Design of Nanostructured Enzyme Immobilized Platforms for Precise Diagnosis and Treatment of Endocrine Disease: An Updated Review

Abstract

Endocrine diseases are a big health problem worldwide, due to their complex pathophysiology, the diversity of clinical phenotypes and the necessity of sensitive, stable, and real‑time monitoring of hormonal and metabolic biomarkers. Conventional endocrine assays are plagued by the need for the presence of large amounts of biomarker, the time needed for the response, poor enzyme stability, matrix effects and lack of continuous or point of care suitability. To overcome these limitations, technical approaches are based on nanostructured enzyme-immobilized platforms that provide high surface to volume ratio for high enzyme loading, tunable surface chemistry for control of immobilization, enhanced electron-transfer pathways for electrochemical signal amplification, porous architecture to confine and stabilize enzyme, and plasmonic effects (eg, localized surface plasmon resonance) for optical signal enhancement. In this review, an updated overview of the design principles, mechanisms and biomedical relevance of enzyme immobilization on nanostructured platforms for the diagnosis and treatment-related management of endocrine diseases is provided. The carbon-based materials, metallic and metal-oxide nanoparticles, polymeric systems, metal-organic frameworks, covalent organic frameworks, and hybrid nanocomposites are discussed in the context of their role in tuning the loading of enzymes, catalytic activity, operational stability, signal transduction and biocompatibility. Particular emphasis is placed on applications in diabetes mellitus, thyroid, adrenal disorders and lipid metabolic imbalance, as well as emerging applications in targeted therapy, enzyme guided drug delivery, oxidative-stress regulation and nanotheranostics. The current problems of reproducibility, long-term stability, scalability, biocompatibility and clinical translation are critically discussed. Potential future trends, such as AI-powered biosensors, next-generation nanozymes, multiplexed platforms, and personalized nanomedicine strategies, are suggested.

Introduction

The endocrine system is a vital network of glands and organs responsible for producing and releasing hormones that regulate nearly every cell, organ, and function in the body. It controls processes such as growth, metabolism, reproduction, mood, and development from conception through old age. The pituitary, thyroid, adrenal glands, pancreas, ovaries and testes are major glands each with specific hormones such as insulin, thyroid hormones, cortisol and sex steroids all of which promote body coordination. Such glands directly secrete the hormones into the blood which is used to find its way to the target tissues and organs. One of the crucial functions of the hypothalamus and pituitary gland is hormonal balance and homeostasis and maintains the secretion of hormones through positive feedback mechanisms.¹

Metabolism is controlled by thyroid gland, and adrenal glands are used to regulate the reaction to stress and electrolyte balance. Other hormones that are secreted by the pancreas include glucagon and insulin which are used to regulate blood sugar. Hormones are crucial in the development of a person, sexual growth, reproductive health, and metabolism. A change in equilibrium or disruption in hormonal production can be the cause of numerous endocrine diseases that have an impact on health and well-being. Overall, the system of complex and interconnected chemical signaling of the endocrine system supports normal functioning and development.² Central endocrine regulation originates in the hypothalamus, which integrates neural and hormonal cues to control anterior and posterior pituitary secretions via releasing and inhibitory hormones or neurohypophyseal pathways.³ Through its trophic hormones, the pituitary gland orchestrates the activity of peripheral endocrine glands, including the thyroid gland, which governs basal metabolic rate, thermogenesis, and protein synthesis, and the parathyroid glands, which maintain calcium-phosphate homeostasis through parathyroid hormone-mediated regulation of bone remodeling, renal calcium reabsorption, and vitamin D activation.⁴

The adrenal glands contribute to both steroid hormones (eg, cortisol and aldosterone) and catecholamines essential for metabolic regulation, immune modulation, blood pressure control, and acute stress responses mediated by the hypothalamic-pituitary-adrenal (HPA) axis. The islets of Langerhans make up the endocrine pancreas which regulate the glucose homeostasis by coordinated release of insulin, glucagon, somatostatin and pancreatic polypeptide, to maintain balanced anabolic and catabolic metabolic conditions.³ The gonads (ovaries and testes) generate sex steroids and gametes, which are under the control of the pituitary and coordinate the reproductive activity, sexual differentiation and secondary sexual traits.⁵ Negative-feedback mechanisms, including the thyroid and adrenal axes, are the main regulators of endocrine signaling, which can measure the levels of circulating hormones and regulate the activity of glands to prevent over- or under-secretion, and maintain long-term systemic homeostasis.³

Global Burden and Impact of Endocrine Disorders

Endocrine, metabolic, blood, and immune disorders (EMBID), of which the key endocrine diseases belong, pose a considerable burden on global health that is increasing even with improved medical diagnosis and treatment. The incidence of these disorders globally was 79.47 million new cases in 2021, with a prevalence of 475.78 million infected individuals across the world. The number of deaths caused by EMBID increased to approximately 175,902 in the same year. The incidence rate that was age-standardized has decreased slightly, but the total figures have grown as a result of an aging population and growth. The age-standardized death rate that decreased slightly after reaching its peak in 2017 will increase once again by 2040, showing the current difficulties in effective management of these diseases.^6,7 Increased incidence and prevalence are attributed to population growth between 81–86% and aging between 26–37%. Although there are certain gains in epidemiological situations that led to a slight decline in the rates of incidence, these gains are overwhelmed by the global demographic trends. Disability-adjusted life years (DALYs) attributed to EMBID have been rising significantly; these are primarily the demographic factors. The situation is disproportionately spread all over the world, with low/middle-income countries, especially in Oceania, South Asia and Sub-Saharan Africa, having the greatest incidence and death rates. These areas have disproportional difficulties because of the lack of healthcare facilities and poor access to preventive and curative measures.⁸

The prevalence of autoimmune endocrine diseases is higher in women, whereas in men, there is a higher rate of mortalities associated with metabolic endocrine diseases. Endocrine diseases pose long-term disability and low-quality life in addition to high mortality rates, which have cost healthcare systems globally billions of dollars. The health impact of these diseases is aggravated by the fact that they cause complications like cardiovascular disease, neurological manifestations and metabolic dysfunctions. To counter this increased global burden, better access to healthcare, prevention, and treatment are essential, particularly in resource-restrained environments, to lessen the disparity and increase patient outcomes.⁹

Limitations of Conventional Diagnostic and Therapeutic Approaches

The main limitation of the traditional diagnostic process of the endocrine disorders lies in the complexity of the hormonal system and the transparency of the method of tests. Numerous endocrine diseases, such as the Cushing syndrome, growth hormone deficiency, polycystic ovary syndrome (PCOS), etc, do not have a simple gold standard of diagnosis. The dynamic test protocols that will be applied in diagnosing various conditions like growth hormone deficiency are many, multiple secretagogues are employed and the biological responses of the secretagogues are different therefore, provide inconsistent interpretation. The assays used are also specific and standard with a great difference between assay procedures, and this can give a misleading or wrong result. This complicates the clinical decision and may result in misdiagnosis or delay in diagnosis.¹⁰ The lack of resources in the environment also contributes to the problem nature of the issues since testing is not cheap, and the special laboratory facilities and qualified personnel are limited. Such restrictions compromise the quality of care and the quality of the laboratory tests results, which subsequently affect the quality of the diagnosis and treatment results. Although useful, traditional immunometric assays and recombinant DNA technologies used in endocrine testing are still marked by some tradeoff and weaknesses inherent in the nature of the technique and that impairs precision and clinical utility. Therapeutic approaches also have a few limitations. Insufficiency of endocrine hormones and hormone replacement therapy, which is the primary treatment in most instances could not be capable of giving the body normal endocrine functions due to the complexity of hormone regulation and feedback processes. Hormone treatments can be fatal with their side effects, and the use of the drugs must be strictly monitored to avoid overdose or underdose of the drugs. The incidences of death may be increased by mitigating the risks of adrenal insufficiency, which may reduce bone mineral density by replacing the glucocorticoids. Moreover, in endocrine tumors like breast cancer in estrogen receptor that is sensitive to tamoxifen, resistance to the sustained use of drugs like tamoxifen and emergence of more risks of secondary complications has necessitated sequential therapy or combination therapy to manage this problem. Another factor that has been contributing to the failure in endocrine therapies is the complex network of different signaling pathways, and it is important to have a more specific and individualized treatment regimen.¹¹

The current review aims to offer an integrated and up-to-date discussion on the design of nanostructured enzyme-immobilized platforms for precise diagnosis and treatment related applications in endocrine diseases. It is different from previous ones which mostly focused on nanomaterial-based biosensors, enzyme immobilization techniques, or each endocrine biomarker separately, and instead encompasses all of these aspects in a single review. It focuses on the importance of the nanostructure, surface chemistry, immobilization strategy, orientation of enzymes, the catalytic stability and the signal transduction as these parameters all play a role in the performance of biosensor and clinical relevance. Most importantly, this review gives a disease oriented and design oriented overview of enzyme immobilization on nanostructured material and its application to the detection of endocrine biomarkers, therapeutic monitoring and future precision medicine applications.

Diseases of Endocrine System

Hyperthyroidism

The incidence of hyperthyroidism in the elderly is estimated at 0.5–4%.¹² Although the disease is mostly etiologized by Graves, multinodular goiter and toxic nodular adenoma are becoming more common with age progression.^13,14 Most elderly patients present symptoms closely resembling those of the younger patients such as tremors, anxiety, palpitations, loss of weight, and heat intolerance, thus displaying sympathetic overactivity. However, a third of patients with older age have apathetic hyperthyroidism.¹⁵ The existing empirical evidence shows that adults of age ≥70 years often show less classical signs, and weight loss, apathy, and tachycardia become the most common manifestations (p < 0.001).^16,17 On a large cross-sectional cohort of 3,049 individuals, Boelaert et al reported increased incidence of weight loss and dyspnea in older adults and that they were more likely to report fewer symptoms compared to younger patients (p < 0.001).¹⁸ Overt hyperthyroidism and, to a smaller extent, subclinical hyperthyroidism have been found to cause cardiovascular morbidity, which includes widened pulse pressure, reduced exercise tolerance, atrial fibrillation, and cardiac mass enlargement.¹⁹ The problems of thyroid dysfunction are not only clinical detection of hyperthyroidism or hypothyroidism, but also the requirement of quickly, sensitively and miniaturising the detection of thyroid-related biomarkers in complex biological fluids. The nanostructured enzyme immobilised platforms can address this challenge by optimizing surface area, bioreceptor loading, electron transfer and optical signal amplification. The use of gold nanoparticles, graphene derivatives, metal oxides, MOF-based systems, and fluorescent nanoprobes can offer opportunities for the detection of thyroid hormone with greater sensitivity, as well as detection of multiple hormones simultaneously.

Overt hyperthyroidism and, to a smaller extent, subclinical hyperthyroidism have been found to cause cardiovascular morbidity, which includes widened pulse pressure, reduced exercise tolerance, atrial fibrillation, and cardiac mass enlargement.²⁰ Subclinical hyperthyroidism has also been linked with left ventricular hypertrophy. Moreover, hyperthyroidism is another risk factor that reduces bone mineral density and osteoporotic fracture, especially in postmenopausal women.²¹ Thyroid hormones stimulate bone turnover by their effect on osteoblasts and osteoclasts.²² Bauer et al reported an increased risk of hip and vertebral fractures among women >65 years with TSH ≤0.1 mIU/L.²³ In one study, more older men than women with subclinical hyperthyroidism had hip fractures (13.8% vs 12%). The initial evaluation of suspected hyperthyroidism should include serum TSH, but hospitalized elderly patients who are acutely ill may have a falsely depressed TSH. If there is no clinical evidence of Graves’ disease, radioactive iodine uptake testing is recommended and a radioactive iodine scan when nodules are present. Management begins with beta-adrenergic blockade to reduce heart rate and systolic blood pressure and improve tremors, irritability, and exercise intolerance. Anticoagulation may be needed in those with atrial fibrillation. Definitive treatments include radioactive iodine ablation, antithyroid drugs, or thyroidectomy. Radioactive iodine is often preferred in older adults due to its efficacy and safety, although hyperthyroidism resolves gradually and more than 80% of patients eventually become hypothyroid. Methimazole is the preferred antithyroid drug, but older adults may be at higher risk of recurrence and medication side effects.²⁴

Parathyroid Adenoma

The parathyroid glands are small, oval structures situated near the thyroid gland. Approximately 85% of individuals possess four glands, arranged as two superior and two inferior glands. The embryologically developed fourth branchial pouch is the superior parathyroid glands, which are usually situated on the posterolateral part of the middle to upper segment of the thyroid lobe. The location of the inferior glands, on the contrary, is much more mobile since they migrate with the thymus – both derivatives of the third branchial pouch. Consequently, the inferior glands could be found in any of the locations in this course of migration, which are most commonly located in the lower third of the thyroid gland. In normal conditions, a parathyroid gland is about the size and weight of an apple seed 0.5 g. The lesion that has a weight of less than 0.1 g is referred to as microadenoma and the one with a weight of more than 2 g is known as a giant adenoma. Given that parathyroid adenoma is a benign tumor, its mean weight is about 1 g.^25,26 In most patients, the etiology of parathyroid adenoma is idiopathic. Other affected genetic alterations, although exhibiting sporadic cases, constitute dysregulation of the cyclin D1/PRAD1 gene, which interferes with the normal functioning of parathyroid hormone (PTH). In sporadic adenomas, overexpression of the D1 of the cyclin has been detected in about 20 to 40%. Environmental factors have also been put forward, and past radiation exposure has been shown as a possible risk factor to further parathyroid pathology. Also, persistent calcium deficiency can lead to the development of the disease by working on the continuous stimulation of PTH secretion.²⁷ Nano-relevance is especially important for parathyroid disease during the operating room decision-making process. Although conventional testing is lab-based and time consuming, rapid PTH testing can help to determine the completeness of the parathyroidectomy. Near-patient or intraoperative readout for PTH could be achieved using an enzyme-assisted electrochemical or fluorescent nanosensor with antibody/enzyme recognition and nanomaterial-based signal amplification.

One parathyroid adenoma compound 80 to 85% of primary hyperparathyroidism, with 4 to 5% and 10 to 12% of the cases being attributed to fiddle hyperthyroid adenoma and parathyroid hyperplasia, respectively. Parathyroid carcinoma is very rare and is less than 1% of the cases. Adenomas are predominant in 50–70 years, but they can be at any age. The prevalence of the disease is approximately threefold higher in women compared to men.²⁸ The parathyroid hormone plays a critical role in controlling the calcium and phosphorus homeostasis by acting on the kidneys, the intestine, and skeletal system. In response to hypocalcemia, PTH enhances calcium reabsorption in the distal renal tubules and decreases phosphate reabsorption. It also increases circulating calcium levels indirectly by stimulating osteoclast-mediated bone resorption. Also, PTH stimulates intestinal calcium absorption by acting on vitamin D, and pathological elevation of PTH causes inappropriate hypercalcaemia and hypophosphatemia, which is the basis of clinical features of parathyroid disorders.²⁹ Historically, bilateral four-gland exploration was the standard surgical approach for primary hyperparathyroidism.

But, as only one adenoma is responsible for most cases, a minimally invasive parathyroidectomy is now the procedure of choice and is now considered the standard of care in treating parathyroid adenoma.^30–32 The short half-life of PTH is helpful in facilitating intraoperative monitoring. The Miami criteria use a reduction in PTH of 50% or more 10 minutes after removal of the adenoma as a good indicator of successful removal. If this threshold is not reached, more surgeries are required for further exploration. The ectopic or missing glands can be found in the retropharyngeal space, the retroesophageal space, the posterior mediastinum, the thymus, the mediastinum in general, inside the thyroid capsule or within the thyroid substance.³³

Diabetes Mellitus

Diabetes is a metabolic condition characterised by consistently raised blood glucose levels (BGLs) and a decreased ability to maintain BGLs. Type 1 diabetes is caused by autoimmunity directed against the pancreatic β-cells, causing an absolute decrease in insulin production.³⁴ Conversely, type 2 diabetes is characterized by insulin resistance whereby there is the insensitivity of peripheral tissues to insulin in the blood.³⁵ In both types of the condition, failure to maintain physiological regulatory processes results in dys-regulated glycaemic conditions, and this is expressed as chronic hyperglycaemia or hypoglycaemia. A prolonged period of hyperglycaemia is linked with a broad spectrum of negative events, such as cardiovascular and neurological issues,³⁶ but hypoglycaemia can trigger fatigue, unconsciousness, or even death.³⁷ Diabetes has now become a significant universal health issue with an estimated number of 25.8 million people in the United States and 382 million around the world currently, and it is projected to increase to 592 million by the year 2035.^37,38 In addition, it is projected to rank as the seventh major cause of death in the world by the year 2030.³⁶ The main therapeutic treatment of patients with type 1 diabetes and those with advanced type 2 diabetes usually includes several injections of subcutaneous insulin daily, as well as regular monitoring of the BGL with frequent capillary blood samples. These procedures are frequently painful, can lead to decreased patient compliance and can lead to insulin overdose. Also, intermittent measurements of glucose might not represent any notable glycaemic excursions that might occur between the time intervals of assessment. Therefore, technologies that improve the precision and sustainability of glucose monitoring, or technologies that combine glucose detection with insulin delivery to provide so-called closed-loop technology, are of significant clinical interest. Precise and regular glucose measurements are a major pillar of new diabetes management. It is however well known that the existing clinical glucose measurement systems presently demand a heavy burden on patients because they require constant and sharp needle pricks. Furthermore, the dominant paradigm of intermittent sampling is likely to overlook clinically relevant changes in the blood glucose concentration, which also introduces the risk to patient safety.³⁹ As a consequence, the creation of glucose sensing platforms that are able to provide accurate, minimally invasive, and high-frequency measurements, which, with time, would allow continuous glucose monitoring, has remained a pivotal issue in diabetes research. The best nanoengineering opportunity in diabetes is to link the diagnosis to the treatment. Glucose can be detected via electrochemical, colorimetric or fluorescent signal generation on GOx-immobilised carbon, gold, metal-oxide, hydrogel or MOF-based platform. These sensors can be used to build closed‑loop systems by combining them with glucose-responsive hydrogels, microneedles, vesicles or polymeric insulin depots, to enable the controlled release of insulin when glucose is detected. This gives a better mechanistic rationale for the emphasis on accurate diagnosis and treatment of endocrine disease.

In a closed-loop diabetes control system, glucose is sensed, the sensing signal is used to make a dosing decision, insulin is released, and glucose is sensed again. In this process, nanostructuring is important because quality control is a function of fast, low‑noise, drift-free transduction. On conductive nanocarbons or metal-nanoparticle/carbon interfaces, GOx is better wired and better loaded, for example by a printed CNT/AuNP/GOx electrode, which was made stable by a porous protein scaffold and reduced leakage of enzymes, and by graphene microneedle electrodes, which allow for compact interstitial sensing.⁴⁰ Ag@TiO₂@ZIF-67 added porous reaction spaces and mass/electron-transport pathways, resulting in a 5-second response, 0.99 μM LOD, and strong sensitivity, as ZIF pores served as diffusion/reaction vessels and Ag@TiO₂ amplified catalysis.⁴¹ Closed-loop stimulus-responsive platforms include glucose-responsive hydrogels, vesicles/micelles that release insulin on detecting glucose via GOx, and temperature/pH/redox-sensitive carriers that release insulin on detecting glucose-induced chemistry changes nearby. In hybrid systems, the electrochemical currents are digitized, then subjected to threshold- or PID/MPC-type logic, and converted to an electroosmotic pump or electrochemically actuated redox-responsive microneedles. The major challenges for translation include tissue-reaction driven drift, response latency as a result of stabilization of the sensor and actuation of the material, precise dose quantitation, long-term biocompatibility and demonstrating fail-safe control for regulatory purposes.⁴²

The last twenty years have seen the emergence of a wide range of modalities of glucose sensing. The two systems typically use Concanavalin A (Con A), phenylboronic acid (PBA) or, most frequently, glucose oxidase (GOx) as their molecular recognition constituent that identifies glucose in solution. Analytical interference still is a widespread problem because biological fluids are composed of a variety of electroactive or chemically similar species, such as uric and acetic acids, and structurally related carbohydrates, eg, fructose, lactose, and sucrose.^43,44 Specificity to glucose is hence often required by using glucose-selective enzymes or implementing chemical or physical exclusion in other detection architectures.⁴⁵ Measures of glucose are broadly divided into two major groups: electrical and optical. One common detection method is based on the use of hydrogen peroxide (H₂O₂) or a similar reduced species as an intermediate, which reduces another chemical species to produce a measurable signal. This signal can be in the form of enhancement or spectral shift in fluorescence 6, 8, 11 or instead a variation in current at the electrode surface of an amperometric sensor.^46–48

Hydrogen-peroxide-based methods of detection have a significant benefit, such as simplicity of operation and simple characterization by amperometric means. Nevertheless, these systems also have severe shortcomings, including the degradative nature of hydrogen peroxide on sensor constituents, the high potential needed to oxidize it electrochemically, or its vulnerability to interference by other electroactive species.⁴⁹ Since the wider scientific significance of hydrogen peroxide concentration in measurements is clear, several electrical H₂O₂ sensors have been designed in related areas and later applied to glucose. Therefore, hydrogen-peroxide-based platforms still comprise a large percentage of electrical and optical glucose sensing systems (47).

Nanotechnology and Enzyme Engineering

The immobilization of enzymes on solid supports is associated with a list of functional benefits that can greatly improve the catalytic efficiency, operational stability and the enzyme reuse possibilities. They are responsible for making immobilized enzymes particularly important in several industries, including in environmental remediation, biomedical engineering and industrial biocatalysis. Immobilization strategies can greatly enhance the performance of enzymes by controlling the local microenvironment of the enzyme, ie, by changing the physicochemical properties of the support surface, the level of structural rigidity and accessibility of the substrate to the enzyme, overcoming the performance limitations that are normally apparent with soluble forms of the enzymes.

The main benefit of enzyme immobilization, particularly in large-scale and industrial biocatalysis processes, is that the enzyme is reused better. In the traditional batch processes, the free enzymes are typically not reused, which makes the operation more expensive. On the other hand, the enzymes that have been immobilized can easily be separated out of the reaction medium and reused without much catalytic activity being lost. This is especially useful in continuous-flow bioreactors, biosensors, among others that require long-term enzymatic activity.⁵⁰

The kind of chemical bond formed between the enzyme and the support is not the only factor that determines enzyme immobilization on nanostructured platforms; surface chemistry, hydrophilicity, antifouling behaviour, pore architecture and nanoscale curvature are also important factors. These variables are especially relevant for endocrine disease applications where low concentration of biomarkers and enzyme instability in these complex biological fluids, along with protein fouling, can lead to unreliable assays.^51,52 Although physical adsorption is simple and does not affect enzyme activity under mild conditions, enzyme leaching and random orientation affect its long-term use in serum-based endocrine sensing. Covalent immobilization guarantees more stable attachment and greater operational stability particularly when introducing the functional groups –COOH, –NH₂, –SH, aldehyde, and epoxy groups onto gold, graphene, carbon nanotubes, metal oxides, or polymeric nanostructures. For instance, stable enzyme anchoring and reduced nonspecific protein adsorption of PEGylated gold nanostructures are attractive for electrochemical or plasmonic sensors of hormones and glucose in biological fluids. But too much cross linking may alter the enzyme shape and diminish its ability to catalyze a reaction.^53,54 When enzymes are entrapped in porous silica, polymeric matrices, MOFs, COFs and hydrogel networks, a different advantage is achieved by simply physically preventing enzyme denaturation with the ability to pass small endocrine biomarkers. Serum stability and reduced direct enzyme exposure to interfering proteins can be achieved with silica-based encapsulation with potential drawback of response kinetics slowed by mass-transfer resistance. In the same way, MOF- and COF-based systems are highly porous and provide tunable microenvironment for enzyme confinement but with some considerations regarding reproducibility, pore accessibility, and biocompatibility.^54–56

The ability to recover immobilized enzymes in an easy manner is strongly controlled by the immobilization method of choice as well as physicochemical characteristics of the support medium. An example is that enzymes immobilized on magnetic nanoparticles can be quickly recovered by passing an external magnetic field, but enzymes embedded in mesoporous networks or hydrogel networks can be recovered by traditional filtration or centrifugation. Successful recovery of immobilized enzymes can minimize the costs of operation besides improving the sustainability and the environmental suitability of biocatalytic industrial processes.^57,58 Enzyme immobilization has been known to produce significant changes in substrate specificity and catalytic capacity and is often associated with improved performance in particular reactions. The support material plays a central role in stabilizing the active site of the enzyme by adjusting the conformational state of the active site, frequently stabilizing a structural arrangement that increases the substrate-binding affinity. This conformational stabilization limits flexibility of the enzyme hence making it easier to interact with the catalyst in a more specific way. The silica nanoparticles in this case have been demonstrated to immobilize glucose oxidase, thus increasing its affinity to glucose, which can be explained by the fact that the active-site architecture of the enzyme is stabilized, and increase its catalytic activity in glucose-sensing applications.^59,60

Moreover, the steric constraint of immobilization in confined space, eg mesoporous supports, can be used to selectively control the accessibility of substrates according to their molecular size and geometry. These diffusion‑mediated effects can be exploited to increase the selectivity of biocatalysts, for example to accept only those substrates that can fit in the pore and to exclude large and/or structurally incompatible molecules. An example in point is the immobilization of lipase in mesoporous silica that selectively enhanced the hydrolysis of short-chain esters because of size-exclusion effects. These steric restrictions not only increase the specificity of catalysis but also reduce undesirable side reactions and ultimately improve the efficiency of the enzymatic reaction.⁶¹ The thermal and pH stability are significantly increased by enzyme immobilization, which is one of the main benefits. Free enzymes are naturally sensitive to temperature and pH changes, which can denature their structures and become catalytically inactive. Immobilization of enzymes on solid supports restricts the conformational change of the enzyme and thus its sensitivity to environmental factors like high temperature and extreme pH.⁶² The enzymes that are immobilized are normally more stable to heat than those that are not immobilized. Immobilization matrix provides the protective microenvironment that helps to preserve native conformation of enzymes under high temperatures. This increased stability is especially useful when biocatalysts are used in industry, eg biofuel production, food processing and pharmaceutical manufacturing, where biocatalysts are required to maintain activity under thermal stress.⁶³ Immobilized enzymes also have improved catalytic performance compared to free enzyme such as increased structural stability, better accessibility of the substrate, and the possibility of controlled and stable reaction conditions. Immobilization matrix is one of the most important factors in avoiding enzyme aggregation and maximizing the amount of catalytically active enzyme molecules. Furthermore, immobilization may affect the enzyme–substrate interaction, and enhance enzyme orientation on the support surface and reduce effects of diffusion limitation that often limit the reaction rate in free enzyme systems.^64,65

Strategies and Methods of Enzyme-Immobilization

Physical Adsorption

Physical adsorption is a simple and commonly used technique of immobilizing enzymes, which is based on the idea of noncovalent forces such as van der Waals forces, hydrogen bonding, and electrostatic interactions, that bind enzymes to the surface of a support matrix.⁶⁶ Since no chemical modification of either the support or the enzyme is required, the technique can be considered a simple, cheap and general immobilization platform. Its major advantages are simplicity of procedure and its versatility. It allows the immobilization of a variety of enzymes on a variety of support materials without the need for complicated chemical reactions. Also, by maintaining the natural structure of the enzyme, the risk of denaturation is reduced, thus preserving the enzyme’s catalytic activity as shown in Figure 1. The nanostructure reacts with the active site of the enzyme under mild conditions which ensures the stability of the enzyme. The weak nature of these interactions results in the possible desorption of the enzyme in the harsh conditions. This is usually done under mild conditions (in or near neutral pH and ambient or low temperatures), thereby also stabilizing the enzyme.^67,68

Figure 1.

A representation of how an enzyme can be adsorbed physically on a nanostructure. (A) The enzyme attaches to the nanostructure by a non-covalent interaction via van der Waals forces, hydrogen bonding, and electrostatic interaction. (B) Covalent attachment of an enzyme to a nanostructure. (C) Representation of site-specific immobilization and control of the orientation of an enzyme on a nanomaterial. The active site of the enzyme is held in an optimal orientation to be interacted with by regulating the attaching sites including amine, carboxyl and hydroxyl groups to the surface of the nanomaterial.

However, physical adsorption has some significant drawbacks. Weak interactions between the enzyme and the noncovalent supports are reversible, which can also help to desorb enzymes from the supports, particularly under harsh conditions such as high ionic strength, extreme pH or high temperature as shown in Figure 1A. Besides, the methodology does not allow a good control of the spatial orientation of the enzyme on the support surface, which could hinder access to the active site and therefore the catalytic efficiency. However, physical adsorption is still beneficial in cases where ease of preparation, low cost and moderate stability is adequate such as batch bioreactors, disposable biosensors and initial screening assays.^69–71

Covalent Bonding

Covalent interaction is the fixed chemical bond between an enzyme and its support matrix, which is facilitated by functional groups such as amino, carboxyl or epoxy functional groups, as shown in Figure 1B. This immobilization technique provides significantly greater stability than physical adsorption since covalent bonds are very stable and do not dissociate even under the most severe operating conditions. One of the main benefits of covalent immobilization is that it forms an active and strong enzyme–support interaction making it especially applicable to processes that require recurrent use or functions in harsh environmental conditions. The covalent attachment increases the rigidity and, in turn, the resistance of the enzyme to denaturation, thus, enabling its functional stability in an industrial application using higher temperatures and/or harsh solvents. Furthermore, immobilization can also be used to control the orientation of the enzymes on the surface and this can be used to maximize catalytic activity by favouring the conformations which are favourable to substrate binding.⁷²

Covalent bonding is done when the enzyme forms fixed chemical bonds with its support matrix, which in the majority of cases are catalyzed by such functional groups as amino, carboxyl or epoxy functional groups. This form of immobilization results in a very high level of stability compared to physical adsorption, as covalent bonds are very tough to dissociate even under the most extreme operating conditions. The key advantage of covalent immobilization is that it creates an effective, strong interaction between the enzyme and support, hence, being particularly useful in process that necessitates repeated utilization or is involved in an extreme environmental setting. Covalent bonding helps in rigidity, thereby enhancing the resistance of the enzyme to denaturation and therefore, helps to enable the enzyme to be stable in its functions by the use of a high temperature or other harsh solvents in industry. Moreover, the orientation of enzymes on the support surface can also be somewhat controlled by the same method, and this can also be exploited to optimize catalytic activity through favouring conception that favour substrate binding.^73,74

Cross-Linking

Cross-linking is a common modification method of enzyme immobilization wherein enzyme molecules are linked by covalent bonds together or between the enzyme and a support medium.⁷⁵ Bifunctional cross-linking reagents, like glutaraldehyde or carbodiimides, are normally used in this methodology to react with amino or carboxyl groups within the enzyme surface, creating insoluble aggregates of the enzyme or networks of the enzyme. One of the prominent results of this method is the production of cross-linked enzyme aggregates (CLEAs), which have also attracted a lot of attention owing to their stability in operation and the ability to be separated easily with respect to reaction media,⁷⁶ as illustrated in Figure 2. The cross-linking reaction makes enzyme molecules more rigid in structure and enables them to be more resistant to denaturation and overall more denaturement-resistant, as well as more tolerant to harsh physicochemical environments. This stabilization can be particularly useful in biocatalytic applications in industry, where it is important that the enzyme retains its activity under nonphysiological conditions of the reaction. In large-scale and continuous processing, the crosslinked enzyme systems are also re-usable and have a long operation life, in addition to good cost-effectiveness. The cross-linking can also be optimized to obtain values like enzyme loading capacity, mechanical vulnerability and catalytic efficiency.⁷⁷

Figure 2.

A schematic illustration of the cross-linking technique where bifunctional cross-linkers (eg, glutaraldehyde or carbodiimides) are applied to cross-link enzyme molecules or to cross-link them to the nanostructure.

Biomedical Applications of Enzyme-Nanostructured Platforms

Diagnostic Devices

Enzymes in immobilized form are a basic ingredient in the development of modern diagnostic technologies, especially those developed to detect disease related biomarkers. The immobilization which contributes to its increased stability, usability and long working life is particularly useful in point-of-care (POC) diagnostic systems, as well as portable analytical systems where reliability, speed of signal generation and resilience in operation are important factors. Nanoengineered support materials are also found to enhance the enzyme performance by enhancing structural stability, high enzyme loading, and acceleration of catalytic turnover, thus helping to produce the outcome of a diagnostic process faster and more sensitive.⁷⁸ Immobilized enzymes are widely used in POC diagnostic platforms to identify various pathological conditions like cancer, infection, metabolic diseases, etc, in a timely manner. These systems often are based on either enzyme-linked immunosorbent assays (ELISAs) or enzyme-based signal amplification systems to identify low-abundance biomarkers in biological fluids (blood, saliva, or urine).^79,80 The incorporation of nanomaterials, such as gold nanoparticles and silica nanoparticles, is very important as it allows the enzymes to be anchored in a larger surface area and to detect signals more effectively by detecting the transfer of electrons or energy. Such enhancements are needed to obtain high diagnostic sensitivity and specificity, especially in decentralized or resource constrained, or field-based clinical environments.⁸¹ Enzymes are still a valuable tool in traditional clinical diagnostics for the measurement of organ system function and disease progression. Biomarkers commonly measured in MI are lactate dehydrogenase (LDH), creatine kinase (CK) and alkaline phosphatase. When these enzymes are covalently immobilized on functionalized nanostructured surfaces such as magnetic nanoparticles, carbon‑based surfaces, and mesoporous silica surfaces, it is possible to develop highly sensitive miniaturized biosensing instruments. The systems allow real-time biochemical analysis, decreased reagent consumption, and effortless incorporation into microfluidic or lab-on-a-chip systems, thus, playing a significant role in the development of personalized diagnostics and accurate therapeutic monitoring.⁸²

Therapeutic Enzyme Delivery

Therapeutic enzyme delivery can be defined as the use of enzymes as bioactive molecules to replace the lack of enzymatic performance or to catalyze a particular biochemical change in a specific tissue. Immobilization of therapeutic enzymes on or in nanostructured carriers offers a number of benefits such as increased enzyme stability, targeted biodistribution, and release kinetics. They are especially helpful to enhance the effect of therapy and reduce the systemic toxicity and off-target effects.⁸³ Enzymes that have been immobilized have also found their way into the cancer treatment modality, with the most notable being enzyme-prodrug therapy. In this strategy, the inactive prodrug is transformed enzymatically into an active cytotoxic molecule by an enzyme, which is targeted to specifically localize in tumor tissue. As an illustration, a localized biotransformation of prodrugs in the tumor microenvironment is enabled by conjugating cytosine deaminase to biocompatible nanoparticles, which can enhance the specificity of the drug and reduce collateral damage to normal tissue.⁸⁴

The use of immobilized enzymes as controlled-release platforms further increases the possibilities of therapy. Enzymes that are trapped in hydrogels, liposomes or polymeric nanocarriers can be engineered to react to endogenous physiological signals like pH, temperature or redox gradients. These systems enable continuous and controlled release of enzymes over long durations which is especially beneficial in the management of chronic diseases or treatment of diseases that demand the localized and constant presence of enzymes. Using the example of pH-responsive hydrogel matrices, it is possible to program them to target therapeutic enzymes only in inflamed or diseased tissues, thus improving treatment specificity and patient compliance.^85–87

Enzyme-Immobilized Nanoplatforms in Diagnosis of Endocrine Diseases

Many clinically relevant hormones and regulatory biomarkers are found in the nano- to picomolar range and are subject to diurnal variation, stress, metabolic processes, drug effects and disease activity, making the diagnosis of endocrine diseases challenging. However, conventional laboratory assays like ELISA, chemiluminescence immunoassays and chromatography-based assays are typically bound by matrix interference, episodic sampling, dependence on centralized laboratory, and delayed turnaround time, as well as being limited for continuous or point of care monitoring. These restrictions are of great significance in cases of diabetes, thyroid disorders, adrenal diseases, pituitary disorders and parathyroid abnormalities, where the ability to detect biomarker changes in a timely manner can directly impact clinical decisions. The enzyme-immobilized nanoplatforms offer solutions to these endocrine specific diagnostic challenges. Nanostructured interfaces offer high surface to volume ratios, controllable surface chemistry, high enzyme loading, high catalytic accessibility and high electrochemical, optical or colorimetric signal generation. Immobilization of enzymes on conductive carbon materials, metallic nanoparticles, metal oxides, porous frameworks, polymers, or hybrid nanocomposites can enhance the stability and catalytic activity of the enzymes, and facilitate miniaturized and rapid detection of biomarkers. These platforms are thus not just alternative biosensors but also a design paradigm for transducing low‑level endocrine signals to measurable outputs for improved sensitivity, quicker response time, and increased potential for continuous, wearable, or point-of-care disease monitoring.^88,89

Nanoparticle-based enzyme-immobilized nanoplatforms have been actively developed in the testing of endocrine diseases, such as diabetes mellitus in which glucose assays continue to be a major clinical need. The general concept of working of enzyme immobilized nanoplatforms is illustrated in Figure 3. Nanostructure as an enzyme is immobilized and reacts with a biomarker, resulting in a catalytic reaction. The result of this reaction is the creation of a detection signal which can be monitored in real-time. In this regard, Kausaite-Minkstimiene et al developed an amperometric enzyme-nanozyme biosensor of immobilized glucose oxidase (GOx) with bimetallic platinum-cobalt (PtCo) nanoparticles to amplify the electrochemical glucose properties. GOx was immobilized on PtCo-modified graphite rod electrode in their platform, in which the nanoparticles acted as peroxidase-mimicking nanozymes, catalyzing reduction of the hydrogen peroxide formed during the enzymatic oxidation of glucose. This nanoplatform had a linear range of 0.04 to 2.18 mM, limit of detection of 0.021 mM, and sensitivity of 19.38 uA mM⁻¹ cm⁻² which was a good analogy of analytical performance capable of detecting glucose in a clinical setting. Stability tests showed that the biosensor did not lose 99% of its original response through 26 consecutive measurements, and 95% through 14 days of in vivo storage, which indicated it was stable in repeated diagnostic operations. Significantly, the biosensor has proven to be reliable in human serum samples with recovery values approaching 100% which favours its use in clinical glucose measurements and detection and management of diabetes mellitus.⁹⁰

Figure 3.

A visual representation of the overall concept of a biosensor based on nanostructure.

Lemmerman et al describe the evolution of nanoparticle-assisted imaging technologies to monitor pancreatic 8-cell mass and immune cell infiltration and provide tools that could aid in the more early detection of disease progression than traditional clinical measures. They also highlight how nanosensor systems like carbon nanotubes, quantum dots, nanogels, and conductive nanocomposites are being developed to be both more sensitive and continuous glucose sensors than traditional point-measurement systems. These nanosensors take advantage of the high surface area and electronic properties of nanomaterials for enhancing the analytical performance and can be incorporated into a wearable or implantable glucose monitoring system. In addition, efforts have been made with nanomaterials to use electrochemical platforms, with structures like multi-walled carbon nanotubes and graphene-polypyrrole composites, which could be used for direct insulin detection and thus a real-time assessment of insulin dynamics and β-cell function.⁹¹

In 2017, Choi et al fabricated a chemiluminescent biosensor that immobilized HRP on magnetic beads and a platinum nanoparticle to detect cancer markers (including thyroid cancer markers) in early stages with high sensitivity and low cost through a signal amplification generated from a reaction catalyzed by the platinum nanoparticles.⁹² In 2021, Fröhlich demonstrated nanoparticles with immobilized enzymes for use in biomarker detection (e.g, thyroglobulin, calcitonin) in thyroid cancer and how this could lead to better imaging and routine lab parameter analysis by releasing the enzymes, and thereby enhancing the imaging signal.⁹³ Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) enzymes have been aggregated together and immobilized onto carbon nanotubes (CNTs) to form a dual-enzyme nanoplatform that can be used for the detection of both biomarker and cytokines. The extremely high surface-to-volume ratio of CNTs allows the stable immobilization of enzymes to create a strong scaffold which increases the catalytic efficiency of the enzyme and can lead to very sensitive fluorescence or colorimetric readout and detection limits below 1 pg/mL. This enzyme-CNT platform (originally developed for multiplex cytokine detection) can be easily adapted for endocrine panels such as cortisol and adrenocorticotropic hormone (ACTH) to aid in differential diagnosis of adrenal axis dysfunction. The system has been verified using clinical plasma samples, and discriminating between biomarker profiles associated with diseases has been found reliable.⁹⁴

Case Studies: Nanostructured Enzyme Platforms for Major Endocrine Diseases

Nanostructured enzyme-based platforms have become potent diagnostic agents of endocrine disorders which are more sensitive, signal stable and the biomarker specific as opposed to traditional assays. In this section, representative case studies are discussed in which immobilization of enzymes that interface with nanomaterials has facilitated clinically significant detection of biomarkers of endocrine importance, some of the examples are also mentioned in Table 1.

Table 1.

Nanostructured Platforms with Immobilized Enzymes for Diagnosis and Treatment of Endocrine Diseases

Nanostructure	Immobilized Enzyme	Target Analyte/Disease	Application	Performance Metrics	Key Advantages	Reference
Au nanoparticles on ITO	Glucose oxidase (GOx)	Glucose/Diabetes	Diagnosis (biosensor)	Linear range 0.1–1.8 mmol/L; LOD 15 µM	Enhanced electron transfer, biocompatibility, reproducibility	[95]
Fe3O4@graphene nanocomposite	Laccase + anti-PDIA3 antibody	Free T3/Thyroid disorder	Diagnosis (biosensor)	Selective fT3 detection via redox catalysis	High selectivity, signal amplification	[96]
Acetalated-dextran nanoparticles	Glucose oxidase + catalase	Glucose/Diabetes	Treatment (insulin delivery)	Rapid/extended insulin release up to 16h glycemic control	Glucose-responsive kinetics in vivo	[97]
SnS2 nanosheet FET	Antibody (AbTSH) or DNA aptamer	TSH, T4/Thyroid disorder	Diagnosis (FET sensor)	Ultrasensitive in serum/PBS	Point-of-care potential, high selectivity	[98]
ZnO nanoparticles on GCE	Cortisol antibody (C-Mab)	Cortisol in saliva /Adrenal stress	Diagnosis (immunosensor)	Range 10–10−7 nM in saliva; high sensitivity	Cost-effective, stable quantification	[99]
GOx@Z n-HHTP	Glucose oxidase (GOx)	Glucose/Diabetes	Diagnosis (ECL sensor)	8.5x sensitivity increase; 25x lower LOD	Improved enzyme stability in sweat	[100]
Tb-MOF nanozyme	Hemin (peroxidase mimic)	17β-estradiol/Endocrine disruption	Diagnosis + degradation	LOD 50 pM; dual catalyst/sensor	Multifunctional, stable, low-cost	[101]
MoS2-graphene composite	Horseradish peroxidase (HRP)	Parathyroid hormone (PTH)	Diagnosis (biosensor)	Sensitive serum PTH detection	Biocompatibility, electron conductivity	[102]
Co-Fe-ZnO modified electrode	Glucose oxidase (GOx)	Glucose/Diabetes	Diagnosis (amperometric)	Sensitivity 32.2 μA mM−1 cm−2; LOD low	Selectivity vs interferents like uric acid	[103]
ZnO nanostructures	Anti-cortisol antibody	Cortisol/Adrenal disorder	Diagnosis (immunosensor)	Sensitivity 11.86 mA/M; LOD 1 pM	Electrostatic immobilization stability	[104]
Au nanoparticle-TiO2 nanotubes	Thiolated HRP	H2O2 (glucose proxy)/Diabetes	Diagnosis (biosensor)	Range 65–1600 μM; LOD 5 μM	Improved direct electron transfer	[105]
Cu@CDs nanozyme	Laccase mimic	Free T4/Thyroid disorder	Diagnosis (immunosensor)	Voltammetric blocking pathway	Bioinspired high-performance	[106]
ZnO nanostructures on electrode	N/A (enzyme-free but nano-enabled)	Cortisol/Stress monitoring	Diagnosis (electrochemical)	Athlete stress tracking	Facile, high-performance	[107]

One typical nanobiosensing example in the field of endocrinology is the detection of prolactin, which is a significant biomarker for the diagnosis of prolactinoma and other endocrine disorders of the pituitary. Clinically, it is important to measure prolactin accurately, as hyperprolactinaemia can occur due to pituitary adenoma, due to endocrine disturbance caused by drugs, in pregnancy, and due to macroprolactinaemia. The conventional immunoassays are useful; however, they can be susceptible to false positive interpretations in the complex serum samples and may require centralised laboratory processing. To meet this diagnostic demand, an enzyme-assisted AuNP-based electrochemical biosensor for quantitative detection of prolactin was constructed. Gold nanoparticles were chosen because they have a high surface area to facilitate loading of bioreceptors, good conductivity to promote increased signal generation, and they can be functionalized to facilitate loading of bioreceptors.¹⁰⁸ The catalytic enzyme label in this platform was horseradish peroxidase (HRP) and selective biomarker recognition was achieved with anti-prolactin antibodies. The immobilization chemistry was based on covalent conjugation via a carbodiimide reaction that allowed the stabilization of HRP and antibody moieties on the AuNP surface and ensured both reliable electrochemical signal transduction and reproducible chemistry. It demonstrated a low detection limit (0.5 ng/mL) for monitoring of prolactin. It also maintained 95% activity after 30 days and demonstrated 98% specificity towards related pituitary hormones FSH and LH. The biosensor was highly accurate in discriminating pathological values (>200 ng/mL) of prolactin in simulated serum samples from prolactinoma patients (n = 20) with 92% of discrimination and decreased false-positive results by 40% when compared with standard ELISA.¹⁰⁸ This is an example of the integration of enzyme catalysis, AuNP‑mediated signal amplification and covalent immobilization to enhance endocrine biomarker detection. However, more validation is needed with larger patient cohorts, real serum samples, long‑term storage conditions, and interference testing with heterophile antibodies, macroprolactin and nonspecific serum proteins before clinical translation.¹⁰⁹

A magnetic graphene oxide (MGO) nanoplatform immobilizing GOx and HRP via π-π stacking and coordination bonds for multiplexed endocrine profiling, extended to prolactinoma via prolactin-specific oxidase mimics. Prolactinoma case involved preoperative monitoring in a 35-year-old female with macroadenoma. The cascade system showed <70% initial activity retention, wide linear range (50–750 μM equivalent for metabolites), and reusability over 10 cycles. It detected prolactin surges post-dopamine agonist withdrawal with 15% higher sensitivity than immunoassays, aiding surgical timing. Postoperative monitoring confirmed 85% prolactin reduction, with the platform recycled magnetically for cost-effective follow-up.¹¹⁰ In a study on functional nanostructures for hormone analysis, researchers developed an immunosensor by covalently immobilizing HRP enzyme on silver nanoparticles (AgNPs) integrated into a chitosan-ionic liquid-graphene nanocomposite. The platform targeted thyroid-stimulating hormone (TSH), an important biomarker in Graves’ disease (hyperthyroidism). HRP has been used as a label for signal amplification for colorimetric and electrochemical detection in serum samples. It was determined to have a detection limit of 0.1 mIU/L and 95% recovery with high selectivity against other interferents such as IgG; it can be used for rapid, point-of-care screening of Graves’ patients with orbital involvement.¹¹¹

Dendritic gold nanostructures (DGNs) onto which glucose oxidase (GOx) was immobilized through covalent bonding electroporation on carbon cloth electrodes reduced the loss of this enzyme during continuous amperometric measurements. This biosensor was characterized by a high sensitivity and stability in stir solution and in the detection of glucose at concentrations of diabetic interest. Its results were characterized by reduced leaching and long-term functionality, and it was appropriate in the management of diabetes by implantable or wearable continuous glucose monitoring.¹¹² GOx was simply immobilized on carboxylic acid-functionalized carbon nanotubes (CNTs) via amide bridges utilizing EDC/NHS chemistry by taking advantage of CNTs high surface area to enhance reaction kinetics via hydrogen peroxide reduction. The nanostructured sensor was found to have a drastic result in detection limit, and sensor sensitivity of 2.8 times higher, and linear range of 3.0 μM-9.0 mM, and response time of less than 5 s than the flat electrodes. This channel solved the problem of enzyme denaturation in continuous in vivo measurements, and it could provide consistent performance to real-time blood glucose level monitoring in diabetic patients, and this system formed the basis of closed-loop insulin delivery systems.¹¹³

Challenges and Limitations

While the enzyme-immobilized and nanozyme-based endocrine sensors may be successful in analytical buffer, they may not be clinical ready. For instance, the glucose sensors that can be developed using the nanozyme PtCo may exhibit better chemical stability, simple storage conditions and less reliance on the delicate biological enzymes, and can thus be used in severe or long-term sensing applications. However, their clinical potential depends on the ability to retain selectivity in serum, where other oxidizable molecules, proteins and salts may interfere with the glucose detection. By contrast, enzyme-based sensors using Au nanoparticles may exhibit strong electrochemical signal amplification and high biomolecular specificity, but these may be compromised by enzyme denaturation, surface fouling, electrode fragility and signal drift upon extended exposure to physiological fluids if natural enzymes or antibodies are immobilized in an appropriate way.^114,115 Thus, no platform can be deemed to be superior for detection limit or linear range only. Clinical applicability depends on the performance in real serum or interstitial fluid, reproducible manufacture, calibration stability, antifouling properties and ability to compare results to reference clinical assays. Many of the published studies are still too limited as they have good performance in buffer or spiked samples but not enough validation work has been done in patient‑derived samples. Systems that failed or did not perform well are correlated with enzyme leaching, nonspecific adsorption of serum proteins, poor storage stability, batch to batch variation in the synthesis of nanomaterials and poor correlation with reference methods based on ELISA, chemiluminescence or chromatography.^116,117

The natural enzymes like glucose oxidase and horseradish peroxidase have high substrate specificity, catalytic efficiency, and are sensitive to pH, temperature, denaturation, and proteolytic degradation. Whereas the easier to integrate into nanostructured devices, having lower catalytic selectivity and lower activity in complex biological matrices, nanozymes are more stable and cheaper.^118,119 Immobilization strategy also has a strong influence on the aspect of translatability. Physical adsorption is simple and cheap, but due to weak enzyme–support interactions, it is leachable in serum, saliva, sweat or interstitial fluid, and thus poor for long‑term wearable or implantable endocrine monitors. The covalent immobilization, cross-linking, encapsulation and anti fouling surface engineering methods are more promising for clinical translation but they need to be optimized to prevent enzyme inactivation and loss of catalytic accessibility.^120,121 Other major challenges include evaluation procedures and accuracy of clinical and laboratory data, as discussed below.

Evaluation Procedures

Quantification of hormone is done using a variety of analytical methods such as bioassays, receptor-based assays and immunoassays. In the 1950s, the major techniques to be used in measuring biological activity were bioassays and radioreceptor assays. Since the 1960s, radioimmunoassays but subsequently, in the 1980s, immunoradiometric assays with the introduction of monoclonal antibodies have been the standard methods of quantification of immunoreactive materials.¹²²

Both the isotopic and the non-isotopic types of immunometric assays have become increasingly considered as methods of choice of most of the hormone measurements. Even though such assays can be performed relatively easily, their design can pose interpretation problems to researchers due to their inherent design limitations.¹²³ Besides that, applications of recombinant DNA-based methods, such as Southern blotting, oligonucleotide-specific hybridization, polymerase chain reaction, and restriction fragment length polymorphism methods, are widely used in the study of endocrine disorders.

Accuracy of Clinical and Laboratory Data

In Nigeria, Anumah reported that less than 60 trained endocrinologists are available to a population of more than 140 million people with most of them being concentrated in tertiary health-care units in major urban centres. As a result, the majority of patients will first visit non-specialists who, despite significant limitations, still make a significant contribution to treatment. The lack of sufficient training of staff to perform accurate clinical diagnosis, the lack of relevant investigative facilities, and the high price of treatment also presents a strong challenge in most centres where endocrinology is practised in Nigeria. The cost of treatment is also prohibitive, and many patients are left with no viable alternatives. Consequently, other people resort to the services of unqualified healthcare facilities or drug dealers, where hormonal preparations can be used improperly, and later on, they find themselves back in hospitals with complications, stop follow-up or in most instances, die.¹²⁴

Adequate endocrine assessment is greatly dependent on staffed and properly functioning endocrinology labs that guarantee not only timely handling, but also precise results of hormone assay. Diagnosis of endocrine disorders cannot be done without a strong concept of basic science and endocrine principles. Sadly, the existing laboratory training programmes do not offer much exposure to the theoretical and practical aspects of laboratory techniques of endocrinology. It is a negative impact on the quality of laboratory data and clinical interpretation, which means that there is a need to incorporate endocrinology-specific laboratory training into current curricula.¹²³ In 1997 Okosieme reported that in most developing countries it was not routine to test thyroid antibody and that little research had been done to test thyroid antibodies in African populations. This was the case mostly up to 2009. Such tests are available only in a few commercial laboratories, usually in prohibitively expensive form. Regular endocrine examinations are usually limited to thyroid screening: total T3, total T4, TSH and reproductive hormones, including FSH, LH, prolactin, testosterone, estradiol and progesterone and cost between $12 and $48.¹²⁵

Future Perspectives

The concept of nanostructured enzyme-immobilized platforms is a pioneering strategy in making an accurate diagnosis and treatment of endocrine diseases and has considerable potential in the future. These systems make use of nanotechnology to entrap enzymes on nanomaterials including silica, gold nanoparticles, magnetic nanoparticles, carbon-based structures and hydrogels, which significantly increase enzyme stability, catalytic activity and reusability. These nanomaterials have high surface area, tunable surface chemistry and biocompatibility, which allow enhanced enzyme substrate interaction, and create a high sensitivity and specificity in biosensing and diagnostic applications, which are essential in detection of endocrine disorders.

Future research efforts should move beyond the conceptual general statement to more specific technology platforms with enzymes. One potential approach is a multi-enzyme cascade reaction on a single nanoparticle, co-immobilization of two or more enzymes within a controlled nanoscale system. These systems could help enhance catalytic efficiency, minimize intermediate diffusion loss and allow more information to be captured about complex endocrine metabolic pathways, especially in diabetes and lipid-related disorders.¹²⁶ Additionally, the development of imaging probes that are activated by enzymes is another paradigm. These systems are designed so that enzymatic activity or biomarker-triggered reactions within the system could activate fluorescent, photoacoustic, magnetic resonance, or electrochemical signals, depending on the type of system used, and linked to an endocrine disease. This would enable the detection of endocrine dysfunction on a spatially resolved basis, and not only by bulk measurement of biomarkers.^127,128 Furthermore, a potential approach is the integration of enzyme-immobilised nanoplatforms into development of long-term monitoring wearable or implantable devices. Stable nanostructured interfaces could be paired with the antifouling materials, flexible electronics, microneedles or implantable sensors, to continuously measure glucose, cortisol, lactate, thyroid related biomarkers and metabolic indicators. Future systems need to be clinically translatable and therefore have long-term stability for the enzyme, reproducible fabrication, reduced biofouling, reliable calibration, biocompatible, and have demonstrated performance in real biological fluids.

Enzyme immobilized nanoplatforms have broad biomedical uses beyond diagnostics, which include targeted delivery of enzymes and therapeutics, eg, enzyme-functionalized scaffolds used in tissue repair and regeneration of endocrine-related organ dysfunctions. Such versatile nanobiocatalysts could minimize side effects and increase the effectiveness of treatment in such diseases as diabetes, thyroid diseases, and adrenal glands dysfunctions. Issues like enzyme leaching, cost, scalability, and biocompatibility are currently being solved to enable clinical translation and sustainable development of healthcare.

Conclusion

The use of nanostructured enzyme-immobilized platforms has progressed beyond a generic biosensing idea to become a viable design approach for enhancing endocrine disease diagnosis and treatment monitoring. The studies surveyed show that greatest advances have been made in the field of diabetes-related sensing, mainly in the context of the immobilization of glucose oxidase on carbon-based materials, metallic nanoparticles, metal oxides, and hybrid nanocomposites; this results in better enzyme loading, electron transfer, catalytic stability, and response speed. Of these, the graphene/CNT-metal nanoparticle hybrids and metal-oxide based interfaces seem to be the most promising, as they possess a combination of high conductivity, large surface area and strong interaction between the nanoparticles and enzymes. The most promising platforms for thyroid related applications are those nanostructures that could be surface functionalized with high affinity and amplified signal, such as gold based, carbon based, MOF/COF and hybrid systems. These platforms can be applied to thyroid biomarker detection in low abundance, where traditional assays are limited in sensitivity and miniaturization, and in real-time monitoring. In the case of adrenal disorders, detection advantages primarily focus on nanoplatforms that are capable of stable enzyme/bioreceptor immobilization, antifouling surfaces, and serum/saliva/wearable platforms. While diabetes sensing is more advanced, thyroid and adrenal applications are less advanced, and need to be better validated in real biological matrices.

The covalent immobilization and hybrid immobilization are the most successful methods for achieving enzyme activity and operational stability; however, physical adsorption can be useful for simple and inexpensive prototypes, but has poorer operational stability. The detected ranges of biomarker levels vary according to the biomarker type, sample type and transduction method, but in general, nanostructured platforms can detect levels of metabolic biomarkers in the micromolar-nanomolar range and levels of several hormone-related targets in the nanomolar-picomolar range. Additionally, carbon/metal hybrids are most developed for diabetes, plasmonic and porous nanostructures are more suitable for thyroid biomarkers, and antifouling wearable-compatible nanostructures are more appropriate for adrenal hormone monitoring. Also, immobilization chemistry is as crucial as nanomaterial choice, as the orientation, binding strength, and microenvironment stability of enzymes can significantly affect the sensitivity and lifespan of the sensor. However, there is a need to advance the future work of sensing from proof of concept to serum validation, repeatable fabrication, long-term stability, multiplexed hormone sensing and clinically relevant wearable or point-of-care formats. Thus, future efforts in this field should aim to establish suitable limits of detection in biological samples, scalable fabrication methods, enhanced biocompatibilities, and incorporation of AI-assisted signal analysis of disease-specific enzyme-immobilized nanoplatforms. These developments will play a key role in bringing nanostructured enzyme-based systems from the laboratory to trustworthy tools for personalized diagnostics, monitoring and therapeutics of endocrine diseases.

Funding Statement

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Data Sharing Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

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Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests financial or non-financial or any other interests that might be perceived to influence the results and/or discussion reported in this paper.