A biosensor can be defined as a compact analytical device or unit incorporating a biological or biologically derived sensitive recognition element immobilized on a physicochemical transducer to measure one or more analytes. Microfluidic systems, on the other hand, provide throughput processing, enhance transport for controlling the flow conditions, increase the mixing rate of different reagents, reduce sample and reagents volume (down to nanoliter), increase sensitivity of detection, and utilize the same platform for both sample preparation and detection. In view of these advantages, the integration of microfluidic and biosensor technologies provides the ability to merge chemical and biological components into a single platform and offers new opportunities for future biosensing applications including portability, disposability, real-time detection, unprecedented accuracies, and simultaneous analysis of different analytes in a single device. This review aims at representing advances and achievements in the field of microfluidic-based biosensing. The review also presents examples extracted from the literature to demonstrate the advantages of merging microfluidic and biosensing technologies and illustrate the versatility that such integration promises in the future biosensing for emerging areas of biological engineering, biomedical studies, point-of-care diagnostics, environmental monitoring, and precision agriculture.

We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin ( ; coenzyme B 12 ) serves as a radical repository for a group of enzymes that catalyze unusual isomerizations, whereby a hydrogen atom (H) is interchanged with an electron-withdrawing group (X) on a neighboring carbon atom (Scheme 1) (1-4). To date, 11 AdoCbl-dependent isomerases have been identified and grouped into three classes: (i) Class I (mutases) that catalyze carbon skeletal rearrangements; (ii) Class II (eliminases) that catalyze, with one exception, the migration and elimination of a heteroatom; and (iii) Class III (aminomutases) that catalyze intramolecular 1,2-amino shifts. Turnover for all AdoCbl-dependent isomerases begins with substrate-induced homolysis of the AdoCbl Co-C bond and formation of two paramagnetic centers: the 5Ј-deoxyadenosyl radical (Ado ⅐ ) and cob(II)alamin. The highly reactive Ado ⅐ species propagates radical formation by abstracting a hydrogen atom from the substrate (or an amino acid side chain in the case of ribonucleotide reductase) (5), generating deoxyadenosine and a substrate radical. The latter carbon-centered radical isomerizes to a product radical intermediate, which then reabstracts a hydrogen atom to form Ado ⅐ . Geminate recombination between Ado ⅐ and cob(II)alamin regenerates AdoCbl and primes the enzyme for another catalytic cycle.Studies on AdoCbl-dependent isomerases have shown that the first step in the catalytic cycle (homolytic rupture of the Co-C bond) is coupled kinetically to hydrogen abstraction by Ado ⅐ (6 -9). Thus, the highly reactive Ado ⅐ species is short lived due to rapid neutralization by hydrogen abstraction, and this species has yet to be observed. The second paramagnetic center, cob(II)alamin, "lingers" in the active site, until product is formed, after which it recombines with Ado ⅐ to form the resting enzyme. In the presence of substrate, cob(II)alamin accumulates at a steady-state concentration, which can be observed by EPR and UV-visible spectroscopy. Class I and Class II isomerases have been extensively studied, and the cob(II)alamin spectroscopic signature has been valuable in studies of mechanism (6,8,10). Lysine 5,6-aminomutase (5,6-LAM) is the only Class III enzyme for which detailed studies of mechanism are reported. Cob(II)alamin does not accumulate in steady-state turnover as the enzyme undergoes rapid "suicide inactivation" involving removal of an electron from cob(II)alamin by a substrate and/or product radical intermediate (11). Irreversible formation of cob(III)alamin results, and cob(II)alamin is unable to recombine with Ado ⅐ to reform AdoCbl in the resting form of the enzyme.

Human methionine synthase reductase (MSR) catalyzes the NADPH-dependent reductive methylation of methionine synthase. MSR is 78 kDa flavoprotein belonging to a family of diflavin reductases, with cytochrome P450 reductase (CPR) as the prototype. MSR and its individual flavin-binding domains were cloned as GST-tagged fusion proteins for expression and purification from Escherichia coli. The isolated flavin domains of MSR retain UV-visible and secondary structural properties indicative of correctly folded flavoproteins. Anaerobic redox titrations on the individual domains assisted in assignment of the midpoint potentials for the high- and low-potential flavin. For the isolated FMN domain, the midpoint potentials for the oxidized/semiquinone (ox/sq) couple and semiquinone/hydroquinone (sq/hq) couple are -112 and -221 mV, respectively, at pH 7.0 and 25 degrees C. The corresponding couples in the isolated FAD domain are -222 mV (ox/sq) and -288 mV (sq/hq). Both flavins form blue neutral semiquinone species characterized by broad absorption peaks in the long-wavelength region during anaerobic titration with sodium dithionite. In full-length MSR, the values of the FMN couples are -109 mV (ox/sq) and -227 mV (sq/hq), and the corresponding couple values for FAD are -254 mV (ox/sq) and -291 mV (sq/hq). Separation of the MSR flavins does not perturb their thermodynamic properties, as midpoint potentials for all four couples are similar in isolated domains and in full-length MSR. The redox properties of MSR are discussed in relation to other members of the diflavin oxidoreductase family and the mechanism of electron transfer.

Oligomeric proanthocyanidins constitute a group of water-soluble polyphenolic tannins that are present in the female inflorescences (up to 5% dry wt) of the hop plant (Humulus lupulus). Humans are exposed to hop proanthocyanidins through consumption of beer. Proanthocyanidins from hops were characterized for their chemical structure and their in vitro biological activities. Chemically, they consist mainly of oligomeric catechins ranging from dimers to octamers, with minor amounts of catechin oligomers containing one or two gallocatechin units. The chemical structures of four procyanidin dimers (B1, B2, B3, and B4) and one trimer, epicatechin-(4beta-->8)-catechin-(4alpha-->8)-catechin (TR), were elucidated using mass spectrometry, NMR spectroscopy, and chemical degradation. When tested as a mixture, the hop oligomeric proanthocyanidins (PC) were found to be potent inhibitors of neuronal nitric oxide synthase (nNOS) activity. Among the oligomers tested, procyanidin B2 was most inhibitory against nNOS activity. Procyanidin B3, catechin, and epicatechin were noninhibitory against nNOS activity. PC and the individual oligomers were all strong inhibitors of 3-morpholinosydnonimine (SIN-1)-induced oxidation of LDL, with procyanidin B3 showing the highest antioxidant activity at 0.1 microg/mL. The catechin trimer (TR) exhibited antioxidant activity more than 1 order of magnitude greater than that of alpha-tocopherol or ascorbic acid on a molar basis.

D-Ornithine 4,5-aminomutase (OAM) fromClostridium sticklandii converts D-ornithine to 2,4-diaminopentanoic acid by way of radical propagation from an adenosylcobalamin (AdoCbl) to a pyridoxal 5-phosphate (PLP) cofactor. We have solved OAM crystal structures in different catalytic states that together demonstrate unusual stability of the AdoCbl Co-C bond and that radical catalysis is coupled to large-scale domain motion. The 2.0-Å substrate-free enzyme crystal structure reveals the Rossmann domain, harboring the intact AdoCbl cofactor, is tilted toward the edge of the PLP binding triosephosphate isomerase barrel domain. The PLP forms an internal aldimine link to the Rossmann domain through Lys 629 , effectively locking the enzyme in this "open" pre-catalytic conformation. The distance between PLP and 5-deoxyadenosyl group is 23 Å , and large-scale domain movement is thus required prior to radical catalysis. The OAM crystals contain two Rossmann domains within the asymmetric unit that are unconstrained by the crystal lattice. Surprisingly, the binding of various ligands to OAM crystals (in an oxygen-free environment) leads to transimination in the absence of significant reorientation of the Rossmann domains. In contrast, when performed under aerobic conditions, this leads to extreme disorder in the latter domains correlated with the loss of the 5-deoxyadenosyl group. Our data indicate turnover and hence formation of the "closed" conformation is occurring within OAM crystals, but that the equilibrium is poised toward the open conformation. We propose that substrate binding induces large-scale domain motion concomitant with a reconfiguration of the 5-deoxyadenosyl group, triggering radical catalysis in OAM.Enzymes use conformational motion, from small molecular vibrations to the reorganization of active site residues, and occasionally through to large-scale domain movement, to achieve catalytic prowess (1-3). The coupling of dynamics to catalysis requires precise timing and control, and this is especially true of enzymes that house highly oxidative radical intermediates such as the adenosylcobalamin (AdoCbl) 4 (coenzyme B 12 )-dependent isomerases. Ornithine 4,5-aminomutase (OAM; EC 5.4.3.5) belongs to this group of enzymes. OAM, from Clostridium sticklandii, functions in the oxidative fermentation of L-ornithine by conversion of D-ornithine to 2,4-diaminopentanoate (4). In addition to AdoCbl, the enzyme contains pyridoxal L-phosphate (PLP), which forms an internal aldimine link to Lys 629 in the resting state of the enzyme (5). The incoming substrate induces transimination, whereby the migrating amine of the substrate forms an external aldimine link to PLP. Homolysis of the AdoCbl Co-C bond is triggered by formation of the external aldimine generating cob(II)alamin and the highly reactive carbon-centered 5Ј-deoxyadenosyl radical (Ado ⅐ ). H ⅐ abstraction by Ado ⅐ from the PLP-substrate complex produces a substrate radical that isomerizes, possibly through a cyclic (azacyclopropylcarbinyl radical) intermediate (6) (...