Physical and chemical characterization of enolase immobilized polydiacetylene Langmuir–Blodgett film

• Published in: Sensors and actuators. B, Chemical Chemical, 2006-05, Vol.115 (1), p.526-533
• Main Authors: Sadagopan, K., Sawant, Shilpa N., Kulshreshtha, S.K., Jarori, Gotam K.
• Format: Article
• Language: English
• Subjects: AFM; Colorimetric response; Covalent immobilization; Enolase; Plasmodium falciparum; Protein–ligand interaction
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2005.10.031

Hexa histidine tagged recombinant Plasmodium falciparum enolase (His6-Pfen) was covalently immobilized on a Langmuir–Blodgett film of a self assembled mixture of 10,12-pentacosadiynoic acid and its N-succinimidyl ester derivative (PDA LB-film). The film was polymerized with a UV-lamp at 254nm to obtain a blue coloured, protein hooked polydiacetylene film. Atomic force microscopy (AFM) was used to characterize the surface morphology of the protein-immobilized film. The colorimetric response (CR) of the His6-Pfen hooked PDA LB-film to 2-phosphoglyceric acid (2-PGA), the substrate of enolase, in the presence of magnesium ions was studied spectrophotometrically. The CR of glutathione S-transferase-Pfen (GST-Pfen) immobilized film prepared by using similar procedure was also examined. The results suggest that binding of Mg (II) to the enzyme facilitates the interaction of the enzyme with 2-phosphoglycerate. This ligand-binding event could be detected by an observed increase in colorimetric response of the film by ∼10%. Thus the incorporation of enolase on a PDA film resulted in the formation of a novel material, which can serve as a biosensor.