Enzyme-based Electrochemical Biosensors Research Articles

A theoretical and computational analysis of the concentration of substrate and product in enzyme-based electrochemical biosensors: Akbari-Ganji’s method

A mathematical model for a two-compartment biocatalytic system has been discussed. The modelling biosensor has two layers, one composed of a membrane and the other of an enzyme layer. This model involves a set of nonlinear differential equations and matching boundary conditions. We can obtain the substrate and product concentration in the enzyme and membrane layer by solving the nonlinear equations analytically using Akbari-Ganji’s method and numerically using compact finite difference schemes for the first time. The impact of the diffusion module, ratio of thicknesses of the enzyme and membrane layers, saturation, and degradation parameters on the biosensor concentration have been discussed. A satisfactory level of agreement is found between our analytical results and the simulation outcomes. These formulae can be employed for monitoring the biosensor response.

An ALP enzyme-based electrochemical biosensor coated with signal-amplifying BaTiO3 nanoparticles for the detection of an antiviral drug in human blood serum.

Tenofovir (TFV) is an antiviral drug used to treat the co-infections of HIV/HBV viruses. Accurate monitoring of TFV drug levels is essential for evaluating patient adherence, optimizing dosage, and assessing treatment efficacy. Herein, we propose an innovative electrochemical sensing approach by using the alkaline phosphatase (ALP) enzyme with the support of BaTiO3 nanoparticles. An attractive sensitivity and selectivity of the developed sensor towards TFV detection were achieved. First, the nanoparticles were synthesized by following a single-step sol-gel method and characterized through various analytical techniques, including SEM, EDX, FT-IR, BET, zeta potential, XRD, and UV-vis and Raman spectroscopy. The suggested mechanism demonstrated the formation of a strong bond between TFV and the ALP enzyme, primarily through the phosphate group, resulting in enzyme inhibition. Various parameters like nanoparticle amount, electrode modification time with enzyme and BaTiO3 nanoparticles, and drug incubation time were optimized. The biosensor demonstrated an outstanding limit of detection (LOD) of 0.09 nM and recovery percentages of 98.6-106% in human blood serum, indicating adequate repeatability and selectivity. The proposed biosensor can be converted into a portable device for measuring small sample volumes and observing patients for immediate medical care or personalized therapies. It achieved better sensitivity compared to existing methods, making it suitable for precise drug detection in microdoses.

Development of Redox-Active Lyotropic Lipid Cubic Phases for Biosensing Platforms.

Enzyme-based electrochemical biosensors play an important role in point-of-care diagnostics for personalized medicine. For such devices, lipid cubic phases (LCP) represent an attractive method to immobilize enzymes onto conductive surfaces with no need for chemical linking. However, research has been held back by the lack of effective strategies to stably co-immobilize enzymes with a redox shuttle that enhances the electrical connection between the enzyme redox center and the electrode. In this study, we show that a monoolein (MO) LCP system doped with an amphiphilic redox mediator (ferrocenylmethyl)dodecyldimethylammonium bromide (Fc12) can be used for enzyme immobilization to generate an effective biosensing platform. Small-angle X-ray scattering (SAXS) showed that MO LCP can incorporate Fc12 while maintaining the Pn3m symmetry morphology. Cyclic voltammograms of Fc12/MO showed quasi-reversible behavior, which implied that Fc12 was able to freely diffuse in the lipid membrane of LCP with a diffusion coefficient of 1.9 ± 0.2 × 10-8 cm2 s-1 at room temperature. Glucose oxidase (GOx) was then chosen as a model enzyme and incorporated into 0.2%Fc12/MO to evaluate the activity of the platform. GOx hosted in 0.2%Fc12/MO followed Michaelis-Menten kinetics toward glucose with a KM and Imax of 8.9 ± 0.5 mM and 1.4 ± 0.2 μA, respectively, and a linearity range of 2-17 mM glucose. Our results therefore demonstrate that GOx immobilized onto 0.2% Fc12/MO is a suitable platform for the electrochemical detection of glucose.

A graphitized carbon@boron carbide-mediated laccase-based amperometric biosensor for epinephrine detection

The development of novel electron transfer mediator is important for laccase-based electrochemical biosensors. Here, graphitized carbon coated boron carbide (GC@B4C) was first used as the mediator to transfer electrons from laccase (Lac) to nickel foam (NF) electrode. The GC shell of GC@B4C particles was characterized and confirmed by HRTEM, XRD and Raman spectroscopy. The fabricated Lac/GC@B4C/NF electrode was applied for amperometric detection of epinephrine (EP). Detection results indicated Lac/GC@B4C/NF electrode has wide linear range (0.1 µM-2.6 mM), low detection limit (40 nM), high sensitivity (76.05 µA mM−1), strong anti-interference ability and favorable stability for EP detection. This work opened up the use of nonmetallic carbide as the mediator for enzyme-based electrochemical biosensors.

Biosensor development for low-level acetaldehyde gas detection using mesoporous carbon electrode printed on a porous polyimide film

Acetaldehyde, which is an intermediate product of alcohol metabolism, is known to induce symptoms, including alcohol flushing, vomiting, and headaches in humans. Therefore, real-time monitoring of acetaldehyde levels is crucial to mitigating these health issues. However, current methods for detecting low-concentration gases necessitate the use of complex measurement equipment. In this study, we developed a low-cost, low-detection-limit, enzyme-based electrochemical biosensor for acetaldehyde gas detection that does not require sophisticated equipment. The sensor was constructed by screen-printing electrodes onto a porous polyimide film, using grafted MgO-templated carbon (GMgOC) as working electrode material, carbon for the counter electrode, and silver/silver chloride for the reference electrode. Pyrroloquinoline-quinone-dependent aldehyde dehydrogenase was immobilized on the working electrode, and a chamber was attached to the electrode chip and filled with 1-methoxy-5-methylphenazinium methyl sulfate solution. The sensor can be used to measure acetaldehyde gas concentrations from 0.02 to 0.1 ppm, making it suitable for monitoring human skin gas. This low detection limit was achieved by delivering the analyte through the porous polyimide film on which the electrodes were printed and accumulating acetaldehyde in the mesoporous GMgOC of the working electrode. This mechanism suggests that this sensor design can be adapted to develop other low-detection limit gas sensors, such as those for screening skin gas biomarkers.

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.

Synergistic catalysis and detection of hydrogen peroxide based on a 3D-dimensional molybdenum disulfide interspersed carbon nanotubes nanonetwork immobilized chloroperoxidase biosensor

Enzyme-based electrochemical biosensors are promising for a wide range of applications due to their unique specificity and high sensitivity. In this work, we present a novel enzyme bioelectrode for the sensing of hydrogen peroxide (H2O2). The molybdenum disulfide nanoflowers (MoS2) is self-assembled on carboxylated carbon nanotubes (CNT) to form a three-dimensional conductive network (3D-CNT@MoS2), which is modified with 1-ethyl-3-methylimidazolium bromide (ILEMB), and followed by anchoring chloroperoxidase (CPO) onto the nanocomposite (3D-CNT@MoS2/ILEMB) through covalent binding to form a bioconjugate (3D-CNT@MoS2/ILEMB/CPO). The ILEMB modified 3D-CNT@MoS2/ILEMB has good hydrophilicity and conductivity, which not only provides a suitable microenvironment for the immobilization of CPO but also facilitates the direct electron transfer (DET) of CPO at the electrode. The 3D-CNT@MoS2/ILEMB/CPO bioconjugate modified electrode has a high catalytic efficiency for H2O2. Through electroenzymatic synergistic catalysis for H2O2 detection by 3D-CNT@MoS2/ILEMB/CPO-GCE, a wide detection range of 0.2 μmol·L−1 to 997 μmol·L−1 and a low detection limit of 0.097 μmol･L−1 with high sensitivity of 1050 µA·mmol·L−1·cm−2 were achieved. Additionally, the 3D-CNT@MoS2/ILEMB/CPO-GCE displayed exceptional stability, selectivity, and reproducibility. Furthermore, 3D-CNT@MoS2/ILEMB/CPO-GCE is suitable for sensing of H2O2 in human urine s with good recovery, suggesting its potential application for the detection of H2O2 in biomedical field.

Gold Nanoclusters with Two Sets of Embedded Enzyme Nanoparticles for Applications as Electrochemical Sensors for Glucose

Enzyme-based electrochemical biosensors have been widely used because of their sensitivity, rapidity, and highly target-specific reactions. However, the immobilized enzymes can hinder electron transfer on the surface of the electrode and can decrease the sensitivity of electrochemical biosensors. To overcome this problem, we synthesized conductive gold nanoclusters with two sets of embedded enzyme nanoparticles, which are called gold nanocluster-embedded dual-enzyme nanoparticles (AuNC-DENPs) composed of a glucose oxidase-stabilized gold nanocluster (GOX-AuNC) and a horseradish peroxide-stabilized gold nanocluster (HRP-AuNC). Moreover, the application of these nanoparticles as an enzyme-based highly sensitive electrochemical sensor was investigated. Owing to the effect of AuNCs, these nanoparticles have good conductivity compared to bare protein nanoparticles. In addition, the synthesized AuNC-DENPs enabled the combination of a two-enzyme cascade reaction, in which the GOX-AuNC component of the nanoparticle oxidized glucose to generate hydrogen peroxide, which then reacted with the adjacent HRP-AuNC component on the nanoparticle. Given the proximity of the two enzyme components in a single nanoparticle, the AuNC-DENPs markedly reduced the diffusion and decomposition of H2O2 during the cascade reaction and showed an enhanced catalytic reaction compared to a mixture of enzymes. As a result, the biosensor exhibited high sensitivity (18,944 μA/mM cm2) and could detect very low concentrations of glucose ranging from 5 to 320 nM and a LOD of 2.58 nM. In addition, by analyzing nontarget materials and serum together with glucose, it was confirmed that the sensor has good selectivity for glucose.

Direct electron transfer kinetics of histamine dehydrogenase at air plasma-treated graphite nanofibers

As histamine is one of the important mediators for allergic reactions, its efficient detection methods and real-time monitoring systems are required for food analyses and drug discoveries to suppress allergic reactions. Although histamine dehydrogenase (HmDH) is a promising candidate for developing enzyme-based electrochemical biosensors, some electron mediators are frequently employed to observe electrocatalytic currents for histamine oxidation. Here, direct electrochemistry of HmDH was studied at the surface of graphite nanofibers (GNFs), which provide active reaction sites for redox species. Air plasma-treated GNFs were used for constructing a three-dimensional network that works both as an electrical nanowire and an enzyme support. Even though the amount of oxygen-containing functional groups didn’t significantly increase at the GNF surface with increase in the air plasma treatment time, direct electron transfer from reduced HmDH by histamine to the GNFs was improved probably due to capped and curvature of the graphite edge sites with oxygen-containing functional groups, which were generated by the air plasma treatment. The air plasma-treated GNFs also allowed enhancement of the complex-formation reaction rate of HmDH with histamine, as the air plasma treatment time increased.

Enzyme-based electrochemical biosensor for antimonite detection in water.

Antimonite (SbIII) is a naturally occurring contaminant demanding on-site ultrasensitive detection. The enzyme-based electrochemical (EC) biosensors are promising, but the lack of specific SbIII oxidizing enzymes hindered the past efforts. Herein, we modulated the specificity of arsenite oxidase AioAB toward SbIII by regulating its spatial conformation from tight to loose using the metal-organic framework ZIF-8. The constructed EC biosensor, AioAB@ZIF-8, exhibited the substrate specificity toward SbIII at 12.8s-1μM-1, an order of magnitude higher than that of AsIII (1.1s-1μM-1). Relaxing AioAB structure in ZIF-8 was evidenced by the break of the S-S bond and the conversion of α helix to the random coil as suggested by Raman spectroscopy. Our AioAB@ZIF-8 EC sensor exhibited a dynamic linear range in 0.041-4.1μMat a response time of 5s, and the detection limit at 0.041μMat a high sensitivity of 1894nAμM-1. The insights into tuning the specificity of an enzyme shed new light on biosensing metal(loid)s without specific proteins.

Effect of dendrimer-based interlayers for enzyme immobilization on a model electrochemical sensing system for glutamate

In this paper, we discuss dendrimer usage in enzyme-based electrochemical biosensors, particularly with respect to biomolecule loading on the sensing surface. A novel approach to design bioactive layers with immobilized enzymes for electrochemical biosensors using the surface plasmon resonance (SPR) method in combination with electrochemical impedance spectroscopy was presented. The gold surface was modified with linear linkers (various mercaptoalkanoic acids and aminoalkanethiols) and poly(amidoamine) dendrimers from the first- to fifth-generation. The best functionalization procedure was established by detailed SPR studies and transferred onto gold electrodes to electrochemically examine the model enzymatic reaction catalysed by glutamate oxidase. In the case of the chronoamperometric method, the determined sensitivity was 3.36 ± 0.08 μA∙mM−1, and the low limit of detection (LOD) was 1.52 μM. Comparing the sensitivity and LOD obtained for CV measurements, the values of these parameters were 2.5 times higher and 4 times lower, respectively, for the fourth-generation dendrimer-based biosensor and the biosensor with a linear linker. The positive impact of the dendrimer interlayer on the long-term enzyme activity was also confirmed. The research results indicate the possibility of a significant increase in the sensor response using the dendrimer itself without enriching it with electrochemical components.

AChE-based electrochemical biosensor for pesticide detection in vegetable oils: matrix effects and synergistic inhibition of the immobilized enzyme

Enzyme-based electrochemical biosensors have been widely deployed for the detection of a range of contaminants in different food products due to their significant advantages over other (bio)sensing techniques. Nevertheless, their performance is greatly affected by the sample matrix itself or by the matrix they are presented with in pretreated samples, both of which can impact the accuracy as well as the sensitivity of the measurements. Therefore, and in order to acquire reliable and accurate measurements, matrix effects and their influence on sensor performance should be taken into consideration. Herein, acetylcholinesterase (AChE)-modified electrochemical sensors were employed for the detection of pesticides in vegetable oils. Sensor interrogation with pretreated oil samples, spiked with carbofuran, revealed the inhibitory potential of the extracted matrix varies between different types of vegetable oil and their fatty acid content. In addition, synergies between the extracted matrix from different types of vegetable oils and the carbamate pesticide, carbofuran, were observed, which led to significant deviations of the sensor’s performance from its anticipated behavior in buffered solution. Taking the aforementioned into consideration, appropriate calibration curves for each type of vegetable oil were drafted, which allowed for the highly reproducible determination of different pesticide concentrations in pretreated real samples. Collectively, a better understanding of AChE inhibition by single or multiple contaminants present in vegetable oils was gained, which can find many applications in numerous fields, ranging from sensor development to the design of new pesticides and medicinal products.

Food Spoilage: Detection of Biogenic Amines in Food Samples by Enzyme-based Electrochemical Biosensors

Food Spoilage: Detection of Biogenic Amines in Food Samples by Enzyme-based Electrochemical Biosensors

In Vivo Detection of Glutamate in Tomatoes by an Enzyme-Based Electrochemical Biosensor.

The in vivo and on-site detection of key physiology parameters in plants will be of great relevance for precision agriculture and food technology. In this work, a sensitive enzymatic glutamate sensor was successfully developed. To enhance the conductivity and catalytic ability and to fix the glutamate oxidase, Au–Pt nanoparticles were first deposited on screen-printed electrodes, and then carboxylated graphene oxide and carboxylated multiwalled carbon nanotubes were fabricated for the synthesis of the electrode. The detection range of the glutamate sensor is widest (2 μM to 16 mM) up to date, and its detection limit is relatively low (0.14 μM). A number of standard curves were built in the pH range of 3.5–7.5, which can be applied in various plants and fruits. Using this sensor, the glutamate level in tomatoes was determined in vivo. This glutamate sensor has important practical value in precision agriculture. Our strategy also provides a way to establish the detection modes for other biomolecules in plants.

A Dual Electrode Biosensor for Glucose and Lactate Measurement in Normal and Prolonged Obese Mice Using Single Drop of Whole Blood.

Understanding the levels of glucose (G) and lactate (L) in blood can help us regulate various chronic health conditions such as obesity. In this paper, we introduced an enzyme-based electrochemical biosensor adopting glucose oxidase and lactate oxidase on two working screen-printed carbon electrodes (SPCEs) to sequentially determine glucose and lactate concentrations in a single drop (~30 µL) of whole blood. We developed a diet-induced obesity (DIO) mouse model for 28 weeks and monitored the changes in blood glucose and lactate levels. A linear calibration curve for glucose and lactate concentrations in ranges from 0.5 to 35 mM and 0.5 to 25 mM was obtained with R-values of 0.99 and 0.97, respectively. A drastic increase in blood glucose and a small but significant increase in blood lactate were seen only in prolonged obese cases. The ratio of lactate concentration to glucose concentration (L/G) was calculated as the mouse’s gained weight. The results demonstrated that an L/G value of 0.59 could be used as a criterion to differentiate between normal and obesity conditions. With L/G and weight gain, we constructed a diagnostic plot that could categorize normal and obese health conditions into four different zones. The proposed dual electrode biosensor for glucose and lactate in mouse whole blood showed good stability, selectivity, sensitivity, and efficiency. Thus, we believe that this dual electrode biosensor and the diagnostic plot could be used as a sensitive analytical tool for diagnosing glucose and lactate biomarkers in clinics and for monitoring obesity.

Cytochrome P450 2B6 Amperometric Biosensor for Continuous Monitoring of Propofol

Despite the growing evidence of improved patient outcomes and of a substantially reduced environmental impact of propofol-based total intravenous anesthesia when compared to volatile-based techniques, the vast majority of general anesthetics still use volatile agents for the maintenance phase. A significant reason for this is the lack of suitable point-of-care, real-time blood propofol measurement techniques. Here we present an enzyme-based electrochemical biosensor for the detection of propofol. Deactivated yeast cells expressing the enzyme cytochrome P450 2B6 are immobilized, alongside gold nanoparticles, within a chitosan film upon the surface of a screen printed electrode. In the presence of the cofactor NADPH, the enzyme converts propofol to a quinone/quinol redox pair that can be detected using simple electrochemistry. This approach avoids the issue of electrode fouling that commonly renders electrochemical propofol sensors impractical. The sensor has a limit of detection of 67 ± 7 ng/ml and a sensitivity of 4.2 ± 0.2 nA/ <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu \text{g}$ </tex-math></inline-formula> /ml/mm <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{\mathbf {2}}$ </tex-math></inline-formula> . It has been successfully demonstrated in a serum-like solution and has shown a linear response across the therapeutic range of propofol (1 – <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10~\mu \text{g}$ </tex-math></inline-formula> /ml). Additionally, the sensor has shown good specificity with regards to potential interfering compounds.

Development of Mlra Enzyme-Based Electrochemical Biosensor for the Direct Detection of MC-LR

Microcystins are potent hepatotoxins and carcinogens produced by cyanobacteria (blue-green algae). In particular, MC-LR, one of the most toxic microcystins, tends to accumulate and pollute water reservoirs around the world, exhibiting extraordinary environmental stability. Microcystinase (MlrA) is a key enzyme in the detoxification pathway of MC-LR expressed by aerobic gram-negative bacteria. MlrA hydrolyzes a peptide bond within the cyclic MC-LR structure thus generating a nearly benign linear product.The substrate-specificity and stability of MlrA makes it an ideal candidate for an enzyme-based electrochemical biosensor. In this study, the enzyme kinetics of partially purified, heterologously expressed recombinant Mlr-A was elucidated. Polypyrrole-modified screen-printed electrodes were constructed and MlrA was incorporated via covalent immobilization. The resulting enzyme-modified chips were used to study the electrochemical properties of MC-LR and MlrA. Within a small potential window, both MC-LR substrate and MlrA enzyme are electrochemically inert. However, it was shown that enzymatic hydrolysis of MC-LR significantly affected the capacitive current measured overtime. These concentration-dependent changes in non-faradic current were observed even at relatively low MC-LR concentration of 10mM/L, and provide a proof-of-concept for the use of MlrA in an enzyme-based electrochemical biosensor. This study may pave the way towards designing biosensors for environmental diagnostics that enable both monitoring and remediation. Figure 1

Nanocluster-assisted protein-film voltammetry for direct electrochemical signal acquisition.

Acquisition of the direct electrochemical response of protein is the cornerstone for the development of the third generation of electrochemical biosensors. In this work, we developed a nanocluster-assisted protein-film voltammetry technique (NCA-PFV) which can achieve the acquisition of the electrochemical signal and maintain the activity without affecting of the protein's structure. With this strategy, a lipid bilayer membrane is used to immobilize the membrane protein so as to maintain its natural state. Copper nanoclusters with a size smaller than most proteins are then used to function at sub-protein scale and to mediate the electron hopping from the electroactive center of the electrode. As a model, the direct electrochemical signal of cyclooxygenase (COX) is successfully obtained, with a pair of well-defined redox peaks located at -0.39mV and -0.31mV, which characterize the heme center of the enzyme. Its catalytic activity towards the substrate arachidonic acid (AA) is also retained. The detection range for AA is 10-1000μM and the detection limit is 2.4μM. Electrochemical monitoring of the regulation of the catalytic activity by an inhibitor DuP-697 is also achieved. This work provides a powerful tool for the fabrication of enzyme-based electrochemical biosensors, and is also of great significance for promoting the development and application of next-generation electrochemical biosensors.

Sensitive and non-invasive cholesterol determination in saliva via optimization of enzyme loading and platinum nano-cluster composition.

An excessive cholesterol level can lead to cardiovascular diseases, such as stroke, hypertension, and myocardial infarction. A non-invasive, painless method of determining the cholesterol level in blood would improve the user's convenience. To provide rapid and accurate determination of cholesterol, we have developed a simple, disposable, enzyme-based electrochemical biosensor that can detect salivary cholesterol. It is possible to detect low concentrations of cholesterol in saliva using the optimized vertical structure of the platinum nano-cluster (Pt-NC) and the immobilization of a proper volume of an enzyme. The biosensor exhibited a linear range from 2 to 486 μM, the limit of detection was about 2 μM, and the sensitivity of the sensor was calculated to be 132 μA mM-1 cm-2. It also showed good specificity for ascorbic acid, uric acid, dopamine, glucose, and lactate. In a test with an actual sample, the performance of the biosensor was confirmed by measuring total cholesterol in the saliva of a patient with hyperlipidemia. The cholesterol levels measured in the saliva of three patients with hyperlipidemia were 520, 460, and 290 μM. Therefore, the Pt-NC based enzyme sensor is a promising candidate for the detection of cholesterol in human saliva.

Review—Pencil Graphite Electrodes as Platform for Enzyme and Enzyme-Like Protein Immobilization for Electrochemical Detection

Carbon-based electrodes are being used widely nowadays for biosensor applications, primarily owing to their good electrical conductivity and ease of functionalization. At the same time, the increasing demand for the low cost, disposable and the ease of availability for the do-it-yourself assemblies have provided an opportunity to look beyond conventional carbon materials for electrochemical analysis. In recent time, the pencil lead, entitled as the pencil graphite has been used as an electrode for the enzyme-based electrochemical biosensors. The review highlights the various aspects involved in using pencil graphite electrode (PGE) as a working electrode. This includes the various pretreatment strategies used, which is the first step toward the effective surface functionalization, followed by strategies used for the immobilization of the functional nanomaterials and the enzymes and finally, the integration of the PGE with different types of sensor assemblies. A comprehensive discussion on the latest development in this area also suggests future perspectives based on PGE to develop low-cost point-of-care diagnostics.