Electrochemical sensors based on molecularly imprinted polymers

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Trends in Analytical Chemistry, Vol. 23, No. 1, 2004

Electrochemical sensors based on molecularly imprinted polymers M.C. Blanco-Lo´pez, M.J. Lobo-Castan˜o´n, A.J. Miranda-Ordieres, P. Tun˜o´n-Blanco Molecularly imprinted polymers (MIPs) are becoming an important class of synthetic materials mimicking molecular recognition by natural receptors. This review examines the literature on non-covalent MIP-based electrochemical sensors over the last 10 years. With insight into the different sensing phases, electrochemical transductions and integration strategies, we evaluate achievements and difficulties to date and assess future prospects. # 2003 Published by Elsevier B.V. Keywords: Electrochemical sensors; Molecularly imprinted polymers (MIPs)

M.C. Blanco-Lo´pez, M.J. Lobo-Castan˜o´n, A.J. Miranda-Ordieres, P. Tun˜o´n-Blanco* Departamento de Quı´ mica Fı´ sica y Analı´ tica, Facultad de Quı´ mica, Universidad de Oviedo, C/ Julia´n Claverı´a 8, E-33006 Oviedo, Spain

*Corresponding author. Tel.: +34 985 103487; Fax: +34 985 103125; E-mail: ptb@fluor.quimica. uniovi.es

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1. Introduction MIPs are becoming an important analytical tool. Non-covalent imprinting, in particular, has a great range of applications because of the theoretical lack of restrictions on size, shape or chemical character of the imprinted molecule. The possibility of tailor-made, highly selective arti¢cial receptors at low cost, with good mechanical, thermal and chemical properties makes these synthetic materials appear ideal chemoreceptors. There are great hopes for development of a new generation of chemical sensors using these novel synthetic materials as recognition elements [1^3]. A review of the literature allows us to observe that, despite the large amount of data available to date on formulae for MIPs, the main applications continue to be in the separation ¢eld, whereas the development of sensors and electrochemical sensors, in particular, is signi¢cantly slower. Electrochemical sensing could o¡er good limits of detection (LODs), at low cost, with the possibility of easy miniaturization and automation. This type of transduction is specially attractive with a view to making

readily available a range of small devices based on recognition by a templating e¡ect in relevant applications, such as biomarkers in clinical chemistry, environmental control in the ¢eld, on-line quality control in the pharmaceutical industry or detection of food fraud. The aim of this article is to evaluate how close we are to that reality by examining data available in the literature on electrochemical sensing using MIP recognition processes. The ¢rst report in this ¢eld was published 10 years ago [4]. Since then, several recognition elements and di¡erent types of electrochemical transduction have been explored, and the number of publications has increased (Fig. 1). Two other reviews covering di¡erent aspects have been published [5,6]. This article is a compilation and an analysis of the literature on non-covalent MIP-based electrochemical sensors, with the aims of highlighting the achievements to date in the development of analytical applications and identifying the problems encountered and strategies developed. Relevant information relating to the preparation of electrochemical sensors is presented in several tables.

2. Recognition elements As most of the work on molecular recognition by imprinting e¡ects was carried out using radical polymerization of acrylic or vinylic type of polymers, these systems were the ¢rst to be tested for sensor applications and the most frequently used (Fig. 2a). The results reported in the literature have been classi¢ed according to the recognition

0165-9936/03/$ - see front matter # 2003 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00102-5

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004

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Figure 1. Growth of MIP-based electrochemical sensing publications in the last 10 years.

Figure 2. Distribution of publications on electrochemical sensors according to: a) nature of sensing phases; and, b) types of transduction.

element. Tables 1 and 2 cover studies using radically polymerized systems; Table 3 electropolymerized polymers; and, Table 4 inorganic sol-gels or monolayers. 2.1. Acrylic or vinylic types of polymers The studies corresponding to the development of MIP sensors based on radical polymerization with acrylic or

vinylic types of monomers have been classi¢ed in two groups:


the MIP is in the form of particles produced after grinding the polymer monolith (Table 1); and,
the MIP is prepared as a ¢lm on the transducer surface (Table 2). http://www.elsevier.com/locate/trac

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Table 1. Electrochemical MIP sensors using particulated radically polymerized acrylate or vinylic polymers Monomers

Transduction/substrate

Initiator

Template

Observations

Binding medium

Measurement medium

Useful conc. range

Ref.

MAA/EDMA

Amperometry/Pt

Thermal, AIBN

Morphine

MIP particles (1–25 mm) immobilized with agarose gel (0.5 mm layer thickness) Competitive binding assay High sensitivity Slow response ( 24 h)

20 mM citrate buffer pH 6+10% EtOH

20 mM citrate buffer pH 6+10% EtOH.

0.1–10 mg/ml

[7]

LOD: 0.05 mg/ml

4-vinylpyridine/ EDMA

DPV/screen printed electrodes 1–1000 mM

Thermal, ADVN

2,4-Dichlorophenoxyiacetic acid (herbicide)

MIP immobilized with agarose gel (1-mm layer thickness) Competitive binding assay

20 mM phosphate buffer pH 7 + 10% MeOH

0.1 M KCl in 20 mM phosphate buffer + 10% MeOH

Styrene/DVB (19:1)

CV, DPV screen printed

Thermal, AIBN

1-hydroxypyrene (PAH metabolite)

35 % aqueous MeOH

50 % MeOH, 0.025 M phosphate buffer pH 12

0.1–1 mM

[9]

MAA/EDMA (1:5) acetonitrile

DPV/graphite powder in solid matrix composite electrode

Thermal, AIBN

Clenbuterol

Particles < 53 mm ink Non competitive assay Single use Competitive assay  45 min response time Electrode renewal by mechanical polishing Reproducible (RSD 3.81 %)

Aqueous solutions

50 mM HClO4+10% EtOH

0.004–25 mM

[10]

Vinylimidazole + DVB (1:9) acetonitrile

Amperometry / Carbon paste electrode

1 M Tris HCl pH 9

1 M Tris HCl pH 9

LOD: 100 mM

[11]

Catalytic MIP, mimicking phosphotriesterase Particles 10–25 mM Measurements at 40 C

MAA, Methacrylic acid; EDMA, Ethylene glycol dimethacrylate; DVB, Divinylbenzene; AIBN, 2,20 azobis 2-isobutyronitrile; ADVN, 2,20 azobis 2,4-dimethyl-valeronitrile

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Paraoxon (organophosphotriester insecticide)

[8]

Monomers

Transduction/substrate

Initiator

Template

Observations

Binding medium

Measurement medium

Useful conc. range

Ref

MAA/EDGMA (1:5) chloroform

ISFET/silicon wafer

Thermal AIBN

L-phenylalanine anilide

Thin polymer membrane (1–3 mm) Flow measurements Silanization Glass plate to press and cover polymer mixture. Bad reproducibility between sensors.

EtOH (95%)

EtOH (95%)

n.a.

[4]

Conductometric/ Glass filter

Thermal AIBN

Atrazine

50 mM Tris pH 8

50 mM Tris pH 8

0.01–0.50 mg/L

[12]

Conductometric/ Glass filter

Thermal AIBN

L-Phenylalanine, 6-amino1-propyluracil, atrazine, sialicic acid

Bad reproducibility 12 hours to full recovery 30 min response time Covalent and non-covalent imprinting 30 min response time

50 mM Tris pH 8

50 mM Tris pH 8

1–50 mM

[13]

Conductometric

Photochemical AIBN

Atrazine

25 mM phosphate buffer pH 7.5+35 mM NaCl

25 mM phosphate buffer pH 7.5+35 mM NaCl

5–100 nM

[14]

DEAEM/EDMA (1:5) DMF

MAA or DEAM+EDMA DMF MAA+TEDMA+ OUA (DMF, chloroform)

MAA/EDMA

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(1:4.5) DMF AMPS/MBA* (1:2) H2O AMPS/MBA (1:2) H2O MAA/EDGMA (1:5) acetonitrile

CV/ITO

Photochemical AIBN

Theophylline

Monomer mixture poured between two quartz plates Thin, flexible and stable RSD <5% 60–120 mm membrane, 6–10 min response time. Silanization on ITO and acrylic film covalently bonded (nm) Gate effect studies only

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Table 2. Electrochemical MIP sensors using radically polymerized acrylate or vinyl polymers prepared as films

[15]

0.1 M KNO3 +5 mM Fe(CN)62

0.1 M KNO3 +5 mMFe(CN)62

n.a.

[16]

Capacitive/ Au coated with SAM of hexadecanethiol

Photochemical benzophenone

Desmetryn

10-nm layer Response time 5 min

100 mM KCl+5 mM phosphate pH 7.2

100 mM KCl+5 mM phosphate pH 7.2

1–7 mM

[17]

Capacitive/Au coated with SAM of haxadecanethiol

Photochemical benzophenone

Creatinine (metabolite)

Photografted MIP Reversible sensor response RSD 10%, reproducible 6 months

100 mM KCl+5 mM phosphate pH 7.2

100 mM KCl+5 mM phosphate pH 7.2.

10–600 mM

[18]

DPV/Glassy carbon

Photochemical acetophenone

Vanillylmandelic acid

Response time 2 min

Acetonitrile

25 mM citrate+10% acetonitrile

0.1–1.5 mM

[19]

*AMPS, 2-acrylamido-2-methyl-1-propanesulphonic acid; MBA, N,N0 -methylenediacrylamide; DEAEM, Diethylaminoethyl methacrylate; OUA, Oligourethane acrylate; n.a., Not available

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Table 3. Electrochemical MIP sensors using electropolymerized films Monomers Pyrrole

Solvent NaNO3 0.1 M

Transduction/ substrate

Template

Potentiometry/Glassy carbon

NO-3

Acetonitrile (TBAP) OPPy pH 7, 0.5 M phosphate buffer

CV/Glassy carbon

Adenosine Inosine ATP

Phenol

50 mM NaH2PO4

Capacitive/ Au /mercaptophenol

Phenylalanine

Protopothyrin IX

Cl2CH2 (TBAP) 10 mM acetate pH 5.18

CV/Glassy carbon

Nitrobenzene

Capacitive/Au

Glucose

o-phenylenediamine

TBAP, Tetrabutylammonium perchlorate; DMSO, Dimethylsulphoxide; n.a., Not available

Chemical recognition is affected by electropolymerization variables Template not removed from the film Good selectivity factors for ClO-4 and IUltra-thin films (0.16 nm) Potential sweeps in the measurement medium Selectivity and sensitivity changes are given by interaction with the film, and not by diffusion (transport) through the film Oxidation: expulsion of template Alkanethiols to fill defects Response time 15 min. 60 min for stationary value RSD 15% Poor temporal stability and reversibility. Single use 45–60 min incubation Concentration >0.01 M required 95-nm film thickness Film unstable Single-use electrode

Binding medium 0.005 M phosphate

Measurement medium 0.005 M phosphate pH 5.7

Useful conc. range

Ref

-5

[25]

5.0  10 –0.5 M LOD 2  1x10-5 M

pH 5.7 0.5 M phosphate buffer pH 7

0.5 M phosphate buffer pH 7

n.a.

[26]

5 mM NaH2PO4 140 mM NaCl pH 7.5

5 mM NaH2PO4 140 mM NaCl pH 7.5

0.5–8 mg/ml

[27]

DMSO

Phosphate pH 0.1 M

0.01M–1 M

[28]

10 mM Tris+ 100 mM NaCl pH 7.14

10 mM Tris+ 100 mM NaCl pH 7.14

0.1–20 mM LOD 0.05 mM

[29]

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Pyrrole (overoxidised)

Observations

Recognition element

Transduction/substrate

Template

Observations

Monolayer (Hexadecylmercaptane)

CV/Au

Cholesterol

Monolayer (Hexanethiol, dodecanthiol)

Capacitive/Au

Barbituric acid

Analytical signal is decrease of the ferricyanide reduction 50% EtOH+50 mM NaClO4 + 5 mM K3Fe(CN)6 15–60 mM peak with increasing concentration of cholesterol RSD < 5% (3 sensors) 5-min response time Spreader-bar approach to avoid distortion of the monolayer 5 mM phosphate buffer + KCl 100 mM (pH 5.5) n.a.

Monolayer (several thiols) SiO2 sol-gel

CV (and QCM)/Au

TiO2 sol-gel

ISFET/SiO2

TiO2 sol-gel

ISFET/Al2O3

TiO2 sol-gel

ISFET/Al2O3

Pre-formed polymer (polyphosphazene/ THF solution)

CV/Glassy carbon

Two-component monolayer Photochemical imprint in two-component monolayers Effect of chain length of the thiols in the template release Dopamine Selectivity study Drop coating (450-nm film thickness) Permeability studies with template in the medium 4-chlorophenoxyacetic acid Drop coating on the gate 2,4 dichlorophenoxyacetic acid 5-min equilibration time Fumaric acid Thickness: 85  10 mm Maleic acid Phenoxynaphthacene quinone

Methoxyferrocenecarboxylic acid CV, DPV/ Glassy Carbon Rifamycine SV

Binding medium/Measurement medium

Useful conc. range Ref [33]

[34]

0.01 Phosphate buffer pH 7.0+0.1 M Na2SO4

n.a.

[35]

0.1 M phosphate pH 7.4

n.a.

[38]

0.1 M phosphate

0.5–6 mM 0.1–9.0 mM 0.08–1 mM LOD:15 mM 0.2–3.0 mM LOD: 0.25 mM 0.05–6.25 mM LOD: 0.10 mM 0.25–6.6  10-6 M

[39] [40] [40]

0.1 M phosphate M

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Chirospecificity towards the imprinted enantiomer

0.1 M phosphate

Drop coating, evaporation 25-uses electrode 32 % RSD - 9 electrodes Fast response time

0.1 M phosphate pH 7

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Table 4. Miscellaneous recognition elements

[41] [42]

n.a., Not available

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With the former, the starting point was very often an already established MIP, tested by batch binding assays with other types of detection. When working with ¢lms, functional monomers used were methacrylic acid (MAA), vinylpyridine (VPy) and acrylamide derivatives. Thermal polymerization was initiated with 2,20 -azobis 2-isobutyronitrile (AIBN) normally, whereas photochemical polymerization was undertaken with AIBN, benzophenones or acetophenone derivatives. Among the porogenic solvents, dimethylformamide (DMF), a polar solvent, is frequently used when the aim is to develop a sensor for aqueous applications, in order to minimize swelling e¡ects [12,14,15]. An aqueous solution of monomers was also used for surface imprinting [17,18]. Perhaps the most relevant conclusion in relation to the preparation of ¢lms for sensors is that their £exibility (controlled by the ratio of functional monomer to cross-linker, or by addition of oligourethanes) directly relates to the performance of the sensor. Traditional formulae include high crosslinker ratios (5:1 mole of functional monomer), which favor the creation of imprinted sites and obtain the mechanical strength required for chromatographic stationary phases. But it was reported that, with as little as 20% of crosslinker, it was still possible to observe memory e¡ects by using batch-binding assays [20]. When using ¢lms for sensors, it was observed in some cases that some degree of £exibility favors the binding of the template and improves the adherence to the electrode surface. This tendency has been followed with optical [21,22] and piezoelectric [23,24] devices, but with few electrochemical sensors [14,15,17,18]. There needs to be a compromise between the minimum cross-linker necessary to obtain template memory and £exibility. 2.2. Electropolymerized ¢lms The possibility of inducing selectivity by the presence of the template during polymerization has been tested with several non-crosslinked electrogenerated polymers (Table 3), such as polypyrrole, polyphenol, polyprotoporphirin IX and poly(o-phenylenediamine) (o-PPD). Three of these polymers (polyphenol, polypyrrole and o-PPD) can be prepared easily by electropolymerization from aqueous solutions of their monomers. Nevertheless, compared with the wellestablished acrylate or vinyl derivatives, there has been little work in MIPs carried out with these polymers. Polypyrrole has recently been studied as a MIP with respect to enantioselectivity towards amino acids [30]. However, only a few applications to electrochemical sensing have been reported [25,26]. The creation of imprinted sites with this system is based on irreversible overoxidation that polypyrrole undergoes at potential values higher than those for its reversible doping and undoping, thus inducing charge and complementary

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structure e¡ects. We consider that these studies are based on an extension of the properties of permselectivity against anions (and selectivity towards cations) of thin, overoxidized polypyrrole ¢lms, which have led to important analytical applications involving the determination of cations in presence of anionic interferents [31,32]. However, during the overoxidation process, the template species is expelled, so this new approach to molecular imprinting avoids the templateextraction procedure and undesired bleeding e¡ects. In general, the great attractions of these systems as recognition elements for the electrochemist are ease of preparation (by cycling potential sweeps from solutions of their monomers and templates) and the possibility of obtaining very thin ¢lms with good reproducibility on many conductive substrates. These are especially attractive in developing applications for analytes of large molecular size, which would otherwise be signi¢cantly impeded in di¡using through the polymeric layer. 2.3. Miscellaneous systems Other systems, such as self-assembled monolayers (SAMs) [33^36], sol-gels [37^41] and pre-formed polymers [42,43] have been explored (Table 4). The modi¢cation of molecular recognition by SAMs has been the object of many studies. In a general sense, the formation of rigid nanostructures organized around the template molecule by SAMs at the electrode surface can be considered as a form of two-dimensional imprinting. The preparation of these systems involves the simultaneous adsorption of template and mercaptan molecules at a metallic surface (usually a gold electrode). The recognition is possible if speci¢c interactions are developed to form a stable complex between the template and the alkylthiol chain forming the monolayer [33]. Antecedents for this templated e¡ect can be found in the preparation of binary monolayers, which have shown selectivity depending on the size and the geometry of several quinone electrochemical probes [36]. In these systems, the long chain component acts as rigid matrix blocking penetration through the layer, and the short chain element acts as a gate, where interactions with the rigid matrix take place [36]. Molecular imprinting in SAMs could be regarded as the optimal nanoscale dimension to minimize di¡usion barriers. However, the main drawback of this form of imprinting is the lack of stability of the non-crosslinked ¢lm, with possible destruction of recognition sites by lateral di¡usion of molecules, especially if the template is removed. This problem has been overcome by developing the spreader-bar approach, whereby a thiol derivative of the target analyte (barbituric acid) was used as template and removal was not needed for recognition [34]. The selectivity and the kinetics of the association are strongly a¡ected by the length of the alkanethiols; if

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004 they are too short, there is no recognition, and, if they are too long, they could block the sites with their £exible chain and higher activation energies for association are required. Optimum matrix-forming systems reported are dodecanethiol [34] and tetradecanethiol [35]. Sol-gel systems might contain micropores with speci¢c sites for interaction with the host molecule, if they have been prepared from a solution of the precursors and the template. To our knowledge, there is only one report on selectivity induced by the presence of dopamine in a SiO2 ¢lm at the surface of the electrode [38], and several studies with TiO2 sol-gel layers tested by electrochemical measurements [39^41]. Finally, the possibility of molecular recognition induced by solidi¢cation of a pre-formed polymer on the transducer surface has also been reported. In these cases, it is more di⁄cult to control the interactions that direct selective recognition. The systems tested with this approach consisted of polyphosphazene for a voltammetric sensor for rifamycine SV [42] and polyacrilonitrile for microgravimetric detection of ca¡eine [43].

Trends e¡ect; amperometry; and, voltammetry (Figs. 2b and 3). Some of these techniques require strong, constant agitation to obtain a stationary regime, so the recognition element should exhibit enough rigidity to prevent distortion of the recognition sites under those operating conditions. 3.1. Conductometry Conductometry is based on the current £ow established by migration of ions of opposite charge, when an electric ¢eld is established between two electrodes immersed in the electrolyte solution. The development of a MIP-based conductometric sensor then relies on the preparation of the MIP as a membrane. Early studies have used glass ¢lters as polymerization support [12,13]. In other works, the polymerization mixture is cast between two quartz plates to obtain a 60^120 mm thick membrane [14,15]. This transduction is the least sensitive of the electrochemical techniques, because conductivity is additive, so it is impossible to discriminate between two ions. The small di¡erences in ionic limiting equivalent conductance are not enough to discriminate species. Moreover, if the concentration of one ion is very high, it could mask others.

3. Types of transduction The transduction techniques that have been used for the preparation of MIP-based electrochemical sensors are: conductometry; capacitive, impedance spectroscopy; potentiometry; chemical (ion-selective) ¢eld

3.2. Capacitance or impedance Interfacial phenomena can be followed by changes in capacitance or impedance of the system. The requirement is to have a totally pore-free, thin, dielectric ¢lm, usually on gold substrates. Electropolymerized poly-

Figure 3. Types of transduction systems used for the preparation of electrochemical MIP-based sensors. Classification according to IUPAC recommendations.

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mers, such as phenol (non-conductive) are a good alternative, since the grow of the ¢lm is easy to control, but an additional step is required to ¢ll pores (with chemisorption of alkylthiols on a gold surface) [27]. However, a photografting procedure with acrylate derivatives as recognition elements has proved to be useful as a sensor for creatinine and the herbicide desmetryn [17,18]. Capacitance measurements have also been carried out with an imprinted SAM of mercaptanes on a gold substrate [33]. 3.3. Potentiometry Potentiometry is based on creating a potential di¡erence across a membrane placed between two solutions with charged species of di¡erent activity. For the development of MIP sensors, it is important to note that the creation of a membrane potential does not require the template to be extracted from the membrane. This is an advantage, because extraction of the template to leave recognition sites ready for binding is very often a source of uncertainty at the determination or a sensitivity-limiting factor. Another unique feature of potentiometry is that species do not have to di¡use through the membrane, so there are no size restrictions on the template compound. Despite all these advantages, this type of transduction has not been explored much and we found only one report in the literature [25]. 3.4. CHEMFETs or ISFETs Potentiometric systems can be applied to the development of chemical-sensitive ¢eld-e¡ect transistors (CHEMFETs) or ion-sensitive ¢eld-e¡ect transistors (ISFETs). A semiconductor substrate can be modi¢ed with a ¢lm, so that it becomes sensitive to a change in surface potential arising from a chemical reaction or a change of charge at that ¢lm on the gate of a ¢eld-e¡ect transducer. As a result, there is a change in the current that £ows between the source and drain electrodes of the transistor. The great interest in these devices derives from their ease of miniaturization, which requires preparation at wafer level, good adhesion to substrates and control of thickness. 3.5. Amperometry Amperometric determinations require a linear relationship to be established between the concentration of electroactive species and the current measured at constant potential. It can also be applied to non-electroactive species that take part in a displacement step coupled with an electrochemical reaction [8]. Di¡usion of species towards the working electrode and the outward di¡usion of reaction products are required for the establishment of a current; otherwise, the surface would be passivated. Porosity in the MIP layer (whatever the nature of the recognition element) is therefore

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essential to provide the channels required for di¡usion of species and electron transfer reactions at a bare electrodic surface. Measurement then involves selective extraction of the template, followed by exchange of electrons with the electrode surface. 3.6. Voltammetry Voltammetry involves monitoring the current generated upon application of a potential sweep. This is the most selective electrochemical technique, since the oxidation or reduction potential of a particular substrate is its intrinsic property. There are several types of voltammetric techniques, depending on the shape of the applied potential function. For linear sweep voltammetry (LSV) and cyclic voltammetry (CV), the potential applied changes linearly with time. When the potential sweep is not a linear function but comprises constant increments on a linear ramp (di¡erential pulse voltammetry, DPV) or a square wave function (square wave voltammetry, SWV), these techniques could o¡er better sensitivity, because they o¡er better signal-tonoise ratios. However, SWV has not yet been used with these type of sensors. CV has the advantage of allowing the density of template units at the surface to be estimated through an easy coulometric analysis of the resulting redox peaks, as it sometimes used to be done to determine the optimum incubation time [35]. In general, the measurement medium for all these techniques is an inert electrolyte bu¡er solution. Tables 1^4 show that there is a tendency to work with low values of ionic strength ( < 50 mM), probably to facilitate ionic interactions between template and polymer. When using an acrylic or vinylic type of polymer, measurements are frequently carried out in mixed media with organic solvents, such as ethanol, to improve the wettability of the polymeric ¢lm. Although establishment of arti¢cial MIP sensors is still at the research level, the greatest challenge is to develop arti¢cial enzymes (i.e. MIP polymers with enzyme-like catalytic activity). We found one report on a catalytic MIP sensor with amperometric detection [11]. With conductometric, impedometric, potentiometric, or ISFET transduction, the binding of the template is enough to generate the analytical signal (similarly to piezoelectric devices). By contrast, with MIP-based amperometric and voltammetric devices, the necessary electron-transfer step gives rise to products that can foul the electrode surface. The signal will not therefore be recovered as long as the transducer surface is not free of adsorbed products, independently of the reversibility of binding at the recognition element. This problem has been taken into account in the applications reported, and, in some cases, the ease of preparing materials and their low cost have allowed the use of disposable, single-use electrodes [8,9]. Another

Trends in Analytical Chemistry, Vol. 23, No. 1, 2004 approach is to prepare a composite bar containing MIP particles in a solid binding matrix, with a surface that can be renewed simply by mechanical polishing [10]. Alternatively, the surface can be washed with a good solvent for the products [19], although full recovery is di⁄cult because of the restricted access associated with the porosity of the polymer.

4. Strategies for MIP-transducer integration The slow development of MIP sensors could indicate that the adaptation of MIP recipes to sensor technology is not straightforward, and that there need to be speci¢c integration strategies to develop these new arti¢cial recognition elements. 4.1. Integration through powder processing Since most MIPs applications require the preparation of a monolith that then has to be ground and sieved, some studies on MIP sensors concentrate on immobilizing the polymeric particles as close as possible to the transducer surface. Thus, agar gel was used to immobilize MIP particles either on a platinum electrode for amperometric detection of morphine [7] and on the surface of screen-printed electrodes for voltammetric determination of herbicides [8]. Alternatively, a plastic membrane with polyvinyl chloride (PVC) could be used to immobilize MIP particles. In this way, several piezoelectric sensors and applications in biological media have been described [44]. However, with electrochemical transduction, unspeci¢c adsorption was observed. As a strategy for developing biosensors, the modi¢cation of carbon pastes by incorporating enzymes or others reagents has been widely used in combination with amperometric transduction. These types of electrode, incorporating MIP particles, could greatly simplify the development of amperometric and voltammetric sensors. In addition, carbon paste is an easy option for single-use electrodes, as it cuts down problems relating to surface renewal. However, the frequent need to use organic solvents restricts applications of this type of electrode, unless the MIP has been tested in aqueous media. We found only one report of an application in aqueous solution [11]. Other forms of immobilization explored included casting a composite ink to produce MIP screen-printed electrodes [9], and forming a composite electrode with the MIP, graphite, and a solid binding matrix, such as n-eicosane [10]. For sensing layers using particulate MIP, the sensor response time is closely related to the particle size. Most reported studies used the fraction up to 25 mm [7,10,11] or 50 mm [9], which generally led to slow kinetics for intraparticle di¡usion and consequently

Trends long response times. There are no data relating response time to particle size, and we did not ¢nd any reports with a polymer of smaller particle size produced by polymerization routes, such as precipitation polymerization [45]. A di¡erent type approach to integration consists of preparing composite materials containing a conducting polymer in the pores of MIP particles, which should exhibit both predetermined molecular recognition and electrical conductivity. A preliminary report showed that the chemical growth of polypyrrole in the pores of a MIP does not alter its recognition abilities but, as far as we are aware, this issue has not been explored further [46]. 4.2. Integration through ¢lms The integration strategy most frequently used to date consists of preparing the recognition element as a ¢lm on the transducer surface (Tables 2^4). In any case, control of layer thickness is necessary to adapt sensor response time and sensitivity. In general, three-dimensional networks are preferred over two-dimensional, because they are more stable and more favorable for anchoring the molecule at several points. Their a⁄nities for the polymer should be higher, although at the cost of a slower response, so thickness-response time must be a compromise. With acrylic polymers, the ideal thickness (10^100 nm) could be achieved by grafting polymerization [17]. Table 5 summarizes strategies for preparing and attaching a ¢lm of a radically polymerized recognition element to the electrode surface.

5. Measurement protocols The measurement protocol relates to every transduction system, and very often relies on successful selective extraction of the analyte into the MIP element, followed by measurements in the same or a di¡erent medium. Alternatively, the selectivity induced by the presence of the template during polymerization can be tested by voltammetry (by carrying out potential sweeps in a medium containing the analyte) [26,38]. This is particularly interesting when new recognition elements are being tested, such as electropolymerized ¢lms, which lack stability for an extraction step. Furthermore, this type of recognition can be useful for in situ applications, since it does not require a separation step. It is important to note that detection with MIP-based electrochemical sensors is not restricted to the protocols and operating procedures developed for separations or binding assays. The measurement protocols that have been used over the past 10 years can be divided in two groups: those involving extraction of template; and, those that do not require extraction of template http://www.elsevier.com/locate/trac

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(Fig. 4). Among the former, the analytical signal can be originated by the analyte (direct measurement [9,14,15,17^19],) an electrochemical probe, such as Fe(CN)26 [16,33], or a competitive species (indirect measurement [7,8,10]). Examples of applications without initial template extraction are potentiometric determination of NO-3 [25] and capacitive determination of barbituric acid with self-assembled monolayers [34].

6. Analytical characteristics 6.1. LODs It has frequently been reported that one of the main problems in developing analytical applications with acrylic MIPs is the di⁄culty of attaining low LODs [23]. This has led to new fabrication routes, aiming to increase the concentration of imprinted sites in particle systems. In the ¢eld of sensors, it is therefore advisable to use the most sensitive types of transduction, (i.e. amperometric or DPV among electrochemical

sensors). In any case, the LOD depends on a combination of the type of transduction and features relating to the recognition phase. 6.2. Selectivity One of the di⁄culties of measurement protocols involving an extraction step is the strong e¡ects of unspeci¢c adsorption on the electrode surface, particularly when carbon electrodes are used [8]. In some cases, selectivity is tested with the recognition element by batch binding assays, but not with the sensor, thus possibly ignoring important interferences [9]. A unique feature of voltammetry is the possibility of improving detectability by separately optimizing the binding medium and the measurement medium. Other techniques of electrochemical transduction use the same medium for binding and detection. In addition, structurally related interferents can be identi¢ed by their electrochemical potential [19]. Enantiomeric resolution, one of the main advantages of using MIPs, has been reported with ISFETs [39,40].

Figure 4. Measurement protocols used for the development of MIP-based electrochemical sensors.

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[17,18] Photografting

Sandwich technique

Silanization

[12—15, 23,47,48]

[19]

An aliquot of the polymerization mixture is deposited on the electrode surface and a rotation speed (< 500 rpm) is applied. The excess of polymerization mixture is eliminated and only a thin layer remains. The metallic substrate was soaked in a solution of with 3-methacryloxypropyl-trimethoxysilane. Then, it was immersed in the acrylic monomer mixture. After polymerization, the weakly adsorbed copolymer is eliminated by washing in water, and only the covalent attached polymer remains on the electrode surface. A drop of the polymerization mixture was deposited on the transducer and covered with a quartz disc, or deposited between two quartz discs. Polymerization is photoinitiated. (This has been applied to the preparation of piezoelectric sensors and to the preparation of membranes for conductometry). The radical initiator is adsorbed at the transducer surface. Then, the monomer mixture is deposited on it, and, after polymerization and removal of the excess, only a thin film remains on the sensing surface. Spin-coating

[4,16]

Example Ref. Procedure Technique

Table 5. Strategies used to prepare and attach a film of acrylic or vinyl polymers to the electrode surface

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Trends 6.3. Reproducibility and reversibility Ideally, these systems should be fully reversible, with alternating binding and washing cycles to recover the recognition properties. Sometimes, full recovery requires long washing times, e.g., 12 hours [13]. Nevertheless, partial recovery of the recognition element is not a problem because MIP-based electrochemical sensors can be incorporated into disposable devices that use a fresh, inexpensive component every time. Applications with screen-printed electrodes have been reported [8,9]. Electropolymerization provides another way of obtaining very reproducible, easily prepared sensors. 6.4. Application to real samples Sensors reported to date have been tested with standard solutions only. Among the literature cited, there is one report on spiked samples (clenbuterol sensing in liver extracts [10]), so there are not yet any data on the in£uence of matrix e¡ects on the response of the sensors with di¡erent types of recognition element, transduction and measurement protocols. 6.5. Response time and long-term stability In general, response times with sensors using MIP particles have been longer than with ¢lms, because of the di⁄culties associated with di¡usion. The shortest response times have involved grafting polymerization [17,18]. Sensors using acrylic or vinyl MIPs are reported to have good stability during prolonged storage (more than 6 months in many cases), as expected for a highly cross-linked polymer.

7. Summary Over the last 10 years, great e¡orts have been made to combine MIP technology with electrochemical sensing. The majority of the sensor systems explored to date have used radically initiated polymerization with acrylic or vinylic polymers as recognition elements, but other phases (electrogenerated polymers, monolayers, sol-gel systems) have also been tested. Conductometric, capacitive, impedance spectroscopy, ISFET, potentiometric, amperometric and voltammetric transduction have been explored. Sensitivity requirements point towards amperometric and voltammetric devices. The slow development of electrochemical sensing devices based on MIPs might indicate problems in integrating these arti¢cial recognition elements with electrochemical transducers. On the basis of the studies undertaken, there is no doubt that these initial di⁄culties will be overcome, and it is very likely that a new generation of MIP-based electrochemical sensors will be established in the decade ahead. http://www.elsevier.com/locate/trac

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Acknowledgements This work was carried out with ¢nancial support from the Spanish Government (Ministerio de Ciencia y Tecnologi¤a, Project BQ-2002-00261).

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