Björn Persson

Amperometric glucose sensors based on immobilized glucose-oxidizing enzymes and chemically modified electrodes

Amperometric biosensors based on two different reaction mechanisms are presented. Common to both types is the combination of a selective enzymatic reaction with a selective mediated reaction that can be followed amperometrically at 0 mV vs. SCE and below. One type is based on the chemical modification of carbon pastes with a dehydrogenase, the necessary cofactor NAD+ and a redox mediator. In the presence of the enzyme substrate NADH will be produced. The high overvoltage for the electrochemical oxidation of the NADH is decreased by the addition of the redox mediator to the paste. The redox mediators used are phenoxazine derivatives, making the electrocatalytic oxidation of NADH possible at 0 mV vs. SCE and below. A glucose sensor based on glucose dehydrogenase is described. Another type is based on the co-immobilization of a hydrogen peroxide-producing oxidase with horseradish peroxidase onto the surface of solid graphite. The detection is based on an apparent direct electron transfer from the electrode to the immobilized peroxidase starting at +600 mV and reaching a maximum at about 0 mV vs. SCE. The enzyme layer is stabilized by the addition of bovine serum albumin and cross-linking with glutaraldehyde. A glucose sensor based on glucose oxidase is presented.

A chemically modified graphite electrode for electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide based on a phenothiazine derivative, 3-β-naphthoyl-toluidine blue O

Abstract The 7-dimethylamino-2-methyl-3-naphthamido-phenothiazinium salt (3-NTB) was obtained by reacting Toluidine Blue O (TBO) and 2-naphthoyl chloride. It was adsorbed on graphite to give a chemically modified electrode (CME) with a formal redox potential of −135 mV (vs. SCE) at pH 7. The 3-NTB CME catalyses the electrochemical oxidation of the reduced co-enzyme nicotinamide adenine dinucleotide (NADH). The reaction rate between 3-NTB and NADH was evaluated from rotating disk electrode measurements. The 3-NTB CME was also mounted in a flow-through amperometric cell in a single-channel FIA system and tested as a sensor for NADH. The calibration graph was linear over almost three decades of concentration at pH 9 with a sensitivity of 13.8 mA M −1 and a detection limit of 0.2 (μM NADH with an injection volume of 50 μl. The NADH sensor showed good stability in alkaline solutions and an improved response in terms of a decrease in pH dependence compared with previously studied sensors based on graphite electrodes modified with phenoxazine derivatives.

FAUNUS: An object oriented framework for molecular simulation

BackgroundWe present a C++ class library for Monte Carlo simulation of molecular systems, including proteins in solution. The design is generic and highly modular, enabling multiple developers to easily implement additional features. The statistical mechanical methods are documented by extensive use of code comments that – subsequently – are collected to automatically build a web-based manual.ResultsWe show how an object oriented design can be used to create an intuitively appealing coding framework for molecular simulation. This is exemplified in a minimalistic C++ program that can calculate protein protonation states. We further discuss performance issues related to high level coding abstraction.ConclusionC++ and the Standard Template Library (STL) provide a high-performance platform for generic molecular modeling. Automatic generation of code documentation from inline comments has proven particularly useful in that no separate manual needs to be maintained.

Protein adsorption and complement activation for di-block copolymer nanoparticles

Four types of nanoparticles with core-diffuse shell structures have been synthesized through self-assembly of PICBA-Dextran block copolymers. These nanoparticles are designed to carry pharmaceutically active molecules into the human body through injection into the blood stream. In this work, we have determined how the characteristics of the diffuse shell influence the adsorption of three types of proteins: Bovine Serum Albumin (BSA), fibrinogen, and a protein from the complement system that triggers recognition and elimination by macrophages. We have determined the structural characteristics of the diffuse shells using Nuclear Magnetic Resonance (NMR), Small Angle Neutron Scattering (SANS) and Quasi-Elastic Light Scattering (QELS). We have measured the adsorption of Bovine Serum Albumin (BSA) through Immunodiffusion methods, and found that it adsorbed in substantial amounts even when the distance between dextran chains at the core-diffuse shell interface is quite short. We have observed the aggregation of the nanoparticles induced by fibrinogen, and found that it was prevented when the density of dextran chains protruding from the core surface was sufficiently high. Finally we have measured the activation of the complement system by the nanoparticles, and found that it was also limited by the surface density of dextran chains that protrude from the core and by their mesh size within the diffuse shell.

Amperometric biosensors based on electrocatalytic regeneration of NAD+ at redox polymer-modified electrodes

Abstract Amperometric biosensors based on redox polymer-mediated electron transfer from NADH to carbon paste electrodes, regenerating the NAD + needed for the dehydrogenase-catalysed reaction, are described. These sensors, operating around 0 mV vs SCE, can drive an unfavourable equilibrium of a dehydrogenase-catalysed reaction to the product side, and are reagentless in that NAD + need not be added to the analyte solution. By covering the sensor with an additional polymer, protection of the electrode surface, inclusion of water soluble components, exclusion of interferents and a diffusion controlled linear response current can be obtained.

Amperometric thin film biosensors based on glucose dehydrogenase and Toluidine blue O as catalyst for NADH electrooxidation

Amperometric glucose sensors were constructed based on solid graphite electrodes, surface-modified with NAD+ dependent glucose dehydrogenase (GDH), Toluidine Blue O (TBO), and protective ionic polymers. The electrocatalytic oxidation of NADH was evaluated from cyclic voltammetry with TBO dissolved, adsorbed, and electrostatically or covalently bound to polymers. The NADH and glucose sensors constructed were investigated and operated at 0 mV vs. Ag/AgCl using single potential step chronoamperometry. The operational stability of the glucose sensors was limited by leakage of NAD+. A glucose sensitivity much higher than carbon paste electrode was found. A sensitivity as high as 25 microA cm-2 mM-1 was achieved.

Biofuel anode based on d-glucose dehydrogenase, nicotinamide adenine dinucleotide and a modified electrode

A biofuel cell anode has been made from a modified graphite electrode and immobilized d-glucose dehydrogenase [β-d-glucose:NAD(P)+ 1-oxidoreductase, EC 1.1.1.4 7] so that energy could be drawn from the conversion of d-glucose to d-gluconic acid. An equivalent amount of dihydronicotinamide adenine dinucleotide (NADH) was formed from NAD+ and reduced the surface groups of the modified electrode. Reoxidationn of the latter produced the electrons necessary for a power output from the cell. Electrode modification was made by adsorption of N,N-dimethyl-7-amino 1,2-benzophenoxazinium onto the graphite. A current density of 0.2 mA cm−2 at a cell voltage of ∼0.8 V was obtained for more than 8 h with a simulated oxygen cathode. The internal resistance in the cell, in particular in the separator, appeared to be the main current-limiting factor.

Charge-Induced Patchy Attractions between Proteins

Static light scattering (SLS) combined with structure-based Monte Carlo (MC) simulations provide new insights into mechanisms behind anisotropic, attractive protein interactions. A nonmonotonic behavior of the osmotic second virial coefficient as a function of ionic strength is here shown to originate from a few charged amino acids forming an electrostatic attractive patch, highly directional and complementary. Together with Coulombic repulsion, this attractive patch results in two counteracting electrostatic contributions to the interaction free energy which, by operating over different length scales, is manifested in a subtle, salt-induced minimum in the second virial coefficient as observed in both experiment and simulations.

Anisotropic Interactions in Protein Mixtures: Self Assembly and Phase Behavior in Aqueous Solution

Recent experimental studies show that oppositely charged proteins can self-assemble to form seemingly stable microspheres in aqueous salt solutions. We here use parallel tempering Monte Carlo simulations to study protein phase separation of lysozyme/α-lactalbumin mixtures and show that anisotropic electrostatic interactions are important for driving protein self-assembly. In both dilute and concentrated protein phases, the proteins strongly align according to their charge distribution. While this alignment can be greatly diminished by a single point mutation, phase separation is completely suppressed when neglecting electrostatic anisotropy. The results highlight the importance of subtle electrostatic interactions even in crowded biomolecular environments where other short-ranged forces are often thought to dominate.

Molecular evidence of stereo-specific lactoferrin dimers in solution.

Gathering experimental evidence suggests that bovine as well as human lactoferrin self-associate in aqueous solution. Still, a molecular level explanation is unavailable. Using force field based molecular modeling of the protein-protein interaction free energy we demonstrate (1) that lactoferrin forms highly stereo-specific dimers at neutral pH and (2) that the self-association is driven by a high charge complementarity across the contact surface of the proteins. Our theoretical predictions of dimer formation are verified by electrophoretic mobility and N-terminal sequence analysis on bovine lactoferrin.