Advances in Enzyme-Based Biosensors: Emerging Trends and Applications ^†

Abstract

Enzyme-based biosensors have emerged as a transformative technology, leveraging the specificity and catalytic efficiency of enzymes across various domains, including medical diagnostics, environmental monitoring, food safety, and industrial processes. These biosensors integrate biological recognition elements with advanced transduction mechanisms to provide highly sensitive, selective, and portable solutions for real-time analysis. This review explores the key components, detection mechanisms, applications, and future trends in enzyme-based biosensors. Artificial enzymes, such as nanozymes, play a crucial role in enhancing enzyme-based biosensors by mimicking natural enzyme activity while offering improved stability, cost-effectiveness, and scalability. Their integration can significantly boost sensor performance by increasing the catalytic efficiency and durability. Additionally, lab-on-a-chip and microfluidic devices enable the miniaturization of biosensors, allowing for the development of compact, portable devices that require minimal sample volumes for complex diagnostic tests. The functionality of enzyme-based biosensors is built on three essential components: enzymes as biocatalysts, transducers, and immobilization techniques. Enzymes serve as the biological recognition elements, catalyzing specific reactions with target molecules to produce detectable signals. Transducers, including electrochemical, optical, thermal, and mass-sensitive types, convert these biochemical reactions into measurable outputs. Effective immobilization strategies, such as physical adsorption, covalent bonding, and entrapment, enhance the enzyme stability and reusability, enabling consistent performance. In medical diagnostics, they are widely used for glucose monitoring, cholesterol detection, and biomarker identification. Environmental monitoring benefits from these biosensors by detecting pollutants like pesticides, heavy metals, and nerve agents. The food industry employs them for quality control and contamination monitoring. Their advantages include high sensitivity, rapid response times, cost-effectiveness, and adaptability to field applications. Enzyme-based biosensors face challenges such as enzyme instability, interference from biological matrices, and limited operational lifespans. Addressing these issues involves innovations like the use of synthetic enzymes, advanced immobilization techniques, and the integration of nanomaterials, such as graphene and carbon nanotubes. These advancements enhance the enzyme stability, improve sensitivity, and reduce detection limits, making the technology more robust and scalable.

1. Introduction

Enzyme-based biosensors represent a widely used class of analytical devices that rely on the specificity and catalytic efficiency of enzymes to detect target analytes with high accuracy. These biosensors combine a biological component—typically an enzyme—with a physicochemical transducer to convert biochemical reactions into measurable signals. Their unique ability to offer rapid, sensitive, and selective responses makes them indispensable in diverse sectors such as food quality control, medical diagnostics, industrial processing, and environmental monitoring [1]. A major advantage of enzyme-based biosensors lies in their high specificity and sensitivity, which are primarily dictated by the enzyme–substrate interaction. This ensures that even trace amounts of target compounds, such as glucose, cholesterol, urea, or pesticides, can be accurately identified [2,3]. Additionally, these biosensors deliver fast response times, making them ideal for applications requiring real-time monitoring or point-of-care testing [4]. They are also cost-effective and can be easily miniaturized, enabling the development of portable biosensing platforms suitable for field and personal use [2,4]. In the food industry, enzyme-based biosensors are utilized for ensuring food safety by detecting contaminants such as heavy metals, pesticides, and microbial metabolites [5,6]. In environmental applications, they help in the assessment of water and soil quality by detecting toxic compounds, making them essential tools in pollution monitoring [7]. Perhaps their most widespread use is in medical diagnostics, particularly in the monitoring of blood glucose levels for diabetes management and the detection of biomarkers for various diseases [8,9]. Enzyme-based glucose sensors, often employing glucose oxidase, remain the gold standard in blood sugar monitoring [10]. There are several types of enzyme-based biosensors, each classified by the transduction method used. Amperometric and potentiometric biosensors detect changes in electrical signals resulting from enzyme-mediated reactions. These are commonly applied for metabolites like glucose and cholesterol [6,11]. Optical biosensors, on the other hand, measure changes in absorbance, fluorescence, or luminescence. Surface plasmon resonance and chemiluminescence are also integrated into optical platforms for enhanced sensitivity [12,13]. Additionally, thermistor and piezoelectric biosensors measure changes in temperature or mass during enzymatic reactions, providing alternative non-electrical detection methods [2]. Despite their strengths, enzyme-based biosensors do face certain challenges. Their performance can be compromised in complex sample matrices due to interference from non-target substances and enzyme instability under varying environmental conditions. Techniques such as enzyme immobilization on nanostructured materials, incorporation of stabilizing agents, and surface functionalization have been developed to improve both the operational stability and selectivity [2,14]. A growing trend involves integrating enzyme biosensors into wearable devices, which allows for continuous, non-invasive health monitoring through bodily fluids like sweat or interstitial fluid [9]. A particularly promising innovation is the use of nanozymes—engineered nanomaterials with enzyme-like catalytic activity. Nanozyme-based biosensors offer advantages such as greater stability, tunable properties, and resistance to denaturation, making them suitable for harsh conditions or long-term use [13]. These developments, alongside advancements in sensor miniaturization, wireless connectivity, and artificial intelligence-driven analysis, are driving the future of enzyme-based biosensing toward more robust, multifunctional, and real-time analytical tools.

2. Key Components and Working Principles of Enzyme-Based Biosensors

Enzyme-based biosensors are powerful analytical devices designed to detect specific chemical substances by leveraging the catalytic activity of enzymes. These biosensors are composed of three primary components—biological recognition elements (enzymes), transducers, and immobilization matrices—that work synergistically to convert biochemical reactions into measurable signals.

Figure 1 illustrates the working principle of enzymatic biosensors for both substrate and inhibitor measurements. In substrate-based biosensors, such as glucose detection using glucose oxidase, the interaction between the substrate and enzyme generates a measurable product signal at the transducer. In contrast, inhibitor-based biosensors, such as acetylcholinesterase systems for pesticide detection, rely on the suppression of enzymatic activity by inhibitors, resulting in reduced or blocked signal generation.

2.1. Biological Recognition Element

The core of an enzyme-based biosensor is the enzyme itself, which specifically interacts with the target analyte (substrate). Enzymes serve as biocatalysts, initiating a reaction with their substrate to produce a detectable byproduct. Commonly used enzymes include glucose oxidase for glucose monitoring, cholesterol oxidase for cholesterol detection, diamine oxidase for biogenic amines, and cholinesterase for pesticide detection [1,5,6,16].

2.2. Transducer

The transducer converts the biochemical signal produced by the enzyme–substrate reaction into a quantifiable electrical or optical signal. Several types of transducers are used, including electrochemical (amperometric and potentiometric), optical (fluorescence, absorbance, bioluminescence), thermistor (detecting temperature change), and piezoelectric (detecting mass or mechanical changes) [16,17]. The choice of transducer often depends on the intended application and required sensitivity.

2.3. Immobilization Matrix

To ensure the enzyme remains in proximity to the transducer and retains its activity over time, it is immobilized using various techniques such as physical adsorption, covalent bonding, entrapment in gels or polymers, or incorporation into nanoparticles. The immobilization method significantly affects the sensor’s stability, reusability, and response time [2,5,12].

Working principles: The functional mechanism of enzyme-based biosensors relies on the specific enzyme–substrate interaction: when the target analyte comes into contact with the enzyme, a catalytic reaction occurs, often resulting in the production or consumption of specific molecules (e.g., hydrogen peroxide, oxygen, protons). This biochemical transformation is the first step in signal generation [6,16].

Signal transduction: The enzymatic reaction causes a change in a physicochemical parameter—such as pH, redox potential, heat, mass, or light emission—which is detected by the transducer. This change is then converted into an electrical or optical signal that corresponds to the analyte concentration [1,16].

Measurement techniques: Electrochemical techniques involve detecting the voltage (potentiometric) or current (amperometric) generated by redox reactions [6]. Optical methods measure changes in light properties caused by the enzymatic activity (e.g., absorbance in UV–visible spectra or fluorescence) [12]. Thermistor sensors register the heat released or absorbed during the reaction. Piezoelectric biosensors detect changes in mass on the sensor surface as a result of the enzymatic binding or conversion process [16].

3. Enzyme Types Used in Enzyme-Based Biosensors

Enzyme-based biosensors employ specific enzymes as biorecognition elements due to their high catalytic efficiency, substrate specificity, and compatibility with various transduction methods. These biosensors have been widely adopted in medical diagnostics, environmental monitoring, and food quality control. Below is an expanded overview of six major enzyme types used in such biosensors.

Glucose oxidase (GOx): Glucose oxidase is one of the most extensively used enzymes in biosensor development. It catalyzes the oxidation of β-D-glucose into gluconic acid and hydrogen peroxide, the latter of which can be detected electrochemically. GOx-based biosensors are central to glucose monitoring in diabetes care, with applications also extending to the food industry for sugar content analysis. Common detection methods include amperometric and potentiometric systems, often enhanced by nanomaterial-based electrode modifications for improved sensitivity and stability [18].

Urease: Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide, causing measurable pH changes. It is extensively used in biosensors for kidney function diagnostics, environmental analysis of urea-based fertilizers, and quality control in the dairy industry. Urease-based biosensors typically employ optical transduction techniques, such as chemiluminescence and surface plasmon resonance [19].

Lactate oxidase (LOx): Lactate oxidase converts L-lactate into pyruvate and hydrogen peroxide. Its primary use is in sports medicine and critical care, where lactate monitoring is crucial for assessing metabolic stress and sepsis risk. LOx-based biosensors have been integrated into wearable devices and “smart tattoos” for real-time, non-invasive monitoring [20].

Cholesterol oxidase (ChOx): Cholesterol oxidase catalyzes the oxidation of cholesterol to cholest-4-en-3-one and hydrogen peroxide. It is applied in biosensors for cardiovascular health monitoring, as well as in food science to measure cholesterol in animal-based products. Detection methods often combine electrochemical and optical transduction for increased accuracy [5].

Acetylcholinesterase (AChE): Acetylcholinesterase hydrolyzes acetylcholine into choline and acetate. Its inhibition by organophosphate and carbamate compounds forms the basis for pesticide detection and neurotoxin monitoring. AChE-based biosensors commonly use electrochemical transducers and measure changes in enzyme activity as an indicator of toxin presence [20].

Tyrosinase: Tyrosinase catalyzes the oxidation of phenolic compounds to quinones. It is widely used in environmental monitoring for phenolic pollutants, in the food industry for antioxidant content determination, and in clinical assays for catecholamines. Tyrosinase-based biosensors use amperometric or optical detection methods, with nanomaterial-modified electrodes improving the performance [6].

4. Transduction Methods in Enzyme-Based Biosensors

Enzyme-based biosensors utilize enzymes as biorecognition elements to selectively detect specific analytes. The fundamental role of the transduction method is to convert biochemical signals generated by enzymatic reactions into measurable physical outputs. The choice of transduction strategy directly impacts the sensitivity, selectivity, response time, and potential applications of the biosensor.

Optical transduction: Optical biosensors detect changes in light properties—such as absorbance, fluorescence, or refractive index—triggered by enzymatic activity. Absorptiometry detects changes in light absorption due to the formation of colored products or consumption of substrates. Fluorescence-based detection employs fluorogenic substrates or cofactors (e.g., flavins, heme groups) to produce measurable emission, which is often enhanced through techniques like Förster resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), and fluorescence correlation spectroscopy (FCS) [16]. Chemiluminescence and bioluminescence rely on light emission from enzyme-catalyzed chemical reactions. Surface plasmon resonance (SPR) measures refractive index changes at a sensor’s surface, enabling label-free and real-time detection.

Electrochemical transduction: Electrochemical biosensors remain the most widely used due to their high sensitivity, cost-effectiveness, and ease of integration into portable devices. Amperometry measures the current produced by redox reactions during enzymatic catalysis [6]. Potentiometry monitors changes in electrode potential caused by ionic species generated or consumed during the enzymatic process [6]. Voltammetry records current changes as a function of the applied potential, providing detailed redox profiles [21]. Electrochemical impedance spectroscopy (EIS) detects variations in the charge transfer resistance or capacitance at the electrode–solution interface [21]. Electrochemiluminescence (ECL) merges electrochemical excitation with luminescent detection, offering high sensitivity and low background interference [21].

Thermal transduction: Thermal or calorimetric biosensors measure heat changes resulting from the exothermic or endothermic nature of enzymatic reactions. Temperature variations are detected using thermistors or other thermal sensors [16]. These devices are universal for enzymes generating measurable enthalpy changes.

Gravimetric transduction: Gravimetric biosensors measure mass changes on a sensing surface due to analyte binding or enzymatic product formation. Devices such as quartz crystal microbalance (QCM) and surface acoustic wave (SAW) sensors detect oscillation frequency shifts proportional to the mass variation [16].

Magnetic and micromechanical transduction: Magnetic biosensors detect changes in magnetic properties due to enzymatic binding or catalysis [6]. Micromechanical biosensors sense nanometer-scale mechanical deflections or stress changes on cantilevers coated with enzymes [6].

Table 1. Comparison of transduction methods in enzyme-based biosensors: principles, advantages, and applications.

Transduction Method	Principle	Advantages	Applications
Optical	Light absorption, fluorescence, and SPR	High sensitivity, real-time, and label-free detection	Environmental monitoring and medical diagnostics [22]
Electrochemical	Current, voltage, or impedance change	Low cost, portable, and miniaturizable	Point-of-care testing and food quality control [6,21,23]
Thermal	Heat change from enzymatic reactions	Universal applicability	Clinical and biochemical assays [16]
Gravimetric	Mass variation on sensor surface	High precision and label-free	Toxin and biomolecule detection [16]
Magnetic/Micromechanical	Magnetic property change or mechanical deflection	High specificity for specialized uses	Advanced biosensing platforms [6]

provides a comparison of transduction methods in enzyme-based biosensors, highlighting their underlying principles, advantages, and applications.

5. Types of Enzyme-Based Biosensors

Enzyme-based biosensors are classified into various types depending on their functional mechanisms, detection technologies, and fields of application. These distinctions allow biosensors to be tailored for specific analytical tasks, including biomedical diagnostics, environmental assessments, and food quality monitoring. By utilizing enzymes as highly selective biological recognition elements, these biosensors convert biochemical reactions into measurable signals, offering rapid, accurate, and versatile detection platforms.

5.1. Classification by Electron Transfer Mechanism

Direct electron transfer (DET) enzymes: DET-type biosensors utilize enzymes that can directly transfer electrons to the electrode without requiring a mediator. This feature results in a simpler sensor design and faster signal response. Such enzymes, including monomeric and oligomeric oxidoreductases, are often used in advanced biosensor systems for their high electron transfer efficiency [24].

Mediator-modified enzyme systems: In contrast, many biosensors employ enzymes that require redox mediators to shuttle electrons between the enzyme’s active site and the electrode. A notable example is glucose dehydrogenase modified with phenazine ethosulfate (PES), which facilitates a quasi-DET pathway. These mediator-assisted systems expand the usability of a broader range of enzymes in biosensing applications [25].

5.2. Classification by Detection Technique

Electrochemical biosensors: Electrochemical enzyme biosensors are among the most commonly used types due to their high sensitivity, fast response times, and adaptability. These devices measure changes in current (amperometric), potential (potentiometric), or impedance (conductometric) that result from enzymatic reactions. Electrochemical biosensors have evolved through several generations, with the third generation emphasizing DET-type systems for improved efficiency and miniaturization [26,27].

Optical biosensors: Optical enzyme biosensors detect analytes through changes in light absorption, emission, or scattering caused by enzyme–substrate interactions. These include techniques such as absorbance spectrophotometry, chemiluminescence, and surface plasmon resonance (SPR), which are especially useful in multiplexed detection formats and non-invasive diagnostics [12,28].

5.3. Classification by Application Area

Biomedical applications: Enzyme-based biosensors play a critical role in medical diagnostics. They are widely used for monitoring glucose levels in diabetes, cholesterol in cardiovascular diseases, and biomarkers of oxidative stress and cancer. Their precision and adaptability make them suitable for point-of-care and wearable healthcare devices [29].

Environmental monitoring: These biosensors are used to detect toxic environmental contaminants, such as pesticides, heavy metals, and endocrine-disrupting chemicals. They offer field-deployable, low-cost solutions for environmental analysis that are often integrated into portable sensing platforms [15].

Food industry: In food technology, enzyme biosensors ensure safety and quality control by detecting components like glucose, lactose, and ethanol, as well as contaminants like organophosphates. They support real-time monitoring in processing lines and post-packaging inspection [5,6].

6. Emerging Enzyme-Based Biosensors: Aggregation-Induced Emission, Core/Shell Nanoparticles, and Inkjet-Printed Biosensors

Core/shell nanoparticles (CSNs): Core/shell nanoparticles represent a class of nanostructures composed of a central “core” material surrounded by a protective or functional “shell” layer. This architecture enables the combination of distinct physical and chemical properties from both components, resulting in improved performance for biosensing applications. In enzyme-based biosensors, CSNs offer improved thermal and chemical stability of immobilized enzymes, reduced cytotoxicity compared with unmodified nanoparticles, enhanced solubility in biological media, and better cell permeability and biocompatibility. Applications of CSNs span from bioimaging and targeted drug delivery to highly selective biosensing platforms [30,31,32]. In diagnostics, enzyme-responsive CSNs have been designed to detect enzymes such as alpha amylase for forensic and medical point-of-care testing. These systems protect the active sensing components from environmental degradation, enabling a longer shelf-life and enhanced sensitivity [33].

Aggregation-induced emission (AIE): AIE is a luminescent phenomenon in which certain fluorophores become more emissive when aggregated. Advantages of AIE-based biosensors include high photostability for prolonged imaging, large Stokes shifts for improved signal clarity, strong biocompatibility for in vivo applications, and high luminescence efficiency in the aggregated state. AIE luminogens (AIEgens) have been used for detecting proteases, phosphatases, glycosidases, cholinesterases, and telomerase using mechanisms such as enzyme-catalyzed hydrolysis, electrostatic adsorption, and biological redox reactions [34,35,36].

Inkjet-printed biosensors: Inkjet printing technology allows the direct, precise deposition of enzymes and other biomolecules onto diverse substrates without compromising activity. Key benefits include low-cost, scalable production of biosensor arrays; high-resolution patterning for micro-scale sensor designs; and multiplexing capabilities to detect multiple analytes on a single device. Applications include reagentless electrochemical biosensors for detecting phosphate [13], glucose and lactate [4], multiplexed analytes [37], and specific proteins like lysozyme [38].

Table 2. Summary of emerging enzyme-based biosensors: advantages, applications, and challenges.

Category	Description	Advantages	Applications	Challenges
Aggregation-Induced Emission (AIE)	AIE-based biosensors utilize molecular probes that emit fluorescence upon aggregation. Examples include tetraphenylethene-substituted pyridinium salt (TPE-Py) and tetra-anionic sulphonyl derivative of tetraphenylethylene (Su-TPE) [39,40,41].	- High sensitivity and selectivity; - Simple and rapid detection; - Excellent photostability and luminescence efficiency [25,39,40].	- Detection of alkaline phosphatase (ALP) activity; - Trypsin and organophosphorus pesticides detection; - Glucose detection [18,39,40].	- Instability and biotoxicity; - Lack of modifiable functional groups [42].
Core/Shell Nanoparticles	Core/shell nanoparticles are used to enhance enzyme-based electrochemical biosensors due to their unique physicochemical properties [30].	- High selectivity and sensitivity; - Tunable surface characteristics; - Improved stability and biocompatibility [30].	- Quantification of environmental pollutants; - Food contaminants; - Clinical biomarkers [30].	- Integration with specific functions; - Development of hybrid nanostructures [30].
Inkjet-Printed Biosensors	The inkjet printing technique is used to functionalize electrodes for enzyme-based biosensors, enabling point-of-care detection [4].	- Low cost; - Rapid response time; - High selectivity and stability [4].	- Detection of phosphate in saliva; - Versatile for various analytes [4].	- Ensuring reproducibility; - User-friendly operation [4].
Self-Assembling AIE Nanoparticles	Self-assembling AIE nanoparticles are fabricated through dispersion of amphiphilic polymers in phosphate-buffered saline [42].	- Fine-tuned particle size and morphology; - Superior sensing performance; - Enhanced signal amplification [42].	- Detection of organophosphorus pesticides; - Cell imaging [42].	- Stability and biosafety; - Functional group modification [42].
Single-Molecule Enzyme Nanocapsules (SMENs)	SMENs provide enhanced stability for enzyme-based biosensors, addressing issues like thermal stability and organic solvent tolerance [43].	- Improved thermal stability; - Long-term operational stability; - Rapid substrate transportation [43].	- Point-of-care diagnostics; - Biomedical detection; - Wearable devices [43].	- Ensuring biocatalytic activity; - Structural dissociation prevention [43].

presents an overview of emerging enzyme-based biosensors, detailing their classification, functional descriptions, benefits, practical applications, and limitations.

7. Applications of Enzyme-Based Biosensors

Enzyme-based biosensors have widespread applications across industries, as shown in Figure 2. They are used for real-time monitoring of food contaminants and quality indicators [5,6]. They facilitate non-invasive monitoring of blood glucose, cholesterol, and biomarkers for various diseases [3,8]. For environmental monitoring, they are instrumental in detecting pollutants such as pesticides, heavy metals, and endocrine-disrupting compounds [3]. By coupling biological recognition via enzymes with sensitive transduction mechanisms, these biosensors have become indispensable in fields such as food safety, clinical diagnostics, environmental monitoring, pharmaceuticals, agriculture, and biotechnology [26,27].

7.1. Food Industry Applications

In the food sector, enzyme-based biosensors play a crucial role in ensuring food safety and maintaining product quality. They are employed to detect hazardous contaminants, such as pesticides, heavy metals, and chemical residues, that could pose health risks to consumers [3,6]. Furthermore, these biosensors aid in process control, allowing real-time monitoring of parameters such as sugar and alcohol contents during fermentation, thus ensuring product consistency and compliance with industry standards [5].

Food safety: Enzyme-based biosensors are used for detecting pesticides, heavy metals, and toxins in raw and processed food products [6,44]. For example, biosensors incorporating acetylcholinesterase are widely used for the detection of organophosphorous and organochlorine pesticides due to their enzyme inhibition properties. These biosensors can rapidly detect foodborne pathogens and allergens, which is crucial for preventing outbreaks [45,46]. Enzyme-linked immunosensors are often employed for specific detection of bacterial toxins and viral antigens.

Food quality: Changes in analyte levels, such as sugars, alcohols, amino acids, and biogenic amines, are indicative of food spoilage. Biosensors that use oxidases (e.g., glucose oxidase, alcohol oxidase) or dehydrogenases allow freshness monitoring in meat, seafood, and dairy products [47,48]. Enzyme-based biosensors can be integrated into quality control workflows to monitor nutrient content, detect adulterants, and ensure compliance with regulatory standards.

Process control: In fermentation and enzymatic processing, biosensors can continuously monitor carbohydrates, alcohols, and pH in real time [48]. Compact, automated biosensor systems enable operators to adjust processing parameters instantly, improving the product consistency and reducing waste.

Table 3. Enzyme-based biosensors in food industry applications, enzymes used, detection targets, advantages, and challenges.

Application Area	Enzymes Used	Detection Targets	Advantages	Challenges
Food Safety	Invertase, diamine oxidase, polyamine oxidase, putrescine oxidase, cholesterol oxidase, and cholinesterase tyrosinase [5,49,50].	Organophosphorous and organochlorine pesticides. Heavy metals. Toxins and pathogens [6,50].	High specificity and sensitivity. Rapid detection time enabling on-site analysis. Cost-effective alternative to chromatography-based methods [5,48].	Limited enzyme stability due to denaturation. Short operational lifetime [50].
Food Quality	Multiple enzymes for detecting sugars, alcohols, amino acids, flavors, and sweeteners [47]. Xanthine oxidase for hypoxanthine detection in fish [51].	Spoilage indicators, such as hypoxanthine in fish. Flavor profile deviations and sugar content in beverages [47,51].	Portable and field-deployable sensors. Potential for miniaturized integrated systems. Faster than microbial culture-based freshness tests [2].	Continuous flow analysis systems required for large-scale operations [48].
Process Control	Glucose oxidase, invertase, and other saccharide-detecting enzymes. Multiple enzymes for ethanol, fructose, lactate, and cholesterol [48].	Real-time monitoring of carbohydrates in fermentation. Bioprocess analytes for brewing, dairy, and bioethanol production [48].	Versatility in analyte detection [48]. Easy integration with industrial automation [5].	Ensuring specificity in complex fermentation matrices [48].
Pathogen Detection	Enzymes targeting bacterial metabolic activity for foodborne pathogen identification [45].	Pathogens such as Salmonella, E. coli, and Listeria monocytogenes. Allergen proteins [45].	Very high sensitivity, allowing early contamination detection [2].	Stability loss under variable storage and transport conditions [51].
Contaminant Detection	Esterase for organophosphate pesticide breakdown. Oxidoreductases for heavy metal detection [44,50].	Pesticide residues, lead, cadmium, and arsenic [44,50].	Suitable for environmental and food safety monitoring [44].	Enzyme inhibition by non-target compounds leading to false positives [44].

presents enzyme-based biosensors within food industry applications, detailing the enzymes applied, analytes detected, advantages offered, and existing challenges.

7.2. Medical and Clinical Diagnostics

Enzyme-based biosensors are foundational tools in medical diagnostics, especially for monitoring chronic diseases such as diabetes, cardiovascular disorders, and cancer. Glucose oxidase-based sensors, for instance, remain the gold standard for glucose monitoring in diabetic patients, offering real-time, precise, and portable diagnostic capabilities [52,53]. Their integration into point-of-care devices further enables timely interventions, particularly in remote or resource-limited settings [2]. Enzyme-based biosensors’ high sensitivity, specificity, portability, and adaptability to point-of-care settings make them a critical component of modern healthcare diagnostics [2,54,55,56]. They can detect target analytes at extremely low concentrations, making them indispensable in early disease detection. Their enzyme–substrate specificity reduces the cross-reactivity and enhances the diagnostic accuracy, which is particularly important for monitoring chronic conditions, such as diabetes, cardiovascular disease, and certain cancers [2,54,56]. Due to their compact design and low production costs, enzyme-based biosensors are ideal for point-of-care testing (POCT). They minimize the need for centralized laboratory facilities, enabling rapid on-site diagnostics in remote and resource-limited settings [2,54,55]. Recent developments have enabled non-invasive detection of biomarkers in saliva, sweat, and tears, offering patient-friendly alternatives to traditional blood tests. This capability supports continuous health monitoring, an increasingly important feature in personalized medicine [57,58]. Electrochemical enzyme-based biosensors dominate the clinical market due to their rapid response, miniaturization potential, and low power requirements. Optical biosensors, particularly fluorescence- and surface-plasmon-resonance-based systems, are expanding applications in multiplexed and label-free diagnostics [2,28]. The incorporation of nanomaterials, such as carbon nanotubes, graphene, and metallic nanoparticles, has significantly enhanced the signal amplification, stability, and detection limits. Lab-on-a-chip and microfluidic integration have further reduced the sample volume requirements and testing time [57]. Advances in flexible electronics have led to wearable enzymatic biosensors capable of real-time monitoring of glucose, lactate, and cholesterol through sweat or interstitial fluid, expanding the possibilities for sports medicine, remote patient monitoring, and chronic disease management [58,59]. Enzyme-based biosensors are employed in diagnosing and monitoring a variety of conditions, including diabetes (glucose-oxidase-based systems), cardiovascular disorders (lactate oxidase), cancer biomarkers, and neurodegenerative diseases [3,8]. Beyond glucose, modern biosensors target uric acid, cholesterol, creatinine, and phenolic compounds, offering real-time data for patient-specific therapy adjustments [58,59,60].

Table 4. Enzyme-based biosensors in various disease diagnostics.

Disease Type	Enzyme-Based Biosensor Application	Advantages	Challenges	Examples
Infectious Diseases	Rapid detection of disease biomarkers (antigens, antibodies, metabolites) using enzymes such as horseradish peroxidase for colorimetric or electrochemical readouts.	High sensitivity and specificity, cost-effective, portable, and rapid diagnostics suitable for field use.	Need for bioengineering and nanomaterial integration to enhance performance; ensuring stability in resource-poor settings.	Tuberculosis and neglected tropical diseases (e.g., leishmaniasis, schistosomiasis).	[57,61]
Metabolic Diseases	Detection of metabolic biomarkers using oxidase or dehydrogenase enzymes; commonly used for glucose and cholesterol monitoring.	High sensitivity, rapid results, and suitability for point-of-care testing.	Challenges in real-world implementation include calibration stability and interference from other biomolecules.	Diabetes and hypercholesterolemia.	[62,63]
Cardiac Diseases	Detection of cardiac biomarkers like troponin, myoglobin, and creatine kinase-MB using enzyme-linked immunosensors.	Improved sensitivity with multienzyme labels; enables rapid diagnosis at the point of care.	Limited signal amplification in conventional designs; need advanced amplification strategies.	Myocardial infarction and heart failure.	[62]
Cancer	Detection of tumor biomarkers (e.g., PSA, AFP, CEA) via enzyme-linked assays integrated with nanomaterials for higher sensitivity.	High specificity, potential for early diagnosis and real-time treatment monitoring.	Limited commercialization from research stage to clinical diagnostics.	Prostate cancer, liver cancer, and colorectal cancer.	[55,63]
Neurodegenerative Diseases	Detection of biomarkers like beta-amyloid, tau protein, and dopamine using enzyme-amplified biosensors for early-stage disease monitoring.	High sensitivity and specificity; potential for early intervention.	Limited adoption in clinical practice; stability and reproducibility issues.	Alzheimer’s disease and Parkinson’s disease.	[55]

provides an overview of enzyme-based biosensors in disease diagnostics, outlining the disease types, biosensor applications, advantages, challenges, and representative examples.

7.3. Environmental Monitoring

Environmental applications of enzyme-based biosensors are expanding rapidly. They are used for the detection of pollutants like pesticides, phenolic compounds, and heavy metals in water, air, and soil samples. This capability allows for on-site, cost-effective monitoring of environmental health, supporting regulatory compliance and pollution mitigation efforts [3,55]. These biosensors combine enzymes with diverse detection techniques, including electrical, chemical, optical, and electrochemical methods, to identify contaminants in environmental samples with high precision [22,55,64].

Detection of pollutants: Enzyme-based biosensors are effective for detecting a wide variety of pollutants in the environment. They are capable of detecting toxic metals such as mercury and lead with high sensitivity [65,66]. Utilization of esterase-based catalytic biosensors enables detection of organophosphorus and carbamate pesticides. Electrochemical enzyme biosensors detect pharmaceuticals such as antibiotics and hormones in natural waters [67,68].

Water and air quality monitoring: Enzyme-based biosensors are valuable tools for monitoring both water and air quality. Used for detecting heavy metals, pesticides, organic pollutants, and microbial contaminants, providing real-time, on-site analysis [69,70]. They also assess the biochemical oxygen demand (BOD) in water systems. Portable biosensor systems, including UAV(Unmanned Aerial Vehicles)-mounted devices, allow for the detection of pollutants and radiation leakage in remote areas [71,72].

Monitoring toxicity effects: Biosensors also provide toxicity assessment capabilities. Fluorescent enzyme-based biosensors measure changes in fluorescence intensity caused by toxic compounds [22]. Real-time monitoring enables rapid detection and mitigation of hazards [73,74].

Table 5. Enzyme-based biosensors in environmental monitoring: aspects, applications, advantages, and challenges.

Aspect	Description	Detection Techniques	Applications	Advantages	Challenges
General Overview	Enzyme-based biosensors use specific enzymes as biorecognition elements to detect environmental contaminants by converting biochemical reactions into measurable signals [64].	Electrical, chemical, optical, fluorescence, electrochemical, and mechanical signal transduction [22,55].	Water quality assessment, air pollution monitoring, toxicity evaluation, and soil contamination analysis [64].	High sensitivity and selectivity, rapid analysis, portability, and low operational cost [64,75].	Limited robustness in extreme environments and susceptibility to environmental matrix effects [76].
Technological Advancements	Integration of nanomaterials, microfluidics, and advanced optical systems to improve biosensor performance [75].	FRET, FLIM, FCS, fluorescence intensity monitoring, amperometry, conductometry, and chemiluminescence [22].	Real-time in situ detection, continuous monitoring systems, and lab-on-a-chip environmental assays [74].	Enhanced sensitivity and detection limits and the ability to detect multiple analytes simultaneously [75].	Higher complexity and cost; dependence on skilled operators for calibration and maintenance [76].
Specific Applications	Detection of organic pollutants, heavy metals, pesticides, endocrine disruptors, and halogenated hydrocarbons [7].	Electrochemical inhibition-based biosensors, fluorescence biosensors, and optical fiber sensors [22].	Environmental protection programs, industrial effluent testing, and regulatory compliance monitoring [74].	High specificity for target contaminants and capability to operate in diverse sample matrices [75].	Performance affected by non-specific binding and sample complexity [74].
Enzyme Immobilization	Critical for stability and reusability; methods include entrapment, adsorption, covalent bonding, cross-linking, and affinity interactions [77].	Enzyme-loaded membranes, sol–gels, carbon nanotube coatings, and polymer matrices [78].	Long-term environmental monitoring and point-of-care environmental diagnostics [64].	Improved electron transfer, enhanced enzyme stability, and reusability [77].	Immobilization method affects enzyme activity and sensor reproducibility [78].
Future Prospects	Use of bio–nano hybrids, graphene, quantum dots, and eco-friendly immobilization strategies [78].	Smart biosensors integrated with wireless and IoT-based environmental monitoring systems [74].	Early detection of pollution events, climate-related environmental changes, and disaster risk management [75].	Ultra-sensitive detection, multi-analyte sensing, minimal sample prep [78].	Need for rugged, field-deployable systems with minimal maintenance [64].

highlights the use of enzyme-based biosensors in environmental monitoring, presenting key aspects, detection techniques, applications, benefits, and challenges.

7.4. Agricultural Applications

In agriculture, enzyme-based biosensors facilitate soil and crop health monitoring by detecting agrochemical residues, such as fertilizers and herbicides. Their use supports precision farming practices, enhancing the crop yield while minimizing environmental harm [6,55]. These sensors enable real-time field diagnostics, contributing to more sustainable and environmentally friendly agricultural operations. Enzyme-based biosensors have demonstrated significant potential in agriculture, offering sensitive, specific, and rapid detection capabilities for a variety of applications. These include pesticide detection, soil and water quality monitoring, and crop health monitoring.

Pesticide detection: Enzyme-based biosensors for pesticide detection typically employ the principle of enzyme inhibition, where enzymes such as acetylcholinesterase (AChE) are inhibited by organophosphorus compounds, resulting in a measurable signal change [6]. These biosensors can detect organophosphates, carbamates, and organochlorines in soil, water, and food [50,78,79]. Recent innovations include ZnO-based nanostructured biosensors immobilized with AChE for enhanced sensitivity and flexible, portable designs [79]. Advantages include rapid, cost-effective, and on-site monitoring capabilities critical for mitigating environmental and health risks [80,81].

Soil and water quality monitoring: These biosensors monitor contaminants such as heavy metals, pesticides, and organic pollutants in soil and water systems by translating biochemical interactions into electrochemical or optical signals [64,80]. Commonly detected metals include Hg²⁺, As³⁺, and Cu²⁺, with enzyme-based systems offering high sensitivity and specificity. Their real-time capabilities enable continuous assessment of environmental quality, aiding in sustainable land and water management [82,83].

Crop health monitoring: Enzyme-based biosensors can track plant health by detecting biochemical markers of nutrient deficiency, disease using enzymes like oxidoreductases and peroxidases [82]. Such devices support early disease detection, optimize fertilization strategies, and improve the yield and resource efficiency.

Table 6. Enzyme-based biosensors in agricultural applications.

Application Area	Enzyme(s) Used	Detection Method	Target Analytes	Key Features/Findings
Food Safety and Quality	Invertase, diamine oxidase, polyamine oxidase, and putrescine oxidase	Electrochemical (potentiometric, amperometric)	Sugars, alcohols, amino acids, amines, organic acids, mycotoxins, and chemical contaminants	High specificity and sensitivity, rapid response times, low cost, and user-friendly operation for routine food quality assessment [84].
Pesticide Detection	Esterase, acetylcholinesterase (AChE), laccase, and catalase	Electrochemical (voltammetric, amperometric)	Organophosphates (e.g., paraoxon, chlorpyrifos) and carbamates.	High sensitivity and stability, low limit of detection, reusability, minimal interference from other compounds, and potential for field applications [50,79,85].
Environmental Monitoring	Cholinesterases, photosynthetic system II, alkaline phosphatase, cytochrome P450A1, peroxidase, tyrosinase, urease, and aldehyde dehydrogenase	Electrochemical (potentiometric, amperometric)	Heavy metals, pesticides, and organic pollutants	Integration with nanomaterials for enhanced sensitivity and cost-effective and rapid analysis suitable for large-scale monitoring [13,82,86].
Soil and Crop Monitoring	Oxidoreductases, amino oxidases, polyphenol oxidases, and peroxidases	Electrochemical, optical	Herbicides, insecticides, plant pathogens, and fertilizers	Enables monitoring of soil nutrient status, pathogen detection, and heavy metal contamination, and supports precision agriculture strategies [82].
Food Traceability	Thermostable esterase-2 (EST2)	Fluorescence-based	Organophosphates (e.g., paraoxon, methyl-paraoxon)	Detects residual pesticides in real food samples, and suitable for high-throughput screening of enzyme variants with altered selectivity profiles [50].

summarizes enzyme-based biosensors in agriculture, including the enzymes used, detection approaches, target analytes, and key findings.

8. Key Limitations of Enzyme Stability in Biosensors

Enzyme stability is a fundamental determinant of the performance, reliability, and commercial viability of enzyme-based biosensors. Despite their widespread use, enzymes are inherently fragile biomolecules, and their catalytic activity is susceptible to a range of physical, chemical, and environmental stresses. These limitations pose significant challenges for developing biosensors that are robust, reusable, and suitable for long-term applications, particularly in medical diagnostics, food safety, and environmental monitoring. One of the most critical limitations is sensitivity to environmental conditions, particularly temperature and pH fluctuations. Enzymes typically function optimally within a narrow temperature range, and exposure to high temperatures can cause irreversible denaturation, while low temperatures may reduce the catalytic efficiency. For example, glucose oxidase (GOX) has demonstrated temperature-dependent instability, which was partially mitigated using silanization at specific concentrations to enhance its thermal resilience [87]. Similarly, pH extremes can impair the enzyme structure and activity, though some electrode systems, such as carbon-paste-based enzyme electrodes, have shown unusual acid resistance, maintaining activity in highly acidic environments [77]. Another key issue is operational stability, which encompasses enzyme activity over time and multiple uses. A significant challenge is enzyme leaching, especially in sensors designed for repeated use. Although immobilization techniques—such as covalent bonding, entrapment in hydrogels, or adsorption onto nanomaterials—can reduce leaching and prolong activity, they may still be insufficient for extended applications [77,88]. Strategies like liposome encapsulation and specialized enzyme membranes have been developed to prevent unfolding and preserve the functional integrity over long durations [89,90]. Material interactions also significantly influence enzyme stability. The choice of immobilization matrix—whether it is nanoparticles, polymer composites, or nanocomposites—can enhance or hinder enzyme function. Random immobilization often results in unfavorable enzyme orientation or steric hindrance, reducing accessibility to the substrate and accelerating degradation [91,92]. Innovations like polymer-based protein engineering (PBPE) and nanocapsule encapsulation have shown promise in creating more protective microenvironments for enzymes, improving resilience to harsh conditions [43]. From a chemical stability perspective, enzymes are vulnerable to oxidative damage and proteolytic degradation during storage or operation. Additives such as electrolytes, polyols, and polyelectrolytes have been used to prevent these degradative processes by stabilizing enzyme conformation and providing hydration shells that reduce structural breakdown [93]. Additionally, exposure to organic solvents, especially in industrial or field settings, can disrupt protein folding. As a response, high-throughput assays have been developed to assess enzyme tolerance to solvents directly from crude extracts, offering a realistic measure of enzyme durability in complex environments [94]. A persistent design challenge lies in balancing the enzyme activity and stability. Enhancing one often comes at the expense of the other. For instance, mutations that improve thermal resistance may reduce the substrate affinity or turnover rate, as highlighted in studies that explored the structural basis of this trade-off [95]. In advanced applications like enzymatic biofuel cells (EBFCs) used in powering wearable sensors, enzyme stability directly affects the power output and miniaturization. These cells face issues such as a low current density and instability over time, impeding their use in continuous biosensing devices [96].

9. Future Directions of Enzyme-Based Biosensors

The future of enzyme-based biosensors is poised for transformative growth, driven by rapid advancements in nanotechnology, materials science, bioengineering, and device miniaturization. These biosensors, which rely on the catalytic and specific recognition properties of enzymes, are evolving to meet the increasing demand for portable, sensitive, and cost-effective diagnostic and monitoring tools across healthcare, environmental monitoring, food safety, and biotechnology. A major trend shaping the future is the integration of nanotechnology, especially the use of nanomaterials and nanozymes, to enhance biosensor performance. Incorporating nanomaterials—such as metal nanoparticles, carbon nanotubes, and zeolites—into biosensor platforms has improved their sensitivity, stability, and response time [97,98]. These innovations have enabled miniaturized, high-throughput, and multiplexed biosensing systems suitable for both clinical and industrial applications. Nanoparticles, nanowires, and nanocomposites are being employed to improve enzyme immobilization, increase the surface area for interaction, and enhance electron transfer, thereby boosting the sensitivity and stability [66,99]. Nanozymes—engineered nanomaterials that mimic natural enzyme activity—are emerging as powerful alternatives to biological enzymes due to their robustness, lower cost, and ability to function under harsh conditions [100,101]. These innovations are expected to redefine the architecture and efficiency of future biosensing platforms. Another key area of innovation is enzyme immobilization. Future biosensors will rely on more sophisticated immobilization techniques such as site-specific covalent binding, encapsulation in functional polymers, and use of biomimetic scaffolds to preserve the enzyme activity and extend the operational lifespan [77,102]. The development of multi-enzyme systems and co-immobilization strategies will enable the detection of multiple analytes or the execution of complex metabolic pathways within a single biosensor, broadening their functionality and diagnostic power [15]. Miniaturization and portability are also shaping the trajectory of enzyme-based biosensors. The emergence of lab-on-a-chip technologies and microfluidic systems allows for the integration of biosensors into compact, handheld devices capable of real-time and on-site analysis [103]. Additionally, paper-based biosensors offer low-cost, disposable platforms ideal for resource-limited settings, especially in point-of-care diagnostics and environmental screening [101]. These developments align with the global shift toward decentralized healthcare and on-demand testing.

Addressing the long-standing challenge of enzyme stability will be central to the future of biosensor design. Advances in protein engineering, chemical cross-linking, and encapsulation techniques are improving enzyme resilience to temperature, pH, solvents, and mechanical stress [104,105]. Moreover, the exploration of genetically engineered enzymes tailored for specific applications will help overcome limitations related to enzyme degradation and reproducibility [106,107].

The future also lies in expanding the application landscape of these biosensors. In healthcare, enzyme-based biosensors will play an increasingly important role in non-invasive diagnostics, continuous monitoring of chronic diseases, and early detection of infections [54,57]. In the environmental domain, their application is expected to broaden to detect emerging pollutants; antibiotic residues; and microplastics in water, soil, and air [7,64]. In the food and agricultural sectors, biosensors will be used to ensure safety, monitor spoilage, and detect contaminants in real time [108]. The advent of wearable biosensors is another revolutionary direction. Integrated into textiles, patches, or wearable devices, these biosensors will allow continuous, real-time health monitoring of key metabolites like glucose, lactate, or cortisol, enabling personalized medicine and preventive care [109]. Furthermore, combining biosensors with smart technologies, such as smartphones, wireless communication, and cloud computing, will create intelligent platforms capable of remote diagnostics, real-time feedback, and integration with IoT-based health systems [39].

Despite their promising capabilities, enzyme-based biosensors face several challenges. A major limitation is enzyme instability, as enzymes are susceptible to denaturation under adverse environmental conditions, such as extreme pH or temperature fluctuations [52]. Furthermore, issues related to reproducibility and high production costs—especially involving enzyme immobilization—have hindered widespread commercialization [27]. Ongoing research is addressing these concerns by developing more robust immobilization strategies, synthetic enzyme mimetics (nanozymes), and integrated sensor systems for enhanced performance and broader usability.

10. Conclusions

Enzyme-based biosensors have evolved into vital tools across a spectrum of applications, including medical diagnostics, environmental monitoring, food safety, biotechnology, and industrial processing. Their high specificity, rapid response times, and adaptability make them indispensable in scenarios requiring real-time, accurate detection of analytes. These biosensors operate through a synergistic interplay between enzymes, transducers, and immobilization strategies, translating biochemical interactions into quantifiable signals. However, despite their remarkable capabilities, several challenges remain—chief among them being enzyme instability, interference from complex sample matrices, and limited operational longevity. Recent innovations have sought to address these issues by introducing synthetic analogs such as nanozymes, which offer enhanced durability, cost-effectiveness, and resistance to denaturation. Similarly, the integration of nanomaterials—including graphene, carbon nanotubes, and metal nanoparticles—has significantly improved the sensor sensitivity and stability, especially when paired with advanced immobilization techniques like site-specific binding and polymer encapsulation. Enzyme-based biosensors are poised to benefit from emerging trends in wearable technology, microfluidics, and artificial intelligence. Wearable biosensors embedded in fabrics or patches offer promising avenues for non-invasive, continuous health monitoring, while microfluidic platforms are enabling ultra-compact, sample-efficient diagnostic systems. AI-driven data processing and wireless connectivity further enhance these biosensors’ diagnostic capabilities and accessibility. Despite lingering barriers—such as cost, manufacturing complexity, and enzyme degradation—ongoing research in bioengineering, nanotechnology, and materials science continues to push the boundaries of what enzyme-based biosensors can achieve. The convergence of these disciplines will likely usher in a new era of biosensors that are not only more robust and multifunctional but also scalable for widespread, real-world deployment.