Wearable sweat sensor for ovulatory hormone monitoring

Abstract

Tracking ovulatory hormone levels is key to detecting fertility windows and providing actionable information for facilitating or preventing conception, as well as assessing and managing infertility and other conditions associated with hormone imbalances. Conventional fertility methods suffer drawbacks associated with invasive sampling, inadequate prediction accuracies, or repetitive user intervention. To overcome these and other limitations, the present Applicants developed a wearable device for contemporaneous monitoring of estradiol, progesterone, and luteinizing hormone and physiological biomarkers (temperature, pH). Flexible field-effect transistors functionalized with biomarker-specific aptamers enabled sensitive and selective determination of the three principal ovulatory hormones across physiological ranges in artificial human sweat. By including an on-demand iontophoretic sweat sampling feature, the present Applicants established an integrated wearable platform for high-accuracy, minimally invasive, rapid, and personalized ovulatory status monitoring.

Description

WEARABLE SWEAT SENSOR FOR OVULATORY HORMONE MONITORINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to United States Provisional Patent Application No. 63 / 560,522, filed March 1, 2024, the contents of which are incorporated herein by reference in their entirety.STATEMENT OF GOVERNMENT SPONSORED RESEARCH

[0002] This invention was made with government support under DA045550 awarded by the National Institute of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present embodiments relate generally to health care, and more particularly to a wearable sensor such as a skin patch that tracks ovulation hormones to predict fertility windows accurately.BACKGROUND

[0004] Women spend more than half their lives managing their reproductive status. This amounts to trying to facilitate or prevent pregnancy for 35-40 years on average (H.-W. Su, Y.-C. Yi, T.-Y. Wei, T.-C. Chang, C.-M. Cheng, Detection of ovulation, a review of currently available methods. Bioeng. Transl. Med. 2, 238-246 (2017)). The recent U.S. Supreme Court decision rescinding constitutional rights to legal and safe abortion and the associated reduction in access to hormonal fertility management underscores the need for nonpharmacologic methods to determine daily fertility in a highly accurate manner for actionable decision-making. In addition to fertility monitoring, ovulatory hormones are measured for clinical indications, including diagnosing endometriosis and polycystic ovary syndrome, detecting and managing perimenopause, and assessing several types of cancers (D. Unuane, H. Tournaye, B. Velkeniers, K. Poppe, Endocrine disorders & female infertility. Best Pract. Res. Clin. Endocrinol. Metab. 25,861-873 (2011)). Gold-standard hormone levels measured by laboratory assays require blood draws and are costly, time-intensive, and inconvenient for repeated measurements rendering them nonideal for personal monitoring applications.

[0005] Nonhormonal approaches for predicting fertility windows include fertility -based awareness methods (i.e., menstrual cycle calendar logs, basal body temperature tracking, and monitoring changes in cervical mucous) (Id.). While accessible, methods that rely on selfreporting and calendar algorithms based on average cycle lengths result in ovulation prediction accuracies of only about 20% (S. Johnson, L. Marriott, M. Zinaman, Can apps and calendar methods predict ovulation with accuracy? Curr. Med. Res. Opin. 34, 1587-1594 (2018)). Adding daily temperature measurements improves accuracy, but factors such as stress, illness, alcohol or drug use, disrupted sleep, and the intrinsic variability of the menstrual cycle still result in less- than-ideal prediction accuracies (R. P. Stephen, R. B. George, Fertility awareness-based methods: Another option for family planning. J. Am. Board Fam. Med. 22, 147-157 (2009)).

[0006] Commercial wearable devices (e.g., Oura Ring, Ava Bracelet) have emerged as tools to track a variety of biophysical markers including heart rate, heart rate variability, skin temperature, blood oxygen level, and respiratory rate. A company-funded longitudinal study of the Ava Bracelet in >200 participants found that biophysical markers might enable ovulation prediction accuracies up to 90% in women with average (24 35-day) cycles when coupled with machine learning algorithms (B. M. Goodale, M. Shilaih, L. Falco, F. Dammeier, G. Hamvas, B. Leeners, Wearable sensors reveal menses-driven changes in physiology and enable prediction of the fertile window: Observational study. J. Med. Internet Res. 21, el 3404 (2019)). Nonetheless, 90% accuracy could result in as many as 40 chances for unplanned pregnancy across the reproductive lifespan. Platforms that integrate biophysical and biochemical sensing modalities, the latter tracking hormonal changes across menstrual cycles, will enable greater ovulation prediction accuracy, particularly in the context of cycle irregularities and other extenuating factors.

[0007] Luteinizing hormone (LH) was initially thought to be the best indicator of ovulation. However, recent studies have cast doubt on its reliability (S. Krotz, L. J. McKenzie, P. Cisneros, J. Buster, P. Amato, S. Carson, Prevalence of premature urinary luteinizing hormone surges in women with regular menstrual cycles and its effect on implantation of frozen-thawedembryos. Fertil. Steril. 83, 1742-1744 (2005)). Surges in LH are variable and the estrogen rise that precedes ovulation may be more universally predictive (S. J. Park, L. T. Goldsmith, J. H. Skurnick, A. Wojtczuk, G. Weiss, Characteristics of the urinary luteinizing hormone surge in young ovulatory women. Fertil. Steril. 88, 684-690 (2007)). In 2013, commercial urinary lateral flow assays, such as Clearblue, incorporated estrogen metabolite detection, in tandem with LH, to detect more fertile days (up to 4) in a given cycle with up to 99% accuracy (Id.). Multiplexed lateral flow assays are now commercially available to measure urinary estradiol, progesterone, LH, and follicle-stimulating hormone (FSH) for fertility hormone tracking (e.g., Initio, Mira).

[0008] Wearable biosensors are attractive for personalized monitoring due to their ability to provide non- or minimally invasive, continuous feedback that does not require daily user intervention. Wearable biochemical sensors have been developed for electrolytes (W. Gao, S. Emaminejad, H. Y. Y. Nyein, S. Challa, K. Chen, A. Peck, H. M. Fahad, H. Ota, H. Shiraki, D. Kiriya, D.-H. Lien, G. A. Brooks, R. W. Davis, A. Javey, Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis. Nature 529, 509-514 (2016); J. R. Sempionatto, T. Nakagawa, A. Pavinatto, S. T. Mensah, S. Imani, P. Mercier, J. Wang, Eyeglasses based wireless electrolyte and metabolite sensor platform. Lab Chip 17, 1834-1842 (2017); A. Koh, D. Kang, Y. Xue, S. Lee, R. M. Pielak, J. Kim, T. Hwang, S. Min, A. Banks, P. Bastien, M. C. Manco, L. Wang, K. R. Ammann, K.-I. Jang, P. Won, S. Han, R. Ghaffari, U. Paik, M. J. Slepian, G. Balooch, Y. Huang, J. A. Rogers, A soft, wearable microfluidic device for the capture, storage, and colorimetric sensing of sweat. Sci. Transl. Med. 8, 366ral65 (2016); L. B. Baker, J. B. Model, K. A. Barnes, M. L. Anderson, S. P. Lee, K. A. Lee, S. D. Brown, A. J. Reimel, T. J. Roberts, R. P. Nuccio, J. L. Bonsignore, C. T. Ungaro, J. M. Carter, W. Li, M. S. Seib, J. T. Reeder, A. J. Aranyosi, J. A. Rogers, R. Ghaffari, Skin-interfaced microfluidic system with personalized sweating rate and sweat chloride analytics for sports science applications. Sci. Adv. 6, eabe3929 (2020); T. R. Ray, M. Ivanovic, P. M. Curtis, D. Franklin, K. Guventurk, W. J. Jeang, J. Chafetz, H. Gaertner, G. Young, S. Rebollo, J. B. Model, S. P. Lee, J. Ciraldo, J. T. Reeder, A. Hourlier-Fargette, A. J. Bandodkar, J. Choi, A. J. Aranyosi, R. Ghaffari, S. A. McColley, S. Haymond, J. A. Rogers, Soft, skin-interfaced sweat stickers for cystic fibrosis diagnosis and management. Sci. Transl. Med. 13, eabd8109 (2021); Y. Song, J. Min, Y. Yu, H. Wang, Y. Yang, H. Zhang, W. Gao, Wireless battery-free wearable sweat sensor powered byhuman motion. Sci. Adv. 6, eaay9842 (2020)), enzymes (B. Ciui, A. Martin, R. K. Mishra, B. Brunetti, T. Nakagawa, T. J. Dawkins, M. Lyu, C. Cristea, R. Sandulescu, J. Wang, Wearable wireless tyrosinase bandage and microneedle sensors: Toward melanoma screening. Adv. Healthc. Mater. 7, 1701264 (2018)), metabolites (J. Kim, J. R. Sempionatto, S. Imani, M. C. Hartel, A. Barfidokht, G. Tang, A. S. Campbell, P. P. Mercier, J. Wang, Simultaneous monitoring of sweat and interstitial fluid using a single wearable biosensor platform. Adv. Sci. (Weinh) 5, 1800880 (2018); S. Imani, A. J. Bandodkar, A. M. V. Mohan, R. Kumar, S. Yu, J. Wang, P. P. Mercier, A wearable chemical-electrophysiological hybrid biosensing system for real-time health and fitness monitoring. Nat. Comm. 7, 11650 (2016); H. Lee, C. Song, Y. S. Hong, M. Kim, H. R. Cho, T. Kang, K. Shin, S. H. Choi, T. Hyeon, D.-H. Kim, Wearable / disposable sweat-based glucose monitoring device with multistage transdermal drug delivery module. Sci. Adv. 3, el601314 (2017); J. Kim, G. Valdes-Ramirez, A. J. Bandodkar, W. Jia, A. G. Martinez, J. Ramirez, P. Mercier, J. Wang, Non-invasive mouthguard biosensor for continuous salivary monitoring of metabolites. Analyst 139, 1632-1636 (2014); H. Y. Y. Nyein, W. Gao, Z. Shahpar, S. Emaminejad, S. Challa, K. Chen, H. M. Fahad, L.-C. Tai, H. Ota, R. W. Davis, A. Javey, A wearable electrochemical platform for noninvasive simultaneous monitoring of Ca2+ and pH. ACS Nano 10, 7216-7224 (2016)), caffeine (L.-C. Tai, W. Gao, M. Chao, M. Bariya, Q. P. Ngo, Z. Shahpar, H. Y. Y. Nyein, H. Park, J. Sun, Y. Jung, E. Wu, H. M. Fahad, D.-H. Lien, H. Ota, G. Cho, A. Javey, Methylxanthine drug monitoring with wearable sweat sensors. Adv. Mater. 30, 1707442 (2018)), synthetic opioids and nerve agents (R. K. Mishra, K. Y. Goud, Z. Li, C. Moonla, M. A. Mohamed, F. Tehrani, H. Teymourian, J. Wang, Continuous opioid monitoring along with nerve agents on a wearable microneedle sensor array. J. Am. Chem. Soc. 142, 5991-5995 (2020)), nutrients (M. Wang, Y. Yang, J. Min, Y. Song, J. Tu, D. Mukasa, C. Ye, C. Xu, N. Heflin, J. S. McCune, T. K. Hsiai, Z. Li, W. Gao, A wearable electrochemical biosensor for the monitoring of metabolites and nutrients. Nat. Biomed. Eng. 6, 1225-1235 (2022)), inflammatory biomarkers (B. Jagannath, M. Pali, K.-C. Lin, D. Sankhala, P. Naraghi, S. Muthukumar, S. Prasad, Novel approach to track the lifecycle of inflammation from chemokine expression to inflammatory proteins in sweat using electrochemical biosensor. Adv. Mater. Technol. 7, 2101356 (2022); J. Tu, J. Min, Y. Song, C. Xu, J. Li, J. Moore, J. Hanson, E. Hu, T. Parimon, T.-Y. Wang, E. Davoodi, T.-F. Chou, P. Chen, J. J. Hsu, H. B. Rossiter, W.Gao, A wireless patch for the monitoring of C-reactive protein in sweat. Nat. Biomed. Eng. 7, 1293-1306 (2023)), and hormones (B. Wang, C. Zhao, Z. Wang, K. A. Yang, X. Cheng, W. Liu, W. Yu, S. Lin, Y. Zhao, K. M. Cheung, H. Lin, H. Hojaiji, P. S. Weiss, M. N. Stojanovic, A. J. Tomiyama, A. M. Andrews, S. Emaminejad, Wearable aptamer field-effect transistor sensing system for noninvasive cortisol monitoring. Sci. Adv. 8, eabk0967 (2022); C. Ye, M. Wang, J. Min, R. Y. Tay, H. Lukas, J. R. Sempionatto, J. Li, C. Xu, W. Gao, A wearable aptamer nanobiosensor for non-invasive female hormone monitoring. Nat. Nanotechnol., (2023). DOI: 10.1038 / s41565-023-01513-0; M. Pali, B. Jagannath, K.-C. Lin, S. Upasham, D. Sankhalab, S. Upashama, S. Muthukumar, S. Prasad, Catch (Cortisol Apta WATCH): ‘Bio-mimic alarm’ to track anxiety, stress, immunity in human sweat. Electrochim. Acta 390, 138834 (2021); N. K. Mintah Churcher, S. Upasham, P. Rice, S. Bhadsavle, S. Prasad, Development of a flexible, sweat-based neuropeptide Y detection platform. RSC Adv. 10, 23173-23186 (2020); B. K. Sarker, R. Shrestha, K. M. Singh, J. Lombardi, R. An, A. Islam, L. F. Drummy, Label-free neuropeptide detection beyond the Debye length limit. ACS Nano 17, 20968-20978 (2023)). Like cortisol, estradiol and progesterone are small-molecule steroid hormones (272.4 g / mol and 314.5 g / mol, respectively). Their low molecular weights along with lipophilicity enable sufficient diffusion through capillary and glandular epithelial cell membranes and thus, interstitial fluid or sweat concentrations that track with blood levels (R. M. Torrente-Rodriguez, J. Tu, Y. Yang, J. Min, M. Wang, Y. Song, Y. Yu, C. Xu, C. Ye, W. W. IsHak, W. Gao, Investigation of cortisol dynamics in human sweat using a graphene-based wireless mHealth system. Matter 2, 921-937 (2020)) for monitoring by wearables.

[0009] Estradiol and progesterone concentrations have been determined in eccrine sweat (C. C. Muir, K. Treasurywala, S. McAllister, J. Sutherland, L. Dukas, R. G. Berger, A. Khan, D. deCatanzaro, Enzyme immunoassay of testosterone, 17P-estradiol, and progesterone in perspiration and urine of preadolescents and young adults: Exceptional levels in men's axillary perspiration. Horm. Metab. Res. 40, 819-826 (2008)). These hormones are found at pM-nM concentrations and exhibit positive correlations with concentrations in urine, in addition to serum, both of which are standard clinically accessed biofluids. In contrast to steroid hormones, LH is a heterodimeric glycoprotein (33 kDa). While no previous studies were identified that determined LH levels in sweat, immunostaining shows the presence of LH receptors in thesecretory and ductal portions of sweat glands (J. E. Pabon, J. S. Bird, X. Li, Z. H. Huang, Z. M. Lei, J. S. Sanfilippo, M. A. Yussman, C. V. Rao, Human skin contains luteinizing hormone / chorionic gonadotropin receptors. J. Clin. Endocrinol. Metab. 81, 2738-2741 (1996); P. Y. Venencie, G. Meduri, S. Pissard, A. Jolivet, H. Loosfelt, E. Milgrom, M. Misrahi, Luteinizing hormone / human chorionic gonadotrophin receptors in various epidermal structures. Br. J.Dermatol. 141, 438-446 (1999)). These findings point to the presence of LH in sweat and the use of sweat as a readily accessible alternative to serum or urine for LH monitoring.

[0010] However, presently there are no wearable solutions for triple hormone monitoring for high-accuracy fertility tracking. Decades of literature support the underlying biology. Moreover, recent over-the-counter urinary lateral flow assays support the idea of quantitative measurement of multiple ovulatory hormones for high-accuracy fertility assessment (e.g., Initio, Mira). Having the same capability in a wearable format would enable users to access data automatically (i.e., without the need for daily urine sampling).

[0011] It is against this technological backdrop that the a technological solution to these and other problems rooted in this technology was sought.SUMMARY

[0012] The present embodiments relate to a wearable aptamer-field-effect transistor (FET) sensing platform that can non-invasively measure three low-abundance ovulatory hormones at physiologically relevant concentrations (nM-pM) in sweat. According to certain aspects, this solution to in situ biosensing is a platform that is generalizable for a wide range of significant targets for clinical and health and wellness applications. An aptamer-FET approach according to embodiments enables high-sensitivity in situ measurements of small-molecule and peptide targets that are inaccessible by other types of biosensors, including electrochemical aptamer biosensors, which do not have the sensitivity or dynamic range needed to detect most endogenous targets. Unlike prior approaches, the present approach does not require complex hybridization strategies. Using biocompatible thin-film I Ch FETs provides a direct route to scalable manufacturing.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] These and other aspects and features of the present embodiments will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures, wherein:

[0014] FIGs. 1 A to 1G illustrate example aspects of noninvasive multiplexed ovulatory hormone, pH, and temperature monitoring according to embodiments.

[0015] FIGs. 2A to 21 illustrate example aspects of flexible polyimide thin-film fieldeffect transistors (FETs) for sweat monitoring according to embodiments.

[0016] FIGs. 3A to 31 illustrate example aspects of electronic hormone sensing using aptamer-functionalized field-effect transistors according to embodiments.

[0017] FIGs. 4A to 4C illustrate example aspects of rearrangements of aptamer structure upon adaptive binding to hormone targets according to embodiments.

[0018] FIGs. 5A to 5D illustrate example aspects of on-demand sweat sampling by iontophoretic (IP) delivery of a sweat agonist according to embodiments.

[0019] FIG. 6 is a graph illustrating temperature sensor calibration over a wide temperature range.

[0020] FIG. 7 illustrates surface functionalization chemistry for covalently linking aptamer sequences to indium oxide surfaces according to embodiments.

[0021] FIG. 8 illustrates predicted secondary structures of three example aptamer sequences based on mfold according to embodiments. The BES.1 aptamer is for estradiol, the PG01 aptamer is for progesterone and the LH aptermater is the luteinizing hormone aptamer.

[0022] FIG. 9 provides optical images of a microfluidic chamber according to embodiments fdling over time after administration of iontophoresis current compared to control with no iontophoretic stimulation (unstimulated).DETAILED DESCRIPTION

[0023] The present embodiments will now be described in detail with reference to the drawings, which are provided as illustrative examples of the embodiments so as to enable those skilled in the art to practice the embodiments and alternatives apparent to those skilled in the art. Notably, the figures and examples below are not meant to limit the scope of the presentembodiments to a single embodiment, but other embodiments are possible by way of interchange of some or all of the described or illustrated elements. Moreover, where certain elements of the present embodiments can be partially or fully implemented using known components, only those portions of such known components that are necessary for an understanding of the present embodiments will be described, and detailed descriptions of other portions of such known components will be omitted so as not to obscure the present embodiments. Embodiments described as being implemented in software should not be limited thereto, but can include embodiments implemented in hardware, or combinations of software and hardware, and vice- versa, as will be apparent to those skilled in the art, unless otherwise specified herein. In the present specification, an embodiment showing a singular component should not be considered limiting; rather, the present disclosure is intended to encompass other embodiments including a plurality of the same component, and vice-versa, unless explicitly stated otherwise herein. Moreover, applicants do not intend for any term in the specification or claims to be ascribed an uncommon or special meaning unless explicitly set forth as such. Further, the present embodiments encompass present and future known equivalents to the known components referred to herein by way of illustration.

[0024] The present embodiments relate to a wearable device to monitor sweat estradiol levels, and more particularly toward high-accuracy fertility tracking and ovulation prediction by developing biosensors for multiple ovulatory hormones (estradiol, progesterone, and LH). The present devices also measure sweat pH and skin temperature. Relatedly, thin-film I Ch FETs have been functionalized with aptamers for label-free, highly sensitive and selective detection of small-molecule targets (e.g., serotonin, dopamine, glucose, cortisol, phenylalanine) in biofluids (K. M. Cheung, K. A. Yang, N. Nakatsuka, C. Zhao, M. Ye, M. E. Jung, H. Yang, P. S. Weiss, M. N. Stoj anovic, A. M. Andrews, Phenylalanine monitoring via aptamer-fi eld-effect transistor sensors. ACS Sens. 4, 3308-3317 (2019); N. Nakatsuka, K. A. Yang, J. M. Abendroth, K. Cheung, X. Xu, H. Yang, C. Zhao, B. Zhu, Y. S. Rim, Y. Yang, P. S. Weiss, M. N. Stojanovic, A. M. Andrews, Aptamer-field-effect transistors overcome Debye length limitations for smallmolecule sensing. Science 362, 319-324 (2018)). Here, the present Applicants characterized previously reported estradiol and LH aptamers (K. A. Yang, R. Pei, D. Stefanovic, M. N. Stojanovic, Optimizing cross-reactivity with evolutionary search for sensors. J. Am. Chem. Soc.134, 1642-1647 (2012); S. Liang, A. B. Kinghorn, M. Voliotis, J. K. Prague, J. D. Veldhuis, K. Tsaneva-Atanasova, C. A. McArdle, R. H. W. Li, A. E. G. Cass, W. S. Dhillo, J. A. Tanner, Measuring luteinising hormone pulsatility with a robotic aptamer-enabled electrochemical reader. Nat. Comm. 10, 852 (2019)), and embodiments include a new progesterone aptamer sequence for such FET -based sensors and rapid electronic sensing on flexible polyimide substrates. The present embodiments thus include an integrated and optimized an on-demand sweat sampling platform that utilizes iontophoretic drug delivery to stimulate eccrine sweat production coupled with a tape-based thin-film microfluidic structure for biofluid routing to produce an integrated wearable device.

[0025] RESULTS and DISCUSSION

[0026] Sensor characterization

[0027] FIGs. 1 A to 1G illustrate example aspects of a noninvasive multiplexed ovulatory hormone, pH, and temperature monitoring according to embodiments. An aspect of the present embodiments is the recognition that 17[3-Estradiol (E2), progesterone (P4), and LH are three key menstrual cycle hormones with respective distinct time-dependent concentration profiles and functions (B. G. Reed, B. R. Carr, The Normal Menstrual Cycle and the Control of Ovulation. K. R. Feingold, Ed., MDText.com, Inc., South Dartmouth, MA, 2015). FIG. IB is a graph illustrating typical changes in skin temperature and plasma hormone concentrations throughout the menstrual cycle. E2, 17 -estradiol; P4, progesterone; LH, luteinizing hormone. As illustrated in FIG. IB, in a model cycle, after the first day of menstruation, an initial steady rise in estradiol stimulates ovarian follicular growth. After day 7 of the cycle, a dominant preovulatory follicle produces a larger rise in estradiol, which in turn, causes a rapid surge in LH. Ovulation occurs ~24 36 h after the onset of this surge and ~12 h after the LH peak (-day 14). The LH surge stimulates the synthesis of progesterone, which prepares the uterine wall for implantation of a fertilized egg. Progesterone levels peak -8 days after ovulation, coinciding with the expected time of implantation and a second peak in estradiol.

[0028] FIG. 1C is a diagram illustrating example aspects of how luteinizing hormone and follicle-stimulating hormone (FSH) are produced by the anterior pituitary gland in the brain for release into the circulatory system and actions in the ovaries and other tissues. Estradiol andprogesterone are produced by the ovaries and have local actions and effects throughout the body. FIG. 1G illustrates the three ovulatory hormones monitored by the present embodiments. Monitoring all three hormones concomitantly enables highly accurate determination of fertile windows to improve facilitating or preventing conception. Including progesterone differentiates the follicular vs. luteal phase rises in estradiol as shown in FIG. IB and confirms ovulation (Id.).

[0029] The present embodiments include highly sensitive aptamer-FET sensors using quasi-2D ImCh as the semiconductor on a variety of stiff (thin-film and patterned) and soft substrates (polyimide, polyethylene terephthalate, and polydimethylsiloxane) (Q. Liu, C. Zhao, M. Chen, Y. Liu, Z. Zhao, F. Wu, Z. Li, P. S. Weiss, A. M. Andrews, C. Zhou, Flexible multiplexed I Ch nanoribbon aptamer-fi eld-effect transistors for biosensing. iScience 23, 101469 (2020); C. Zhao, K. M. Cheung, I. W. Huang, H. Yang, N. Nakatsuka, W. Liu, Y. Cao, T. Man, P. S. Weiss, H. G. Monbouquette, A. M. Andrews, Implantable aptamer-field-effect transistor neuroprobes for in vivo neurotransmitter monitoring. Sci. Adv. 7, eabj7422 (2021); C. Zhao, T. Man, Y. Cao, P. S. Weiss, H. G. Monbouquette, A. M. Andrews, Flexible and implantable polyimide aptamer-field-effect transistor biosensors. ACS Sens. 7, 3644-3653 (2022); C. Zhao, Q. Liu, K. M. Cheung, W. Liu, Q. Yang, X. Xu, T. Man, P. S. Weiss, C. Zhou, A. M. Andrews, Narrower nanoribbon biosensors fabricated by chemical lift-off lithography show higher sensitivity. ACS Nano 15, 904-915 (2021); C. Zhao, X. Xu, S. H. Bae, Q. Yang, W. Liu, J. N. Belling, K. M. Cheung, Y. S. Rim, Y. Yang, A. M. Andrews, P. S. Weiss, Large-area, ultrathin metal-oxide semiconductor nanoribbon arrays fabricated by chemical lift-off lithography. Nano Lett. 18, 5590-5595 (2018); Y. S. Rim, S. H. Bae, H. Chen, J. L. Yang, J. Kim, A. M. Andrews, P. S. Weiss, Y. Yang, H. R. Tseng, Printable ultrathin metal oxide semiconductor-based conformal biosensors. ACS Nano 9, 12174-12181 (2015)).

[0030] FIG. 1A is a photograph of an example skin-mounted flexible sensor array 102 with integrated iontophoretic electrodes 104 for sweat stimulation and FIG. ID is a schematic representation of an example aptamer-FET sensing mechanism according to embodiments. In contrast to prior FET biosensors, the present embodiments relate to flexible polyimide aptamer- FET sensor arrays to sense estradiol, progesterone, and LH. FIG. IE is a schematic representation of an example mechanism of iontophoretic induced sweat generation, includingdelivery of the cholinergic agonist pilocarpine to stimulate local sweat production. P+, pilocarpine nitrate.

[0031] FIG. IF is a schematic diagram illustrating an example sensor patch according to embodiments and fabrication method for the same. As shown, the fabricated patch in this example is formed on a flexible polyimide substrate 132. A patterned double-sided adhesive 134 includes patterned iontophoretic electrodes 104. Patterned on the substrate 132 are a temperature sensor 138, FET source / drain electrodes 140, FET gate electrode 142 and flat cable connections 144 for the sensor 138 and electrodes 140 / 142. An example process (e.g. steps 112 to 122 in FIG. IF) for fabricating an example sensor patch will be described in more detail below.

[0032] FIGs. 2A to 21 illustrate example aspects of Flexible polyimide thin-film fieldeffect transistors (FETs) for sweat monitoring according to embodiments.

[0033] FIG. 2A provides example transfer curves and FIG. 2B provides example output curves for gate-voltage modulation and saturation behavior, respectively, in artificial sweat. In these examples, source-drain currents (IDS) were monitored over a range of drain (0 to 100 mV) and gate (0 to 500 mV) voltages using an on-device Ag / AgCl electrode for solution gate-voltage biasing. Accordingly, FIG. 2A provides example transfer curves (IDS-VGS) with VDS varied from 0-100 mV in 20 mV increments demonstrating characteristic FET gate-voltage modulation and FIG. 2B) provides example output curves (IDS-VDS) with VGS varied from 0-500 mV in 100 mV increments demonstrating saturation behavior. As further shown in FIG. 2C, negligible gate leakage currents at Ag / AgCl pseudo-reference electrodes were detected compared to FET source-drain currents.

[0034] Mechanical resilience is an important aspect of wearable sensors. FIG. 2D is a photograph of flexible sensor arrays on polyimide before and after crumpling to determine electrical stability. The thin-film polyimide substrate (approx.. 7 pm), the ImCh semiconductor layer (3 to 4 nm), and the Ti / Au metal electrodes (10 nm / 50 nm) are inherently flexible. As such, these multimodal devices exhibited reproducible FET behavior after crumpling as shown in FIG. 2D. FIG. 2E provides example overlaid transfer curves from a representative FET after increasing numbers of consecutive crumpling cycles and FIG. 2F provides a graph of IDS VS. crumpling cycles (VGS=400 mV). As shown in these examples, even after 50 consecutive crumpling cycles, the transfer curves shown in FIG. 2E and source-drain currents shown in FIG.2F remained stable. These findings illustrate the robustness of I Ch FETs on flexible polyimide substrates for wearable applications on the skin and are similar to the present Applicants’ previous findings on E12O3 FETs on 1.4 pM-thick polyethylene terephthalate (Id ).

[0035] The present embodiments include functionalized I C channels of FETs with (3 aminopropyl)triethoxysilane (APTES) diluted with trimethoxy(propyl)silane (PTMS) (1 :9 v / v) via chemical vapor deposition that create pH-sensitive FETs. Changes in hydrogen ion concentrations alter the protonation state of the APTES amine tail groups producing increases in source-drain currents with decreasing pH (for n type semiconductors such as I Ch) (Id.). Monitoring the pH of sweat is of interest because changes in sweat pH are indicative of exercise intensity and hydration status (W. Dang, L. Manjakkal, W. T. Navaraj, L. Lorenzelli, V. Vinciguerra, R. Dahiya, Stretchable wireless system for sweat pH monitoring. Biosens. Bioelectron. 107, 192-202 (2018)). An integrated pH sensor can also be used for self-referencing during hormone monitoring (Id.).

[0036] The present Applicants employed a previously reported calibration method (i.e., calibrated response), which considers both the current change (IDS) and the slope of the individual FET transfer curves to minimize device-to-device variations and to compensate for current drift over time (F. N. Ishikawa, M. Curreli, H. K. Chang, P. C. Chen, R. Zhang, R. J. Cote, M. E. Thompson, C. Zhou, A calibration method for nanowire biosensors to suppress device-to-device variation. ACS Nano 3, 3969-3976 (2009)). FIG. 2G is an example calibration curve for FET pH sensing across the sweat physiological range. As shown, the pH sensors of embodiments demonstrated linear (R2=0.99) and reproducible responses over the physiological sweat range from pH 7.4 to 4.5 (see L. B. Baker, Physiology of sweat gland function: The roles of sweating and sweat composition in human health. Temperature 6, 211-259 (2019)). It should be noted that the error bars in FIGs. 2F and 2G are the standard deviations of N=3 FETs and N=3 thermistors. In I, *P<0.05.

[0037] FIG. 2H illustrates example real time IDS changes of a representative pH FET sensor upon exposure to increasingly acidic and basic phosphate buffered saline. As shown, raw current changes were visualized in real-time when FETs were exposed to increasingly acidic phosphate-buffered saline. The pH sensors demonstrated rapid response times as well as reversible behavior when basic phosphate-buffered saline solutions were introduced.

[0038] For multimodal sensing, the present Applicants integrated a gold thermistor into arrays for continuous skin temperature monitoring. Basal body temperature measurements remain one of the most prevalent biophysical methods for determining ovulation timing. Although basal temperature is typically measured orally, ovulation-associated changes in body temperature can be detected with high sensitivity in wearable-friendly skin locations, such as the wrist or finger (Y. Uchida, M. Izumizaki, The use of wearable devices for predicting biphasic basal body temperature to estimate the date of ovulation in women. J. Therm. Biol. 108, 103290 (2022); T. Y. Zhu, M. Rothenbiihler, G. Hamvas, A. Hofmann, J. Welter, M. Kahr, N. Kimmich, M. Shilaih, B. Leeners, The accuracy of wrist skin temperature in detecting ovulation compared to basal body temperature: Prospective comparative diagnostic accuracy study. J. Med. Internet Res. 23, e20710 (2021)). Skin temperature measurements have been incorporated into commercial wearables including the Oura Ring and Ava bracelet.

[0039] FIG. 21 illustrate example thermistor responses to detect the typical 0.3 °C change in body temperature associated with ovulation. As shown in FIG. 21, the present temperature sensor demonstrated linear (R2=0.99) and reproducible responses over a broad temperature range from 25 °C to 50 °C (FIG. 6). Moreover, the present temperature sensors had sufficient sensitivity to detect the subtle 0.3 °C temperature increase typically associated with ovulation. It should be noted that in FIG. 21, *P<0.05.

[0040] Aptamer-FET biosensors for ovulatory hormone monitoring

[0041] Recent advances in solution-phase systematic evolution of ligands by exponential enrichment (SELEX) to identify DNA receptors have yielded aptamers with high affinity and selectivity to previously underexplored targets (K. A. Yang, H. Chun, Y. Zhang, S. Pecic, N. Nakatsuka, A. M. Andrews, T. S. Worgall, M. N. Stojanovic, High-affinity nucleic-acid-based receptors for steroids. ACS Chem. Biol. 12, 3103-3112 (2017)). In an example embodiment, the aptamer sequence BES. l was immobilized on FET surfaces for estradiol sensing (Kd=1.4 pM) (Id.). The present Applicants isolated a new high-affinity aptamer to sense progesterone (Kd=4 nM) (Table SI). For luteinizing hormone detection, some embodiments utilize a recently reported aptamer (B23; Kd=300 nM) that had been subjected to counter- sei ection against FSH as the latter has the same p-subunit as LH (Id.). Each aptamer sequence was thiolated at the 5’ endand covalently linked to amino-silanized I Ch semiconductor channels using 3 maleimidobenzoic acid N hydroxysuccinimide ester as a coupling reagent (FIG. 7) (Id.).TABLE SIAptamer SequenceEstradiol (E2) 5’- / 5ThioMCD6-D / CTC TCG GGA CGA CAT GGA TTT TCC ATC AAC GAA GTG CGT CCG TCC CG-3’Progesterone (P4) 5’- / 5ThioMCD6-D / CGA CGG GGG TGG CTT GTT AAT ACT CTG TAT GGC ATA GGG TAG GCT AGGCTC ACT GTC G-3’Luteinizing hormone (LH) 5’- / 5ThioMCD6-D / TAT GGT ATG CTG TGT GGT ATG GGG TGG CGT GCT CT-3’

[0042] The present Applicants spiked artificial sweat samples containing physiologically relevant metabolites, including uric acid, urea, lactic acid, ammonia, and 19 amino acids, with ovulatory hormones to investigate sensor responses and generate calibration curves (K. Midander, A. Julander, J. Kettelarij, C. Liden, Testing in artificial sweat-is less more? Comparison of metal release in two different artificial sweat solutions. Regul. Toxicol. Pharmacol. 81, 381-386 (2016)). All solutions were buffered at an average sweat pH of 5 to mimic the physiological environment.

[0043] FIGs. 3A to 31 illustrate example aspects of electronic hormone sensing using aptamer-functionalized field-effect transistors according to embodiments. For example, FIG. 3A illustrates the transfer curves from a representative estradiol-aptamer FET in response to increasing estradiol concentrations from 1 pM to 1 pM. FIG. 3B is a graph illustrating example calibrated responses to estradiol for FETs functionalized with the estradiol aptamer (302- black curve) according to embodiments versus FETs lacking aptamer modification (304 -red curve). As shown in FIG. 3B, the calibrated responses sufficiently covered the physiological range of estradiol levels in serum (~30 pM to 3 nM) for healthy, nonpregnant women (S. Tsepelidis, F. Devreker, I. Demeestere, A. Flahaut, C. Gervy, Y. Englert, Stable serum levels of anti-mullerian hormone during the menstrual cycle: A prospective study in normo-ovulatory women. Hum. Reprod. 22, 1837-1840 (2007); O. J. Ginther, M. A. Beg, E. L. Gastal, M. O. Gastal, A. R. Baerwald, R. A. Pierson, Systemic concentrations of hormones during the development offollicular waves in mares and women: A comparative study. Reproduction 130, 379-388 (2005)) and during the first trimester of pregnancy (-3 9 nM) and recently reported eccrine sweat estradiol levels in healthy human women (1 pM 30 pM) (Id.). Control experiments using FETs with all surface chemistry except functionalization of the estradiol aptamer sequence produced negligible FET-calibrated responses upon estradiol additions (shown by 304 in FIG. 3B).

[0044] Target selectivity was investigated by monitoring source-drain currents (IDS) in real-time upon exposure to similarly structured nontarget steroid hormones at or above their physiological concentrations. FIG. 3C is a graph illustrating real-time sensing responses of an estradiol-aptamer-functionalized FET to the nontarget steroids progesterone and cortisol versus increases in source-drain current (IDS) to estradiol. As shown by 312, exposing estradiol- aptamer-functionalized FETs to 100 nM progesterone or cortisol produced negligible IDS responses comparable to exposure to artificial sweat with no target or counter-target. Conversely, as shown by 314, upon the addition of estradiol (100 pM to 10 nM), IDS increased accordingly.

[0045] FIG. 3D shows transfer curves from a representative progesterone-aptamer-FET according to embodiments upon exposure to increasing progesterone concentrations ranging from 1 pM to 1 pM covering the physiological range in serum (-1 nM to 50 nM) (Id.).Progesterone has been detected in eccrine sweat (facial perspiration) in women by ELISA at ~1.5 nM (Id.). FIG. 3E illustrates responses to progesterone for FETs functionalized with the progesterone aptamer (shown by 322) versus FETs lacking aptamer modification. As shown by 324, control FETs lacking the progesterone-aptamer showed no target-dependent changes in calibrated responses. The progesterone aptamer was further evaluated to determine its selectivity against other steroid hormones, namely estradiol and cortisol. Both nontargets produced negligible changes in IDS that were comparable to exposure to artificial sweat alone. Large changes in FET current responses were produced by the addition of 1 1000 nM progesterone. FIG. 3F is a graph illustrating real-time sensing responses of a representative progesterone- aptamer-functionalized FET to nontarget steroids estradiol and cortisol (illustrated by 332) vs. increases in IDS in response to progesterone (illustrated by 334).

[0046] FIG. 3G illustrates the transfer characteristics upon exposure of LH-aptamer- FETs to increasing LH concentrations in the pM pM range. This dynamic range covers the physiological plasma LH surge (-200 nM) that typically precedes ovulation (Id.). FIG. 3Hillustrates responses to LH for FETs functionalized with the LH aptamer (342) versus FETs lacking the LH aptamer (344). As shown in FIG. 3H, the LH aptamer-modified FETs demonstrated large concentration-dependent increases in calibrated responses compared to control FETs lacking LH aptamers. To investigate the selectivity of LH-aptamer-FETs, sensors were exposed to high concentrations of bovine serum albumin (5 pM) and FSH (500 nM), As shown in FIG. 31, the nontarget proteins induced negligible FET current changes (352) compared to subsequent additions of LH (354).

[0047] It should be noted that in FIGs. 3B, 3E, and 3H, the error bars are standard deviations for A- 3 FETs with aptamer (Apt) or N=2 FETs for the no aptamer condition.

[0048] Aptamer-target conformation changes

[0049] FIG. 4A to 4C illustrate example aspects of rearrangements of aptamer structure upon adaptive binding to hormone targets according to embodiments. In these examples, the present Applicants used circular dichroism (CD) spectroscopy to investigate the target-induced conformational changes of aptamer secondary structures to provide additional insight into FET sensing mechanisms (N. Nakatsuka, J. M. Abendroth, K.-A. Yang, A. M. Andrews, Divalent cation dependence enhances dopamine aptamer biosensing. ACS Appl. Mater. Inter. 13, 9425- 9435 (2021)). As shown by FIG. 4A, the estradiol aptamer in the absence (402) and presence (404) of its target had an absorbance maximum at -280 nm and an absorbance minimum at -245 nm indicating the presence of double- stranded B DNA segments. The aptamer secondary structure was predicted using mfold, which corroborated double-stranded regions in the putative structure (FIG. 8). While the CD spectra were not indicative of the formation of G quadruplex- like secondary structure upon target addition (i.e., no wavelength shifts in peak maxima or minima), the ellipticity increased. The latter indicates that target binding induced spatial rearrangement of existing aptamer secondary structure and aptamer-associated solution ions.

[0050] As shown by FIG. 4B, the binding of progesterone to its aptamer (406) caused a small red shift in the CD absorbance maximum from 280 nm to -290 nm with the minimum remaining at -260 nm, characteristic of a target-related shift to an antiparallel G quadruplex-like structure. The large increase in ellipticity associated with the aptamer-progesterone complex indicates a large influence of target binding on secondary structure.

[0051] As shown by FIG. 4C, recognition of LH by the LH aptamer (408) produced a small decrease in ellipticity and a small blue shift in the maximum absorbance from -270 nm to -260 nm with no change in the minimum absorbance at -240 nm. These findings suggest target- induced formation of a parallel G quadruplex-like motif. Previous CD spectroscopy with this LH aptamer was interpreted as indicating a structure-switching binding mechanism (Id.).

[0052] Estradiol and progesterone are neutral molecules. As such, target charge does not play a role in their aptamer-sensing mechanisms similar to previous findings with glucose- aptamer-FET sensing (Id.). The increases in source-drain current observed upon estradiol or progesterone addition to the corresponding aptamer-FETs are associated with the reorientation of negatively charged phosphodiester aptamer backbones (or some portion thereof) away from the n type ImCh semiconductor channels. This reduces the electrostatic repulsion of the semiconductor charge carriers causing an increase in source-drain current. The increase in source-drain current associated with LH addition can be explained by analyzing the protonation states of the amino acid side chains of the a and subunits of LH. At pH=5, an effective positive charge of Z=+20 was calculated for LH using the Prot pi bioinformatics toolbox. Therefore, when LH aptamers bind LH, the local charge near the n type semiconductor surface becomes more positive. This decreases the electrostatic repulsion of semiconductor charge carriers and increases the drainsource current. The large target-associated increases in IDS further suggest that the backbone of the LH aptamer also predominantly reorients away the semiconductor surface for an additive effect with charged target binding.

[0053] Characterization of an integrated on-demand sweat sampling system

[0054] Integrated biofluid sampling is a critical requirement for developing practical wearable devices. Iontophoresis can be used for on-demand sweat sampling via transdermal delivery of cholinergic agonists that stimulate the production of eccrine sweat. Various compounds such as acetylcholine, pilocarpine, and carbachol have been loaded into hydrogels and investigated in different systems to assess their sweat generation profiles (P. Simmers, S. K. Li, G. Kasting, J. Heikenfeld, Prolonged and localized sweat stimulation by iontophoretic delivery of the slowly-metabolized cholinergic agent carbachol. J. Dermatol. Sci. 89, 40-51 (2018); S. Emaminejad, W. Gao, E. Wu, Z. A. Davies, H. Yin Yin Nyein, S. Challa, S. P. Ryan,H. M. Fahad, K. Chen, Z. Shahpar, S. Talebi, C. Milla, A. Javey, R. W. Davis, Autonomous sweat extraction and analysis applied to cystic fibrosis and glucose monitoring using a fully integrated wearable platform. Proc. Natl. Acad. Sci. U.S.A. 114, 4625-4630 (2017)). The emergence of commercial iontophoretic systems (e.g., Maroduct and Nanoduct) for sweat sampling affirms the safety and efficacy of this approach.

[0055] FIGs. 5A to 5D illustrate example aspects of on-demand sweat sampling by iontophoretic (IP) delivery of a sweat agonist according to embodiments. To integrate on- demand sweat sampling, the present embodiments include flexible polyimide sensor arrays coupled with thin-film tape-based microfluidic devices. For example, FIG. 5A is a schematic diagram of an example integrated microfluidic module comprised of a sensor array 502, IP hydrogels 504, and tape-based fluidic structures 506. The microfluidic structures 506 served as an adhesive for attachment to the skin 508 and defined the sensing chamber, iontophoresis gel chambers, and the inlet / outlet channels for biofluid routing. Optimal iontophoresis conditions are required to maximize sweat generation while minimizing off-target effects such as skin irritation and local heating (H. Hojaiji, Y. Zhao, M. C. Gong, M. Mallajosyula, J. Tan, H. Lin, A. M. Hojaiji, S. Lin, C. Milla, A. M. Madni, S. Emaminejad, An autonomous wearable system for diurnal sweat biomarker data acquisition. Lab Chip 20, 4582-4591 (2020)).

[0056] FIG. 5B is a graph illustrating current density optimization for sufficient filling of the sensing chamber with sweat. As shown in the example of FIG. 5B, the present embodiments can optimize the applied current density by fixing the iontophoresis duration at 5 min, which has been demonstrated to yield sufficient quantities of sweat in similar systems with minimal discomfort (Id.). FIG. 5C is a schematic representation of the chamber filling over time after administration of IP current compared to control with no IP stimulation. Representative photos of an example filling of a chamber of embodiments are provided in FIG. 9 (outlined portions show the extent of the filling). As shown in FIG. 5C, the sweat volumes extracted were estimated after iontophoresis at various times based on the filling percentage of each microfluidic sensing chamber (total volume 25 pL). FIG. 5D provides optical images of unstimulated 512 versus IP stimulated 514 sensors according to embodiments.

[0057] The present Applicants investigated the amount of sweat generated (e,g, in uL) after the application of current densities ranging from 0.25-0.45 mA / cm2as illustrated in FIG.5B. In one example investigation, the entire sensing chamber volume was filled by current densities at or above 0.4 mA / cm2, which was determined to be the minimum current density required. FIG. 5C and FIG. 9 depict an example chamber filling over time for a representative device. Sweat entered from the pilocarpine-loaded gel side of the device and the center inlet. The sensing chamber was filled after 20 min (illustrated in FIG. 5D), with excess sweat (after 25 min) directed towards the outlet microchannel, which opens to the skin surface. A control experiment was conducted with the iontophoresis gels in contact with the skin for 1 h but without current stimulation wherein no sweat was observed in the sensing chamber (FIG. 9). These results show the effective attachment of the microfluidic module to the skin and sensor array, with no apparent sweat loss due to leakage.

[0058] CONCLUSIONS AND FUTURE PROSPECTS

[0059] The present disclosure provides a wearable FET -based sensor device capable of hormonal (estradiol, progesterone, and LH) and physiological (skin temperature and sweat pH) monitoring in human sweat. The devices demonstrated excellent electro-mechanical stability, even after many severe mechanical deformations. The present embodiments integrate and optimize an on-demand sweat sampling platform via iontophoretic drug delivery and biofluid routing using microfluidic adhesive structures. The aptamer-FET sensing mechanisms highlight a key advantage of using oligonucleotide bioreceptors coupled with highly sensitive, nonlinear semiconductor signal transduction. Together, they enable direct sensing under high ionic strength physiological conditions without the need for chemically conjugated redox probes or complex complementary hybridization schemes (J. Gerson, M. K. Erdal, M. H. McDonough, K. L. Ploense, P. Dauphin-Ducharme, K. M. Honeywell, K. K. Leung, N. Arroyo-Curras, J. M.Gibson, N. A. Emmons, W. Meiring, I. P. Hespanha, K. W. Plaxco, T. E. Kippin, High-precision monitoring of and feedback control over drug concentrations in the brains of freely moving rats. Sci. Adv. 9, eadg3254 (2023)). Aptamer-FETs simplify the fabrication and sensing mechanisms for electronic biosensors. The present use of biocompatible thin-film I Ch FETs is amenable to scalable manufacturing (W. S. Liao, S. Cheunkar, H. H. Cao, H. R. Bednar, P. S. Weiss, A. M. Andrews, Subtractive patterning via chemical lift-off lithography. Science 337, 1517-1521 (2012)) while straightforwardly achieving the high sensitivity needed for in situ sensing.

[0060] A wearable device having continuous skin contact and the capability to detect ovulatory hormone levels twice a day over weeks would offer several advantages compared to single-use urinary lateral flow assays recommended for twice-daily use for several days before ovulation. Continuous monitoring across monthly cycles would obviate uncertainty associated with trying to time ovulation to know when to begin using lateral flow assays. Moreover, human sperm survive up to five days after intercourse (S. S. Suarez, A. A. Pacey, Sperm transport in the female reproductive tract. Hum. Reprod. Update 12, 23-37 (2005); A. J. Wilcox, C. R. Weinberg, D. D. Baird, Timing of sexual intercourse in relation to ovulation — effects on the probability of conception, survival of the pregnancy, and sex of the baby. N. Engl. J. Med. 333, 1517-1521 (1995)), whereas the LH surge only occurs -24-36 hours before ovulation. As such, lateral flow assays for LH neglect to identify a 4-day fertility window before ovulation to assist those trying to facilitate pregnancy. Fertile days would also go undetected when unprotected intercourse could lead to unplanned pregnancy. Most conventional lateral flow assays only provide qualitative data to determine whether ovulation has occurred and are not recommended by their manufacturers for use in preventing pregnancy, even when estradiol metabolites are included. Aptamer-FET measurements are quantitative and could be used to provide information for managing contraceptive timing, as well as for diagnosing and managing various medical conditions where changes in hormone levels need to be quantified.

[0061] Wearable devices have a lower impact on daily routines compared to urinary lateral flow assays, which users have to take responsibility for implementing, portending higher use compliance and therefore, better quality and more actionable data from wearable devices. Having the capability to monitor all three key ovulatory hormones instead of one or even two hormones, in conjunction with body temperature over the entire menstrual cycle will enable highly accurate detection of fertility windows combined with ease of use for wearers. Likewise, cycle-tracking over time at the individual level, when combined with machine learning algorithms and a smartphone app, is expected to provide highly personalized and accurate information to wearers, even for those with irregular cycles and / or extenuating medical conditions. Data can also be sent directly to healthcare providers.

[0062] To advance and validate the technology described for commercial and clinical use, several items will need to be addressed. First, although there are promising findingscorrelating estradiol and progesterone levels in serum vs. sweat (Id.), larger studies to confirm ovulatory hormone correlations should be conducted to ascertain reliability across subjects and time points of the menstrual cycle. Notably, LH sweat concentrations and correlations with serum have yet to be investigated.

[0063] Second, the present Applicants used amine-functionalized surface chemistries to conjugate individual aptamers to polyimide FET arrays and tested each hormone individually (i.e., one aptamer and one hormone per array). Multiplexed, addressable surface chemistries and custom hardware will be needed to monitor all three hormones simultaneously and in real time. Others and the present Applicants have already demonstrated wireless, wearable electronics for wearable sweat sensing applications (J. Min, S. Demchyshyn, J. R. Sempionatto, Y. Song, B. Hailegnaw, C. Xu, Y. Yang, S. Solomon, C. Putz, L. E. Lehner, J. F. Schwarz, C. Schwarzinger, M. C. Scharber, E. Shirzaei Sani, M. Kaltenbrunner, W. Gao, An autonomous wearable biosensor powered by a perovskite solar cell. Nat. Electron. 6, 630-641 (2023)). Third, sweat induction via iontophoresis is mainly used for single-point determinations. Emaminejad and colleagues reported an autonomous wearable platform for repeated and programmable sampling of low volumes of sweat (2 pL) (Id.). Alternately, Prasad and colleagues have used passive sweat collection involving small volumes (~l-5 pL) for repeated biomarker monitoring (M. Pali, B. Jagannath, K.-C. Lin, D. Sankhala, S. Upasham, S. Muthukumar, S. Prasad, Tracking metabolic responses based on macronutrient consumption: A comprehensive study to continuously monitor and quantify dual markers (cortisol and glucose) in human sweat using watch sensor. Bioeng. Transl. Med. 6, el 0241 (2021); R. D. Munje, S. Muthukumar, A. Panneer Selvam, S. Prasad, Flexible nanoporous tunable electrical double layer biosensors for sweat diagnostics. Sci. Rep. 5, 14586 (2015); A. Ganguly, K. C. Lin, S. Muthukumar, S. Prasad, Autonomous, real-time monitoring electrochemical aptasensor for circadian tracking of cortisol hormone in submicroliter volumes of passively eluted human sweat. ACS Sens. 6, 63-72 (2021)). Thus, a path forward can be delineated for a wearable aptamer-FET -based platform for multiplexed ovulatory hormone and physiological monitoring with repeated sweat sampling for high-accuracy fertility tracking. This type of platform can be readily expanded for non-invasive monitoring of a wide variety of biomarkers by conjugating target-selective aptamer sequences (K. Yang, N. M. Mitchell, S. Banerjee, Z. Cheng, S. Taylor, A. M. Kostic, I. Wong, S. Sajjath, Y. Zhang, J.Stevens, S. Mohan, D. W. Landry, T. S. Worgall, A. M. Andrews, M. N. Stojanovic, A functional group-guided approach to aptamers for small molecules. Science 380, 942-948 (2023)) to different FETs in sensing arrays.

[0064] EXAMPLE MATERIALS and METHODS

[0065] Example Materials

[0066] (3 Aminopropyl)trimethoxysilane (APTES), trimethoxy(propyl)silane (PTMS), 1 dodecanethiol, ethanol, dimethyl sulfoxide (DMSO), maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and pilocarpine nitrate were purchased from Sigma Aldrich. Other materials were poly dimethylsilane (PDMS) (Sylgard), artificial sweat (I2BL-0011, Pickering Laboratories), phosphate-buffered saline (Gibco# 10010023, Thermo Fisher Scientific) supplemented with 2 mM MgCb, 3M double-sided tape (-150 pm, 3M Science), polyimide (PI-2611, HD Microsystems), indium (III) nitrate hydrate [In(NO3)3»xH2O, 99.999%] (Alfa Aesar), 4-inch Si wafers, thickness 500 pm with 100 nm thermally grown SiO2 (Silicon Valley Microelectronics), Ag / AgCl ink (Creative Materials), and deionized water (Milli-Q, Millipore). Aptamers were obtained from Integrated DNA Technologies. The progesterone aptamer was identified using an insertion-reselection approach to evolve existing aptamers as described in the above mentioned publications. Aptamer sequences were thiol ated at the 5’ ends for functionalization on FET surfaces (Table SI).

[0067] Example Sensor Fabrication

[0068] Returning to the example shown in FIG. IF, polyimide films can be fabricated by spin coating as-received polyimide solutions on Si wafers at 112 at 3000 rpm for 30 s to produce ~7-pm-thick polyimide films per the manufacturer’s instructions. In 114, the films can be baked in a convection oven at 150 °C for 90 s, followed by a 4 °C / min ramp to 350 °C. The temperature can be held at 350 °C for 30 min before cooling (e.g. thereby forming substrate 132). In 116, aqueous solutions (0.1 M) of indium (III) nitrate can be prepared in deionized water and spin-coated at 3000 rpm for 30 s (thickness -3 4 nm) on the polyimide films (e.g. thereby forming the I Ch channels for the aptamer FETs of embodiments). The coated substrates can then be prebaked at 150 °C for 10 min followed by thermal annealing at 350 °Cfor 4 h. In 118, the Ti / Au (e.g. 10 nm / 50 nm) source, drain, gate, iontophoretic, and thermistor electrodes (e.g. 104, 138, 140, 142) were patterned by photolithography followed by metal deposition via electron beam evaporation. In 120, flexible Ag / AgCl ink can be screen printed on top of Au gate electrodes to produce pseudoreference electrodes. After fabrication in 122, the polyimide films can be delaminated from the Si wafers by cutting the outside edges with a razor blade.

[0069] All FETs can be functionalized for biosensing using previously reported protocols (Id.). Briefly, APTES and PTMS (1 :9 v / v) can be self-assembled on the I Ch channels using vapor deposition. Devices and solutions can be placed in a water bath at 40 °C for 1 h followed by baking on a hot plate at 80 °C for 10 min. The devices can then be dried with N2 gas and incubated with 1 mM 1 dodecanethiol for 1 h to passivate the exposed Au source and drain electrodes. The FETs for pH sensing can be used without further modification.

[0070] To functionalize FETs for hormone sensing, silanized FETs can be rinsed with ethanol, dried under N2, and immersed in 1 mM MBS (dissolved in 1 :9 (v / v) DMSO:phosphate- buffered saline) for 30 min. Meanwhile, 1 pM solutions of thiolated aptamers can be prepared by heating at 95 °C for 5 min followed by rapid cooling to room temperature in an ice bath. The MBS-modified I Ch channels can be immersed in the corresponding aptamer solutions and incubated overnight (~12 h). Before measurements, aptamer-modified FET sensors can be rinsed with deionized water and dried under N2 gas. Control FETs without aptamer modification can be silanized with PTMS.

[0071] FET Measurements

[0072] In experiments using devices fabricated according to embodiments, a PDMS well was used to cover each FET for solution-sensing. The sensor array had inter-FET distances of ~3 mm, which were large enough to isolate individual FETs for testing. Either phosphate-buffered saline (pH 7.4) or artificial sweat was used as the electrolyte as described. Measurements were performed using a manual analytical probe station (Signatone, Filroy, CA) equipped with a Keithley 4200A-SCS semiconductor parameter analyzer (Tektronix, Beaverton, OR). Five consecutive sweeps were averaged for each transfer curve. Calibrated responses were calculatedby dividing the absolute sensor response (Al) by the change in source-drain current with voltage sweep ( IDS / AVG) (Id.).

[0073] Circular Dichroism Spectroscopy

[0074] Aptamers were prepared by heating (without target) for 5 min at 95° C in phosphate-buffered saline followed by slow cooling to room temperature (Id.). Samples contained 2 pM aptamers with or without 2 uM targets. Circular dichroism spectra were collected on a JASCO-J815 spectrophotometer, with Peltier temperature control and autotitration attachments (Oklahoma City, OK). Measurements were taken at room temperature. Four scans were obtained for each sample, with 0.5 nm resolution, 1.0 nm bandwidth, a 20 nm / min scan rate, and a 4 s response time. Two replicates were prepared for each sample, and the average of eight scans was calculated and plotted. Spectra from phosphate-buffered saline samples without aptamers or targets were subtracted from each aptamer / target spectrum to correct for background absorbance.

[0075] Example lontophoretic Sweat Stimulation

[0076] lontophoretic hydrogels were prepared according to their respective placement on the anode or cathode (Id.). Hydrogels were prepared by dissolving 4% agarose in 0.1 M phosphate-buffered saline at 120 °C. Solutions were stirred until clear. For cathodes, the solution temperature was lowered to 60 °C, and 2% pilocarpine was added under continuous stirring. The anode and cathode solutions were pipetted into 3D-printed molds matching the areas of the iontophoresis electrodes to produce hydrogels with thicknesses of ~1 mm. The iontophoresis gels were allowed to cool at room temperature and stored at 4 °C until use.

[0077] Iontophoresis optimization was carried out by placing the appropriate hydrogel on its corresponding electrode before attachment to the surface of the skin of a volunteer subject. Iontophoresis current was then applied at various densities across the two electrodes for 5 min. After iontophoresis, the volume of sweat extracted was measured by estimating the filling% of the sensing chamber.

[0078] The herein described subject matter sometimes illustrates different components contained within, or connected with, different other components. It is to be understood that such depicted architectures are illustrative, and that in fact many other architectures can be implemented which achieve the same functionality. In a conceptual sense, any arrangement of components to achieve the same functionality is effectively "associated" such that the desired functionality is achieved. Hence, any two components herein combined to achieve a particular functionality can be seen as "associated with" each other such that the desired functionality is achieved, irrespective of architectures or intermedial components. Likewise, any two components so associated can also be viewed as being "operably connected," or "operably coupled," to each other to achieve the desired functionality, and any two components capable of being so associated can also be viewed as being "operably coupleable," to each other to achieve the desired functionality. Specific examples of operably coupleable include but are not limited to physically mateable and / or physically interacting components and / or wirelessly interactable and / or wirelessly interacting components and / or logically interacting and / or logically interactable components.

[0079] With respect to the use of plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0080] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as "open" terms (e g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.).

[0081] Although the figures and description may illustrate a specific order of method steps, the order of such steps may differ from what is depicted and described, unless specified differently above. Also, two or more steps may be performed concurrently or with partial concurrence, unless specified differently above. Such variation may depend, for example, on the software and hardware systems chosen and on designer choice. All such variations are within the scope of the disclosure. Likewise, software implementations of the described methods couldbe accomplished with standard programming techniques with rule-based logic and other logic to accomplish the various connection steps, processing steps, comparison steps, and decision steps.

[0082] It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation, no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations).

[0083] Furthermore, in those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to "at least one of A, B, or C, etc." is used, in general, such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate thepossibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0084] Further, unless otherwise noted, the use of the words “approximate,” “about,” “around,” “substantially,” etc., mean plus or minus ten percent.

[0085] Although the present embodiments have been particularly described with reference to preferred examples thereof, it should be readily apparent to those of ordinary skill in the art that changes and modifications in the form and details may be made without departing from the spirit and scope of the present disclosure. It is intended that the appended claims encompass such changes and modifications.

Claims

WHAT IS CLAIMED IS:

1. A device comprising: a wearable FET-based sensor device capable of ovulatory hormonal and physiological monitoring in human sweat.

2. The device of claim 1, further comprising an integrated and optimized an on-demand sweat sampling platform via iontophoretic drug delivery and biofluid routing using microfluidic adhesive structures.

3. The device of claims 1 or 2, wherein the sensor device includes an aptamer-FET sensing mechanism.

4. The device of claim 3, wherein the sensing mechanism uses oligonucleotide bioreceptors coupled with highly sensitive, nonlinear semiconductor signal transduction.

5. The device of claim 3, wherein the sensing mechanism includes biocompatible thin-film I Ch FETs.

6. The device of claim 1, wherein ovulatory hormonal monitoring includes one or more of estradiol, progesterone, and LH monitoring.

7. The device of claim 1, wherein physiological monitoring includes one or more of skin temperature and sweat pH monitoring.

8. A method comprising: performing, using a wearable FET-based sensor device, ovulatory hormonal monitoring and physiological monitoring in human sweat.

9. The method of claim 8, further comprising iontophoretic drug delivery and biofluid routing using microfluidic adhesive structures.

10. The method of claims 8 or 9, wherein performing includes sensing biomarkers in the human sweat via an aptamer-FET sensing mechanism.

11. The method of claim 10, wherein the sensing mechanism uses oligonucleotide bioreceptors coupled with highly sensitive, nonlinear semiconductor signal transduction.

12. The method of claim 10, wherein the sensing mechanism includes biocompatible thin- film ImCh FETs.

13. The method of claim 8, wherein ovulatory hormonal monitoring includes one or more of estradiol, progesterone, and LH monitoring.

14. The method of claim 8, wherein physiological monitoring includes one or more of skin temperature and sweat pH monitoring.

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