Cortisol is a steroid hormone released by the adrenal glands in response to adrenocorticotropic hormone (ACTH) stimulation from the anterior pituitary gland. It is a monovalent antigen due to its low molecular weight of 362.47 g/mol. Cortisol has significant effects, especially on carbohydrate metabolism. Its level in the circulation normally peaks at in the morning hours, drops in the evening and reaches a nadir around midnight. Cortisol can be obtained from the blood, saliva and urine to diagnose Cushing Syndrome (hypercortisolism), Addison disease (hypocortisolism), monitoring treatment for hypercortisolism or hypocortisolims and evaluating stress-related conditions. When testing for conditions involving excessive release of cortisol, the peak serum level may be normal, but the diurnal drop may not be observed. As a result, levels should be drawn at 8 AM, 4 PM and sometimes 8 PM. When testing for conditions involving decreased cortisol release, a single morning level may be drawn. To distinguish Cushing syndrome from simple obesity, a 24 h urine free cortisol level may be appropriate. Urine free cortisol is proportional to serum free cortisol and is also the most sensitive and specific test for the initial screening for Cushing syndrome. Cortisol is also present in saliva and is proportional to unbound cortisol in the serum, including diurnal variations. Thus, in adrenal insufficiency (as can be found in Addison disease among others), the morning salivary cortisol is decreased, while evening salivary cortisol levels (drawn between 11 PM and midnight) are increased in Cushing syndrome. Of note, cortisol reference ranges may vary by time drawn (whether in adults, adolescents or children), by site (serum, saliva or urine), by methodologies (radioimmunoassay or high-performance liquid chromatography). Extremely high cortisol levels in the morning in addition to a lack of diurnal variation may suggest the presence of carcinoma. Also, certain drugs are associated with increased cortisol levels, such as cocaine, amphetamines, alcohol, nicotine and naloxone, while lithium, levodopa, phenytoin (in women), morphine, etomidate and ketoconazole can cause decreased cortisol levels. Cortisol levels may also be increased in bright light exposure, ectopic ACTH production, high stress (thermal, traumatic or physiological), metabolic syndrome (with hypertension and obesity), burns, shock, post-surgical states, severe liver or kidney disease, acute infections or inflammatory disease, hyperpituitarism, hyper thyroidism, pregnancy, strenuous exercise, hypoglycemia, and drugs (coticotropin, estrogens, oral contraceptives, yohimbine and vasopressin). Decreased cortisol levels may also be seen in chromophobe adenoma, craniopharyngioma, hypothyroidism, liver disease, postpartum pituitary necrosis, rheumatoid arthritis and drugs (dexamethasone). Organophosphate exposure on the central nervous system can cause alterations in the endocrine system that can lead to imbalances in cortisol secretion which may then be measured by immunosensors and immunoassays. In psychiatry and behavioral science, cortisol has a regulatory effect on serotonin function and a stimulant-like effect on dopamine neurotransmission. In general, excessive cortisol activity may contribute to symptoms of psychotic mood disorders and schizophrenia, while cortisol levels may be elevated in brief or sustained stress and major depression. In contrast, studies have shown that cortisol levels are low in post-traumatic stress syndrome (PTSD). Furthermore, stress and cortisol elevation have been implicated in studies of relapse in substance-dependent patients [100
Unlike with some other biomarkers, with cortisol (which is linked with many stress-induced diseases and requires near real-time detection), the model where the biological fluid samples are first collected from patients with later processing and analysis in centralized laboratories with relatively long reporting times and a possibility of several points where quality control could fail, does not work well. Therefore, much effort in the recent years has gone towards the development of easy-to-use biosensors for on-site measurement of cortisol levels and related diagnosis.
Nanomaterials, such as CNTs and conducting polymer-metal nanocomposites have been used in cortisol sensing as the chemiresistive transducer and as the antibody immobilization matrix respectively, to enhance the sensitivity and selectivity of electrochemical cortisol immunosensors. Tilli et al.
] fabricated a label-free immunosensor for cortisol using SWCNTs as the chemiresistive transducer. The SWCNTs were functionalized with cortisol-3-O-carboxymethyloxime-N-hydroxysuccinimide (3-O-CMO-NHS) ester, a cortisol analog, and a monoclonal anti-cortisol antibody was ligated to this receptor. Cortisol from artificial saliva displaced the anti-cortisol antibody at the receptor producing corresponding decreases in the resistance/conductance of the nanotube-biomolecule hybrid biosensor. The immunosensor was selective and sensitive with detection limit of 1 pg/mL, a sensitivity of 14.9 ng/mL and a linear range from 1 pg/mL to 10 ng/mL.
Arya et al.
] prepared a mediator and label free cortisol biosensor by depositing polyaniline (PANI) protected gold nanoparticle composite electrophoretically onto a gold electrode. Monoclonal antibodies specific and selective for cortisol were then covalently immobilized onto the surface of the conducting polymer/Au electrode using N-ethyl-Nʹ-(3-dimethylaminopropyl) carbodiimide and N
-hydroxysuccinimide (EDC/NHS) chemistry. Using cyclic voltammetry as the electrochemical detection method, they obtained a detection limit of 1 pM, a sensitivity of 1.63 μA/M and a linear range from 0.36 pg/mL to 36 ng/mL (1 pM–100 nM). They also explored a similar biosensor design with the PANI polymer nanocomposite containing silver/silver oxide (Ag/AgO) (core/shell with diameter = 5 nm). This immunosensor had a detection limit of 0.64 pM/mL, a sensitivity of 0.66 μA/M and a linear range from 0.36 pg/mL to 3.6 μg/mL.
Yamaguchi et al.
] designed a reusable immunosensor for noninvasive and quantitative analysis of cortisol in saliva in about 35 minutes. The immunosensor has a fluid control mechanism with both a vertical and a lateral flow. The immune reaction, a competitive immunoassay, occurs in the vertical flow component of the sensor while the lateral flow component removes proteins, other steroid hormones, and any unreacted enzyme-labeled cortisol-conjugate from the sample thereby minimizing cross-reactivity of the anti-cortisol Abs and ultimately improving the sensitivity of the biosensor. A flat electrode consisting of three Pt electrodes (a working electrode, a reference, and an auxiliary electrode) detects the current produced by the oxidation of hydrogen peroxide which is previously produced by the glucose oxidase enzyme label in the presence of glucose substrate. This immunosensor with fluid control mechanism had a dynamic range from 0.1–10 ng/mL cortisol in saliva [122
]. Typical cortisol concentration range in the saliva of healthy adults is 1–8 ng/mL [123
3.2.2. Ceruloplasmin (Cp)
Serum ceruloplasmin (along with serum copper and calculated free serum copper) is used to screen for the autosomal recessive inherited disorder of copper metabolism in which circulating free copper levels are elevated leading to excess copper being deposited in the brain, eyes, liver and kidneys known as Wilson disease (Hepatolenticular degeneration). In Wilson disease, serum Cp is decreased, while the free copper level is elevated (>25 μg/dL) [100
]. Cp is a copper-binding protein which is synthesized in the liver with a molecular weight of 132 kiloDaltons (kDa) and consists of a single polypeptide chain. Serum levels are 20–40 mg/dL in normal adults which may be elevated twofold in oral contraceptive therapy, pregnancy or as an acute phase reactant (a marker of inflammation, infection, tissue injury, malignancy and cardiovascular disease) [99
]. Drugs associated with elevated Cp levels include anticonvulsants, estrogens, methadone and nicotine, while drugs associated with elevated copper levels include anticonvulsants and estrogens. A molecule of Cp can bind six atoms of copper imparting a blue color to the protein and combined with the yellow of other chromogens of plasma imparts a greenish color to plasma with elevated Cp [99
]. Screening for Wilson disease is indicated if there is clinical suspicion of Wilson disease, family history of Wilson disease, early onset hepatitis or cirrhosis and neuropsychiatric symptoms consistent with Wilson disease. Diagnosi of Wilson disease is confirmed based on physical findings (liver disease, neurologic signs, Kayser-Fleischer ring in the cornea), low serum Cp level, increased serum free copper, increased serum copper and increased urine copper [99
]. Neuropsychiatric symptoms of Wilson disease may precede other disease manifestations and present as psychosis (delusions and hallucinations), depression, mild cognitive impairment, behavioral problems (personality changes), and abnormal motor system movements (parkinsonism, dystonia, chorea, tremor (the classic “wing-beating” type), lack of coordination, hypokinetic speech, dysphagia, bulbar and pseudobulbar palsies [100
Ojeda et al.
] prepared and described the first electrochemical immunosensors for determination of Cp in 2013. Two configurations involving magnetic beads (MBs) functionalized with Protein A or Streptavidin for immobilization of Cp Abs were compared using competitive immunoassay format with synthesized alkaline phosphatase (ALP)-Cp conjugate. ALP is the enzyme label responsible for catalyzing the formation of electrochemically active 1-naphthol from 1-naphthylphosphate substrate. Upon capturing MBs-immunoconjugates onto screen-printed electrodes (SPEs), quantification of Cp was completed by differential pulse voltammetry (DPV) detection of 1-naphthol produced in response to 1-naphthylphosphate addition. Linear range of calibration curve for Protein A-MBs was 0.1–1000 µg/mL with a detection limit of 0.040 µg/mL. The corresponding linear range for Streptavidin immobilized anti-Cp Abs on MBs was 0.025–20 µg/mL with a 0.018 µg/mL detection limit [124
]. Good results were also obtained when using the immunosensor for the determination of Cp in spiked human serum samples.
Recently, Garcinuño et al.
] of the same research group, described a novel amperometric immunosensor for the determination of Cp in human serum and urine based that is based on covalent binding to CNT-modified screen-printed electrodes. This is the first reported immunosensor for Cp with amperometric detection. The immunosensor configuration includes an indirect competitive immunoassay with covalent immobilization of Cp, the analyte, on activated carboxylic acid groups at CNT-modified screen-printed electrodes (SPE). After Cp immobilization, an immunoreaction is allowed to take place between the free Cp from the sample and anti-Cp Abs in solution and the remaining non-conjugated anti-Cp Ab is attached onto the Cp-CNTs modified SPE. Ultimately, the determination of Cp is done by adding a secondary antibody labeled with horseradish peroxidase (HRP) anti-IgG and measuring the amperometric current resulting from the addition of hydrogen peroxide (H2
) in the presence of hydroquinone as the redox mediator. The immunosensor for Cp had good reproducibility, a linear range between 0.07 and 250 μg/mL and the detection limit was 21 ng/mL [125
]. The analytical performance of the immunosensor should allow the fast determination of Cp in spiked human serum and urine samples.
3.2.3. Thyroid-Stimulating Hormone (TSH)
TSH is a glycoprotein hormone produced by thyrotrope cells in the anterior pituitary gland. TSH stimulates the thyroid gland to release thyroxine (T4
) and stored triiodothyroxine (T3
) which are hormones that determine basal metabolic rate, protein synthesis, carbohydrate, lipid and vitamin metabolism and calcium release from bones. In addition, T4
is converted to the more potent T3
. The circulating T4
influence the release of TSH and thyrotropin-releasing hormone (TRH) from the hypothalamus via a feedback inhibition. Thyroid function can be tested by: (1) measuring free thyroxine (free T4
); or (2) by measuring TSH (sensitive TSH assay/highly sensitive TSH assay or thyrotropin assay). FT4
is measured in preference to total T4
because its measurement is not affected by blood protein levels. Blood is usually required for the test and FT4
is usually ordered after an abnormal TSH test or when clinical suspicion of thyroid disease persists even though TSH is within normal levels. In fact, variations in levels of TSH and FT4
together suggest the following disease states:
TSH ↑, FT4 ↓: hypothyroidism;
TSH ↑, FT4 normal: subclinical hypothyroidism;
TSH ↓, FT4 ↑: hyperthyroidism;
TSH ↓, FT4 normal: subclinical hyperthyroidism;
TSH ↓, FT4 ↓: nonthyroidal illness.
is measured when there is an abnormal TSH result or signs/symptoms of thyroid disease (in the presence of normal TSH). Reference range for TSH in adult human serum is usually between 18 pg/mL and 0.23 ng/mL. The exact linear detection range depends on the assay methodology, therefore it is recommended to consult laboratory reference values. However, if patients are already being treated for hypothyroidism using levothyroxine, the upper limit of normal is 0.05 ng/mL. Increased levels of FT4
suggest hyperthyroidism or treated hypothyroidism, while decreased FT4
levels suggest primary hyperthyroidism (due to thyroid gland dysfunction), secondary hypothyroidism (due to a pituitary cause), tertiary hypothyroidism (due to a hypothalamic cause), hypothyroidism treated with triiodothyronine (T3
) and late pregnancy. Hyperthyroidism, as defined by the low TSH and elevated FT4
levels, may present with psychiatric symptoms such as mood disorders, psychosis and delirium. In the elderly, a syndrome of apathetic hyperthyroidism may be present and is characterized by lethargy, tachycardia, atrial fibrillation, heart failure and severe cognitive impairment which may be diagnosed as dementia [100
]. On the other hand, hypothyroidism, as defined by low FT4
with elevated TSH levels, is associated with fatigue, depression, and memory impairment, while low FT4
with low TSH is seen in nonthyroidal illness in acutely ill patients. A normal FT4
with an elevated or low TSH level suggests subclinical hypothyroidism or hyperthyroidism respectively which may manifest into fully developed or attenuated thyroid disease. As noted earlier, FT4
can vary in pregnancy, severe or chronic illness. In fact, dated FT4
may also be caused by certain drugs such as aspirin, furosemide, heparin, carbamazepine, phenytoin, propranolol, valproate and contrast material used in medical imaging, while drugs that cause low FT4
levels include phenytoin, methadone, phenobarbital, estrogen, lithium, carbamazepine and oral contraceptives [100
Measuring thyroid function via TSH (sensitive TSH assay/highly sensitive TSH assay or thyrotropin assay) is useful when pituitary (secondary) hypothyroidism is suspected. In this case, TSH should be tested along with FT4
. If the thyroid itself (primary disease) is suspected to be the source of hyper or hypothyroidism, then the sensitive TSH is the best initial screening test. If the assay is normal, no further testing is required. However, if the assay is not normal, FT4
is measured. TSH and FT4
together suggest the following diagnoses:
TSH ↑, FT4 ↓: hypothyroidism;
TSH ↑, FT4 normal: subclinical hypothyroidism (and check thyroid antibodies);
TSH ↓, FT4 ↑: hyperthyroidism (thyroid-stimulating immunoglobin, thyroid peroxidase;
(TPO) antibody, and TSH receptor antibody are checked);
TSH ↓, FT4 normal: subclinical hyperthyroidism;
TSH ↓, FT4 ↓: nonthyroidal illness.
Indications for testing, include: diagnosing hypo- or hyperthyroidism in a symptomatic patient, screening for thyroid disease in at-risk patients (such as women over 50 years old), patients with newly diagnosed type 2 diabetes, pregnant women, postpartum women and patients treated with lithium, monitoring the efficacy of thyroid replacement therapy in a patient with hypothyroidism, standard component of the workup for patients with depression, memory impairment, or dementia and assisting in the work up of female infertility. Reference ranges vary by age, type of hyper- or hypothyroidism (borderline and probable). Treatment for hyper- or hypothyroid states may cause TSH to remain abnormal for up to 6 weeks after a euthyroid state has been reached. Critical values of <0.1 μU/mL indicates primary hyperthyroidism or exogenous thyrotoxicosis and risk of atrial fibrillation (a major risk factor for stroke). Increased levels of TSH can be caused by primary hypothyroidism (up to 100 times normal), TSH-producing tumor (e.g., breast or lung), Hashimoto thyroiditis, the recovery phase of subacute thyroiditis or nonthyroidal illness, insufficient thyroid replacement or thyroid hormone resistance treated hypothyroid patients. Decreased levels of TSH may be due to primary hyperthyroidism, secondary hypothyroidism (pituitary disease), tertiary hypothyroidism (hypothalamic disease), subclinical hyperthyroidism (due to toxic mutlinodular goiter or treated Graves disease), euthyroid sick syndrome or over-replacement of thyroid hormone in treated hypothyroid patients. Other factors that can affect TSH levels include old age (>80 years old, upper limit of normal is 10 μU/mL), treatment with atenolol, carbamazepine, aripiprazole, estrogen, chlorpromazine, ferrous sulfate, haloperidol, lithium, metoclopramide, iodine-containing food or drugs, morphine, lovastatin, phenothiazines, prednisone, phenytoin, sumatriptan, valproate, and amphetamine (abusers). Decreased TSH levels can be caused by acute illness, extreme stress, aspirin use, corticosteroids, carbamazepine, interferon-α2, hydrocortisone and the first trimester of pregnancy. Of relevance to psychiatry and behavioral health, low TSH with elevated FT4
indicates hyperthyroidism which can be associated with mood disorders, psychosis, and delirium. In the elderly, a syndrome of apathetic hyperthyroidism may be present and is characterized by lethargy, tachycardia, atrial fibrillation, heart failure and severe cognitive impairment which may be diagnosed as dementia [100
]. Elevated TSH with low FT4
indicates hypothyroidism which is associated with fatigue, depression, and memory impairment. Low TSH with low FT4
is commonly seen in acutely ill patients, while abnormal TSH with normal FT4
is consistent with subclinical thyroid disease which may develop into full-blown hyper- or hypothyroidism or an attenuated form of these two entities. Of note, numerous psychotropic drugs affect TSH level (as detailed above) without causing disease [100
Zhang et al.
] designed a novel electrochemical immunosensor for sensitive determination of TSH utilizing nanogold-functionalized magnetic beads (GoldMag) on a gold nanoparticle-dispersed graphene nanosheets immunosensing platform. The authors prepared polyethyleneimine-functionalized magnetic beads by a wet chemical method onto which electroactive thionine molecules and gold nanoparticles were alternately immobilized using and opposite-charged adsorption technique and an in situ
synthesis method, respectively. The GoldMag nanostructures served as signal trace tags for the enzyme label horseradish peroxidase (HRP)-anti TSH antibody conjugates in a sandwich immunoassay. The conjugated signal tags increased with increasing TSH concentration which was also determined in human serum specimens. This electrochemical immunosensor had detection limit of 0.23 pg/mL and a linear range from 0.45 pg/mL to 0.91 ng/mL [126
]. The results indicated that the GoldMag nanostructures, conjugated with HRP, enhanced the sensitivity of the electrochemical immunosensor. The signal amplification was partially achieved by high HRP-loading on the nanostructures and in part the high conductivity of the nanomaterials.
3.2.4. Luteinizing Hormone (LH)
In response to stimulation by gonadotropin-releasing hormone (GHR) from the hypothalamuc, LH is secreted by the anterior pituitary which in turn stimulates the testes to produce testosterone and the ovaries to produce estradiol. These hormones then feed back to the hypothalamus and pituitary to help regulate LH release. In females, a surge of LH in mid-menstrual cycle causes ovulation of the follicle-stimulating hormone (FSH)-ripened ovarian follicle. Therefore, in women, LH and FSH levels can help distinguish failure of the ovaries themselves (primary ovarian failure) from ovarian failure due to pituitary or hypothalamic dysfunction (secondary ovarian failure). LH and FSH may also be utilized in the diagnoses of virilization/hirsutism, polycystic ovary syndrome, reduced sex drive, infertility, and precocious or delayed puberty. In primary ovarian failure, both LH and FSH levels are high. In males, LH and FSH are needed for sperm development and maturation. In addition in males, LH and FSH may be indicated for testing in hypogonadism, testicular tumors, cryptorchidism, reduced sex drive, infertility and erectile dysfunction. It is important to note that LH and FSH levels fluctuate throughout the day; therefore a single sample may give inaccurate hormone activity.
Reference ranges differ by laboratories and by stage of puberty, gender, and menstrual cycle phase. As stated earlier, increased levels are seen in primary ovarian failure, primary testicular failure, polycystic ovary syndrome, menopause, precocious puberty and liver disease, while decreased levels are seen in pituitary or hypothalamic dysfunction resulting in secondary gonadal (ovarian or testicular) failure, anorexia nervosa, depression, severe stress, malnutrition, severe illness and delayed puberty. LH levels can also be increased with the use of naloxone, spironolactone, clomiphene or certain anticonvulsants, while decreases may be noted with the use of phenothiazines, oral contraceptives, digoxin, or hormone treatments. Of note, LH levels are used in the work up of certain psychiatric and behavioral conditions. For example, LH levels (as stated earlier), may be decreased in anorexia nervosa, severe stress, phenothiazine use or depression. LH levels may be increased with naloxone use [100
Lillie et al.
] fabricated a simple immunosensor for LH measurement between 1 and 800 IU/L using impedance spectroscopy detection. The sensor was prepared by polymerizing pyrrole loaded with antibody for LH on a gold inter-digitated electrode. Farace et al.
] also prepared an immunosensor for LH with impedance spectroscopy detection. The reagentless sensor had intergrated biorecognition and transduction system with the antibodies immobilized by entrapping them in a conducting polypyrrole matrix.