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Recent advances in micro/nano-particles for clinical detection of cancer

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DOI: 10.1039/c3ay40791h

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Analytical
Methods
www.rsc.org/methods Volume 5 | Number 21 | 7 November 2013 | Pages 5839–6248

ISSN 1759-9660

CRITICAL REVIEW
Li et al.
Recent advances in micro/nano-particles for clinical detection of
cancer biomarkers 1759-9660(2013)5:21;1-K
Analytical
Methods
CRITICAL REVIEW

Recent advances in micro/nano-particles for clinical

detection of cancer biomarkers
Cite this: Anal. Methods, 2013, 5, 5862

Jianping Li,*a Cong Fua and Catherine F. Yangb

Cancer is a major threat to public health and is still one of the leading causes of death worldwide. Cancer
biomarkers are extremely important in the process of early detection of cancer, diagnosis, judgment of the
curative effect and the prognosis. They also play an important role in mechanistic research into
carcinogenesis. Detection methods based on cancer biomarkers for the determination of cancer can
directly affect the diagnosis and treatment. Therefore, the development of highly sensitive and selective
approaches for the detection of cancer biomarkers is critical. Micro- and nanoparticles play an important
role in the clinical detection of cancer biomarkers. In this review, we provide an overview of the
Received 11th May 2013
Accepted 25th July 2013
commonly used clinical detection methods for cancer biomarkers, such as enzyme-linked
immunosorbent assay, chemiluminescence, electrochemiluminescence, as well as their technical
DOI: 10.1039/c3ay40791h
characteristics. Additionally, various applications of recent advances in microparticles and nanoparticles
www.rsc.org/methods in the clinical detection of cancer biomarkers are also reviewed.

1 Simple introduction to micro/nano- 2 Simple introduction to cancer biomarkers

particles
Microparticles and nanoparticles (micro/NPs) are on the micron
to nanometer scales and they exhibit several unique character-
istics due to their micro-scale effects and large specic surface
area.1 There have been many reports over the last decade about
their applications in the disciplines of physics, chemistry,
biology, medicine and drugs.2–4 There are many ways to prepare
micro/NPs,5,6 among which chemical synthesis is the most
commonly used method in practice.7,8 Many instrumental
analysis methods have been employed for the characterization
of micro/nanoparticles, frequently used ones include scanning
electron microscopy (SEM),9,10 scanning tunnelling microscopy
(STM),11 transmission electron microscope (TEM),12 X-ray
diffraction (XRD) test,13 X-ray photoelectron spectroscopy
(XPS)14 and so on.15–17
The most effective way to reduce the threat of cancer is early
detection, diagnosis and treatment. Cancer biomarkers (CBs),
also known as tumor biomarkers, are excretions or antigens of
tumors when they are developing in the organisms. Presently,
more than 100 CBs have been discovered and over 30 of them
have been used in clinical examination. In clinical utility,
several CBs need to be evaluated for the diagnosis of one type of
tumor. Some of them are biomarkers for several kinds of
tumors. Commonly used CBs include alpha fetoprotein (AFT),
carcinoembryonic antigen (CEA), carbohydrate antigen 125
(CA125), carbohydrate antigen 153 (CA153), carbohydrate
antigen 199 (CA199). Several methods, such as enzyme-linked
immunosorbent assay (ELISA), chemiluminescence immuno-
assay, microsphere enzyme immunoassay and time-resolved
uoroimmunoassay, have been used in practical assays for the
detection of CBs.18–20

a
College of Chemistry and Bioengineering, Guilin University of Technology, Guilin, 3 The role of micro/NPs in bioanalysis
541004, P. R. China. E-mail: likianping@263.net; Fax: +86-773-8990404; Tel: +86-
773-8990404 The micro/NPs consistently used in bioanalytical chemistry
b
Department of Chemistry and Biochemistry, Rowan University, Glassboro, NJ 08028, usually include noble metal NPs (such as gold, silver, etc.),
USA. E-mail: yang@rowan.edu; Fax: +1-856-2565455; Tel: +1-856-2565455 quantum dots (QDs), carbon nanomaterials (such as carbon

Jianping Li is a professor at the College of Chemistry and Bioen-
gineering, Guilin University of Technology. He received his bach-
elors and masters degrees from Jilin University in 1988 and 1991,
and a Ph.D. degree from Zhejiang University in 2003. His research
is focused on chemical sensors and biosensors.
Cong Fu has been studying for his masters degree at Guilin
University of Technology since 2011, and he works on the fabri-
cation of highly sensitive electrochemical sensors.

5862 | Anal. Methods, 2013, 5, 5862–5874 This journal is ª The Royal Society of Chemistry 2013
Critical Review
nanotubes (CNTs), and graphene), magnetic microspheres and
NPs, inorganic carriers of silica and calcium carbonate NPs, and
a variety of micro/nanocomposite materials.21–23 For example,
some micro/NPs can be labeled by analytes and act as the signal
source. They amplify the response signals. They also act as
carriers for reagents or analytes to avoid biological or physio-
logical interference in vivo. With the use of magnetic materials,
analytes in complicated matrices can be separated and detected
simultaneously. The micro/NPs will also improve the targeting
effects of the reagents towards certain analytes. The selectivity
of immunosensors can also be enhanced by sandwich type
immunoassays, recently established based on the specic
combination of the antigen–antibody on the sensor.24,25 Au NPs,
magnetic NPs and magnetic composite NPs could all be used for
this method.
3.1 Labels for analyte
The sensitivity depends largely on the signal intensity produced
by the labels, so highly sensitive labels are the key factor of the
assay. Commonly used labels include uorescent light emitting
agents, enzymes, electrochemical species, etc. The application
of nanomaterials will likely improve the performance of the
biosensor. As mentioned above, commonly used micro/NPs
labels in CBs detection include metal NPs, QDs, carbon nano-
materials and composite micro/NPs etc.
3.1.1 Au NPs. Au NPs are widely used in bioanalytical
chemistry. Since Au NPs have advantages of good biocompati-
bility, non-toxic effects and nano-size effect, they have been
used a great deal in the fabrication of nano-biosensors and
nano-biochips.
3.1.2 Quantum dots. Semiconductor QDs are semi-
conductor nanocrystals composed of metals of IIB-VIA (such as
CdSe, CdTe, ZnSe) or IIIA-VA (such as InAs, InP). QDs have
unique quantum-size related optical and electrical properties for
applications in biology, catalysis, and nanotechnology.26 QDs
have several photophysical advantages over organic dyes such as
a high uorescence yield, tunable spectra, stability, and low
toxicity, which make them good biomarkers in biomedical
applications. QDs are widely used as labels of analytes.
Composite particles of QDs and metal NPs have spurred a great
deal of interest among researchers.27–29 For example, Liu et al.30
developed an Au/TiO2 hybrid mesoporous lm sensitized by CdSe
QDs for photoelectrochemical applications. The CdSe QDs were
deposited on the TiO2 NP lm of the Au/TiO2 hybrid structures by
chemical bath and the results showed that the photocurrent of
the TiO2 lm was signicantly enhanced by the Au NPs.
Catherine F. Yang is a professor at Rowan University. She obtained
her Ph.D. degree from Tus University in 1993 and then undertook
Postdoctoral work at Harvard Medical School in 1995. During her
tenure at Rowan, she received many academic awards including
Cottrell College Award of Research Corporation, National Institute
of Health AREA Grant Award, Innovative Research Award of
National Applied Chemical Laboratory, and National Science
Foundation MRI Grant Award.
This journal is ª The Royal Society of Chemistry 2013
Analytical Methods
3.1.3 Carbon nanomaterials. Carbon nanomaterials,
including carbon nanotubes, fullerenes and other graphitic
nanomaterials (carbon blacks and carbon ber), have attracted
special attention in preparing biosensors.31 CNTs are classied
as single-walled carbon nanotubes (SWCNTs) and multi-walled
carbon nanotubes (MWCNTs). The micro-tubular and porous
structure of the CNTs maintains the activity of biological
molecules and improves the immobilization efficiency. The
biological performance was also improved. In addition, CNTs
have excellent surface chemical properties and good electrical
properties. Therefore, they are ideal materials for the prepara-
tion of biosensors.32 Compared with conventional bulk carbon-
based sensors, the biosensors produced from CNTs are more
sensitive and they respond faster.
3.2 Nanomaterials as carriers
NPs have been used extensively for drug delivery to the tumor as
an efficient carrier. These nanocarriers, with small particle
diameters and narrow particle size distributions, can be used
for targeting specic localities, and protecting and stabilizing
the drug molecules aer surface modication. They could assist
in combining detection and treatment to enable real-time
monitoring of the healing efficacy. Based on these advantages
and the use of a nanocarrier for drug delivery, a growing
number of researchers began to focus their research on NPs as a
versatile carrier for clinical tests. The materials usually used as
carriers were silica, CaCO3, and TiO2 NPs.33 These materials
exhibit excellent biocompatibility and bioactivity, resulting in
intensication of the enzyme’s performance aer immobiliza-
tion in the immunoassay.
3.3 Signal magnication
There have been many reviews about micro/NPs used in analysis
for signal magnication.34–36 The enhanced performance of
nanomaterial-based devices could provide a new opportunity
for amplifying the signal of those biosensors by incorporating
the analyte with nanomaterials.37 This is due to their unique
chemical and physical properties caused by their quantum
connement, surface, small size, and macro quantum tunnel
effects.38
3.3.1 Au/Ag NPs. Nano Au and Ag particles show advan-
tages including good conductivity, catalysis effects, photoelec-
tric properties and biological compatibility, and they have been
widely used in the bioanalytical eld. In particular, Au NPs are
very attractive materials because they can be easily prepared
with narrow size distributions and combined with a large
number of signaling molecules leading to high sensitivity in
immunoassays.39 Au NPs are also more stable and can be
combined with various biomolecules (such as antigens,
hormones and proteins) in a convenient way. Nanoscale struc-
tures of Au NPs on conductive surfaces combined with high
electrical conductivity can facilitate fast electron transfer to and
from redox enzymes, which provides a sensitive platform for
biosensors.
3.3.2 Magnetic NPs. Magnetic NPs can be used to label
biomolecules for molecular recognition and detection by the
Anal. Methods, 2013, 5, 5862–5874 | 5863
Analytical Methods
complex operation of sample mixing, separation, and detection
technique. Liu et al.40 developed a highly sensitive protein
detection method based on a novel enzyme-labeled Au NP probe
for the determination of CEA. The enzyme-labeled Au NP probe
was prepared by coating Au NPs with antibody, single-stranded
DNA, and HRP. Magnetic NPs functionalized with another
antibody were used as a capture probe. Then, the target protein
was sandwiched by the enzyme-labeled Au NP probe. The probe
was subsequently captured through immunoreaction. The
target immunoreaction event can be sensitively transduced via
the enzymatically amplied optical signal.
3.3.3 Carbon nanomaterials. CNTs are an ideal material for
the fabrication of biosensors for their excellent electrical and
surface chemical properties. Compared with common carbon
sensors, biosensors made with CNT have a higher sensitivity
with a wider detection temperature. The response time is also
shorter. Ou's group41 immobilized MWCNT, chitosan and Au
NPs using a layer-by-layer self-assembly method on the elec-
trode surface for adsorption of antibodies to construct a highly
sensitive, stable amperometric CEA immunosensor. Due to the
addition of the Au NPs, MWCNT and other materials, the
sensitivity of the sensor was greatly improved. Shiddiky et al.42
developed a sensitive electrochemical immunosensor for the
detection of epithelial cell adhesion molecule antigen by
employing bionanoconjugates as signal-transduction labels.
The bionanoconjugates were fabricated by carboxylation of
graphene oxide nanosheets and immobilizing streptavidin and
amine-functionalized CdSe QDs on the carboxylated graphene
sheet, followed by immunoreaction with the biotinylated
secondary antibodies. The sensitivity of the immunosensor was
greatly enhanced. Yuan et al.43 proposed an approach to prepare
an immunosensor based on CNTs decorated with Au NPs for
signal amplication in the simultaneous detection of three CBs.
Neither non-specic adsorption nor cross reaction were found
in the experiment, showing consistent selectivity and
reproducibility.
3.4 Combining separation and detection
The components of samples in clinical examination are very
complex. In order to avoid interference and inuence on the
determination of trace and ultra-trace analytes from the high
content matrix composition in samples, it is highly desirable to
develop a system of combining separation and detection
together. There have been many techniques and nanomaterials
newly developed for this purpose.
Capillary electrophoresis and capillary electro-
chromatography (CE/CEC) are the most rapidly developing
techniques which can separate and analyze very small quanti-
ties of bioanalytes. The functionalized NPs modied by a
different matrix were added into the electrophoresis running
buffer. The NPs can be dynamically adsorbed on the wall of the
capillary to change (or reverse) the electroosmotic ow, and they
can also act as a pseudostationary phase to participate in the
allocation and reservation procedures of the analytes in
the column, thereby improving the selectivity and the separa-
tion efficiency of the column.44,45 The recently developed
5864 | Anal. Methods, 2013, 5, 5862–5874
Critical Review
microuidic chip-based technology can complete the whole
analytical process of sample preparation, reaction, and detec-
tion in biological, chemical, and medical areas. Fan's group
developed a CEA microuidic chip based on Au NPs and
microuidic technology.46 A sandwiched reaction among Au
NPs labeled secondary CEA antibody, CEA, and monoclonal
antibody was adopted aer CEA was separated by and captured
on the chip in a microuidic channel.
3.4.1 Magnetic NPs. In recent years magnetic NPs have
been widely used in biomedicine and biotechnology research.47
In magnetic separation technology, analytes are enriched by the
magnetic NPs and then separated by a foreign magnetic eld.
They also have specic magnetic guidance qualities. In
magnetic separating methods, the surface of the magnetic NPs
has to be modied by ligands (such as a specic antibody) that
can be recognized by the targeting biological molecules. Then
the targeting molecules can be enriched by combining with the
NPs and then separated together with them by an applied
magnetic eld. Biosensors fabricated with magnetic materials,
including enzyme sensors, immunosensors and nucleic acid
biosensors, which enrich and detect the target analytes simul-
taneously, are constructed based on this mechanism.48
Magnetic particles include organic polymer magnetic
microspheres and inorganic magnetic metal NPs. Polymeric
microspheres were frequently used in early research on sensors.
For example, Wang's team49 utilized magnetic beads as a
capture for eliminating non-specic adsorption effects which
hampered the detection of DNA hybridization. With the aid of
magnetic separation, interferences were eliminated. The
protocol offered assays of DNA sequences related to the breast
cancer BRCA1 gene. Our group50 has proposed an ECL sensor
for the detection of CA125 based on enzyme amplication and
magnetic nanoparticle enrichment. The sandwich-type immu-
noassay was performed on the magnetic force-controlled
carbon paste electrode via the immunoreactions among glucose
oxidase-labeled anti-CA125, CA125, and anti-CA125 on the
surface of magnetic nanoparticles. Inorganic magnetic particles
have received growing interest for use in electrochemical and
luminescence sensors because of their small particle size, good
biocompatibility, good conductive properties, etc. They are
suitable for trace antigen detection by immunoassay tech-
niques. Tang and his colleagues51 reported a series of immu-
noassay methods based on magnetic NPs. An electrochemical
magnetic controlled microuidic device was developed using
magnetic core–shell NiFe2O4–SiO2 beads for the detection of
different kinds of CBs. On the working electrode, the antibody
was immobilized on the NiFe2O4–SiO2 NPs surface. Another
immunoassay method52 developed was based on a sandwich
type complex on the electrode with thionine-doped magnetic Au
NPs for the labeling of HRP-bound anti-CEA as secondary
antibodies. The bound bionanospheres may catalyze the
reduction of H2O2 with the help of the doped thionine as a
mediator resulting in the signal output being dramatically
amplied.
Amorphous calcium phosphate, calcium carbonate and
silica are biologically relevant minerals. They can not only
improve the biological compatibility of the NPs, but also act as
This journal is ª The Royal Society of Chemistry 2013
Critical Review
carriers of sensitive agents. In order to improve the biological
compatibility of the NPs and enhance the load of the biological
species, the surface of the MNPs was modied with these
inorganic materials.
Magnetic uorescence composite NPs (MFCNP) have the
characteristics of both magnetic materials and uorescent
materials. Therefore, they have better performance and a wider
application range compared with traditional NPs. The MFCNP
may be used for imaging while other procedures such as
magnetic separation or targeting movement can be accom-
plished at the same time. The MFCNP may also enable the
magnetic manipulation of the particles by an external magnetic
eld under the uorescent visualizing conditions. Conse-
quently, they can be used in circumstances such as the sepa-
ration and detection of proteins and DNA, drug transportation
and tumor treatment.53
3.4.2 QDs. As previously discussed, QDs are nano-sized
particles having a semiconductor core. They have also been
applied to the separation and detection of analytes. For
example, Lin's group54 reported a sensitive portable uores-
cence biosensor for protein detection based on QDs and a
lateral ow test strip. They introduced QDs as uorescence
probes into a portable dry-reagent strip biosensor system. The
QDs had superior signal brightness and high photostability
which resulted in high sensitivity and selectivity of the sensor.
Immunoassays combined with highly sensitive anodic stripping
voltammetry (ASV) can obtain high sensitivity by measuring the
stripping currents of the NPs. Wang's team55 presented multiple
DNA target hybridization and simultaneous assays based on the
use of three QDs. The oligonucleotide labeled QDs were
immobilized on magnetic particles. Aer the hybridization
reaction with DNA targets, resolved stripping peaks of Zn, Cd,
Pb were recorded (Fig. 1).
3.5 Test strip
Many immunochromatographic test systems for hand-held
diagnostics have been developed owing to their convenience,
specicity and sensitivity, and they are also used in real-time
monitoring. These strip tests have found widespread use in the
detection of narcotics, toxins, highly dangerous infections, and
urogenital diseases. The immunochromatographic assay is
Fig. 1 Multi-target electrical DNA detection protocol based on different inor-
ganic colloid nanocrystal tracers (adapted from ref. 55, Copyright 2003, with
permission from American Chemical Society).
This journal is ª The Royal Society of Chemistry 2013
Analytical Methods
based on eluent movement along the membrane (lateral diffu-
sion), giving rise to specic immune complexes at different
membrane sites. The complexes are visualized as colored
bands. There have been many reports about micro/NPs used in
test strips as labels. For example, Du's group56 developed a
uorescence test strip based on QDs and ZrO2 NPs for the
detection of phosphorylated acetylcholinesterase. They also
proposed a strip-based biosensor for direct detection of tri-
chloropyridinol in saliva using Au NPs as the label.57 Stayton58
proposed a stimuli-responsive NP system for multiplexed
magneto-enrichment and non-instrumented lateral ow strip
detection of model antigens from spiked pooled plasma. The
integrated reagent system allows purication and enrichment
of the Au NPs-labeled biomarker half-sandwich that can be
applied directly to lateral ow test strips.
3.6 Imaging
Imaging plays a critical role in cancer diagnosis, radiation
planning and evaluation of treatment efficiency. Computed
tomography (CT) is one of the most useful diagnostic tools
among commonly used biomedical imaging techniques. There
are many reports of the applications of NPs in CT, which can
help avoid the disadvantages of traditional contrast agents.59
Besides traditional SEM and TEM, NPs can also be used for
imaging or signal amplication in other imaging techniques
such as uorescence, CL, ECL, nuclear magnetic resonance
(NMR) etc. As biological probes, NPs have attracted great
attention for their unique optical and controllable surface
chemical characteristics as diagnosis and bio-imaging tools. Au
NPs modied with ligands can specically mark the receptor of
the tumor cell. They can also provide information on specic
molecules in cancer detection and bio-imaging assay.60 El-
Sayed's group61 synthesized Au nanorods and conjugated them
to anti-epidermal growth factor receptor monoclonal antibodies
and incubated them in cell cultures. A strongly scattered red
light from gold nanorods was observed using a laboratory
microscope. Haam et al.62 located human skin cancer cells
using cetuximab-labeled uorescent magnetic NPs in NMR and
uorescence spectral imaging, and then detected skin cancer
cells by NMR and spectral images.
Surface plasmon resonance (SPR) is a powerful label-free
optical detection technique for studying biomolecular interac-
tions in real-time. It offers a new generation biomolecular
analysis with label-free, real-time molecular detection. SPR in
nanometer-sized structures is called localized surface plasmon
resonance. Precious metals, especially Au NPs, have gained
great potential in the detection of CBs in SPR, due to their
strong absorption and emission characteristics of plasma
resonance.63,64 Surface plasmon resonance imaging (SPRI) has
been established as one of the primary optical methods for the
direct detection of bioaffinity adsorption onto DNA, protein,
and RNA microarrays. The absorption spectrum of the particles
is not only related to the material, but also to the shape and the
symmetry of the particles, the surrounding medium, and the
distance between the particles. Therefore, NPs of different sizes
have different absorption peaks.65 Corn’s group employed SPRI
Anal. Methods, 2013, 5, 5862–5874 | 5865
Analytical Methods
for the detection of protein adsorption onto RNA aptamer
microarrays and enzymatically amplied for the analysis of
some biomarkers.66,67 The distances between the Au NPs can be
regulated by DNA. Based on this principle, Lu et al.68 developed
a visual detection method for nucleic acid aptamers, ribozymes,
and other new bio-recognition systems. Some researchers have
also investigated tumor imaging using QDs. Gao's team69
prepared multifunctional NPs probes based on semiconductor
QDs for cancer targeting and imaging in living animals. The
designed luminescent QDs were covered with an amphiphilic
polymer and linked to tumor-targeting ligands. Han et al.70
presented a method for an aqueous one-pot synthesis of bright
and ultra-small core–shell CdTe–CdS QDs with tunable near-
infrared uorescence and used them for the application of
tumor targeting and imaging in vivo. Near-infrared-emitting
QDs were directly synthesized in the aqueous phase through a
facile one-step strategy.
In recent years, many emerging magnetic resonance contrast
agents (MRCA) have contributed to new development for
magnetic resonance imaging (MRI).71 The magnetic NPs used in
MRI are superparamagnetic iron oxide (SPIO), which is a new
type of negative contrast medium for magnetic resonance. The
SPIO is usually constructed by Fe3O4 NPs wrapped by glucosan,
and they can be enriched in certain parts of the human body.
Kircher et al.72 designed a brain tumor molecular imaging
strategy to combine magnetic resonance imaging, photo-
acoustic imaging and Raman imaging (MPRs). The triple-
modality strategy can be used to characterize the margins of
brain tumors in living mice both preoperatively and intra-
operatively (Fig. 2).
3.7 Bio-barcode assay
The bio-barcode assay is an emerging diagnostic tool, based on
advances in nanotechnology, and is used for enzyme-free
ultrasensitive detection of various protein and nucleic acid
targets. The bio-barcode assay can be much more sensitive than
conventional ELISA-based assays, depending on the target and
sample complexity. This increase in sensitivity offers the
Fig. 2 Triple-modality MPR concept. MPRs are injected intravenously into a
mouse bearing an orthotopic brain tumor (adapted from ref. 72, Copyright 2012,
with permission from Nature Publishing Group).
5866 | Anal. Methods, 2013, 5, 5862–5874
Critical Review
opportunity to monitor existing biomarkers at levels not
possible with conventional assays. Mirkin et al.73 developed a
multiplexed protein detection method with bio-barcode assay.
In the assay, the Au NP probes can bind to the target–magnetic
particle complex through antigen–antibody interactions and
the detection involves silver amplication effect. Nam and his
collaborators74 proposed a highly sensitive protein detection
method of bio-barcode colorimetric assay. SiO2, Fe3O4 and Au
NPs were used in this assay, and the red-to-blue color change
was subsequently measured aer the barcode DNA was released
using DNA-modied Au NPs probes. The micro/NPs increased
the number of attached barcode DNA molecules on the
particles.
3.8 Analysis of molecular structure and conformation
The surface-enhanced Raman scattering (SERS) phenomenon is
commonly described as the enhancement of Raman signals of
certain adsorbents by 104 to 106 times on the surface of some
metals.75 SERS has become a powerful tool for the study of metal
NP structure and conformation of a system because it provides
not only spectroscopy of the electron transfer on the surface,
but also the structure and conformation information. Qian
et al.76 described biocompatible and nontoxic nanoparticles for
in vivo tumor targeting and detection based on pegylated gold
nanoparticles and SERS. They showed that large optical
enhancements can be achieved under in vivo conditions for
tumor detection in live animals. Joseph et al.77 provided a novel
way to study the kinetics of a catalytic reaction in situ by NPs
enhanced SERS. The authors attached separate Au and Pt NPs
on the same glass surface. This approach provides the struc-
tural characterization of the reactant and products, as well as
the rate constants in the experiment.
4 The application of micro/NPs in CB assays
The early detection and diagnosis of cancer is very meaningful
for treating cancer and decreasing cancer mortality, and thus
there is a critical need to determine CBs at trace or even ultra-
trace levels. Nanotechnology, combined with detection
methods, can help achieve the detection, observation and
analysis of cancer cells and tissues, and make the assay for the
early diagnosis of cancer more sensitive, accurate, simple and
convenient. It may also assist in detecting the internal biolog-
ical characteristics during the development of the cancer and
provide early detection and diagnosis of cancer at the cellular
level.78,79 Biosensors, such as electrochemical biosensors,
luminescent immunosensors and quartz crystal microbalance
integrated with NPs could enhance their performance. It is
necessary to label CBs molecules with external reagents to
produce detectable signals for highly sensitive detection.
4.1 Biosensors based on electrochemical analysis
Nanoparticles can display four unique advantages over macro-
electrodes when used for electroanalysis: enhancement of mass
transport, catalysis, highly effective surface area and control
over electrode microenvironment. Therefore, much work has
This journal is ª The Royal Society of Chemistry 2013
Critical Review
been done on their formation, characterization and employ-
ment for the detection of many electroactive species. These
electrochemical sensors can be classied as amperometric,
potentiometric and impedimetric sensors. The amperometric
type is the most widely used.
4.1.1 Amperometric sensors. The application of ampero-
metric immunosensors for the detection of biomarkers has
gained much attention.80–82 The method is based on the
recording of current produced by the redox materials at the
working electrode under constant voltage. The research topic
recently focused primarily on the improvement of the sensitivity
of the amperometric immunosensors.
There are many ways to fulll this purpose. For example,
many reports of CBs amperometric sensors based on the cata-
lytic reduction of PB as a tag have been presented.83–85 Kong
et al.86 presented a novel strategy based on a branched elec-
trode. The electrochemical signal of sandwich type electro-
chemical immunoassays can also be improved by the label
method.
It is evident that the use of multilayered Au NPs in the
construction of biosensors increases the specic surface area
and the loading of immune reagents, as well as the electron
transfer efficiency. Our group87 has developed an electro-
chemical immunosensor using magnetic core–shell NPs to coat
with self-assembled multilayer of Au NPs. High sensitivity and
response features were gained, due to the huge surface area,
large loading amount of the antibody and the biological
compatibility of the multilayered Au NPs. Combining this
immunosensor with microuidic techniques, Rusling and his
collaborators88 designed an inexpensive disc with gold array
featuring microwells surrounding 8-electrodes. These dispos-
able gold array chips with microuidics provided amperometric
immunoarrays that were used to measure the CB interleukin-6
in diluted serum. They also prepared a densely packed Au NPs
platform combined with sandwich type immunoassay with
magnetic bead bioconjugate (Fig. 3).89 Du et al.90 used colloidal
Au NPs labelled CA199 antibody to modify a carbon paste
electrode. HRP enzyme-labelled CA199 was selected as a second
antibody. Lin et al.91 developed a CB immunosensor using
graphene to accelerate electron transfer. Prostate-specic
antigen (PSA)-microbead carried Au NPs act as a tracing tag to
Fig. 3 The mechanism of the Au NPs film electrodes and multi-enzyme-particle
amplification (adapted from ref. 89, Copyright 2009, with permission from
American Chemical Society).
This journal is ª The Royal Society of Chemistry 2013
Analytical Methods
label the signal antibody and the Au NPs can also induce the
silver deposition. Ju's group92 also developed an electro-
chemical immunoassay method for simultaneous determina-
tion of CBs based on electrochemical stripping analysis of Ag
NPs, which were deposited with a reduction reaction catalyzed
by antibody-functionalized Au NPs labels.
4.1.2 Potentiometric biosensors. Potentiometric immuno-
sensors combined with the characteristics of high-sensitivity of
enzyme immunoassay and high-selectivity of ion-selective
electrodes can be used for real-time detection. When the
immobilized antibody combined with the corresponding
antigen, the charge density changes on the electrode surface
result in changes to the membrane potential on the electrode
surface. Since the antigen in the solution is dissociative, the
potential difference between the indicator electrodes immobi-
lized with antibody and the reference electrode depends on the
concentration of the antigens in the solution and the relation-
ship between the concentration and the potential follows the
Nernst equation. Fu et al.93 designed a potentiometric immu-
noassay protocol for the determination of CEA by using a
transparent immune affinity reactor of Fe3O4 bionanorods
immobilized with the conjugation of anti-CEA antibodies. The
potential response can be changed aer CEA binding onto
the immobilized anti-CEA.
4.1.3 Impedance-based biosensors. Micro/nano materials
can also be used to prepare impedance immunosensors. The
conductivity change induced by the adsorption or desorption of
various bioactive compounds (such as antigens/antibodies,
aptamers, barcodes etc.) on the electrode surface can lead to a
change in the interfacial electron transfer performance. As an
effective means of interfacial properties characterization, elec-
trochemical impedance spectroscopy (EIS) is oen used to
measure the changes of chemical and electrical properties, due
to the specic immunointeraction.94 As a frequency domain
measurement method, EIS is used to study the electrode system
by measurement of the frequencies over a wide range. This
assists in obtaining more information on the kinetics of elec-
trode reaction and the electrode interface structure than using
the other conventional electrochemical methods. It has the
advantages of high sensitivity, short detection time and the
method is simple. Tang et al.95 presented a simple impedance
immunoassay strategy for sensitive detection of AFP by using a
target-induced release of Au NPs-labeled anti-AFP antibodies
from polyvinylpyrrolidone-coated magnetic carbon nanotubes.
Pan's team96 developed a magneto-controlled strategy using
ow-injection EIS for CEA immunosensing by immobilizing
anti-CEA on epoxysilane-modied core–shell magnetic Fe3O4–
SiO2 NPs which can be attached on a carbon paste electrode
surface to form an immunosensor for CEA detection.
4.2 Optical analysis
4.2.1 Imaging and Raman scattering spectroscopy. As
previously mentioned, there have been many analyses of micro/
NPs used in imaging analysis. Kopelman's group97 established a
targeted molecular imaging platform for CBs detection at the
cellular and molecular level with standard clinical CT using Au
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Analytical Methods
NPs as probes targeted to tumor antigens. Jon et al.98 reported
multifunctional NPs for targeted molecular CT imaging and
therapy of prostate cancer. Law et al.99 proposed a NPs-
enhanced biosensor by integrating both the NPs and immu-
noassay into a SPR system for detecting tumor necrosis factor
alpha (TNF-a) antigen. An antibody-functionalized sensing lm,
together with antibody-conjugated Au NPs, served as a plas-
monic coupling partner used as a sandwich immunoassay for
TNF-a detection at a concentration as low as the fmol range. Cui
et al.100 developed a novel SERS probe for distinguishing breast
cancers with different epidermal growth factor receptor 2
(HER2) statuses. In such a probe, anti-HER2 antibody-conju-
gated silver NPs have been synthesized for specic targeting of
HER2-positive breast cancer cells. This type of SERS probe holds
the potential for a direct detection of living breast cancer cells
with the advantages of easy fabrication, high SERS sensitivity,
and biocompatibility.
SERS is a new technique developed on the basis of Raman
scattering, which has a high specicity. Raman imaging allowed
for guidance of intraoperative tumor resection. Histological
correlation validated that Raman imaging was accurately
delineating the brain tumor margins. This new triple-modality
NPs approach has promise for enabling more accurate brain
tumor imaging. The nanometer particles of gold and silver can
produce an enhancement effect on the Raman scattering signal.
Therefore, gold and silver NPs were used as the probes for CBs
detection by surface-enhanced Raman scattering techniques.
For example, Gong et al.101 prepared Ag–SiO2 core–shell NPs and
linked with polyclonal AFP antibody; then embedded it with
rhodamine B isothiocyanate dye molecules as Raman tags. They
also prepared amino group modied silica-coated core–shell
magnetic NPs which were modied with anti-AFP.
4.2.2 Fluorescence analysis. Fluorescence analysis is
generally highly sensitive, selective and simple. Fluorescent
biosensors play an important part in biological detection. Bio-
logical probes marked by uorescent materials and uores-
cence analysis methods based on the uorescence resonance
energy transfer (FRET) principle have been widely used in
bioassays, such as the recognition of pathogens, the detection
of genetic mutations and single nucleotide polymorphisms and
the determination of proteins. The commonly used uores-
cence donors/acceptors are usually uorophores, or uores-
cence quenchers. The usage of organic quenchers, which are
usually used as acceptors, is limited because the efficiency is
low. Au NPs have high molar absorption and wide adsorption
spectrum range. Compared with conventional organic
quenchers, the coefficient is much higher. Dubertret et al.102
found that Au NPs of 1.4 nm diameter could quench all the
uorescent dyes efficiently.
Fluorescence immunoassays have long been developed.103
The concentration of the analyte can be determined by
comparing the concentrations of the analytes and the relative
intensity of the uorescence. Labeled immunoassays are an
important means of quantifying bioactivators such as the
antigen–antibody system. Jokerst et al.104 fabricated a module
with semiconductive QDs, and the sandwich type module which
was used in a biosensor for multichannel detection of three
5868 | Anal. Methods, 2013, 5, 5862–5874
Critical Review
CBs. The antibody marked by QDs may be used to enhance the
uorescent signals and catch the antigen simultaneously. Li
et al.105 described a protocol for simultaneous immunoassay of
two lung CBs based on dual-color QDs. During the procedure,
two captured antibodies labeled with biotin tags, two antigens
and two antibodies were mixed together to form sandwich
complexes and then streptavidin coated polystyrene beads were
added, and a detection limit of 1.0 ng mL 1 was obtained.
Upconversion micro/NPs are excited at long wavelengths in
the near infrared region with a shorter wavelength emission in
the visible or near infrared regions. Owing to their small phys-
ical dimensions and good biocompatibility, upconversion NPs,
as a new class of uorescent probes, can be easily coupled to
proteins or other biological macromolecular systems and used
for in vivo imaging, detection, and diagnosis of cancers.106 Xu
et al.107 synthesized strong uorescence upconversion NPs. The
particles were coated with a thin layer of SiO2 outside, which
were further modied with amino groups and then covalently
linked with rabbit anti-CEA antibodies. The antibody composite
NPs were used as uorescent biolabels for the detection of CEA.
The immunoparticles gave a bright green emission of upcon-
version uorescence aer excitation, and enabled the uores-
cent imaging and detection of HeLa cells.
4.2.3 Chemiluminescence and electrochemiluminescence.
CL immunoassays are highly selective and sensitive. The
equipment is simple and the assay can be nished in a short
time. Currently CL immunoassays are commonly used for the
detection of CBs by NPs. ECL is a technique that combines CL
and electrochemistry. ECL immunoassay is a new generation of
immunoassay technology, by which CBs can be detected
sensitively.108,109 For example, Jie's group110 synthesized
magnetic Fe3O4/CdSe–CdS composite NPs with 3-amino-
propyltriethoxysilane polyelectrolyte shell and used them to
fabricate ECL immunosensors for the detection of CEA. Anti-
CEA was immobilized on the composite NPs. Aer immuno-
reactions, a change in steric hindrance occurred, which led to a
decrease in ECL intensity. Guo et al.111 developed an ECL sensor
for AFP detection. AFP was immobilized on a glassy carbon
electrode by forming a complex of graphene–CdS QD–alginate,
which acted as the immobilizing support of AFP, and CdSe/ZnS
QDs as the label. Aer the sandwich reaction, CdSe/ZnS QDs
can effectively scavenge the ECL of the composite. Our group
proposed a novel protocol of ECL immunoassay based on
enzyme amplication and magnetic NPs enrichment for CA125
assay.112 Anti-CA125 was rst loaded onto Fe3O4 magnetic NPs.
The sandwich-type immunoassay was performed via the
immunoreactions among glucose oxidase (GOD)-labeled anti-
CA125, CA125, and anti-CA125 on the surface of magnetic NPs.
ECL was generated by the reaction between luminol and GOD-
derived hydrogen peroxide (Fig. 4). The sensitivity of CA125
determination was signicantly enhanced, due to the ampli-
cation effect of enzymatic reaction. The proposed ECL method
provided a simple selective immunoassay strategy, which was
applied in the determination of CA125 in clinical serum
samples. Jie also designed an ultrasensitive ECL immunosensor
for the detection of CEA.113 Because of the hybrid nano-
structures constructed, the ECL signal was amplied and the
This journal is ª The Royal Society of Chemistry 2013
Critical Review
Fig. 4 Scheme of CA125 electrochemiluminescence immunoassay detection
(adapted from ref. 112, Copyright 2012, with permission from Springer).
sensitivity was enhanced by 17 times compared with the system
in which only QD were adopted (without Au NPs). Sardesai
et al.114 fabricated an ECL immunosensor array for the detection
of PSA and IL-6. The array was fabricated on a conductive
pyrolytic graphite chip by forming capture-antibody-decorated
SWCNT forests.
Within the last decade, simple and rapid CB detection
methods based on Au NPs have been established. Among these
methods, optical detection is still the primary method.115 The
uorescence enhancing and quenching effects of Au NPs loaded
with uorescent reagents can be used in bioanalysis.116 Zetter
et al.117 developed target-activated uorogenic probes based on
Au NPs functionalized with a self-assembled heterogeneous
monolayer of dye-labeled peptides and PEG to visualize proteo-
lytic activity in vivo. Au NPs probes targeted to trypsin and
urokinase-type plasminogen activator achieved 5–8 fold signal
amplication. Raman spectroscopic determination of bio-
substrates using Au NPs has drawn much attention in recent
years.118 Huo's team119 proposed a one-step homogeneous
immunoassay for the detection of free-PSA using Au NPs probes
coupled with dynamic light scattering measurements. Au NPs
and nanorods were rst conjugated with two different primary
anti-PSA antibodies and then used as optical probes for the
immunoassay. The change of particle size results in the change of
their wavelengths and light scattering intensities in Raman
spectroscopy, and the formation or enlargement of metal NPs
caused by metal deposition produces the change of SERS signals.
4.3 Mass-sensitive biosensor (piezoelectric biosensors)
Piezoelectric sensors may transfer the signal of quality changes
into a frequency variation of the oscillation circuit.120 The typical
piezoelectric material applied to piezoelectric sensors is quartz
crystal. The oscillating electric eld generated by quartz crystals
is based on its resonant frequency, which is decided by its
chemical properties, size, shape and mass. As a new sensing
technology with the properties of high sensitivity, label-free,
simple in operation and fast real-time detection, piezoelectric
sensors have shown rapid development in recent years, and have
been widely used in the elds of biomedicine, environmental
monitoring and clinical assays. As the most important piezo-
electric sensor, quartz crystal microbalance (QCM) has many
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Analytical Methods
advantages such as high sensitivity, low detection limits, real-
time monitoring etc. Therefore it has a wide application range in
clinical diagnosis. As early as 2002, Chen et al.121 developed a
QCM immunosensor for human ferritin (FER) assay. Au NPs also
exhibit a great potential application in the mass-type sensing for
bioassays based on the mass changes aer immunoreactions.
Haick and his collaborators proposed a new method by using an
array of sensors based on Au NPs.122 It was simply performed by
detecting the electro-resistances of the Au NP based sensors to
distinguish the breath of lung cancer patients and the breath of
healthy individuals in an atmosphere of high humidity. The Au
NPs in the lm of the sensor can provide sites for the absorption
of the analyte molecules. In addition, some research has indi-
cated that multiple marks can be achieved by combining
different metal NPs in a suitable manner.123
The larger cluster masses of Au NPs provide high sensitivity
for mass-sensitive sensors. For a piezoelectric immunosensor, it
is important to increase specic binding to biomarkers and
decrease nonspecic adsorption to the others. Zhang et al.124
developed a multi-channel model of a QCM microarray immu-
nosensor for human chorionic gonadotropin (hCG) detection.
Every crystal unit of the microarray can oscillate independently
without interfering with the others. Compared with a one-
channel immunosensor, it can provide eight times faster
detection speeds for the hCG assay.
Other examples of the micro/NPs used in the detection of
CBs are shown in Table 1.
5 Prospects
Due to the micro/nano-scale effect and large specic surface
areas, micro/NPs have unique and excellent electrical, optical
and thermal properties. There are numerous reports of the
application of micro/NPs in the areas of catalysis, sensors and
biomedicine etc. In this paper, many highly sensitive CB
immunosensors based on micro/NPs, including Au/Ag NPs,
magnetic NPs, CNTs and some other inorganic compound NPs,
as well as some of their composite NPs, have been reviewed.
These micro/NPs are believed to be an important analytical tool
in bioassays. By successfully addressing the major issues of
sensitivity and selectivity in CBs detection, the new micro/NPs-
based protocols will have an important inuence on clinical
assays and diagnosis.163 Currently, relevant research for CBs
detection based on micro/NPs is focused on improving the
fabrication techniques, so as to improve their sensitivity and
other performance characteristics.164 However, there are still
some problems to be solved before wide adoption and appli-
cation. For example, the complexities and challenges involved
in the synthesis process of homogeneous particles. Some
methods mentioned above have poorer stability and reproduc-
ibility compared with conventional methods. This is probably
due to the complicated procedures involved during the
synthetic process. Furthermore, some protocols used still
require expensive equipment and proper training for the oper-
ators. It is expected that future research on micro/NPs will result
in effective, easy to use portable devices for the real-time
monitoring of CBs,165 such as test strips or lab-on-chip.166 The
Anal. Methods, 2013, 5, 5862–5874 | 5869
Analytical Methods Critical Review

Table 1 Examples of the micro/NPs used for CBs detection

Micro/nano-
materials Target Detection methods Detection limits Ref.

Au NPs CEA SERS 1–10 pg mL 1 119

Au NPs CEA Amperometric 3 fg mL 1 120
Au NPs CEA Fluorescence — 121
Au NPs CA125 Amperometric 1.8 U mL 1 122
Au NPs AFP Spectrometry 0.1 ng mL 1 123
Au NPs CEA Amperometric 4 pg mL 1 124
AFP 7 pg mL 1
Au NPs AFP ECL 0.33 pg mL 1 125
Au NPs CA125 Amperometric 1.73 U mL 1 126
Ag NPs CA125 ECL 0.03 U mL 1 127
Au NPs PSA SPRI 1 ng mL 1 128
Au NPs PSA Amperometric 0.5 pg mL 1 129
Au NPs CA153 Amperometric 0.0015 U mL 1 130
CA125 0.00034 U mL 1
CEA 0.0012 ng mL 1
QDs CEA ECL 0.002 pg mL 1 131
QDs CEA Fluorescence 1.0 ng mL 1 132
QDs AFP ECL — 133
QDs AFP Amperometric 10 pg mL 1 134
QDs AFP Fluorescence 0.4 ng mL 1 135
QDs AFP ECL 0.2 fg mL 1 136
QDs PSA Fluorescence — 137
QDs CA125 Fluorescence 0.02 ng mL 1 138
CNT AFP ESA 0.018 ng mL 1 139
CNT AFP DPV 0.02 ng mL 1 140
CNT CEA Amperometric 0.1 ng mL 1 141
CNT PSA Amperometric 4 pg mL 1 142
Fullerenes AFP Amperometric 5.77 pg mL 1 143
Fullerenes CEA ECL 3.3 pg mL 1 144
Fe3O4 CEA Amperometric 0.07 ng mL 1 145
Fe3O4 CEA Potentiometric 0.9 mg L 1 146
Fe3O4@QDs CEA Fluorescence 3.5 ng mL 1 147
ADP 3.9 ng mL 1
Graphene@Au CEA Amperometric 1.0 pg mL 1 148
1
CNT@Au HCG Amperometric 0.0026 mIU mL 149
Pt@Au CEA Amperometric 0.11 pg mL 1 150
SiO2@Au AFP Amperometric 0.02 ng mL 1 151
SiO2@Ag CEA Amperometric 1 fg mL 1 152
SiO2@p-acid CA125 Fluorescence 0.04 ng mL 1 153
SiO2@QDs PSA ECL 0.72 pg mL 1 154
Pt/Ru@QDs HCG ECL 0.8 pg mL 1 155
Au@QDs AFP ECL 0.2 pg mL 1 156
Graphene@Ag AFP Amperometric 3 pg mL 1 157
CaCO3 NSE Fluorescent 2.0 pg mL 1 158
DPV 0.1 pg mL 1
TiO2 PSA ECL 1.0 fg mL 1 159
Magnetic bead PSA SPRI 10 fg mL 1 160
Au structure screen CA125 Impedance 6.7 U mL 1 161
Au colloid CEA Amperometric 0.22 ng mL 1 162

new research direction will likely grow into areas such as Natural Science Foundation of Guangxi Province (no.
multichannel and multianalyte detection, as well as label-free 2012GXNSFAA053032).
detection.167 We also expect the future emergence of more
types of micro/NPs as well as novel synthetic methods for References
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