Introduction Insufficient or elevated hormone cortisol levels triggered by physical or psychological stress can cause several health problems, such as Addison's disease, Cushing's syndrome, and chronic fatigue syndrome. High serum cortisol levels are also related to increased mortality in COVID-19 patients. Thus, sensitive cortisol detection can provide an early indication for critical patients and monitor treatment efficacy. The physiological range of salivary cortisol fluctuates in a circadian rhythm between sub-nanomolar to nanomolar range. Late-night salivary cortisol concentration in the range of 2.76-166 nM was determined for clinically confirmed Cushing’s patients. A proper cortisol test requires mapping a patient’s cortisol levels over cycle of 24 hours. Most of the reported cortisol immunosensors involve biological receptors, such as antibodies, which are expensive, require refrigerated transportation and storage, and suffer from steric hindrance arising from antibody-small molecule interactions [1]. Cortisol immunoassays also suffer from a slow response (30 min to 5 hr) and cross-reactivity with structurally similar hormones, such as prednisolone and progesterone.Alternatively, the biorecognition can be realized using molecularly imprinted polymer (MIP), which provides artificial binding sites and cavities in polymer for target-specific binding. MIP holds numerous advantages over antibodies, including longer lifetimes, higher stability, and cost-effectiveness [2]. Yet, there remain several issues in the MIP-based biosensors. For instance, conventional particle-based MIP requires a time-consuming and tedious polymerization process. The coating of such MIP tends to result in an uncontrolled thickness, slow diffusion process for targets, and inefficient signal transduction and limited sensitivity. We overcome these shortcomings by creating controllable MIP thin films with target selective binding sites through electropolymerization. We also dope the MIP with nano gold to enhance the sensitivity of cortisol detection. Method The MIP was formed directly onto a gold nanoparticle-deposited glassy carbon electrode (GCE) through one-step voltammetric electropolymerization (inset to Figure 1a) with a mixture of o-phenylenediamine (o-PD) and HAuCl4 in the presence of cortisol template. Imprinted cortisol molecules were eluted by washing in an ethanol solution to create cortisol specific imprinting sites into the Au-poly-o-PD composite film. For the control experiment, a non-imprinted polymer (NIP) film was also prepared under the same process conditions in the absence of templates. We controlled the process conditions (e.g., the ratio of monomer/ HAuCl4/ template, number of polymerization cycles, pH, elution buffer, elution, and reaction time) to optimize the performance of the MIP sensor. The sensor's electrochemical responses were performed with cyclic voltammetry (CV) and square wave voltammetry (SWV). Results and Discussion The MIP film can be produced by electropolymerization within 20 min to achieve reproducible and controllable characteristics. During electropolymerization, AuNPs grow and anchor to the polymeric framework, possibly through the abundant amine groups on o-PD. This hybrid metal-polymer composite film could increase the electron transfer rate for the reaction with the redox reagents and thus enhance the sensitivity of cortisol detection. During electrodeposition, cortisol molecules were imprinted into composite film, which can be attributed to the H-bond between the hydroxyl groups on cortisol and amine groups on o-PD. The sensing mechanism relies on the change in redox current peak measured in the presence of the redox reagents, K3Fe(CN)6 and K4Fe(CN)6. The current decreases with the increase of the target cortisol binding on the MIP that hinders the redox reagents' access to the electrode. The CV curves in Figure 1a characterize the sensor electrode during MIP fabrication and cortisol sensing. The proposed sensor responds to cortisol detection within 8 min. The peak currents decrease with the increase in cortisol concentration (Figure 1b) due to the reduced number of imprinted cavities. The sensor exhibits a dynamic range of detection covering the physiological cortisol level from 10 pM to 100 nM with a detection limit of 1.5 pM, while the NIP shows negligible change under various cortisol concentrations (Figure 1c). Figure 1d shows that the cortisol MIP sensor also exhibits a highly selective detection against several structurally similar hormones, including estriol, estrone, progesterone, and β-estradiol. Conclusions We demonstrate nano gold-doped MIP electrode for sensitive, rapid, and selective detection of the hormone cortisol. The cost-effective, robust sensor provides an alternative to the conventional immunoassay and holds great potential for point-of-care cortisol detection.