Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device.

Monitoring the periodic diurnal variations in cortisol from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. Current detection assays are immunologically based and require cumbersome antibody immobilization chemistries, thereby limiting the assay versatility, kinetics, and reproducibility. We present a quantitative aptamer-based detection methodology for cortisol that does not require target labeling, capture probe immobilization on the detection surface or wash steps prior to readout. Using a recognition system of aptamer functionalized gold nanoparticles pre-bound with electro-active triamcinolone, the cortisol level is detected based on its competitive binding to the aptamer by following signal from the displaced triamcinolone using square wave voltammetry at patterned graphene-modified electrodes in a microfluidic or nanoslit device. Due to the 3D analyte diffusion profile at the aptamer interface and the ability to enhance the surface area for cortisol capture, this assay shows signal linearity over a five-log analyte concentration range (10 µg/mL to 30 pg/mL) and exhibits rapid binding kinetics with cortisol versus other glucocorticoids, as apparent from the absence of interferences from estradiol, testosterone and progesterone. The assay is carried out within the biologically relevant range for glucocorticoids in serum and saliva matrices, and benchmarked versus ELISA and radioimmunoassays. Based on absence of cumbersome surface immobilization and wash steps for carrying out this assay, its quantitative signal characteristics and its ability to resist interferences from other glucocorticoids, we envision its application towards routine monitoring of cortisol within bio-fluids.