New insights into the construction of a µPAD for cortisol detection in human saliva samples freshly extracted without previous treatment

Stress is a disease of modern life generated by excess cortisol hormone levels. A paper-based microanalytical device (µPAD) was developed to analyze cortisol in human saliva samples without prior purification treatment. The design includes optimized flow channels to use freshly extracted human saliva samples. The density, viscosity, and pH properties of human saliva and artificial saliva models were characterized. These properties helped to establish optimal theoretical dimensions for our µPAD design, which works with untreated human saliva. A microfluidic engineering analysis was performed to understand the dynamics of the sample flow in the sensor, obtaining a design that allows free and rapid transport of freshly extracted human saliva, covering the surface of the µPADs in approximately three minutes. Its characteristic design allows for the direct use of human saliva without pretreatment, making it an effective tool as a universal device with multiple applications in different research fields, specifically as a point-of-care (POC) device. After the design was developed, a proof-of-concept method based on colorimetric detection of salivary cortisol, similar to enzyme-linked immunosorbent assay (ELISA) analysis, was performed using gold nanoparticles. The colorimetric response was obtained after 7 min of adding the fresh human saliva sample and the corresponding feedback after UV light lamp illumination. The authors designed a paper-based sensor that works with freshly extracted human saliva without any previous treatment for the detection of hormonal stress, generating an advance in biomolecular detection for use as a POC tool.