Enzyme-modified Electrode Research Articles

Glucose Biosensors - State of the Art and Prospects

Rapid glucose concentration detection in technical and biological systems is an important scientific and technical task of modern chemistry, engineering and technology. The article provides an overview of the last technical solutions in this area. The issues of developing first generation biosensors are considered. However, the main disadvantage of such systems was the significant influence of ascorbic and uric acids on the generated signal, which significantly reduced their selectivity and accuracy. To solve this problem, it is possible to use ion-selective membranes such as Nafion and polycarbonate. The second generation of glucose biosensors uses artificial mediators to facilitate electron transfer between the enzyme and the electrode. These mediators can be immobilized directly by the enzyme or introduced into an enzyme-modified electrode. Suitable mediators include conducting organic salts, ferrocene, quinone compounds, ferricyanide, transition metal complexes, phenothiazine and foxazine compounds. Effective interactions between enzymes and mediators are critical for efficient electron transport. Various approaches have been proposed to tailor mediators, such as the use of Os complexes, noncovalent functionalization of carbon nanotubes, and stabilization of artificial mediators. The third generation of enzyme glucose biosensors uses direct electron transfer to perform electrochemical reduction. Various approaches have been considered, including reassembling apoproteins on cofactor-modified enzymes and electrically coupling enzymes to electrode surfaces using redox polymers or nanomaterials such as gold nanoparticles. Such approaches ensure the formation of an effective enzyme-electrode bond. In addition, the thickness of the enzymatic layer can affect the performance of the biosensor. External factors such as temperature, pH and humidity can have a significant impact on the performance of such electrodes.

Electrochemical Biosensor Based on Chitosan- and Thioctic-Acid-Modified Nanoporous Gold Co-Immobilization Enzyme for Glycerol Determination

An electrochemical biosensor based on chitosan- and thioctic-acid-modified nanoporous gold (NPG) co-immobilization glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) was constructed for glycerol determination in wine. The NPG, with the properties of porous microstructure, large specific surface area, and high conductivity, was beneficial for protecting the enzyme from inactivation and denaturation and enhancing electron transfer in the modified electrode. The co-immobilization of the enzyme by chitosan-embedding and thioctic-acid-modified NPG covalent bonding was beneficial for improving the catalytic performance and stability of the enzyme-modified electrode. Ferrocene methanol (Fm) was used as a redox mediator to accelerate the electron transfer rate of the enzyme-modified electrode. The fabricated biosensor exhibited a wide determination range of 0.1–5 mM, low determination limit of 77.08 μM, and high sensitivity of 9.17 μA mM−1. Furthermore, it possessed good selectivity, repeatability, and stability, and could be used for the determination of glycerol in real wine samples. This work provides a simple and novel method for the construction of biosensors, which may be helpful to the application of enzymatic biosensors in different determination scenarios.

Paper-Based Competitive Immunochromatography Coupled with an Enzyme-Modified Electrode to Enable the Wireless Monitoring and Electrochemical Sensing of Cotinine in Urine

This paper proposes a combined strategy of using paper-based competitive immunochromatography and a near field communication (NFC) tag for wireless cotinine determination. The glucose oxidase labeled cotinine antibody specifically binds free cotinine in a sample, whereas the unoccupied antibody attached to BSA-cotinine at the test line on a lateral flow strip. The glucose oxidase on the strip and an assistant pad in the presence of glucose generated H2O2 and imposed the Ag oxidation on the modified electrode. This enabled monitoring of immunoreaction by either electrochemical measurement or wireless detection. Wireless sensing was realized for cotinine in the range of 100–1000 ng/mL (R2 = 0.96) in PBS medium. Undiluted urine samples from non-smokers exhibited an Ag-oxidation rate three times higher than the smoker’s urine samples. For 1:8 diluted urine samples (smokers), the proposed paper-based competitive immunochromatography coupled with an enzyme-modified electrode differentiated positive and negative samples and exhibited cotinine discrimination at levels higher than 12 ng/mL. This novel sensing platform can potentially be combined with a smartphone as a reader unit.

Diffusion-limited electrochemical d-fructose sensor based on direct electron transfer-type bioelectrocatalysis by a variant of d-fructose dehydrogenase at a porous gold microelectrode

Abstract An electrochemical d -fructose sensor was fabricated by immobilizing a variant of d -fructose dehydrogenase on a porous gold microelectrode. The enzyme-modified electrode bioelectrocatalytically oxidizes d -fructose via direct electron transfer. The catalytic current reaches a steady-state limiting value at 0.05 V vs. Ag|AgCl (sat. KCl). The temperature dependence of the sensor suggests that its response is limited by the diffusion of d -fructose. Therefore, the sensor requires no calibration. The sensitivity of the sensor, (2.0 ± 0.2) × 102 μA cm−2 mM−1, is reproducible. The upper limit of detection is ~2.0 mM and the kinetics of the bioelectrocatalytic reaction is determined the limitation. The fabricated sensors were applied in the determination of d -fructose in commercial beverages as well as honey, and compared favorably with a more commonly used photometric method.

Electrochemical enzyme biosensor for carnitine detection based on cathodic stripping voltammetry

Recently, the amount of carnitine in saliva has been considered a biomarker for pancreatic cancer. Traditional analytical instrument such as liquid chromatography and mass-spectroscopy (MS) has been employed for carnitine detection. Only FET based biosensor was reported for carnitine, but the limit of detection was far above the required concentration. Here, we describe a highly sensitive and selective enzyme biosensor that we have developed for carnitine by using an enzyme-modified electrode combined with cathodic stripping voltammetry. The biosensor exhibits high sensitivity because of the preconcentration on the gold electrode of CoA, which was generated by the enzymatic reaction between carnitine and acetyl coenzyme A (acetyl-CoA) with carnitine acetyltransferase. Although both acetyl-CoA and CoA can adsorb on the gold electrode, the potential difference of reductive desorption detects CoA selectively. Under optimized conditions, the biosensor exhibited a linear range from 0.025 to 25 μM with a detection limit (S/N = 3) of 0.025 μM for carnitine determination. Moreover, the recovery experiments determination results show a stable range from 96.6 to 97.2 %. This developed carnitine biosensor has a potential in clinical detection due to the excellent performance.

Epidermal Microfluidic Electrochemical Detection System: Enhanced Sweat Sampling and Metabolite Detection.

Despite tremendous recent efforts, noninvasive sweat monitoring is still far from delivering its early analytical promise. Here, we describe a flexible epidermal microfluidic detection platform fabricated through hybridization of lithographic and screen-printed technologies, for efficient and fast sweat sampling and continuous, real-time electrochemical monitoring of glucose and lactate levels. This soft, skin-mounted device judiciously merges lab-on-a-chip and electrochemical detection technologies, integrated with a miniaturized flexible electronic board for real-time wireless data transmission to a mobile device. Modeling of the device design and sweat flow conditions allowed optimization of the sampling process and the microchannel layout for achieving attractive fluid dynamics and rapid filling of the detection reservoir (within 8 min from starting exercise). The wearable microdevice thus enabled efficient natural sweat pumping to the electrochemical detection chamber containing the enzyme-modified electrode transducers. The fabricated device can be easily mounted on the epidermis without hindrance to the wearer and displays resiliency against continuous mechanical deformation expected from such epidermal wear. Amperometric biosensing of lactate and glucose from the rapidly generated sweat, using the corresponding immobilized oxidase enzymes, was wirelessly monitored during cycling activity of different healthy subjects. This ability to monitor sweat glucose levels introduces new possibilities for effective diabetes management, while similar lactate monitoring paves the way for new wearable fitness applications. The new epidermal microfluidic electrochemical detection strategy represents an attractive alternative to recently reported colorimetric sweat-monitoring methods, and hence holds considerable promise for practical fitness or health monitoring applications.

(Invited) Plasma Membrane Cholesterol as a Diagnostic

Cholesterol is a tightly regulated major structural component of the cell plasma membrane (PM) where it forms stoichiometric complexes with phospholipids and sphingolipids. The amount of cholesterol in the PM exhibits a regulatory role in basal activity of several biomolecular processes by direct binding to proteins and by indirect local environmental effects within the PM that are also coupled to overall cellular cholesterol homeostasis. The term “active cholesterol” refers to PM cholesterol not complexed to lipids, a cholesterol state that arises above a threshold mole fraction of cholesterol in the PM. Active cholesterol level in the PM provides a control mechanism for cellular cholesterol homeostasis through its recognition by membrane bound proteins that activate genes of cholesterol synthesis enzymes. Uptake of LDL, production and release of HDL as well as reversible storage of cholesterol in the cytosol by covalent modification are also regulated and dependent on PM cholesterol (thermodynamic) activity: active cholesterol. A number of human disease states have been found to have associated alterations in PM cholesterol. Thus, an experimental aim of this research group has been the ability to monitor PM cholesterol thermodynamic activity at live cells and tissues without significant perturbation of the native physiological state. In particular, the active cholesterol pool is easily depleted leading to its physiological determination by traditional assays being out of reach. Our approach is to measure the rate of cholesterol diffusion from the PM at the point-of-contact of a microelectrode. The enzyme-modified electrode consumes the sparingly-soluble aqueous-cholesterol adjacent to the PM thus perturbing the equilibrium between aqueous- and PM-cholesterol resulting in diffusion of cholesterol from the PM only at the site of electrode contact. That is, the enzyme modified electrode creates a demand for cholesterol at the site of contact and measures the diffusional response. The rate of cholesterol diffusion (micro-extraction) is related to the thermodynamic activity of cholesterol (which is controlled by the active cholesterol concentration) in the PM by classic kinetic theory. The transition state is cholesterol that has exited the PM and has not yet formed a solvent cage. Through Collaboration with Minchul Shin at Georgia Southern University, ongoing work for human cholesterol sensing is using nano/micro manufacturing to produce sensor chips containing well-spaced regions of SiO2 that are circular with a 10-20 µm diameter for attachment of oxidase. This general approach to noninvasive human sensing could serve as a platform for screening children for hypercholesterolemia and for other markers such as glucose.

A novel amperometric inhibition biosensor based on HRP and gold sononanoparticles immobilised onto Sonogel-Carbon electrode for the determination of sulphides

ABSTRACTA novel inhibition biosensor used for the detection of sulphides (Na2S) has been developed. The biosensor is based on the immobilisation of horseradish peroxidase (HRP) on the Sonogel-Carbon (SNGC) electrode using glutaraldehyde, Poly(4-vinylpyridine) and gold sononanoparticles (AuSNPs). The Poly(4-vinylpyridine) was used due to its high affinity for sulphide anions, while the presence of gold sononanoparticles enhances the electron transfer reaction and improves the analytical performance of the biosensor. The amperometric measurements were performed at an applied potential of −0.15 V vs. Ag/AgCl in 50 mM sodium acetate buffer solution pH = 6.0. The apparent kinetic parameters (Kmapp, Vmax) of immobilised HRP were calculated in the absence of inhibitor (sulphide) using caffeic acid as substrate. Under the optimal experimental conditions, the determination of sulphide can be achieved in a dynamic range of 0.4–2.8 µM with a low limit of detection of 0.15 µM. The electrochemical impedance spectroscopy (EIS) was also used to characterise the interactions of substrate and inhibitor with the enzyme-modified electrode. The developed biosensor exhibited high sensitivity, selectivity and stability, and can be successfully applied to the detection of sulphide in water.

Catalase‐Modified Carbon Electrodes: Persuading Oxygen To Accept Four Electrons Rather Than Two

We successfully exploited the natural highly efficient activity of an enzyme (catalase) together with carbon electrodes to produce a hybrid electrode for oxygen reduction, very appropriate for energy transformation. Carbon electrodes, in principle, are cheap but poor oxygen reduction materials, because only two-electron reduction of oxygen occurs at low potentials, whereas four-electron reduction is key for energy-transformation technology. With the immobilization of catalase on the surface, the hydrogen peroxide produced electrochemically is decomposed back to oxygen by the enzyme; the enzyme natural activity on the surface regenerates oxygen, which is further reduced by the carbon electrode with no direct electron transfer between the enzyme and the electrode. Near full four-electron reduction of oxygen is realised on a carbon electrode, which is modified with ease by a commercially available enzyme. The value of such enzyme-modified electrode for energy-transformation devices is evident.

Detection of 25-Hydroxyvitamin D3 with an Enzyme modified Electrode

Since testing for circulating vitamin D concentrations is relatively expensive and time consuming, rapid means of measurement are desired. As a step towards this goal, enzyme-modified electrodes responsive to 25-hydroxyvitamin D3 (25(OH)D3) have been developed. To make the enzyme, a synthetic gene encoding a human cytochrome P450 27B1 (CYP27B1) enzyme, which is a mitochondrial type heme-thiolate monooxygenase that converts 25(OH)D3 into 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), was expressed and purified. The CYP27B1 enzyme was combined with NADPH-adrenodoxin reductase (ADR) and adrenodoxin (ADX) and activity was characterized using liquid chromatography mass spectrometry (LC-MS/MS). It was found that dihydroxyvitamin D3 isomers were produced in addition to 1,25(OH)2D3. The enzyme was immobilized on glassy carbon electrodes using pH-adjusted Nafion® along with cobalt sepulchrate trichloride (Co(sep)3+) as a redox mediator and electrode performance was characterized using cyclic and square wave voltammetry. The results demonstrate detection of 25(OH)D3 in buffer within the physiological range (5-200 ng/ml).

The reductive amination of α-ketoisocaproic acid using a leucine dehydrogenase-modified gold electrode

ABSTRACTIn the present paper, the reductive amination of α-ketoisocaproic acid to L-leucine was monitored using a new modified electrode based on gold nanoparticles (AuNPs) and leucine dehydrogenase (LeuDH). The modified electrode was prepared by self-assembling of AuNPs with thiocytosine followed by the immobilization of the enzyme. The enzyme-modified electrode response was investigated by chronoamperometry for indirect quantification of ammonium ion () involved in the reaction. This reaction is catalyzed by LeuDH and takes place in the presence of ammonium chloride, as nitrogen source, and nicotinamide adenine dinucleotide (NADH), as cofactor. The enzyme-modified electrode was also used for in situ electrochemical NADH regeneration. After one step NADH regeneration the conversion of ammonium ion was improved. The modified electrode has good sensitivity (0.91 × 10−3 A/M) and reproducibility with an RSD of 8.4% (n = 4). The experimental determination of ammonium ion was confirmed by standard Berthelot spectrophotometric method.

A Highly Sensitive and Selective Hydrogen Peroxide Biosensor Based on Gold Nanoparticles and Three-Dimensional Porous Carbonized Chicken Eggshell Membrane.

A sensitive and noble amperometric horseradish peroxidase (HRP) biosensor is fabricated via the deposition of gold nanoparticles (AuNPs) onto a three-dimensional (3D) porous carbonized chicken eggshell membrane (CESM). Due to the synergistic effects of the unique porous carbon architecture and well-distributed AuNPs, the enzyme-modified electrode shows an excellent electrochemical redox behavior. Compared with bare glass carbon electrode (GCE), the cathodic peak current of the enzymatic electrode increases 12.6 times at a formal potential of −100mV (vs. SCE) and charge-transfer resistance decreases 62.8%. Additionally, the AuNPs-CESM electrode exhibits a good biocompatibility, which effectively retains its bioactivity with a surface coverage of HRP 6.39×10−9 mol cm−2 (752 times higher than the theoretical monolayer coverage of HRP). Furthermore, the HRP-AuNPs-CESM-GCE electrode, as a biosensor for H2O2 detection, has a good accuracy and high sensitivity with the linear range of 0.01–2.7 mM H2O2 and the detection limit of 3μM H2O2 (S/N = 3).

Detection of hydrogen peroxide and glucose by enzyme product precipitation on sensor surface

AbstractBiocatalysed precipitation of an insoluble product accumulated on the enzyme-modified electrode surface was applied as the amplification path for low concentration sensing of hydrogen peroxide and glucose. Sensitive electrochemical and quartz-crystal microbalance (QCM) biosensors based on biocatalytic precipitation were developed. A horseradish peroxidase (HRP) monolayer-modified electrode was used to sense H

Estimation of Tea Polyphenols by Differential Pulse Voltammetry with Electrodes Modified by Tyrosinase Extracted from Crude Sources

Tea contains various polyphenols exhibiting antioxidant activities. To measure total polyphenol content quantitatively, we purified tyrosinase extracted from crude source such as potato, banana and modified platinum working electrode with this enzyme. Recovery of tyrosinase from potato and banana was achieved by extraction of crude enzyme with phosphate buffer solution (PBS) at pH = 7.4 followed by ammonium sulfate precipitation (20–60%), dialysis against 10 mM PBS at pH = 7.4 and ion exchange chromatography in DEAE-cellulose -52 colunm. The activity of the enzyme was assayed at an optimum pH of 6.8 and a Km of 3.46–11.68 mM was obtained. Differential pulse voltammetry (DPV) was then performed on the enzyme-modified electrode to exploit the oxidation of polyphenols by tyrosinase. Various grades of tea were tested for total polyphenols (TP) and the results were compared with those obtained using commercial enzyme and by HPLC. A very good correlation index is 0.98 was obtained between the TP content by HPLC and that determined by DPV with purified tyrosinase from potato. The methods exhibited very low limits of detection (0.579–0.635 mg/l). The results ensured that DPV could be a useful technique for determination of TP in tea for its quality evaluation.

Structure effect on graphene-modified enzyme electrode glucose sensors

Using structural characterizations and electrochemical measurements, we explored and investigated the effect of the structure of enzyme electrodes with glucose oxidase (GOD) that were modified by reduced graphene oxide (rGO) sheets . The rGO sheets with different defect density, layers, and oxygen concentrations were chosen to modify the enzyme electrode, and all the modified enzyme electrodes exhibited excellent electrocatalytic activities and performances towards glucose. The abundant defects in rGO induce easy absorption of GOD. At a low oxygen concentration, rGO sheets help to induce the direct electron transfer (DET) on the rGO-modified electrode, and at a higher oxygen concentration, the reduction of H2O2 occurred instead of DET on the surface of the rGO-modified electrode. When rGO modified the enzyme electrode under the working model of H2O2 reduction, an increase in the number of the oxygen functional groups could lead to an increase in the absorption of GOD, resulting in the improvement of the affinity and sensitivity of the biosensor. The rGO-modified enzyme electrode can provide faster response, higher sensitivity, and better affinity by optimizing and controlling the structure of graphene and its derivatives.

One-pot preparation of glucose biosensor based on polydopamine–graphene composite film modified enzyme electrode

Abstract A green, facile and low cost approach was developed to fabricate a glucose biosensor based on polydopamine–graphene hybrid film modified glucose oxidase enzyme electrode. Dopamine, graphene oxide and glucose oxidase were mixed and casted on Au electrode, and after electrochemical processing, polydopamine, glucose oxidase and graphene modified electrode was obtained. The as-synthesized enzyme electrodes were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared and X-ray diffraction spectra. The biosensor showed the excellent amperometric biosensing performances, e.g., a high detection sensitivity (28.4 μA mM−1cm−2), a low limit of detection (0.1 μm), a short response periods (

Amperometric carbon paste enzyme electrodes with Fe3O4 nanoparticles and 1,4-Benzoquinone for glucose determination

Two new amperometric carbon paste enzyme electrodes including Fe3O4 nanoparticles with and without 1,4-benzoquinone were developed for glucose determination. Electron transfer properties of unmodified and Fe3O4 nanoparticles and/or 1,4-benzoquinone modified carbon paste electrodes were investigated in 0.1 M KCl support electrolyte containing Fe(CN)63−/4− as redox probe by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) methods. Fe3O4 nanoparticles increased electron transfer at solution/electrode interface. The parameters affecting the analytical performance of the enzyme electrode have been investigated in detail and optimized for Fe3O4 nanoparticle modified enzyme electrode (Fe3O4–CPEE). Fe3O4 nanoparticles and 1,4-benzoquinone modified enzyme electrode (BQ‐Fe3O4‐CPEE) exhibited linear response from 1.9 × 10‐7 M to 3.7 × 10‐6 M, from 7.2 × 10‐6 M to 1.5 × 10‐4 M and from 1.3 × 10‐3 M to 1.2 × 10‐2 M with an excellent detection limit of 1.9 × 10‐8 M. BQ‐Fe3O4‐CPEE was used for determination of glucose in serum samples and results were in good agreement with those obtained by spectrophotometric method.

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM(-1) cm(-2)), low detection limit (25 nM), and fast response time (t(95%) ≈ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH-biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β-anomer of cello-oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real-time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose.

Highly sensitive amperometric detection of bilirubin using enzyme and gold nanoparticles on sol–gel film modified electrode

We describe the development of a simple and highly sensitive electrochemical (amperometric) sensing of bilirubin based on bilirubin oxidase (BOx) incorporated into the gold nanoparticles (AuNPs). This nanoelectrode platform with self-assembled enzyme is highly sensitive toward the electrochemical oxidation of bilirubin and increased the bilirubin concentration linearly from 1 to 5000μM with a correlation coefficient of 0.9960, and an apparent Michaelis constant (KM,app) of 44±0.4μM. Using an amperometric method, the detection limit for bilirubin at the enzyme-modified electrode was 1.4nM (signal-to-noise ratio=3). The modified electrode retained a stable response for 2 days while losing only ca. 3.4% of its initial sensitivity during a 10 days storage period in 0.2M phosphate buffer solution (pH=8.4) at ≤4°C. The practical application of the modified electrode was demonstrated by measuring the concentration of bilirubin in blood serum sample.

Fabrication of CNT/Ferrocene/Glucose Oxidase/Chitosan-Layered Bioanode for Glucose/Oxygen Biofuel Cells

An enzyme-modified electrode was fabricated by entrapping glucose oxidase (GOx) and ferrocene (Fc) onto a multiwall carbon nanotube (MWCNT)-coated electrode. The MWCNT, Fc, GOx, and chitosan (CHI) were sequentially coated on a glassy carbon electrode. The MWCNT/Fc/GOx/CHI electrode was characterized by scanning electron microscopy (SEM), and cyclic voltammetry (CV). The prepared electrode exhibited good electrochemical performance for the glucose analysis with a linear range of 0–60 mM glucose. It was found that the MWCNT film on the electrode remarkably enhanced the performance of the electrode. The MWCNT/Fc/GOx/CHI electrode was integrated with a bilirubin oxidase-immobilized cathode for a biofue cell application. The maximum power density at a glucose concentration of 10 mM was 13 μW/cm2 at a cell voltage of 0.19 V. The results of this study indicate that the MWCNT/Fc/GOx/CHI electrode could be applied in the development of biofuel cells and bisensors.