101
provide relevant complementary information about the binding interactions that
these spectrophotometric techniques do not provide (Skogsberg et al. 2007; Bompart
et al. 2009).
Static binding studies can also be performed by radioligand binding assays, in
which the polymer is incubated with the template analyte and its radiolabeled variant and free radiolabeled analyte is quantified by scintillation counting (Paniagua
González et al. 2006).
The characterization of the binding properties by chromatographic assays is
commonly developed by zonal and frontal chromatography. Zonal chromatography
has been applied mainly for the evaluation of selectivity. For this approach, MIP is
packed in a chromatography column and the analyte and structural analogues are
injected into the system. Longer retention of the analyte compared to analogues
means the polymer’s preference for the target analyte.
In frontal chromatography, the analyte is dissolved in the mobile phase and flows
continuously through the packed MIP column. The analyte binds to the binding site
of the MIP and when it reaches saturation, the analyte elutes from the column. As a
result, a sigmoidal chromatogram is obtained where the inflection point reflects the
retention volume (V R ). Here, the concentration of the analyte in the mobile phase
corresponds to the free concentration of the analyte (F), while the bound concentration (B) is calculated from Eq. 5.1:
B mol g
F V
m
R
m
/
(
)=
(5.1)
where m is the mass of polymer packed in the column. The experimental data
obtained by this way give binding isotherms that are fitted to a mathematical model
in order to elucidate the binding mechanism (Gutierrez-Climente et al. 2016).
Isothermal titration calorimetry (ITC) has proven adequate to study thermodynamics of slow binding processes in imprinted materials (Kirchner et al. 2002;
Salian and Byrne 2013). It measures the heat produced over time when the target
analyte is added to the imprinted polymer. When the measured heat is plotted against
added template/polymer molar ratio, a decreasing profile is observed, which allows
to determine binding enthalpy and, consequently, to assess the efficiency of the
imprinting process (Weber et al. 2002).
Apparent affinity constants can also be calculated with other techniques, such as
surface plasmon resonance (SPR) and QCM. SPR is an optical method that measures the refractive index of a thin layer of a material adsorbed on a metal. When
applied for MIP characterization, the NPs are immobilized on the surface of the
metal film while the ligand in the mobile phase runs along the flow cell. If binding
occurs, the refractive index changes leads to a change in SPR angle. The apparent
association and dissociation constants can be derived from the change rates of SPR
signals (Taniwaki et al. 2003; Li and Husson 2006; Lépinay et al. 2012).
The QCM is a mass-sensitive piezoelectric device capable of detecting nanogram changes in mass by measuring the change in frequency of a quartz crystal
5 Plastic Receptors Developed by Imprinting Technology as Smart Polymers Imitating…
provide relevant complementary information about the binding interactions that
these spectrophotometric techniques do not provide (Skogsberg et al. 2007; Bompart
et al. 2009).
Static binding studies can also be performed by radioligand binding assays, in
which the polymer is incubated with the template analyte and its radiolabeled variant and free radiolabeled analyte is quantified by scintillation counting (Paniagua
González et al. 2006).
The characterization of the binding properties by chromatographic assays is
commonly developed by zonal and frontal chromatography. Zonal chromatography
has been applied mainly for the evaluation of selectivity. For this approach, MIP is
packed in a chromatography column and the analyte and structural analogues are
injected into the system. Longer retention of the analyte compared to analogues
means the polymer’s preference for the target analyte.
In frontal chromatography, the analyte is dissolved in the mobile phase and flows
continuously through the packed MIP column. The analyte binds to the binding site
of the MIP and when it reaches saturation, the analyte elutes from the column. As a
result, a sigmoidal chromatogram is obtained where the inflection point reflects the
retention volume (V R ). Here, the concentration of the analyte in the mobile phase
corresponds to the free concentration of the analyte (F), while the bound concentration (B) is calculated from Eq. 5.1:
B mol g
F V
m
R
m
/
(
)=
(5.1)
where m is the mass of polymer packed in the column. The experimental data
obtained by this way give binding isotherms that are fitted to a mathematical model
in order to elucidate the binding mechanism (Gutierrez-Climente et al. 2016).
Isothermal titration calorimetry (ITC) has proven adequate to study thermodynamics of slow binding processes in imprinted materials (Kirchner et al. 2002;
Salian and Byrne 2013). It measures the heat produced over time when the target
analyte is added to the imprinted polymer. When the measured heat is plotted against
added template/polymer molar ratio, a decreasing profile is observed, which allows
to determine binding enthalpy and, consequently, to assess the efficiency of the
imprinting process (Weber et al. 2002).
Apparent affinity constants can also be calculated with other techniques, such as
surface plasmon resonance (SPR) and QCM. SPR is an optical method that measures the refractive index of a thin layer of a material adsorbed on a metal. When
applied for MIP characterization, the NPs are immobilized on the surface of the
metal film while the ligand in the mobile phase runs along the flow cell. If binding
occurs, the refractive index changes leads to a change in SPR angle. The apparent
association and dissociation constants can be derived from the change rates of SPR
signals (Taniwaki et al. 2003; Li and Husson 2006; Lépinay et al. 2012).
The QCM is a mass-sensitive piezoelectric device capable of detecting nanogram changes in mass by measuring the change in frequency of a quartz crystal
5 Plastic Receptors Developed by Imprinting Technology as Smart Polymers Imitating…