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Title page for ETD etd-03072016-162510

Type of Document Master's Thesis
Author Kammer, Michael Nolan
Author's Email Address michael.n.kammer@vanderbilt.edu
URN etd-03072016-162510
Title Characterization of aptamer-small molecule interactions with backscattering interferometry
Degree Master of Science
Department Biomedical Engineering
Advisory Committee
Advisor Name Title
Frederick Haselton Committee Chair
Darryl Bornhop Committee Member
  • Interferometry
  • Aptamers
Date of Defense 2016-03-04
Availability unrestricted
Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, and therefore produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule – aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need to use a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM – aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofover, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R2 values > 0.95 and excellent signal to noise at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.
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