We present a methodology for quantitative sensing of the contents of a target material (TM) in a given sample which employs biosensing bioluminescent bacteria. These bacteria are genetically engineered to respond to the presence of a specific TM in their microenvironment by producing bioluminescence. Herein, we extend this methodology to include quantitative sensing of the TM content in the inspected sample by exploiting the dependence of the bioluminescence produced by the bacteria on the content of the TM in the inspected sample. However, employing bacteria as precise measurement devices is inherently problematic, as the signal they produce varies between different batches of bacteria, and changes as the batch ages. Moreover, As the methodology is designed for outdoor operation, the sensitivity of the bacteria response to changes in the environmental conditions needs to be taken into account. These hurdles are overcome in a special optoelectronic sensor which measures in parallel the responses produced by the inspected sample, and a standard sample containing a known quantity of the TM. Both measurements are conducted by identical sensing channels using bacteria from the same batch, and under the same environmental conditions. The “standard ratio” (SR) defined as the ratio between the maximum responses of the inspected sample and the standard sample was found to be independent of the batch and environmental conditions. A calibration curve of the SR vs. the TM concentration in a set of preprepared samples is used to gauge SR at the sensor output to the TM concentration in the inspected sample.
We describe a biosensing module in which live bacteria, genetically “tailored” to respond to the presence of a specific target material, constitute the core sensing element, reporting their response by bioluminescence. The module is constructed of two channels: an ‘induced’ channel that measures the bioluminescent light emitted by bacteria exposed to the inspected area, and a ‘reference’ channel that measures in parallel the bioluminescent light emitted spontaneously by bacteria of the same batch. This enables to overcome signal variations generated by different batches of bacteria, and due to varying environmental operating conditions. A special low-noise optoelectronic circuit was constructed to detect the bioluminescence emitted by the bacteria in both channels. The bacteria are encapsulated in polymer beads that also contain nutrients and water, enabling long-term maintenance-free operation. The beads are packaged in special cassettes at the bottom of the module, so that the induced channel cassette is in direct contact with the ground underneath the module, whereas the reference channel cassette is isolated from the ground. The module contains, in addition, a digital signal processing unit, and a wireless communication unit. The module is designed to operate outdoors as an autonomous network element designed for large scale in-situ deployment. The module described herein was developed for the detection of buried landmines, by sensing the presence of 2,4-dinitrotoluene (DNT) vapors released by the mine, accumulating in the ground above it. Detection of DNT in the sub-ppm range is demonstrated.
Current landmine detection methodologies are not much different in principle from those employed 75 years ago, in that they require actual presence in the minefield, with obvious risks to personnel and equipment. Other limitations include an extremely large ratio of false positives, as well as a very limited ability to detect non-metallic landmines. In this lecture a microbial-based solution for the remote detection of buried landmines described. The small size requirements, rapid responses and sensing versatility of bacterial bioreporters allow their integration into diverse types of devices, for laboratory as well as field applications. The relative ease by which molecular sensing and reporting elements can be fused together to generate dose-dependent quantifiable physical (luminescent, fluorescent, colorimetric, electrochemical) responses to pre-determined conditions allows the construction of diverse classes of sensors. Over the last two decades we and others have employed this principle to design and construct microbial bioreporter strains for the sensitive detection of (a) specific chemicals of environmental concern (heavy metals, halogenated organics etc.) or (b) their deleterious biological effects on living systems (such as toxicity or genotoxicity). In many of these cases, additional molecular manipulations beyond the initial sensor-reporter fusion may be highly beneficial for enhancing the performance of the engineered sensor systems. This presentation highlights several of the approaches we have adopted over the years to achieve this aim, while focusing on the application of live cell microbeads for the remote detection of buried landmines and other explosive devices.
A novel water chemical toxin sensor has been successfully developed and evaluated as a working portable
laboratory prototype. This sensor relies on a disposable plastic biochip prepared with a 4x4 micro-laboratory (μLab)
chambers array of Escherichia coli reporter cells and micro-fluidic channels for liquids translocation. Each bacterial
strain has been genetically modified into a bioluminescent reporter that responds to a pre-determined class of chemical
agents. When challenged with a water sample containing a toxic chemical, the sensor responds with an increased
bioluminescent signal from the biochip that is monitored over time. The signal is received by a motorized
photomultiplier-based analyzer and interpreted by signal processing software. We have performed several levels of
analysis: (i) the change in the bioluminescent signal from the sensor bacteria serves as a rapid indication for the
presence of toxic chemicals in the water sample; (ii) the intensity of the change indicates the toxin concentration level;
and (iii) the pattern of the responses for the different members of the bacterial panel on the biochip characterizes the
biological origin of the toxin. The analyzer contains housing mechanics, electro-optics for signal acquisition, motorized
readout calibration accessories, hydro-pneumatics modules for water sample translocation into biochip micro
laboratories, electronics for overall control and communication with the host computer. This prototype has a
demonstrated sensitivity for broad classes of water-borne toxic chemicals including naladixic acid (a model genotoxic
agent), botulinum and acetylcholine esterase inhibitors. This work has initiated an investigation of a novel handheld
field-deployable Water Toxicity Analysis (WTA) device.
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