A MIP-based Impedimetric Sensor

for the Detection of low-MW Molecules

18. Thoelen*1, R. Vansweevelt1, J. Duchateau1, F. Horemans1, J. DHaen1,2, L. Lutsen2,
1,2

D. Vanderzande , M. Ameloot , M. vandeVen , T.J. Cleij , and P. Wagner

1 Hasselt University, Institute for Materials Research, Wetenschapspark 1, 3590 Diepenbeek,

Belgium 2 IMEC, Division IMOMEC, Wetenschapspark 1, B-3590 Diepenbeek, Belgium
3 Biomedical Research Institute, Hasselt University and transnationale Universiteit
Limburg, Agoralaan, B-3590 Diepenbeek, Belgium
ronald.thoelen@uhasselt.be

Abstract
Mimicking the selectivity and sensitivity of biological systems for sensor
devices is of increasing interest in biomedical, environmental and chemical analysis.
Synthetic materials with imprinted nanocavities, acting as highly selective artificial
receptors, are a tailor-made solution in obtaining such a sensor. Incorporation of
such molecularly imprinted polymers (MIPs) in a platform suitable for
electrochemical measurements, can offer high sensitivity together with device
miniaturization and an electronic read-out. As a proof of principle, a MIP-based
sensor for L-nicotine has been developed. To this end, the molecular structure of Lnicotine was imprinted in a polymer matrix of PMAA. Subsequently, microparticles
of the imprinted polymer were immobilized on thin films of the conjugated polymer
OC1C10-PPV. These films were incorporated in an impedimetric sensing device.
Using electrochemical impedance spectroscopy, the real part of the impedance was
monitored for various concentrations. This setup allows for the detection of Lnicotine from 1 to 10 nM and is insensitive for the resembling molecule L-cotinine.
Keywords: Molecular imprinting, L-nicotine sensor, impedance spectroscopy,
conjugated polymers
1

1. Introduction
Molecularly imprinted polymers (MIPs) are an important class of synthetic
materials mimicking the molecular recognition of low-weight molecules by natural
receptors (Cormack and Elorza 2004; Haupt and Mosbach 1998; Piletsky et al.
2001). The focus areas in which MIPs are being implemented are mainly materials
science and separation technology (Ansell et al. 1996). However, these materials
become also increasingly popular for sensor purposes. The technology provides a
wide pallet of sensing devices for different kinds of target templates, such as
bacteria (Harvey et al. 2006), low molecular weight molecules (Piletsky et al. 2006;
Dickert et al. 2004) and proteins (Bossi et al. 2007). The detection of the binding can
be achieved by electrochemical transducers with MIP membranes, piezo-electric or
optical transducers (Blanco-Lopez et al. 2004; Ebarvia and Sevilla 2005; Liao et al.
2004; Merkoci and Alegret 2002; Panasyuk-Delaney et al. 2001).
The imprinting technique is based on the development of either non-covalent
or reversible covalent complexes formed between the target molecule and suitable
functional monomers. Subsequently, a cross-linker is added to this mixture to form a
matrix in which the complexes are fixed. After extraction of the target molecule
from this matrix, a tailor-made, highly selective synthetic receptor is created.
In this work, the aim is to develop an approach for a fast, inexpensive, MIPbased analyte-sensor. The fast readout is achieved by using impedance spectroscopy
in a basic electrochemical cell configuration and monitoring the time resolved
capacitance. To enhance the signal, a conjugated polymer is used to immobilize the
non-conducting MIP particles onto the electrode surface. The conjugated polymer,
OC1C10-PPV, which is stable in aqueous and a variety of organic solvent solutions,
has also successfully been used as transducer layer in immunosensors (Cooreman et
al. 2005). As proof of principle, a MIP-based sensor for L-nicotine, C 10H14N2, has
been developed. L-nicotine has previously been employed as a template for
molecular imprinting in chromatography. In 2001 the first successful attempt was
made to implement this material in a QCM device (Tan et al. 2001), which achieved
a detection limit of 25 nM. The MIP particles in Tans work were immobilized in a
PVC matrix and the analyte was dissolved in double-distilled water. The relevant
medical concentration for nicotine in urine for non-smokers is 0.3 M and for
smokers 6.3 M.
2

2. Experimental
The PPV derivative, OC1C10-PPV, which was used as the base polymer for the
immobilization layer was synthesized via the sulphinyl route (Louwet et al. 1995). All
chemical and physical properties were in agreement with previously reported data. This
polymer behaves as a p-type semiconductor with a bandgap of 2.1 eV. The imprinted
polymer was synthesized using ethylene glycol dimethacrylate (EGDM) as the crosslinker, the monomer methacrylic acid (MAA) and chloroform as the porogen. Prior to
polymerization, the stabilizers in the MAA and EGDM were removed. For the initiator
azobisisobutyronitrile (AIBN) was used. All chemicals used in the polymerization were
of analytical grade and were obtained from various commercial sources. The target
molecule L-nicotine, C10H14N2, was obtained from Acros. A similar molecule, Lcotinine, C10H12N2O, which was obtained from Sigma-Aldrich, was used to test the
specificity. Cotinine is a byproduct of the human metabolism of nicotine. Both
molecules are shown in Fig. 1.

Fig. 1. molecule structure of L-nicotine (MW: 162.23 Da

) and L-cotinine (MW: 176.22 Da ).
For the preparation of the L-nicotine molecularly imprinted polymer (MIP), a
mixture of MAA (12.5 mmol), EGDM (25.2 mmol) and AIBN (0.66 mmol) was
dissolved in 7 ml chloroform together with the template molecule L-nicotine (6.41
mmol). To exclude oxygen in the mixture, it was degassed for 10 min with N 2.
Subsequently, for polymerization the solution was sealed and kept in a thermostatic
water bath at 60C for 72 hours. After polymerization the solid MIP was grounded with
a mechanical mortar for 24 hours and sieved through a 25-m sieve. Only particles with
a size smaller than 25 m were used. Next, the L-nicotine was removed

from the MIP powder by extensive washing with methanol (48 hours), followed by a
mixture of acetic acid/acetonitrile (1/1) (48 hours) and finally again methanol (X
hours) using a continuous extraction setup. A non-imprinted polymer (NIP) was
synthesized in the same way but without the presence of the target molecule.
The binding constant and quality of the molecularly imprinted polymer were
evaluated with UV-Vis spectroscopy using the Varian Cary 500 UV-Vis-NIR
spectrophotometer. The morphology of the sensor surface was investigated using
optical microscopy with a Zeiss Axiovert 40 MAT, and scanning electron
microscopy, with a FEI Quanta 200 FEG-SEM. Microscope images were processed
using the image analysis program ImageJ 1.37v from the National Institute of
Health, USA. In order to detect the L-nicotine recognition electronically, the
electrochemical impedance principle was used. The activated electrodes, together
with the reference electrodes, were placed in an impedimetric addition setup
allowing time-resolved, differential measurements. The impedance spectra were
measured with zero bias and an oscillating voltage of 50 mV in a frequency range
from 100 Hz to 1 MHz with a HP4194A Gain-Phase analyzer. The time resolved
measurements were performed at a fixed frequency of 213 Hz, in order to be
sensitive to changes at the interface between the sensor and the electrolyte.
3. Results and discussion
3.1 Characterization of the MIPs
For a refined analysis of the binding mechanisms between MIPs/NIPs and
nicotine/cotinine, batch rebinding experiments were performed using UV-Vis absorption
spectroscopy by measuring the nicotine absorption at 260 nm. To test the specificity of
the L-nicotine imprinted polymers, the MIP particles were also exposed to several
concentrations of L-cotinine. The same experiments were performed with the NIPparticles. A fixed amount of MIP particles, 20 mg, was added to different concentrations
of L-nicotine Ci from 0.2 to 1 mM. After stirring these solutions for one hour at room
temperature, the concentration of target molecules remaining in solution (C f) was
measured. Subsequently, the amount of target molecules bound to the MIP (S b) and the
amount of bound molecules per gram MIP were calculated. Binding isotherms and
corresponding Scatchard plots for the imprinted and non4

imprinted polymers are shown in Fig. 2. Due to the high affinity of the specific binding
sites in the MIP, the amount of L-nicotine bound to the MIP is higher than in the NIP.
The amounts of L-cotinine bound to MIP and NIP are equal and significantly lower as
compared to the L-nicotine binding characteristics. The binding of L-cotinine is nonspecific and may involve the free acid groups at the surface of the polymer.

Fig. 2. Graph of binding isotherms (a) and corresponding Scatchard plots (b) for
imprinted and non-imprinted polymers exposed to L-nicotine and L-cotinine.
The binding sites in imprinted polymers have a large heterogeneous distribution
due to different organization of the complementary functional groups and different
shape-selective cavities (Umpleby et al. 2000; Spivak 2005). For a single class of
binding sites, the Langmuir isotherm would be a straight line (Langmuir 1918). The
Scatchard plots, Fig. 2b, indicate however the existence of a non-linear relationship,
thus implying a hetergenous distribution of the binding sites, each with different affinity.
For this type of distributions the binding isotherm can be better analyzed using the
Freundlich model (Freundlich 1926), see Equation 1. This model describes an
adsorption system with emphasis on two factors, namely the lateral interaction between
the

adsorbed molecules and the

Sb = A C

energetic

surface

heterogeneity.

(1)
Where Sb is the amount of target molecule bound per gram of MIP/NIP, A is the
Freundlich constant and the Freundlich heterogeneity parameter. The binding

isotherms in Fig. 2a were fitted using Equation 1. The parameters are presented in
Table 1 and can be used to plot the affinity distribution according to Equation 2,
where N(K) is the amount of binding sites available for each association constant K.
This can be seen in Fig. 3.

N (Ki ) = A

sin()

Ki

(2)

Using the parameters the total number of binding sites, N tot, fore the average
association constant, Kav, within the range from 1 100 mM

-1

can be calculated

using Equation 3 and 4 (Spivak 2005). These values are also listed in Table 1.

Ntot = A

sin() (K

min

Kmax )

(3)

K =
av

(Kmax1 Kmin1 )
1 (Kmin Kmax )

(4)

Table 1. Freundlich parameters from fit in Fig. 2a together with the number-average
association constant and the total number of binding sites for the range of binding
-1

constants from 1 100 mM .

MIP L-nicotine
NIP L-nicotine
MIP L-cotinine
NIP L-cotinine

A
217.9
88.8
1.6
0.4

0.45
0.64
0.58
0.68

Kav
-1
(mM )
10.8
7.9
8.7
7.4

Ntot
(mole/g)
59.8
24.3
0.46
0.11

Fig. 3. The affinity distribution for imprinted and non-imprinted polymers based
on the Freundlich model, the graphs were calculated using Eq. 2 with the
parameters from Table 1, error bars are smaller than symbol size.
The results in Table 1 confirm the higher affinity of the MIP for L-nicotine than for Lcotinine. In addition, it can be seen that the MIP particles have twice as many binding
sites for L-nicotine as the NIP, while the number of binding sites for L-cotinine is even
lower. For the region between 1 and 100 mM

-1

the amount of binding sites for L-

cotinine is only 0.46 mole/g for the MIP and 0.11 mole/g for the NIP.

3.2 Sensing concept and results

The sensing devices consisted of a glass cover slip substrate, 20 mm by 12 mm,
on which eight platina electrodes were sputtered, resulting in a device consisting of four
sensitive regions. A 200 nm thick film of the conjugated polymer OC 1C10-PPV, was
spincoated on top of the electrodes from a chlorbenzene solution. Subsequently, the MIP
particles

were

transferred

to

the

OC 1C10-PPV covered

electrode

using

poly(dimethylsiloxane) (PDMS) stamp, which was pressed on the electrode with a small
force of about 100 Pascal during a few seconds. After removal of the stamp, a thin film
of micro particles of MIP remained on the surface. Subsequently, the polymer surface
was heated to 110 C, which is above glass transition temperature OC 1C10-PPV, during
ten minutes causing the powder particles to sink into the

conjugated polymer matrix. The sample was packaged into an addition setup with
four sensing sites, shown in Fig. 4, and the impedance was measured time resolved
at a fixed frequency of 213 Hz. The addition setup allowed addition of the target
molecule by adding of few droplets in the buffer solution to reduce artifacts from
replacing the buffer during the measurement. The four sensing sites allowed
simultaneous monitoring of a bare Pt electrode, a Pt electrode covered with OC 1C10PPV and a Pt electrode covered with OC 1C10-PPV-MIP layer and OC1C10-PPV-NIP
layer. Before and after each measurement a complete impedance spectrum was
measured.

Fig. 4. Sensing device with four sensing channels with 500 m electrode spacing,
Pt / Pt-OC1C10 PPV / Pt- OC1C10 PPV-MIP / Pt- OC1C10 PPV-NIP.
After immobilizing the MIP particles on top of the electrode surface, the density
was investigated using optical microscopy. It could be seen that the macroscopic
covering percentage of the particles was about 23.5 %. The average size of the clusters
of particles was 49.3 m. Fig. 5 shows the optical microscope image.

Fig. 5. Optical microscope images of MIP particles on the transducer layer

(a) and the size distribution graph (b), the solid line is a guide to the eye.

To study the coverage at the sub-micron scale, scanning electron microscopy

(SEM) was used. In between the large clusters, a coverage of 6.1 % of small
fragments was found with an average size of 0.047 m. With the electron
microscope we can distinguish the separate MIP particles on top of the electrodes as
seen in Fig. 6a. To investigate the sinking of the MIP particles into the polymer
layer, a cross-section was taken of the same device, seen in Fig. 6b. The sunken
splinter has a diameter of 300 nm.

Fig. 6. SEM image of MIP particles seen from above (a) and the cross-section (b),
using an accelerating voltage of 15.0 keV and a large-field detector (LFD).

Electrical measurements were performed by using electrochemical impedance

spectroscopy. Simultaneous a bare Pt electrode and OC 1C10-PPV coated electrodes
without and with MIP and NIP particles were measured in phosphate buffered saline
(PBS) solution before addition of nicotine. PBS is used to mimic the ionic strength
of body liquids and to maintain a constant pH. The latter may be important since Lnicotine is known to have different protonations depending on the acidity of the
solution. (Troje et al. 1997). To investigate the recognition event, the real part of the
impedance is tracked in time at the lower frequency regime (213 Hz). At this regime
the signal represents events that take place at the interface between the electrolyte
and the sensor surface. Binding of nicotine effects the formation of the double layer
at the electrode surface, which is reflected in the capacitive changes at the lower
frequency range. In addition, it influences the charge transfer from the electrolyte to
the electrode.

Fig. 7a shows the dose-response curves of the MIP- and NIP-based sensor
electrode after addition of L-nicotine to the PBS solution. The black curve, which
corresponds to the signal of the MIP coated electrode, shows an increase of 45%
when 10 nM of L-nicotine is introduced in the setup, whereas the signal of the NIP
coated electrode only increased with 30%. The change of the bare OC 1C10-PPV is in
same order of magnitude as the NIP sample. Also the bare OC 1C10-PPV and NIP
electrodes react on addition of L-nicotine, which is probably due to adsorption at the
interface. In the case of the NIP, this adsorption may involve the free acid groups,
whereas in the case of OC 1C10-PPV this may be the result of - interactions. The
impedance change is measured 20 minutes after each addition of L-nicotine.
Subsequently, the influence of L-cotinine is investigated in the same way as
was done for L-nicotine. L-cotinine differs only by one oxygen atom from Lnicotine (cf. Fig. 1). Fig. 7b shows the response of the four different channels. A
signal increase is observed due to adsorption of L-cotinine to the polymer surface.
The response is not specific while both responses are in the same order and
markedly lower than the reaction of L-nicotine of the sensor. It can be expected that
this adsorption has a similar origin as discussed above for the non-specific
adsorption of L-nicotine.

Fig. 7. Impedance change due to L-nicotine exposure to the sensor surface in

the concentration range of 1 10 nM (a) and due to L-cotinine exposure in the
concentration range of 1 12 nM (b).
1
0

To eliminate the contribution of non-specific adsorption of L-nicotine to the

surface of the MIP, it is better to plot the signal in a differential way. This is
achieved by subtracting the normalized impedance signal of the NIP from the
normalized MIP signal. Fig. 8 shows a clear difference between the reaction of the
sensor to L-nicotine and L-cotinine. A linear response is found from 2 to 5 nM with
a response of 4.3% increase of the differential signal per nanomolar. For
concentrations higher than 5 nM the impedance change is similar for MIP and NIP.
This is due to full occupation of the specific binding places in the MIP and in this
way the MIP reacts in the same way as the NIP.

Fig. 8. Differential signal, NIP subtracted from the MIP.

4. Conclusion
The microscopy results show a good immobilization of the powdered MIP to the
OC1C10-PPV covered electrode surface using the PDMS stamping technique. UV-Vis
Spectroscopy has been used to characterize the MIPs before implementing them in the
sensing device. Binding properties have beene investigated using the Freundlich model,
which indicates the occurrence of specific recognition of L-nicotine and insensitivity
towards L-cotinine. The measured values of the binding parameters are comparable to
literature values. It is demonstrated that by using impedimetric detection, it is possible to
detect low-MW molecules using molecularly imprinted polymers, which are
immobilized on a conjugated polymer thin film. This technique
11

offers the possibility to detect 10 nM with a response increase of 45 percent, which

is much lower than relevant medical concentrations. The reference measurements
with L-cotinine, which only differs one oxygen atom from L-nicotine, indicates a
high specificity. As a fast, easy-to-use sensor with electric readout, the MIP-based
impedimetric sensor could be useful for the detection of small molecules in the
pharmaceutical, environmental, diagnostic and biotechnological sector.
Acknowledgements
This work is supported by the School for Life Sciences of transnational University
Limburg,

by

Hasselt

University

via

the

BOF-project

Development

and

Characterisation of Fluorescent Molecularly Imprinted Polymers for Nanotechnology

and Applications in Advanced Electronic Devices, Chemo- and Bio-sensors and the
Fund for Scientific Research Flanders via the Scienctific Research Community FWOWOG (W0.035.04N) Hybrid systems at nanometer scale. Technical assistance by J.
Sogen and J. Baccus (Hasselt University) is gratefully acknowledged.

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