93 ng/mL no real-time/a few yes parallel multiplexing 31
AFP 1.0 × 10−3−100 ng/mL 2.7 × 10−5 ng/mL no >1 h seconds yes 32
β-hCG 2.0 × 10−4−20 IU/L 1.1 × 10−5 IU/L no >1 h yes
CA125 (1.0 × 10−4)−10 U/mL 1.7 × 10−5 U/mL no >1 h yes
CEA (1.0 × 10−4)−10 ng/mL 2.0 × 10−5 ng/mL no >1 h yes
human prealbumin 0.05−1000 μg/L 0.01 μg/L no 35 min yes 33

fluorescence NSE 75.0 pM no 45 min real-time/a few yes simultaneous and 34

7913
CEA 0.5 pM no 45 min seconds yes parallel multiplexing
Cyfra21-1 14.3 pM no 45 min yes
SCC 6.7 pM no 45 min yes
CA15-3 0.03 U/mL no 45 min yes
PSA 1.6 ng/mL no yes 35
EGFR 0.12 nM or 23 ng/mL in buffer, 0.18 no 1h yes 36
nM or 34 ng/mL in serum

colorimetry pathogen of Salmonella, 6.7 aM no real-time no parallel multiplexing 37

Listeria, and E. coli O157
IgG/hepatitis B virus IgG, 1 ng/mL to 10 μg/mL; HBsAg, IgG, 1.6 ng/mL; HBsAg, 1.3 ng/mL no 1h no 38
0.34 ng/mL to 340 μg/mL

SERS AFP, GPC3 0.1 pM no 4h 10−60s (usual yes simultaneous and 39

EGFR 1 ng/mL cetuximab no <1 h acquisition time) yes parallel multiplexing 40
SEB 2−100 pg/mL 1.3 pg/mL no 1h yes 41
NT-proBNP 1 fg/mL to 1 ng/mL 0.75 fg/mL no >1 h yes 42

SPR lysozyme dimer 1.7−35 nM 1.4 nM yes 10 min real-time yes parallel multiplexing 43
gluten peptides 1.12−19.2 ng/mL 0.33 ng/mL yes 20 min yes 44
CA125 0.26 U/mL 1h yes 45
β2-M 0.55 ng/mL 1h yes 45
ApoA1 7.7 ng/mL 1h yes 45
Review

DOI: 10.1021/acs.chemrev.7b00027
Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews Review

staphylococcal enterotoxin B (SEB), N-terminal pro-brain natriuretic peptide (NT-proBNT), β2-microglobulin (β2-M), apo-lipoprotein A1(ApoA-I), Plasmodium falciparum histidine-rich protein 2 (Pf
antigen (CA15-3), α-fetoprotein (AFP), C-reactive protein (CRP), human chorionic gonadotrophin-β (β-hCG), carcinoma antigen 125 (CA125), hepatitis B virus (HBsAg), glypican-3 (GPC3),
Carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), prostate specific antigen (PSA), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC), carbohydrate
ref

46
47
multiplexing ability

parallel multiplexing
instrument
read-out

yes
yes
read-out speed

real time/a few

seconds
operation
time

>2 h
>2 h
label-
free

no
no

Figure 1. Schematic illustration of the SERS-based immunoassay

developed by Ni et al. in 1999. Reprinted with permission from ref 59.
Copyright 1999 American Chemical Society.
limit of detection

competitive and noncompetitive ones (the latter is also known

as the “sandwich” or “two-site” immunoassays) (Figure 3).
Typical noncompetitive immunoassays are performed using a
sandwich structure, in which the targets are sandwiched
between the capture antibodies and the SERS probes. Thus,
4 ng/mL

the concentration of targets is quantified by the SERS probes

2.5 pM

bound to the detection antibodies, which is positively

correlated to the intensity of the readout signal. However, in
competitive SERS immunoassays, the antigens are labeled to
act as the immunoprobes, in which the targets compete with
the SERS probes for binding to the antibodies. Hence, the
intensity of detected signal is negatively correlated to the
dynamic range

concentration of the targets. Generally, the competitive

immunoassays are suitable for the detection of small molecules
with only one binding site to antibodies (such as peptides, ions,
10−1000 ng/mL
0.001−70 nM

and hormones), while noncompetitive immunoassays are

usually employed for analyzing macromolecules (e.g., proteins)
with at least two binding sites for antibodies. Compared to the
competitive immunoassays, the noncompetitive ones usually
perform better in consideration of sensing sensitivity, dynamic
range, and reproducibility.
On the other hand, both the labeled and label-free SERS
techniques could be used to develop immunoassays (Figure 4).
analyte

While labeled SERS immunoassays require the Raman reporter-

labeled immunoprobes to detect the target molecules, label-free
vancomycin

SERS immunoassays directly measure the intrinsic Raman

Pf HRP2

spectra of the target analytes. Compared to labeled SERS

immunoassays, label-free SERS detection greatly simplifies the
Table 1. continued

detection procedures. However, it requires highly efficient

SERS substrates and strong nanoparticle−molecule binding.
One major obstacle is that not all kinds of analytes could be
methods

discriminated from their intrinsic SERS spectra. It is not easy to

analyze chemicals with a small scattering cross-section. On the
HRP2).
ELISA

contrary, labeled SERS immunoassay is a universal technique

that could be used to analyze almost all kinds of analytes. The
a

7914 DOI: 10.1021/acs.chemrev.7b00027

Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews
Figure 2. Principles of the heterogeneous and homogeneous SERS immunoassay.
Figure 3. Principles of noncompetitive and competitive SERS immunoassay.
high sensitivity and standardized protocol make it popular in
clinical diagnosis, biochemical analysis, and environmental
monitoring.
2.2. Design and Fabrication of SERS Probes
In a SERS-based immunoassay, the immunoprobes play a
critical role in specific and quantitative detection. Thus, the
structure of SERS nanoprobes should be carefully designed to
improve the performance of immunoassays. Typical SERS
nanoprobes contain four main parts: metal nanoparticles,
Raman reporters, protection layers, and targeting molecules
(Figure 5). In such nanoprobes, the Raman signal could be
enhanced by attaching the reporter molecules to the surfaces of
metal nanoparticles. To obtain a large enhancement factor and
high stability of Raman signals, the structure of metal
7915
Review
nanoparticles needs to be carefully designed, and Raman
reporters with intrinsic high-scattering cross sections are usually
selected as the labels. Additionally, a protection layer wrapped
around the metal nanoparticles is often introduced to avoid the
loss of Raman reporters, which also facilitates the conjugation
of targeting molecules. Then, the analytes can be specifically
recognized by the targeting molecules. Generally, the aim for
designing SERS probes is to improve their sensitivity, chemical
and physical stability, and targeting efficiency, as well as their
multiplexing ability. Since there are already some wonderful
reviews focusing on the design and fabrication of SERS
probes,7,72 we will only briefly provide some basic points here.
2.2.1. Optical Label: Organic and Inorganic Raman
Reporters. Raman reporters act as the signal source in SERS
DOI: 10.1021/acs.chemrev.7b00027
Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews
Figure 4. Principles of label-free SERS detections: (a) adsorption-mediated label-free SERS detection and (b) antibody/ligand-mediated label-free
SERS detection.
Figure 5. General procedure for the fabrication of a typical SERS probe.
probes. There are several general requirements for selecting the
reporter molecules: (1) The reporter molecules can be easily
and strongly anchored to the surfaces of metal nanoparticles.
(2) The scattering cross-section of the reporters should be
intrinsically high enough to obtain a strong intensity of Raman
signals. (3) Their Raman spectra should have few characteristic
peaks, which can minimize the band overlap with other
reporters in multiplex detection. Up to now, various molecules
have served as Raman reporters, ranging from small organic
molecules [such as 4-mercaptobenzoic acid (4MBA)73 and
rhodamine 6G (R6G)74] to inorganic materials (such as
graphene oxide75 and carbon nanotube76). The diversity of
Raman spectra offers wide choices for fabricating SERS
nanoprobes.
2.2.2. Signal Amplifiers: Metal Nanoparticles. As is
well-known, metal nanoparticles can enhance Raman signals of
reporter molecules through the local electromagnetic field and
electron transfer. Traditionally, Au or Ag nanopheres are the
most commonly used SERS substrates.77,78 In recent decades,
metal nanoparticles with different sizes and shapes were
synthesized. Technologies have been developed for the
fabrication of nanorods,79 nanowires,80 nanostars,81 and
nanoprisms,82 which show a higher SERS enhancement factor
compared to that of the nanospheres. Unlike nanospheres with
only one SPR band, the nanorods show a longitudinal SPR
band and a transverse SPR band. The tunable longitudinal SPR
band and strong enhancement at the tips of the nanorods make
them one of the excellent candidates for SERS applications.
Besides, the multibranched metal nanostars and nanoprisms
have also attracted much attention as SERS substrates due to
the following two reasons. One is that they own increased
surface-to-volume ratios, allowing for more Raman reporters to
attach onto their surfaces. The other reason is that the
hybridization of the individual tips of the nanostars or
7916
Review
nanoprisms could further enhance SERS signals. In addition,
it has also been reported that closely spaced nanowires could
also generate hot spots, resulting in an intense SERS
enhancement. In addition to selecting metal nanoparticles
with different shapes, another important method to improve
the SERS activity is to create “hot spots” through controlled
aggregation of nanoparticles.83,84 Previously, we have fabricated
a kind of SERS probe based on aggregated Au nanoparticles
with greatly enhanced SERS signals, in which the Raman
molecules were used to induce the aggregation of gold
nanoparticles. Afterward, a layer of silica shell was coated
onto the Au aggregates to improve the stability of the
nanoaggregates. Alternatively, SERS hot spots could be
generated through the formation of “nanogaps”.85 These
nanogaps can be created as nanosphere dimers,86 nanorod
dimers,87 nanowire dimers,88 nanostar dimers,89 nanosphere
trimers,90 nanopheres/nanodisk trimers,91 etc. The greatly
enhanced local electromagnetic field in the nanogap leads to
strongly enhanced Raman signals. Especially, several groups
have reported the fabrication of SERS nanoparticles containing
interior nanogaps.92−94 For example, Lim et al. presented a type
of DNA-tailorable nanoparticle with interior nanogaps. This
kind of nanoparticle with an excellent SERS enhancement is
able to provide reproducible SERS signals.94 Recently, core−
shell-type metal nanoparticles have also been widely used for
designing SERS probes. In one of our previous works, Au@Ag
nanorods (NRs) were used to fabricate SERS nanoprobes for
immunoassays.17,73 By combining the high stability of gold and
the large enhancement factor of silver, Au@Ag NRs were found
to be very useful in the applications of SERS-based immuno-
assay,61,95 cell imaging,96 label-free chemical detection,79,97 and
drug delivery.98 More recently, graphene-enhanced Raman
scattering (GERS) using graphene as the substrate for Raman
enhancement has been presented.99 The novel enhancing
DOI: 10.1021/acs.chemrev.7b00027
Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews Review

Figure 6. (a) High-throughput protein detection on glass slides using OMP NPs. Reprinted with permission from ref 17. Copyright 2012 American
Chemical Society. (b) Multiplex DNA detection on glass slides using encoded SERS nanoprobes. Reprinted with permission from ref 60. Copyright
2012 Royal Society of Chemistry. (c) Multiplex detection of serological cancer markers on a quartz array. Reprinted with permission from ref 112.
Copyright 2015 Royal Society of Chemistry. (d) Glycoprotein detection on an array of boronate-affinity molecularly imprinted polymers (MIPs)
using SERS probes. Reprinted with permission from ref 113. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

substrate could produce clean, reproducible, and increased
Raman signals. Compared to conventional Raman enhance-
ment techniques, GERS mainly relies on a chemical mechanism
and therefore shows unique molecular selectivity. It has been
employed for analytical applications from graphene-veiled
SERS to graphene-based SERS for quantitative analysis.100
2.2.3. Protection Layer: Silica, Polymers, Etc. A large
percentage of SERS probes contain one or more protection
layers, which aim to protect Raman reporters from the outside
environment and provide anchor spots for targeting molecules.
Silica,61 titanium dioxide,101 polymers [such as polyethylene
glycol, poly(acrylic acid), polyallylamine hydrochlor-
ide],73,102,103 mesoporous silica,98 and liposomes104 are
commonly introduced materials to form the protection layers,
which improve the stability of SERS probes. In 2010, Tian’s
group developed a novel SERS method named shell-isolated
nanoparticle-enhanced Raman spectroscopy, in which the
Raman signal amplification was provided by gold nanoparticles
coated with an ultrathin silica or alumina shell.105 The use of
the shell coating around the Au nanoparticle protects the SERS-
active nanostructure from contacting with whatever is being
probed and serves as “smart dust” over the surface that is to be
probed. Recently, Jiang et al. synthesized a Ag@mesoporous
silica (MSiO2)@Ag core−shell architecture,106 in which the
silica shell improved the stability while the inner Ag core and
outer Ag shell can form SERS hot spots. Here, the silica shell
not only serves as a protection layer but also acts as a spacer to
control the plasmonic coupling effects for an improved SERS
enhancement.
2.2.4. Targeting Molecules: Antibodies and Aptamers.
In general, the specificity of SERS-based immunoassay is
achieved through the specific antibody (or aptamer)−antigen
interactions. Hence, SERS probes should be conjugated with
antibodies, peptides, or aptamers for binding to the target
7917
molecules specifically. Among these biomolecules, antibodies
are the most commonly used ones to recognize specific
antigens. The purity, affinity, and orientation of antibodies
attached to the nanoparticles significantly influence the
targeting efficiency. Even though antibodies are relatively
expensive and not quite stable, the mature fabrication and
modification technique still make them the most popular
targeting molecules used in SERS probes.
Except antibodies, aptamers have become attractive mole-
cules in recent years, due to the properties such as high affinity
and specificity to their targets, easy chemical synthesis and
modification, and rapid tissue penetration. Aptamers are
peptides or oligonucleotides that can bind to their specific
molecules by folding into distinct secondary and tertiary
structures. The aptamer is usually selected from a large pool of
random sequences. Although aptamers generally have a lower
affinity to target molecules as compared to antibodies, the high
stability, low cost, high reproducibility, and small molecular
weight still make them an excellent alternative to antibod-
ies.107−109
3. PLATFORMS FOR SERS-BASED IMMUNOASSAY
SERS-based immunoassay has been performed on various
platforms, ranging from solid substrates to liquid microbeads,
microfluidic chips, optical fibers, papers, optical waveguides, etc.
Each of these platforms has its own characteristics regarding the
fabrication, human operation, detection performance, and
overall costs. In this section, the design, fabrication, and
characteristics of each platform are highlighted, together with
the comparison and discussion of the advantages and
disadvantages of these platforms.
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Figure 7. (a) Tuberculosis antigen detection on 1D magnetic plasmonic nanoparticles substrate using grapheme quantum dots. Reprinted with
permission from ref 115. Copyright 2015 American Chemical Society. (b) Multiplex Raman shift immunoassay on silver nanofilms using
microcontact printing. Reprinted with permission from ref 117. Copyright 2016 American Chemical Society.
3.1. SERS-Based Immunoassay on Solid Substrate
In the early years, SERS-based immunoassays were usually
performed on solid surfaces, including metallic and nonmetallic
substrates. In general, antibodies or aptamers are first
immobilized onto the solid substrates through chemical
bonding. Then the target molecules are captured by the
antibodies or aptamers on the substrate. Finally, SERS
nanoprobes are used for a quantitative analysis of the analytes
captured by the substrate. The solid substrates serve as sensing
platforms for specific biomolecular interactions, target analyte
separation, and signal transduction.
3.1.1. Nonmetallic Substrate. Nonmetallic immune
substrates include glass slides,60 quartz slides,110 multiwell
plates,111 etc. These commercially available solid substrates
could be modified with antibodies or aptamers and used as the
immune substrates. Although most of them are flat substrates
without special nanostructures, their ease of use still makes
them very popular among researchers, medical doctors, and
scientists.
Among the above-mentioned substrates, chemical-modified
glass slides are one of the most popular substrates for antibody
immobilization. Previously, our group has performed SERS-
based immunoassays on glass slides using SERS nanoprobes
composed of different kinds of nanoparticles, including gold@
silver nanorods,73 and organic−metal−quantum dots compo-
site nanoparticles (Figure 6a).17 Li et al. realized multiplex
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DNA detection with Au@Ag nanoparticles on DNA-attached
glass slides (Figure 6b).60 The benefits of using glass slides as
the immune substrates include facile preparation, high
uniformity, and low cost. Alternatively, Zhang and Du used a
quartz slide to develop an immunoassay with carbon-nanodot-
decorated Ag@SiO2 SERS probes.110 Li et al. performed a
SERS-based sandwich immunoassay on a quartz array to
achieve simultaneous detection of multiple serological cancer
markers in real serum samples (Figure 6c).112 Another high-
throughput solid substrate for immunoassays is commercially
available multiwell plates. Liang et al. employed 96-well
polystyrene plates to create a novel platform for high-
throughput analysis, in which Raman scattering light was
used as the signal-generating system of an ELISA.111
Interestingly, Ye et al. developed an array of boronate-affinity
molecularly imprinted polymers (MIPs) for the determination
of glycoproteins with a sandwich structure (Figure 6d).113 The
MIP ensures a high specificity while the SERS probes improve
the sensitivity. The boronate-affinity SERS assay exhibited
significant advantages over a conventional immunoassay in
terms of cost efficiency, stability, and speed. The method can be
easily extended to the analyses of other glycoprotein
biomarkers.
3.1.2. Metallic Substrate. Except the above-mentioned
nonmetallic substrates, metallic substrates have also been
employed widely for SERS-based immunoassays, in which the
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Figure 8. (a) Homogeneous immunoassay on 3D ordered silver nanoshell silica photonic crystal beads using SERS nanotags. Reprinted with
permission from ref 125. Copyright 2016 Royal Society of Chemistry. (b) “Lab on a bubble” immunoassay developed with silica microspheres.
Reprinted with permission from ref 118. Copyright 2012 American Chemical Society. (c) Phage−SERS sensing platform developed with silica
microparticle immune substrates and filamentous M13 bacteriophage-assisted SERS probes. Reprinted with permission from ref 126. Copyright 2014
Wiley-VCH Verlag GmbH & Co. KGaA. (d) SERS-based biomarker immunoassay developed with core−satellite nanoassembly. Reprinted with
permission from ref 127. Copyright 2015 Royal Society of Chemistry.

coupling of surface and resonance enhancements can further
improve the detection sensitivity. Genrally, the metallic SERS
substrates are fabricated either by microelectronics fabrication
technology or through the assembly of nanoparticles. The
former route requires sophisticated instruments and laborious
human work, but the obtained SERS substrates are usually
highly uniform and reproducible. The nanoparticle-assisted
fabrication is sometimes easy to perform, but it requires
elaborate design and careful operation.
In a study by Zheng et al., an Au nanohole array was
prepared by using polystyrene microbeads as the template,114
which was then used in combination with gold nanostar-based
SERS nanoprobes for the quantitative detection of Hg2+ and
Ag+ in human saliva. In this platform, the coupling between the
local surface plasmon resonance of the nanohole and nanostar
enhanced the local electromagnetic field, subsequently
improving the detection sensitivity. Besides, Zou et al.
developed a sensing platform based on linearly aligned
magnetoplasmonic nanoparticles (NPs) (Figure 7a).115 The
one-dimensional alignment of magneto-plasmonic NPs sim-
plified the immunoassay process and enabled a fast, enhanced
signal transduction. They used graphene quantum dots as the
SERS−fluorescence dual-mode labels to detect tuberculosis
antigen. Similarly, a silver nanoparticle assembled film was
produced to develop an ultrasensitive, multiplex Raman
frequency shift immunoassay (Figure 7b).39 In this method,
microcontact printing was employed to define the ordered
domains of chemisorbed Raman reporters on the substrate,
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which greatly reduced the interferences when sensing multiple
biomarkers. Recently, Liu et al. presented a plasmonic
immunosandwich assay for detecting low-copy-number protein
in single living cells.116 The combination of gold layer substrate
with SERS probes enhanced the Raman signal by 9 orders of
magnitude, which enabled unltrasensitive detection of intra-
cellular proteins. Interestingly, different from the typical
sandwich immunoassay format, a SERS quantitative bioassay
was created on aptamer-functionalized nanopillars.117 As shown
in Figure 7c, an ultrasensitive and specific detection of 5-
carboxytetramethylrhodamine (TAMRA)-labeled vasopress in
molecules was achieved by collecting highly enhanced SERS
signals emanating from nanojunctions formed between the
leaning nanopillars and aptamer-functionalized surfaces. The
SERS hot spots created by aptamer-target binding on a solid
substrate provide an alternative tool for immunoassays.
3.2. SERS-Based Immunoassay in Liquid Phase (Suspension
Array)
To create a SERS-based immunoassay, the solid substrates can
be replaced by microbeads in liquid phase. The homogeneous
environment in liquid immunoassays greatly reduces the
diffusion distance between antibodies and antigens, resulting
in faster immune reactions. In general, microspheres are
employed for the enrichment of target molecules in liquid
immunoassays, including silica beads,118 polystyrene beads,119
magnetic beads,120,121 and so on. For high-throughput
detections, multiple microbeads modified with specific anti-
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Figure 9. (a) A multiplex and straightforward aqueous immunoassay using silica-coated magnetic nanobeads. Reprinted with permission from ref 61.
Copyright 2013 Elsevier B.V. (b) Multiplex cancer cell detection and separation with silica-coated magnetic beads and SERS−fluorescence dual-
mode nanoprobes. Reprinted with permission from ref 135. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA. (c) S. aureus detection with a
MnFe2O4@Ag nanoparticle-based immunoassay. Reprinted with permission from ref 139. Copyright 2015 American Chemical Society. (d) High-
performance CTC enrichment with biomimetic immuno-magnetosomes and magnetic nanoclusters. Reprinted with permission from ref 140.
Copyright 2016 Wiley-VCH Verlag GmbH & Co. KGaA.

bodies or aptamers are used for simultaneous detection of
multiple analytes in the liquid phase. As a large crowd of
microbeads are readily suspended and drifted around in
solutions, this high-throughput platform is also known as a
suspension array.122,123 Moreover, magnetic beads are usually
introduced in such arrays for a more easily achieved separation
or enrichment of the nanoprobes. Here, we will discuss some
examples of the substrates, like nonmagnetic and magnetic
ones, respectively.
3.2.1. Nonmagnetic Substrate. Frequently used non-
magnetic beads include silica spheres, 118 polystyrene
spheres,119 metal nanoparticles124 and photonic crystal
beads,62 which are then modified with antibodies or aptamers
to form the immune substrate. Through the specific antibody−
antigen binding, SERS immunoprobes can be captured by the
microbeads. The presence and concentration of target
molecules can be characterized by SERS spectra.
For example, SERS-based homogeneous immunoassay with a
sandwich structure could be created by antibody-functionalized
polystyrene (PS) microbeads.119 SERS nanoprobes can be
collected onto the PS microspheres through the recognition of
the target molecules, forming a typical sandwich immunocom-
plex for quantitative analysis. Yan et al. employed aptamer-
functionalized Au@Ag nanostars (NSs) for ultrasensitive
determination of chloramphenicol in milk samples.124 Chlor-
amphenicol could bind to the aptamers on Au@Ag NSs, which
greatly reduced SERS signals. Here, the metal nanoparticles
served simultaneously as the immune substrate and the SERS
signal amplifier. Besides, as shown in Figure 8a, 3D ordered
silver nanoshell silica photonic crystal beads or other kinds of
photonic crystal beads have also been used for creating such
SERS arrays.16,62,125 When combined with the SERS-encoding
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technique, these immune substrates are capable of conducting a
simultaneous detection of multiple biomarkers or cells in the
liquid phase. Interestingly, a kind of “lab on a bubble”
immunoassay platform was developed by Schmit et al. using
buoyant silica microspheres (Figure 8b).118 In such an
immunoassay structure, the analyte could be rapidly detected
without an additional magnetic field or a centrifugation
procedure. In addition, there are some studies focusing on
signal amplification by using novel biomaterials in immuno-
assays. Lee et al. utilized filamentous M13 bacteriophage with a
high surface area as a signal amplifier. As shown in Figure 8c,
SERS nanoparticles were deposited on a single phage via a
layer-by-layer approach in their experiments, leading to
exponential gains in Raman intensities.126 The phage−SERS
sensing platform was demonstrated as a highly sensitive, facile,
and rapid antigen detection system.
Another kind of homogeneous immunoassay is achieved
through the self-assembly of nanoparticles. The self-assembly of
nanostructures exploits the enhancement of the local electro-
magnetic field by creating SERS hot spots, greatly improving
the sensitivity of immunoassays. On the basis of this concept,
Feng et al. developed satellite nanoassemblies for the detection
of mucin-1 (MUC1) (Figure 8d),127 prostate-specific antigen
(PSA), 128 aflatoxin B1 (AFB1),129 folic acid (FA),130
biomarkers,131 etc. The self-assembly of metal nanostructures
assisted by target biomarkers created “SERS hot spots” with
enhanced Raman signals, which can be used for sensitive
quantitative analyses.
In terms of high-throughput detection, the microbeads-based
immunoassay is compatible with the flow cytometer. Walker’s
group developed a SERS-flow cytometry for triplex detection of
leukemia and lymphoma cells in combinaion with SERS-
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Figure 10. (a) A gold-array-embedded gradient microfluidic chip for SERS-based immunoassay. Reprinted with permission from ref 63. Copyright
2012 Royal Society of Chemistry. (b) A magnetic-assisted immunoassay on a microfluidic chip. Reprinted with permission from ref 145. Copyright
2015 Elsevier B.V. (c) “Magnetic focusing” microfluidic immunoassay for the detection of biomarkers. Reprinted with permission from ref 146.
Copyright 2015 American Chemical Society. (d) A SERS-assisted 3D barcode immunoassay for high-throughput immunoassay. Reprinted with
permission from ref 66. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA.
encoded nanoprobes.132 This platform is expected to promote
the development of SERS immuno-phenotyping for improving
the sensitivity and specificity of blood cancer diagnosis.
3.2.2. Magnetic Substrate. The excellent properties of
magentic nanoparticles, such as high surface-to-volume ratio,
easy fabrication, low toxicity, and flexible bioconjugation, make
them popular in immunoassays. Magnetic nanoparticles with
sizes from nanometers to micrometers can be synthesized using
various chemical methods. In a typical magnetic immunoassay,
the repetitive washing processes can be easily and rapidly
fulfilled with a magnet without tedious human work. Addition-
ally, magnetic beads help to enrich the target molecules in
samples, which improves the sensitivity of SERS-based
immunoassays.
Commonly used magnetic beads are Fe3O4 and Fe2O3
nanospheres.120,121 Although these magnetic nanoparticles
can be directly used for biomodification due to the rich
functional groups on their surfaces, it is recommended that a
protective layer (e.g., silica, metal or polymer) is first coated on
the magnetic beads. For instance, the presence of hydroxy
groups on the surfaces of these magnetic nanospheres enables
the easy reaction with silane reagents. Hence, a silica shell can
be coated onto the surfaces of magnetic nanoparticles.133 The
coating of a silica nanoshell not only improves the stability and
biocompatibility of magnetic beads but also makes it more
convenient for the bioconjugation on magnetic beads. As a
result, silica-coated magnetic nanoparticles are widely used in
various research fields. In the case of SERS-based immuno-
assays, magnetic beads are usually wrapped with a gold or silver
nanoshell to form the magnetic/metal nanocomposites.134 The
metal shell helps to enhance the SERS signal upon antibody−
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antigen binding. This kind of nanocomposite is useful in
enormous applications in SERS-based immunoassays.
Previously, we have developed a multiplex and straightfor-
ward aqueous immunoassay using silica-coated magnetic
nanobeads and SERS nanoprobes (Figure 9a).61 The magnetic
beads were coated with a silica nanoshell to provide anchor
spots for antibodies. After the sandwich immunocomplex was
formed through the specific interactions between biomolecules,
the concentration of analytes can be quantified by SERS
intensities. The use of magnetic beads avoids tedious washing
steps during immune reactions, while the SERS nanoprobes
provide excellent optical properties (Figure 9b).135 Similarly,
work by Wang et al. used the magnet-assisted SERS-based
immunoassay to achieve a highly sensitive detection of the
hormone estradiol E2.136 Li et al. fabricated multifunctional
magnetic beads [Fe3O4@polymethacrylic acid (PMAA)-SS-FA]
to capture, detect, and release circulating tumor cells.27 The FA
molecules can target cancer cells while the disulfide bond is
sensitive to glutathione. Hence, the platform could be used for
the separation and recovery of cancer cells. On the other hand,
as mentioned above, metal-nanoshell-coated magnetic beads
were usually used for SERS enhancement. Many structures
have been proposed to obtain such nanoparticles, among which
Fe3O4@Au is one of the popular employed nanocomposites.137
Recently, Jun et al. prepared a multimodal tagging material (M-
SERS dots, Fe3O4@SiO2@Ag NPs) which has strong magnetic
properties as well as Raman signatures for imaging, separating,
and targeting specific cancer cells.138 Wang et al. developed a
MnFe 2O4 @Ag nanoparticle-based immunoassay for the
detection of Staphylococcus aureus using aptamers as the
recognition units (Figure 9c).139 The presence of a metal
nanoshell helps to enhance the Raman signals and improves the
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Figure 11. (a) A microfluidic SERS sensor based on nanopillar forests that are produced through the oxygen-plasma-stripping-of-photoresist
technique. Reprinted with permission from ref 148. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA. (b) A plasmonic nanoarray embedded
microfluidic chip fabricated by combining photolithography and nanosphere lithography. Reprinted with permission from ref 149. Copyright 2015
Royal Society of Chemistry.
sensitivity for SERS immunoanalysis. Very recently, Xiong et al.
fabricated biomimetic immuno-magnetosomes for the enrich-
ment of circulating tumor cells (CTCs) from blood samples
(Figure 9d).140 Coating of a leukocyte membrane on the
prepared magnetic nanoclusters ensured the high specificity of
CTC detection.
3.3. Immunoassays on a SERS−Microfluidic Chip
With the rapid development of microfluidic (lab-on-a-chip)
technologies in the 21st century, SERS has been combined with
microfluidics to create optofluidic immunoassays with an
improved performance.141,142 The microfluidic platform
exhibits numerous advantages, including fast response, low
sample consumption, high-throughput screening ability, and
portability.143,144 The high surface-to-volume ratio of micro-
meter-sized channels could accelerate the antibody−antigen
binding. Consequently, the reaction rate of immunoassays
performed on microfluidic devices can be further accelerated
compared to those on solid substrates or in the aqueous phase.
In addition, the continuous flowing environment helps to
reduce nonspecific adsorption of SERS nanoprobes, which in
turn increases the sensitivity and accuracy for a quantitative
detection. Therefore, SERS-based immunoassay has become
more and more popular in the last 5 years. Typically, there are
two basic kinds of SERS−microfluidic platforms: the micro-
fluidic channels and the droplet microfluidics. Here, the
characteristics of each of the two types will be discussed,
respectively.
3.3.1. Microfluidic Channels. The most common micro-
fluidic immunoassays are performed in microfluidic channels
with a dimension ranging from 10 to 1000 μm. The
microfluidic channels enable a parallel analysis of multiple
samples simultaneously. Combined with the SERS technique,
the microfluidic immunoassays can improve the performance of
immunoassays in terms of sensitivity, response time,
throughput, and overall costs.
In the past 5 years, a variety of SERS-based immunoassays
have been performed in microfluidic channels. Importantly, the
bottoms of the microfluidic channels are designed to contain a
film of metal or metal nanoparticles to generate SERS signals.
Choo’s group reported a programmable and fully automatic
gold-array-embedded gradient microfluidic chip for SERS-based
immunoassay, in which sandwich SERS immunoassays were
performed within microfluidic channels embedded with gold
arrays. The platform was able to simultaneously detect multiple
α-fetoprotein (AFP)-containing samples within 60 min (Figure
10a).63 Later, they also implanted magnetic nanoparticles into
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microfluidic chips to assist SERS immunoassays for the fast and
sensitive detection of an anthrax biomarker (Figure 10b).145 All
of the immunoassay processes can be automatically performed
in the microfluidic channels, avoiding tedious human operation
and control. Recently, Li et al. developed a “magnetic focusing”
microfluidic immunoassay (Figure 10c).146 The enrichment of
hot spots in microfluidic channels for SERS detection enabled a
high sensitivity. Besides, Kaminska et al. employed an Au−Ag-
coated GaN substrate as the novel immune solid platform to
increase the efficiency of SERS-based microfluidic immuno-
assay. They realized the detection of hepatitis B virus antigen in
human blood.147
The microfluidic chip could be combined with the SERS-
encoding technique for high-throughput biosensing. Previously,
a kind of SERS-assisted barcode chip for immunoassays was
developed by our group (Figure 10d).66 Multiple parallel
microfluidic channels were used for the patterning of antibody
barcodes. Further, by combining the SERS-spectral-encoding
technique with the microfluidic spatial encoding, a simulta-
neous detection of multiple targets in multiple samples could
be achieved within 30 min.
In addition, many research groups have reported that
plasmonic arrays with high SERS activity could be implemented
into microfluidic chips for a highly sensitive and reproducible
biochemical analysis. For example, Mao et al. introduced
nanopillar forests into a microfluidic SERS sensor with a high
sensitivity (Figure 11a).148 In their study, the SERS substrate
was prepared by employing noble-metal-covered Si nanopillar
forests as a substrate, in which the nanopillar forests were
produced on the basis of the oxygen-plasma-stripping-of-
photoresist technique. Under optimized fabrication parameters,
the enhancement factor could reach an order of 1.5 × 106 and
the SERS signal variation was within the range of ±13%, which
makes the devices more reliable for chemical analysis. Similarly,
a plasmonic nanopillar array embedded microfluidic chip was
developed by Zhao et al. for in situ SERS analysis (Figure
11b).149 By combining photolithography and nanosphere
lithography, the chip is well integrated with highly efficient
SERS substrates. Highly reproducible SERS signals can be
obtained in the presence of microfluids. The wide enhancement
field and sufficient inter-nanopillar spacing allow the reprodu-
cible SERS detection of dsDNA, which indicates the potential
of this platform for the detection of biomolecules. Yick et al.
fabricated Au-nanodot-decorated carbon nanotube arrays using
a cold atmospheric-pressure microplasma jet, which required no
expensive vacuum equipment and could be operated in the
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Figure 12. (a) Multiplex bacteria detection with SERS−microfluidic platform. Reprinted with permission from ref 155. Copyright 2016 American
Chemical Society. (b) A wash-free droplet SERS immunosensor for PSA detection. Reprinted with permission from ref 156. Copyright 2016 Royal
Society of Chemistry.

Figure 13. (a) Multiplex cancer biomarker detection combining SERS with hollow core photonic crystal fiber. Reprinted with permission from ref
160. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA. (b) A tip-coated multimode fiber with a double-substrate “sandwich” structure for
protein detection. Reprinted with permission from ref 438. Copyright 2011 American Chemical Society. (c) Intracellular pH sensing in a single cell
with a plasmonic SERS optical fiber. Reprinted with permission from ref 161. Copyright 2009 Springer-Verlag. (d) Quantitative molecular
phenotyping with topically applied SERS nanoparticles for intraoperative guidance of breast cancer lumpectomy. Reprinted with permission from ref
162. Copyright 2016 Nature Publishing Group.

open air and at room temperature.150 The 3D hybrid
nanostructure could serve as a reliable SERS sensing platform.
In addition, De Angelis et al. showed that hollow plasmonic
nanostructures can accumulate the plasmonic field into
nanofluidic channels, thus providing a straightforward approach
to combine microfuidics with plasmonics.151 The same
7923
approach can be used to investigate biochemical processes
occurring on the cell membrane152 and to perform broadband
SERS analysis.153
3.3.2. Droplet Microfluidics. Unlike the continuous
microfluidic flow systems, the droplet microfluidics focuses
on creating discrete volumes by utilizing immiscible phases.
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Figure 14. (a) A plasmonic paper-based analytical platform with functional versatility and a sub-attomolar detection limit using SERS as a
transduction method. Reprinted with permission from ref 64. Copyright 2013 American Chemical Society. (b) A lateral flow assay biosensor for
highly sensitive detection of HIV-1 DNA. Reprinted with permission from ref 165. Copyright 2013 Elsevier B.V.
Droplet microfluidic systems enable miniaturized reactions in
compartmentalized droplets of femtoliters to microliters. It
enables rapid and high-throughput analysis without increasing
the complexity and size of the microfluidic chip. This kind of
platform provides advantages including rapid mixing of the
reagents, controlled timing of the reaction, and the ability to
synthesize highly uniform micro/nanostructures.154 Droplet
microfludics could be combined with the SERS technique for
rapid and reproducible analysis. The turbulent flow inside each
droplet greatly accelerates the mixing between nanoparticles
and analytes, which improves the efficiency of reactions. To
date, the droplet SERS platform has been used for various
biochemical applications. Previously, our group developed a
droplet SERS−microfluidic chip for the rapid and sensitive
detection of thiocyanate in human saliva and serum.79 Au@Ag
NRs were mixed with saliva and serum samples that contain
SCN− in microdroplets. The peak intensity at 2100 cm−1,
which originates from the −CN bond, was used to
characterize the concentration of thiocyanate. Muhlig et al.
employed a lab-on-a-chip (LOC) platform for the differ-
entiation of six species of mycobacteria using SERS spectros-
copy (Figure 12a).155 The spectral information was derived
from the vibrational signals of the cell-wall component mycolic
acid. This platform was successfully applied to identify
Mycobacterium tuberculosis complex and non-tuberculous
mycobacteria. Moreover, similar to the SERS immunoassay in
the liquid phase, droplet microfluidics have also been combined
with magnetic immunoassays for the ease of separation or wash
of SERS probes. For example, Choo and coauthors presented a
novel wash-free immunosensor for PSA detection (Figure
12b).156 The magnetic bars enabled the separation of free and
bound SERS tags, so that the SERS intensity could be used to
characterie the concentration of PSA.
3.4. SERS-Based Immunoassay on Optical Fibers
While for immuno-detection, SERS-based immunoassays can
also be conducted based on optical fibers, especially in the
applications using portable SERS spectrometers and endos-
copies, due to their long-distance optical conduction ability and
flexibility.157−159 Combining the SERS technique with a hollow
core photonic crystal fiber (HCPCF), Dinish et al. developed
an ultrasensitive platform for multiplex detection of cancer
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biomarkers (Figure 13a).160 In this platform, immunoassay was
performed in the hollow core of the fiber. HCPCF provides
better light confinement and a larger interaction length for the
guided light and the analyte, resulting in an improved sensitivity
of bioanalytes at extremely low sample volume. Gu and
coauthors fabricated two structures for SERS−optical fiber
biosensors. One is based on a tip-coated multimode fiber with a
double-substrate “sandwich” structure, which achieved a limit of
detection (LOD) of 0.2 μg/mL in protein detection (Figure
13b).438 The other one is based on a liquid core photonic
crystal fiber with a detection limit of 106 cells/mL for the
detection of bacteria. Both of their SERS biosensors showed
great potential for highly sensitive biosensing. Vo-Dinh’s group
focued on a SERS-based plasmonic biosensor using an optical
fiber, in which the pH-sensitive 4MBA molecules were attached
onto silver nanoisland films coated onto the fiber (Figure
13c).161 This sensor can be used to measure the intracellular
pH in human mammary epithelial cells and PC-3 human
prostate cancer cells.
Recently, Wang et al. combined SERS nanoprobes with
optical fibers for a rapid (15 min) quantitative molecular
prototyping (QMP) (Figure 13d).162 The results of QMP
imaging of excised tissues from fresh human breast specimens
agree well with those of flow cytometry and immuno-
histochemistry, which may find potential applications for
guiding breast-conserving surgeries.
3.5. Paper-Based SERS Platforms
More recently, paper has emerged as a potential replacement
for conventional substrates. It is quite simple and cheap to
produce, easy to take, and has an intrinsic capacity for fluid
transportation.163,164 Generally, the paper-based devices
provide the advantages of extremely low cost, high flexibility,
and ease of use. Importantly, some of these devices provide an
excellent detection sensitivity comparable to traditional
substrates, making them a promising choice for SERS-based
immunoassays.
As an example, a plasmonic paper-based analytical platform
for a quantitative SERS detection was developed by
Singamaneni and coauthors (Figure 14a).64 The analytical
platform was prepared using a simple cut and drop method
without lithography. The fluids could transport through the
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Figure 15. A SERS sensing platform combining a plasmonic array with a super-hydrophobic substrate for unltrasensitive detection. Reprinted with
permission from ref 168. Copyright 2011 Nature Publishing Group.
paper with a rapid capillary-driven flow. There is no need for
microchannel patterning. The combination of this concept with
SERS analysis pushes the detection limit down to the attomolar
level, making microfluidic paper-based analytical devices
competitive with the traditional sensors. Commercial inkjet
printers can also be employed for fabricating SERS substrates.
Hoppmann et al. developed an inkjet-printed paper-based
SERS devices for 1,2-bis(4-pyridyl)ethylene (BPE) detection
(Figure 14b).165 The low-cost fabrication, ease of use, and high
sensitivity make it a potentially practical SERS substrate.
Another kind of novel SERS substrate is fabricated through the
in situ synthesizing method. Liu et al. was able to synthesize
gold nanoparticles on flexible silk fabrics directly through redox
reaction.166 The simple SERS substrates were used for trace
analysis of p-aminothiophenol (pATP), 4-mercaptopyridine (4-
MPy), and crystal violet (CV) molecules. One limitation of the
inkjet printing is that the functional inks must be suitable for
printing. To overcome this shortcoming, Große et al. combined
laser printing with paper substrates for peptide patterning. They
exploited material-specific adsorption of peptides and enabled
the selective functionalization of laser-printed patterns and
nonprinted paper regions.167 In this device, no specific toners
are needed for printing. The peptides could be specifically
captured on the printed or nonprinted areas. This kind of
substrate could potentially be used for the patterning of other
biomolecules, like antibodies and aptamers, which could be
used as the immune substrates for SERS-based immunoassays.
3.6. Hydrophobic SERS Substrates
The detection of a few molecules in diluted solutions has
attracted much attention in the field of biomedicine, security,
and environmental monitoring. To achieve ultrasensitive
detections, various hydrophilic SERS substrates have been
developed. The hydrophobic SERS substrates also belong to
the solid substrate. However, due to their unique properties of
enriching the hydrophilic target molecules, we specially discuss
these kinds of novel solid substrates here.
In general, most of the traditional SERS substrates are
hydrophilic; hence, the analyte molecules are easily spread over
the surfaces (since most bioanalytes are also hydrophilic). One
of the most promising routes to improve the sensitivity is to
fabricate super-hydrophobic SERS substrate to overcome the
diffusion limit of analyte molecules. To overcome the limit
dictated by diffusion, De Angelis et al. combined super-
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hydrophobic surfaces with nanoplasmonics for detection
(Figure 15).168,169 They achieved an ultrasensitive SERS
detection under an attomolar concentration. Besides, a method
using Ag/ZnO surface modified with organic stearic acid and
polyvinylpyrrolidone was demonstrated by Meng and co-
authors.170 The hydrophobic substrate enhanced the SERS
signals by 3 times compared to the hydrophilic surfaces, which
improved the sensitivity of SERS-based detections. Leiterer et
al. presented a straightforward way to obtain the monolayer
assembled by silver nanoparticles, in which Ag NPs were coated
with a hydrophobic shell to induce self-assembly without any
other surfactants.171 The SERS hot spots generated with the
assistance of hydrophobic nanoparticles could enhance the
SERS signals. Similarly, Hou et al. synthesized hydrophobic Ag
NPs in the presence of tri-n-octylphosphine sulfide.172 They
demonstrated that the hydrophobic AgNPs had a higher SERS
enhancement factor than that of the hydrophilic ones. On the
basis of the above results, the sensitivity of label-free SERS
detection could be improved with the phase transfer from
hydrophobic to hydrophilic. As another interesting example,
Wallace et al. realized the super-hydrophobic analyte
concentration utilizing SERS substrates made of colloid-pillar
arrays.173 The pillars provide a route to concentrate target
molecules via super-hydrophobic droplet evaporation effects. It
was estimated that the concentration was over 100 times
increased, resulting in a detection limit down to 2.9 × 10−12 M
for mitoxantrone dihydrochloride. Despite the tremendous
advancement, it is still challenging to create a platform for
detecting analyte molecules in all phases with a high specificity
and sensitivity. To overcome this limitation, Yang et al.
developed a universal platform named slippery liquid-infused
porous SERS.174 The platform enables the enrichment and
delivery of analytes originating from solid, liquid, and gas
phases. The analyte molecules could be delivered to SERS-
active sites for a highly sensitive detection at a concentration
down to the sub-femtomolar level. Tabakman et al. fabricated
plasmonic substrates for multiplex protein microarrays with a
femtomolar sensitivity and a broad dynamic range.175 The gold
on gold nanoisland film, prepared by uniform, solution-phase
growth method, affords near-infrared fluorescence enhance-
ment (NIR-FE) of up to 100-fold, which is useful for the
significant improvement of protein microarray detection assays.
Later, Zhang et al. employed the developed plasmonic sensor
for biomarker discovery and diagnosis of type 1 diabetes, which
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Table 2. Comparison of Different Immunoassay Platforms on Fabrication, Human Operation, Sample Consumption, Time
Consumption, and Detection Sensitivity
solid
liquid: nonmagnetic and
nonmetallic metallic magnetic
fabrication commercially microfabrication commercially available/
available and self-assembly chemical synthesis
human operation repeated washing and reagent adding repeated washing and
mixing
sample and reagent 10−30 10−30
consumption (μL)
time consumption (h) 2−4 2−4
sensitivity level μM to pM nM to fM μM to fM
showed a comparable sensitivity and specificity to radio-
immunoassays.176
3.7. Comparison between Different Platforms for
SERS-Based Immunoassay
In summary, different platforms have their own characteristics.
Here, we list the advantages and disadvantages of each platform
in Table 2 for a clear comparison.
4. SERS ENCODING FOR MULTIPLEX DETECTION
In practical biomedical applications (e.g., clinical diagnosis),
high-throughput sensing technologies have become increasingly
important with the growing demands in which millions of
synthetic reactions and biological tests are performed
simultaneously. One of the most popular ways to conduct a
multiplex assay is to use encoding microbeads or nanoprobes
with a unique code to identify the attached ligand
molecules.177,178 Up to now, various encoding strategies have
been proposed and demonstrated, including spectrometric
encoding,179−181 graphical enciphering,182,183 electronic en-
crypting,184,185 physical encoding,186,187 chemical encipher-
ing,188,189 and magnetic encrypting,190,191 generating a great
number of accurate codes for multiplex detections. Since
nonoptically based enciphering methods have been well
described elsewhere,192,193 we only discuss the optical (i.e.,
spectroscopic) encoding methods with a focus on the SERS-
encoding technique, which is widely used owing to its
convenient enciphering and noninvasive deciphering operation.
Optical encoding, also known as spectrometric encoding, can
be divided into four main families according to the light-
emitting mechanisms: fluorescence, SERS, reflection, and
luminescence lifetime.194,195 Among these, fluorescence- and
SERS-based encoders occupy a dominant role owing to their
relatively simple encoding process and high encoding
capacity.195 Fluorescence is popular for its high brightness
and easy readout, while SERS is advantageous for its narrow
spectral bands and high resistance toward fluorescence
backgrounds.70 However, when typical fluoresent dyes are
used, the limited numbers of available fluorescent agents with
the same excitation wavelength and the overlapping of the
emission bands caused by broad emission profiles restrict the
number of available distinct codes. In the SERS encrypting
method, distinct emission signatures can be obtained by
selecting different Raman reporters or their combinations.196
Additionally, ratiometric codes can be created by tuning the
intensity ratio between different characteristic SERS peaks,
which can greatly increase the enciphering capacity.197 Further,
by combining SERS with other optical-encoding methods, the
encoding capacity can even be increased exponentially.135 To
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microfluidic optical fiber paper hydrophobic
microfabrication commercially commercially microfabrication
available available
highly automated highly repeated repeated washing and
automated reagent reagent adding
adding
0.1−5 0.01−5 2−20 10−30
0.25−1 2−3 0.25−1 2−3
nM to fM nM to fM μM to pM nM to aM
realize the detection of multiple analytes in an immumoassay,
fabrication of SERS-encoded nanoparticles (i.e., the SERS
encoders) is a prerequisite. Up to now, increasing attention has
been paid to the creation of hybrid nanoparticles for generating
SERS encoders with a high encoding accuracy and capacity.
4.1. Design and Fabrication of SERS Encoders
Efficient SERS-encoding strategies depend directly on the
design and fabrication of SERS encoders. Although SERS
probes for immunoassay have already been introduced in the
previous section, the specific requirements of encoded SERS
probes (denoted as SERS encoders) still deserve to be
demonstrated. Up to now, various kinds of SERS encipherors
have been reported. To simplify the following discussion and
better demonstrate the spectral-encoding technique, we
propose a concise classification method for SERS encryptors
according to their structures. Usually, in a SERS encoder,
Raman reporters are assembled onto metal nanoparticles to
produce SERS signals, forming a signal-generating layer
composed of reporter molecules. Accordingly, if the SERS
encipherors contain only one signal-generating layer, they can
be classified as monolayer ones. Meanwhile, if the SERS
encryptor contains more than one signal-generating layer, they
can be referred to as multilayer ones (Figure 16).65,198,199 Here,
the “signal” is not limited to SERS, as other kinds of optical
signals (e.g., fluorescence) can also be included.
As shown in the left panel of Figure 16, typical monolayer
SERS encoders are obtained by assembling a layer of Raman
reporters on the surfaces of metal nanoparticles. Since the size
of the reporter molecules is quite small as compared with that
of the inner metallic nanoparticles (typically around 10−100
nm), the overall size of such monolayer SERS encipherors is
determined by the metal nanoparticles (i.e., on the nanoscopic
scale). The nanoscale monolayer SERS encryptor can be
denoted as nanoencoders for simplicity.196 By choosing
different kinds of Raman reporters assembled in the signal-
generating layer, nanoencoders with distinguished SERS codes
can be acquired. However, the limited nanoscale surface area
and the complexity of the reporter adsorption process severely
hinder the realization of reproducible nanoencoders, especially
when several Raman reporters are involved together. Thus, the
biggest obstacle in fabricating nanoencoders is how to produce
strong and reproducible SERS signals.
To solve this problem, another kind of monolayer SERS
encoder is proposed, which is illustrated in the middle pannel
of Figure 16. This type of SERS encoder (denoted as
microencoders according to their overall sizes) also contains
one signal-generating layer. The difference as compared with
the SERS nanoencoders is that, in a nanoencoder, Raman
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Figure 16. Classification of SERS encoders regarding their
composition and structure. For clarity, only two or three coding
elements (Raman reporters or quantum dots) are shown. Images are
not to scale.
molecules are directly used as the signal generators and
assembled on a nanoscale metallic core. However, in the
microencoder, individual SERS nanoprobes (or SERS dots) are
used as the SERS-generating units and assembled on a larger
microscale scaffold (e.g., polystyrene spheres).92,200 To sum up,
both SERS nanoencoders and microencoders contain a single
signal-encoding layer, but their overall sizes and signal-
generating units are different.
In microencoders, since the signal-generating layer consists
of SERS nanoprobes/dots, various SERS codes can be facilely
obtained by changing the SERS nanoprobes/dots. Typically, to
fabricate such microencoders, metal nanoparticles are only
Figure 17. Typical TEM image of SERS labels (AuNPs−TFMBA@SiO2). (b) SEM image of a single PS bead encoded with SERS nanoparticle
labels. (c) SERS spectra of AuNPs−TFMBA@SiO2 (left) and molecular structures of Raman reporters employed in this study (right). (d) Typical
Raman spectra (left) of microbeads with different types of SERS label using different Raman reporters (10000, 01000, 00100, 00010, 00001, and
11111) and their corresponding spectral barcodes (right). Reprinted with permission from ref 204. Copyright 2015 The Royal Society of Chemistry.
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decorated with one kind of Raman reporter to form the raw
SERS nanoprobes/dots. Different SERS nanoprobes/dots can
be produced by changing the Raman reporters. Then, the as-
prepared SERS nanoprobes/dots with different codes self-
assemble onto the microscale scaffold, forming a larger
microencoder. Commonly employed scaffolds are silica and
polystyrene microspheres due to their abundant size choices
and easy surface modification.201,202 This kind of microencoder
exhibits a high feasibility in achieving different codes, which can
be straightforwardly accomplished by changing the type or the
amount of SERS nanoprobes/dots on their surfaces. The SERS
nanoprobes/dots can be fabricated in realitively large scale with
acceptable signal variations, thereby microencoders usually
exhibit a better reproducibility as compared with nanoencoders.
In addition to the monolayer SERS encoders discussed
above, another type of SERS encoder is the multilayer one,
which refers to the encoders containing more than one signal-
generating layer (see the right pannel of Figure 16). In the
monolayer encoders, signal generators are all incorporated in a
single layer, making the encoding procedures sometimes
chaotic since the signal generators can compete with each
other when adsorbing to the cores. The advantage of multilayer
encoders is that different layers contain different kinds of signal
generators, which are separated from each other. Thus, the
interference between different signal generators can be greatly
reduced and the encoding procedures become more control-
lable.
A representative multilayer encoder is the SERS−fluores-
cence dual-encoded nanoprobe presented by our group. The
nanoprobe contains two signal-generating layers, a SERS layer
consisting of Raman reporters and a fluorescent layer
composed by quantum dots (QDs). To be specific, the
nanoprobe was fabricated by a layer-by-layer assembling
strategy using metal nanoparticles, organic molecules, and
semiconductor QDs. Raman reporters and fluorescent QDs
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Figure 18. (a) Schematic illustration of the preparation of the encoded SERS nanoprobes. (b) Color-coded SERS spectra of individual Raman
reporters: BDT (red), 2-NAT (green), and 4MT (blue). (c) Codes, structures, and measured spectra of the synthesized 19 SERS encoders. The
spectra are only shown from wavenumber 600 to 900 for clarity. Reprinted with permission from ref 65. Copyright 2014 The Royal Society of
Chemistry.
were assembled onto two different layers and separated with an
inert isolating layer. Such a structure can well-prevent the signal
cross-talk and guarantee a high stability. The two-layer-type
nanoprobe can fulfill SERS−fluorescence joint spectral
encoding (SFJSE), which is dicussed in detail in section 4.3.1.
4.2. SERS Spectral Encoding Strategy
4.2.1. Raman-Frequency-Based Encoding. So far, SERS-
encoding approachs depending on either Raman frequency or
signal intensity have both been reported. Due to the narrow
bandwidths in Raman spectra, the SERS-encoding method
using Raman frequency (i.e., Raman wavelength) has attracted
great attention. Su et al. proposed spectroscopically distin-
guished SERS codes by using three different Raman tags in
composite organic−inorganic nanoparticles (COINs).203 Re-
cently, with the investigation of wavelength-based-encoding
strategy, practically available codes with more Raman reporters
were accomplished in reality. For example, Wang and co-
workers developed a kind of SERS microencoder that was
fabricated using five types of silica-encapsulated SERS nano-
encoders (AuNPs-Ra@SiO2) (Figure 17).204 These five types
of SERS nanoencoders were composed of Raman reporters
such as 4MBA, 2,7-mercapto-4-methylcoumarin (MMC), 2-
mercapto-4-methyl-5-thiazoleacetic acid (MMTAA), 5-thio-2-
nitrobenzoic acid (MNBA), and 2,3,5,6-tetrafluoro-4-mercap-
tobenzoic acid (TFMBA), respectively. Thus, the correspond-
ing distinct peaks at 1080, 1172, 1290, 1337, and 1380 cm−1
were used for encoding in the SERS microencoder (Figure
17c). Since the most intensive peak of each Raman reporter was
separated by at least 35 cm−1, the SERS signature can be easily
distinguished, resulting in a straightforward decoding process. A
binary notation was introduced to characterize possible signal
combinations, where “1” represents the presence of the
corresponding SERS peaks while “0” represents the absence
(Figure 17d). Consequently, 31 spectrally distinct SERS
signatures (i.e., barcodes) can be obtained by mixing five
different Raman reporters together.
Definitely, the potential number of codes (denoted as N) can
be increased simply by using more kinds of Raman reporters,
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which can be expressed with an easy combination calculus as
follows
n
N= ∑ Cni = 2n − 1
i=1
where N is the total possible number of codes and n is the
number of different kinds of Raman reporters.204 According to
the equation, more than 100 barcodes can be generated with 7
different Raman reporters.
4.2.2. Signal-Intensity-Based Encoding. According to
the above-mentioned encoding method using Raman fre-
quency, an enormous number of Raman reporters have to be
involved with the increasing number of required codes. This
makes the fabrication procedures rather complicated and limits
the actual encoding capacity. Although the amount of
commercially available Raman reporters is relatively abundant,
their similar chemical structures make them difficult to be well
distinguished by Raman frequency when lots of reporters are
used together. Thus, as one of the solution, another SERS-
encoding method has been developed by using signal intensity
as an additional code element. As presented by our group, an
enormous number of codes could be generated using the
intensity-based SERS-encoding approach.65 We initially se-
lected three kinds of Raman reportersbenzenedithiol (BDT),
2-naphthalenethiol (2NAT), and 4-methoxythiophenol
(4MT)to fabricate SERS nanoencoders, which were
commercially available aromatic compounds with different
functional groups. Correspondingly, Raman peaks at 730, 767,
and 794 cm−1 were used as the encoding bands. A ternary
notation was introduced to describe the intensity of character-
istic peaks, where “0” represented the absence of the SERS peak
and “1” or “2” stood for a low- or high-intensity level of the
corresponding peak, respectively. According to the information
on both the wavenumber and intensity of the SERS signals, a
total of 19 codes were achieved experimentally with distinguish-
able spectral signatures. The components, structures, and
measured SERS spectra of different codes are listed in Figure
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Chemical Reviews
Figure 19. (a) Schematic illustration of the preparation of organic−metal−QD hybrid nanoparticles (OMQ NPs). (b) STEM image and the
corresponding EDX elemental distribution of Au, Ag, Si, and Cd in OMQ NPs. The merged mapping is also shown. (c) Composition, measured
spectra, and codes of the synthesized 15 nanoparticles. Reprinted with permission from ref 17. Copyright 2012 American Chemical Society.
18. It is worth noting that only 7 codes can be generated in a
sole wavenumber-encoding system.
Similarly, the groups of Lee and Wu further explored the
feasibility of a joint SERS-encoding approach using Raman
shifts and intensities.197,200 For instance, two kinds of Raman
reporters, such as 4-methylbenzenethiol (4MT) and 2-
naphthalenethiol (2NAT), were mixed at different molar ratios
(4MT:2NAT = 3:1, 7:1, 12:1, 19:1, 39:1) to generate different
codes. Raman shifts and intensities of representative peaks of
each chemical can be clearly resolved. Under this circumstance,
the maximum number of potential codes (N) was determined
by both the number of selected reporters (M) and the number
of intensity levels (i)
M
L!
N= ∑ (i k − i + 1)
(L − k) ! k!
k=1
where L is the number of available Raman reporters.200,203 For
example, when two SERS reporters and five intensity levels
were selected (in this case, L referred to 35), more than 10 000
types of SERS nanoencoders with specific signatures can be
created.
4.3. SERS-Included Joint Encoding
As is well-known, the spectral fingerprint region of most
organic Raman reporters is in the range of 500−2000 cm−1 (i.e.,
only ∼100 nm with excitation wavelengths in the visible
region). Although Raman spectra have quite narrow bands,
such a narrow spectral window (∼100 nm) still gets quite
crowded when multiple reporters are used simultaneously,
resulting in inevitable spectral overlap. A brilliant solution is to
introduce another kind of optical signal (e.g., fluorescence) to
SERS encoders. Such encoders based on joint optical signals
can remarkably increase the encoding capacity compared with
those tagged solely with SERS signals.16,17 Besides, combining
SERS signals with a spatial address for hybrid encoding offers
another choice for increasing the number of generated codes.
4.3.1. SERS−Fluorescence Joint Spectral Encoding. As
mentioned above, although multiplex optical sensing could be
achieved with either SERS or fluorescence encoding, the
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encoding capacity is limited by the spectral overlap when a sole
SERS or fluorescence signal is used, which is far less than that
predicted theoretically. Specifically, the Raman fingerprint
region of most organic SERS reporters is in the range of
500−2000 cm−1 (i.e., only ∼100 nm for excitation wavelengths
at visible region) and the commonly used SERS reporters have
similar chemical structures. Therefore, decoding the Raman
spectrum of a plurality of reporters may become difficult owing
to the spectral overlap. All of these factors restrict the number
of codes that can be realized practically.
To solve this problem, we have presented a new concept for
optical encoding, the SERS−fluorescence joint spectral
encoding method (SFJSE) (Figure 19).17 This method has
two characteristics to ensure a greatly enlarged encoding
capacity. First, the spectral range available for encoding is
broadened through the utilization of both photoluminescence
(PL) and SERS spectral regions, allowing for more spectrally
distinguishable codes. For example, when two kinds of Raman
reporters (4MBA and DTNB) and two kinds of QDs (CdTe
515 and CdTe 591) were employed, a total of 15 codes were
achieved experimentally (Figure 19c). In constrast, in an
encoding system based solely on fluorescence or SERS signals,
only three codes can be generated using two fluorescence or
two SERS agents, which was far fewer than those based on
SFJSE. When more fluorescence (m) or SERS agents (n) are
introduced, the potential encoding capacity of OMQ NPs (N)
can be estimated using the following equations
m+n m+n
(m + n)!
N= ∑ Cm+ ni = ∑ = 2m + n − 1
i=1 i=1
i ! (m + n − i )!
N /N1 = (2m + n − 1)/(2m − 1)
N /N2 = (2m + n − 1)/(2n − 1)
where N1 and N2 are the number of codes in fluorescence and
SERS-encoding systems, respectively.17 Moreover, the number
of available codes can be further increased when the intensity-
encoding mode is also considered.
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Figure 20. (a) Label-free SERS immunoassay of IgGs. IgGs are captured and analyzed using antibody-modified Fe3O4@Ag nanocomposites.
Reprinted with permission from ref 239. Copyright 2014 American Chemical Society. (b) SERS-based gradient optofluidic sensor for fast and
sensitive immunoassay. Hollow gold nanospheres are used as the SERS nanoprobes. Reprinted with permission from ref 232. Copyright 2010
American Chemical Society. (c) SERS immunoassays based on frequency shifts. Binding of the target antigen can cause a frequency shift in the
Raman bands, which can subsequently be used to quantify the targets. Reprinted with permission from ref 238. Copyright 2012 American Chemical
Society. (d) Labeled SERS immunoassay of IgGs using gold mesoflowers as the SERS nanoprobes and gold-coated magnetic nanoparticles as the
capturing substrates. Reprinted with permission from ref 243. Copyright 2014 American Chemical Society.

Second, to create such an encoder, organic−metal−quantum
dot hybrid nanoparticles (OMQ NPs) were achieved using a
core−shell structure with multiple layers, in which Raman
reporters and fluorescent QDs were assembled onto different
layers (Figure 19a).17 It makes the fabrication process easier
and more controllable for increasing the number of involved
agents. Therefore, using the proposed SFJSE method, the
number of the codes that can be generated practically is much
more than that through a traditional fluorescence- or SERS-
based encoding method, which makes the SFJSE approach
particularly promising for multiplex bioanalysis at high levels.
Later, Li and co-workers further developed this SERS−
fluorescence joint encoding system with material displacement.
They used bipyramid gold nanocrystals as the SERS substrates
instead of Au/Ag nanorods for a more sensitive detection.
Moreover, gold nanoclusters served as fluorescent agents for a
better biocompatibility.198
4.3.2. Spectral−Spatial Joint Encoding. As discussed
above, there are many different types of SERS-based encoding.
However, they are restricted to spectroscopic encoding, which
also can be referred to as 1D encoding. Interestingly, by
combining SERS spectroscopic encoding (1D) with spatial
encoding (2D), a 3D encoding strategy can be accomplished.
Our group proposed the concept of a “3D SERS barcode”,
which adds SERS fingerprints as the third dimension to the
widely used 2D barcode (Figure 12d).66 To make the 3D SERS
barcode easier to understand, a multiplex immunoassay was
conducted on the basis of a microfluidic chip. First, multiple
proteins were spatially separated using a patterned antibody
barcode substrate, forming a 2D hybridization array. Then
SERS nanoencoders were used to identify and quantify the
proteins. As different SERS nanoencoders carry different kinds
of Raman reporters, they can incorporate spectroscopic
information (1D) into the 2D spatial barcode, resulting in a
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3D barcode. The SERS-assisted 3D barcode greatly enhances
the information storage capacity. All necessary information,
including the species and amount of target analytes, can be
simultaneously read out during the detection process. To
summarize, the SERS-based joint encoding strategy is practical
and feasible for the easy and rapid identification of large
chemical and biological libraries. A beneficial aspect of the joint
encoding strategy is the diversification of encoding elements.
Besides fluorescent or spatial signals, extinction or other optical
signals can also be combined with SERS. Moreover,
substantially more codes can be acquired when multimodes
or the intensity-encoding mode is also considered.
5. APPLICATIONS OF SERS-BASED IMMUNOASSAY
So far, SERS-based immunoassays have been well-employed in
biochemical and biomedical applications for the detection of
biological targets, such as proteins,110,205,206 nucleic acids,207,208
virus,209 cells,210,211 and toxins.212 Up to now, there are a series
of well-established traditional biological detection strategies for
these targets [such as ELISA,213 Western blot,214 flow
cytometry,215 mass spectroscopy,216 electrophoresis,217 and
polymerase chain reaction (PCR)218]. They have played a quite
important role in the development and investigation of
sophisticated biological and physiological issues. However,
they usually suffer from some unsatisfactory shortcomings,
including limited sensitivity, complicated working procedures,
and a high risk of artificial contaminants. Compared with
traditional biological detection methods, SERS-based immuno-
assays stand out due to some unique and excellent character-
istics, for example, negligible background noise, low spectral
overlap, high sensitivity, large dynamic range, and fine
multiplexing ability.8,130,219,220
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Table 3. SERS-Based Immunoassays for IgG Detection

refs IgGs recognition unit detection platform dynamic range LOD
83 human IgG goat anti-human IgG Ag glass substrate 100 ng/mL to 100 fg/mL 100 fg/mL
239 human IgG anti-IgG antibody liquid phase 100 ng/L to 500 mg/L 600 fg/mL
246 human IgG anti-human IgG gold patterned substrate 1 μg/mL to 10 fg/mL 10 fg/mL
247 human IgG goat anti-human IgG fingerprints on glass slides 1 × 10−7 to 1 × 10−1 mg/mL 1 × 10−7 mg/mL
248 human IgG goat anti-human IgG gold triangle nanoarray 0.1 pg/mL to 10 ng/mL 7 fg/mL
81 human IgG goat anti-human IgG gold nanostar substrate (1 × 10−4)−10−14 g/mL 10 fg/mL
249 human IgG goat anti-human IgG nanoporous waveguide (PAA) film (1 × 10−10)−(1 × 10−6) g/mL 0.1 ng/mL
250 human IgG goat anti-human IgG bowl-shaped silver cavity array 0.5−5 ng/mL 0.1 ng/mL
206 human IgG anti-human IgG polystyrene substrate 45−500 pM 45 pM
251 mouse IgG goat anti-mouse IgG liquid phase 50 ng/mL to 1.9 pg/mL 1.9 ng/mL
252 mouse IgG goat anti-mouse IgG Ag + diatom biosilica substrates 1 pg/mL to 10 μg/mL 10 pg/mL
253 mouse IgG goat anti-mouse IgG antibody Ag colloid/PVP/paper 1−500 ng/mL 0.33 ng/mL
244 mouse IgG goat anti-mouse IgG liquid phase 1−(5 × 105) fg/mL 1 fg/mL
61 mouse IgG goat anti-mouse IgG liquid phase 0.1 pg/mL to 1 μg/mL 0.1 pg/mL
254 mouse IgG goat anti-mouse IgG gold-plated filters 10−1−(1 × 105) ng/mL 0.84 ng/mL
84 mouse IgG goat anti-mouse IgG glass substrate 100 pg/mL to 100 μg/mL 100 pg/mL
110 mouse IgG anti-β-actin mouse antibody quartz slide 0.01−1000 μg/mL 2.5 ng/mL
255 rabbit IgG goat anti-rabbit IgG bowl-shaped silver cavity thin film 0.1−20 ng/mL 5 pg/mL
232 rabbit IgG polyclonal antibody microfluidic chips 0−100 ng/mL 1−10 ng/mL
66 mouse IgG goat anti-mouse IgG microfluidic chips 10−8−10−14g/mL 10 fg/mL
human IgG goat anti-human IgG
242 mouse IgG anti-mouse IgG Ag substrate 5 ng/mL to 5 fg/mL 800 ag/mL
human IgG anti-human IgG 5 ng/mL to 500 fg/mL 5 fg/mL
238 H1 protein anti-H1 antibody Au/Ag bimetallic glass 0−100 nM 2.2 nM
p53 protein anti-human p53 substrate 2.5 nM

5.1. Analysis of IgGs
IgGs play a vital role in the immune system of an organism,
helping to protect the organism from external invasions of
bacteria and viruses. Hence, detection of IgGs is important for
the determination of infectious or immune-system-related
diseases (e.g., rheumatoid arthritis, hepatitis, and lupus
erythematosus). In addition to Western blot and mass
spectrometry, the most representative method for analyzing
the species and concentrations of IgGs is immunoassay, which
is usually realized by the immune recognition between IgGs
and their specific antibodies. According to different signal
readout schemes, immunoassays for IgGs can be divided into
several categories, including ELISA,221−223 chemilumines-
cence,224,225 colorimetry,226,227 fluorescence,228,229 SPR,230,231
SERS,83,232 etc. The other immunoassays have been reviewed
well elsewhere.1,2,233−236 Here, we focus on SERS-based
immunoassays (Figure 20). SERS-based immunoassays can be
divided into two kinds. The first one uses the intensity of the
SERS signal as the indicator for IgGs,232,237 while the second
one uses the frequency shift of SERS bands to detect the
targets.39,238 The former kind can be subsequently divided into
two types, i.e., label-free SERS immunoassay239 and labeled
SERS immunoassay.83,240 Label-free SERS immunoassays are
relatively simple as compared with labeled SERS immuno-
assays, since they do not need a specially designed SERS
nanoprobe. However, labeled SERS immunoassays can offer a
higher sensitivity as compared with label-free SERS immuno-
assays due to the strong Raman signals offered by Raman
reporter molecules. These features make SERS-based immuno-
assays suitable for IgG identification applications, such as
immuno-phenotyping.
Usually, SERS-based immunoassay can achieve quite low
LODs and large dynamic ranges.241−243 For example, by using
magnetic beads and SERS nanobioprobes, Xiao et al. realized
7931
the analysis of mouse IgG.244 The detection limit is 1 fg/mL
and the dynamic range is from 1 to 5 × 105 fg/mL, which is 5
orders of magnitude. In their work, the SERS nanoprobe was
fabricated by attaching antibodies onto gold-core−silver-shell
nanorods, and Raman reporter molecules were embedded in
the silver shell to provide SERS signals. Mouse IgGs were
anchored on the surfaces of the magnetic beads. Hence, the
immune recognition between mouse IgGs and the antibodies
on the SERS nanoprobes allows the formation of a sandwich-
type immune complex. The fine performance of the presented
assay is attributed to the 3D structure of magnetic beads, which
can reduce the steric effect, as well as the structure of SERS
nanoprobes, which can generate strong SERS enhancement.
Analyzing of IgGs can be largely automated and simplified
with the assistance of microfluidic chips.142,245 Chon et al.
developed a SERS-based gradient optofluidic sensor for fast and
sensitive immunoassay.232 The LOD for rabbit IgG was
estimated to be 1−10 ng/mL, and the assay took less than
30 min to achieve the results because the experimental
procedures were automatically controlled with the microfluidic
chip. Recently, our group reported the detection of IgGs using
a 3D-barcode microfluidic chip.66 The LOD was found to be 10
fg/mL with a dynamic range from 10−8 to 10−14 g/mL. The
most outstanding advantage of this chip-based assay is its
excellent multiplexing ability offered by spatial and SERS
encoding, which is well-discussed in detail in section 4. Typical
SERS-based immunoassays of IgGs are listed in Table 3.
5.2. Study of Disease Biomarkers
Early diagnosis of malignant disease (e.g., cancer) is important
for choosing appropriate therapeutic strategies and improving
the overall survival rates. Formerly, diagnosis of cancer had
been strongly dependent on traditional approaches, such as X-
ray, computer tomography, endoscopy, ultrasonography,
pathological section, and so on.256−261 However, these
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Figure 21. SERS immunoassay of disease biomarkers. (a) Rapid detection of HER2 using an ac-EHD microfluidic chip. Bifunctional fluorophore-
integrated gold/silver (F-SERS) nanoshells are used as the SERS probes. Reprinted with permission from ref 280. Copyright 2016 American
Chemical Society. (b) Aptamer-guided Ag−Au nanostructures for targeting of MUC1-overexpressing cancer cells. Reprinted with permission from
ref 287. Copyright 2012 American Chemical Society. (c) SERS-based immunoassay of tumor marker VEGF using DNA aptamers and silica-
encapsulated hollow gold nanospheres. Reprinted with permission from ref 271. Copyright 2013 Royal Society of Chemistry.

approaches are mainly focused on the anatomic form of tissues,
which usually lacks sensitivity toward early-stage tumors.
Besides, these traditional diagnosis approaches are often
accompanied by surgical procedures, which are harmful to the
patients and hinder repetitive and timely examinations. With
the development of molecular and medical biology, researchers
have found that tumor cells (or cells sensitive to tumors) can
secret special biomarkers that can enter body fluids (e.g., blood,
urine, ascites, and saliva). The presence of biomarkers in body
fluids is a valuable and prompt warning of health conditions.
Hence, analyzing and screening of biomarkers is a powerful tool
for early-stage diagnosis. Up to now, the discovered biomarkers
related to cancers include proteins (e.g., receptors, cancer
antigens), microRNAs (miRNAs), circulating DNA, and
CTCs.262−267 The most well studied biomarker species are
proteins. Typical protein biomarkers include carcinoembryonic
antigen (CEA), AFP, human epidermal growth factor receptor
2 (HER2), vascular endothelial growth factor (VEGF), PSA,
tumor suppressor p53, and epidermal growth factor receptor
(EGFR). Similar to IgG analysis, SERS-based immunoassays
have found prosperous applications in the detection of disease
biomarkers63,125,158,248,268−276 (Figure 21 and Table 4). More-
over, the detection of biomarkers using SERS-based immuno-
assay platforms only requires a little amount of body fluids and
thus strongly reduces the injury toward patients and allows
repetitive examinations during therapy. This is crucial for
prognosis evaluations and adjusting therapeutic strategies in
time.
5.2.1. Detection of Protein Biomarkers. The detection
of protein biomarkers follows a similar principle as compared
with the detection of IgGs, that is, using immuno-functionalized
SERS nanoprobes to recognize these biomarkers.16,145,277,278 By
combining ELISA with SERS, Liang et al. presented a novel
immunoassay protocol for the evaluation of PSA in whole
serum and urine.111 The introduction of aggregated Ag NPs
resulted in a quite impressive sensitivity in the ultralow
concentration range (10−9−10−6 ng/mL). The high sensitivity
of this protocol is attributed to multistage signal amplifications,
7932
including efficient Raman signal enhancing provided by
aggregated Ag NPs. Wang et al. utilized an alternating current
electro-hydrodynamic (ac-EHD) microfluidic chip and SERS
nanoprobes to realize the fast and sensitive detection of
HER2.279 Interestingly, in their design, ac-EHD-induced
nanoscaled surface shear forces enhanced the capture kinetics
and thus greatly shortened the overall reaction time. The LOD
was found to be 10 fg/mL and the detection only took 40 min.
Similarly, Zhou et al. also employed a typical sandwich-type
SERS immunoassay for the detection of cancer biomarkers.280
The silica-coated silicon NPs were used as the SERS
nanoprobes, which were functionalized with specific antibodies.
The immune substrate was fabricated by depositing Ag film
onto SiC sandpapers. Capturing antibodies were immobilized
onto the Ag films. With the presence of target biomarkers, the
SERS nanoprobes linked to the Ag film via the formation of
sandwich-type immune complexes. Hence, the Raman signal of
the silicon NPs was enhanced and thus can be used as the
indicator of biomarkers. Separate detection of three biomarkers
in human serum sample was realized, including PSA, AFP, and
carbohydrate antigen 19-9 (CA19-9). The LODs were found to
be 1.79 fg/mL (PSA), 0.46 fg/mL (AFP), and 1.3 × 10−3 U/
mL (CA19-9), respectively.
Aside from antibodies, the recognition of biomarkers can also
be accomplished by aptamers. Since their discovery, aptamers
have been well-known for excellent binding affinity toward their
targets (e.g., thrombin, peptides, and cells).281 DNA aptamers
provide a powerful candidate for the design of novel immune
sensors with high performance. Up to now, aptamers have been
employed as the recognition unit in immunoassays based on
electrochemistry, SPR, and SERS. Electrochemistry- and SPR-
based assays using aptamers have been reviewed else-
where.282−284 For SERS-based immune reactions using
aptamers, the detection scheme is quite similar to those using
antibodies.285,286 Wu et al. fabricated an aptamer-guided Ag−
Au nanostructure using photoreduction.287 To generate SERS
signals, Rh6G is attached to the nanostructure. The aptamer
they used is S2.2, which is a 25 base oligonucleotide specific for
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Table 4. SERS-Based Immunoassays for Biomarkers

recognition
refs biomarkers unit detection platform dynamic range LOD
305 A1AT antigen antibody 10−1000 ng/mL 10 ng/mL
129 aflatoxin B1 (AFB1) DNA Au NS 1−1000 pg/mL 0.48 pg/mL
63 α-fetoprotein (AFP) antibody gold array 0−10 ng/mL 0.1 ng/mL
269 angiogenin antibody gold-patterned microarray chip 10−4−10−12g/mL 0.1 pg/mL
145 anthrax biomarker poly-γ-D- antibody magnetic beads 100 pg/mL to 100 μg/mL 100 pg/mL
glutamic acid (PGA)
137 antigen gp51 anti-gp51 MNP-Au 0.95 mg/mL
112 CA15-3 antibody 0.1−500 U/mL for CA15-3 antigens 0.99 U/mL
CA27-29 0.1−500 U/mL for CA27-29 antigens 0.13 U/mL
CEA 0.1−500 ng/mL for CEA antigen 0.05 ng/mL
40 cetuximab antibody MDA-MB-468 breast cancer cells 0−2000 ng/mL 1 ng/mL
115 CFP-10 antibody 1 pg/mL to 1 μg/mL 0.0511 pg/mL
118 cholera toxin (CT) antibody silica microspheres 1100 ng
303 ctDNA DNA single-walled carbon nanotubes 10 fM to 1 nM 0.3 fM
306 detection of polar antibiotics Ag−graphene/electrode
304 DNA DNA magnetic NPs
307 dopamine aptamer 0.01−10pM 0.006 pM
308 EGF, ErbB2, IGF-1 antibody
309 EGFR antibody photonic crystal fiber (PCF) 100 pg in 10 nL
310 EGFR antibody
279 EGFR2 antibody gold-coated glass chips 1 fg/mL to 1 ng/mL 10 fg/mL
130 folic acid antibody Ag NPs 0.005−1 ng/mL 0.86 pg/mL
113 glycoproteins boronate- 0.1 ng/mL to 100 μg/mL (HRP), 1 3 nM (HRP), 0.7 nM
affinity ng/mL to 10 μg/mL (AFP) (AFP)
311 heparin protamine 0.5−150 ng/mL 0.5 ng/mL
147 hepatitis B virus anti-HBsAg SERS-active substrate 0−625 IU/mL 0.01 IU/mL
312 HIV-1 DNA DNA lateral flow assay 64 ng/mL ∼ 8 pg/mL 0.24 pg/mL
136 hormone estradiol E2 anti-E2 magnetic NPs 0.1−1000 pg/m 0.65 pg/mL
antibody
248 human IgG antibody gold triangle nanoarray 0.1 pg/mL to 10 ng/mL 7 fg/mL
313 IL6 antibody Au 10 ng to 1 μg 10 ng
270 IL6/TNFα antibody nitrocellulose membrane 0−5 ng/mL 7.15 pg/mL
0−14 ng/mL 10.7 pg/mL
288 latent fingerprints aptamer glass substance
39 liver cancer biomarkers α- antibody Ag island film 5.0 pM
fetoprotein and glypican-3
314 lung cancer markers antibody
315 microcystin-LR (MC-LR) DNA Au NFs 0.01−100 nM 8.6 ± 0.4 pM
300 miR-203 DNA 10−14 −10−7 M 6.3 fM
294 mir-21 miRNA gold slide 4.44−1480 nM 0.36 nM
7.40−1480nM 0.85 nM
291 miRNA miRNA silver nanorod arrays
297 miRNA miRNA Silica microbeads 1 fM to 10 pM 0.3 fM
268 MMP-7 and CA19-9 antibody Au/glass and mask to generate 2.28 and 34.5 pg/mL
array
316 mouse double minute peptide 1.5 nM
(MDM2) protein
287 MUC1 aptamer breast cancer cell
274 N-terminal pro-brain antibody CoFe2O4 1 fg/mL to 1 ng/mL 0.75 fg/mL
natriuretic peptide
273 platelet-derived growth factor DNA Au film (1.0 × 10−12)−10−8 M 0.42 pM
B-chain (PDGF-BB)
317 prostate-specific antigen (PSA) DNA 0−4 ng/mL 0.5 ng
318 proteases peptides AuNPs 0−100 nM 85 fM
319 protein antibody 0−(3.4 × 105) ng/mL 10 zM
320 protein antibody 146 ng/L
74 PSA antibody silicon (microcontact printing) 1 pg/mL 1 pg/mL
111 PSA and the adrenal stimulant antibody 96-well plates 10−9 and 10−6 ng/mL
ractopamine (Rac)
280 PSA/AFP/CA19-9 antibody silver NPs 1.79 fg/mL, 0.46 fg/mL,
1.3 × 10−3 U/mL
276 RGG9 domain of nucleolin AS1411 G- split-ring resonators 10−13−10−5 M 10−13 M
quadruplex

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Table 4. continued
recognition
refs biomarkers unit detection platform dynamic range LOD
321 telomerase DNA electrodes 5−1000 Hela cells 5 cells
322 telomerase DNA sensor surface 1 tumor cell/1000
background cells
323 telomerase DNA TS-modified AuNPs 0−100 Hela cell/μL 1 Hela cell/μL
324 telomerase DNA porphyrin graphene nanocomposite 0−5000 cells/mL 10 Hela cells/mL
electrode
78 telomerase TS primers 1 cell/mL
325 telomerase TS primers MB@Au@TS 0−1000 cells/mL 1 cell/mL (SERS), 10 cell/
mL (colorimetry)
326 telomerase DNA Ag substrate
327 telomerase DNA
328 telomerase DNA silica microbeads (SiMBs) single HeLa cell
329 thrombin peptides 160 fM
330 thrombin ATP MB 0.27 pM
117 vasopressin molecules ATP gold-decorated silicon nanopillars 1 pM to 1 nM 1 pm/mL
271 VEGF ATP/VEGF gold microarray 0.1−1000 ng/mL 1−10 pg/mL
antibody
331 vitellogenin (Vg) antibody nanosculptured thin films (nSTFs) 5 pg/mL to 25 ng/mL 5 pg/mL
of silver on Si substrates
332 WNV and RVFV antigen antibody magnetic NPs 5 fg/mL (PBS), 25 pg/mL
(FBS)

MUC1 mucin. MUC1 is a transmembrane glycoprotein and is
overexpressed in primary and metastatic breast cancers. By
measuring the SERS signals on cells, it was found that the
aptamer-guided nanostructure can specifically bind to MUC1-
overexpressing cancer cells. Moreover, the nanostructures
bound on cancer cell membranes can be subsequently used
as the NIR absorber and heat generator for photothermal
therapy. Ko et al. reported a SERS-based immunoassay of
tumor marker VEGF using DNA aptamers.271 They used a
gold-patterned microarray chip as the immune substrate, and
capturing antibodies were immobilized on the chip. They also
fabricated silica-encapsulated hollow gold nanospheres as the
SERS nanoprobes. VEGF-specific aptamers were decorated on
the SERS nanoprobes. Consequently, a sandwich-type immune
complex can be formed by the capturing antibody, target
VEGF, and SERS nanoprobes. As compared with conventional
ELISA, the dynamic range of the aptamer-based assay was 3−4
orders of magnitude larger and the detection sensitivity was 2−
3 orders of magnitude higher. Zhao et al. fabricated a SERS
nanoprobe for the imaging of fingerprints.288 Aptamers were
decorated on the surface of the nanoprobes to recognize and
bind lysozyme in the fingerprints. Owing to the fine sensitivity
of SERS and the excellent selectivity of aptamers, high-
resolution imaging of fingerprints was realized. Moreover, such
an aptamer-based SERS imaging technique does not require
complicated pre- or post-treatment and can be used to detect
various types of fingerprints.
5.2.2. Detection of miRNAs and Circulating DNAs.
miRNAs are short (19−25 nucleotides) noncoding RNAs that
are important for the regulation of genes. miRNAs are
considered as an important kind of biomarker since they are
closely related with the development of cancerous dis-
eases.289,290 Detection of miRNAs can be accomplished by
traditional methods such as PCR, Northern blotting, and
microarrays. However, these methods suffer from some
shortcomings, such as large sample amount, complicated
primer design, and cross hybridization. Recently, SERS has
also been applied to realize the facile and sensitive detection of
miRNAs. Generally, SERS-based detection of miRNAs can be
7934
divided into three kinds: label-free detection,291−293 direct
hybridization detection,294−296 and signal amplification detec-
tion.297−299 Label-free detection of miRNAs is conducted by
enhancing the Raman signals of RNA strands via SERS
substrates, which is relatively simple and thus will not be
discussed in detail here. Direct hybridization detection of
miRNAs is realized by using complementary DNA capturing
strands. Signal amplification detection of miRNAs is accom-
plished by the addition of enzymes or complementary strands
to exaggerate the signals.
Guven et al. reported direct hybridization detection methods
for mir-21.294 They employed two detection schemes. The first
one is to immobilize the target mir-21 on a gold slide, then
SERS nanoprobes decorated with mir-21 complementary
strands can hybridized with the mir-21. The second detection
scheme is a sandwich-type assay. A DNA strand that is
complementary to one end of mir-21 is immobilized on the
gold slide. This DNA strand is used to capture mir-21. Then a
SERS nanoprobe decorated with another DNA strand that is
complementary to the rest of the mir-21 can hybridize with it.
Thus, a sandwich-type structure is formed by the capturing
strand, mir-21, and SERS nanoprobe, which is similar to the
immune complex formed in sandwich-type immunoassays. In
both detection schemes, the SERS intensity of the hybridized
nanoprobes can be used to report the concentration of mir-21.
The LOD of the first scheme was 0.36 nM and that of the
second scheme was 0.85 nM. Besides, the assay only took less
than 40 min with a final performance comparable to that of
PCR. As an example of signal amplification assays of miRNAs,
Zheng et al. presented an enzyme-free quadratic SERS signal
amplification approach for the detection of circulating miRNAs
in human serum.297 Signal amplification was realized through
the combination of a miRNA-triggered hybridization chain
reaction and Ag+-mediated cascade amplification. An ultrahigh
sensitivity (LOD is 0.3 fM) toward miRNA was achieved with
this approach. Moreover, this approach can also be used for the
direct detection of miRNAs in serum collected from patients
with different stages of chronic lymphocytic leukemia. The
results correspond well with those determined by qPCR. Zhang
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Chemical Reviews
Figure 22. SERS-based telomerase activity detection and telomere length measurement. (a) TEC-SERS method for ultrasensitive detection of
telomerase. Reprinted with permission from ref 78. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA. (b) In situ sensing of cellular
telomerase using SERS. Reprinted with permission from ref 340. Copyright 2016 WILEY-VCH Verlag GmbH & Co. KGaA. (c) SERS based in situ
hybridization (SISH) for telomere length measurement. Reprinted with permission from ref 327. Copyright 2016 American Chemical Society.
et al. reported a cascade amplification and SERS-based
detection method for miR-203, which is an important
biomarker for tumor growth and progression.300 The method
was realized with the assistance of a specially designed
multifunctional nucleic acid probe as well as the nicking
endonuclease and polymerases. The detection limit of miR-203
was found to be 6.3 fM.
Circulating DNAs are found in peripheral blood of patients
and can reflect the genomic state of cancerous disease.301 In the
past year, SERS has just begun to be employed for the analysis
of circulating DNAs.302 Zhou et al. presented a SERS-based
approach for the detection of circulating DNAs in human
blood.303 A triple-helix molecular switch (THMS) was used as
the recognition element for circulating DNAs. With the aid of
RNase HII, numerous T-rich strands of the THMS will be
released in the presence of target circulating DNAs and
subsequently be absorbed on single-walled carbon nanotubes
(SWNTs) via π−π stacking interactions. Then the T bases on
SWNTs can facilitate the in situ growth of copper nano-
particles, which can enhance the Raman signals of SWNTs.
Hence, the intensity of SERS signals of SWNTs indicates the
concentration of target circulating DNAs. Wee et al. reported a
multiplex detection of mutations in circulating tumor DNA
using SERS.304 They first amplified three melanoma mutations
with PCR, and then mutation-specific SERS nanotags were
hybridized with the PCR products. Finally, the SERS-nanotag-
labeled PCR products were captured by magnetic beads. The
presence or absence of melanoma mutations in circulating
DNAs can be determined by measuring the SERS spectra. This
SERS-assisted method can reproducibly detect as few as 0.1%
(10 copies, CV < 9%) of target sequences.
5.2.3. Detection of Telomerase Activity and Telomere
Length Measurement. Aside from the above-mentioned
common biomarkers, telomerase is another important bio-
marker. The overexpression of telomerase and abnormal
variation in telomere length are closely related with carcino-
genesis, which makes telomerase and telomere new therapeutic
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Review
targets for the design of anticancer drugs.333,334 Telomerase
activity detection and telomere length measurement can
provide important information for the diagnosis and
therapeutics of cancerous diseases. Telomerase activity
detection methods include telomere repeat amplification
protocol (TRAP),335,336 electrochemical method,321 SPR,322
colorimetry,323 and chemiluminescence.324 Telomere length
measurement methods include Southern blotting, hybridization
protection assay,337 qPCR,338 and fluorescent in situ hybrid-
ization (FISH).339
SERS-based detection of telomerase activity is usually based
on the fact that telomerase can enlongate telomere repeats and
thus induce a SERS signal variation. Our group was the first to
use a SERS technique for the detection of telomerase activity
and telomere length measurement (Figure 22). We reported
the telomerase elongation controlled SERS (TEC-SERS)
method for ultrasensitive detection of telomerase.78 TEC-
SERS utilizes the fact that SERS is highly sensitive to the
aggregation state of metal nanoparticles. Telomerase-sensitive
SERS nanoprobes were fabricated by decorating spherical gold
nanoparticles with telomerase substrate primer (TS primer)
and Raman molecules. Then the SERS nanoprobes were used
to detect the telomerase activity of tumor cells. The detection
limit of TEC-SERS is 2 orders of magnitude lower than that of
other methods, which is beneficial for the ultraearly diagnosis of
malignant tumors. Besides, no PCR amplification is needed in
the TEC-SERS method, which greatly simplifies the detection
procedure, avoids false negative results, and improves reliability.
Later, we also presented a colorimetry- and SERS-based
telomerase activity screening method.325 Magnetic capturing
nanoparticles with TS primer anchored on their surfaces and
SERS nanoprobes with telomere complementary strand
attached to their surfaces were synthesized. By monitoring
the color and SERS signal of the final sediments, both
colorimetry- and SERS-based telomerase activity detection was
achieved. This method simultaneously holds the merits of
colorimetry and SERS, i.e., fast screening and high sensitivity.
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Chemical Reviews
Figure 23. SERS assays of bacteria and viruses. (a) Label-free SERS assay of bacteria using vancomycin-coated Ag-nanoparticle arrays. Reprinted
with permission from ref 345. Copyright 2011 Macmillan Publishers Ltd. (b) Microfluidic dielectrophoresis device for rapid online detection of a
single bacterium. Bacterial cells are recognized by SERS nanoprobes decorated with antibodies. Reprinted with permission from ref 353. Copyright
2014 Wiley-VCH Verlag GmbH & Co. KGaA. (c) Schematic illustration of SERS enzyme-catalyzed immunoassay of respiratory syncytial virus.
Reprinted with permission from ref 355. Copyright 2016 Elsevier B.V.
Thus, efficacious and sensitive screening of numerous samples
can be realized. The detection limits for SERS and colorimetry
were 1 tumor cell/mL and 10 tumor cells/mL, respectively. In
addition, two other research groups also started to work on
SERS-based telomerase activity detection. Xu et al. reported in
situ sensing of cellular telomerase using SERS and multigap-
embedded nanoassemblies.340 Shi et al. presented a quadratic
SERS signal amplification based method for telomerase
detection with LOD down to the single-cell level.328
For measuring telomere length, we presented two SERS-
based strategies.326,327 The typical one is called SERS-based in
situ hybridization (SISH).327 The SISH method uses two kinds
of SERS nanoprobes to hybridize in situ with telomeres and
centromeres, respectively. Telomere length is evaluated using a
redefined telomere-to-centromere ratio (T/C ratio). The
calculation method for the T/C ratio in the SISH method is
more reliable than that in FISH. The SISH method can be used
to analyze single telomeres. These features make SISH an
excellent alternative strategy for telomere length measurement.
5.3. Identification of Bacteria and Viruses
Detection of bacteria and viruses is crucial in food safety and
surveillance of epidemic diseases.9,341,342 The complexity of the
samples makes SERS quite suitable for the identification of
bacteria and viruses, since SERS holds a fine endurance toward
biological background interferences (Figure 23). Label-free
SERS strategy has been well applied in the identification of
bacteria and viruses, which is realized by directly analyzing the
SERS spectra of bacteria and viruses.102,343−345 Here, we mainly
focus on labeled SERS immunoassays of bacteria and viruses,
which usually rely on the specific recognition of proteins of
bacteria and viruses by SERS nanoprobes.147,346−352 Lin et al.
reported a microfluidic dielectrophoresis device for a rapid
online detection of single bacterium353 (Figure 23b). They
fabricated SERS nanoprobes by immobilization of bacteria-
specific antibodies onto the surfaces of nanoaggregate-
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Review
embedded beads. Hence, these SERS nanoprobes can be
bound to the target bacteria via the immune reaction between
the antibodies and proteins on the bacteria’s surface. With the
assistance of a microfluidic chip, the total assay time was
significantly reduced (<2 h). Besides, the amount of antibodies
used in the SERS-based assay was 100-fold less than that used
in the traditional ELISA. The LOD was found to be 70 cfu/mL.
Neng et al. reported a multiplex immunoassay for the surface
envelope and capsid antigens of the viral zoonotic viruses.332
Detection was mediated by immune recognition using SERS
nanoprobes and paramagnetic nanoparticles conjugated with
antibodies. The LOD was 5 fg/mL in PBS and 25 fg/mL in
fetal bovine serum. Li et al. presented a SERS enzyme-catalyzed
immunoassay of respiratory syncytial virus.125 The combination
of ELISA and SERS produced an impressive LOD of 0.05 pg/
mL, which is 20 times lower than that of a colorimetric assay.
In addition to the identification of whole virus particles,
researchers also focused on the investigation of virus DNAs
using SERS techniques. Here, the recognition of target virus
DNAs is fulfilled by their complementary DNA strands
modified on the surfaces of SERS nanoprobes or nanotags.354
For example, Fu et al. developed a SERS-based lateral flow
assay biosensor for a highly sensitive detection of HIV-1
DNA312 (Figure 24). The SERS nanotags were fabricated by
attaching Raman reporters and target complementary DNA
strands onto gold nanoparticles. Capturing DNA strands were
immobilized on the lateral flow strip. With the target HIV-1
DNA present, a sandwich structure was formed by the
capturing strand, HIV-1 DNA, and SERS nanoprobe. Hence,
SERS signals can be used to identify the HIV-1 DNA. The
detection limit of the target DNA was found to be 0.24 pg/mL.
5.4. Detection of Ions and Toxins
SERS-based immunoassays can also be used to detect metal
ions and toxins (Figure 25). Especially, monitoring of heavy
metal ions and toxins is important for environmental
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Chemical Reviews
Figure 24. SERS-based lateral flow assay biosensor for a highly
sensitive detection of HIV-1 DNA. (a) Schematic illustration of the
lateral flow biosensor. (b) Working principle of the SERS-based lateral
flow chip. Reprinted with permission from ref 312. Copyright 2016
Elsevier B.V.
maintenance and public health. Specific SERS-based immune
detection of ions and toxins is commonly realized by antibodies
Figure 25. SERS immunoassays for ions and toxins. (a) SERS-based competitive immunoassay for mercury ions. Reprinted with permission from ref
365. Copyright 2014 The Royal Society of Chemistry. (b) Gold nanohole array based SERS biosensor for detection of silver and mercury ions in
human saliva. Reprinted with permission from ref 114. Copyright 2015 The Royal Society of Chemistry. (c) Competitive SERS immunoassay for
accurate and sensitive detection of chloramphenicol with the assistance of magnetic beads and SERS probes. Reprinted with permission from ref 369.
Copyright 2016 Elsevier B.V.
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Review
or DNAs/aptamers.118,356−360 Li et al. presented a strategy for
the detection of aflatoxin B1.129 They constructed a SERS
nanosensor using aflatoxin B1 aptamer-modified Ag satellites
and complementary-strand-modified Au nanospheres. Aflatoxin
B1 can destroy the core−satellite structured nanosensor due to
its strong binding affinity toward the aptamer and induce a
significant reduction in SERS intensity. The linear detection
range was from 1 to 1000 pg/mL with a detection limit of 0.48
pg/mL. Yang et al. reported a magnetic-separation-assisted
competitive SERS immunoassay for chloramphenicol.369 A
competitive immune reaction happened between free chlor-
amphenicol and chloramphenicol-modified SERS nanotags.
The dynamic range of this assay was from 1 to 1 × 104 pg/mL
and the LOD was 1 pg/mL.
Up to now, SERS has been used to detect ions including
Hg2+, Ag+, Pb2+, and Cd2+. As a representative, mercury ion
(Hg2+) has been thoroughly investigated using SERS-based
immunoassays.361−364 It can be recognized with antibodies or
T−Hg2+−T base pairs. Wang et al. presented a SERS-based
competitive immunoassay of Hg2+ on a glass substrate.365 The
LOD was 8 × 10−5 ng/mL. Later, She et al. developed a similar
competitive immunoassay for an ultrasensitive detection of
Hg2+ in water, human serum, and urine samples.366 The assay
was conducted on an immuno-chromatographic test (ICT)
strip. The immunoprobe was fabricated by attaching Raman
reporters and Hg2+ antibodies onto the surfaces of gold
nanoparticles. The competitive binding of immunoprobes to
free Hg2+ or immobilized Hg2+ can cause a difference in the
SERS signals on the ICT strip; thus, SERS signals can be used
to determine the concentration of Hg2+ with a LOD of 0.45 pg/
mL.
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Table 5. SERS-Based Immunoassay of Ions and Toxins

refs ions recognition unit detection platform dynamic range LOD
361 Hg2+ ATP Au@Ag 10 pM
362 Hg2+ DNA GNS dimer 0.002−1 ng/mL 0.8 pg/mL
364 Hg2+ DNA Au NR oligomer 5−5000 pM 4.3 pM
366 Hg2+ antibody nitrocellulose (NC) membrane 0.12 ng/mL to 0.45 pg/mL 0.45 pg/mL
370 Hg2+ DNA AuNPs/rGO/SiO2/Si 0.1 nM
371 Hg2+ DNA Ag@AuNPs 5 pM to 50 nM 5 pM
368 Hg2+ DNA AuNPs@SiNWs array 1 pM to 100 nM 7.3 × 10−13 M
365 Hg2+ antibody GNP 10−5−100 ng/mL 8 × 10−5 ng/mL
114 Hg2+, Ag+ DNA a gold nanohole array 1.7 pM
372 Hg2+, Ag+ DNA MSS@Au/Au 0.1−1000 nM (Hg2+), 10−1000 nM 2 nM (Hg2+), 10 nM
(Ag+) (Ag+)
363 Hg2+, Cu2+ DMT and TMT AuNR on fiber 10−2−10−6 M Hg2+, 1−100 μM Cu2+ 1 μM
373 Pb2+ DNA AuNP 16.7−666.7 nM 16.7 nM
374 Pb2+ none DNAzyme 20 nM to 1 μM 20 nM
375 Ag+ 2MNA AuNP (5 × 10−8)−(1.7 × 10−5) M 2.5 × 10−8 M
376 Cd2+ ATRP AuNP 1.0−25 μM 1.0 μM
377 Zn2+ 4- AgNP 0.1−10 mM 0.1 mM
mercaptopyridine
378 malachite green label free Fe3O4/Ag core/shell 10−350 ppb 10 ppb
379 aromatic molecules label free Au/AgNP-rGO 1−10 nM 1 nM
380 BPE pyridyl units paper substrate adsorbed with 0.1−5 nM 0.1 nM
nanorods
381 CV molecules label free wrinkled nanoporous gold films
129 aflatoxin B1 DNA AuNS 1−1000 pg/mL 0.48 pg/mL
369 chloramphenicol antibody magnetic NPs 1−(1 × 104) pg/mL 1.0 pg/mL
382 ochratoxin-A ATP gold nanotriangles
359 zearalenone antibody Au 1−1000 pg/mL 1 pg/mL
383 kanamycin antibody Fe3O4 2 pg/mL to 80 ng/mL 2 pg/mL
384 melamine DNA AuNP 1.0 pg/mL to 10 ng/mL 1.0 pg/mL
356 staphylococcal aptamer Fe3O4@Au 2.5 fM to 3.2 nM 224 aM
enterotoxin B

Besides antibodies, the detection of ions can also be achieved
by DNA strands.367 For example, silver ion can be detected via
the formation of the C−Ag+−C pair, and mercury ion can be
detected via the formation of the T−Hg2+−T pair. Zheng et al.
reported a gold nanohole array based SERS biosensor to detect
Ag+ and Hg2+.114 They fabricated SERS nanoprobes using
silica-coated gold nanostars. Two different kinds of single-
strand DNAs were attached on the nanohole array and
nanoprobe, respectively. If Ag+ or Hg2+ is present, the two
kinds of single-strand DNAs will hybridize through the
formation of C−Ag+−C or T−Hg2+−T pairs. As a result, the
SERS nanoprobe will get very close to the nanohole array, and
a huge Raman enhancement will be generated, leading to a
strong SERS signal. Sun et al. used gold-nanoparticle-decorated
silicon nanowires as the SERS substrate for the detection of
Hg2+.368 A Raman-reporter-tagged single-strand DNA was used
as the Hg2+ recognition unit. Hg2+ can bend the single-strand
DNA via T−Hg2+−T pairs and pull the Raman reporter close
to the SERS substrate, resulting in strong SERS signals. The
LOD was 7.3 × 10−13 M. There are quite a few works using
similar working principles to detect ions and they are
summarized in Table 5.
5.5. Sorting of Cells
Sorting of cells is another prosperous application of SERS-
based immunoassays, especially in the detection and classi-
fication of tumor-derived cells (e.g., CTCs) (Figure
26).310,385−387 Similar to the detection of other targets, cells
are differentiated by various proteins on their surfaces.388−394 A
necessity for SERS-based immune sorting of cells is SERS
7938
nanoprobes/nanotags, which are decorated with cell-specific
recognition units (e.g., peptides, antibodies, or aptamers).
Considering aptamers, Tan’s group has performed serial studies
on how to generate aptamers for cell recognition.108,109,395
Usually, when selecting aptamers, purified proteins are used as
the targets. Tan et al. found that it was more accurate to directly
use the whole cell as the target when selecting aptamers for
cells (cell-SELEX), since free proteins might take different
steric configurations as compared to proteins on cell
membranes.
With cell-specific ligands, the SERS nanoprobes/nanotags
help to determine the species and expression levels of proteins
on the cell surfaces, which eventually contributes to the
identification of cells. For example, Wang et al. reported the
successful detection of CTCs in human peripheral blood using
SERS nanoparticles.396 The CTCs specific SERS nanoparticles
were constructed by linking EGF peptides and Raman reporters
onto gold nanoparticles. With EGF peptides on their surfaces,
the SERS nanoparticles can bind to EGFR-overexpressing
CTCs via immune recognition. They found that the SERS
nanoparticles can rapidly detect CTCs from peripheral blood of
patients with squamous cell carcinoma of the head and neck.
Similarly, Bhana et al. fabricated an iron oxide−gold nano-
particle for the detection of SKBR3 cells from whole blood.397
The magnetic and SERS-active nanoparticle is decorated with
SKBR3-cell-specific antibodies to allow capturing of these cells.
The LOD of this strategy was 1−2 cells/mL of blood. Song et
al. developed a SERS-encoded nanogapped plasmonic nano-
particle for the identification of cancer cells.93 The nanoparticle
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Chemical Reviews
Figure 26. SERS immunoassays for cells. (a) Detection of CTCs in
human peripheral blood using SERS probes. Reprinted with
permission from ref 396. Copyright 2011 American Association for
Cancer Research. (b) Identification of rare cancer cells assisted by a
microfluidic channel and SERS. Reprinted with permission from ref
401. Copyright 2015 American Chemical Society.
possesses a sub-2-nm inner gap, which can generate a strong
Raman enhancement. By modifying the nanoparticle’s surfaces
with MUC-1 aptamers, the nanoparticle can target MUC-1-
overexpressing cancer cells and realize SERS imaging of these
cancer cells.
By combining SERS-based immunoassays with microfluidic
chips, efficient cell sorting can be realized.398−400 For instance,
Pallaoro et al. reported a rapid identification of prostate cancer
cells using microfluidic channel and SERS biotags.401 Neuro-
pilin-1-specific peptides were attached on the SERS biotags,
making these biotags specific for neuropilin-1-overexpressing
cancer cells. When incubated with cancer cells, the SERS
biotags can bind to the target cells. These cancer cells together
with adsorbed biotags were flowed through a microfluidic
channel, and the cancer cells were identified by the SERS
signatures. This microfluidic-chip-based assay can detect one
target cancer cell out of 100 control cells.
In addition to detecting protein biomarkers in solutions or
on cell membranes, SERS probes can also be employed for in
vivo targeting of tumors. Up to now, in vivo tumor targeting
experiments were all performed on animals (e.g., mouse
xenografts).402−406 SERS probes are decorated with tumor-
specific ligands (e.g., antibodies) and injected into the animal
models for “active” tumor targeting. That is, the injected SERS
probes can target and bind to tumor sites via recognition
between the targeting ligands and tumor biomarkers.407
7939
Review
Another kind of in vivo tumor targeting using nanoparticles
(with no specific ligands) is “passive” tumor targeting, which is
usually realized by the enhanced permeability and retention
(EPR) effect. Passive tumor targeting is well-reviewed else-
where and will not be discussed in detail in this review.408,409
Dinish et al. reported in vivo active targeting of three breast
cancer biomarkers (i.e., EGFR, CD44, and TGFβRII) using
SERS nanoprobes decorated with antibodies.410 Wang et al.
presented an endoscopic imaging strategy for detecting
gastrointestinal cancers. In their strategy, the molecular
phenotype of tumor tissues is visualized by SERS nanoprobes
decorated with anti-EGFR or anti-HER2 antibodies.411 Besides,
SERS nanoprobes (passively or actively targeted) can also
facilitate surgical tumor resection by identifying and imaging
millimeter/submillimeter-sized tumors using SERS sig-
nals.412−414 In vivo targeting of tumors using SERS nanoprobes
is quite beneficial, since the fingerprint characteristic of SERS
enables their precise tracking in complicated biological
environments. However, the complex in vivo circumstances
bring more rigorous requirements for the SERS nanoprobe’s
targeting ability. This is because proteins in blood or other
body fluids tend to adsorb on the SERS probes, forming a
protein “corona”, which can severely disrupt the recognition
between targeting ligands and biomarkers.415,416 To avoid the
formation of a protein corona, SERS nanoprobes for in vivo
applications are usually decorated with an outer passivation
layer using polyethylene glycol, which can effectively resist
protein adsorption as well as improve the stability of SERS
nanoprobes.417
5.6. Multiplex Biochemical Analysis Using SERS Encoders
The most extraordinary advantage of the SERS technique is its
excellent multiplexing ability.7,70,418−422 The sharp bands of
Raman spectra make SERS an ideal candidate for optical
encoding, which can further facilitate multiplex and high-
throughput immuno-detection. SERS-based optical encoding
has been explained in detail in section 4. Here, we will provide
several typical immuno-detections based on SERS-encoded
nanoprobes and platforms, which can better prove the excellent
multiplexing ability of SERS (Figure 27, 28). Up to now, SERS-
encoded nanoprobes (i.e., SERS encoders) have been
employed in the analyses of several different kinds of biological
or chemical targets, including DNAs, proteins, bacteria, cells,
and ions.15,60,160,423−426
5.6.1. Multiplex Immuno-Detection of Biomolecules
and Cells. In the past few years, our group has been working
on SERS-based multiplex bioanalyte detection. We introduced
a SFJSE method using specially designed nanoencoders.17
These SFJSE-based nanoencoders have been employed for the
immuno-detection of proteins and cells.61,135 For example, we
fabricated SERS−fluorescence dual-encoded magnetic nano-
beads and used them to separate rare cancer cells from
enormous normal cells.135 By decorating the surfaces of the
dual-encoded nanobeads with cancer-cell-specific antibodies,
we can efficiently capture four different kinds of cancer cells
from phosphate-buffered saline or human blood samples. We
also developed a 3D SERS barcode microfluidic chip and
realized the simultaneous detection of multiple kinds of
proteins.66
Zhao et al. fabricated SERS-encoded nanotags for the
detection of three different kinds of virus genes.92 The
nanotags that contain nanogaps can generate SERS enhance-
ment. Raman reporter molecules are placed inside these
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Chemical Reviews Review

Figure 27. Multiplex SERS immunoassays. (a) Multiplex immuno-phenotyping and imaging of cancer cell receptors using Au@pNIPAM SERRS
tags. Reprinted with permission from ref 429. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA. (b) Sensitive multiplex biomarker detection
using SERS-encoded silver pyramids. Reprinted with permission from ref 131. Copyright 2015 Wiley-VCH Verlag GmbH & Co. KGaA. (c)
Multiplex virus DNA detection using gold nanostructures containing nanogaps. Reprinted with permission from ref 93. Copyright 2014 American
Chemical Society. (d) Multiplex protein detection using antibody-functionalized SERS-encoded nanoparticle assemblies. Reprinted with permission
from ref 430. Copyright 2014 Wiley-VCH Verlag GmbH & Co. KGaA.

nanogaps to provide SERS signals. By using different reporter

Figure 28. Multiplex cell imaging using SERS probes. The Raman
reporters are alkyne-containing molecules. The SERS probes are
decorated with ligands that target cancer cells. Reprinted with
permission from ref 431. Copyright 2016 American Chemical Society.
7940
molecules, different SERS signatures can be obtained, thus
realizing SERS encoding. The surfaces of the SERS tags are
decorated with target-specific DNA strands. Hepatitis A virus
(HAV) Vall7 polyprotein gene, hepatitis B virus (HBV) surface
antigen gene, and human immunodeficiency virus (HIV)
antigen gene are simultaneously detected using the SERS
nanotags and magnetic microparticles. The LODs of HAV,
HBV, and HIV were 0.39, 0.18, and 0.51 pM with a good
dynamic range of 5 orders of magnitude. Similar applications in
multiplex DNA detections have also been reported by other
groups.
Xu et al. reported SERS-encoded silver pyramids for the
simultaneous and ultrasensitive detection of multiple protein
biomarkers, including MUC1, PSA, and thrombin.131 Recog-
nition units for these biomarkers are aptamers. With target
biomarkers presented, the specific biorecognition between the
biomarker and the aptamer will alter the 3D geometries of the
pyramids and cause Raman signal enhancement. LODs were
0.96, 85, and 9.2 aM with detection ranges of 1−500 aM, 0.1−
50 fM, and 0.01−5 fM for PSA, thrombin, and MUC1,
respectively. Wang et al. presented a multiplex homogeneous
immunoassay for the sensitive detection of proteins.427 The
SERS nanoparticles are attached with Raman reporters and
antibody fragments. The immune recognition between anti-
body fragments and target proteins closely assembles the SERS
nanoparticles and results in strong SERS signals. Reproducible
detection and quantification of three cytokines (i.e., interferon
γ, interleukin-2, and tumor necrosis factor α) are achieved.
Tang et al. presented a SERS-frequency-shift immunoassay for
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 Table 6. Multiplex SERS Assays
multiplexing recognition
refs targets ability detection platform unit dynamic range LOD
92 virus genes HAV, 3 magnetically separated DNA 5 orders of LOD 0.39 pM
HBV, (liquid phase) 0.18 pM
HIV 0.51 pM
Chemical Reviews

268 biomarkers MMP-7, 2 metal substrate antibody 2.28 pg/mL

CA19-9 34.5 pg/mL

434 biomarkers p53, p21 2 glass antibody 5 ng/mL to 500 fg/mL 1 pg/mL

131 biomarkers PSA, 3 Ag pyramids in liquid aptamer 1−500 aM 0.96 aM

thrombin, phase 0.1−50 fM 85 aM
mucin-1 0.01−5 fM 9.2 aM

435 thrombin, PDGF BB, Ig E 3 magnetically separated aptamer 10 pg/mL to 5.0 ng/mL 3.2 pg/mL
(liquid phase)

428 plant pathogens Botrytis cinerea, Pseudomonas 3 magnetically separated DNA

syringae, Fusarium oxysporum (liquid phase)

436 pathogens 2 magnetically separated antibody 103−106 cfu/mL 1 × 103 cfu/mL

7941
(liquid phase)

437 pathogen antigens EHI_115350, 2 metal substrate antibody 0−10 ng/mL 1 pg/mL
EHI_182030 10 pg/mL

429 proteins EGFR, EpCAM, CD44 3 cell membrane antibody

273 phenotypic markers 3 cell membrane antibody

438 proteins 2 liquid phase 0.2−2 μg/mL 0.2 μg/mL

17 IgG 2m+n − 1 glass antibody 1 μM to 1 fM 1 fM

61 IgG 3 magnetically separated antibody 1 μg/mL to 0.1 pg/mL 0.1 pg/mL

(liquid phase)

135 cancer cells 4 magnetically separated antibody 2 × 105 MRC-5 (SBR3/HeLa), 5 × 104 MRC-5 (Jurkat T), a single cancer cell
(liquid phase) 9 × 104 MRC-5 (LNCaP)

433 Hg2+ 2 liquid phase DNA 0.002−0.5 ng/mL 1.69 pg/mL

Ag+ 1.71 pg/mL

372 Hg2+ 2 magnetically separated DNA 0.1−1000 nM 2 nM

Review

(liquid phase)

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 Table 6. continued
multiplexing recognition
refs targets ability detection platform unit dynamic range LOD
Ag+ 10−1000 nM 10 nM

114 metal ions (Ag+/Hg2+) 2 metal substrate DNA 1.7 pM for the Au nanohole array chip, 5 pM
Chemical Reviews

for the Au film chip

113 glycoproteins HRP, 2 liquid phase boronate 0.1 ng/mL to 100 μg/mL 0.7 nM
AFP affinity 1 ng/mL to 10 μg/mL 3 nM

112 CA15-3 3 glass antibody 0.1−500 U/mL 0.99 U/mL

CA27-29 0.1−500 U/mL 0.13 U/mL
CEA 0.1−500 ng/mL 0.05 ng/mL

125 CEA 2 liquid phase antibody 0.01 pg/mL to 1000 ng/mL 6.6 × 10−6 ng/mL
AFP 0.1 pg/mL to 1000 ng/mL 7.2 × 10−5 ng/mL

439 CEA 2 liquid phase antibody 1 ng/mL to 1 fg/mL 1.48 pg/mL

NSE 2.04 pg/mL

272 CA125 4 microfluidic antibody 1 aM to 1 pM 15 fM

HER2 17 fM

7942
HE4 21 fM
eotaxin-1 6.5 fM

304 melanoma DNA 3 magnetically separated DNA 10 copies

(liquid phase)

440 Staphylococcus aureus 2 magnetically separated aptamer 102−107 cfu/mL 35 cfu/mL

Salmonella typhimurium (liquid phase) 15 cfu/mL
Review

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Chemical Reviews
Figure 29. (a) Assembly of a single nanowire on graphene to be used as SERS substrate. Reprinted with permission from ref 441. Copyright 2016
Royal Society of Chemistry. (b) Ordered SERS microsphere array obtained by assembling Fe3O4@Ag@SiO2 microparticles. Reprinted with
permission from ref 442. Copyright 2016 American Chemical Society. (c) Evaporation-based assembly of silver nanowires. Reprinted with
permission from ref 444. Copyright 2016 Wiley-VCH Verlag GmbH & Co. KGaA.
multiplex detection of liver cancer biomarkers AFP and
glypican-3.39 The sensitivity is down to sub-picomolar
concentrations in saline solution.
Lau et al. reported a multiplex point-of-care diagnostic
platform for plant pathogens.428 This platform employs SERS
and recombinase polymerase amplification (RPA) techniques.
They utilized DNA-strand-modified SERS nanotags and
magnetic nanoparticles. Multiplex detection of three pathogens
was successfully demonstrated. Experimental results proved
that the RPA−SERS method was faster and more sensitive than
PCR.
Bodelon et al. synthesized surface-enhanced resonance
Raman scattering (SERRS)-encoded colloids for the identi-
fication and imaging of proteins on cell membranes.429 In their
experiments, simultaneous imaging of three tumor biomarkers
on the cancer cell membrane was realized, including EGFR,
epithelial cell adhesion molecule (EpCAM), and homing cell
adhesion molecule (CD44). The SERRS-encoded colloids can
discriminate tumor and nontumor cells.
5.6.2. Multiplex Ion Detection. Aside from the above-
mentioned biological targets, platforms based on SERS can also
be used to analyze other chemical targets, such as metal ions
and small molecules. For example, Kang et al. reported a
simultaneous detection of three metal ions (i.e., Hg2+, Ag+, and
Pb2+) using a gold-nanowire-on-chip SERS sensor.432 Our
group has presented a magnetic-nanobead-assisted SERS-based
method for the simultaneous detection of Hg2+ and Ag+.372 Li
et al. reported a SERS-encoded nanoplatform for the
simultaneous detection of Hg2+ and Ag+.433 With the excellent
multiplexing ability, SERS holds a great potential in developing
highly efficient and informative biochemical detection platforms
(Table 6).
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Review
6. CHALLENGES AND PERSPECTIVES
SERS has been reported to realize single-molecule detection
from more than 20 years. Despite the excellent optical-sensing
capacity, SERS has not yet been used for practical applications
such as clinical diagnosis and biochemical detection. To
promote SERS-based immunoassay from a research focus to
practical detection method, future attention should be focused
on the improvement of reproducibility, the reduction of costs,
the ease of use for point-of-care (POC) applications, etc. In this
section, we will focus on these issues by discussing practical
routes and future prospects toward the optimization of SERS-
based immunoassays.
6.1. Improving the Reproducibility of SERS-Based
Immunoassay
The relatively poor reproducibility of SERS-based immuno-
assay is a common problem that hinders the promotion of
SERS to practical applications. To improve the reproducibility
of SERS-based biosensing, the uniformity and stability of SERS
substrates and nanoprobes should be improved.
6.1.1. Uniformity of the Immune Substrates. In
addition to the previously mentioned plasmonic arrays (the
last paragraph of section 3.3.1), there are some other substrates
for reproducible SERS analysis. Kim et al. assembled a single
nanowire on graphene as a novel SERS substrate (Figure
29a).441 Gold nanowires (Au NWs) with well-defined single-
crystal geometry were coated onto a monolayer graphene,
forming a well-defined and continuous nanogap that provides
extremely reproducible and stable SERS signals. The in-
nanowire (in-NW) and nanowire−nanowire (NW−NW)
reproducibility was verified through SERS mapping and
cumulative curves. Moreover, the platform showed an improved
stability and long-term oxidation immunity. The high
reproducibility and stability of this substrate make it a
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Chemical Reviews
Figure 30. (a) A 3D-printed supercapacitor-powered electrochemiluminescent protein immunoarray. Reprinted with permission from ref 450.
Copyright 2016 Elsevier B.V. (b) A portable and microcapsule array chip fabricated by 3D ice printing for visual target detection. Reprinted with
permission from ref 451. Copyright 2015 American Chemical Society.
potentially practical SERS substrate. Zheng et al. employed self-
assembled polystyrene microbeads as templates to fabricate a
gold nanohole array as a reproducible SERS substrate.114 Niu et
al. assembled monodisperse Fe3O4@Ag@SiO2 microspheres
onto hierarchical surfaces (Figure 29b).442 Perfect integration
of ordered microsphere arrays has been achieved with the
assistance of a magnetic field. The simple manipulation and low
cost make it a promising candidate for a SERS substrate.
Another route to fabricate highly uniform substrate is
evaporation-based self-assembly. Self-aligned nanorods and
nanowires could be attached onto the surface and used as the
SERS substrates (Figure 29c).443,444 Recently, Zhu et al.
fabricated a hierarchically ordered array of silver nanorod
bundles with a large SERS enhancement factor (108) and a high
reproducibility of SERS-signal (RSD 10%).445 The great SERS
enhancement was due to the strong EM field coupling among
those neighboring Ag nanorods with nanogaps of around 2 nm.
Currently, there are also some commercial SERS substrates
with a high reproducibility, such as gold (or silver) coated
nanostructured silicon like Klarite or those by produced
Silmeco, nanosponge gold/silver SERS substrates developed
by Ocean Opotics, and gold nanorods substrates developed by
Horiba Scientific. It is expected that all the above substrates
might be used for SERS-based immunoassays to improve the
reproducibility.
Another popular technique for substrate fabrication is 3D
printing. Using this new technology, the desired materials could
be assembled under computer control to fabricate certain 3D
structures.446−448 Since the 1990s, remarkable progress has
been made in 3D printing technology. It enables a rapid
fabrication of personalized products at very low cost. Up to
now, 3D printing have been employed for printing tissues and
organs, microfluidic chips,446 electrochemical devices,449 etc.
Recently, Kadimisetty et al. developed a low-cost 3D-printed
immunoarray for the detection of three proteins (Figure
30a).450 The system costs around ∼€0.50 for each protein and
each detection can be completed in 35 min. This device is
suitable for resource-limited clinical environments. Zhang et al.
7944
Review
used the cost-effective 3D ice printing technology to fabricate
ice structures for the rapid visual detection of nitrite and
glucose (Figure 30b).451 A variety of ice structures with
different geometries and sizes can be easily achieved with 3D
ice printing. The platform has a potential for POC diagnosis
without the requirement of medical specialists. Currently, the
3D printing technology has not been combined with SERS
detection yet. The common materials for SERS substrates,
including gold and silver, are seldom used as the printing
materials due to the intrinsic properties of metal. However, 3D
printing has already been used to fabricate various nanostruc-
tures with nonmetallic materials. The combination of 3D
printing with traditional metallic nanofabrication technologies
might be promising for producing highly reproducible and
SERS-active 3D substrates at low costs.
On the other hand, there are some other uniform
nanosubstrates that broaden the dynamic range of detection.
In a typical immunoassay, the intensity of readout signal often
undergoes a linear zone (commonly known as the dynamic
range) and a saturation plateau. So far, the majority of
immunoassays still suffer from a narrow dynamic range. Thus,
ways to broaden the dynamic range are urgently required in
diagnostics applications. Since the concentration of target
molecules is usually very low in the practical applications, one
typical method for improving the dynamic range is to decrease
the LOD value. For example, Miele et al. fabricated the sensing
surfaces by using regular arrays of microdomains of different
wettabilities, self-assembling analyte molecules in the more
wettable domains.169 Thus, the detecting sensitivity was
increased to ∼10 aM due to the concentrated molecules in
the active domains, leading to a remarkable improvement in the
dynamic range as 6 orders of magnitude (10 aM to 1 pM).
Besides, Dai and co-workers presented protein microarrays on a
novel nanostructured, plasmonic gold film with near-infrared
fluorescence enhancement of up to 100-fold, which shows a
detecting limit to the 5 fM level with a dynamic range over 6
orders of magnitude.452 In the case of a high concentration of
targets, the dilution operation can be carried out to obtain
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Chemical Reviews
Figure 31. (a) Multilayered shell nanotags with highly uniform single-particle Raman readout. Reprinted with permission from ref 458. Copyright
2012 Royal Society of Chemistry. (b) Ag shell−Au satellite hetero-nanostructure SERS probes. Reprinted with permission from ref 459. Copyright
2014 American Chemical Society.
accurate results. Thus, a lower detection limit and a broader
dynamic range are two key factors for developing SERS
immunoassays for practical uses.
6.1.2. Stability of SERS Nanoprobes. On the other hand,
the structure of the nanoprobes should be carefully designed to
improve the stability of the nanoprobes.453,454 The famous
shell-isolated nanoparticle-enhanced Raman spectroscopy
(SHINERS) developed by Li et al. employed an ultrathin silica
or alumni shell to protect metal nanoparticles from the
aggregation and direct contact with the surrounding environ-
ment, while maintaining a high SERS activity.105 The versatility
of SHINERS has been demonstrated through the detection of
yeast cells, pesticide residues on food/fruits, and hydrogen
adsorption on single-crystal flat surfaces of Pt and Si. SHINERS
expands the flexibility of SERS for various applications in
biology, chemistry, medicine, and environmental monitoring.
Another popular strategy to fabricate stable SERS nanoprobes
is using DNA as the template to assemble nanoparticles.455−457
Complementary hybridization between specially designed DNA
strands can facilitate the formation of stable and sophisticated
nanostructures with fine SERS-enhancing abilities.
To improve the SERS activity, “hot spots” are frequently
used for signal amplification in SERS nanoprobes. However, the
creation of SERS hot spots often leads to uncontrollable
aggregation of the nanoparticles. There is a balance between
the stability of SERS nanoprobes and high SERS activity. The
anisotropic structure with uncontrollable hot spots might lead
to signal fluctuation and dependency on incident light
polarization. To overcome these problems, Liu et al. fabricated
SERS nanotags with highly uniform and reproducible signals
using layer-by-layer assembly and cross-linkage of small silver
nanoparticles (Figure 31a).458 The problem of reproducibility
could be solved to a large extent by the assembly of small metal
nanoparticles into a highly controllable multilayered shell. The
SERS enhancement is within the narrow range from 1.1 × 107
to 1.4 × 107. Chang et al. developed a Ag shell−Au satellite
nanostructural composite as a NIR-active SERS probe (Figure
31b).459 The assembly of gold nanospheres onto silver
nanoparticles leads to the formation of uniform 3D hot spots.
The hetrometallic shell−satellite nanoparticles produced
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intense and uniform SERS signals with an EF of 1.4 × 106
and a relative standard deviation of 11%. These novel
nanostructures open an avenue for reliable SERS immunoa-
nalysis.
6.2. Real Sample Detection
Commercialization of SERS-based immunoassay requires a
precise quantification of biomolecules in real samples (e.g.,
human blood). Currently, it is still challenging to minimize the
interference from other components in real sample and to
realize precise quantification of biomarkers.
6.2.1. Serum and Plasma. Human plasma is extracted
from whole blood by removing the blood cells and clotting
factors, while human serum is the plasma without fibrinogens.
The components of human plasma include proteins, hormones,
ions, glucose, etc. To predict and validate the ability of SERS-
based immunoassay in real sample detection, the proof-of-
concept experiment is usually performed by quantifying the
concentration of target molecules in serum or plasma. Li et al.
developed a multiplex immunoassay for the simultaneous
detection of three serological cancer biomarkers using the SERS
technique.112 The platform was able to realize the detection of
CA15-3, CA27-29, and CEA in human serum with LODs down
to 0.99 U/mL, 0.13 U/mL, and 0.05 ng/mL, respectively. The
3D hierarchical plasmonic nanoarchitecture enhanced SERS
immunosensor developed by Li et al. realized EGFR detection
in human plasma samples from clinical breast cancer
patients.248 The detection results were comparable with those
obtained from commercial ELISA kits.
Besides the above examples, a great number of reports have
claimed successful quantification of biomolecules in human
serum or plasma. However, to the best of our knowledge, there
are no commercial SERS kits for immunoassays to date.
Possible reasons might arise from the low reproducibility
between different tests. Although the reproducibility of SERS-
based immunoassay in a single test (using the same materials
and platform in the same day) could be improved by
optimizing the stability and uniformity of immune substrates
and SERS probes, as discussed in the previous section, the
reproducibility of detection results from multiple tests are not
easy to keep in accordance. This is primarily due to the low
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Chemical Reviews
Figure 32. (a) An integrated barcode chip for the rapid and multiplex protein detection in human blood. Reprinted with permission from ref 461.
Copyright 2008 Nature Publishing Group. (b) An “mChip” HIV assay done in Rwanda on hundreds of locally collected human blood samples.
Reprinted with permission from ref 462. Copyright 2011 Nature Publishing Group.
reproducibility of SERS immunoprobes: (1) Since the SERS
probes are usually fabricated by chemical methods, the size and
shape of the nanoparticles could be varied due to the
differences in the quality of original materials, the mixing
speed, the temperature, the pH value of surrounding environ-
ment, etc. This could become more complicated with respect to
the chemical and biological modification process. As a result, it
is not easy to maintain the high uniformity of SERS probes.
Calibration should be carried out before each test to improve
the precision of detection. (2) The complex components in the
environment could lead to the aggregation of SERS probes,
which is almost uncontrollable and consequently leads to
significant variations in SERS signals and the nonspecific
adsorption of immunoprobes. Additionally, in physiological
conditions, the fraction of target molecules is usually miniscule
in relation to the other molecules present. Nonspecific
adsorption of nontarget molecules becomes one of the most
pressing concerns, which will raise the detection limit of the
strategy and thereby decrease the sensitivity of the assay.
Generally, the interfering molecules tend to interact with the
surface materials mainly through electrostatic interactions or
van der Waals forces, which is far weaker than that between the
target molecules and the recognition probes. Thus, one typical
way to solve the problem of nonspecific adsorption is to reduce
the reaction time. Driskell et al. introduced the concept of a
rotating capture substrate instead of a conventional static
substrate.460 This facile method increased the flux of target
antigen and SERS labels to the solid substrate, resulting in a
lower nonspecific adsorption. Similarly, an accelerated SERS
immunoassay based on a syringe pump was proposed to
actively transport the analyte and extrinsic Raman labels to the
capture surface, which avoided the nonspecific binding resulted
from the passive diffusion.254 Recently, Wang et al. utilized ac-
EHD-induced nanoscaled surface shear forces to remove the
nonspecific adsorption, which also enhanced the capture
kinetics simultaneously.279 Therefore, how to construct the
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surfaces with high resistance to nonspecific adsorption in
complex matrixes is another important issue to consider in the
future research.
6.2.2. Whole Blood. Whole blood contains a large quantity
of blood cells, including red blood cells and white blood cells.
Immunoassays for whole blood without preprocessing greatly
reduce human work, which would be critical for POC diagnosis
in resource-limited areas. Currently, SERS-based immunoassay
has already been used for detecting CTCs in whole blood. For
example, Wu et al. employed SERS nanoprobes for the direct
detection of CTCs in whole rabbit blood with a LOD down to
5 cells/mL.25 Wang et al. employed EGF−SERS nanoparticles
to rapidly detect CTCs in peripheral blood specimens from
patients.396 These platforms have demonstrated the possibility
of using SERS-based immunoassay for cancer diagnosis at the
cellular level. On the other hand, it would be more meaningful
to analyze biomarkers at the molecular level. Up to now, there
are a few platforms which have been used for direct
identification of protein or DNA biomarkers from the whole
blood using optical techniques other than SERS. Fan et al. have
developed an integrated barcode chip for rapid (10 min) and
multiplex protein detection in human blood (Figure 32a).461
The microfluidic chip enables the isolation of blood cells and
the measurement of plasma protein biomarkers at the same
time on a single chip using fluorescence immunoassays. The
device holds potential for inexpensive, noninvasive, and
informative clinical diagnoses. Chin et al. performed an
“mChip” assay in Rwanda on hundreds of locally collected
human blood samples, which enabled a rapid diagnosis of HIV
using unprocessed whole blood (Figure 32b).462 Unlike
benchtop ELISA assays, the mChip can perform these
immunoassays with minimal equipment and can be completed
within 20 min using only several microliters of blood sample,
which is an ideal choice for diagnoses in resource-limited areas.
We envision that the introduction of SERS to this platform
might improve the immunoassay performance in terms of the
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Chemical Reviews
Figure 33. (a) Scheme of the flow-focusing microfluidic device used for controlled Ag-NP aggregation. (b) At the flow-focusing junction, the sample
stream is enveloped by the side streams and diffusion drives lateral mass transport between the laminar flows, here visualized with a fluorescent dye.
(c) Schematic of the reaction. (d) Latent variables calculated for the calibration data after varimax rotation of the principal components. Reprinted
with permission from ref 464. Copyright 2013 American Chemical Society.
sensitivity and multiplexing ability, as long as the reproducibility
of the SERS technique is improved.
6.2.3. Other. Besides human blood, there are other human
body fluids containing various biomarkers, such as saliva, urine,
and sputum. SERS-based immunoassays have been used for the
detection of Hg2+ in human saliva and urine,366 pyocyanin in
sputum,463 drugs in human saliva and urine (Figure 33),464 etc.
In addition to body fluids, SERS has been applied to analyze
targets in food samples, such as Escherichia coli O157 in apple
juice,465 zearalenone in feed samples,359 kanamycin in milk
samples,383 and Salmonella typhimurium and Staphylococcus
aureus in pork samples.441 All of these works make SERS an
excellent candidate for environmental monitoring and food
quality and safety evaluation. Similar to the blood tests, the
quantification of other biochemical substances in real samples is
not an easy job due to the interference from other species in
the complex environment. The testing parameters should be
carefully adjusted to reach the best performance.
6.3. Point-of-Care Testing (POCT)
POCT refers to the test at or near the point-of-care, that is, at
the time and place of patient care. POCT is critical to
healthcare in resource-limited areas. An ideal POCT device
should fulfill the following requirements: small size, low sample
consumption, low costs, easy operation, and intuitive readout
without complicated instrumentation. So far, extensive efforts
have been made toward POC SERS immunoassays. Worsley et
al. developed a lateral flow SERS immunoassay for the
multiplex detection of cardiac and inflammation biomarkers.270
The combination of the SERS technique with the lateral flow
platform enables an easy and rapid detection of biomarkers,
which would become one potential candidate for POC
diagnosis. Saha et al. performed SERS immunoassay on a
paper-based microfluidic platform, in which both the plasmonic
nanoparticles and analytes were in mobile phase.466 The
analyte-induced controlled particle aggregation produced
reproducible and much enhanced SERS signals, which resulted
in a sensitivity beyond picomolar concentration. The presented
approach is simple, rapid, and cost-effective and requires
microliter sample volume. In terms of the SERS measurement,
portable Raman spectrometers have been widely used for in situ
SERS analysis.105,467 Mohs et al. developed a hand-held
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spectroscopic device for in vivo and intraoperative cancer
detection that provides promising routes for real-time tumor
detection and image-guided surgery.468 On account of the high
sensitivity and excellent multiplexing ability, SERS-based
immunoassay is promising for POC applications. To further
promote practical and commercial applications, the overall
costs should be reduced. First, the fabrication of SERS
nanoprobes requires many more antibodies than the
fluorescence method. The bioconjugation efficiency should be
improved to reduce the antibody consumption. Second, the
POC SERS measurement requires a sophisticated portable
instrument, which increases the financial burden, especially for
rural areas. Consequently, advanced techniques for the design
and fabrication of portable Raman spectrometer should be
developed. Third, POC diagnosis requires a simple readout of
SERS spectra, which becomes more sophisticated for SERS
decoding in multiplex analysis. Therefore, new software
programs integrated with data processing, analysis, and readout
should be developed, so that the end-point users could easily
obtain the detection results. All the above issues should be
considered for turning the proof-of-concept research into
commercial POC systems.
6.4. Practical SERS-Encoding Technique
SERS-encoding technique has been widely used for multiplex
biosensing.70 The narrow Raman bands endow SERS with an
excellent coding capacity. In principle, SERS encoding would
enable the simultaneous detection of more than 100 kinds of
targets, which could be differentiated from each other by
Raman frequencies. However, realistically, simultaneous
detection of only a few (two to five) kinds of biomolecules
has been achieved by SERS encoding to date. There are two
main practical problems to be settled. On the one hand, the
actual kinds of Raman reporters without spectral band overlap
are still limited. Commonly used Raman reporters with large
scattering cross sections are usually similar in chemical
structures, which inevitably leads to band overlaps. On the
other hand, nonspecific antibody−antigen interaction, physical
adsorption, and cross-reactions among multiple encoded
nanoprobes reduce the specificity of SERS, making it difficult
to distinguish different analytes.
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Chemical Reviews
Figure 34. (a) “Switch-off” biosensing for chymotrypsin-catalyzed reaction by SPR−SERS spectroscopy. Reprinted with permission from ref 472.
Copyright 2013 Royal Society of Chemistry. (b) Mercury ion detection by optical waveguide and surface-enhanced Raman spectroscopy. Reprinted
with permission from ref 473. Copyright 2012 Elsevier B.V.
To minimize band overlaps, it is ideal that the Raman
molecules only have one strong Raman band, which can greatly
minimize possible band overlaps between different codes. One
practical solution is to synthesize a group of Raman reporters
with similar but different chemical structures. The characteristic
Raman bands of these reporters should be close to each other
and without obvious spectral overlaps to obtain the maximum
kinds of different codes.431 Another solution is to combine
different encoding techniques together. For example, the
SERS−fluorescence joint spectral encoding technique proposed
by our group greatly increased the number of different codes
that can be practically obtained.17,135 Similarly, Zhang et al.
developed dual-encoded microbeads through a host−guest
nanostructure, in which fluorescent dyes and quantum dots
were used for joint encoding.469 The idea of joint encoding
provides an alternative for high-throughput multiplex detection.
Future work in this field should focus on simplifying the
structure and fabrication protocol of the nanoparticles
incorporated with different labels. Finally, complex Raman
spectra could be analyzed with data processing methods like
principle component analysis (PCA) and support vector
machine (SVM), so that the merged Raman bands from
different reporters could be further extracted for simultaneous
quantitative analysis of multiple targets.399,470,471
Another critical problem that limits the practical application
of the SERS-encoding technique is nonspecific interactions. To
reduce the nonspecific adsorption of SERS nanoprobes,
researchers should pay much attention to experimental details,
including the orientation of antibodies, the monodispersity of
nanoparticles, and the blocking of nonspecific adsorption sites.
On the other hand, antibody−antigen cross-reactions are
common in immune reactions, which could be reduced by
improving the quality of antibodies. This relies heavily on the
synthetic technology of highly purified antibodies. In general,
all the above issues related to biomolecular interactions would
limit the available number of SERS codes that can be used for
high-throughput biosensing. To fully exploit the excellent
encoding capacity of the SERS technique, the above problems
should be carefully concerned for future researches.
6.5. Combining SERS with Other Techniques for
Multimodal Immunoassay
SERS has shown its advantage in ultrasensitive detection,
structural analysis, and high stability. However, SERS-based
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immunoassay has some intrinsic disadvantages. First, the typical
sandwich immunoassay format fails to provide time-dependent
information on the analytes in one sensing unit. Dynamic
analysis requires repetitive measurements at different time
points. Second, SERS usually has a relatively slow response and
readout speed, making it relatively unsuitable for real-time
analysis. Third, it is challenging to obtain reproducible SERS
signals due to the instability of nanoprobes in complex
microenvironments. Therefore, the combination of SERS with
other detection techniques is expected to overcome some
intrinsic shortcomings of SERS-based immunoassays.
6.5.1. SERS−SPR Immunoassay. SERS has been
combined with SPR for dual mode biosensing. SPR could be
used for real-time monitoring of antibody−antigen interactions,
while SERS enables the analysis of the chemical structures of
the analytes. By combining SERS with SPR, the relatively low
reproducibility of SERS detection could be compensated by
SPR detection. Fu et al. proposed a “switch-off” biosensing
strategy for chymotrypsin detection based on the combined use
of the SERS and SPR techniques (Figure 34a).472 This
approach could be used for real-time monitoring of the
reaction process, and the sensitivity reached down to 0.4 nM.
Similarly, Ma et al. developed a SERS−SPR dual-mode sensor
by assembling Au−pATP multilayers onto an optical waveguide
(Figure 34b).473 The integrated chip was used for Hg2+
detection with a low concentration of 1 nM. The combination
of SERS with SPR provides a novel idea for real-time, specific,
and sensitive detection of various target molecules.
6.5.2. SERS−Fluorescence Immunoassay. A variety of
nanoprobes that simultaneously incorporate SERS and
fluorescence signals into a composite nanoparticle have been
used for immunoassays. The fluorescence mode could be used
for fast screening of biomolecules, while the SERS mode can be
used for sensitive quantitative analysis. Previously, we have
designed a SERS−fluorescence dual-mode nanoprobe based on
multilayer core−shell nanoparticles.61 In the nanoprobe,
Raman-reporter-labeled nanoparticles provide SERS signals,
while quantum dots provide fluorescence signals. The dual-
mode nanoprobe could be used for rapid and sensitive
bioanalysis and imaging. Recently, Zou et al. employed
graphene quantum dots as both the SERS and fluorescence
labels, which simplifies the structure of the immunoprobes.115
Dual-mode nanoprobes partially overcome the slow readout
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Chemical Reviews
Figure 35. (a) A SERS−electrochemical biosensos for monitoring DNA−drug interactions. Reprinted with permission from ref 474. Copyright 2016
Elsevier B.V. (b) A SERS−electrochemistry dual-mode immunosensor for simultaneous detection of multiple biomarkers. Reprinted with permission
from ref 475. Copyright 2015 Royal Society of Chemistry.
speed of Raman spectroscopy and thus hold a potential for
high-throughput detection.
6.5.3. SERS−Electrochemistry Immunoassay. Electro-
chemical immunoassay is a label-free and sensitive detection
technique that has attracted much attention. The fast response
and ease of integration make it suitable for POC applications.
Ilkhani et al. designed a SERS−electrochemical biosensor for
chemotherapeutic drug screening, in which the DNA−drug
interaction was monitored through SERS−electrochemical
transduction (Figure 35a).474 The variation of SERS spectra
indicated the binding between the DNA and drug, while the
electrochemical function realized quantitative detection with a
LOD down to 8 μg/mL. Lu et al. developed a SERS−
electrochemistry dual-mode immunosensor for simultaneous
detection of multiple biomarkers (Figure 35b).475 Simultaneous
detection of CEA and cytokeratin-19 (CK-19) showed high
sensitivity, selectivity, long-term stability, and reliability.
6.5.4. Immunoassay with Hyperbolic Metamaterials.
Recently, Sreekanth et al. developed an extremely sensitive
biosensing platform based on hyperbolic metamaterials that
allows the detection of low molecular weight biomolecules in
highly diluted solutions.476 The platform can support highly
confined bulk plasmon guided modes over a broad wavelength
range from visible to near IR. They reported the ability of this
metamaterial platform to detect ultralow molecular weight (244
Da) biomolecules at picomolar concentrations using an affinity
model based on streptavidin−biotin interaction. This novel
platform could potentially be combined with SERS for
multimodal immunoassay with extreme sensitivity.
7. CONCLUSION
SERS offers many unique advantages over traditional immuno-
assay techniques, including ultrasensitivity and excellent
multiplexing ability. The diversity of SERS-active platforms,
from traditional solid substrates and liquid microbeads to
microfluidic chips, paper substrates, optical fibers, and hydro-
phobic substrates, provides flexible choices for various
applications. Specifically, the standard sandwich immunoassay
on solid substrate has laid the foundation for the development
of other platforms; to simplify the assay protocols, microbeads
are employed to establish immunoassays in liquid phase; for
parallel analysis of large amounts of samples with low volumes,
the microfluidic platform offers one of the best choices; to
enable on-site detection with remote control, immunoassays
with optical fibers could be used; with the purpose of reducing
the assay expenses, the paper-based substrate, which requires
minimum cost for fabrication, might become an option; and in
order to achieve ultrasensitive analysis, superhydrophobic
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substrates could be developed to enrich the target molecules.
With these platforms, SERS-based immunoassay has found
enormous applications in clinical diagnosis, environmental
monitoring, and biochemical analysis. Furthermore, combined
with the optical-encoding technique, SERS shows a great
promise for high-throughput assays.
Currently, researchers have tried to apply these SERS
platforms for detecting real samples. Some of the detection
results showed a comparable performance to those using ELISA
kits, which is the current employed golden standard. However,
up to now, none of these SERS-based immunosensing
platforms have been approved for clinical uses. Looking into
the future, there is a great demand for developing highly
reproducible SERS-active immunoassay platforms for practical
applications, such as clinical diagnosis. Fortunately, the highly
uniform nanosubstrates fabricated by the lithography, template,
and self-assembly technology have provided solutions to
improve the reproducibility of SERS substrates (section
6.1.1). Meanwhile, various SERS-active nanoprobes constructed
by the assembly methods based on templates, electrostatic
adsorption, and surface chemistry help to maintain the
monodispersity of the nanoparticles in complex environments
(section 6.1.2). For real sample detection, novel surface
chemical engineering should be designed to minimize the
nonspecific adsorption of biofluids (section 6.2).
Additionally, it would be beneficial to develop SERS-based
immunoassay platforms with small size, low sample con-
sumption, reduced cost, easy operation, and intuitive readout.
In this respect, the microfluidic chips show the highest potential
for POC applications, while the paper-based test stripes could
become an excellent choice for household assay devices
(section 6.3). At present, some commercially available
immunoassay systems based on microfluidics and paper have
been developed. For example, the FDA-approved i-STAT hand-
held microfluidic device enables the detection of cardiac
markers with easy operation, and the paper-based pregnancy
test strips have been widely used for household early pregnancy
diagnosis. Summarily, great efforts are still needed to promote
SERS-based immunoassay from proof-of-concept research
works to commercially available analytical tools.
AUTHOR INFORMATION
Corresponding Author
*E-mail: cyp@seu.edu.cn.
ORCID
Yiping Cui: 0000-0002-4648-2506
DOI: 10.1021/acs.chemrev.7b00027
Chem. Rev. 2017, 117, 7910−7963
Chemical Reviews Review

Notes Foundation of Jiangsu Province (No. BK20150623), the

The authors declare no competing financial interest. Science Foundation for The Excellent Youth Scholars of
Southeast University, and the Fundamental Research Funds for
Biographies the Central Universities.
Zhuyuan Wang received her B.S. in electronic science and engineering
in 2001 and her Ph.D. degree in physical electronics in 2005, both ABBREVIATIONS USED
from Southeast University, Nanjing, China. From 2005 to 2006, she 2-NAT = 2-naphthalenethiol
worked as a postdoctoral fellow in the State University of New York at 4-MPy = 4-mercaptopyridine
Buffalo, Buffalo, NY. From 2006 to 2011, she was an associate 4-MT = 4-methoxythiophenol
professor in the Advanced Photonics Center at Southeast University, ac-EHD = alternating current electrohydrodynamic
where she became a full professor in 2011. She has published over 120 AFB1 = aflatoxin B1
papers in key journals. Her research interests cover a number of optical AFP = α-fetoprotein
spectroscopic techniques in biological applications, such as surface- BDT = benzenedithiol
enhanced Raman scattering (SERS), fluorescence, and nonlinear CA19-9 = carbohydrate antigen 19-9
optical spectroscopy. CEA = carcinoembryonic antigen
Shenfei Zong received her B.S. in electronic science and engineering in CTC = circulating tumor cell
2010 and her Ph.D. degree in optical engineering in 2014, both from CV = crystal violet
Southeast University, Nanjing, China. Since then, she has been a DTNB = 5,5′-dithiobis(2-nitrobenzoic acid)
lecturer at the Advanced Photonics Center of Southeast University. EGFR = epidermal growth factor receptor
She is now focusing on cell imaging and biological analyte detection EIA = enzyme immunoassay
using optical spectroscopic techniques including SERS and fluo- ELISA = enzyme-linked immunosorbent assay
rescence. EpCAM = epithelial cell adhesion molecule
Lei Wu received his B.S. in electronic science and engineering from FA = folic acid
Southeast University, Nanjing, China, in 2012. He is currently a Ph.D. FISH = fluorescent in situ hybridization
candidate at the Advanced Photonics Center under the supervision of GERS = graphene-enhanced Raman scattering
Profs. Yiping Cui and Zhuyuan Wang. His research interests include HAV = hepatitis A virus
the development and application of SERS−microfluidic immunosen- HBV = hepatitis B virus
sors for the detection of disease biomarkers. HCPCF = hollow core photonic crystal fiber
HER2 = human epidermal growth factor receptor 2
Dan Zhu received her B.S. in electronic science and engineering from HIV = human immunodeficiency virus
Southeast University, Nanjing China, in 2011. She is currently a Ph.D. ICT = immuno-chromatographic test
candidate at the Advanced Photonics Center under the supervision of LOC = lab-on-a-chip
Profs. Yiping Cui and Zhuyuan Wang. Her research focuses on the LOD = limit of detection
interaction between liposomal nanohybrids and tumor cells. MBA or 4MBA = 4-mercaptobenzoic acid
Yiping Cui received his B.S., M.S., and Ph.D. degrees from the MIP = molecularly imprinted polymer
Department of Electronic Engineering, Southeast University, Nanjing, miRNA = microRNA
China in 1982, 1984, and 1994, respectively. Since 1993, he has been a MMC = 2,7-mercapto-4-methylcoumarin
professor in the Department of Electronics (now the School of MMTAA = 2-mercapto-4-methyl-5-thiazoleacetic acid
Electronic Science and Engineering), Southeast University, Nanjing, MUC1 = mucin-1
China. From 1995 to 1996, he was a visiting professor at the State NPs = nanoparticles
University of New York at Buffalo. He established the Advanced NRs = nanorods
Photonics Center of Southeast University and has been engaged in NSs = nanostars
research on polymeric nonlinear optics, nanophotonic materials, and OMQ NPs = organic−metal−quantum dot hybrid nano-
biophotonics. He has published over 470 papers in key journals and particles
coauthored two book chapters in monographs“Optics of Intense pATP = p-aminothiophenol
Light and Its Applications” and “Physics of Nonlinear Optics” (English PCR = polymerase chain reaction
version)and a graduate student textbook, Laser Physics. For his POC = point-of-care
pioneering contributions to the understanding of nonlinear and PSA = prostate-specific antigen
luminescent properties of polymeric, organic, and nanophotonic QDs = quantum dots
materials, he was elected as a fellow of the Optical Society of America.
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7963 DOI: 10.1021/acs.chemrev.7b00027

Chem. Rev. 2017, 117, 7910−7963