Beatriz Arévalo a , V. Serafín a , J. F. Beltrán-Sánchez b , J. Aznar-Poveda b , A. J. García-Sánchez b , J. García-Haro b , S. Campuzano a , P. Yáñez-Sedeño a and J. M. Pingarrón a . a Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid, Ciudad Universitaria S/N, 28040, Madrid, Spain. E-mail: [email protected] b Department of Information and Communication Technologies (ICT), Technical University of Cartagena, ETSIT, Campus Muralla del Mar, 30202, Cartagena, Spain. Fertility-related hormones are the main components that regulate the reproductive cycle Fertility-related hormones are involved in ovulation and pregnancy HYPOTHALAMUS PITUITARY GLAND PRL LH FSH E2 P4 Gonadotropic hormones Sex steroid hormones GnRH OVARIES UTERUS PROLACTIN ( PRL) stimulates milk production in mammary glands and progesterone synthesis in the corpus luteum PROGESTERONE ( P4) prepares the uterus for pregnancy LUTEINIZING HORMONE ( LH) stimulates the formation of the corpus luteum and ovulation ESTRADIOL ( E2) keeps the oocyte in the ovaries and acts as a growth hormone for reproductive organs + + ProtG-MBs cAb P4/ P4-HRP + + PRL NA-MBs dAb-HRP b-cAb + + LH NA-MBs dAb-HRP b-cAb + + ProtG-MBs cAb E2/ E2-HRP e- BQ HRPox H2O2 HQ HRPred H2O i, nA t, s -14 -12 -10 -2 0 2 4 6 0 50 100 150 200 250 300 -i, nA Log([P4], ng mL -1 ) 0 20 40 60 80 100 0 200 400 600 800 1000 -i, nA [LH], mIU mL -1 -14 -12 -10 -2 0 2 4 0 200 400 600 800 1000 -i, nA Log([E2], ng mL -1 ) 0 20 40 60 80 100 0 200 400 600 800 1000 1200 -i, nA [PRL], ng mL -1 ANALYTICAL CHARACTERISTIC P4, pg mL -1 E2, pg mL -1 LH, mIU mL -1 PRL, ng mL -1 Linear range 38.1 250.0 23.3 50,000.0 1.0 100.0 1.0 100.0 R 2 0.999 0.998 0.999 0.999 EC 50 6 1 1.8 0.2 - - LOD 2.85/1.7* 5.88/6.88* 0.30/0.10* 0.32/0.32** p (Hill Slope) 0.48 0.04 0.88 0.09 - - RSD, (n = 10,[hormone]), % 2.20 (10 ng mL -1 ) 2.26 (5 ng mL -1 ) 1.80 (50 ng mL -1 ) 2.66 (25 ng mL -1 ) DIRECT COMPETITIVE FORMAT SANDWICH FORMAT ANALYTICAL CHARACTERISTICS SELECTIVITY and CROSS - REACTIVITY 0 200 400 600 800 1000 1200 1400 SPCE SP 4 CE SPCE SP 4 CE SPCE SP 4 CE SPCE SP 4 CE P4 LH E2 PRL 0 200 400 600 800 1000 1200 1400 0 2 4 6 8 10 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 0 2 4 6 8 10 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 0 2 4 6 8 10 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 -i, nA 0 2 4 6 8 10 0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 0 2 4 6 8 10 B/S (P4, E2); S/B (LH, PRL) Man Woman Breastfeeding woman Menopausal woman 0 20 40 60 -i, nA ESTRADIOL Man Woman Breastfeeding woman Menopausal woman 0 20 40 60 80 100 -i, nA PROLACTIN Man Woman Breastfeeding woman Menopausal woman 0 5 10 15 20 -i, nA LUTEINIZING HORMONE Man Woman Breastfeeding woman Menopausal woman 0 20 40 60 80 100 120 140 -i, nA PROGESTERONE CHI potentiostat SALIVA SAMPLES SP4CE Magnet * at SPdCE/ ** at SPCE First multiplexed immunoplatform for the amperometric multidetermination of four fertility-related hormonoes: P4, E2, LH and PRL Methodology based on the modification of the MBs’ surface with competitive (P4, E2) or sandwich (LH, PRL) immunocomplexes. Attractive analytical characteristics and potential to determine simultaneously the target hormones in saliva samples. The developed immunoplatform, coupled to a custom designed quadruple potentiostat fabricated using commercial off-the-shelf components (COTS-based potentiostat), can be easily exploited to analyze a larger number of samples or hormones as a POC device for the monitoring of fertility processes CONCLUSIONS ACKNOWLEDGEMENTS ARISE2 Project “Future IoT Networks and Nano-networks (FINe)” ((AEI)/ (FEDER), (UE)) under ID PID2020-116329GB-C22; ATENTO project (Fundación Séneca, Región de Murcia), under grant 20889/PI/18; LIFE project (Fondo SUPERA COVID-19 funded by Consejo Superior de Investigaciones Científicas CSIC, Universidades Españolas and Banco Santander); the financial support of RTI2018- 096135-B-I00 (Spanish Ministerio de Ciencia, Innovación y Universidades) and PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación); Research Projects, and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT- 4349) are gratefully acknowledged. B. Arévalo and J. Aznar-Poveda acknowledge Predoctoral contracts from the Spanish Ministerio de Economia y Competitividad (PRE2019-087596 and BES-2017-081061, respectively). Block diagram showing the different electronic components employed (a), three-dimensional rendering of the designed and own-manufactured acquisition board (b), and an overall view of the constructed device for the simultaneous determination of P4, LH, E2 and PRL hormones in saliva (c). SPCE SP 4 CE WE1: P4 WE2: LH WE3: E2 WE4: PRL COTS-based potentiostat ELISA E=-0.2 V vs. Ag pseudo-reference electrode