Electrochemical Enzyme Biosensors Research Articles

Electrochemical enzyme biosensors based on calcium phosphate materials for tyramine detection in food samples

Electrochemical tyrosinase biosensors for tyramine determination were developed by the immobilization of the enzyme in calcium phosphate materials (CaPs) followed by cross-linking with glutaraldehyde. Tyramine was detected by the electrochemical reduction at −0.1V of the o- enzymatically-formed dopaquinone. Three different CaPs were explored as immobilization systems, monetite, brushite and brushite cement. Biosensors based on brushite matrices provide better analytical properties than the monetite one. Compared to brushite, a 10-fold increase of sensitivity was obtained with the brushite cement-based biosensor, which highlights the effect of brushite crystal formation in the presence of the enzyme in the biosensor performance. Several variables involved in the enzyme immobilization method such as glutaraldehyde cross-linking time, PPO/brushite ratio and thickness of the brushite-enzyme film were investigated. Furthermore, the effects of pH and temperature on biosensor performance were also optimized.Brushite cement-PPO-GA biosensor resulted in a reliable, highly sensitive, fast, inexpensive and easy analytical method for tyramine detection. Under optimal conditions (time of 15min, a ratio of 1.0 and 50μg of the brushite-enzyme mixture, 20°C and pH 6,0), a linear range of 5.8 × 10−7 to 1.6 × 10−5, sensitivity 1.50 × 103mAM−1 cm−2, detection limit, 4.85 × 10−8M and a response time, 6s were obtained. The suitability of the proposed biosensor to determine the tyramine content in cheese samples has been explored. The mean analytical recovery of added tyramine in gouda and brie cheeses were found to be 95.5±5.8 and 96.9±7.5 respectively. A study of the tyramine content evolution over the course of a week under inadequate storage showed the importance of monitoring the degradation of certain foods.

An origami paper device for complete elimination of interferents in enzymatic electrochemical biosensors

Many efforts have been made to prevent interferences in enzymatic electrochemical biosensors by permselective membranes or mediators with low redox potential. However, it is difficult to completely eliminate interferents without compromised sensitivity by these traditional procedures. We propose here a method based on an origami paper device that separates the electrochemical reactions of interferents and substrates for complete depletion of interferents and precise analysis of substrates. Interferents such as ascorbate, urate and paracetamol were completely consumed by a simple electrolysis step, while substrates were quantitatively analyzed by coulometry. With GOx as a model enzyme, an interference-free and calibration-free coulometric glucose biosensor has been demonstrated successfully. The proposed origami paper device provides a facile and easy-controlled approach to eliminate the electroactive interferents completely for enzymatic electrochemical biosensors.

Facile fabrication of electrochemical ZnO nanowire glucose biosensor using roll to roll printing technique

A three electrode electrochemical enzymatic biosensor consisting of ZnO nanowires was successfully fabricated using flexographic printing technique. The incorporation of ZnO nanowires at the working electrode provides advantages such as simple functionalization and high surface area for enhanced sensitivity. The flexographic printing technique allows ultra-high throughput and low cost mass production of devices due to the roll-to-roll nature of the technique. Therefore, the techniques developed here are prudent to the development of technologies capable of meeting the vast market demand for biosensing. Carbon electrodes, silver/silver chloride reference electrodes and ZnO seed layer precursors were directly printed onto a flexible plastic substrate through flexographic printing. The printing process was optimised to allow a suitable seed layer to be formed on the porous printed-carbon electrode to allow selective growth of ZnO nanowires using a hydrothermal growth method. The ZnO nanowires were subsequently functionalised with glucose oxidase, which was used in this work to form a glucose sensor as an exemplary use of the device. The fabricated nanowire electrochemical biosensing devices showed a typical sensitivity of 1.2±0.2μAmM−1cm−2 with a linear response to the addition of glucose over a concentration range of 0.1mM to 3.6mM.

Synthesis of gold nanoparticles supported on functionalized nanosilica using deep eutectic solvent for an electrochemical enzymatic glucose biosensor.

Engineering of nanoparticle (NP) surfaces offers an effective approach for the development of enzymatic biosensors or microbial fuel cells with a greatly enhanced direct electron transport process. However, lack of control over the surface functionalization process and the operational instability of the immobilized enzymes are serious issues. Herein, we demonstrate a facile and green deep eutectic solvent (DES)-mediated synthetic strategy for efficient amine-surface functionalization of silicon dioxide and to integrate small gold nanoparticles (AuNPs) for a glucose biosensor. Owing to the higher viscosity of the DES, it provides uniform surface functionalization and further coupling of the AuNPs on the SiO2 support with improved stability and dispersion. The amine groups of the functionalized Au-SiO2NPs were covalently linked to the FAD-center of glucose oxidase (GOx) through glutaraldehyde as a bifunctional cross-linker, which promotes formation of "electrical wiring" with the immobilized enzymes. The Au-SiO2NP/GOx/GC electrode exhibits direct electron transfer (DET) for sensing of glucose with a sensitivity of 9.69 μA mM-1, a wide linear range from 0.2 to 7 mM and excellent stability. The present green DES-mediated synthetic approach expands the possibilities to support different metal NPs on SiO2 as a potential platform for biosensor applications.

Decoration of reduced graphene oxide with rhodium nanoparticles for the design of a sensitive electrochemical enzyme biosensor for 17β-estradiol

A novel nanocomposite material consisting of reduced graphene oxide/Rh nanoparticles was prepared by a one-pot reaction process. The strategy involved the simultaneous reduction of RhCl3 and graphene oxide with NaBH4 and the in situ deposition of the metal nanoparticles on the 2D carbon nanomaterial planar sheets. Glassy carbon electrode coated with this nanocomposite was employed as nanostructured support for the cross-linking of the enzyme laccase with glutaraldehyde to construct a voltammperometric biosensor for 17β-estradiol in the 0.9–11 pM range. The biosensor showed excellent analytical performance with high sensitivity of 25.7AµM−1cm−1, a very low detection limit of 0.54pM and high selectivity. The biosensor was applied to the rapid and successful determination of the hormone in spiked synthetic and real human urine samples.

New CNT/poly(brilliant green) and CNT/poly(3,4-ethylenedioxythiophene) based electrochemical enzyme biosensors

A combination of the electroactive polymer poly(brilliant green) (PBG) or conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) with carbon nanotubes to obtain CNT/PBG and CNT/PEDOT modified carbon film electrodes (CFE) has been investigated as a new biosensor platform, incorporating the enzymes glucose oxidase (GOx) as test enzyme, alcohol oxidase (AlcOx) or alcohol dehydrogenase (AlcDH). The sensing parameters were optimized for all biosensors based on CNT/PBG/CFE, CNT/PEDOT/CFE platforms. Under optimized conditions, both GOx biosensors exhibited very similar sensitivities, while in the case of AlcOx and AlcDH biosensors, AlcOx/CNT/PBG/CFE was found to give a higher sensitivity and lower detection limit. The influence of dissolved O2 on oxidase-biosensor performance was investigated and was shown to be different for each enzyme. Comparisons were made with similar reported biosensors, showing the advantages of the new biosensors, and excellent selectivity against potential interferents was successfully demonstrated. Finally, alcohol biosensors were successfully used for the determination of ethanol in alcoholic beverages.

Detection of Volatile Plant Chemicals Using Novel Enzymatic Electrochemical Biosensor

Volatile organic compounds released by pathogen-infected and pest-infested plants can be used as fingerprints for plant disease and infestation detection via electronic nose technologies 1,2. p-ethylphenol (EP) and methyl salicylate (MeSA) were discovered as two most important volatile organic compounds. In this work, enzymatic based electrochemical biosensors were developed for the detection of EP and MeSA. For EP detection, a tyrosinase (TYR)-immobilized amperometric biosensor was developed. Cyclic voltammetry (CV) was used to study the electrochemical reaction. CV indicated that the sensitivity reached to 8.528 µA·cm-2·µM while limit of detection (LOD) reached to 0.210 µM. Steady state constant potential amperometry displayed lower sensitivity (4.052 µA·cm-2·µM), but better LOD (0.095 µM) than CV. For MeSA detection, alcohol oxidase and horseradish peroxidase were applied for detection of methanol that produced after hydrolysis of MeSA. CV and constant potential amperometry techniques were used to determine sensitivity of 0.112 and 0.283 µA·cm-2·µM, and LOD of 23 and 1 µM for the CV and DPV respectively. In order to obtain better sensitivity and higher specificity for MeSA detection, salicylate hydroxylase (SH) and TYR were immobilized on the multiwalled carbon nanotube modified glassy-carbon electrode. After hydrolysis of MeSA, salicylate reacts with SH and TYR to generate amperometric signal. The sensitivity and LOD generated from CV were reported to be 21.29 µA·cm-2·µM and 0.137 µM respectively. Constant potential amperometry was also applied for MeSA detection. The results display that sensitivity of 30.61 µA·cm-2·µM and LOD of 0.013 µM can be achieved. Interference and simulated analyte based studies indicate that the biosensor is suitable for application in agricultural sensing. Reference: T. Baldwin, A. Kessler and R. Halitschke, Curr. Opin. Plant Biol., 2002, 5, 351.P. W. Par´e and J. H. Tumlinson, Plant Physiol., 1999, 121, 325. Figure 1

An intimately bonded titanate nanotube–polyaniline–gold nanoparticle ternary composite as a scaffold for electrochemical enzyme biosensors

In this work, titanate nanotubes (TNTs), polyaniline (PANI) and gold nanoparticles (GNPs) were assembled to form a ternary composite, which was then applied on an electrode as a scaffold of an electrochemical enzyme biosensor. The scaffold was constructed by oxidatively polymerising aniline to produce an emeraldine salt of PANI on TNTs, followed by gold nanoparticle deposition. A novel aspect of this scaffold lies in the use of the emeraldine salt of PANI as a molecular wire between TNTs and GNPs. Using horseradish peroxidase (HRP) as a model enzyme, voltammetric results demonstrated that direct electron transfer of HRP was achieved at both TNT-PANI and TNT-PANI-GNP-modified electrodes. More significantly, the catalytic reduction current of H2O2 by HRP was ∼75% enhanced at the TNT-PANI-GNP-modified electrode, compared to that at the TNT-PANI-modified electrode. The heterogeneous electron transfer rate constant of HRP was found to be ∼3 times larger at the TNT-PANI-GNP-modified electrode than that at the TNT-PANI-modified electrode. Based on chronoamperometric detection of H2O2, a linear range from 1 to 1200 μM, a sensitivity of 22.7 μA mM−1 and a detection limit of 0.13 μM were obtained at the TNT-PANI-GNP-modified electrode. The performance of the biosensor can be ascribed to the superior synergistic properties of the ternary composite.

Recent advances in electrospun metal-oxide nanofiber based interfaces for electrochemical biosensing

Synthesis of various electrospun metal-oxide nanofibers and their application towards electrochemical enzymatic and enzyme-free biosensor platforms has been critically discussed.

Enzymatic electrochemical biosensor for urea with a polyaniline grafted conducting hydrogel composite modified electrode

A new conducting polymer hydrogel (CPH) comprising polyaniline grafted polyvinyl alcohol–polyacrylamide ensured high enzyme loading and urea sensitivity.

Enzyme Functionalized Metal Nanostructures for Enhanced Electrochemical Detection of Lactate

Lactate is a key molecule in the anaerobic energy system of humans, primarily considered as the biomarker for tissue oxidative stress. Heavy workout, sepsis, trauma and cancer cause excess lactate production in bio-fluids including sweat. Therefore, lactate measurement is integral for clinical emergencies, sports and general medicine. An enzymatic electrochemical biosensor comprising lactate oxidase immobilized on metal nanostructures is developed here for continuous monitoring of lactate. The sensing mechanism entails detection of H2O2 produced during enzymatic oxidation of lactate. Three kinds of metal nanostructures, viz. gold, copper, and platinum were tested as catalysts for electrochemical oxidation of H2O2to increase the electro-activity and detection sensitivity. Scanning electron microcopy and cyclic voltametry were used to characterize the nano-structured biosensors. Results show that the platinum nanoflakes decorated electrode provides a better sensitivity and detection limit towards lactate detection by providing favorable microenvironment to retain the bioactivity of the enzyme.

Mesoporous Materials‐Based Electrochemical Enzymatic Biosensors

AbstractThis review (239 references) is dealing with the use of mesoporous materials in the field of electrochemical enzymatic biosensors. After a brief discussion on the interest of mesoporous materials for electrochemical biosensing, a presentation of the various types of inorganic and organic‐inorganic hybrid mesostructures used to date in the elaboration of enzymatic bioelectrodes is given and the main bioelectrode configurations are described. The various biosensor applications are then summarized on the basis of a comprehensive table and some representative illustrations. The main detection schemes developed in the field are based on amperometry and mediated electrocatalysis, as well as some on spectroelectrochemistry.

A Layer‐by‐Layer Biosensing Architecture Based on Polyamidoamine Dendrimer and Carboxymethylcellulose‐Modified Graphene Oxide

AbstractA novel nanostructured architecture for the construction of electrochemical enzyme biosensors is here described. It implies the electrostatic layer‐by‐layer assembly of four‐generation ethylenediamine core polyamidoamine G‐4 dendrimers on glassy carbon electrodes coated with a graphene oxide‐carboxymethylcellulose hybrid nanomaterial. This modified surface was further employed for the covalent immobilization of the model enzyme tyrosinase through a glutaraldehyde‐mediated cross‐linking. The prepared enzyme electrode allowed the amperometric detection of catechol in the 2–400 nM range. The biosensor showed excellent analytical performance with high sensitivity of 6.3 A/M and low detection limit of 0.9 nM. The enzyme electrode retained over 93 % of the initial activity after 40 days at 4 °C.

An enzymatic glucose biosensor based on a glassy carbon electrode modified with cylinder-shaped titanium dioxide nanorods

We describe a highly sensitive electrochemical enzymatic glucose biosensor. A glassy carbon electrode was modified with cylinder-shaped titanium dioxide nanorods (TiO2-NRs) for the immobilization of glucose oxidase. The modified nanorods and the enzyme biosensor were characterized by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The glucose oxidase on the TiO2-NRs displays a high activity and undergoes fast surface-controlled electron transfer. A pair of well-defined quasi-reversible redox peaks was observed at −0.394 and −0.450 V. The TiO2-NRs provide a good microenvironment to facilitate the direct electron transfer between enzyme and electrode surface. The biosensor has two linear response ranges, viz. from 2.0 to 52 μM, and 0.052 to 2.3 mM. The lower detection limit is 0.5 μM, and the sensitivity is 68.58 mA M−1 cm−2. The glucose biosensor is selective, well reproducible, and stable. In our perception, the cylindrically shaped TiO2-NRs provide a promising support for the immobilization of proteins and pave the way to the development of high-performance biosensors.

Gold nanoparticles coated zinc oxide nanorods as the matrix for enhanced l-lactate sensing

In this study, an enzymatic electrochemical biosensor for l-lactate detection was proposed. The device was developed based on gold nanoparticles (Au NPs) modified zinc oxide nanorods (ZnO NRs). The sensing performance of the device was examined by cyclic voltammetry and amperometry. Compared with pristine ZnO based biosensor, Au/ZnO based sensor exhibited higher sensitivity of 24.56μAcm−2mM−1, smaller KMapp of 1.58mM, lower detection limit of 6μM and wider linear range of 10μM–0.6mM for l-lactate detection. The introduction of Au NPs enhances electro-catalytic ability and electron migration, which contributes to the improvement of the sensing performance. Hence, the results confirm the essential character of Au NPs in such semiconductor based electrochemical biosensing system.

Enzymatic electrochemical glucose biosensors by mesoporous 1D hydroxyapatite-on-2D reduced graphene oxide.

A novel hydrothermal process was used for the preparation of hydroxyapatite (HAp) nanorods on two-dimensional reduced graphene oxides (RGO). The hydrothermal reaction temperature improves the crystallinity of HAp and partially reduces graphene oxide (GO) to RGO. The crystalline structure, chemical composition and morphology of the prepared nanocomposites were characterized by using various analytical techniques. Nanorods of HAp with a diameter and length of ∼32 and 60-85 nm were grown on basal planes and edges of the layered RGO sheets. The estimated specific surface area and pore-size distribution are 120 m2 g-1 and 5.6 nm, respectively. We also report the direct electrochemistry of glucose oxidase (GOx) on 1D HAp-on-2D RGO nanocomposite-modified glassy carbon electrode (GCE) for glucose sensing. The electrocatalytic and electroanalytical applications of the proposed RGO/HAp/GOx-modified GCE were studied by cyclic voltammetry (CV) and amperometry. The increased electron rate constant of 3.50 s-1 was obtained for the modified GCE. The reported biosensor exhibits a superior detection limit and higher sensitivity ca. 0.03 mM and 16.9 μA mM-1 cm-2, respectively, with a wide linear range of 0.1-11.5 mM. The tremendous analytical parameters of the reported sensor surpass those of related modified electrodes and are promising for practical industrial applications.

Decorating graphene oxide/nanogold with dextran-based polymer brushes for the construction of ultrasensitive electrochemical enzyme biosensors.

A novel strategy was employed to prepare a water-soluble graphene derivative by using dextran-based polymer brushes as solubilizing agents. Graphene oxide was grafted with (3-mercaptopropyl) trimethoxysilane and further decorated with Au nanoparticles. This hybrid nanomaterial was then reduced and anchored with polysaccharide-based polymer brushes by chemisorption of an end-group thiolated dextran derivative on the Au nanoparticles. The resulting hybrid nonmaterial allowed highly stable aqueous dispersions to be obtained, which were used to coat glassy carbon electrodes for the preparation of a model tyrosinase electrochemical biosensor for catechol. The enzyme electrode showed excellent electroanalytical performance with fast response in about 5 s, a linear range of 100 pM-120 nM, a very high sensitivity of 45.9 A M-1 and a very low detection limit of 40 pM for catechol.

Electrochemical enzymatic biosensors based on metal micro-/nanoparticles-modified electrodes: a review

AbstractThe functions of metal structures of micro- or nano-dimensions in the sensing mechanisms of amperometric enzyme-based biosensors are considered in the light of the principles of detection of the latter. The applications of metal mono- or bimetallic nanoparticles-modified materials as catalytic electrodes in the fabrication of first-generation and the role which metal nanoparticles play in promoting or enhancing the electron transfer rates in third-generation electrochemical biosensors are reviewed. Some examples of gold NPs functionalised with enzymes via gold-thiol chemistry as a strategy for enzyme immobilisation and spatial orientation when developing amperometric biosensors are also discussed.

Electronically type-sorted carbon nanotube-based electrochemical biosensors with glucose oxidase and dehydrogenase.

An electrochemical enzyme biosensor with electronically type-sorted (metallic and semiconducting) single-walled carbon nanotubes (SWNTs) for use in aqueous media is presented. This research investigates how the electronic types of SWNTs influence the amperometric response of enzyme biosensors. To conduct a clear evaluation, a simple layer-by-layer process based on a plasma-polymerized nano thin film (PPF) was adopted because a PPF is an inactive matrix that can form a well-defined nanostructure composed of SWNTs and enzyme. For a biosensor with the glucose oxidase (GOx) enzyme in the presence of oxygen, the response of a metallic SWNT-GOx electrode was 2 times larger than that of a semiconducting SWNT-GOx electrode. In contrast, in the absence of oxygen, the response of the semiconducting SWNT-GOx electrode was retained, whereas that of the metallic SWNT-GOx electrode was significantly reduced. This indicates that direct electron transfer occurred with the semiconducting SWNT-GOx electrode, whereas the metallic SWNT-GOx electrode was dominated by a hydrogen peroxide pathway caused by an enzymatic reaction. For a biosensor with the glucose dehydrogenase (GDH; oxygen-independent catalysis) enzyme, the response of the semiconducting SWNT-GDH electrode was 4 times larger than that of the metallic SWNT-GDH electrode. Electrochemical impedance spectroscopy was used to show that the semiconducting SWNT network has less resistance for electron transfer than the metallic SWNT network. Therefore, it was concluded that semiconducting SWNTs are more suitable than metallic SWNTs for electrochemical enzyme biosensors in terms of direct electron transfer as a detection mechanism. This study makes a valuable contribution toward the development of electrochemical biosensors that employ sorted SWNTs and various enzymes.

The electrochemical enzymatic glucose biosensor based on mesoporous carbon fibers activated by potassium carbonate

Glucose-sensing electrodes have fabricated using mesoporous electro-spun carbon fibers, which have mesopores mainly and more oxygen functional groups on the surface, activated by potassium carbonate (K2CO3) with different concentrations. These porosity and the functional groups on the carbon fibers affected the immobilization of glucose oxidase on the electrode. The sensitivity of the electrode is determined based on enzymatic activity increasing glucose concentrations and calculated by enzyme kinetics using the Michaelis–Menten equation. The electrode prepared from carbon fibers activated with 4MK2CO3, which has the highest mesopore volume and specific surface area, has the sensitivity 3.4μAmM−1cm−2 and the broad linear range of up to 20mM.