Microfluidic immunosensor for rapid and highly-sensitive salivary cortisol quantification

This paper presents a novel poly(dimethylsiloxane) (PDMS) microfluidic immunosensor that integrates a complementary metal-oxide-semiconductor (CMOS) optical detection system for a rapid and highly-sensitive quantification of salivary cortisol. The simple and non-invasive method of saliva sampling provides an interesting alternative to the blood, allowing a fast sampling at short intervals, relevant for many clinical diagnostic applications. The developed approach is based on the covalent immobilization of a coating antibody (Ab), a polyclonal anti-IgG, onto a treated PDMS surface. The coating Ab binds the capture Ab, an IgG specific for cortisol, allowing its correct orientation. Horseradish peroxidase (HRP)-labelled cortisol is added to compete with the cortisol in the sample, for the capture Ab binding sites. The HRP-labelled cortisol, bonded to the capture Ab, is measured through the HRP enzyme and the tetramethylbenzidine (TMB) substrate reaction. The cortisol quantification is performed by colorimetric detection of HRP-labelled cortisol, through optical absorption at 450 nm, using a CMOS silicon photodiode as the photodetector. Under the developed optimized conditions presented here, e.g., microfluidic channels geometry, immobilization method and immunoassay conditions, the immunosensor shows a linear range of detection between 0.01-20 ng/mL, a limit of detection (LOD) of 18 pg/mL and an analysis time of 35 min, featuring a great potential for point-of-care applications requiring continuous monitoring of the salivary cortisol levels during a circadian cycle.FCT with the reference project UID/EEA/04436/2013, by FEDER funds through the COMPETE 2020 – Programa Operacional Competitividade e Internacionalização (POCI) with the reference project POCI-01-0145-FEDER-006941. V C Pinto thanks the FCT for the SFRH/BD/81526/2011 PhD grant. P J Sousa thanks the FCT for the SFRH/BD/81562/2011 PhD grant. S.O. Catarino thanks the FCT for the SFRH/BPD/108889/2015 grant, supported by national funds from Ministérios da Ciência, Tecnologia e Ensino Superior and by FSE through the POCH - Programa Operacional Capital Humano