Single-molecule DNA biosensors for rapid microbial detection

The health of human, animal and plant populations is under constant threat due to infections by a large variety of pathogenic microorganisms, such as bacteria and viruses. The fact that pathogens are able to spread rapidly and harm human populations has also led to the development of a variety of biological weapons which threaten not only soldiers in the field of operations, but even civilian populations in benign public environments such as a tube stations or airports. Threats to civilians also include those from non malicious sources such as within healthcare (hospital acquired infections, e.g. the superbug MRSA) or from the food industry (contaminated foods with pathogenic E. coli). The ability to diagnose such infections rapidly would dramatically aid patient survival and outcome and prevent further spreading of the disease.

In our proposal we describe a diagnostic solution based on 'single-molecule' fluorescence for the rapid identification of multiple pathogens. We have already established a basic test for the presence of DNA specific to a particular bacterial strain and aim to build on this to develop a range of 'intelligent' biosensors based on Boolean logic and signal amplification. Essentially, such sensors will be able to provide a yes/no answer on pathogen threat level given a combination of target inputs e.g. if (pathogen 1 AND pathogen 2) but (NOT pathogen 3). Our sensors aim to produce a time-to-result on the order of 10-15 min, compared with current technologies that vary between hours and days due to the requirement of sample amplification - either bacterial culturing or DNA amplification.

In parallel, we propose to further develop a compact and affordable single-molecule fluorescence microscope to perform such tests. Currently single-molecule microscopes are prohibitive in terms of size and cost; we aim to produce a cut-down version with a footprint of approximately 30cmx50cmx20cm (suitable for benchtop operation) and a small fraction of the cost of the full-size microscopes.

Rapid and sensitive detection of pathogenic microbes is vital for public protection in terms of disease outbreak or bioterrorist attack. Current technologies involve a time-to-result from several hours to days, far from the ideal 'real-time' analysis.

We propose a solution based on synthetic biology for rapid identification of nucleic acids specific to pathogens of interest; the combination of multiplexed detection with basic Boolean logic (AND, OR and NOT) allows us to create a modular molecular 'computational toolkit' to design biosensors that create a defined output given a distinct combination of target inputs. Such a system simplifies downstream analysis by providing a yes/no response on pathogen threat level. The output is a combination of fluorescence observables collected from immobilised DNA molecules.

Further, performing fluorescence assays on the single-molecule level facilitates extremely sensitive and rapid detection. Experimental apparatus for single-molecule detection is currently high-cost and not user-friendly. We are therefore developing a simplified and economical single-molecule setup for use with our synthetic-biology biosensor.

Successful completion of the project will provide a synthetic-biology biosensing platform and accompanying instrumentation that can be extended to offer fast, sensitive and versatile sensing of microbial pathogens, and fuel applications in health-related fields, including defense and biosecurity. An extension will develop more robust biosensor versions for use in operational environments. A further extension should target microbial pathogens associated with potential bioterrorism attacks, such as B. anthracis and Y. pestis; this extension will require DNA probes that target these pathogens under laboratory conditions and simulated operational environments. Further extensions to other bioterrorism bacteria and viruses are easily envisaged.

An important aim of this proposal is to develop an inexpensive, robust and compact instrument for single-molecule measurements. This aim is related to a close collaboration with SME Chelsea Technologies Group (CTG), a commercial partner with extensive experience in several defense and homeland security areas. This relation with CTG will help us focus our biosensors and instruments on specific biosecurity threats; CTG also benefits from our interactions, since they are alerted of opportunities in ultrasensitive detection and biological assays. A straight-forward extension may be a portable instrument for clinical lab or theaters of military operations. Future miniaturization that could lead to handheld instruments, which can be invaluable for first respondents in potential incidents in the urban rail networks, in inspections at ports of entry in the UK, and in monitoring the safety of water resources. The drive towards simpler and affordable single-molecule detection should also drive the development of similar instruments for the academic market, allowing non-physicists to gain access to ultrasensitive microscopes that detect single molecules.

The biosensor platform should also offer excellent opportunities for improved health care through rapid diagnosis of infections. Applications may involve detection of microbes responsible for hospital-acquired infections (e.g., MRSA and C. difficile) or rapid detection (within 15 min) of influenza strains in the doctor's office. The biosensor platform can improve food safety by providing means to screen for food-borne pathogens, such as enterohemorrhagic E.coli strains O157:H7 and O104:H4. The potential applications in agriculture also include better monitoring of pathogens afflicting animal populations by detecting devastating viruses such as the Bluetongue virus and the virus that causes foot-and-mouth disease. Successful biosensors along these lines may be pursued by a spin-off company, a option that can be assisted by Isis Innovation, the technology transfer arm of Oxford University.

Our development of the biosensor platform and compact instrument will be communicated through the usual academic channels such as scholarly journals, departmental and group websites, press releases, and outreach efforts. The timing of publication will ensure that any potential intellectual property and rights have been evaluated and, if necessary, secured.

Our synthetic biology efforts for rapid biosensing should encourage colleagues from physical sciences to use synthetic biology for many biological and non-biological applications. The need for a theoretical modeling to describe the biosensor will also stimulate interactions with theoreticians that can improve our sensor design and operation. Finally, the post-doctoral fellows involved in this work will interact with researchers from many backgrounds, and work on a complex problem that requires tools from many disciplines, thus acquiring important transferable skills and highly interdisciplinary training essential for a successful career in the biotech and pharmaceutical industries.