A DNA aptamer with affinity and specificity for human osteopontin (OPN), a potential breast cancer biomarker, was selected using the SELEX process, considering its homology rate and the stability of its secondary structures. This aptamer exhibited a satisfactory affinity towards OPN, showing dissociation constants lower than 2.5 nM. It was further used to develop a simple, label-free electrochemical aptasensor against OPN. The aptasensor showed good sensitivity towards OPN in standard solutions, being the square wave voltammetry (SWV), compared to the cyclic voltammetry, the most sensitive technique with detection and quantification limits of 1.4 ± 0.4 nM and 4.2 ± 1.1 nM, respectively. It showed good reproducibility and acceptable selectivity, exhibiting low signal interferences from other proteins, as thrombin, with 2.6-10 times lower current signals-off than for OPN. The aptasensor also successfully detected OPN in spiked synthetic human plasma. Using SWV, detection and quantification limits (1.3 ± 0.1 and 3.9 ± 0.4 nM) within the OPN plasma levels reported for patients with breast cancer (0.4-4.5 nM) or with metastatic or recurrent breast cancer (0.9-8.4 nM) were found. Moreover, preliminary assays, using a sample of human plasma, showed that the aptasensor and the standard ELISA method quantified similar OPN levels (2.2 ± 0.7 and 1.7 ± 0.1 nM, respectively). Thus, our aptasensor coupled with SWV represents a promising alternative for the detection of relevant breast cancer biomarkers.
The authors acknowledge the financial support from the Strategic
funding of UID/BIO/04469/2013 unit and COMPETE 2020
(POCI-01-0145-FEDER-006684), and from project BioTecNorte
(project number NORTE-01-0145-FEDER-000004). This work was
also financially supported by Project POCI-01e0145-FEDER-006984
e Associate Laboratory LSRE-LCM and by Project UID/QUI/00616/
2013 e CQ-VR both funded by FEDER - Fundo Europeu de Desenvolvimento
Regional through COMPETE2020 - Programa Operacional
Competitividade e Internacionalização (POCI) e and by
national funds through FCT - Fundaç~ao para a Ciência e a Tecnologia,
Portugal. S. Meirinho also acknowledges the research grant
provided by Project UID/EQU/50020/2013.
