This paper focuses on the immobilization of a proteolytic enzyme, trypsin, on plasma polymerized allylamine (ppAA) films. The later have been deposited onto silicon substrate by means of radiofrequency glow discharge. The covalent attachment of the enzyme was achieved in three steps: (i) activation of the polymer surface with glutaraldehyde (GA) as a linker, (ii) immobilization of trypsin and (iii) imino groups reduction treatment. The effects and efficiency of each step were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Fluorescent spectroscopy was used to evaluate the change of the biological activity following the immobilization steps. The results showed that enzyme immobilization on GA-modified substrate increases the enzyme activity by 50% comparing to adsorbed enzymes, while the imino reduction treatment improves the enzyme retention by about 30% comparing to untreated samples. In agreement with XPS and AFM data, UV-vis absorption spectroscopy, used to quantify the amount of immobilized enzyme, showed that allylamine plasma polymer presents a high adsorption yield of trypsin. Although the adsorbed enzymes exhibit a lower activity than that measured for enzymes grafted through GA linkers, the highest catalytic activity obtained was for the enzymes that underwent the three steps of the immobilization process.
Bibliographical noteFunding Information:
We gratefully acknowledge the support of the Agence Nationale de la Recherche under contract ANR-05-NT05-2_46562 and the Interdisciplinary Research Program of Lille1 University (PPF BioMEMS 2006, no. 1803). The authors would like to thank Sylvie Godet and Dominique Deresmes for their technical assistance and comments.
- Covalent immobilization
- Immobilized enzyme activity
- Plasma polymerized allylamine
- Trypsin adsorption