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Development and Characterization of Aptamers for the use in Surface Plasmon Resonance Sensors for the Detection of Glycated Blood Proteins

Reaver, Nathan George Frederick
http://rave.ohiolink.edu/etdc/view?acc_num=toledo1373319138

Abstract Details

2013, Master of Science in Bioengineering, University of Toledo, Bioengineering.
The concentration ratio of glycated to non-glycated forms of various blood proteins can be used as a diagnostic measure in diabetes to determine a history of glycemic compliance. Depending on a protein's half-life in blood, compliance can be assessed from a few days to several months in the past, which can be used to provide additional therapeutic guidance. The most commonly studied glycated protein for assessing glycemic compliance is glycated hemoglobin. Current glycated protein concentration detection methods are limited in their ability to measure multiple proteins, and are susceptible to interference from other blood pathologies. In this study, DNA aptamers were developed and characterized for use in Surface Plasmon Resonance (SPR) sensors to assess the percentage of hemoglobin that is glycated in a patient. The aptamers were developed by way of a modified Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process which selects DNA sequences that have a high binding affinity to a specific protein. DNA products resulting from this process were sequenced, and identified aptamers were synthesized. The SELEX process was performed multiple times. One process produced aptamers specific to a glycated form of hemoglobin, another produced aptamers specific to non-glycated hemoglobin and aptamers specific to all forms of hemoglobin. A final SELEX process produced aptamers specific to fibrinogen. Equilibrium dissociation constants for the affinity of the identified aptamers to glycated hemoglobin, hemoglobin, and fibrinogen were calculated from fitting a Langmuir binding model to experimental, real time, binding data obtained through SPR. It was determined that two aptamers, GHA1 and GHA2, were selective to glycated hemoglobin, with equilibrium dissociation constants of 11.5 nM and 51.8 nM, respectively; one aptamer, HA2, was selective to non-glycated hemoglobin, with an equilibrium dissociation constant of 187 nM; and one aptamer was selective to both glycated and non-glycated hemoglobin, with equilibrium dissociation constants for the two proteins of 36.9 nM and 35.2 nM, respectively. It was also demonstrated how these aptamers could be used in an SPR sensor. The aptamers identified in this study can potentially be used in a SPR aptamer-based biosensor for detection of glycated hemoglobin, a technology that has the potential to deliver low-cost and immediate glycemic compliance assessment in either a clinical or home setting.
Brent Cameron, PhD (Advisor)
Patricia Relue, PhD (Committee Member)
Ronald Fournier, PhD (Committee Member)
252 p.
Biochemistry; Biomedical Engineering; Biomedical Research; Chemistry; Engineering; Health Care; Health Sciences; Medicine; Molecular Chemistry; Optics; Organic Chemistry; Technology
aptamer; aptamers; surface plasmon resonance; SPR; diabetes mellitus; glycated protein; biosensor; hemoglobin; glycated hemoglobin; SELEX; Systematic Evolution of Ligands by Exponential Enrichment; fibrinogen; non-enzymatic glycosylation; plasmonics

Recommended Citations

Citations

  • Reaver, N. G. F. (2013). Development and Characterization of Aptamers for the use in Surface Plasmon Resonance Sensors for the Detection of Glycated Blood Proteins [Master's thesis, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1373319138

    APA Style (7th edition)

  • Reaver, Nathan. Development and Characterization of Aptamers for the use in Surface Plasmon Resonance Sensors for the Detection of Glycated Blood Proteins. 2013. University of Toledo, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=toledo1373319138.

    MLA Style (8th edition)

  • Reaver, Nathan. "Development and Characterization of Aptamers for the use in Surface Plasmon Resonance Sensors for the Detection of Glycated Blood Proteins." Master's thesis, University of Toledo, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1373319138

    Chicago Manual of Style (17th edition)

toledo1373319138
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© 2013, all rights reserved.
This open access ETD is published by University of Toledo and OhioLINK.