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 SiO2 surface is a robust, very straightforward, and inexpensive method for preparing biosensors.
Johnathan Kiel, Wes Walker, Carrie Andrews, Amy De Los Santos, Roy Adams, Matthew Bucholz, Shelly McBurnett, Vladimir Fuentes, Karon Rizner, Keith Blount
Pathogenic ecology is the natural relationship to animate and inanimate components of the environment that support
the sustainment of a pathogen in the environment or prohibit its sustainment, or their interactions with an introduced
pathogen that allow for the establishment of disease in a new environment. The anthrax bacterium in the spore form
has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to determine
the environmental reservoir of Bacillus anthracis between outbreaks of anthrax and to examine the potential factors
influencing the conversion of the Bacillus anthracis from a quiescent state to the disease causing state. Here we
provide environmental and laboratory data for the cycling of Bacillus anthracis in plants to reconcile observations that
contradict the soil borne hypothesis of anthrax maintenance in the environment.
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 Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.
KEYWORDS: Current controlled current source, Simulation of CCA and DLA aggregates, Biological weapons, Chemical elements, Biological detection systems, Molecules, Computed tomography, Defense and security, Patents, In vitro testing
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 Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. 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 E.coli, 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 E. coli to direct binding to whatever microbe or cell desired and to produce DALM attached to the plasmid DNA. Using two other vectors pSV2neoNR101 or pSV2neoNR8005 (ATCC # 69617 and 69618, respectively), both propagated in the E. coli 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.
Anthrax has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to design a culture technique to rapidly isolate and identify `live' anthrax. In liquid or solid media form, 3AT medium (3-amino-L-tyrosine, the main ingredient) accelerated germination and growth of anthrax spores in 5 to 6 hours to a point expected at 18 to 24 hours with ordinary medium. During accelerated growth, standard definitive diagnostic tests such as sensitivity to lysis by penicillin or bacteriophage can be run. During this time, the bacteria synthesized a fluorescent and thermochemiluminescent polymer. Bacteria captured by specific antibody are, therefore, already labeled. Because living bacteria are required to generate the polymer, the test converts immunoassays for anthrax into viability assays. Furthermore, the polymer formation leads to the death of the vegetative form and non-viability of the spores produced in the medium. By altering the formulation of the medium, other microbes and even animal and human cells can be grown in it and labeled (including viruses grown in the animal or human cells).
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