| Abstract [eng] |
The direct electrochemical technique for lipase activity detection was developed using synthetic enzyme substrate containing ferrocene reporting and ester target groups - (9-(5'-ferrocenylpentanoyloxy) nonyl disulfide (FPONDS); a simple drip-and-dry sensor surface preparation method was suggested. Using SPR and ATR-FT-IR methods we detected the rearrangement of the Thermomyces lanuginosus lipase (TLL) layer on hydrophobic surfaces, which is followed by a considerable increase of TLL activity, thus presenting a direct evidence of the importance of conformational configuration on esterase activity. The utility of the proposed electrochemical technique to assess the stereospecificity of esterases, specifically TLL, was demonstrated. A rotating disk electrode method was developed, which could be utilized to TLL activity in micellar substrate systems. The adsorption equilibrium phenomenon between the OPNQ micells and glassy-carbon surface was utilized to develop a TLL assay in micellar systems. The technique is based on direct micelle concentration detection, therefore, allows avoiding complications related to the polymorphism of the substrates of esterases. We present sum frequency generation spectroscopy based proof-of-a-concept TLL activity assay; the method allows to overcome complications common in classical esterase activity assays related to the polymorphism of substrate, and measures enzyme activity at air/water interface. The tethered bilayer membrane based methodology for the detection of phospholipases by the electrochemical impedance spectroscopy was proposed. |
