Real-time high sensitivity detection of biological agents

For security and defence purposes, for example in battlefield settings or at airports or other public buildings, it is important to be able to detect harmful biological substances such as proteins and viruses and bacteria. These types of biological agents can often be detected by using complex time-consuming tests based on antibodies. Antibodies are large proteins produced by humans and other mammals as part of their immune system to recognise invading agents such as viruses and bacteria to protect the body. They are therefore very good at recognising other molecules, but their large size and complex structure make them difficult to produce to the same quality all the time, and they are not very stable to temperature or harsh treatment. This means they are not ideal for building biosensors for long shelf life and use in a wide range of difficult environments.

Our work focuses on an area called synthetic biology, which the BBSRC defines as "Linking bioscience, engineering and computer sciences to develop rationally designed biological parts, devices and systems". We are proposing to integrate engineered biological molecules with electronic devices to provide efficient and simple methods for the sensitive detection of biological agents such as proteins, viruses and bacteria. We will generate a molecular signal amplification process which is only switched on upon successful and specific detection of the harmful biological substances.

We will produce small proteins that mimic the binding properties of antibodies, but which are simple to produce and are robust and highly stable. We will produce two of these that bind to a target protein and will link these to other biological parts including something we call a split enzyme which will only be able to reform once both the antibody-type protein bind to the target protein. This reformed enzyme will lead to an amplification of the binding signal allowing sensitive detection of the target protein. We will test how well the new device works and in future can develop biosensors that detect multiple different target proteins.

Biosensor devices for defence and security applications must meet a number of specific requirements including multiplex detection, ease of use and low power usage. In addition it is important that such devices display high selectivity and sensitivity. This synthetic biology application integrates biomolecular engineering with electronic detection to develop proof-of-concept biosensors capable of high sensitivity detection of target molecules. The targets indicated by defence related industry as testbeds of such technology are the protein ovalbumin, the bacteriophage MS2 and the bacterial spores of Bacillus globigii. In this study we will focus upon ovalbumin. We will screen phage display antibody-mimetic libraries based on small stable scaffold proteins. Two binders with distinct non-overlapping epitopes will be identified by sequencing and SPR. We will use a split galactose oxidase (GO) to provide a redox enzyme system with each parts linked to one of the two binding proteins. The enzyme will only reform a functional enzyme upon binding of ovalbumin by both binding proteins resulting in an amplification of the binding signal by allowing multiple turnover of substrate galactose. For each catalytic cycle two electrons will be transferred to the electrode surface via a mediator. This redox detection system is based upon the well established amperometric approach used for glucose oxidase sensors. The binding protein/split enzyme components will then be immobilised onto the electrode surface and the response of the electrode to various concentrations of ovalbumin in solution measured to assess the sensitivity of the biosensor. We will also test the system under conditions that introduce contaminants to determine the selectivity of the system. There are many future applications including multiplexing options for development of such biosensors which can be built with many thousands of individually addressable electrodes per square centimetre.

The outputs of this project potentially impact not only on defense and security application but also on a wide and diverse range of other applications including clinical diagnostics, regenerative medicine, environmental monitoring, forensics, as well as research tools. Consequently it has relevance for a range of beneficiaries.
Potential stakeholders include:
1) Public sector organisations including Ministry of Defense; Home Office; Environment Agency/DEFRA; Department of Health; NHS.
2) Business/industry including Ministry of Defense suppliers; Forensic services; Diagnostics companies; Pharmaceutical/Biotech companies; Water companies; Research equipment companies;
3) Third Sector - including overseas agencies, voluntary organisations and charities in developing countries.
4) General public.
5) Academia/Research organizations.
The above stakeholders are likely to benefit as follows:

Security: This project directly addresses a specific defence capability issue, that of detection of biological warfare agents in theatre or security-sensitive civilian settings with high specificity and sensitivity. The three main criteria for biosensor performance in biological warfare and security applications are multiplexed detection, low power consumption, and ease-of-use. The output of this project will be a proof-of-concept device which delivers these requirements. This will enable improved identification of biohazards with positive implications for homeland security, defense and the safety of the general public both in the UK and overseas.

Healthcare Benefits: The emergence of personalised and stratified medicine with their inherent requirement for sophisticated diagnostics, together, ideally, with the transfer of clinical testing to point-of-care including rapid access to primary care services, are putting increasing pressure on current diagnostics services. These would benefit from more accessible and more accurate health monitoring and diagnostics tests. Furthermore, highly sensitive diagnostics tests will support the identification of new disease biomarkers, discovery of new drugs and development of targeted treatments and more rapid patient profiling. For the Department of Health/NHS this will lead to improved and more cost-effective healthcare service. The general public will benefit through early diagnosis and hence increased chances of full recovery, as well as easier access to diagnostics tool. Also, indirectly through improved and targeted drugs.

Environmental Benefits: The availability of low-cost, easy-to-use, and highly sensitive biosensors for environmental monitoring will enable better and more frequent testing, and will, for example, enable early detection compounds in the environment which pose a direct risk to human health. This is also of particular benefit to the third-world where contaminated water poses a great risk to human health. Organisations such as Environment Agency/DEFRA will benefit from being able to make informed decisions regarding environmental risk and consequently in the formulation and implementation of environmental policy.

Commercial and Economic: Obvious commercial/economic beneficiaries from this research are suppliers of the Ministry of Defense and Security Services. Furthermore, clinical diagnostics companies will benefit from the novel technology that will enable the development of low-cost, highly sensitive and specific disease test kits which can be disposable, easy to use and applicable at point of care. This has the potential to expand significantly the current clinical diagnostics market even to over-the-counter diagnostics.

Third World Overseas agencies, voluntary organisations, charities and populations in developing countries have the potential to benefit from affordable test kits for disease and water quality monitoring and analysis.