Procedure 52 Analysis of the activity of β-galactosidase from E. Coli by scanning electrochemical microscopy (SECM)

Abstract

This chapter discusses the immobilization of biotin-labeled b-galactosidase on streptavidin-coated paramagnetic beads and analysis of the activity of b-galactosidase by scanning electrochemical microscopy (SECM). Ultramicroelectrode (UME) was prepared by sealing Pt wires into soft glass, exposing a gross section by grinding on polishing paper, beveling the edges at an angle of 45˚ to obtain an RG≈10 and polishing the front end with Al 2 O 3 suspension. Data evaluation was performed with an in-house developed software package MIRA. Analysis of the galactosidase activity includes mounting of SECM setup, addition of p-aminophenyl-b-D-galactopyranoside (PAPG) in buffer, and positioning of UME over the sample. The current should decrease as the UME approaches the surface. When the current does not decrease anymore, stop the positioning system and retract the UME by 65 mm. Further UME is used to detect p-aminophenol (PAP), it moved in horizontal direction to find the microbeads microspot with a translation speed of 10 μms­ –1 . After finding the microspot, run an SECM image. To quantify the flux of PAP generated at the bead mound, a current profile was extracted from the image, including the highest current value that corresponds to the center of the bead spot. The result value confirms that only a small fraction of the enzyme substrate PAPG provided is converted. Therefore, the enzyme reaction follows the regime of substrate saturation.