100
5.4.3 Characterization of Binding Properties
It is widely accepted that binding behavior of MIPs relies on 3D specific nanocavities created during the course of polymerization because of the addition of
a template. After proper removal of the template, the molecular memory of the
template itself remains in those cavities, being complementary in functionality, shape and size. The non-covalent imprinting approach should not be understood as a method that provides identical imprinted sites throughout the polymer
matrix, since prepolymerization interactions between functional monomers and
the template are non- covalent. The strength of the established prepolymerization
complex will be responsible for the similarity of imprinted sites, and therefore,
strong monomer- template complexes will result in greater homogeneity of the
binding site.
The binding properties of MIPs are usually determined through an isotherm that
measures the concentration-dependent recognition behavior of a system (Shimizu
2005). Here, the analyte bound to the imprinted sites is plotted vs free analyte concentration in solution. The limit level is usually given as a function of the polymer
mass (μmol/mg), while free concentration is expressed in concentration units (mM
or even μM).
Experimental binding isotherms must subsequently conform to a non-linear
mathematical model to obtain the values of the different binding parameters, such
as the number of binding sites and binding affinity. The most commonly used models for MIP characterization are Langmuir, Bi-Langmuir, Freundlich-Langmuir and
Freundlich. The Langmuir isotherm is the simplest model and assumes a homogeneous distribution of the binding site, and therefore, bound analyte concentration
can be calculated according to a unique binding or association constant. Oppositely,
the other models are based on a heterogeneous distribution of the binding site,
assuming that there are two (Bi-Langmuir model) or more types of binding sites
(Freundlich and Freundlich-Langmuir models). The latter can be used for almost
any MIP, regardless of whether it shows a heterogeneous or almost homogeneous
distribution of the binding site, while the former is accurate within a limited concentration range.
The empirical data plotted on binding isotherms are obtained through binding
studies, which have been carried out mainly by batch experiments or frontal chromatography. In batch assays, a certain mass of a MIP is incubated in a solution having a known analyte concentration until equilibrium is reached. The MIP is then
removed from the solution and the remaining unbound free analyte concentration is
quantified. This assay should cover a wide range of analyte concentration in order
to visualize the saturation of the isotherm (Ansell 2015).
UV-Vis and fluorescence spectrophotometry with or without previous chromatographic separation has been successfully used by Cai and Gupta (2004) to perform
quantitative measurements of unbound analyte before and after incubation of MIPs
with a series of analyte concentrations. FTIR, NMR and Raman spectroscopies
A. Gómez-Caballero et al.
5.4.3 Characterization of Binding Properties
It is widely accepted that binding behavior of MIPs relies on 3D specific nanocavities created during the course of polymerization because of the addition of
a template. After proper removal of the template, the molecular memory of the
template itself remains in those cavities, being complementary in functionality, shape and size. The non-covalent imprinting approach should not be understood as a method that provides identical imprinted sites throughout the polymer
matrix, since prepolymerization interactions between functional monomers and
the template are non- covalent. The strength of the established prepolymerization
complex will be responsible for the similarity of imprinted sites, and therefore,
strong monomer- template complexes will result in greater homogeneity of the
binding site.
The binding properties of MIPs are usually determined through an isotherm that
measures the concentration-dependent recognition behavior of a system (Shimizu
2005). Here, the analyte bound to the imprinted sites is plotted vs free analyte concentration in solution. The limit level is usually given as a function of the polymer
mass (μmol/mg), while free concentration is expressed in concentration units (mM
or even μM).
Experimental binding isotherms must subsequently conform to a non-linear
mathematical model to obtain the values of the different binding parameters, such
as the number of binding sites and binding affinity. The most commonly used models for MIP characterization are Langmuir, Bi-Langmuir, Freundlich-Langmuir and
Freundlich. The Langmuir isotherm is the simplest model and assumes a homogeneous distribution of the binding site, and therefore, bound analyte concentration
can be calculated according to a unique binding or association constant. Oppositely,
the other models are based on a heterogeneous distribution of the binding site,
assuming that there are two (Bi-Langmuir model) or more types of binding sites
(Freundlich and Freundlich-Langmuir models). The latter can be used for almost
any MIP, regardless of whether it shows a heterogeneous or almost homogeneous
distribution of the binding site, while the former is accurate within a limited concentration range.
The empirical data plotted on binding isotherms are obtained through binding
studies, which have been carried out mainly by batch experiments or frontal chromatography. In batch assays, a certain mass of a MIP is incubated in a solution having a known analyte concentration until equilibrium is reached. The MIP is then
removed from the solution and the remaining unbound free analyte concentration is
quantified. This assay should cover a wide range of analyte concentration in order
to visualize the saturation of the isotherm (Ansell 2015).
UV-Vis and fluorescence spectrophotometry with or without previous chromatographic separation has been successfully used by Cai and Gupta (2004) to perform
quantitative measurements of unbound analyte before and after incubation of MIPs
with a series of analyte concentrations. FTIR, NMR and Raman spectroscopies
A. Gómez-Caballero et al.