are shown in Figure 3. It can be seen that the nanocomposite gels have a rapid increase in adsorption over 8 h, and then it increased slowly with prolonged time. After 10 h, the adsorption process reach the equili-
Figure 1. FT-IR Spectra of a) nanosized silica particles and b) Mb imprinted NIPAM nanocomposite gel.
Figure 2. Surface Morphology of Mb imprinted PNIPAM nanocomposite gels.
Figure 3. Adsorption Kinetics of Mb imprinted PNIPAM Nanocomposite gels.
brium. In this case, the first part of the kinetic plot shows the Mb molecules first adsorbed on the surface of the nanogels. The second part of the plot with a smaller slope indicates a slow adsorption rate. This has been observed for both the non-imprinted and imprinted nanocomposite gel. As the adsorption rate does not remain constant, a certain type of rate must be selected for quantitative investigation of the experimental results. Instead of arbitrarily taking the rate measured at a certain adsorption, we have preferred to determine the initial slope of the adsorption curves. This enabled us to determine the actual initial adsorption rates directly.
This adsorption equilibrium is most probably a result of the geometric shape affinity between Mb molecules and Mb cavities in the nanocomposite gels. Molecular imprinting generates binding cavities that are complementary to the original template in both shape and functionality.
3.4. Adsorption Isotherm of PNIPAM
Figure 4 shows the dependence of the adsorbed amount of Mb on the initial Mb concentration. As seen this figure, the adsorption values increased with increasing concentration of Mb and a saturation value is achieved at Mb concentration of 1.5 ng∙mL−1, which represents saturation of the active binding and recognition sites of the nanocomposite gels. The adsorption capacity of the imprinted nanocomposite gels was found to be strongly dependent the initial Mb concentration.
The adsorption behaviors of the nanocomposite gels were evaluated by the Langmuir isotherm, the experimental data are fitted to the Langmuir equation. The calculated results are listed in Table1
As shown in Table 1, the non-linear Langmuir fits well for the Mb adsorption on the imprinted and nonimprinted nanocomposite gels prepared at different Mb concentration (correlation coefficient, rL > 0.943). The maximum adsorption and the Langmuir adsorption equilibrium constant of the imprinted hydrogels were higher than those of the non-imprinted hydrogels according to the adsorption isotherms. The rL values show that favorable adsorption of Mb on both the imprinted and nonimprinted hydrogels takes place; therefore, the imprinted nanocomposite gels are favorable adsorbers.
The other well known isotherm is the Freundlich adsorption isotherm which is a special case for heterogeneous surface energy in which the energy term in the Langmuir equation varies as a function of surface cover-
Figure 4. The adsorption isotherms of Mb on nonimprinted and imprinted nanocomposite gels.
Table 1. Langmuir isotherm constants of the nanocomposite gels.
age strictly because of variation of the sorption. The isotherms obtained are well fit by the Freundlich isotherm. The slope of the straight line fit yields n, which is a measure of the heterogeneity of a system. A more homogeneous system has an n value that approaches unity and a more heterogeneous system has an n value that approaches zero. The Freundlich isotherm is useful to produce a direct measurement of the adsorption properties. The calculated results from the Freundlich isotherm are also listed in Table2
The magnitude of the exponent 1/n gives an indication of the favorability of the adsorption . The values, n > 1 represents a favorable adsorption condition for Mb adsorption on the hydrogels, and the high correlation coefficients (rF > 0.915) showed that the Freundlich isotherm also agrees well with experimental data. However, rL > rF and, therefore, the non-linear Langmuir isotherm fits the experimental data better than the Freundlich one. A good fit of this isotherm reflects monolayer adsorption.
3.5. Selectivity of Nanocomposite Gels
The special selectivity tests of non-imprinted and imprinted hydrogels prepared with different temperatures were carried out using Mb as substrate. The amount of Mb adsorbed to the imprinted and non-imprinted nanocomposite gels were determined with the equilibrium adsorption method (Figure 5).
The selectivity, α1 Mb/Hb, α2 Mb/Fbg and separation, β imprinted/ non-imprinted, factors were calculated and are given in Table3 The data in Table 3 show that the imprinted hydrogels exhibit high selectivity for the imprinting Mb molecule compared to Hb and Fbg. However, the non-imprinted hydrogels exhibit low a values under the same conditions. Although Mb and Hb have almost the same isoelectric point (6.9 - 7.3), Hb has a larger molecular mass (MW 65 kDa) than the template Mb (MW 17 kDa). Moreover, Hb is tetrameric protein composed of pairs of two different polypeptides and has a biconcave shape; Mb consists of one polypeptide and has an ellipsoidal shape . Since the cavities formed by
Figure 5. Adsorption capacities of Mb, Hb and Fbg.
Table 3. Selectivity (α1, α2) and seperation (β) factors of the nanocomposite gels.
the imprinted hydrogels are matched to the size of Mb, it is very difficult for molecules with a different dimension or molecular mass to enter the cavities
Temperature sensitive imprinted poly(N-isopropylacrylamide) nanocomposite gels were syntheses via in-situ, free radical crosslinking polymerization of corresponding monomer in nano-sized silica and five different concentrations of myoglobin solution by using the molecular imprinting method. Nanocomposite gels imprinted with Mb showed a higher adsorption capacity for Mb than nanocomposite gels prepared by the usual procedure. The highest Mb adsorption was observed via the imprinted nanocomposite gels with 12.5% Mb. In addition, selectivity studies were also performed by using two reference molecules as fibrinogen and hemoglobin. The imprinted nanocomposite gels exhibited good selectivity for Mb and high adsorption rate depending on the number of Mb sized cavities.