Smart, Soft Contact Lens For Wireless Immunosensing of Cortisol

Abstract

Despite various approaches to immunoassay and chromatography for monitoring cortisol concentrations, conventional methods require bulky external equipment, which limits their use as mobile health care systems. Here, we describe a human pilot trial of a soft, smart contact lens for real-time detection of the cortisol concentration in tears using a smartphone. A cortisol sensor formed using a graphene field-effect transistor can measure cortisol concentration with a detection limit of 10 pg/ml, which is low enough to detect the cortisol concentration in human tears. In addition, this soft contact lens only requires the integration of this cortisol sensor with transparent antennas and wireless communication circuits to make a smartphone the only device needed to operate the lens remotely without obstructing the wearer’s view. Furthermore, in vivo tests using live rabbits and the human pilot experiment confirmed the good biocompatibility and reliability of this lens as a noninvasive, mobile health care solution.

INTRODUCTION

The steroid hormone, cortisol, which is known as a stress hormone, is secreted by the adrenal gland when people are stressed psychologically or physically (1). This secretion occurs when the adrenal gland is stimulated by adrenocorticotropic hormone, which is secreted by the pituitary gland when it is stimulated by the corticotropin-releasing hormone secreted by the hypothalamus. This serial cortisol secretion system is referred to as a hypothalamus–pituitary gland–adrenal gland axis, which is affected by chronic stress, resulting in abnormal secretion of cortisol (23). The accumulation of cortisol caused by the abnormal secretion of cortisol increases the concentrations of fat and amino acid, which can result in diverse severe diseases (e.g., Cushing’s disease, autoimmune disease, cardiovascular complications, and type 2 diabetes) and neurological disorders (such as depression and anxiety disorders) (27). In contrast, abnormally low cortisol levels can lead to Addison’s disease, which results in hypercholesterolemia, weight loss, and chronic fatigue (8). In addition, it was recently reported that plasma cortisol can be correlated to the prognosis of traumatic brain injury (9). Furthermore, the extent of cortisol secretion varies from person to person, and it changes continuously (1011). Thus, developing health care systems for real-time monitoring of the cortisol level has been explored extensively over the past decade as the key to the quantitative analysis of stress levels. Although various efforts have led to the development of cortisol sensors that can measure the concentration of cortisol in blood, saliva, sweat, hair, urine, and interstitial fluid (1217), the accurate measurement of cortisol concentrations has been limited because of the difficulties associated with the transportation and storage of cortisol as well as the instability of the biologically active cortisol in these body fluids at room temperature. In addition, these conventional sensing methods require bulky equipment for the extraction and analysis of these body fluids, which is not suitable for mobile health care systems (1218). Therefore, the development of noninvasive and wearable sensors that can monitor cortisol concentration accurately is highly desirable for a smart health care solution. For example, the immunoassay method, which uses an antigen-antibody binding reaction, has been used extensively for electrochemical cortisol immunosensors using saliva and interstitial fluid, except tears (121419). However, these immunosensors still require the use of bulky impedance analyzers for the analysis of the Nyquist plot from electrochemical impedance spectroscopy. Although the cyclic voltammetry (CV) technique can be used as an alternative approach for sensing cortisol, additional bulky electrochemical instruments still are necessary for analyzing the CV curves (131419). Recently, wearable forms of cortisol sensors that use sweat were developed (15), but they still required bulky measurement equipment (1516). Therefore, portable and smart sensors that can monitor the accurate concentration of cortisol in real time are highly desirable for use in mobile health care.

Among the various body fluids, tears, in particular, contain important biomarkers, including cortisol (2021). Thus, the integration of biosensors with contact lenses is a potentially attractive candidate for the noninvasive and real-time monitoring of these biomarkers from tears (2225). However, an approach for fabricating a smart contact lens for sensing the cortisol in tears has not been demonstrated previously. Thus, here, we present an extraordinary approach for the formation of a smart, soft contact lens that enables remote, real-time monitoring of the cortisol level in the wearer’s tears using mobile phones. This smart, soft contact lens is composed of a cortisol sensor, a wireless antenna, capacitors, resistors, and integrated circuit chips that use stretchable interconnects without obstructing the wearer’s view. The components of this device (except the antenna) were protected from mechanical deformations by locating each of the components on discrete, rigid islands and by embedding these islands inside an elastic layer. A graphene field-effect transistor (FET; with the binding of monoclonal antibody) was used as this cortisol immunosensor, which exhibited a sufficiently low detection limit, i.e., 10 pg/ml, for its sensing of cortisol in human tears in which the cortisol concentration ranges from 1 to 40 ng/ml (26). This sensor was integrated with a near-field communication (NFC) chip and antenna inside the soft contact lens for the real-time wireless transmission of the data to the user’s mobile device (e.g., a smart phone or a smart watch). The antenna occupies a relatively large area of this soft lens, so it requires its high stretchability, good transparency, and low resistance for operating a standard NFC chip at 13.56 MHz. In our approach, the hybrid random networks of ultralong silver nanofibers (AgNFs) and fine silver nanowires (AgNWs) enabled high transparency and good stretchability of this antenna and its low sheet resistance for reliable standard NFCs (at 13.56 MHz) inside this smart contact lens. Thus, the fully integrated system of this smart contact lens provided wireless and battery-free operation for the simultaneous detection and transmission of the cortisol concentration from tears to a mobile phone using standard NFC. In addition, a human pilot trial and in vivo tests conducted using live rabbits demonstrated the biocompatibility of this lens, and its safety against inflammation and thermal/electromagnetic field radiation suggests its substantial usability as a noninvasive, mobile health care solution.

RESULTS

Cortisol immunosensor

A graphene FET sensor was fabricated by binding the cortisol monoclonal antibody (C-Mab) to the surface of graphene for the immunosensing of cortisol. Here, graphene acts as a transducer that converts the interaction between cortisol and C-Mab into electrical signals. Figure 1A shows the immobilization process of C-Mab to graphene. Immobilization proceeds through amide bonding of the C-Mab onto the carboxyl group of the graphene surface via the EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride]/NHS (N-hydroxysulfosuccinimide) coupling reaction. A chemical vapor deposition–synthesized graphene layer was transferred onto a desired substrate and exposed to ultraviolet ozone (UVO) to activate the surface of the graphene with the carboxylate group. Figure S1 shows the contact angle between this surface of the graphene and a droplet of deionized (DI) water. Longer exposure time to UVO can decrease the hydrophobicity of graphene with decreasing the contact angle. Table S1 shows the increase in the electrical resistance of graphene that resulted from this UVO treatment. In our experiment, 2 min of exposure time to UVO decreased the contact angle from 70° to 38° without increasing the resistance of the graphene notably. UVO exposure times longer than this threshold time degraded the resistance of the graphene excessively, so the time of exposure of our samples to UVO was limited to 2 min. Figure S2A illustrates the process of immobilizing C-Mab through the EDC/NHS coupling reaction. This two-step coupling reaction of EDC and NHS can mediate the amide bonding between the carboxylate group of the UVO-exposed graphene and the amine group of the protein (12172728). Here, EDC forms reactive O-acylisourea ester, thereby making the surface unstable. This O-acylisourea ester reacts with the NHS to form amine-reactive NHS ester with the surface still remaining semistable. Then, C-Mab with the amine group reacts with the amine-reactive NHS ester, thereby forming stable amide bonding that can immobilize C-Mab to the NHS on the surface of the graphene. Figure S2B shows the Fourier transform infrared (FTIR) spectroscopy spectra of the DI water after the cortisol sensor had been immersed for 24 hours. The spectra of the DI water in which the sensor was immersed were not significantly different from those of the pristine DI water. However, the C-Mab solution that had a concentration of 1 μg/ml had a significant peak intensity in the range of 3000 to 2800 cm−1, representing the N-H bonding in the C-Mab. These results indicated that C-Mab formed stable bonding on the carboxylated graphene and was negligibly detached by exposure to water.

From https://advances.sciencemag.org/content/6/28/eabb2891

Gender-related Differences in the Presentation and Course of Cushing’s Disease

2003 Apr;88(4):1554-8.  doi: 10.1210/jc.2002-021518.

Abstract

Cushing’s disease (CD) presents a marked female preponderance, but whether this skewed gender distribution has any relevance to the presentation and outcome of CD is not known.

The aim of the present study was the comparison of clinical features, biochemical indices of hypercortisolism, and surgical outcome among male and female patients with CD. The study population comprised 280 patients with CD (233 females, 47 males) collected by the Italian multicentre study.

Epidemiological data, frequency of clinical signs and symptoms, urinary free cortisol (UFC), plasma ACTH and cortisol levels, responses to dynamic testing, and surgical outcome were compared in female and male patients.

Male patients with CD presented at a younger age, compared with females (30.5 +/- 1.93 vs. 37.1 +/- 0.86 yr, P < 0.01), with higher UFC and ACTH levels (434.1 +/- 51.96 vs. 342.1 +/- 21.01% upper limit of the normal range for UFC, P < 0.05; 163.9 +/- 22.92 vs. 117.7 +/- 9.59% upper limit of the normal range for ACTH, P < 0.05).

No difference in ACTH and cortisol responses to CRH, gradient at inferior petrosal sinus sampling, and cortisol inhibition after low-dose dexamethasone was recorded between sexes. In contrast, the sensitivity of the high-dose dexamethasone test was significantly lower in male than in female patients.

Of particular interest, symptoms indicative of hypercatabolic state were more frequent in male patients; indeed, males presented a higher prevalence of osteoporosis, muscle wasting, striae, and nephrolitiasis. Conversely, no symptom was more frequent in female patients with CD.

Patients with myopathy, hypokalemia, and purple striae presented significantly higher UFC levels, compared with patients without these symptoms. Lastly, in male patients, pituitary imaging was more frequently negative and immediate and late surgical outcome less favorable.

In conclusion, CD appeared at a younger age and with a more severe clinical presentation in males, compared with females, together with more pronounced elevation of cortisol and ACTH levels.

Furthermore, high-dose dexamethasone suppression test and pituitary imaging were less reliable in detecting the adenoma in male patients, further burdening the differential diagnosis with ectopic ACTH secretion. Lastly, the postsurgical course of the disease carried a worse prognosis in males. Altogether, these findings depict a different pattern for CD in males and females.

From https://pubmed.ncbi.nlm.nih.gov/12679438/

New way to study pituitary tumors holds potential for better diagnoses and treatments

Houston Methodist neurosurgeons and neuroscientists are looking at a new way to classify pituitary tumors that could lead to more precise and accurate diagnosing for patients in the future.

Found in up to 10% of the population, pituitary tumors, also called adenomas, are noncancerous growths on the pituitary gland and very common. Although these pituitary tumors are benign in nature, they pose a major health challenge in patients.

The new tests being investigated at Houston Methodist not only have the potential to lead to better diagnoses for patients with pituitary adenomas, but also for many other types of brain tumors in the future. The findings, which were published Jan. 28 in Scientific Reports, an online journal from Nature Publishing Group, describe a new way being looked at to study the blood of patients with pituitary tumors to determine exactly what tumor type they have and whether they might respond to medical treatment rather than surgery.

“Often called the ‘master gland,’ the pituitary gland controls the entire endocrine system and regulates various body functions by secreting hormones into the bloodstream to control such things as metabolism, growth and development, reproduction and sleep,” said corresponding author Kumar Pichumani, Ph.D., a research physicist at the Houston Methodist Research Institute. “When pituitary adenomas occur, they may secrete too much of one or more hormones that could lead to a variety of issues, ranging from infertility and sexual dysfunction to vision problems and osteoporosis, among many other health problems.”

Neurosurgeon David S. Baskin, M.D., director of the Kenneth R. Peak Center for Brain and Pituitary Tumor Treatment and Research in the Department of Neurosurgery at Houston Methodist Hospital, collaborated with Pichumani on this study. He said some pituitary tumors can be treated with medication rather than surgery, but a precise diagnosis of the type of tumor someone has and what  it’s secreting is essential for proper treatment. This is sometimes very difficult to do based on standard endocrine hormone testing.

“To guide our decisions on diagnosis and treatment, we currently rely on a blood-based hormone panel test that measures the levels of hormones in the blood to determine which hormones are overproducing in the tumor,” Baskin said. “However, some tumors secrete too much of more than one hormone, making this test ambiguous for diagnosis.”

Led by Pichumani and Baskin, a team of researchers from the Peak Brain and Pituitary Tumor Treatment and Research Center and Houston Methodist Neurological Institute studied 47 pituitary adenoma patients of different subtypes by collecting blood during surgery to remove their tumors. They confirmed that elevated blood levels of a non-hormonal compound called betahydroxybutyrate, also known as BHB, was found only in patients with the prolactinoma subtype of noncancerous pituitary gland brain tumor that overproduces the hormone prolactin. This compound is known to supply energy to the brain during starvation, which led the researchers to speculate that BHB might be providing non-hormonal energy to these prolactinoma tumors causing them to grow and spread. The discovery could be further developed into a diagnostic lab test.

This study is part of a developing field called metabolomics in which researchers study small molecules in tumors to see what’s unique about their metabolism and how they’re used as nutrients to supply energy. This contributes to better diagnoses and discovering new ways to kill tumors by poisoning the specific energy they use without causing damage to normal cells.

The researchers are now enrolling more patients in a larger study currently underway to validate the results of their pilot study. If successful, they say BHB could be used as a non-hormonal metabolic biomarker for prolactinoma pituitary tumor diagnosis and prognosis to supplement the current hormone panel tests. They’re also looking for biological reasons why only prolactin-secreting tumors have elevated BHB blood levels to inform therapeutic intervention.

From https://medicalxpress.com/news/2020-02-pituitary-tumors-potential-treatments.html

BIPSS Diagnostic Method May Cause False Positive in Some Cases of Cyclic Cushing’s Syndrome

A diagnostic technique called bilateral inferior petrosal sinus sampling (BIPSS), which measures the levels of the adrenocorticotropic hormone (ACTH) produced by the pituitary gland, should only be used to diagnose cyclic Cushing’s syndrome patients during periods of cortisol excess, a case report shows.

When it is used during a spontaneous remission period of cycling Cushing’s syndrome, this kind of sampling can lead to false results, the researchers found.

The study, “A pitfall of bilateral inferior petrosal sinus sampling in cyclic Cushing’s syndrome,” was published in BMC Endocrine Disorders.

Cushing’s syndrome is caused by abnormally high levels of the hormone cortisol. This is most often the result of a tumor on the pituitary gland that produces too much ACTH, which tells the adrenal glands to increase cortisol secretion.

However, the disease may also occur due to adrenal tumors or tumors elsewhere in the body that also produce excess ACTH — referred to as ectopic Cushing’s syndrome.

Because treatment strategies differ, doctors need to determine the root cause of the condition before deciding which treatment to choose.

BIPSS can be useful in this regard. It is considered a gold standard diagnostic tool to determine whether ACTH is being produced and released by the pituitary gland or by an ectopic tumor.

However, in people with cycling Cushing’s syndrome, this technique might not be foolproof.

Researchers reported the case of a 43-year-old woman who had rapidly cycling Cushing’s syndrome, meaning she had periods of excess cortisol with Cushing’s syndrome symptoms — low potassium, high blood pressure, and weight gain — followed by normal cortisol levels where symptoms resolved spontaneously.

In general, the length of each period can vary anywhere from a few hours to several months; in the case of this woman, they alternated relatively rapidly — over the course of weeks.

After conducting a series of blood tests and physical exams, researchers suspected of Cushing’s syndrome caused by an ACTH-producing tumor.

The patient eventually was diagnosed with ectopic Cushing’s disease, but a BIPSS sampling performed during a spontaneous remission period led to an initial false diagnosis of pituitary Cushing’s. As a result, the woman underwent an unnecessary exploratory pituitary surgery that revealed no tumor on the pituitary.

Additional imaging studies then identified a few metastatic lesions, some of which were removed surgically, as the likely source of ACTH. However, the primary tumor still hasn’t been definitively identified. At the time of publication, the patient was still being treated for Cushing’s-related symptoms and receiving chemotherapy.

There is still a question of why the initial BIPSS result was a false positive. The researchers think that the likely explanation is that BIPSS was performed during an “off phase,” when cortisol levels were comparatively low. In fact, a later BIPSS performed during a period of high cortisol levels showed no evidence of ACTH excess in the pituitary.

This case “demonstrates the importance of performing diagnostic tests only during the phases of active cortisol secretion, as soon as first symptoms appear,” the researchers concluded.

From https://cushingsdiseasenews.com/2020/01/02/cushings-syndrome-case-study-shows-drawback-in-bipss-method/

Imaging Agent Effectively Detects, Localizes Tumors in Cushing’s Syndrome

Wannachalee T, et al. Clin Endocrinol. 2019;doi:10.1111/cen.14008.
May 20, 2019

A radioactive diagnostic agent for PET imaging effectively localized primary tumors or metastases in most adults with ectopic Cushing’s syndrome, leading to changes in clinical management for 64% of patients, according to findings from a retrospective study published in Clinical Endocrinology.

As Endocrine Today previously reported, the FDA approved the first kit for the preparation of gallium Ga-68 dotatate injection (Netspot, Advanced Accelerator Applications USA Inc.), a radioactive diagnostic agent for PET scan imaging, in June 2016. The radioactive probe is designed to help locate tumors in adult and pediatric patients with somatostatin receptor-positive neuroendocrine tumors. Ga-68 dotatate, a positron-emitting analogue of somatostatin, works by binding to the hormone.

In a retrospective review, Richard Auchus, MD, PhD, professor of pharmacology and internal medicine in the division of metabolism, endocrinology and diabetes at the University of Michigan, and colleagues analyzed data from 28 patients with ectopic Cushing’s syndrome who underwent imaging with gallium Ga-68 dotatate for identification of the primary tumor or follow-up between November 2016 and October 2018 (mean age, 50 years; 22 women). All imaging was completed at tertiary referral centers at Mayo Clinic, University of Michigan and The University of Texas MD Anderson Cancer Center. Researchers assessed patient demographics, imaging modalities, histopathological results and treatment data. Diagnosis of Cushing’s syndrome was confirmed by clinical and hormonal evaluation. The clinical impact of gallium Ga-68 dotatate was defined as the detection of primary ectopic Cushing’s syndrome or new metastatic foci, along with changes in clinical management.

Within the cohort, 17 patients underwent imaging with gallium Ga-68 dotatate for identification of the primary tumor and 11 underwent the imaging for follow-up. Researchers found that gallium Ga-68 dotatate identified suspected primary ectopic Cushing’s syndrome in 11 of 17 patients (65%), of which seven tumors were solitary and four were metastatic. Diagnosis was confirmed by pathology in eight of the 11 patients: Five patients had a bronchial neuroendocrine tumor, one patient had a thymic tumor, one had a pancreatic neuroendocrine tumor, and one metastatic neuroendocrine tumor was of unknown primary origin. One patient had a false positive scan, according to researchers.

Among the 11 patients with ectopic Cushing’s syndrome who underwent gallium Ga-68 dotatate imaging to assess disease burden or recurrence, the imaging led to changes in clinical management in seven cases (64%), according to researchers.

“Our study demonstrates the high sensitivity of [gallium Ga-68 dotatate] in the localization of [ectopic Cushing’s syndrome], for both occult primary tumors and metastatic lesions,” the researchers wrote. “Importantly, the use of [gallium Ga-68 dotatate] impacted clinical management in 64% of patients with [ectopic Cushing’s syndrome] overall.”

The researchers noted that the high cost and limited availability of PET/CT imaging might preclude the widespread use of gallium Ga-68 dotatate for imaging in patients with suspected ectopic Cushing’s syndrome, and that experience with the scans remains limited vs. other imaging studies.

“Nevertheless, combing the experience of three large referral centers, our study gathers the largest number of [patients with ectopic Cushing’s syndrome] imaged with [gallium Ga-68 dotatate] to date and provides a benchmark for the utility of this diagnostic modality for this rare but highly morbid condition,” the researchers wrote. – by Regina Schaffer

DisclosuresThe authors report no relevant financial disclosures.

From https://www.healio.com/endocrinology/adrenal/news/online/%7B69e458a8-e9a0-4567-a786-00868118b435%7D/imaging-agent-effectively-detects-localizes-tumors-in-cushings-syndrome

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