A homogenous bimodal (immuno/PCR) pathogen detection system based on a bio-nanoparticle

Since ancient times armies have attempted to employ biological weapons in order to defeat a foe. Early examples involved tainting water supplies with dead animals or catapulting plague ridden corpses over the walls of besieged cities. In more recent times biological and chemical weapons have become the focus of rogue states and terrorist organisations. Perhaps most high profile of these was the anthrax attacks that occurred in the US in 2001 killing 5 people. Since the UK renounced the use of biological weapons in 1975 it has increased efforts to improve detection of such biowarfare agents. In this project we aim to develop a simple rapid system that will detect biological weapons more quickly and effectively, improving the defence and security of the UK.

The system that we will develop is an addition to an existing pathogen detection system that we have pioneered in Birmingham. The existing system relies on viruses to detect the pathogen of choice. These viruses have a spaghetti like structure which means they align with respect to one another when solutions containing them are stirred or forced through narrow tubes. We have modified these viruses so that they are also able to adhere specifically to pathogens; when bound to pathogens the viruses are no longer able to align. The modification of these viruses and their use in an assay is a core part of a new area of science called synthetic biology. This new discipline aims to use biological materials to enhance the performance of everyday devices. In this case we are using synthetic biology to enhance biowarfare detection.

The assay involves detecting the changes in the alignment of the viruses and translating this information into a signal that can be read by a soldier or security operative. This area of synthetic biology poses a significant challenge as we need to translate a signal from the virus (which is less 1,000,000th of a metre long) into a signal that can be read. To achieve this we use a simple, yet underused, method (linear dichroism spectroscopy) that is able to detect whether the viruses are aligned or not. If they are aligned then there is no pathogen present, if they are not aligned then the pathogen is present.

The method for carrying out the assay has a number of useful characteristics that makes it ideally suited for use by the security services. The assay is simple and very quick allowing the it to be carried out without the need for laboratory equipment. The assay can also be configured so that more than one pathogen can be detected in a single assay. This is important as there are a wide range of biowarfare agents that need to be detected at the same time. Unfortunately, however our current method is only able to use one of the available pathogen detection modes, based on using antibodies, in order to carry out detection.

In this project we aim to address this insufficiency by adding a second detection mode to our system. This mode involves directly detecting and identify the DNA from the biowarfare agent. To add this method to our system we have to add small pieces of DNA that match those in biowarfare agent to the surface of the virus. These DNA fragments specifically stick to the DNA in the biowarfare agent preventing the virus from aligning and hence giving us a signal. If this is successful we also aim to add a second step that uses an enzyme to amplify the signal (in a method called PCR) which will make the whole system much more sensitive. In the final stage of the project we will combine the DNA based assay with the antibody based assay to produce a system that is more effective, more flexible and less prone to false results than any current system. In this way we will also demonstrate one of the first uses of synthetic biology in a detection application.

No single methodology has been successfully developed that can detect, without error, all dangerous pathogens. In many cases identification involves carrying out a number of assay types (e.g. culture based, immuno assays and PCR) which together provide certainty in detection. This approach requires the use of multiple instruments and is not suited to detection in the field. In this project we aim to add to our recent development of a rapid immuno-assay system based on a synthetic biology reagent by developing the reagent further to carry out DNA based assays.

The new method is based on the observation that long molecules align in fluid flow. When aligned, chromophores within the molecule have a linear dichroism signal that can be measured. We have shown that the filamentous bacteriophage M13 aligns to an exceptionally high degree in flow and hence has a very high LD signal. This signal dominates even in solutions containing contaminants, allowing the alignment of the M13 to be specifically monitored without interference. We have engineered M13 to bind specifically to pathogens, such as E. coli O157, by the adding antibodies to the surface of the M13. These M13-conjugates bind to antigens on the surface of the pathogen, disrupting of the alignment of M13 and hence the LD signal. By measuring this change in LD it is possible to quantify bacterial titres with a sensitivity better than 10^5 cells/ml.
In this project we will extend this approach by further modifying the M13 scaffold to make it detect DNA interactions, providing the basis for use in SNP and PCR type assays. To achieve this we will attach DNA oligonucleotides to M13 and then develop protocols for using these nano-structures for DNA based assays. We will also apply our multiplexing methodology to provide an assay platform that can perform multiple PCR and immunoassays in the same sample using the same instrumentation.

Biowarfare detection.
Implicit in this project is an aim to develop a system that addresses the need to detect and identify biowarfare threats. Our system will provide detection at or near the front line, carried out by personnel with minimum training, ensuring that defensive responses occur more quickly and efficiently thereby reducing casualties. We have enlarged on the benefits of our system in the pathways to impact section.

Healthcare
Detection of microbes including pathogens has a very wide application in a number of areas other than defence. Perhaps most obvious of these is the application in the healthcare industry. We have already built extensive collaborations with key opinion leaders in the NHS including consultant clinicians in acute care and respiratory medicine and leading clinical biochemists and microbiologists. We would expect that results from this project will be used to develop assay systems to address needs in sepsis and routine infection diagnosis. This in turn will have a profound effect on the way infections are treated in clinical practice. For example the new instrument will provide an opportunity for more point of care testing, providing quick and accurate diagnosis and hence more appropriate treatment choice and clinical outcome. In some sectors, for example sexually transmitted infection control rapid diagnosis is closely linked to effective detection, our instrument will offer a new tool in such situations. In other more proactive screening scenarios, for example preoperative MRSA screening our rapid detection system would provide near instant results removing the risk that samples and/or results get lost during transit to and from centralised laboratories. Our discussions with clinical microbiologists at QE Birmingham have also indicated that our instrument would have a place in existing clinical laboratories as a sample pre-screen method. Current methods rely on an overnight culture step which is costly and time consuming. Clinical experts felt that our system would provide an excellent pre-screening system identifying common pathogens without need for the culture step. This will reduce costs and again increase the speed of diagnosis

Environmental and industrial process monitoring
The versatility and portability of our instrument combined with the ability to identify microbial type makes it ideally suited to use in environmental health monitoring. In the past simple luciferase-ATP assays have allowed cleanliness to be assessed. However, in processes where the product includes some form of microbial based process (these could range from cheese making to biotherapeutics) it is more important that contamination is both detected AND identified. Our system provides an ideal solution allowing contamination to be identified and remedied more quickly reducing process disruption and resultant financial loss.

Bioassay industry
Our system will benefit the industry directly by providing a new method with a wide range of applications. In addition it will provide a new market for suppliers, including those for antibodies and oligonucleotides as well as instrument and consumable manufacturers

UKPLC
Success in our endeavour will benefit UKPLC in a wide range of areas. Successful commercialisation of the technology will improve the UK's standing in the diagnostics market bringing direct financial benefit to the UK. The improvement in infection diagnostics and treatment will reduce healthcare costs and decrease the number of days of sickness for UK citizens. While, better contamination control in industrial production will help important UK industries, particularly those involved in bioprocessing and biotherapeutic development. Finally, The development of the technology for UK defense and security will ensure bio-terrorism remains a very low likelihood.