Date of Award

12-2006

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biological Engineering

Advisor

Paul J. Millard

Second Committee Member

Mauricio Pereira da Cunha

Third Committee Member

David J. Neivandt

Abstract

There is a growing need for portable biosensors that may be reused repeatedly by gentle means of removal and regeneration of biomolecular sensing layers. Novel methods for regenerating biosensors have been developed in which the highly specific detection of nucleic acid sequences is carried out using molecular padlock probe (MPP) technology and surface-associated rolling circle amplification (RCA). This technique has a low occurrence of false positive results when compared to polymerase chain reaction, and is an isothermal reaction, which is advantageous in systems requiring low power consumption such as remote field sensing applications. Unique base sequences derived from RNA of both infectious hematopoietic necrosis virus (IHNV) and infectious salmon anemia virus (1SAV) were detected and identified using MPP in combination with surface-associated RCA on both gold and glass surfaces. Gold-sputtered 96-well polystyrene microplates and a fluorescent label were used to explore the detection limits of the surface-associated RCA technique, specificity for different MPP, conditions for regeneration of the biomolecular sensing surface, and reproducibility of measurements on regenerated surfaces. The technique was used to create highly selective biomolecular surfaces capable of discriminating between DNA oligonucleotides with sequences identical to RNA from 1SAV and IHNV. As little as 0.6 femtomole of circularized MPP was detectable with this fluorimetric assay. The sensing layers could be reused for at least four cycles of amplification using thermal denaturation, with less than 33% decrease in RCA response over time. Because the nucleic acid product of the test is attached to a surface during amplification, the technique is directly applicable to a variety of existing sensing platforms, including acoustic wave and optical devices. Methods of regeneration of biomolecular sensing layers without heating were investigated, using the noncovalent binding of NeutrAvidin™ to surfaces derivatized with desthiobiotin (DSB). Release of NeutrAvidin from the surface upon introduction of D-biotin, for which NeutrAvidin has 20-fold higher affinity, was dependent on the concentration of D-biotin, and the incubation time. Optimal release of NeutrAvidin (~98%) was achieved with >20mM D-biotin in aqueous solution incubated for 1 hr at 37°C, and ~3 hrs at room temperature. RCA products were observed to bind nonspecifically to surfaces, necessitating an additional elevated temperature rinse with >90°C buffer containing polyoxyethylenesorbitan monolaurate (Tween-20) for release. Circularized MPP formed to ISAV or IHNV were also captured selectively on the inner surfaces of glass microcapillary tubes by immobilized DNA oligonucleotide primers. Extension of the immobilized primers by isothermal RCA was measured using a fluorescent label and microfluorimetry. The measurement of RCA products within microcapillary tubes is a step toward miniaturized, arrayable sensors for the multiplex detection of bacterial, viral, and protozoan pathogens.

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