Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation
if the cells are first exposed to 2.88 J/cm<sup>2</sup> of red light 24 hr prior to the lethal laser exposure. Changes in expression of
various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene
expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized
absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO)
by O<sup>2</sup> in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important
regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from
l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we
measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a
fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible,
increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest
overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.
Data showing what appears to be nonthermal inactivation of M13 bacteriophage (M13), Tobacco mosaic virus, Escherichia coli (E. coli), and Jurkatt T-cells following exposure to 80-fs pulses of laser radiation have been published. Interest in the mechanism led to attempts to reproduce the results for M13 and E. coli. Bacteriophage plaque-forming and bacteria colony-forming assays showed no inactivation of the microorganisms; therefore, model systems were used to see what, if any, damage might be occurring to biologically important molecules. Purified plasmid DNA (pUC19) and bovine serum albumin were exposed to and analyzed by agarose gel electrophoresis (AGE) and polyacrylamide gel electrophoresis (PAGE), respectively, and no effect was found. DNA and coat proteins extracted from laser-exposed M13 and analyzed by AGE or PAGE found no effect. Raman scattering by M13 in phosphate buffered saline was measured to determine if there was any physical interaction between M13 and femtosecond laser pulses, and none was found. Positive controls for the endpoints measured produced the expected results with the relevant assays. Using the published methods, we were unable to reproduce the inactivation results or to show any interaction between ultrashort laser pulses and buffer/water, DNA, protein, M13 bacteriophage, or E. coli.
Silicon dioxide surfaces are commonly used in photonic microsensors for bioreceptor attachment. Functionalization of
sensor surface with aptamer receptors provides the opportunity to develop low cost, robust, field deployable sensors.
Most aptamer sensors are constructed by covalently linking modified aptamers to a derivatized surface. There have been
reports of using UV crosslinking to directly immobilize DNA with sequences that end with poly(T)10-poly(C)10 on an
unmodified glass surface for hybridization. We have expanded this strategy using thrombin-binding aptamers (TBAs)
with three different tail modifications. TBA with PolyT20 tail showed the best performance in terms of sensitivity and
dynamic range. PolyTC tailed aptamers did not bind thrombin well, which may be due to that the interactions between
the C bases and G-quadruplex affect their target binding capability. When compared to biotinylated aptamer
immobilized on a streptavidin surface, polyT aptamer printed directly on plain glass showed comparable affinity. Direct
immobilization of TBA on nonfunctionalized silicon dioxide wafer and its binding towards thrombin has also been
demonstrated. Our results showed that using polyT-tagged aptamer probes directly immobilized on unmodified glass
and SiO<sub>2</sub> surface is a robust, very straightforward, and inexpensive method for preparing biosensors.
We were unable to reproduce published inactivation results, or show any interaction, between 90 femtosecond (fs) pulses
of 850 nm or 425 nm laser radiation and buffer/water, DNA, protein, M13 bacteriophage or <i>E. coli</i>. Using agarose
electrophoresis and polyacrylamide gel electrophoresis, we examined purified plasmid DNA (pUC19), bovine serum
albumin, and DNA and coat proteins extracted from M13 following exposures to irradiances of up to 120 MW/cm<sup>2</sup>. We
measured M13 viability using an assay for plaque-forming ability in soft agar after exposure to the same irradiances used
for the protein and DNA experiments. Exposures of up 1 GW/cm<sup>2</sup> at 850 nm had no effect on the viability of <i>E. coli </i>as
measured by a colony forming assay in soft agar. Peroxynitrite, known to be toxic, to cause single strand breaks in
DNA, and fragment proteins in vitro gave positive results in all assays.
The preliminary data presented here suggests that direct coating of biological agent with DNA capture elements and organic semiconductor (DALM) with chelated rare earths such as scandium, europium or neodymium can be used to track the agent, even when the biological components have been subsequently destroyed. The use of these three taggant components in conjunction with each other affords the opportunity to determine the presence of the biological agent by several methods---laser induced plasma spectroscopy, thermochemiluminescence, mass spectroscopy, polymerase chain reaction (PCR; if the primers are left on the DCEs or the agent's own DNA is used as the source of the amplicon). The specific DCE-labeling or PCR allows for confirmation of physical measurement results as specific to the agent.
Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to <i>Bacillus anthracis</i> spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and <i>Francisella tularensis</i>. DCEs can easily distinguish <i>Bacillus anthracis</i> from its nearest relatives, <i>Bacillus cereus</i> and <i>Bacillus thuringiensis</i>. Development of a high through-put process is currently being investigated.
Proc. SPIE. 5416, Chemical and Biological Sensing V
KEYWORDS: Defense and security, Patents, Molecules, Computed tomography, Biological detection systems, Chemical elements, In vitro testing, Biological weapons, Simulation of CCA and DLA aggregates, Current controlled current source
DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to <i>Bacillus anthracis</i> spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and <i>Francisella tularensis</i>. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.
Biosynthetic semiconductor, diazoluminomelanin (DALM), is a polymer of tyrosine, luminol, and nitrite. DALM has a very large cross section of absorption for light from ultraviolet to radio frequencies. This polymer can be made efficiently in a genetically engineered <i>E.coli</i>, JM109/pIC2ORNR1.1 (ATCC# 69905). We have been pursuing ways to couple electromagnetic radiation to vectors using this polymer. DNA capture elements (DCEs; formerly aptamers) have made this possible. We incorporated DCEs into the plasmid of this <i>E. coli</i> to direct binding to whatever microbe or cell desired and to produce DALM attached to the plasmid DNA. Using two other vectors pSV<sub>2</sub>neoNR10<sub>1</sub> or pSV<sub>2</sub>neoNR800<sub>5</sub> (ATCC # 69617 and 69618, respectively), both propagated in the <i>E. coli</i> host HB101, we have also inserted genes necessary for DALM production into animal and human cell lines (mouse monocytic leukemia: ATCC # CRL- 11771, -11772, -1173, mouse mammary adenocarcinoma: ATCC# CRL-12184, -12185; and human carcinoma of the cervix: ATCC # CRL-12510). The DCE/DALM vectors can be used to tag target cells, detectable by broad-spectrum light absorbance, luminescence, or fluorescence. DCE/DALM can further be activated with light, microwave energy, or by oxidative chemistry to kill the targeted microbes or other cells.
In developing high temperature incendiary weapons, the temperature and duration required to inactivate spores is needed information. Three common biowarfare simulants, Bacillus anthracis var Sterne, Bacillus thuringiensis var Kurstaki and Bacillus globigii var niger have been studied for their susceptibility to heat. The spores of all three simulants lose viability when exposed to temperatures between 250 and 300 degree(s)C for 1 second. Bacillus globigii is perhaps the most heat resistant of the three simulants studied, with Bacillus anthracis and Bacillus thuringiensis having similar susceptibilities to heat. Low temperature experiments requiring longer durations were also conducted; over a period of days at 90 degree(s)C. Bacillus anthracis spores can be inactivated. Thermodynamic and kinetic analysis were also performed. An important implication for any high temperature incendiary is the amount of heat or energy the spores absorb between ambient temperatures and 100 degree(s)C. A phase transition occurs centered at 184 degree(s)C for Bacillus thuringiensis. This is also the beginning of a massive weight loss from the spores, as well as a point at which the kinetics of the kill seem to change.