INSTITUTO DE QUIMICA, FISICA DE LOS MATERIALES, MEDIOAMBIENTE Y ENERGIA
Unidad Ejecutora - UE
Endotoxin detection in a competitive electrochemical assay: Synthesis of a suitable endotoxin conjugate
GRACIELA PRIANO; DIEGO PALLAROLA; FERNANDO BATTAGLINI
Año: 2007 vol. 362 p. 108 - 108
A biotinlipopolysaccharide (biotinLPS) conjugate was synthesized from LPS smooth from Salmonella minnesota, yielding a conjugate with a biotin/LPS ratio equal to 1:1 and endotoxic activity of 0.08 EU ng-1. The conjugate was used in an amperometric competitive assay to determine endotoxins with endotoxin-neutralizing protein (ENP) as the recognition element. The assay is performed on a modified electrode, involving the covalent binding of carboxymethyl dextran (CMDex) to a cystamine-modified gold electrode and then the covalent binding of the recognition protein, ENP, to CMDex. The assay is carried out by incubating the modified electrode in an LPS sample to which biotinLPS was added. Both species compete for the recognition sites on the modified surface. After the incubation stage and a careful rinsing, the electrode is immersed in a solution containing neutravidinhorseradish peroxidase conjugate (N-HRP), which binds to the sites containing biotinLPS on the electrode. The system is rinsed and a current signal is generated by the addition of hydrogen peroxide and a redox mediator. The assay is able to detect LPS from Salmonella minnesota at concentrations as low as 0.1 ng ml-1, equivalent to 0.07 EU ml-1.