Biosensores Opticos Song2011

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REVIEW
Colorimetric Biosensing Using Smart Materials
Yujun Song, Weili Wei, and Xiaogang Qu*
polymers.[1,6,11,14–17] The robust physical

In recent years, colorimetric biosensing has attracted much attention because or chemical properties of these materials
of its low cost, simplicity, and practicality. Since color changes can be read make them promising candidates for
out by the naked eye, colorimetric biosensing does not require expensive colorimetric biosensing. Gold nanopar-
ticles display unique size-, shape-, and
or sophisticated instrumentation and may be applied to field analysis and composition-dependent optical proper-
point-of-care diagnosis. For transformation of the detection events into color ties. Aggregation or redispersion of the
changes, a number of smart materials have been developed, including gold nanoparticles will result in color changes
nanoparticles, magnetic nanoparticles, cerium oxide nanoparticles, carbon of the colloidal solution.[1,2] Some of the
nanotubes, graphene oxide, and conjugated polymers. Here, we focus on metal oxide nanoparticles, such as Fe3O4
and CeO2, can catalyze the reaction of
recent developments in colorimetric biosensing using these smart materials.
the peroxidase substrate to produce color
Along with introducing the mechanisms of color changes based on different change.[14,15] Also, carbon nanotubes and
smart materials, we concentrate on the design of biosensing assays and their graphene oxide possess intrinsic per-
potential applications in biomedical diagnosis and environmental monitoring. oxidase activity and can catalyze the color
reaction.[16,17] Some of the conjugated poly-
mers, such as polythiophene derivatives or
1. Introduction polydiacetylene, can change color upon conformational transi-
tion or aggregation.[6,11]
Over the past decades, biosensing technology has made signifi-
The biological applications of these materials are currently
cant progress by taking advantage of a large number of novel
under intense investigation.[1,5,9] The aim of this review is to
materials designed and fabricated for the applications.[1–13]
summarize recent developments in colorimetric biosensing
These new materials provide a particularly useful platform and
using these smart materials. We will introduce the mechanism
have been used in a wide range of biosensing. The newly devel-
of color changes based on different smart materials and con-
oped methods have shown great advantages over conventional
centrate on the design of biosensing assay and their potential
assays, particularly in sensitivity, selectivity, and practicality.
applications in biomedical dignosis and environmental moni-
Among these assays, colorimetric biosensing has attracted
toring (Scheme 1).
much attention because of its low cost, simplicity, and practi-
cality. Since color changes can be read out by the naked eye,
colorimetric biosensing does not require expensive or sophisti-
cated instrumentation and can be applied to field analysis and
point-of-care diagnosis.[1–7,11]
The key challenge for colorimetric biosensing is trans-
forming the detection events into color changes. To this end,
a number of smart materials have been developed, including
gold nanoparticles, magnetic nanoparticles, cerium oxide nano-
particles, carbon nanotubes, graphene oxide, and conjugated
Y. Song, W. Wei, Prof. X. Qu

Division of Biological Inorganic Chemistry
State Key Laboratory of Rare Earth Resource Utilization
Laboratory of Chemical Biology
Changchun, Jilin 130022, China
E-mail: xqu@ciac.jl.cn
Y. Song
Graduate School of the Chinese Academy of Sciences
Changchun Institute of Applied Chemistry
Chinese Academy of Sciences
Changchun, Jilin 130022, China
Scheme 1. Representation of the colorimetric biosensing used for dis-
DOI: 10.1002/adma.201101853 ease diagnosis and environmental monitoring.
Adv. Mater. 2011, 23, 4215–4236 © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com 4215
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2. Gold Nanoparticles (AuNPs)

Yujun Song received his
Small AuNPs (e.g., 13 nm in diameter) appear red in color B.S. from the College of
because the nanoparticles have an optical absorption around Chemistry and Molecular
520 nm due to surface plasmon resonance.[2,4,18] For small Sciences at Wuhan University
AuNPs, surface electrons are made to oscillate by the incoming in 2005. He obtained his
light in a dipole mode. As the size of the AuNP increases, light Ph.D. under the supervi-
can no longer polarize the nanoparticles homogeneously, and sion of Prof. Xiaogang Qu
higher order modes at lower energy dominate. This causes a from the Chinese Academy
red-shift and broadening of the surface plasmon band.[18] There- of Sciences in 2011. He is
fore, small AuNPs aggregates will make their surface plasmons currently working as a post-
combine and result in the color change from red to purple. doctoral researcher at Cornell
The color change of AuNPs provides an elegant platform for Medical College (USA). His
absorption-based colorimetric detection with AuNPs as signal research interests include biofunctional materials and
reporters.[1,3,4] On the one hand, the interparticle plasmon coup- biosensing.
ling leads to a huge absorption band shift (up to ≈300 nm).[4,18]
Therefore, the color change can be observed by the naked eye Weili Wei received his B.S.
and can be used for qualitative analysis without sophisticated from Chongqing University
instruments. On the other hand, due to the extremely high in 2004 and his Ph.D. from
extinction coefficient, AuNP-based colorimetric assays have the Research Center of
high sensitivity, which is comparable to bioassays based on Eco-Environmental Sciences,
fluorescence.[4] The color change or aggregation process can Chinese Academy of
be quantitatively analyzed by detecting the absorption spectra Sciences (CAS). In the fall
using a standard spectrophotometer. Generally, the absorbance of 2010 he joined the group
at 520 nm corresponds to dispersed particles while a longer of Prof. Xiaogang Qu at
wavelength (e.g., 600 nm) relates to aggregated particles.[4] the Changchun Institute of
AuNP aggregation in these assays can be induced by an inter- Applied Chemistry, CAS as a
particle crosslinking mechanism in which the enthalpic ben- research assistant professor.
efits of interparticle bonding formation overcome interparticle His research work includes fabrication of chiral sensors
repulsive forces. Alternatively, AuNP aggregation can be guided using smart materials.
by the controlled loss of colloidal stability in a non-crosslinking-
aggregation mechanism. Such mechanisms are also extended Xiaogang Qu received his
to other noble metal nanomaterials, such as silver nanoparti- Ph.D. from the Chinese
cles and gold nanorods.[19–21] Academy of Sciences
(CAS) in 1995 with the
2.1. Crosslinking Aggregation CAS President’s Award. He
worked with Professor J. B.
Chaires at the Mississippi
2.1.1. Assembly of Gold Nanoparticles
Medical Center and
Interparticle crosslinking aggregation is the most common Professor A. H. Zewail
approach in AuNP-based colorimetric biosensing. DNA can be at California Institute of
covalently attached to AuNPs via thiol-gold chemistry, and DNA- Technology. Since late 2002,
functionalized AuNPs were used for highly sensitive and selec- he has been a professor at
tive crosslink colorimetric detection.[4] The pioneering work for the Changchun Institute
using DNA as a platform to detect a target analyte was devel- of Applied Chemistry, CAS. From December 2006 to May
oped by Mirkin and co-workers (Figure 1a).[1,22,23] In presence 2007, he visited Professor A. J. Heeger’s group at the
of target DNA molecules, AuNP aggregation will be induced University of California Santa Barbara. His current research
by hybridizing two complementary DNA strands on AuNPs. is focused on ligand-nucleic acids or related protein inter-
Therefore, a red-to-purple color change is observed in the col- actions, and biofunctional materials for advanced medical
loid solution. Upon increasing the temperature, the hybridized technology.
DNA duplex will be denaturated above the melting tempera-
ture (Tm), which causes the dissociation of aggregates into
dispersed AuNPs. Moreover, the melting profile suggests the
melting transition is extremely sharp, which might enhance the by measuring absorbance (or looking at the color) as a func-
selectivity of perfectly matched target DNA strands over those tion of temperature. This technique offered several advantages
with mismatches.[22,23] Further exploration of the potential over other techniques such as arrays probed by fluorescence
of these materials in DNA detection showed that by virtue of in that 1) it exhibited a high degree of discrimination between
these sharp melting transitions target DNA could be differ- perfectly matched target oligonucleotides and targets with
entiated from DNA with single base-pair mismatches simply single base-pair mismatches, 2) it was quick and easy, and
4216 wileyonlinelibrary.com © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Mater. 2011, 23, 4215–4236
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REVIEW
can be easily adapted to high-throughput screening. The assay
has a number of advantages over conventional systems that
monitor molecule–DNA interactions directly or with molecular
fluorophore probes. The sharp melting transition associated
with network materials made from DNA-interlinked gold nano-
particles, their strong absorbance at 520 nm, and the large per-
turbations in the Tm values upon analyte binding allow signifi-
cantly better discrimination between weak, intermediate, and
strong DNA-binding molecules.
By using aptamers as the molecular recognition element,
AuNPs can also be assembled for sensitive colorimetric detec-
tion of a wide range of analytes. Chang and co-workers func-
tionalized platelet derived growth factor (PDGF) aptamers
onto AuNPs to detect of target proteins (Figure 1d).[35] As each
PDGF can bind two aptamers in the presence of PDGF, the
crosslinking of AuNPs was formed and the solution turned
to a purple color. They also found that too much PDGF could
Figure 1. Schematic representation of the interparticle crosslinking aggre- inhibit the color change. At high concentration, AuNPs were
gation and color change of AuNPs induced by a) target DNA, b) Hg2+, completely covered by PDGF and no aptamers were available
c) coralyne, and d) PDGF. for crosslinking. Furthermore, a competitive binding assay for
the PDGF receptor was developed as well.
In addition to the AuNP-DNA system, other biological
3) its optical read-out did not require expensive sophisticated recognition events (e.g., antibody–antigen interactions,[36]
instrumentation.[1] streptavidin–biotin interactions,[37] and lectin–sugar interac-
A coordinate bond between DNA bases is another way to tions[38]) and some chemical reactions[39] can also be used in the
form duplex crosslinkers. Mirkin and co-workers reported a systems. Furthermore, biologically relevant organic molecules
colorimetric sensor for Hg2+ detection based on a DNA–AuNPs (such as peptides) that have multiple gold surface binding tags
system (Figure 1b).[24] It is well known that Hg2+ can coordinate (such as thiol) can also directly crosslink AuNPs by chemical
selectively to the bases that consist of thymidine–thymidine interactions (e.g., Au–S).[4,40]
(T–T) mismatches.[25,26] Therefore Hg2+ could selectively bind
to the T–T sites and raise the Tm of the mismatched structures.
2.1.2. Disassembly of Gold Nanoparticles
As the temperature was raised just above the melting tempera-
ture of the AuNPs, the solution containing Hg2+ maintained a Instead of using target analytes to form or stabilize AuNP
purple color while all other aggregates turned red. By using this aggregates, the reverse process that uses target analytes to break
method, a detection limit of 100 nM was reached.[24] However, the crosslinkers to disassemble AuNPs was also proven to be a
during the detection process, this detection system requires general strategy for colorimetric sensing.[4] To break the DNA
careful heating and monitoring of thermal denaturation tem- crosslinkers, the direct and effective way is to use the endonu-
perature. To overcome this drawback, Liu and co-workers have clease to hydrolyze the DNA-duplex interconnects. Mirkin and
made significant improvements in the design, allowing one- co-workers first reported an enzyme-responsive nanoparticle
step, room-temperature colorimetric detection of Hg2+.[27] system that uses a DNA-AuNP assembly as the substrate to
Small ligands can recognize and regulate unique nucleic evaluate enzymatic activity and to screen enzyme inhibitors.[41]
acid structures, which can also be used for formation of DNA In this method, the dispersion of the aggregates of AuNPs inter-
crosslinkers.[28] Meanwhile, the AuNP-DNA conjugate offers an connected by a DNA duplex leads to significant color changes
ideal system for screening nucleic acid drug candidates. AuNP when the endonuclease degrades the DNA duplex. Although
probes are ideal for this purpose due to their intense optical highly selective and more sensitive than conventional methods,
properties, enhanced binding properties, and sharp melting this visual inspection assay is limited by the need to prepare
transitions.[1] Mirkin and co-workers have developed colori- probes by functionalizing two separate batches of AuNPs with
metric assays for screening the duplex or triplex binders by two different thiol-modified oligonucleotide strands. To over-
monitoring the color of nanoparticle network materials exposed come this limitation, Ren and co-workers take advantage of the
to the DNA binders with increasing temperature.[29,30] Ren palindromic recognition sequence of the restriction nucleases
and co-workers used homoadenine DNA as a model system to and the unique optical properties of AuNPs to monitor restric-
develop a potentially high-throughput and simple AuNP-based tion endonucleases in real time (Figure 2a).[42] It is well known
assay for screening small molecules that trigger the formation that the majority of restriction endonucleases are type II restric-
of non-Watson–Crick homoadenine duplexes (Figure 1c).[31] It tion enzymes.[43] The recognition sites of type II endonucleases
has been reported that coralyne can strongly bind to single- are typically short, palindromic sequences of double-stranded
stranded homoadenine-containing nucleic acids.[32–34] There- DNA. This detection system is composed of two elements: a
fore, homoadenine-DNA-modified AuNPs can be used for single type of DNA-functional AuNP probe and an appropriate
detection of coralyne. As the method involves only one type of oligonucleotide linker that can hybridize with the probe DNA.
ssDNA modified AuNP, the assay is simplified in format and The linker was designed to contain a self-complementary region,
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Figure 2. Schematic illustration of the colorimetric assay for a) endonuclease and methyltransferase activity and inhibition, b) lead, c) adenosine, and
d) cysteine. Panel (a) reproduced with permission.[42] Copyright 2009, American Chemical Society. Panel (b) reproduced with permission.[44] Copy-
right 2003, American Chemical Society. Panel (c) reproduced with permission.[48] Panel (d) reproduced with permission.[50] Copyright 2008, American
Chemical Society.
which can form a duplex structure with a base-pair overlap aggregation at a constant temperature was observed.[46] Mean-
containing the recognition sites and overhanging 3´-ends. The while, using 42 nm AuNPs instead of 13 nm AuNPs, a distinct
overhanging portion was complementary to the oligonucle- color change could be as fast as 5 min.
otide probe, and a DNA-AuNPs assembly formed based on the In addition to cleavage of nucleic acid, target-induced DNA
hybridization of oligonucleotide linkers with the probe DNA structure switching can also be used to disassemble the com-
immobilized on surface of the AuNPs. The designed AuNPs plex. Niemeyer and co-workers employed DNA base-pairing
assembly could be used as the substrate for the endonuclease interactions to disassemble nanoparticle aggregates.[47] The
detection, as disassembly of AuNPs can be achieved by incu- results clearly demonstrate the feasibility of using fueling oli-
bation with an endonuclease that cleaves double-stranded (ds) gomers for the reversible switching of nanoparticle aggregation.
DNA in a site-specific manner. This new design could also Lu and co-workers introduced the structure-switching aptamer
be applied to the assay of methyltransferase activity since the into the system to disassemble gold nanoparticles and designed
methylation of DNA inhibits its cleavage by the corresponding the new biosensors for colorimetric sensing of adenosine and
restriction endonuclease, and thus, this new methodology can cocaine (Figure 2c).[48] In the presence of adenosine or cocaine,
be easily adapted to high-throughput screening of methyltrans- the aptamer switched its structure to bind the target and only
ferase inhibitors.[42] a few bases were left to hybridize, making the hybridization
Another approach is to introduce the DNAzyme cleavage reac- unstable at room temperature. As a result, the aggregates were
tion into the nanoparticle system. Lu and co-workers reported a dissociated and a color change from purple to red was observed.
colorimetric lead sensor based on the disassembly of gold nano- Unlike the relatively slow AuNPs assembly process, disas-
particles by a Pb2+-dependent DNAzyme (Figure 2b).[3,44] In their sembly can be finished in seconds. Similarly, sensors respon-
sensor design, they used a Pb2+-specific DNAzyme composed of sive to potassium ions have also been obtained.[49]
a catalytic and a substrate strand. With the addition of Pb2+, the Another example is to remove the coordinate bond between
substrate strand cleaves into two pieces, resulting in head-to-tail the DNA crosslinkers to reduce their stability. Mirkin and co-
or tail-to-tail aggregation with a concomitant red to blue color workers report the development of a highly sensitive and selec-
shift. The detection limit of the sensor was ≈100 nM, which was tive colorimetric detection method for cysteine based upon
able to detect lead extracted from paint. However, in the above oligonucleotide-functionalized gold nanoparticle probes that
system, the head-to-tail alignment of AuNP suffered from high contain strategically placed T–T mismatches complexed with
steric effects. Therefore, an annealing step was needed to form Hg2+ (Figure 2d).[50] The cysteine analyte can bind Hg2+ and
AuNP aggregates.[45,46] By changing the alignment to tail-to-tail, remove it from the network structure thereby lowering the
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temperature at which the DNA duplexes dissociate and the cor- an appropriate salt concentration (e.g., 200 mM NaCl), citrate-
responding purple-to-red color change takes place. By using this capped AuNPs are stabilized in the presence of ssDNA, but
assay, concentrations as low as 100 nM cysteine can be detected aggregate in the presence of dsDNA. This approach provides
in a colorimetric format.[50] a means to detect the presence of target DNA or monitor DNA
hybridization.
Besides salt, conjugated polyelectrolyte has also been
2.2. Non-Crosslinking Aggregation found to lead to the ready aggregation of such nanoparticles
(Figure 3b). Recently, Xia et al. demonstrated a novel sensing
2.2.1. Label-Free Non-Crosslinking Detection strategy employing single-stranded probe DNA, unmodified
AuNPs, and a positively charged, water-soluble conjugated poly-
In the above colorimetric sensors, the color change was based
electrolyte to detect a broad range of targets including nucleic
on assembly or disassembly the crosslinking of AuNPs. Intrigu-
acid (DNA) sequences, proteins, small molecules, and inorganic
ingly, it has been shown that non-crosslinking-based AuNPs
ions.[52] This biosensor approach is based on the observation
aggregation can also be used for developing colorimetric sen-
that, while the conjugated polyelectrolyte specifically inhibits
sors.[3,4] In preparation of AuNPs, the surface-tethered capping
the ability of ssDNA to prevent the aggregation of AuNPs, no
ligands or species are often used to control AuNPs growth and
such inhibition is observed with double-stranded or otherwise
to stabilize AuNPs against van der Waals attraction-induced
“folded” DNA structures. Colorimetric assays employing this
aggregation. With respect to electrostatic stabilization, the sur-
mechanism for the detection of hybridization are sensitive and
face charges, together with the counter ions in the medium,
convenient picomolar concentrations of target DNA are readily
form a repulsive electric double layer that stabilizes colloids
detected with the naked eye and the sensor works even when
against van der Waals attractive forces.[4] A characteristic feature
challenged with complex sample matrices such as blood serum.
of electrostatic repulsion is its high sensitivity to the bulk ionic
Likewise, by employing the binding-induced folding or associa-
strength; the force of electrostatic repulsion diminishes sig-
tion of aptamers, they have generalized the approach to the spe-
nificantly at high salt concentrations, when the electric double
cific and convenient detection of proteins, small molecules, and
layer is highly suppressed. Therefore, citrate-capped AuNPs are
inorganic ions.
stabilized in water but undergo aggregation when salt concen-
Based on the salt-induced non-crosslinking AuNPs aggre-
trations are increased.[4] Based on this principle, citrate-capped
gation, Ren and co-workers have demonstrated a pH sensing
AuNPs have been used for biodetection. For example, Li and
assay that using i-motif DNA structural conformational transi-
Rothberg found that single-stranded (ss) DNA can bind to
tion when the pH changes (Figure 4a).[53] I-motif DNA forms a
citrate-capped AuNPs through DNA base–gold interactions and
four-stranded DNA structure with stretches of cytosine bases.
stabilize AuNPs electrostatically (Figure 3a).[51] However, dsDNA
At low pH, the C residues are partially protonated and the DNA
is not as robust as ssDNA and shows little binding affinity to
folds into the closed i-motif structure; when the pH is increased
citrate-capped AuNPs. Therefore, in the presence of the com-
to basic, the C+ residues are deprotonated and it unfolds to a
plementary DNA target, the formed duplex structure provides
single-stranded form.[54,55] Therefore, the i-motif undergoes a
little stabilization, because, once hybridized, the DNA bases are
precise structural change driven by a pH change. This method
not free to bind to the AuNP surface. Under this condition, at
took advantage of the observed non-crosslinking AuNP aggre-
gation phenomenon and the conformational switch of DNA to
fabricate a sensitive colorimetric pH sensor with high sensi-
tivity and accuracy.
A new concept for achieving a potential high-throughput
and unmodified AuNP-based assay for the fast and colorimetric
screening of triplex specific binders/inducers (Figure 4b) was
presented by Ren and co-workers.[56] The assay consists of a
dsDNA and ssDNA, which has the proper sequence to form a
triplex with the dsDNA. In the absence of ligand, the ssDNA
adsorbed onto AuNP and prevented the individual red AuNPs
from forming blue aggregations under high-ionic strength
conditions due to the low stability of the triplex structure.
However, introducing a triplex binder/inducer, stabilizes tri-
plex formation through Hoogsteen-type hydrogen bonds.
Upon addition of AuNPs, the triplex would not be able to bind
and stabilize individual red AuNPs, resulting in purple or
blue AuNP aggregations. This concept can also be extended
to the detection of Hg2+, K+, Pb2+, thrombin, cocaine, and ATP
by taking advantage of the different effects of DNA conforma-
tion change with and without target analytes on citrate-capped
Figure 3. Schematic representation of different aggregation behavior AuNPs.[57–62]
of AuNPs-DNA upon addition of a) salt or b) positively conjugated A similar strategy can also be applied to screen of Alzheimer’s
polymer. disease β-amyloid (Aβ) inhibitors. Our group reported the
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Figure 4. a) pH sensor based on conformational switch of i-motif DNA and non-crosslinking AuNP aggregation. b) Structure and color change of the
AuNP-based approach for screening triplex DNA binders. c) AuNP-based strategy for screening Aβ aggregation inhibitors. d) Colorimetric detection
of glucose in rat brain based on glucose-induced color change of gold nanoparticles. Panel (a) reproduced with permission.[53] Copyright 2008, Royal
Society of Chemistry. Panel (b) reproduced with permission.[56] Copyright 2010, Elsevier. Panel (c) reproduced with permission.[63] Copyright 2010, Royal
Society of Chemistry. Panel (d) reproduced with permission.[66]
first example that using Aβ-AuNPs to screen small molecules bind to the Aβ peptide and inhibit the transition into a β-sheet
that can inhibit Aβ aggregation and designed a simple and secondary structure and amyloid formation. Consequently, the
potentially high-throughput AuNP-based assay for screening of peptide cannot induce AuNPs aggregation.
Aβ inhibitors (Figure 4c).[63] At pH 5.0, Aβ (12–28) has been Once the DNA was cleaved into small pieces, the small frag-
reported to transform from mainly random-coiled monomers ments could not prevent the salt-induced aggregation. Recently,
to β-sheet oligomers,[64] and the peptide (isoelectric point Mao and co-workers have successfully demonstrated the appli-
pI ≈ 6) is positive-charged. The AuNPs are negatively charged cation of an AuNP-based colorimetric assay for the simple and
due to the surface-adsorbed citrate anions. The neutralization effective detection of glucose in the rat brain (Figure 4d).[66]
of the surface charge will lower the ξ potential of the AuNPs In the sensor design, they took advantage of the cascade reac-
and cause aggregation. It is very likely that although their struc- tions of glucose oxidase catalyzed oxidation of glucose and the
tures are stabilized by noncovalent interactions the oligomers Fenton reaction of H2O2 to generate ·OH radicals to oxidatively
can be assumed to act as polycations and can cause the adja- cleave the ssDNA. The formed DNA fragments could not effec-
cent AuNPs to undergo aggregation.[65] While in the presence tively stabilize AuNPs at certain salt concentration and resulted
of Aβ aggregation inhibitors, the inhibitor molecules would in a change in the agglomeration state of AuNPs from a
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REVIEW
well-dispersed state to an aggregated one, which essentially originates from the different aggregation mechanisms. On the
forms the basis for the colorimetric detection of glucose. one hand, in this non-crosslinking system, the aggregation is
driven by the London–van der Waals attractive force between the
2.2.2. AuNP-Modified Non-Crosslinking Detection nanoparticles, when the repulsive interaction is greatly reduced
by formation of duplexes on their surfaces. The attractive force
DNA covalently modified AuNPs provide another platform works from a distance and leads to the rapid aggregation. On
for an effectively non-crosslinking colorimetric assay. In the the other hand, in the crosslinking system, the kinetics of the
non-crosslinking aggregation system, van der Waals attrac- aggregation are dominated by random collisions between the
tion dominates aggregation once interparticle repulsive forces nanoparticles with relatively slow Brownian motion.[67]
are insufficient to stabilize AuNPs. The loss of colloidal stabi- Li and co-workers also functionalized AuNPs with short
lization, often modulated by salt, can be realized by removing thiol-modified DNA and investigated DNA aptamer folding
surface charges and surface-grafted (charged) polymers and by upon binding to a non-nucleic acid target molecule (adenosine
target-induced (charged) polymer conformational transitions.[4] or K+) on the AuNP surface and its effect on AuNP colloidal
Maeda and co-workers reported that AuNPs covalently modified stability (Figure 5b).[69] Intriguingly, they observed a unique col-
with ssDNA are more stable against salt-induced aggregation loidal stabilization effect that AuNPs attached to folded aptamer
than dsDNA-tethered AuNPs formed by the hybridization of the structures are more stable against salt-induced aggregation
complementary target DNA strand with DNA probes on AuNPs than those tethered to unfolded aptamers. Moreover, distinct
(Figure 5a).[67] The aggregation process of this non-crosslinking AuNP aggregation and redispersion stages can be reversibly
system is much more rapid than that of the crosslinking sys- controlled by manipulating aptamer folding and unfolding
tems, which takes several tens of minutes to hours at room states with adenosine and adenosine deaminase. This finding
temperature.[67,68] The authors suppose that this difference is different from the system that the formation of rigid DNA
Figure 5. a) Aggregation behaviors of the AuNPs-DNA in the presence of the complementary target or a target containing a single base mismatch
in NaCl solution. b) Different stability of AuNPs with folded and unfolded adenosine binding DNA aptamer. c) Colorimetric assay for screening of
quadruplex binders and evaluating quadruplex–duplex selectivity. Panel (a) reproduced with permission.[67] Copyright 2003, American Chemical Society.
Panel (b) reproduced with permission.[69] Copyright 2008, American Chemical Society. Panel (c) reproduced with permission.[71]
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reduce their toxicity and to produce a color change.[14,73] Peroxi-

REVIEW
duplexes on AuNP surface resulted in colloidal destabiliza-

tion. The conformation that aptamers adopt on AuNP surfaces dase substrates, such as 3,3´,5,5´-tetramethylbenzidine (TMB),
appears to be a key factor that determines the relative stabilities di-azo-aminobenzene (DAB) and o-phenylenediamine (OPD),
of different AuNPs.[69] They performed dynamic light scattering can be used to evaluate the catalytic activity of peroxidase.[14]
experiments that revealed the height of the folded aptamer In the presence of peroxidase, these substrates undergo dis-
layer on the AuNP surface was larger than that of the unfolded tinguishable color change when enzymatically oxidized with
(but largely collapsed in salt solution) aptamer layer on the sur- hydrogen peroxide (H2O2). The catalytic color reaction provides
face. From both the perspective of electrostatic and steric stabi- another means for colorimetric biosensing.
lization, folded aptamers are more extended from the surface For a long time, natural horseradish peroxidase (HRP) has
and therefore have a higher stabilization effect on AuNPs than been used for these purpose.[77–80] However, the natural peroxi-
unfolded aptamers. dase enzyme bears some serious disadvantages: 1) it is unstable
Additionally, Li and co-workers have demonstrated the use and easily denatured by environmental changes or digested
of enzymatic cleavage of DNA on well-dispersed AuNPs for by proteases and 2) its preparation and purification is usually
designing simple colorimetric biosensors.[70] The method relies time-consuming and expensive.[16] To overcome these draw-
on reducing AuNP stability by the removal of colloid stabi- backs, much effort has been devoted to developing peroxidase
lizers through enzymatic cleavage at a pretuned salt concentra- mimetics. For instance, peroxidase mimetics including haemin,
tion. The assays are performed at salt concentrations such that haematin, porphyrin, haemaglobin, and cyclodextrin have been
DNA-modified AuNPs are barely stabilized by the electrostatic studied and applied in biosensing and water treatment.[81–90]
and steric stabilization. Enzymatic cleavage of DNA chains on Magnetic nanoparticles (MNPs) are of great interest for
the AuNP surface destabilizes the AuNPs, resulting in a rapid researchers due to their wide application in magnetic resonance
aggregation driven by van der Waals attraction and a red-to- imaging, drug delivery, biological separation, and biological
purple color change. Two different systems are chosen, DNase catalysis.[91–95] For increasing their functionality, MNPs are often
I and a Pb2+-dependent RNA-cleaving DNAzyme, to demon- coated metal catalysts or conjugated with enzymes, antibodies,
strate the utility of the assay for the detection of metal ions or nucleic acids as they are usually thought to be chemically
and sensing enzyme activities. This approach did not require and biologically inert.[93,95–97] For example, biological catalytic
the extra steps to prepare AuNP aggregates and was conducted and magnetic separation immunoassay systems have been fab-
on well-dispersed AuNPs, therefore the accessibility of DNA by ricated using HRP-entrapped magnetite-containing spherical
enzymes appeared to be better in comparison to AuNP aggre- silica nanoparticles.[97] Intriguingly, Yan and co-workers have
gates where the DNA strands are embedded inside.[70] made a surprising discovery that Fe3O4 MNPs in fact possess
Based on the similar strategies, Ren and co-workers demon- an intrinsic enzyme mimetic activity similar to HRP.[14] They
strated a new concept to simultaneously evaluate G-quadruplex carefully investigated the catalytic activity of the Fe3O4 MNPs,
stabilization and selectivity by small ligands, using enzymatic which like HRP, shows dependence on pH, temperature, and
manipulation of DNA modified AuNPs (Figure 5c).[71] The assay H2O2 concentration. The optimal pH and temperature for the
uses a quadruplex-forming and a non-quadruplex-forming peroxidase-like activity of 300 nm Fe3O4 MNPs were approxi-
oligomer as substrates. Exonuclease I, which can hydrolyze mately pH 3.5 and 40 °C, which are very similar to the values
nucleotides from the 3´ end of single-stranded but not double- for HRP. However, Fe3O4 MNPs required a H2O2 concentration
stranded DNA,[72] was used to interrogate the effect of ligands. two orders of magnitude higher than HRP to reach the maximal
Before enzymatic cleavage, DNA-modified AuNPs are stable at level of peroxidase activity. Furthermore, further increase in the
certain salt concentrations because of the electrostatic and steric H2O2 concentration inhibited the peroxidase-like activity of the
stabilization provided by tethered negatively charged DNA poly- Fe3O4 MNPs, as is observed for the enzyme catalyzed reaction.
mers. However, for a random sequence, the removal of sur- The catalytic properties of MNPs are dependent on size and
face, colloid-stabilized DNA chains by enzymatic cleavage will structure. Different sizes (300, 150, and 30 nm) of Fe3O4 MNPs
destabilize the AuNPs, resulting in a rapid aggregation, driven were studied and the nanoparticles with the smallest sizes show
by van der Waals attraction, and a red-to-purple color change. the highest catalytic activity (Figure 6a–c).[14] This phenomenon
On the other hand, formation of G-quadruplex would inhibit may be due to the smallest nanoparticles having the greatest
the enzymatic hydrolysis and G-quadruplex stabilization would surface-to-volume ratio to interact with substrates. In addition,
enhance the inhibition. Therefore, by comparing independent Zhu and co-workers investigated the catalytic activity of three
degradation rates of the two types of AuNPs, the assay can distinct structures of Fe3O4 nanocrystals, cluster spheres, octa-
determine the relative binding affinity of quadruplex-binding hedra, and triangular plates (Figure 6d–f).[98] The results showed
molecules and discriminate the inhibitory effect from different that the peroxidase-like activities of the Fe3O4 nanocrystals were
sources with the naked eye. structure-dependent and followed the order cluster spheres >
triangular plates > octahedra; this order was closely related to
their preferential exposure of catalytically active iron atoms or
crystal planes.
3. Iron Oxide Nanoparticles
Based on these findings, Yan and co-workers developed two
Peroxidase activity has a great potential for practical application immunoassays using the intrinsic dual functionality of the
and has been used in the bioremediation of waste water or as Fe3O4 MNPs as a peroxidase and magnetic separator.[14] They
diagnostic kits.[14,73–76] These wide applications are attributed used dextran to modify the Fe3O4 MNPs before immobilizing
to the ability to catalyze the oxidation of organic substrates to the antibody or protein of interest on the modified surface of
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REVIEW
colored product, which provides a colorimetric detection of
H2O2. They combined the catalytic reaction of glucose with glu-
cose oxidase (GOx) and the Fe3O4 MNPs catalytic reaction, and
a colorimetric method for glucose detection was developed in
this work. The developed method exhibited sensitive and selec-
tive response toward glucose detection.
Recently, Chen and co-workers established a simple and
rapid colorimetric method for the determination of melamine
in dairy products.[100] In the sensor design, they firstly mix mela-
mine and H2O2 to generate an addition compound, which is
stable at 100 °C according to the thermal analysis results.[101–104]
Based on this reaction and the system described above (ABTS-
H2O2), they demonstrated a new method for the determination
of trace amounts of melamine in dairy products by measuring
the wastage of H2O2.[100] Therefore, the existence of melamine
can cause a color change of the reaction system, and the color
change can be visually observed. This method could detect con-
centrations as low as 0.25 ppm (2.0 μM) melamine in raw milk
and milk powder.
Very recently, Park and co-workers demonstrated a new
method to detect of nucleic acids based on target-induced
Figure 6. Size- and structure-dependent peroxidase-like activity of Fe3O4
nanocrystals. Small nanoparticles show high catalytic activity; for different
shielding against the peroxidase-mimicking activity of magnetic
structures, the activity follows the order: cluster spheres > triangular nanoparticles (Figure 7b).[105] In the experiment, the sample
plates > octahedra. Panels (a–c) reproduced with permission.[14] Copy- being analyzed is first subjected to polymerase chain reaction
right 2007, Nature Publishing Group. Panels (d–f) reproduced from.[98] (PCR) amplification and then mixed with unmodified MNPs.
Finally, the colorimetric substrate and H2O2 were added to the
solution to give a color reaction. They found the PCR prod-
the Fe3O4 MNPs. In the first immunoassay, Fe3O4 MNPs conju- ucts can shield the peroxidase activity of MNPs and reduce the
gated with protein A was used in place of an enzyme-conjugated rate of the peroxidase-catalyzed reaction. On the one hand, the
secondary antibody (Figure 7a). Similar to conventional ELISA, amplified target DNA causes a decrease in the accessibility of
a plate coated with hepatitis B virus surface antigen (preS1) was the positively charged substrate o-phenylenediamine (OPD) to
first incubated with anti-HBV preS1 antibody. After thoroughly the MNPs through its electrostatic interactions with the nega-
wash, the Fe3O4 MNPs modified with protein A and the sub- tively charged phosphate backbone of DNA. On the other hand,
strate TMBmolecules were added. Finally, protein A bound to DNA molecules can directly adsorb on the surface of MNPs,
the primary anti-preS1 antibody and Fe3O4 MNPs catalyzed a leading to significant inhibition of the substrate binding to the
color reaction in the presence of H2O2. In the second immu- MNPs. However, in the absence of target DNA, there were no
noassay, the magnetism and intrinsic peroxidase activity of amplified nucleic acids formed and MNPs exhibit their normal
Fe3O4 MNPs were combined in a novel capture–detection peroxidase activity. As a result, assay samples without target
immunoassay. Firstly, an antibody to cardiac troponin I (TnI), DNA display an intense colorimetric response while those con-
a well-known biomarker for myocardial infarction, was immo- taining target DNA show a significantly reduced colorimetric
bilized on the Fe3O4 MNPs. The antibody-labeled Fe3O4 MNPs signal. This difference can be easily detected with the naked
were then mixed with serum, allowing capture of the target TnI eye.
in the sample. The TnI captured by Fe3O4 MNPs was easily sep-
arated from the sample using a magnet. After several washes,
the MNPs with target bound were transferred onto a plate
4. Cerium Oxide Nanoparticles
coated with another anti-TnI antibody. After washing off non-
bound MNPs, in the presence of TMB and H2O2, the bound Cerium oxide has been extensively used in electrolytes for solid
Fe3O4 MNPs catalyzed a color reaction, which was measured oxide fuel cells (SOFC), ultraviolet absorbents, oxygen sensors,
using an ELISA reader at 652 nm. In compared with traditional and automotive catalytic converters.[106–111] It is reported that
magnetic-ELISA, these methods did not require an additional cerium oxide nanoparticles (nanoceria) possess antioxidant
step to introduce a secondary antibody carrying HRP to allow activity at physiological pH values, indicating potential use in
detection. biomedical applications, such as protection against radiation
Besides the use in immunoassays, the application of this damage, oxidative stress, and inflammation.[111–115] The anti-
novel property of Fe3O4 MNPs can be extended to other biosen- oxidant activity of these nanoparticles was attributed to their
sors. Wang and co-workers make use of the novel properties of ability to reversibly switch from Ce3+ to Ce4+.[114] Recently, Perez
Fe3O4 MNPs as peroxidase mimetics to colorimetric detection and co-workers demonstrated that nanoceria has an intrinsic
of glucose.[99] Fe3O4 MNPs were able to catalyze the oxidation of oxidase-like activity at acidic pH values, as it can quickly oxidize
a peroxidase substrate 2,2´azino-bis(3-ethylbenzothiazoline-6- a series of organic substrates even without any oxidizing agent
sulfonic acid) diammonium salt (ABTS) by H2O2 to the oxidized (e.g., H2O2).[15] They found that the observed activity is not only
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REVIEW
of molecules to and from the nanoceria core

surface faster than a thicker coating.[15]
The oxidase-like activity of ceria nanopar-
ticles shows promising application in the
design of more robust and reliable TMB-
based immunoassays. Perez and co-workers
attached folic acid to cerium oxide nanopar-
ticles via click chemistry and performed an
immunoassay to probe cancer cells instead of
traditional ELISA (Figure 8).[15] In traditional
ELISA, an HRP-labeled secondary antibody
is utilized to assess the binding of a spe-
cific primary antibody to a particular target
or surface receptor.[77–80] This binding event
is assessed by the ability of HRP to oxidize
a chromogenic substrate, such as TMB, in
the presence of hydrogen peroxide. In tradi-
tional ELISA, the high rate of negative results
is mainly attributed to 1) the instability of
the antibodies that, when denatured, do not
bind effectively to their target; 2) the insta-
bility of HRP that, when denatured, loses its
peroxidase activity; and 3) the instability of
hydrogen peroxide, which, upon prolonged
storage, decomposes and losses its ability to
oxidize the substrate TMB in the presence of
HRP. As neither hydrogen peroxide nor anti-
body is required in this assay, this method
is more robust than current HRP-antibody
based assays.
Figure 7. a) Schematic representation of Fe3O4 MNPs-based immunoassay (top) or Fe3O4

MNPs-based capture–detection immunoassay (bottom). b) Strategy for label-free, colorimetric
5. Carbon Nanotubes (CNTs)
detection of target DNA by using Fe3O4 MNPs. Panel (a) reproduced with permission.[14] Copy- Carbon nanotubes have been considered as
right 2007, Nature Publishing Group. Panel (b) reproduced with permission.[105]
the leading candidate for nanodevice appli-
cations because of their 1D electronic band
pH dependent but is also dependent on the size of the cerium structure, biocompatibility, controllable property of conducting
oxide nanoparticles and the thickness of the polymer coating. electrical current, and reversible response to biochemical rea-
In the experiment, they synthesized biocompatible dextran- gents.[9,116–120] CNTs are tubular materials with a high aspect
coated nanoceria (DNC) and investigated their ability for facili- ratio and a diameter in the nanoscale range.[121] According to
tating the oxidation of a series of organic dyes at low pH values. their structure, CNTs can be classified into two main types:
In the peroxidase-catalyzed reactions, H2O2 is required as the 1) single-walled carbon nanotubes (SWNTs), which consist
electron acceptor or oxidizing agent. However, at pH 4.0, addi- of a single layer of graphene sheet seamlessly rolled into a
tion of peroxidase substrates (TMB or ABTS) to the solution cylindrical tube,[122,123] and 2) multiwalled carbon nanotubes
of DNC will give the characteristic color within minutes. This (MWNTs), which consist of multiple layers of concentric cyl-
result suggests DNC can catalyze the oxidation of both TMB inders with the space of about 0.34 nm between the adjacent
and ABTS in the absence of H2O2. Further studies showed that layers.[116] Since the pristine CNTs contain variable amounts of
DNC-catalyzed oxidation is pH-dependent. As the buffered solu- impurities, such as amorphous carbon and metallic nanoparti-
tion increases from pH 4.0 to 7.0, the ability of DNC to oxidize cles, many efforts has been devoted to selectively remove of the
the dye decreases. At pH 7.0, no significant oxidation of TMB impurities.[16,124–126] Additionally, as pristine CNTs are almost
or ABTS was observed, even in the presence of H2O2 or upon insoluble in all solvents, the development of efficient methodol-
overnight incubation. In addition, they studied the effect of the ogies for the chemical modification of CNT has stimulated the
coating thickness and nanoparticle size on the catalytic activity preparation of soluble CNTs that can be employed in several
of nanoceria. Results show that the nanoparticles with a thin biological applications.[121,127–130] Among them, the most com-
poly-(acrylic acid) coating (isPNC) have a higher catalytic activity monly used method is oxidized treatment using strong acids,
than those with a thicker dextran coating (swDNC). This result which leaves an open hole in the tube side and functionalizes
might be attributed to the fact that nanoceria with a thin and the open end of SWNTs with carboxyl group to increase their
permeable poly(acrylic acid) coating can facilitate the transfer solubility in aqueous solution.[119,120,131]
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REVIEW
be attributed to their intrinsic properties rather than metal resi-
dues. We found that the treatment of pristine SWNTs by soni-
cating in mixed-acid can efficiently remove the metal residues.
After 36 h treatment, there was no cobalt signal observed in
SWNTs evident from energy dispersive X-ray (EDX) analysis.
However, this sample still shows a significant activity, which
suggests that SWNTs possess intrinsic peroxidase-like activity.
Therefore, the catalytic effect we observed is not due to cobalt
residues. It can be related to the electronic structures of SWNTs
and their interactions with H2O2 and TMB. Previous studies
have shown that the high reduction potential of H2O2 induces
the spectral changes of SWNTs, thus indicating that H2O2 can
withdraw electrons from the SWNT valance band by electron
transfer. Moreover, a direct interaction between H2O2 and
SWNTs has been reported and could be responsible for SWNT
catalytic activity.[134,135]
Based on this finding, we designed a label-free colorimetric
detection system for disease-associated single nucleotide poly-
morphism (SNPs) in human DNA (Figure 9a).[16] ssDNA is
flexible and can interact non-covalently with SWNTs. It wraps
around individual SWNTs, forming stable complexes by means
of π-stacking interactions between the nucleotide bases and the
side walls of the tube, as well as hydrophobic and van der Waals
interactions.[16,136] dsDNA has also been proposed to interact
with SWNTs, but its affinity is significantly weaker than that
of ssDNA. The DNA structure of B-form, the normal right-
handed double-helical conformation, dsDNA is more rigid than
that of ssDNA. When SWNTs bind to the dsDNA major groove,
the DNA is destabilized and can even become condensed.[120]
Therefore, at a high salt concentration, ssDNA can strongly
adsorb on the SWNT surface by wrapping, the negatively
charged DNA backbone on the SWNT surface will increase
individual SWNT electrostatic repulsion and resist salt-induced
SWNT aggregation. However, unlike ssDNA, dsDNA cannot
inhibit salt-induced SWNT aggregation. Therefore, SWNTs in
solution with dsDNA are easily precipitated by centrifugation,
whereas SWNTs with ssDNA can inhibit precipitation. The pre-
Figure 8. Comparison of traditional ELISA (a) and nanoceria-based cipitate can be collected and redispersed in phosphate buffer. In
ELISA (b). In traditional ELISA, an HRP antibody is utilized as a sec- the presence of TMB and H2O2, the obtained SWNTs will cata-
ondary antibody that, upon hydrogen peroxide treatment, facilitates the lyze a color reaction that can be judged by the naked eye and
oxidation of TMB, resulting in color development. In nanoceria-based
easily be monitored by the absorbance change at 652 nm. By
ELISA, the oxidase-like activity of nanoceria facilitates the direct oxidation
of TMB without the need of HRP or hydrogen peroxide. Reproduced with using our simple, label-free, colorimetric detection system, the
permission.[15] direct repeatable detection limit for target DNA can be as low
as 1 nM.[16] Furthermore, this label-free colorimetric detection
method can be used to distinguish disease-associated single
Recent studies have shown that SWNTs have catalytic activity nucleotide polymorphism in human DNA.
even in the absence of catalytic factors.[132,133] Our group discov- Building on the peroxidase-like activity of carbon nano-
ered that SWNTs possess intrinsic peroxidase-like activity.[16] In tubes and magnetic nanoparticles, our group developed
the presence of H2O2, SWNTs catalyze the reaction of the per- a turn-on, highly sensitive and selective copper sensor by
oxidase substrate TMB thereby producing a color change. The combining click chemistry and the peroxidase-like catalytic
process can be detected by monitoring the absorbance change reaction together (Figure 9b).[137] As copper poses serious
at 652 nm. Furthermore, SWNT catalytic activity is dependent environmental problems and is potentially toxic for all
on pH, temperature, and H2O2 concentration. Under the exper- living organisms, highly sensitive and selective detection of
imental conditions, the optimal pH, temperature, and H2O2 copper (Cu2+ or Cu+) has received much attention in recent
concentration are 4.0, 40 °C, and 125 mM, respectively; these years.[138–140] In our experiment, magnetic silica nanopar-
are very similar to the values for the natural enzyme HRP. More ticles (MSNs) are clicked on MWNTs, and the hybrid nano-
importantly, we compared the catalytic efficiency of SWNTs material intrinsic peroxidase-like activity and its catalytic
containing different amounts of cobalt residues. The results enzyme-substrate color reaction are used for copper detection.
clearly show that the observed “catalytic” effect of SWNTs can By using click chemistry, MSNs and MWNTs are assembled to
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REVIEW
Figure 9. a) Protocol for SNP detection of complementary (top) and mismatched duplex DNA (bottom); b) Schematic representation of a combination
of click chemistry and peroxidase-like catalytic color reaction for turn-on selective sensing of copper ions. Panel (a) reproduced with permission.[16]
Copyright 2010, Wiley-VCH. Panel (b) reproduced with permission.[137] Copyright 2010, Royal Society of Chemistry.
mimic natural enzyme; MSNs and the substrates (TMB) are trace amount of metal catalyst in the sample but instead is
confined on the surface of MWNTs. Due to the high electrical caused by its own intrinsic property.[17] In the experiment, nei-
conductance of MWNTs, electron communications between ther H2O2 nor GO-COOH alone can efficiently oxidize TMB,
MSNs and substrates are electrically ‘‘wired’’.[141] These which indicates that the interactions between GO-COOH and
synergetic effects might lead to the observed high catalytic H2O2 and TMB are important for the catalytic reaction. The
activity. This approach shows high sensitivity and selectivity, different absorption spectra show that a bathochromic shift
and allows simple detection of copper by the naked eye. The (≈19 nm) occurs for GO-COOH after the addition of 88.2 mM
direct detection limit can be as low as 1 mM.[137] Furthermore, H2O2, consistent with a previously suggested mechanism that
glutathione (GSH) can be used to reduce Cu(II) to Cu(I) and electron transfer occurs from the top of the valence band of
form a stable complex with Cu(I), which is the catalyst for graphene to the lowest unoccupied molecular orbital (LUMO)
the Huisgen 1,3-dipolar cycloaddition reaction.[139] Given the of H2O2.[134,149] Additionally, TMB is absorbed on the surface of
natural abundance of glutathione, this approach has potential graphene and donates lone-pair electrons in the amino groups
to detect Cu(I) in biological media. to graphene, which confers an increase in electron density and
mobility in graphene. This charge-transfer n-type doping of
graphene increases the Fermi level and therefore the electro-
chemical potential from the LUMO of H2O2.[149] This acceler-
6. Graphene Oxide (GO)
ates the electron transfer from graphene to H2O2. As a result,
As a novel one-atom-thick planar sheet of sp2-bonded carbon nitrogen enrichment in this way might provide a higher den-
atoms, graphene has received much attention in recent sity of catalytically active centers with low stereohindrance for
years in materials science and biotechnology.[142–146] Produc- binding redox species.
tion of graphene sheets in bulk quantity and its modifica- In comparison with HRP, GO-COOH is low-cost, easy to
tion with functional groups to improve water solubility have obtain, more stable to biodegradation, and less vulnerable to
been well developed.[147,148] All these achievements provide denaturation.[17] Therefore, GO-COOH shows great advantages
new insights into the application of this nanomaterial in over HRP for practical application and can be used as a diag-
medical diagnosis and biosensing. Similar to carbon nano- nostic kit for H2O2 and glucose. For control of diabetes mel-
tubes, carboxyl-modified graphene oxide (GO-COOH) has litus, it is important for minimizing diabetic complications
peroxidase-like activity that can catalyze the reaction of per- to maintain blood glucose concentrations within the normal
oxidase substrate TMB in the presence of H2O2 to produce physiological range.[150] Using GO-COOH to fabricate sensors
a blue color reaction.[17] This finding provides new insights for detection of the products of the glucose oxidase, a colori-
into application of this nanomaterial to medical diagnosis metric method for glucose detection has been developed by
and biosensing. our group (Figure 10a). This method can be used to detect
GO-COOH catalytic activity is dependent on pH, tem- glucose in buffer solution and diluted blood and commercial
perature, and H2O2 concentration. The optimal pH, tempera- fruit juices. Glucose in buffer solution can be detected as low
ture, and H2O2 concentration are pH 4.0, 35 °C, and 150 mM, as 1 × 10−6 mol L−1 with a linear range from 1 × 10−6 to 2 ×
respectively, which are close to the values for natural enzyme 10−5 mol L−1. According to the calibration curve, the concentra-
HRP.[17] Kinetic studies indicate that GO-COOH has even tion of glucose in the normal blood sample is about 4.89 mM.
higher catalytic activity to TMB than the natural enzyme, HRP. The general range of blood glucose concentration in healthy
Like HRP, the catalytic reaction follows a ping-pong mecha- and diabetic persons is about 3–8 mM and 9–40 mM, respec-
nism. We have carefully investigated the mechanism and found tively.[151] Therefore, this colorimetric method is applicable to
that the observed peroxidase-like activity is not related to the real samples to determine glucose concentration.
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REVIEW
corresponding to different concentrations of
PSA can be directly detected with the naked
eye.
Although GO-COOH possesses intrinsic
peroxidase-like activity, no catalytic activity is
observed at pH 7.0, which may limit their use
under physiological conditions.[17] Hemin is a
well-known natural metalloporphyrin and has
been used as a mimetic enzyme for labeling
antigen and antibody reactions. Hemin shows
its optimal catalytic activity at pH 7.0.[17] As a
flattening molecule, hemin can be assembled
onto the surface of graphene through π–π
stacking.[153] We have reported a folic acid
(FA) conjugated graphene–hemin composite
(GFH) for colorimetric detection of cancer
cells based on the color reaction catalyzed
by GFH synergetic peroxidase-like activity
(Figure 10b).[154] This system using graphene
shows several advantages over other mate-
rials (i.e., mesoporous silica or carbon nano-
tubes): 1) the large size of GFH suggests that
this complex not only can be visualized under
an optical microscope, but also cannot easily
internalize into cells, so they can be visual-
ized by a binding agent to surface receptors
or other biomarkers for detection of cancer
cells; 2) the strong π–π stacking interaction
between graphene and hemin, which makes a
large number of hemin molecules well adsorb
on both sides of the graphene sheet and each
active site (hemin) completely exposed to
the substrates to exhibit high activity; and
3) the abundant groups (hydroxyl-, epoxy-, and
carboxyl-groups) on GO serve as versatile
substrates to immobilization of biomolecules,
therefore our system shows many promising
applications.
Because of the specificity and high affinity
of folic acid, GFH can selectively bind to the
Figure 10. a) Colorimetric detection of glucose by using glucose oxidase (GOx) and GO-COOH-
catalyzed reactions. b) Cancer cell detection by using folic acid (FA) conjugated graphene–
surface of human cervical cancer cells (HeLa)
hemin composite. c) Protocol for SNPs detection based on hemin-graphene hybrid nanosheets. and human breast cancer cells (MCF-7) by
Panel (a) reproduced with permission.[17] Panel (b) reproduced with permission.[154] Copyright targeting folate receptors. Due to the large
2011, Royal Society of Chemistry. Panel (c) reproduced with permission.[157] Copyright 2011, size and peroxidase-like activity of GFH,
American Chemical Society. selective GFH binding not only can be visual-
ized under bright field microscopy, but also
The intrinsic peroxidase-like activity has also been used can be quantitatively determined by a colorimetric method. As
for colorimetric immunoassay for the detection of cancer graphene has high affinity for hemin and TMB, the active sites
biomarker prostate specific antigen (PSA).[152] GO can cata- (hemin) and the substrates (TMB) were confined in a nanoscale
lyze the reaction of hydroquinone in the presence of H2O2 region. This mechanism is similar to that of natural enzymes in
to produce a brown color solution. Secondary antibody (Ab2) which the extraordinarily high catalytic efficiency is largely due
functionalized GO (GO-Ab2) was used as label for the immu- to the ability to bring substrates into proximity with their active
noassay, while magnetic bead (MB) was selected to immobilize sites.[155] Meanwhile, because of the high electrical conductance
primary anti-PSA antibody (Ab1). In the presence of PSA, an of graphene, the electron communication between hemin and
immunocomplex, sandwiching the antigen protein, is formed substrates is electrically connected.[141] These synergetic effects
between the GO-Ab2 and MB-Ab1. With the separation of the significantly enhance hemin catalytic activity. This method
immunocomplex using an external magnetic field, different can detect as low as 1000 cells. Since folate receptors are over-
amounts of GO-Ab2 in the solution were mixed with hydroqui- expressed for different types of cancer cells, this method can be
none and H2O2 solution and displaying colors. Different colors general for cancer cell detection.[156]
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REVIEW
Recently, Dong and co-workers prepared hemin-graphene (a)

hybrid nanosheets (H-GNs) by a simple wet-chemical strategy, O
which exhibit high solubility and stability in water.[157] The N
absorption of hemin makes H-GNs exhibit the peroxidase-like N
activity. Due to the charge screening effects, the high salt con-
centration could induce H-GNs to coagulate. Moreover, they
S n
are able to differentiate ss and dsDNA in an optimum salt con-
centration owing to the different affinities of ss and dsDNA
to graphene (Figure 10c). ssDNA can stably be adsorbed by (b)
graphene oxide, due to the π–π stacking interaction between O
the ring structure in the nucleobases and the hexagonal cells N
of the graphene oxide.[158] In contrast, dsDNA cannot stably
adsorb on H-GNs and retain its helical structure because the
ring structure of nucleobases is effectively shielded within the S n
densely negatively charged phosphate backbone of dsDNA. So
at the optimum ion concentration, the H-GNs can distinguish Figure 11. Chemical structures of some cationic chromic polythiophenes
ss and dsDNA. By combining the peroxidase activity and ss and a) polymer 1 and b) polymer 2.
dsDNA differentiating ability of H-GNs, they developed a label-
free colorimetric detection method for DNA sequence spe- polymer. A general behavior of the polyelectrolytes is that any
cificity. H-GNs solution with dsDNA was easily precipitated by anionic polyelectrolytes (e.g., DNA) can form strong electrostatic
adding electrolyte, whereas H-GNs with ssDNA could inhibit complexes with a cationic polyelectrolyte, and the formed neu-
precipitation. In the presence of TMB and H2O2, the H-GNs tral complexes are known to form stable aggregates. In 2002,
supernatant will catalyze a color reaction that can be judged Leclerc and co-workers reported new water-soluble cationic poly-
by the naked eye and easily be monitored by the absorbance thiophene derivatives (polymer 1) (Figure 11), which can easily
changes at 652 nm. Using this method, they can detect as low transduce oligonucleotide hybridization with a specific 20-mer
as 2 nM complementary target DNA. Furthermore, this DNA capture probe into a clear colorimetric output.[163] The cationic
sensor was of sufficient selectivity to easily differentiate single polythiophene derivative is soluble in aqueous solutions with
mismatches, which offered the opportunity to determinate a maximum absorption at 397 nm. This maximum absorption
SNPs. As the concentration of mismatched target increased at short wavelength is related to a random-coil (nonplanar or
from 20 to 200 nM, the color difference could be distinguished non-conjugated) conformation of the polythiophene derivative,
even by the naked eye. as any twisting of the conjugated backbone leads to a decrease
of the effective conjugation length. However, in the solid state,
the maximum absorption wavelength is located at 540 nm that
7. Conjugated Polymers (CPs) is attributed to an aggregated (planar or conjugated) form.[163]
Similar to other water-soluble cationic polyelectrolytes, this
7.1. Polythiophenes Derivatives polythiophene derivative can form strong electrostatic complexes
with negatively charged oligomers and polymers (Figure 12a).
Water-soluble conjugated polymers (CPs) have large, delo- In the experiment, they used four different negatively charged
calized molecular structures and exhibit unique optical oligonucleotides: a capture probe sequence (X1), a perfect com-
characteristics.[10–13,159] Due to ultrafast energy transfer of plementary target (Y1), a two-mismatch complementary target
excitons along the conjugated backbones, sensors based on (Y2), and a one-mismatch complementary target (Y3). In the
conjugated polymers have been shown to be very sensitive to presence of one equivalent of capture oligonucleotide X1, the
minor perturbations due to amplification by a collective system yellow polymeric solution turn to red color (absorption max-
response and therefore offer a remarkable advantage over imum, λmax = 527 nm), which is attributed to the formation of a
small-molecule-based sensors.[10,160,161] This collective response so-called duplex between the polythiophene and the oligonucle-
influences optoelectronic properties, such as Förster resonance otide probe. In the medium, the stoichiometric polyelectrolyte
energy transfer (FRET), electrical conductivity, and fluorescence complexes tend to be insoluble and these red-violet aggregates
efficiency, properties which can be used to report, or “trans- have an absorption spectrum similar to that obtained in the solid
duce”, target analyte presence.[10,162] Some polythiophenes are state. With the addition of the complementary oligonucleotide
known to exhibit interesting chromic features (change of color Y1, the solution again becomes yellow (λmax = 421 nm) after
induced by a conformational change of the conjugated back- 5 min, which may be caused by the formation of a new more
bone) in the presence of different stimuli (Figure 11).[11] The soluble complex between the cationic polymer and hybridized
binding of target molecules makes a change of the conforma- oligonucleotides. This new triplex structure was characterized
tion of the conjugated polymer, which results in a modification by circular dichroism measurements, which revealed a right-
of its optical property and can also been developed for colori- handed helical (twisted) orientation of the polythiophene back-
metric biosensing. bone compatible with the binding of the polymer to the nega-
An example is colorimetric sensing of DNA based on water- tively charged phosphate backbone of double-stranded DNA.[163]
soluble conjugated polymers. DNA is an ionic polymer and To verify the specificity of this polymeric optical transducer in
could serve as the template for the attachment of the conjugated the presence of imperfect or incomplete hybridizations, two
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REVIEW
of potassium ions, the solution exhibits dif-
ferent optical properties. The formation of a
quadruplex state of the aptamers stabilized
by K+ allows polythiophene to wrap this
folded structure through electrostatic interac-
tions. Furthermore, when the chloride coun-
terion was replaced by a bromide or an iodide
anion, similar results were observed in the
solution, suggesting the specificity of the
detection toward potassium cations.
A similar method can be used to detect
the human α-thrombin (Figure 12b).[164] It
is well known that oligonucleotide X2 (5´-
GGTTGGTGTGGTTGG-3´) is an aptamer
of human α-thrombin and a conformational
change occurs when the aptamer binds to
the thrombin molecule.[165,166] Therefore,
the specific detection of human α-thrombin
could be realized due to the formation of a
quadruplex structure of the aptamer (X2).
The 1:1:1 complex formed between cationic
polymer, aptamer X2, and thrombin has the
same orange color and UV visible absorp-
tion spectrum as that induced by K+. In the
presence of thrombin, the thrombin aptamer
will form into a quadruplex structure and
bind to the protein, while the cationic poly-
thiophene wraps around this quadruplex
structure. This wrapping seems to partially
hinder the aggregation and planarization of
the positively charged polymer in the pres-
ence of ssDNA X2 (path A). To verify the spe-
cificity of the detection, two control experi-
Figure 12. a) Formation of polythiophene/single-stranded nucleic acid duplex and poly- ments with a nonbinding sequence and BSA
thiophene/hybridized nucleic acid triplex. b) Detection of human α-thrombin using ssDNA (bovine serum albumin) were carried out
thrombin aptamer and cationic polythiophene. Panel (a) reproduced with permission.[163] Copy- under identical conditions. In both cases, an
right 2002, Wiley-VCH. Panel (b) reproduced with permission.[164] Copyright 2004, American important red shift towards the lower energy
Chemical Society.
(λmax = 505 nm) was observed, and the color
of these solutions was red-violet, a typical
different 20-mer oligonucleotides differing by only one or two color of the planar and highly conjugated structure of the
nucleotides were investigated. A very distinct, stable, and repro- polythiophene backbone when mixed with unfolded ssDNA
ducible UV-vis absorption spectrum is observed in the case of (path B).
oligonucleotide target with two mismatches Y2 when compared Leclerc and co-workers also describe an easy and rapid
to perfect hybridization. In some cases, it is even possible to methodology for the enantiomeric resolution of d- and
distinguish only one mismatch. l-adenosine.[159] This approach is based on the fact that two
Aptamers are known to exhibit high affinity and selectivity stacked G-quartets are formed by mixing d-adenosine to its
against a variety of targets, including ions, small organic mol- aptamer, while l-adenosine does not have the effect.[159,167,168]
[ 3 ]
ecules, amino acids, proteins, etc. By using aptamers as the Since l-adenosine does not have the ability to induce a confor-
detection elements, this method shows more widely applica- mational change of the aptamer, the cationic polythiophene
tion. Due to the fold-inducing properties for potassium cation will bind to the aptamer and lead to the formation of a duplex.
specific aptamer, Leclerc and co-workers used this method for Therefore, when the polymer is put in the presence of both the
colorimetric detection of monovalent potassium cation.[164] aptamer and l-adenosine, the aqueous solution becomes red
The aqueous solution of the polythiophene alone shows with a maximum of absorption at 500 nm. However, in the pres-
λmax ≈ 400 nm, which is in agreement with the above results. ence of d-adenosine and its aptamer, a maximum of absorption
In the presence of LiCl, NaCl, RbCl, and ssDNA (aptamer of at 410 nm is observed.
potassium cation), the solution changed into red color (λmax = By using another water-soluble polythiophene (polymer 2,
527 nm). This red shift is attributed to the formation of a stoi- Figure 11), an adenosine triphosphate (ATP) sensor method
chiometric complexation between unfolded anionic ssDNA and was also recently described.[169] As the concentration of ATP
the cationic polythiophene derivative. However, in the presence is increasing in water, polymer 2 exhibits a red shift in the
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REVIEW
through electrostatic interaction in which

polymer 2 adopts a highly conjugated or
planar conformation and thus the positively
charged polymer exhibits a relatively red-
shifted absorption. In contrast, when ssDNA
is digested by S1 nuclease or hydroxyl radical
into small fragments, the polymer 2/ssDNA
duplex is not formed. In this case, polymer 2
remains in a random-coil or non-conjugated
conformation and shows a relatively short
absorption wavelength. The nuclease diges-
tion or oxidative damage by hydroxyl radical
of DNA can be observed by UV-vis absorp-
tion spectroscopy or just visualized by the
naked eye.
Another example is label-free detec-
tion of mercury ion detection (Figure 13b)
reported by Fan, Wang, and co-workers.[171]
A mercury-specific oligonucleotide (MSO)
probe was used in the experiment, which
is rich in thymines (T) and readily forms a
stem-loop structure in the T–Hg2+–T con-
figuration in the presence of target Hg2+ ion.
As PMNT binds to the Hg2+-free MSO and
the Hg2+–MSO complex in different ways,
the solution exhibits distinguishable optical
properties in response to the target-induced
conformational change. Based on this obser-
vation, a simple “mix-and-detect” optical
mercury sensor was developed for use in
aqueous solutions. Even with the naked eye,
micromolar Hg2+ ion concentrations could
be identified within minutes. By using the
fluorometric method, the detection limit was
improved to the nanomolar range (42 nM).
Using similar strategies, a rapid colorimetric
detection strategy for pH-driven conforma-
tional conversion of DNA i-motif structure
was also developed.[172]
Figure 13. a) Schematic representation of the assay for nuclease. b) Description of the optical Recently, Liu and co-workers
mercury sensing mechanism based on target-induced conformational change of MSO and designed two conjugated polymers for
resultant optical change of PMNT. Panel (a) reproduced with permission.[170] Copyright naked-eye detection and quantification
2006, American Chemical Society. Panel (b) reproduced with permission.[171] Copyright 2007, of heparin.[173,174] The first is a cationic
Wiley-VCH. poly(fluorene-co-phenylene) derivative with
a small fraction of benzothiadiazole, which
absorbance spectra, changing the solution color from yellow changes its fluorescent color from blue to orange upon
to pink-red. The absorption changes of the polythiophene interaction with heparin in solution.[173] The second is cati-
were attributed to the formation of an electrostatic complex onic poly(1-methyl-3-[2-[(4-methyl-3 -thienyl)oxy]ethyl]-1H-
between the cationic polymer and anionic ATP, which resulted imidazolium) (P4Me-3TOEIM), which can be used for visual
in increasing planarity of the conjugated polymer backbone. heparin detection and quantification in blood serum.[174] The
This approach shows selectivity for ATP over other anions negatively charged heparin interacts with positively charged
such as Cl−, HPO42−, and HCO32−, adenosine diphosphate polythiophene through electrostatic interaction, which leads
(ADP), adenosine monophosphate (AMP), or uridine triphos- to polymer conformation and color change from yellow to
phate (UTP). orange in solution. Quantification of heparin is also demo-
Wang and co-workers developed a new method for the label- nstrated by correlating the changes in polymer absorbance
free and real-time monitoring of the cleavage of ssDNA by to the heparin concentration. A linear calibration curve
single-strand specific S1 nuclease and hydroxyl radical based is observed in the 0–6.7 U mL−1 and 0–2.2 U mL−1 ranges
on polymer 2 (Figure 13a).[170] The cationic polymer 2 can form for heparin quantification in pure water and in fetal bovine
an interpolyelectrolyte complex with ssDNA, called duplex, serum, respectively.
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REVIEW
organization within PDA systems affect the conjugated frame-
R1
work and contribute to the structural and colorimetric trans-
polydiacetylene formations. There are three different approaches to introduce
receptor in the biosensing system based on PDA: 1) introducing
R2 recognition units through direct chemical modification of the
n PDA head group, which has employed for detection of chem-
Figure 14. Chemical structure of polydiacetylene. ical and biological toxins, viruses, and bacteria (Figure 15a);
2) the PDA framework can act as “scaffolding” for the stabi-
lization of additional lipophilic dyes and/or recognition ele-
7.2. Polydiacetylene (PDA) ments that can be non-covalently incorporated into the vesicles
(Figure 15b); and 3) the lipid/PDA assemblies further facilitate
Polydiacetylene (PDA) is a remarkable polymeric system that the non-covalent incorporation of various molecular markers
exhibits unique chromatic properties (Figure 14).[6,175–178] and recognition modules (Figure 15c).[6]
PDA is generally synthesized through 1,4- addition of aligned
diacetylenic monomers, initiated by UV irradiation.[6,175] Since
7.2.1. Detection of Viruses and Microorganisms
no chemical initiators or catalysts are required for the polymeri-
zation process, the polymers are not contaminated with impuri- Most of the applications involved chemical modification of the
ties, and consequently, purification steps are not required.[176] PDA head groups for incorporation of functional and biomo-
The produced polymer exhibits an intense blue color, due to lecular recognition units at the PDA surface. The pioneering
electronic delocalization within the conjugated framework, work was done by Charych and co-workers to demonstrate that
giving rise to absorption at around 650 nm in the visible region PDAs serve as fascinating sensor matrices for the detection of
of the electromagnetic spectrum.[6] Interestingly, PDA has biologically interesting target molecules.[187] In the experiment,
the ability to undergo a blue-to-red color change in response they prepared a PDA Langmuir–Blodgett (LB) film, function-
to a variety of environment perturbations, such as tempera- alized with sialic acid, and showed that the film undergoes a
ture changes,[179–182] pH, and surface pres-
sure.[183] The mechanism of the color change
is related to the irreversible stress-induced
structural transition of the conjugated back-
bone of the polymer.[184,185] This externally
induced conformational transformation of
the PDA backbone essentially shortens the
effective conjugation network, giving rise to
an appearance of the polymer as a red sub-
stance. The structural transition of the back-
bone is believed to be primarily affected by
surface perturbations to the pendant side
chains of the PDA assemblies.[182,185]
Since the discovery of PDAs, a number
of biosensors have been developed based on
PDA vesicles and films. Bilayer configura-
tion is one of the self-assembled structures
utilized for exploiting the chromatic proper-
ties of PDA for biosensing applications. The
lipidomimetic structural features of PDA,
i.e., hydrophobic tail (terminated by a methyl
group) and hydrophilic head group (carboxy-
late), result in the formation of biomimetic
membrane assemblies, such as monolayers
at the air/water interface and vesicular aggre-
gates in aqueous solutions.[6] These organi-
zations have facilitated utilization of the
unique optical properties of PDA for varied
biological sensing applications. Addition-
ally, PDA bolaamphiphiles (containing polar
head groups at both sides) were also shown
to form stable vesicles.[186] Furthermore, Figure 15. Colorimetric detection of molecular recognition by introducing recognition units
these vesicles exhibited dramatic colorimetric through a) chemical modification of the PDA head group, b) inserting other functional lipid
responses to varied external stimuli, sug- into the PDA matrix, and c) inserting various molecular markers and recognition modules into
gesting that both head-group and side-chain the lipid/PDA assemblies.
www.advmat.de
REVIEW
blue-to-red color change when exposed to an influenza virus. exclusively at the 2-acyl position in glycerophospholipids to
In addition, they also described colorimetric detection of influ- yield a free fatty acid and a lysophospholipid.[193] In the detec-
enza virus by using sialic acid residues functionalized PDA ves- tion system, hydrophobic products of enzyme-catalyzed reac-
icles.[188] In this biomolecular material, the sialic acid ligands tions of hydrophilic substrates perturb the ordered structures of
provide a recognition function for viral binding while PDA PDAs thus causing the color transition.
backbones provide a detection element for color change. The In addition, colorimetric PDA sensor systems based on
multivalent nature of viral binding at the interface triggered the specific antibody–antigen interactions have been developed.
conformational changes in the polymer side chains followed Gill and co-workers reported to fabricate rugged and sensitive
by disruption of conjugation in the chromophoric polymer chromatic solid-state materials by encapsulation of bichromic
backbone. In the presence of influenza virus, the solution of polydiacetylene-phospholipid liposomes appended with immu-
PDA vesicles immediately undergoes a color change. The color noglobulins in hybrid silica–siloxane sol–gel polymers.[194] The
change is readily visible with the naked eye and can be quanti- resulting composites undergo dramatically and visible blue-
fied by visible absorption spectroscopy. The titration experiment to-red color transitions upon exposure to the corresponding
shows that as little as 11 × 107 virus particles can be detected by antigens that can be used as sensors. Jelinek and co-workers
using this method. have developed a novel molecular assembly, which facilitates
Instead of ligands directly attached to the diacetylene lipids, rapid biomolecular recognition of peptides, displayed at a
Jiang and co-workers reported a modified approach to insert the biomimetic membrane interface, by antibodies in aqueous
receptor molecules into the PDA matrix by using a glycolipid solutions.[195] The colorimetric sensor is based upon vesicular
such as dioctadecyl glyceryl ether-β-glucosides (DGG), which is particles of PDA, incorporating membrane-like phospholipids
inexpensive and easily obtained.[189] This system can be used to domains, as well as the epitopes, displayed at the N-termini of
colorimetric detection of Escherichia coli (E. coli). As the receptor hydrophobic amino acid sequences. Polymerized diacetylenes
glycolipids were inserted by physical force, this method shows exhibit unique chromatic properties. They showed that the
great advantages over direct modification by the chemical combined epitopepeptide/phospholipid/polydiacetylene mole-
method. Subsequently, Jiang and co-workers investigated the cular aggregates undergo rapid colorimetric transitions when
influences of the spacer length of the glycolipid receptors as they come in contact with antibodies that specifically recognize
well as the different diacetylenic lipid matrixes in color change- the epitopes.
able polydiacetylenic vesicles on the detection of E. coli.[190] The Recently, Kim and co-workers described a PDA-based colori-
experimental results demonstrated that the glycolipids with metric biosensor to detect the biotin–streptavidin (STA) interac-
longer spacer would be benefit to detecting E. coli. tions in solution.[196] The high affinity between soluble strepta-
vidin and the PDA–biotin complex gave rise to the blue–red
7.2.2. Detection of the Interaction between Proteins and Ligands transition, accompanied by vesicle aggregation due to the
multimeric interactions involving streptavidin and four biotin
The PDA detection system also shows potential application units.
for detection of the interactions between proteins and target
molecules. PDAs can be made into artificial cell membranes
7.2.3. Detection of DNA
to mimic membrane processes of molecular recognition and
signal transduction. Charych and co-workers incorporated Color sensing of DNA strains through PDA functionalized
monosialotetrahexosylganglioside (GM1) into PDAs liposomes with oligonucleotides was also illustrated. Ma and co-workers
and studied the molecular recognition of cholera toxin at the reported PDA vesicles attached with probe DNA molecules
interface of the liposome.[191] A color change was observed due undergo the blue-to-red colorimetric transition upon binding
to conformational changes in the conjugated polymer backbone. with complementary strands of DNA, enabling them to be
The “colored liposomes” might be used as simple colorimetric used as colorimetric DNA sensors (Figure 16a).[197] The design
sensors for drug screening or as new tools to study membrane– utilized the structural/chromatic transformations of PDA as
membrane or membrane–receptor interactions. a vehicle for amplification of the oligonucleotide recognition
The PDA detection system also shows potential application signal. Recently, Park and co-workers envisaged a distinctly dif-
for detection of the specific interactions between enzyme and ferent PDA-based strategy in which ionic interactions between
substrates. Stevens and co-workers reported a novel biosensor positively charged PDA and negatively charged dsDNA would
for detection of glucose by utilizing the ligand-induced confor- be utilized to develop a universal PDA sensor for the detec-
mational changes of a hexokinase immobilized on the sensor tion of different PCR amplicons regardless of their sequences
surface.[192] The large conformational changes in the protein are (Figure 16b).[198] Interestingly, the procedure would avoid com-
coupled to the chromatic polymer backbone. The colorimetric plex chemical modifications of the diacetylene monomer and a
change is induced by the enzyme conformational changes, denaturation step. To probe the design of a PDA-based universal
caused by the binding of a small molecule to the enzyme DNA sensor, modified diacetylene monomers, having two dif-
active site. This color change mechanism is different from ferent amine moieties (primary amine and quaternary amine),
the mechanical stress provoked by the toxin or virus directly were synthesized and used to generate amine-functionalized
disrupts the polymer film.[187,191] Another biosensor based on PDA liposomes. Tests with the resulting PDA sensors demon-
enzyme can detect the action of phospholipase A2 (PLA2), an strated that DNA molecules, amplified by PCR, can be detected
enzyme that can catalyze the hydrolysis of an acyl ester bond at concentrations of ≈100 nM.
www.advmat.de
REVIEW
to combine with other useful properties, such
as magnetic and near infrared characteristics.
Therefore, in combination of their unique
properties and inherent increase in signal-
to-noise ratio provided by miniaturization,
colorimetric biosensing based on these smart
materials is promising for future biomedical
diagnosis and environmental monitoring.
Acknowledgements
The authors thank members of the Qu group
for helpful discussions and technical assistance.
Financial support was provided by 973 Project
(2011CB936004), NSFC (20831003, 90813001,
Figure 16. a) Schematic diagram of the colorimetric detection of DNA using polydiacetylene 20833006, 90913007) and Funds from the Chinese
vesicles functionalized with probe DNA. b) Color transition of the PDA sensor induced by Academy of Sciences.
ionic interactions between negatively charged phosphate groups of nucleic acids and positively
charged amine headgroups of PDA. Received: May 18, 2011
Published online: July 29, 2011
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polymers.[1,6,11,14–17] The robust physical

Y. Song, W. Wei, Prof. X. Qu

2. Gold Nanoparticles (AuNPs)

reduce their toxicity and to produce a color change.[14,73] Peroxi-

duplexes on AuNP surface resulted in colloidal destabiliza-

of molecules to and from the nanoceria core

Figure 7. a) Schematic representation of Fe3O4 MNPs-based immunoassay (top) or Fe3O4

Recently, Dong and co-workers prepared hemin-graphene (a)

through electrostatic interaction in which

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