Monitoring yoctomole alkaline phosphatase by capillary electrophoresis with on-capillary catalysis-electrochemical detection

An electrophoretically mediated microanalysis method for detection of yoctomole (ymol) alkaline phosphatase (ALP) was developed by a combination of on-capillary enzyme-catalyzed reaction and electrochemical detection. In this method, ALP molecules were electrokinetically injected into a capillary of 10 μm i.d. and then electromigrated to the section of the capillary immersed in a warm water bath of 37 °C, where ALP reacted for a certain time with disodium phenyl phosphate as the enzyme substrate. ALP could be measured through determining the electroactive product phenol of the enzyme-catalyzed reaction by using electrochemical detection. The phenol concentration was proportional to the mass of ALP. As a catalyst, ALP was not consumed during the reaction, which provided amplification of signal with prolonged the reaction time. In order to enhance the signal-to-noise ratio, the detection end of the capillary was etched to a horn-shape and a single carbon fiber microcylinder electrode of 6 μm in diameter as the working electrode was inserted into the detection end of the capillary. Under these conditions, the mass of ALP as low as 1.2 × 10⁻²² mol (72 molecules) or 4.0 × 10⁻²³ mol (24 molecules) could be detected for the on-capillary reaction time of 15 min or 2 h.