A label-free, fluorescence based assay for microarray

dc.contributor.authorNiu, Sanjunen
dc.contributor.committeechairSaraf, Ravi F.en
dc.contributor.committeememberOyama, Shigeo Teden
dc.contributor.committeememberCox, David F.en
dc.contributor.committeememberMarand, Evaen
dc.contributor.committeememberEnglish, Samen
dc.contributor.departmentChemical Engineeringen
dc.date.accessioned2011-08-22T19:03:47Zen
dc.date.adate2004-08-23en
dc.date.available2011-08-22T19:03:47Zen
dc.date.issued2004-08-12en
dc.date.rdate2004-08-23en
dc.date.sdate2004-08-12en
dc.description.abstractDNA chip technology has drawn tremendous attention since it emerged in the mid 90 s as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges.. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same intensity of excitation light.. The fluorescence contrast is used to quantify the amount of probe-target hybridization. A mathematical model that considers multiple reflections and scattering is developed to explain the mechanism of the fluorescence contrast which depends on the thickness of the PS film. Scattering is the dominant factor that contributes to the contrast. The potential of this assay to detect single nucleotide polymorphism is also tested.en
dc.description.degreePh. D.en
dc.format.mediumETDen
dc.identifier.otheretd-08122004-113221en
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-08122004-113221en
dc.identifier.urihttp://hdl.handle.net/10919/11229en
dc.publisherVirginia Techen
dc.relation.haspartdissertation_final.pdfen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectSNPsen
dc.subjectfluorescenceen
dc.subjectlabel-freeen
dc.subjectmicroarrayen
dc.subjectDNA chipen
dc.subjectpolystyreneen
dc.titleA label-free, fluorescence based assay for microarrayen
dc.typeDissertationen
thesis.degree.disciplineChemical Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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