Enhancing DNT Detection by a Bacterial Bioreporter: Directed Evolution of the Transcriptional Activator YhaJ.

Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli-based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter's molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated.

An autonomous bioluminescent bacterial biosensor module for outdoor sensor networks, and its application for the detection of buried explosives.

We describe a miniaturized field-deployable biosensor module, designed to function as an element in a sensor network for standoff monitoring and mapping of environmental hazards. The module harbors live bacterial sensor cells, genetically engineered to emit a bioluminescent signal in the presence of preselected target materials, which act as its core sensing elements. The module, which detects and processes the biological signal, composes a digital record that describes its findings, and can be transmitted to a remote receiver. The module is an autonomous self-contained unit that can function either as a standalone sensor, or as a node in a sensor network. The biosensor module can potentially be used for detecting any target material to which the sensor cells were engineered to respond. The module described herein was constructed to detect the presence of buried landmines underneath its footprint. The demonstrated detection sensitivity was 0.25 mg 2,4-dinitrotoluene per Kg soil.

Detection of buried explosives with immobilized bacterial bioreporters.

The unchecked dispersal of antipersonnel landmines since the late 19th century has resulted in large areas contaminated with these explosive devices, creating a substantial worldwide humanitarian safety risk. The main obstacle to safe and effective landmine removal is the identification of their exact location, an activity that currently requires entry of personnel into the minefields; to date, there is no commercialized technology for an efficient stand-off detection of buried landmines. In this article, we describe the optimization of a microbial sensor strain, genetically engineered for the remote detection of 2,4,6-trinitrotoloune (TNT)-based mines. This bioreporter, designed to bioluminescence in response to minute concentrations of either TNT or 2,4-dinitotoluene (DNT), was immobilized in hydrogel beads and optimized for dispersion over the minefield. Following modifications of the hydrogel matrix in which the sensor bacteria are encapsulated, as well as their genetic reporting elements, these sensor bacteria sensitively detected buried 2,4-dinitrotoluene in laboratory experiments. Encapsulated in 1.5 mm 2% alginate beads containing 1% polyacrylic acid, they also detected the location of a real metallic antipersonnel landmine under field conditions. To the best of our knowledge, this is the first report demonstrating the detection of a buried landmine with a luminescent microbial bioreporter.

Standoff detection of explosives and buried landmines using fluorescent bacterial sensor cells.

A standoff detection scheme for buried landmines and concealed explosive charges is presented. The detection procedure consists of the following: Live bacterial sensor strains, genetically engineered to produce a dose-dependent amount of green fluorescent protein (GFP) in the presence of explosives' vapors, are encapsulated and spread on the suspected area. The fluorescence produced by the bacteria in response to traces of the explosive material in their microenvironment is remotely detected by a phase-locked optoelectronic sampling system. This scheme enables fast direct access to a large minefield area, while obviating the need to endanger personnel and equipment. Moreover, the employment of phase locking detection efficiently isolates the bacterial sensors' fluorescent output from the background optical signals. This facilitates the application of bacterial sensors in an outdoor environment, where control of background illumination is not possible. Using this system, we demonstrate standoff detection of 2,4-DNT both in aqueous solution and when buried in soil, by sensor bacteria either in liquid culture or agar-immobilized, respectively, at a distance of 50 m in a realistic optically noisy environment.

Electro-optical modulation with immunity to optical damage by bipolar operation in potassium lithium tantalate niobate.

A method for suppressing the formation of optical damage in quadratic electrooptic devices operated at short wavelengths is presented. Formation of optical damage is attributed to the generation of a trapped space charge induced by photoionization of impurity ions by the propagating beam. It is shown that in potassium lithium tantalate niobate where the electrooptic effect is quadratic, operating the electrooptic device by a bipolar driving voltage prevents the space charge from accumulating, which inhibits the formation of the optical damage. A 6 hours continuous operation of electrooptic modulator for a 30 W/cm(2) at λ = 445 nm input beam is demonstrated.

Simultaneous quantification of the fluorescent responses of an ensemble of bacterial sensors.

Bacterial bioreporters are genetically engineered microbial strains capable of detecting specific chemicals, groups of chemicals or global biological effects such as toxicity or genotoxicity. A scheme for simultaneous selective detection of the fluorescent signals emitted by a bacterial biosensor array, able to detect four different types of toxicants, using a single photodetector (photomultiplier) is presented. The underlying principle of the scheme is to convert the spatially distributed signals from all the elements in the array to temporally distributed frequency multiplexed signals at the output of the photodetector. Experimental proof of this concept is demonstrated in a four-channel system, in which low power (a few tens of picowatts) fluorescent signals produced by the bacterial sensors are measured, while maintaining a wide dynamic range of detection (more than 3 orders of magnitude). Simultaneous monitoring of concentrations down to a few mg/l of different chemicals in a liquid sample is demonstrated.