Optical Detection of Foodborne Bacterial Pathogens using Nanosensors

This programme of research involves the development of a new tool based on the use of innovative bionanosensors with superior performance for the detection of bacterial pathogens in a sensitive, quantitative and multiplexed manner. This will involve developing nanoparticle based analytical technology for the simultaneous detection of multiple bacterial pathogens associated with food poisoning. Current methods for detecting bacteria are time consuming (1-2 days in the case of bacteria culturing on selective media), expensive and require specialised personnel and equipment. Therefore, there is a strong need for faster, simpler and more reliable isolation and detection of bacterial pathogens that can be carried out in the field and can simultaneously detect multiple bacteria within a single test. Therefore, development of a simple, portable detection platform is proposed which can carry out multiplexed point of care (POC) detection.

Successful pathogen detection is crucial for the health of the general public as the threat of infectious disease is dramatically increasing as a result of bacteria developing resistance to antimicrobial drugs. Major threats to human health from bacterial infections such as E. coli have led to urgent demands to develop highly efficient strategies for isolating and detecting microorganisms in connection with food safety, medical diagnostics, water quality, and counter terrorism. Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis and Salmonella attacks the stomach lining and intestines and in severe cases can result in blood poisoning.

The research involves the use of an optical detection technique called Raman scattering which will be developed for the POC detection of bacterial pathogens. If light of a particular wavelength is directed onto a molecule then some of the scattered light will change wavelength. This change in wavelength is related to the structure of the molecules and provides a molecular fingerprint that can be used for definitive identification. However Raman scattering is an intrinsically weak process and the signal can be greatly enhanced if the molecule is coloured and is adsorbed onto a roughened metal surface (surface enhanced resonance Raman). The metal can be thought of as essentially amplifying the Raman scattering from a molecule on the surface and in this case the metal will take the form of metal nanoparticles. Since a fingerprint unique to the molecule is produced, the composition of mixtures can easily be identified without separation.

A novel diagnostic tool will be developed for the detection of multiple bacterial pathogens, namely Escherichia coli, Salmonella typhimurium and Campylobacter jejunii in a single assay combined with enhanced Raman detection. However, this technology will not be limited to these organisms and can readily be applied to other pathogens. This will involve using magnetic nanoparticles which have a biomolecule on the surface known as a lectin which will bind to the surface of bacterial cells. This will allow isolation and separation of bacteria from the surrounding medium upon application of a magnetic. Additionally, silver nanoparticles which are functionalised with a coloured molecule or label, resulting in intense surface enhanced Raman signals, and a biomolecule which will bind specifically to a particular strain of bacteria (antibody or aptamer) will be added. When the correct bacteria are present binding will occur resulting in magnetic isolation of the bacteria from the matrix as well as it now having a SERS response. By using a different label for each biomarker, a unique spectrum will be achieved for each biomarker allowing multiple biomarkers to be detected simultaneously. A portable Raman spectrometer will then be used to detect the bacteria present.

Successful pathogen detection is crucial for the health of the general public as the threat of infectious disease is dramatically increasing due to bacteria developing resistance to antimicrobial drugs. Major threats to human health from bacterial infections such as E. coli have led to urgent demands to develop highly efficient strategies for detecting microorganisms. Therefore, there is a strong need for faster, simpler, and more reliable isolation and detection of multiple bacterial pathogens using novel point of care/use (POC) technology. The use of a novel tool is proposed for the multiplexed detection of foodborne bacterial pathogens. The technology is based upon the use of surface enhanced Raman scattering (SERS) due to its high sensitivity and multiplexing capabilities. SERS active silver coated magnetic nanoparticles will be functionalised with lectins which are capable of specifically recognising and binding to carbohydrate constituents on the surface of bacteria. These lectin functionalised magnetic nanoparticles will be used to selectively capture bacteria from the sample matrix. Silver nanoparticles will then be functionalised with a Raman reporter and a biorecognition molecule (antibodies/aptamers) which is specific towards a bacterial strain. A SERS response will only be obtained when the SERS active nanoparticle binds specifically to its bacterial target. The magnetic 'plug' will then be interrogated using Raman spectrometers that are field deployable. In this way the detection strategy will be fully portable and allow for rapid, point of use detection. Once the multiplexed quantitative SERS signal is generated it needs to be analysed such that the unequivocal detection of a pathogen is made and/or the concentration of the bacteria predicted. We shall build on our considerable chemometric expertise where we shall use discriminant analysis approaches for bacterial identification and multivariate regression-based methods for quantification.

One only needs to open the newspaper and be reminded of the problems within our food. The recent food poisoning outbreaks throughout the world have included, for example, the German E. coli outbreak in 2011 that infected nearly 4000 people and resulted in 53 deaths, and it is well accepted that the incidence of Salmonella (~12k p.a.; www.hpa.org.uk) and Campylobacter (~60K p.a.; www.hpa.org.uk) implicated in food poisoning is under reported in the UK. Therefore there is an on-going and urgent need for the rapid detection and enumeration of bacterial pathogens.

Thus developing rapid, new technology for the multiplexed POC detection of bacterial pathogens in food before they are consumed by the general public will be of huge benefit in terms of preventing illness. This will have implications in terms of economic cost savings due to more information per test being obtained but also on the cost burden on the NHS by reducing the amount of cases of food poisoning outbreaks.

The approach being developed will be a portable, POC platform where bacterial identification can be rapidly carried out in the field. The worldwide need for such rapid, POC diagnostic devices is huge and the outcome of the research will be a world leading position which could lead to company formation and considerable opportunity for wealth generation and employment in the UK. This proof of concept approach can be extended in the future to the detection of other bacterial strains as well as related biomarkers for other conditions. Therefore, this POC platform has the capability for extension into third world detection strategies for example for pathogen detection and disease diagnosis and for security/military applications for chemical, biological threats. Therefore the impact of this research is immense and far reaching.

There are a large number of beneficiaries in both the academic and industrial communities. Any protectable outcome from the research will be covered through IP protection in the form of a patent. Faulds and Goodacre have an extensive network of potential end users that will allow this technology to be evaluated with a view to assessing industrial interest. The main benefit from this research is the generation of a new tool which will contribute to the food security and health sectors and in particular to the sustainability of SERS technology as an emerging detection technique. The PI already has a relationship with Dstl who are already funding several PhD studentships in the area of SERS and they will have great interest in the outcomes from this research. There remains a continuing threat of terrorist/insurgent attack on military/civilian personnel and key strategic infrastructures both within the UK mainland and in operational theatres. To counter these threats reliable, low cost, widely available, screening capability is required that would provide a step change in the current in-service test kits and protocols. A simple procedure to test for numerous threats simultaneously which has an unambiguous result, is affordable and can be widely deployed as an essential tool in advancing our current capability. The technology could be adapted for detection of other analytes of interest to the MoD, e.g. detection of biological species such as proteins or toxins or chemical warfare agents. Therefore this research could have great impact in protecting the general public as well as the military.

The applicants have a strong track record of entrepreneurial activities arising from research and there is an ethos within the research collaboration that fosters this activity through direct mentoring and engagement with industry but also the availability of specific courses run by the Strathclyde Programme in Research and Leadership (SPIRAL) and University of Manchester Intellectual Property (UMIP). In addition, opportunities for further engagement with prospective industrial and academic partners through TIC at the UoS and the MIB at UoM.