An ultrasensitive protein array based on electrochemical enzyme immunoassay

An ultrasensitive electrochemical enzyme immunoassay (EEIA) for the detection of proteins on an 8x8 array is described. The assay is based on wired enzyme technology. Briefly, capture antibody was covalently immobilized on a self-assembled 11-mercaptoundecanoic acid (MUA) monolayer coated gold electrode. After incubating with a target protein (antigen), the gold electrode was treated sequentially in biotinylated detection antibody solution and in avidin-horseradish peroxidase conjugate (A-HRP) solution. A cationic redox polymer (electrochemical mediator) overcoating was applied to the gold electrode through layer-by-layer electrostatic self-assembly. The formation of a bilayer brought the HRP in electrical contact with the underlying electrode, making the bilayer an electrocatalyst for the reduction of hydrogen peroxide where the redox polymer acts as an artificial mediator. Consequently, the concentration of protein could be quantified amperometrically. This electrochemical immunoassay combined the specificity of the immunological reaction with the sensitivity of the electrochemical detection. The applicability of the system in protein detection was demonstrated with a snake toxin, beta-bungarotoxin, a neurotoxin from the venom of the snake Bungarus multicinctues. Under optimized experimental conditions, the assay allowed the detection of beta-bungarotoxin in the range of 20 pg/mL to 1.5 ng/mL with a detection limit of 10 pg/mL (20 fg). A higher detection limit of 25 pg/mL was obtained in serum.