DNA as a support for glucose oxidase immobilization at Prussian blue-modified glassy carbon electrode in biosensor preparation

An amperometric glucose biosensor has been developed using DNA as a matrix of Glucose oxidase (GOx) at Prussian-blue (PB)-modified glassy carbon (GC) electrode. GC electrode was chemically modified by the PB. GOx was immobilized together with DNA at the working area of the PB-modified electrode by placing a drop of the mixture of DNA and GOx. The response of the biosensor for glucose was evaluated amperometrically. Upon immobilization of glucose oxidase with DNA, the biosensor showed rapid response toward the glucose. On the other hand, no significant response was obtained in the absence of DNA. Experimental conditions influencing the biosensor performance were optimized and assessed. This biosensor offered an excellent electrochemical response for glucose concentration in micro mol level with high sensitivity and selectivity and short response time. The levels of the relative standard deviation (RSDs), (<4%) for the entire analyses reflected a highly reproducible sensor performance. Through the use of optimized conditions, a linear relationship between current and glucose concentration was obtained up to 4 x 10(-4) M. In addition, this biosensor showed high reproducibility and stability.     

A comparative study of enzyme immobilization strategies for multi-walled carbon nanotube glucose biosensors

This work addresses the comparison of different strategies for improving biosensor performance using nanomaterials. Glucose biosensors based on commonly applied enzyme immobilization approaches, including sol-gel encapsulation approaches and glutaraldehyde cross-linking strategies, were studied in the presence and absence of multi-walled carbon nanotubes (MWNTs). Although direct comparison of design parameters such as linear range and sensitivity is intuitive, this comparison alone is not an accurate indicator of biosensor efficacy, due to the wide range of electrodes and nanomaterials available for use in current biosensor designs. We proposed a comparative protocol which considers both the active area available for transduction following nanomaterial deposition and the sensitivity. Based on the protocol, when no nanomaterials were involved, TEOS/GOx biosensors exhibited the highest efficacy, followed by BSA/GA/GOx and TMOS/GOx biosensors. A novel biosensor containing carboxylated MWNTs modified with glucose oxidase and an overlying TMOS layer demonstrated optimum efficacy in terms of enhanced current density (18.3 ± 0.5 μA mM(-1) cm(-2)), linear range (0.0037-12 mM), detection limit (3.7 μM), coefficient of variation (2%), response time (less than 8 s), and stability/selectivity/reproducibility. H(2)O(2) response tests demonstrated that the most possible reason for the performance enhancement was an increased enzyme loading. This design is an excellent platform for versatile biosensing applications.     

Enzyme biosensor based on plasma-polymerized film-covered carbon nanotube layer grown directly on a flat substrate

We report a novel approach to fabrication of an amperometric biosensor with an enzyme, a plasma-polymerized film (PPF), and carbon nanotubes (CNTs). The CNTs were grown directly on an island-patterned Co/Ti/Cr layer on a glass substrate by microwave plasma enhanced chemical vapor deposition. The as-grown CNTs were subsequently treated by nitrogen plasma, which changed the surface from hydrophobic to hydrophilic in order to obtain an electrochemical contact between the CNTs and enzymes. A glucose oxidase (GOx) enzyme was then adsorbed onto the CNT surface and directly treated with acetonitrile plasma to overcoat the GOx layer with a PPF. This fabrication process provides a robust design of CNT-based enzyme biosensor, because of all processes are dry except the procedure for enzyme immobilization. The main novelty of the present methodology lies in the PPF and/or plasma processes. The optimized glucose biosensor revealed a high sensitivity of 38 μA mM(-1) cm(-2), a broad linear dynamic range of 0.25-19 mM (correlation coefficient of 0.994), selectivity toward an interferent (ascorbic acid), and a fast response time of 7 s. The background current was much smaller in magnitude than the current due to 10 mM glucose response. The low limit of detection was 34 μM (S/N = 3). All results strongly suggest that a plasma-polymerized process can provide a new platform for CNT-based biosensor design.     

A disposable amperometric sensor screen printed on a nitrocellulose strip: a glucose biosensor employing lead oxide as an interference-removing agent

A new type of disposable amperometric sensor is devised by screen printing thick-film electrodes directly on a porous nitrocellulose (NC) strip. The chromatographic NC strip is then utilized to introduce various sample pretreatment layers. As a preliminary application, a glucose biosensor based on hydrogen peroxide detection is constructed by immobilizing glucose oxidase (GOx) on the NC electrode strip and by formulating a strong oxidation layer (i.e., PbO2) at the sample loading area, placed below the GOx reaction band. The screen-printed PbO2 paste serves as a sample pretreatment layer that removes interference by its strong oxidizing ability. Samples applied are carried chromatographically, via the PbO2 paste, to the GOx layer, and glucose is catalyzed to liberate hydrogen peroxide, which is then detected at the electrode surface. The proposed NC/PbO2 strip sensor is shown to be virtually insusceptible to interfering species such as acetaminophen and ascorbic and uric acids and to exhibit good performance, in terms of the sensor-to-sensor reproducibility (standard deviation, +/-0.026 - +/-0.086 microA), the sensitivity (slope, -0.183 microA/mM), and the linearity (correlation coefficient, 0.994 in the range of 0-10 mM).     

Preparation of multiwalled carbon nanotubes-platinum-Nafion-glucose oxidase nanobiocomposite and its application to glucose biosensor

Platinum (Pt) nanoparticles were electrodeposited within multiwalled carbon nanotubes-Nafion-glucose oxidase (MWNTs-Nafion-GOx) nanobiocomposite by a potentiostatic method. The morphology and nature of the resulting MWNTs-Pt-Nafion-GOx nanobiocomposite were characterized by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS). The electrocatalytic properties of the MWNTs-Pt-Nafion-GOx nanobiocomposite film modified glassy carbon electrode were characterized by cyclic voltammetry and amperometry in the presence of hydrogen peroxide. The glucose biosensor sensitivity was strongly influenced by the deposits of Pt nanoparticles and amount of GOx concentration within the MWNTs-Pt-Nafion-GOx nanobiocomposite film. The optimized glucose biosensor displayed a sensitivity of 640 nA mM(-1), a linear range of up to 4 mM, a detection limit of 4 microM, and a response time of less than 4 s at an operating potential of +500 mV versus Ag/AgCl (3 M KCl).     

A bioconjugated polyglycerol dendrimer with glucose sensing properties

In this work, the biological and electrochemical properties of glucose biosensor based on polyglycerol dendrimer (PGLD) is presented. Streptokinase (SK), glucose oxidase (GOx) and phosphorylcholine (PC) were immobilized onto PGLD to obtain a blood compatible bioconjugate with glucose sensing properties. The bioconjugated PGLD was entrapped in polyaniline nanotubes (PANINT's) through template electrochemical polymerization of aniline. PANINT's were used as electron mediator due to their high ability to promote electron-transfer reactions involving GOx. Platelet adhesion, fibrinolytic activity and protein adsorption were studied by in vitro experiments to examine the interaction of blood with PGLD biosensor. The PGLD biosensor exhibits a strong and stable amperometric response to glucose. The enzyme affinity for the substrate (K (M) (app) ) indicates that the enzyme activity was not significantly altered after the bioconjugation of GOx with PGLD dendrimer. The bioelectrochemical properties suggest that the bioconjugated PGLD developed in this work appears to be a good candidate for providing interfaces for implantable biosensors, especially oxidoreductase-based sensors.     

Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films

A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.     

Modified gold surfaces by 6-(ferrocenyl)hexanethiol/dendrimer/gold nanoparticles as a platform for the mediated biosensing applications

An electrochemical biosensor mediated by using 6-(Ferrocenyl) hexanethiol (FcSH) was fabricated by construction of gold nanoparticles (AuNPs) on the surface of polyamidoamine dendrimer (PAMAM) modified gold electrode. Glucose oxidase (GOx) was used as a model enzyme and was immobilized onto the gold surface forming a self assembled monolayer via FcSH and cysteamine. Cyclic voltammetry and amperometry were used for the characterization of electrochemical response towards glucose substrate. Following the optimization of medium pH, enzyme loading, AuNP and FcSH amount, the linear range for the glucose was studied and found as 1.0 to 5.0 mM with the detection limit (LOD) of 0.6 mM according to S/N = 3. Finally, the proposed Au/AuNP/(FcSH + Cyst)/PAMAM/GOx biosensor was successfully applied for the glucose analysis in beverages, and the results were compared with those obtained by HPLC.     

Nitric oxide-releasing sol-gel particle/polyurethane glucose biosensors

A hybrid sol-gel/polyurethane glucose biosensor that releases nitric oxide is developed and characterized. The biosensor consists of a platinum electrode coated with four polymeric membranes including the following: (1) sol-gel with immobilized glucose oxidase (GOx); (2) polyurethane to protect the enzyme; (3) NO donor-modified sol-gel particle-doped polyurethane; and (4) polyurethane. This configuration was developed due to the drastic reduction in sensitivity observed for NO donor-modified sol-gel film-based glucose sensors. For the hybrid sol-gel/polyurethane biosensor, sol-gel particles are first modified with the NO donor and then incorporated into a polyurethane layer that is coated onto the preimmobilized GOx electrode. In this manner, the GOx layer is not exposed to the harsh conditions necessary to impart NO release ability to the biosensor, and only a minimal decrease in sensitivity due to the NO release is observed. The glucose response of the NO-releasing glucose biosensor and its NO generation profiles are reported. In addition, the stability of the sol-gel particles in the supporting polyurethane membrane is discussed.     

Titanium dioxide–cellulose hybrid nanocomposite and its glucose biosensor application

This paper investigates the fabrication of titanium dioxide (TiO₂)–cellulose hybrid nanocomposite and its possibility for a conductometric glucose biosensor. TiO₂ nanoparticles were blended with cellulose solution prepared by dissolving cotton pulp with lithium chloride/N,N-dimethylacetamide solvent to fabricate TiO₂–cellulose hybrid nanocomposite. The enzyme, glucose oxidase (GOx) was immobilized into this hybrid nanocomposite by physical adsorption method. The successful immobilization of glucose oxidase into TiO₂–cellulose hybrid nanocomposite via covalent bonding between TiO₂ and GOx was confirmed by X-ray photoelectron analysis. The linear response of the glucose biosensor is obtained in the range of 1–10 mM. This study demonstrates that TiO₂–cellulose hybrid nanocomposite can be a potential candidate for an inexpensive, flexible and disposable glucose biosensor.     

Gold nanoparticle-polyaniline composite films for glucose sensing

Gold nanoparticles (AuNPs) have been self-assembled onto electrochemically deposited polyaniline (PANI) films on indium-tin-oxide (ITO) coated glass plates to fabricate glucose biosensor. The covalent immobilization of glucose oxidase (GOx) in the near vicinity of gold nanoparticles has been obtained using N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS), chemistry between amino groups of PANI and COOH groups of GOx. These AuNPs-PANI/ ITO and GOx/AuNPs-PANI/ITO composite films have been characterized using Fourier transform infra red (FTIR) and cyclic voltammetry (CV) techniques, respectively. The fast electron transfer from the modified PANI surface to electrode is indicated by the observed increase in amperometric response current of these GOx/AuNPs-PANI/ITO bioelectrodes. These GOx/AuNPs-PANI/ITO bioelectrodes exhibit response time of 10 s, linearity from 50 to 300 mg/dl and show value of apparent Michaelis-Menten constant (Km(app)) of 2.2 mM.     

Electrophoretically deposited nano-structured polyaniline film for glucose sensing

Electrophoretically deposited nano-structured polyaniline (NS-PANI) film has been utilized for fabrication of glucose biosensor by covalent immobilization of glucose oxidase (GOx) using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide chemistry. This GOx/NS-PANI/ITO bioelectrode has been characterized using scanning electron microscopy, FT-IR, UV-Visible spectroscopy and differential pulse voltammetry (DPV) techniques. The response studies carried out on GOx/NS-PANI/ITO bioelectrode using DPV and photometric studies reveal linearity up to 400 mgdL^− 1 with sensitivity as 1.05 × 10^− 4 mA mg^− 1 dL and 3.887 × 10^− 5 Abs mg^− 1 dL, respectively. The lower value of Michaelis-Menten constant obtained for immobilized GOx (2.1 mM) compared with that of free GOx (5.85 mM) suggests high affinity of enzyme to this matrix.     

Electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes

Platinum nanoparticles with a diameter of 2-3 nm were prepared and used in combination with single-wall carbon nanotubes (SWCNTs) for fabricating electrochemical sensors with remarkably improved sensitivity toward hydrogen peroxide. Nafion, a perfluorosulfonated polymer, was used to solubilize SWCNTs and also displayed strong interactions with Pt nanoparticles to form a network that connected Pt nanoparticles to the electrode surface. TEM and AFM micrographs illustrated the deposition of Pt nanoparticles on carbon nanotubes whereas cyclic voltammetry confirmed an electrical contact through SWCNTs between Pt nanoparticles and the glassy carbon (GC) or carbon fiber backing. With glucose oxidase (GOx) as an enzyme model, we constructed a GC or carbon fiber microelectrode-based biosensor that responds even more sensitively to glucose than the GC/GOx electrode modified by Pt nanoparticles or CNTs alone. The response time and detection limit (S/N = 3) of this biosensor was determined to be 3 s and 0.5 microM, respectively.     

ENFET glucose biosensor produced with dendrimer encapsulated Pt nanoparticles

The biosensors based on ENFETs for direct glucose concentration analysis have been fabricated by introducing dendrimer encapsulated Pt nanoparticles and glucose oxidase (GOx) via a layer-by-layer self-assembly method. The free amine groups located on each poly(amidoamine) dendrimer molecule were exploited to immobilize enzyme through covalent attachment. Depending on metal nanoparticles within dendrimers and biocompatibility of dendrimers, the fabricated glucose sensitive ENFET shows obviously enhanced sensitivity and extended lifetime compared with the conventional ones. The fabricated sensor has a linear range of 0.25–2.0 mM, and a detection limit of ca. 0.15 mM. The influence of buffer concentration, ionic strength and pH was discussed. The biosensor also has good stability, which response could be used for detecting of glucose samples at intervals for at least 1 month when it stored in dry state at 4 °C.     

Fabrication of a glucose biosensor based on citric acid assisted cobalt ferrite magnetic nanoparticles

A novel and practical glucose biosensor was fabricated with immobilization of Glucose oxidase (GOx) enzyme on the surface of citric acid (CA) assisted cobalt ferrite (CF) magnetic nanoparticles (MNPs). This innovative sensor was constructed with glassy carbon electrode which is represented as (GOx)/CA-CF/(GCE). An explicit high negative zeta potential value (-22.4 mV at pH 7.0) was observed on the surface of CA-CF MNPs. Our sensor works on the principle of detection of H2O2 which is produced by the enzymatic oxidation of glucose to gluconic acid. This sensor has tremendous potential for application in glucose biosensing due to the higher sensitivity 2.5 microA/cm2-mM and substantial increment of the anodic peak current from 0.2 microA to 10.5 microA.     

Multilayer membranes via layer-by-layer deposition of glucose oxidase and Au nanoparticles on a Pt electrode for glucose sensing

We prepared multilayer membranes by the layer-by-layer deposition of glucose oxidase (GOx) and Au nanoparticles (5, 10, or 50 nm φ) on sensor substrates, such as a Pt electrode and a quartz glass plate, to prepare glucose sensors. The enzyme activity of GOx remained even in alternate assemblies, and the activity increased with the increasing number of depositions. The apparent K_m values of the deposited GOx were 28–32 mM, while a reported value in a solution is 33 mM. These results suggest that Au nanoparticles can be used as binders for the deposition of GOx without significant change in the affinity between GOx and glucose.     

Optimal environment for glucose oxidase in perfluorosulfonated ionomer membranes: improvement of first-generation biosensors

An optimal environment for glucose oxidase (GOx) in Nafion membranes is achieved using an advanced immobilization protocol based on a nonaqueous immobilization route. Exposure of glucose oxidase to water-organic mixtures with a high (85-95%) content of the organic solvent resulted in stabilization of the enzyme by a membrane-forming polyelectrolyte. Such an optimal environment leads to the highest enzyme specific activity in the resulting membrane, as desired for optimal use of the expensive oxidases. Casting solution containing glucose oxidase and Nafion is completely stable over 5 days in a refrigerator, providing almost absolute reproducibility of GOx-Nafion membranes. A glucose biosensor was prepared by casting the GOx-Nafion membranes over Prussian Blue-modified glassy carbon disk electrodes. The biosensor operated in the FIA mode allows the detection of glucose down to the 0.1 microM level, along with high sensitivity (0.05 A M(-1) cm(-2)), which is only 10 times lower than the sensitivity of the hydrogen peroxide transducer used. A comparison with the recently reported enzyme electrodes based on similar H2O2 transducers (transition metal hexacyanoferrates) shows that the proposed approach displays a dramatic (100-fold) improvement in sensitivity of the resulting biosensor. Combined with the attractive performance of a Prussian Blue-based hydrogen peroxide transducer, the proposed immobilization protocol provides a superior performance for first-generation glucose biosensors in term of sensitivity and detection limits.     

Amperometric biosensor based on carbon nanotubes coated with polyaniline/dendrimer-encapsulated Pt nanoparticles for glucose detection

A novel amperometric glucose biosensor based on the nanocomposites of multi-wall carbon nanotubes (CNT) coated with polyaniline (PANI) and dendrimer-encapsulated Pt nanoparticles (Pt-DENs) is prepared. CNT coated with protonated PANI is in situ synthesized and Pt-DENs is absorbed on PANI/CNT composite surface by self-assembly method. Then Glucose oxidase (GOx) is crosslink-immobilizated onto Pt-DENs/PANI/CNT composite film. The results show that the fabricated GOx/Pt-DENs/PANI/CNT electrode exhibits excellent response performance to glucose, such as low detection limit (0.5 µM), wide linear range (1 µM–12 mM), short response time (about 5 s), high sensitivity (42.0 µA mM^? 1 cm^? 2) and stability (83% remains after 3 weeks).     

Optimizing the functionalization process for nanopillar enhanced electrodes with GOx/PPY for glucose detection

In this study, the functionalization process for nanopillar enhanced electrodes (NEEs) using glucose oxidase (GOx) with polypyrrole (PPY) is optimized for the purpose of achieving enhanced sensing performances for these electrodes in glucose detection. Specifically, an optimal roughness factor for the NEEs and an optimal set of electro-polymerization/deposition parameters for their functionalization using GOx/PPY are identified. Results show that NEEs with a roughness factor of about 60 are optimal for enhancing the amperometric current responses and that for such electrodes an electro-functionalization/deposition process at a deposition current of 50?μA?cm(-2) and a total charge of 150?mC?cm(-2) will give rise to a high sensing performance with a sensitivity as high as 36?μA?cm(-2)?mM(-1).     

Investigation of carbon nanofibers as support for bioactive substances

In this paper we have studied the adsorption properties of various bio-active systems onto the surface of carbon nanofibers (CNF) synthesized by chemical vapor deposition (CVD). Amino acids (alanine, aspartic acid, glutamic acid) and glucose oxidase (GOx) were adsorbed on CNF and the results were compared with those obtained when activated carbon (AC) was used as support. CNF and AC properties (hydrophilic or hydrophobic properties) were characterized by the pH value, the concentration of acidic/basic sites and by naphthalene adsorption. CNF with immobilized GOx was additionally investigated as a highly sensitive glucose biosensor. An amperometric method was used in an original manner to detect the changes in the specific activity of GOx, immobilized longer time on CNF. The method demonstrates that not the whole enzyme adsorbed onto CNF can catalyze the oxidation of glucose from the solution.