Visible radiation effects on flavocytochrome b2 in dilute aqueous solution: a steady-state and laser flash photolysis study

Irradiation of flavocytochrome b2 by visible radiation at 450 nm in dilute aqueous solution is found to have a devastating effect not only on its activity but also on the important flavin mononucleotide (FMN) constituents. The active site and the substrate binding site are also found to be largely modified on exposure to visible radiation. This has a telling effect on the constituent aromatic amino acids, tryptophan and tyrosine, and therefore justifies the role of FMN as a very potent photosensitizer. Partial unfolding of the irradiated enzyme molecule is also observed. Damage is much greater in deaerated conditions, which indicates that molecular oxygen plays a protecting role in this particular system. The inactivation is mediated through rapid electron transfer from tryptophan and tyrosine to excited flavin, forming flavin semiquinone and tryptophanyl and tyrosinyl radicals, which in turn cause permanent damage at the molecular level.     

Polarity dependence of the radiative and nonradiative rates of flavone derivatives comprising structurally similar amino moieties: change in the nature of the emitting state

Photophysical behavior of several structurally related electron donor-acceptor flavone derivatives, which have been synthesized and characterized, has been studied as a function of the polarity of the media. Significant variation of the absorption and fluorescence response of the systems has been observed with change in the polarity of the medium. The results show an increase in the radiative rate constant and a decrease in the nonradiative rate constant of the systems with increase in the polarity of the media. This finding has been attributed to the change in the nature of the emitting state from a mixed n-pi* and pi-pi* state to a dominant pi-pi* state with increase in the polarity of the medium. The results of single-crystal diffraction studies and theoretical calculations based on density functional method support the idea of close proximity of the n-pi* and pi-pi* states and the change in their relative contributions toward the emission process with the polarity of the medium. Laser flash photolysis studies show that the triplet state is not involved in the variation of the fluorescence response of the systems.     

Unexpectedly large binding constants of azulenes with fullerenes

In spite of having small pi-conjugation systems, azulenes show large binding constants (10(4)-10(5)) to C(60) and C(70), which are larger than those of monoporphyrins and alternant aromatic hydrocarbons.     

PCR-based detection in a micro-fabricated platform

We present a novel, on-chip system for the electrokinetic capture of bacterial cells and their identification using the polymerase chain reaction (PCR). The system comprises a glass-silicon platform with a set of micro-channels, -chambers, and -electrodes. A platinum thin film resistor, placed in the proximity of the chambers, is used for temperature monitoring. The whole chip assembly is mounted on a Printed Circuit Board (PCB) and wire-bonded to it. The PCB has an embedded heater that is utilized for PCR thermal cycle and is controlled by a Lab-View program. Similar to our previous work, one set of electrodes on the chip inside the bigger chamber (0.6 microl volume) is used for diverting bacterial cells from a flowing stream into to a smaller chamber (0.4 nl volume). A second set of interdigitated electrodes (in smaller chamber) is used to actively trap and concentrate the bacterial cells using dielectrophoresis (DEP). In the presence of the DEP force, with the cells still entrapped in the micro-chamber, PCR mix is injected into the chamber. Subsequently, PCR amplification with SYBR Green detection is used for genetic identification of Listeria monocytogenes V7 cells. The increase in fluorescence is recorded with a photomultiplier tube module mounted over an epifluorescence microscope. This integrated micro-system is capable of genetic amplification and identification of as few as 60 cells of L. monocytogenes V7 in less than 90 min, in 600 nl volume collected from a sample of 10(4) cfu ml(-1). Specificity trials using various concentrations of L. monocytogenes V7, Listeria innocua F4248, and Escherichia coli O157:H7 were carried out successfully using two different primer sets specific for a regulatory gene of L. monocytogenes, prfA and 16S rRNA primer specific for the Listeria spp., and no cross-reactivity was observed.