Electrochemical Enzyme Biosensors Research Articles

Sensitive Electrochemical Sequential Enzyme Biosensor for Glucose and Starch Based on Glucoamylase- and Glucose Oxidase- Controllably Co-Displayed Yeast Recombinant

Sensitive Electrochemical Sequential Enzyme Biosensor for Glucose and Starch Based on Glucoamylase- and Glucose Oxidase- Controllably Co-Displayed Yeast Recombinant

Core‐shell Hierarchical Enzymatic Biosensor Based on Hyaluronic Acid Capped Copper Ferrite Nanoparticles for Determination of Endocrine‐disrupting Bisphenol A

AbstractA novel enzymatic electrochemical biosensor (mCuF/PANI‐nf/HA/Lacc/GCE) was designed for detection of bisphenol A (BPA). The copper ferrite nanoparticles was obtained by co‐precipitation and its surface was modified with ‐NH2 functional organosilane. Polyaniline nanofibers were also synthesized by cyclic voltammetry and characterized by FTIR, XRD, TGA, SEM and TEM, respectively. Then, it was crosslinked with hyaluronic acid as an immobilization matrix for Laccase to adhere to surface of the modified copper ferrites. Cyclic and differential pulse voltammetries were used to evaluate the electrochemical performances of the biosensor, which has a LOD value of 5.40 nM and a LOQ value of 16.20 nM in the 0.01–7.50 μM linear working range. The biosensor was successfully applied for determination of BPA in seawater, canned water and milk samples with recoveries ranging from 96.0 % and 100.7 %. In addition, accuracy of the voltammetric determination method in the real samples was carried out by HPLC and spike/recovery test. The layer‐by‐layer surface modification strategy of the designed mCuF/PANI‐nf/HA/Lacc/GCE biosensor opens a new perspective on both BPA determination and using biopolymer in the structure of enzymatic electrochemical biosensors.

High-Throughput Strategy for Glycine Oxidase Biosensor Development Reveals Glycine Release from Cultured Cells.

Glycine is an important biomarker in clinical analysis due to its involvement in multiple physiological processes. As such, the need for low-cost analytical tools for glycine detection is growing. As a neurotransmitter, glycine is involved in inhibitory and excitatory neurochemical transmission in the central nervous system. In this work, we present a 10 μM Pt-based electrochemical enzymatic biosensor based on the flavoenzyme glycine oxidase (GO) for localized real-time measurements of glycine. Among GO variants at position 244, the H244K variant with increased glycine turnover was selected to develop a functional biosensor. This biosensor relies on amperometric readouts and does not require additional redox mediators. The biosensor was characterized and applied for glycine detection from cells, mainly HEK 293 cells and primary rat astrocytes. We have identified an enzyme, GO H244K, with increased glycine turnover using mutagenesis but which can be developed into a functional biosensor. Noteworthy, a glycine release of 395.7 ± 123 μM from primary astrocytes was measured, which is ∼fivefold higher than glycine release from HEK 293 cells (75.4 ± 3.91 μM) using the GO H244K biosensor.

A novel electrochemical biosensor for bisphenol A detection based on engineered Escherichia coli cells with a surface-display of tyrosinase

Electrochemical enzyme biosensors based on various modified electrodes have been developed for bisphenol A detection. However, enzyme activity loss and high cost limit their application. In this study, an electrochemical biosensor was developed for bisphenol A detection by displaying tyrosinase on the surface of Escherichia coli cells, followed by adsorption onto a glassy-carbon electrode. The biosensor exhibited a linear relationship between the concentration range of 0.01–100 nM BPA (R2 =0.9967) and the current peak. The detection limit was 0.01 nM, which is higher than that of other chemically modified tyrosinase biosensors. For the detection of bisphenol A in tea and juice samples, the fabricated biosensor presented respectable accuracy comparable to that of high-performance liquid chromatography. Furthermore, high recovery rates (97.78%−100.32%) were obtained. Therefore, this biosensor could be a promising and reliable analytical tool for the detection of bisphenol A.

Boronic Acid Substituted Polyaniline Based Enzymatic Biosensor System for Catechol Detection

AbstractIn this research, an electrochemical enzymatic biosensor system for sensitive and selective catechol detection was developed with using boric acid substituted Polyaniline film coated two different electrodes; gold screen‐printed electrode and glassy carbon electrode. Electropolymerization of 3‐aminophenylboronic acid is carried out through various electropolymerization conditions, and the best conditions for biosensors systems are concluded. The developed tyrosinase enzyme‐based biosensor system is used for the determination of catechol. The limit of detections(LOD) of the developed biosensor was 0.25 μM and 1.8 μM with Au SPE and GCE systems, respectively. Additionally, real sample analysis was also performed for controlled catechol added green tea samples.

An Electrochemical Enzyme Biosensor for Ammonium Detection in Aquaculture Using Screen-Printed Electrode Modified by Gold Nanoparticle/Polymethylene Blue.

A SPEC/AuNPs/PMB modified electrode was prepared by electrodeposition and electro-polymerization. The electrochemical behavior of reduced nicotinamide adenine dinucleotide (NADH) on the surface of the modified electrode was studied by cyclic voltammetry. A certain amount of substrate and glutamate dehydrogenase (GLDH) were coated on the modified electrode to form a functional enzyme membrane. The ammonia nitrogen in the water sample could be calculated indirectly by measuring the consumption of NADH in the reaction. The results showed that the strength of electro-catalytic current signal was increased by two times; the catalytic oxidation potential was shifted to the left by 0.5 V, and the anti-interference ability of the sensor was enhanced. The optimum substrate concentration and enzyme loading were determined as 1.3 mM NADH, 28 mM α-Ketoglutarate and 2.0 U GLDH, respectively. The homemade ceramic heating plate controlled the working electrode to work at 37 °C. A pH compensation algorithm based on piecewise linear interpolation could reduce the measurement error to less than 3.29 μM. The biosensor exhibited good linearity in the range of 0~300 μM with a detection limit of 0.65 μM NH4+. Compared with standard Nessler’s method, the recoveries were 93.71~105.92%. The biosensor was found to be stable for at least 14 days when refrigerated and sealed at 4 °C.

Enzymatic electrochemical biosensor for glyphosate detection based on acid phosphatase inhibition.

A novel enzymatic electrochemical biosensor was fabricated for the indirect detection of glyphosate-based acid phosphatase inhibition. The biosensor was constructed on a screen-printed carbon electrode modified with silver nanoparticles, decorated with electrochemically reduced graphene oxide, and chemically immobilized with acid phosphatase via glutaraldehyde cross-linking. We measured the oxidation current by chronoamperometry. The current arose from the enzymatic reaction of acid phosphatase and the enzyme-substrate disodium phenyl phosphate. The biosensing response is a decrease in signal resulting from inhibition of acid phosphatase in the presence of glyphosate inhibitor. The inhibition of acid phosphatase by glyphosate was investigated as a reversible competitive-type reaction based on the Lineweaver-Burk equation. Computational docking confirmed that glyphosate was the inhibitor bound in the substrate-binding pocket of acid phosphatase and that it was able to inhibit the enzyme efficiently. Additionally, the established method was applied to the selective analysis of glyphosate in actual samples with satisfactory results following a standard method.

Efficient acetylcholinesterase immobilization for improved electrochemical performance in polypyrrole nanocomposite-based biosensors for carbaryl pesticide

• Optimized surface conditions for the immobilization of an expressive amount of active acetylcholinesterase. • The carbaryl pesticide detection follows a Langmuir isotherm with high affinity adsorption constant. • Simple and straightforward methodology to immobilize a high density of active acetylcholinesterase. • Acetylcholinesterase with the active site close to the interface enabling a fast direct electron transfer. • Enzymatic electrochemical biosensor with low LOD and high sensitivity toward the determination of carbaryl pesticide. Efficient and portable sensors are required to monitor and evaluate pesticide contaminants in the environment, food supply and biological fluids. In this work, a study of indigo carmine and dodecyl sulphate doped polypyrrole - gold nanoparticle nanocomposite films revealed changes during the polymerization that optimized the surface conditions for the immobilization of acetylcholinesterase enzyme (from electric eel ), leading to improved catalytic performance. The results suggest a similar behavior to AChE from electric ray , as previously reported in a theoretical study, where the active site is orientated closer to the electrode when the enzyme is immobilized on a positively charged surface, promoting faster direct electron transfer. In addition, this interaction allows the immobilization of an expressive amount of active enzymes. The lack of hindrance led to a high binding interaction of the analyte (carbaryl) towards the adsorption sites of the enzyme (Langmuir isotherm constant of 7.39 × 10 8 ). This electrochemical enzymatic biosensor presented linear responses ranging from 0.05 to 0.25 ng mL −1 for carbaryl (a wide spectrum insecticide), with a detection limit of 0.033 ng cm 2 mL −1 , quantification limit of 0.11 ng cm 2 mL -1 and sensitivity of -59.5 × 10 3 A cm -2 mL g −1 . The biosensor showed good intra-electrode repeatability and inter-electrode reproducibility with the relative standard deviation of 1.8 % and 3.7 % towards thiocholine oxidation and carbaryl pesticide detection, respectively. The biosensor stability improved when stored at lower temperature (-15 °C).

Ruthenium‐based Conjugated Polymer and Metal‐organic Framework Nanocomposites for Glucose Sensing

AbstractMany studies have focused on effective ways to exploit enzyme immobilization on an electrode surface to help improve the performance of enzymatic electrochemical biosensors. Herein, a novel glucose sensor was fabricated by immobilizing glucose oxidase (GOx) onruthenium‐based conjugated polymer (CP) and metal‐organic framework (MOF) nanocomposites. This has not only reduced the applied potential to 0.2 V (vs. Ag/AgCl), but also improved the effective surface area for enzyme immobilization.PPG@Ru@UiO‐66‐NH2 was tailored by controlled chemical synthesis from a pre‐synthesized water‐soluble conjugated polymer (poly(N‐phenylglycine)) and metal‐organic framework (UiO‐66‐NH2). The resulting nanocomposites were characterized using Fourier transform infrared spectroscopy, X‐ray fluorescence, scanning electron microscopy, and cyclic voltammetry. The PPG@Ru@UiO‐66‐NH2/GOx coated electrodedisplayed a linear measurementrange for glucose from 1 mM to 10 mM, with a sensitivity of 45.92 μA ⋅ mM−1cm−1 and limit of detection of5 μM( ). Furthermore, the practical application of the fabricatedglucosesensor was tested in simulative blood samples with satisfactoryaccuracy. This approach alsoopens a new door for applications regarding both enzymatic electrochemical biosensors and enzymatic biofuel cells (EBFCs).

Nitrogen-Doped Graphdiyne as a Robust Electrochemical Biosensing Platform for Ultrasensitive Detection of Environmental Pollutants.

Owing to its unique chemical structure, natural pores, high structure defects, good surface hydrophilicity and biocompatibility, and favorable electrical conductivity, nitrogen-doped graphdiyne (NGDY) has been attracting attention in the application of electrochemical sensing. Taking advantage of these fascinating electrochemical properties, for the first time, two types of electrochemical enzymatic biosensors were fabricated for the respective detection of organophosphorus pesticides (OPs) and phenols based on the immobilization of acetylcholinesterase or tyrosinase with NGDY. Results revealed that the sensitivities of the NGDY-based enzymatic biosensors were almost twice higher than that of the matching biosensor in the absence of NGDY, proving that NGDY plays a vital role in immobilizing the enzymes and improving the performance of the fabricated biosensors. The effects of nitrogen doping on improving the biosensing performance were studied in depth. Graphitic N atoms can enhance the electrical conductivity, while imine N and pyridinic N can help to adsorb and accumulate the substance molecules to the electrode surface, all of which contribute to the significantly improved performance. Furthermore, these two types of biosensors also demonstrated excellent reproducibility, high stability, and good recovery rate in real environmental samples, which showed a valuable way for the rapid detection of OPs and phenols in the environment. With these excellent performances, it is strongly anticipated that NGDY has tremendous potential to be applied to many other biomedical and environmental fields.

Natural Molybdenite- and Tyrosinase-Based Amperometric Catechol Biosensor Using Acridine Orange as a Glue, Anchor, and Stabilizer for the Adsorbed Tyrosinase.

To develop a natural mineral-based electrochemical enzyme biosensor, natural molybdenite (MLN), tyrosinase (TYR), and acridine orange (AO) were coadsorbed onto a glassy carbon electrode (GCE). The developed TYR/AO/MLN-GCE-based amperometric TYR biosensor exhibited excellent performance for highly sensitive determination of catechol (linear range, 0.1–80 μM; sensitivity, 0.0315 μA/μM; LOD, 0.029 μM; response time, <4 s) with good reproducibility and good operational and storage stabilities. The electrochemical impedance spectroscopy (EIS) and quartz crystal microbalance with dissipation (QCM-D) revealed interesting roles of AO: (1) an efficient glue for enhancing the amount of the adsorbed TYR on the MLN-GCE, (2) an anchor for efficient orientation of the adsorbed TYR on the MLN-GCE, and (3) a stabilizer providing a suitable microenvironment for the adsorbed TYR on the MLN-GCE surface. This physical adsorption-based AO-coupled enzyme-modification strategy onto natural MLN would be a versatile strategy to develop cost-effective and environment-friendly natural mineral-based electrochemical biosensors and bioelectronic devices.

Recent advances in electrochemical enzymatic biosensors based on regular nanostructured materials

Due to the emergence of nanomaterials, enzymatic biosensors have achieved fast and unprecedented development in biomedicine, agriculture, food and other fields. Recently, an increasing number of studies have verified that regular nanostructures enable the effective promotion of enzymatic reactions and electron transfer because of their superior surface area and conductivity. Therefore, nanostructure control of biosensing materials has become a new research hotspot in the past decade. In this review, we will focus on the recent progress of electrochemical enzymatic biosensors based on regular nanostructured materials, especially emphasizing the preparation methods and advances of these regular nanostructures. In addition, we carefully discuss the advantages and disadvantages of these regular nanostructured materials during the enzymatic detection of various analytes in different fields. Finally, we conclude by identifying the main challenges and prospective research directions for regular biosensing nanomaterials, as well as providing suggestions for the development of high-performance biosensors in the future.

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives.

Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on possible solutions involving the use of nanomaterials to ensuring the biosensors’ selectivity. Overall, the examples presented illustrate the various tools available when developing enzyme biosensors for new applications and stress the necessity to more comprehensively investigate their selectivity and validate the biosensors versus standard analytical methods.

Charge Transfer and Biocompatibility Aspects in Conducting Polymer-Based Enzymatic Biosensors and Biofuel Cells.

Charge transfer (CT) is a very important issue in the design of biosensors and biofuel cells. Some nanomaterials can be applied to facilitate the CT in these bioelectronics-based devices. In this review, we overview some CT mechanisms and/or pathways that are the most frequently established between redox enzymes and electrodes. Facilitation of indirect CT by the application of some nanomaterials is frequently applied in electrochemical enzymatic biosensors and biofuel cells. More sophisticated and still rather rarely observed is direct charge transfer (DCT), which is often addressed as direct electron transfer (DET), therefore, DCT/DET is also targeted and discussed in this review. The application of conducting polymers (CPs) for the immobilization of enzymes and facilitation of charge transfer during the design of biosensors and biofuel cells are overviewed. Significant attention is paid to various ways of synthesis and application of conducting polymers such as polyaniline, polypyrrole, polythiophene poly(3,4-ethylenedioxythiophene). Some DCT/DET mechanisms in CP-based sensors and biosensors are discussed, taking into account that not only charge transfer via electrons, but also charge transfer via holes can play a crucial role in the design of bioelectronics-based devices. Biocompatibility aspects of CPs, which provides important advantages essential for implantable bioelectronics, are discussed.

Cascaded Biocatalysis and Bioelectrocatalysis: Overview and Recent Advances.

Enzyme cascades are plentiful in nature, but they also have potential in artificial applications due to the possibility of using the target substrate in biofuel cells, electrosynthesis, and biosensors. Cascade reactions from enzymes or hybrid bioorganic catalyst systems exhibit extended substrate range, reaction depth, and increased overall performance. This review addresses the strategies of cascade biocatalysis and bioelectrocatalysis for (a) CO2 fixation, (b) high value-added product formation, (c) sustainable energy sources via deep oxidation, and (d) cascaded electrochemical enzymatic biosensors. These recent updates in the field provide fundamental concepts, designs of artificial electrocatalytic oxidation-reduction pathways (using a flexible setup involving organic catalysts and engineered enzymes), and advances in hybrid cascaded sensors for sensitive analyte detection.

Fabrication and application of a novel electrochemical biosensor based on a mesoporous carbon sphere@UiO-66-NH2/Lac complex enzyme for tetracycline detection.

The overuse of tetracycline results in a threat to human, poultry and livestock health. An enzymatic electrochemical biosensor is an ideal alternative method for accurate and rapid tetracycline detection, while the unstable and easily deactivated nature of the enzyme limits its development. To overcome these limitations, a highly sensitive enzymatic electrochemical biosensor for the determination of tetracycline is developed in this work based on a complex enzyme which was constructed using a mesoporous carbon sphere@UiO-66-NH2 (MCS@UiO-66-NH2) core-shell composite with embedded laccase (Lac). Compared to pure MCS and UiO-66-NH2, the MCS@UiO-66-NH2 core-shell composite has an advantageous mesoporous structure (pore diameter >8 nm), which is suitable for the immobilization of small laccase. The biosensor based on the complex enzyme exhibits a superior activity and enhanced stability as compared with that made using a pure enzyme because the mesoporous structure of the MCS@UiO-66-NH2 composite can effectively protect the laccase against inactivation and denaturation. Besides, its high specific surface area and good conductivity are beneficial to enzyme immobilization and electron transfer in the modified electrode. The biosensor based on this complex enzyme exhibits a relatively low detection limit of 8.94 × 10-7 mol L-1 and a detection range of 1.0 × 10-6-6.0 × 10-5 mol L-1 for tetracycline detection. Furthermore, the developed biosensor possesses good long-term stability, selectivity and reproducibility, indicating its potential application for tetracycline determination in actual food. This research work provides a prospective solution to resolve the stability and inactivation problems of enzymatic electrochemical biosensors in different application scenarios.

Development of enzymatic electrochemical biosensors for organophosphorus pesticide detection

The enzymatic electrochemical biosensor has the advantages of simple operation, speed, and integration in the detection of organophosphorus pesticide (OPs) residues. It has the potential to become the best alternative to the traditional OP detection technology. This article introduces the OP identification principle of different enzymes, the OP detection mechanism of several common sensors, and the enzyme assembly method. In addition, the article discusses application of nanomaterials in sensor preparation and sensor performance parameters in the past decade. The related content of early sensors is outside the scope of this article.

An Enzymatic Electrochemical Biosensor for Real-Time Detection of Physiologically Relevant Nicotine Concentrations

The glucose biosensor, built upon the redox enzyme glucose oxidase, is the most commercially successful and studied enzymatic biosensor. However, the lack of available and functionally validated enzymes is prohibiting the development of redox-based sensors for other important analytes. Herein, we present the development and assessment of an electrochemical nicotine biosensor, using genomic screening to identify the gene for a known nicotine catabolizing redox enzyme. The resulting nicotine biosensor demonstrated a specific, sensitive, and stable operational profile with a limit of detection of 27 μM over the range of 0-200 μM. This range is well within the physiological concentrations of nicotine present in smoker urine. Specificity and cross-reactivity were measured against structurally similar compounds to nicotine as well as to known physiological by-products. Our results highlight that this novel enzymatic electrochemical nicotine biosensor possesses operational capabilities for monitoring of nicotine in physiologically relevant conditions. The screening methodology can be generalized for the discovery of enzymes for novel sensor development.

Cellulose acetate–chitosan based electrospun nanofibers for bio-functionalized surface design in biosensing

Production of multifunctional biomaterials in nanoscale is one of the most popular topics in nanotechnology related to biological applications because of its biocompatibility and biodegradability. Herein, cellulose acetate (CA) was blended with chitosan (CS) for the formation of non-soluble electrospun nanofibers in aqueous medium. Amine-functionalized nanofiber surfaces were obtained using carbohydrates, which is an advantage for biomolecule immobilization by covalent bonds. Different solvent systems and CA–CS feeding ratio were examined to obtain ultrafine non-beaded nanofibers. After showing the surface characteristics of CA–CS nanofibers, glucose oxidase (GOx) was immobilized on CA–CS coated electrode by covalent conjugation. Cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy measurements were carried out to prove the surface modification. Finally, CA–CS/GOx platform was investigated to detect glucose in samples and some potential interfering compounds were tested to find out the performance of CA–CS/GOx. CA–CS electrospun nanofibers were well-characterized and applied successfully to fabricate repeatable electrochemical enzymatic biosensor for the first time in this paper.

Electrochemical Enzymatic Biosensing of Neotame Supported by Computational Methods

AbstractHerein a highly sensitive electrochemical enzymatic biosensor for the detection of neotame in soft drinks was developed based on multiwalled carbon nanotubes (MWCNTs) decorated with aloe vera‐derived gold nanoparticles (AuNPs) and carboxyl esterase (CaE) enzyme to support the wave strengthening strategy. The electrochemical biosensor revealed high sensitivity with a limit of detection (LOD) and limit of quantification (LOQ) of 27 μg L−1 and 83 μg L−1 respectively. The calibration curve showed a linear dependence of the cathodic peak current on the neotame concentration profile with a correlation factor of 0.9829, signifying an improved electrocatalytic property of the electrode. The good feasibility of the proposed strategy was confirmed by assessing the interactions between the analyte and the enzyme using computational tools. The density functional theory (DFT) calculations of neotame reveal a HOMO‐LUMO energy gap of −0.46618 eV, suggesting a great tendency to act as an electron donor. Furthermore, adsorption and analyte‐enzyme docking studies were carried out to better understand the redox mechanisms. These results revealed that neotame form hydrogen bonds with LEU 249 and GLU251 and other amino acids of the hydrophobic channel of the binding sites, thereby facilitating the redox reaction for the detection of neotame. These outcomes confirm that the aloe vera‐derived AuNPs are good platforms for immobilizing CaE because of its high surface area, promoting the electron transfer from neotame to form an enzyme‐substrate complex, contributing to an enhanced biosensing signal. Furthermore, the enhanced catalytic activity of this enzymatic biosensor for the detection of neotame in soft drinks has huge potential in the food and beverage industry.