Sensors and Actuators B 117 (2006) 286–294

A novel capacitive immunosensor for transferrin detection based on

ultrathin alumina sol–gel-derived films and gold nanoparticles
Tanji Yin, Wanzhi Wei ∗ , Liu Yang, Xiaohua Gao, Yanping Gao
State Key Laboratory of Chemical, Biological Sensing Technologies & Chemometrics, Hunan University, Changsha 410082, PR China
Received 22 July 2005; received in revised form 21 November 2005; accepted 21 November 2005
Available online 27 December 2005

Abstract
A novel capacitive immunosensor was successfully developed for the direct detection of transferrin based on an ultrathin ␥-alumina sol–gel-
derived film and gold nanoparticles. The thin film was formed and air-dried by dripping Al2 O3 sol with a microliter syringe on a gold electrode
modified with a self-assembled mercaptoacetic acid (MAA) monolayer. After defects in the film were blocked with a long-chain alkylthiol, gold
nanoparticles were deposited on the surface of the modified film via a potential step from +1.1 to 0.0 V (versus Ag|AgCl|KCl) for 15 s in a 0.5 M
H2 SO4 solution containing 0.1 mM HAuCl4 . Finally, the antibody was immobilized on the gold nanoparticles under the optimized experimental
conditions. The capacitive sensor prepared by the present method can provide high sensitivity because of the ultrathin inorganic film with high
permittivity and good biocompatibility of gold nanoparticles. Compared with a capacitive immunoassay based on antibody-embedded ultrathin
␥-alumina sol–gel-derived films, the novel immunosensor presented a lower detection limit of 0.05 ng/ml and a wider linear response range of
1–75 ng/ml for transferrin detection. The prepared procedure of the novel immunosensor also provided a new approach to fabrication of capacitive
immunosensors.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Alumina sol–gel-derived films; Gold nanoparticles; Capacitive immunosensor; Transferrin

1. Introduction
Recently, capacitive immunosensors have attracted consid-
erable interests for high sensitivity, specificity, label-free, and
ordinary instrumentations [1–12].
Based on the electrical double-layer theory, the principle
of capacitive immunosensors was that when an electrode cov-
ered with the dielectric layer was immersed in an electrolyte
solution, it could be considered as a resemblance to a capaci-
tor. And any adsorption or binding of reagents onto the sens-
ing layer surface would change the thickness and/or dielectric
behavior. The changes of the recognition layer could be sensi-
tively detected with numerous methods, such as potential pulse
method [8], lock-in amplifier method [9], field-effect capacitor
[13], and electrochemical impedance method [14–18]. Electro-
chemical impedance spectroscopy (EIS) was regarded as an
effective technique for probing the features of the interfacial
∗ Corresponding author. Tel.: +86 731 8821861; fax: +86 731 8821967.
E-mail address: weiwz2003@126.com (W. Wei).
state [19], which could provide a rapid method for real-time, in
situ monitoring the kinetic process of biomolecule interactions,
such as antigen–antibody [20], oligonucleotide–DNA [21], and
biotin–avidin [22].
When an immunosensor based on a capacitive transducer
was constructed, the immobilization of a receptor (antibody or
antigen) on the electrode surface was significant. Firstly, the
activity of the receptor specific to the analyte of interest must be
readily available, and there must be an efficient transduction of
antibody–antigen interaction into an analytical signal. Secondly,
the formed layer should be insulating to prevent interferences
from redox couples in the electrolyte solution and high faradic
background currents. Or else, it equals to a resistor in paral-
lel with the capacitor, which will partially short-circuit. This
might increase the resistance current and decrease the capaci-
tance response. Thirdly, the sensitive layer should be as thin as a
nanometer one to obtain high sensitivity. Otherwise, the capac-
itance change caused by antibody–antigen interaction would be
not effectively detected [8].
Many different immobilization techniques have been repor-
ted in previous literature, such as self-assembled monolayer

0925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2005.11.041
 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294
(SAM) technique [5–10], electro-polymerization technique
[11,12], etc. However, the immunosensors based on these tech-
niques still have some potential limits in practical application.
Lately, the low-temperature porous sol–gel-derived films mod-
ified electrochemical biosensors were reported as an alternative
effective method applied to the immunosensors [23–28], owing
to their numerous advantages, including physical rigidity, chem-
ical inertness, high photochemical property, biodegradation, and
thermal stability. Jiang et al. [1] demonstrated a label-free capac-
itive immunosensor based on antibody-embedded ultrathin ␥-
alumina sol–gel films, which was employed to directly detect
the liver fibrosis makers in a multichannel capacitance analysis
system. The thickness of the ultrathin film was only 20–40 nm,
and the sensitivity was greatly improved. Nevertheless, most
of the sol–gel-derived films modified biosensors were based
on biomolecules trapped in a sol–gel matrix. Therefore, there
were two potential limits: (1) the protein embedded in the film
was not effectively utilized, and the quantity of the protein was
largely consumed and (2) when the sol–gel-derived films were
dried, the protein embedded would be inevitably denatured.
Therefore, developing a higher sensitive, effective response
immunoassay employing sol–gel-derived immunosensors was
necessary.
Additionally, gold nanoparticles have provoked much inter-
est in recent years for their unique properties, such as a very large
surface area and excellent biocompatibility [29]. Gold nanopar-
ticles have been successfully applied in immobilizing various
kinds of biomolecules, such as DNA, enzyme. and other pro-
teins [30–32]. When such biomolecules were adsorbed on gold
nanoparticles, their bioactivity still remained [33]. However, to
the best of our knowledge, the study of combining ultrathin ␥-
alumina sol–gel-derived films with gold nanoparticles has been
unexplored.
Transferrin is a very important ␤-globulin and the carrier
of ferric ions in vertebrate. Many diseases are associated with
the changes in transferrin content in serum. Moreover, serum
transferrin has been well adopted for a certain clinical maker
of protein-calorie malnutrition, and urinary transferrin has been
used as an underlying marker for prostaticcarcinoma. Thus, the
determination of transferrin gained much interest for clinical
analysis [34]. Presently, numerous techniques for transferrin
detection were applied, such as chromatographic methods [35]
and amperometric immunoassay [36]. However, most of these
methods needed expensive instruments and could not obtain
real-time experimental data.
In this paper, a novel, sensitive, label-free, real-time moni-
toring capacitive immunosensor with a low detection limit will
be established for transferrin detection by using a gold elec-
trode modified with MAA monolayer/alumina sol–gel-derived
film/1-dodecanethiol/Au nanoparticles/antibody.
2. Experimental
2.1. Chemicals
Human transferrin, anti-human transferrin (developed in
goat-fractionated antiserum), 1-dodecanethiol, mercaptoacetic
287
acid (MAA), and aluminum isopropoxide (Al(i-PrO3 )) were
purchased from Sigma–Aldrich (USA). The reconstitution and
storage of the antibody were based on the instructions of Sigma.
Human serum albumin (HSA) was obtained from Shanghai San-
gon Biological Engineering Technology & Services Co. Ltd.
Hydrochloroauric acid (AuCl3 ·HCl·4H2 O) was purchased from
National Pharmacy Group Chemical Reagent Ltd. Co. (Shang-
hai, China).
All other reagents were of analytical grade and were used
without further purification. Double distilled water was used in
all experiments.
A 0.01 M phosphate buffer saline solution (PBS) of pH 7.4
was used as the background electrolyte. The probe solution
was prepared by using the mixture of 1 mM K3 [Fe(CN)6 ] and
K4 [Fe(CN)6 ], containing the 0.01 M PBS (pH 7.4).
2.2. Apparatus
The electrochemical measurements were carried out on a CHI
660A electrochemical workstation (Shanghai Chenhua Appara-
tus Co.) with CHI software, connecting to a personal computer.
All experiments were performed at room temperature in a con-
ventional three-electrode system comprising a gold disk elec-
trode (2 mm in diameter), a platinum wire auxiliary electrode
and an Ag|AgCl|KCl (3 M) reference electrode (all from CH
Instruments, USA). All potentials were relative to the reference
electrode.
A magnetic stirrer (Model 79-1, Shanghai Hexin Technique
and Education Equipment Co., China) was employed to stir the
test solution during the interaction between an antibody and an
antigen.
The surface morphology of modified electrodes was ana-
lyzed by Sirion200 field emission scanning electron microscope
(SEM, FEI Co., USA).
2.3. Preparation of Al2 O3 sols
According to the previous literature [37], preparation of alu-
mina sols was briefly described as below. The mixtures of
Al(i-PrO3 ) and distilled water with certain ratios of Al/H2 O
(mol/mol) were stirred for 1 h at 80 ◦ C and hydrolyzed at 90 ◦ C
by adding a certain volume of 1 M HCl. Then, the glassware was
opened for several hours to volatilize i-PrOH. Finally, the mix-
tures were refluxed for 8 h at 90 ◦ C, and a stable and homogenous
boemite sols (␥-AlOOH) were obtained.
2.4. Modification of the electrodes
2.4.1. Pretreatment of gold electrodes
Gold electrodes were polished carefully with 1.0, 0.3, and
0.05 ␮m ␣-Al2 O3 power slurries on a 1200 grit Carbimet disk,
until a mirror shiny surface appeared, and then was sonicated in
ethanol and distilled water for 3 min to remove trace alumina and
possible contamination. The polished electrodes were dipped in
a 0.5 M H2 SO4 solution, and were cycled between 0 and 1.5 V,
until reproducible cyclic voltammograms were obtained. Then,
the electrodes were dried with N2 .
288 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294
2.4.2. Self-assembling of MAA
The cleaned and surface-activated gold electrodes were
immersed in a 0.1 M HClO4 solution containing 5 mM MAA
for 30 min to obtain a self-assembled monolayer, and then rinsed
with distilled water and dried in a N2 stream.
2.4.3. Fabrication of the ultrathin alumina sol–gel-derived
films
Electrodes coated with Al2 O3 sol–gel-derived films were pre-
pared by dripping 0.5 ␮l of the prepared boemite sols on the
MAA monolayer modified gold electrodes with a microliter
syringe, and air-dried at room temperature. When the solvents
were volatilized, the alumina sols were converted into gels and
the ultrathin films were formed.
2.4.4. Blocking film defects
The gold electrodes with coupled layers were immersed in
1-dodecanethiol for 30 min to block the unblocked pinholes, and
then rinsed with distilled water thoroughly to remove physically
adsorbed 1-dodecanethiol, and dried with purified N2 .
2.4.5. Deposition of gold nanoparticles
The electrochemical deposition procedure of Au nanocrystals
was adopted from the reported literatures [38,39]. Gold nanopar-
ticles were deposited via a potential step from +1.1 to 0.0 V
(versus Ag|AgCl|KCl) for 15 s by immersing the modified elec-
trodes into a 0.5 M H2 SO4 solution containing 0.1 mM HAuCl4 .
2.4.6. Antibody immobilization
After rinsed with distilled water, the electrodes coated with
Au nanoparticles were immerged in a 0.01 M PBS solution (pH
7.4) containing anti-human transferrin (450 ng/ml) for 30 min at
4 ◦ C, and then were washed with an abundant amount of doubly
distilled water and PBS (pH 7.4) and placed in a refrigerator for
further experiments.
2.5. Capacitance measurement
The immunosensors were constructed in the cell of 10 ml PBS
(pH 7.4) with the prepared electrodes as the working electrodes.
The capacitive changes of the immunosensors were measured
by electrochemical impedance technique. After the base line
leveled off, a certain amount of human transferrin in a 0.01 M
PBS solution (pH 7.4) was added into the electrochemical cell.
The background solutions were slowly stirred. The working fre-
quency was decided by Bode plots. The working voltage and the
amplitude of the ac signal applied to the working electrode were
an open circuit potential and 0.005 V, respectively.
3. Results and discussion
3.1. Construction of the capacitive immunosensor
3.1.1. Preparation of Al2 O3 sols
As mentioned above, the thickness of the modified film was a
key factor for the design of a capacitive sensor. For alumina sols,
the Al/H2 O ratio (mol/mol) with different water contents would
change the thickness of the gels and sequentially influenced the
response of the immunosensors. According to the previous work
[1], the thickness of the Al2 O3 gel films with Al/H2 O ratios
from 1:100 to 1:300 was 40–20 nm, which was much thinner
than those of the prepared SiO2 gel films. Furthermore, the per-
mittivity of the inorganic alumina films (∼9.2) was higher than
those of SiO2 films (∼3.9). This would increase the capacitance
of the film and make the high RC time constant, which decreased
the current-sampling noise [1]. Therefore, the nanometer thick-
ness of alumina films with high permittivity provided the ideal
performance of the capacitance measurement. In the paper, the
molar ratio of Al/H2 O = 1:100 was adopted for the preparation
of the Al2 O3 sol.
Moreover, before coated with the Al2 O3 sol–gel-derived film,
the gold electrode was modified with a self-assembled MAA
monolayer, with the aim at: (1) reducing the apparent area of
the electrode to obtain the isolation of the sensitive layer and
(2) improving the adhesion strength of the sol–gel-derived film
based on the strong bonding between sulfur groups of the alkane
chain thiols and the Au substrate (bond energy; 44 kcal/mol), and
thus enhanced the used life.
3.1.2. Deposition of Au nanoparticles
Gold nanoparticles were available for adsorbing and immobi-
lizing the proteins during the construction of biosensors, because
of large surface area and good biocompatibility. Generally, Au
nanoparticles were adsorbed on the electrode surface by dipping
the electrode into the solution containing Au colloidal particles.
However, there were two potential limits: (1) the preparing pro-
cess of Au colloid was complicated, consuming time and the
magnitude of Au nanoparticles was difficult to be controlled and
(2) the procedure of Au nanoparticles adsorbed on the electrode
also took time, and the nanoparticles might be not uniformly
distributed on the surface of the electrode.
In the experiment, after the defects in the alumina sol–gel-
derived film were blocked with a long-chain alkylthiol, the
ultrafine pores still existed, because of the inherent high sur-
face area of the ␥-alumina sol–gel matrix. Therefore, owing to
electron transfer, Au nanoparticles were directly deposited and
tumbled in the alumina sol–gel-derived film. The results showed
that the method was not only simple and time-saving, but also
could obtain well-proportioned Au nanoparticles.
3.1.3. Immobilization conditions for antibody
Anti-human transferrin adsorbed on the gold nanoparticles
would increase the thickness of the modified layers and fur-
ther decrease the capacitance value of the system. Therefore,
selecting the proper adsorbed time was necessary to reduce
the non-specific response of the capacitive immunosensors and
improve the measuring sensitivity. The capacitive measurement
showed that after adsorbed at 4 ◦ C for 30 min, the capacitance of
the immunosensor reached a stable value. Therefore, the adsorb-
ing time of 30 min was adopted in the experiment.
The influence of the concentration of the antibody was inves-
tigated under the same condition. Fig. 1 showed that the capac-
itance decreased gradually with increasing the quantity of the
antibody, and ultimately leveled off when the concentration of
 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294
Fig. 1. Capacitance vs. the concentration of anti-human transferrin. The error
bars represent the relative standard deviation for three separate measurements.
Fig. 2. SEM images of: (a) the electrode surface modified with a MAA self-assembled monolayer/alumina sol–gel-derived film, (b) Au nanoparticles deposited
electrode surface, and (c) the surface of antibody-coated electrode.
289
anti-human transferrin exceeded 450 ng/ml, suggesting that the
sensitive layer was saturated. Consequently, the optimal concen-
tration (450 ng/ml) was chosen for further antibody immobiliza-
tion.
3.2. SEM characterization
SEM was employed to investigate the surface morphology
of the sensitive layers. The SEM image of the electrode mod-
ified with a MAA monolayer/alumina sol–gel-derived film is
shown in Fig. 2(a), which displayed a three-dimensional porous
open structure. After the defects of the film were blocked with a
long-chain alkylthiol, gold nanoparticles were deposited on the
surface and homogeneously distributed, as shown in Fig. 2(b).
The mean diameter of the uniform nanoparticles was 20–40 nm.
Then, anti-human transferrin was adsorbed and immobilized on
the Au nanoparticles under the optimized experimental condi-
tions. As can be seen in Fig. 2(c), the antibody was distributed
regularly throughout the electrode surface. Therefore, when the
prepared electrode was exposed to the solution containing a spe-
cific antigen, the specific interaction would occur.
290 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294

3.3. Cyclic voltammetry characterization

In accordance with the plate capacitor model, the modified

layers of capacitive sensors should be insulated. Cyclic voltam-
metry (CV) of electroactive species is a valuable approach to
probing the insulating property of a layer on the electrode sur-
face. To characterize the insulation of the different modified
layers, CV was carried out in the 1 mM K3 Fe(CN)6 /K4 Fe(CN)6
redox couples and 0.1 M KCl supporting electrolyte. Curve (a)
in Fig. 3 showed that the redox couple was oxidized and reduced
at a bare gold electrode surface and a pair of typical redox peaks
was observed. When the surface was covered with a MAA self-
assembled monolayer, a sharp decrease of the redox peak current
in curve (b) was observed. After the defects of the electrode
coated with the alumina sol–gel-derived film were blocked with
1-dodecanethiol, the redox peak was disappeared in curve (c) of
Fig. 3. However, the peak current could be still observed, which Fig. 4. The capacitive change of the immunosensor exposed to 0.01 M PBS (pH
suggested that the ultrafine pores of Al2 O3 sol–gel-derived film 7.4).
were still existent, and electron transfer was imaginable. There-
fore, when Au nanoparticles were directly deposited on the decreased continuously in the initial 60 min and then tended to
surface of the film, the faradaic current decreased slightly in become constant after the immunosensors were exposed to the
curve (d). Afterward, when the antibody protein was adsorbed buffer solution, consistently with the literature [1]. This result
and immobilized on the gold nanoparticles, the redox peak dis- was critical for the stability of the prepared gel films. Therefore,
appeared again in curve (e). Therefore, the prepared electrode the pretreatment step, immerged in the buffer solution for 60 min,
was insulating and could be utilized as a capacitive immunosen- was necessary to obtain a stable gel film and a reproducible
sor. response. In the experiment, the prepared immunosensors were
laid in the buffer solution prior to the experiment to save the
3.4. The swelling effect of alumina sol–gel-derived films mentioned pretreatment.

When the prepared capacitive immunosensors were dipped
into the buffer solution, swelling of the gel films should be
concerned due to the adsorption of water. This event would
increase the thickness of the sensitive layer and thus decrease the
capacitance of the sensors. Fig. 4 showed that the capacitance
3.5. The parameters of capacitive detection
According to Randles equivalent circuit model (Fig. 5), the
following equation was necessary for the capacitive measure-
ments:
j
Rf  − > Rs
ωC
where ω is the angular frequency of ac impedance, ω = 2πf, and
f is the working frequency (Hz). The left of the formula ensured
that Rf was so great that the circuit could be reasonably consid-
ered as an open circuit, which required a good insulation of the
layers and a higher working frequency. The right part ensured
that there was the maximal voltage between the two sides of
the insulating film, which would improve the sensitivity of the
sensors. To achieve the aim, it was necessary to decrease the

Fig. 3. Cyclic voltammograms recorded in the presence of 1 mM

K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1) mixture containing 0.1 M KCl, using (a) a
bare Au electrode, (b) a gold electrode modified with a MAA self-assembled
monolayer, (c) an electrode coated with a ␥-Al2 O3 sol–gel-derived film/1-
dodecanethiol and treated as (b), (d) an electrode further modified with gold
nanoparticles, and (e) an antibody-immobilized electrode treated as (d). Scan
rate was 50 mV/s.
Fig. 5. Equivalent circuit of electrochemical impedance measurement. Rs is
resistance of the solution, C is the capacitance of the dielectric layer, Rf is the
ohmic resistance, and Zw is the Warburg impedance. RE, CE, and WE represent
reference, counter, and working electrode, respectively.
 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294 291

Fig. 6. Bode plots for determination of the frequency dependence of the

Fig. 7. Real-time capacitive response curves of: (a) the test solution with
impedance and phase angle for the capacitive immunosensor.
75 ng/ml transferrin and (b) the test solution of HAS at the same concentration
as transferrin used in (a). The arrow indicates the point of injection of samples.
resistance of the solution and choose a lower working frequency.
Therefore, selecting the proper working frequency was needed
Verifying the response of unspecific adsorption of proteins
for measuring the capacitance of the immunosensors with elec-
was a significant factor for the immunosensors. In the experi-
trochemical impedance technique.
ment, human serum albumin (HSA) was selected as the non-
In the experiment, the working frequency (1 Hz–100 kHz)
specific adsorbed protein, which was added to the measuring
was determined by measuring the effect of frequency on the elec-
cell under the same condition as transferrin. Curve (b) of Fig. 7
trochemical impedance. Fig. 6 gave a Bode plot of impedance
showed the relative capacitance change of the non-specific
magnitude and phase angle versus working frequency. As shown
adsorption of HAS was less 5%, which could be considered
in the plot, when the frequency was less than 10 kHz, the slope of
as the physical adsorption on the sensitive layer of the sensor.
the impedance curve was close to −1, which suggested that the
The results also implied that the capacitive change was caused
device was an analogous Randle cell type [16]. The phase angle
by a specific interaction between the antibody and the antigen,
was nearly −90◦ between 100 and 1000 Hz, which indicated
not by unspecific adsorption of the protein on the electrode sur-
that the equivalent circuit displayed a nearly ideal capacitive
face in curve (a) of Fig. 7. Therefore, the prepared capacitive
characteristic. Therefore, the frequency of 962 Hz was chosen
immunosensor was selective for the detection of the target anti-
as the working frequency for detecting the capacitance of the
gen.
immunosensors.
Samples of 1–100 ng/ml transferrin were successively
injected into the measuring cells, and when the relative capac-
3.6. Detection of transferrin antigen itance changes reached the maximum value and kept constant,
the data were recorded. Fig. 8 shows the curves of the relative
The interaction between transferrin and anti-human transfer-
rin was real-time monitored with the impedance versus time
technique without additional label procedure. The imaginary
part of the impedance (Zim ) was used to calculate a value for the
capacitance (C) by the following equation [40]:
1
C=−
2πfZim
where f is the working frequency, 962 Hz.
In view of the deviations of the electrode surface and the
thickness of the sensitive films, the relative capacitance change
(C/C0 ) was defined to minimize the effect of the initial capac-
itance between different sensors. Here, C is the capacitance
change of the receptor film during the process of immunoreac-
tion and C0 is the initial capacitance of the receptor film. Curve
(a) of Fig. 7 showed an example of such capacitance measure-
ment. The capacitance of the immunosensor decreased because
of the specific interaction of antigen with the antibody on the sen- Fig. 8. Real-time capacitive response curves to different transferrin concentra-
sitive layer, that is, because of the additional thickness and/or tions. From top to bottom, the concentration was 1, 5, 10, 20, 30, 40, 50, 60, and
the changes of dielectric behavior. 75 ng/ml, respectively. The arrow indicates the points of injection of samples.
292 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294

were determined with the same electrode or different electrodes.

The relative standard deviations (R.S.D.) (n = 5) were less than
10% in the concentration range of 1–75 ng/ml. The reason for
the good reproducibility may be that gold nanoparticles homo-
geneously distributed in the sol–gel-derived film could steadily
immobilize the transferrin antibody and protect the bioactivity
and specific ability of the antibody protein.

3.7. Recovery test of transferrin

To test the application of the immunosensors, the samples

with various concentrations of transferrin were measured, and
the recoveries of the sensors varied from 94.0 to 104.8%, as
shown in Table 1. The good recovery indicated that the prepared
capacitive immunosensor might be promising in clinical testing.
Fig. 9. Calibration curve for transferrin detection transformed from Fig. 8.
3.8. Activity restoration of the immunosensor
capacitance changes versus time for the different concentrations
of transferrin. A linear relationship between the relative capac- Generally, the exhausted immunosensor can be restored by
itance change and the transferrin concentration was obtained in simple rinsing with a subacidity solution. In the experiment,
the range of 1–75 ng/ml. The detection limit was 0.05 ng/ml, as the final immunosensors were carefully washed in a stirring
calculated from the smallest measurable value of the capacitance glycine–hydrochloric acid solution (pH 3.5) for 15 min [11,12],
variation under the optimal experimental conditions, which was and the human transferrin combined with the modified layer
more sensitive than that of the reported capacitive immunosen- was released. Then, the regenerated immunosensors were put to
sor based on antibody-embedded ultrathin ␥-alumina sol–gel- the background solution of a PBS buffer solution (pH 7.4) for
derived films (1 ng/ml) [1]. The calibration curve for transferrin the next capacitance measurement to check whether their initial
detection under the optimum conditions, as shown in Fig. 9, capacitance of the sensors was restored or not. The six parallel
was transformed from the results of Fig. 8. The linear regression restoring experiments, as shown in Table 2, suggested that the
equation was: initial capacitance of the sensors could be restored to over 80%

 of their original values, and the activity-restored immunosen-
C
(%) = −0.3602Cx (ng/ml) − 4.474 sors could provide approximately the same capacitance changes,
C0
compared to the fresh fabricated ones, without any noticeable
The correlation coefficient was 0.9962. Here, Cx is the con- loss of sensitivity.
centration of transferrin.
To investigate the reproducibility of the sensors fabricated 4. Conclusions
with the present method, the different transferrin concentrations
A sensitive, label-free, specific capacitance immunosensor
Table 1 was successfully fabricated for transferrin detection by com-
Recovery results of transferrin immunosensor bining an ultrathin ␥-alumina sol–gel-derived film with gold
Added transferrin C/C0 (%) Found transferrin Recovery (%) nanoparticles. The nanometer thickness of the alumina film with
(ng/ml) (ng/ml) high permittivity presented the ideal performance for capaci-
5 −6.16 4.68 94.0 tance measurement. Moreover, the immobilization density of the
10 −8.25 10.48 104.8 antibody was high and the protein retained their immunoactivity,
30 −15.35 30.19 100.6 because of the large surface area and excellent biocompatibil-
60 −26.12 60.10 100.2 ity of gold nanoparticles. The proposed sensor was selective for
All data were obtained in a 0.01 M PBS solution (pH 7.4) by electrochemical the target antigen and the non-specific adsorption of the pro-
impedance technique. tein did not present much interference. The activity restoration

Table 2
Evaluation of the activity regenerated immunosensor
Fresh sensor Regenerated sensor

1 2 3 4 5 6

C/C0 (%) −18.6 −17.9 −17.3 −16.8 −16.0 −15.2 −14.9

Recovery (%) 96.2 93 90.3 86 81.7 80

All data were obtained from detection in the 40 ng/ml of transferrin after the transferrin antibody and antigen configuration on the surface of the sensor was washed
by acidic solution (glycine–HCl, pH 3.5).
 T. Yin et al. / Sensors and Actuators B 117 (2006) 286–294
of the sensor was also imaginable. Compared with traditional
immunoassays, the novel method achieved the real-time mon-
itoring the specific interaction between an antibody and an
antigen without labeling. The immobilization procedure in the
present work provided a new approach to fabrication of capaci-
tive immunosensors.
Acknowledgements
The authors gratefully acknowledge the financial support
of Doctorate Fund (No. 20020532007) and Key Sci/Tech Res.
Project (No. 03123) of Education Department, China.
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Biographies
Tanji Yin graduated from Hunan University in 2004 and received her bache-
lor degree. In 2004, she became a graduate student at State Key Laboratory of
Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical
Engineering of Hunan University, Changsha, China. Her research interests
cover chemical and biosensors.
Wanzhi Wei obtained his PhD degree from Hunan University in 1992. He is
currently a professor of the College of Chemistry and Chemical Engineering
in Hunan University, China. His research concentrates on the new analyti-
cal techniques in life science and environment science, and biological and
chemical sensing.
Liu Yang graduated and obtained his PhD degree from Hunan University
in 2005, majoring in chemistry. He is currently in employment in Hongta
Group of Yunnan, China.
Xiaohua Gao graduated from Henan Normal University in 2003 and received
her bachelor degree. In 2003, she became a graduate student of Hunan Uni-
versity majoring in chemistry. Her research interests cover chemical and
biosensors.
Yanping Gao graduated from Hunan University in 2004 and received her
bachelor degree. In 2004, she became a graduate student of Hunan Uni-
versity majoring in chemistry. Her research interests cover chemical and
biosensors.