Uniform molecularly imprinted microspheres and nanoparticles prepared by precipitation

Analytica Chimica Acta584(2007)112–121Uniform molecularly imprinted microspheres and nanoparticlesprepared by precipitation polymerization:The control of particlesize suitable for different analytical applicationsKeiichi Yoshimatsu a,b,Kristina Reimhult c,Anatol Krozer c,Klaus Mosbach a,Koji Sode b,Lei Ye a,∗a Pure and Applied Biochemistry,Chemical Center,Lund University,Box124,S-22100Lund,Swedenb Department of Biotechnology,Tokyo University of Agriculture and Technology,2-14-16,Naka-cho Koganei,Tokyo184-8588,Japanc The Imego Institute,Arvid Hedvalls Backe4,S-41133G¨o teborg,SwedenReceived29June2006;received in revised form24October2006;accepted2November2006Available online10November2006AbstractMolecularly imprinted polymers(MIPs)are being increasingly used as selective adsorbents in different analytical applications.To satisfy the different application purposes,MIPs with well controlled physical forms in different size ranges are highly desirable.For examples,MIP nanoparticles are very suitable to be used to develop binding assays and for microfluidic separations,whereas MIP beads with diameter of 1.5–3␮m can be more appropriate to use in new analytical liquid chromatography systems.Previous studies have demonstrated that imprinted microspheres and nanoparticles can be synthesized using a simple precipitation polymerization method.Despite that the synthetic method is straightforward,thefinal particle size obtained has been difficult to adjust for a given template.In this work,we initiated to study new synthetic conditions to obtain MIP beads with controllable size in the nano-to micro-meter range,using racemic propranolol as a model template.Varying the composition of the cross-linking monomer allowed the particle size of the MIP beads to be altered in the range of130nm to2.4␮m,whereas the favorable binding property of the imprinted beads remained intact.The chiral recognition sites were further characterized with equilibrium binding analysis using tritium-labeled(S)-propranolol as a tracer.In general,the imprinted sites displayed a high chiral selectivity:the apparent affinity of the(S)-imprinted sites for(S)-propranolol was20times that of for(R)pared to previously reported irregular particles, the chiral selectivity of competitive radioligand binding assays developed from the present imprinted beads has been increased by six to seven folds in an optimized aqueous solvent.©2006Elsevier B.V.All rights reserved.Keywords:Molecular imprinting;Precipitation polymerization;Propranolol;Assay;Nanoparticles1.IntroductionMolecularly imprinted polymers(MIPs)are tailor-made synthetic materials having selective molecular recognition capability[1–5].Imprinted binding sites are generated by co-polymerization of functional monomer with cross-linking monomer in the presence of a template molecule.The functional monomer interacts with the template to form a stable complex during the cross-linking reaction.After polymerization,the tem-plate is removed to afford binding sites complimentary to the ∗Corresponding author.Tel.:+46462229560;fax:+46462224611.E-mail address:lei.ye@tbiokem.lth.se(L.Ye).template structure.A wide range of molecules can be used as templates to prepare MIPs with binding affinity and speci-ficity comparable to biological antibodies[6–8].Due to their favorable molecular recognition capability and stability,poten-tial applications of MIPs have been investigated in broad areas, such as ligand binding assays[9],liquid chromatography[10], solid-phase extraction[11],sensors[1],and catalytic chemical reactions[2].Traditionally,MIPs were synthesized as porous monolith, which after grinding and sieving,gave irregular particles with different sizes in the range of5–100␮m.Although this method allows easy preparation of small amount of MIPs,it is time-consuming and yields only moderate amount of useful MIPs (yield typically less than50%).The irregularity of size and shape0003-2670/$–see front matter©2006Elsevier B.V.All rights reserved. doi:10.1016/j.aca.2006.11.004K.Yoshimatsu et al./Analytica Chimica Acta584(2007)112–121113of such MIP particles also made sample handling difficult,and chromatography efficiency reduced.For new analytical applica-tions,the irregular particles are inferior to well defined polymer beads,especially in developing MIP-based assays,sensor arrays and separation modules.In addition to improving binding perfor-mance of MIPs,new physical formats of MIPs and more efficient synthetic methodologies were important research topics in the past years.Previously,we described a simple method to prepare molecularly imprinted polymer beads using a precipitation poly-merization method[12,13].The imprinting reaction was carried out in a near-θsolvent(as a rule of thumb,the Hildebrand solu-bility parameter of a near-θsolvent should be3–5(MPa)0.5away from that of a polymer),leading to cross-linked polymer beads containing specifically imprinted binding sites.The formation of polymer microspheres was achieved by entropic precipitation of nanogel particles and continuous capture of nascent oligomers [14].As no interfering reagent(i.e.surfactant or stabilizer)was used during polymer synthesis,the method turned out to be gen-erally applicable to a broad range of template structures,and purification of the imprinted polymer beads was easily achieved. MIP nanoparticles and microspheres prepared by precipitation polymerization have been used in ligand binding assays[15,16], liquid chromatography[17],capillary electrochromatography [18–20],solid-phase extraction[21]and chemical sensing[22].To satisfy different analytical applications,MIPs with well controlled physical forms in different size ranges are highly desirable.For examples,MIP nanoparticles are very suitable to use in homogeneous binding assays and in microfluidic sep-aration modules,whereas monodispersed MIP beads in the range of1.5–3␮m may be used as new stationary phases in liquid chromatography systems to afford very fast sepa-ration.Despite the straightforward synthesis offered by the precipitation polymerization,for a given imprinting system it has been difficult to adjust and control thefinal particle size without deteriorating the imprinting effect.In this work,we intended to study new precipitation polymerization conditions to obtain MIP beads with controllable size in the range of 100nm to3␮m in diameter.The small MIP nanoparticles are ideal to use in the well established non-separation assay for-mats,for example using measurements based onfluorescence polarization–depolarization orfluorescence resonance energy transfer(FRET)techniques[23],whereas the1.5–3␮m MIP microspheres may be more appropriate to use with new analyt-ical chromatography instruments(e.g.capillary LC and UPLC) to provide very fast analytical separation[24].In this work,we started to investigate the precise control of the size of MIP beads that can be synthesized by the precip-itation polymerization ing propranolol as a model template,we demonstrate that,as a major reaction component, the cross-linking monomer has a profound effect on thefinal particle size and polymer product yield.Varying the ratio of two different cross-linkers used,we were able to synthesize monodisperse MIP beads with different sizes in the100nm to 2.4␮m range.The polymer beads obtained were characterized by elemental analysis,Fourier transform infrared spectroscopy (FT-IR)to study the conversion of the different monomers.The particle size and morphology were analyzed using scanning elec-tron microscopy(SEM)and photon correlation spectroscopy(PCS).As confirmed by radioligand binding analysis,all the MIPbeads obtained maintained excellent imprinting effect,and canbe readily employed to develop binding assays for complicatedsamples.2.Experimental2.1.MaterialsDivinylbenzene(DVB,technical grade,55%,mixture of iso-mers)and trimethylolpropane trimethacrylate(TRIM,technicalgrade)were obtained from Aldrich(Dorset,UK).Prior to use,DVB was passed through an aluminum oxide column to removethe polymerization inhibitor.Acetic acid(glacial,100%),ace-tonitrile(99.7%)and azobisisobutyronitrile(AIBN,98%)usedfor polymer synthesis were purchased from Merck(Darmstadt,Germany).AIBN was re-crystallized from methanol before use.Methacrylic acid(MAA,98.5%)was purchased from ACROS(Geel,Belgium)and used as received.(R,S)-Propranololhydrochloride(99%),(S)-propranolol hydrochloride(99%)and(R)-propranolol hydrochloride(99%)supplied by Fluka(Dorset,UK)were converted into free base form before use.(S)-[4-3H]-Propranolol(specific activity555GBq mmol−1,66.7␮M solution in ethanol)was purchased from NEN Life Science Prod-ucts Inc.(Boston,MA).Scintillation liquid,Ecoscint A wasfrom National Diagnostics(Atlanta,GA).Other solvents wereof analytical grade.2.2.ApparatusScanning electron microscopy(SEM)imaging was carriedout on a JEOL JSM-6700F Field Emission Scanning ElectronMicroscope(Tokyo,Japan)unless otherwise stated.Polymermicrospheres were sputter coated with gold prior to the SEMmeasurement.Photon correlation spectroscopy measurementwas performed on a Zetasizer Nano ZS instrument equipped witha software package DTS Ver.4.10(Malvern Instruments Ltd.,Worcestershire,UK).Polymer particles(2mg)were mixed withacetonitrile(1mL),sonicated in a benchtop ultrasonic cleanerfor20min until no particle aggregate could be observed.Thecolloidal sample was diluted with acetonitrile to afinal concen-tration of20␮g mL−1prior to particle size measurement.Thehydrodynamic size of the particles was measured in acetonitrileat25◦C.The size statistics graph was plotted from the resultsoffive measurements for each sample.2.3.Polymer synthesesMolecularly imprinted microspheres and nanoparticles weresynthesized using precipitation polymerization under theconditions described in Table1.The template molecule,(R,S)-propranolol was dissolved in40mL of acetonitrile ina150mm×25mm borosilicate glass tube equipped with ascrew cap.The functional monomer(MAA),the cross-linkingmonomer(DVB or TRIM)and the initiator(AIBN)were then114K.Yoshimatsu et al./Analytica Chimica Acta584(2007)112–121 Table1Preparation of(R,S)-propranolol-imprinted polymer microspheresPolymer Template(mmol)MAA(mmol)DVB(mmol)TRIM(mmol)Agitation Size(␮m)a Yield(%)C mip50(mg mL−1)bCross-reactivity(%)cmipD1d0.53 1.31 5.250No0.6–3.1330.4 5.2 mipD20.53 1.31 5.250Yes 2.4330.4 4.2 mipT20.53 1.310 2.02No0.13910.7 4.2 mipDT10.53 1.31 1.31 1.52Yes 1.277 1.4 4.7 mipDT20.53 1.31 2.63 1.01Yes 1.8560.8 4.7 mipDT30.53 1.31 3.940.51Yes 1.8400.5 3.0a Average diameter measured by SEM.b The concentration of polymer particles that bound50%of the radioligand.c The cross-reactivity of the assay towards(R)-propranolol,defined as the IC50of(S)-propranolol divided by the IC50of(R)-propranolol.d The corresponding non-imprinted control polymer is named as refT1,etc.added.The solution was purged with a gentleflow of Ar for5min and sealed under Ar.Polymerization was carried out byinserting the borosilicate glass tube in a water bath pre-set to60◦C for24h.When agitation was used,the borosilicate glasstube wasfixed horizontally in a Stovall HO-10HybridizationOven(Greensboro,NC,USA),and rotated at a speed of20rpm.The temperature was ramped from20◦C to60◦C within20min,thereafter kept for24h.After polymerization,particles werecollected by centrifugation.The template was removed by batch-mode solvent extraction with methanol containing10%aceticacid(v/v),until no template could be detected from the wash-ing solvent by spectrometric measurement.Polymer particleswerefinally washed with acetone and dried in a vacuum cham-ber.Non-imprinted reference polymers were synthesized underidentical conditions except for omission of the template(R,S)-propranolol.2.4.Radioligand binding analysis2.4.1.Saturation experimentIn a series of polypropylene microcentrifuge tubes,increas-ing amounts of polymer particles were suspended in a mixtureof25mM citrate buffer(pH6.0):acetonitrile(50:50,v/v).Afteraddition of(S)-[4-3H]-propranolol(246fmol),the mixture wasincubated at room temperature overnight.A rocking table wasused to provide gentle mixing.After the incubation,sam-ples were centrifugated at14000rpm for10min.Supernatant(500␮L)was taken from each microcentrifuge tube and mixedwith10mL of scintillation liquid(Ecoscint A),from whichthe radioactivity was measured using a model2119Rackbeta ␤-radiation counter from LKB Wallac(Sollentuna,Sweden). The amount of labeled(S)-propranolol bound to polymer par-ticles was calculated by subtraction of the free fraction fromthe total amount added.Data are mean values of duplicatemeasurements.2.4.2.Displacement experimentIn a series of polypropylene microcentrifuge tubes,afixed amount of polymer particles and(S)-[4-3H]-propranolol(246fmol)were mixed in a mixture of25mM citrate buffer(pH6.0):acetonitrile(50:50,v/v).To the tubes were added increas-ing amounts of(R)-and(S)-propranolol dissolved in the samesolvent.Afterwards,the samples were incubated and processed in the same way as in the saturation experiment.Data are mean values of triplicate measurements.3.Results and discussionIn a previous study,Andersson used(R,S)-propranolol as template to prepare imprinted polymer monolith.MAA was used as a functional monomer and ethylene glycol dimethacry-late(EDMA)as a cross-linker.The polymer was synthesized using toluene as a porogenic solvent.Irregular particles were obtained by grinding the polymer monolith.Despite that the template used was a racemate mixture,the imprinted polymer particles displayed chiral selective response in a competitive radioligand binding assay when tritium-labeled(S)-propranolol was used as a tracer[25].In aqueous buffer the cross-reactivity of(R)-propranolol was approximately35%.The results sug-gested that the imprinted binding sites were isolated,allowing selective probing of the(S)-propranolol-imprinted cavities with the labeled tracer.In this work,(R,S)-propranolol was used as template because of its relatively low cost.The imprinted bind-ing sites were studied by radioligand binding analysis using tritium-labeled(S)-propranolol as a tracer.Considering future applications,all the binding experiments were carried out in an aqueous buffer modified with a polar organic solvent.The selected solvent composition has been optimized in a previous study on(S)-propranolol-imprinted microspheres to minimize non-specific adsorption[16].3.1.Controlling particle size and size distribution of propranolol-imprinted polymer beadsIn previous studies,we found that when DVB was used as cross-linker,scintillation microspheres obtained displayed bind-ing affinity and specificity for(S)-propranolol superior to that prepared in toluene using TRIM as cross-linker[15,16].This was presumably due to that DVB provided additional␲–␲interac-tion with the aromatic moiety of(S)-propranolol in acetonitrile during the imprinting reaction.Although the imprinted micro-spheres had excellent binding performance,they had a relatively broad size distribution in the range of0.6–2␮m[16].In the present study,reducing the cross-linking density using the tech-nical DVB-55resulted in similar particle morphology and sizeK.Yoshimatsu et al./Analytica Chimica Acta 584(2007)112–121115distribution (mipD1,Fig.1a).The overall yield of the polymer microspheres (as calculated from the total amount of monomer added)was relatively low,around 30–40%regardless of the dif-ferent cross-linking densities.For the preparation of DVB-based microspheres,it has been found that appropriate agitation dur-ing the precipitation polymerization can lead to monodisperse beads [26].Using DVB as cross-linker and a mixture of acetoni-trile and toluene as solvent,Wang et al.adopted similar agitation in precipitation polymerization to achieve monodisperse beads that were successfully imprinted against theophylline [17].The use of the special solvent composition resulted in larger polymer beads (5␮m),which are appropriate to use as stationary phases in HPLC but not ideal for binding assays.To develop new affinity materials for high speed HPLC separation,we are more inter-ested in MIP beads with diameter in the range of 1.5–3␮ing neat acetonitrile as solvent and applying a gentlerotationFig.1.Scanning electron micrographs of mipD1(a),mipD2(b and c),mipT2(d and e),refT2(f),mipDT1(g),mipDT2(h),mipDT3(i)and refDT1(j).The scale bar corresponds to 10␮m in (a)and (b);1␮m in (c),(d),and (f–j);100nm in (e).All images were obtained with a JEOL JSM-6700F microscope except for (a),which was obtained with an earlier JEOL JSM-840A microscope.116K.Yoshimatsu et al./Analytica Chimica Acta 584(2007)112–121Fig.1(Continued ).to the reaction vessel,we indeed obtained very uniform poly-mer microspheres with an average diameter of 2.4␮m (mipD2,Fig.1b and c).Unlike the poly(DVB-co-MAA)microspheres,the use of TRIM as cross-linker resulted in much smaller MIP beads with diameter of around 100–300nm (Fig.1d and e),with approx-imately 90%yield.When TRIM was used as cross-linker,imprinted uniform nanospheres could be synthesized without using agitation during the precipitation polymerization.Interest-ingly,the size of the non-imprinted reference particles (refT2,Fig.1f)in the dry state was about two folds of the imprinted particles (mipT2),suggesting that the template compound had an important influence on the particle growth during the pre-cipitation polymerization.This may not be surprising given that the functional monomer MAA existed in different forms in the two reaction systems.In the absence of template,MAA can form hydrogen-bonded dimers in the non-imprinted system.The pre-polymerization solution contains both free MAA and MAA dimers.In the imprinted system,there is an additional molecular interaction between MAA and propranolol,which might somehow affect the growth of the cross-linked polymer nuclei to result in smaller polymer beads.The effect of template feeding on the final particle size will be studied in our further investigation.It order to study the particle sizes of the poly(TRIM-co-MAA)nanoparticles in solution,mipT2and refT2were re-suspended in acetonitrile,and characterized with photon cor-relation spectroscopy (PCS).The PCS measurements provided valuable information about the hydrodynamic radius of the col-loidal particles.Fig.2shows the different size distribution of the imprinted and the non-imprinted reference polymer beads.Clearly,the MIP beads are about half of the size of the reference pared to the SEM images in Fig.1d–f,the size of the poly(TRIM-co-MAA)nanoparticles was found to increase by approximately 50%after being suspended in acetonitrile.We should point out that,in accordance with the SEM images in Fig.1d and e,the PCS results suggest that the imprinted poly-mer mipT2has a very narrow particle size distribution.In fact the size spread of mipT2is as narrow as (or narrower than)that of standard latex particles routinely used for instrument calibration.Depending on the cross-linkers used,the propranolol-imprinted polymer beads had drastically different e of DVB cross-linker resulted in large particles (2.4␮m)with rela-tively low yield,whereas TRIM gave sub-micron beads (130nm)with high polymer yield.To explore if combined use of DVB and TRIM will give satisfactory imprinted beads,we prepared a series of propranolol-imprinted polymers containing different proportion of the two cross-linkers (Table 1),of which the totalK.Yoshimatsu et al./Analytica Chimica Acta584(2007)112–121117Fig.2.Particle size distribution of mipT2(a)and refT2(b)measured by photon correlation spectroscopy.weight of the cross-linkers was kept constant.As we expected, the average particle size of the imprinted poly(DVB-co-TRIM-co-MAA)microspheres had an intermediate size in the range of1.2–1.8␮m.Although there was not a simple linear corre-lation between particle size and the amount of DVB used,the size of the microspheres indeed increased with the fraction of the DVB cross-linker(Fig.1g–i).When a TRIM/DVB ratio of75/25(w/w)was used,the imprinted polymer(mipDT1) contained a small amount of secondary particles with diame-ter of around130nm(Fig.1g).The amount of the secondary particles was estimated to be less than2%,based on gravi-metric measurement following several sedimentation steps to Table2Elemental microanalysis of the cross-linked polymer beadsPolymer Experimental value(%)Theoretical value(%)C O C OmipD286.0 6.186.5 5.1refD285.6 5.986.5 5.1mipT262.629.162.828.8refT262.129.662.828.8mipDT169.321.968.722.9mipDT274.017.174.617.0mipDT378.213.780.511.0remove the secondary particles.It is interesting to note that under the same synthetic condition,the non-imprinted refer-ence polymer refDT1contained only one type of microspheres (Fig.1j),suggesting again that the template propranolol had a profound influence on particle nucleation and growth during the precipitation polymerization.3.2.Elemental analysis:carboxyl content in cross-linked polymer beadsThe carbon and oxygen content of mipD2and refD2were obtained by elemental microanalysis(Table2).The oxygen content can be used to estimate the actual ratio of DVB and MAA incorporated in the cross-linked particles.As theexper-Fig.3.FT-IR spectra of polymers mipD2(a),mipDT3(b),mipDT2(c),mipDT1 (d)and mipT2(e).118K.Yoshimatsu et al./Analytica Chimica Acta 584(2007)112–121Fig.4.FT-IR spectra of polymers mipD2and refD2(a);mipT2and refT2(b);mipDT1and refDT1(c).imental oxygen content for both mipD2and refD2was 18%higher than the theoretical value,the final carboxyl functional-ity in the polymer beads is obviously higher than what would be expected from the monomer composition.The actual molar ratio of DVB and MAA that are incorporated into the cross-linked polymer beads is therefore 3.3:1,which is 18%lower than the theoretical value of 4:1.The apparently low effi-ciency of DVB incorporation into cross-linked particles may explain the relatively low yield for both the imprinted and the non-imprinted poly(MAA-co-DVB)beads [26].For poly-mers containing TRIM cross-linker,the yield of the cross-linked microspheres increases when more TRIM is incorporated into polymer particles (Table 1),which is in agreement with the increased oxygen content from mipDT3to mipDT2to mipDT1(Table 2).For polymers prepared using both TRIM and DVB,the com-patibility between the two cross-linkers is ideally judged from their reactivity ratios (r 1and r 2)[29].However,the values of r 1and r 2for co-polymerization of TRIM and DVB are not available from the literature.If we suppose the reactivity ratios between TRIM and DVB are similar to that between methyl methacry-late and styrene (r 1≈r 2≈0.5)[30],the product of the reactivity ratios (r 1r 2≈0.25)implies that co-polymerization of the two cross-linkers would result in more or less alternating TRIM and DVB units along the polymer chain,at least at the early poly-merization stage.In such a case,one may expect that the core of the polymer beads would contain evenly distributed TRIM and DVB units,and the outer layer of the beads would con-tain preferentially the more abundant cross-linker added in the pre-polymerization mixture.3.3.FT-IR analysis:investigation of self-association of carboxyl groups in imprinted and non-imprinted polymer beadsThe FT-IR spectra of the imprinted poly(MAA-co-DVB),poly(MAA-co-TRIM)and poly(MAA-co-DVB-co-TRIM)beads are shown in Fig.3.For all the samples,characteristic peaks at around 1700cm −1(C O stretch of carboxylic acid)and 3440cm −1(OH stretch)were observed.For poly(MAA-co-DVB),three bands at 831cm −1,798cm −1and 708cm −1corresponding to the out-of-plane bending of the aromatic C HK.Yoshimatsu et al./Analytica Chimica Acta584(2007)112–121119Fig.5.Uptake of[3H]-(S)-propranolol(246pM)in citrate buffer(25mM,pH6.0)/acetonitrile(50/50,v/v)with increasing amount of imprinted and non-imprinted polymer beads:(a)mipD1( ),refD1( ),mipD2( )and refD2( );(b)mipT2( )and refT2( );(c)mipDT1( ),refDT1( ),mipDT2( ),refDT2( ),mipDT3 (᭹)and refDT3( ).are clearly seen(Fig.3a).Symmetric and asymmetric ester C O stretch bands at1264cm−1and1154cm−1were observed for poly(MAA-co-TRIM)(Fig.3e).For polymer beads pre-pared using simultaneously the two different cross-linkers (mipDT1,mipDT2and mipDT3),IR bands characteristic for both DVB and TRIM were observed.The relative intensity of the characteristic bands is qualitatively in agreement with the ratio between the two cross-linkers used(Fig.3b–d).For imprinted polymers prepared using MAA as a functional monomer,it is possible that some carboxylic acids form dimers during the imprinting reaction.This means that even though the final imprinted and non-imprinted reference polymers may con-tain the same level of functional groups,more carboxylic acids in non-imprinted reference polymers may exist as hydrogen bonded dimers,so that the amount of“free”carboxyl groups in non-imprinted reference polymers becomes lower than the cor-responding imprinted polymers.The status of carboxylic acids in the imprinted and non-imprinted poly(MAA-co-DVB)beads can be studied by focusing on IR bands around1735cm−1and 1707cm−1,which can be assigned to the C O stretch signal of the“free”and the hydrogen-bonded carboxylic acid dimers, respectively[27,28].As shown in Fig.4a,for both mipD2and refD2,substantial amount of carboxylic acid exists as hydrogen-bonded dimers.In fact,the fraction of“free”carboxylic acid in refD2is marginally higher than in mipD2.For all the polymers containing TRIM cross-linker,the situation became more com-plicated because the C O stretch signal of the ester overlapped with that of the“free”carboxylic acid.In general,no appreciable120K.Yoshimatsu et al./Analytica Chimica Acta584(2007)112–121band corresponding to the acid dimer can be easily distinguished (Fig.4b and c).3.4.Specific propranolol binding to the imprinted polymer beads in aqueous bufferResults of elemental analysis and IR spectroscopy suggest that the carboxyl contents in the imprinted and non-imprinted reference polymer beads are approximately the same.For more, the fractions of the“free”carboxyl groups in the imprinted and non-imprinted polymer systems are also similar.Based on this,specific binding provided by the imprinted sites can be estimated by measuring the difference of propranolol uptake between the imprinted and non-imprinted beads.When analyzed by radioligand binding experiments in citrate buffer:acetonitrile, all the MIP beads showed much higher propranolol binding than the corresponding non-imprinted control beads.In fact,the non-specific adsorption of propranolol,as measured by the pro-pranolol uptake with the non-imprinted beads,was negligible (Fig.5).For the DVB-based polymers,regardless of the differ-ent particle size distribution observed for mipD1and mipD2, the amount of the MIP beads needed to bind50%of the radioli-gand(C mip50)was approximately the same(0.4mg)(Fig.5a), indicating that the gentle agitation used during the precipitation polymerization did not affect target binding properties of the MIP microspheres.Although refD2has at least the samelevel Fig.6.Displacement of radioligand binding to0.5mg of:(a)mipD1(square)and mipD2(triangle)with increasing amount of(R)-propranolol(open)and(S)-propranolol(filled);(b)mipT2with increasing amount of(R)-propranolol( )and(S)-propranolol(᭹);(c)mipDT1with increasing amount of(R)-propranolol( ) and(S)-propranolol(᭹).。