Spelling suggestions: "subject:"DNA monolayer""
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Electrochemical in situ investigation of thiolate DNA monolayers on gold with fluorescence imagingMurphy, Jeffrey N. 11 1900 (has links)
DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods.
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Electrochemical in situ investigation of thiolate DNA monolayers on gold with fluorescence imagingMurphy, Jeffrey N. 11 1900 (has links)
DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods.
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Electrochemical in situ investigation of thiolate DNA monolayers on gold with fluorescence imagingMurphy, Jeffrey N. 11 1900 (has links)
DNA-modified surfaces have been widely studied for microarray and biosensor applications, in particular sequence-specific detection of DNA, for which electrochemical and optical signs can be produced. Variations in the organization and surface density of adsorbed DNA are known to affect the sensitivity and reliability of assays performed using such surfaces, however most measurements of such surfaces to date have little to no spatial resolution, limiting the information that can be gathered regarding the heterogeneity of the organization of adsorbed DNA molecules. We have applied in situ epi-fluorescence microscopic imaging in conjunction with electrochemical measurements to fluorescently labelled thiolate DNA, adsorbed on polycrystalline gold electrodes with a mercaptohexanol (MCH) passive layer. Spatially resolved information on the organization of adsorbed DNA on the surface is gathered within an area measuring 520by 730micrometres with a 0.96 micrometre resolution. The technique has enabled us to investigate "hotspots" (regions of anomalously bright fluorescence) and regional variation in fluorescence; since molecular fluorescence is quenched as a function of distance from the metal substrate, potential modulation with consequent DNA reorientation or layer specificity of the adsorption. Furthermore, an alternative means to the conventional preparation of thiolate-DNA / MCH monolayers has been developed. In this new method, a gold substrate passivated with MCH is subsequently immersed in an aqueous solution of 5'hexylthiol modified DNA. Through a ligand exchange process, DNA is immobilized forming a mixed MCH / DNA monolayer. Samples prepared via the new method display fewer hotspots and improved fluorescence switching of the DNA during electromodulation for samples made with single stranded (ss) DNA and with double stranded (ds) DNA. Measurement of the DNA surface concentration using ruthenium (III) hexaammine chloride with cyclic voltammetry for self assembled monolayers (SAMs) prepared via the new method are on the order of 1% of the maximum grafting density obtainable for both ssDNA and dsDNA by conventional methods. / Science, Faculty of / Chemistry, Department of / Graduate
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Localization of metal ions in DNADinsmore, Michael John 28 April 2008
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-bidi-font-weight:bold'>M-DNA is a novel complex formed between DNA
and transition metal ions under alkaline conditions.<span
style='mso-spacerun:yes'> </span>The unique properties of M-DNA were
manipulated in order to rationally place metal ions at specific regions within
a double-stranded DNA helix.<span style='mso-spacerun:yes'>
</span>Investigations using thermal denaturation profiles and the ethidium
fluorescence assay illustrate that the pH at which M-DNA formation occurs is
influenced heavily by the DNA sequence and base composition.<span
style='mso-spacerun:yes'> </span>For instance, DNA with a sequence consisting
of poly[d(TG)d(CA)] is completely converted to M-DNA at pH 7.9 while DNA consisting
entirely of poly[d(AT)] remains in the B-DNA conformation until a pH of 8.6 is
reached.<span style='mso-spacerun:yes'> </span>The pH at which M-DNA formation
occurs is further decreased by the incorporation of 4-thiothymine (s<sup>4</sup>T).<span
style='mso-spacerun:yes'> </span>DNA oligomers with a mixed sequence composed
of </span>half d(AT) and the other half d(TG)d(CA)<span style='mso-bidi-font-weight:
bold'> showed that only 50% of the DNA is able to incorporate Zn<sup>2+</sup>
ions at pH 7.9.<span style='mso-spacerun:yes'> </span>This suggests that only
regions corresponding to the tracts of <span class=GramE>d(</span>TG)d(CA) are
being transformed.<span style='mso-spacerun:yes'> </span><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-fareast-language:ZH-CN'>Duplex DNA monolayers were self-assembled on
gold through <span class=GramE>a</span> Au-S linkage and both B- and M-DNA
conformations were studied using X-ray photoelectron spectroscopy (XPS) in
order to better elucidate the location of the metal ions.<span
style='mso-spacerun:yes'> </span>The film thickness, density, elemental
composition and ratios for samples were analyzed and compared.<span
style='mso-spacerun:yes'> </span>The DNA surface coverage, calculated from
both XPS and electrochemical measurements, was <span class=GramE>approximately
1.2 x 10<sup>13 </sup>molecules/cm<sup>2</sup></span><sub> </sub>for
B-DNA.<span style='mso-spacerun:yes'> </span>All samples showed distinct peaks
for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA.<span
style='mso-spacerun:yes'> </span></span><span style='mso-bidi-font-weight:
bold'>On addition of Zn<sup>2+</sup> to form M-DNA the C 1s, P 2p and S 2p
showed only small changes </span><span style='mso-fareast-language:ZH-CN'>while
both the N 1s and O 1s spectra changed considerably.<span
style='mso-spacerun:yes'> </span>This result is consistent with Zn<sup>2+</sup>
interacting with oxygen on the phosphate backbone as well as replacing the
imino protons of thymine (T) and guanine (G) in M-DNA.<span
style='mso-spacerun:yes'> </span>Analysis of the Zn 2p spectra also
demonstrated that the concentration of Zn<sup>2+</sup> present under M-DNA
conditions is consistent with Zn<sup>2+</sup> binding to both the phosphate
backbone as well as replacing the imino protons of T or G in each base
pair.<span style='mso-spacerun:yes'> </span>After the M-DNA monolayer is
washed with a buffer containing only Na<sup>+</sup> the Zn<sup>2+</sup> bound
to the phosphate backbone is removed while the Zn<sup>2+</sup> bound internally
still remains. </span><span style='mso-bidi-font-weight:bold'>Variable angle x-ray
photoelectron spectroscopy (VAXPS) was also used to examine monolayers
consisting of mixed sequence oligomers.<span style='mso-spacerun:yes'>
</span>Preliminary results suggest that under M-DNA conditions, the zinc to
phosphate ratio changes relative to the position of the <span class=GramE>d(</span>TG)d(CA)
tract being at the top or bottom of the monolayer.<span
style='mso-spacerun:yes'> </span><span style='mso-spacerun:yes'> </span><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-bidi-font-weight:bold'>Electrochemistry was also used to investigate
the properties of M-DNA monolayers on gold and examine how the localization of
metal ions affects the resistance through the DNA monolayer.<span
style='mso-spacerun:yes'> </span>T</span>he effectiveness of using the IrCl<sub>6</sub><sup>2-/3-
</sup>redox couple to investigate DNA monolayers and the potential advantages
of this system over the standard Fe(CN)<sub>6</sub><sup>3-/4-</sup> redox
couple are demonstrated.<span style='mso-spacerun:yes'> </span>B-DNA
monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn<sup>2+</sup>
at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance
spectroscopy (EIS) and chronoamperometry (CA) with IrCl<sub>6</sub><sup>2-/3-</sup>.<span
style='mso-spacerun:yes'> </span><sup><span style='mso-spacerun:yes'> </span></sup>Compared
to B-DNA, M-DNA showed significant changes in CV, EIS and CA spectra.<span
style='mso-spacerun:yes'> </span>However, only small changes were observed
when the monolayers were incubated in Mg<sup>2+ </sup>at pH 8.6 or in Zn<sup>2+</sup>
at pH 6.0.<span style='mso-spacerun:yes'> </span>The heterogeneous
electron-transfer rate (<i style='mso-bidi-font-style:normal'>k</i><sub>ET</sub>)
between the redox probe and the surface of a bare gold electrode was determined
to be 5.7 x 10<sup>-3</sup> cm/s.<span style='mso-spacerun:yes'> </span>For a
B-DNA modified electrode, the <i style='mso-bidi-font-style:normal'>k</i><sub>ET</sub>
through the monolayer was too slow to be measured.<span
style='mso-spacerun:yes'> </span>However, under M-DNA conditions, a <i
style='mso-bidi-font-style:normal'>k</i><sub>ET</sub> of 1.5 x 10<sup>-3</sup>
cm/s was reached.<span style='mso-spacerun:yes'> </span>As well, the percent
change in resistance to charge transfer (R<sub>CT</sub>), measured by EIS, <span
class=GramE>was</span> used to illustrate the dependence of M-DNA formation on
pH.<span style='mso-spacerun:yes'> </span>This result is consistent with Zn<sup>2+</sup>
ions replacing the imino protons on thymine and guanine residues.<span
style='mso-spacerun:yes'> </span>Also, at low pH values, the percent change in
R<sub>CT</sub> seems to be greater for <span class=GramE><span
style='mso-bidi-font-weight:bold'>d(</span></span><span style='mso-bidi-font-weight:
bold'>TG)<sub>15</sub>d(CA)<sub>15</sub> compared to oligomers with mixed
d(AT) and d(TG)d(CA) tracts.<span style='mso-spacerun:yes'> </span></span>The
IrCl<sub>6</sub><sup>2-/3- </sup>redox couple was also effective in
differentiating between single-stranded and double-stranded DNA during
dehybridization and rehybridization experiments.<span
style='mso-spacerun:yes'> </span><span style='mso-bidi-font-weight:bold'><o:p></o:p></span></p>
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Localization of metal ions in DNADinsmore, Michael John 28 April 2008 (has links)
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-bidi-font-weight:bold'>M-DNA is a novel complex formed between DNA
and transition metal ions under alkaline conditions.<span
style='mso-spacerun:yes'> </span>The unique properties of M-DNA were
manipulated in order to rationally place metal ions at specific regions within
a double-stranded DNA helix.<span style='mso-spacerun:yes'>
</span>Investigations using thermal denaturation profiles and the ethidium
fluorescence assay illustrate that the pH at which M-DNA formation occurs is
influenced heavily by the DNA sequence and base composition.<span
style='mso-spacerun:yes'> </span>For instance, DNA with a sequence consisting
of poly[d(TG)d(CA)] is completely converted to M-DNA at pH 7.9 while DNA consisting
entirely of poly[d(AT)] remains in the B-DNA conformation until a pH of 8.6 is
reached.<span style='mso-spacerun:yes'> </span>The pH at which M-DNA formation
occurs is further decreased by the incorporation of 4-thiothymine (s<sup>4</sup>T).<span
style='mso-spacerun:yes'> </span>DNA oligomers with a mixed sequence composed
of </span>half d(AT) and the other half d(TG)d(CA)<span style='mso-bidi-font-weight:
bold'> showed that only 50% of the DNA is able to incorporate Zn<sup>2+</sup>
ions at pH 7.9.<span style='mso-spacerun:yes'> </span>This suggests that only
regions corresponding to the tracts of <span class=GramE>d(</span>TG)d(CA) are
being transformed.<span style='mso-spacerun:yes'> </span><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-fareast-language:ZH-CN'>Duplex DNA monolayers were self-assembled on
gold through <span class=GramE>a</span> Au-S linkage and both B- and M-DNA
conformations were studied using X-ray photoelectron spectroscopy (XPS) in
order to better elucidate the location of the metal ions.<span
style='mso-spacerun:yes'> </span>The film thickness, density, elemental
composition and ratios for samples were analyzed and compared.<span
style='mso-spacerun:yes'> </span>The DNA surface coverage, calculated from
both XPS and electrochemical measurements, was <span class=GramE>approximately
1.2 x 10<sup>13 </sup>molecules/cm<sup>2</sup></span><sub> </sub>for
B-DNA.<span style='mso-spacerun:yes'> </span>All samples showed distinct peaks
for C 1s, O 1s, N 1s, P 2p and S 2p as expected for a thiol-linked DNA.<span
style='mso-spacerun:yes'> </span></span><span style='mso-bidi-font-weight:
bold'>On addition of Zn<sup>2+</sup> to form M-DNA the C 1s, P 2p and S 2p
showed only small changes </span><span style='mso-fareast-language:ZH-CN'>while
both the N 1s and O 1s spectra changed considerably.<span
style='mso-spacerun:yes'> </span>This result is consistent with Zn<sup>2+</sup>
interacting with oxygen on the phosphate backbone as well as replacing the
imino protons of thymine (T) and guanine (G) in M-DNA.<span
style='mso-spacerun:yes'> </span>Analysis of the Zn 2p spectra also
demonstrated that the concentration of Zn<sup>2+</sup> present under M-DNA
conditions is consistent with Zn<sup>2+</sup> binding to both the phosphate
backbone as well as replacing the imino protons of T or G in each base
pair.<span style='mso-spacerun:yes'> </span>After the M-DNA monolayer is
washed with a buffer containing only Na<sup>+</sup> the Zn<sup>2+</sup> bound
to the phosphate backbone is removed while the Zn<sup>2+</sup> bound internally
still remains. </span><span style='mso-bidi-font-weight:bold'>Variable angle x-ray
photoelectron spectroscopy (VAXPS) was also used to examine monolayers
consisting of mixed sequence oligomers.<span style='mso-spacerun:yes'>
</span>Preliminary results suggest that under M-DNA conditions, the zinc to
phosphate ratio changes relative to the position of the <span class=GramE>d(</span>TG)d(CA)
tract being at the top or bottom of the monolayer.<span
style='mso-spacerun:yes'> </span><span style='mso-spacerun:yes'> </span><o:p></o:p></span></p>
<p class=MsoNormal style='text-align:justify;text-indent:.5in;line-height:150%'><span
style='mso-bidi-font-weight:bold'>Electrochemistry was also used to investigate
the properties of M-DNA monolayers on gold and examine how the localization of
metal ions affects the resistance through the DNA monolayer.<span
style='mso-spacerun:yes'> </span>T</span>he effectiveness of using the IrCl<sub>6</sub><sup>2-/3-
</sup>redox couple to investigate DNA monolayers and the potential advantages
of this system over the standard Fe(CN)<sub>6</sub><sup>3-/4-</sup> redox
couple are demonstrated.<span style='mso-spacerun:yes'> </span>B-DNA
monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn<sup>2+</sup>
at pH 8.6 and studied by cyclic voltammetry (CV), electrochemical impedance
spectroscopy (EIS) and chronoamperometry (CA) with IrCl<sub>6</sub><sup>2-/3-</sup>.<span
style='mso-spacerun:yes'> </span><sup><span style='mso-spacerun:yes'> </span></sup>Compared
to B-DNA, M-DNA showed significant changes in CV, EIS and CA spectra.<span
style='mso-spacerun:yes'> </span>However, only small changes were observed
when the monolayers were incubated in Mg<sup>2+ </sup>at pH 8.6 or in Zn<sup>2+</sup>
at pH 6.0.<span style='mso-spacerun:yes'> </span>The heterogeneous
electron-transfer rate (<i style='mso-bidi-font-style:normal'>k</i><sub>ET</sub>)
between the redox probe and the surface of a bare gold electrode was determined
to be 5.7 x 10<sup>-3</sup> cm/s.<span style='mso-spacerun:yes'> </span>For a
B-DNA modified electrode, the <i style='mso-bidi-font-style:normal'>k</i><sub>ET</sub>
through the monolayer was too slow to be measured.<span
style='mso-spacerun:yes'> </span>However, under M-DNA conditions, a <i
style='mso-bidi-font-style:normal'>k</i><sub>ET</sub> of 1.5 x 10<sup>-3</sup>
cm/s was reached.<span style='mso-spacerun:yes'> </span>As well, the percent
change in resistance to charge transfer (R<sub>CT</sub>), measured by EIS, <span
class=GramE>was</span> used to illustrate the dependence of M-DNA formation on
pH.<span style='mso-spacerun:yes'> </span>This result is consistent with Zn<sup>2+</sup>
ions replacing the imino protons on thymine and guanine residues.<span
style='mso-spacerun:yes'> </span>Also, at low pH values, the percent change in
R<sub>CT</sub> seems to be greater for <span class=GramE><span
style='mso-bidi-font-weight:bold'>d(</span></span><span style='mso-bidi-font-weight:
bold'>TG)<sub>15</sub>d(CA)<sub>15</sub> compared to oligomers with mixed
d(AT) and d(TG)d(CA) tracts.<span style='mso-spacerun:yes'> </span></span>The
IrCl<sub>6</sub><sup>2-/3- </sup>redox couple was also effective in
differentiating between single-stranded and double-stranded DNA during
dehybridization and rehybridization experiments.<span
style='mso-spacerun:yes'> </span><span style='mso-bidi-font-weight:bold'><o:p></o:p></span></p>
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