Development of DNA detection assays using functionalised nanoparticles

The ability to detect DNA relating to a specific disease is at the forefront of current research in molecular diagnostics. The detection techniques utilised for such work need to afford excellent selectivity as well as sensitivity. Current methods commonly involve the use of fluorescence; however there has been a heightened interest in the use of SERRS for these purposes in recent years due to the numerous advantages SERRS offers over fluorescence. Nanoparticles are often selected as the SERRS substrate of choice. These nanoparticles can undergo a number of functionalisation steps in order for them to either act as labels for detection or to detect specific target molecules, or perform both tasks simultaneously. This research details the preparation of a number of biofunctionalised SERRS active nanoparticle conjugates and their incorporation into a DNA detection assay. The assay is based on a split-probe principle, with one of the probes becoming attached to a SERRS active particle giving rise to a SERRS signal from the assay, whilst the other probe is tethered to a magnetic bead to allow immobilisation of the completed assay. The nanoparticle conjugates were functionalised with a Raman reporter and stabilised by a polyethylene glycol layer on the surface of the nanoparticles. A biomolecule of choice was then used to functionalise the nanoparticle, allowing it to either bind to an oligonucleotide probe or to the target DNA strand directly. After the assay protocol has been completed the samples were measured by SERRS. The assay systems developed were tested to assess their ability to selectively and sensitively detect DNA with a specific sequence. The assay protocol was optimised in order to improve these aspects of the assay performance with a view to making the assay more suitable for testing of genomic samples. A method was developed for the synthesis of gold-shelled magnetic nanoparticles for incorporation into the assay system. Iron oxide core nanoparticles were synthesised and coated with a metallic gold shell. The shelling protocol was optimised and a method for the assessment of the shelling protocol developed. Using analysis of SEM images, zeta potential measurements and SERRS, the gold-shelling of the magnetic nanoparticles was verified. A proof of concept assay demonstrated the suitability of the resultant magnetic nanoparticles for use in the DNA detection assay.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2013

Author dc:creator

• Clark, Natalie

DOI

10.48730/s446-g697

Identifier

T13492

OAI identifier oai:identifier

oai:strathclyde:ht24wj48w