The aim of this project was to develop a highly sensitive, closed-tube, multiplexed assay for the detection of DNA using surface enhanced Raman scattering (SERS) analysis. This was achieved through the development of a novel molecular diagnostic assay based on nanoparticle assembly combined with a magnetic manipulation step, consisting of oligonucleotide-functionalised silver nanoparticles and silver-coated magnetic nanoparticles. Hybridisation with target DNA leads to a controlled aggregation of the nanoparticles resulting in strong signal enhancement from a Raman reporter attached to the silver nanoparticle probe. Application of an external magnet allows the hybridised network of nanoparticles to be focussed within the interrogation volume of the laser, thus concentrating the target and removing much of the background matrix. Sensitive detection of four different 24-base synthetic DNA sequences was demonstrated, using four different Raman reporters. Typically, ≤ 20 fmol of target was detected at > 3:1 level of discrimination compared with a blank sample, and the assay was also successfully demonstrated in a duplex format. The rate and extent of hybridisation between target and probes is strongly influenced by the length of target DNA, and an investigation was carried out into the factors affecting this. Increasing the length of probe sequences and using a tail-to-tail orientation, combined with the use of a polymer in the buffer to act as a hybridisation accelerant, resulted in a significant improvement in hybridisation with long target sequences. With these improved conditions, SERS discrimination of > 3:1 compared with blank samples has been demonstrated for just 5 fmol of two different target sequences of ≥ 144 bases in length, and the developed assay was also used to successfully detect two different types of PCR-amplified DNA sequence from plasmid DNA. The work presented here represents a step forward in capability within the field of nanoparticle assembly, and moves this technology closer to the point where it can be used in a multiplexed commercial platform.
|Date of Award||29 Apr 2016|
- University Of Strathclyde
|Sponsors||EPSRC (Engineering and Physical Sciences Research Council)|
|Supervisor||Duncan Graham (Supervisor) & Karen Faulds (Supervisor)|