Sensing Floss—Saliva Diagnostics on a Thread

Stress significantly impacts a patient's overall health; when stressed, the adrenal gland produces corticosteroids (such as cortisol) and adrenaline into the bloodstream. Cortisol negatively affects the regulation of various physiological processes such as carbohydrate metabolism and blood glucose levels. Long-term stress can disrupt homeostasis in the cardiovascular, renal, skeletal, and endocrine systems, leading to the development of chronic diseases. Moreover cortisol levels vary throughout the day. Because of its central role, continuous monitoring of cortisol levels in the human body is critical for health and wellness. Cortisol levels are often measured using immunochemical and analytical methods in blood, plasma, serum, oral fluid, sweat, and hair samples. Of these methods, saliva provides the most abundant, non-invasive source of cortisol biomarker. Towards the goal of routine stress-level examination, we engineer a thread-based electrochemical sensor for quantifying cortisol directly in saliva. The thread platform resembles a floss, which we define here as “sensing floss”. Threads offer the most flexible option for tissue-embedded sensing, microfluidics, and bioelectronics compared to other flexible platforms. This work demostartes the design of thread-based sensing floss both as a saliva sampler and for the realization of ultrasensitive and label-free electrochemical detection of cortisol in saliva using differential pulse voltammetry (DPV). To fabricate the cortisol sensing floss, a commercial gold-coated thread was used as a conductive base electrode surface to capture and strengthen the signal. The gold-coated thread was initially cleaned with isopropyl alcohol (sonication, 15 minutes) and plasma treatment (5 minutes), resulting in a smoother and cleaner surface for immobilization of the recognition element. The gold thread surface was then treated with dithiobis(succinimidyl propionate) (DSP, 4 mM in DMSO), formed a self-assembled monolayer, and rinsed with DMSO and phosphate buffer. Later, it was incubated with a designated concentration of cortisol-specific monoclonal antibody, mAb-cortisol (10 ngmL^-1) at 4°C for 4 hrs under a humid environment, and further incubated with bovine serum albumin (BSA) to incorporate an antifouling layer, and finally washed with buffer. The detection of the target analyte (cortisol) is based on the binding event between the cortisol molecule and the covalently immobilized mAb-Cortisol on the gold electrode surface, where an adopted conformation of the antigen-antibody complex alters the electron transfer rate at the electrode, resulting in a drop in measured current level. The surface topology and electrochemical properties of a cortisol-biosensor were studied using Fourier transform infrared spectroscopy, scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Under optimum experimental circumstances, the devised cortisol-biosensor demonstrated a dynamic range from 1.0 pg mL^-1 to 1.03 ng mL^-1 with a linear range of 1.0 pg mL^-1 to 0.52 ng mL^-1 and a limit of detection (LOD) of 1.0 pg mL^-1 (S/N = 3) in cortisol spiked saliva samples. DPV measurements are carried out in the presence of a redox probe containing 1.0 mM Ferri/Ferro cyanide in PBS (0.10 M, pH 7.2, 0.10M KCl). Our results show that the sensing floss is capable of detecting cortisol in both spiked buffer media and saliva samples. The analytical figures of merit of the cortisol-sensing platform confirm that the designed sensing floss could be particularly successful for low-level detection and continuous monitoring of salivary cortisol. The sensing floss can be extended to detection of other biomarkers in saliva for variety of health indicators and disease conditions.