Advertisement

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 Campuzano
  • María Pedrero
  • J.-Pablo Salvador
  • M.-Pilar Marco
  • José M. Pingarrón
Research Paper

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-[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.

Keywords

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

Notes

Acknowledgments

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)

References

  1. 1.
    Klingenberg R, Hansson GK (2009) Treating inflammation in atherosclerotic cardiovascular disease: emerging therapies. Eur Heart J 30:2838–2844CrossRefGoogle Scholar
  2. 2.
    Ross R (1999) Atherosclerosis is an inflammatory disease. Am Heart J 138:S419–S420CrossRefGoogle Scholar
  3. 3.
    Malaguarnera M, Vacante M, Russo C, Malaguarnera G, Antic T, Malaguarnera L, Bella R, Pennisi G, Galvano F, Frigiola A (2013) Lipoprotein(a) in cardiovascular diseases. BioMed Res Int 2013:1–9Google Scholar
  4. 4.
    Marcovina SM, Koschinsky ML, Albers JJ, Skarlatos S (2003) Report of the national heart, lung, and blood institute workshop on lipoprotein(a) and cardiovascular disease: recent advances and future directions. Clin Chem 49:1785–1796CrossRefGoogle Scholar
  5. 5.
    Riches K, Porter KE (2012) Lipoprotein(a): cellular effects and molecular mechanisms. Cholesterol. doi: 10.1155/2012/923289, Article ID 923289, p 10Google Scholar
  6. 6.
    Dubé JB, Boffa MB, Hegele RA, Koschinsky ML (2012) Lipoprotein(a): more interesting than ever after 50 years. Curr Opin Lipidol 23:133–140CrossRefGoogle Scholar
  7. 7.
    Kamstrup PR, Tybjaerg-Hansen A, Steffensen R, Nordestgaard BG (2009) Genetically elevated lipoprotein(a) and increased risk of myocardial Infarction. JAMA 301:2331–2339CrossRefGoogle Scholar
  8. 8.
    Erqou S, Kaptoge S, Perry PL, Di AE, Thompson A, White IR, Marcovina SM, Collins R, Thompson SG, Danesh J (2009) Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA 302:412–423CrossRefGoogle Scholar
  9. 9.
    Etingin OR, Hajjar DP, Hajjar KA, Harpel PC, Nachman RL (1991) Lipoprotein(a) regulates plasminogen activator inhibitor-1 expression in endothelial cells: a potential mechanism in thrombogenesis. J Biol Chem 266:2459–2465Google Scholar
  10. 10.
    Tsimikas S, Hall JL (2012) Lipoprotein(a) as a potential causal genetic risk factor of cardiovascular disease. J Am Coll Cardiol 60:716–721CrossRefGoogle Scholar
  11. 11.
    Kostner KM, Oberbauer R, Hoffmann U, Stefenelli T, Maurer G, Watschinger B (1997) Urinary excretion of apo(a) in patients after kidney transplantation. Nephrol Dial Transplant 12:2673–2678CrossRefGoogle Scholar
  12. 12.
    Suk DJ, Rifai N, Buring JE, Ridker PM (2006) Lipoprotein(a), measured with an assay independent of apolipoprotein(a) isoform size, and risk of future cardiovascular events among initially healthy women. JAMA 296:1363–1370CrossRefGoogle Scholar
  13. 13.
    Gazzaruso C, Garzaniti A, Giordanetti S, Falcone C, Fratino P (2002) Silent coronary artery disease in type 2 diabetes mellitus: the role of lipoprotein(a), homocysteine and apo(a) polymorphism. Cardiovasc Diabetol 1:5CrossRefGoogle Scholar
  14. 14.
    Fan J, Sun H, Unoki H, Shiomi M, Watanabe T (2001) Enhanced atherosclerosis in Lp(a) WHHL transgenic rabbits. Ann N Y Acad Sci 947:362–365CrossRefGoogle Scholar
  15. 15.
    Imhof A, Rothenbacher D, Khuseyinova N (2003) Plasma lipoprotein Lp(a) and risk of coronary heart disease: what are the mechanisms involved? Eur J Cardiovasc Prev Rehabil 10:362–370CrossRefGoogle Scholar
  16. 16.
    Motta M, Giugno I, Bosco S (2001) Serum lipoprotein(a) changes in acute myocardial infarction. Panminerva Med 43:77–80Google Scholar
  17. 17.
    Nordestgaard BG, Chapman MJ, Ray K, Borén J, Andreotti F, Watts GF, Ginsberg H, Amarenco P, Catapano A, Descamps OS, Fisher E, Kovanen PT, Kuivenhoven JA, Lesnik P, Masana L, Reiner Z, Taskinen M-R, Tokgözoglu L, Tybjærg-Hansen A (2010) Lipoprotein(a) as a cardiovascular risk factor: current status. Eur Heart J 2844–2853Google Scholar
  18. 18.
    Torres MB, Álvarez FH, Rodríguez Hernández A, Sorell-Gómez L, Cabalé MB (1998) Evaluación de un método rápido para la detección de niveles elevados de Lipoproteína (a): Aubiodot Lp (a). Rev Cubana Cardiol Cir Cardiovasc 24:29–35Google Scholar
  19. 19.
    Wang J, Zhang C, Gong J, Zhu Y, Fu L, Wang X, Li K (2007) Development of new enzyme-linked immunosorbent assay for oxidized lipoprotein(a) by using purified human oxidized lipoprotein(a) autoantibodies as capture antibody. Clin Chim Acta 385:73–78CrossRefGoogle Scholar
  20. 20.
    Gómez M, Valle V, Arós F, Sanz G, Sala J, Fiol M, Bruguera J, Elosua R, Molina L, Martí H, Covas MI, Rodríguez-Llorián A, Fitó M, Suárez-Pinilla MA, Amezaga R, Marrugat J (2009) Oxidized LDL, lipoprotein (a) and other emergent risk factors in acute myocardial infarction (FORTIAM study). Rev Esp Cardiol 62:373–382CrossRefGoogle Scholar
  21. 21.
    Hayat A, Barthelmebs L, Sassolas A, Marty J-L (2011) An electrochemical immunosensor based on covalent immobilization of okadaic acid onto screen printed carbon electrode via diazotization-coupling reaction. Talanta 85:513–518CrossRefGoogle Scholar
  22. 22.
    Huy TQ, Hanh NTH, Chung PV, Anh DD, Nga PT, Tuan MA (2011) Characterization of immobilization methods of antiviral antibodies in serum for electrochemical biosensors. Appl Surf Sci 257:7090–7095Google Scholar
  23. 23.
    Vallina-García R, García-Suárez MM, Fernández-Abedul MT, Méndez FJ, Costa-García A (2007) Oriented immobilisation of anti-pneumolysin Fab through a histidine tag for electrochemical immunosensors. Biosens Bioelectron 23:210–217CrossRefGoogle Scholar
  24. 24.
    Valiokas R, Klenkar G, Tinazli A, Reichel A, Tampe′ R, Piehler J, Liedberg B (2008) Self-assembled monolayers containing terminal mono-, bis-, and tris-nitrilotriacetic acid groups: characterization and application. Langmuir 24:4959–4967CrossRefGoogle Scholar
  25. 25.
    Esteban-Fernández de Ávila B, Escamilla-Gómez V, Campuzano S, Pedrero M, Pingarrón JM (2013) Disposable electrochemical magnetoimmunosensor for the determination of troponin T cardiac marker. Electroanalysis 25:51–58CrossRefGoogle Scholar
  26. 26.
    Conzuelo F, Gamella M, Campuzano S, Martínez-Ruiz P, Esteban-Torres M, de las Rivas B, Reviejo AJ, Muñoz R, Pingarrón JM (2013) Integrated amperometric affinity biosensors using Co2+–tetradentate nitrilotriacetic acid modified disposable carbon electrodes: application to the determination of β-lactam antibiotics. Anal Chem 85:3246–3254CrossRefGoogle Scholar
  27. 27.
    Huang Y, Bell MC, Suni II (2008) Impedance biosensor for peanut protein Ara h 1. Anal Chem 80:9157–9161CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Berta Esteban-Fernández de Ávila
    • 1
  • Susana Campuzano
    • 1
  • María Pedrero
    • 1
  • J.-Pablo Salvador
    • 2
    • 3
  • M.-Pilar Marco
    • 2
    • 3
  • José M. Pingarrón
    • 1
  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

Personalised recommendations