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Electrochemical Immunoassay Detection of Bacillus Anthracis Protective Antigen

A self-contained microelectrochemical immunoassay detection of Bacillus anthracis protective antigen (PA) has been developed. A microcavity array (VEG4x1TM) of 50-μm diameter cavities with self-contained microelectrodes were used for the detection of as low as 2.5 pg of B. anthracis Protective Antigen in a less than 12 min total assay time from capture to signal generation. The microelectrochemical sandwich enzyme-linked immunoassay involved covalent immobilization of the capture antibody on the free end of mercaptoundecanol self-assembled monolayers at the bottom Aumicrodisk electrode that also serves as the bottom of the microcavities. Signal generation involved alkaline phosphatase hydrolysis of the enzyme substrate p-aminophenyl phosphate to paminophenol that is detected with the wall middle layer gold as the detecting electrode and the top layer gold as the pseudoreference/auxiliary electrodes. The microelectrodes in the cavity exhibited background signals lower than signal for the lowest concentration detected that was 2.5 pg.