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Development of a Fe3O4 nano-particle modified silica
monolith in centrifugal spin column format for enrichment
of phosphorylated compounds.
Ali Alwya, Sarah P. Clarkeb, Dermot F. Broughamb, Brendan Twamleyc, Brett
Paulld, Blanaid Whitea,c, and Damian Connolly*a,e
aIrish
Separation Science Cluster, National Centre for Sensor Research, Dublin City
University, Glasnevin, Dublin 9, Ireland.
bNational Institute for Cellular Biotechnology, School of Chemical Sciences, Dublin
City University, Dublin 9, Ireland.
cSchool of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland.
dAustralian Centre for Research on Separation Science, School of Chemistry,
University of Tasmania, Hobart 7001, Tasmania, Australia.
ePharmaceutical and Molecular Biotechnology Research Centre, Department of
Chemical and Life Sciences, Waterford Institute of Technology, Waterford, Ireland.
Telephone: +353 (51) 845509, Email: dconnolly@wit.ie
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Electronic Supplementary Information
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Figure S1: Dynamic light scattering data for determination of size distribution of
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prepared iron oxide nano-particles.
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Figure S2: Digital photograph (looking down the sample well) of an aminated silica
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monolith after coating with citrate-stabilised iron oxide nano-particles.
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Figure S3: EDX spectrum of an aminated silica MonoSpin with immobilised iron
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oxide nano-particles (single layer).
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Figure S4: (a): Molecular structure of poly(diallyldimethylammonium) chloride. (b):
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Schematic representation of layer-by-layer assembly of negatively charged nano-
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particles using a positively charged linear polymer.
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Figure S5: EDX spectrum of an aminated silica MonoSpin with immobilised iron
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oxide nano-particles (layer-by-layer coating).
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Chromatographic method development and method validation for LC
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separation of selected nucleotides.
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In order to evaluate the selectivity of the nano-structured monolith for
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phosphorylated compounds, a range of nucleotides, namely adenosine, AMP,
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ADP and ATP were selected for study. It was anticipated that adenosine would
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display little or no selectivity for the iron oxide surface during a solid phase
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extraction protocol since it bears no phosphate groups. Therefore, a liquid
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chromatographic ion-pair assay was developed for the separation of
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adenosine, AMP, ADP and ATP using TBAP as ion-pair reagent and a Waters
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Symmetry C18 column. The optimised gradient separation of a 5 µM standard
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of adenosine and the three nucleotides is shown below (Fig. S6). The linear
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range of the method was 100 nM to 250 µM for all four analytes with R2 values
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≥ 0.9980. Concentrations of nucleotides at the low, mid-level and high end of
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the linear range were used for a precision study; namely 100 nM, 1 µM and 50
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µM. Six consecutive injections of each standard were made and %RSD was ≤
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0.4 % for retention time and ≤ 0.4 % for peak area at each concentration level
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(except at 100 nM, which was ≤ 3.9 %). Sensitivity (limit of detection, LOD) of
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the method was taken as the concentration of each analyte which gave a
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signal to noise ratio of 3, and determined to be 10 nM for each analyte. The
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limit of quantitation (LOQ) was 100 nM.
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Figure S6: Optimised separation of a 5 µM standard (a) of adenosine, AMP, ADP
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and ATP overlaid with a blank injection (b). Chromatographic conditions: Column:
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4.6 mm x 75 mm Waters Symmetry C18, 3.5 µm. Mobile phase A: 5 mM TBAP (pH
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7.0) in 5 % ACN. Mobile phase B: 5 mM TBAP (pH 7.0) in 80 % ACN. Gradient
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programme: 0-5 mins: 0% B, 5-15 mins: 0-50% B, 15-20 mins: 50 % B, 20.0-20.5
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mins: 50-0% B and 20.5-30 mins: 0 % B. Flow rate: 0.4 mL/min. Injection volume: 40
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µL. Column temperature: 40
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adenosine, (2): AMP, (3): ADP, (4): ATP.
oC.
Detection wavelength: 254 nm. Peaks: (1):
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Figure S7: (A): HPLC analysis of the water rinse fraction from a Fe3O4 nanoparticle
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modified MonoSpin (a) and a blank MonoSpin (b). (B): HPLC analysis of the eluted
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fraction phosphate buffer from a Fe3O4 nanoparticle modified MonoSpin (a) and a
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blank MonoSpin (b). Chromatographic conditions and peak assignments as in Figure
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S6.
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