Advertisement

Microchimica Acta

, 185:323 | Cite as

Amperometric immunoassay for the obesity biomarker amylin using a screen printed carbon electrode functionalized with an electropolymerized carboxylated polypyrrole

  • Gonzalo Martínez-García
  • Esther Sánchez-Tirado
  • Araceli González-Cortés
  • Paloma Yáñez-Sedeño
  • José M. Pingarrón
Original Paper
  • 166 Downloads

Abstract

Amylin (the islet amyloid polypeptide) is a hormone related to adiposity, hunger and satiety. It is co-secreted with insulin from pancreatic B-cells. An amperometric immunosensor is presented here for the determination of amylin. It is making use of a screen printed carbon electrode (SPCE) functionalized with electropolymerized poly(pyrrole propionic acid) (pPPA) with abundant carboxyl groups that facilitate covalent binding of antibody against amylin. A competitive immunoassay was implemented using biotinylated amylin and streptavidin labeled with horse radish peroxidase (HRP-Strept) as the enzymatic tracer. The amperometric detection of H2O2 mediated by hydroquinone was employed as an electrochemical probe to monitor the affinity reaction. The variables involved in the preparation and function of the immunosensor were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The calibration graph for amylin, obtained by amperometry at −200 mV vs Ag pseudo-reference electrode, showed a range of linearity extending from 1.0 fg∙mL−1 to 50 pg∙mL−1, with a detection limit of 0.92 fg∙mL−1. This is approximately 7000 times lower than the minimum detectable concentration reported for the ELISA immunoassays available for amylin. The assay has excellent reproducibility and good selectivity over potential interferents.

Graphical abstract

Schematic of an amperometric competitive immunoassay for the obesity biomarker amylin using a poly(pyrrole propionic acid)-modified screen-printed electrode. The detection limit is 0.92 fg∙mL-1 amylin. The method provides excellent reproducibility for the measurements, good selectivity and successful applicability to human urine and serum samples.

Keywords

Screen-printed carbon electrodes Electrochemical biosensor Conducting polymer Mix&Go™ Urine Serum 

Notes

Acknowledgements

The financial support of projects CTQ2015-70023-R (Spanish Ministry of Economy and Competitivity Research Projects), and S2013/MT-3029 (NANOAVANSENS Program from the Comunidad de Madrid) are gratefully acknowledged.

Compliance with ethical standards

The authors declare that they have no competing interest.

References

  1. 1.
    Cooper G, Willis A, Clark A, Turner R, Sim R, Reid K (1987) Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci 84:8628–8632.  https://doi.org/10.1073/pnas.84.23.8628 CrossRefGoogle Scholar
  2. 2.
    Zhang J, Chen Y, Li D, Cao Y, Wang Z, Li G (2016) Colorimetric determination of islet amyloid polypeptide fibrils and their inhibitors using resveratrol functionalized gold nanoparticles. Microchim Acta 183:659–665CrossRefGoogle Scholar
  3. 3.
    Konarkowska B, Aitken JF, Kistler J, Zhang S, Cooper GJ (2006) The aggregation potential of human amylin determines its cytotoxicity towards islet β-cells. FEBS J 273:3614–3624.  https://doi.org/10.1111/j.1742-4658.2006.05367.x CrossRefGoogle Scholar
  4. 4.
    Percy AJ, Trainor DA, Rittenhouse J, Phelps J, Koda JE (1996) Development of sensitive immunoassays to detect amylin and amylin-like peptides in unextracted plasma. Clin Chem 42:576–585Google Scholar
  5. 5.
    Castillo MJ, Scheen AJ, Lefèbre PJ (1995) Amylin/islet amyloid polypeptide: biochemistry, physiology, patho-physiology. Diabete Metab 21:3–25Google Scholar
  6. 6.
    Koda JE, Fineman M, Rink TJ, Dailey GE, Muchmore DB, Linarelli LG (1992) Amylin concentrations and glucose control. Lancet 339:1179–1180CrossRefGoogle Scholar
  7. 7.
    Sanke T, Hanabusa T, Nakano Y, Oki C, Okai K, Nishimura S, Kondo M, Nanjo K (1991) Plasma islet amyloid polypeptide (amylin) levels and their responses to oral glucose in type-2 (non-insulin-dependent) diabetic patients. Diabetologia 34:129–132.  https://doi.org/10.1007/BF00500385 CrossRefGoogle Scholar
  8. 8.
    Kautzky-Willer A, Thomaseth K, Pacini G, Clodi M, Ludvik B, Streli C, Waldhäusl W, Prager E (1994) Role of islet amyloid polypeptide secretion in insulin-resistant humans. Diabetologia 37:188–194.  https://doi.org/10.1007/s001250050092 CrossRefGoogle Scholar
  9. 9.
    Harter E, Svoboda T, Ludvik B, Schuller M, Lell B, Kuenburg E, Brunnbauer M, Woloszcruk W, Prager E (1991) Basal and stimulated plasma levels of pancreatic amylin indicate its co-secretion with insulin in humans. Diabetologia 34:52–54CrossRefGoogle Scholar
  10. 10.
    Butler PC, Chou J, Bradford Carter W, Wang Y-N, Bu B-H, Chang D, Chang J-K, Rizza RA (1990) Effects of meal ingestion on plasma amylin concentration in NIDDM and nondiabetic humans. Diabetes 29:752–756CrossRefGoogle Scholar
  11. 11.
    Ooi HW, Cooper SJ, Huang C-Y, Jennins D, Chung E, Maeji NJ, Whittaker AK (2014) Coordination complexes as molecular glue for immobilization of antibodies on cyclic olefin copolymer surfaces. Anal Biochem 456:6–13.  https://doi.org/10.1016/j.ab.2014.03.023. CrossRefGoogle Scholar
  12. 12.
    Ojeda I, Barrejón M, Arellano LM, González-Cortés A, Yáñez-Sedeño P, Langa F, Pingarrón JM (2015) Grafted-double walled carbon nanotubes as electrochemical platforms for immobilization of antibodies using a metallic-complex chelating polymer: application to the determination of adiponectin cytokine in serum. Biosens Bioelectron 74:24–29.  https://doi.org/10.1016/j.bios.2015.06.001 CrossRefGoogle Scholar
  13. 13.
    Muir W, Barden MC, Collett SP, Gorse A-D, Monteiro R, Yang L, McDougall NA, Gould S, Maeji NJ (2007) High-throughput optimization of surfaces for antibody immobilization using metal complexes. Anal Biochem 363:97–107.  https://doi.org/10.1016/j.ab.2007.01.015 CrossRefGoogle Scholar
  14. 14.
    Serafín V, Torrente-Rodríguez RM, Batlle M, García de Frutos P, Campuzano S, Yáñez-Sedeño P, Pingarrón JM (2017) Electrochemical immunosensor for receptor tyrosine kinase AXL using poly(pyrrolepropionic acid)-modified disposable electrodes. Sensors Actuators B Chem 240:1251–1256.  https://doi.org/10.1016/j.snb.2016.09.109 CrossRefGoogle Scholar
  15. 15.
    Eguílaz M, Moreno-Guzmán M, Campuzano S, González-Cortés A, Yáñez-Sedeño P, Pingarrón JM (2010) An electrochemical immunosensor for testosterone using functionalized magnetic beads and screen-printed carbon electrodes. Biosens Bioelectron 26:517–522.  https://doi.org/10.1016/j.bios.2010.07.060 CrossRefGoogle Scholar
  16. 16.
    Dong H, Cao X, Li CM, Hu W (2008) An in situ electrochemical surface plasmon resonance immunosensor with polypyrrole propylic acid film: comparison between SPR and electrochemical responses from polymer formation to protein immunosensing. Biosens Bioelectron 23:1055–1062.  https://doi.org/10.1016/j.bios.2007.10.026 CrossRefGoogle Scholar
  17. 17.
    Ball GM, in "Vitamins in foods: analysis, Bioavalaibility, and stability", CRC Taylor and Francis, Boca Raton FL (2006) p. 223.Google Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Analytical Chemistry, Faculty of ChemistryComplutense University of MadridMadridSpain

Personalised recommendations