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Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling
The 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...
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2008
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| Online Access: | https://hdl.handle.net/2134/4641 |
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| _version_ | 1818176140500533248 |
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| author | Samantha Louise Kerr |
| author_facet | Samantha Louise Kerr |
| author_sort | Samantha Louise Kerr (7165235) |
| collection | Figshare |
| description | The 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. |
| format | Default Thesis |
| id | rr-article-9395957 |
| institution | Loughborough University |
| publishDate | 2008 |
| record_format | Figshare |
| spelling | rr-article-93959572008-01-01T00:00:00Z Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling Samantha Louise Kerr (7165235) Other chemical sciences not elsewhere classified Oligonucleotides DNA Phosphorus Gold Nano-particles Labelling ICPMS Collision/reaction cell HPLC-ICP-MS ESI-MS Platinum Metallodrugs Cisplatin Oxaliplatin Chemical Sciences not elsewhere classified The 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. 2008-01-01T00:00:00Z Text Thesis 2134/4641 https://figshare.com/articles/thesis/Enhancing_nucleic_acid_detection_using_inductively_coupled_plasma_mass_spectrometry_by_means_of_metal_and_nano-particle_labelling/9395957 CC BY-NC-ND 4.0 |
| spellingShingle | Other chemical sciences not elsewhere classified Oligonucleotides DNA Phosphorus Gold Nano-particles Labelling ICPMS Collision/reaction cell HPLC-ICP-MS ESI-MS Platinum Metallodrugs Cisplatin Oxaliplatin Chemical Sciences not elsewhere classified Samantha Louise Kerr Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| title | Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| title_full | Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| title_fullStr | Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| title_full_unstemmed | Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| title_short | Enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| title_sort | enhancing nucleic acid detection using inductively coupled plasma mass spectrometry, by means of metal and nano-particle labelling |
| topic | Other chemical sciences not elsewhere classified Oligonucleotides DNA Phosphorus Gold Nano-particles Labelling ICPMS Collision/reaction cell HPLC-ICP-MS ESI-MS Platinum Metallodrugs Cisplatin Oxaliplatin Chemical Sciences not elsewhere classified |
| url | https://hdl.handle.net/2134/4641 |