Nanosensors for closed tube molecular diagnostics

This proposal aims to investigate a nanoparticle assembly approach for the detection of specific DNA sequences which relate to DNA markers which are indicative of infection. The approach is based on a nanoparticle conjugate made from silver metal nanoparticles functionalised with a dye molecule and a specific DNA sequence which correlates to an infection found in cerebral spinal fluid. When the DNA functionalised metal nanoparticles recognise the target DNA they will hybridise and self-assemble into discrete nanoclusters which have unique optical signatures. The optical signal obtained
will be dependent on the dye, and hence the specific DNA sequence, which was used to label the nanoparticle, therefore a unique signal will be obtained for each DNA target that is present.

The optical response obtained will be based on a technique called surface enhanced Raman scattering (SERS). If light of a particular wavelength is directed onto a molecule, then some of the light scattered by the molecule will have changed
wavelength. This change in wavelength is related to the molecular structure and provides a vibrational fingerprint that can be used for identification. This is known as Raman scattering however it is an extremely weak effect. By attaching a molecule to a metal nanoparticle the scattering that is observed is greatly increased. This is known as surface enhancement and the metal is used to effectively amplify the Raman effect and can be used to study a single molecule. These signals are further increased if the molecule being analysed has a chromophore i.e. is a coloured molecule.

Therefore this nanoparticle assembly approach will be designed to give a SERS response as a positive indication of the presence of the target DNA sequence. In this case the metal nanoparticles surface will be functionalised with a SERS active dye label such that the SERS signal will be 'switched on' when the hybridisation to the target DNA sequence has taken place. The major benefit of the SERS technique is that each dye label, and hence DNA sequence, will have a unique fingerprint spectrum which has sharp, easily identifiable peaks which can be used to discriminate between multiple species in one sample. Therefore it is possible to identify up to ten different DNA sequences which relate to ten different infectious agents in one sample.

The attraction of this approach is the combination of extreme sensitivity and the multiplexing capacity i.e. the ability to detect multiple DNA targets at once. The ultimate aim is to achieve PCR-less detection of a specific DNA sequence from a
clinically relevant sample in a closed tube format. This is extremely important when considering secondary infections. Once the primary infection has been identified, secondary infections are not normally tested for unless the situation arises where the primary infection is not responding to treatment as expected. This is a growing problem in the healthcare environment where patients often present with multiple infections or acquire a secondary hospital acquired infection. The main benefits of using this nanosensing approach is therefore the ability to look at multiple DNA sequence detection events simultaneously from very small volumes of samples and without using numerous detection technologies. This project will focus on cerebral spinal fluid samples where the sample size is very small and repeat patient sampling is not always possible therefore there is a clear need to get as much information from one test as possible as from the limited sample and in a short timeframe.

The main beneficiaries from this research will ultimately be the general public. In providing benefit to the general public the company Renishaw Diagnostics will benefit from this research as will the academic and researchers involved in this proposal. Initially this will have immediate benefit to those situated in the UK but as this is an international market that is being proposed we see the beneficiaries expanding and onto the international scene. The way in which they will benefit will be mainly from the translation of the research into a commercial product for rapid testing of infections of cerebral spinal fluid. The general public will benefit from the availability of tests to rapidly diagnose the presence or absence of specific infectious agents within CSF and then inform on the appropriate therapeutic application. One of the main drivers here is
time to decision and also the ability for multiple testing from one sample. This of course benefits any patient by reducing the amount of sample that is required or by eliminating the need for multiple samples being required. This will result in a reduction in cost and time of the testing which in turn means that diagnosis could be carried out much more rapidly, reducing the time between sampling and result. Ultimately this will lead to earlier detection and treatment of infection or disease, allowing the correct treatment to be prescribed as early as possible. This leads to more efficient disease
management with a greater benefit to the patient and healthcare sector.

The company will benefit as they will have a high value product which has unique selling points and will be able to form part of the next generation of diagnostic products based on their nano healthcare application range. This in turn will provide
employment and security to employees within the company and also potential expansion as this product gains traction in the market place. In opening up a new product line for a small company more employees will be required and as such this will generate employment for Ph.D. level scientists in the UK, as well as contributing to the overall wealth of the nation.

The academics involved in this project and the researchers will benefit from being involved in the translation of research into a commercial environment and from this experience gain greater expertise in taking forward similar such research projects in the future. They will also gain through the interaction with the company and the end users.