CONCLUSIONES Y FUTUROS DESARROLLOS

8.1.1 BW Agent Detection using Direct Methods

The work described in chapter 3 lays the foundations for an automated assay for toxin detection, using a waveguide biosensor. It was demonstrated that luciferase could be covalently coupled to anti-ricin antibodies and used to signal the binding of the proteinaceous toxin ricin to the surface o f a fibre-optic waveguide. Using non-optimised instrumentation, lOng ml'^ ricin was

detected. The light output was very low and subject to a high degree o f noise. However these initial experiments demonstrate that it is possible to monitor, in real time, the binding o f the labelled antibody to the waveguide surface. By careful optimisation of the optical arrangement of the biosensor, it should be

possible to reduce the detection limit to sub microgram levels, whilst retaining a rapid speed o f response.

8.1.1.1 In Practice...

The waveguide biosensor employing direct detection of luciferase labelled antibodies would lend itself very well to use as a fieldable device for airborne toxin BW agents.

By pre-coating the waveguide surface with anti-toxin antibodies, and incorporating luciferase-labelled secondary antibody into the sample fluid, continuous monitoring for the presence o f toxin should be possible. Such an assay would interface well to a cyclone aerosol sampler, which can provide a continuous sample o f liquid containing particles collected from the aerosol environment. Response time should be rapid, within a minute or so o f the toxin becoming present in the environment around the detector. However, to be really useful, a multi-analyte capability would be required.

As mentioned in chapter 3, the use o f planar waveguides, rather than optic fibres, may prove more suitable for multi-analyte detection. Planar waveguides have been used, either with arrays o f antibodies on a single waveguide surface, (Rowe et al, 1999) or as patterned arrays o f waveguides, each with a single antibody (Flanagan & Sloper, 1992). The use o f a bioluminescent rather than fluorescent label would offer a simpler optical arrangement for biosensor design. This is discussed in more detail below.

8.1.2 Implications for Optical Biosensor Design.

Evanescent field optical biosensors have most commonly exploited either the evanescent excitation of fluorescent labels, (in sandwich immunoassays), or the change in refractive index, e.g. o f a surface plasmon resonance, on the binding o f an analyte within the evanescent field. Sensors exploiting the latter

analyte cannot be distinguished from any other large molecule resting on the surface. Consequently much research has been devoted to the former where the fluorescently labelled secondary antibody specifically attaches itself to the analyte-capture antibody complex within the evanescent field, (Hale et al,

1996). The non-specific binding is now limited to those labelled antibodies and antibody-analyte complexes that stick to the surface. Despite the considerable amount o f research in this area, only one such sensor is currently commercially available.

The Raptor™ is produced by Research International in the US. According to the marketing literature it is a “portable, four channel fluorometric assay system that can be used for high sensitivity monitoring o f biological agents, toxins, explosives and other chemical contaminants”. It has been developed from research work carried out at the Naval Research Laboratories (NRL), with the aim o f producing a detector for airborne BW agents (Ligler et a/,1998). The device is sold for $45,000 and has been available since m id-1998.

There are several reasons why optical evanescent biosensors have not been more widely exploited. Initially work was aimed at the incorporation o f all the reagents within the sensor device, in an attempt to eliminate problems

associated with the irreproducible delivery o f reagents, (Bradley et al, 1987). The enhancement o f flow injection analysis techniques brought about by the fusion o f biosensors and automated analytical instrumentation has substantially reduced this problem. However, still to be resolved are the optical interferences encountered and the optical noise, both o f which reduce sensitivity.

Optical interference is encountered because the excitation light not only excites the fluorophore attached to the antibody but also fluorescent and

phosphorescent impurities in the material o f the waveguide, in other optical components and even in filters. Additionally excitation light is scattered, with the scattered component being much larger than the fluorescent emission being monitored. This places exceptional demands on the quality of the filters, (Sloper & Flanagan, 1993). Even if these interferences were to be absent the fluctuations in the detected signal would still limit overall sensitivity. Clearly

the elimination o f an excitation beam would be a major advance, removing both sources o f interference and simplifying the optics. This has been achieved with the development o f the bioluminescent assay presented in this thesis.

8.1.3 Reagents for Optical Biosensors

Bioluminescence assays reagents are becoming more generally available

because o f the use o f luciferase in reporter gene assays, in microbial testing and in other applications. This technology base will help in the development o f luciferase as a label for immunoassays and also gene probe assays.

The number and variety o f mutant firefly luciferase enzymes available is increasing steadily. As well as thermostable enzymes and enzymes with altered kinetic properties, a number o f colour mutants have also been identified (White et al, 1996, Kutuzova et al, 1997). These range from red to yellow-green and appear very different when viewed with the human eye. However, the spectral variation is actually very small, with 60nm, separating the spectral peaks o f the most red from the most yellow-green, (Arslan et al, 1997). Colour mutants are easy to generate, however, and it may be possible to identify those with

emission spectra more suited to the optimum spectral bandwidth of conventional photomultiplier tubes.

Another favourable factor which may encourage the adoption o f firefly

luciferase-based detection methods is that the conditions and reagents used for bioluminescent assays are benign, which is not always the case for the

chemiluminescent alternatives.