Use of an antimicrobial protein for endotoxin detection in a competitive electrochemical assay

Endotoxins, also referred to as pyrogens, are part of the cellular walls of Gram-negative bacteria and are capable of inducing fever when entering into the blood stream. Current methods of detection involve the use of either amoebocytes from horseshoe crab that clots upon exposure to endotoxin or a live assay on rabbits. In this work, the detection of endotoxins by an electrochemical competitive assay is presented. Two configurations of modified electrodes were constructed using a recombinant endotoxin neutralizing protein (ENP) as recognition element. A modified lipopolysaccharide with horseradish peroxidase was used for the competitive assay. Modified electrodes constructed by electrostatic interaction of ENP and an electroactive polymer can detect the presence of endotoxins in concentrations as low as 0.2EU mL(-1), below the limit imposed for water in injectable drugs in the American Pharmacopoeia. Modified electrodes constructed by covalent linking of ENP to a carboxymethyl dextran matrix bound to the electrode show a better dynamic range but a higher detection limit.