Implantable devices, biosensor devices, systems and methods

Abstract

The present invention is directed to biosensor devices, portable instruments and systems, including glass, that use aptamers and are more specifically battery-less, and to methods for manufacturing or operating instruments and devices, multiple instruments and systems.

Description

Technical Field

[0001] The present invention is directed to biosensor devices, implantable devices and systems including devices and / or implantable devices, and methods of manufacturing or operating devices / devices, devices and systems.

Background Art

[0002] A biosensor is a device that combines a biological component and a physicochemical detector, or a biochemical sensor, respectively, in order to detect and analyze a specific biological or biochemical analyte (hereinafter referred to as a biomarker or biological quantity). It is designed to convert a biological response, such as the interaction between a biomolecule and a target analyte, into a measurable signal.

[0003] The biological components of a biosensor can be various biological elements including enzymes, antibodies, nucleic acids, or whole cells. These components are selected based on their ability to recognize and interact with the target analyte of interest. The target analyte causes a specific biochemical or biophysical change when bound to the biological component.

[0004] The physicochemical detector (hereinafter also referred to as a biosensor device) is often called a transducer and converts a biological response into a measurable signal. This can be achieved through various mechanisms such as electrical, optical, electrochemistry methods. The transducer generates a signal proportional to the concentration or presence of the target analyte, enabling quantitative or qualitative analysis.

[0005] Biosensors have a wide range of applications in various fields, including medicine, environmental monitoring, food safety, and biotechnology. They can be used to detect and monitor biomarkers for disease diagnosis, measure blood glucose levels in diabetes management, detect environmental pollutants, or analyze DNA sequences. Offering advantages such as rapid detection, sensitivity, selectivity, portability, and real-time monitoring, biosensors have become a valuable tool in many industries.

[0006] The present invention relates, in particular, to an implantable device for detecting at least one biological quantity within the body of an animal or a human.

[0007] In this context, an implantable device is a device that is implanted into the body of an animal or a human, more specifically subcutaneously, or more specifically, configured to be fully implantable.

[0008] Implantable devices used to detect biological quantities within the body of an animal or human are also called implantable biosensors. Implantable biosensors are designed to monitor and measure specific biological quantities, or biological parameters, within the body.

[0009] Implantable biosensors can be used for a wide range of applications, including medicine, biomedical research, and veterinary medicine. Implantable biosensors can collect and / or provide real-time data on various biological quantities, namely physiological or biochemical parameters, enabling patients, healthcare professionals, and researchers to monitor physical conditions, diagnose diseases, and optimize treatment strategies.

[0010] Examples of implantable biosensors used to detect biological quantities include: Implantable glucose sensors used to continuously monitor blood glucose levels in diabetic patients. These sensors provide accurate and frequent blood glucose measurements, eliminating the need for regular finger-prick tests. Implantable pressure sensors used to measure pressure within different parts of the body. For example, implantable intracranial pressure sensors monitor pressure within the brain, aiding in the diagnosis and management of physical conditions such as hydrocephalus and traumatic brain injury. Implantable temperature sensors used to measure body temperature from within the body. These sensors can be used for a variety of purposes, including monitoring core body temperature during surgery and tracking temperature changes in specific tissues. Implantable sensors that can measure the acidity or alkalinity of solutions and monitor pH levels in various body fluids and tissues, providing useful information for the diagnosis and management of physical conditions such as acidosis and alkalosis. Implantable oxygen sensors that measure oxygen levels within body tissues and fluids. These sensors can be used to monitor tissue oxygenation in intensive care settings, assess wound healing, and optimize oxygen delivery during surgery.

[0011] There are also wearable and implantable biosensors that are not directly related to medicine. These types of biosensors are designed for a variety of purposes outside of the traditional medical field. Implantable biosensors can be used to monitor the physiological parameters of athletes during training and competition. These sensors can track metrics such as heart rate, body temperature, hydration levels, and lactate concentration. The collected data can help athletes optimize their training, prevent overtraining, and improve performance. Another area of ​​application relates to biohacking and quantified self. That is, some people engage in biohacking and quantified self-assessment, which involves tracking and optimizing various aspects of their own biology. It is possible to monitor parameters such as body temperature, sleep patterns, hormone levels, and even brain activity using implantable biosensors. This data can provide insights into an individual's health, habits, and performance optimization. Another area of ​​application relates to industrial monitoring. In industrial environments, implantable biosensors can be used for worker safety and environmental monitoring. For example, sensors implanted in workers can monitor exposure to hazardous substances, radiation levels, and other occupational hazards. This helps ensure a safe working environment and enables the early detection of potential health risks. Another application area relates to animal tracking and research. In wildlife and animal research, implantable biosensors are used for tracking, behavioral monitoring, and physiological data collection. These sensors can be implanted in animals to track migration patterns, habitat use, reproductive behavior, or monitor specific biological parameters. The collected data supports conservation efforts, understanding animal behavior, and ecological research. While these examples involve implantable biosensors for non-medical purposes, they still involve monitoring and collecting biological / physiological quantities, including physical parameters directly related to biology, such as radiation exposure. Although the application areas differ, the underlying principle of using biosensors for the collection of biological data (specifically real-time data) remains consistent.

[0012] Implantable biosensors offer the advantages of simplified monitoring of biological quantities, specifically continuous real-time monitoring, enabling early detection and timely intervention of abnormalities. These biosensors are designed to minimize tissue damage and discomfort due to their small size and biocompatibility. Furthermore, these biosensors can transmit data to external devices using wireless communication technology for analysis and interpretation.

[0013] Access to and use of assisted reproductive technologies (ART) is remarkably low in the global population. The European Society for Human Reproduction and Embryology estimates that the optimal utilization rate of in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) is 1,500 couples per million people per year, and that a large number of ART cycles are necessary for a successful outcome (Andersen AN, et al.; European IVF-monitoring program (EIM), European Society of Human Reproduction and Embryology (ESHRE). Assisted reproductive technology in Europe, 2001. Results generated from European registers by ESHRE. Hum Reprod. 2005 May;20(5):1158~76. doi:10.1093 / humrep / deh755.Epub (January 21, 2005. PMID:15665021.) However, a 2011 study reported that the global ART utilization rate was only 477 cycles per million people, and that an estimated 2 million cycles of ART resulted in 500,000 births (Adamson GD, et al., V. International Committee for Monitoring Assisted Reproductive Technology: World Report on Assisted Reproductive Technology, 2018.)

[0014] The low success rate of IVF is understood to be a result of implanting IVF-derived embryos into a uterus that is not receptive. One reason for this low success rate is thought to be that high estrogen levels resulting from the administration of gonadotropins to retrieve a large number of eggs rendered the uterus unreceptive. Therefore, uterine receptivity, regulated by the interaction of progesterone and estrogen, is extremely important for successful implantation and pregnancy outcomes. The results of experiments using a mouse delayed implantation model by Ma et al. provide evidence that estrogen levels within a very narrow range are a decisive factor in converting uterine receptivity to a refractory state, suggesting that the uterus is highly sensitive to estrogen levels in relation to implantation (Ma WG, Song H, Das SK, Paria BC, Dey SK. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc Natl Acad Sci USA. 2003 Mar 4;100(5):2963~8. doi:10.1073 / pnas.0530162100. Epub 2003 Feb 24. PMID:12601161;PMCID:PMC151449).

[0015] Furthermore, ART carries numerous risks for women, including ovarian hyperstimulation and the pregnancy itself. In Denmark, 1.2% of women develop ovarian hyperstimulation syndrome (OHSS), and 75% of them require hospitalization for more than 24 hours. This has led to increased efforts in the industry to ensure greater accuracy in treatment. A study of 2,700 women showed that individualized ovarian stimulation leads to better outcomes. However, reliable and accurate biomarkers are not frequently identified (Roudebush, WE, Kivens, WJ and Mattke, JM (2008), "Biomarkers of ovarian reserve", Biomarker Insights, 2008 Apr 16;3:259~268.doi:10.4137 / bmi.s537.PMID:19578510;PMCID:PMC2688347.). The process is not standardized across clinics, and there is still variation in whether clinics recommend monitoring with blood markers or rely solely on ultrasound scans.

[0016] Currently, IVF treatment requires women to undergo blood tests every other day. While this is a significant inconvenience, it is necessary to obtain more accurate results and outcomes of the procedure and to adapt clinical protocols during treatment to enhance treatment safety. Women also use other techniques to track hormone levels, such as urine strips to measure the presence of luteinizing hormone (LH), saliva samples to measure progesterone levels, and wearable devices to measure body temperature to track ovulation cycles. Depending on the woman's progress, monitoring is performed two to three times a week, sometimes more frequently. However, there are few alternatives to accurately monitor hormone levels, and none are performed in real time. The lack of more precise data has similarly limited the optimization of precision medicine in ART cycles and fertility treatments. Women's hormone levels are not understood in real time over long periods, and the impact this is likely to have on the women's community is revolutionary not only for ART but also for many other fertility-related issues, such as assisted reproductive technologies and the detection of certain ovarian disorders. Measuring estrogen, progesterone, and luteinizing hormone levels in real time is expected to allow for easy and accurate identification of the fertile period. Since estradiol and progesterone are both direct markers originating from the ovaries, they are among the most reliable indicators of ovarian events (Blackwell, L., Cooke, D., and Brown, S. (2018), "Self-Monitoring of Fertility Hormones: A New Era for Natural Family Planning?" Linacre Q. 2018 Feb;85(1):26~34. doi:10.1177 / 0024363918756387. Epub 2018 Mar 28. PMID:29970935; PMCID:PMC6027114).

[0017] Regarding in vitro fertilization (IVF), baseline hormone levels can be used to determine a woman's natural reproductive profile and make decisions about specific ovarian stimulation methods. This technology can also be used to monitor an individual's hormonal response and determine appropriate drug dosages and administration times. One example is human chorionic gonadotropin (hCG) administered on day 6 when serum estradiol levels are persistently elevated (Liu Y, Li J, Zhang W, Guo Y. Association between serum estradiol level on the hCG administration day and neonatal birthweight after IVF-ET among 3659 singleton live births. Sci Rep. 2021 Mar 16;11(1):6084. doi:10.1038 / s41598-021-85692-7. PMID:33727635; PMCID:PMC7966761).

[0018] Furthermore, while assessment of LH pulsetility is crucial for the clinical diagnosis of reproductive disorders, current methods are hindered by frequent blood sampling and the associated, expensive, sequential immunochemical analysis. Normal reproductive function is controlled by a highly coordinated hormonal feedback pattern across the hypothalamic-pituitary-gonadal (HPG) axis (Barbieri, RL. The endocrinology of the menstrual cycle. Methods Mol. Biol. 1154, 145~169 (2014) doi:10.1007 / 978-1-4939-0659-8_7. PMID:24782009). Pulsed release of LH, estradiol, and progesterone are key components for downstream regulation of sex steroid hormone synthesis and mature oocyte production. Alterations in hormone pulse secretion patterns have been associated with hypothalamic dysfunction, resulting in numerous reproductive disorders, including polycystic ovary syndrome (PCOS) (Bachelot, A. et al. Luteinizing hormone pulsatility in patients with major ovarian hyperandrogenism. J. Endocrinol. Invest. 30, 636~646 (2007) doi:10.1007 / BF03347443. PMID:17923794), hypothalamic amenorrhea (Touraine, P. et al. Resumption of luteinizing hormone pulsatility and hypogonadotropic hypogonadism after endoscopic ventriculocisternostomy in a hydrocephalic patient. Fertil. Steril. 76, 390~393 (2001)), and late or precocious puberty. Measuring hormonal pulsetility to determine modifications of secretion patterns is currently impractical in routine clinical practice due to its extremely resource-intensive nature. It requires collecting peripheral blood every 10 minutes for at least 8 hours, and continuous analysis using immunochemical assays is expensive.However, there are human studies conducted by specialized clinical research groups (Prague, JK et al. Neurokinin 3 receptor antagonism as a novel treatment for menopausal hot flushes: a phase 2, randomized, double-blind, placebo-controlled trial. Lancet 389, 1809~1820 (2017), doi:10.1016 / S0140-6736(17)30823-1. Epub April 3, 2017. PMID:28385352; PMCID:PMC5439024. Dhillo, WS et al. Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males. J. Clin. Endocrinol. Metab. 90, 6609~6615 (2005) Epub 2005 Sep 20.PMID:16174713.) indicates that there is a potential therapeutic benefit for women undergoing in vitro fertilization (IVF) and women in menopause in gaining the ability to more accurately monitor fluctuations in hormone levels. Currently, there are three main problems hindering widely known clinical hormone concentration profiling and its pulseability analysis: (1) The resolution of hormone concentration profiles is limited by sampling protocols and immunochemical analysis - no method exists that has the ability to monitor LH pulseability in real time. (2) Measuring hormone concentration profiles using continuous clinical chemiluminescence immunoassay is costly (approximately £20 per sample, requiring 50 samples per patient). (3) Hormone pulseability analysis is difficult because it typically requires sophisticated algorithms that can appropriately and efficiently account for the physiological factors (including excretion) that influence the typically inherent biological fluctuations, pulse-to-pulse variability, and hormone secretion and computation. There is a clear, unmet medical need for better translational technologies that would enable routine clinical LH pulsetility analysis in patients with reproductive disorders.

[0019] A biosensor device is a device that uses biological components such as enzymes, antibodies, and living cells to detect the presence or concentration of specific substances, such as chemicals or biomolecules. A biosensor typically consists of three components: a biological recognition element, a transducer, and a signal processing system.

[0020] A biological recognition element is a component of a biosensor that interacts with a target molecule and generates a signal. For example, an enzyme biosensor is thought to use an enzyme to catalyze a reaction that produces a detectable product, while an antibody biosensor is thought to use an antibody to bind to a specific antigen and generate a signal. A transducer is a component of a biosensor that converts the signal generated by the biological recognition element into a measurable output signal such as an electrical signal, optical signal, or acoustic signal. A signal processing system is a component of a biosensor that interprets the output signal and provides information about the concentration or presence of the target molecule. According to a preferred embodiment of the present invention, the biological recognition element is an aptamer, more specifically a biosensor membrane containing an aptamer layer.

[0021] In vivo monitoring provides a direct connection to bodily processes that occur at the cellular and tissue levels. Implantable biosensors will be able to precisely detect localized levels of different biomarkers.

[0022] Implantable devices are significantly established in contraception; intrauterine devices have been on the market since the 19th century and have been widely available since the 1970s. Furthermore, subcutaneous contraceptive implants were launched in the United States in 2006. While conventional implantable devices have been used in this market, biosensor devices, specifically subcutaneous biosensor devices, have never been used for infertility treatment. [Prior art documents] [Non-patent literature]

[0023] [Non-Patent Document 1] Andersen AN, et al.; European IVF-monitoring programme (EIM), European Society of Human Reproduction and Embryology (ESHRE). Assisted reproductive technology in Europe, 2001. Results generated from European registers by ESHRE. Hum Reprod. 2005 May;20(5):1158 - 76. doi:10.1093 / humrep / deh755. Epub 2005 Jan 21. PMID:15665021. [Non-Patent Document 2] Adamson GD, et al., V. Monitoring Assisted Reproductive Technology: World Report on Assisted Reproductive Technology, 2018. [Non-Patent Document 3] Ma WG, Song H, Das SK, Paria BC, Dey SK. Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation. Proc Natl Acad Sci USA. 2003 Mar 4;100(5):2963 - 8. doi:10.1073 / pnas.0530162100. Epub 2003 Feb 24. PMID:12601161; PMCID:PMC151449 [Non-Patent Document 4] Roudebush, W.E., Kivens, W.J. and Mattke, J.M. (2008), "Biomarkers of ovarian reserve", Biomarker Insights, 2008 Apr 16;3:259 - 268. doi:10.4137 / bmi.s537. PMID:19578510; PMCID:PMC2688347. [Non-Patent Document 5] Blackwell, L., Cooke, D. and Brown, S. (2018), "Self-Monitoring of Fertility Hormones: A New Era for Natural Family Planning?" Linacre Q. 2018 Feb;85(1):26 - 34. doi:10.1177 / 0024363918756387. Epub 2018 Mar 28. PMID:29970935; PMCID:PMC6027114 [Non-Patent Document 6] Liu Y, Li J, Zhang W, Guo Y. Association between serum estradiol level on the hCG administration day and neonatal birthweight after IVF - ET among 3659 singleton live births. Sci Rep. 2021 Mar 16;11(1):6084. doi:10.1038 / s41598-021-85692-7. PMID:33727635; PMCID:PMC7966761 [Non-Patent Document 7] Barbieri, R.L. The endocrinology of the menstrual cycle. Methods Mol. Biol. 1154, 145 - 169 (2014) doi:10.1007 / 978-1-4939-0659-8_7. PMID:24782009 [Non-Patent Document 8] Bachelot, A. et al. Luteinizing hormone pulsatility in patients with major ovarian hyperandrogenism. J.Endocrinol.Invest. 30, 636 - 646 (2007) doi:10.1007 / BF03347443. PMID:17923794 [Non-Patent Document 9] Touraine, P. et al. Resumption of luteinizing hormone pulsatility and hypogonadotropic hypogonadism after endoscopic ventriculocisternostomy in a hydrocephalic patient. Fertil. Steril. 76, 390~393 (2001) [Non-Patent Document 10] Prague, JK et al. Neurokinin 3 receptor antagonism as a novel treatment for menopausal hot flushes: a phase 2, randomised, double-blind, placebocontrolled trial. Lancet 389, 1809~1820(2017), doi:10.1016 / S0140-6736(17)30823-1.Epub April 3, 2017.PMID:28385352;PMCID:PMC5439024.Dhillo, WS et al.Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal axis in human males.J.Clin.Endocrinol.Metab.90,6609~6615(2005)Epub 2005 Sep 20.PMID:16174713 [Overview of the project] [Problems that the invention aims to solve]

[0024] Therefore, there is a need for improved technologies that enable hormone detection, specifically real-time detection. [Means for solving the problem]

[0025] In one aspect, the present invention relates to a biosensor device for detecting at least one hormone, (a) with at least one metal electrode surface; (b) A biosensor film comprising at least one aptamer attached to the surface of a metal electrode, wherein the aptamer is: (i) Having the ability to bind to at least one hormone; and (ii) Modified with one or more functional groups for attaching at least one aptamer to the surface of a metal electrode, Biosensor membrane and; This relates to biosensor devices, including those mentioned above.

[0026] Preferably, the biosensor device is configured to detect at least one hormone in a body fluid, more specifically in interstitial fluid.

[0027] Preferably, the biosensor device is an implantable device configured to be completely implantable in the body of an animal or human, more specifically subcutaneously. Preferably, the biosensor device is a wearable device configured to be used in contact with the skin of an animal or human.

[0028] Preferably, at least one hormone is selected from the group consisting of estradiol, luteinizing hormone (LH), progesterone, and any combination thereof.

[0029] Preferably, the biosensor device further includes a carrier, preferably transparent or preferably opaque, more specifically a biocompatible glass carrier.

[0030] Preferably, the aptamer is a single-stranded nucleic acid molecule, and / or preferably a DNA or RNA molecule that binds specifically with high affinity and specificity.

[0031] Preferably, the aptamer has a length of about 25 to 70 nucleotides, preferably about 30 to about 65 nucleotides.

[0032] Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group.

[0033] Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is located at the end of the aptamer.

[0034] Preferably, the aptamer is a single-stranded nucleic acid molecule, preferably a DNA or RNA molecule, and the functional group for attaching at least one aptamer to the metal electrode surface is located at the 3' or 5' end, preferably the 5' end.

[0035] Preferably, the aptamer has the ability to bind to at least one hormone selected from the group consisting of estradiol, luteinizing hormone (LH), and progesterone.

[0036] Preferably, the aptamer is one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

[0037] Furthermore, the present invention relates to a biosensor device according to the present invention for use in diagnosis, more particularly in the detection of at least one hormone, and more particularly in monitoring hormones during assisted reproductive technology (ART), during menopause, in hormonal disorders such as polycystic ovary syndrome (PCOS), in endometriosis, and / or during hormone therapy.

[0038] Furthermore, the present invention relates to the use of a biosensor device according to the present invention for in vitro diagnostics, specifically for the detection of at least one hormone.

[0039] Furthermore, the present invention relates to an implantable device for implantation in the body of an animal or a human, more specifically for subcutaneous implantation, - A biosensor device according to the present invention; - An electronic control device for controlling a biosensor device, more specifically, an electronic control device for generating, collecting, and preferably encrypting measurement data obtained by detection by the biosensor device; This includes information about portable devices.

[0040] Furthermore, the present invention relates to a system for detecting at least one hormone, - Furthermore, the portable device according to the present invention includes a communication device for wireless data communication with an external computer device separate from the portable device, in particular an NFC (Near Field Communication) device; - Communication devices for wireless data communication with portable devices, specifically external computer devices including NFC devices; Regarding systems that include this.

[0041] Furthermore, the present invention relates to a method for operating a biosensor device according to the present invention: (a) The step of bringing the surface of a metal electrode into contact with body fluid, more specifically interstitial fluid; (b) The step of enabling at least one aptamer on the surface of a metal electrode to bind at least one hormone; (c) Preferably, the step of detecting the binding of at least one hormone by electrically controlling the surface of at least one metal electrode via differential pulse voltammetry; Regarding methods including

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains.

[0043] It should be noted in detail that the use of the indefinite article "a" or "an" implies "one or more." Therefore, for example, the term "a hormone" includes "one" and "two or more" hormones.

[0044] As used herein, the terms “comprising” or “comprises” mean “non-restrictively including.” These terms are intended to be open-ended to define the presence of any of the described features, elements, integers, steps, or components, and are not intended to exclude the presence of one or more additional features, elements, integers, steps, components, or groups thereof. Thus, the terms “comprising” or “comprises” include the more restrictive terms “consisting of” and “consisting essentially of.” In one embodiment, the terms “comprising” or “comprises” as used throughout this application, and more specifically within the claims, may be replaced with the terms “consisting of” or “consisting essentially of.”

[0045] In a preferred embodiment, the present invention relates to a biosensor device for detecting at least one hormone, comprising at least one metal electrode surface and a biosensor film, wherein the biosensor film comprises at least one aptamer attached to the metal electrode surface.

[0046] The biosensor device according to the present invention improves IVF technology and current therapeutic administration methods by enabling real-time monitoring. Furthermore, this device brings innovation to other areas of women's health where frequent and accurate hormone level capture is important, such as polycystic ovary syndrome, endometriosis, and menopause. Applications in these areas will assist clinicians in early diagnosis and deliver more accurate treatment. Moreover, the data collected by the inventive biosensor will enable the construction of better predictive and personalized machine learning algorithms with superior capabilities and more accurate results, which are essential in clinical practice.

[0047] The biosensing membrane of a biosensor device (also called the “biosensor membrane”) is also called an “aptasensor”. The aptasensor can accurately and repeatedly detect the target analyte, in particular, a biological quantity. The aptasensor is preferably prepared electrochemically in several steps as outlined in the examples herein. These steps preferably include: activation of the metal electrode surface acting as the working electrode; preferably: addition of a nanoparticle layer for increased sensitivity; addition of a functional sensing layer formed by an aptamer; and preferably: addition of a porous protective membrane that is ultimately biocompatible. While the biosensor is in contact with the analyte, even in trace amounts, the aptamer binds to it, and the measurement signal is detected and recorded. Using this approach, it is possible to measure precise quantities, in particular the amount or concentration of the analyte, or modifications, in particular time-dependent modifications. The aptasensor is preferably calibrated and / or otherwise validated prior to its deployment.

[0048] In principle, biosensing of analytes via electrochemical sensors can be achieved using antibodies, aptamers, and protein receptors. The advantage of aptamers lies in their ability to bind to the analyte in a repeatable manner, while antibodies bind to the target irreversibly. Compared to other biosensors using enzymes or antibodies, the chemical simplicity, stability, specificity, and selectivity of aptamers enable continuous in vivo monitoring. Advantages including equivalent or superior affinity and specificity to the target, relatively small size, easier modification and immobilization, superior stability, and higher reproducibility have enabled the development of the highly specific biosensors of the present invention. The inventors have found that using aptamers provides a significant and unexpected advantage in terms of biosensor device lifespan compared to other biological recognition elements, for the purpose of detecting biomarkers, particularly hormones, in vivo.

[0049] Integrating aptamers into microelectrodes small enough to facilitate implantation results in challenges such as limited current signals due to low aptamer density on the electrode surface. This, in turn, leads to a low signal-to-noise ratio (SNR), which in embodiments of the present invention can be further exacerbated by the limitations of low-power communication devices, particularly NFC devices. To enhance the SNR, the inventors have developed strategies involving the use of nanoparticles, particularly gold and / or platinum nanoparticles, carbon nanotubes, or carbon nanoparticles. As a result of using these nanoparticles, the overall sensing surface area is significantly increased and the SNR is improved. Incorporating nanoparticles into the sensing layer significantly enhances the sensitivity of electrochemical sensors. These nanoparticles can be used for a variety of purposes, including modification of the electrode surface, labeling of specific target molecules for detection, signal amplification, and catalysis of ongoing chemical reactions. Furthermore, nanoparticles possess excellent biocompatibility, providing a larger surface area on the electrode, thereby improving electron transfer capability, facilitating the immobilization of bioactive materials on the electrode surface, and reducing the time required for detection. The nanoparticles used can vary in size (within the nanometer range), shape (spherical, cylindrical, planar, etc.), and composition, including precious metal nanomaterials such as gold and silver, semiconductor materials such as quantum dots, carbon-based nanomaterials such as carbon nanotubes and graphene oxide, and composite nanomaterials. Depending on the specific nanomaterial and substrate surface, numerous methods exist for depositing the nanoparticles, such as dip coating, spin coating, solvent evaporation, chemical vapor deposition, and transfer printing.

[0050] Preferably, the biosensor device is configured to detect physiological fluids or body fluids, such as blood, serum, saliva, urine, sweat, tears, mucus, vaginal secretions, uterine wall and menstrual blood, or interstitial fluid, preferably at least one hormone in the interstitial fluid.

[0051] As described above, in vivo monitoring provides a direct link to bodily processes performed at the cellular and tissue levels. Implantable biosensors can accurately detect local levels of different biomarkers. Thus, in one embodiment, the biosensor device is an implantable instrument configured to be fully implantable within the body of an animal or human, specifically subcutaneously.

[0052] As used herein, "aptamer" refers to a short single-stranded ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or xenonucleic acid (XNA) that binds a target molecule with a specific affinity. High affinity is primarily achieved through complex and diverse secondary and tertiary structures via intramolecular partial double-stranding of the single-stranded nucleic acid, resulting in a defined three-dimensional structure. Typically, aptamers are about 20 to 100 nucleotides in size. Target molecules can include small molecules, heavy metal ions, larger ligands such as proteins, and even entire cells. Aptamers may also be modified with specific functional groups to enhance their function or compatibility with larger, engineered molecular systems. Aptamers have applications in a wide range of fields, including, but not limited to, detection, therapy, reagent production, and engineering. For example, aptamers are used as detection molecules to bind certain biomolecules, such as hormones, present in bodily fluids, particularly interstitial fluid.

[0053] In relation to the present invention, any aptamer capable of binding to the hormone to be detected can be used. Those skilled in the art know how to develop aptamers that specifically bind to a given hormone, including, but not limited to, the systematic evolution of ligands by exponential enrichment (SELEX). SELEX is a multi-step process aimed at identifying and optimizing short single-stranded DNA or RNA molecules, i.e., aptamers, that can bind to a specific target molecule with high affinity and specificity. The SELEX process is known to those skilled in the art and may vary slightly depending on the specific end goal, target molecule, and resources available to the researcher.

[0054] For example, the SELEX process may include the following steps: 1) Target identification: The first step is to identify the target molecule to which the aptamer binds. This can be a small molecule, a protein, or even an entire cell. 2) Library generation: A large library of random DNA or RNA sequences is synthesized, typically containing billions of different sequences. These sequences serve as a starting point for aptamer selection. 3) Selection: The actual SELEX process begins. A library of random sequences is incubated with the target molecule to allow aptamers with binding affinity to the target to bind. 4) Separation: The bound aptamers are separated from the unbound ones. Various techniques can be used for this purpose, such as filtration, affinity chromatography, or magnetic separation, and are well known to those skilled in the art. 5) Amplification: The bound aptamers are eluted and amplified using a process called polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR). This step increases the concentration of the selected aptamers for the subsequent selection round. 6) Iterative Rounds: Repeat steps 3-5 for several rounds, typically 8-15 rounds, to enrich the pool of aptamers with high affinity and specificity for the target. Gradually increase the strictness of the selection criteria in each round to favor the selection of the best aptamer. 7) Sequencing and Analysis: After a desired number of selection rounds, the enriched aptamer pool is sequenced to determine the DNA or RNA sequences of the selected aptamers. Bioinformatics analysis is then performed to identify common motifs or structural features among the aptamers. 8) Aptamer optimization: The selected aptamer sequence is synthesized and further optimized to improve its binding properties, stability, and specificity. This can be achieved through chemical modification, cleavage, and mutagenesis. 9) Verification and Characterization: The optimized aptamers are tested to confirm their binding affinity, specificity, and functionality using various techniques such as surface plasmon resonance, fluorescence assays, and cell-based assays. These methods are known to those skilled in the art. 10) Applications: Ultimately, the validated aptamers can be used in a wide range of applications, including diagnostics, therapeutics, biosensing, drug delivery, and biomarker discovery.

[0055] Preferably, the aptamer is a single-stranded nucleic acid molecule, preferably a DNA molecule. For example, the aptamer is an oligonucleotide.

[0056] Preferably, the aptamer has a length of about 25 to 70 nucleotides, and more specifically, about 30 to about 65 nucleotides.

[0057] As described herein, aptamers can be modified with functional groups to enhance their functionality. There are various methods for modifying aptamers with functional groups, generally depending on the specific functional group and / or the attachment site of the functional group on the aptamer. These methods involve basic chemistry and are generally known in the art. In one embodiment, an aptamer is modified with a functional group to allow at least one aptamer to adhere to a metal electrode surface. In another embodiment, an aptamer is modified with one or more functional groups to allow at least one aptamer to adhere to a metal electrode surface. In principle, any functional group that enables the adhesion of an adapter to a metal electrode surface is usable, and the functional group may depend on the properties and composition of the metal electrode surface. In a particular embodiment, the functional group is a thiol group (-SH). The thiol group on the aptamer enables the adhesion of the aptamer to the metal electrode surface, particularly to a gold electrode surface. This is called a self-assembled monolayer (SAM). In principle, functional groups can be located at any site within an aptamer, as long as the aptamer's binding activity to the target analyte is not impaired or compromised. The precise location of the functional group may depend on the specific aptamer and the metal electrode surface.

[0058] A "self-assembled monolayer (SAM)" refers to a single layer of molecules that spontaneously assembles into an orderly pattern on a solid substrate. Without being bound by a specific theory, this organization occurs through intermolecular forces such as van der Waals interactions, hydrogen bonds, or electrostatic interactions. SAMs are generally formed by immersing a substrate in a solution containing the desired molecules, allowing them to adsorb and self-assemble on the surface. SAMs play a crucial role in crosslinking processes by providing templates for the adhesion of crosslinking agents and polymers. Functional groups present on the SAM surface react with crosslinking agents, enabling the formation of intermolecular covalent bonds. The crosslinking process enhances the stability and mechanical properties of the resulting material. SAMs provide a clear and controllable surface architecture available for crosslinking reactions, enabling the development of advanced materials with tuned properties and improved performance.

[0059] Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is located at the end of the aptamer. For example, the aptamer is a single-stranded nucleic acid molecule, preferably a DNA molecule. In this case, the functional group for attaching at least one aptamer to the metal electrode surface may be located at the 3' end and / or the 5' end. Preferably, the aptamer is a single-stranded nucleic acid molecule, preferably a DNA molecule, and the functional group for attaching at least one aptamer to the metal electrode surface is located at the 3' end. Preferably, the aptamer is a single-stranded nucleic acid molecule, preferably a DNA molecule, and the functional group for attaching at least one aptamer to the metal electrode surface is located at the 5' end. Attachment at the 5' end can be achieved, for example, by substituting the hydroxyl group of the phosphate group of the 5' terminal nucleotide with a thiol group. Attachment at the 3' end can be achieved, for example, by substituting the hydroxyl group of the ribose unit of the 3' terminal nucleotide with a thiol group.

[0060] Preferably, the biosensor of the present invention is configured for the detection of at least one hormone. This allows the aptamer to have the ability to bind to at least one hormone. The hormones to be detected include, but are not limited to, estradiol, luteinizing hormone, and progesterone. Preferably, the hormone to be detected is selected from the group consisting of estradiol, luteinizing hormone (LH), progesterone, and any combination thereof. Furthermore, the hormone to be detected may be selected from the group consisting of estradiol, luteinizing hormone (LH), progesterone, cortisol, FSH, testosterone, serotonin, triiodothyronine T3, tetraiodothyronine T4, calcitonin, insulin, melatonin, TSH, hGH, AmH, glucagon, and any combination thereof.

[0061] Therefore, preferably, the aptamer has the ability to bind at least one hormone selected from the group consisting of estradiol, luteinizing hormone, and progesterone. For example, if the biosensor is for detecting estradiol, the biosensor membrane includes at least one aptamer having the ability to bind estradiol. For example, if the biosensor is for detecting luteinizing hormone, the biosensor membrane includes at least one aptamer having the ability to bind luteinizing hormone. For example, if the biosensor is for detecting progesterone, the biosensor membrane includes at least one aptamer having the ability to bind progesterone. In another example, if the biosensor is for detecting, for example, a combination of estradiol and progesterone, the biosensor membrane includes at least one aptamer having the ability to bind estradiol and at least one aptamer having the ability to bind progesterone. In another example, the biosensor is for detecting a combination of estradiol, luteinizing hormone, and progesterone. Therefore, the biosensor membrane includes at least one aptamer capable of binding estradiol, at least one aptamer capable of binding luteinizing hormone, and at least one aptamer capable of binding progesterone. Preferably, the aptamers of the biosensor membrane consist of at least one aptamer to be detected or a combination of aptamers to be detected. Preferably, the biosensor membrane includes multiple aptamers to which a specific hormone to be detected is bound. In one embodiment, the multiple aptamers to be detected are arranged as layers on the surface of a metal electrode.

[0062] The amount of individual aptamers capable of binding to specific hormones, or the relative amount of aptamers capable of binding to different hormones present on the biosensor membrane, is variable and may depend on the desired stability and mechanical properties of the resulting material, as well as the desired architecture that enables the development of advanced materials with tuned properties and improved performance.

[0063] Preferably, the aptamer is one or more selected from the following group of aptamers: LH aptamer: 5'-TATGGTATGCTGTGTGGTATGGGGTGGCGTGCTCT-3' (Sequence ID 1) Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which may be present at the 3' and / or 5' ends of SEQ ID NO: 1. In a preferred embodiment, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which is present at the 5' end. β-estradiol aptamer: 5'-TTTTTTTTTTTTTTTGCTTCCAGCTTATTGAATTACACGCAGAGGGTA-3'(Sequence ID 2) Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which may be present at the 3' and / or 5' ends of SEQ ID NO: 2. Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which is present at the 5' end. 5'-GCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGCTTTTTTTTTTTT-3'(Sequence ID 3) Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which may be present at the 3' and / or 5' ends of SEQ ID NO: 3. In a preferred embodiment, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which is present at the 3' end. Progesterone aptamer: 5'-GCATCACACACCGATACTCACCCGCCTGATTAACATTAGCCCACCGCCCACCCCCGCTGC-3' (SEQ ID NO: 4) Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which may be present at the 3' and / or 5' ends of SEQ ID NO: 4. Preferably, the functional group for attaching at least one aptamer to the metal electrode surface is a thiol group, which is present at the 5' end.

[0064] Preferably, the present invention relates to a biosensor device for use in diagnosis. Preferably, the biosensor device of the present invention is for detecting hormones. For example, the biosensor can be used to determine the properties and / or concentration of at least one hormone. Hormone detection may be useful for monitoring hormones during assisted reproductive technology (ART) or menopause, or for monitoring the menstrual cycle. Hormone detection may also be useful for studying the progression and treatment of hormonal disorders such as polycystic ovary syndrome (PCOS) or endometriosis, or for diagnosing hormonal disorders. Hormone detection may also be useful during sex reassignment, menopause, and hormone therapy during ART.

[0065] Preferably, the present invention relates in detail to in vitro diagnostics for detecting at least one hormone. For example, in vitro diagnostics may be useful for diagnosing hormones from any sample derived from a subject, e.g., human or animal, including, but not limited to, body fluids, e.g., blood, serum, urine, interstitial fluid, or saliva. In vitro diagnostics may also be useful in basic research, e.g., for calibrating biosensors (see also the examples herein).

[0066] Furthermore, the present invention relates to a method for operating a biosensor device according to the present invention, comprising the following steps: (a) A step of bringing the biosensor membrane into contact with body fluids, specifically interstitial fluid; (b) a step of enabling at least one aptamer of the biosensor membrane to bind to at least one hormone; and (c) A step of detecting the binding of at least one hormone by electrically controlling the surface of at least one metal electrode.

[0067] Preferably, step (b) is carried out under physiological conditions.

[0068] Preferably, step (c) is carried out by differential pulse voltammetry (DPV). DPV is an electrochemical technique used for signal detection and quantification in various application fields. This involves applying a series of voltage pulses to an electrochemical cell and measuring the resulting current response.

[0069] Preferably, the biosensor device includes at least one electrode, which includes a metal electrode surface supporting a biosensor membrane. Preferably, the biosensor device further includes a working electrode, preferably a reference electrode, preferably a counter electrode. Preferably, the biosensor device includes an electronic control device for controlling at least one metal electrode surface. Preferably, the electronic control device is programmed in detail to perform an electrical measurement of at least one biological quantity by controlling at least one metal electrode surface. Preferably, the electrical measurement is voltammetry, preferably DPV (differential pulsed voltammetry) measurement. DPV measurement will be described in more detail below.

[0070] The present invention also relates to a method of using the biosensor device or implantable device or system of the present invention for the treatment of hormonal disorders in subjects such as humans or animals. Hormone detection in a therapeutic context is thought to be useful for monitoring during assisted reproductive technology (ART), during menopause, or for monitoring irregular menstrual cycles. Hormone detection is also thought to be useful in the context of treating hormonal disorders such as polycystic ovary syndrome (PCOS) or endometriosis. Hormone detection is also thought to be useful in the context of sex reassignment. For example, it is possible to determine the properties and / or concentration of at least one hormone using a biosensor and set up hormone replacement therapy for the patient according to the measurement results. In one embodiment, the method includes the treatment of a hormonal disorder, such as infertility, and includes the following steps: (a) A step of bringing the target body fluid, more specifically interstitial fluid, into contact with the biosensing membrane; (b) A step that allows at least one hormone to bind to at least one aptamer of the biosensing membrane; (c) A step of detecting the binding of at least one hormone by electrically controlling the surface of at least one metal electrode, more specifically via voltammetry, e.g., DPV; (d) A step of determining the properties and / or concentration of at least one hormone; (e) If the detected progesterone level is above the threshold level, preferably 1.5 ng / mL, the subject is treated with progesterone; and / or if the detected estradiol level is in the range of approximately 1,000 to 2,000 pg / mL, the subject is treated with hCG.

[0071] Example 1 The following steps describe the method used to prepare the aptamer prior to the self-assembling coating of the electrode surface: 1) Spin down the lyophilized powder vial to reach the dried aptamer pellet inside the vial (obtained as thiol-modified aptamers for estrodiol, LH, and progesterone as indicated in SEQ ID NOs. 1-4, obtained from Basepair Bio). 2) Resuspend the adapter pellet in 10 mM Tris, pH 7.5, 0.1 mM EDTA (0.6057 g Tris, 0.0184 g EDTA, 500 ml DI water; resuspension buffer) in nuclease-free water. 3) Add resuspension buffer to the aptamer pellet until a concentration of 100 μM is reached. 4) Prepare a folding buffer (1 mM MgCl2, 1x PBS, pH 7.5) and dilute the aptamer solution in the folding buffer to a 10-fold working concentration. Heat the solution to 90-95°C for 5 minutes. 5) Cool the solution to room temperature for 15 minutes. 6) Prepare a reduced DTT buffer: 20 mM DTT in 1 × TE buffer (10 mM Tris, pH 8; 1 mM EDTA), or 20 mM DTT in Tris-HCl buffer, or 1 M dithiothreitol (DTT). This step should be performed immediately before use. 7) The thiol-modified aptamer is reduced with 20 mM DTT in 1 × TE buffer, and the same volume of folded aptamer (1:1) is used with aptamer reduction buffer and incubated at room temperature (20-25°C) for 1 hour. 8) Remove DTT using a desalting column or a buffer exchange column. 9) Dilute the aptamer to the final working concentration using a buffer containing 1 mM MgCl2 (in PBS, which does not contain nucleases). 10) The aptamer is ready to coat the electrodes at any time.

[0072] Example 2 The following steps describe the method used to prepare functionalized gold electrodes for hormone measurement: 1) Simultaneously with the preparation of the aptamer (Example 1), a gold electrode on glass is prepared. 2) The electrodes are activated using cyclic voltammetry on 45 segments in 50 mM sulfuric acid with three electrodes (counter electrode, reference Ag / Cl electrode, and gold working electrode). 3) Following this step, the sensor was gently washed with 10 mM sulfuric acid. 4) The DPV (differential pulsed voltammetry) signal of the gold electrode was recorded before aptamer addition. 5) A 10 μl aptamer solution at a concentration of 5 mM was added to the gold working electrode, and the electrode was incubated for 16 hours to immobilize the aptamer onto the gold electrode via a self-assembled monolayer. 6) Afterward, any excess aptamer is washed away from the surface using sterile PBS. 7) Add 2 mM MCH (6-mercapto-1-hexanol) as a blocking agent to the electrode and incubate for 1 hour. 8) Record the DPV signal for dropcasting of the hormone concentrate (1 pg / ml to 1 μg / ml) in the ISF (interstitial fluid). 9) Finally, the sensitivity is measured by analyzing the peak current of each DPV signal. 10) The experiment was repeated at least three times, and the results are shown in Figure 11.

[0073] In a preferred embodiment, the present invention relates to an implantable device / implantable biosensor device.

[0074] Implantable biosensors carry the risk of rejection and adverse reactions due to various factors, including the body's immune response and the materials used within the implant.

[0075] When a foreign object is introduced into the body, the immune system may recognize it as non-self and initiate an immune response to eliminate or neutralize it. This immune response can manifest as inflammation, swelling, pain, or even immune-mediated rejection of the implant. The severity of the immune response varies from person to person and may be influenced by factors such as genetic characteristics and overall health. When using implantable biosensors, there is a need to reduce the risk of a severe immune response. Therefore, implantable devices are made from biocompatible materials, meaning they are designed to minimize adverse effects on living tissues. The selection of materials used for implants plays a crucial role in determining compatibility with the body. Some materials may be more likely to trigger an immune response or cause adverse reactions than others. For example, some metals or polymers may induce hypersensitivity reactions or release toxic substances when in contact with bodily fluids or tissues.

[0076] Transplantation procedures carry the risk of introducing bacteria or other microorganisms into the body, which can lead to infection. Infection can cause localized inflammation, tissue damage, and may even necessitate the removal of the implant. There is a need for implantable biosensors with attributes that minimize the risk of bacteria or other microorganisms entering the body.

[0077] The skills and techniques of the surgeon performing the implantation procedure also influence the risk of adverse reactions. Improper implant placement, intraoperative trauma, or insufficient sterilization increase the likelihood of complications and subsequent rejection. There is a need for implantable biosensors that are easily administered by surgeons.

[0078] Implantable devices involve significant financial investment, including the cost of the device itself, surgical procedures, and post-implantation care. Therefore, maximizing the lifespan of implantable devices is crucial to optimizing the cost-effectiveness of treatment. A longer lifespan for implantable devices reduces the need for frequent replacements and modifications, ultimately leading to a reduction in overall healthcare costs.

[0079] Implantable devices are often intended to provide long-term solutions for medical conditions and disabilities. The longer the lifespan of the device, the longer the patient can expect improved functionality, convenience, and quality of life. Avoiding frequent replacement or maintenance procedures minimizes disruption to daily life and reduces the burden on patients and caregivers.

[0080] Implantation procedures inherently carry risks, including the possibility of complications such as infection, bleeding, and tissue damage. Minimizing the need for additional surgeries or corrective procedures due to implant failure or deterioration can reduce the cumulative risks associated with multiple surgeries. This also reduces the likelihood of additional complications and the associated medical costs.

[0081] Implantable devices often play a crucial role in monitoring and managing medical conditions. For example, implantable sensors for monitoring blood glucose levels in diabetic patients provide valuable data for adjusting treatment. Extending the lifespan of such devices ensures continuous and reliable data collection, enabling healthcare professionals to make informed decisions about treatment plans.

[0082] Implantable devices should be designed to maintain their functionality and biocompatibility throughout their intended lifespan to minimize the risk of complications, adverse reactions, or device failure. Regular monitoring, follow-up care, and periodic evaluations are necessary to ensure the continued efficacy and safety of implantable devices.

[0083] Therefore, a fundamental object of the present invention, according to one aspect of the present invention, is to overcome one or more of the problems of the above-mentioned implantable biosensors, and more specifically, to provide an implantable device that has a sufficient lifespan and provides long-term safety.

[0084] This objective is addressed by an implantable device for detecting at least one biological quantity within the body of an animal or human, more specifically for implantation within the body of an animal or human, more specifically for subcutaneous implantation, comprising a biosensor device for detecting a biological quantity in vivo and an electronic control device for controlling the biosensor device, wherein the implantable device includes an encapsulation device that at least partially houses at least one of the biosensor device and the electronic control device, and the encapsulation device is at least partially made of glass.

[0085] The encapsulation device may be a single-part device or may include two or more encapsulation components that form the encapsulation device. Using a large number of encapsulation components may facilitate the assembly of portable devices. Preferably, the encapsulation device includes two encapsulation components, together capable of enclosing a cavity for housing an electronically controlled device and preferably other functional devices, such as a communication device or a biosensor device. Preferably, each encapsulation device includes exactly one, two, three, four, five, or six, or more, encapsulation components capable of enclosing the cavity together. Preferably, at least one, two, three, four, five, or more of the encapsulation components include glass or are made partially or entirely from glass, and more specifically substantially entirely from glass. The glass is preferably transparent or preferably opaque.

[0086] Preferably, the encapsulation device comprises at least one wafer substrate containing glass or made partially or entirely of glass, where “made entirely of glass” includes cases where these wafers are made substantially entirely of glass, meaning that the wafer may contain a low fraction of non-glass portions. The glass is preferably partially or entirely transparent and / or preferably partially or entirely opaque. Preferably, the glass wafer comprises a plurality of open or preferably non-glass material, preferably filled with metal, the vias being cylindrical in particular and having a height of about 100 to 1000, preferably 200 to 800, preferably 300 to 700, preferably 400 to 600, preferably 500 micrometers, a diameter of preferably 20 to 500, preferably 40 to 200, preferably 50 to 150 micrometers, and a pitch of preferably 1000 to 20, preferably 800 to 30, preferably 500 to 50 or preferably 300 to 50 micrometers. For example, HermeS® glass wafer substrates, available from Schott AG in Germany, are glass wafers made substantially of glass. These include hermetically sealed solid glass through-vias (TGVs). Fine-pitch vias allow for reliable conduction of electrical signals and power into and out of portable devices using such glass, as the vias are completely filled with conductive metal. A wafer substrate is generally understood to mean a solid, planar plate- or disc-shaped element that acts as a substrate for at least one electrode or other sensing element. Preferably, the wafer substrate is an element cut from a larger wafer, more specifically from a glass wafer.

[0087] Preferably, the wafer substrate includes at least one metal-filled via connected to a device of a portable device, more specifically: an electronic control device, a biosensor device, more specifically at least one electrode of a biosensor device, or a communication device. Preferably, the wafer substrate includes at least one metal-filled via connected to at least one sensing element, more specifically at least one electrode of a biosensor device, on one side of the wafer substrate and connected to an electronic control device on the other side of the wafer substrate. Preferably, the wafer substrate includes at least one metal-filled via connected to a communication device on one side of the wafer substrate and connected to an electronic control device on the other side of the wafer substrate.

[0088] A wafer substrate is understood to be a thin, substantially flat, preferably substantially rectangular or substantially circular piece of material. Preferably, the wafer substrate provides a solid and uniform surface on which various electronic structures, circuits, or electrodes that may form part of an electronically controlled device and / or communication device can be built.

[0089] The wafer substrate is preferably made of glass. The wafer substrate may also be constructed from another amorphous or crystalline material, such as a semiconductor material like silicon (Si). Glass wafers are preferred due to their desirable electrical and mechanical properties, availability, and compatibility with standard lithography processes. Furthermore, glass offers excellent biocompatibility even without a biocompatible coating. However, for encapsulation devices or wafer substrates made of partially or entirely glass or non-glass material, a biocompatible coating is also a preferred option. The coating may be a glass coating.

[0090] The inventors of this invention have discovered that glass can be used to create a safe and reliable encapsulation device that includes one or more hermetically sealed cavities having a long lifecycle.

[0091] The glass used in the encapsulation device may preferably be one of the following: bioactive glass, borosilicate glass, aluminosilicate glass, phosphate glass, silica glass, or chalcogenide glass. Bioactive glass has the ability to bond with biological tissues. These typically contain calcium, phosphorus, and silica as their main components. Borosilicate glass, such as Pyrex®, has high chemical resistance and thermal stability. Aluminosilicate glass is a family of glasses containing aluminum and silicon. These have excellent mechanical strength. Phosphate glass typically contains phosphorus pentoxide (P2O5) as its main component. These exhibit high solubility in aqueous solutions. Silica glass, such as fused quartz, is mainly composed of silicon dioxide (SiO2). These have excellent light transmittance, low thermal expansion coefficient, and excellent chemical stability. Chalcogenide glass contains chalcogen group elements, including sulfur, selenium, and tellurium. These have unique optical and electrical properties. Preferably, the glass is Borofloat® 33, AF32® eco33, or D263® Teco, all of which are available from Schott AG in Germany. Preferably, the encapsulation device is made from one type of glass material, which facilitates the assembly of the encapsulation device.

[0092] In a preferred embodiment, at least one wafer substrate forms part of a biosensor device, and preferably supports one or more metal electrodes that support a biosensor film.

[0093] In a preferred embodiment, at least one wafer substrate supports an electronically controlled device, more specifically, used to control a measurement process using a biosensor device.

[0094] In a preferred embodiment, at least one wafer substrate supports a communication device, more specifically an RFID device, and more specifically an NFC device.

[0095] In a preferred embodiment, one wafer substrate supports two or all of the following: an electronic control device, at least one sensing element of a biosensor device, more specifically at least one electrode, and a communication device.

[0096] In a preferred embodiment, the encapsulation device comprises two wafer substrates stacked on top of each other, arranged in parallel in particular, and preferably spaced apart to encapsulate a cavity capable of housing one or more electronic control devices, biosensor devices, and communication devices. The spacing may preferably be 200 to 5000, preferably 300 to 3000, and preferably 500 to 2500 micrometers. The sides of the cavity may be integral with either wafer substrate or closed by bar members, which are separate components bonded to either substrate wafer, preferably in particular by welding or bonding.

[0097] In a preferred embodiment, at least two glass components, more specifically two glass wafer substrates, of the encapsulation device for the implantable device are preferably aligned in a stacked configuration and bonded by femtosecond laser stitching. This technique has unexpectedly proven to guarantee complete airtightness and, in itself, protect the electronic components and internal cavities from the aforementioned exposure to physiological fluids, even under stress conditions and during long-term test use of the implantable device.

[0098] In a preferred embodiment, the encapsulation device preferably includes a stack of two, three, four, or more wafer substrates spaced apart to enclose a cavity.

[0099] The wafer substrate may have flange portions that form a trough-shaped substrate member. The hollow portion of such a trough can act as a cavity for housing or covering an electronic control device, a biosensor device, or a communication device.

[0100] The encapsulation device may have a capsule shape when assembled. This facilitates, for example, the implantation of an implantable device using a syringe.

[0101] The encapsulation device, when assembled, has a box shape, and may be more specifically a rectangular parallelepiped, preferably a rectangular parallelepiped. The edges and / or corners are rounded, and more specifically, have a rounding radius of preferably 0.05 to 5 mm, preferably 0.1 to 3 mm.

[0102] In a preferred embodiment, the encapsulation device includes a main wafer substrate and a trough-shaped top cover wafer substrate. The top cover wafer substrate covers one side of the main wafer substrate, and a cavity can be defined between the side of the main wafer substrate and the hollow space defined by the trough-shaped top cover wafer substrate.

[0103] Preferably, the sealing device includes a trough-shaped bottom cover wafer substrate. The bottom cover wafer substrate covers another side of the main wafer substrate, and a cavity can be defined between the other side of the main wafer substrate and the hollow space defined by the trough-shaped bottom cover wafer substrate.

[0104] An encapsulation device, more particularly at least one wafer substrate, more particularly at least one trough-shaped wafer substrate, may include one or more pores. The pores may be configured to allow aqueous solutions, more particularly bodily fluids, to flow into cavities covered by at least one wafer substrate. A suitable wafer substrate for forming such a porous surface is CoralPor® porous glass, commercially available from Schott AG in Germany. The geometry of the pores may be cylindrical, the pore height may be preferably in the range of 200 to 800 μm, more preferably 300 to 600 μm, more preferably 360 to 580 μm, the pore diameter may be preferably 10 to 500 μm, more preferably 10 to 300 μm, the pore size may be preferably 20 to 150 μm, more preferably 32 to 80 μm, and the porosity may be preferably 40 to 80%, more preferably 60 to 70%, more preferably 60 to 75%.

[0105] Preferably, the encapsulation device completely encloses the electronic control device, or the encapsulation device completely encloses the electronic control device and the communication device, or the encapsulation device completely encloses the electronic control device, the communication device and the biosensor device, where complete enclosure means that when the implantable device is implanted in the body of a human or animal, the completely enclosed electronic control device and / or communication device are not in contact with bodily fluids, and / or more specifically, when the implantable device is implanted in the body of a human or animal, the biosensor device is in contact with bodily fluids.

[0106] In general, the encapsulation device may consist partially or completely of at least one non-glass material, such as a polymer. In another embodiment, the biosensor device may be encapsulated by embedding it in a encapsulation compound, which may be biocompatible or contain a biocompatible coating layer.

[0107] Each electronically controlled device preferably includes one or more of the following components: a microcontroller, more particularly a low-power or ultra-low-power microcontroller; one or more integrated circuits (ICs), more particularly an ASIC (Application-Specific Integrated Circuit); one or more electronic amplifiers for amplifying electrical measurement signals, more particularly currents, received from the biosensor device; a light source, preferably an LED, for visually transmitting information to the user of the portable device, or another output device for transmitting signals (e.g., acoustic signals) to the user. The electronically controlled devices may be part of the biosensor device or connected to the biosensor device.

[0108] An ASIC is a type of integrated circuit (IC) designed and manufactured to perform a specific set of functions within a particular application or system. Unlike general-purpose ICs designed to perform a wide range of functions, ASICs are customized and optimized for specific applications, resulting in higher performance, lower power consumption, and often lower cost compared to other solutions. However, an integrated circuit can also be a general-purpose IC programmed to perform a specific set of functions, for example.

[0109] A specific set of functions may include, in detail, controlling one or more electrodes of a biosensor device to perform a measurement process by measuring the current or impedance between at least two electrodes of the biosensor device as the potential applied to at least two electrodes fluctuates. The measurement is preferably DPV. A specific set of functions may include, receiving a measurement signal from the biosensor device and deriving measurement data therefrom. A specific set of functions may include transmitting the measurement data to an external data processing device (e.g., a mobile phone, tablet PC, or PC) via a communication device. A specific set of functions may include, via a communication device, in detail an RFID device, in detail an NFC device, receiving electrical energy from radio waves reaching an externally portable device, and preferably using said electrical energy to power the measurement process.

[0110] Preferably, the electronically controlled device is programmed to implement a specific set of functions.

[0111] DPV stands for Differential Pulse Voltammetry, an electrochemical technique used for signal detection and quantification in various application fields. It involves applying a series of voltage pulses to an electrochemical cell and measuring the resulting current response. A preferred electrochemical cell setup for DPV is as follows: a working electrode, a reference electrode, and a counter electrode are placed within the electrochemical cell. The DPV technique applies a series of voltage pulses to the working electrode. The pulses typically consist of a step potential, a holding potential, and a pulse width. The step potential provides the driving force for the electrochemical reaction, and the holding potential gives the system time to reach equilibrium before the next pulse. Current measurement: After each voltage pulse, the resulting current response is measured using a potentiostat. The current response corresponds to the electrochemical reaction occurring at the working electrode. The obtained current response is typically plotted against the applied voltage pulses. The resulting DPV curve exhibits a characteristic peak shape that can facilitate electrochemical reactions, providing information about the concentration of the analyte, the redox reaction, and other electrochemical processes with the analyte occurring in the system containing the analyte.

[0112] An electronically controlled device may contain at least one piece of program code to implement a specific set of functions, which may include one or more of the following functions: The electronically controlled device is preferably programmed to implement one or more of the following functions: • Processing measurement data acquired by a biosensor device. This measurement data is derived from the measurement process performed by the biosensor device. • Encryption algorithm, specifically a lightweight encryption algorithm, to encrypt the measurement data; Establishing a data connection between at least one biosensor device or communication device of at least one portable device and a communication device of an external computer device, for example, by using a data communication protocol, such as a known communication protocol like TCP / IP, or a custom-made communication protocol; • Data is exchanged between at least one biosensor device or at least one portable device's communication device and an external computer device's communication device, for example, by using a data communication protocol, such as a known communication protocol like TCP / IP, or a custom-made communication protocol; • Storing data received from at least one biosensor device or at least one communication device of a portable device in data storage which may be part of or connected to the biosensor device or portable device.

[0113] The communication device is preferably a programmable device, and more specifically, is programmed to transmit measurement data via a wireless connection to an external computer device, more specifically by RFID, preferably NFC or WLAN, to receive measurement data from an electronically controlled device, preferably in an encrypted format.

[0114] RFID stands for Radio Frequency Identification, and an RFID device refers to a system or device that uses radio waves to wirelessly exchange data using radio frequency electromagnetic fields. An RFID system for data exchange typically consists of two main components: an RFID transponder and an RFID reader, which may be an external computer device, or more specifically, part of a mobile phone. An RFID transponder is a small electronic device that includes a microchip and an antenna. The microchip stores measurement information / measurement data generated by a biosensor device. The antenna enables communication with the RFID reader using radio waves. RFID transponders are preferably passive, but can also be active. Passive RFID transponders have no internal power supply and rely on energy transmitted from the RFID reader to power them. When the reader emits radio waves, the transponder's antenna captures the energy and uses it to activate the microchip, preferably activating the measurement process and transmitting the stored measurement data to the reader. Active RFID transponders, which may be part of a portable device, have their own internal power source, usually a battery, which allows them to transmit signals continuously or on demand, providing a longer reading range and the ability to store more data. Active transponders are typically used in application areas that require longer distance or real-time tracking. An RFID reader is a device that emits radio waves and receives signals from an RFID transponder. It consists of an antenna, a transceiver for transmitting and receiving radio signals, and a decoder for interpreting the data received from the tag. The reader communicates with the tag wirelessly and can read many tags simultaneously within its range. When an RFID transponder enters the range of an RFID reader, the reader's radio waves activate the tag. The transponder responds by sending stored data to the reader, which then decodes and processes the information. The reader can be connected to a computer or backend system to further process and utilize the data collected from the transponder.

[0115] NFC stands for Near Field Communication, and an NFC device is a type of device that incorporates NFC technology, enabling it to communicate wirelessly with other NFC-enabled devices or transponders in close proximity. NFC is a short-range wireless communication technology that operates at a frequency of 13.56 MHz. Typical NFC devices include smartphones, tablets, smartwatches, and other electronic devices with embedded NFC chips. These devices can interact with each other by bringing them close together or by tapping them together.

[0116] NFC enables contactless communication between devices. Two NFC-enabled devices can establish a connection and exchange data simply by placing them together or tapping them. NFC allows for the transfer of various types of data, such as measurement data, between devices. This data exchange can be initiated by the user or triggered by a specific application or service. NFC technology provides a convenient and secure method for short-range wireless communication and data transfer.

[0117] The communication device is preferably an RFID device, more specifically an NFC device.

[0118] A biosensor device includes at least one sensing element, more specifically at least one electrode, which may carry a biosensor membrane, such as an aptamer-containing membrane, for measuring a biomarker of an aptamer during the measurement process, preferably the binding of a hormone, or a surface concentration on the electrode surface. The biosensor membrane is configured to come into contact with body fluids to measure biological quantities in body fluids. Preferably, the electrodes include a reference electrode and one or more working electrodes, where one working electrode carries the biosensor membrane. The biosensor device may be a standalone device. The biosensor device may preferably include an electronically controlled device and / or communication equipment. The biosensor device may be understood to include the same components as an implantable device, except for the encapsulation device. If the encapsulation device is omitted, the biosensor device may be used as an in vitro solution or in contact with the body of a human or animal, for example, with skin tissue.

[0119] The biosensor device may be a passive device powered by energy derived from radio waves received via a communication device to query the biosensor device from an external computer device. The external computer device, such as a mobile phone, may include a communication device capable of emitting the radio waves to query the biosensor device. The communication device generally has the capability to wirelessly exchange data with at least one biosensor device. In detail, the biosensor device and / or portable device is a battery-less device / device.

[0120] However, it is also preferable that the biosensor device includes a battery for the purpose of providing electrical energy to perform a measurement process using the biosensor device and / or for providing electrical energy to exchange data with at least one external computer device. In this context, “external” means that the computer device is separate from the biosensor device and the implantable device. If the implantable device is implanted in the body of a human or animal, the implantable device is separate from the external computer device.

[0121] At least one sensing element may be an electrode. In this case, at least one biological quantity is measured using an electrical measurement process, specifically voltammetry or capacitance.

[0122] At least one sensing element is an optical sensing element which may include a radiation source, more specifically a light source and / or a radiation sensor, such as a photodetector. In this case, at least one biological quantity is measured using an optical measurement process, more specifically a measurement that is sensitive to a change in the refractive index of the test material, or a measurement that is sensitive to a change in the amplitude and / or emission spectrum (light spectrum) of the sensing radiation (light) emitted from the radiation source. The optical sensing element may be a photochemical sensing element which has the ability to detect the presence or change in the concentration of at least one chemical or biological compound attached to the measuring surface of the photochemical sensing element, which occurs when the measuring surface is brought into contact with the fluid to be measured, more specifically a bodily fluid.

[0123] At least one electrode may be made of gold. Furthermore, it may support at least one layer of commercially available metal nanoparticles, particularly gold nanoparticles. This increases the measuring surface area of ​​the biosensor film—the surface may not be planar but surround the nanoparticles. A layer of platinum nanoparticles may be provided on top of the gold nanoparticle layer. The inventors unexpectedly discovered that a composite layer of gold and platinum nanoparticles efficiently extends the lifespan of the biosensor device and, consequently, the implantable device. The gold nanoparticles are preferably attached to one or more electrodes, particularly the working electrode. The platinum nanoparticles are preferably attached to the gold nanoparticle layer.

[0124] The present invention also relates to a system for detecting at least one biological quantity in the body of an animal or human, the system comprising: • At least one biosensor device according to the present invention, or at least one portable device according to the present invention • At least one external computer device separate from at least one biosensor device or at least one portable device, the external computer device including a communication device for wirelessly exchanging data, more specifically measurement data, with the communication device of the at least one biosensor device or at least one portable device, more specifically an RFID, NFC or WLAN device.

[0125] The system may also include program code to be executed by an external computer device, configured to implement data exchange between at least one biosensor device or communication device of at least one portable device and a communication device of the external computer device. More specifically, the external computer device may be programmed using the program code to implement one or more of the following functions: Establishing a data connection between a communication device of at least one biosensor device or at least one portable device and a communication device of an external computer device, for example, by using a data communication protocol, such as a known communication protocol like TCP / IP, or a custom-made communication protocol; • Exchanging data between a communication device of at least one biosensor device or at least one portable device and a communication device of an external computer device, for example, by using a data communication protocol, such as a known communication protocol like TCP / IP, or a custom-made communication protocol; • Decrypting encrypted data, specifically encrypted measurement data, received from at least one biosensor device or at least one portable device's communication equipment; • Processing data, specifically decoded data, specifically evaluating the data to obtain measurements from the data; • Storing data received from at least one biosensor device or at least one portable device communication device in data storage that is part of or can be connected to an external computer device.

[0126] It is pointed out that the method for preparing gold electrodes on a glass wafer substrate is an invention independent of other inventive embodiments of implantable devices and biosensor devices. This method includes the following steps: - Step of providing a glass wafer substrate; - A step of depositing an electrode structure made of an adhesion-enhancing layer for improving the adhesion of gold onto a glass wafer substrate, wherein the adhesion-enhancing layer is preferably made of NiCr or chromium by using deposition with a NiCr or Ni source, and more specifically by implementing a lithography process; - Specifically, the step involves depositing gold onto the electrode structure using gold atom deposition.

[0127] Preferably, a method for preparing a gold electrode on a glass wafer includes one of the following steps: • A step to clean all contaminants from the glass wafer substrate. • A step of creating a pattern for electrodes on a glass wafer substrate using a suitable photoresist. • A step of creating a photomask with the desired electrode pattern. This mask will be used to expose a photoresist and create the desired pattern. • Step of preparing the developer according to the manufacturer's instructions. • A step of using a suitable NiCr, Ni, or gold source for deposition, for example, a (gold) deposition apparatus. • A step in which the glass wafer substrate is immersed in a cleaning solution to remove surface contaminants. • A step in which the glass wafer substrate is thoroughly rinsed with distilled water and dried. • A step of uniformly applying photoresist to the surface of a glass wafer substrate. • A step of ensuring uniform coverage and a smooth surface using a spin coater or other suitable method. • A step to dry the photoresist according to the manufacturer's instructions. • A step of placing a photomask on a glass wafer substrate. • A step in which the glass wafer substrate is exposed to ultraviolet light, following the manufacturer's instructions for the photoresist. Upon exposure, the photoresist becomes soluble in the exposed area, while remaining insoluble in the masked area. • The step of immersing the exposed cover glass in the developer solution, according to the manufacturer's instructions. The developer removes the soluble photoresist, revealing the electrode pattern. The step of placing a prepared glass wafer substrate with an exposed electrode pattern in a vacuum chamber with a source of NiCr, Ni, or gold. • A step of creating a vacuum in a chamber and evaporating gold from a source of NiCr, or Ni, or gold. NiCr, Ni, or gold atoms condense on the surface of the glass wafer substrate, forming a thin layer of NiCr, Ni, or gold that covers the electrode pattern. Depending on specific requirements, post-treatment such as annealing or surface modification may be necessary to enhance the adhesion and properties of NiCr, Ni, or gold electrodes. By following these procedures, NiCr, Ni, or gold electrodes can be lithographically applied to a glass wafer substrate using gold atom deposition.

[0128] Each electrode has a complex layered structure to ensure sufficient electrical contact with the biosensor. Each layer contributes to the performance of the single atomic layer that binds to the biosensor to generate an electronic signal. Nanolayers are deposited onto the wafer to maximize electronic contact with the aforementioned biosensor. This process is carried out by atomic layer deposition to achieve critical contact with the adhesive layer.

[0129] Preparation of back / outer surface masks / etching of wafers in preferred manufacturing steps: Biocompatible glass wafers with a cutting diameter of approximately 100 mm (up to 300 mm) are prepared and cleaned in a preparation solution to improve the adhesion of the initial substrate.

[0130] The wafers are prepared using positive and negative masks during the manufacturing process to enable the application of a thin-film metallization layer. Advantageously, a glass adhesive layer is used to bond and provide adequate tensile strength (50N) to allow for the metallization of an additional layer on the prepared surface.

[0131] The wafer is then ready for either additional metallization to increase the layer thickness to 50,000 nm by electroplating, or activation of the Au surface to allow the biosensor to be attached to the electrode.

[0132] The biosensor device can be prepared in the following three steps. 1) Activation of the deposited gold layer on the glass. This step is performed by connecting the working electrode (gold) to a potentiostat and performing continuous cyclic voltammetry (CV) in 50 mM sulfuric acid. While CV is being performed, the electrode is cleaned with ethanol and DI water. 2) Deposition of gold nanoparticles onto the gold layer on the glass surface. This step is performed to increase the surface area of ​​the gold surface. And finally: 3) Activation of the biosensor membrane. This biosensor membrane is composed of self-assembled monolayers (SAMs) of aptamers, and this SAM layer plays a role in permanently attaching the aptamers to the gold platform.

[0133] The biosensor membrane may contain molecules for binding biomarkers derived from bodily fluids. These molecules may be aptamers or antibodies. Preferably, the hormone is one of estradiol, LH, or progesterone.

[0134] The present invention also relates to a method for operating an implantable device or biosensor device according to the present invention. a. A step of bringing the biosensor membrane into contact with bodily fluids; b. A step that allows a biomarker, specifically a hormone, to bind to a biosensor membrane; c. A step of detecting the binding of a hormone to a biosensor membrane by electrically controlling the surface of at least one metal electrode; It can also be defined by methods that include this.

[0135] The present invention relates, in particular, to an implantable device for implantation in the body of an animal or human, more particularly for subcutaneous implantation, comprising a biosensor device for detecting biological quantities in vivo, more particularly the biosensor device described in the claims, and an electronic control device for controlling the biosensor device, and relating to each of the following preferred embodiments of the implantable device, where each embodiment of the implantable device may be claimed by a claim, or an implantable device relating to a combination of multiple embodiments may be claimed by the patent claims of paragraph 1.

[0136] According to Embodiment 1, the implantable device is, More specifically, an encapsulation device, as described in many embodiments herein, for at least partially encapsulating at least one of a biosensor device and an electronic control device, and which is at least partially made of glass; Includes.

[0137] According to Embodiment 2, the portable device configured in detail according to Embodiment 1 further includes a communication device connected to an electronically controlled device for wireless data communication with an external device, in detail an NFC device.

[0138] According to Embodiment 3, in more detail, a portable device configured according to Embodiment 1 or Embodiment 2 is further: The encapsulation device completely encloses the electronically controlled device, or The enclosed device completely encloses the electronic control device and the communication device, or The enclosed device completely encapsulates the electronic control device, communication device, and biosensor device. This complete encapsulation ensures that, when the implantable device is implanted in a human or animal body, the completely encapsulated electronic control device and / or communication device do not come into contact with bodily fluids, and / or, more specifically, that the biosensor device may come into contact with bodily fluids when the implantable device is implanted in a human or animal body. Defined by:

[0139] According to Embodiment 4, more specifically, a portable device configured according to Embodiment 1, preferably according to any embodiment of Embodiments 2 to 3, is further defined by including at least one wafer substrate for which the encapsulation device is substantially made of glass.

[0140] According to Embodiment 5, more specifically, a portable device configured according to Embodiment 4, preferably according to any embodiment of Embodiments 2 to 3, is further defined by the fact that the encapsulated device includes at least one wafer substrate supporting one or more electrodes on one side of the wafer substrate and preferably supporting at least one electronic component, more specifically an electronic control device, on the other side of the wafer substrate, wherein preferably one or more electrodes are connected to at least one electronic component.

[0141] The present invention, in particular, is: a. The step of bringing the biosensor membrane of the biosensor device into contact with bodily fluids; b. A step that allows a biomarker, preferably a hormone, to bind to a biosensor membrane; c. A step of detecting the binding or surface concentration of a hormone on a biosensor membrane by electrically controlling the surface of at least one metal electrode; The present invention relates to a method for operating a portable device or a biosensor device according to any one of embodiments 1 to 5, including the above.

[0142] According to aspect 6, the present invention relates to a system for detecting at least one biological quantity in the body of an animal or human: • At least one biosensor device according to the present invention, or at least one biosensor device according to any embodiment of Embodiments 1 to 5, or other embodiments described herein; • At least one external computer device separate from at least one biosensor device or at least one portable device, the external computer device including a communication device for wireless exchange of data, more specifically measurement data, between the at least one biosensor device or at least one portable device, more specifically an RFID, NFC or WLAN device; It is intended for systems that include this.

[0143] According to Embodiment 7, the present invention is directed to program code to be executed by an external computer device, configured to implement data exchange between a communication device of at least one biosensor device or at least one portable device and a communication device of the external computer device. More specifically, the external computer device may be programmed, more specifically by using the program code, to implement one or more of the following functions: Establishing a data connection between a communication device of at least one biosensor device or at least one portable device and a communication device of an external computer device, for example, by using a data communication protocol, such as a known communication protocol like TCP / IP, or a custom-made communication protocol; • Exchanging data between a communication device of at least one biosensor device or at least one portable device and a communication device of an external computer device, for example, by using a data communication protocol, such as a known communication protocol like TCP / IP, or a custom-made communication protocol; • Decrypting encrypted data, specifically encrypted measurement data, received from at least one biosensor device or at least one portable device's communication equipment; • Processing data, specifically decoded data, specifically evaluating the data to obtain measurements from the data; • Storing data received from at least one biosensor device or at least one portable device communication device in data storage that is part of or can be connected to an external computer device.

[0144] According to aspect 8, the present invention relates to a method for manufacturing a biosensor device and / or an implantable device according to the present invention, The steps include: preparing at least one metal electrode surface, more specifically an electrode, preferably made of gold or containing gold, on at least one surface of at least one wafer substrate, which is preferably a glass wafer substrate; The steps include: preparing a biosensor film on the surface of at least one metal electrode; Optionally: the step of placing an electronically controlled device on at least one wafer substrate; Optionally: the step of manufacturing an encapsulation device using at least one wafer substrate to encapsulate a cavity in a biosensor device or an implantable device; It is directed towards methods that include this.

[0145] According to aspect 8, the method relating to aspect 8 is: The step of providing a number of wafers (two or more) including multiple wafer substrates for the parallel manufacture of a number of portable devices, wherein the number of wafers is preferably aligned in a stacked state; Preferably, to form at least one cavity, multiple wafer substrates, more specifically multiple wafers, are welded at stitching points or areas, and the resulting laminate of welded glass wafer substrates, more specifically, at least one cavity, will surround at least one of the components of a biosensor device or portable device, namely electrodes, electronic control devices, more specifically microcontrollers, amplifiers, RFID (preferably NFC) chipsets; and an antenna; Optionally: the step of cutting one or more laminated and welded wafer substrates from a laminated and welded wafer substrate, in particular, one or more of the components to support and / or surround; Includes.

[0146] Currently available implantable sensor devices rely on batteries to power their operation for a limited period, after which the batteries need to be replaced or wirelessly charged. In either case, there are many drawbacks to the patient's health and well-being. Due to the need for surgical removal to remove the battery, there are various associated health risks and potential surgical site infections. In the case of charging, the time required for charging may inconvenience the patient's movement and position during wireless power transmission.

[0147] Therefore, according to this particular aspect of the present invention, it is an object of the present invention to provide a biosensor device or an implantable device that avoids the aforementioned drawbacks such as long charging times for recharging the device or extraction procedures.

[0148] This problem is solved by the biosensor device according to Embodiment 1 and the method according to Embodiment 15. Further embodiments are described by Embodiments 2 to 14 and 16, and further preferred configurations are described by the entire patent application document. Any embodiment of the biosensor device described can be used alone or in combination with any aspect of the present invention, as with one or more of the further embodiments disclosed herein.

[0149] A biosensor device according to Embodiment 1 of the present invention is configured to be implanted in the body of a human or animal, more specifically to be implanted subcutaneously. Such a biosensor device configured to be implanted is also called an “implantable device” and may include an encapsulation device for at least partially enclosing at least one of the biosensor device and an electronic control device. According to Embodiment 1, the biosensor device (1i) or implantable device (1i) includes at least one metal electrode surface, preferably a portion of an electrode, more specifically an electrochemical sensing electrode, for detecting voltage equivalent data that quantifies a biological quantity, and Includes an electronically controlled device (2i), where the electronically controlled device (2i) is • At least one amplifier (3i), • A microcontroller (5i) that converts voltage equivalent data into digitized measurement data, A communication device (6i), more specifically an NFC device, wherein the NFC device (6ii) is configured to transmit (uplink) digitized measurement data of an electrochemical sensor to an external device (28i) and to receive (downlink) specific data (100i) from an external device (128), and is further configured to be powered on by an external computer device (108) so that the biosensor device (101) can operate fully. It includes.

[0150] Preferably, the microcontroller (5i) is programmed to encrypt the digitized data into encrypted data, which is then transmitted to a (short-range) communication device (6i) and further transmitted to an external computer device (8i) by a short-range antenna (7i).

[0151] Preferably, the biosensor device includes an electrochemical sensor for detecting at least one biological / biochemical analyte (biological quantity), and an electronic circuit used to monitor the body's hormone levels, more specifically hormone levels related to the female reproductive cycle or hormone levels during in vitro fertilization, wherein the electronic circuit includes at least one amplifier (4i) for converting and amplifying a current derived from the analyte into voltage equivalent data in relation to a reference voltage level, at least one amplifier for generating the reference voltage level, and preferably an ultra-low power microcontroller (5i) for digitizing the voltage equivalent data into digital data, and preferably a short-range antenna. The system includes an NFC communication device, which is configured to transmit digital data from an electrochemical sensor to an external computer device (8i; uplink) and to receive specific data from the external computer device (downlink), and more preferably is configured to be powered on by the external computer device so that the biosensor device can operate fully, wherein the microcontroller (5i) is preferably programmed to encrypt the digitized data into encrypted data, the encrypted data is transmitted to the communication device, and the encrypted data is further transmitted to the external computer device by a (near-field) antenna (7i).

[0152] In this context, an electrochemical sensor is understood as a device that measures and detects chemical substances by converting chemical reactions occurring at electrodes into electrical signals. It utilizes the principles of electrochemistry to detect and quantify various analytes or target substances in a sample, i.e., at implantation sites within the human or animal body. The biosensor device according to the present invention preferably generally includes an electrochemical sensor comprising at least one electrode or at least one metal electrode surface.

[0153] For measuring the concentration of an analyte, also known as measuring or detecting an analyte, the sensor preferably includes a reference electrode. The reference electrode provides a stable potential on which the reaction of the analyte is compared, ensuring accurate measurement. By monitoring the change in current or potential as a result of the reaction of the analyte, the sensor can determine, or participate in, the concentration of the target substance in the sample.

[0154] In this context, biological quantities or biological / biochemical analytes are understood, for example, as hormones in the human or animal body, more specifically, as hormone levels related to the female reproductive cycle, or as hormone levels during in vitro fertilization.

[0155] In this context, an amplifier that converts and amplifies the current derived from the analyte into voltage equivalent data is understood as a current-to-voltage converter, such as a transimpedance amplifier, which is implemented almost exclusively using one or more operational amplifiers. The voltage equivalent data corresponds to the voltage equivalent level of the data generated using a resistor.

[0156] An electrochemical sensor is connected to an electronic circuit via a working electrode. Therefore, the electronic circuit includes at least one working electrode to connect to an amplifier, specifically a transimpedance amplifier, in order to electrically connect the electrochemical sensor to the electronic circuit, in order to convert the current supplied from the electrochemical sensor into a voltage, i.e., a voltage equivalent, also called voltage equivalent data, according to the current data as data from the electrochemical sensor.

[0157] In this context, the reference voltage corresponds to a voltage reference level generated by an operational amplifier in combination with a voltage divider circuit topology that is followed by signal buffering by an amplifier.

[0158] In this context, an ultra-low power microcontroller is understood as a microcontroller that enables data processing with the minimum necessary system power.

[0159] In this context, a Near Field Communication (NFC) device is understood as an NFC chip tag, also called an NFC tag or simply an NFC chip, which is a small electronic device containing an integrated circuit (IC) with an NFC antenna, i.e., a near-field antenna. This tag is designed to store and transmit data wirelessly using NFC technology. An NFC chip tag is understood as a passive device, which means it does not have its own power source. Instead, it relies on power supplied by an external device, for example. When an external device approaches an NFC tag, specifically within a few centimeters, the external device generates an electromagnetic field that induces a small current within the tag's near-field antenna. This current powers the NFC chip, which enables it to communicate with the external device.

[0160] NFC chip tags can be embedded in or mounted on printed circuit boards and / or flexible circuit boards. The data stored on NFC tags can range from simple text or URLs to more complex information such as biosensor device details, product information, and commands for specific actions.

[0161] NFC chip tags are highly versatile, can be programmed and reprogrammed multiple times, and offer flexible use. NFC chip tags can be read and written to by NFC-enabled devices such as smartphones, tablets, and dedicated NFC readers. By bringing an NFC-enabled device, such as a mobile device, close to or tapping an NFC tag, users can read the information stored on the tag or initiate specific actions based on the tag's programming.

[0162] A biosensor device is fully functional when it can convert the current from the electrochemical sensor to an operational amplifier into a corresponding voltage, pass it to an ultra-low-power microcontroller for digitization and encryption, transmit it to an NFC device, and then transmit it from the NFC device to an external device that decrypts the transmitted encrypted data. A biosensor device is not fully functional if it is simply powered on at the NFC frequency by an external device, for example, without authentication to allow decryption.

[0163] The ultra-low power microcontroller is programmed to encrypt the digitized data into encrypted data, which is then transmitted to a short-range wireless communication device, and further transmitted to an external device.

[0164] An Inter-Integrated Circuit (I2C) interface is used to transmit encrypted data from a microcontroller to an NFC device. The I2C interface is a widely used serial communication protocol that allows multiple devices to communicate with each other over a shared bus. While I2C and NFC are different protocols, the I2C protocol is combined with the NFC protocol. For example, an NFC-enabled device can connect to a microcontroller or other peripherals using the I2C interface. This allows the NFC device to communicate with the microcontroller, which can then control other components or perform additional processing based on the NFC data. In such a setup, the NFC device handles NFC-specific communication, and the I2C interface facilitates communication between the NFC device and the microcontroller. In this example, the microcontroller also includes an encryption algorithm and is programmed to encrypt the provided digitized data. The encrypted data is then transmitted to the NFC device via the I2C interface. This feature has the effect of preventing the ultra-low power microcontroller from starting any operation until it receives a valid command sequence from an authenticated external device, such as an NFC-enabled mobile phone running the respective application, while the power to generate the voltage level required to operate the biosensor device, i.e., the harvested voltage level, can be generated without authentication by an NFC-enabled external device or any external device having the same radio frequency (RF) as the NFC frequency.

[0165] While the I2C communication protocol specifications for transmission baud rate and byte format are general values, the data packets, i.e., the byte streams exchanged between the ultra-low power microcontroller and the NFC chip tag, are custom-specified. Thus, for example, some of the transmission bytes that the microcontroller needs to perform, such as acquiring voltage equivalent data and transmitting data to NFC, or control parameters for the operation of the biosensor device itself, are encoded. Preferably, the byte stream or data packet generated for NFC transmission includes encrypted data acquired from the biosensor / temperature channel, control parameters, and a security encryption scheme for data decryption, in addition to a byte field occupied by the unique ID number of the portable device, with a total data payload of 1kB in each NFC transmission (downlink / uplink).

[0166] Since the biosensor device is preferably battery-less, it avoids the aforementioned drawbacks and can remain in the human body (i.e., subcutaneously) for a period not determined by the stored energy level, and each cycle of device operation only requires the presence of a nearby NFC-enabled mobile phone. From the patient's perspective, for the intended use of this device, namely monitoring hormones in the body, readings can be performed sporadically rather than continuously, thus minimizing the impact of this reading procedure on the patient's daily life, and therefore not being particularly inconvenient. Furthermore, the implantable electronic circuitry described here does not actively stimulate underlying body tissues by any physical conversion mechanism, which may be electrical, optical, or ultrasonic, as other commercially available devices do, making it safer for use in the body and meeting the standards of regulatory authorities worldwide.

[0167] In a preferred embodiment 2, the external computer device is an NFC-enabled portable PC, tablet PC, or mobile phone, and the external computer device is programmed to decrypt the transmitted encrypted data into decrypted data. This feature has the effect of preventing any other mobile device from interacting with the biosensor device. Furthermore, this makes data retrieval extremely easy because the patient always carries their mobile device and no additional device is required. In addition, the data can be stored on the mobile device and easily transmitted to a physician, for example, for evaluation.

[0168] In a preferred embodiment 3 of the present invention, the decoded data is visualized on the display, specifically on a touchscreen, of an external computer device.

[0169] Visualization of transmitted data allows patients to quickly understand, for example, the magnitude of relevant hormone concentrations without requiring additional technical equipment.

[0170] In a preferred embodiment 4 of the present invention, the external device is positioned at a distance of less than 2 cm or 2 cm from the biosensor device for data transmission, i.e., to establish an uplink / downlink. Such a distance is particularly suitable for a biosensor device implanted subcutaneously. Both the uplink / downlink and power supply lines are operable up to a gap distance of 2 cm between the implantable device and the external communication device, particularly between the NFC source, and do not affect the electronic performance metrics set for the implantable device (i.e., nominal harvest voltage level and transmission quality).

[0171] In a preferred embodiment 5 of the present invention, the sampling rate for digitization includes a rate of 24 samples per second and a resolution of 10 bits, or occurs at a rate of 24 samples per second and a resolution of 10 bits. Such values ​​are useful because the resolution in terms of bits / temperature for the data collected from the biosensor / temperature sets the accuracy limit for the portable device, while the number of samples per second is preferably limited to 1 kB for each NFC transmission.

[0172] In a preferred embodiment 6 of the present invention, the digitized data is temporarily stored in the microcontroller. In order to perform data encoding, both the data derived from the logical cryptographic operation (XOR) and the intermediate result must be temporarily stored in the microcontroller's limited RAM memory before the processed encrypted data is embedded in the NFC data packet for transmission.

[0173] In a preferred embodiment 7 of the present invention, a reference voltage level is generated during the detection of at least one biochemical analyte. This feature allows for the consideration of changes in the reference voltage level for any reason, thereby resulting in higher measurement accuracy. By fine-tuning the reference voltage level, different chemical processes more relevant to the analyte (i.e., selection of specific chemical functional groups and / or improvement of the geometric arrangement of specific molecular structures) are activated, thereby achieving better accuracy of the measurement signal without the need to increase the resolution (number of bits) of the digitized electronic circuit.

[0174] In a preferred embodiment 8 of the present invention, the electronic circuit includes at least three working electrodes configured to detect at least three analytes, more specifically, different analytes, of the electrochemical sensor. In a more preferred embodiment of the present invention, the electronic sensor includes two, four, five, or more working electrodes configured to detect one, two, three, four, five, or even more analytes. This is advantageous because it is possible to measure the same analyte using several working electrodes to improve the accuracy of the biosensor device.

[0175] In a preferred embodiment 9 of the present invention, the detection of chemical analytes is performed simultaneously, and the simultaneously generated currents provided by the electrochemical sensor are processed in parallel by an electronic circuit. This feature advantageously allows for simultaneous monitoring of the concentrations of different analytes, for example, by visualization on a mobile phone display.

[0176] In a preferred embodiment 10 of the present invention, the digitized data further includes the temperature of the biosensor device. Since the microcontroller advantageously includes an internal temperature indicator built into the chip die, providing information on the temperature level via an uplink communication channel enables, for example, a more detailed analysis of the provided patient-specific data.

[0177] In Embodiment 11 of the present invention, the specific data may include user parameters, such as data for setting the ID number of the biosensor device, the operating state of the biosensor device, or control data for the detection process of the biosensor device. This feature enables further individualization of the biosensor device, and more specifically, patient-specific implantable sensors.

[0178] In a preferred embodiment 12 of the present invention, the biosensor device includes an optical indicator, more particularly a light-emitting diode, configured to optically indicate the operating status of the biosensor device to the user. For example, a small, embedded light-emitting diode (LED), when properly powered on by the NFC interface of a mobile phone, provides visual feedback of the operation occurring on the side of the implantable device. This makes troubleshooting the biosensor device easier, for example, without having to remove the implanted biosensor device.

[0179] In a preferred embodiment 13 of the present invention, the biosensor device includes a rigid and / or flexible printed circuit board (PCB). More specifically, a flexible substrate can be used to avoid mechanical stress in the structure of the biosensor device and / or to achieve a particular advantageous shape of the biosensor device. However, more specifically, it is equally possible to assemble the components directly onto a wafer substrate without using a PCB.

[0180] In a preferred embodiment 14 of the present invention, the (short-range) antenna is wound around a rigid PCB in N loops, where N is preferably selected from 15 to 25 loops, more preferably from 18 to 22 loops, or N=20, and / or the antenna includes an enameled copper wire with a thickness of 0.15 mm. The internal NFC antenna is wound within the external limits of the circuit board, while allowing AC radio frequency waves generated, for example, by a mobile phone, to be continuously captured by the NFC antenna and converted by the NFC chip tag itself to an equivalent DC level called the harvest voltage level, which can then be utilized by the rest of the embedded electronic device.

[0181] This problem is further addressed in Embodiment 15 of the present invention in a method for monitoring the levels of analytes, more specifically, the levels of bodily hormones inside the body of a human or animal, and more specifically, the levels of hormones during the female reproductive cycle or in vitro fertilization: - A step of providing a biosensor device, preferably including an electrochemical sensor, for detecting at least one chemical analyte; and - A step of providing an electronically controlled device, more specifically an electronic circuit, wherein the electronically controlled device is: At least one amplifier, more specifically an amplifier configured to convert and amplify the current derived from the object under analysis into voltage equivalent data in relation to a reference voltage level, and Preferably: at least one amplifier for generating a reference voltage level, and A preferably ultra-low power microcontroller for digitizing voltage equivalent data into digital data, and A communication device, more specifically a near-field communication device (NFC) including, for example, a near-field communication antenna, configured to transmit (uplink) digitized data from a biosensor device or electrochemical sensor to an external computer device and to receive specific data (downlink) from the external computer device, more preferably configured to be powered on by the external computer device so that the biosensor device is in a fully operational state, wherein the microcontroller is programmed to encrypt the digitized data into encrypted data, the encrypted data is transmitted to the communication device, for example an NFC device, and the encrypted data is further transmitted to the external computer device, Steps that include; - Optionally: the step of connecting the electrochemical sensor to an electronic sensor means, specifically at least one working electrode; - Preferably: the step of supplying energy to operate the biosensor device by an external computer device, preferably using a wireless connection of the device, and more specifically using radio waves; - Optionally: the step of visualizing the transferred and decrypted data on the display of an external computer device; This is resolved by a method that includes [a specific method].

[0182] In a preferred embodiment 16 of a biosensor device, more specifically an electronically controlled device, more specifically an electronic circuit, the acquisition and amplification of ion currents is performed simultaneously by a total of three analytes (or species) stored in a physiological solution and / or fluid, which are then digitized in an ultra-low power microcontroller, and the final transfer of the collected digital samples is performed via a near-field communication (NFC) interface to an external mobile phone located at a certain distance, preferably less than 2 cm, from the electronic circuit. The same mobile phone is responsible for delivering the power necessary for the implantable electronic device (energy harvesting) through the radio frequency electromagnetic field generated by NFC (13.56 MHz = standard), thereby enabling the electronic device to reach a fully operational state for performing the aforementioned electrochemical acquisition task, without relying on any stored internal energy or battery. The implantable electronic circuit is intended to be used to monitor in vivo hormone levels directly related to the reproductive cycle in women in a normal physiological state or undergoing in vitro fertilization (IVF) treatment.

[0183] Amperometric measurement refers to a type of electrochemical analysis that involves measuring the electric current flowing through a system. Specifically, it involves the detection and quantification of the analyte or chemical species based on changes in current resulting from electrochemical reactions at electrodes. In amperometric measurement, electrodes are immersed in an electrolyte solution containing the analyte. An electrical potential is applied to the electrodes, initiating an electrochemical reaction involving the analyte. As the reaction progresses, electrons are transferred between the electrodes and the analyte, resulting in a measurable current. This current is directly proportional to the concentration of the analyte in the solution.

[0184] Further preferred configurations of the implantable devices, biosensor devices and systems, and methods according to the present invention will become apparent from the following description of exemplary embodiments in conjunction with the drawings and their description. Unless otherwise stated or indicated by the context, identical components of the exemplary embodiments are characterized by substantially the same reference numerals. [Brief explanation of the drawing]

[0185] [Figure 1] Figure 1 shows a schematic cross-sectional view of a portable device according to one embodiment of the present invention. [Figure 2a] Figure 2a shows a schematic perspective view of the portable device shown in Figure 1. [Figure 2b] Figure 2b is a top view showing the upper surface of an electrode-supported wafer substrate for a portable device according to one embodiment of the present invention. [Figure 2c] Figure 2c is a top view showing the upper surface of another electrode-supported wafer substrate of a portable device according to another embodiment of the present invention. [Figure 3] Figure 3 shows a top view of one cross-section of a circular wafer containing a plurality of electrode assemblies for cutting from the wafer in order to manufacture a plurality of electrode-supported wafer substrates as shown in Figure 2b, each for a portable device according to one embodiment of the present invention. [Figure 4a] Figure 4a shows a perspective side view of a portable device according to one embodiment of the present invention, comprising an upper wafer substrate, an intermediate wafer substrate, and a bottom wafer substrate. [Figure 4b] Figure 4b is a perspective side view of the intermediate wafer substrate of the portable device shown in Figure 4a, where the intermediate wafer substrate is shown upside down. [Figure 4c] Figure 4c shows a perspective side view of the transplantable device shown in Figure 4a, where the transplantable device is shown upside down. [Figure 5]Figure 5 shows, on the left, a perspective view of the intermediate wafer substrate of the portable device according to Figure 4a, and on the right side of the page, a collection of five vertically bound drawings, from top to bottom, namely: a top view of the first (upper) wafer substrate of the portable device according to Figure 4a, and a cross-sectional view cut along line AA; a top view of the second (intermediate) wafer substrate of the portable device according to Figure 4a, a cross-sectional view cut along a length L that vertically penetrates the center of the upper surface of the wafer substrate of the portable device according to Figure 4a, and a cross-sectional view cut along a line running along the through hole 32; a bottom view of the second (intermediate) wafer substrate of the portable device according to Figure 4a, a bottom view of the bottom side of the third (bottom) wafer substrate of the portable device according to Figure 4a, and a cross-sectional view cut along line BB. [Figure 6] Figure 6 shows a system according to the present invention, using an implantable device and a biosensor device according to the present invention, each according to one embodiment. [Figure 7] Figure 7 shows a schematic diagram of one embodiment of the electronic circuit of a biosensor device according to one embodiment of the present invention. [Figure 8] Figure 8 illustrates the operating principle of a biosensor device according to one embodiment of the present invention. [Figure 9] Figure 9 schematically shows the layout of the back surface of the rigid PCB of a biosensor device according to one embodiment of the present invention. [Figure 10] Figure 10 schematically shows the layout of the rigid PCB surface of a biosensor device according to one embodiment of the present invention. [Figure 11] Figure 11 shows three diagrams with measurement data measured by a biosensor device according to one embodiment of the present invention.

[0186] Figure 1 shows a schematic cross-sectional view of an implantable device 1 according to one embodiment of the present invention. The implantable device 1 includes a biosensor device 2 and an encapsulation device 3. The biosensor device 2 is specialized to provide long-term operation of the biosensor device within living tissue and includes all functional components necessary to provide a working implantable device, except for the encapsulation device 3, which provides biocompatibility to prevent or reduce the risk of rejection or adverse reactions caused by the release of harmful substances due to further factors including the body's immune response or degradation of the implantable device. In this example, the outer layer of the encapsulation device is made substantially entirely of glass, except for electrodes and conductive traces (not shown) running on the surface of the upper wafer substrate 7a. The upper wafer substrate 7a is part of the biosensor device 2 and further forms part of the encapsulation device 3. In detail, the encapsulation device is made substantially entirely of glass, and the electrodes are provided on the upper surface 3a of the encapsulation device 3. The encapsulation device 3 is made substantially entirely of glass, specifically Borofloat® from Schott AG of Germany. The upper glass wafer 7a and the intermediate glass wafer substrate 7b, and optionally the bottom glass wafer substrate 7c as well, include vias 12 filled with a metallic conductive material, such as tungsten or NiFe. Such glass wafer substrates may be cut from larger wafers commercially available from Schott AG in Germany as HermeS® wafers. The three glass wafer substrates 7a, 7b, and 7c are connected here by a glass weld seam 8, which hermetically seals the cavities 9a and 9b inside the encapsulation device.

[0187] Electrodes 4a, 4b, and 4c are sensing elements of the biosensor device 2 and are configured to perform pontammetry measurements, specifically DPV, when the implantable device is implanted in tissue. The electrodes include a counter electrode 4b, which is a planar gold electrode mounted on the upper surface 3a of the glass wafer substrate 7a; a reference electrode 4c, which is also a planar gold electrode mounted on the upper surface 3a of the glass wafer substrate 7a; and a working electrode 4a, which is also a planar gold electrode having a functionalized surface and mounted on the upper surface 3a of the glass wafer substrate 7a. The functionalized surface is shown in a simplified form as a laminate 10 of layers. However, the actual three-dimensional atomic surface structure (not shown) is more complex. Alternatively, a number of additional electrodes may be provided to enable electrical measurements, preferably four electrodes.

[0188] Furthermore, generally, two or more electrodes can be provided on different surfaces of an implantable device, or encapsulation device, or biosensor device, particularly on a wafer substrate. For example, one or more electrodes can be provided on a first (outer) surface 3a of the encapsulation device, and one or more electrodes can be provided on a second (outer) surface 3b of the encapsulation device, the other second surface 3b which may face the first surface 3a.

[0189] The layered structure 10 is obtained by layering or stepwise depositing different nanoparticles or molecules onto an existing surface. This layered structure 10 is also called a biosensor film 10.

[0190] The biosensor membrane 10 includes a layer 10a of gold nanoparticles deposited on the planar gold surface of the electrode 4a. While the gold nanoparticle layer 10a can generally be omitted, this layer is beneficial for improving the detection signal. The diameter of the gold nanoparticles is approximately 60 nm, but other particle sizes in the range of 10 nm to 200 nm, or 30 nm to 100 nm, are equally preferred. The effect of using the gold layer is that the total volume of the gold surface interacting with the bodily fluids is generally increased compared to the planar surface of the electrode 4a. As a result, the surface area provided by the nanoparticles and available for binding aptamers to the electrode 4a is significantly increased. Consequently, the number of sites for binding biomarkers (hormones or other targets in bodily fluids) is significantly increased, improving the signal quality of the measurement, specifically the signal-to-noise ratio.

[0191] In this example, an additional nanoparticle layer 10b is provided on top of the gold nanoparticle layer 10a. In this example, this additional metal nanoparticle layer 10b, which is platinum nanoparticles, was found to improve the lifespan and reliability of the biosensor film 10.

[0192] The aptamer 10c is deposited onto the nanoparticle layers 10a and 10b and bonded to the nanoparticles, thus completing the biosensor film 10. The thiolated aptamer tends to form a self-assembled monolayer (SAM) on the metal surface of the nanoparticles, which helps improve the binding stability of the aptamer and its permanent adhesion to the gold / platinum platform.

[0193] The biosensor was prepared in the following three steps: 1) Activation of the deposited gold layer on the glass. This step is performed by connecting the working electrode (gold) to a potentiostat and applying continuous cyclic voltammetry (CV) in an acidic solution: This optional step of activating the gold electrode usually involves a cleaning or conditioning process to remove any contaminants or oxide layers, ensuring better electrical contact and performance. 2) Adhesion of gold nanoparticles onto a gold layer on glass, and optionally provision of platinum nanoparticles; 3) Provision of aptamer layers.

[0194] The electronic control device 11 here includes a printed circuit board 11a carrying an integrated circuit 11b for controlling electrodes 4a, 4b, and 4c, and for controlling the electrical measurement of at least one biological quantity, each of which is controlled. Traces (not shown) on the printed circuit board 11a leading to the electronic control device 11, in particular to the integrated circuit 11b, are connected by wires 13 to vias 12 leading to the electrodes. Furthermore, the electronic control device 11 here includes a communication device 11c, which in this example is an RFID device, in particular to an NFC chipset. The NFC chipset is connected to the integrated circuit 11b by traces (not shown) on the PCB 11a. Furthermore, the NFC chipset is connected to an antenna 15 by wires and vias 12 on the PCB 11a in this example. The antenna 15 may be assembled anywhere in the biosensor 2 or the implantable device 1, preferably within the encapsulation device 3. Preferably, the antenna 15 is assembled on a wafer substrate or on a portion of the encapsulation device 3, preferably on the same wafer substrate that also carries the electronic control device 11.

[0195] Electrodes 4a, 4b, and 4c are connected to the electronically controlled device using vias 12. The advantage of using a substrate wafer with vias 12 is that the setup of the biosensor device 2, and thus the portable device 1, becomes much more compact. Each electrode 4a, 4b, and 4c may be connected to at least one via 12 by a gold trace provided on the gold surface, which can lead from the gold electrode to a gold connection pad. See Figure 2b. In Figure 1, electrode 4a is mounted directly to a via located below electrode 4a, and similar connection paths are provided for electrodes 4b and 4c.

[0196] The electronic control device 11 is mounted here on a wafer substrate, in this case on an intermediate wafer substrate 7b. However, it is equally preferable to mount the electronic control device 11 on the same wafer substrate that also supports at least one electrode, on the side 3c of the wafer substrate opposite to the side 3a that supports the electrode.

[0197] In this example, the integrated circuit 11b is an ASIC.

[0198] The electronically controlled device 11, more specifically the integrated circuit 11b, is configured, more specifically programmed, to provide an electrometric constant measurement for the purpose of obtaining a measurement signal, or to repeatedly measure the concentration of a biomarker in a body fluid, or a time-dependent change in a signal in a body fluid, where the signal represents the amount of a biomarker bound to the biosensor membrane 10. The measurement may be triggered by a time schedule, or by an external computer device, such as a smartphone with the control app installed, which is operable by the user to initiate the measurement itself (e.g., by touching the "start" area in a graphical user interface provided by a control app on the screen of a mobile phone device).

[0199] The electrical measurement is preferably voltammetry, more specifically DPV measurement.

[0200] Generally speaking, DPV is an abbreviation for Differential Pulse Voltammetry, an electrochemical technique used for signal detection and quantification in various application fields. This involves applying a series of voltage pulses to electrodes in contact with electrolytes that form body fluids (e.g., interstitial fluid) and measuring the resulting current response.

[0201] Electrode setup: The apparatus comprises a working electrode 4a, a reference electrode 4c, and a counter electrode 4b. Alternatively, a number of additional electrodes may be provided to enable electrical measurement, preferably four electrodes.

[0202] DPV technology applies a series of voltage pulses to the working electrode 4a. The pulses typically consist of a step potential, a holding potential, and a pulse width. The step potential provides the driving force for the electrochemical reaction, and the holding potential gives the system time to reach equilibrium before the next pulse.

[0203] Current measurement: After each voltage pulse, a potentiostat is used to measure the resulting current response. This current response corresponds to the electrochemical reaction occurring at the working electrode.

[0204] The obtained current response is typically plotted against the applied voltage pulse. The resulting DPV curve exhibits a characteristic peak shape that can provide information about the analyte concentration, redox reactions, and other electrochemical processes occurring within the system with the analyte that facilitates the electrochemical reactions.

[0205] Figure 2a shows a schematic perspective view of the implantable device 1 shown in Figure 1, which gives an impression of the actual dimensions of the implantable device 1, which has a width W=2.0 mm, a height H=2.0 mm, and a length L=16 mm. This implantable device has rounded edges and corners (not shown) and can be implanted by a surgeon using a syringe.

[0206] Figure 2b shows a top view of the upper surface of the electrode-supported wafer substrate 7a' of a portable device 1 according to an embodiment of the present invention that can be used in Figure 1a, the wafer substrate 7a' having vias 12. Electrodes 4a', 4b', 4c', and 4d' are deposited on the upper surface 3a of the wafer substrate. Four electrodes are provided in this figure, each electrode connected to a connecting pad 14b by a conductive trace 14a, both of which are deposited on the same surface 3a as the gold electrodes in the same manufacturing steps and made of the same gold.

[0207] Figure 3a shows a partial top view of a cross-section of a circular wafer 40 containing a plurality of electrode assemblies to be cut from the wafer in order to manufacture a wafer substrate 7a' supporting a plurality of electrodes as shown in Figure 2b, each for a portable device according to one embodiment of the present invention. The wafer substrate 40 is provided with an assembly of vias 12 that run perpendicularly through the wafer surface and are filled with a conductive metal, such as tungsten. The wafer 40 is a HermeS® glass wafer manufactured by Schott AG of Germany.

[0208] Figure 2c shows a top view of the upper surface of another electrode-mounted wafer substrate 7a'' of a portable device according to another embodiment of the present invention. The wafer 40 is a HermeS® glass wafer manufactured by Schott AG in Germany. Four electrodes 4a'', 4b'', 4c'', and 4d'' are deposited on the upper surface 3a'' of the wafer substrate 7a''. The four wafer substrates cover almost the entire surface of the wafer substrate or a fraction of the surface, which is selected from the range of 80-100%, preferably 90-100%, and preferably 90-99%. Using a larger surface area for the electrodes provides more binding sites for binding biomarkers, thereby improving signal quality when performing electrical measurements.

[0209] Figure 4a shows a perspective side view of a portable device 21 according to one embodiment of the present invention, comprising an upper wafer substrate 27a, an intermediate wafer substrate 27b, and a bottom wafer substrate 27a. The upper wafer substrate 27a and the bottom wafer substrate 27a are configured to have a trough shape and to be arranged as covers for covering components positioned on the upper and lower sides of the intermediate wafer substrate 27b, respectively.

[0210] Figure 4b shows a perspective side view of the intermediate wafer substrate 27b of the portable device shown in Figure 4a, where the intermediate wafer substrate is shown upside down. On the bottom side of the intermediate wafer substrate 27b, an integrated circuit (ASIC) 31b for electrically controlling numerous components of the electronic control device of the biosensor device of the portable device 21, namely electrodes 24a, 24b, 24c, and 24d, and a communication device 31c including an NFC device and an NFC antenna 35 are mounted.

[0211] Similar to the diagram of the intermediate wafer substrate 27b in Figure 4b, the left-hand diagram of Figure 5 shows a perspective top view of the intermediate wafer substrate 27b of the portable device according to Figure 4a, having four gold electrodes 24a, 24b, 24c, and 24d aligned parallel to each other and at regular intervals along the length L of the intermediate wafer substrate 27b. The electrodes occupy almost the entire surface of the intermediate wafer substrate 27b. The intermediate wafer substrate 27b is a HermeS® glass wafer manufactured by Schott AG of Germany and has vias 32 that run vertically through the wafer substrate to the top surface and are filled with a conductive metal, such as tungsten. Each electrode is in contact with a via 32 and connected via the vias to an ASIC 31b that may be directly attached to the bottom surface of the intermediate wafer substrate 27b without using a PCB for electronic component placement, or by using a PCB for electronic component placement (not shown). The use of a wafer with vias allows for the implementation of a compact solution for portable devices according to this embodiment of the present invention. The features of the portable device 21 can be readily modified by those skilled in the art using other preferred features and configurations disclosed herein.

[0212] Figure 5 shows, on the right side of the page, a collection of five vertically bound drawings, arranged from top to bottom: i. A top view of the first (upper) wafer substrate 27a of the implantable device 21 shown in Figure 4a, and a cross-sectional view cut along line AA indicating through holes or pores 39. The first (upper) wafer substrate 27a acts as a covering lid to cover the surface 23a supporting four gold electrodes 24a, 24b, 24c, and 24d. One or two of the electrodes support a biosensor film, such as the biosensor film 10 as described with reference to Figure 1, for example. The other electrodes are gold electrodes used as reference or counter electrodes for voltammetry (in detail: DVP) measurements. By using the covering lid 27a, the biosensor film and electrodes are protected from mechanical damage that may result from the implantation process or long-term use in the patient's body. The porous membrane formed by the main wall of the upper wafer substrate and the covering lid 27a allows for easy intake of bodily fluids into the internal volume contained by the trough-shaped covering lid 27a and the upper surface of the intermediate wafer substrate 27b to which the covering lid 27a is welded, thereby firmly and irrevocably connecting the covering lid to the intermediate wafer substrate. The main wall of the covering lid, and possibly the flange surrounding the outer rim of the main wall, are also made of CoralPor® porous glass, commercially available, for example, from Schott AG in Germany. Welding is achieved by laser welding, specifically femtosecond laser welding, at the weld line and weld area indicated by number 28, once all wafer substrates are precisely aligned with each other, equipped with, for example, electronic components and electrodes, as well as biosensor membranes. ii. Top view of the second (intermediate) wafer substrate 27b of the portable device shown in Figure 4a: Vias 32 are shown as black dots here for illustrative purposes, but are actually covered with gold electrodes and are therefore not visible. iii. In detail, a cross-sectional view along a length L perpendicular to the center of the upper surface of the wafer substrate of the portable device relating to Figure 4a, showing vias 32 that run through the entire height of the intermediate wafer substrate to electrically connect the upper and lower sides. Similarly, on the right side, a cross-sectional view cut along a line running along the through-hole 32 is also shown. iv. Bottom view of the second (intermediate) wafer substrate of the portable device shown in Figure 4a. Electronic components 35, 31b and 31c are mounted thereon; and v. Bottom view of the bottom side of the third (bottom) wafer substrate 27c of the implantable device according to Figure 4a, and a cross-sectional view cut along line BB. The wafer substrate 27c is a glass substrate formed in a trough shape to cover (electronic) components mounted on the bottom surface of the intermediate wafer substrate 27b, with a covering lid 27c welded at the welding area 28 using a femsecond tracer welding process. This welding creates an hermetically sealed cavity between the covering lid 27 and the bottom surface of the intermediate wafer substrate 27b. This hermetically sealed cavity prevents any bodily fluids from entering the cavity, reliably protecting the electronic components from corrosion, and the patient's body is reliably protected from any non-biocompatible materials of the biosensor for the entire lifespan of the implantable device, which is reliably sealed by the encapsulation device formed from the three wafer substrates 27a, 27b, and 27c. In this embodiment, no part of the patient's body comes into direct contact with any material other than the biocompatible glass forming the encapsulation device.

[0213] Description of preferred embodiments of the method for manufacturing a portable device according to the present invention: Each biocompatible glass layer is prepared and cleaned under cleanroom conditions. The encapsulation device preferably uses one or more, preferably two or more, more specifically three or four, or up to four wafer substrates to layer electrodes, a biosensor film, more specifically an electronically controlled device, and preferably an NFC antenna, with each wafer aligned with a reference point and calibrated to match the aligned stack. A femtosecond laser is used to scan within a controlled phase plane to enable internal stitching and welding of the glass layer, with the aim of creating at least one cavity that is sealed to a tight seal. This sealed cavity is preferably capable of withstanding 5 atmospheres (approximately 550 kPa) for 4 hours with a residence time of 2 hours, and the leakage measured by mass spectrometer is 5 × 10⁻⁶ -8ATM / CM 3 It is preferable that the number of seconds does not exceed / s.

[0214] Femtosecond lasers create stress microzones, and each wafer design and chip geometry requires a unique boundary layer to allow for the sealing of numerous cavities on a single chip. This improves the overall yield of biocompatible glass wafers and enhances manufacturing efficiency. This process and design guideline is suitable for glass wafer manufacturing processes to ensure that the single-piece chips cut from wafers with sealed cavities are not affected by stress induced by thermally influenced working zones within the stitched regions.

[0215] Preferably, a number of wafers (two or more) containing multiple wafer substrates for the parallel manufacture of multiple portable devices are aligned in the form of a laminate, then welded at post-stitching points or regions, and the resulting welded glass wafer substrate laminate, which includes at least one of the components enclosed in one or more cavities (electrodes, electronic control devices, in particular microcontrollers, amplifiers, RFID (preferably NFC) chipsets; antennas), is cut from the laminated and welded wafers.

[0216] Once the layers are welded, a quality assessment is completed for each chip to ensure that both the airtightness and alignment of the device's internal operating components are within performance requirements.

[0217] This laser seal allows a biocompatible, sealed cavity, implantable for full internal operation of electronic devices, to amplify signals generated by a biosensor.

[0218] A preferred embodiment of a method for preparing a glass-encapsulated device includes one or more of the following steps: - Each biocompatible glass layer is prepared and cleaned under cleanroom conditions. - The device uses up to four wafers to layer electronics, biosensors, NFC antennas, and biomembranes, with each wafer aligned to a reference point and calibrated to match the aligned stack. - A femtosecond laser is used to scan within a controlled phase plane to enable internal stitching and welding of the glass layer, with the aim of creating a cavity that is sealed to an airtight degree. - This sealed space must withstand 5 atmospheres (approximately 550 kPa) for 4 hours with a residence time of 2 hours, and the leakage rate measured by mass spectrometer was 5 × 10⁻⁶ -8 ATM / CM 3 The / s limit must not be exceeded. - Femtosecond lasers create stress microzones, and each wafer design and chip geometry requires a unique boundary layer to allow for the sealing of numerous cavities on a single chip. This improves the overall yield of biocompatible glass wafers and enhances manufacturing efficiency. This glass wafer manufacturing process ensures that the single-piece chips (laminated wafer substrates) cut from wafers with sealed cavities are not affected by stress induced by thermally affected zones within the stitched regions. - The chip separation process is performed after laser welding of individual cavities and is completed by a standard glass cutting operation in which the glass is cut along the scribing lines and heat-affected zones. - Once the layers are welded, a quality assessment is completed for each chip to ensure that both the airtightness and alignment of the device's internal operating components are within performance requirements. - This laser seal allows a biocompatible, sealed cavity, which is implantable for the full internal operation of electronic devices, to amplify signals generated by a biosensor. - The laser welding process preferably completely seals the electronic device inside the glass cavity. This cavity has exemplary dimensions of 16×1.65×0.55 mm. The electronic device is wired through vias, also known as TGVs, using trace wiring on the glass surface, up to the aforementioned biosensor. The TGVs are hermetically sealed, enabling electrical contact with the biosensor electrodes on the external surface.

[0219] Detailed Figure 6 shows, in a system 1000 for detecting at least one hormone in a body fluid inside a patient's body 999: - A subcutaneously implantable device 1 or 21, further comprising a communication device 11c or 31c, specifically an NFC (Near Field Communication) device, for wireless data communication with an external computer device 500 separate from the implantable device, and - An external computer device 500, specifically a portable computer device, preferably a smartphone or a tablet PC, comprising a CPU 501 for executing computer program code, specifically the computer program code according to the present invention, and a communication device 511, specifically an NFC device, for wireless data communication with the implantable device; showing a system including.

[0220] Data exchange is preferably initiated when program code, such as an application executed by the CPU on the computer device, receives user input via the touchscreen (not shown) of the computer device 500, which sends a request to the NFC devices 11c, 31c to transfer measurement data to the computer device 500, preferably in an encrypted form. Preferably, the measurement performed by the biosensor device 2 of the portable device 1 is preferably initiated when program code, such as an application executed by the CPU on the computer device, receives user input via the touchscreen (not shown) of the computer device 500, which sends a request to the NFC devices 11c, 31c to start the measurement. Since the portable device is preferably a battery-less device, the radio waves of the communication device 501 are preferably used to provide the energy necessary for the measurement and / or encryption of the measurement data and / or transfer of the measurement data to the computer device via the wireless connection 800.

[0221] Figure 7 shows a schematic diagram of one embodiment of the electronic circuit. The electronic circuit embedded in the portable device consists of four off-the-shelf components as shown in Figure 7. These four components include a central microcontroller 5i (MCU), which is an ultra-low-power microcontroller with an 8-bit architecture such as the ATTiny20, and the PIC12LF device family from Microchip, Inc. of Chandler, Arizona, USA; an NFC chip tag 6i, such as the NTAG I2C plus family from NXP, Inc. of Eindhoven, Netherlands; and finally, two operational amplifiers 3 with dual amplification channels, such as the AD8506 family from Analog Devices, Inc. of Wilmington, Massachusetts, USA, which have an extremely low input bias current of, for example, 1 pA and an ultra-low current consumption of, for example, 20 μA. The maximum current consumption of the electronic circuit 2i is set to a level of 1 mA, and the minimum voltage level is 1.8 V, which results in a power consumption of 1.8 mW. In relation to the reference voltage level imposed by the reference electrode 28i, as shown in the schematic diagram in Figure 7, three electrochemical analytes or species can be simultaneously monitored by available working electrode pads 9i connected to three independent amplifiers 3i assembled in a transimpedance circuit topology 29i, which use resistors 15i to convert the ion current to a voltage equivalent level and amplify it. At this time, the output voltage signal of each amplifier 3i is digitized in three separate analog-to-digital converters (ADCs) inside the microcontroller 5i at a sampling rate of 24 samples per second and a resolution of 10 bits. An additional amplifier 4i is responsible for generating the aforementioned voltage reference level during amperometric measurement and subsequent signal buffering by amplifier 4i, using a voltage divider circuit topology centered on resistors 16i and 17i. A similar voltage divider topology is used at the positive electrode of each transimpedance amplifier 29i to generate an internal bias voltage that level-shifts the amplified baseline higher than the ground signal, achieved by resistors 18i and 19i.

[0222] Data samples generated during the digitization phase are temporarily stored in the RAM memory of the MCU5i, digitally processed, encrypted using a lightweight security algorithm, and then digitally transmitted to the NFC chip tag 6i using the standard I2C interface 20i protocol at a baud rate of 9600bps, where they are internally stored in non-volatile memory.

[0223] The I2C communication protocol was originally developed by Philips Semiconductors and covers the definition of communication signals (SDA and SCL) and specifications for transmission baud rate and byte format. However, the data packets (byte streams) exchanged between the MCU and NFC chip tag are entirely custom-made here, with some of the transmitted bytes encoding sequence actions that the MCU 5i needs to perform, such as biosignals, i.e., current acquisition, data transmission, or control parameters for the operation of the implant 1i itself. The harvest voltage level 13i can be generated by any NFC-enabled device 8i or any RF source with the same frequency, but the MCU 5i will not initiate any operation until it receives a valid command sequence from an authenticated mobile phone running its respective application.

[0224] Figure 2 illustrates the operating principle of the biosensor device 1i. The biosensor device 1i is a portable device and includes an encapsulated device according to the present invention, which is substantially made of glass. The biosensor device 1i is shown positioned on a coin 21i for size comparison. The NFC antenna 7i is shown wrapped around the PCB substrate. A small embedded light-emitting diode (LED) 11i provides visual feedback of the operation occurring on the portable side when properly powered on by the NFC interface of the mobile phone 8i. If an external NFC field 22 is present, internally stored and encrypted data samples are wirelessly transmitted (uplink) to the external mobile phone 8i and visualized on dedicated application software 31i installed on the mobile phone 8i. Since the microcontroller 5i also has an internal temperature indicator built into the chip die, information regarding the temperature level is also provided via the uplink communication channel. By the same process, AC high-frequency radio waves 22i generated by the mobile phone 8 are continuously captured by the internal NFC antenna 7i. Antenna 7i consists of 20 turns of 0.15mm thick enameled copper wire wound around the outer edge of a circuit board 12i having dimensions of, for example, 6mm x 12mm, and is equivalent to a DC level of 13(V) by the NFC chip tag 6i itself. HARVEST After being converted to ), the rest of the embedded electronic device becomes available. In the opposite communication direction (downlink), specific data 10i, such as user parameters, can be transmitted from the same mobile phone application software 31ii to set an ID number for the portable biosensor device 1i, for example, to control the operation of the device and the amperometric measurement process.

[0225] Figure 9 schematically shows the layout of the back surface of the rigid PCB of the biosensor device, and Figure 10a shows the 3D schematic layout of the front surface of the rigid PCB of the biosensor device. Physically, the electronic circuit diagram of the described portable device is represented in the form of printed circuit board (PCB) technology with protected wiring conductors 24i and through-layer vias 25i, which are conductive paths or holes that connect different layers such as PCBs or ICs together and thus provide a means for current to pass between layers of the circuit board, carrying electrical signals between different chipsets, resistors 15i-19i, 30i, capacitors 14i and NFC antennas 7i, while exposed electronic pads 23i made with a Ni / Au surface finishing process are used to solder components to the PCB substrate 12i, as shown in both Figures 9 and 10, or are left exposed 27i to enable connections to programming pins for the MCU 5i or electrochemical functionization of the electrochemical sensor. Copper wiring 24i is placed on both the top and bottom layers of PCB12i, but component assembly is performed only on the top layer. For the manufacture of the described PCB8i, a rigid 12i, e.g., laminated copper or epoxy resin in an FR-4 circuit board, or a flexible 12i, e.g., a polyimide film substrate, can be used.

[0226] Any method disclosed herein includes one or more steps or actions to carry out the described method. The steps and / or actions of a method are interchangeable with one another. In other words, the order and / or use of any particular steps and / or actions are modifiable unless a specific order of steps or actions is required for the proper operation of the embodiment. Furthermore, only a subroutine or portion of a method described herein may constitute a separate method that falls within the scope of this disclosure. In other words, some methods may include only a portion of the steps described in a more detailed method.

[0227] Throughout this specification, any reference to “an embodiment,” “that embodiment,” or “preferred configuration” means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment. Therefore, not all quotations or variations thereof found throughout this specification necessarily refer to the same embodiment.

[0228] The use of the term "first" in relation to a feature or element in a claim does not necessarily imply the presence of a second or additional such feature or element. Modifications to the details of the embodiments described above may be made without departing from the fundamental principles of this disclosure.

[0229] Furthermore, those skilled in the art will understand, along with the merits of this disclosure, that various features are sometimes combined into a single embodiment, drawing, or description thereof for the purpose of streamlining the disclosure in the above description of embodiments. However, this method of disclosure should not be interpreted as reflecting an intention that any claim requires more features than those expressly described in that claim. Rather, as reflected in the following claims, the inventive embodiments lie in combinations of fewer features than all the features of any single disclosed embodiment described above. Thus, the claims are expressly incorporated herein, with each claim standing independently as a distinct embodiment. This disclosure includes all permutations of independent claims and their dependent claims.

Claims

1. In a biosensor device for detecting at least one hormone, (a) with at least one metal electrode surface; (b) A biosensor film comprising at least one aptamer attached to the surface of the metal electrode, wherein the aptamer is: (i) Having the ability to bind to at least one of the hormones; and (ii) The at least one aptamer is modified with one or more functional groups for attaching to the metal electrode surface, Biosensor membrane and; Biosensor devices, including those mentioned above.

2. A biosensor device according to claim 1, configured to detect at least one hormone in a body fluid, more specifically in interstitial fluid.

3. The biosensor device according to any one of claims 1 to 2, wherein the at least one hormone is selected from the group consisting of estradiol, luteinizing hormone (LH), progesterone, and any combination thereof.

4. Furthermore, the biosensor device according to any one of claims 1 to 3, further comprising a carrier for supporting the at least one metal electrode surface, more particularly a glass carrier, such as a wafer substrate.

5. The biosensor device according to any one of claims 1 to 4, wherein the aptamer is a single-stranded nucleic acid molecule, preferably a DNA or RNA molecule.

6. The biosensor device according to any one of claims 1 to 5, wherein the aptamer has a length of about 25 to 70 nucleotides, preferably about 30 to about 65 nucleotides.

7. The biosensor device according to any one of claims 1 to 6, wherein the functional group for attaching the at least one aptamer to the metal electrode surface is preferably a thiol group present at the end of the aptamer.

8. The biosensor device according to any one of claims 1 to 7, wherein the aptamer is a single-stranded nucleic acid molecule, preferably a DNA or RNA molecule, and a functional group for attaching at least one aptamer to the metal electrode surface is present at the 3' end or 5' end, preferably the 5' end.

9. The biosensor device according to any one of claims 1 to 8, wherein the aptamer has the ability to bind to at least one hormone selected from the group consisting of estradiol, luteinizing hormone (LH), and progesterone.

10. The biosensor device according to any one of claims 1 to 9, wherein the aptamer is one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO:

4.

11. A biosensor device according to any one of claims 1 to 10 for use for the purpose of diagnosis, more specifically detection of at least one hormone, more specifically monitoring hormones during assisted reproductive technology (ART), during menopause, in hormonal disorders such as polycystic ovary syndrome (PCOS), in endometriosis and / or during hormone therapy.

12. An implantable device for implantation into the body of an animal or human, more specifically for subcutaneous implantation, - A biosensor device according to any one of claims 1 to 11; - An electronic control device for controlling the biosensor device, more specifically, an electronic control device for generating, collecting, and preferably encrypting measurement data obtained by detection by the biosensor device; A portable device, including a transplantable device.

13. A system for detecting at least one hormone, Furthermore, the portable device according to claim 12 includes a communication device for wireless data communication with an external computer device separate from the portable device, more particularly an NFC (Near Field Communication) device; A communication device for wireless data communication with the aforementioned portable device, more specifically the external computer device including an NFC device; A system that includes this.

14. Use of the biosensor device according to any one of claims 1 to 11, in particular in the detection of at least one hormone for in vitro diagnosis.

15. A method for operating a biosensor device according to any one of claims 1 to 11, a portable device according to claim 12, or a system according to claim 13: (a) The step of bringing the biosensor membrane into contact with a body fluid containing the at least one hormone, more specifically, interstitial fluid; (b) the step of enabling the at least one hormone to bind to the at least one aptamer of the biosensor membrane; (c) The step of detecting the binding of the at least one hormone by electrically controlling the surface of the at least one metal electrode; A method that includes this.

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