Cabon Nanotube

Quantitative lateral flow immunosensor using carbon nanotubes as label
Adeyabeba Abera and Jin-Woo Choi*
Received 28th June 2010, Accepted 26th August 2010
DOI: 10.1039/c0ay00412j
A novel immunosensor utilizing multi-walled carbon nanotube as label on a lateral flow system for
simple and quantitative electrical detection was investigated. Multi-walled carbon nanotubes
(MWCNTs) were first modified with polyvinylpyrrolidone (PVP) for uniform dispersion in aqueous
solution. Then, the MWCNTs were conjugated with human immunoglobulin G (IgG) using 1-(3-
(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC) coupling chemistry. The lateral
flow immunosensor was made of nitrocellulose membrane that transports sample and reagents through
a porous structure by capillary action. The performance of the immunosensor was demonstrated using
human IgG as a model analyte competing with the conjugate (MWCNT-labeled human IgG) at the
capture zone. As a result of binding reaction between the conjugate and the immobilized Protein A at
the capture zone, the conjugated MWCNTs formed a conducting network at the capture zone
providing conductance measurement corresponding to the amount of captured conjugate. Quantitative
immunoassay response for the target human IgG was demonstrated in the range of 25 to 200 μg mlÀ1
without an additional amplification step. The presented immunosensing technique could be expanded
for the detection of various analyte-specific biomolecules with a potential for simple and rapid tests
suitable for point-of-care diagnostics.
1. Introduction
Lateral flow (LF) immunoassay is one of the most successful
point-of-care diagnostic tools commercially available for detecting
various health and environmental agents.1–6
A lateral flow
immunosensor is made of a porous membrane for both immobi-
lizing biological receptors and transporting sample reagents.
Sample reagents are transported in the lateral direction through
the porous membrane by capillary force and captured by the
biological receptors immobilized at the capture zone. LF immu-
nosensors are simple to use, low-cost, rapid, and portable with
relatively long shelf life. Examples of lateral flow diagnostics
include home pregnancy tests, detection of drugs of abuse, diag-
nosis of infectious diseases, and cancer markers. However, such
systems often suffer from low sensitivity, and are mostly restricted
to qualitative or semi-quantitative measurements.
Generally, LF immunosensors are based on optical detection
where tagged particles or enzymes provide visible signal generation
or amplification. Typically, colloidal gold is used as a label for
colorimetric detection traced by the visible color. Several efforts
have been made towards signal quantification using enhanced
colorimetric labels or conducting materials such as phosphorescent
nanoparticles,7 polyaniline,8 and polyaniline with colloidal gold.9
Colorimetric techniques are often limited by low sensitivity while
fluorescent techniques suffer from instability, photobleaching,
activation step, and requirement for a complicated reader device.
This work addresses such limitations by utilizing carbon
nanotubes (CNTs) as labeling material for direct electrical signal
measurement. CNTs have remarkable one-dimensional elec-
tronic conduction properties and large surface area, and allow
the adsorption of biomolecules for sensing.10–12 In order to utilize
these properties of carbon nanotubes for biosensing applications,
surface functionalization is necessary to uniformly disperse
CNTs in aqueous solution and immobilize biorecognition
molecules on the surface of the nanotubes. Adsorption of
surfactants and polymers on the surface of CNTs is considered as
an effective method to enhance the dispersion of CNTs without
affecting their properties.13–15 Compared to CNTs, other con-
ducting materials such as colloidal gold and polymer nanowires
do not possess the unique material properties suitable for surface
functionalization or maintain high electrical conduction after
binding reaction.9,16
In this work, a new immunoassay technique using carbon
nanotube labels on lateral flow system is introduced. The illus-
tration of this technique is shown in Fig. 1. MWCNTs are
dispersed in water with PVP using sonication. Adsorption of
PVP provides a thin and rough layer of organic coating by
wrapping around the MWCNTs. Human IgG is immobilized on
the bare (not covered by PVP) surface of the MWCNTs. The
MWCNT–human IgG conjugate is then applied on a lateral flow
immunosensor where the MWCNT–human IgG conjugate is to
be captured by immobilized Protein A at the capture zone. The
conjugated MWCNTs form a conducting network at the capture
zone corresponding to the amount of binding. Consequently,
quantitative signal can be obtained by simple resistance
measurement of the capture zone. Blocking schemes were also
implemented on the MWCNTs and the lateral flow strips to
address non-specific interaction.
2. Methods and experiments
2.1 Materials and reagents
Carboxylated multi-walled carbon nanotubes (purity > 95 wt%)
were obtained from Cheaptubes, Inc. (Brattleboro, VT). Hi-flow
Department of Electrical and Computer Engineering, Louisiana State
University, Baton Rouge, LA, 70803, USA; Web: www.ece.lsu.
edu/biomems/. E-mail: choi@ece.lsu.edu; Tel: +1-225-578-8764
This journal is © The Royal Society of Chemistry 2010
Anal. Methods, 2010, 2, 1819–1822 | 1819
TECHNICAL NOTE
www.rsc.org/methods | Analytical Methods
Downloaded on 30 December 2012
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J
View Article Online / Journal Homepage / Table of Contents for this issue
Page 2
Plus nitrocellulose membranes (HF13502), glass fiber pads,
cellulose membranes, and a supporting card for lateral flow assay
were obtained from Millipore (Bedford, MA). Protein A sus-
pended in solution was obtained from BioVision, Inc. (Mountain
View, CA). Purified human immunoglobulin G (IgG, 6.2 mg mlÀ1)
suspended in 0.01 M sodium phosphate (0.15 M NaCl, pH 7.4)
was obtained from Sigma-Aldrich (St Louis, MO). Poly-
vinylpyrrolidone (PVP, Mw 10 000), bovine serum albumin (BSA),
Tween-20, glutaraldehyde, and 2-(N-morpholino)ethanesulfonic
acid (MES) were from Sigma-Aldrich. 1-(3-(Dimethylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-
hydroxysulfosuccinimide (sulfo-NHS) were obtained from
Thermo Fisher Scientific, Inc. (Rockford, IL). All immunore-
agents were diluted in pH 7.4 phosphate saline buffer (PBS) unless
noted otherwise.
2.2 MWCNT–antibody conjugation
In order to start the conjugation process, 1.5 mg of carboxylated
MWCNTs were dispersed in 2 ml of deionized (DI) water with 0.6
mg of PVP. The solution was sonicated for 4 hours in an ultra-
sonic bath at 27 C to exfoliate the MWCNTs and accelerate the
dispersion. The dispersed solution was mixed with 1 ml of EDC
(0.4 M) and sulfo-NHS (0.1 M) in pH 6.1 MES buffer. The
solution was left to react for 30 minutes with gentle agitation at
room temperature. The mixture was microcentrifuged at 13 000
rpm for 3 minutes with PBS buffer and the supernatant was dis-
carded. The washing and re-suspending process was repeated
three times to remove excess reagents. Human IgG solution was
prepared at 200 μg mlÀ1 by diluting with PBS buffer for conju-
gation. The solution containing functionalized MWCNTs was
then mixed with 100 μl of the human IgG solution and left to react
overnight at 4 C. This mixture was centrifuged at 13 000 rpm for
3 minutes and the supernatant was discarded. The washing and re-
suspending process was repeated three times to remove unbound
human IgG, and the resulting MWCNT–antibody conjugate
solution was kept at 4 C until use.
2.3 Preparation of lateral flow immunosensors
A nitrocellulose membrane was used as a detection pad with
designated capture zone for immobilizing Protein A. Fiber glass
and cellulose membranes were used for sample application and
for absorption of excess reagents by placing them at the two ends
of the nitrocellulose membrane, respectively. The LF immuno-
strips were prepared following general procedure reported else-
where.7,9 First, a nitrocellulose membrane was saturated with
10% (v/v) methanol in deionized (DI) water for 30 minutes to
remove any residue and dried in air at room temperature. The
membrane was then treated with 0.5% (v/v) glutaraldehyde
solution to strengthen the immobilization of Protein A on the
nitrocellulose membrane and rinsed with DI water. After drying,
Protein A diluted in PBS buffer to 1 mg mlÀ1 was immobilized at
a designated capture zone by dragging a dispenser with manual
control. The membrane was then incubated with 2% BSA and
0.05% (v/v) Tween-20 in PBS for 1 hour in order to block the
membrane from making unwanted binding. The membrane was
rinsed with PBS containing 0.05% (v/v) Tween-20 to remove the
excess blocking reagent and left to dry at room temperature.
3. Results and discussion
A lateral flow immunosensor was developed by assembling
a lateral flow strip with sample application, detection, and
absorbent pads. The specific binding reaction was based on
immobilized Protein A and human IgG that was detected by the
conjugated MWCNT labels. Since CNTs have inherent affinity
to biological elements causing non-specific binding, control tests
were conducted at different functionalization steps. First, the
PVP treated MWCNTs were applied to the LF immunosensors
to bind with immobilized Protein A. As a result, unwanted
binding of the MWCNTs with Protein A at the capture zone was
observed as shown in Fig. 2(a). This non-specific binding could
have been caused by the affinity of the exposed MWCNTs
surface to the Protein A. Next, the crosslinking agents, EDC and
sulfo-NHS, were added to the PVP treated carbon nanotubes
and the same test was repeated. In this case, no binding reaction
was observed between the MWCNTs and Protein A at the
capture zone as shown in Fig. 2(b). This result showed that the
surfaces of the MWCNTs were completely covered by the PVP
and the amide coupling agents limiting unoccupied sites from
non-specific binding. Finally, MWCNT–IgG conjugate solution
was applied, where binding between human IgG and Protein A
was evident at the capture zone as shown in Fig. 2(c). Scanning

electron microscopy (SEM) was used to examine the capture
zone, where the MWCNTs formed a conducting network as
a result of binding. Fig. 3 shows SEM image of the nitrocellulose
membrane without MWCNT labels and with varying concen-
tration of MWCNT labels corresponding to the amount of
binding.
Prior to the immunoassay test, varying concentration of the
conjugate solutions ranging from 0 to 200 μg mlÀ1 was prepared
by dilution in PBS buffer in order to determine the detection limit
of the conjugate solution. 50 μl of each solution were applied to
the LF immunosensors where the conjugated human IgG was
captured by the Protein A at the capture zone resulting in
colorimetric signal as shown in Fig. 4. As depicted in Fig. 4, the
color intensity of the signal was stronger for higher concentration
of the conjugated human IgG. For quantitative analysis of the
binding reaction, the resistance across the capture line was
measured after the complete development of the visible signal
($15 minutes). The measured electrical resistance decreased as
the concentration of the MWCNT–human IgG conjugate
increased due to the increased amount of MWCNTs forming
a conducting network. Three replications of the immunosensors
with same conjugate concentration were tested for consistency of
the measurements. The electrical resistance was measured using
electrodes with 3 mm spacing and while maintaining same
contact resistance.
The developed immunosensor was used with increasing
concentration of human IgG as target analyte in solution.
Concentration of the human IgG target analyte ranged from 25
to 200 μg mlÀ1 and the concentration of the conjugate solution
was fixed at 200 μg mlÀ1. Each target analyte solution was briefly
mixed with the conjugate and then the mixture was applied to the
LF immunosensor as a one-step immunoassay process. As the
sample solution moved across the capture zone, a competitive
immunoassay reaction between the conjugated human IgG and
those in the solution occurred. This produced a detection signal
by the MWCNTs across the capture line varying in color
intensity that was inversely proportional to the amount of
human IgG target analyte captured as shown in Fig. 5(a). The
detection signals were visible with the application of only 20 μl,
and saturation occurred with the application of 50 μl of total
sample solutions within 15 minutes. The visibility of the capture
zone decreased with increasing target human IgG concentration.
As the concentration of the target analyte increased, it occupied
more binding sites limiting the binding reaction between the
conjugated human IgG and the Protein A. The corresponding
measured results in Fig. 5(b) clearly show that the electrical
resistance increased with increasing concentration of the target
human IgG. Although the visibility of capture lines decreased with
increasing target IgG concentration (100, 150, and 200 μg mlÀ1)

Quantitative lateral flow immunosensor using carbon nanotubes as label
Adeyabeba Abera and Jin-Woo Choi*
Received 28th June 2010, Accepted 26th August 2010
DOI: 10.1039/c0ay00412j
A novel immunosensor utilizing multi-walled carbon nanotube as label on a lateral flow system for
simple and quantitative electrical detection was investigated. Multi-walled carbon nanotubes
(MWCNTs) were first modified with polyvinylpyrrolidone (PVP) for uniform dispersion in aqueous
solution. Then, the MWCNTs were conjugated with human immunoglobulin G (IgG) using 1-(3-
(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC) coupling chemistry. The lateral
flow immunosensor was made of nitrocellulose membrane that transports sample and reagents through
a porous structure by capillary action. The performance of the immunosensor was demonstrated using
human IgG as a model analyte competing with the conjugate (MWCNT-labeled human IgG) at the
capture zone. As a result of binding reaction between the conjugate and the immobilized Protein A at
the capture zone, the conjugated MWCNTs formed a conducting network at the capture zone
providing conductance measurement corresponding to the amount of captured conjugate. Quantitative
immunoassay response for the target human IgG was demonstrated in the range of 25 to 200 μg mlÀ1
without an additional amplification step. The presented immunosensing technique could be expanded
for the detection of various analyte-specific biomolecules with a potential for simple and rapid tests
suitable for point-of-care diagnostics.
1. Introduction
Lateral flow (LF) immunoassay is one of the most successful
point-of-care diagnostic tools commercially available for detecting
various health and environmental agents.1–6
A lateral flow
immunosensor is made of a porous membrane for both immobi-
lizing biological receptors and transporting sample reagents.
Sample reagents are transported in the lateral direction through
the porous membrane by capillary force and captured by the
biological receptors immobilized at the capture zone. LF immu-
nosensors are simple to use, low-cost, rapid, and portable with
relatively long shelf life. Examples of lateral flow diagnostics
include home pregnancy tests, detection of drugs of abuse, diag-
nosis of infectious diseases, and cancer markers. However, such
systems often suffer from low sensitivity, and are mostly restricted
to qualitative or semi-quantitative measurements.
Generally, LF immunosensors are based on optical detection
where tagged particles or enzymes provide visible signal generation
or amplification. Typically, colloidal gold is used as a label for
colorimetric detection traced by the visible color. Several efforts
have been made towards signal quantification using enhanced
colorimetric labels or conducting materials such as phosphorescent
nanoparticles,7 polyaniline,8 and polyaniline with colloidal gold.9
Colorimetric techniques are often limited by low sensitivity while
fluorescent techniques suffer from instability, photobleaching,
activation step, and requirement for a complicated reader device.
This work addresses such limitations by utilizing carbon
nanotubes (CNTs) as labeling material for direct electrical signal
measurement. CNTs have remarkable one-dimensional elec-
tronic conduction properties and large surface area, and allow
the adsorption of biomolecules for sensing.10–12 In order to utilize
these properties of carbon nanotubes for biosensing applications,
surface functionalization is necessary to uniformly disperse
CNTs in aqueous solution and immobilize biorecognition
molecules on the surface of the nanotubes. Adsorption of
surfactants and polymers on the surface of CNTs is considered as
an effective method to enhance the dispersion of CNTs without
affecting their properties.13–15 Compared to CNTs, other con-
ducting materials such as colloidal gold and polymer nanowires
do not possess the unique material properties suitable for surface
functionalization or maintain high electrical conduction after
binding reaction.9,16
In this work, a new immunoassay technique using carbon
nanotube labels on lateral flow system is introduced. The illus-
tration of this technique is shown in Fig. 1. MWCNTs are
dispersed in water with PVP using sonication. Adsorption of
PVP provides a thin and rough layer of organic coating by
wrapping around the MWCNTs. Human IgG is immobilized on
the bare (not covered by PVP) surface of the MWCNTs. The
MWCNT–human IgG conjugate is then applied on a lateral flow
immunosensor where the MWCNT–human IgG conjugate is to
be captured by immobilized Protein A at the capture zone. The
conjugated MWCNTs form a conducting network at the capture
zone corresponding to the amount of binding. Consequently,
quantitative signal can be obtained by simple resistance
measurement of the capture zone. Blocking schemes were also
implemented on the MWCNTs and the lateral flow strips to
address non-specific interaction.
2. Methods and experiments
2.1 Materials and reagents
Carboxylated multi-walled carbon nanotubes (purity > 95 wt%)
were obtained from Cheaptubes, Inc. (Brattleboro, VT). Hi-flow
Department of Electrical and Computer Engineering, Louisiana State
University, Baton Rouge, LA, 70803, USA; Web: www.ece.lsu.
edu/biomems/. E-mail: choi@ece.lsu.edu; Tel: +1-225-578-8764
This journal is © The Royal Society of Chemistry 2010
Anal. Methods, 2010, 2, 1819–1822 | 1819
TECHNICAL NOTE
www.rsc.org/methods | Analytical Methods
Downloaded on 30 December 2012
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J
View Article Online / Journal Homepage / Table of Contents for this issue
Page 2
Plus nitrocellulose membranes (HF13502), glass fiber pads,
cellulose membranes, and a supporting card for lateral flow assay
were obtained from Millipore (Bedford, MA). Protein A sus-
pended in solution was obtained from BioVision, Inc. (Mountain
View, CA). Purified human immunoglobulin G (IgG, 6.2 mg mlÀ1)
suspended in 0.01 M sodium phosphate (0.15 M NaCl, pH 7.4)
was obtained from Sigma-Aldrich (St Louis, MO). Poly-
vinylpyrrolidone (PVP, Mw 10 000), bovine serum albumin (BSA),
Tween-20, glutaraldehyde, and 2-(N-morpholino)ethanesulfonic
acid (MES) were from Sigma-Aldrich. 1-(3-(Dimethylamino)-
propyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-
hydroxysulfosuccinimide (sulfo-NHS) were obtained from
Thermo Fisher Scientific, Inc. (Rockford, IL). All immunore-
agents were diluted in pH 7.4 phosphate saline buffer (PBS) unless
noted otherwise.
2.2 MWCNT–antibody conjugation
In order to start the conjugation process, 1.5 mg of carboxylated
MWCNTs were dispersed in 2 ml of deionized (DI) water with 0.6
mg of PVP. The solution was sonicated for 4 hours in an ultra-
sonic bath at 27 C to exfoliate the MWCNTs and accelerate the
dispersion. The dispersed solution was mixed with 1 ml of EDC
(0.4 M) and sulfo-NHS (0.1 M) in pH 6.1 MES buffer. The
solution was left to react for 30 minutes with gentle agitation at
room temperature. The mixture was microcentrifuged at 13 000
rpm for 3 minutes with PBS buffer and the supernatant was dis-
carded. The washing and re-suspending process was repeated
three times to remove excess reagents. Human IgG solution was
prepared at 200 μg mlÀ1 by diluting with PBS buffer for conju-
gation. The solution containing functionalized MWCNTs was
then mixed with 100 μl of the human IgG solution and left to react
overnight at 4 C. This mixture was centrifuged at 13 000 rpm for
3 minutes and the supernatant was discarded. The washing and re-
suspending process was repeated three times to remove unbound
human IgG, and the resulting MWCNT–antibody conjugate
solution was kept at 4 C until use.
2.3 Preparation of lateral flow immunosensors
A nitrocellulose membrane was used as a detection pad with
designated capture zone for immobilizing Protein A. Fiber glass
and cellulose membranes were used for sample application and
for absorption of excess reagents by placing them at the two ends
of the nitrocellulose membrane, respectively. The LF immuno-
strips were prepared following general procedure reported else-
where.7,9 First, a nitrocellulose membrane was saturated with
10% (v/v) methanol in deionized (DI) water for 30 minutes to
remove any residue and dried in air at room temperature. The
membrane was then treated with 0.5% (v/v) glutaraldehyde
solution to strengthen the immobilization of Protein A on the
nitrocellulose membrane and rinsed with DI water. After drying,
Protein A diluted in PBS buffer to 1 mg mlÀ1 was immobilized at
a designated capture zone by dragging a dispenser with manual
control. The membrane was then incubated with 2% BSA and
0.05% (v/v) Tween-20 in PBS for 1 hour in order to block the
membrane from making unwanted binding. The membrane was
rinsed with PBS containing 0.05% (v/v) Tween-20 to remove the
excess blocking reagent and left to dry at room temperature.
3. Results and discussion
A lateral flow immunosensor was developed by assembling
a lateral flow strip with sample application, detection, and
absorbent pads. The specific binding reaction was based on
immobilized Protein A and human IgG that was detected by the
conjugated MWCNT labels. Since CNTs have inherent affinity
to biological elements causing non-specific binding, control tests
were conducted at different functionalization steps. First, the
PVP treated MWCNTs were applied to the LF immunosensors
to bind with immobilized Protein A. As a result, unwanted
binding of the MWCNTs with Protein A at the capture zone was
observed as shown in Fig. 2(a). This non-specific binding could
have been caused by the affinity of the exposed MWCNTs
surface to the Protein A. Next, the crosslinking agents, EDC and
sulfo-NHS, were added to the PVP treated carbon nanotubes
and the same test was repeated. In this case, no binding reaction
was observed between the MWCNTs and Protein A at the
capture zone as shown in Fig. 2(b). This result showed that the
surfaces of the MWCNTs were completely covered by the PVP
and the amide coupling agents limiting unoccupied sites from
non-specific binding. Finally, MWCNT–IgG conjugate solution
was applied, where binding between human IgG and Protein A
was evident at the capture zone as shown in Fig. 2(c). Scanning
Fig. 1 Illustration of the detection principle using carbon nanotubes as label for quantitative lateral flow immunosensing in competitive format. Carbon
nanotubes form a conducting network at the capture zone corresponding to the amount of binding reaction. Consequently, quantitative signal can be
obtained by simple resistance measurement of the capture zone.
1820 | Anal. Methods, 2010, 2, 1819–1822
This journal is © The Royal Society of Chemistry 2010
Downloaded on 30 December 2012
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J
View Article Online
Page 3
electron microscopy (SEM) was used to examine the capture
zone, where the MWCNTs formed a conducting network as
a result of binding. Fig. 3 shows SEM image of the nitrocellulose
membrane without MWCNT labels and with varying concen-
tration of MWCNT labels corresponding to the amount of
binding.
Prior to the immunoassay test, varying concentration of the
conjugate solutions ranging from 0 to 200 μg mlÀ1 was prepared
by dilution in PBS buffer in order to determine the detection limit
of the conjugate solution. 50 μl of each solution were applied to
the LF immunosensors where the conjugated human IgG was
captured by the Protein A at the capture zone resulting in
colorimetric signal as shown in Fig. 4. As depicted in Fig. 4, the
color intensity of the signal was stronger for higher concentration
of the conjugated human IgG. For quantitative analysis of the
binding reaction, the resistance across the capture line was
measured after the complete development of the visible signal
($15 minutes). The measured electrical resistance decreased as
the concentration of the MWCNT–human IgG conjugate
increased due to the increased amount of MWCNTs forming
a conducting network. Three replications of the immunosensors
with same conjugate concentration were tested for consistency of
the measurements. The electrical resistance was measured using
electrodes with 3 mm spacing and while maintaining same
contact resistance.
The developed immunosensor was used with increasing
concentration of human IgG as target analyte in solution.
Concentration of the human IgG target analyte ranged from 25
to 200 μg mlÀ1 and the concentration of the conjugate solution
was fixed at 200 μg mlÀ1. Each target analyte solution was briefly
mixed with the conjugate and then the mixture was applied to the
LF immunosensor as a one-step immunoassay process. As the
sample solution moved across the capture zone, a competitive
immunoassay reaction between the conjugated human IgG and
those in the solution occurred. This produced a detection signal
by the MWCNTs across the capture line varying in color
intensity that was inversely proportional to the amount of
human IgG target analyte captured as shown in Fig. 5(a). The
detection signals were visible with the application of only 20 μl,
and saturation occurred with the application of 50 μl of total
sample solutions within 15 minutes. The visibility of the capture
zone decreased with increasing target human IgG concentration.
As the concentration of the target analyte increased, it occupied
more binding sites limiting the binding reaction between the
conjugated human IgG and the Protein A. The corresponding
measured results in Fig. 5(b) clearly show that the electrical
resistance increased with increasing concentration of the target
human IgG. Although the visibility of capture lines decreased with
increasing target IgG concentration (100, 150, and 200 μg mlÀ1),
Fig. 2 Non-specific adsorption on the nitrocellulose membrane: (a)
MWCNTs partially coated with PVP were captured by Protein A due to
non-specific adsorption which must be addressed to avoid false signal; (b)
almost no detection signal was observed at the capture zone indicating
that the surface of the MWCNTs were completely covered eliminating
non-specific adsorption, and (c) selective binding between the MWCNT–
human IgG conjugate and immobilized Protein A.
Fig. 3 SEM images of the MWCNT network at the capture zone: (a) before sample application; (b) lower MWCNT coverage due to a smaller number
of binding; and (c) higher MWCNT coverage due to a larger number of binding. The scale bar is 1 μm.
Fig. 4 Calibration results using conjugate solutions in different
concentrations. MWCNTs as label provided a quantitative detection
capability.
This journal is © The Royal Society of Chemistry 2010
Anal. Methods, 2010, 2, 1819–1822 | 1821
Downloaded on 30 December 2012
Published on 29 September 2010 on http://pubs.rsc.org | doi:10.1039/C0AY00412J
View Article Online
Page 4
the electrical measurements clearly showed differences corre-
sponding to the various concentrations at all levels.
4. Conclusions
A lateral flow immunosensing technique using carbon nanotubes
as a label was effectively demonstrated for quantitative antibody
detection based on simple electrical measurements. The surface
of the MWCNTs was partially modified with PVP for uniform
dispersion in aqueous solutions. Further functionalization steps
were conducted to attach antibodies on the exposed surface of
MWCNTs utilizing EDC/sulfo-NHS chemistry. The modified
MWCNTs successfully provided both direct colorimetric and
conductimetric measurements for antibody binding without addi-
tion amplification step. SEM images showed the MWCNT
network formed as a result of antibody binding at the capture zone.
The lateral flow system facilitated the binding reaction and reagent
handling in a simple and convenient manner. This immunosensing
technique is easy for integration and requires ordinary tools for
signal measurement. Therefore, it holds the potential for quanti-
tative determination of various antibodies and antigen in a point-
of-care setup. The detection limit and time could be improved
further with proper optimization of the conjugating process and
the lateral flow system