molecular imprinting technology: challenges and prospects for the future

Molecular Imprinting Technology: Challenges andProspects for the Future

OLOF RAMSTROM* AND RICHARD J. ANSELLPure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University,

Lund, Sweden

ABSTRACT Molecular imprinting is a technique for the fabrication of biomimeticpolymeric recognition sites or ‘‘plastic antibodies/receptors’’ which is attracting rapidlyincreasing interest. By this technology, recognition matrices can be prepared whichpossess high substrate selectivity and specificity. In the development of this technology,several applications have been foreseen in which imprinted materials may be exchangedfor natural recognition elements. Thus, molecularly imprinted polymers have been usedas antibody/receptor binding mimics in immunoassay-type analyses, as enzyme mimicsin catalytic applications and as recognition matrices in biosensors. The best developedapplication area for imprinted materials, though, has been as stationary phases forchromatography, in general, and chiral chromatography, in particular. This review seeksto highlight some of the more intriguing advantages of the technique as well as pointingout some of the difficulties encountered. The prospects for future development will alsobe considered. Chirality 10:195–209, 1998. © 1998 Wiley-Liss, Inc.

KEY WORDS: molecular imprinting; molecular recognition; chirality; chromatography;catalysis; biosensor; immunoassay; antibody mimic

Molecular Imprinting Technology (MIT) is an attractivesynthetic approach to mimic natural molecular recogni-tion.1–7 In this technology macromolecular (usually syn-thetic) entities are prepared by a polymerisation process inwhich sites are introduced by use of a ligand as a templatein a casting procedure. The technique is schematically de-picted in Figure 1. The selected ligand or imprint antigen isfirst allowed to establish binding interactions with polymer-isable chemical functionalities and the resulting complexesor adducts are subsequently copolymerised with crosslink-ers into a rigid polymer. After extraction of the antigen,specific recognition sites are left in the polymer in whichthe spatial arrangement of the functional groups in thepolymer network together with the shape are complemen-tary to the imprinted molecule. Currently, two basic ap-proaches to molecular imprinting may be distinguished:viz. 1) the self-assembly approach, where the pre-arrangement between the imprint antigen and the func-tional monomers is formed by noncovalent or metal coor-dination interactions, and 2) the pre-organised approach,where the complexes in solution prior to polymerisationare maintained by (reversible) covalent bonds. By use of ahigh percentage of crosslinker, completely insoluble poly-mers of substantial rigidity are obtained.

ApplicationsA wide range of compounds have been used as imprint

antigens to investigate the feasibility of various practicalapplications.1 Thus, compounds such as drugs,8–13 aminoacids,14–16 carbohydrates,17–22 proteins,23–25 nucleotide

bases,26 hormones,27,28 pesticides,29–31 and coenzymes,32

have been used successfully for the preparation of selectiverecognition matrices. In addition to studies where the na-ture of the recognition events per se have been the majorissue, several areas of application have been envisaged forimprinted matrices (Table 1), viz. 1) the use of molecularlyimprinted polymers in separation and isolation,2,21,33–35 2)the use of molecularly imprinted polymers as antibodyand receptor mimics in immunoassay-type analy-ses,3,10,11,27,30,36 3) the use of molecularly imprinted poly-mers as enzyme mimics in catalytic applications,37–42 and4) the use of molecularly imprinted polymers in biosensor-like devices.43–46 Of these, Molecular Imprinting Chroma-tography (MIC) has been the most extensively studied ap-plication area and several intriguing separations have beenperformed, which have exhibited high separation factorsand resolutions (cf. Fig. 2). More recently, applications ofmolecularly imprinted polymers as antibody mimics in im-munoassays have also attracted increasing interest.

Characteristics of Molecularly Imprinted PolymersApart from the more obvious recognition properties of

molecularly imprinted polymers, their physical and chemi-cal characteristics are highly appealing. These materials

*Correspondence to: Olof Ramstrom, Pure and Applied Biochemistry, Cen-ter for Chemistry and Chemical Engineering, Lund University, PO Box124, S-221 00 Lund, Sweden. E-mail: [email protected] for publication 14 January 1997; Accepted 4 March 1997

CHIRALITY 10:195–209 (1998)

© 1998 Wiley-Liss, Inc.

exhibit high physical and chemical resistance towards vari-ous external degrading factors. Thus, molecularly im-printed polymers are remarkably stable against mechanicalstress, elevated temperatures and high pressures,43,47 re-sistant against treatment with acid, base, or metal ions andstable in a wide range of solvents.43 The storage life of thepolymers is also very high: storage for several years atambient temperature leads to no apparent reduction in per-

Fig. 1. Schematic representation of imprint formation following the self-assembly approach. The functional monomers are arranged around theimprint antigen as a result of the interactions between complementarychemical functionalities. After polymerisation, the imprint antigen is re-moved by extraction, exposing recognition sites possessing a ‘‘memory’’for the shape and chemical functionality of the imprint antigen.

Fig. 2. Chromatographic enantioseparation of N-Ac-L-Phe-L-Trp-OMe(1) from its enantiomer N-Ac-D-Phe-D-Trp-OMe (2) by a molecularly im-printed polymer against 1. Attenuation was increased 10-fold after 13 minto get a clearer view of the peak resulting from the print species, (separa-tion factor a = 17.8). 200 × 4.6 mm column, isocratic elution with chloro-form/acetic acid (99:1 v/v) at 1.0 mL/min; 10 µg racemate injected in20 µl mobile phase.65

TABLE 1. Applications of molecularly imprinted polymerswith reference examples

Present Developments Reference exampleRecognition studies 16, 79, 85, 87, 97Separations, isolations

● Chiral separations 21, 59, 60● Substrate-selective separations 17, 29, 31, 98

Antibody/receptor binding mimics● competitive ligand binding assays 11, 29, 30, 36● diagnostic applications 10

Enzyme mimics/catalysis 37, 39–42, 99Biosensor-like devices 43–46Site-mediated synthesis 73, 100Future DevelopmentsPreparative scale separationsControlled-release matricesEquilibrium shifting

196 RAMSTROM AND ANSELL

formance. Further, the polymers can be used repeatedly, inexcess of 100 times during periods of years without loss ofthe ‘‘memory effect.’’8 In comparison with natural biologi-cal recognition sites, which are often proteins, these prop-erties are highly advantageous.

RECOGNITION IN MOLECULARLYIMPRINTED POLYMERS

Molecular Recognition

The study of complex interactions between molecularspecies, molecular recognition, and the ability to mimicnatural binding phenomena has intrigued scientists for along time.48–50 The recognition events taking place in en-counters between ligands and receptors are highly depen-dent on the additive effect of a number of binding forces.Optimal combination of the potential binding forces maylead to strong binding. Thus, in order to achieve efficientcomplexation between the host molecule and the guestspecies, several factors such as shape complementarity,functional complementarity, and contributions from thesurrounding pool of solvent have to be considered.

In biological systems, molecular complexes are oftenformed by a plethora of noncovalent interactions such ashydrogen bonds and ion pairing. Although these interac-tions, when considered individually, are weak in compari-son with covalent bonds, the concerted action of several ofthese bond types often leads to complexes with very highstability. The high degree of specificity that can beachieved, in combination with the dynamic properties ofthe interactions, make these bond types prerequisite formany biological processes.

Methods for Observing Recognition

The recognition properties of molecularly imprintedpolymers have been evaluated in two principal ways—chromatographic retention analysis and batch-rebindinganalysis. The benefit of the former is that a high degree ofsensitivity can be obtained by the chromatographic pro-cess if a small quantity of analyte is injected on to a largechromatography column. Good resolutions result from thelarge number of theoretical plates. The selectivities of theimprinted matrices may then be expressed by the retentionvolumes of different ligands: Non or poorly interacting spe-cies are eluted before the imprint antigen. Another advan-tage with this technique is that the binding performancemay be estimated by frontal zone analysis from which ap-parent affinity constants (KD) and site population densities(Bmax) may be obtained.51 However, although chromato-graphic analysis is a useful and reliable tool for the evalu-ation of recognition properties, the (flow) kinetics of theprocess may interfere with the interpretation of the results.As a consequence of the mobile phase flow through thematrix, only the sites with fast binding kinetics are de-tected and evaluated. This is clearly demonstrated whencomparing the affinity constants determined with eitherfrontal zone chromatography or radioligand batch analysis(see below). In the former case KD-values in the mM rangeare normally acquired,51 whereas in the latter, KD-values in

the nM-range may be recorded.36 This 106-fold differencesuggests that only sites with sufficiently fast binding kinet-ics, association and dissociation, are detected in the chro-matographic mode. The chromatographic flow processthus shields the effects of the best sites showing the high-est affinities in the materials.

For studies of the recognition process occurring underequilibrium conditions, batch-analysis may be performed.In this case, all the sites in the matrix are taken into con-sideration and no external kinetic process have to be re-garded. However, the sensitivity enhancement given bythe chromatographic process is not available since onlyone equilibrium step is involved. Several different ap-proaches may be used in equilibrium studies depending onthe level of sensitivity required. To observe small differ-ences in binding, or binding of very small quantities ofligand, methods with very low detection limits are re-quired. In recent years, radioligand binding assays havebeen used extensively due to the very high sensitivity ofthis method, which makes it possible to study the verysmall population of sites with strongest binding.3,10 Therecognition properties are estimated either by monitoringthe direct binding of the ligands of interest to the imprintedpolymers, or by using a competition assay technique,where the ligands compete for the binding sites with the(radiolabelled) imprint antigen. By these techniques, theapparent affinity constants and binding site populations, aswell as the cross-selectivities, of the imprinted polymersmay be obtained. A useful way of describing the selectivi-ties displayed by the matrices is to relate the observedvalue for 50% displacement of the imprint species (IC50) bya certain ligand to the value estimated for the actual im-print antigen.

Chiral RecognitionIn nature, chirality is often an intrinsic property of the

binding sites of macromolecular recognition elements,such as receptors, enzymes and antibodies. The way inwhich a natural binding site is formed, starting from build-ing elements with inherent chirality, obviously renders theconstruction chiral. The pursuit of efficient mimics of natu-ral binding sites requires the introduction of chirality in thesynthetic sites. By means of sophisticated chemical con-siderations, starting from optically pure building blocks,synthetic binding sites may be created which possess chi-ral discrimination properties.52,53 Other approaches involvethe use of natural binding elements, such as cyclodextrins,which are fundamentally chiral.54

Molecular imprinting technology offers a means for theintroduction of chirality in macromolecular matrices start-ing from achiral building blocks. The chirality is a conse-quence, and a print negative, of the original asymmetrycarried by the imprint antigen. If the optical antipode to theantigen is administered to the sites, much lower bindingstrengths are observed compared to the original antigen.On the other hand, the use of achiral imprint antigens willnot lead to any detectable chiral discrimination by the mo-lecularly imprinted polymer.

Although the imprinting technique, in its most basic for-mat, makes use of simple achiral monomer units, this is

MOLECULAR IMPRINTING TECHNOLOGY 197

certainly not a prerequisite for an efficient binding matrix.The use of chiral building blocks in the imprinting proce-dure may in fact enhance the chiral discrimination effect.This was reflected in a study where a chiral polymerisablemetal-coordination complex was used in the imprinting ofamino acids.55 Polymers prepared in the absence of imprintantigens showed some discrimination for the chosen en-antiomers due to the inherent chirality of the monomer.This discrimination could, however, be enhanced when thepolymers were prepared in the presence of antigen. How-ever, since other imprinting investigations have indicatedthat no additional discrimination may be obtained whenstarting from chiral monomers,56,57 it is obvious that everysystem has to be designed and optimised individually forthe outcome of specific binding matrices.

In all applications presently developed for molecularlyimprinted polymers, chirality has been one issue of inves-tigation. Originally, though, the use of chirality in the stud-ies was a means for monitoring and validating the imprint-ing effect rather than a goal in itself. Thus, in several im-printing studies selective discrimination of enantiomericpairs has been the issue, since their geometrical structureis the principal differentiating quality between them.Amino acid derivatives were often chosen as model com-pounds because they are inexpensive, stable, possess arange of functional groups and are soluble in convenientsolvents. By the employment of a single enantiomer asimprint antigen, the imprinting effect exhibited by the poly-mers is easily monitored since chiral discrimination wouldnot be acquired if the separation was based only on ion-exchange or size-exclusion processes.

Using self-assembly imprinting protocols, highly effi-cient chirally discriminating sites have been prepared andlarge separation factors between enantiomers have beenrecorded when the resulting molecularly imprinted poly-mers have been used as chiral stationary phases in chro-matography. Characteristic of these stationary phases isthe pre-determined elution order of the enantiomers, de-pending only on which enantiomeric form was used asimprint antigen. For instance, when the R-enantiomer isused as imprint antigen, the S-form will be eluted first, andvice versa if the S-enantiomer is used as template (cf. Fig.3).58

The recognition mechanism in molecularly imprintedpolymers prepared by non-covalent interactions againstchiral templates has been discussed in several studies.59,60

The main factors responsible for selective rebinding of li-

gands to the imprinted sites have been shown to be ionicinteractions and hydrogen bonding.61–63 The three-dimensional arrangement of these interaction points, ar-rested by the polymerisation step, leads to an inherentchirality of the formed sites. Although this arrangementanswers for most of the selectivity, the shape of the sitealso plays an important role. The configuration of the sur-rounding polymer backbone, as moulded in place aroundthe imprint antigen, contributes to ligand specificity over-all. This effect is sometimes less pronounced and substan-tial freedom in ligand structure can be observed.17,64 How-ever, often the effect is considerable and minute differ-ences in size can be distinguished. In several cases, theposition of a single methyl group is crucial for recogni-tion.9,10,64 In this perspective, the shape effect of the site isparticularly pronounced for parts of an imprinted moleculein close proximity to ionic or hydrogen bonds. For struc-tural features of the analyte more distal from such bonds,shape is less important (cf. Fig. 4).

For imprint antigens containing two chiral centres, allfour stereoisomers may be selectively recognised by theimprinted polymers. Thus, with a polymer imprintedagainst the dipeptide N-Ac-L-Phe-L-Trp-OMe, using primar-ily hydrogen bonding interactions, the LL-form could se-lectively be distinguished from the DD-, DL,- and LD-isomers (separation factors: a = 17.8, 14.2, and 5.21, respec-tively) (cf. Fig. 2).65 Similar effects were recorded forpolymers imprinted against ephedrine and pseudoephed-rine, respectively, although these effects were less promi-nent.64 In systems where more than two chiral centres areinvolved, such as carbohydrates, these properties of mo-lecularly imprinted polymers become even more signifi-cant. In polymers imprinted against a glucose derivative,very high selectivities between various stereoisomers wererecorded.17

IMPRINTED MATERIALSDifferent Matrices

Several polymer systems have been developed for use inmolecular imprinting technology. At present, primarily ac-rylate-based, styrene-based or silane-based polymeric ma-terials have been used in imprinting protocols (Table 2). Ofthese, polyacrylate-based matrices have been the most ex-tensively studied. The free-radical polymerisation mecha-nism used in these systems is advantageous for severalreasons. The polymerisation reaction is normally veryrapid, and may be initiated by several factors (thermo- or

Fig. 3. Schematic demonstration of the pre-determined elution order achieved using molecu-larly imprinted chiral stationary phases. If, e.g.,the R-enantiomer of a chiral compound is used asimprint antigen, it will be retained longer than theS-enantiomer when a racemic mixture is adminis-tered. If, on the other hand, the S-enantiomer isused as antigen, the opposite elution order will beobserved.


photolytic initiation are most commonly used). Further,polymers may be prepared successfully in a wide range ofsolvents at ambient temperature and pressure. Finally, themechanism does not interfere severely with most imprintantigens, although in certain cases radical quenching mayoccur.

Molecularly imprinted polymers have been used in sev-eral configurations (Table 3). By far the most frequentlyused technique involves the preparation of bulk polymermonoliths. After fragmentation and particle sieving (givingparticles of usually about 25 µm) the resulting powdersmay be used in several applications. For chromatographicapplications other configurations have been developed.Thus, polymers have been prepared in situ in chromatog-raphy columns,66,67 and in capillary electrophoresis sys-tems.68 Since the flow properties in chromatography aredependent on particle size and shape, attempts have alsobeen made to acquire molecularly imprinted polymer par-ticles homogeneous in dimensions and morphology. Thishas been accomplished following two different routes: 1)grafting/coating of the imprinted polymer on pre-formedparticles, such as silica or poly-(trimethylolpropane tri-methacrylate) particles,69–72 and: 2) preparation of beadsby suspension, emulsion or dispersion polymerisation (cf.Fig. 5).73–76 In this manner, spherical molecularly im-printed polymer particles with narrow size distribution canbe obtained, providing good flow performances in chroma-tography. For analytical or sensor device applications, thinlayers or polymer membranes have been developed. In thiscase, the polymer is either directly cast as a thin layer ona surface or chip,45,46 or alternatively, molecularly im-printed polymer particles are glued together using a par-

ticle binding agent obtaining, e.g., coated glass plates simi-lar to those used in thin layer chromatography.77 The ob-vious advantages with the direct approaches (i.e., grafting,beads, and membranes) are that particle sizing is unnec-essary and that the sites left in the polymer are undamagedfrom any fragmentation or sieving process.

A further technique, which may be denoted surface im-printing,24,78–82 has been developed particularly for ‘‘large’’imprint antigens. The imprint antigen is first allowed toform adducts with functional monomers in solution and theformed complexes are subsequently allowed to bind to anactivated surface such as silica wafers or glass surfaces.Thus, with this technique, a designed imprinted, or im-aged, surface is obtained. Potentially this approach shouldbe valuable for creating specific cell binding surfaces.When preparing molecularly imprinted polymer monolithsagainst large imprint antigens, there is a risk of permanententrapment of the template in the polymer after polymeri-sation. When using thin polymeric layers or imprinted sur-faces this drawback may be overcome.

Solvent EffectsThe solvent plays an immense role in the outcome of a

molecular imprinting process. As porogen in the polymeri-sation, the solvent governs the strength of non-covalentinteractions in addition to its influence on the polymer mor-phology. Generally, the more polar the porogen, the worsethe resulting recognition effect becomes, as a consequenceof the influence of the solvent polarity on noncovalent in-teractions. All noncovalent interactions are dependentupon the polarity of the solvents, as reflected by its dielec-tric constant, electron donating/accepting properties and

Fig. 4. Molecular recognition by imprinted polymers. Left: For a poly-mer imprinted against (+)-(S,S)-pseudoephedrine, a single methyl group iscrucial for recognition. The polymer can resolve the enantiomers of pseu-doephedrine (racemic separation factor aRac = 3.19) and halostachine (aRac= 2.12), but is noneffective in the resolution of a racemic mixture of ephed-

rine (aRac = 1.0).64 Right: Ligand binding by a polymer imprinted againstoctyl-a-D-glucoside. The methyl-derivative binds approximately as effec-tively (IC50 = 1.3 µM) to the polymer as the original imprint antigen (IC50= 1.7 µM) demonstrating the tolerance exhibited by the recognition sitesfor parts of the structure not in close proximity to ionic- or hydrogen bonds.17


hydrogen donating/accepting capability. Of the differentnoncovalent bond types commonly used in molecular im-printing, Coulombic charge-charge interactions display theleast solvent dependence, inversely weakened by solventpolarity. For other electrostatic bond types, a greater de-pendence is found. In order to optimise the bindingstrengths, the best imprinting porogens are solvents ofvery low dielectric constant such as toluene and dichloro-methane. The use of more polar solvents weakens the in-teraction forces formed between the print species and the

functional monomers resulting in potentially poorer recog-nition. On the other hand, the influence of the porogen onthe structure of the prepared polymers may compensatefor this apparent drawback; the specific surface area andthe mean pore diameter are dramatically dependent on thetype of porogen used. Thus, acetonitrile (a fairly polar sol-vent) leads to more macroporous polymers than chloro-form.83 A lower surface area and a lower macroporositymay lead to diminished recognition, because of lower ac-cessibility to the sites.

In the recognition step similar questions about thechoice of solvent arise. Since all noncovalent forces areinfluenced by the properties of the solvent, non-polar sol-vents normally lead to the best recognition. When applyingthe polymers to gradually more polar solvents, the recog-nition is diminished. Also, the morphology is affected sincethe swelling of the polymers is dependent on the surround-ing medium. Thus, the swelling is most pronounced inchlorinated solvents, such as chloroform and dichloro-methane, as compared to solvents like acetonitrile and tet-rahydrofuran.84 This swelling behaviour may lead tochanges in the three-dimensional configuration of the func-tional groups taking part in the recognition in the sitesresulting in poorer binding capability.

TABLE 3. Configurations of molecularlyimprinted materials

ConfigurationReferenceexample

Fragmented polymer monoliths 10, 26, 35, 79, 103Composite polymer beads 69–72Polymer beads from suspension, emulsion,

or dispersion polymerisation 73–76In situ polymerisation 66, 68Polymer particles bound in thin layers 77Polymer membranes 45, 46, 104, 105Surface-imprinted polymer phases 24, 78–82

TABLE 2. Polymer systems

Functional monomer class Example of crosslinker Reference example

acrylateethylene glycol dimethacrylate 89, 101, 102

acrylamide

N,N8-phenylene bisacrylamide 69

vinylbenzenep-divinylbenzene 4

vinylheteroaromaticethylene glycol dimethacrylate 16, 58

silanetetraethoxysilane 23


Fig. 5. Scanning electron micrographs of molecularlyimprinted polymers. Samples were placed on aluminiumpegs and sputter coated with 15 nm of gold using a polaronE5150 coater. The images were obtained using an ISI 100ASEM at 25 kV. A: Particles obtained from a ground andsieved bulk trimethylolpropane trimethacrylate/methacrylic acid imprinted polymer. Magnification 500×.B: Beads of trimethylolpropane trimethacrylate/methacrylic acid imprinted polymer obtained by suspen-sion polymerisation in perfluorocarbon liquid. Magnifica-tion 200×. C: Thin imprinted ethylene glycol dimethacry-late/methacrylic acid polymer membrane prepared on aglass substrate. Magnification 30,000×.


In biological systems, water plays the dominant role assurrounding media. Therefore, mimics of natural bindingevents are most effectively demonstrated in aqueous sys-tems. However, the influence of water as porogen in theimprinting process or recognition media severely dimin-ishes the interaction energy of non-covalent interactions.With the techniques currently in use, where small, oftenmonofunctional, monomers are employed in the recogni-tion of the imprint antigen in the solution prior to polymeri-sation, and where ionic interactions, hydrogen bonds, andvan der Waals interactions come into play, the disruptingeffect of water intrinsically leads to very weak recognitioneffects. Also, the solubility of the necessary ingredients,such as crosslinkers and monomers, may be too low toform solid polymers. These properties of water have beendifficult to overcome and, until now, no real exampleswhere molecularly imprinted polymers have been pre-pared following the self-assembly approach in aqueous me-dia (other than metal coordination) have been reported.Nevertheless, several studies where recognition has beendemonstrated in aqueous systems have been pre-sented.11,36,63,85 In order to overcome the bond-breakingeffects of water, molecularly imprinted polymers have firstbeen prepared in organic media, where the interactions arestrong, and subsequently been used in aqueous environ-ments (Fig. 6). With this protocol, the designed sitesformed in the imprinting process, may lead to the con-certed action of participating functional groups to selec-tively recognise the ligands.

The recognition properties of molecularly imprintedpolymers in water are spectacularly changed in compari-son to an organic phase. For recognition matrices wherehydrogen bonding is the major factor for recognition, theligand binding is dramatically reduced. Even for a recog-nition system showing as high enantioseparation as shownfor the dipeptide N-Ac-Phe-Trp-OMe imprint (separationfactor, a = 17.8 in organic phase, Fig. 2), addition of only 8%water reduces the enantioseparation to a level where itbecomes barely detectable (a = 1.02 in acetonitrile/8% wa-ter).63 For recognition systems based on ionic interactions,the water effect is less pronounced, and in several systemspresented the recognition is maintained, albeit weakened,in aqueous phase (Fig. 6). Furthermore, for ionic interac-tions, the pH-dependence of the recognition is substantial86

(Ramstrom and Gustavsson. 1996. Unpublished data). Se-lective rebinding occurs primarily at a pH where the poly-mer functional groups and the analyte functional groupsare ionised indicating a simple ion-exchange relationship.

Another feature of aqueous phase analysis, using mo-lecularly imprinted polymers prepared in organic solvents,is the recognition mechanism. When analysed in organicmedia, hydrogen bonding, and ionic interactions have beenshown to be the major driving force in the complex forma-tion between the polymeric sites and the ligands. In aque-ous phase, however, a change in the binding regime oc-curs, and the principal interaction mechanisms appear tobe hydrophobic interactions in conjunction with ionic in-teractions (cf. Fig. 7).36 In this perspective, it is importantto distinguish between real aqueous phases containing wa-ter for the most part, and water-containing organic sol-vents. The role of water in mixtures of water/buffer withorganic solvents is different depending on the proportionof aqueous phase content. In systems comprising mostlyorganic solvent, where water/buffer is added as a modifier,the action of the aqueous phase is to control pH and reducethe binding strength of noncovalent interactions leading toless binding of the analytes to the polymer. On the otherhand, in ‘‘real’’ aqueous phases, the hydrophobic effect be-comes apparent and here, an increase in organic phasecontent will lead to diminished binding.

TECHNICAL CHALLENGESCapacity

One aspect to be considered in molecular imprinting isthe need for substantial amounts of imprint antigen, whichin itself may be expensive. Thus, normally 50–500 µmolimprint antigen per gram dry polymer is used. However,this can be compensated for, as up to 99% can be subse-quently recovered from the polymer by extraction and po-tentially reused after purification.37,87 Another drawbackthat has been pointed out is the low capacity of molecularlyimprinted polymers for the imprint antigen. For molecu-larly imprinted polymers prepared using the self-assemblyprotocol, a low imprinting yield is generally achieved.Thus, usually 10–15% of the loaded imprint antigen resultin efficient binding site formation. Following the recogni-tion mechanism used with most molecularly imprinted

Fig. 6. Chiral separation in aqueous media of a racemic mixture ofCbz-tyrosine by a molecularly imprinted polymer against the L-enantiomer.100 × 4.6 mm column, isocratic elution with 10 mM phosphate buffer, pH4.0/acetonitrile (75:25 v/v) at 0.5 ml/min; 10 µg racemate injected in 20 µlmobile phase.87


polymers, e.g., when methacrylic acid is used as functionalmonomer, this result is expected. Since the pre-arrangement complex is formed in equilibrium with vari-ous other aggregates in solution, such as carboxylic groupdimerisation between the methacrylic acids and imprintantigen complexation, an excess of functional monomer isnecessary for the formation of sufficient recognition sites.

One way to increase the capacity of the polymers wouldbe to make use of stronger interaction mechanisms be-tween the imprint antigen and the functional monomers.By going to the extreme and using covalent interactions, ahigher yield of imprinted recognition sites may beachieved.4 By this protocol, 70–90% of the imprint antigenmolecules result in available binding sites. Since the for-mation of the sites in molecularly imprinted polymers pre-pared by the preorganised approach is specified to a greatextent by the structure of the imprint antigen-monomeradducts, fewer nonspecific sites are formed. Also, thethree-dimensional polymeric morphology may be lessprone to rearrange upon removal of the imprint antigenposterior to polymerisation. Stronger interactions may thuslead to higher capacities of the polymers. In many applica-tions, though, it is desirable to maintain a noncovalent in-teraction mechanism, because of kinetic considerations.An intermarriage between the two protocols may, in someapplications, be a possible compromise.88

The capacity is not a consequence of the imprinting pro-tocol alone. Another factor that comes into play is thephysical strength exerted by the structure of thecrosslinker. Recent developments of polymeric systems,

using novel branched crosslinkers, have led to consider-able improvements. Optimised systems using trimethylol-propane trimethacrylate (TRIM) or pentaerythritol triacry-late (PETRA)-based polymers are capable of resolving,with base-line separation in chromatographic systems, be-tween 0.1–1 mg of racemic mixtures of peptides using onegram of dry polymer, which is towards the same capacityas found with other chiral stationary phases (Fig. 8).89

In this perspective it has to be mentioned that there is adifference between chromatographic separations and ap-plications where the recognition reaches equilibrium, suchas immunoassay-type analyses. In the former, the dynamicprocess of separation is dependent on ‘‘easily’’ accessibleimprinted sites with fast binding kinetics. Equilibrium as-says, on the other hand, utilise essentially all the sites inthe polymer and thus, the capacity may be regarded ashigher. However, when used for immunoassay-type appli-cations, only a small proportion of the total sites areutilised; those exhibiting the highest binding affinities.10

Heterogeneity

The inherent nature of the pre-arrangement process inself-assembly systems may give rise to the formation ofdifferent interaction modes between the imprint antigenand the various functional monomers prior to polymerisa-tion. Thus, the imprint antigen may interact with one orseveral functional monomers, with other imprint antigenmolecules, with solvent molecules or with crosslinker mol-ecules in the complicated liquid situation prior to polymeri-sation. Functional monomers may also form a multitude ofdifferent binding modes and interact with, e.g., other func-

Fig. 7. Phase diagram displaying the role of water in the binding ofligands by molecularly imprinted polymers prepared by self-assembly. Aswitch in binding regime occurs when going from systems containing alow degree of water, where water acts as a potent breaker of polar inter-actions, to systems containing mostly water, where the hydrophobic effectcomes into play.

Fig. 8. HPLC chromatogram showing separation of Boc-D,L-phenylalanine on Boc-L-phenylalanine imprinted polymer beads producedby suspension polymerisation using 1,2-dichloroethane as porogenic sol-vent. 100 × 4.6 mm column, isocratic elution with dichloromethane/aceticacid (99:1 v/v) at 0.1 ml/min; 400 µg racemate injected in 20 µl mobilephase.


tional monomers. In addition, the polymerisation process isnot instantaneous and will not preserve the pre-polymerisation situation. Rather, the kinetics of the poly-merisation process allows the reformation of the equilibriaduring the rigid polymer formation. It is, however, notclear how this mechanism operates, and it is highly desir-able that more studies on the polymerisation mechanism,in conjunction to molecular imprinting, are performed. Theoutcome of the process will inevitably be reflected in acorresponding number of different recognition sites in theformed polymer.10 This heterogeneity of the site distribu-tion, or ‘‘polyclonality,’’ is reflected by the estimated bind-ing strengths of the polymers. Scatchard analyses of thebinding strength distribution will result in non-linearcurves, ranging from sites with very high binding strengthall the way down to sites with scarcely any binding capa-bility at all (Fig. 9). In MIC applications, this heterogeneityresults in severe ‘‘tailing.’’90 The imprint antigen will, onadministration, be retained more or less strongly by thedifferent sites leading to an extended elution profile. Thisphenomenon is, however, not observed with ligands notselectively bound to the sites. A possible means to reduceor avoid the heterogeneity would therefore be great ofvalue in chromatographic analyses. Also, in order to in-crease the linearity range in immunoassay-type analyses,such as drug monitoring, a less polyclonal binding matrixis required.

Nonspecific InteractionsThe specificity, and the resolution efficiency that can be

achieved in chromatographic separations, are also affectedby the number and strength of the nonspecific interactionsthat create a ‘‘noise level’’ in the discrimination process. Inorder to sharpen the selectivities of the molecularly im-

printed polymers a means is required by which these non-specific binding modes are cancelled. Preliminary studiesof molecularly imprinted polymers, prepared with meth-acrylic acid as functional monomer, treated with diazo-methane prior to maximal rebinding of the antigen, re-vealed that the noise can be reduced in the polymer. Fur-ther development of efficient nonspecific site-blockers maylead to molecularly imprinted polymers with substantiallyhigher selectivities.

FUTURE PROSPECTSApplications

Molecular imprinting chromatography (MIC). Thereis significant potential for applications of imprinted poly-mers in molecular separations and isolation. Since mostchiral drugs on the market are administered as racemicmixtures, racemic resolution of drugs is a major potentialapplication.91,92 In light of recent guidelines from officialauthorities concerning drug preparation and administra-tion,93 forcing moves towards enantiomerically pure com-pounds, new and efficient techniques for enantiosepara-tions are needed.

One advantage of imprinting for separations is that itallows the preparation of tailor-made supports with pre-determined selectivity. Thus, the need to screen a range ofchiral stationary phases to find one that effects a givenseparation is obviated, and one knows that the imprintedenantiomer is retained longest on the support. A potentialdrawback is the fact that imprint material is required in theinitial step. If the latter is valuable, then the fact that theobtained polymers can be used more than one hundredtimes following extraction of the original imprint materialcan compensate for initial inconvenience. With optimisedrecovery of the print species, this drawback may be over-come. Another possibility to master this obstacle, would bethe use of a low-cost substitute compound, that closelyresembles the original target substance, in the imprintingstep, resulting in polymeric separation materials suffi-ciently selective for the desired compound.

To date, most applications in MIC have been performedon an analytical scale. The low capacity of these materialshas reduced the expectation for productive scale-up efforts.The format of molecularly imprinted matrices, though,makes the technique very attractive for scale-up. Thephysical nature of the materials obtained may easily beadjusted to suit preparative size columns and reactors, andthe physical and chemical endurance of the materials areattractive for use in harsh environments. In view of therecent developments in bead-technology, there exist greatopportunities for up-scaling the parameters of the separa-tion process successfully. Other approaches, such as insitu polymerisation and perfusion chromatography may beeffective as well.

Enantiopolishing

An interesting application area for molecularly imprintedpolymers may be in the selective removal of enantiomericimpurities. Normally, after asymmetric synthetic protocols,the enantiomeric yield is less than 100%. In such cases andfor other sources of optical impurities, such as racemisa-

Fig. 9. Scatchard plot for corticosterone binding to an anti-corticosterone polymer indicating the range of different binding sites in thematerial. B denotes amount bound ligand and F amount free ligand.27


tion reactions, the tailor-made properties of molecularlyimprinted polymers may be used. The strategy for such‘‘enantiopolishing’’ would be to run the sample over a chiralstationary phase prepared against the wanted enantiomer,leading to faster elution of any optical impurity. In thismanner, the collection of the retained enantiomer wouldlead to a sample of higher enantiomeric purity. An earlyexample of this strategy, although only on the analyticalscale, was the purification of an enantiomerically impuresample of phenylalanine anilide (5% D-enantiomer) using amolecularly imprinted polymer against the L-enantiomer. Aperfectly clear separation of the D-contamination from theL-enantiomer was obtained showing the usefulness of thisprinciple.94

Antibody receptor binding mimics. Molecularly im-printed polymers for use as artificial antibodies/receptorshave potential for rapid development, notably in immuno-assay-like diagnostic techniques. The physical and chemi-cal resistance of imprinted polymers together with the pos-sibility of sterilising the polymers, the stability of the rec-ognition properties, the low cost of preparation andobviation of the need for host animals are all obvious ad-vantages of imprinted polymers in comparison to naturalantibodies and receptors. In addition to immunoassays,screening applications may be a future prospect for im-printed polymers. In combination with combinatorialchemistry techniques, such polymers may be used as toolsfor the selection of lead molecules from large libraries ofcompounds. In particular, if a natural receptor desired forscreening is difficult to isolate, initial screening may be

accomplished by the use of molecularly imprinted receptormimics.

Catalytic/synthetic applications. This is an area inwhich considerable effort has been invested in recentyears. Although some intriguing results have been pre-sented, the area still awaits a real breakthrough. For indus-trial synthetic schemes, though, polymeric catalysts havethe potential to become a real complement to natural cata-lysts. The cost-efficiency with which such materials may beproduced is a great advantage for catalytic applications.

In addition to ‘‘real’’ catalysis, synthetic schemes wheremolecularly imprinted polymers are used to guide theproduct formation, either directly or indirectly, may be-come interesting. Condensation of specific substrates inimprinted polymer sites is one approach which may pro-duce very efficient syntheses. The use of molecularly im-printed polymers for the control or elimination of by-products, or for the shifting of unfavourable equilibria arealso plausible applications of these materials.

Biosensor like devices. The use of molecularly im-printed polymers in biosensor devices seems fairly close athand (Fig. 10). In the short term, effort should be devotedto obtaining imprinted structures that can be placed indirect contact with the transducer. The chances are goodthat biosensor-like devices can be obtained using imprintsthat are robust and which have binding specificities notfound with biological molecules. Furthermore, the devel-opment of molecularly imprinted polymers capable ofrecognising analytes carrying auxiliary markers, such as

Fig. 10. Biomimetic sensor. Left: Schematic representation of a biosensor. A recognition element, selective for a certain analyte, is connected to atransducer, which converts the recognition signal to an electronic signal. Right: Example where a molecularly imprinted polymer was used as recognitionelement. A polymer against the fluorescently labelled amino acid dansyl-phenylalanine was applied as a layer at the tip of a fibre-optic sensing device.44


fluorescence labels, similarly to the non-labelled specieswill evidently lead to a broadening of this area.

Controlled release matrices. Depending on the condi-tions, the binding affinities for the imprint antigens exhib-ited by the molecularly imprinted polymers can be reason-ably high. This renders such polymers potentially useful ascontrolled release matrices. If the binding strength of thecompounds may be sufficiently regulated in an on-off-manner, such that the binding can be reduced, either to-tally in one step or gradually, by means of simple externalinfluences such as pH, light, or heat, then polymers can bemade to release a certain compound at a certain positionand time. Thus, if the ligand bound is a drug compoundand the polymeric matrix is biocompatible, such matricesmay be employed to control the drug delivery in vivo.Other applications of this technique could be the con-trolled release of antibiotics in solutions sensitive to bacte-rial degradation. By the addition of targeting elements tosuch molecularly imprinted controlled-release matrices,control of the release position may also be achieved.

New Imprinted Materials

It is likely that a widening of the range of imprintedmaterials will occur. Although polymeric systems based onacrylates, vinylbenzenes, and silanes have been the mostsuccessful, the development of other types of polymericmaterials is highly desirable. Given the hydrophobic na-ture of many of the imprinted materials produced today, itis of great importance to develop materials that are morehydrophilic and easily compatible with aqueous systems.One option in this direction would be to make use of aminoacids as monomers in the imprinting process. With thisapproach, protein mimics more closely resembling thenatural macromolecules would be acquired.

Versatility. The potential versatility of the molecularimprinting technology is virtually unlimited. To date,though, the majority of imprint antigens have been smallmolecules such as monosaccharides, amino acids and drugcompounds. The obvious reason for this apparent restric-tion is the current preparation protocol to molecularly im-printed polymers. The high degree of crosslinking maylead to permanent entrapment of large molecules. The pur-suit of new, predetermined recognition materials for mac-romolecules such as proteins, polysaccharides, and poly-nucleotides, will certainly result in new materials suited forthese particular types of imprint antigens. Materials with alarger degree of accessibility to the sites for large mol-ecules, and more compatible with the hydrophilic nature ofthese antigens have to be produced. Following the tech-nique of surface imprinting, new core matrices may bedeveloped that are suitable for the directed attachment offunctional groups capable of binding the antigens. Also, theintense research efforts currently devoted to structuredthin layers and mono-layers offer an opportunity for thedesign of efficient macromolecular architectures. In thelonger term, these materials may even result in imprintedmaterials directed towards viruses and cells.95

Imprinting in Aqueous Systems

To make a real impact in biotechnology, high fidelityimprints that can perform well in aqueous environmentshave to be developed. Since water has a weakening effecton noncovalent interactions, molecularly imprinted poly-mers are normally prepared in organic phase where theseinteractions are stronger. However, imprinted polymersprepared in organic media are often suitable for use inaqueous media as stated above. The forces between thefunctional monomers and the imprint antigen are main-tained in organic media prior to polymerisation. After poly-mer formation, the recognition between the polymer andimprint antigen is stronger in organic than aqueous media.However, the coordinated actions of several, albeit weakinteractions in concerted may lead to sufficiently stronginteractions in aqueous systems.

For imprint antigens which are insoluble in organic sol-vents, imprinting can only be done in cosolvent mixtures,or, ideally, pure aqueous systems. Then, the forces be-tween the imprint antigen and monomers must be strongenough to be maintained during polymerisation. One wayto achieve this may be to employ multifunctional mono-mers/crosslinkers.8,13 If isolated interaction points were tobe employed individually in the presence of water, mostlikely no effect would be seen. Given the additive effect onbinding strength exerted e.g., by ion-pairs,96 the concertedaction of several interaction points between the monomerand the imprint antigen may be of adequate strength to bemaintained in water. Such additional and possibly syner-getic effects may be a useful means to produce well-designed recognition sites.

In this perspective, the use of the hydrophobic effectmay be a useful tool in the search for systems workingdirectly in water. Although hydrophobic forces are poten-tially more difficult to master, being less specific and lessdirectional, combinations of solvents may enable the con-trol of hydrophobicity so as to achieve strong binding. Incombination with other, more specific, interaction forcessuch systems may be successful. Such systems would bemore analogous to natural binding systems, where nonco-valent bonds are formed and maintained in a hydrophobicmicroenvironment in the protein binding site.

Yet another possible solution to this problem would be toutilise strong, covalent, bonds in the aqueous imprintingstep and subsequently use noncovalent interactions in theapplications of the material. In this way, the binding siteformation in water is secured by the strength of the cova-lent bonds in the imprinting step, whereas the dynamicsand versatility of the noncovalent interactions may be em-ployed during ligand rebinding. A potential problem withthis protocol is that the binding site is changed by thechemical modification of the site after cleavage of the im-print antigen-monomer adducts. There is thus a risk ofreducing the site selectivity.

Complexity

Until now, the attraction of molecular imprinting tech-nology has lain very much in the ease and simplicity of the


preparation process. As a consequence of the quest formore versatile materials with efficient recognition capabili-ties in aqueous environments, more complex systems willmost likely be developed. Combinations of covalent, non-covalent, and metal-coordination interactions may provideways to solve some of the obstacles encountered today.Furthermore, more complex monomer species, maintain-ing concerted functionalities (as already stated), will prob-ably be employed. Molecular imprinting using the self-assembly approach has until now mainly been based onionic interactions and hydrogen bonds. It is highly prob-able, and desirable, that other bond types will be used inthe technology. The more tools become available for de-signing imprinted materials, the better the resulting matri-ces may perform. We believe that when the technology hasdeveloped further, it will be possible to produce real tailor-made recognition matrices for any given imprint antigen,by judiciously choosing from a tool-box of different inter-action types and monomers. In combination with currentdevelopments in combinatorial chemistry, it is likely thatnew, previously unknown, binding elements will be addedto the tools presently at hand.

CONCLUSIONS

Molecular imprinting technology has seen rapidly accel-erated research activity during the last few years. In addi-tion to the use of imprinted materials for basic recognitionstudies, there has been enhanced activity in the search fornew and potentially useful applications. In conclusion, mo-lecular imprinting technology is likely to undergo furtherrapid growth in the near future. Already, some fruits of thetechnique are on the edge of commercialization and, withintensified efforts from different interested parties in thearea, new and commercially attractive applications willemerge.

ACKNOWLEDGMENTS

The authors wish to express their sincere gratitude toProfessor Klaus Mosbach for his interest and fruitful com-ments in the preparation of this work, and to Dr. Lars I.Andersson for valuable discussions.

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molecular imprinting technology: challenges and prospects for the future

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