Detection of B-type natriuretic peptide (BNP) at screen printed carbon electrodes modified with gold nanoparticles grafted through aryl diazonium salt chemistry

Trabajo presentado en la XXI Reunión de la Sociedad Española de Química Analítica, celebrada en Valencia, del 5 al 7 de septiembre de 2017A key step in the proper functioning of an electrochemical biosensor is the surface architecture connecting the biological sample to the transducer. The type of materials involved, its geometry and size strongly affect the response of the biosensor. Furthermore, gold nanoparticles (AuNPs) have a strong capacity for biomolecules adsorption without losing of biological activity and facilitate the electron transfer between redox biomaterial and the electrode surface. Thus, immobilization of AuNPs on bulk electrodes using covalently attached organic layers containing thiol groups and amino groups on their distal end offers a simple, fast, and versatile approach for preparing nanoparticle electrode arrays with low background capacitance. On the order hand, since the signs and symptoms of heart failure (HF) are not very specific and the availability of echocardiography is limited, a very interesting alternative approach to diagnosis HF is to determine accurately the blood concentration of natriuretic peptides (NPs). Between them, the B-type natriuretic peptide (BNP) is a 32-amino acid polypeptide secreted by the ventricles of the heart in response to excessive stretching of heart muscle cells. Interestingly, it has been found that BNP concentration in serum is elevated in HF patients compared to controls, establishing a cut-off value of 400 pg mL-1 . With the purpose to develop a competitive electrochemical strategy for the accurate determination of clinically relevant levels of BNP in serum samples, in this work, we have prepared a sensitive electrochemical immunosensor for this polypeptide determination based on the immobilization of capture antibodies onto gold nanoparticles grafted through aryl diazonium salt chemistry onto screen-printed carbon electrodes (SPCEs) by means of a sandwich type immunoassay involving a peroxidase-labeled detector antibody. This novel nanostructured platform resulted in an ordered layer of gold nanoparticles adsorbed onto 4-aminothiophenol which combines the advantages of the high conductivity and the stability of immobilized biomolecules. To monitor the extension of the affinity reactions the amperometric transduction at disposable screen-printed carbon electrodes was performed at -200 mV (vs the Ag pseudo-reference electrode) upon addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. After optimizing all the experimental variables affecting the operation and response of this immunosensing approach its analytical performance was characterized and their practical applicability successfully demonstrated by analysis of real human serum samples. The promising analytical performance exhibited by this new amperometric immunosensor of simple operation, disposable SPCE format and possibility to use pocket-size electrochemical transducers makes it very attractive alternative to commonly used ELISAs for the development of automated POC systems for on-site determination of this powerful biomarker associated with HF.The financial support of projects: Retos Colaboración RTC-2015-4184-1 (cofinanced by the Ministry of Economy and Competitivity and FEDER “una manera de hacer Europa”), CTQ2015-70023-R and CTQ2015-64402-C2-1-R (MINECO Research Projects) and S2013/MT-3029 (NANOAVANSENS Program from the Comunidad de Madrid) are gratefully acknowledged.Peer reviewe