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The development and application of a sequence-selective DNA sensor

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The development and application of a sequence-selective DNA sensor

Millan, Kelly M (1997) The development and application of a sequence-selective DNA sensor. PhD thesis, Concordia University.

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Abstract

DNA has been covalently immobilized onto glassy carbon electrodes and stearic acid modified carbon paste electrodes using water soluble carbodiimide and hydroxysuccinimide reagents. This promotes coupling between surface carboxylic acid groups and guanine bases. Preliminary results with model compounds suggest the coupling occurs at the N1 imino position of the guanine base. Octadecylamine modified carbon paste electrodes (ODACPEs) have been modified with DNA via a phosphoramidate bond between the 5$\sp\prime$ phosphate and the primary amine of octadecylamine. Immobilized DNA was detected voltammetrically with either Co(bpy)$\sb3\sp{3+},$ Co(phen)$\sb3\sp{3-},$ Os(bpy)$\sb3\sp{2+},$ three complexes which exhibit quasireversible one electron redox activity and associate selectively with double-stranded DNA, or with a daunomycin-glucose oxidase conjugate with a ferrocene mediator. Voltammetric peak currents obtained with a poly(dG)poly(dC)-modified GCE depend on Co(bpy)$\sb3\sp{3+}$ concentrations in a nonlinear fashion and indicate saturation binding with immobilized DNA. Ferrocene derivatives which do not bind selectively to dsDNA yield linear calibration curves (i$\sb{\rm p}$ vs concentration) at both DNA-modified and unmodified GCEs. Voltammetric peak currents for Co(phen)$\sb3\sp{3+}$ reduction were used to estimate the constant local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of $1.74\times 10\sp3$ M$\sp{-1}$ yield $8.6\pm 0.2$ mM base pairs. A prototype sequence-selective DNA sensor was developed by immobilizing a 20-mer oligo(dT)$\sb{20}$ following its enzymatic elongation with dG residues, which yielded the species oligo(dT)$\sb{20}$(dG)$\sb{99}.$ An increase in peak current was obtained after hybridization with target DNA. The single-stranded probe at the GCE surface is regenerated after rinsing the sensor in hot distilled water. These results demonstrate that this sensor can be used as a reusable sequence-selective biosensor for DNA. A comparison of batch and in situ hybridization rates of an immobilized poly(dT) demonstrated that hybridization occurred at a faster rate under high ionic strength in the absence of a hybridization indicator and yielded a detection limit of 100 ng/mL. This technique was applied to DNA sensors selective for the $\Delta$F508 deletion region of human DNA. These sensors were capable of selectively detecting their target sequences when hybridization occurred at 43$\sp\circ$

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Millan, Kelly M
Pagination:xxi, 233 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D
Program:Dept. of Chemistry and Biochemistry
Date:1997
Thesis Supervisor(s):Mikkelsen, Susa
ID Code:5579
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 17:01
Last Modified:08 Dec 2010 10:45
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