Application of silicon photonic microring resonators to the study of surface bioconjugation and protein capture agent binding kinetics

aniline-catalyzed hydrazone ligation;thrombin–binding aptamers;binding kinetics;ricin;single domain antibodies

Byeon, Ji-Yeon

关键词: aniline-catalyzed hydrazone ligation;    thrombin–binding aptamers;    binding kinetics;    ricin;    single domain antibodies;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/24088/Byeon_Ji-Yeon.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The completion of the Human Genome Project has led us to the next frontier of proteomics-understanding functions and structures of the proteome. With an estimated one million functionally distinctive proteins, a means for high-throughput analysis, such as protein microarray, is required for the identification and quantification of proteins from biological samples. Even though protein microarrays have been shown to be a powerful technique for proteomics, there are still many challenges involved in building a protein array chip. Some of the challenges include finding good protein capture agents with high affinity and specificity, choosing an appropriate surface chemistry to immobilize proteins of interest efficiently, employing sensitive, fast, and reproducible detection methods, and reducing the amount of non-specific protein-sensor interactions. To address some of these issues, we employ silicon photonic microring resonators, an emerging surface-sensitive analysis method that our group has recently utilized for monitoring biomolecular binding and detection in an array-based, label-free biosensor format.First, aniline-catalyzed hydrazone ligation between surface immobilized hydrazines and aldehyde-modified antibodies is shown to be an efficient method to functionalize model oxide-coated sensor substrates with protein capture agents, allowing derivatization of substrates with reduced incubation time and sample consumption. The efficiency of this surface bioconjugation at various pHs and in the presence or absence of catalytic aniline was directly evaluated using silicon photonic microring resonators. It is found that aniline significantly increases the net antibody loading for surfaces functionalized over a pH range from 4.5 to 7.4, resulting in more sensitive antigen detection when functionalized microrings are utilized in a label-free immunoassay. Furthermore, these experiments also reveal an interesting pH-dependent non-specific binding trend of antibody that plays an important role in dictating the amount of antibody attached onto the substrate, highlighting the competing contributions of the bioconjugate reaction rate and the dynamic interactions that control opportunities for a solution-phase biomolecule to react with a substrate-bound reagent.Some of new emerging capture agents, aptamers and single domain antibodies, are also investigated using silicon photonic microring resonators. Binding kinetics of thrombin–binding DNA aptamers and a conventional monoclonal antibody against the same target antigen are simulatenously evaluated in a multiplexed manner. It is found that the thrombin-binding aptamer has a slightly higher affinity constant (KD) than its antibody counterpart. However, we suggest that, despite having a ―worse‖ affinity, the thrombin-binding aptamer can be used to quantitate the target antigen with a lower limit of detection due to its faster association rate. In a similar way, kinetic association and dissociation constants of two single domain antibodies targeting ricin toxin are evaluated simultaneously. In this preliminary study, single domain antibodies are found to be much more highly specific to ricin as compared to the antibody against the same target. Optimization of experimental conditions is still needed to obtain more precise kinetic values of ricin capture agents; however, more importantly, we envision that the ability to monitor in parallel the binding kinetics of multiple biomolecular interactions on a scalable and cost-effective platform will be of great utility in evaluating protein binding agents for a range of biomedical applications.Lastly, perfluoropolymer surfaces‘ wetting properties are controlled using nanostructuring of the surface for applications in creating non-fouling surfaces. The wetting properties of the perfluoropolymer, CYTOP, can be easily controlled by changing a few key parameters of the reactive ion etching process. With SF6 plasma, even superhydrophobic (contact angle higher than 150°) surfaces can be obtained. The Wenzel model and the measured contact angles are in good agreement when the surface roughness factors are relatively small. Unlike static contact angles, however, contact angle hysteresis is not improved and furthermore, protein non-specific adsorption experiments show that plasma treating CYTOP does not significantly improve the material‘s non-fouling properties.