MINIREVIEW
Piezoelectric sensors based on molecular imprinted
polymers for detection of low molecular mass analytes
Yildiz Uludag
˘
1,2
, Sergey A.Piletsky
1
, Anthony P. F. Turner
1
and Matthew A. Cooper
2
1 Cranfield Health, Cranfield University, Silsoe, UK
2 Akubio Ltd, Cambridge, UK
Introduction
Biosensors are analytical devices that comprise a sam-
ple-delivery mechanism with a biological recognition
element and a suitable transducer, usually coupled to
an appropriate data-processing system (Fig. 1). The
biological recognition element is typically an enzyme,
microorganism, cell, tissue or other bioligand [1] and
the transducer is required to convert the physico-chem-
ical change resulting from the interaction of molecules
with the receptor into an electrical signal. Over the
past decade the benefits of label-free analysis have
begun to gain a foothold as a mainstream research
tool in many laboratories [2,3]. These techniques do
not require the use of detection labels (fluorescent,
radio or colorimetric) to facilitate measurement; hence
detailed information on an interaction can be obtained
during analysis while minimizing sample processing

(Received 6 July 2007, accepted 24 August
2007)
doi:10.1111/j.1742-4658.2007.06079.x
Biomimetic recognition elements employed for the detection of analytes are
commonly based on proteinaceous affibodies, immunoglobulins, single-
chain and single-domain antibody fragments or aptamers. The alternative
supra-molecular approach using a molecularly imprinted polymer now has
proven utility in numerous applications ranging from liquid chromatogra-
phy to bioassays. Despite inherent advantages compared with biochemi-
cal ⁄ biological recognition (which include robustness, storage endurance
and lower costs) there are few contributions that describe quantitative ana-
lytical applications of molecularly imprinted polymers for relevant small
molecular mass compounds in real-world samples. There is, however, sig-
nificant literature describing the use of low-power, portable piezoelectric
transducers to detect analytes in environmental monitoring and other appli-
cation areas. Here we review the combination of molecularly imprinted
polymers as recognition elements with piezoelectric biosensors for quantita-
tive detection of small molecules. Analytes are classified by type and
sample matrix presentation and various molecularly imprinted polymer
synthetic fabrication strategies are also reviewed.
Abbreviations
MIP, molecularly imprinted polymer; QCM, quartz crystal microbalance.
FEBS Journal 274 (2007) 5471–5480 ª 2007 Akubio Ltd. Journal compilation ª 2007 FEBS 5471
glucose dehydrogenase and rank order binding to the
enzyme has been determined [7], and biotin has been
detected with a high-frequency microfluidic acoustic
biosensor using an anti-biotin serum [8]. Real-time
detection of 4-aminobutyrate (one of the main inhibi-
tory neurotransmitters) was achieved with an anti-
(4-aminobutyrate) serum with a minimum detection limit

molecules and the analysis of binding events. In gen-
eral, they are based on quartz crystal resonators, which
are found in electronic devices such as watches, com-
puters and televisions, with over one billion units
mass-produced each year. Quartz crystal is a piezoelec-
tric material which mechanically oscillates if an alter-
nating voltage is applied. A QCM consists of a thin
quartz disc sandwiched between a pair of electrodes.
The mode of oscillation depends on the cut and geom-
etry of the quartz crystal. If mass is applied to the sur-
face of the quartz resonator, the frequency of the
oscillation decreases. By measuring the change of fre-
quency, it is possible to determine the change in mass.
Measurement of mass using quartz crystal resonators
was first examined by Sauerbrey [11], who showed that
the frequency change of the crystal resonator is a linear
function of the mass per area m
s
, or absolute mass Dm:
Df
m
¼À
f
2
0
F
q
q
q
m

the mass den-
sity, and A
el
the electrode size of the crystal resonator.
Equation (1) is valid only for thin, solid layers depos-
ited on the resonator.
Initially, the QCM system was used for dry measure-
ments, later when suitable oscillator circuits were
developed, it was possible to carry out measurements
under liquid conditions [12]. This led to the use of
QCM systems as biosensors to detect molecular inter-
actions (Fig. 2). A new equation was derived by
Kanazawa and Gordon to explain the relationship
between density (q
l
) and viscosity (g
l
) of the liquid and
the frequency of the quartz crystal resonator:
Df ¼Àf
3=2
q
ffiffiffiffiffiffiffiffiffiffiffiffi
q
1
g
1
pq
q
l

1
f
0
pl
q
q
q
r
!
ð3Þ
In addition to the frequency shift, there also exists a
dampening of the resonator caused by the viscous
Fig. 2. Schematic representation of quartz crystal resonance
sensing.
Antibody/Protein
Enzyme
Microorganism
Cell
Recognition elements
Transducer
Optical
Piezoelectric
Electrochemical
Calorimetric
Electric
signal
Analyte
Antibody/Protein
Enzyme
Microorganism

not strictly accurate for all applications. The device is
also known as thickness-shear mode resonator or a
bulk acoustic wave sensor, because the bulk of the
crystal oscillates at a resonance frequency in a thick-
ness shear mode of vibration.
Surface acoustic wave sensors are also based on the
piezoelectric properties of quartz crystal. In this case
only a surface wave is generated by the electrodes and
the frequency of the surface waves is $ 100 MHz to
1GHz [14]. These frequencies are much higher than
thickness-shear mode resonators, and this is the reason
for the higher sensitivity of surface acoustic wave sen-
sors. However, higher sensitivity also means higher
response to viscosity changes and this problem causes
difficulties when surface acoustic wave sensors are used
in liquids [15]. By monitoring the change in resonant
frequency and motional resistance that occurs upon
adsorption of a ligand to the surface, quartz crystal
resonators can be used to characterize interactions
with peptides [16], proteins and immunoassay markers
[17], oligonucleotides [18], viruses [19], bacteria [20]
and cells [21]. The technology can thus be applied to
an extremely wide range of biological and chemical
entities with a molecular mass range from < 200 Da
through to an entire cell.
Application of acoustic sensors to small molecule
detection
The detection limit of many affinity biosensors is clo-
sely linked to the molecular mass of the analyte. Many
researchers prefer to immobilize the small molecule

alone. For example, Carmon et al. [26] immobilized a
glucose ⁄ galactose receptor on a QCM sensor surface
and exposed the receptor to 180 Da sugars. A repro-
ducible frequency change was observed which was
ascribed to the conformational change of the receptor
upon ligand binding. Similarly conformational changes
have been invoked in the binding of ions and peptides
to calmodulin due to ion or peptide binding [27], and
the insertion of an Ad-2a model peptide onto glyco-
lipid monolayers [28]. In the latter case, the 2a-helix
structure of the peptide in the bulk solution is known
to convert to a b-structure upon association with a
lipid monolayer. This conformational change was man-
ifested as a frequency decrease for the piezoelectric
sensor.
This approach has been extended further in a
dynamic electropolymerization study in which imped-
ance data were acquired during polymerization at the
fundamental and third harmonic modes of a 10 MHz
thickness shear mode resonator [29]. At a critical
thickness, the system exhibited mechanical resonance,
a special condition in which the mechanical shear
deformation across the polymer film corresponded to
one quarter of the acoustic wavelength. At this point,
the resonant frequency and admittance data showed
dramatic changes with polymer coverage. Several
groups have also extended full impedance analysis
incorporating shear modulus modelling to protein films
[30] and phage binding [31].
Y. Uludag

and density q
L
). For these layered compo-
nents, it is possible to derive the surface mechanical
impedances:
Z
M
¼ jxq
s
ð4Þ
for an ideal ⁄ rigid mass layer, where r
s
is the mass per
area contributed by the interfacial layer;
Z
F
¼
ffiffiffiffiffiffiffiffi
q
f
G
q
tanhðch
f
Þð5Þ
for a viscoelastic film (MIP), where c is the shear wave
propagation constant (c ¼ j2pf
o
(r
f

ffiffiffiffiffiffiffiffi
q
f
G
p
sinhðch
f
Þ
ffiffiffiffiffiffiffiffi
q
f
G
p
cosh hðch
f
ÞþZ
L
sinhðch
f
Þ
ð7Þ
where Z is the impedance for the composite system.
Note that the impedance measured by the piezoelectric
sensor is not simply the sum of those for individual
layers, as for each layer there will be an acoustic phase
shift, which causes a transformation of the impedance
contributed by layers more distant from the resonator.
In addition, this model does not accommodate the typ-
ically inhomogeneous layers that exist in reality in
MIPs exposed to biological systems, or changes in dis-

where g
p
and q
p
are the liquid viscosity and density,
respectively, and v
p
is the fraction of the volume
within the penetration depth occupied by protein. This
could be extended to encompass both a receptor layer
and analyte layer if necessary. Integrating the mole
fraction of MIP ⁄ water in combination with the defini-
tion of a composite impedance above, we can derive:
Z ¼ v
p
j2pf
0
ffiffiffiffiffiffiffiffi
q
f
G
q
ð1 À v
p
ÞZ
L
cosh hðch
f
Þþ
ffiffiffiffiffiffiffiffi

tionary phases for chromatographic methods. Later,
the application of imprinted polymers was extended to
the biosensors area, where MIPs have been used as
recognition elements as an alternative to biological
materials such as antibodies and proteins. Similarly,
synthetic receptors formed by molecular imprinting
can be used to recognize biological or nonbiological
molecules on QCM sensors. Here the imprint of a tem-
plate molecule is formed on a synthetic polymer that
has cavities resembling the geometric shape of the tem-
plate and also has binding sites for template recogni-
tion [33]. MIPs as synthetic receptors have several
advantages over biological receptors [35]. The main
advantage of MIPs is their stability to harsh condi-
tions, in contrast to natural biomolecules that are sen-
sitive to environmental changes and can denature
easily. Because of the robust nature of MIPs, biosen-
sors that use MIP surfaces in general have a longer
shelf life than analogous biological sensors. MIPs are
simple to prepare, and their adaptation to a variety of
Detection of low molecular mass analytes Y. Uludag
˘
et al.
5474 FEBS Journal 274 (2007) 5471–5480 ª 2007 Akubio Ltd. Journal compilation ª 2007 FEBS
practical applications has been widely demonstrated.
In addition, molecular interaction studies with MIPs
can be performed in organic solvents as well as aque-
ous solvents.
Template molecules can be imprinted to a polymer
with covalent- [36], noncovalent- [37] or metal-ion-

cess should be optimized. Template design, monomer
selection, solvent selection and polymerization condi-
tions all require attention. In general, the performance
of MIPs in aqueous solutions is poor, therefore, if
water-soluble templates need to be used the polymeri-
zation method needs to be carefully considered. High
nonspecific binding and heterogeneity of binding sites
needs to be addressed for successful application. If,
after polymerization, there are still embedded template
molecules remaining in the polymer, this will reduce
the capacity and invalidate analysis. Therefore extra
care needs to be taken to remove the template from
the polymer and the 3D structure of the polymer
should allow for easy regeneration of the template
from the polymer for repeated bindings. Reproducible
fabrication of MIPs is essential for gathering reliable
results from each assay.
MIP–QCM sensors
Every year many new studies are published involving
MIP–QCM sensors. In these applications, MIP synthe-
sis is performed either in situ on the sensor surface or
via preprepared MIP particles ⁄ beads that are immobi-
lized on the sensor surface using a PVC matrix. The
thickness of the imprinted polymers varies between
18 nm and 5 lm. To obtain a reproducible and reli-
able MIP–QCM sensor, it is essential to control the
thickness and properties of the polymer coating on the
sensor surface.
Monomers and target
Target & monomers complex

Two approaches to synthesize in situ imprinted poly-
mers for QCM sensing have been reported. In one
approach, the gold surface of the QCM was treated
with a thiolated molecule to create active groups on
the sensor surface and improve the adhesion of the
imprinted polymer on the gold electrode (Table 2).
Allyl mercaptan [40,41] (N-Acr-l-Cys-NHBn)
2
[42],
mercaptoethanol [43], thioctic acid-modified glycidyl
methacrylate [44], and mercaptoundecanoic acid [45]
have been used to activate the gold electrode. In the
other approach, polymer synthesis was performed
directly on to the gold surface without any activation.
MIPs could be deposited by surface grafting, spin
coating, sandwich casting, or electro-polymerization
methods.
Surface grafting method
Although it is difficult to control MIP film thickness
during polymer synthesis, in a sensing device it is
essential to reduce batch-to-batch variation. For this
purpose, Piacham et al. investigated a possible route to
prepare ultra-thin MIP films (< 50 nm) specific for
(S)-propranolol [45]. The imprinting process was per-
formed directly on to the quartz surface after coating
the gold-coated crystal surface with mercaptoundeca-
noic acid. The carboxyl groups of mercaptoundecanoic
acid were then activated with initiators, 2-ethyl-5-phen-
ylisoxazolium-3-sulfonate and 2,2-azobis(2-amidino-
propane) hydrochloride. The sensor was dipped into a

[42]
(S)-Propranolol Mercaptoundecanoic
acid
Surface grafted ABAH TRIM Methacrylic acid [45]
Bilirubin Allyl mercaptan Surface grafted Benzophenone Divinylbenzene 4-Vpy [41]
Indole-3-acetic acid Allyl mercaptan and
1-butanethiol
Sandwich casting AIBN EGDMA N,N-dimethylaminoethyl
methacrylate
[40]
Sialic acid Allyl mercaptan Sandwich casting – EGDMA 4-Vpy and AMVN [40]
Dansylphenylalanine Thioctic acid-modified
GMA and thioctic
acid dodecane ester
Sandwich casting AIBN EGDMA Methacrylic acid, 4-Vpy [46]
L-Tryptophan Thioctic acid-modified
GMA
Sandwich casting – TRIM Acrylamide [44]
Sialic acid – Sandwich casting – EGDMA N,N,N-trimethylaminoethyl
methacrylate, HEMA and
AMVN
[40]
L-serine – Sandwich casting AIBN EGDMA Methacrylic acid [17]
L-menthol – Sandwich casting AIBN EGDMA Methacrylic acid [48]
Sorbitol – Electro-polymerization AIBN – m-Aminophenol [51]
Tegafur – Electro-polymerization – – m-Aminophenol [52]
Benzene, toluene
and xylene
– Spin coating AIBN Divinylbenzene Styrene [62]
Detection of low molecular mass analytes Y. Uludag

and, 0.03 for norepinephrine and 0.02 for epinephrine).
Electro-polymerization method
An electro-polymerization method was applied to pre-
pare imprinted polymers on quartz crystal sensor sur-
faces to detect sorbitol, poly(o-phenylenediamine),
tegafur and nucleotides [50–52]. Feng et al. synthesized
an o-phenylenediamine film for sorbitol on a QCM
crystal by cyclic voltammetry [51]. After MIP deposi-
tion the binding assays with sorbitol, glucose, fructose,
mannitol and glycerol were performed using a QCM
device. Glycerol could bind to the sorbitol-imprinted
surface, however, the binding of other compounds was
very limited. The detection limit of sorbitol binding
was found to be 1 mm.
Polymerization prior to sensor coating
MIPs have be prepared using a bulk polymerization
method; after grinding and sieving, the resulting parti-
cles are mixed with PVC and coated on the sensor
surface with spin coating. Imprinted polymers for
microcystin-LR, nandrolone, phenacetin, nicotine and
paracetamol were prepared using this method on
QCM sensor surfaces [17,53–55].
Application areas of MIP–QCM biosensors
The three most common application areas for MIP–
QCM sensors are clinical diagnosis, environmental
monitoring and control of enantiomeric separation.
Most studies describe detection in buffer or organic
solvents indicating the early stage in development of
these devices with respect to real applications
[17,40,43,51,52,56,57]. Although it is possible to detect

Summary
The inherent robustness, ease of manufacture and
high capacity of MIPs make them a potentially useful
alternative for small molecule detection using piezo-
electric biosensors. Although the majority of applica-
tions involve the use of buffered pure solutions rather
than real clinical or environmental samples for detec-
tion, this perhaps simply reflects the early stage of
development of the technology. Selectivity is still a
significant issue for imprinted polymers and this can
hamper specific, sensitive detection of analytes in
Y. Uludag
˘
et al. Detection of low molecular mass analytes
FEBS Journal 274 (2007) 5471–5480 ª 2007 Akubio Ltd. Journal compilation ª 2007 FEBS 5477
complex fluids. Polymerization methods are critical
determinants of selectivity and overall assay perfor-
mance of MIPs and as there is no general procedure
for MIP preparation, each template requires optimiza-
tion of several parameters to fabricate reproducible,
high-performance sensor surfaces. It is expected that
the improvements to polymerization techniques should
greatly enhance the selectivity and binding capacity of
the MIP–QCM sensors.
In many ways, the stage of development of MIP
interfaces is reflected in the state of development of
robust piezoelectric biosensors compared with analo-
gous robust electrochemical and optical biosensors
that have benefited from more than two decades and
several billion dollars of research and development.

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