Báo cáo khoa học: Piezoelectric sensors based on molecular imprinted polymers for detection of low molecular mass analytes potx

However, there are relatively few examples of small-molecule detection using traditional immunoassay formats: niacinamide detection has been achieved in serum and urine [6], the endogeno

Piezoelectric sensors based on molecular imprinted

polymers for detection of low molecular mass analytes Yildiz Uludag˘1,2, Sergey A.Piletsky1, Anthony P F Turner1and Matthew A Cooper2

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 requirements and assay run times [4] Unlike label-and reporter-based technologies that simply confirm the presence of the detector molecule, label-free tech-niques can provide direct information on analyte bind-ing to target molecules typically in the form of mass addition or depletion from the surface of the sensor substrate [5]

Of the various label-free detection modalities, piezo-electric sensing has become popular with researchers because of the low barriers to entry, inherent simp-licity, ease-of-use, low cost, and speed to result However, there are relatively few examples of small-molecule detection using traditional immunoassay formats: niacinamide detection has been achieved in serum and urine [6], the endogenous cofactors NAD+ and NADP+ have been titrated against the enzyme

Keywords

drug; hapten; label-free; molecularly

imprinted polymer; QCRS; quantification;

quartz crystal microbalance; screening;

small molecule

Correspondence

M A Cooper, Akubio Ltd, 181 Cambridge

Science Park, Cambridge CB4 0GJ, UK

Fax: +44 1223 225 336

Tel: +44 1223 225 326

E-mail: mcooper@akubio.com

(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.

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

of 38 lm [9] Kurosowa et al [10] reported a portable

dioxin sensor able to detect

2,3,7,8-tetrachlorodibenzo-p-dioxin Dioxin is well-known as a highly toxic

com-pound that poses a threat to the environment The

quartz crystal microbalance (QCM) sensor surface

was prepared by immobilizing

anti-(2,3,7,8-tetrachlo-rodibenzo-p-dioxin) serum and a linear calibration

curve was created using 100–0.1 ngÆmL)1

2,3,7,8-tetra-chlorodibenzo-p-dioxin before detection of the

com-pound in fly ash samples

Hence mass-sensitive acoustic immunoassays can

provide a label-free method for detecting and analysing

molecules However, biological materials are expensive,

sensitive to harsh conditions and their shelf life on the

sensor surface can be limited The use of molecularly

imprinted polymers (MIPs) as synthetic receptors

pro-vides an attractive alternative to biological receptors

MIP recognition elements provide sensor surfaces that

have a long shelf life, are robust and simple to prepare,

and provide a 3D interfacial matrix layer with high

binding capacity for analytes Such binding capacity is

crucial when the molecular mass of the analyte is

< 500 Da This review summarizes the key approaches

to incorporating imprinted polymers as recognition

ele-ments in piezoelectric sensors for detection of small

molecules

Acoustic biophysics

Acoustic biosensors allow the label-free detection of

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 ms, or absolute massDm:

Dfm¼  f

2

Fqqqms¼  f

2

Fqqq

Dms

Ael

ð1Þ

where f0 is the resonance frequency of the unperturbed quartz resonator, Fq the frequency constant of the crystal (Fq¼ f0.dq), dq the thickness, qq the mass den-sity, and Aelthe 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 (ql) and viscosity (gl) of the liquid and the frequency of the quartz crystal resonator:

Df ¼ f3=2 q

ffiffiffiffiffiffiffiffiffiffiffiffiq

1g1

pqqlq

r

ð2Þ

where qqand lqare the quartz density and shear mod-ulus, respectively [13] In a two-layer system these frequency shifts simply add up to an overall shift:

Df ¼ Dfmþ Dfl ¼ f02 Dms

FqqqAel

þ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffig

1q1

f0plqqq

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

Cell

Recognition elements Transducer

Optical Piezoelectric Electrochemical Calorimetric

Electric signal Analyte

Fig 1 Schematic representation of a biosensor.

liquid layer There are numerous examples in the

liter-ature on the applications of Eqns (2) and (3) and more

sophisticated algorithms that incorporate the

measure-ment of complex shear modulus in addition to mass,

viscosity and density, but a detailed analysis of this

approach lies beyond the scope of this review

How-ever, it is possible to further optimize sensor sensitivity

by appropriate matching of the interfacial polymer

chemistry physical properties with the acoustic sensor

design, and a few seminal examples in this regard are

noted below

It can been seen from the above that a quartz crystal

resonator is thus sensitive not only to mass, but also

to viscosity and density changes on the resonator

sur-face Therefore the term quartz crystal microbalance is

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

analyte on the sensor surface and measure the binding

of a larger molecule [22] or to perform a competitive

displacement assay with a hapten–carrier conjugate

[23] Another option is to conjugate the small molecule

to a bead or other additional mass load to increase the

molecular mass of the complex detected [24] Other

approaches utilize a coupled assay format in which direct binding then results in capture of an enzyme that can convert a soluble substrate to a precipitate to effect signal amplification For example, organophos-phorous and carbamate pesticides have been detected using a two-enzyme system to produce peroxide, which combined with peroxidase and benzidine formed an insoluble product that absorbed to the sensor surface [25] Nonionic surfactants were reported to enhance the surface deposition of suspended precipitate enabling detection of carbaryl and dichlorvos pesti-cides down to 1 p.p.m This group also published an assay for 4-aminophenol which involved precipitation

of indophenol in an amount proportional to the 4-aminophenol concentration in the sample [25]

In addition to the above strategies, the intrinsic sen-sitivity of QCM to shear modulus, viscosity and den-sity changes manifested at the surface interfacial layer allows for the development of novel small-molecule detection assays In this case, the binding of a small molecule that induces conformational changes in the interfacial layer results in a modulated shear modulus (related to rigidity and⁄ or flexibility) Such effects can significantly amplify the response due to mass binding 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]

For all the examples cited above, the binding

capacity of the sensor surface is critical to maximize

the sensitivity of the sensor for the detection of small

molecules Here the synergies between robust, stable

MIPs with a mass and shear-modulus sensitive sensor

become apparent The meso⁄ microporous 3D matrix

structures formed by MIPs, not only increase the total

number of binding sites acoustically coupled to the

sensor, but also result in additional frequency changes

manifested in analyte- and dose-dependent modulation

of the surface-associated shear modulus of the

poly-mer layer in the polypoly-meric structure In other words,

binding of analyte to a MIP can result a larger

change in the total acoustic load on the sensor and

hence enable more robust detection of small

mole-cules It is possible to model this phenomena as a

finite viscoelastic layer (of thickness hf, density rf and

shear modulus G¼ G¢ + jG¢¢ [32] The latter value,

G, is the complex shear modulus where G¢ and G¢¢ are

the layer storage and layer loss moduli, respectively

These layers are then exposed to a bulk liquid (of

vis-cosity gL and density qL) For these layered

compo-nents, it is possible to derive the surface mechanical

impedances:

for an ideal⁄ rigid mass layer, where rs is the mass per

area contributed by the interfacial layer;

ZF¼ ffiffiffiffiffiffiffiffi

qfG

q

for a viscoelastic film (MIP), where c is the shear wave

propagation constant (c¼ j2pfo(rf⁄ G)1 ⁄ 2) and j¼ -1;

ZL¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2pf0qLgL 2

r

for a liquid layer (semi-infinite Newtonian liquid), and

finally;

Z¼ j2pf0

ffiffiffiffiffiffiffiffi

qfG

q ZLcosh hðchfÞ þ ffiffiffiffiffiffiffiffi

qfG

p sinhðchfÞ ffiffiffiffiffiffiffiffi

qfG p cosh hðchfÞ þ ZLsinhðchfÞ 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-tribution of mass induced by varying matrix conditions

and analyte binding

When layers of a material of differing density and viscosity to the liquid (such as a MIP) are deposited at the surface–liquid interface, there is a contribution from both the bulk liquid and the added material that has displaced liquid and mechanically coupled to the interface In this case, the penetration depth can be defined as:

d¼ ð1  vpÞ

ffiffiffiffiffiffiffiffiffiffiffig

L

pf0qL

r

þ vp

ffiffiffiffiffiffiffiffiffiffiffi

gL

pf0qf

s

ð8Þ

where gp and qp are the liquid viscosity and density, respectively, and vp 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:

Z¼ vpj2pf0

ffiffiffiffiffiffiffiffi

qfG

q ð1  vpÞZLcosh hðchfÞ þ ffiffiffiffiffiffiffiffiq

fG

p sinhðch

fÞ ffiffiffiffiffiffiffiffi

qfG

p cosh hðchfÞ þ ð1  vpÞZLsinhðchfÞ

ð9Þ

Molecularly imprinted polymers

The history of MIPs can be traced to experiments per-formed by Polyakov and his group in 1931 Silica gels prepared by Polyakov’s group showed selective binding towards one of the solvents used for the gel prepara-tion Later, studies by Wulf [33] and Haupt and Mos-bach [34] helped to establish this technique in relation

to organic polymers Initially, MIPs were used as sta-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 temtem-plate 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

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-mediated [38] interactions, followed by appropriate

cross-linking agents Imprinting consists of three steps;

first, one or several functional monomers are mixed

with the template molecule in a solvent Second,

poly-merization of monomers occurs in the presence of a

cross-linker Third, the template is removed from the

polymer using basic, acidic or detergent solutions

(Fig 3) The performance and selectivity of MIPs

towards a target molecule depend on many factors

These include the molecular diversity of the monomer

units employed, the geometry of the imprinted cavity,

the rigidity of the cavity and associated implications

for enthalpy⁄ entropy compensation These factors all

govern the affinity and selectivity of the MIP

recogni-tion elements towards the analyte of interest A

discus-sion of the importance of hydrogen bonds, van der

Waal’s forces, ionic interactions, ion dipole

interac-tions and hydrophobic interacinterac-tions and other

molecu-lar phenomena between the template and the monomer

lies beyond the scope of this minireview and the reader

is referred to recent reviews and books in this area

[35,39]

To prepare MIPs with good affinity and selectivity,

polymerization conditions and the selection of

mono-mer and cross-linking agent are important parameters

for optimization (Table 1) There is no general

proce-dure for MIP preparation; therefore, depending on the

application, procedures need to be examined thor-oughly before a decision is made After the selection of the polymerization procedure, the variables of the pro-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

Polymerisation

-T arget

+ Target

Imprinted polymer Monomers and target

Target & monomers complex

Polymerisation

-T arget

+ Target

Imprinted polymer Fig 3 Schematic representation of

molecular imprinting.

Table 1 Variables that need to be optimized for the preparation imprinted polymers.

Imprinting

mechanism Polymerization format Monomer selection Medium selection Polymerization conditions

Covalent Bulk polymerization Combinatorial screening Organic solvent Cross-linking agent selection

Noncovalent MIP beads Thermodynamic calculations Aqueous solvent Ratio of template ⁄ monomer ⁄ cross-linking

agent Metal-mediated Films on bead ⁄ particles

or sensor surface

Pressure Time

In situ polymerization

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 mercaptoundecamercaptoundeca-noic 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 solution containing template, monomer (methacrylic acid) and cross-linker (trimethylolpropane trimethacry-late) UV irradiation resulted in a surface-grafted poly-mer film on the quartz resonator Although the detection limit of this MIP–QCM sensor was too high for practical application, Piacham et al succeeded in producing sensors that generated  30 Hz response on the injection of 19 mm (S)-propranolol

Sandwich casting method Alternatively, a sandwich casting method can be used either after surface activation of the QCM sensor sur-face [40,46,47], or directly on to the sensor sursur-face [17,40,48,49] In this method, a polymerization mixture

is dropped on to the quartz crystal and a microcover glass is pressed on to the sensor while UV irradiation

is applied The aim is to distribute the polymer

Table 2 Some examples of MIP-QCM studies 2,2¢-azobis, (2 amidinopropane) hydrochloride; ABAH, 2-ethyl-5-phenylisoxazolium-3-sulfonate; AIBN, azobis-(isobutyronitrile); AMVN, 2,2¢-azobis (dimethylvaleronitrile); EGDMA, ethylene glycol dimethacrylate; GMA, glycidyl methacry-late; HEMA, 2-hydroxyethyl methacrymethacry-late; MAH, methacrylamidohistidine; MUDA, mercaptoundecanoic acid; NBAA, N-benzylacrylamide; TRIM, trimethylolpropane trimethacrylate; 4-Vpy, 4-vinylpyridine.

Polymerization

acrylamide

[42] (S)-Propranolol Mercaptoundecanoic

acid

Indole-3-acetic acid Allyl mercaptan and

1-butanethiol

methacrylate

[40]

Dansylphenylalanine Thioctic acid-modified

GMA and thioctic acid dodecane ester

L -Tryptophan Thioctic acid-modified

GMA

methacrylate, HEMA and AMVN

[40]

Benzene, toluene

and xylene

solution evenly on the quartz and thus obtain a

uni-form polymer layer

Kugimiya and Takeuchi synthesized a MIP on a

quartz crystal sensor surface using a plant hormone,

indole-3-acetic acid, as a template [40] Platinum

coated 9 MHz quartz crystals were treated with allyl

mercaptan and 1-butanethiol to introduce vinyl groups

to the sensor surface The monomers and the template

were dissolved in chloroform and dropped onto the

QCM sensor and held with a glass microcover

Poly-merization was initiated by UV irradiation Assays for

indole-3-acetic acid binding to the MIP-coated surface

were performed using three crystals and a linear

rela-tionship was obtained from 10 to 200 nmol

indole-3-acetic acid The coefficient of variation between the

three sensors was 9.0%

Spin coating method

The spin coating approach was used by Ling et al [43]

to prepare MIPs for catecholamines Ling et al first

activated the gold-coated crystal with mercaptoethanol,

and then the homogeneous phase MIP solution was spin

coated Using this method, dopamine-,

epinephrine-and norepinephrine-imprinted resonators were prepared

and binding assays were performed The results show

that dopamine-specific MIP-coated resonators have

bet-ter selectivity than the other MIPs prepared (relative

binding selectivity of dopamine–MIP is 1 for dopamine

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 small molecules in buffered or organic solutions, it is also important to determine the amount of a particular drug or any other chemical in body fluids For instance, Tan et al determined the amount of nicotine and paracetamol in human serum and urine [55,58] The detection limit of nicotine was found to be 25 nm using an imprinted polymer-coated sensor Wu and colleagues fabricated bilirubin specific imprinted mers on a QCM sensor using a photo-graft poly-merization method [41] Bilirubin concentration is considered an important index to identify liver dis-eases A bilirubin-specific sensor was challenged with both bilirubin and its analogue biliverdin, cross-reac-tivity of bilirubin versus biliverdin binding was 31 : 20 Yan et al [59] developed a MIP–QCM sensor for daminozide (a potentially carcinogenic chemical the detection of which is important in food safety) with a detection limit of 50 pgÆmL)1 MIP–QCM sensors for acetaldehyde, monoterpenes and bisphenol A have been prepared for environmental pollution detection

by different groups [48,60,61] Synthesis of enantiomer-ically pure organic compounds is very important for industrial production MIP–QCM sensors capable of enantiomeric discrimination are very useful tools for process end-point analysis and various groups have discriminated between R- and S-propranolol, l- and

d-tryptophan, l- and d-serine and l- and d-dansylphe-nylalanine enantiomers [17,44,49]

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 soluapplica-tions 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

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

Many researchers rely on piezoelectric devices built

in-house that are generally very sensitive to artefacts

including temperature drift, humidity and pressure

effects, resulting in less reliable and less reproducible

measurements Commercially available systems that

minimize these effects with physical compensation on

in-line referencing are appearing, but they are still far

from the idealized portable device for near patient

testing, point of care or remote environmental

moni-toring in a handheld device However, the

fundamen-tal elements of acoustic sensors, i.e cost of goods,

simplicity and ease of manufacture, clearly have the

potential to be exploited in an integrated portable

device for use in quantitative detection of low

molec-ular mass analytes It is expected that recent advances

in understanding acoustic biosensing technology will

generate such devices that can be combined with the

benefits of MIPs as the recognition element to

pro-vide a new generation of sensors for use in routine

measurements outside the controlled laboratory

envi-ronment

References

1 Turner APF (2007) Encyclopedia of Chemical

Technology Wiley, New York, NY

2 Myszka DG, Jonsen MD & Graves BJ (1998)

Equilib-rium analysis of high affinity interactions using

BIA-CORE Anal Biochem 265, 326–330

3 Myszka DG & Rich RL (2000) Implementing surface

plasmon resonance biosensors in drug discovery Pharm

Sci Techn Today 3, 310–317

4 Cooper MA (2002) Optical biosensors in drug

discov-ery Nat Rev Drug Discov 1, 515–528

5 Richter RP, Him JLK, Tessier B, Tessier C & Brisson

AR (2005) On the kinetics of adsorption and

two-dimensional self-assembly of annexin A5 on

sup-ported lipid bilayers Biophys J 89, 3372–3385

6 Long YM, Li WF, Nie LH & Yao SZ (2001) Ion-selective piezoelectric sensor for niacinamide assay

in serum and urine J Pharmaceut Biomed Anal 24, 361–369

7 Godber B, Thompson KS, Rehak M, Uludag Y, Kelling

S, Sleptsov A, Frogley M, Wiehler K, Whalen C & Cooper MA (2005) Direct quantification of analyte con-centration by resonant acoustic profiling Clin Chem 51, 1962–1972

8 Li X, Thompson KS, Godber B & Cooper MA (2006) Quantification of small molecule-receptor affinities and kinetics by acoustic profiling Assay Drug Dev Technol

4, 565–573

9 Zhou AH & Muthuswamy J (2004) Acoustic biosensor for monitoring antibody immobilization and neurotrans-mitter GABA in real-time Sens Actuators B Chem 101, 8–19

10 Kurosawa S, Park JW, Aizawa H, Wakida SI, Tao H & Ishihara K (2006) Quartz crystal microbalance immuno-sensors for environmental monitoring Biosens Bioelec-tron 22, 473–481

11 Sauerbrey G (1959) Verwendung von Schwingquarzen zur Wagung dunner Schichten und zur Microwagang

Z Phys 155, 206–212

12 Nomura T & Okuhara M (1982) Frequency-shifts of piezoelectric quartz crystals immersed in organic liquids Anal Chim Acta 142, 281–284

13 Kanazawa KK (1997) Mechanical behaviour of films on the quartz microbalance Faraday Discussions, 77–90

14 Dickert FL, Forth P, Bulst WE, Fischerauer G & Knauer U (1998) SAW devices-sensitivity enhancement

in going from 80 MHz to 1 GHz Sens Actuators B Chem 46, 120–125

15 Janshoff A, Galla HJ & Steinem C (2000) Piezoelectric mass-sensing devices as biosensors – an alternative to optical biosensors? Angew Chem Int Ed 39, 4004–4032

16 Furtado LM, Su HB, Thompson M, Mack DP & Hay-ward GL (1999) Interactions of HIV-1 TAR RNA with Tat-derived peptides discriminated by on-line acoustic wave detector Anal Chem 71, 1167–1175

17 Percival CJ, Stanley S, Braithwaite A, Newton MI & McHale G (2002) Molecular imprinted polymer coated QCM for the detection of nandrolone Analyst 127, 1024–1026

18 Hook F, Ray A, Norden B & Kasemo B (2001) Characterization of PNA and DNA immobilization and subsequent hybridization with DNA using acoustic-shear-wave attenuation measurements Langmuir 17, 8305–8312

19 Zhou XD, Liu LJ, Hu M, Wang LL & Hu JM (2002) Detection of hepatitis B virus by piezoelectric biosensor

J Pharmaceut Biomed Anal 27, 341–345

20 Fung YS & Wong YY (2001) Self-assembled monolay-ers as the coating in a quartz piezoelectric crystal

immunosensor to detect salmonella in aqueous solution.

Anal Chem 73, 5302–5309

21 Richert L, Lavalle P, Vautier D, Senger B, Stoltz JF,

Schaaf P, Voegel JC & Picart C (2002) Cell interactions

with polyelectrolyte multilayer films Biomacromolecules

3, 1170–1178

22 Liu Y, Yu X, Zhao R, Shangguan D-H, Bo Z & Liu G

(2003) Real time kinetic analysis of the interaction

between immunoglobulin G and histidine using quartz

crystal microbalance biosensor in solution Biosens

Bioelectron 18, 1419–1427

23 Halamek J, Hepel M & Skladal P (2001) Investigation

of highly sensitive piezoelectric immunosensors for

2,4-dichlorophenoxyacetic acid Biosens Bioelectron 16,

253–260

24 Nakamura C, Song S-H, Chang S-M, Sugimoto N &

Miyake J (2002) Quartz crystal microbalance sensor

tar-geting low molecular weight compounds using

oligopep-tide binder and pepoligopep-tide-immobilized latex beads Anal

Chim Acta 469, 183–188

25 Karousos NG, Aouabdi S, Way AS & Reddy SM

(2002) Quartz crystal microbalance determination of

organophosphorus and carbamate pesticides Anal Chim

Acta 469, 189–196

26 Carmon KS, Baltus RE & Luck LA (2004) A

piezoelec-tric quartz crystal biosensor: the use of two single

cyste-ine mutants of the periplasmic Escherichia coli

glucose⁄ galactose receptor as target proteins for the

detection of glucose Biochemistry 43, 14249–14256

27 Wang X, Ellis JS, Lyle EL, Sundaram P & Thompson

M (2006) Conformational chemistry of surface-attached

calmodulin detected by acoustic shear wave

propaga-tion Mol Biosystems 2, 184–192

28 Ban T, Morigaki K, Yagi H, Kawasaki T, Kobayashi

A, Yuba S, Naiki H & Goto Y (2006) Real-time and

single fibril observation of the formation of amyloid

beta spherulitic structures J Biol Chem 281,

33677–33683

29 Saraswathi R, Hillman AR & Martin SJ (1999)

Mechanical resonance effects in electroactive

polycar-bazole films J Electroanalyt Chem 460, 267–272

30 Hook F, Kasemo B, Nylander T, Fant C, Sott K &

Elwing H (2001) Variations in coupled water, viscoelastic

properties, and film thickness of a Mefp-1 protein film

during adsorption and cross-linking: a quartz crystal

microbalance with dissipation monitoring, ellipsometry,

and surface plasmon resonance study Anal Chem 73,

5796–5804

31 Bu¨ttgenbach S, Rabea J, Zimmermann B & Hauptmann

P (2001) High-frequency thickness-shear mode resonators

for sensor application in liquids, Proceedings of the

SPIE Photonics East Conference, Volume 207

Boston, MA

32 Brown MJ, Hillman AR, Martin SJ, Cernosek RW &

Bandey HL (2000) Manipulation of electroactive

polymer film viscoelasticity: the roles of applied poten-tial and frequency J Mater Chem 10, 115–126

33 Wulff G (1986) Molecular recognition in polymers pre-pared by imprinting with templates ACS Symposium Series, pp 186–230 American Chemical Society, Washington, DC

34 Haupt K & Mosbach K (1998) Plastic antibodies: devel-opments and applications Trends Biotechnol 16, 468–475

35 Piletsky SA, Turner NW & Laitenberger P (2006) Molecularly imprinted polymers in clinical diagnostics) future potential and existing problems Med Eng Phys

28, 971–977

36 Shiomi T, Matsui M, Mizukami F & Sakaguchi K (2005) A method for the molecular imprinting of hemo-globin on silica surfaces using silanes Biomaterials 26, 5564–5571

37 Piletsky SA, Matuschewski H, Schedler U, Wilpert A, Piletsky EV, Thiele TA & Ulbricht M (2000) Surface functionalization of porous polypropylene membranes with molecularly imprinted polymers by photograft copolymerization in water Macromolecules 33, 3092–3098

38 Wu L & Li Y (2004) Metal ion-mediated molecular-imprinting polymer for indirect recognition of formate, acetate and propionate Anal Chim Acta 517, 145–151

39 Yan M & Ramstrom O (2005) Design and analysis of molecular imprinting, in Molecularly imprinted materi-als: science and technology, pp 363–394 Marcel Dekker, New York, NY

40 Kugimiya A & Takeuchi T (2001) Surface plasmon res-onance sensor using molecularly imprinted polymer for detection of sialic acid Biosens Bioelectron 16, 1059–1062

41 Wu AH & Syu MJ (2006) Synthesis of bilirubin imprinted polymer thin film for the continuous detection

of bilirubin in an MIP⁄ QCM ⁄ FIA system Biosens Bio-electron 21, 2345–2353

42 Lin CY, Tai DF & Wu TZ (2003) Discrimination of peptides by using a molecularly imprinted piezoelectric biosensor Chem Eur J 9, 5107–5110

43 Ling TR, Yau ZS, Tasi YC, Chou TC & Liu CC (2005) Size-selective recognition of catecholamines by molecu-lar imprinting on silica–alumina gel Biosens Bioelectron

21, 901–907

44 Liu F, Liu X, Ng SC & Chan HSO (2006) Enantioselec-tive molecular imprinting polymer coated QCM for the recognition of 1-tryptophan Sens Actuators B Chem

113, 234–240

45 Piacham T, Josell A, Arwin H, Prachayasittikul V & Ye

L (2005) Molecularly imprinted polymer thin films on quartz crystal microbalance using a surface bound photo-radical initiator Anal Chim Acta 536, 191–196

46 Cao L, Zhou XC & Li SFY (2001) Enantioselective sensor based on microgravimetric quartz crystal

microbalance with molecularly imprinted polymer film.

Analyst 126, 184–188

47 Liu K, Wei WZ, Zeng JX, Liu XY & Gao YP (2006)

Application of a novel electrosynthesized

polydop-amine-imprinted film to the capacitive sensing of

nico-tine Anal Bioanalyt Chem 385, 724–729

48 Percival CJ, Stanley S, Galle M, Braithwaite A, Newton

MI, McHale G & Hayes W (2001) Molecular-imprinted,

polymer-coated quartz crystal microbalances for the

detection of terpenes Anal Chem 73, 4225–4228

49 Haupt K, Noworyta K & Kutner W (1999) Imprinted

polymer-based enantioselective acoustic sensor using

a quartz crystal microbalance Anal Commun 36,

391–393

50 Sallacan N, Zayats M, Bourenko T, Kharitonov AB &

Willner I (2002) Imprinting of nucleotide and

monosac-charide recognition sites in acrylamidephenylboronic

acid–acrylamide copolymer membranes associated with

electronic transducers Anal Chem 74, 702–712

51 Feng L, Liu Y, Tan Y & Hu J (2004) Biosensor for the

determination of sorbitol based on molecularly

imprinted electrosynthesized polymers Biosens

Bioelec-tron 19, 1513–1519

52 Liao HP, Zhang ZH, Li H, Nie LH & Yao SZ (2004)

Preparation of the molecularly imprinted

polymers-based capacitive sensor specific for tegafur and its

characterization by electrochemical impedance and

piezoelectric quartz crystal microbalance Electrochim

Acta 49, 4101–4107

53 Chianella I, Piletsky SA, Tothill IE, Chen B & Turner

AP (2003) MIP-based solid phase extraction cartridges

combined with MIP-based sensors for the detection of

microcystin-LR Biosens Bioelectron 18, 119–127

54 Tan Y, Peng H, Liang C & Yao S (2001) New assay

system for phenacetin using biomimic bulk acoustic

wave sensor with a molecularly imprinted polymer

coat-ing Sens Actuators B Chem 73, 179–184

55 Tan Y, Yin J, Liang C, Peng H, Nie L & Yao S (2001)

A study of a new TSM bio-mimetic sensor using a molecularly imprinted polymer coating and its applica-tion for the determinaapplica-tion of nicotine in human serum and urine Bioelectrochemistry 53, 141–148

56 Ersoz A, Denizli A, Ozcan A & Say R (2005) Molecu-larly imprinted ligand-exchange recognition assay of glucose by quartz crystal microbalance Biosens Bioelec-tron 20, 2197–2202

57 Pogorelova SP, Bourenko T, Kharitonov AB & Willner

I (2002) Selective sensing of triazine herbicides in imprinted membranes using ion-sensitive field-effect transistors and microgravimetric quartz crystal micro-balance measurements Analyst 127, 1484–1491

58 Tan YG, Zhou ZL, Wang P, Nie LH & Yao SZ (2001)

A study of a bio-mimetic recognition material for the BAW sensor by molecular imprinting and its applica-tion for the determinaapplica-tion of paracetamol in the human serum and urine Talanta 55, 337–347

59 Yan S, Fang Y & Gao Z (2007) Quartz crystal micro-balance for the determination of daminozide using molecularly imprinted polymers as recognition element Biosens Bioelectron 22, 1087–1091

60 Hirayama K, Sakai Y, Kameoka K, Noda K & Naganawa R (2002) Preparation of a sensor device with specific recognition sites for acetaldehyde by molecular imprinting technique Sens Actuators B Chem 86, 20–25

61 Tsuruoka M, Yano K, Ikebukuro K, McNiven S, Takeuchi T & Karube I (1996) Rapid detection of com-plementary- and mismatched DNA sequences using flu-orescence polarization Anal Lett 29, 1741–1749

62 Dickert FL, Lieberzeit P, Miarecka SG, Mann KJ, Hay-den O & Palfinger C (2004) Synthetic receptors for chemical sensors – subnano- and micrometre patterning

by imprinting techniques Biosens Bioelectron 20, 1040–1044