UC Merced

Isothermal titration calorimetry (ITC) is a powerful tool for investigating interactions between biomolecules – which are especially pertinent to many biosensing platforms. Interactions between nucleic acid aptamers and protein targets are of particular interest as aptamers offer a versatile alternative to traditionally used molecular recognition elements. ITC has been used to characterize interactions between protein targets and aptamer molecular receptors for a better understanding of the target binding process.

ITC experiments often consist of interacting biomolecules in dilute solutions. However, many aptamer-based applications require immobilization to a solid support. Interactions between neighboring immobilized aptamers change the local environment around each aptamer. Furthermore, as the solid support surface becomes increasingly crowded, aptamer / target binding is likely to deviate from what is known about binding in a dilute solution. Several barriers exist that complicate incorporating aptamer functionalized gold nanoparticles into the ITC platform. Barriers stemming from low signals produced from interacting biomolecules require unique resolutions. Overcoming these barriers is necessary for accurate characterization of binding events.

This dissertation details how ITC was enabled for investigating target binding between immobilized aptamers on gold nanoparticle surfaces and target proteins – a novel approach for characterizing target binding of immobilized aptamer-based systems. By precisely accounting for reaction stoichiometry, matching titrant and titrate buffers, and countering non-specific adsorption, we demonstrate that ITC can be used to characterize systems that feature immobilized aptamers on colloidal gold nanoparticles and protein targets – unveiling ITC as an additional means for guiding biosensor design. Additionally, an investigation into how aptamer loading density affects target binding reveals an improvement to binding affinity as loading density increases. ITC analysis reveals that entropic compensation stemming from prepaid entropic penalties from aptamer immobilization explains the counterintuitive trend of binding affinity improvements as loading density increases. Of important note, at sufficiently high loading densities, a precipitous decline of target binding is observed, likely from increased steric hindrance from neighboring aptamers. Limitations of ITC on aptamer functionalized gold nanoparticle systems are discussed as well as future directions for using ITC to better understand target binding in crowded environments.