Reconstitution Of Apo-glucose Oxidase Research Articles

Molecular simulation of flavin adenine dinucleotide immobilized on charged single-walled carbon nanotubes for biosensor applications

The reconstitution of apo-glucose oxidase (apo-GOx) on single-walled carbon nanotubes (SWNTs) functionalized with the cofactor, flavin adenine dinucleotide (FAD), greatly improved electron transfer turnover rate of the redox reactions in glucose sensing with glucose sensors. The research reported here is aimed to better understand molecular details of affection of the charging SWNT to the conformational changes of FAD, in order to find a rational design and selection scheme of SWNT which is suitable for the FAD and apo-GOx to perform their reconstitution. In this report, molecular simulations of FAD functionalized differently charged SWNTs were carried outin an aqueous environment, with counterions to maintain total charge neutrality. The conformation and orientation changes were observed by both trajectory and quantitative analyses. The simulation results showed that in both uncharged and positively charged SWNT situations, FAD adsorbed onto SWNT at the end of the simulations, which increased the steric resistance of molecules and hindered the reconstitution of apo-GOx and FAD to some degree. By contrast, FAD functionalized negatively charged SWNT maintained its original conformation largely. In addition, negatively charged SWNT may be the best choice for electron transfer mediator for the reconstitution of apo-GOx on relay-cofactor units associated with electrodes.

Switchable photochemical/electrochemical wiring of glucose oxidase with electrodes

Reconstitution of apo-glucose oxidase on a photoisomerizable-FAD monolayer linked to a Au electrode leads to the photonic wiring of the enzyme with the electrode and to the optical/electrochemical switching of the bioelectrocatalytic functions of the enzyme.

Reconstitution of apoglucose oxidase with FAD conjugates for biosensoring of progesterone

The reconstitution of Aspergillus niger apoglucose oxidase (apoGOx) with FAD conjugates for biosensoring of progesterone was investigated. ApoGOx prepared by partial unfolding of the protein under acidic conditions consisted of reconstitutable monomers (50 ± 10%), reconstitutable dimers (20 ± 10%) and irreversibly aggregated oligomers (30 ± 20%). Incubation of monomeric apoGOx with FAD or N 6-(6-aminohexyl)-FAD (ahFAD) restored glucose oxidase (GOx) activity and induced dimerization with stoichiometric incorporation of FAD. N 6-(6-aminohexyl)-FAD progesterone conjugates also induced dimerization. However, holoenzyme reconstitution required relatively high concentrations of apoprotein and was dependent on the type of conjugate. Restoration to 25–50% of the original enzyme activity was obtained. Binding of the FAD-progesterone conjugates might hinder the closure of a protein lid needed for dimer formation. Our results illustrate the prospects of FAD conjugates in sensitive detection of progesterone in biological matrices in a biosensor based on the recombination of apoGOx with progesterone-conjugated FAD.

Electrical contacting of glucose oxidase by DNA-templated polyaniline wires on surfaces

Poly(aniline–aniline boronic acid) wires are generated on ds-DNA templates, and the resulting wires exhibit redox functions at neutral pH aqueous solutions. The association of flavin adenine dinucleotide (FAD) to the boronic acid ligand followed by the reconstitution of apo-glucose oxidase on the cofactor units yield an integrated enzyme-electrode where the biocatalyst reveals direct electrical contact with the electrode.

Heterogeneous Electron-Transfer Kinetics for Flavin Adenine Dinucleotide and Ferrocene through Alkanethiol Mixed Monolayers on Gold Electrodes

The heterogeneous electron-transfer properties of flavin adenine dinucleotide (FAD) were studied at self-assembled monolayer (SAM)-modified gold electrodes and compared to ferrocene-modified SAMs as a reference system. A modified form of FAD, N6-(2-aminoethyl)-FAD, was covalently immobilized onto a mixed self-assembled monolayer (SAMs) formed by two series of alkanethiols (HS−(CH2)n−COOH with a diluent HS−(CH2)n−CH3 where n = 5, 7, 10, or 15 and HS−(CH2)n−COOH with a diluent HS−(CH2)n−CH2OH where n = 5, 7, 10, or 11). The electron-transfer (ET) rates followed an exponential decay with the increasing thickness of the SAM (ket = A exp(−βn); n = number of bonds) with an attenuation factor (β) of 1.0 per bond for FAD with both diluents. Despite the similarity in β the apparent rate constant for electron transfer was 2 orders of magnitude greater with the alcohol-terminated diluent relative to the methyl-terminated SAM. In comparison the ferrocenyl SAMs also had β values of 1.0−1.2 per bond, depending on the composition of the diluent layer, with the apparent rate constant for electron transfer being significantly faster with the alcohol-terminated diluent layer. The reconstitution of apo-glucose oxidase (apo-GOx) onto a FAD-modified gold electrode was also carried out; however, no biocatalytic activity was observed. Based on the β value for the FAD-modified electrodes and the magnitude of the apparent rate of electron transfer, rate constants for direct electron transfer to glucose oxidase at carbon nanotube-modified electrodes and graphite electrodes suggest the enzyme has been partially denatured from its native configuration, thus bringing the redox-active center of the enzyme closer to the electrode and allowing appreciable ET to be observed.

Electrical Contacting of Flavoenzymes and NAD(P)+-Dependent Enzymes by Reconstitution and Affinity Interactions on Phenylboronic Acid Monolayers Associated with Au-Electrodes

The preparation of integrated, electrically contacted, flavoenzyme and NAD(P)(+)-dependent enzyme-electrodes is described. The reconstitution of apo-glucose oxidase, apo-GOx, on a FAD cofactor linked to a pyrroloquinoline quinone (PQQ) phenylboronic acid monolayer yields an electrically contacted enzyme monolayer (surface coverage 2.1 x 10(-)(12) mol cm(-)(2)) exhibiting a turnover rate of 700 s(-)(1) (at 22 +/- 2 degrees C). The system is characterized by microgravimetric quartz-crystal microbalance analyses, Faradaic impedance spectroscopy, rotating disk electrode experiments, and cyclic voltammetry. The performance of the enzyme-electrode for glucose sensing is described. Similarly, the electrically contacted enzyme-electrodes of NAD(P)(+)-dependent enzymes malate dehydrogenase, MalD, and lactate dehydrogenase, LDH, are prepared by the cross-linking of affinity complexes generated between the enzymes and the NADP(+) and NAD(+) cofactors linked to a pyrroloquinoline quinone phenylboronic acid monolayer, respectively. The MalD enzyme-electrode (surface coverage 1.2 x 10(-)(12) mol cm(-)(2)) exhibits a turnover rate of 190 s(-)(1), whereas the LDH enzyme-electrode (surface coverage 7.0 x 10(-)(12) mol cm(-)(2)) reveals a turnover rate of 2.5 s(-)(1). Chronoamperometric experiments reveal that the NAD(+) cofactor is linked to the PQQ-phenylboronic acid by two different binding modes. The integration of the LDH with the two NAD(+) cofactor configurations yields enzyme assemblies differing by 1 order of magnitude in their bioelectrocatalytic activities.

Integration of polyaniline/poly(acrylic acid) films and redox enzymes on electrode supports: an in situ electrochemical/surface plasmon resonance study of the bioelectrocatalyzed oxidation of glucose or lactate in the integrated bioelectrocatalytic systems.

Electropolymerization of aniline in the presence of poly(acrylic acid) on Au electrodes yields a polyaniline/poly(acrylic acid) composite film, exhibiting reversible redox functions in aqueous solutions at pH = 7.0. In situ electrochemical-SPR measurements are used to identify the dynamics of swelling and shrinking of the polymer film upon the oxidation of the polyaniline (PAn) to its oxidized state (PAn(2+)) and the reduction of the oxidized polymer (PAn(2+)) back to its reduced state (PAn), respectively. Covalent attachment of N(6)-(2-aminoethyl)-flavin adenin dinucleotide (amino-FAD, 1) to the carboxylic groups of the composite polyaniline/poly(acrylic acid) film followed by the reconstitution of apoglucose oxidase on the functional polymer yields an electrically contacted glucose oxidase of unprecedented electrical communication efficiency with the electrode: electron-transfer turnover rate approximately 1000 s(-1) at 30 degrees C. In situ electrochemical-SPR analyses are used to characterize the bioelectrocatalytic functions of the biomaterial-polymer interface. The current responses of the bioelectrocatalytic system increase as the glucose concentrations are elevated. Similarly, the SPR spectra of the system are controlled by the concentration of glucose. The glucose concentration controls the steady-state concentration ratio of PAn/PAn(2+) in the film composition. Therefore, the SPR spectrum of the film measured upon its electrochemical oxidation is shifted from the spectrum typical for the oxidized PAn(2+) at low glucose concentration to the spectrum characteristic of the reduced PAn at high glucose concentration. Similarly, the polyaniline/poly(acrylic acid) film acts as an electrocatalyst for the oxidation of NADH. Accordingly, an integrated bioelectrocatalytic assembly was constructed on the electrode by the covalent attachment of N(6)-(2-aminoethyl)-beta-nicotinamide adenine dinucleotide (amino-NAD(+), 2) to the polymer film, and the two-dimensional cross-linking of an affinity complex formed between lactate dehydrogenase and the NAD(+)-cofactor units associated with the polymer using glutaric dialdehyde as a cross-linker. In situ electrochemical-SPR measurements are used to characterize the bioelectrocatalytic functions of the system. The amperometric responses of the system increase as the concentrations of lactate are elevated, and an electron-transfer turnover rate of 350 s(-1) between the biocatalyst and the electrode is estimated. As the PAn(2+) oxidizes the NADH units generated by the biocatalyzed oxidation of lactate, the PAn/PAn(2+) steady-state ratio in the film is controlled by the concentration of lactate. Accordingly, the SPR spectrum measured upon electrochemical oxidation of the film is similar to the spectrum of PAn(2+) at low lactate concentration, whereas the SPR spectrum resembles that of PAn at high concentrations of lactate.

Electrical contacting of glucose oxidase by surface-reconstitution of the apo-protein on a relay-boronic acid-FAD cofactor monolayer.

A new methodology for the surface-reconstitution of apo-flavoenzymes on a relay unit (pyrroloquinoline quinone, PQQ) functionalized with a boronic acid ligand as a linker to native FAD was developed. The reconstitution of apo-glucose oxidase (apo-GOx) on the PQQ-FAD monolayer yields an electrically contacted enzyme-electrode.

Glucose oxidase electrodes via reconstitution of the apo-enzyme: tailoring of novel glucose biosensors

Reconstitution of an apo-flavoenzyme with a relay-FAD-cofactor dyad yields an electrically contacted enzyme, “electroenzyme”. This is exemplified by the reconstitution of apo-glucose oxidase, apo-GOx, with a ferrocene–tethered FAD-cofactor. The resulting semisynthetic protein stimulates the bioelectrocatalyzed oxidation of glucose. Kinetic analysis of the ferrocene–FAD reconstituted enzyme reveals that its electrical communication with an electrode support is superior as compared to a protein randomly functionalized by ferrocene relay units. A pyrroloquinolinoquinone (PQQ)–FAD cofactor monolayer is assembled on a Au-electrode. Apo-GOx is reconstituted on the solid support to yield an aligned enzyme electrode of unprecendently efficient electrical contact. The electron-transfer turnover rate with the electrode is estimated to be 900±150 s −1, a value close to the electron transfer rate between native GOx and molecular oxygen. The effective electrical communication of the integrated enzyme electrode stimulates the efficient bioelectrocatalyzed oxidation of glucose, and results in high current densities and high sensitivity for glucose. The effective electrical contact of the enzyme-electrode yields also a specific glucose sensing electrode that is not perturbed by oxygen or interferrants. The possible application of the enzyme-electrode as an invasive biosensor is addressed.

Reconstitution of Apo-Glucose Oxidase on Nitrospiropyran and FAD Mixed Monolayers on Gold Electrodes: Photostimulation of Bioelectrocatalytic Features of the Biocatalyst

N6-(2-Aminoethyl)-FAD is covalently linked to a thioctic acid monolayer associated with an Au-electrode. The surface coverage of the FAD units is 2.2 × 10-11 mol cm-2. Apo-glucose oxidase (apo-GOx) reconstituted onto the FAD monolayer, yields a bioelectrocatalytically active enzyme layer, GOx, at a surface coverage of 2 × 10-12 mol cm-2. In the presence of N-dimethyl-2-aminoethyl ferrocene (I) as diffusional electron mediator, the enzyme electrode stimulates the bioelectrocatalyzed oxidation of glucose. A photoisomerizable nitrospiropyran−FAD mixed monolayer is assembled on an Au-electrode. The surface coverage of the FAD units is 2 × 10-11 mol cm-2, and the molar ratio of the nitrospiropyran and FAD units is 3:4. Reconstitution of apo-GOx onto the FAD sites of the photoisomerizable nitrospiropyran−FAD mixed monolayer yields a photoswitchable enzyme electrode for the light-stimulated oxidation of glucose in the presence of I as electron mediator. The photoswitchable properties of the enzyme electrode orig...

Electrical contact of redox enzymes with electrodes: novel approaches for amperometric biosensors

Electrical communication of the redox-active center of enzymes with an electrode surface is a fundamental element for the development of amperometric biosensor devices. Different methods to assemble enzyme-electrodes exhibiting electrical contact between the redox protein and electrode surface are discussed with specific examples for tailoring glucose sensing electrodes. By one approach, a multilayer array of glucose oxidase is assembled on a Au-electrode. The number of enzyme layers is controlled by the synthetic methodology to assemble the electrode. Electrical contact between the enzyme array and the electrode is established by chemical modification of the protein layer with N-(2-methyl-ferrocene)-caproic acid, acting as an electrode is established by chemical enzyme layers associated with the electrode allows one to control the sensitivity of the resulting enzyme electrode. A further means of enhancing the sensitivities of enzyme electrodes involves the application of rough Au-electrodes as base-support to assemble the enzyme network. The high surface area of these electrodes (roughness factor ≈ 20) allows the increase of the biocatalyst content in a single monolayer, and the resulting amperometric responses of the electrodes are ca. 8-fold enhanced compared to enzyme layers assembled on smooth electrodes of identical geometrical areas. A novel method to electrically wire flavoenzymes with electrode surfaces was developed by reconstitution of the apo-flavoenzyme with a ferrocene-tethered FAD diad. Reconstitution of apo-glucose oxidase with the ferrocene-FAD diad yields an active bioelectrocatalyst of direct electrical communication with the electrode, ‘electroenzyme’. The reconstitution methodology was further applied to tailor enzyme-electrodes of superior properties for electrical contact with the electrodes. A pyrroloquinoline quinone-FAD diad monolayer was assembled on a Au-electrode. Apo-glucose oxidase was reconstituted on the surface with the FAD-cofactor site to yield the aligned biocatalyst on the electrode. The pyrroloquinoline quinone. PQQ, redox unit acts as an electron relay that electrically contacts the FAD redox-site of the enzyme with the electrode. The surface reconstituted enzyme exhibits direct electrical communication with the electrode and acts as bioelectrocatalyst for the oxidation of glucose. The electrical communication of the reconstituted glucose oxidase on the PQQ-FAD monolayer is extremely efficient. The experimental current density at a glucose concentration of 80 mM is 300 ± 100 μA · cm −2. This value overlaps the theoretical current density of glucose oxidase electrode (290 ± 60 μA · cm −2) taking into account the limiting turnover-rate of the enzyme, 900 ± 150 s −1 (at 35°C). The extremely efficient electrical contact of the reconstituted enzyme and the electrode yields an enzyme-electrode that is insensitive to oxygen and is not affected by glucose-sensing interferants such as ascorbic acid. The application of the different enzyme-electrode configurations as bioelectronic devices for the determination of glucose is addressed.

Assembly of functionalized monolayers of redox proteins on electrode surfaces: novel bioelectronic and optobioelectronic systems

Functionalized monolayer electrodes provide the grounds for bioelectronic and optobioelectronic devices. Reconstitution of apo-glucose oxidase, apo-GOx, onto a pyrroloquinoline quinone-FAD diad, assembled as a monolayer on a Au-electrode, yields an aligned bioelectrocatalytically active enzyme on the electrode surface. The resulting reconstituted enzyme electrode exhibits superior electrical contact with the electrode surface and acts as an amperometric glucose sensing electrode. The enzyme electrode operates under oxygen and is unaffected by interfering substrates such as ascorbic acid. Photoswitchable redox proteins integrated with electrode surfaces act as active systems for the amplified amperometric transduction of recorded optical signals. Chemical modification of glucose oxidase by photoisomerizable nitrospiropyran units or reconstitution of apo-GOx with a photoisomerizable nitrospiropyran-FAD diad, yield photoisomerizable glucose oxidase with photoswitchable biocatalytic features. Assembly of the photoactive enzymes as monolayers on the Au-electrode results in functionalized surfaces for the cyclic ‘ON-OFF’ amplified amperometric transduction of recorded optical signals. A further method to photostimulate the electrical contact between a redox protein and an electrode involves the functionalization of the electrode with a photoisomerizable monolayer interface. A mixed monolayer consisting of pyridine and nitrospiropyran units was used to photoregulate the association and dissociation of cytochrome c to and from the monolayer assembly. The photostimulated electrical contact of cytochrome c with the monolayer electrode was employed to mediate the bioelectrocatalyzed reduction of oxygen in the presence of cytochrome oxidase, COx. The latter system provides an assembly for the cyclic amperometric transduction of recorded optical signals.

Electrical Wiring of Glucose Oxidase by Reconstitution of FAD-Modified Monolayers Assembled onto Au-Electrodes

Electrical Wiring of Glucose Oxidase by Reconstitution of FAD-Modified Monolayers Assembled onto Au-Electrodes