Electrochemical Enzyme Immunoassay of a Peptide Hormone at Picomolar Levels

Abstract

A novel electrochemical enzyme immunoassay system with a 10 ng L(-1) level detection limit was developed for the determination of B-type natriuretic peptide (BNP), an important marker for the diagnosis of heart failure. Sample BNP was added to a solution containing a certain concentration of acetylcholinesterase(AChE)-labeled anti-BNP antibody to undergo an immunological reaction. After the immunological reaction, we proposed two assay schemes. One involves measuring the amount of antibody-enzyme conjugate that reacted with two BNP molecules (reacted conjugate). The other involves measuring the amount of antibody-enzyme conjugate with at least one free binding site (unreacted conjugate). Then the amount of reacted or unreacted conjugate was determined by measuring the AChE activity after the recovery of each conjugate from the immunological reaction mixture. To determine the trace level of the recovered antibody-enzyme conjugate, the AChE activity was determined with high sensitivity on the basis of the chemisorption/electrochemical desorption process of thiocholine, which was produced through the enzymatic reaction, on a silver surface. The thiocholine chemisorption (i.e., accumulation) on the silver electrode surface resulted in a sensitivity for the electrochemical determination of the AChE activity that was 2 orders of magnitude greater than that obtained when using direct measurement without accumulation. The procedure for determining the AChE activity of unreacted conjugate after its recovery on a BNP-modified disk was applied to the determination of BNP in serum samples. This procedure involves the removal of the immunological reaction mixture before the enzymatic reaction process, which allows the AChE activity to be measured without any interference from endogenous pseudocholinesterase, which exists with high activity in serum. With both procedures, the BNP could be measured within an hour. The detection limits were 20 and 40 ng L(-1) using the reacted and unreacted conjugate measuring procedures, respectively.