Modification of a capillary for electrophoresis by electrostatic self-assembly of an enzyme for selective determination of the enzyme substrate

The integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reaction for selective determination of the enzyme substrate is described. Enzyme immobilization is achieved by electrostatic assembly of poly (diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the GOx produces hydrogen peroxide which migrates the length of the capillary and is detected amperometrically at the capillary outlet. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre- or post-separation chemical modification of the analyte. The enzyme reactor is found to have an optimal response to glucose when a 5:1 mixture of PSS:GOx is used. Under these conditions the limit of detection for glucose is found to be between 5.0 x 10(-4) and 1.3 X 10(-3) M, dependent upon the inner-diameter of the capillary. The apparent Michaelis-Menten constant for the enzyme reaction was determined to be 0.047 (+/- 0.001) M and 0.0037 (+/- 0.0007) M for a 50 and 10 pm inner-diameter capillaries, respectively. These results indicate that the enzyme reaction is efficient, having enzyme kinetics similar to that of a reaction occurring in solution. This enzyme immobilization method was also applied to another enzyme, glutamate oxidase, yielding similar results.