Joint Synthetic Biology Initiative - Biological Amplification of Chemical Warfare Agent Sensors - Towards 'Deviceless Devices'

Since WW1 the regrettable use of chemical warfare agents (CWAs) has resulted in a global need to efficiently detect, identify and decontaminate these supertoxic agents. Chemical warfare agents are now classified as weapons of mass destruction. Of prime concern are the G- and V-type nerve agents (e.g. GB - Sarin and VX) and the mustard vesicants (e.g. HD, mustard gas). Concerns over CWAs have recently increased due to their potential use by terrorist organisations. In addition, there are a number of highly toxic industrial chemicals (TIC) such as hydrogen cyanide and hydrogen fluoride which are used in a range of different industries. If there were to be a deliberate or accidental release of such toxic chemicals, the ability to accurately detect and identify the presence of CWA or TICs would become extremely important in determining the best course of action and preventing loss of life.

In the proposed work we wish to develop a system for the detection of CWAs for use by security personnel with little operator training. The central theme here is to exploit previous work undertaken in our laboratories that has resulted in highly selective 'tests' for the main classes of CWA. These systems are very effective in identifying CWAs in liquid form and we wish to adapt this work to detection of toxic gases. Of the CWA agents of interest, it is agents such as Sarin that pose greatest threat as vapours. The high volatility, and extreme toxicity, of this agent means that the ability to detect it in the gas phase is essential for military and civilian authorities.

We propose to adapt our existing G-agent chemical sensors to gas phase detection by using a biological 'amplification' mechanism. Hence the presence of GB will promote the release of fluoride anions which in turn will bind to a synthetic sensor molecule. This event triggers the release of biochemicals that are subsequently acted upon by series of naturally occurring catalysts (enzymes). The final reaction in the sequence is based upon the same biochemistry that causes bioluminescence in Fireflies, and thus the primary output of the system will be light. This last reaction is part of a cycle so that one molecule of Sarin triggers the release of many photons of light. In essence the analyte GB triggers a cascade of reactions that results in the amplification of an initially small event. It is hoped that approach can be generalised to many types of target compound and allow the sensitive detection of dangerous materials without the need to use complex and expensive instruments. This will be great benefit in providing low cost sensor systems and obviate the need for external power sources. A further benefit is that security personnel will require little or no training to use the proposed 'devices'.

In this proposal we wish to develop a sensor system capable of detecting gas phase chemical warfare agent (CWA) analytes. In previous work we have developed highly selective colorimetric tests for the fluoride and cyanide containing CWA threats (G-agents, eg. Sarin) based on ferrocene boronic esters. In these systems analyte cleavage by a nucleophile affords fluoride or cyanide that subsequently binds to the weakly Lewis acidic boronic esters. This binding causes as shift in the oxidation potential of the adduct such that an external oxidants, e.g. oxygen or a redox indicator, may irreversibility oxidise the complex to yield an observable response. The weak Lewis acidity of these receptors renders this chemistry highly selective towards fluoride, and in some cases cyanide. This work is the basis of a selective test for liquid G-agent samples.

In the current work we wish to adapt this selective chemistry to the detection of gas phase analytes. The artificial receptor chemistry will be coupled to an amplification cascade of enzyme driven reactions. Thus fluoride binding to the ferrocene boryl receptor triggers the release of AMP (or ATP) from kinetically inert cobalt(III) or chromium(III) species. The released AMP is then converted by pyruvate orthophosphate dikinase (PPDK) to ATP which subsequently promotes a luciferase catalysed conversion of luciferase to oxyluciferase with the emission of visible light. The AMP generated is cycled back to ATP and the reaction continues until the luciferin substrate is consumed. Thus a single receptor binding event is greatly amplified with a multi-photon output. The overall reaction is irreversible and, since oxyluciferin is moderately intensely coloured, may also be considered as colorimetric. Thus the system may be regarded as a bi-modal chemiluminescent-colorimetric dosimeter.

The proposed work aligns with the RCUK's 'Pathways to Impact' policy documentation under the following headings; "Commercialisation and exploitation," "Improving national security," and "Improving health and well being". This is to be facilitated by the development of rapid, on-site, low cost, low operator skill devices for chemical warfare agent detection CWAs. The goal of the work is to prepare sensitive sensors for CWAs that operate without the need of complicated equipment. In their first generation these devices would be portable, lightweight, and require simple equipment to generate a signal output. The work also includes an explore the potential of amplified colorimetric responses - this would ultimately permit the formulation of 'devices' that are highly selective towards the target agent(s) whilst requiring no external power sources and be inexpensive to manufacture. We believe that our work, in seeking to provide easy-to-use devices for CWA identification for use military and civilian personnel, will provide a safer living environment and improved security, as well as helping in the detection of crime and identification of criminals and scenes of crimes. Such devices also offer the possibility for a significant pre-emptive anti-terrorist impact, minimization of potential damage in the event of a terrorist attack, peace-of-mind exposure monitoring for emergency personnel and forensic scene-of-crime analysis of suspicious substances. The technology would also provide evidence that would aid the prosecution of terrorists involved in chemical weapons manufacture and use. The development of improved detection technologies may potentially save lives and reduce the disruption to civilian population being kept to a minimum.