Characteristics of a miniature compartment-less glucose-O2 biofuel cell and its operation in a living plant

We report the temperature, pH, glucose concentration, NaCl concentration, and operating atmosphere dependence of the power output of a compartment-less miniature glucose-O(2) biofuel cell, comprised only of two bioelectrocatalyst-coated carbon fibers, each of 7 micro m diameter and 2 cm length (Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2002, 124, 12962). The bioelectrocatalyst of the anode consists of glucose oxidase from Aspergillus niger electrically "wired" by polymer I, having a redox potential of -0.19 V vs Ag/AgCl. That of the cathode consists of bilirubin oxidase from Trachyderma tsunodae "wired" by polymer II having a redox potential of +0.36 V vs Ag/AgCl (Mano, N.; Kim, H.-H.; Zhang, Y.; Heller, A. J. Am. Chem. Soc. 2002, 124, 6480. Mano, N.; Kim, H.-H.; Heller, A. J. Phys. Chem. B 2002, 106, 8842). Implantation of the fibers in the grape leads to an operating biofuel cell producing 2.4 micro W at 0.52 V.     

A membrane-less enzymatic fuel cell with layer-by-layer assembly of redox polymer and enzyme over graphite electrodes

Layer-by-layer (LBL) assembly of alternate osmium redox polymers and glucose oxidase, at anode, and laccase, at cathode, using graphite electrodes form a membrane-less glucose/O(2) enzymatic fuel cell providing a power density of 103 μW cm(-2) at pH 5.5.     

A biofuel cell based on pyrroloquinoline quinone and microperoxidase-11 monolayer-functionalized electrodes

A pyrroloquinoline quinone (PQQ) monolayer-functionalized-Au-electrode and a microperoxidase-11 (MP-11)-modified Au-electrode are used as catalytic anode and cathode in a biofuel cell element, respectively. The cathodic oxidizer is H₂O₂ whereas the anodic fuel-substrate is 1,4-dihydronicotinamide adenine dinucleotide, NADH. The PQQ-monolayer electrode catalyzes the oxidation of NADH in the presence of Ca²⁺ ions. The MP-11-functionalized electrode catalyzes the reduction of H₂O₂. The biofuel cell generates an open-circuit voltage, V_oc, of ca. 320 mV and a short-circuit current density, I_sc, of ca. 30 μA·cm⁻². The maximum electrical power, W_max, extracted from the cell is 8 μW at an external load of 3 kΩ. The fill factor of the biofuel cell, f=W_max·I_sc⁻¹·V_oc⁻¹, is ca. 25%.     

A glucose/oxygen enzymatic fuel cell based on redox polymer and enzyme immobilisation at highly-ordered macroporous gold electrodes

Highly ordered macroporous electrodes are prepared by electro-deposition of gold through a polystyrene sphere template. Drop-coating redox polymer and either glucose oxidase, for the anode, or Melanocarpus albomyces laccase, for the cathode on the macroporous gold provides film-coated electrodes for assembly of membrane-less glucose/oxygen enzymatic fuel cells (EFC) in pH 7.4 buffer containing 10 mM glucose and 0.15 M NaCl. Under these conditions the maximum power density of 17 μW cm(-2) for EFCs using films adsorbed to planar gold electrodes increased to 38 μW cm(-2) for films adsorbed to 2? sphere gold macroporous electrodes.     

Evaluation of an amperometric glucose biosensor based on a ruthenium complex mediator of low redox potential

An amperometric glucose ring-disk biosensor based on a ruthenium complex mediator of low redox potential was fabricated and evaluated. This thin-layer radial flow microsensor (10 μl) with ring-disk working electrode displayed remarkable amperometric sensitivity. For Ru₃(μ₃-O)(AcO)₆(Py)₃(ClO₄) (Ru-Py), a trinuclear oxo-acetate bridged cluster, a reversible redox curve of low redox potential and narrow potential window (redox potentials were −0.190 and −0.106 V versus Ag/AgCl wire, respectively) was observed, which is comparable to many reported mediators such as ferrocene derivatives and other ruthenium complexes. The glucose and hydrogen peroxide assays were carried out with this complex-modified electrode Ru-Py-HRP-GOx/Nafion. The sensitivity was obtained 24 nA (15.4 mA M⁻¹ cm⁻²) for 10 μM glucose and 126 nA (160 mA M⁻¹ cm⁻²) for 5 μM H₂O₂, respectively with a working potential at 0 V versus Ag/AgCl. Ascorbic acid was studied as interference to the glucose assay. The application of 0 V potential versus Ag/AgCl did not avoid the occurrence of the oxidation of ascorbic acid, however, the pre-coating of ascorbate oxidase on the disk part of the ring-disk working electrode efficiently pre-oxidized the ascorbic acid and hence eliminated its interference on the glucose response. The practical reliability was also evaluated by assaying the dialysate from the prefrontal cortex of Wistar rats.     

Preparation and characterization of a new enzyme electrode based on solid paraffin and activated graphite particles

A new electrochemical biosensor was developed by incorporating an enzyme into a solid-paraffin-graphite-particle matrix. Tyrosinase served as model enzyme and the biosensor response was characterized with respect to its response to dopamine. The influence of different experimental parameters (tyrosinase loading, flow rate, oxygen dependence, pH, etc.) was investigated in order to optimize the biosensor performance. The electrode response was fast, reversible and linear in a large concentration domain (0.1 muM-1 mM). The enzyme-solid paraffin carbon paste electrode (CPE) showed markedly improved stability in flow injection analysis compared to the classical liquid paraffin-graphite-based biosensors. The biosensor allowed a sampling rate of 79 samples per hour, the repeatability of the injections was improved with respect to the classical CPE with a relative standard deviation of 2.2% (N = 63), and the detection limit for dopamine was 50 nM. The biosensor response to some phenol and catechol derivatives was also investigated.     

Visualisation of the local bio-electrocatalytic activity in biofuel cell cathodes by means of redox competition scanning electrochemical microscopy (RC-SECM)

Optimisation of biocatalytic systems for the electroreduction of molecular O₂ in biofuel cell cathodes implies screening of the catalytic activity of enzyme/redoxpolymer assemblies. Os-complex modified electrodeposition polymers are suggested for linking bilirubin oxidase catalysed O₂ reduction via an electron hopping sequence along the redox polymer to the electrode. They can be non-manually precipitated on electrode surfaces by electrochemically induced pH modulation. Cyclic voltammetry provides a good estimation of the electrocatalytic activity of a redox polymer/enzyme modified electrode surface. In addition, scanning electrochemical microscopy operating in redox competition mode (RC-SECM) supplies images of the spatial distribution of the biocatalytic activity.     

Electrochemically triggered Michael addition on the self-assembly of 4-thiouracil: generation of surface-confined redox mediator and electrocatalysis

Generation of a surface-confined redox mediator (RM) by an electrochemically triggered Michael addition reaction and the electrocatalytic properties of the mediator are described. Electrogenerated o-quinone undergoes Michael addition reaction with the self-assembled monolayer (SAM) of 4-thiouracil (4-TU) on a gold (Au) electrode and yields a surface-confined RM, 1-(3,4-dihydroxyphenyl)-4-mercapto-1H-pyrimidin-2-one (DPTU). The Michael addition reaction depends on the electrolysis potential and time, solution pH, and concentration of catechol (CA) used in the reaction. The redox mediator, DPTU, exhibits reversible redox response, characterstic of a surface-confined species at approximately 0.22 V in neutral pH. The anodic peak potential of DPTU shifts by 58+/-2 mV while changing the solution pH by one unit, suggesting that protons and electrons taking part in the redox reaction are in the ratio of 1:1. The apparent rate constant (ksapp) for the heterogeneous electron-transfer reaction of the RM was determined to be 114+/-5 s-1. The surface coverage (Gamma) of DPTU on the electrode surface was 8.2+/-0.1x10(-12) mol/cm2. DPTU shows excellent electrocatalytic activity toward oxidation of reduced nicotinamide adenine dinucleotide (NADH) with activation overpotential, which is approximately 600 mV lower than that observed at the unmodified Au electrode. The dipositive cations in the supporting electrolyte solution amplify the electrocatalytic activity of DPTU. A 2.5-fold enhancement in the catalytic current was observed in the presence of Ca2+ or Ba2+ ions. The sensitivity of the electrode toward NADH in the presence and absence of Ca2+ ions was 0.094+/-0.011 and 0.04+/-0.0071 nA cm-2 nM-1, respectively. A linear increase in the catalytic current was obtained up to the concentration of 0.8 mM, and the electrode can detect amperometrically as low as 25 nM of NADH in neutral pH.     

A self-powered "sense-act-treat" system that is based on a biofuel cell and controlled by boolean logic

Bio-logic-al: an autonomous, integrated "sense-act-treat" system that is based on an enzymatic biofuel cell has been developed. The system couples a biocomputing logic-detection method with a drug-release system to provide a logic-activated therapeutic intervention in response to a simulated abnormal physiological state, without the need for an external power source, control electronics, or microelectromechanical actuators.     

Evaluating cell migration in vitro by the method based on cell patterning within microfluidic channels

Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.     

Biofuel cell anode based on the Gluconobacter oxydans bacteria cells and 2,6-dichlorophenolindophenol as an electron transport mediator

A basic scheme of the use of the Gluconobacter oxydans bacteria cells as a biocatalyst at an anode of a biofuel cell with air-based cathode is raised up. The anode and cathode of the cell are made of graphite; 2,6-dichlorophenolindophenol serves as an electron transport mediator; and glucose is the substrate to be oxidized. The open-circuit voltage is 55 mV, for the bacteria cell, the mediator, and glucose concentrations of 3 mg/ml (raw weight), 34 mM, and 10 mM, respectively. The voltage and current of the biofuel cell loaded with an external resistance of 10 kohm are 5.6 mV and 0.56 mA. The cell’s internal resistance is 88 kohm.     

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.     

An electric cyclophane: cavity control based on the rotation of a paraphenylene by redox switching

Although a free rotation around a single bond gives no mechanical output, it has let us imagine a molecular motor. A para-substituted aromatic ring can be regarded as a rotator with the para-rotation axis. When it is incorporated in a wider pi-conjugated system, a quinoidal structure is generated accompanied by oxidation on the substituted groups at the 1,4-position, and the axis is fixed. A paraphenylenediamine was selected as the nanomechanical molecular module capable of locking and releasing the free rotation using an electrode in solution. We inserted the module into a simple molecular system, cyclophane. It was clarified that the cyclophane was able to open and close its cavity in a reversible redox process repeatedly.     

An alternative analysis of enzyme systems based on the whole reaction time: evaluation of the kinetic parameters and initial enzyme concentration

This work presents an alternative analysis of the integrated rate equations corresponding to the simple Michaelis-Menten mechanism without product inhibition. The suggested new results are reached under a minimal set of assumptions and include, as a particular case, the classical integrated Michaelis–Menten equation. Experimental designs and a kinetic data analysis are suggested to the estimation of the maximum steady-state rate, V _max, the Michaelis–Menten constant, K _m, the initial enzyme concentration, [E]₀, and the catalytic constant, k ₂. The goodness of the analysis is tested with simulated time progress curves obtained by numerical integration.     

The design of dehydrogenase enzymes for use in a biofuel cell: the role of genetically introduced peptide tags in enzyme immobilization on electrodes.

The immobilization of the mutants of L-lactate dehydrogenase (LDH) on poly(aniline) (PANi) composite films has been investigated. Mutants possessing peptide tags of varying charge and nucleophilicity were created to probe the nature of the interaction between the protein and PANi. These results are significant for the development of a 'generic' approach to the immobilization of enzymes and other proteins.     

An enzyme electrode based on electromagnetic entrapment of the biocatalyst bound to magnetic beads

A new type of biosensor is described. It is based on the use of a Clark-type oxygen electrode placed in an electromagnetic field. The biomolecules used in this sensor are immobilized on small magnetic beads which are added to the system when needed and removed as required. By varying the strength of the magnetic field, a homogeneous distribution of particles at the electrode tip is achieved. The electrode is used for the determination of glucose with immobilized glucose oxidase as well as cells of Saccharomyces cerevisiae.     

An enzyme electrode based on immobilized arylsulfatase for the selective assay of sulfate ion

An enzyme electrode has been constructed for the assay of sulfate ion based on inhibition of the reaction

The steady-state current arising from oxidation of the product, 4-nitrocatechol, is measured at +0.8 V vs. S. C. E. The competitive inhibition of this reaction by added sulfate ion causes a decrease in this steady-state current in a linear relationship to pSO₄ in the range 2–4. The enzyme arylsulfatase (arylsulfate sulfohydrolase, EC 3.1·6.1) is chemically immobilized in a layer on a platinum electrode. This enzyme electrode also gives linear calibration plots for phosphate ion (10^-2–10^-4 M) based on its competitive inhibition of the above reaction, and for fluoride ion (10^-2–10^-4 M) based on its activation of the reaction. The assay of 4-nitrocatechol sulfate (NCS) in the range 10^-6–10^-4 M is possible. By proper control of the NCS concentration the electrode can be made almost completely specific for sulfate: only molybdate interferes. To establish the best operating conditions for the electrode, the effect of pH on the V_m and K_m were determined.     

Design of a bioelectrocatalytic electrode interface for oxygen reduction in biofuel cells based on a specifically adapted Os-complex containing redox polymer with entrapped Trametes hirsuta laccase

The design of the coordination shell of an Os-complex and its integration within an electrodeposition polymer enables fast electron transfer between an electrode and a polymer entrapped high-potential laccase from the basidiomycete Trametes hirsuta. The redox potential of the Os^3+/2+-centre tethered to the polymer backbone (+ 720 mV vs. NHE) is perfectly matching the potential of the enzyme (+ 780 mV vs. NHE at pH 6.5). The laccase and the Os-complex modified anodic electrodeposition polymer were simultaneously precipitated on the surface of a glassy carbon electrode by means of a pH-shift to 2.5. The modified electrode was investigated with respect to biocatalytic O₂ reduction to H₂O. The proposed modified electrode has potential applications as biofuel cell cathode.