Lipoprotein(a) determination in human serum using a nitrilotriacetic acid derivative immunosensing scaffold on disposable electrodes.

Berta Esteban-Fernández de Ávila, Susana Campuzano, María Pedrero, J-Pablo Salvador, M-Pilar Marco, José M Pingarrón

Index: Anal. Bioanal. Chem 406(22) , 5379-87, (2014)

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Abstract

A novel strategy for the construction of a disposable integrated amperometric immunosensor for the sensitive and rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum is reported. The approach uses a sandwich format involving the covalent immobilization of selective capture antibodies (antiLp(a)) on the surface of N-[Nα,Nα-bis(carboxymethyl)-lysine]-12-mercaptododecanamide (HS-NTA)-modified screen-printed carbon electrodes (SPCEs). After a blocking step with skimmed milk, the modified antiLp(a)-SPCEs were incubated with a mixture solution containing the target analyte and a fixed concentration of a specific biotinylated antibody (biotin-antiLp(a)) and a streptavidin-horseradish peroxidase (HRP) (Strep-HRP) conjugate. The amperometric responses of the resulting immunosensor at -0.10 V (vs an Ag pseudo-reference electrode), upon addition of 3,3',5,5'-tetramethylbenzidine (TMB) as electron transfer mediator and H2O2 as the enzyme substrate, were used to monitor the extent of the immunoreactions. The developed methodology exhibited a wide range of linearity between 0.02 and 10 μg mL(-1), a low detection limit (LOD) of 8 ng mL(-1), and a great selectivity against other serum components. The usefulness of the Lp(a) immunosensor was demonstrated by analyzing spiked serum samples as well as a reference serum containing a certified Lp(a) content.