Sensitive amperometric immunosensing using polypyrrolepropylic acid films for biomolecule immobilization

Article abstract of DOI:10.1021/ac060657o

An electrochemical immunosensor was constructed using an electropolymerized pyrrolepropylic acid (PPA) film with high porosity and hydrophilicity. A high density of carboxyl groups of PPA was used to covalently attach protein probes, leading to significantly improved detection sensitivity compared with conventional entrapment methods. As a model, anti-mouse IgG was covalently immobilized or entrapped in the PPA film and used in a sandwich-type alkaline phosphatase-catatyzing amperometric immunoassay with p-aminophenyl phosphate as the substrate. With covalent binding, the detection limit for IgG in PBS buffer, pH 7.4, was 100 pg/mL with a dynamic range of 5 orders of magnitude. The covalent bonding mode in the carbonate-bicarbonate buffer, pH 9.6, further brought down the detection limit to 20 pg/mL with remarkable selectivity.

Full text of DOI:10.1021/ac060657o

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Anal. Chem. 2006, 78, 7424-7431
Sensitive Amperometric Immunosensing Using
Polypyrrolepropylic Acid Films for Biomolecule
Immobilization
Hua Dong,† Chang Ming Li,*,† Wei Chen,† Qin Zhou,† Zhao Xian Zeng,‡ and John H. T. Luong†,§
School of Chemical and Biomedical Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798,
Watson Pharma, Inc., 620 North 51st Avenue, Phoenix, Arizona 85043, and Biotechnology Research Institute, National
Research Council Canada, Montreal, Quebec, Canada H4P 2R2
Although high sensitivity (femtomoles) has been reported,7 this
approach cannot provide reproducible and addressable deposition
of relevant immunoreagents with controlled spatial resolution, thus
limiting its application in microarray and biochips. Another
alternative method is to employ an electropolymerized conducting
polymer as a matrix to immobilize immunoreagents. After the
pioneering work of Foulds and Lowe,8 the immobilization of
biomolecules such as enzymes, DNA, antibodies, and even whole
cells in conducting polymers has been widely studied to fabricate
biosensors, including immunosensors. Compared with the bead-
based method, the electrosynthesis of conducting polymers allows
for precise control of probe immobilization on surfaces regardless
of their size and geometry.9 Since the polymerization occurs on
the electrode surface, the probes are essentially entrapped in
proximity to the electrode. This feature is of particular importance
toward the development of sensing microelectrodes and micro-
electrode arrays to shorten the response time and alleviate
interference from the bulk solution. Furthermore, the amount of
immobilized probes can be easily controlled either by changing
their concentration or by adjusting the thickness of the polymer
matrix through the electrode potential, electropolymerization time,
or both.
An electrochemical immunosensor was constructed using
an electropolymerized pyrrolepropylic acid (PPA) film
with high porosity and hydrophilicity. A high density of
carboxyl groups of PPA was used to covalently attach
protein probes, leading to significantly improved detection
sensitivity compared with conventional entrapment meth-
ods. As a model, anti-mouse IgG was covalently im-
mobilized or entrapped in the PPA film and used in a
sandwich-type alkaline phosphatase-catalyzing ampero-
metric immunoassay with p-aminophenyl phosphate as
the substrate. With covalent binding, the detection limit
for IgG in PBS buffer, pH 7.4, was 100 pg/mL with a
dynamic range of 5 orders of magnitude. The covalent
bonding mode in the carbonate-bicarbonate buffer, pH
9.6, further brought down the detection limit to 20 pg/
mL with remarkable selectivity.
Immunosensing, a combination of specific immunoreaction
with sensitive optical or electrochemical transduction, has attracted
much attention since the 1970s.1-4 Owing to the adaptability of
electrochemical sensing to miniaturization, considerable research
efforts have focused on electrochemical arrayed immunosensors
and biochips with high sensitivity and specificity. One of the key
steps in the construction of electrochemical immunosensors is
to select a pertinent method for probe immobilization. The most
widely used method is the bead-based immobilization technique
where probes are physically adsorbed or covalently bound to the
surface of polystyrene microbeads with a magnetic iron core.5-6
Among all of the conducting polymers studied up to date,
polypyrrole (PPy) can be considered as one of the most attractive
materials due to its excellent conductivity, stability, and biocom-
patibility even in neutral pH medium.10,11 At present, most PPy-
based immunosensors are performed with impedance or potential-
step methods by measuring the changes in capacitance or
resistance induced in antigen-antibody binding events, offering
real-time and label-free measurement.12-16 However, there are few
* To whom correspondence should be addressed. E-mail: ecmli@ntu.edu.sg.
† Nanyang Technological University.
‡ Watson Pharma, Inc.
§ Biotechnology Research Institute.
(7) Bange, A.; Halsall, H. B.; Heineman, W. R. Biosen. Bioelectron. 2005, 20,
2488-2503.
(8) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473-2478.
(9) Ionescu, R. E.; Gondran, C.; Gheber, L. A.; Marks, R. S. Anal. Chem. 2004,
76, 6808-6813.
(10) Ramanavicius, A.; Habermu¨ller, K.; Cso¨regi, E.; Laurinavicius, V.; Schuh-
mann, W. Anal. Chem. 1999, 71, 3581-3586.
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A.; Halsall, H. B.; Heineman, W. R.; Scheller, F. W. Anal. Chem. 1996, 68,
2453-2458.
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T.; Saito, M.; Mizuno, J.; Wada, Y. Biosen. Bioelectron. 2005, 20, 2306-
2309.
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3329.
(4) Wang, J.; Tian, B.; Rogers, K. R. Anal. Chem. 1998, 70, 1682-1685.
(5) Kim, S. K.; Hesketh, P. J.; Li, C. M.; Thomas, J. H.; Halsall, H. B.; Heineman,
W. R. Biosen. Bioelectron. 2004, 20, 887-894.
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(12) Li, C. M.; Chen, W.; Yang, X.; Sun, C. Q.; Gao, C.; Zheng, Z. X.; Sawyer, J.
Frontier Biosci. 2005, 10, 2518-2526.
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1992, 64, 997-1003.
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2, 27-30.
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144-156.
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7424 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006
10.1021/ac060657o CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/22/2006

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Scheme 1. Chemical Synthesis of
Pyrrolepropylic Acid (PPA)
Human serum was purchased from Sigma (Madison, WI). The
enzyme substrate, PAPP, was synthesized as described in the
literature.17 In brief, 1 mL of p-nitrophenyl phosphate (PNPP;
Sigma, St. Louis, MO) and 10 mL of 0.05 M H2SO4 were added to
the cathode chamber, separated in the electrolytic cell by a Nafion
membrane (Aldrich), where Pt foils were used as the cathode and
anode electrodes. Electrolysis was carried out using the galvano-
static mode at 8 mA/cm2 for 5 h, and the resulting solution was
adjusted to pH 9.0 with sodium hydroxide. The deionized water
(18.2 MΩ‚cm) was obtained from a Millipore Milli-Q water
purification system. All other reagents were of analytical grade
with highest purity.
Figure 1. Schematic of the amperometric enzyme immunosensor
based on the polypyrrolepropylic acid (PPA) film. The electroactive
product, p-aminophenol (PAP), was converted from the enzymatic
conversion of PAPP by alkaline phosphatase (ALP): (a) entrapment
mode; (b) covalent bonding mode using EDC as the cross-linker.
Synthesis of Pyrrolepropylic Acid. Different methods to
electrochemically synthesize polypyrrole have been extensively
studied and reported.18 PPA was prepared according to the
following protocol: 3.8 mL of pyrrole (55 mmol) and 0.3 mL of
benzyltrimethylammonium hydroxide were admixed, and then 2.9
mL of acrylonitrile (55 mmol) was added gradually to keep the
temperature below 40 °C. After overnight stirring, the resulting
mixture was hydrolyzed by adding 50 mL of potassium hydroxide
(10 M) and refluxed overnight. The solution was cooled, and HCl
was gradually added to acidify the solution to pH 3. The aqueous
layer was extracted with ethyl acetate four times, each time with
30 mL of solvent. The combined organic layer was washed with
75 mL of brine and then dried with anhydrous magnesium sulfate.
After solvent evaporation, the crude product was crystallized by
methylene chloride and hexane. About 5 g of pure pyrrolepropylic
reports on PPy-based amperometric immunosensors using a
labeled enzyme to catalyze a substrate into a detectable electro-
active product. The lack of widespread applications of PPy for
amperometric immunosensing can be attributed to the poor
permeability of PPy that hinders the diffusion of the electroactive
product in and out of the polymer film.9
This paper describes the construction of a new amperometric
immunosensor using electrosynthesized polypyrrolepropylic acid
(PPA) as immobilization matrix. Probes can be attached to the
abundant carboxyl groups on the polymer surface or entrapped
in the polymer network, leading to fast and reliable amperometric
responses. In our studies, mouse IgG is selected as a model
analyte and its detection is realized in the presence of p-
aminophenyl phosphate (PAPP) using the sandwich mode with
rat anti-mouse IgG and sheep anti-mouse IgG (conjugated to
alkaline phosphatase) as the primary and second antibodies,
respectively (Figure 1). The performance of immunosensing with
respect to detection sensitivity and reliability is presented and
discussed in detail.
1
acid was obtained (65% yield). NMR (CDCl3): 2.83 (t, 2H, J )
7.2 Hz), 4.20 (t, 2H, J ) 7.2 Hz), 6.14 (t, 2H, J ) 2.1 Hz), 6.67 (t,
2H, J ) 7.2 Hz), 9.00 ppm (s, bd peak, 1H). The reaction can be
summarized in (Scheme 1).
Electrochemical Instrumentation. Electropolymerization,
cyclic voltammetry and differential pulse voltammetry were
performed with an Autolab potentiostat/galvanostat (PGSTAT30,
Eco Chemie B.V., Utrecht, The Netherlands). Before measure-
ment, glassy carbon (GC) electrodes were polished with 0.3-µm
γ-alumina powder, rinsed with deionized water, and then polished
with 0.05-µm powder. After polishing, the electrodes were ultra-
sonically cleaned for 10 min in deionized water and finally soaked
in acetone for 5 min, followed by vigorously washing with
deionized water. Cyclic voltammograms of the polished electrodes
EXPERIMENTAL SECTION
Chemicals and Materials. Pyrrole was purchased from
Aldrich (Madison, WI) and distilled prior to use. Glassy carbon
electrodes (3 mm in diameter) were purchased from CH Instru-
ments (Austin, TX). Alkaline phosphatase-conjugated Affinipure
rat anti-mouse IgG (H+L), Chrompure mouse IgG, and biotin-SP
conjugated Affinipure sheep anti-mouse IgG F(ab′)2 were obtained
from Jackson ImmunoResearch Laboratories (West Grove, PA).
The cross-linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) was purchased from Pierce (Rockford, IL).
4-
were measured in 0.05M Fe(CN)63-/Fe(CN)6 to confirm that
the potential difference between cathodic and anodic peaks was
not more than 65 mV (the theoretical value is ∼59 mV).
(17) Dong, H.; Li, C. M.; Zhou, Q.; Sun, J. B.; Miao, J. M. Biosens. Bioelectron.
In press.
(16) Ramanaviciene, A.; Ramanavicius, A. Biosen. Bioelectron. 2004, 20, 1076-
(18) Li, C. M.; Sun, C. Q.; Chen, W.; Pan, L. Surf. Coat. Technol. 2005, 198,
474-477.
1082.
Analytical Chemistry, Vol. 78, No. 21, November 1, 2006 7425

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Otherwise, the electrode was reconditioned until the desired result
was obtained. All experiments were conducted using a three-
electrode system, with a Pt wire and Ag/AgCl (saturated KCl) as
counter and reference electrodes, respectively.
Electrochemical impedance spectroscopy (EIS) was used to
characterize the PPA film formation, probe immobilization, and
antigen-antibody interaction using an Autolab potentiostat/
galvanostat (PGSTAT30) equipped with a frequency response
analyzer module. All impedance data were recorded at an open
circuit voltage with an amplitude of 10 mV over a frequency range
of 1 Hz-1 MHz in 0.1 M PBS solution containing 10 mM Fe-
4-
(CN)63-/Fe(CN)6
.
AFM Characterization. To substantiate the difference be-
tween the electrochemical behavior of PPy and PPA, the surface
morphology of the PPy and PPA films, respectively was character-
ized by AFM (Nanoman, Veeco, Santa Barbara, CA) using tapping
mode and operating in air.
FT-IR Characterization. To identify the main functional
groups on the surface of electropolymerized PPA films, attenuated
total reflection Fourier transform infrared (ATR-FT-IR) spectros-
copy was performed by using the Nicolet Magna IR 560 ESP
spectrophotometer (Thermo Nicolet, Madison, WI) incorporated
with a “Golden Gate” ATR module (Graseby Specac, Smyrna, GA).
The IR data show that the strongest peak appears at 1730 cm-1
,
which can be assigned to CdO in carboxyl groups, suggesting
the abundance of carboxyl groups in the PPA film.
Immunosensor Construction. Two methods were used for
probe immobilization. For covalent bonding, PPA monomers were
first electropolymerized on the GC electrode to form a PPA film
by the galvanostatic method at 0.1 mA for 500 s. The coated
electrode was washed three times with 0.1 M PBS and dried under
a gentle nitrogen stream. The electrode was then immersed in
150 µL of acetonitrile (ACN) containing 1.5 wt % EDC for 1.5 h at
room temperature. After careful rinsing with ACN, the electrode
was exposed to a solution containing 100 µg/mL sheep anti-mouse
IgG for 2 h, followed by washing and 1% BSA treatment for 2 h to
alleviate nonspecific protein adsorption. The resulting electrode
was incubated with different concentrations of mouse IgG (0.1
ng/mL to 1 µg/mL) for 2 h followed by washing with PBS.
Thereafter, 100 µL of 20 µg/mL rat anti-mouse IgG (H+L)
conjugated alkaline phosphatase (ALP) was dropped onto the
electrode surface and incubated for 2 h. Finally, all the modified
electrodes were rinsed with PBS before electrochemical measure-
ment. In the entrapment method, 0.3 M PPA was mixed with 100
µg/mL sheep anti-mouse IgG and then electrochemically polym-
erized to form a protein-doped PPA film. Incubation of antigen
and binding of the second antibody were similar to that described
above.
4-
Figure 2. Cyclic voltammograms of 50 µM Fe(CN)63-/Fe(CN)6
on polymer-coated GC electrodes with the scan range of -0.2 to
+0.65 V vs Ag/AgCl. (A) PPy electropolymerized with the charge of
0 (a), 1 (b), 2 (c), and 3 mC (d). (B) PPA electropolymerized with the
charge of 0 (a), 10 (b), 20 (c), and 30 mC (d). Scan rate, 50 mV/s.
RESULTS AND DISCUSSION
Electrochemical Behavior of the PPy and PPA Films.
Figure 2 compares the cyclic voltammograms (-0.2 to +0.65 V
vs Ag/AgCl) of Fe(CN)63-/Fe(CN)6 obtained on the PPy- and
4-
PPA-coated GC electrodes in 0.1 M PBS at pH 7.4. The film
thickness was controlled by the charge passing during the
electropolymerization step. Replacement of HN in pyrrole by the
propylic acid group (CH2CH2COOH) resulted in different elec-
trochemical behavior. During the growth of the PPy film, the
redox peak current of Fe(CN)63-/Fe(CN)6 declined sharply
4-
whereas the peak potential separation between anodic and
cathodic waves increased drastically. Indeed, the 3-mC-thick PPy
film did not display defined redox waves for Fe(CN)63-/Fe(CN)64-
,
indicating both the deactivation of PPy and the poor permeability
of Fe(CN)63-/Fe(CN)6 through the polymer film. Since most
4-
Amperometric Measurement. The sandwich immunoassay
was performed by dropping 150 µL of PAPP solution to cover the
modified immunosensor. Differential pulse voltammetry (DPV)
was used to detect amperometric responses because of its high
signal-to-noise (S/N) ratio. In the DPV measurement, the pulse
amplitude, pulse width, sampling width, and pulse period were
50 mV and 0.06, 0.02, and 0.2 s, respectively, and the potential
range was -0.4 to +0.3 V versus Ag/AgCl.
of electroactive products such as H2O2 converted from the enzyme
catalytic reaction exhibit higher positive oxidation potentials
compared to the PPy film, the continual detection of these
electroactive species will keep the polymer in an oxidized state
for a long time and cause the loss of polymer conductivity, namely,
deactivation.19-25 Even the oxidation potential of electroactive
(19) Wang, J.; Musameh, M. Anal. Chim. Acta 2005, 539, 209-213.
(20) Gao, M.; Dai, L.; Wallace, G. G. Synth. Met. 2003, 137, 1393-1394.
7426 Analytical Chemistry, Vol. 78, No. 21, November 1, 2006

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the loss of conductivity (or, namely, deactivation) even if held at
high positive potential for a long period. Therefore, the decay in
the redox peaks (Figure 2B) was mainly caused by the limited
4-
diffusion of Fe(CN)63-/Fe(CN)6 into a thick PPA membrane.
The same experiment was also carried out with PAP, the
enzyme-catalytic product of PAPP, as an indicator. Similarly, that
is, the PPy film drastically hindered the permeation of PAP while
the PPA film appeared much more permeable. The CV curves of
4-
PAP and Fe(CN)63-/Fe(CN)6 recorded on PPA-coated elec-
trodes both exhibited the increasing peak currents with the
growth of cycles, which proved the mass-transfer process of PAP
4-
and Fe(CN)63-/Fe(CN)6 diffusion through the polymer film.
However, the time required for PAP to reach its maximum peak
current was much shorter compared to that for Fe(CN)63-/Fe-
(CN)64-, indicating a faster diffusion rate of PAP. A rationale
behind such behavior could be explained as the electrostatic
3-
repulsion between the PPA film (negative charge) and Fe(CN)6
/
Figure 3. (A) Comparison of polymer conductivity between PPy
(curve a) and PPA (curve b) in 0.1 M PBS solution (pH 7.4) using
cyclic voltammetry; (B, inset) amplified CV curve of PPA. Scan rate,
50 mV/s.
Fe(CN)64-. As a consequence, the permeation of Fe(CN)63-/Fe-
(CN)64- was reduced. Obviously, the fast diffusion rate of PAP in
the PPA film would lead to the reduced response time of the
immonosensor.
Structural Characteristics of the PPA Film. Under positive
potential and with long exposure, PPy gradually loses its conduc-
tivity due to the destruction of long-range conjugation in the
polymer.24 Thus, amperometric responses from electroactive
species in the solution cannot be detected on the deactivated PPy
surface. In the case of PPA with poor conductivity, the signal
response was always observed regardless of the duration in which
the polymer was kept at a positive potential. This result could
only be explained by the difference in the polymer structures. In
our experiment, AFM was used to probe the PPA film. The AFM
micrographs of the bare GC electrode (Figure 4a,b) displayed
some scrapping traces, caused by 0.05-µm alumina powder during
electrode cleaning and polishing. When the electrode was covered
with the polypyrrolepropylic acid film, the AFM micrograph
illustrated a porous structure without any appearance of scrapping
traces (Figure 4c,d). The aperture size was mainly distributed over
0.2-0.5 µm, which was sufficient for the permeation of ions. The
formation of the porous structure was not totally unexpected since
electrons could not efficiently transfer through the PPA film.
Hence, a long-chain polymer could not be synthesized in the
absence of free radicals, indicating a self-limiting process. From
Figure 4c,d, the PPA film thickness was estimated to be ∼100
nm after charging with 0.1 mA for 500 s. For comparison, the
AFM images of the PPy film prepared under the same condition
was measured (Figure 4e,f). Obviously, the structure of PPy was
very compact without any observable pore on the polymer surface.
products such as PAP is close to that of PPy; the electrooxidation
of PPy (doping) in a broad potential range will increase the
background current and thus decreases the S/N ratio. In addition,
the dense structure and hydrophobic property of the PPy film
after polymerization restrict the permeation of electroactive species
to the electrode surface. Without reconditioning (overoxidation),19
the signal response of the PPy-coated electrode is very weak and
undetectable, particularly at low analyte concentrations.
In contrast, the redox waves of Fe(CN)63-/Fe(CN)64- (curves
a and b in Figure 2B) were almost identical even if the PPA film
thickness increased from 0 to 10 mC. The peak current and
potential separation were only slightly changed compared to the
bare GC electrode (Figure 2B, curve a). For the 30-mC film
thickness, the redox peaks (curve d in Figure 2B) were still
observable but decreased gradually with the growth of the film
thickness. This behavior was reasoned to result from the deactiva-
4-
tion of PPA or the retarded diffusion of Fe(CN)63-/Fe(CN)6
through the polymer film. To decipher this behavior, cyclic
voltammograms of PPA and PPy measured in the PBS buffer were
used to estimate their conductivity. A pair of redox peaks in the
PPA polymer was observed at ∼0.0 and -0.2 V versus Ag/AgCl
(Figure 3, curve b), implying that the PPA film might also possess
a doping and undoping process like PPy (Figure 3, curve a).
However, the peak current of PPA was much lower compared
with that of PPy, indicative of poor conductivity of the PPA film.
Besides, it was found that the electropolymerization potential of
PPA was ∼2.3 V versus Ag/AgCl, compared to ∼0.82 V for PPy
under the same conditions. In other words, the deposition of PPA
film required higher overpotential than the level required for the
PPy film. On this basis, the PPA film could not be subjected to
4-
As a result, ions such as Fe(CN)63-/Fe(CN)6 and PAP cannot
penetrate this film. Nevertheless, PPy could also become porous
after overoxidation at a very high positive potential, resulting in
detectable signals.26,27 However, the activity of the immobilized
biomolecules might be affected at high anodic potential when a
single electropolarization step was used to entrap such biomol-
ecules.
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mobilization/Binding Events on the Electrode Surface. Abun-
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(25) Rangamani, A. G.; McTigue, P. T.; Verity, B. Synth. Met. 1995, 68, 183-
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