Biotechnol. Prog.

2007, 23, 517−531 517

REVIEWS
Development of Immunosensors Using Carbon Nanotubes
Jithesh V. Veetil and Kaiming Ye*
Biomedical Engineering Program, College of Engineering, University of Arkansas, Fayetteville, Arkansas 72701

With increasing reports on bioterrorism, avian flu, and other bio-threats, rapid and real time
detection methods are highly warranted. Studies on developing highly sensitive immunosensors
aiming at the early detection and clinical diagnoses of various diseases including cancer are
undertaken all over the globe. Carbon nanotubes (CNTs) have been widely discussed as materials
with enormous potential for a wide range of in ViVo and in Vitro bioapplications, ranging from
drug delivery to highly sensitive biosensors, owing to their superior electronic and mechanical
properties along with nanoscale dimensions. Though a lot of attention has been drawn toward
carbon nanotubes for the past 15 years in academia and to a certain extent in industry, CNT-
based immunosensors and other applications are still in the nascent stage, and there are many
challenges to be overcome for the successful commercialization of the concepts. This article
highlights on the recent developments and the possible impacts of carbon nanotube based
immunosensors.

Contents by bioassays include methods such as ELISA (enzyme-linked

Introduction
Integration of Molecular Recognition
Functions into CNT for Immunosensing
Solubilization of CNTs by Surface
Modification
Noncovalent Attachment of the Antibodies
to the CNTs
Covalent Linkage of Antibodies to the
CNTs
Methods To Prevent the Nonspecific
Binding of Proteins to CNTs
Visualizing the Antibodies on the Surface of
CNTs
Development of Immunosensors Using
Functionalized CNT
FET Immunosensors
Other Electrochemical Immunosensors
CNT Alignment and Nanoarray
Immunosensor Development
Sensitivity and Stability of CNT-Based
Immunosensors
Future Directions and Challenges in
CNT-Based Immunosensors
Disposable CNT Immunosensors
Miniaturized, Multiplexed Sensors for
Point-of-Care Testing
Implantable Immunosensors
Introduction
Developing methods and devices for identification of specific
proteins are one of the major challenges in biosensor develop-
ment and clinical diagnosis. The traditional detection of proteins
* To whom correspondence should be addressed. Phone: 479-575-5315.
Fax: 479-575-2846. Email: kye@uark.edu..
10.1021/bp0602395 CCC: $37.00
immunosorbent assay), RIA (radioimmunoassay), and electro-
517
phoretic immunoassay. However, most of these techniques
518 involve sophisticated instrumentation and tedious and time-
consuming steps. Hence, search for new, simple, sensitive,
518 reliable, and inexpensive diagnostic methods with real-time
output is of considerable interest. As the outcome of extensive
519 research in proteomics, many new techniques have been put
forward that are essential to diagnose various disease states, to
519 defend against biological threats, and to improve drug discovery.
Immunosensors, a type of biosensors, can be defined as
520 compact analytical devices incorporating antibody or antigen
or its fragment, either integrated within or intimately associated
520 with a physicochemical transducer (Figure 1). On the basis of
a specific reaction of the antibody and antigen, immunosensors
521 provide a sensitive and selective tool for determining proteins.
Immunosensors can help in direct monitoring of the molecular
521 recognition event on the surface of a chip. A large number of
523 immunosensors have been developed using different transducers
523 that exploit changes in mass, heat, electrochemical, or optical
properties (1). Most of the reagents employed in immunoassays
526 such as antibody, enzymes, and fluorescence labels are very
expensive, and also the analytes such as blood from a neonate
527 or spinal fluid are precious commodity. Hence, miniaturization
of the diagnostic devices without affecting their sensitivity or
527 detection limits is highly desired. With advancement in the field
527 of microfluidics and lab-on-chip concepts, novel high-throughput
immunosensors that offer decreased analysis time and ease for
528 automation, integration, and portability are explored.
However, it was found that with lower amounts of analytes
employed/desired in the immunosensor, a detection limit
comparable to that of the conventional ELISA-based immu-
noassays could not be realized most often. Hence it is desirable
to optimize the sensitivity and detection limits (below picomolar
levels) of the immunosensor along with its miniaturization,
which can be reused several times to compete with the
conventional assays. One of the major methods for improving
the sensitivity of the immunosensor is by incorporating nano-
© 2007 American Chemical Society and American Institute of Chemical Engineers
Published on Web 04/26/2007
518
Figure 1. Schematic representation of the working principle of
immunosensors.
particles in it. Attempts have been made to improve biosensor
performance by incorporating various nanoparticles such as latex
(2), gold (3, 4), and carbon nanotubes (CNTs) (5, 6).
CNT can be described as a sheet of carbon atoms rolled up
into a tube with a diameter of around tens of nanometers. Since
their discovery in 1991 (7), numerous attempts have been made
all over the world to develop technologies that can yield highly
purified single- or multiwalled carbon nanotubes. Multiwalled
carbon nanotubes (MWCNTs) are made up of several concentric
cylinders of graphite sheets, with about 3.4 Å spacing between
the layers. MWCNTs usually have 2-100 nm diameter (typi-
cally 2-10 nm in internal diameter) compared to the single layer
cylindrical graphite sheet of single-walled carbon nanotubes that
is usually (SWCNTs) about 0.2-2 nm in diameter. The length
of the CNTs can vary from micrometers to centimeters with a
very high aspect ratio (length/diameter). The nanotubes usually
have a high surface area to weight ratio of 300 m2/g, and most
of this surface area is accessible to both electrochemistry and
immobilization of biomolecules. Mainly, three techniques, i.e.,
carbon arc-discharge (8, 9), laser ablation (10), and chemical
vapor deposition (11), have been developed to synthesize CNTs.
Various methods have also been established to purify CNTs
(12), since nanotubes produced in all of these methods come
out with a number of impurities. Sensing applications represent
one of the first three fields of nanotubes research expected to
deliver their promises in commercial products. The other two
are displays and memory devices (13, 14)
The superior mechanical and conductive properties (high
actuating stresses, low driving voltages, and high energy
densities) of CNTs make them very promising for developing
ultrasensitive and miniaturized immunosensors for disease
diagnosis. It is expected that ELISA-based immunosensors can
be miniaturized and fabricated in the form of carbon nanotube
array-based sensor chips through NEMS (nano-electro mechan-
ical system) techniques. However, CNTs themselves do not
provide any measurable signals for sensing biomolecules. It is,
therefore, of critical importance to develop techniques that can
endow the CNTs with both a molecular recognition and a signal
transduction function. In this Review, we intend to track the
development of these techniques and their applications in the
fabrication of CNT-based immunosensors.
Integration of Molecular Recognition Functions into
CNT for Immunosensing
As we have mentioned above, CNTs cannot be directly used
to detect biomolecules in an immunoassay because they cannot
Biotechnol. Prog., 2007, Vol. 23, No. 3
specifically recognize biomolecules. In order to make an
immunosensor using CNTs, we must introduce a molecular
recognition function, as well as a signal transduction function,
into the CNTs so that they can specifically recognize and detect
biomolecules from biological samples. A number of technologies
have been established to endow CNTs with a molecular
recognition function through a process called functionalization.
The basic idea for CNT functionalization is to immobilize
proteins, peptides, enzymes, antibodies, or antigens onto the tip,
inner, or outer sidewalls of the CNTs (15). We will discuss
these techniques in the following sections.
Solubilization of CNTs by Surface Modification. The lack
of solubility in physiological solutions is one of main difficulties
in integrating CNTs in biological systems. To overcome these
difficulties, extensive studies have been conducted to search
for ways to solubilize CNTs (16-18). As applied in solubili-
zation of many nanoparticles, the surface property of a CNT
can be modified to convert it from being hydrophobic to
hydrophilic. This can be achieved in different ways including
chemical, electrochemical, thermal, or plasma oxidation treat-
ment (19). CNT tips and sidewalls can be modified by covalent
and noncovalent methods. Covalent modification was initially
used for solubilization of the nanotubes, but currently, it has
been employed in many bioapplications such as antibody
attachment on the tips of the nanotubes in developing immu-
nosensors. Treatment of CNTs with sulfuric and nitric acid
mixtures under oxidative conditions helps open the end and to
introduce functional groups such as carboxylic acids on the
nanotubes, but the acid treatment along with sonication usually
shortens the tube length and thereby reduces the high aspect
ratio of the SWCNTs. Another method of solubilizing the CNT
involves generating acyl chloride on the nanotube surface by
reacting with thionyl chloride (SOCl2) and dimethyl formamide
(DMF), followed by the addition of glucosamine (20). The
solubility of the SWCNTs prepared by this methodology was
found to be 0.1-0.3 mg/mL depending on the temperature. The
replacement of the carboxyl group on the CNT with an amine
group can be followed by the DRIFTS spectroscopy results. A
more highly soluble CNT with stability at about 20 mg/mL was
reported by Georgakilas et al. (21) by attaching N-protected
amino acids to the side wall of nanotubes.
The direct sidewall functionalization of SWCNTs can be
achieved by reduction reactions by using reactive species such
carbenes (22), nitrenes (23), or aryl radicals (24). Acid oxidation
is the most common method for the tip modification of the
CNTs, whereas fluorination is used to functionalize the side-
walls. However, the covalent binding of the fluorine resulted
in drastic changes in the electrical conductivity of the nanotubes.
In order to maintain the electronic properties (sp2 nanotube
structure), the noncovalent method is usually preferred. Hence
one common approach to increase the solubility of CNT in water
is to treat CNT with detergents or surfactant such as sodium
dodecyl sulfate (SDS) and Triton-X 100 (25). Panhuis et al.
(26) studied the interactions of an enzyme with Triton-X 100
treated CNT in water and reported that the hydrophilic surface
of the CNT-Triton conjugate interacted with the hydrophilic
surface of the enzyme. Using Raman spectroscopy, they
confirmed that the enzyme interacted with treated rather than
untreated CNTs. Polymers such as poly(vinylpyrrolidone) (PVP)
and polystyrene sulfonate (PSS) can be used for wrapping CNTs
and thereby reversible solubilization (27). Bifunctional mol-
ecules such as 1-pyrenebutanoic acid succinimidyl ester can
irreversibly adsorb by π-stacking on the hydrophobic surface
of the SWCNTs in organic solvents such as methanol or
Biotechnol. Prog., 2007, Vol. 23, No. 3
dimethyl formamide. This mode of functionalization is highly
preferred in the immunosensor developments, as the succinim-
idyl ester on the nanotube surface is highly reactive to
nucleophillic substitution by primary and secondary amines that
exist on the surface of the antibodies (28). Another method of
noncovalent CNT solubilization is by sonicating the nanotube
in the presence of phosphotungstic acid (29). Recent studies
indicated that CNTs can be dissolved in a starch-iodine
complex, although they are not soluble in aqueous solutions of
starch (30). Other molecules that can disperse CNTs in water
by noncovalent methods include amylase (31), gum arabic (32),
cyclodextrins (33, 34), Nafion (18), etc. A number of different
functionalization and solubilization strategies for CNTs are
extensively classified and reviewed elsewhere (16, 35-37).
All of these efforts have made CNTs more useful in the
fabrication of immunosensors using nanotechnologies. Follow-
ing the solubilization of CNTs, researchers started to think about
adding molecular recognition functions to the CNTs so that they
can specifically recognize a target molecule from a complex
biological sample. The easiest way to integrate a molecular
recognition capability is to adsorb an antigen or an enzyme onto
the surface of the CNT, so that a target molecule can be
specifically recognized through the interaction between the target
molecule and CNT surface-attached antigens or enzymes. A
number of other techniques have also been developed to improve
the efficiency of protein attachment. Here we first discuss the
surface noncovalent adsorption and then CNT protein covalent
conjugation in the subsequent sections.
Noncovalent Attachment of the Antibodies to the CNTs.
The next major challenge in the fabrication of immunosensors
is to attach antibodies or antigens to the CNTs. This can be
done by using either covalent or noncovalent methods.
A simple nonspecific binding scheme can be used to form
noncovalent protein-nanotube conjugates. Numerous lines of
evidence revealed that proteins can adsorb spontaneously on
the sidewalls of acid-oxidized CNTs (38). Though reports on
the direct adsorption of the antibodies on the CNTs are relatively
fewer, considerable studies have been carried out on the
adsorption of enzymes and other proteins on the nanotube
surface (39-41). Since the noncovalent adsorption of antibodies
on the surface is devoid of harsh chemical treatment otherwise
employed in covalent bond formation, the possibility of loss of
activity or antigen capturing ability is comparatively less. Studies
with enzymes adsorbed on the nanotube surface indicates that
immobilization of proteins onto the nanotube did not hinder
the partitioning of the substrate to the active site of the protein
(40). O’Connor et al. (42) reported a method of antibody
adsorption onto SWCNTs on basal plane graphite disk electrode
surface. At pH 7.2, anti-biotin antibody was incubated on
SWCNT surface for 3 h, followed by washing the excess
antibody with PBS containing 0.05% Tween 20. Saturation of
the nanotube surface was found to be with 0.5 mg/mL antibody.
In other studies, Naguib et al. (43) reported a successful
adsorption of monoclonal CD3 antibodies onto carbon nanofi-
bers. They found that the protein binding to the nanofibers can
be enhanced by poly-L-lysine (PLL) and improved with increas-
ing disorder and hydrophilicity of the nanofibres’ surface.
Oxidized and disordered surfaces of pyrolytically stripped
nanofibres showed the improvement of the wetting and attach-
ment of PLL and proteins compared to the hydrophobic and
well-ordered surfaces of heat-treated nanofibres. It can be
assumed that this method can be used for the nanotubes also,
but there are inherent difficulties in attaching the antibodies to
the SWCNTs by noncovalent methods. The relative size of the
519
immunoglobulins (7-8 nm, human IgG, 150 kDa) compared
to the diameter of SWCNTs (1-2 nm) may affect the binding
and stability of the antibody adsorbed on CNT (38). For
example, proteins such as ferritin were found not to be able to
directly adsorb on the SWCNT surface (28). Wang et al. (44)
employed a phage-display technique to determine if there was
any selective affinity for the protein sequences toward the CNTs
under noncovalent conditions. It was found that histidine and
tryptophan residues play a significant role during the protein-
CNT interaction. This reinstates the fact that investigating the
structure and function of proteins immobilized onto nanotubes
is critical for developing a better understanding of how proteins
interact with nanotubes and eventually for the design of
functional carbon nanotube-protein systems. In contrary, it is
possible to synthesize monoclonal antibodies that have specific
affinity toward CNTs and other forms of carbon such as
fullerenes. It was demonstrated that anti-fullerene monoclonal
IgG adsorbs specifically on the surface of SWCNTs (45). Such
antibody-coated nanotubes can be used to develop cellular
probes. The amount of antibodies adsorbed per unit weight of
nanotube is a critical parameter when they are employed for
various applications. In the case of proteins such as peroxidase
and chymotrypsin, these values were found to be about 0.5-
0.6 mg protein/mg of SWCNT (39).
Covalent Linkage of Antibodies to the CNTs. As mentioned
above, noncovalent absorption of proteins onto the CNTs can
be problematic when the CNTs-based immunosensors are
desired, as the nonspecific binding of proteins to the CNTs will
remarkably deteriorate the specificity of the sensors. To address
this issue, various techniques have been developed to specifically
conjugate CNTs with proteins that can recognize target mol-
ecules from complex biological samples. There are a number
of ways to achieve this goal. One way to do this is to covalently
cross-link proteins to CNTs after attaching them with some
functional groups (46). For example, a carboxylic acid group
can be added to either sidewall or the tips of the CNTs by acid
oxidization. These groups can be further activated using N-ethyl-
N′-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)
as a coupling agent (47). The EDC treatment leads to the
formation of a highly reactive and unstable acylurea derivative.
It then forms a more stable active ester in the presence of
N-hydroxysulfosuccinimide (NHS). As a result, a protein can
be cross-linked to the CNT through a nucleophilic substitution
reaction between the active ester and the amine group of the
protein, which forms an amide bond between CNT and the
protein (Figure 2). Carboxyl groups at the open end of acid-
oxidized, shortened SWCNT can also be converted into active
carbodiimide esters by treating with N,N′-dicyclohexyl carbo-
diimide (DCC) along with the organic solvent DMF (48).
In another studies, Bianco’s group (49) applied two different
strategies to prepare peptide-conjugated CNTs, on the basis of
the fragment condensation and the selective chemical ligation.
A model pentapeptide and an antigenic epitope from the foot-
and-mouth disease virus (FMDV) were covalently linked to
SWCNT using this method. A detailed structural characterization
has been performed, using transmission electron microscopy
(TEM) and 2D NMR spectroscopy. The antigenicity of the
FMDV peptide conjugated nanotubes was characterized by a
surface plasmon resonance analysis and the ELISA assay. The
results from both experiments suggested that the peptide bound
to the nanotubes and adopted a correct secondary conformation
necessary for antibody recognition when employed in immun-
osensors. Moreover, an in ViVo study showed that the FMDV
520
Figure 2. Most commonly used methods of antibody immobilization on CNTs: (left) covalent attachment of proteins to CNTs using diimide
activated imidation coupling using N-ethyl-N′-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS)
(46); (right) irreversible, noncovalent adsorption of CNTs with 1-pyrene butanoic acid, succinimidyl ester (28).
peptide-CNT conjugates are immunogenic. These findings
highlight the potential use of peptide-CNT conjugates for
diagnostic purposes.
It is advantageous to develop specific binding methods that
are devoid of the extensive chemical treatment of antibodies
employed in covalent binding. One of the modified methods
involves the covalent binding of a protein, such as streptavidin,
on the surface of functionalized nanotubes by the EDC coupling
chemistry, followed by the addition of biotynylated antibodies
(50). The specificity of streptavidin-biotin conjugation leads
to the immobilization of antibodies on the CNT surface, without
affecting the inherent properties of the antibody to capture the
antigen. Biomolecules such as BSA can also be coupled to the
CNTs using EDC chemistry followed by a simple adsorption
of the antibodies. Sun’s group adopted this strategy to prepare
immuno-carbon nanotubes E. coli O157 immunodetection (51,
52). Another method that draws on the advantage of noncovalent
side wall of functionalized SWCNTs followed by a covalent
linkage of the antibodies was developed by Park et al. (53).
Here, with the linker molecule, carbonyldiimidazole-Tween20
(CDI-Tween 20) surfactant, the hydrocarbon chain binds with
the nanotubes with strong hydrophobic interactions, while the
active imidazole group of the linker molecule reacts with the
NH2 groups on the surface of the target antibodies to form
covalent bonds. Another linker molecule used to covalently
attach the antibodies to the CNT is 1-pyrenebutanoic acid
succinimidyl ester (54) (Figure 2). Antibodies attached to the
CNTs by this method show a better affinity and stability in the
immunosensors.
Methods To Prevent the Nonspecific Binding of Proteins
to CNTs. Biological samples are usually complex in nature with
numerous types of proteins and other molecules. Nonspecific
binding (NSB) of undesired molecules from the sample is a
major obstacle in the development of biosensors. CNT-based
immunosensors also must be designed in such a way that NSB
of molecules other than the desired analyte to the nanotubes is
minimized. Hence, research on minimizing protein nonspecific
binding draws a lot of special attention. NSB, mainly by the
spontaneous adsorption of the protein, to the CNT surface is
usually attributed to hydrophobic interactions between the
protein and the nanotube surface. To reduce this type of
nonspecific binding, various surfactants including poly(ethylene
oxide) (PEO) and poly(ethylene glycol) (PEG) have been tested.
PEO-containing molecules are known for their protein-resisting
abilities (55). Of the 10 PEO-containing molecules investigated
by Chen et al. (56), Tween 20 and a series of pluronic triblock
copolymers were found to be able to strongly adsorb onto the
CNT surface and impart high protein resistance. PEG is another
Biotechnol. Prog., 2007, Vol. 23, No. 3
biocompatible polymer well-known for its protein-repelling
properties. PEG can be attached to the matrix covalently or
noncovalently onto metals (57) or glass (58), where it acts an
effective barrier against nonspecific protein adsorption by
forming a highly hydrophilic, neutrally charged, sterically
hindering layer at the surface. Shim et al. (40) reported the use
of PEG to prevent protein nonspecific binding to CNTs. It was
found that the coadsorption of Triton and PEG on SWCNTs is
highly effective in preventing nonspecific adsorption of strepta-
vidin to nanotubes. Streptavidin (60 kDa) is a relatively small
protein that can adsorb more readily to the sidewall of SWCNT.
Nevertheless, with a large protein such as fibrinogen (340 kDa),
which is well-known to strongly adsorb onto a hydrophobic
surface, the nonspecific adsorption to SWCNT was found to
be less owing to the much lager size of fibrinogen relative to
the diameter of SWCNTs (1-2 nm). Streptavidin coating of
the CNT surface thus benefits as a molecular adaptor for the
antibody binding as well as to minimize the nonspecific binding
from the sample mixtures (41). BSA, a common blocking agent
used in ELISA tests, can also be used in reducing the NSB of
proteins to CNTs (41, 42).
Visualizing the Antibodies on the Surface of CNTs
It is of utmost importance to characterize the antibodies
attached to the CNTs, because the success of the immunosensor
depends on the effective immobilization of the antibody. To
visualize the antibody on CNTs, techniques such as atomic force
microscopy, scanning electron microscopy (SEM), and confocal
microscopy can be used. Labeling the antibodies with fluorescent
dyes such as FITC can aid in the observation of the nanotubes
under the microscope. To this end, Sirdesmukh et al. (59) studied
the interaction between CNTs and antibodies through confocal
microscopy and electron microscopy with negative staining
using phosphotungstic acid as the staining reagent. In their
experiment, CNTs prepared by agitating with sodium dodecyl
benzene sulfonate and dihexyloxacarbocyanine iodide were
linked to a fluorescent dye that emits fluorescence at 488 nm.
The labeled CNTs were mixed with prelabeled polyclonal goat
anti-mouse IgG prelabeled with Alexa 546 (emission maxima
at 543 nm). They observed the interaction between the antibodies
and CNTs. Confocal microscopy and SEM revealed that the
nanotubes were initially well dispersed and co-localized after
the adsorption of antibodies. The high localization could be due
to the separation of the CNTs with surfactants, which enabled
a very high surface area to volume ratio.
SWCNTs can be covalently modified with BSA by EDC
treatment as discussed in previous sections. These nanotubes
can be further modified with antibodies against a specific antigen
Biotechnol. Prog., 2007, Vol. 23, No. 3
Figure 3. SWCNTs were visualized under confocal microscopy with red fluorescent rhodamine labeled antibody (on the left, bottom) and E. coli
cells labeled with green fluorescent protein (on the middle). The yellow spots (on the right) indicate that the nanotubes are bound to the cells.
Corresponding SEM images of the cells are on the top panel (51)
for development of immunodetection methods. Elkin et al. (51)
reported that the anti-E. coli antibody adsorbed on the CNT
retains its activity as visualized by the capture of E. coli on the
SWCNTs using SEM images and also by confocal microscopy
(Figure 3). This also indicates that CNT-based immunosensors
for the detection of microorganisms can be explored.
Development of Immunosensors Using Functionalized
CNT
Though significant progress has been made in exploring CNTs
as electrodes and further as biosensors, there are no reports yet
on commercialized devices. There are many fundamental
problems that need to be carefully studied before CNT-based
sensors can be commercialized. One of the major challenges in
the commercialization of CNT sensors is the fabrication.
Although many works have been done, we still do not have a
good approach to fabricate CNT sensors because of the lack of
adequate ways to detect the signals where the ultrasensitivity
is required. Majority of the sensors are in the nascent stage and
can be considered only as “immunoelectrodes” rather than
immunosensors. Most of the prototype CNT biosensors reported
so far mainly focused on glucose detection employing im-
mobilized glucose oxidase (GOD) (60-63). Obviously, many
different CNT-based sensors can be developed using the same
approaches mentioned above for the detection of various
clinically significant biomolecules.
Based on the mechanism of transduction, the CNT-based
immunosensors reported so far can be grouped into (1) field
effect transistor (FET) immunosensors and (2) other electro-
chemical immunosensor such as amperometric immunosensors.
FET Immunosensors. A FET is a type of transistor com-
monly used for weak-signal amplification. In the FET, current
flows along a semiconductor path called the channel. At one
end of the channel, there is an electrode called the source. At
the other end of the channel, there is an electrode called the
drain. The physical diameter of the channel is fixed, but its
effective electrical diameter can be varied by the application of
a voltage to a control electrode called the gate. The conductivity
of the FET depends, at any given instant in time, on the electrical
diameter of the channel. A small change in gate voltage can
cause a large variation in the current from the source to the
drain by which the FET amplifies signals. Because of its high
sensitivity, FET has been widely used in immunobiosensors.
521
Interestingly, Tans et al. have discovered that SWCNT can
serve as a FET for signal transduction (64). On the basis of
their discovery, SWCNT-based FET has been fabricated through
patterned catalyst growth techniques combined with conven-
tional photolithography and lift-off methods (65). These FETs
were built by positioning the semiconductive SWCNTs con-
necting the two metal electrodes. Many of the CNT-based
immunodetection systems currently reported are based on this
design where CNTs act as single conducting or network
conducting channels. SWCNTs on these transistors are usually
modified by attaching specific antibodies in developing the
immunosensors. In a recent report, Park et al. (53) immobilized
monoclonal antibody against carcinoembryonic antigen (CEA)
on the sidewalls of SWCNT on the FET using CDI-Tween 20
as the linker (Figure 4). Attachment of the linker followed by
the protein addition led to decrease in the conductance.
Incubation of the sensor with pure samples of the CEA resulted
in further reduction the current values and was used to report
the detection limits of the device (53). More investigations on
the actual electronic sensing of the protein binding on the CNT-
based FET sensors were carried out by Chen et al. (66). Schottky
barrier modulation at the metal-nanotube contact region due
to the protein binding on the CNTs dominates the conductance
change. Hence, improvements in sensitivity of SWCNT-FET
immunosensors can be achieved by modifying the geometry of
the devices. Evaporating metals for source and drain electrodes
using a shadow mask at a tilted angle during the device
fabrication will increase the Schottky contact area, leading to
better sensitivities (67).
On the other hand, attempts have been made to fabricate
sensitive FET immunosensors for the biomarker detection, with
major focus on early diagnosis of cancer. It is believed that
nanotechnologies have greater potentials for developing the next
generation of highly sensitive and selective sensors. To prove
these concepts, Li et al. demonstrated a novel FET immun-
osensor device for detecting prostate specific antigen (PSA) at
levels suitable for prostate cancer clinical detection (68). In their
design, the surface of SWCNTs that connects the source and
drain electrodes was functionalized with 1-pyrenebutanoic acid
succinimidyl ester and then treated with anti-PSA monoclonal
antibodies (Figure 4). Measurements conducted before and after
incubation of PSA on this sensor indicated that the current
through the nanodevice increased significantly upon the captur-
522 Biotechnol. Prog., 2007, Vol. 23, No. 3

Figure 4. Prototype FET-immunobiosensors. Anti-PSA antibodies were covalently attached to the SWCNT by depositing 1-pyrenebutanoicacid
succinimidyl ester on the nanotube surface (68) (left). Addition of PSA results in reduction of conductance in the device specifically (right). A
similar device, wherein monoclonal antibody against Carcino embryonic antigen was covalently immobilized on the CNT-FET device by using the
linker carbonyldiimidazole-Tween20 (CDI-Tween 20) and used for the detection of CEA antigen (right). CNT-FET immunosensor can also be
fabricated with protein functionalized on the reverse side of the silicon wafer (bottom) (97)

ing of antigen onto the functionalized CNTs. This can be
attributed to the fact that SWCNTs are p-type semiconductors.
A reduction in the conductance of the CNT devices with the
attachment of primary antibody on the CNT-FET was also
reported by Sirdeshmukh et al. (59). The amine groups present
in the antibodies donate electrons to the to the p-type nanotube,
thereby recombining the holes in the nanotubes, which leads to
an increase in the resistance at positive bias voltage (69). Also,
this reduction in conductance is independent of the net charge
(or pI) of the proteins (56). Extensive exploratory studies on
the such FET immunosensor were carried out by Chen et al.
(56). Fabrication of the CNT-based FET device on quartz
followed by the QCM measurements also revealed that signals
can be generated specifically when the immunosensor captures
the analyte (Figure 5). A FET immunosensor was fabricated
for detecting the human autoimmune disorder. The CNT in the
FET device was modified with a recombinant human auto
antigen U1A (a prominent auto antigen in systemic lupus
erythematosus (SLE) and mixed connective tissue disease, 33
kDa; detection of autoantibodies against the U1A antigen is the
common clinical assay for systemic SLE). Experiments by
incubating the device with monoclonal antibody 10E3 against
the U1A antigen also resulted in a decrease in conductance,
and the specific recognition occurred even at concentrations
close to 1 nM (56). The binding process was also monitored
by microgravimetric QCM analysis by fabricating the CNT
electrodes on a quartz surface. Using similar strategies, a few
other CNT-FET immunosensors are also reported. In one
immunosensor, human chorionic gonadotrophin (hCG, a hor-
mone produced during pregnancy) was immobilized on CNT-
FET and the decrease in conductance when monoclonal antibody
against hCG specifically bound to it was monitored (66).
Immunoglobulin E (IgE) concentration in the solution can be
determined by using a CNT-FET where monoclonal antibody
against IgE is immobilized on the device (70).
Another novel approach for a CNT-based FET detection
system was reported by Takeda et al. (71). In this case, neither
the antigens nor the antibodies were directly immobilized on
the CNTs. The CNTs were grown on the silicon substrate, and
on the reverse side of the substrate the hemagglutinin antigen
was immobilized (Figure 4). Various concentrations of anti-
HA antibodies were added to the sensor, and the current-
voltage (I-V) curves between the source and drain electrode
of the FET were measured while changing the potential by
employing the back gate potential control system. Though it is
not as good as the FET having proteins directly attached to the
Biotechnol. Prog., 2007, Vol. 23, No. 3
Figure 5. Schematic representation of a CNT-FET immunosensor for the detection of SLE antibodies (56) (A). U1A antigen was covalently linked
to the sidewall of SWCNTs through a linker (B). Only the specific antibody binds to the immunosensor when incubated with the sample, which
resulted in decrease in conductance (C). The specific binding of antibody was also monitored by QCM analysis (D) where change in mass generate
the signal (E).
nanotubes, it can be boasted of having the advantage of ease of
regeneration of the sensor, since comparatively vigorous wash-
ing methods can be employed as the antigen-antibody reaction
happens on the reverse side of the nanotubes.
CNT-FET can be considered as a robust technology for
immunosensing. The main advantage of the CNT-FET immu-
nosensor is that it can lead to a “reagentless”, single-stage
analysis, but it must also be remembered that adequate care
must be taken to avoid nonspecific binding, which can result
in erratic outputs.
Other Electrochemical Immunosensors. The electronic and
structural properties make CNT an attractive candidate for
bioelectrochemical applications. Most of CNT-based electro-
chemical biosensors focus on glucose estimation devices with
immobilized GOD enzyme. General principles and classification
of electrochemical CNT biosensors have been reviewed widely
(72, 73). Simple CNT electrochemical sensors can be fabricated
by depositing the suspension of single-/multiwall CNTs on the
glassy carbon electrode (GCE) or other screen printed electrode
surfaces. Incubation of such CNT-modified electrodes in a
protein suspension leads to adsorption on the nanotube surface,
but it is difficult to control the parameters affecting the
measurements in such electrodes. There are two different types
of detection patterns reported for CNT-based electrochemical
immunosensors: (a) label-free immunosensors and (b) immu-
nosensors that employ labels and mediators. These systems
usually include a CNT-modified electrode, a reference electrode
(e.g., Ag/AgCl) and a counter electrode (e.g., Pt wire). A label-
free, electrochemical microelectrode in which a Pt working
microelectrode was modified with SWCNT was reported by
Okuno et al. (54) (Figure 6). Monoclonal antibodies against total
prostate specific antigen (T-PSA) were immobilized covalently
onto the nanotubes on the working microelectrode. PSA samples
of varying concentrations were added to the microelectrode, and
electrochemical signals were recorded using differential pulse
voltammograms (DPV). In this case, the label-free electro-
chemical signals rely on the detection of intrinsic oxidation
signal of the protein deriving from their electroactive amino
acid residues tyrosine and tryptophan. With increase in T-PSA
concentration, the current was found to increase. It was
523
postulated that increase in current might be arising from the
conformational changes of T-PSA during the binding process
on to the attached antibody on the SWCNT surface, which in
turn affects the packing density of the antibody, causing a defect
in the monolayer coverage on electrode surface. These defects
can lead to a higher electron transfer through the electrolyte
and CNTs.
Electrochemical luminescence (ECL)-based immunosensors
have also been developed using CNTs. Wohlstadter et al.
compounded CNT with poly ethylene vinyl acetate (EVA) to
produce a composite and extruded it into sheets that were etched
with acids to introduce carboxyl groups on the surface of CNTs
(50). Streptavidin was coated onto the nanotubes by covalent
coupling and biotinylated anti-AFP (anti-R fetoprotein) antibod-
ies were immobilized on the surface of streptavidin-coated
CNTs. Upon exposure to samples containing AFP, a sandwich
complex formed through the immune reaction between AFP and
anti-AFP-antibody conjugated with ruthenium(II) tris(2,2′-
bipyridine) (Ru(bpy)32+). The complexes were attached to CNT,
leading to a detectable signal for sensing AFP. Ru(bpy)32+ is a
label that can be excited by electron-transfer reactions near the
electrode. It emits a photon (electrochemiluminescence, ECL)
when it relaxes from its excited state to its ground state. It was
found that the value of the ECL signal increased with an increase
in AFP concentration. The sensor can detect as low as 0.1 nM
(7 ng/mL) of AFP.
CNT Alignment and Nanoarray Immunosensor
Development
CNTs usually exist in a randomly entangled state. Flat mat-
like layers of CNTs are utilized in many aforementioned
prototype immunosensors. However, the sensing efficiency
could be relatively low as a result of the random alignment.
Immune reactions, especially those reactions that occur between
antigens and antibodies, require that these two complementary
molecules be orientated appropriately to allow the binding event.
Therefore, it is highly desirable to prepare ordered CNTs such
as the one shown in Figure 7. It is expected that forests or arrays
of CNTs can remarkably enhance the performance of the
sensors. A number of techniques have been developed to align
524 Biotechnol. Prog., 2007, Vol. 23, No. 3

Figure 6. Label-free electrochemical immunosensor based on CNTs (54). Monoclonal antibodies against the total prostate antigen (T-PSA-mAb)
were covalently linked to SWCNTs. The peak current for the intrinsic oxidation of proteins increases when antigen-antibody complex is formed.
Differential pulse voltammogram (left, bottom) when PSA (a), phosphate buffer solution (b), and BSA were incubated, indicating the specific
response of the CNT immunosensor. Calibration curve for T-PSA based on the electrochemical signal from the sensor (bottom, right)

CNTs in a certain type of pattern. CNTs can be arranged by
postsynthesis fabrication or by synthesis-induced alignment
procedures (74, 75). A detailed account on horizontally aligned
and vertically aligned micropatterned CNTs can be found in
Dai et al.’s review (76). Dai and colleagues developed a novel
method for photolithographic generation of perpendicularly
aligned SWCNT arrays with resolution on a micrometer scale
(77). Meyyappan et al. (78) also demonstrated the synthesis of
SWCNT into a large-scale vertically aligned electrode array.
These nanoarrays are further chemically modified, as described
in previous sections, to attach the antibodies to develop
immunosensors.
In addition, certain chemical methods can be employed to
fabricate aligned SWCNT arrays. A wet chemical approach for
organizing SWCNTs on a gold surface using a cystamine self-
assembling technique was developed by Liu et al. (79). Gooding
et al. (48) also reported a technique to vertically align SWCNTs
on the polycrystalline gold surface via a self-assembled mono-
layer of cysteamine. A slightly modified method for SWCNT
alignment using a thioethanol/cystamine mixed monolayer on
a gold surface was developed by Patolsky et al. (80). They used
the EDC-based method for covalently coupling SWCNTs to the
thioethanol/cysteamine mixed monolayer on the gold electrode
surface and attached the proteins to SWCNTs. It was reported
that the length of the aligned nanotubes controls the electron
transfer between the protein and the electrode. The incorporation
of 2-thioethanol on the gold surface was anticipated to prevent
the nonspecific adsorption of surfactant-protected SWCNTs onto
the electrode surface and also to prevent lying of the SWCNT
pipes on the gold surface. SWCNT can act as a nanoconnector
that electrically contacts the active site of enzymes such as
glucose oxidase, peroxidase, and alkaline phosphatase and the
electrode. The electrons are transported along distances greater
than 150 nm, and the rate of electron transport is controlled by
the length of the SWCNT, indicating the compatibility of
SWCNT with the preparation of novel biomaterial hybrid
systems that may have fascinating new properties.
Though there are many reports on CNT vertical array
biosensors for glucose estimation (63, 81, 82), immunosensors
based on nanotube arrays are only a few. O’Connor et al. (42)
reported a method of fabricating vertical array immunosensors
using SWCNTs. Self-assembly of nanotubes was performed on
glass or silicon wafers with native oxide or quartz crystal
microbalance resonators having gold electrodes. Dipping the
electrodes in nafion solution followed by ferric chloride and
then the DMF suspension of SWCNTs led to forest assemblies
of nanotubes by metal-assisted chelation and electrostatic
interactions (42, 47, 83, 84). They demonstrated that a nanosen-
sor can be fabricated using highly oriented SWCNT forests in
a configuration with horseradish peroxidases (HRP) and myo-
globin (Mb) linked to the ends of the nanotubes. It was found
that the SWCNT forest behaved similar to a metal electrically,
conducting electrons from the external circuit to the enzyme.
The sensitivity to the changes in the concentration of H2O2 was
0.049 and 0.033 µA/µM with detection limits of 50 and 70 nM
for SWCNT/HRP and SWCNT/Mb, respectively. Building upon
this approach, they fabricated a protein immunosensor for
amperometric enzyme-linked immunoassays of human serum
albumin (84) (HSA), which can be used in the diagnosis of
kidney function abnormalities, microalbuminuria in cancer
patients, and diabetic nephrosis, where HSA is found to vary
from the normal level (85). Immunosensors were then made
by chemically attaching antibodies to the carboxylated ends of
nanotube forests. A sandwich assay method with secondary
Biotechnol. Prog., 2007, Vol. 23, No. 3 525

Figure 7. Schematic representation of the aligned nanotube with exposed functional groups which can be used to immobilize the antibodies (top,
left). CNTs can be aligned on substrates as forests or arrays. SEM micrograph of carbon nanotubes aligned perpendicular to the substrate (74) (top,
middle) and the CNT arrays (75) (top, right). Similar to the protein microarrays, CNT based antibody arrays can also be fabricated. Schematic
representation of CNT-antibody array fabrication (a), SEM images of CNTs attached with IgG (b, d). Fluorescent labeled immunoarray chip is also
shown (c) (88).

antibodies linked with HRP enzyme was used to generate
electrochemical signal (Figure 8). Using a similar method, PSA
can be detected at levels from 0.02 nM. In this case, bovine
serum albumin and detergent worked best for blocking non-
specific binding. Linearity of response was obtained for 0.6-
30 ng/mL of PSA. The PSA encompasses a range of 4-10 ng/
mL, commonly accepted in clinical practice for prostate
diagnosis and risk assessment. In another experiment, using
similar strategies, the same group immobilized anti-biotin
antibodies on the SWCNT forests by adsorption (42). After
blocking the surface with BSA, a competitive binding step was
performed by incubating biotin and biotin-HRP conjugates
simultaneously on the antibody-immobilized and BSA-blocked
SWCNT surface. It was found that, in the presence of the
soluble mediator hydroquinon (1 mM), unlabeled biotin can be
detected by using this competitive assay with a detection
limit of 16 µM. Hydroquinon is one of the efficient electron
donors to HRP with a reaction rate as high as 1.2 × 107 M-1
s-1. However, such methods, though found to have good
sensitivity in laboratory studies, suffer the major set back that
soluble mediators need to be incorporated in the assays, which
will be an obstacle in commercial development of the immu-
nosensors.
Another threshold area for the development of immunosensors
is based on cantilevers; CNTs have the ability to act as the
cantilevers (86). Upon binding of molecules to the CNT
cantilever, there will be transduction of the molecular binding
event into an electrical signal based on the difference in
oscillating frequency between the fundamental mode of the
cantilever and the one corresponding to the molecule attached
cantilever. By measuring the frequency shift, one could obtain
precise information about the mass of the attached molecule.
Using CNT, mass measurements in femtograms could be
achieved. Figure 9 shows the resonance of a carbon particle
that is attached to the end of a nanotube. Likewise, with the
antibody (Ab) attached to a substrate and another attached to
the tip of a cantilever above it, antigen (Ag) floating through
the solution can bind to one of the Ab, and the cantilever can
bend down, piezoelectrically, in order to sandwich the three
together. When Ab’s and Ag’s bind to one another, it takes a
certain amount of force to pull the pair apart, depending on the
Ag-Ab in study. V-Shaped cantilevers are being researched for
the detection of antigens such as small pox, anthrax, HIV, etc.
(87). This nanobalance method can be applied to other particles
of similar dimensions, such as viruses.
Microarray-like chips modified with CNTs can be fabricated
for various bioapplications (Figure 8). Byon et al. (88) have
reported a BSA-free protein chip with a carbonyldiimidazole
activated Tween 20 (CDI-Tween20) functionalized SWCNT
film. A simple one-step treatment with CDI-Tween 20 on high
yield CVD-grown SWCNT film allowed both the immobiliza-
tion of proteins and the suppression of nonspecific binding.
Protein microspots on such chips were reproducible and
homogeneous. The optimal concentration for the immobilization
of probe protein was determined using the specific binding of
Cy3-IgG (20 µg/mL) to the Protein A spotted at various
concentrations, and it was reported that saturation occurs at
around 250 µg/mL.
526 Biotechnol. Prog., 2007, Vol. 23, No. 3

Figure 8. Electrochemical immunosensors with enzyme labels: schematic representation of a sandwich electrochemical CNT immunosensor
(A) (47). Amplification of sensitivity on such sensors can be achieved by employing CNT-based immuno-nanotubes, which contain both the
secondary antibody and the enzyme label (B). Immunosensor employing this amplification strategy (C) was used for the detection of PSA antigen
(D) (91).

Antibody bound to the carbon nanotube can also help in
nanofabrication of devices. In one such design, by Matsui’s
group (89), an array of antigens were immobilized onto the
shaved regions of the alkyl thiol self-assembled monolayer using
the AFM tip on gold substrates. CNTs with antibody (IgG)
adsorbed on surface were selectively attached on to the antigen
regions in the gold device (Figure 10). By this technique,
multiple blocks with a variety of protein functions can be
directed to specific locations on a chip easily, as in development
of multiplex immunosensors.
Sensitivity and Stability of CNT-Based Immunosensors
The relatively higher sensitivity of the CNT immunosensor
can be attributed manly to the higher antibody loading ability
of these devices. Whereas tremendous amount of researches
have been carried out in this field, most of the immunodiagnostic
devices, mainly the biosensors, are yet to be demonstrated
successfully on a commercial platform. It should be noted
that most of the research works published in these years fail to
give adequate information on the stability, repeatability, and
reusability of the CNT biosensors. Though the biomolecules
employed in the development of the sensor are ligand-specific,
one of the challenges in the development of a successful
diagnostic device is posed by the highly viscous nature of
biological samples such as blood and the vast number of
interferents present in samples. Hence, it is very crucial that
studies are performed in this direction.
Wang et al. (90) developed a CNT-based amplified bioelec-
tronic protocol involving antigen-antibody binding along with
magnetic separation of the analyte-linked magnetic bead-CNT
Biotechnol. Prog., 2007, Vol. 23, No. 3
Figure 9. Resonance vibrations of a nanotube loaded with a spheroidal
carbon particle. From the resonance frequency, the mass of the particle
can be detected (86).
assembly followed by enzyme amplification and chronopoten-
tiometric stripping detection of the product at the CNT-modified
electrode. Glossy carbon electrodes were modified using carbon
nanotubes that were linked to alkaline phosphatase (ALP) using
EDC coupling chemistry. The capture of ALP-loaded CNT tags
to the streptavidin modified magnetic beads was carried out by
an antibody-antigen-antibody interaction, which was quanti-
tated as an enzymatic electrochemical reaction on the electrode
surface (Figure 11). There was almost 104-fold improvement
in the sensitivity of CNT-based dual amplification compared
to the conventional biosensors based on a single enzyme. The
high sensitivity is attributed to the high alkaline phosphatase
loading per CNT, which was estimated to be 9,600 molecules
of ALP per CNT. This method had a detection limit of 500
fg/mL and reproducible signals.
In the case of electrochemical immunosensors, the sensitivity
can be affected by soluble mediators such as hydroquinon
employed during the assay. The non-mediated sandwich am-
perometric immunosensor developed by Yu et al. (47) was found
to have a detection limit of 75 nM, and its sensitivity was about
0.004 nA/pmol/mL HSA, whereas these values were 1 nM and
46 nA/pmol/mL, respectively, for the hydroquinon-mediated
assay. The electron mediation of the immunosensors by water-
soluble hydroquinone (HQ) dramatically lowered the detection
limit to 1 nM, providing significantly better performance than
alternative methods. To improve the sensitivity without using
the mediators, Yu et al. recently came up with another strategy
by using immunonanotubes as one of the reagents added during
the detection step (91). In a sandwich assay on a CNT forest
electrode, the labeled secondary labeled antibody was replaced
with MWCNT loaded with multiple labels and secondary
antibody molecules (Figure 8). Amplification of the signal in
this case improves the sensitivity, and such a system could detect
PSA at 4 pg/mL level. At that level of sensitivity, nanotube
sensors can be used to detect rare biomarkers that are now
beyond the ability of current sensors.
Improved fabrication of these SWNT forests on pyrolytic
graphite surfaces utilized aged nanotube dispersions provided
higher nanotube density and conductivity. Raman spectroscopy
of the forests suggested fewer sidewall defects on the aged
nanotubes allowing them to form more densely packed forests.
Enhanced performance with aged nanotubes was monitored
using SWCNT forests with bound peroxidase
Most of the published articles discussed so far mainly focused
on the development of prototype devices for bioapplications,
527
but studies on the effect of interferents, stability of the device,
reusability, and repeatability are scarce. Table 1 lists a number
of CNT-based prototype immunosensors and their lower detec-
tion limits reported in recent years. We have not come across
any reports on any commercial CNT-based immunosensors for
clinical diagnosis or lab-on-chip applications.
Future Directions and Challenges in CNT-Based
Immunosensors
Although CNT-based immunosensors have been extensively
explored, there are a number of challenges that need to be
addressed in order to fabricate immunosensors using CNTs.
First, an increase in sensitivity and lower detection limits
reported must be realized in real-time applications. To achieve
these goals, some soluble mediators (such as hydroquinon) can
be used to further increase the sensitivity of the electrochemical
immunosensors. Exploring conductive polymers for fabricating
immunosensors can also help to replace the soluble mediators
and to manufacture reagent-less immunosensors. On the other
hand, the complex nature of biological samples warrants rugged
sensing systems, for samples such as whole blood or sputum
involve many components. Hence, apart from the specificity
of the binding, due consideration must be given to the
nonspecific binding, the effect of varying viscosity, and tem-
perature of the samples during sensing.
Most of the research in recent times focused on single-walled
CNTs, but multiple-walled CNTs can also be useful for smart
material and sensor application. They might be advantageous
with its larger diameter and stiffness.
Disposable CNT Immunosensors. Regeneration of the
antigen capturing site on the CNT immunosensors and recali-
bration is usually a difficult task. Removal of previously bound
antigen and the percentage availability of active sites after each
step of analysis needs to be assessed with each sensor as the
variation from case to case cannot be standardized. Considering
this situation, the major focus for the CNT immunosensors must
be on disposable immunochips. The bulk production of nano-
tubes and other reagents at a cheaper rate can open up the
possibility of disposable CNT immunosensors. Another area that
needs to be explored for commercial success of the immun-
osensors is the ready availability of specific antibodies and
antigens to be immobilized. Research in protein science and
antibody engineering can lead to the production of more stable
antibodies that can tolerate harsh immobilization treatments,
exhibit stability at solid phase on the CNT electrodes, and retain
the antigen binding ability for longer time periods. Alternative
methods are being explored in immunosensor fabrication.
Semisynthetic antibodies or antibody fragments, aptamers
(single-stranded DNA or RNA oligonucleotide sequences with
the capacity to recognize various target molecules with high
affinity and specificity), and molecular imprinted polymers
(MIP) can find application as the biocomponent in the next
generation of sensors. Recent reports indicate that aptamers that
have specific affinity toward the antigens can be advantageous
over monoclonal antibodies in developing the CNT-based
immunosensors (70). Another field of interest is the detection
of bioterrorism. The current strategy for detection includes fiber-
optic tubes lined with antibodies coupled to light-emitting
molecules. Development of CNT-based immunosensors can lead
to an alternative technology for highly sensitive and fast
detection of hazardous pathogens such as anthrax, botulin,
Salmonella, and ricin.
Miniaturized, Multiplexed Sensors for Point-of-Care Test-
ing. CNTs allow for miniaturization of the immunosensors.
528 Biotechnol. Prog., 2007, Vol. 23, No. 3

Figure 10. Fabrication of gold substrate with antibody-nanotube assembly on the complementary antigen substrates via biological recognition.
Self-assembled monolayer (SAM) of alkylthiol on gold (Au) substrates was shaved off by using an AFM tip to form trenches, and antigens are
deposited on the trenches. Nanotube with surface adsorbed antibody was immobilized onto the complementary antigen regions via the biological
recognition (89).

Figure 11. Amplification of biosensor detection by CNT. Capture of ALP-loaded CNT to the streptavidin-labeled magnetic beads (MB) by Ab-
Ag-Ab interaction and the consecutive electrochemical detection of the enzymatic reaction on the CNT modified glassy carbon electrode leads to
better sensitivity (90).

Table 1. Summary of Significant Analytes Detected Using CNT Immunosensors

analyte sensor details detection limit ref
anti-hCG SWCNT with hCG, FET 1.0 pM (67)
U1A-specific monoclonal antibodies SWCNT-FET with recombinant U1A autoantigen 1.0 nM (56)
AFP CNT with anti-AFP antibody, electrochemical 0.1 nM (50)
HSA SWCNT forest with anti-HSA antibody, amperometric 1.0 nM (47)
IgG SWCNT, with protein A, FET 1.0 pM (67)
PSA SWCNT with anti-PSA antibody, electrochemical, label-free 0.25 ng/mL (54)
PSA SWCNT with anti-PSA antibody, FET 5.0 ng/mL (68)
CEA SWCNT with anti-CEA monoclonal antibody, FET 54 pg/mL (53)
anti-hemagglutinin SWCNT with hemagglutinin, FET 0.05 ng/mL (96)
IgG CNT, electrochemical with ALP label 500 fg/mL (90)
Currently, there is great interest in high-throughput multiplex This can replace the time-consuming ELISA-based tests by
assays. Advances in CNT synthesis and fabrication of CNT eliminating multiple washing steps, thereby significantly short-
arrays open up new possibilities of developing the microelec- ening the sample analysis time. Furthermore, CNT immunosen-
trodes wherein many antibodies with adequate concentration sors can be an integral part of the “lab-on-chip” devices for
can be “spotted” on CNT array chips. The CNTs contribute to point-of-care testing. Designing the lab-on-chip CNT immun-
the large surface area for antibody immobilization per unit area osensors can be further explored for online sample monitoring.
of the chip. In such antibody microarrays, each antibody spot In addition, miniaturized portable designs can be employed for
allows simultaneous detection and quantitation of analytes from the development of wearable immunosensors that can detect
a complex mixture of sample. Such devices might find applica- specific molecules during field experiments and in the battlefield
tions in the detection of drugs of abuse or disease biomarker as well. Most of the rapid immunoassays currently available in
screening as in cancer, etc. Miniaturization of the CNT-based market are either qualitative or semiquantitative in nature. CNT-
immunosensors can allow the integration of the various steps based immunosensors have the potentials to bridge this gap.
of the immunoassays into one compact single sensor. In the Implantable Immunosensors. The development of implant-
current scenario, the majority of the clinical assays are tedious able sensors capable of real-time monitoring of clinically
and time-consuming and require trained laboratory personnel important molecules remains a major challenge. CNT-based
to carry out the tests. Doctors and patients can avoid delays in immunosensors offer new opportunities for developing these
assessing the condition and prescribing treatment if the results implantable sensors for disease control and closed-loop drug
of clinical assays can be made available with point-of-care delivery. Apart from the fluorescence-based SWCNT optical
testing devices. Integration of the CNT immunoelectrodes with biosensors, no other research in this direction has been reported
microelectronics is another threshold area in developing robust (92, 93). Though CNTs are projected as an ideal candidate for
sensors. Recent developments in microfluidics and nanofabri- the immunosensors, their suitability as implantable miniaturized
cation give options for sophisticated immunoassays being sensors can be realized only after establishing the cytotoxic
performed with relatively lower sample volume and reagents. effects, stability, and biocompatibility. Current reports on the
Biotechnol. Prog., 2007, Vol. 23, No. 3
cytotoxic effects are contradictory in nature. Carbon nanotubes
and other nanoparticles possess a small size, large surface area,
and the ability to generate reactive oxygen species (ROS). Thus,
as the particle size reduces, its tendency for pulmonary toxicity
increases. However, coating with various materials, surface
excitation by UV radiation, particle aggregation, etc. can modify
the effects due to particle size. Pharyngeal aspiration of pristine
SWCNT can induce robust acute inflammatory reaction with
the very early onset of a fibrinogenic response and the formation
of granulomas in mice (94). Even 2 months after aspiration,
the presence of nanotubes was observed in the pulmonary
granulamatous lesion. On the contrary, functionalized SWCNTS
are reported to be less toxic than pristine SWCNTs (95).
Pantarotto’s group recently reported that ammonium-function-
alized single- and multiwalled CNTs followed a rapid first-order
clearance from the blood compartment through renal excretion
route without any toxic side effects or mortality (95). Hence, a
cautious approach is necessary in case of implantable biosensors
until the in ViVo effects are unambiguously characterized.
Biofouling of implantable sensors and the effect of complex
tissue architecture under in ViVo conditions also need to be
considered here. Fabrication and integration of the miniaturized
signal transmission devices along with implantable CNT im-
munosensors is also a major engineering challenge. Neverthe-
less, we can anticipate that CNTs will emerge as the prime
materials for making miniaturized immunosensors in the near
future.
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Received August 8, 2006. Accepted April 6, 2007.
BP0602395