Real-Time Cortisol Detection Using Electrocatalytic Polymer-Based Sensor

Abstract

Cortisol is a steroid hormone produced by the adrenal gland in response to stress or agitate states. Deficiency or excess cortisol level can be triggered by physical or psychological stress, which is considered a biomarker for many diseases, such as Cushing syndrome [1]. In the human body, cortisol concentrations fluctuate in a circadian rhythm, being the highest in the early morning and the lowest in the night. A reliable cortisol test cannot depend on a single or one-day average measurement. Instead, screening a patient's dynamic cortisol levels over the circadian cycle is expected. The existing clinical cortisol measurements require blood, urine, or saliva samples sent to a lab to await results. The detection methods are mainly based on immunoassay that requires labeled antigens for a competitive assay and time-consuming incubation time (15–30 min), which hinders continuous detection needed for real-time stress management. To enable accurate time course measurements, a new detection approach is required to allow real-time determination of cortisol without costly reagents, labeling processes, additional redox reagents, and cumbersome laboratory equipment.Nanomaterials that mimic the catalytic behaviors of enzymes have great potential for continuous monitoring of cortisol levels through the direct electrochemical reading from cortisol redox reaction. There are very few reports on artificial cortisol catalytic sensors which require complicated fabrication or extremely non-neutral pH conditions for sensing [2]. Copper phthalocyanine (CuPc), a macrocycle with transitional copper (Cu) ion, holds electrocatalytic behavior and can be utilized for electrochemical redox reactions of various biomolecules, including H2O2, phenol, dopamine and serotonin. However, the detection of steroid hormones using CuPc has not been demonstrated yet. Compared to similar compounds based on porphyrin, phthalocyanine has N atoms in the meso positions and rich π-electrons that permit the formation of stable metallic phthalocyanine complexes. In this work, we develop copper phthalocyanine tetrasulfonate doped polypyrrole (PPy:CuPcTS) nanocomposite onto disposable screen-printed electrodes (SPEs) and demonstrate its electrocatalytic activity for real-time cortisol sensing. The incorporation of water-soluble CuPcTS into the PPy film forms an electrochemically conducting polymer in the negative potential window and catalyzes the reduction reaction of cortisol. The PPy:CuPcTS/SPE sensor can directly determine cortisol concentrations in real-time.The PPy:CuPcTS film was formed through one-step anodic electropolymerization on SPEs. The morphology and elemental composition of the film were characterized by scanning electron microscopy (SEM) and energy dispersive X-Ray analysis (EDX), respectively. Electrochemical responses were obtained with cyclic voltammetry (CV) and chronoamperometry. Cortisol detection was performed with 1x PBS solution.During the electropolymerization of PPy with CuPcTS, parts of the pyrrole rings hold a positive charge and form a complex with the sulfonated group in the CuPcTS. The CuPcTS and the PPy rings can also bond through π-π stacking. The stacked arrangement creates more conduction pathways due to highly delocalized electrons. The chelated Cu in CuPcTS catalyzes the reduction of ketone group in cortisol to an alcohol group [3], as schematically shown in Fig. 1(a). The SEM images in Fig.1 (b) show that the polymerization of PPy in the presence of CuPcTS results in a rough surface, very different from the smooth surface morphology on an undoped PPy film. The successful doping of CuPcTS in the PPy film is further confirmed by the presence of Cu peaks in the EDX (Fig.1 (b)). The CV curves in Fig. 1(c) indicate that cortisol reduction can be achieved using PPy:CuPcTS/SPE, evidenced by the increase in cathodic current at -1 V vs. Ag/AgCl upon adding cortisol. The phenomenon was not observed with PPy/SPE. The cathodic current increases with cortisol concentrations. The proposed sensor provides a dynamic detection range from 1 nM to 500 nM (Fig. (d)). Fig. 1(e) shows that the cortisol sensor also exhibits a selective detection against common interfering molecules, including glucose, ascorbic acid, and dopamine.PPy:CuPcTS nanocomposite was successfully demonstrated for electrocatalytic cortisol sensing. The sensor was achieved by a facile fabrication method and can operate at neutral pH without redox probes and labeling steps. The new sensor possesses great potential for low-cost, real-time monitoring of stress levels.