Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance.

Sensors--an effective approach for the detection of explosives

The widespread exploitation of biosensors in the analysis of molecular recognition has its origins in the mid-1990s following the release of commercial systems based on surface plasmon resonance (SPR). More recently, platforms based on piezoelectric acoustic sensors (principally 'bulk acoustic wave' (BAW), 'thickness shear mode' (TSM) sensors or 'quartz crystal microbalances' (QCM)), have been released that are driving the publication of a large number of papers analysing binding specificities, affinities, kinetics and conformational changes associated with a molecular recognition event. This article highlights salient theoretical and practical aspects of the technologies that underpin acoustic analysis, then reviews exemplary papers in key application areas involving small molecular weight ligands, carbohydrates, proteins, nucleic acids, viruses, bacteria, cells and lipidic and polymeric interfaces. Key differentiators between optical and acoustic sensing modalities are also reviewed.

A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions.

Electropolymerized films have received considerable attention in the development of biosensors and biochips, and are advancing rapidly. This paper reviews recent advances and scientific progress in electrochemical immobilization procedures for biological macromolecules on electrodes via electrogenerated polymer films. Biomolecule immobilization is classified as covalent linkage, attachment by affinity interactions, and physical entrapment. The last approach entails the use of conducting and non-conducting films, composite polymer films, and templates for the electropolymerization process. Some advances in the electrochemical transduction of biological events (enzymatic reaction, immunoreaction, or oligonucleotide hybridization) involving the redox properties or the conductivity of electropolymerized films are also presented.

Biosensors based on electropolymerized films: new trends

A new molecularly imprinted polymer (MIP)-based electrochemical sensor for monitoring 2,4,6-trinitrotoluene (TNT) in natural waters and soil samples.

The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP−NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridine (PPB) are described. The MBP domain of MBP−NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 × 104 and 7.1 × 104 M-1 s-1 were obtained for TNT and DNT, respectively. The apparent Michaelis−Menten constants (KM) for MBP−NR in solution and on the surface, using TNT as substrate, were determi...

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low μM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.

Assessing the Impact of Denitrifier-Produced Nitric Oxide on Other Bacteria

The affinity of the maltose binding protein−nitro reductase fusion (MBP−NR) to electropolymerized films of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridinium (PPB) has been studied in aqueous medium by the quartz crystal microbalance (QCM) technique. It was found that the MBP domain of MBP−NR exhibits specific binding toward PPB films through the maltose binding site and that the immobilized MBP−NR retains its enzymatic activity toward trinitrotoluene (TNT) reduction. Although some MBP−NR was nonspecifically adsorbed onto a maltose-covered PPB film as well as to a bare Au electrode, in both cases the adsorbed enzyme exhibited no catalytic activity. Nitro reductase (NR) also appeared to adsorb onto the PPB films but did not exhibit any enzymatic activity. The electropolymerization of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridinium (PPB) onto a Au electrode in acetonitrile solution was studied by electrochemical quartz crystal microbalance (EQCM). It was found that the extent of polymerization upon pyrrole-centered oxidat...

Study of Specific Binding of Maltose Binding Protein to Pyrrole-Derived Bipyridinium Film by Quartz Crystal Microbalance

Although the electropolymerization of N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridinium (PPB) was anticipated to proceed through the 2 and 4 positions of the pyrrole ring, the emergence of two new peaks at -0.14 and -0.56 V versus Ag/AgCl in addition to those expected at -0.89 and -1.60 V upon continuous potential scanning, indicated the existence of a competitive coupling reaction. Using cyclic voltammetry and electrochemical quartz crystal microbalance techniques, a plausible mechanism for the polymerization reaction is proposed. The proposed mechanism involves quaternization of the second pyridine nitrogen, resulting in polymerization through a bipyridinium-pyrrole bond formation. Consequently, four types of pyridines would be formed: type 1, pyridinium next to pyrrole; type 2, pyridinium next to type 1; type 3, pyridinium between propyl chain and nonquaternized pyridine; type 4, nonquaternized pyridine. The surface coverage values of the nonquaternized nitrogens, type 4, which had been assumed to be the sites that bind specifically to the maltose binding protein (MBP) domain of MBP enzyme fusions was found to be sufficiently high (lower limit ofca. 5 x 10 - 1 0 mol cm - 2 ) to ensure that the MBP fusions can easily bind.

Zeki Naal

Papers

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

Assessing the Impact of Denitrifier-Produced Nitric Oxide on Other Bacteria

Study of Specific Binding of Maltose Binding Protein to Pyrrole-Derived Bipyridinium Film by Quartz Crystal Microbalance

Detailed study of N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridinium (PPB) electropolymerization