Electrochemical Enzyme Biosensors Research Articles

Impact of microelectrode geometry and surface finish on enzymatic biosensor performance

Food production, environmental protection, biotechnology, and medicine, all depend on biosensors for measuring specific biological molecules. Among biosensors, enzymatic electrochemical biosensors have received particular attention due to their simple operation and enzymes’ inherent specificity. Researchers have extensively explored novel materials and biological designs to improve performance (i.e., sensitivity, linearity, limit of detection, and response time). However, in comparison, physical and geometrical design has not received the same attention. To this end, we compared platinum (Pt) microelectrodes with circular or fractal geometry with the same surface area (2D geometry). We also studied the effect of 3D geometry by nanostructuring both circular and fractal microelectrodes via electroplating Pt black. Fractal Pt black microelectrodes displayed the highest current density, charge storage capacity, and sensitivity towards H2O2. Next, we immobilized glucose oxidase onto various microelectrode geometries by microcontact stamping. Fractal Pt and Pt black glucose biosensors were 91.7 % and 83.3 % more sensitive than circular counterparts. Circular and fractal Pt black glucose biosensors were 63.0 % and 55.9 % more sensitive than Pt counterparts. Fractal geometry also provided better linearity and limit of detection. We modified enzyme layer thickness through multiple layer stamping and found a trade-off whereby increasing thickness increased sensitivity but also increased response time. Lastly, we developed a COMSOL Multiphysics numerical model to interpret the amperometric data and the impact of physical design on critical parameters. The work here will serve as a guideline for improving enzymatic electrochemical and other biosensors via physical design, which is simpler to modify than material or biological design.

RECENT ADVANCEMENT IN ELECTROCHEMICAL BIOSENSOR BASED ON REDOX ENZYME COMPOSITE MODIFIED ELECTRODE FOR THE DETECTION OF BIOMOLECULES

Recent developments in electrochemical biosensors have resulted in new applications in a variety of disciplines, such as clinical diagnosis, food processing quality control, and environmental monitoring. The electrochemical biosensor is a widely used sensing device that transmits an electrical signal from biological activities. Electrochemical biosensors have led to significant advancements in detecting various biomolecules such as uric acid, glucose, cholesterol, lactate, and DNA, including cancer biomarkers, viruses, and antibodies. An electrode is a vital part of this kind of sensor since it serves as a strong support for immobilizing biomolecules (enzyme, antibody, and nucleic acid) and allowing electron flow. Redox enzymes are the foundation of enzymatic electrochemical biosensors, which use oxidation or reduction reactions to detect a substrate, which is then converted into an electrical signal. Immobilizing enzymes onto solid supports is a common method to enhance the performance of an enzymatic electrochemical biosensor. However, there are some challenges, such as potential loss of enzyme activity, difficulties in maintaining enzyme stability, and issues related to the reproducibility and consistency of enzyme immobilization. This review covers all challenges in enzymatic electrochemical biosensors. Numerous new biosensor platforms are announced each year as researchers look for various electrode materials. Additionally, the researchers should familiarize themselves with the practical methods used in electrode production in order to build a successful biosensor. This review highlights current developments in modifying enzyme-immobilized biosensors for detection of different biomolecules. The article further describes methods of enzyme immobilization and various enzymes with different nanomaterials for the development of novel enzymatic electrochemical biosensors.

Electrochemical enzyme biosensors for medical application

Electrochemical enzyme biosensors for medical application

Ti3C2 MXene/MoS2@AuNPs ternary nanocomposite for highly sensitive electrochemical detection of phoxim residues in fruits

Phoxim, extensively utilized in agriculture as an organothiophosphate insecticide, has the potential to cause neurotoxicity and pose human health hazards. In this study, an electrochemical enzyme biosensor based on Ti3C2 MXene/MoS2@AuNPs/AChE was constructed for the sensitive detection of phoxim. The two-dimensional multilayer structure of Ti3C2 MXene provides a robust framework for MoS2, leading to an expansion of the specific surface area and effectively preventing re-stacking of Ti3C2 MXene. Additionally, the synergistic effect of self-reduced grown AuNPs with MoS2 further improves the electrical conductivity of the composites, while the robust framework provides a favorable microenvironment for immobilization of enzyme molecules. Ti3C2 MXene/MoS2@AuNPs electrochemical enzyme sensor showed a significant response to phoxim in the range of 1 × 10−13 M to 1 × 10−7 M with a detection limit of 5.29 × 10−15 M. Moreover, the sensor demonstrated excellent repeatability, reproducibility, and stability, thereby showing its promising potential for real sample detection.

Laser-Induced Graphene for Electrochemical Analysis of Food and Agricultural Contaminants

Carbon-nanomaterial sensors offer promising routes toward low-cost, high-throughput electrochemical sensors. Carbon nanomaterials, such as graphene and carbon nanotube (CNT), have been studied with various fabrication methods to produce sensors, including high-quality thin film graphene and CNT by chemical vapor deposition. However, the complexity and cost associated with their fabrication have hindered their implementation into single-use test strip sensors. Even high-yield mechanical and/or chemical exfoliation of graphene from graphite followed by solution phase printing of electrodes can be costly due to the complexity of ink formulation and the need for post-print annealing requirements. However, laser-induced graphene (LIG) avoids the need to exfoliate graphene from graphite or to create a solution-phase graphene ink by converting sp3 carbon found in carbon-rich substrates into conductive sp2-hybridized carbon found in graphene through a laser scribing technique. The resulting LIG also exhibits a high surface area and is porous with a high density of edge plane sites, which are beneficial for biofunctionalization and heterogeneous charge transport during electrochemical sensing. Furthermore, we have demonstrated that the surface area and wettability of the LIG can be tuned to greatly improve the sensitivity of electrochemical enzymatic biosensors (detection limits down to the picomolar range) and reduce the water layer buildup between the ion-selective membrane and the electrode, which improves the accuracy of the sensor readings. The fabricated LIG electrodes have been used in a wide variety of electrochemical sensing applications, including in food safety (foodborne pathogens and other contaminants), environmental monitoring (agrochemicals), and health monitoring sensors (salivary potassium and lactate, and urinary potassium and ammonium). Different electroanalytical methods are used, including amperometric, potentiometric, coulometric, and impedimetric, to quantify the analytes. We demonstrate how the ISE (ion-selective electrodes) can be functionalized with PVC-based ion-selective membranes for potassium, nitrate, nitrite, ammonium, sodium, and pH in various biological solutions, including soil and water for nutrient/fertilizer monitoring in farm fields. Additionally, we demonstrate how the hydrophilic LIG graphene can be used to create other highly sensitive electrochemical biosensors including enzymatic pesticide biosensors and Salmonella spp. immunosensors. All these electroanalytical sensors were validated in real-world samples and demonstrated to have limits of detection and linear sensing ranges that are relevant to their sensing applications. Results demonstrate the utility of LIG-based sensors for rapid and sensitive electrochemical measurement of contaminants as a readily modified platform for scalable production of electroanalytical devices applied toward point-of-service environmental and food monitoring.

A Theoretical Approach to Understand the Nonlinear Processes in Enzymatic Electrochemical Biosensors.

The enzymatic biosensors' response can be monitored based on the results of nonlinear differential equations. The nonlinear reaction-diffusion equations proposed for this enzyme-based electrochemical biosensor include a nonlinear term associated with Michaelis-Menten kinetics. Herein, the system of nonlinear reaction-diffusion equations is solved using a modified homotopy perturbation method. For all values of the rate constants, the approximate analytical expressions for the concentration profiles, current, sensitivity, and gradient of biosensor have been determined. Performance factors of an enzymatic electrochemical biosensor, such as response time, sensitivity, accuracy, and resistance, are discussed. The analytical results and numerically simulated outcomes using Matlab software have been compared.

A Sensitive Enzymatic Electrochemical Biosensor for Cholesterol Based on Cobalt Ferrite@Molybdenum Disulfide/Gold Nanoparticles.

Cholesterol is essential in biological systems, and the level of cholesterol in the body of a person acts as a diagnostic marker for a variety of diseases. So, in this work, we fabricated an enzymatic electrochemical biosensor for cholesterol using cobalt ferrite@molybdenum disulfide/gold nanoparticles (CoFe2O4@MoS2/Au). The synthesized composite was used for the determination of cholesterol by voltametric methods. The electroactive material CoFe2O4@MoS2/Au was successfully verified from the physiochemical studies such as XRD, Raman, FT-IR, and XPS spectroscopy along with morphological FESEM and HRTEM characterization. CoFe2O4@MoS2/Au showed outstanding dispersion in the aqueous phase, a large effective area, good biological compatibility, and superior electronic conductivity. The microflower-like CoFe2O4@MoS2/Au was confirmed by scanning electron microscopy. The image of transmission electron microscopy showed decoration of gold nanoparticles on CoFe2O4@MoS2 surfaces. Furthermore, a one-step dip-coating technique was used to build the biosensor used for cholesterol detection. In addition to acting as an enabling matrix to immobilize cholesterol oxidase (ChOx), CoFe2O4@MoS2/Au contributes to an increase in electrical conductivity. The differential pulse voltammetry method was used for the quantitative measurement of cholesterol. The calibration curve for cholesterol was linear in the concentration range of 5 to 100 μM, with a low limit of detection of 0.09 μM and sensitivity of 0.194 μA μM-1 cm-2. Furthermore, the biosensor demonstrates good practicability, as it was also employed for identifying cholesterol in real samples with acceptable selectivity and stability.

Low-interference and sensitive electrochemical detection of glucose and lactate using boron-doped diamond electrode and electron mediator menadione.

To minimize background interference in electrochemical enzymatic biosensors employing electron mediators, it is essential for the electrochemical oxidation of electroactive interfering species (ISs), such as ascorbic acid (AA), to proceed slowly, and for the redox reactions between electron mediators and ISs to occur at a low rate. In this study, we introduce a novel combination of a working electrode and an electron mediator that effectively mitigates interference effects. Compared to commonly used electrodes such as Au, glassy carbon, and indium tin oxide (ITO), boron-doped diamond (BDD) electrodes demonstrate significantly lower anodic current (i.e., lower background levels) in the presence of AA. Additionally, menadione (MD) exhibits notably slower reactivity with AA compared to other electron mediators such as Ru(NH3)63+, 4-amino-1-naphthol, and 1,4-naphthoquinone, primarily due to the lower formal potential of MD compared to AA. This synergistic combination of BDD electrode and MD is effectively applied in three biosensors: (i) glucose detection using electrochemical-enzymatic (EN) redox cycling, (ii) glucose detection using electrochemical-enzymatic-enzymatic (ENN) redox cycling, and (iii) lactate detection using ENN redox cycling. Our developed approach significantly outperforms the combination of ITO electrode and MD in minimizing IS interference. Glucose in artificial serum can be detected with detection limits of ~ 20μM and ~ 3μM in EN and ENN redox cycling, respectively. Furthermore, lactate in human serum can be detected with a detection limit of ~ 30μM. This study demonstrates sensitive glucose and lactate detection with minimal interference, eliminating the need for (bio)chemical agents to remove interfering species.

Sol-gel synthesized RexBi1-x-O thin films for electrochemical creatinine sensing: A facile fabrication approach

The escalating predominance of renal diseases has obliged vigorous research on developing efficient and cost-effective biosensors for creatinine, an aspirant biomarker reflecting renal health. Herein, we fabricated a highly sensitive and selective Rhenium (Re)-doped Bismuth oxide (Bi2O3) (RexBi1-x-O) based enzymatic electrochemical creatinine biosensor. Various concentrations of Re (5%, 10%, and 20%) were mixed with Bi2O3 solution via facile sol-gel synthesis and deposited on an Indium Tin oxide (ITO) substrate. The detailed physicochemical and electrochemical performance of the thin film was investigated and compared at different annealing temperatures (400 °C, 500 °C, and 600 °C). The optimized RexBi1-x-O thin film (Re0.10Bi0.90-O at 500 °C annealing temperature) depicted a limit of detection (LOD) as low as 28 μM in a concentration range of 0.05 mM–2 mM with excellent selectivity. To ensure the feasibility of the proposed creatinine sensor, its sensing performance was verified in human serum contaminated with altered creatinine concentrations, which manifested a satisfactory recovery rate. Eventually, the outstanding performance of RexBi1-x-O-based enzymatic creatinine sensor indorsed it as an accountable candidate for even more complex markers detection.

Sulfonated-polypyrene aniline/polyaniline composite fortified with Cu-GQD@ZIF8 as an electrochemical enzymatic urea biosensor.

The determination of urea concentration is essential for human health owing to its crucial role in the ability to metabolize nitrogen-containing substances. This study developed new electrochemical enzymatic detection systems via the synergistic effect of the superior features of novel electropolymerizable pyranine-aniline (PA, 4), polyaniline (PANI) compounds, graphene quantum dots (GQDs) and zeolitic imidazolate framework-8 (ZIF8). The novel compound 4 was characterized via1H-NMR, 13C-NMR, FTIR, and MALDI-TOF mass spectroscopies. Furthermore, Cu-GQD@ZIF8 hybrid materials containing GQD and integrated electroactive Cu metal were prepared in this study. The surface morphology of the prepared Cu-GQD@ZIF8 hybrid material was investigated through microscopic methods such as SEM and TEM, and chemical characterizations were performed using FTIR, XPS, XRD, and TGA analyses. After the characterization of the novel materials, the urease (Urs) enzyme was bound to the new modified electrode surface. Next, the enzymatic biosensor properties of the Urs/Cu-GQD@ZIF8/PANI/PA/GCE sensor electrode for urea detection via reduction of PANI were investigated by DPV and CV techniques. The LOD and LOQ values of the presented sensor were calculated to be 0.77 μM and 2.31 μM, respectively, in the linear range of 1.0-80.0 μM, based on DPV measurements. The presented biosensor system determined the amount of urea in an artificial serum sample, and its accuracy was confirmed via the recovery test and GC-MS analysis.

Probing the influence of crosslinkers on the properties, response, and degradation of enzymatic hydrogels for electrochemical glucose biosensing through fluorescence analysis.

Drop-cast crosslinked hydrogels are a common platform for enzymatic electrochemical biosensors. Despite the widespread use of these complex systems, there are still several questions about how their physicochemical properties affect their performance, stability, and reproducibility. In this work, first-generation faradaic biosensors composed of glucose oxidase and branched polyethyleneimine (BPEI) are prepared using either glutaraldehyde (GA) or ethylene glycol diglycidyl ether (EGDGE) as crosslinkers. While EGDGE gels present an increasing electrochemical response with increasing crosslinker concentration, the current of GA gels decreases at high crosslinker concentration probably due to the hampered diffusion on tightly networked gels. We compared different strategies to use fluorescence microscopy to gain insight into the gel structure either by labeling the gel components with fluorophores or taking advantage of the intrinsic fluorescence of the imines formed upon crosslinking with GA. By monitoring the fluorescence of the crosslinking bonds and the electrochemical response, we demonstrate that hydrolysis, a common hydrogel degradation mechanism, is not responsible for the loss of electrical current over time in gels prepared with glutaraldehyde. Most hydrogel-based electrochemical biosensor studies do not perform specific experiments to determine the cause of the degradation and instead just infer it from the dependence of the current on the preparation conditions (most commonly concentrations). We show that, by taking advantage of several analytical techniques, it is possible to gain more knowledge about the degradation mechanisms and design better enzymatic biosensors.

A new strategy to build electrochemical enzymatic biosensors using a nanohybrid material based on carbon nanotubes and a rationally designed schiff base containing boronic acid

We report a nanohybrid material obtained by non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with the new ligand (((1E,1′E)-(naphthalene-2,3-diylbis(azaneylylidene))bis(methaneylylidenedene)) bis(4-hydroxy-3,1-phenylene))diboronic acid (SB-dBA), rationally designed to mimic some recognition properties of biomolecules like concanavalin A, for the development of electrochemical biosensors based on the use of glycobiomolecules as biorecognition element. We present, as a proof-of-concept, a hydrogen peroxide biosensor obtained by anchoring horseradish peroxidase (HRP) at a glassy carbon electrode (GCE) modified with the nanohybrid prepared by sonication of 2.0 mg mL−1 MWCNTs and 0.50 mg mL−1 SB-dBA in N,N-dimethyl formamide (DMF) for 30 min. The hydrogen peroxide biosensing was performed at −0.050 V in the presence of 5.0 × 10−4 M hydroquinone. The analytical characteristics of the resulting biosensor are the following: linear range between 0.175 μM and 6.12 μM, detection limit of 58 nM, and reproducibility of 2.0 % using the same nanohybrid (6 biosensors), and 9.0 % using three different nanohybrids. The sensor was successfully used to quantify hydrogen peroxide in enriched milk and human blood serum samples and in a commercial disinfector.

Zwitterionic hydrogel for preserving stability and activity of oxidase enzyme for electrochemical biosensor

Enzymatic electrochemical biosensor is the most common analytical platform for medical diagnosis. To mimic the biological environment of the enzyme for maintaining the function of biosensor, zwitterionic hydrogels have been recognized as effective matrices for enzymatic immobilization. Herein, a zwitterionic hydrogel derived from a copolymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-N-methacryloyloxyethyl tyrosine methylester (MAT)] (PMM) was firstly applied as versatile coating to preserve stability and activity of oxidase enzymes, glucose oxidase (GOx) and lactate oxidase (LOx) for enzymatic electrochemical sensor. A screen-printed carbon electrode (SPCE) was sequentially coated with nitrogen-doped graphene (NDG), oxidase enzyme, and PMM mixed with Ru(II)bpy32+ and (NH4)2S2O8 followed by visible light irradiation for 3 min to induce PMM gelation. Electrochemical detection of glucose and lactate using the modified SPCE was performed via amperometry in the presence of hydrogen peroxide. The activity of both GOx and LOx immobilized on the modified SPCE was well maintained for 49 days at 87 and 80 %, respectively. Additionally, two different electrodes, a screen-printed graphene electrode (SPGE), and a screen-printed silver electrode (SPAgE), similarly modified gave the same satisfactory detection of spiked glucose and lactate in human plasma and sweat with 93–118 % recovery. This indicates the potential of the PMM hydrogel as a universal platform for preservation of enzymes which can be easily fabricated without the need for specific chemical modification of the electrode.

A catalase enzyme biosensor for hydrogen peroxide at a poly(safranine T)-ternary deep eutectic solvent and carbon nanotube modified electrode

A new electrochemical enzyme biosensor has been developed to monitor hydrogen peroxide. The sensor uses a multiwalled carbon nanotube modified glassy carbon electrode covered by a poly(safranine T) polymer film prepared by potential cycling electropolymerization in ternary deep eutectic solvent (DES), prepared from choline chloride (ChCl), ethylene glycol (EG) plus malonic acid (MA) or lactic acid (LA) with composition optimization. It is shown that this leads to greater polymer growth and improved film properties compared to binary DES; the process was monitored by an electrochemical quartz crystal microbalance. The best ternary DES composition was found to be 16 % ChCl:MA / 84 % ChCl:EG. Electrochemical characterisation was done by cyclic voltammetry and electrochemical impedance spectroscopy, and the morphology of the synthesized polymer films was examined by scanning electron microscopy. Catalase enzyme was immobilized on the modified electrode and the enzyme biosensor used for measurement of hydrogen peroxide, with a very low detection limit of 34 nM. The selectivity was found to be excellent in relation to common interferents and the biosensor showed very good recovery in peroxide measurements in commercial samples.

An electrochemical AChE-based biosensor for organophosphate pesticides using a modified CuNWs/rGO nanocomposite on a screen-printed carbon electrode

An enzymatic electrochemical biosensor was built for the indirect detection of organophosphates (OPs), based on acetylcholinesterase inhibition. The biosensor was fabricated for enhanced performance on a screen-printed carbon electrode (SPCE), modified with copper nanowires (CuNWs) composited with reduced graphene oxide (rGO). The oxidation current was measured using the cyclic voltammogram (CV) method, as generated by the enzymatic interaction between acetylcholinesterase (AChE) and its substrate, acetylthiocholine (ATCh). The biosensing response is the reduction in signal caused by the inhibition of acetylcholinesterase in the presence of an organophosphate inhibitor. Benchmarking shows that the CuNWs/rGO nanocomposite enhanced the signal current considerably and decreased the oxidation potential for electrochemical detection of the OP chlorpyrifos, exhibiting a wide linear detection rangefrom 10 µg/L–200 µg/L, with a limit of detection of 3.1 µg/L and limit of quantification of 12.5 µg/L. This sensor is useful for the analysis of chlorpyrifosin drinking water and orange juice, with high recovery rates and no interference effects.

Enzymatic Electrochemical Biosensors for Neurotransmitters Detection: Recent Achievements and Trends

Neurotransmitters (NTs) play a crucial role in regulating the behavioral and physiological functions of the nervous system. Imbalances in the concentrations of NT have been directly linked to various neurological diseases (e.g., Parkinson’s, Huntington’s, and Alzheimer’s disease), in addition to multiple psychotic disorders such as schizophrenia, depression, dementia, and other neurodegenerative disorders. Hence, the rapid and real-time monitoring of the NTs is of utmost importance in comprehending neurological functions and identifying disorders. Among different sensing techniques, electrochemical biosensors have garnered significant interest due to their ability to deliver fast results, compatibility for miniaturization and portability, high sensitivity, and good controllability. Furthermore, the utilization of enzymes as recognition elements in biosensing design has garnered renewed attention due to their unique advantages of catalytic biorecognition coupled with simultaneous signal amplification. This review paper primarily focuses on covering the recent advances in enzymatic electrochemical biosensors for the detection of NTs, encompassing the importance of electrochemical sensors, electrode materials, and electroanalytical techniques. Moreover, we shed light on the applications of enzyme-based biosensors for NTs detection in complex matrices and in vivo monitoring. Despite the numerous advantages of enzymatic biosensors, there are still challenges that need to be addressed, which are thoroughly discussed in this paper. Finally, this review also presents an outlook on future perspectives and opportunities for the development of enzyme-based electrochemical biosensors for NTs detection.

Development of a Redox-Polymer-Based Electrochemical Glucose Biosensor Suitable for Integration in Microfluidic 3D Cell Culture Systems.

The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H2O2 diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 μA mM-1 cm-2.

Damage-free remote SF6 plasma-treated CNTs for facile fabrication of electrochemical enzyme biosensors

Remote SF6 plasma treatment of CNTs is performed to graft hydrophobic fluorine on the surface of CNTs. Fluorinated CNTs (F-CNTs) allow for the facile adsorption of enzyme molecules via hydrophobic interaction between F-CNTs and the hydrophobic patches of enzyme molecules. The loading of glucose oxidase (GOx) on F-CNTs correlates well with plasma treatment time. Interestingly, the Rct value of GOx/F-CNTs decreases when CNTs are treated with plasma for a short time such as 1 and 5 min, which is due to weakly-bound chemisorbed fluorine molecules on F-CNTs (semi-ionic C-F). As an example, the Rct value of GOx/F-CNT electrode with 1 min plasma treatment is 0.57 times lower than that of GOx/CNTs, while increasing its sensitivity by 2.5 times. Upon extending the treatment time such as 30, 45 and 60 min, the XPS results reveal the dominance of covalently-attached fluorine, leading to marginally-increased resistance and sensitivity. It is worthy of noting this work is the first report of simple enzyme adsorption on remote-plasma treated CNT surfaces together with its detailed mechanism studies, especially for the binding of enzyme molecules semi-ionic fluorine.

Recent Prospects of Carbonaceous Nanomaterials-Based Laccase Biosensor for Electrochemical Detection of Phenolic Compounds.

Phenolic compounds (PhCs) are ubiquitously distributed phytochemicals found in many plants, body fluids, food items, medicines, pesticides, dyes, etc. Many PhCs are priority pollutants that are highly toxic, teratogenic, and carcinogenic. Some of these are present in body fluids and affect metabolism, while others possess numerous bioactive properties such as retaining antioxidant and antimicrobial activity in plants and food products. Therefore, there is an urgency for developing an effective, rapid, sensitive, and reliable tool for the analysis of these PhCs to address their environmental and health concern. In this context, carbonaceous nanomaterials have emerged as a promising material for the fabrication of electrochemical biosensors as they provide remarkable characteristics such as lightweight, high surface: volume, excellent conductivity, extraordinary tensile strength, and biocompatibility. This review outlines the current status of the applications of carbonaceous nanomaterials (CNTs, graphene, etc.) based enzymatic electrochemical biosensors for the detection of PhCs. Efforts have also been made to discuss the mechanism of action of the laccase enzyme for the detection of PhCs. The limitations, advanced emerging carbon-based material, current state of artificial intelligence in PhCs detection, and future scopes have also been summarized.

Application of NAD+-Dependent Electrochemical Dehydrogenase Biosensors in Human Physiological Fluids: Opportunities and Challenges

Electrochemical enzymatic biosensors represent a promising, low-cost technology for point-of-care (POC) diagnostics that allows fast response and simple sample processing procedures. In this review, we summarize up-to-date literature on NAD+/NADH (β-nicotinamide adenine dinucleotide)-dependent electrochemical dehydrogenase biosensors and highlight their applications in human physiological fluids. A brief comparison of various enzyme immobilization procedures is first presented, discussing preparation processes and principal analytical performance characteristics. In the following section, we briefly discuss classes of biosensors based on redox mediators-mediated electron transfer systems (METs). Finally, the conclusion section summarizes the ongoing challenges in the fabrication of NAD+-dependent electrochemical dehydrogenase biosensors and gives an outlook on future research studies.