Voltammetric sensor for vanillylmandelic acid based on molecularlyimprinted polymer-modified electrodes

M.C. Blanco-Lopez, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P. Tunon-Blanco *

Departamento de Quımica Fısica y Analıtica, Facultad de Quımica, Universidad de Oviedo, C/Julian Claverıa 8, Oviedo 33006, Spain

Received 4 February 2002; received in revised form 12 July 2002; accepted 22 July 2002

Abstract

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction

of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for

vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin

coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and

solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the

imprinted sensor after rebinding was linear with VMA concentration in the range 19�/350 mg ml�1, whereas the response of the

control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted

sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other

closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-

dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid

was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the

same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by

the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the

development of highly selective analytical sensors.

# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Molecularly imprinted polymers; Voltammetric sensor; Vanillylmandelic acid; Biomimetics

1. Introduction

During the last decade, molecular imprinting technol-

ogy has become a well-established analytical tool

(Haupt and Mosbach, 2000; Haupt, 2001; Ensing and

de Boer, 1999; Owens et al., 1999). It is used to produce

artificial recognition elements, mimicking the highly

selective recognition features of biological receptors,

but with the added advantage of lower price, and higher

thermal and chemical stability (Svenson and Nicholls,

2001). Moreover, molecularly imprinted polymers

(MIPs) offer the possibility of tailor make a polymer

for a given analyte for which a natural receptor may not

exist. They find direct application at solid-phase extrac-

tions (Haupt, 2001; Ensing and de Boer, 1999; Owens et

al., 1999; Stevenson, 1999; Masque et al., 2001), with the

aim of preconcentrating the analyte or separating it

from interferents. Examples can be found in many areas

of interest such as environmental, pharmaceutical, and

biomedical analysis (Masque et al., 2001; Owens et al.,

1999; Andersson, 2000a). They are also widely used as

stationary phases for affinity chromatography or capil-

lary electrophoresis methods (Haupt, 2001; Andersson,

2000a,b; Mullett and Lai, 1998; Chen et al., 2001;

Kempe and Mosbach, 1995) allowing sometimes resolu-

tion of racemic mixtures. In other cases, ligand-binding

assays were designed and their affinities, limits of

detection and selectivity reached values similar to those

obtained with biological receptors (Andersson, 1996,

2000a; Vlatakis et al., 1993; Takeuchi et al., 2000;

Surugiu et al., 2000; Sellergren, 1997; Ramstrom et al.,

1996).

* Corresponding author. Tel.: �/34-985-103-487; fax: �/34-985-103-

125

E-mail address: ptb@fluor.quimica.uniovi.es (P. Tunon-Blanco).

Biosensors and Bioelectronics 18 (2003) 353�/362

www.elsevier.com/locate/bios

0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 1 5 1 - 3

The high selectivity and affinity of MIPs for the

template molecule make them ideal candidates as

recognition elements for sensors (Haupt and Mosbach,

2000; Dickert and Hayden, 1999; Kriz et al., 1997). The

large majority of the MIP-based sensors reported use

the mass accumulation in MIP coating as the transduc-

tion principle in either a quartz microbalance (Percival

et al., 2001; Tan et al., 2001a; Kugimiya and Takeuchi,

1999; Dickert et al., 2001; Kobayashi et al., 2001) or a

surface acoustic wave resonator (Peng et al., 2000; Liang

et al., 1999, 2000; Tan et al., 2001b). Reports can also be

found on sensors with molecular imprinted polymers,

which use a physicochemical property of the analyte for

its detection, such as fluorimetry (Dickert et al., 1999;

Suarez-Rodrıguez and Dıaz-Garcıa, 2000; Piletsky et al.,

1996), absorption spectrometry (McNiven et al., 1998),

luminescence (Jenkins et al., 1999), induced scintillation

(Ye and Mosbach, 2001) or surface plasmon resonance

(Lai et al., 1998).In contrast with the increasing number of MIP

reports on separations and mass-transduction sensors,

it is surprising that the scarce reports on the design of

electrochemical sensors is based on molecular imprint-

ing technology. Table 1 summarises the MIP-based

electrochemical sensors reported in the literature, and

includes the studies of MIP systems using this type of

detection too.

Capacitive (Panasyuk et al., 1999; Panasyuk-Delancy

et al., 2001; Mirsky et al., 1999), conductometric

(Piletsky et al., 1995; Sergeyeva et al., 1999a,b), field-

effect (Lahav et al., 2001a,b), amperometric (Kriz and

Mosbach, 1995), and voltammetric (Kroger et al., 1999;

Pizzariello et al., 2001; Gutierrez-Fernandez et al., 2001;

Kirsch et al., 2001) systems are the transduction

principles that have been used. All of them, with the

exception of voltammetry (and amperometry to some

extent) rely exclusively on the MIP element for the

selectivity of the determination, as it is the case of the

piezoelectric transducers. However, selectivity would be

enhanced if voltammetry is used, since the potential

necessary for generation of the analytical signal (current

intensity) is characteristic of the species that is oxidised

or reduced. Despite all, it could be said that the

analytical potential of voltammetry in the development

of molecular imprinting-based chemical sensors is still

relatively unexplored.In this work, the sensors were prepared by spin

coating the polymerisation mixture on the surface of a

glassy carbon electrode (GCE) and subsequent photo-

chemical polymerisation. This procedure is reported to

yield very reproducible layers 100 nm�/2 mm thick

(Dickert et al., 2001).

When a non-conductive polymer such as the highly

cross-linked MIP is grafted on the electrode surface,

detection of a molecule contained in the polymeric layer

is possible if its pore size and pore density are appro-

priate for the analyte diffusion towards the electrode

surface (Scheme 1), and the measurement is carried out

in a medium favourable to the release of the analyte.The polymeric layer should contain an important

concentration of recognition sites close enough to the

surface and well connected by a network of pores of

appropriate size. If layer thickness is excessive, recogni-

tion sites far away from the electrode surface would

result only in strong diffusion impediments. From the

point of view of molecular imprinting technology,

control of porosity of the resulting polymer can beachieved by means of the volume (and nature) of solvent

used in the monomer mixture (Sellergren, 1999).

This work is an investigation of the limitations and

scope of the application of molecular imprinting tech-

nology to the development of a voltammetric sensor for

vanillylmandelic acid (VMA) using a GCE coated with

an MIP layer. VMA is a metabolite of the catechola-

mines epinephrine and norepinephrine (Krstulovic,1986). Its excretion above normal values is related to

neural crest tumours, such as pheochromocytoma and

neuroblastoma, and it is usually determined by clinical

laboratories in urine samples for the diagnosis of these

pathologies. The methods used include HPLC with

electrochemical (Radjaipour et al., 1994; Parker et al.,

1986) or fluorimetric detection (Tokuda et al., 1990), gas

chromatography with mass spectroscopy (Fauler et al.,1997), and immunoassays (Taran et al., 1997; Mellor

and Gallacher, 1990). The actual demand of mass

screening methods for neuroblastoma in childhood has

encouraged the development of low-cost methods with a

rapid response. For this purpose, a capillary electro-

phoretic method has been recently reported (Garcıa et

al., 2000). An electrochemical sensor based on molecular

imprinted polymers would be a good alternative. Arequirement would be the ability to discriminate be-

tween VMA and other metabolites structurally similar

present in urine such as homovanillic acid (HVA) or 3-

methoxy-4-hydroxyphenylethylene glycol (MHPG). In

this study, we present initial results investigating the

interactions that control selectivity to determine VMA

without the use of a competitive ligand, by using an MIP

film deposited on the surface of a carbon electrode. Thespecies used to check cross-reactivity are shown in

Scheme 2.

2. Experimental

2.1. Reagents and chemicals

VMA, HVA, MHPG, 3,4-dihydroxyphenylacetic acid

(DOPAC), and 2,5-dihydroxyphenylacetic acid (homo-

gentistic acid, HGA) were purchased from Sigma

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362354

(Aldrich, Spain). All chemicals were of analytical grade,and solvents were of HPLC quality (Fluka, Spain).

2.2. Apparatus

Differential pulse voltammetry was performed using a

m-Autolab (Eco Chemie, B.V. Utrech, The Nether-

lands), using a three-electrode system. The MIP-coated

electrode was acting as working electrode. The ampli-

tude of the pulse applied was 50 mV and the scan rate

was 6.25 mV s�1. The reference electrode used was Ag/AgCl/KCl sat. Peak currents were measured by subtrac-

tion of the base line. Unless otherwise stated, potential

sweeps were applied between 0.2 and 1 V.

2.3. Preparation of polymer-coated electrodes

The surface of GCEs was polished with aqueous

alumina slurries on a metallographic polishing cloth,

with successive decrease in particle size (1�/0.3 mm) and

the remaining particles on the surface were removed by

ultrasonic treatment in Mili-Q water. Then the electrode

was oxidised from 0 to 1 V vs. Ag/AgCl.

Methacrylic acid (MAA) and divinylbenzene (DVB)

or ethyleneglycol dimethacrylate (EDMA) were mixed

in a 5:1 molar ratio of cross-linking agent (DVB or

EDMA) to functional monomer (MAA). The template

molecule was added in a 1:8 ratio to functional

monomer; acetonitrile was the porogenic solvent used

and 2,2-dimethoxy-2-phenylacetophenone (DPP) was

Table 1

Summary of information on MIP-based electroanalytical sensors and related studies

Type of electroche-

mical transduction

Recognition system Form Analyte Reference

Capacitance Electropolymerised phenol Film (on gold) Phenylalanine Panasyuk et al.

(1999)

Dodecanethiol Monolayer (on gold) Barbituric acid Mirsky et al.

(1999)

2-Acrylamido-2-methyl-1-propa-

nesulphonic acid/N ,N ?-methyle-

nediacrylamide

Film (on gold) Desmetryn Panasyuk-De-

lancy et al.

(2001)

Conductometric Acrylic and polyurethanes MIPs Membrane Triazine Sergeyeva et al.

(1999a,b)

MAA/EDMA/diethyl aminoethyl

methacrylate

Membrane Atrazine Piletsky et al.

(1995)

Field effect devices TiO2 sol�/gel Film (on Al2O3) (R )- and (S )-2-Methylferrocene carboxylic

acids, (R )-1 or (S )-1; (R ) or (S )-2-phenylbuta-

noic acids; (R ) or (S )-2 propanoic acid, (R )-3 or

(S )-3

Lahav et al.

(2001a)

TiO2 sol�/gel Film (on SiO2) Chloroaromatic acids Lahav et al.

(2001b)

Amperometric Acrylic MIP particles Composite film with

agarose gel (on Pt)

Morphine Kriz and Mos-

bach (1995)

Voltammetric 4-Vinylpyridine/EDMA MIP par-

ticles

Composite film with

agarose gel (on

screen-printed elec-

trodes)

2,4-Dichlorophenoxyacetic acid Kroger et al.

(1999)

MAA/EDMA MIP particles Composite bar (with

graphite and n -eico-

sane)

Clenbuterol Pizzariello et

al. (2001)

Polyphosphazenes Film on glassy car-

bon

Rifamycine Gutierrez-Fer-

nandez et al.

(2001)

Styrene/divinylbenzene MIP par-

ticles

Modified carbon ink

(screen-printed elec-

trodes)

1-Hydroxypyrene Kirsch et al.

(2001)

Acrylic Film (on ITO plate) Theophylline Yoshimi et al.

(2001)

SiO2 sol�/gel Film (on glassy car-

bon)

Dopamine Makote and

Collinson

(1998)

Hexadecyl mercaptan Self-assembled

monolayer (on gold)

Cholesterol Piletsky et al.

(1999)

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362 355

used to initiate photopolymerisation. The monomer

mixture was degassed with He for 2 min in an ice bath

before and after addition of the initiator. Ten microlitres

were pipetted onto the electrode surface, and the excess

of polymer was eliminated by spin coating (1000 rpm).

The modified electrode was left to polymerise under N2

atmosphere, at 4 8C, under UV radiation (l�/350 nm).

A control electrode was prepared in every case

following the same procedure, but in the absence of

template molecule. The control (or non-imprinted poly-

mer-modified electrode) had at any time the same

treatment as the imprinted electrode, to ensure that

the effects observed are only due to the imprinting

features and not to the subsequent treatments under-gone by the electrode.

2.4. Measurement

The electrochemical measurements were carried out in

a three-electrode cell, in aqueous citrate buffer (0.025 M

and pH 3) with 10% (v/v) of acetonitrile, to ensure good

wettability of the polymer layer. The procedure followed

in this study involves:

1) Preparation of the electrode by spin coating of themonomers mixture and in situ photopolymerisation.

2) Extraction of the template molecule. This was

carried out in dioxane�/MeOH mixture at a ratio

of 9:1.

3) Binding in the solution of interest for a fixed period

of time. The time interval which led to the highest

ratio between the intensity current of the imprinted

modified electrode to that recorded with the non-imprinted modified electrode was 25 min. Electro-

des were subsequently immersed in clean acetoni-

trile for 20 s before being transferred to the

electrochemical cell in order to remove the weakly

adsorbed molecules.

4) Quantification in a medium where the release of

analyte from binding sites is possible (i.e. 0.025 M

citrate solution of pH 3 with 10% (v/v) acetonitrile).The intensity of the current detected under these

conditions can be related to the concentration of

analyte present in the binding step.

Scheme 1. Schematic representation of the recognition process in the MIP-modified electrodes used in this study.

Scheme 2. Structures of VMA and the compounds chosen to test

cross-reactivity of the VMA-imprinted sensors.

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362356

5) The electrode is washed prior to further uses in the

dioxane�/MeOH solution used in step (2). The

oxidation products are soluble in this medium.

3. Results and discussion

3.1. Voltammetric monitoring and evaluation of electrode

preparation and the extraction of the template molecule

To the author’s knowledge, there are no previous

reports on the electrochemical oxidation of VMA. Fig. 1

shows a cyclic voltammogram of VMA in a citrate

solution of pH 3. VMA is irreversibly oxidised at this

pH, showing two oxidation peaks at 676 and 854 mV vs.

Ag/AgCl. The backwards sweep shows a reduction peak

(R1) at 391 mV and two new oxidation processes (O3and O4) of lower intensity can be observed at the second

potential sweep, at 459 and 547 mV, respectively. At

pH�/5 the main oxidation peaks, O1 and O2 overlap,

whereas at pH 12 two peaks are distinguishable again.

Other 4-hydroxy-3-methoxy-related compounds de-

scribed in the literature, undergo a single 2e� oxidation

in aqueous solution followed by an irreversible chemical

reaction (Hernandez et al., 1996). The mechanism forthe electrochemical oxidation of VMA seems to be

different and it is at present under study.

The peak potentials of O1 and O2 decrease as the pH

increases. For O2, the variation of the oxidation

potentials with pH can be described by a straight line

in the pH range 1.34�/7.8 that corresponds to the

equation y�/�/ 0.0518x�/1.0049. pH 3 was used for

the measurements because both peaks appear welldifferentiated. The effect of the scan rate on both peak

currents was studied at this pH-value. A linear relation-

ship with the square root of the scanning rate was found

for both peaks, indicating that both processes were

diffusion-controlled in the range of concentrations and

rates investigated (i.e. from 10 to 100 mV s�1 at

concentration 2�/10�4 M for O1 and O2, and for O1

at concentration 10�5 M, and up to 80 mV s�1 at

concentration 10�5 M for O2). Our results indicate that

a possible mechanism involves two one-electron oxida-tion steps, and an intermediate chemical dimerisation

step.

GCEs were chosen for the preparation of the sensors

because of their good adhering features. It was observed

that solution of VMA was favoured when EDMA was

used, allowing higher template to functional monomer

ratio to be used, and therefore EDMA was the cross-

linker monomer preferred. Several parameters such astime before spinning (controlling the viscosity of the

monomer mixture and therefore the thickness of the

non-conductive layer) and volume of porogen (respon-

sible for porosity) were modified in order to achieve an

electrode with good electrochemical features. In this

case, spinning was started after 5 min of initiation of the

photopolymerisation, and the volume of solvent equiva-

lent to a fifth of the total volume of monomers was theratio which lead to the best results.

The measurements with the modified electrodes were

carried out in citrate buffer (pH 3, 0.025 M) with 10%

(v/v) of acetonitrile to ensure good wettability of the

polymer. No further improvements were observed with

higher concentration of organic solvents in solution or

higher ionic strengths. Fig. 2a shows that the presence of

acetonitrile in the measuring medium does not alter thepeak potentials of VMA by DP voltammetry (615 and

820 mV, as measured in a concentration of 10�5 M).

All the sensors prepared with acrylic molecular

imprinted polymer are able to detect the two waves

characteristic of the imprinted molecule during the first

potential sweep. Fig. 2b compares the behaviour of an

imprinted electrode and a control electrode. It can also

be observed a shift on the electrochemical potential ofthe maximum towards higher values with successive

washing steps (Fig. 2c) which might be related to the

difference of strength of the interactions with the

binding sites, being the most energetic ones the most

difficult to oxidise. Dioxane�/MeOH (9:1) was chosen as

washing solution because this solution is a good solvent

for the analyte, and could induce the template molecule

to abandon the imprinted sites in the polymer by dipoleinteractions.

3.2. Binding step

Previous test indicated that the rebinding in aqueous

media is not favoured in this case, possibly due to

competence of H2O for binding sites through hydrogen

bonds. It has been reported that non-covalent MIPs

undergone swelling in polar solvents and this effect,responsible for the access to the imprinted sites, results

in maximum affinity when the polymers are used in the

porogen solvent (Sellergren, 1999; Spivak and Shea,

Fig. 1. Cyclic voltammogram of VMA on a bare GCE. Dashed lines

correspond to the background current and solid lines to the first (1)

and second (2) scans, respectively. Measurements were carried out in a

2�/10�4 M VMA solution at pH 3 with a scan rate of 50 mV s�1.

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362 357

2001). Therefore, binding tests were carried out in

acetonitrile, the porogen used in this case.

The electrodes were incubated in a solution 4.76�/

10�4 M of VMA in acetonitrile. After 5 min, the

electrodes were rinsed and transferred to the electro-

chemical cell as described in Section 2. Then, the

electrodes were washed in a solution of dioxane�/

MeOH (9:1), and a measurement was taken to make

sure that the analyte signal has disappeared and hence

the electrode has been effectively washed. The incuba-

tion procedure was repeated for increasing periods of

time. The second oxidation process was more sensitive

to concentration and was used for quantification. It was

observed that the ratio of the intensity current obtained

with the imprinted electrode to that obtained with the

control electrode was increasing with the time, reaching

a fivefold maximum value for that concentration when

the incubation time was 25 min. Therefore, this was the

time interval chosen to test the electrode response to the

concentration and its selectivity.

Fig. 3 shows the selective response obtained with an

electrode modified with the imprinted polymer as

compared with the response obtained with a control

polymer after incubation in a 9�/10�4 M solution of

the template molecule in acetonitrile. This test was

carried out in parallel with a bare glassy electrode, and

no current was observed when applying a potential

sweep after the incubation procedures, indicating that

the analyte is not adsorbed under these conditions.

Hence, we have to conclude that the currents observed

with the electrodes modified with the polymer film are

due to adsorption in the polymer, and the differences

between them (the increased uptake observed with the

imprinted electrode) must be due to the imprinting

effect. In this case, the interaction seems to be driven by

hydrogen bonds, since acetonitrile is a poor hydrogen

bonding solvent, and it does not compete with the

template for the bonding sites. The important role of

this type of interaction in molecular recognition by non-

covalent MIPs has been frequently reported (Chen et al.,

2001). On the other hand, the results indicate that the

detection in the electrochemical cell is possible because

measurements are carried out in an aqueous media; H2O

competes strongly for the binding sites, and its great

polarity is favourable to the release of the template

towards the solution�/electrode interface.Moreover, by comparing the electrochemical poten-

tials for the maxima corresponding to the imprinted and

non-imprinted polymers, it might be possible to extract

some information about the nature of the interactions in

each case. The maxima were observed at 674 and 840

Fig. 2. (a) Comparison of the DP voltammogram obtained for VMA

in a pH 3, 0.025 M citrate buffer solution (solid line) and the DP

voltammogram obtained with the same solution containing 10% (v/v)

acetonitrile (dashed line). (b) First DP voltammogram obtained with

the imprinted electrode (solid line) and control electrode (dashed line)

in citrate buffer (0.025 M) at pH 3, with 10% (v/v) acetonitrile, prior

template extraction and further uses of the electrodes. (c) Monitoring

the successive extractions of the template (B, C, D curves) at VMA-

imprinted electrode by DP voltammetry.

Fig. 3. DP voltammogram showing the detection of VMA rebinded to

the imprinted layer after 25 min incubation in a 9�/10�4 M solution

in acetonitrile (thick line) and the voltammogram obtained with the

control electrode (thin line) under the same conditions. Dashed lines

correspond to the respective background currents.

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362358

mV for the imprinted electrode, and 649 and 845 mV for

the non-imprinted electrode. The difference of 25 mV

higher for the first signal of VMA might indicate a

stronger interaction with the specific recognition sites,whereas sites in the non-imprinted case would be less

specific and therefore of lower energy.

3.3. Sensor response to concentration of VMA

The sensor response to the concentration of incuba-

tion is shown in Fig. 4. There is a region at concentra-

tion B/10�4 M where response of the imprinted sensoris not observed. When the incubation solution concen-

tration increases from ca. 19 to 350 mg ml�1, the

intensity recorded with the imprinted electrode can be

adjusted to a straight line. The response of the control

electrode is independent of the concentration of incuba-

tion solution, and it is kept at very low values for any

concentration within that range. The current measured

after incubation in 346.8 mg ml�1 with the imprintedelectrode is 10-fold than that of the control electrode.

The high concentration required for response can be

due to a poor affinity of the polymer for the analyte,

since the study did not include an optimisation of MIP

preparation in terms of affinity for VMA. If a long-

range attractive interaction such as electrostatic forces

could be superimposed, the affinity of the polymer for

the template molecule would be enhanced. With thisaim, the MIP synthesis was attempted using vinylpyr-

idine instead of MAA as functional monomer, or a

mixture of both, but solubilisation of the template was

not possible.

Responses along this concentration range or even

higher, in the mM range, were reported for some novel

MIP sensors, which exhibit, nonetheless, excellent

recognition features (Panasyuk-Delancy et al., 2001;Lahav et al., 2001a,b; Kirsch et al., 2001).

3.4. Selectivity

Incubation of the three electrodes (imprinted, control,

bare glassy) was carried simultaneously in 9�/10�4 Msolution of VMA or one of the four compounds chosen

for their structural similarity with VMA (Scheme 2). The

intensity currents measured after transferral to the

electrochemical cell are shown in Table 2.

HGA and mainly DOPAC are significantly adsorbed

on the bare glassy surface. The fact that the currents

recorded with the imprinted and control electrodes in

these cases are lower than that of the bare glassy carbonsurface allows us to conclude that the detection of these

molecules is not due to their retention in the polymer,

but to the adsorption on GCE surface. It might be

possible to reduce the adsorption of these catechols to

some extent by choosing another media for the incuba-

tion.

For MHPG, there is no difference between the

response for the electrode with the MIP film and thatfor the control electrode, indicating that its adsorption is

unspecific. Anyway, the signal is not significant com-

pared with the intensities recorded following an incuba-

tion in VMA solution under the same conditions (Fig. 5)

and this operating procedure. This is important to

evaluate the functional groups that take part in the

adsorption, since the only difference between MHPG

and VMA is the OH group in the place of the carboxylicgroup at the end of the chain. Formation of hydrogen

bonds in the imprinted sites seems therefore to be more

favourable to VMA retention. It is also possible that the

observed weak adsorption in MHPG takes place on the

bare glassy carbon surface, because the current inten-

sities measured are lower than those obtained with the

control glassy electrode.

The structure of HVA in turn differs from that ofVMA in the absence of the OH group in the lateral

chain. HVA, like VMA, is not adsorbed at the glassy

surface, and the current observed for the imprinted

sensor has to be attributed to adsorption at the

imprinted sites, since no current was recorded with the

control polymeric electrode. Both species can be differ-

entiated nevertheless with the electrochemical potential.

In conclusion, these results reveal the necessity of thepresence of both a methoxy group and the carboxylic

group in the same position as in VMA for effective

binding in the imprinted polymer.

3.5. Outlook

One of the great advantages of a voltammetric sensor

is the possibility of combining the selectivity of the MIP

element with the selectivity of the voltammetric deter-mination.

The spin coating procedure followed by in situ

photopolymerisation is a quick and simple preparationFig. 4. Sensor response to the incubation concentration of VMA: (2)

imprinted electrode; (j) control electrode.

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362 359

procedure, and does not require time-consuming opera-

tions such as grinding and sieving. This is an advantage

over particulate coatings when intrinsic characteristics

to the sensor such as thickness of the film and porosity

of MIP need to be optimised. On the other hand, the

response time for the MIP film-based sensor used here is

faster than that reported for an electrode coated with a

particulate composite layer; 25 min was the incubation

time used here, whereas 2 h was required with an

amperometric sensor to achieve a limit current (Kriz and

Mosbach, 1995), and 1 to 2 h was used to monitor the

response of the MIP screen-printed electrodes described

(Kroger et al., 1999; Kirsch et al., 2001).

The adherence properties of the polymer to the

substrate could be optimised through the cross-linking

monomer to functional monomer ratio, which is re-

ported to have an influence also on the molecular

recognition of a membrane for a conductometric sensor

(Sergeyeva et al., 1999a,b), or by electrodic treatments.A drawback of the sensor design used here is the

difficulty for outward diffusion of the electrochemical

products through the polymer layer, and the renewal of

the electrode surface. Nevertheless, the electrodes used

here could be used at least 25 or 30 times with

subsequent binding, washing, and measuring opera-

tions. Alternatively, single use screen-printed electrodescould be easily prepared with this procedure.

This preparation is compatible with the actual trends

in sensor technology, which points towards mass-

produced low-cost devices, easy to miniaturise and

with possibilities for automatisation (Kricka, 1998).

4. Conclusions

It is possible to monitor the response of an MIP

coating on the electrode surface by using DP voltam-metry. The MIP-based sensors used here are able to give

responses 5�/10 times higher than those of the non-

imprinted electrodes in a non-excessive time lapse of 25

min. Unlike the other voltammetric sensors reported in

the literature, a competitive agent is not required in this

case to obtain the sensor response, provided that

measurements are carried out in a solvent favouring

the release of the analyte from the binding sites.Both the methoxy and carboxylic groups are required

for binding in the specific sites. HVA has the capacity to

rebind, but MHPG (where a hydroxyl group has

replaced the carboxylic group) is not detected under

the conditions used here. Absence of the methoxy in

position 3 is enough for not observing any rebinding

effect.

Voltammetry transduction adds the capacity of dis-criminating between species and identifying adsorption

at bare electrode surface or imprinted sites, since it does

not rely totally on the MIP element for the identifica-

tion. The electrochemical potential is additional infor-

mation enhancing the selectivity of the sensor.

Therefore, it might be concluded that MIP-based

voltammetric electrodes are very promising elements

for highly selective analytical sensors.

Acknowledgements

This work was financially supported by DGICYT

(Spain) project PB97-1304.

Table 2

Intensity currents measured with the VMA-imprinted sensor after incubation in a 9�10�4 M acetonitrile solution of the compounds indicated in

Scheme 2

E (V) I (imprinted electrode) (nA) I (control electrode) (nA) I (GCE) (nA)

VMA 0.845 61.2 12.2 �/

DOPAC 0.425 322.9 375.3 1324.1

HGA 0.434 35.3 80.7 207.9

MHPG 0.708 15.4 15.9 38.9

HVA 0.674 48.9 �/ �/

Fig. 5. DP voltammogram obtained with the imprinted electrode

(solid line) and the control electrode (dashed line) after incubation in a

9�/10�4 M solution of MHPG in acetonitrile. The signals obtained

after incubation in a solution of the template molecule under the same

conditions are included for comparison purposes (thin lines).

M.C. Blanco-Lopez et al. / Biosensors and Bioelectronics 18 (2003) 353�/362360

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