Advanced optical waveguide biosensors for the detection of illicit drugs and explosives

Lead Research Organisation: University of York
Department Name: Biology

Abstract

Heroin, cocaine and 'crack' cocaine are now considered to be the most powerfully addictive drugs Western society has ever had to confront. In the inner city areas of the United States, and now in Europe, illicit drugs are generating an unprecedented wave of violence and social disruption. The impact of the dramatic increase in the importation of concealed illicit drugs into the UK over the last few years has prompted a critical appraisal of current technology for the detection of such drugs of abuse. The proliferation of illicit drugs, explosives, new technologies, and expertise increases the potential for drug smugglers and terrorists to evade our existing countermeasures at points of entry to and exit from the UK. Present methods for the detection of illicit drugs and explosives leave much to be desired. To allow analysis to occur in the 'field' there is an urgent need for the development of improved on-site testing. To address this challenge, we are aiming to develop novel inexpensive sensors to rapidly and effectively detect particulate drugs and explosives. We have identified microbial enzymes that have high activity and specificity towards illicit drugs and explosives and have shown that these enzymes can be used as recognition components in sensors. Enzyme catalysed processes naturally occur in aqueous (water-based) environments; however, the presence of water is far from ideal in a sensor that has to be exposed to air for long periods of time, because it tends to evaporate. We are therefore proposing to explore the potential for ionic liquids (salts that are molten at room temperature) as alternative media for optical waveguide sensors. Room temperature ionic liquids (RTILs) possess a range of properties that make them desirable solvents, such as zero vapour pressure and being classified as environmentally friendly. We recently designed and created a new generation of functionalized ionic liquids that have increased hydromimetic (water-like) properties yet retain all the advantages of traditional RTILs. Using these RTILs we have shown that it is possible to obtain enzyme catalysis with complex (co-factor requiring) enzymes at very low levels of water (less than 100 ppm); which was previously impossible using the traditional BMIm PF6-related RTILs. The great potential for the use of these designer RTILs has aroused much excitement from biotechnology, pharmaceutical and chemical industries, leading to the commercialization of their production. These novel funtionalized ionic liquids, along with the appropriate enzymes and reagents will be deposited as thin film wave guides on low-cost moulded polymeric devices. We propose to utilize a unique collaboration between Dstl, PSDB, RTIL producers Bioniqs Ltd., and a multidisciplinary team of academics with expertise in enzymology, ionic liquid chemistry and sensor technologies to develop a commercially viable enzyme-based, prototype handheld biosensor for the detection of particulate illicit drugs and high explosives.

Technical Summary

Enzyme catalysed processes naturally occur in aqueous environments; however, the need for the presence of water for activity can hamper the use of enzymes as biorecognition components in sensors for the detection of drug and explosives particulates in air. We propose to overcome this limitation by developing colorimetric assays in a variety of novel room temperature ionic liquids (RTILs) based upon functionalized ammonium nuclei that are optimized for enzyme-catalyzed reactions. We have a diverse range of enzymes that possess activity towards illicit drugs and explosives. The enzymes will be investigated in ionic liquids that exhibit differing physical properties, such as hydrophilicity, polarity and proticity, in order to establish broad correlations between solvent structure and enzyme activity. Colorimetric detection systems for the drugs and explosives breakdown products in the RTILs will be developed. These functionalized RTILs, enzymes and reagents will be deposited as thin film wave guides on moulded polymeric devices. The effectively zero vapour pressure of the RTILs will provide a stable thin film. To achieve high sensitivity in a colorimetric assay requires a long optical path length. Since the RTIL films will be approximately one micron thick, the change in absorbance will be very small when viewed normal to the film thickness. We can overcome this drawback by launching a leaky waveguide mode in the RTIL film. This means that the light propagates along the film, giving much longer optical path lengths and hence higher sensitivity. Light is partially confined in the waveguide film by total internal reflection at the waveguide/air interface and by Fresnel reflection at the waveguide/substrate interface. Significant enhancements of absorbance can be obtained in this way depending on the thickness and refractive index of the waveguide film.
 
Description York achievements
1. Biochemical and structural characterisation of the explosive degrading cytochrome P450 XplA. This unusual explosive degrading P450 has a unique structural organisation. The characterisation of the enzyme has facilitated the development of a reagent less assay system for the detection of RDX (published in PNAS and J. Biol. Chem.).
2. Engineering a stable truncated form of XplA has permitted the development of a reagent less assay system where the binding of RDX in the active site of XplA could be detected by a waveguide device that monitors a shift in the absorbance spectrum from 420 nm to 389 nm.
3. Engineering PETN reductase (PETNR) for the specific detection of TNT and PETN. PETNR has activity against PETN, nitroglycerin and TNT. In order to distinguish between nitrate ester and nitroaromatic explosives the specificity of PETNR was successfully altered through standard directed evolution techniques followed by site directed mutagenesis (paper submitted to Chem. Comm.).

Additional project achievements
1. Two phase enzyme detection systems compatible for use with waveguide devices were developed for the opiate drug morphine and the explosives RDX and TNT. These comprised of PETNR, XplA or MDH immobilised in agarose gel containing colorimetric reagents (for MDH) overlayed with ionic liquid.
2. Development of a two phase colorimetric detection system for heroin using heroin esterase and morphine dehydrogenase immobilised in polyacrylamide or sol-gels with ionic liquids as the solvent system for solubilising drug particles.
3. We demonstrated the activity of MDH dissolved in protic ionic liquids (PILs). During this project we adapted and developed a range of analytical techniques to make them suitable for studying proteins in alkanolammonium ionic liquids. This enabled achievement of the characterisation of MDH activity and structure in PILs, across the whole range of water contents, and investigation of the solvation environment in PILs (Paper in preparation).
4. Construction of a versatile low-cost waveguiding instrument and associated software to replace expensive optical tables and motion stages.
5. Formation of stable sub-micron films of ionic liquids that were shown to act as leaky waveguides. These films were stable for longer than a week in air.
6. Formation of stable hydrogel waveguiding films containing enzymes. These could be dried and reconstituted very rapidly simply by the addition of water or buffer.
Exploitation Route We engineered the specificity of the enzyme PETN reductase so that the modified enzyme has activity against the explosive PETN but not TNT. The altered specificity is important as it allows the specific identification of TNT and PETN in a sample. A truncated stable form of the cytochrome P450 XplA was engineered that permits the reagent less detection of the explosive RDX.
Sectors Security and Diplomacy

 
Description Use of PETN Reductase as a biocatalyst 
Organisation BASF
Country Germany 
Sector Private 
PI Contribution The enzyme PETN reductase was isolated for its ability to catalyse the transformation of explosives; however, in addition to these activities and its potential use in biosensors for the detection of explosives, PETN reductase has been found to catalyse enatiospecific reactions of value to the pharmaceutical and chemical industries. As a result of this BBSRC project, we entered into a collaboration with BASF (Ludwishafen, Germany) and Prof Kurt Faber at the University of Graz. A number of chemical transformations were evaluated and published.
Start Year 2006
 
Description Use of PETN Reductase as a biocatalyst 
Organisation University of Graz
Country Austria 
Sector Academic/University 
PI Contribution The enzyme PETN reductase was isolated for its ability to catalyse the transformation of explosives; however, in addition to these activities and its potential use in biosensors for the detection of explosives, PETN reductase has been found to catalyse enatiospecific reactions of value to the pharmaceutical and chemical industries. As a result of this BBSRC project, we entered into a collaboration with BASF (Ludwishafen, Germany) and Prof Kurt Faber at the University of Graz. A number of chemical transformations were evaluated and published.
Start Year 2006