Soluble molecularly imprinted nanoparticles were synthesised via iniferter
initiated polymerisation and separated by size via gel permeation
chromatography. Subsequent fractionation of these particles by affinity
chromatography allowed the separation of high affinity fractions from the
mixture of nanoparticles. Fractions selected this way possess affinity similar
to that of natural antibodies (Kd 6.6 × 10−8) M and were also able to
discriminate between related functional analogues of the templ

Chianella, Iva

Guerreiro, A.

Piletska, Elena V.

Piletsky, Sergey A.

Whitcombe, M. J.

Biosensors and Bioelectronics

English

Soluble molecularly imprinted nanoparticles were synthesised via iniferter initiated polymerisation and separated by size via gel permeation chromatography. Subsequent fractionation of these particles by affinity chromatography allowed the separation of high affinity fractions from the mixture of nanoparticles. Fractions selected this way possess affinity similar to that of natural antibodies (Kd 6.6 × 10−8) M and were also able to
discriminate between related functional analogues of the template

Guerreiro, Antonio R.

Whitcombe, Michael J.

Cranfield CERES

Biosensors and Bioelectronics, Volume 24, Issue 8, 15 April 2009, Pages 2740-2743Selection of Imprinted Nanoparticles by Affinity ChromatographyAntónio R. Guerreiro*, Iva Chianella, Elena Piletska, Michael J. Whitcombe, Sergey A.PiletskyCranfield Health, Cranfield University, MK43 0AL, UK* E-mail address: a.guerreiro@cranfield.ac.ukAbstractSoluble molecularly imprinted nanoparticles were synthesised via iniferter initiatedpolymerisation and separated by size via gel permeation chromatography. Subsequent fractionationof these particles by affinity chromatography allowed the separation of high affinity fractions fromthe mixture of nanoparticles. Fractions selected this way possess affinity similar to that of naturalantibodies (Kd 6.6  10-8) M and were also able to discriminate between related functionalanalogues of the template.Keywords: molecularly imprinted polymer; nanoparticle; affinity chromatography1. IntroductionThe development of synthetic materials which mimic biological systems by design or by functionhas become a hot topic in modern science (Mosbach et al., 1996, Zimmerman et al., 2003, Wulff etal., 2006). Among the typical representatives of synthetic receptors are molecularly imprintedpolymers (MIPs) which possess enormous potential for the development of a new generation ofstable biomimetic materials (Holthoff et al., 2007). However, the main drawback of this technologyis the formation of a diverse population of binding sites, ranging from low to high affinity, mainlydue to thermodynamic reasons (Wulff et al., 1977).Nanoparticles are practically always formed during the early stages of MIP synthesis, which isperformed using a relatively high percentage of cross-linkers (60 - 90%). Typically, followinginitiation and primary chain growth, the polymerization reaction proceeds through several stages(Figure 1). Small branched polymers are formed by monomer addition from solution (a). Highlybranched molecules (b) are then formed during the early stages by intra-molecular reaction ofgrowing polymer chains and by chain extension due to addition of new monomers or polymer fromsolution. As reaction progresses, larger chains are formed and more reactive groups are trappedinside the molecule, favouring intramolecular cross-linking. At this stage highly cross-linkedmacromolecular clusters with stable network structure (c) are formed (Li et al., 2004). If thereaction is allowed to proceed, then molecular clusters react with each other forming large globules(d). Continuing association process together with chain growth will lead to the formation ofinsoluble polymer (e). Termination of the reaction at stage (c) leads to the formation of solublenanoparticles. This has been achieved in the past by dilution of monomer mixture prior to gelation,using free radical initiators with thermal initiation (Wulff et al., 2006). At this stage howeverreaction progresses very quickly and precise control over the size of particles is difficult to achievewhen using UV initiation, which was used here to prepare the nanoparticles. Better control over thereaction kinetics can be attained using slower iniferter-mediated polymerization. However thefundamental advantage of using this form of living polymerization, as compared with free radicalpolymerization, lies in the possibility to subsequently re-initiate polymerization by exposure to UVradiation, where the polymer nanoparticles will act as a macroinitiator molecule. The terminatinggroups dissociate to form polymeric radicals (Peppas et al., 2004) which can attack vinyl groupsresulting in extra polymeric growth or covalent immobilization of the particle on a surfacecontaining these groups. This simple approach can be used to easily immobilize the nanoparticleson sensor surfaces or to induce sequential functionalization (e.g. signaling functionality) of theparticles, increasing the scope for practical applications of this type of materials. The nanoparticleapproach can offer a unique solution to the problems frequently encountered in MIP technologyassociated with the presence of low affinity binding sites. Each MIP microparticle (polymer bead orobtained by grinding a monolith) contains many binding sites, with a range of different affinities. Ifhowever we start to shrink particle size, then in theory we should be able to select the ones with“good” binding sites. For this type of material we can use affinity chromatography withimmobilised template to capture particles containing a greater number of relatively high affinitysites. Here we demonstrate the synthesis of MIP nanoparticles combined with affinitychromatography. The product of this process has affinity comparable to that of natural antibodies,whilst allowing for the removal of particles with low affinity.2. Materials and MethodsDue to its length, this section is presented as Supplementary Material.3. Results and Discussion3.1. Preparation of naoparticlesFor proof of concept we have chosen a model based on polymers imprinted with 2,4-diamino-6-methyl-1,3,5-triazine, common name acetoguanamine (see Supplementary Material). The affinityadsorbent for separation of high affinity nanoparticles was prepared using a polymerisable analogueof the template, 2,4-diamino-6-(methacryloyloxy)ethyl-1,3,5-triazine (DMET), see structures onFigure 3. Triazines were successfully imprinted before using similar compositions (Muldoon et al.,1997, Sergeyeva et al., 1999). Optimal results were obtained using a mixture of two cross-linkers:ethylene glycol dimethacrylate (EGDMA) and trimethylolpropane trimethacrylate (TRIM), whichprovided the right balance of flexibility/rigidity which is crucially important for the subsequentmolecular recognition. Control polymers were prepared in the absence of template. Thenanoparticles produced were characterized by size exclusion chromatography (SEC) as describedpreviously (Sun et al., 1998) calibrated with polystyrene standards. Under the present conditionspractically no polymer formation was detected during first 20 s of irradiation (data not shown), withgelation (assessed visually) occurring at around 250 s. Typical electron microscopy images of thematerials synthesized for different periods are presented in Figure 1. Maximum yield of polymerwith Mw 3-100 kDa was achieved just prior to gelation at 170 s irradiation. The yield of the requiredfraction was relatively low, approximately 3% (w/w) of the initial mass of monomers. Higher yieldswere obtained with dilution of the polymerization mixture prior to gelation as reported elswhere(Wulff et al., 2006) (up to 99 %), although the method relies on thermal polymerisation, andincreased temperature during synthesis can have adverse effects on the molecular recognition abilityof polymers as demonstrated previously (Piletsky et al., 2002). For that reason UV initiation wasused to ensure the formation of high affinity binding sites. Also, the use of high monomerconcentration (minimum amount of solvent that enabled template dilution) ensures the formation ofstable monomer-template complexes which in turn should result in high affinity imprints, asopposed to precipitation methods which require high dilution (Haupt, 2003). Subsequentfractionation of the nanoparticles (both imprinted and blank particles) by size was performed bySEC, and four fractions collected, with average apparent molecular weights (Mw) of 10-13 kDa, 60-70 kDa, 90-100 kDa and 120 > kDa.3.2. Presence of residual template.No amino residues were detected by the sensitive o-phthaldialdehyde (OPA) method (Paramas etal., 2006) in the purified materials, demonstrating complete removal of the template, which could bederivatised and detected via fluorescent emission at 440 nm. In the present system, template couldbe detected down to 9 × 10-8 M. Template leaching is a common problem associated with imprintedpolymers synthesized via bulk polymerization, and it is a considerable obstacle when attempting touse MIPs in systems with very low limits of detection (Olsen et al., 1999; Chassaing et al., 2004).3.3. Recognition properties of nanoparticlesThe affinity of polymer fractions was analysed by affinity chromatography on HPLC mode. Thetype of affinity material used (porous ethylene glycol dimethacrylate-co-DMET) has randompolymer branches, some of these will be terminated by a DMET molecule, acting as spacer armsbetween the polymer network and the affinity ligand. The highest retention time was found forparticles with apparent Mw of 90-100 kDa (15.41 ± 0.43 min), particles with 60-70 and 10-13 kDahad retention times 10.85 ± 0.07 and 9.78 ± 0.12 min respectively. Blank particles with apparent Mwof 90-100 kDa were retained for 7.01 ± 0.15 min. The same tests were carried out using a non-affinity phase prepared in the absence of the template analogue, where blank and imprintednanoparticles had the same retention time (3.93 ± 0.04 min) showing no variation in non-specificbinding. This also confirms that both imprinted and blank particles have the same hydrodynamicradius, due to the SEC selection step, so the difference in retention times on the affinity column canbe assigned to specific interactions with the immobilised template. Tests were performed intriplicate. As the fraction with 90-100 kDa demonstrated higher affinity it was used in subsequenttests, after purification by affinity chromatography (see Supplementary Material). Possibly smallerparticles do not have sufficient bulk to accommodate a stable three-dimensional structure of theimprints. This also seems to suggest that the high afinity binding sites are located on the periphery,or on the exterior polymer layers, as smaller particles have retention times which are closer to thatof blank particles. Larger nanoparticle fractions (Mw > 120kDa) had lower retention times than the90-100 kDa fraction. Possibly these are too bulky to interact properly with the affinity phase, and socannot be properly selected via affinity chromatography. For this reason larger fractions were notused on subsequent experiments. Figure 2 demonstrates the efficiency of the affinitychromatography in selecting a population of particles with high affinity binding sites. There is alarge fraction of particles with low affinity in the mixture after the SEC step as can be seen onFigure 2 on left side. When a high affinity fraction collected from the affinity column is injectedagain it is clear that the low affinity fractions are not present. For comparison, a chromatogram ofblank nanoparticles injected on the affinity column after affinity separation is also displayed. Thepeaks present at the beginning of the chromatograms (approx. from 1.5 to 3 min) can be assigned tothe presence of nanoparticle clusters due to mechanical entanglement of their three-dimensionalnetwork as discussed elsewhere (Li et al., 2004). These clusters tend to form in solution and as aconsequence, entangled particles might not have their binding sites accessible and so will not beretained.Further tests were performed with nanoparticles (size range 90-100 kDa) immobilised ontopoly(trimethylolpropane trimethacrylate) poly(TRIM) particles by UV irradiation (seeSupplementary Material). This approach exploits the living nature of the iniferter-preparednanoparticles, which allows easy immobilization of the polymer to surfaces containing vinylgroups. Immobilization phase was synthesized by polymerizing cross-linker for the minimumamount of time necessary to obtain a rigid material, but still with a large number of unreacteddouble bonds. Approximately 36 % of initial double bonds (when compared with liquid TRIM) arestill present in the poly(TRIM).As expected, after 5 minutes of UV irradiation of a solution of nanoparticles mixed with theimmobilization phase only 32.9 % of the initial amount of nanoparticles remained in solution,decreasing to 11.2 % after 8 minutes of UV exposure, further irradiation did not considerablydecrease the amount of nanoparticles in solution. In total, 0.35mg imprinted nanoparticles wereimmobilised per gram of TRIM polymer. The same procedure was repeated with non-imprintedmaterial with similar results (with 11.0 % remaining in solution).The composite material with immobilised nanoparticles offers the possibility of measuring theirbinding properties in solution using the double reciprocal method (Chianella et al., 2003). Thismethod was selected as the solvent used in the rebinding experiments was not entirely compatiblewith techniques such as dialysis, and unspecific adsorption of the template to membranes could alsoinfluence the results. Rebinding experiments were only carried out for the high affinity fraction (ca.56 % of the total fraction of particles with 90-100 kDa) as the remaining fractions had retentiontimes similar to that of the blank material. Binding experiments were carried out for imprinted andnon-imprinted nanoparticles. For comparison purposes the same tests were carried out for bulkimprinted polymer prepared with the same composition used to synthesise the imprintednanoparticles. Template concentration ranged from 8 nM to 1.5 mM. According to the doublereciprocal plots the dissociation constant (Kd) for imprinted material and the concentration ofbinding sites in the immobilised fraction (Bmax) were 6.6  10-8 M and 8.6  10-7 mol/g respectively.For the non-imprinted polymer, Kd and Bmax were 7.9  10-6 M and 1.0  10-5 mol/g respectively.For the imprinted polymer prepared by bulk polymerization Kd and Bmax were 14  10-6 M and 178 10-6 mol/g. The apparent density of binding sites corresponds to approximately 0.6 bindingsites/immobilized imprinted particle, although some binding sites might not be accessible due to theimmobilisation. The affinity of the immobilised imprinted nanoparticles is in line with that reportedfor monoclonal antibodies for atrazine: Kd of 3.87 × 10 -7 M (Grant et al., 1999) and 9.20 × 10 -9 M.(Kramer, 2002).For the specificity tests the immobilised nanoparticles were incubated with a solution of eithertemplate or structural analogues (atrazine, metribuzin and simazine) in acetonitrile. The amount ofeach compound adsorbed by the polymer is depicted in Figure 3. Whilst the blank nanoparticlesshow some preferential binding towards acetoguanamine, this is negligible when compared with theimprinted nanoparticles which have clearly higher affinity and specificity.4. ConclusionsIn conclusion, the products synthesized using this approach are a viable and robust alternative tonatural receptors, with comparable affinity and specificity, but with much higher stability and easeof preparation. The findings reported here might significantly increase the scope and chances forpractical applications of MIP technology.Acknowledgement. SP would like to acknowledge receiving Royal Society-Wolfson ResearchMerit Award. EP would like to acknowledge Leverhulme Trust for financial support.ReferencesChassaing, C., Stokes, J., Venn, R. F., Lanza, F., Sellergren, B., Holmberg, A., Berggren, B., 2004.Journal of Chromatography B 804 (1) 71-81.Chianella, I., Piletsky, S. A., Tothill, I. E., Chen, B., Turner, A. P. F., 2003. Biosensors &Bioelectronics 18 (2-3), 119-127.Grant, S. D., Porter, A. J., Harris, W. J., 1999. Journal of Agricultural and Food Chemistry 47 (1),340-345.Haupt, K., 2003. Analytical Chemistry 75 (17), 376A-383A.Holthoff, E. L. and Bright, F. V., 2007. Analytica Chimica Acta 594 (2), 147-161.Kramer, K., 2002. Environmental Science & Technology 36 (22), 4892-4898.Li, F. X., Lu, Z. F., Qian, H. T., Rui, J. M., Chen, S. N., Jiang, P., An, Y. L., Mi, H. F., 2004.Macromolecules 37 (3), 764-768.Mosbach, K. and Ramstrom, O., 1996. Bio-Technology 14 (2), 163-170.Muldoon, M. T. and Stanker, L. H., 1997. Analytical Chemistry 69 (5), 803-808.Olsen, J., Martin, P., Wilson, I., Jones, G. R., 1999. The Analyst 124, 467-471.Paramas, A. M. G., Barez, J. A. G., Marcos, C. C., Garcia-Villanova, R. J., Sanchez, J. S., 2006.Food Chemistry 95 (1), 148-156.Peppas, N. A. and Ward, J. H., 2004. Advanced Drug Delivery Reviews 56 (11), 1587-1597.Piletsky, S. A., Piletska, E. V., Karim, K., Freebairn, K. W., Legge, C. H., Turner, A. P. F., 2002.Macromolecules 35 (19), 7499-7504.Sergeyeva, T. A., Piletsky, S. A., Brovko, A. A., Slinchenko, E. A., Sergeeva, L. M., El'skaya, A.V., 1999. Analytica Chimica Acta 392 (2-3), 105-111.Sun, S. S., Zhang, C., Yang, Z. X., Dalton, L. R., Garner, S. M., Chen, A., Steier, W. H., 1998.Polymer 39 (20), 4977-4981.Wulff, G., Chong, B. O., Kolb, U., 2006. Angewandte Chemie-International Edition 45 (18), 2955-2958.Wulff, G., Grobeeinsler, R., Vesper, W., Sarhan, A., 1977. Makromolekulare Chemie-Macromolecular Chemistry and Physics 178 (10), 2817-2825.Zimmerman, S. C., Zharov, I., Wendland, M. S., Rakow, N. A., Suslick, K. S., 2003. Journal of theAmerican Chemical Society 125 (44), 13504-13518.Figure CaptionsFigure 1. On the left - schematic representation of chain-growth process, adapted from Li et al.,2004. On the right – images of polymer growth representing stages c to e on the scheme. c - TEMimage of nanoparticles formed by 170 s UV irradiation, magnification 340 k; d and e - SEM imageof polymer formed by aggregation of molecular clusters achieved during 180 and 250 s irradiationrespectively. For full details and polymer composition see Supplementary Material.a)c)b)d)e)Figure 2. Chromatograms for the blank and imprinted nanoparticles (fraction 90-100 kDa) on theaffinity column (A). The chromatograms for the blank and high affinity imprinted nanoparticles(fraction 90-100 kDa) after affinity purification and injected on the affinity column are depicted inFigure B, the first 25 minutes of these chromatograms are magnified and presented as an inset.024681012141618200 50 100 150Time (min)AUBlankMIP-0.10.40.91.41.92.42.93.43.94.40 5 10 15 20 25Time (min)AUBlankMIP A B01230 10 20Time (min)AU00.10.20.30.40.50.60.70.80.911.11.21.31.41.51.61.71.8acetoguanam ine atrazine metribuzin s im azinemmol/gFigure 3. Binding of the template (acetoguanamine) and structural analogues – atrazine, metribuzinand simazine to MIP (light gray bars) and blank (dark gray bars) nanoparticles immobilized on solidsupport. The structure of the polymerisable template analogue, (DMET) is depicted on the left.Error bars represent the standard deviation (n = 3). For these tests, solid phase with the immobilisednanoparticles (70 mg) was placed in 1.5 ml sealed vials with 1 ml solution (40 ng/ml) of thetemplate (acetoguanamine) or one of its analogues - atrazine, metribuzine or simazine inacetonitrile. Vials were incubated for 12 hours, and the concentration of either chemical thatremained in solution was measured by HPLC-MS. See Supplementary Material for full details.

Selection of imprinted nanoparticles by affinity chromatography

http://dspace.lib.cranfield.ac.uk/bitstream/1826/6919/1/Selection%20of%20Imprinted%20Nanoparticles%20by%20Affinity%20Chromatography-2009.pdf

Selection of imprinted nanoparticles by affinity chromatography

Abstract

Similar works

Full text

Available Versions

Cranfield CERES