Thesis-2008-Kerr.pdf (3.73 MB)
Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling
thesis
posted on 2009-05-13, 15:29 authored by Samantha Louise KerrThe application of ICP-MS to the fields of proteomics and genomics has arisen in part due
to its ability to detect and quantify trace levels of S and P, which are major constituents in
proteins and nucleic acids respectively. The development of collision/reaction cell
technology and high resolution instruments has enabled these biologically important
elements to be measured and quantified at the pg - ng ml-1 level. Despite these advances,
the detection limits of P and S are still inferior compared to other elements.
Oligonucleotides containing biotin functionality were labelled with Au nano-particles
attached to a streptavidin protein to achieve site specific labelling, with 100% labelling
efficiency. Each nano-particle contained ~86 Au atoms, resulting in an 882 fold signal
enhancement for 24 base length oligonucleotides. However, this enhancement factor was
only observed when one oligonucleotide bound to one nano-particle in a 1:1 ratio. Much
lower Au labelling efficiencies and signal enhancements were observed when thiolated
oligonucleotides were labelled with maleimide functionalised gold nano-particles. This was
attributed to the extensive and difficult sample preparation steps that were required prior to
labelling.
The detection and quantification of adducts formed between DNA and the Pt anti-cancer
drugs cisplatin and oxaliplatin were also investigated with ICP-MS. Acid digestion of the
carbon based DNA matrix enabled Pt adducts to be quantified at low dose rates of 1 Pt
atom per 1 500 000 nucleotides in ~12 μg DNA. Such sensitive mass spectrometric
determinations could be employed in clinical tests to detect and quantify low level adducts
formed in patients in-vivo. To complement ICP-MS analysis, electrospray ionisation linear
ion trap mass spectrometry was employed to study the interaction of oxaliplatin with the
four DNA nucleobases. Multiple stage mass spectrometry enabled detailed Pt-nucleobase
adduct fragmentation pathways to be established.
The method of DNA detection using P in conjunction with the collision cell, or cool plasma
to form PO+ was also demonstrated and the limitations of the method, namely, polyatomic
interferences and severe matrix effects were highlighted.
History
School
- Science
Department
- Chemistry
Publisher
© Samantha Louise KerrPublication date
2008Notes
A Doctoral Thesis submitted in partial fulfilment of the requirements for the award of degree of Doctor of Philosophy of Loughborough University.EThOS Persistent ID
uk.bl.ethos.515599Language
- en