Biosensors and Bioelectronics 20 (2005) 21632167

Short communication
A molecularly imprinted polymer on indium tin oxide and silicon

Lisa M. Kindschy
1
, Evangelyn C. Alocilja

Department of Biosystems and Agricultural Engineering, Michigan State University, 204 Farrall Hall, East Lansing, MI 48824, USA
Received 21 May 2004; received in revised form 17 August 2004; accepted 20 August 2004
Available online 3 October 2004
Abstract
Molecular imprinting is a technique for creating articial receptor sites in a polymer. Molecularly imprinted polymers (MIPs) are produced
by forming a polymer around a molecule that is used as the template. Upon removal of the template, molecular holes remain which are specic
in shape and size to the target molecule. In this research, a MIP was formed for theophylline using a copolymer of methacrylic acid and
ethylene glycol dimethacrylate. The theophylline MIP was formed on two platforms: indium tin oxide (ITO) and silicon, which were used
as the working electrode for cyclic voltammetry measurements. The presence of theophylline was measured using cyclic voltammetry and
corresponded to the peak current on the cyclic voltammograms. The results of this research agreed with previous results of MIPs immobilized
on an ITO platform. The peak currents of the MIP in the presence and absence of theophylline were compared to the blank polymer for each
platform. The ratio of peak currents on ITO increased by a factor of 9.5 for the MIP compared to the non-imprinted polymer. Similarly, the
ratio of peak currents on silicon increased by a factor of 6 compared to the non-imprinted polymer. This research demonstrated a procedure
for evaluating a MIP layer on two different platforms.
2004 Elsevier B.V. All rights reserved.
Keywords: Molecularly imprinted polymer; Molecular imprinting; Cyclic voltammetry; Indium tin oxide; Silicon; Theophylline
1. Introduction
The development of novel formats for the detection of sub-
stances continues to be a critical area of interest for scientists.
The limitations of current sensors have attracted researchers
to a growing class of articial receptors, called molecularly
imprinted polymers (MIPs) that can be used in place of bio-
logical receptors, such as antibodies. MIPs are produced by
growing a polymer around a template molecule. Once the
polymer has formed, the template is removed and molecular
holes remain that are specic in size and shape to the tem-
plate. Upon rebinding of the target molecule, a measurable
signal is produced that can be quantied. MIPs offer high

Presented at the Eighth World Congress on Biosensor 2004

(abstract BS395).

Corresponding author. Tel.: +1 517 355 0083; fax: +1 517 432 2892.
E-mail addresses: kindschl@msu.edu (L.M. Kindschy),
alocilja@egr.msu.edu (E.C. Alocilja).
1
Tel.: +1 5174328673; fax: +1 5174322892.
specicity and selectivity to the molecules that are used for
their formation.
Briey, the method of molecular imprinting involves the
polymerization of a functional monomer and a cross-linker
around a molecular template. In the rst step, the func-
tional monomers are assembled around the template by
non-covalent bonds. Following this pre-assembly step, the
monomer-template complex is combined with the cross-
linker, an initiator, and usually a porogenic solvent. Poly-
merization is started either thermally by the addition of heat
or photochemically by exposure to UV radiation. The poly-
merization step xes the position of the functional monomers
around the template by creating chemical bonds between the
cross-linkers and functional monomers, such that a memory
of the receptor site is frozen in place upon removal of the
template. After polymerization is complete, the template is
separated from the MIP by washing with an organic solvent.
This paper investigated a well-documented MIP and tem-
plate. In this research, a MIP was produced for theophylline
composed of the monomers methacrylic acid and ethylene
0956-5663/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2004.08.028
2164 L.M. Kindschy, E.C. Alocilja / Biosensors and Bioelectronics 20 (2005) 21632167
glycol dimethacrylate. The rebinding of theophylline to the
MIP layer was measured with cyclic voltammetry. This MIP
was tested on two different platforms: indium tin oxide and
silicon. The responses of the MIPs on the two platforms were
evaluated and compared.
2. Literature review
MIPs are well recognized for their sensor properties
and are frequently used as articial receptors in sensors
(Haupt and Mosbach, 1998, 2000; Piletsky and Turner,
2002; Ramstr omet al., 2001; Takeuchi and Haginaka, 1999).
Methacrylic acid (MAA) and ethylene glycol dimethacrylate
(EGDMA) have been used in several MIP procedures (Kriz
et al., 1995; Siemann et al., 1996; Tan et al., 2001). Further-
more, the bronchodilator theophylline has been successfully
used as the template and target analyte in numerous MIPs
(Hong et al., 1998; Lai et al., 1998; Mullett and Lai, 1998;
Vlatakis et al., 1993; Yoshimi et al., 2001). Theophylline
is an inexpensive, small molecule that has a high number
of polar groups that are able to interact with the functional
monomer. Therefore, the MAA-EDGMApolymer system, in
combination with the template theophylline, was chosen in
this research.
The rebinding of the analyte to the MIP is measured us-
ing cyclic voltammetry. Cyclic voltammetry is not one of the
more prevalent methods for the determination of template re-
binding. Other amperometric measurement techniques have
been successfully applied to MIPs (Blanco-L opez et al.,
2003; Kriz and Mosbach, 1995; Kroger et al., 1999; Yoshimi
et al., 2001).
The objective of the present study was two-fold to (1) ex-
pand on the research performed by Yoshimi et al. (2001) by
varying the imprinting procedure and (2) explore the use of
silicon as a MIP platform. Yoshimi et al. used cyclic voltam-
metry to measure the rebinding of theophylline to a MIP on
indium tin oxide. They observed an increase in current for
imprinted MIP compared to the blank polymer before and
after the addition of theophylline.
The immobilization of MIPs on silicon has not been
widely researched. Hedborg et al. (1993) attached a MIP for
l-phenylalanine to the surface of an oxidized p-type silicon
wafer. The MIP was sandwiched between silicon and glass
to measure the capacitance over a range of potentials. Using
this setup, Hedborg et al. were able to develop a sensor on
silicon that could detect the presence of l-phenylalanine.
3. Materials and methods
3.1. Chemicals
Methacrylic acid (MAA), ethylene glycol dimethacry-
late (EGDMA), 2,2

-azobisisobutyronitrile (AIBN), 3-
(trimethoxysilyl)propyl methacrylate (3-MPS) were pur-
chased fromSigma. p-Type silicon wafers (oxide layer 1 m,
conductivity 1667 S/cm) were obtained from the microfabri-
cation lab at Michigan State University. Indium tin oxide
(ITO)-coated glass slides were acquired from Sigma (ITO
coating 300600

A; resistance 3060 ). All other chemicals
were of ACS grade and used as received.
3.2. Preparation of electrodes
3.2.1. ITO electrode
The ITO-coated glass slides were cut into 1.3 cm squares.
Each electrode was rinsed twice with methanol followed by
rinsing twice with deionized water, and dried under nitrogen
environment.
3.2.2. Silicon electrode
The silicon electrodes were cleaned prior to use to remove
the surface oxide. Silicon was rst placed in a 1:1 (v/v) solu-
tion of methanol and hydrochloric acid for 30 min, and then
thoroughly rinsed twice with deionized water. Silicon was
next boiled in water for 30 min and rinsed twice with deion-
ized water. The silicon electrodes were dried under nitrogen
environment.
3.3. Formation of MIP
The process of preparing the platforms and forming the
MIP is illustrated in Fig. 1. The formation of the MIP fol-
lowed the procedure by Yoshimi et al. (2001) with some
modications. The electrode was silanized in a 10% solu-
tion (v/v) of 3-MPS in toluene for 6 h at 80

C under ni-
trogen atmosphere. Silanization activated the surface of the
substrate allowing the MIP to be covalently bonded to the
surface. Following silanization, the electrode was rinsed with
methanol anddriedunder nitrogenenvironment. The polymer
was prepared using methacrylic acid (MAA) as the functional
monomer, ethylene glycol dimethacrylate (EGDMA) as the
cross-linker, 2,2

-azobisisobutyronitrile (AIBN) as the ini-

tiator, and theophylline as the template. Chemical inhibitors
were removed from MAA and EGDMA by passing them
through an inhibitor removal column (Aldrich 30,631-2) im-
mediately before use. MAA (0.119 ml), EGDMA (1.20 ml),
AIBN (0.036 g), and theophylline (0.063 g) were added to
3.31 ml of N,N-dimethylformamide (DMF). The silanized
electrode was immersed in the above solution and placed un-
der nitrogen atmosphere. The electrode was allowed to poly-
merize for 12 h at 60

C. A reference non-imprinted polymer

(blank) to be referred to as B-ITOor B-Si, was similarly made
by omitting the theophylline template.
3.4. Extraction of template
Both the MIP and blank reference polymer on the
platforms were washed in a 9:1 (v/v) solution of methanol
and acetic acid for 1 h. This washing removed the template
and any excess polymer. The MIP and blank polymer were
L.M. Kindschy, E.C. Alocilja / Biosensors and Bioelectronics 20 (2005) 21632167 2165
rinsed twice with methanol followed by rinsing twice with
water and twice more with methanol. The MIPs were stored
in methanol until measurement to prevent drying or cracking
of the polymer.
3.5. Rebinding and measurement of analyte
The rebinding of theophylline to the MIP and blank
polymer was measured by conventional cyclic voltamme-
try in a three-electrode cell using a Versastat II Potentio-
stat/Galvanostat (Princeton Applied Research). The working
electrode was the treated platform, the reference electrode
was the Ag/AgCl, and the counter electrode was the untreated
material (ITO or silicon). The potential was cycled between
1 and +1 Vfor ITO, and between 2 and +2 Vfor silicon at
a scan rate of 200 mV/s. The resulting current was measured
and plotted against the potential.
Before rebinding of the analyte, a baseline was obtained
for each polymer using a blank electrolyte solution. The blank
was an aqueous solution of 0.1 Mpotassiumnitrate and 5 mM
potassium ferrocyanide. Blank solution of 25 ml was added
to the electrochemical cell, and cyclic voltammetry was per-
formed with the blank solution. After measurement, the blank
was discarded.
Fig. 1. Procedure for the formation of MIP on platform.
The rebinding of the analyte was performed in the elec-
trochemical cell by cyclic voltammetry measurements. The
analyte solution was the blank (0.1 M potassium nitrate and
5 mM potassium ferrocyanide) with the addition of 5 mM
theophylline. Analyte solution of 25 ml was added to the elec-
trochemical cell, and cyclic voltammetry was performed.
4. Results and discussion
4.1. MIP on ITO and silicon
The cyclic voltammograms of the MIP-ITO and B-ITO in
the presence and absence of theophylline are shown in Fig. 2.
The addition of theophylline in the MIP-ITO resulted in an
increase in the peak current of 0.036 mA. Addition of theo-
phylline in the B-ITOresulted in a slight decrease in the peak
current of 0.006 mA. The ratio of the changes in peak current
between the blank and after the addition of theophylline for
the MIP-ITO increased by 6 compared to the B-ITO.
In a previous study, Yoshimi et al. (2001) used ultraviolet
radiation to induce polymerization. In the present research,
the MIP was polymerized thermally by heating. A bulk poly-
mer was formed on the platform and removed by breaking
and subsequent washing steps. For removal of the template,
Yoshimi et al. utilized sonication in water, while the present
research employed rinsing in methanol and acetic acid solu-
tion. Earlytrials inthese experiments foundthat sonicationre-
moved all visible polymer and subsequent testing conrmed
the removal.
Currently, the washing steps are being explored for their
effectiveness at removing the template from a thin MIP layer
attached to the platform. The contribution of the thickness of
the MIP layer on the platform is also under investigation to
relate to the obtained signal and washing process. A proce-
dure to obtain a uniform layer is being considered such as a
spin coating method.
This research also presents some initial results of a thin
MIP layer directly attached to silicon as a sensor. The cyclic
voltammograms for the MIP-Si and B-Si are shown in Fig. 3.
The change in the peak current before and after the addition
of theophylline was 0.058 mA for the B-Si and 0.078 mA for
the MIP-Si. The ratio of the changes in the peak current of
the MIP-Si and the B-Si was only a factor of 1.3 and was
not as marked as the six-fold increase of the MIP-ITO to the
B-ITO.
The shapes of the curves for the silicon platform in Fig. 3
are comparable to those on ITOshown in Fig. 2. As in Fig. 2a
(B-ITO), the current response curve of B-Si before addition
of theophylline is not at compared to that of the MIP-Si.
However, the response curve after addition of theophylline
follows the curve before addition. This indicates that the re-
sponse fromB-Si is non-positive since the difference between
B-Si before and after addition is small.
2166 L.M. Kindschy, E.C. Alocilja / Biosensors and Bioelectronics 20 (2005) 21632167
Fig. 2. Cyclic voltammograms of (a) B-ITO and (b) MIP-ITO in the absence of theophylline (1) and 5 mM theophylline (2).
4.2. Statistical analysis
The cyclic voltammograms inFigs. 2and3showthe signal
before the analyte is added to the electrochemical cell and the
signal after addition of the analyte. Each cyclic voltammo-
gramwas an average of three trials. Each trial was performed
on a separate day using fresh solutions and new polymers to
account for slight variations in the solutions and day-to-day
error. The peak currents on the cyclic voltammograms were
selected for further analysis. Since the base signal was that
before analyte addition, the ratio of the current after analyte
addition to before analyte addition was used to compare be-
tween cyclic voltammograms. Single factor analysis of vari-
ance (ANOVA) was performed on the mean current ratios to
determine if the ratio of the MIP to the non-imprinted poly-
mer (blank) was signicant. The difference in the means was
considered to be signicant when the P-value was less than
0.05, representing a 95% condence level.
4.3. Comparison of peak currents
The ratios of the peak currents of the MIP and non-
imprinted polymer in the presence of theophylline to the ab-
sence of theophylline for ITO and silicon at the maximum
and minimum potentials are shown in Table 1. The corre-
sponding standard deviations of three trials are included in
the table at the minimum and maximum potentials.
The increase in the ratio of the peak currents of the MIP-
ITO compared to the B-ITO at a given potential showed that
the MIP on ITO was successful. The mean ratios at the min-
imum and maximum was 68.4 for the MIP-ITO and 7.15 for
the B-ITO. In this research, the presence of theophylline en-
hanced the peak current by a factor of 9.5. The ratios at the
maximum potential were 74.4 and 14.1 for the MIP-ITO and
B-ITO, respectively. The factor of increase between the MIP-
ITO and B-ITO is 5.3 when only considering the maximum
current. Yoshimi et al. (2001) found these ratios to be 2.94 for
the MIP-ITOand 0.99 for the B-ITOwith a factor of increase
between the MIP and blank of 3. The increase in the ratio of
MIP to blank for this research compared to previous results
might be attributed to modication of the template removal
procedure. The removal of the template by Yoshimi et al.
was performed in water while this research used a mixture of
methanol and acetic acid. The alcoholacid mixture was used
in this research because of its prevalence for the removal of
theophylline from the MAA-EGDMA copolymer (Hong et
al., 1998; Mullett and Lai, 1998; Vlatakis et al., 1993). The
template removal procedure is under investigation for its ef-
fectiveness as well as other factors that may contribute to the
MIP signal, such as the thickness and uniformity of the MIP
layer.
Similar ratios of the peak currents for silicon are shown in
Table 1 for the MIP-Si and B-Si. These initial results show
the potential for MIPs to be produced on a silicon platform.
Fig. 3. Cyclic voltammograms of (a) B-Si and (b) MIP-Si in the absence of theophylline (1) and 5 mM theophylline (2).
L.M. Kindschy, E.C. Alocilja / Biosensors and Bioelectronics 20 (2005) 21632167 2167
Table 1
Ratio of peak currents for 5 mM theophylline to absence of theophylline at
maximum and minimum potentials for ITO and silicon
Platform Ratio of current
Minimum potential Maximum potential
MIP-ITO 62.4 30.3 a 74.4 29.0 a
B-ITO 0.192 1.04 b 14.1 23.2 b
MIP-Si 12.8 3.51 a 8.87 7.03 a
B-Si 2.13 1.30 b 1.17 0.377 a
For a given platform(ITOor Si), means with different letters are signicantly
different (P < 0.05).
The ratio of the peak currents increased by a factor of 6 for the
MIP-Si compared to the B-Si at the minimum potential. The
maximumcurrents were not considered since the means were
not signicantly different. The variation between different
batches of MIPs on silicon merits further investigation to
obtain a consistent response. Additional studies are being
conducted to increase the success of the theophylline MIP on
silicon.
MIPs immobilized on silicon show potential for research.
Silicon is a relatively inexpensive material whose proper-
ties can be uniquely and precisely controlled. Previous lit-
erature has shown difculty forming a thin MIP layer on
silicon (Hedborg et al., 1993). Methods to obtain a consis-
tent response on silicon as well as the formation of MIPs on
various forms of silicon (oxide layers, porous structures) are
being investigated. The potential for research into MIPs at-
tached to silicon will likely increase since few studies have
been conducted and many possibilities for sensors still exist.
5. Conclusions
This research demonstrated a procedure for evaluating
the properties of a molecularly imprinted polymer (MIP)
sensor on two distinct substrates. A MIP was formed for
theophylline using a copolymer of methacrylic acid and
ethylene glycol dimethacrylate on two platforms: indium tin
oxide (ITO) and silicon. It was shown that the MIP could be
formed using thermal polymerization compared to ultraviolet
radiation, as used by Yoshimi et al. The results of the theo-
phylline MIP on ITO supported the ndings of the previous
study of Yoshimi et al. using the same MIP and template.
The presence of theophylline increased the peak current by
a factor of 9.5 for the MIP compared to the non-imprinted
polymer (omission of theophylline). The increase in the peak
current in the presence of the analyte (a rise of 0.006 mA for
B-ITO and 0.036 mA for MIP-ITO) allowed the system to
act as a qualitative sensor to the presence of theophylline.
The ratio of the currents on the MIP-Si increased by a factor
of 6 compared to the B-Si. Studies are being conducted on
the sensitivity of the MIP on ITO, as well as into increasing
the success and repeatability of the MIP on silicon.
Acknowledgement
The authors would like to thank Dr. Dean Aslam, Depart-
ment of Electrical Engineering, Michigan State University,
for providing the silicon wafers.
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