Improvement of the homogeneity of protein-imprinted polymer films by orientated immobilization of the template

A method for preparing homogeneous protein-imprinted polymer films with orientated immobilization of template is described. The template methyl parathion hydrolase (MPH) was modified with a peptide linker (Gly-Ser)5–Cys and was immobilized on a cover glass with a fixed orientation via the linker. The activity of the fusion enzyme (MPH-L) was evaluated by determining the product's absorbance at 405 nm (A405). Both the free and the immobilized MPH-L showed higher retention of the bioactivity than the wide type enzyme (MPH-W) as revealed by the A405 values for MPH-Lfree/MPH-Wfree (1.159/1.111) and for MPH-Limmobilized/MPH-Wimmobilized (0.348/0.118). The immobilized MPH-L also formed a more homogeneous template stamp compared to the immobilized MPH-W. The molecularly imprinted polymer films prepared with the immobilized MPH-L exhibited high homogeneity with low Std. Deviations of 80 and 200 from the CL intensity mean volumes which were observed for batch-prepared films and an individual film, respectively. MPH-L-imprinted polymer film also had a larger template binding capacity indicated by higher CL intensity mean volume of 3900 INT over 2500 INT for MPH-W-imprinted films. The imprinted film prepared with the orientated immobilization of template showed an imprinting factor of 1.7, while the controls did not show an imprinting effect.

Figure optionsDownload as PowerPoint slideHighlights
► MPH was genetically modified at its C-terminal with (Gly-Ser)5–Cys.
► MPH-L was immobilized with fixed orientation via disulfide chemistry.
► The immobilized MPH-L retained the activity of MPH.
► MPH-L formed a homogeneous template.
► Homogeneous MIP film was obtained with orientated immobilization of the template.