Analytical and Bioanalytical Chemistry

, Volume 406, Issue 22, pp 5379–5387 | Cite as

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

  • Berta Esteban-Fernández de Ávila
  • Susana CampuzanoEmail author
  • María Pedrero
  • J.-Pablo Salvador
  • M.-Pilar Marco
  • José M. PingarrónEmail author
Research Paper


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-[N α,N α-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.


Lp(a) Screen-printed electrode Integrated amperometric immunosensor Human serum 



The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are gratefully acknowledged. B. Esteban-Fernández de Ávila acknowledges a FPI fellowship from the Spanish Ministerio de Ciencia e Innovación. The authors would like to acknowledge Audit Diagnostics Company for kindly providing the Lp(a) and its specific antibodies.

Supplementary material

216_2014_7964_MOESM1_ESM.pdf (28 kb)
ESM 1 (PDF 27.6 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Berta Esteban-Fernández de Ávila
    • 1
  • Susana Campuzano
    • 1
    Email author
  • María Pedrero
    • 1
  • J.-Pablo Salvador
    • 2
    • 3
  • M.-Pilar Marco
    • 2
    • 3
  • José M. Pingarrón
    • 1
    Email author
  1. 1.Departamento de Química Analítica, Facultad de CC. QuímicasUniversidad Complutense de MadridMadridSpain
  2. 2.Nanobiotechnology for Diagnostics (Nb4D)BarcelonaSpain
  3. 3.CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN)BarcelonaSpain

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