Characterization of MIPs Using Heterogeneous Binding Models
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Characterization of MIPs Using Heterogeneous Binding Models Ken D. Shimizu Department of Chemistry and Biochemistry University of South Carolina Columbia, SC 29208, U.S.A. ABSTRACT New methods are presented for characterizing MIPs. These methods address the problems of quantitatively comparing the binding properties of different MIPs. Heterogeneous binding models were applied to MIPs based on an exponentially decaying distribution known as the Freundlich isotherm. The Freundlich isotherm was found to accurately model the binding isotherm of the majority of non-covalently imprinted MIPs. Using this model the experimental binding isotherm can be fit in log-log form to a linear equation from which the fitting parameters can be used to plot a quantitative affinity distribution which is a plot of the number of sites with respect to the binding constant of those sites. Comparison of MIPs using this methodology allowed for simpler and more accurate assessment of the binding properties than by previous methods such as the limiting slopes analysese of curved Scatchard plots INTRODUCTION Molecularly imprinted polymers (MIPs) are highly crosslinked polymers that are synthesized in the presence of a template molecule.1,2 Upon removal of the molecular template, a binding cavity is formed with affinity and selectivity for the original template molecule. MIPs compare favorably to other synthetic materials that can be tailored with recognition properties including synthetic molecular receptors and designer antibodies. For example, MIPs have high thermal and chemical stability, are easily and inexpensively synthesized, and are easily tailored with binding properties for almost any molecule of interest. Clearly, the binding properties of molecularly imprinted polymers (MIPs) are their most important characteristic. The comparison of the binding properties of MIPs, however, is complicated by the heterogeneity in MIPs. Unlike antibodies or enzymes that have a single type of binding site with high affinity and selectivity, MIPs contain a wide variation of binding sites that span the range in terms of binding affinity and selectivity. This binding site heterogeneity diminishes the utility of MIPs in chromatographic applications by leading to poor resolutions, highly concentration dependent selectivity, and severe peak asymmetry.3 Heterogeneity also complicates the quantification and comparison of the binding properties of MIPs. For example, a common method to characterize MIPs is using a Scatchard plot (Figure 1). A homogenous system would yield a straight line in a Scatchard plot. MIPs, however, typically have a curved Scatchard plot due to the heterogeneity of the underlying binding sites. The heterogeneity in MIP1 can be modeled by the limiting slopes method by two straight lines to the curve, yielding two sets of binding parameters corresponding to the high- and low-affinity sites. Complications quickly arise when using this analysis to compare two polymers. For example, the high-affinity sites of a second polymer (MIP2) were
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