Introduction Progesterone also known as

Introduction Progesterone, also known as P4, is a steroid hormone acting as a key component in the complex regulation of female reproductive function. P4 plays major physiological roles in the uterus and ovary by releasing mature oocytes helping in implantation and maintenance of pregnancy, as well as in the mammary gland in preparation for milk secretion and suppression of milk protein synthesis before parturition. In the brain, this hormone mediates signals required for sexually responsive behavior, and also supports modulation of bone mass [1]. The ovary is the main site of synthesis and secretion of P4 in the mammal and gives rise to cyclic fluctuations in the levels of circulating hormone. Measurement of P4 in serum or plasma constitutes a routine feature in the clinical investigation of reproductive function, where the changes in its concentration can be used to detect the occurrence of ovulation and to indicate the end of the fertile period. However, an accurate assessment of hormonal status often needs repetitive venipuncture and, thus, development of less invasive methods are strongly encouraged. In this context, saliva is an optimal diagnostic material because its non-invasive and cost-efficient method of sample collection in sufficient amounts for disease screening without the need for highly trained professionals [2]. P4 content in saliva has been measured as an index of ovarian function. The level rarely exceeds 150 pmol L−1 (47 pg mL−1) in the follicular phase and increases in the post-ovulatory period up to peak values from 300 to 750 pmol L−1 (94–235 pg mL−1) six days after ovulation [3]. Common methods for P4 determination use colorimetric ELISA immunoassays involving direct competitive formats where the target analyte competes with a labeled hormone for the limited number of immobilized antibody binding sites. Some of the available commercial kits are applicable to saliva samples. For example, Abcam\'s ab178652-Progesterone ELISA Kit [https://www.abcam.com/progesterone-elisa-kit-ab178652.html] involving wells coated with anti-P4 capture Selonsertib and peroxidase-labeled progesterone (HRP-P4), provides a non-linear calibration plot between 10 and 5000 pg mL−1 P4 with a minimum detectable concentration (MDC) of 3.8 pg mL−1. Precision levels for intra- and inter-assay are 14 and 10%, respectively, with an assay time of 75 min. Similar analytical characteristics are claimed by Salimetrics\' Salivary Progesterone Enzyme Immunoassay Kit [https://salimetrics.com/wp-content/uploads/2018/03/progesterone-saliva-elisa-kit.pdf], with the calibration plot ranging between 10 and 2430 pg mL−1 P4, and a MDC of 5 pg mL−1. Regarding electrochemical immunosensors, few configurations have been reported in the literature and they are mainly applied to the determination of the hormone in cow\'s milk by competitive immunoassay. Strategies involving electrodes modified with gold nanoparticles provide linear calibration plots for P4 ranging between 0 and 40 ng mL−1 [4] and 0.08–7 ng mL−1 [5] with limits of detection (LODs) of 430 pg mL−1 and 80 pg mL−1, respectively. Recently, an electrochemical platform prepared with a thionine-graphene oxide composite offers a linear current vs. log [P4] calibration plot in the 0.02–20 ng mL−1 range with a limit of detection of 6.3 pg mL−1 [6]. In this paper, the construction of a simple and accurate device for the amperometric determination of P4 in raw undiluted saliva is reported. Magnetic microbeads (MBs) were used as solid support to perform the immunoreactions. A direct competitive immunoassay using anti-P4 capture antibodies immobilized onto commercial Protein G-modified MBs was implemented followed by amperometric detection at disposable screen-printed carbon electrodes using the HRP/H2O2/hydroquinone (HQ) system. After optimization of the methodology for the determination of P4, it was applied to the quantification of the endogenous P4 content in saliva samples, and successfully compared with data supplied by the conventional ELISA methodology. The successful coupling of the immunosensor with a low-cost and portable potentiostat developed with COTS components allows sending data readings wirelessly to a remote system for further study and a stand-alone operation with no need for any external control system.