Electrocatalytic interconversion of NADH and NAD(+) by Escherichia coli flavohemoglobin.

E. coli flavohemoglobin, oriented at electrodes via amphiphilic polymyxin B, electrocatalytically interconverts NADH and NAD(+) at its heme potentials operating as an electron transfer relay between the electrode and the protein FAD, where NADH/NAD(+) is transformed. The results are crucial for the development of NAD(+)-dependent bioelectrodes for biosynthesis, biosensors and biofuel cells.

Electronically addressable nanomechanical switching of i-motif DNA origami assembled on basal plane HOPG.

Here, a pH-induced nanomechanical switching of i-motif structures incorporated into DNA origami bound onto cysteamine-modified basal plane HOPG was electronically addressed, demonstrating for the first time the electrochemical read-out of the nanomechanics of DNA origami. This paves the way for construction of electrode-integrated bioelectronic nanodevices exploiting DNA origami patterns on conductive supports.

Electrochemical control of a DNA Holliday Junction nanoswitch by Mg2+ ions.

The molecular conformation of a synthetic branched, 4-way DNA Holliday junction (HJ) was electrochemically switched between the open and closed (stacked) conformers. Switching was achieved by electrochemically induced quantitative release of Mg(2+) ions from the oxidised poly(N-methylpyrrole) film (PPy), which contained polyacrylate as an immobile counter anion and Mg(2+) ions as charge compensating mobile cations. This increase in the Mg(2+) concentration screened the electrostatic repulsion between the widely separated arms in the open HJ configuration, inducing switching to the closed conformation. Upon electrochemical reduction of PPy, entrapment of Mg(2+) ions back into the PPy film induced the reverse HJ switching from the closed to open state. The conformational transition was monitored using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes each located at the terminus of one of the arms. The demonstrated electrochemical control of the conformation of the used probe-target HJ complex, previously reported as a highly sequence specific nanodevice for detecting of unlabelled target [Buck, A.H., Campbell, C.J., Dickinson, P., Mountford, C.P., Stoquert, H.C., Terry, J.G., Evans, S.A.G., Keane, L., Su, T.J., Mount, A.R., Walton, A.J., Beattie, J.S., Crain, J., Ghazal, P., 2007. Anal. Chem., 79, 4724-4728], allows the development of electronically addressable DNA nanodevices and label-free gene detection assays.

Molecular recognition with DNA nanoswitches: effects of single base mutations on structure.

This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 A) for the perfectly matched case, whereas larger separations (up to 50 A) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.

Electrochemically induced oxidative damage to double-stranded calf thymus DNA adsorbed on gold electrodes.

Electrochemically induced oxidative damage to DNA was studied with double-stranded calf thymus DNA immobilized directly on a gold electrode surface. Pre-polarization of the DNA-modified electrodes at +0.5 V versus Ag/AgCl reference electrode, in a free from DNA blank buffer solution, pH 7.4, allowed for subsequent detection of direct electrochemical oxidation of adsorbed on gold DNA, in the potential range from +0.7 to +0.8 V. The redox potential of the process corresponded to the potentials of the oxidation of guanine bases in DNA. It is shown that with increasing potential scan rate, v, the mechanism of electrochemical oxidation of DNA changes from the irreversible 4e(-) oxidative damage of DNA at low v to reversible 1e(-) oxidation at high v, keeping the electrochemical activity of the adsorbed DNA layer virtually the same.

P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes.

Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase.

Mediatorless biosensor for H(2)O(2) based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold.

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4-1.5 A M(-1) cm(-2) at -50 mV versus Agmid R:AgCl. The response to H(2)O(2) in the concentration range 0.1-40 microM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H(2)O(2) detection was 10 nM at the electrodes modified with recombinant HRPs.