Exposing explosives: novel synthetic gene circuits for explosive detection via innovative waveguide sensing

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Whole cell biosensor constructs are intrinsically modular lending themselves to a synthetic biology approach. Our proposed research involves screening mutated expression libraries of the genetic regulatory element XylR to identify recognition elements displaying high binding affinity and specificity towards TNT, then use optical waveguide sensing to develop a proof-of principle whole-cell-biosensor for the recognition of TNT. To achieve this XylR will we will create populations of mutated xylR sequences using methods of directed evolution and use fluorescence-activated, cell sorting techniques to identify XylR variants with improved TNT binding. We propose to use secreted alkaline phosphatase (SEAP) as the reporter, followed by an amplification stage where the SEAP converts non-detectable small molecule substrates into a detectable form. The substrates we will test are fluorescein diphosphate and p-nitrophenyl phosphate.

We have developed a new concept in optical waveguide sensing, the leaky waveguide sensor which we will use to detect the TNT induced fluorescent products. In these devices, the waveguide layer, is a low index material (which can be a gel or involatile liquid) instead of a high-index, solid material. When the product molecules diffuse into the gel they can be detected by their fluorescence. Because the waveguide has a lower refractive index than the substrate but a higher refractive index than the sample layer, light is confined by Fresnel reflection at the waveguide/substrate interface and by total internal reflection at the waveguide/sample interface. The optical mode propagates through this low index medium. This provides the maximum overlap between the light and the chemistry, thus maximising sensitivity to changes in fluorescence caused by SEAP in the waveguide layer. These devices are especially efficient at exciting fluorescence, as the light intensity in the waveguide is higher than the external intensity.

The global scope and capability of terrorism to affect extensive
disruptions is an ever-present threat. The use of devices for the
identification of explosive compounds has a direct impact in preventing
terrorism. The development of biosensors will progress this by providing
systems that detect specific types of explosives, with increased
sensitivity. Future research on biosensors has the potential to optimise
biosensors for portable threat detection, a necessity component of
effective defence against terrorism. This research will be a step-change
towards the production of biosensors with the ability to detect specific
types of explosive compounds with high sensitivity where the main
beneficiaries will be the UK MoD and the Home Office. Such devices will
improve national defence and security capabilities. Additionally, this
research has potential commercial, private sector beneficiaries in the
civil sector for example aviation and other transportation industries
where high-throughput, low cost screening techniques for explosives or
other analytes are desirable.