Nanosensors for closed tube molecular diagnostics

Project: Research

Description

"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."

Key findings

We showed that nanoparticles functionalised with specific DNA sequences could be used to detect target DNA relating to fungal infections using surface enhanced Raman scattering.
StatusFinished
Effective start/end date1/06/1230/06/15

Funding

  • EPSRC (Engineering and Physical Sciences Research Council): £898,862.00

Fingerprint

Nanosensors
DNA sequences
Raman scattering
Metal nanoparticles
Molecules
Nanoparticles
DNA
Coloring Agents
Labels
Wavelength
Fluids
Nanoclusters
Chromophores
Multiplexing
Silver
Molecular structure
Metals
Scattering
Sampling