Elsevier

Analytica Chimica Acta

Volume 584, Issue 1, 12 February 2007, Pages 112-121
Analytica Chimica Acta

Uniform molecularly imprinted microspheres and nanoparticles prepared by precipitation polymerization: The control of particle size suitable for different analytical applications

https://doi.org/10.1016/j.aca.2006.11.004Get rights and content

Abstract

Molecularly 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 μm 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, the final 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 of 130 nm to 2.4 μm, 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 was 20 times that of for (R)-propranolol. Compared 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.

Introduction

Molecularly imprinted polymers (MIPs) are tailor-made synthetic materials having selective molecular recognition capability [1], [2], [3], [4], [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 template is removed to afford binding sites complimentary to the template structure. A wide range of molecules can be used as templates to prepare MIPs with binding affinity and specificity comparable to biological antibodies [6], [7], [8]. Due to their favorable molecular recognition capability and stability, potential 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 of 5–100 μm. 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 than 50%). The irregularity of size and shape of such MIP particles also made sample handling difficult, and chromatography efficiency reduced. For new analytical applications, 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 performance 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 polymerization method [12], [13]. The imprinting reaction was carried out in a near-θ solvent (as a rule of thumb, the Hildebrand solubility parameter of a near-θ solvent should be 3–5 (MPa)0.5 away 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 generally 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], [19], [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 separation modules, whereas monodispersed MIP beads in the range of 1.5–3 μm may be used as new stationary phases in liquid chromatography systems to afford very fast separation. Despite the straightforward synthesis offered by the precipitation polymerization, for a given imprinting system it has been difficult to adjust and control the final 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 100 nm to 3 μm in diameter. The small MIP nanoparticles are ideal to use in the well established non-separation assay formats, for example using measurements based on fluorescence polarization–depolarization or fluorescence resonance energy transfer (FRET) techniques [23], whereas the 1.5–3 μm MIP microspheres may be more appropriate to use with new analytical 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 precipitation polymerization method. Using propranolol as a model template, we demonstrate that, as a major reaction component, the cross-linking monomer has a profound effect on the final 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 the 100 nm to 2.4 μm 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 electron microscopy (SEM) and photon correlation spectroscopy (PCS). As confirmed by radioligand binding analysis, all the MIP beads obtained maintained excellent imprinting effect, and can be readily employed to develop binding assays for complicated samples.

Section snippets

Materials

Divinylbenzene (DVB, technical grade, 55%, mixture of isomers) and trimethylolpropane trimethacrylate (TRIM, technical grade) were obtained from Aldrich (Dorset, UK). Prior to use, DVB was passed through an aluminum oxide column to remove the polymerization inhibitor. Acetic acid (glacial, 100%), acetonitrile (99.7%) and azobisisobutyronitrile (AIBN, 98%) used for polymer synthesis were purchased from Merck (Darmstadt, Germany). AIBN was re-crystallized from methanol before use. Methacrylic

Results and discussion

In 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 dimethacrylate (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

Conclusion

In this work we have developed a new method to gain precise control of molecularly imprinted nanoparticles and microspheres in the range of 100 nm to 2.5 μm. The change of particle size, while maintaining the excellent recognition property, was achieved by varying the ratio of two different cross-linking monomers in basically the same precipitation polymerization system. The combined use of two different cross-linking monomers in precipitation polymerization opened new possibilities of fine

Acknowledgements

We thank the European Commission for financial support to the NANOIMPRINT project (NMP4-CT-2005-516981), and Gunnel Karlsson (Chemical Center, Lund University) for the SEM measurements. KY is a recipient of the Scandinavia-Japan Sasakawa Foundation fellowship.

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