Bis-Aniline-Crosslinked Enzyme–Metal Nanoparticle Composites on Electrodes for Bioelectronic Applications

The electrical contacting of redox enzymes with electrodes is the most fundamental requirement for the development of amperometric biosensors and biofuel cell elements. We describe a novel method to prepare electrically contacted metallic nanoparticles (NPs) or carbon nanotubes (CNTs)/enzyme hybrid composites on electrodes that act as amperometric biosensors or as the constituents of biofuel cells. Au NPs or Pt NPs were modified with thioaniline electropolymerizable groups, and so were the enzymes glucose oxidase (GOx) or bilirubin oxidase (BOD). Electrochemical polymerization of the thioaniline-functionalized Pt NPs and GOx on a thioaniline monolayer-modified Au surface led to the formation of a bis-aniline-bridged Pt NPs/GOx composite electrode that enabled the analysis of glucose through the electrocatalyzed reduction of H₂O₂. Similarly, a Pt NPs/BOD composite-functionalized electrode showed electrocatalytic activity toward the reduction of O₂ to H₂O. Also, a Au NPs/GOx composite-functionalized electrode revealed direct electrical contacting between the enzyme and the electrode through the electrocatalytic reduction of the bis-aniline bridges, and this enabled the bioelectrocatalytic oxidation and the amperometric sensing of glucose. Finally, a biofuel cell consisting of an anode modified with Nile blue/NAD⁺/alcohol dehydrogenase on carbon nanotubes, and a cathode composed of the bis-aniline-crosslinked Pt NPs/BOD composite was constructed. The biofuel cell operates with a power output corresponding to 200 μW cm^-2.     

Dendrimer‐encapsulated Pt nanoparticles/polyaniline nanofibers for glucose detection

A novel amperometric glucose biosensor based on self‐assembling glucose oxidase (GOx) and dendrimer‐encapsulated Pt nanoparticles (Pt‐DENs) on nanofibrous polyaniline (PANI) was described. PANI nanofibers were synthesized via an interfacial polymerization method. A sulfonated polyelectrolytes‐poly(sodium 4‐styrenesulfonate) (PSS) was used to form the negative PANI/sulfonated polyelectrolyte complex, which had good disperse in aqueous solution. GOx was immobilized on the PANI/PSS surface by alternatively assembling a cationic Pt‐DENs layer and an anionic GOx layer. The unique sandwich‐like layer structure (Pt‐DENs/GOx/Pt‐DENs/PANI/PSS) formed by self‐assembling provides a favorable microenvironment to keep the bioactivity of GOx and to prevent enzyme molecule leakage. The fabricated Pt‐DENs/GOx/Pt‐DENs/PANI/PSS electrode exhibited excellent response performance to glucose with a detection limit of 0.5 μM, wide linear range from 10 μM to 4.5 mM, short response time within 5 s, improved sensitivity of 39.63 μA/(mM cm²), and good stability (85% remains after 20 days). © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Continuous glucose monitoring using enzyme-immobilized platinized diamond microfiber electrodes

A sensitive and stable glucose biosensor for in vivo monitoring has been developed using boron-doped diamond microfiber (BDDMF) electrodes. The electrodes were modified with platinum nano-particles to detect H₂O₂, which was enzymatically produced by glucose oxidase (GOx) immobilized on the electrode surface. The platinum-modified BDDMF (Pt-BDDMF) electrodes exhibited much higher sensitivity compared to Pt-microfiber electrodes, Pt electrodes and Pt-modified diamond thin film electrodes. Deposition conditions for Pt nano-particles on the BDDMF electrodes and immobilization of GOx were optimized. GOx/overoxidized polypyrrole (OPPy)/Pt-modified BDDMF electrodes were applied for continuous interference-free glucose monitoring. Amperometric measurements of glucose showed a linear response in the range of 1-70 mM, with an R.S.D. of 3.7% for five injections of 100 μM glucose. The electrodes exhibited good stability over 3 months with no detected anodic current for ascorbic acid (AA), which is an interfering compound.     

Electropreparation of (±)‐10‐camphorsulfonate‐doped poly(o‐phenylenediamine) in acetonitrile and its use in determination of glucose

Poly(o‐phenylenediamine) (PoPD) film has been electrochemically prepared on Pt electrode in an acetonitrile–water medium containing o‐phenylenediamine (oPD) monomer and (±)‐10‐camphorsulfonic acid (HCSA) by using the cyclic voltammetry (CV). The PoPD film (PoPD–CSA) has been characterized by FTIR, CV, EIS, FESEM, and conductivity measurement. The glucose biosensor (Pt/PoPD–CSA/GOx) has been prepared from the PoPD coated electrode by immobilizing glucose oxidase (GOx) enzyme using glutaraldehyde. The biosensor shows a low detection limit and wide linear working range, a good reusability, long‐term stability, and anti‐interference ability. The Pt/PoPD–CSA/GOx has possesses higher sensitivity (2.05 μA/mmol L^?1) and affinity to glucose due to the use of CSA ion as dopant. The linear concentration ranges of Pt/PoPD–CSA/GOx have been found to be 9.6 × 10^?3 to 8.2 mmol L^?1 from calibration curve and 4.6 × 10^?2 to 100 mmol L^?1 from the relationship between the (1/glucose concentration) and (1/current difference). © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 39864.     

Fabrication of bioanode by using electrically conducting polythiophene via entrapment technique

A glassy carbon electrode (GCE) was tailored with conducting polymer polythiophene and further immobilized by an enzyme glucose oxidase (GOx). A thin film of polymer was developed by electrochemical polymerization of thiophene monomer. During electrochemical polymerization of the monomer the enzyme GOx and the redox active mediator ferritin (Frt) were entrapped within this polymer matrix. In this novel approach, the entrapment of enzyme and mediators within a polymer matrix occurs without chemical reaction that could affect their activity. The entrapment of enzyme and mediator within the conducting polymer matrices increases the surface area of the electrode. The tailored GCE/Ptp/Frt/GOx electrode showed a high catalytic activity. The increased surface area causes a high rate of electron transfer between the electrode and Frt engaged as an electron transfer mediator. The electrochemical properties of the electrode were determined by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The fabricated bioanode showed a current density of 3.9mA cm^?2 at 1.0 V vs. Ag/AgCl in a 45 mM glucose solution and suggests proficient chances in biofuel cells (BFCs) applications.     

Properties of the enzyme electrode fabricated with a film of polythiophene derivative and its application to a glucose fuel cell

A polymer electrode in the form of a thin film was prepared by electrochemical copolymerization of 3‐methylthiophene and thiophene‐3‐acetic acid. Glucose oxidase (GOx) was immobilized by covalent binding to the carboxyl groups on the electrode, and the GOx‐immobilized electrode (GOx‐electrode) was used as an anode in a glucose fuel cell. It was demonstrated by cyclic voltametry that in the presence of p‐benzoquinone (BQ), which was adopted as an electron mediator, the GOx‐electrode generated a significant glucose‐oxidation current depending on the concentrations of both glucose and BQ. A large surface area of the GOx‐electrode was considered to afford effective environment for the enzyme reaction and electron transfer. The fuel cell using the GOx‐electrode as an anode gave a power output of 42 μW/cm²‐anode at 30°C, when its anodic compartment contained 100 mM glucose and 10 mM BQ. The performance of the cell was influenced by the concentrations of glucose and BQ in the anodic compartment. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Facile direct electron transfer in glucose oxidase modified electrodes

Glucose oxidase (GOx) is widely used in the glucose biosensor industry. However, mediatorless direct electron transfer (DET) from GOx to electrode surfaces is very slow. Recently, mediatorless DET has been reported via the incorporation of nanomaterials such as carbon nanotubes and nanoparticles in the modification of electrodes. Here we report GOx electrodes showing DET without the need for any nanomaterials. The enzyme after immobilization with poly-l-lysine (PLL) and Nafion^® retains the biocatalytic activities and oxidizes glucose efficiently. The amperometric response of Nafion-PLL-GOx modified electrode is linearly proportional to the concentration of glucose up to 10 mM with a sensitivity of 0.75 μA/mM at a low detection potential (−0.460 V vs. Ag/AgCl). The methodology developed in this study will have impact on glucose biosensors and biofuel cells and may potentially simplify enzyme immobilization in other biosensing systems.     

Polystyrene Attached Pt(IV)�CAzomethine, Synthesis and Immobilization of Glucose Oxidase Enzyme

Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two optimum pH values that were pH = 4.0 and pH = 7. The insertion of stable Pt(IV)–azomethine spacers between the polystyrene backbone and the immobilized GOx, (APS–Sch–Pt–GOx), increases the enzymes’ activity and improves their affinity towards the substrate even at pH = 4. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme was investigated. The storage stability of the immobilized glucose oxidase was shown to be eleven months in dry conditions at +4 °C.     

Poly(2,5‐dimethoxyaniline) films on mild steel for application to glucose biosensor

Poly(2,5‐dimethoxyaniline) (PDMA) films were electrochemically synthesized on mild steel from an aqueous oxalic acid solution using galvanostatic mode. These films were characterized by potential–time curve, UV‐visible absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The enzyme glucose oxidase (GOx) was entrapped into the PDMA film by a physical adsorption method. The resulting PDMA–GOx films were characterized by UV‐visible absorption spectroscopy, FTIR, and SEM. The amperometric response of the PDMA–GOx films was measured as a function of glucose concentration in phosphate buffer solution (pH 7.3). The PDMA–GOx films exhibit a fast and linear amperometric response in the range of 2–20 mM glucose. The maximum current density and Michaelis–Menten constant of PDMA/GOx films are found to be ～483 μA/cm² and 1.12 mM, respectively. The shelf stability and thermal stability of these films were also investigated. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2008     

An electrocatalyst for detection of glucose in human blood: synergy in Pd–AuNPs/GOx/C surfaces

Glucose oxidase (GOx)-based amperometric enzyme electrodes have been the target of substantial research. In this study, new amperometric biosensor for determination of glucose was developed. GOx enzyme was immobilized at bovine serum albumin via entrapment method. For this reason, the optimum conditions of Pd–Au NPs/GOx/C-modified glassy carbon electrode were determined. The electron is directly transferred from glucose to the electrode via the active site of the enzyme. The absence of mediators is the main advantage of such third-generation biosensors. The resulting materials were characterized employing scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction. In addition, the effects of glucose concentration, scan rate, temperature, electroactive interference, stability, reusability of the biosensors were discussed. The applicability to blood analysis was also evaluated. The biosensor has a limit of detection for the determination of glucose 0.0014?mM.     

A new metal chelate sorbent for glucose oxidase: Cu(II)- and Co(II)-chelated poly(N-vinylimidazole) gels

Poly(N-vinylimidazole) (PVIm) gels were prepared by irradiating a binary mixture of N-vinylimidazole (VIm)–water in a ⁶⁰Co-γ source having 4.5 kGy/h dose rate. In the glucose oxidase (GOx) adsorption studies, affinity gels with a swelling ratio of 1100% for PVIm and 40 and 55% for Cu(II)- and Co(II)-chelated PVIm gels, respectively, at pH 6.5 in phosphate buffer were used. FTIR spectra were taken for PVIm and Cu(II)- and Co(II)-chelated PVIm, and glucose oxidase adsorption on these gels, to characterize the nature of the interactions in each species. The results show that PVIm–glucose oxidase interaction is mainly electrostatic and metal ion–chelated PVIm gel–glucose oxidase interaction is of coordinate covalent nature. Cu(II) and Co(II) ions were chelated within the gels via amine groups on the imidazole ring of the gel. Different amounts of Cu(II) and Co(II) ions [maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)] were loaded on the gels by changing the initial concentration of Cu(II) and Co(II) ions at pH 7.0. GOx adsorption on these gels from aqueous solutions containing different amounts of GOx at different pH was investigated in batch reactors. GOx adsorption capacity was further increased when Cu(II) and Co(II) ions were attached [up to 0.53 g GOx/g dry Co(II)-chelated PVIm gels]. More than 90% of the adsorbed GOx was desorbed in 5 h in desorption medium containing 1.0M KSCN at pH 7.0 for plain gel and 0.05M EDTA at pH 4.9 for metal-chelated gel. Nonspecific glucose oxidase adsorption on/in the metal ion–chelated PVIm gel was investigated using 0.02M of phosphate buffer solution. The nonspecific GOx adsorption was determined to be about 18% for PVIm and 8% for the metal ion–chelated PVIm gels. The ionic strength effect was investigated both on PVIm and on the metal ion–chelated PVIm gels for the glucose oxidase adsorption. It was found that ionic strength was more effective on the PVIm gel because of the electrostatic interaction between protonated gel and the deprotonated glucose oxidase side chain. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 446–453, 2001     

Polyaniline synthesis catalysed by glucose oxidase

A novel method for synthesis of polyaniline (PANI) in aqueous media based on application of oxidizing-enzyme glucose oxidase (GOx) is reported. Hydrogen peroxide was produced during catalytic reaction of oxidizing-enzyme glucose oxidase from Penicillium vitale and initiated the polymerization of aniline. The increase in optical absorbance in the range of 340-700 nm was exploited for the monitoring of PANI polymerization process. The role of GOx in the formation of PANI, influence of the initial concentrations of GOx, and glucose and aniline monomer on the aniline polymerization rate was studied. The study of pH influence on polymerization rate showed that PANI polymerization was occurring in a broad pH range from the pH 2.0 to 9.0. Optimal polymerization/oligomerization temperature was found to be at 37 °C, which is also optimal for GOx-catalysed enzymatic reaction. After 10 days of continuous GOx-catalysed polymerization PANI appeared as colloid-microparticles visible by an optical microscope.     

Electrogeneration kinetics of polyaniline in the presence of lithium perchlorate

Summary Polyaniline (Pani) was electrogenerated potentiostatically (1 V vs Ag/Ag⁺) in acid aqueous medium (2 M HNO₃) in the presence of LiClO₄ on Pt electrodes. The kinetic equation of the polymerization process was calculated by means of the electrochemical data of the polymerization charge (Q_pol), determined as R_p= [E]^0.14 [M]². Furthermore, the number of electrons consumed per monomer unit incorporated in the oxidated polymer was determined. The results show that the electrolyte has practically no effect on the reaction kinetics at the concentrations tested, but that it is, moreover, the triggering factor for fewer electrons to be incorporated into the polymer chain. Received: 17 January 2001/Revised version: 13 May 2001/Accepted: 26 July 2001     

A comparative study on the electrochemical properties of ring‐substituted polyanilines

The redox behavior of polyanilines with ring‐substituted groups synthesized by chemical polymerization, poly‐2,5‐dimethoxyaniline (PDMAn), poly‐m‐chloroaniline (PmClAn) and poly‐o‐toluidine (POT), was studied and the morphology and crystal orientation of platinum particles deposited on these polymer membranes was compared. The oxidation of isopropanol on platinized polyaniline‐modified electrodes as a model reaction was also investigated to examine the electrocatalytic properties of the polymers. The results show that the first oxidation potential of the polymers increases in the following sequence: PDMAn, POT and then PmClAn, which can be explained in terms of the electronic and steric effects of ring‐substituted groups. The growth of Pt particles electrodeposited on doping‐state POT and PDMAn polymer membranes takes place by a ‘progressive nucleation mechanism’, but by an ‘instantaneous nucleation mechanism’ on PmClAn. Platinum crystallites trend towards preferred‐orientation and Pt (200) is the preferred face, but the degree of preferred orientation depends on the polymer. The oxidation potentials of isopropanol are located near 0.3 V, and the oxidation currents increase on platinized polymer‐modified electrodes, indicating that the interaction of polymer with Pt particles might improve the catalytic activity of Pt. Polyanilines act not only as dispersion media but also change the electronic properties of Pt crystalline grains. Copyright © 2005 Society of Chemical Industry     

Electrosynthesis and characterization of a conductive polythiophene deriving from a terthiophene monomer

The electrochemical polymerization of the 3,3″-di[(S)-(+)-2-methylbutyl]-2,2′:5′,2″-terthiophene (DMBTT) monomer was carried out potentiodynamically on indium tin oxide (ITO), Pt or glassy carbon (GC) electrodes in anhydrous CH₃CN with TBAPF₆ as supporting electrolyte. Films with good adhesion were obtained for all the tested substrates and they were characterized by various analytical techniques including UV–Vis–NIR absorption spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM), and gel permeation chromatography (GPC). The most outstanding properties of the electrochemically synthesized polymer were compared with those of the same polymer obtained by a chemical method. The film obtained on ITO displayed a reversible electrochromic behavior under potential switching between −0.50 and +1.00 V vs. SCE.     

Poly[tris((p-aminophenoxy)phosphineoxide)-3,3′,4,4′-benzophenonetetracarboxylicdiimide] as a New Polymeric Membrane for the Fabrication of an Amperometric Glucose Sensor

Poly[tris((p-aminophenoxy)phosphineoxide)-3,3′,4,4′-benzophenonetetracarboxylicdiimide] (PAB) was used as an immobilized enzyme membrane for the fabrication of an amperometric glucose sensor. Enzyme immobilization was performed by two different methods and structural morphology of immobilized enzyme membrane was studied by SEM. The amperometric response of the sensor to hydrogen peroxide, formed as the product of the enzymatic reaction, was measured at a potential of 0.7 V in phosphate buffer solution by means of the TB technique. The influence of preparation conditions (polyimide film thickness, enzyme amount) on electrode performance was examined to obtain the optimal experimental parameters. Sensor characteristics (pH, response time, selectivity, stability) of the PAB/GOx/Pt electrode prepared under optimal conditions has been investigated. Results showed that this sensor exhibited a linear range up to 6 mM and a response time of about 60 s. with good stability. When glucose was injected into the PBS solution in the presence of fouling substances (lactose, sucrose, and urea), excellent amperometric responses were observed. Also, it was seen that the PAB/GOx-Pt electrode blocked the signal current of electroactive oxalic acid.     

m‐Chloroaniline emulsion polymerization,macromolecular chain structure and electrochemical properties

Poly(m‐chloroaniline) (PmClAn) was synthesized by emulsion polymerization. The influences of reaction temperature and initiator concentration on polymerizations were studied. It was found that PmClAn with number‐average molecular weight of 1.85 × 10³ g mol^?1 was obtained by the following conditions: 80 °C, [monomer] = 0.187 × 10^?3 mol l^?1, [sodium lauryl sulfate] = 4.8 × 10^?2 mol l^?1, [potassium peroxydisulfate] = 5.6 × 10^?2 mol l^?1, reaction period = 2.0 h. ¹H NMR, FTIR, and transmission and scanning microscopy were used for structural characterization of PmClAn. It was shown that the ratio of benzoid to quinoid units in the macromolecular chain was respectively 3:2, and that PmClAn has a typical crystalline monoclinic form. A PmClAn molecular chain configuration was also proposed on the basis of crystallographic data. Cyclic voltammetry experiments revealed the PmClAn membrane electrode electroactivity. This electroactivity increased when the polymer was proton‐doped. When Pt particles were electrodeposited onto the polymer membrane electrode, they presented a preferred orientation. Isopropanol oxidation intensities with platinized PmClAn modified electrodes were larger than with a platinized Pt electrode. We also found that oxidation occurred mainly on the Pt particles deposited on the polymer, and that the anodic peak potential changed with polymer and its doping level. These results indicated that the Pt particles interacted with the polymer and that catalytic properties could be observed. © 2002 Society of Chemical Industry     

Development of a POA/DBS/GOx Biosensor for the Determination of Glucose

In the present work, a simple technique is described for constructing a poly(o-anisidine) (POA)-dodecylbenzene sulphonic acid (DBS)-glucose oxidase (GOx) (POA-DBS-GOx) electrode. The enzyme glucose oxidase (GOx) was immobilized by crosslinking via glutaraldehyde on the POA-DBS film. The POA-DBS films were synthesized electrochemically on platinum substrate. The synthesized films were characterized by using electrochemical technique, conductivity measurement, UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The conductivity of the polymer films was found to be about 7.61 × 10^?2 S/cm. The crosslinking of enzyme and the porous morphology of the polymer film lead to good stability and good response time of the enzyme electrode. The stability and lifetime of the POA-DBS-GOx electrode have been studied. It shows very good stability and response for 3–4 weeks at 4°C. The results of this study reveal that a phosphate buffer gives better response than acetate buffer in amperometric measurements.     

Hierarchically assembled ZnO nanosheets microspheres for enhanced glucose sensing performances

Nanostructures with higher surface specific area has great potential applications in sensing devices because higher surface specific area not only improve protein/enzyme immobilization efficiency, but also enhances charge transport and sensing performances. Herein, hierarchically assembled ZnO nanosheets microspheres (HAZNMs) were synthesized by facile one-pot solution process at low-temperature. Results showed that as-synthesized HAZNMs possessing higher specific surface area, significantly increased the enzyme loading efficiency which in turn improved the sensing performances. The as-fabricated biosensors showed a remarkably high sensitivity (210.8 μA/mM cm²) in the wide-linear response range of 0.05–23 mM, favorable stability for long-term storage, excellent anti-interference ability and high reliability for glucose detection in human blood serum samples. The improved sensing performances can be ascribed to the high glucose oxidase (GOx) enzyme immobilization on HAZNMs that provides a favorable microenvironment for the maintenance of GOx enzyme bioactivity.     

Immobilization of glucose oxidase and electron-mediating groups on the film of 3-methylthiophene/thiophene-3-acetic acid copolymer and its application to reagentless sensing of glucose

Enzyme electrodes were prepared by covalent immobilization of glucose oxidase (GOx) on the films of conducting copolymer obtained by electrochemical copolymerization of 3-methylthiophene and thiophene-3-acetic acid. Onto the enzyme electrodes, 2,5-dihydroxyphenyl (DHP) groups were introduced as redox mediators by the reaction of 2,5-dihydroxybenzaldehyde using alkylenediamines as linker molecules. The mediator-carrying enzyme electrodes were applied to reagentless glucose sensing system. It was found that response current to glucose was increased by introducing DHP groups, and the longer the alkylene chain of used alkylenediamine was, the higher the response current became. For a comparison, poly-l-lysine was linked onto the films of the conducting copolymer in advance, and immobilization of GOx and then introduction of DHP groups were carried out. The mediator-carrying electrode prepared thus gave higher amperometric response than those prepared with alkylenediamines, suggesting that amino groups of poly-l-lysine functioned effectively as the sites for binding GOx and DHP groups.